WO2025217543A1 - Virus adéno-associé thérapeutique utilisant un acide nucléique à codon optimisé codant lamp2b - Google Patents

Virus adéno-associé thérapeutique utilisant un acide nucléique à codon optimisé codant lamp2b

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Publication number
WO2025217543A1
WO2025217543A1 PCT/US2025/024314 US2025024314W WO2025217543A1 WO 2025217543 A1 WO2025217543 A1 WO 2025217543A1 US 2025024314 W US2025024314 W US 2025024314W WO 2025217543 A1 WO2025217543 A1 WO 2025217543A1
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sequence
nucleic acid
seq
lamp2b
vector
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Scott Hammond
Anna Tretiakova
Ferzin SETHNA
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AskBio Inc
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AskBio Inc
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the present invention relates to methods to treat Danon disease by administering adeno- associated virus (AAV) particles, virions and vectors for expression of a LAMP2B polypeptide, where the nucleic acid encoding LAMP2B is codon optimized.
  • AAV adeno- associated virus
  • virions virions
  • vectors for expression of a LAMP2B polypeptide, where the nucleic acid encoding LAMP2B is codon optimized.
  • BACKGROUND Danon disease is an X-linked dominant disease caused by mutations in the lysosome- associated membrane 2 (LAMP2) gene and is characterized by the clinical triad of cardiomyopathy, skeletal myopathy and mask disability (D’Souza R. et al (2023); Brambati M et al (2019)).
  • LAMP2 codes for three LAMP2 protein isoforms – LAMP2A, LAMP2B and LAMP2C. While most LAMP2 mutations affect al 3 isoforms, isoform-specific mutations in Danon have been reported only for LAMP2B which is expressed in the heart, skeletal tissue and the brain indicating that LAMP 2B deficiency is central to the pathogenesis of Danon (D’Souza R et al (2014). Within celular systems, LAMP2 is required for the fusion of autophagosomes and lysosomes – a crucial step for the degradation of celular waste (D’Souza R et al (2014) 4. Alcali R. et al (2021). Endo Y et al (2015)).
  • nucleic acid encoding a LAMP2B polypeptide
  • the nucleic acid comprises the nucleic segememt (e.g., a nucleic sequence) set forth in any one of SEQ ID NOs 1-3, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NO: 1, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NO: 2, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NO: 3, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the encoded polypeptide is a LAMP2B polypeptide or a functional variant of the LAMP2B polypeptide having the amino acid sequence shown in SEQ ID NO: 9, or at least 60%, or 70%, or 80%, 85% or 90% or 95%, or 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 9.
  • the codon-optimized nucleic acid is comprised within a nucleic acid construct that further comprises viral or non viral sequence elements that facilitate integration and/or expression.
  • described herein is an expression cassete containing a codon-optimized nucleic acid as described herein, operably linked to a muscle-specific promoter.
  • the muscle-specific promoter targets skeletal and cardiac muscle.
  • the muscle-specific promoter targets cardiac muscle.
  • the muscle-specific promoter targets skeletal muscle.
  • the muscle-specific promoter comprises a nucleic acid sequence selected from the group consisting of SP0497, SP0498, SP0499, SP0500, SP0508, SP0509, SP0510, SP0511, SP0512, SP0513, SP0522, SP0524, Syn100, and Spc5- 12 and a nucleic acid having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto. Aty. Dkt.
  • the expression cassete further comprises one or more additional regulatory elements and/or a poly A sequence.
  • the one or more additional regulatory elements is selected from the group consisting of an enhancer, a 5’ untranslated region (5’UTR), an intron, a reverse RNA pol I terminator sequence, and combinations thereof.
  • the intron is an IVS intron.
  • the intron is an IVS intron comprising a nucleic acid sequence of SEQ ID NO: 7, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • rAAV recombinant adeno-associated virus
  • An expression cassete of the invention comprises codon-optimized nucleic acid encoding a LAMP2B polypeptide, wherein the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NOs 1-3, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the expression cassete of the invention further comprises a muscle specific promoter that is operably linked to codon optimized nucleic acids encoding LAMP2B polypeptide.
  • the mucle specific promoter of the invention comprises a nucleic acid sequence selected from the group consisting of SP0497, SP0498, SP0499, SP0500, SP0508, SP0509, SP0510, SP0511, SP0512, SP0513, SP0522, SP0524, Syn100, and Spc5-12.
  • the mucle specific promoter of the invention comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the promoters selected from the group consisting of SP0497, SP0498, SP0499, SP0500, SP0508, SP0509, SP0510, SP0511, SP0512, SP0513, SP0522, SP0524, Syn100, and Spc5-12.
  • the expression constructs of the invention further comprises regulatory elements such as intron sequences, UTR sequences and additionaly comprises poly A sequences.
  • a recombinant adeno-associated virus (rAAV) vector comprising in its genome: (a) 5’ and 3’ AAV inverted terminal repeats (ITR) sequences; and (b) located between the 5’ and 3’ ITRs, the expression cassete specified as described herein.
  • the AAV genome further comprises at least one of: (a) a 5’ ITR; (b) an intron; (c) a poly A sequence; and (d) a 3’ ITR.
  • the AAV genome comprises, in the 5’ to 3’ direction: (a) a 5’ ITR; (b) a muscle-specific promoter; (c) an intron; (d) a codon- optimized nucleic acid as described herein; (e) a poly A sequence; (f) a 3’ ITR.
  • the intron is selected from the group consisting of an IVS sequence, a MVM sequence, a HBB2 sequence, an CMVIE intron sequence, a UBC intron sequence, and a SV40 sequence.
  • At least one of the 5’ ITR or Aty. Dkt. No.046192-000118WOPT 3’ITR comprises an insertion, deletion or substitution.
  • one or more CpG islands in the ITR are removed.
  • the poly A sequence is a ful length SV40 polyA sequence or HGF poly A sequence.
  • poly A sequence is selected from SEQ ID NO: 8, or a nucleic acid sequence having at least 80% sequence identity thereto.
  • the rAAV vector is a chimeric AAV vector, haploid AAV vector, a hybrid AAV vector or a rational polyploid AAV vector.
  • the rAAV vector comprises a rational haploid capsid, a mosaic AAV capsid, a chemicaly modified AAV capsid, or a AAV capsid from any AAV serotypes known in the art.
  • the rAAV capsid is a capsid from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9- PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6i, AAV1P5i, AAV4A1i, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2i, AAVS10P1i, AAV JEA,
  • the rAAV capsid is not AAV9.
  • described herein is a pharmaceutical composition comprising an rAAV vector as described herein in a pharmaceuticaly acceptable carrier.
  • described herein is a method for treating a subject in need of LAMP2B, the method comprising administering rAAV vectors as described herein or a pharmaceutical composition as described herein, or an expression cassete as described herein or a codon-optimized nucleic acid as described herein, to the subject.
  • described herein is a method for treating danon disease, the method comprising administering rAAV vectors as described herein or a pharmaceutical composition as described herein, or an expression cassete as described herein or a codon-optimized nucleic acid as described herein, to the subject.
  • administering to the subject is by systemic administration.
  • systemic administration is by intravenous administration.
  • administering to the subject is by local administration. Aty. Dkt.
  • the local administration is by injection to the heart or by intracoronary administration.
  • the rAAV vector is administered at a dosage range of between 1.0E10 vg to 5.0E14vg.
  • described herein is a use of a rAAV vector in the preparation of a medicament for treating subject in need of LAMP2B, the medicament comprising an rAAV vector as described herein.
  • described herein is a use of a rAAV vector in the preparation of a medicament for treating danon disease, the medicament comprising an rAAV vector as described herein.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 1, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 4, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 1, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 1, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 6, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 2, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 4, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 2, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 2, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 6, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 3, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 4, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 3, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • described herein is an expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 3, operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 6, or a sequence having at least 80% sequence identity thereto.
  • rAAV recombinant adeno-associated virus
  • No.046192-000118WOPT comprising in its genome an expression cassete as described herein.
  • described herein is an expression cassete comprising spc5-12 promoter sequence, wherein the expression cassete is used to treat Danon disease.
  • described herein is an expression cassete comprising SP0524 promoter sequence, wherein the expression cassete is used to treat Danon disease.
  • described herein is an expression cassete comprising any of the nucleic acid sequences selected from the group consisting of SEQ ID NO:s 60, 61, 63, 79, 81 and 82, or a sequence having at least 80% sequence identity thereto.
  • the expression cassete further comprises nucleic acid sequence as set forth by SEQ ID NO: 1-3, each of which encode a LAMP2B polypeptide sequence.
  • the expression cassete further comprises an intron sequence, wherein the intron is selected from the group consisting of an IVS intron sequence, a MVM sequence, a HBB2 sequence, an CMVIE intron sequence, a UBC intron sequence, and a SV40 sequence.
  • the expression cassete further comprises a poly A sequence.
  • the expression cassete is delivered to a subject in need thereof by using a non-viral vector.
  • the expression cassete is delivered to a subject in need thereof by using a viral vector.
  • the viral vector is a recombinant adeno associated virus (rAAV) vector.
  • the viral vector is an AAV2i8 vector.
  • transgenic mouse that does not produce Lamp2 polyeptide or functional form thereof, e.g., Lamp2B polypeptide or functional form thereof, the transgenic mouse’s cels comprising deletions of exons 2-6 of the Lamp2 gene, and a premature stop codon upstream of exon 7 of the Lamp2 gene.
  • the premature stop codon results in an out-of- frame shift of exons 7-9 of the Lamp2 gene.
  • the premature stop codon results in nonsense-mediated mRNA decay of the Lamp2 gene.
  • the transgenic mouse exhibits a complete loss of the Lamp2 gene or protein expression.
  • the transgenic mouse can serve as a mouse model to assess diseases or disorders resulting fom loss of Lamp2, e.g., a Danon disease transgenic mouse model.
  • Another aspect provided herein describes a method of making a transgenic mouse with having no expression of Lamp2, for example, a method of making transgenic mouse having a genome that Aty. Dkt.
  • No.046192-000118WOPT cannot express Lamp2 gene or a functional form thereof, the method comprising contacting a mouse embryo or a plurality of mouse embryonic cels or a mouse embryonic cel with at least one sgRNAsthat target exons 1 and 6 of the Lamp2 gene, and a Cas nuclease.
  • the transgenic mice disclosed herein does not produce Lamp2B polypeptide or a functional form thereof.
  • the contacting is performed by electroporation or by via a viral vector or non-viral vector.
  • the viral vector of any of the aspect described herein of the invention is a cardiotropic AAV vector.
  • the viral vector of any of the aspect described herein is a AAV2i8 vector.
  • the AAV2i8 vector can be used to encapsidate any Lamp2B nucleic acid that is discussed in the invention or is known in the art.
  • the mouse embryo is a C57BL/6J mouse embryo.
  • the at least one sgRNA is selected from TAGTCGATCCTTGATGCGGA (SEQ ID NO: 119) and AACAGTGGTAGGTGTATGCG (SEQ ID NO: 120).
  • the contacted mouse embryos are implanted in pseudopregnant recipient female mice.
  • FIGs 1A and 1B shows intron designed and selection.
  • FIG.1A shows schematic and table describing principal structural components of the exemplary construct of the invention .
  • FIG 1B show optimal designed and selected intron to drive high expression of transgene protein without other non- self-proteins.
  • SV40 poly A was used as a strong bi-directional terminator to prevent 3’ ⁇ 5’ transcript and double-stranded RNA formation.
  • FIG.2 show schematic of assay to identify vector of interest. Twenty-five constructs with diferent combinations of elements were designed and packaged into AAV 2i8, see, e.g., SEQ ID NOs 59-82.
  • FIG.3 shows LAMP2B protein expression normalized to total protein in heart. Bar graph shows signal intensity of indicated construct. LAMP2B protein expression is high in some constructs (Syn1002prob, Syn1007pref, Syn1007prob, SP524 cDNA) and low to medium in others including control constructs (Syn100 RS5 and RS10).
  • the terms ‘prob’, and ‘pref’ indicate codon optimized LAMP2B coding sequences, also referred to as coding sequence variants.
  • cDNA or LAMP2B cDNA refered to as wild type LAMP2B coding sequence. Asterisks indicate selected constructs.
  • FIG.4 shows recovered genome copies/ug DNA from heart for indicated construct. Asterisks indicate selected constructs. Blue bars indicate control coding sequence in the context of Syn100 promoter (Syn100 RS5) and control ITR-ITR construct (RS10). Recovered vector genomes are similar among al the groups indicating uniform transduction of the administered vector.
  • FIG.5 shows RNA expression in heart measured as copies/ug RNA for indicated construct. Asterisks indicate selected constructs. Blue bars indicate control coding sequence in the context of Syn100 promoter (Syn100 RS5) and control ITR-ITR construct (RS10). RNA expression is mostly uniform among the different groups. Notably some constructs (example: Syn100 immunostim dep) have high RNA expression but low protein expression.
  • FIGs 6A and 6B show constructs used in up-selection and study design. FIG.6A show 7 constructs were chosen for up-selection studies. See, e.g., methods for study protocols (Studies 6-10).
  • FIG.6B show the control A vector with control ITR-ITR sequence packaged in AAV9 as wel as AAV2i8. Each vector was manufactured at 2 scales to test for vector lot reproducibility and mice were injected at 2 doses to validate dose dependent protein expression. LAMP2B protein expression and vector genome copies were measured from al 4 chambers of the heart. RNA expression was measured from both ventricles due to insuficient tissue availability from atria.
  • FIGs 7A-7D show LAMP2B protein expression normalized to total protein in heart from vectors manufactured at two liter (2L) scale.
  • FIGs 8A-8D show recovered genome copies/ug DNA in heart from vectors manufactured at 2L scale. Expression is shown in the left ventricle (FIG.8A), left atrium (FIG.8B), right ventricle (FIG.8C), or right atrium (FIG.8D). Dose dependent increase in vector genomes is observed in al groups across the different chambers of the heart.
  • FIGs 9A and 9B show RNA expression in heart measured as copies/ug RNA from vectors manufactured at 2L scale. Expression is shown in the left ventricle (FIG.9A) and right ventricle (FIG.9B) Dose dependent increase in RNA is observed in al groups across the diferent chambers of the heart. Most down-selected constructs have similar RNA expression.
  • FIGs 10A-10D show LAMP2B protein expression normalized to total protein in heart from vectors manufactured at 20L scale. Expression is shown in the left ventricle (FIG.10A), left atrium (FIG.10B), right ventricle (FIG.10C), or right atrium (FIG.10D).
  • FIG.11 shows tissue biodistribution of LAMP2B protein from top selected constructs and control construct. Highest LAPM2B protein expression is observed in the heart, with lower expression in skeletal muscles and very low expression in other organs. Top selected constructs (DA004, DA022 and DA023) display similar tissue biodistribution. Aty. Dkt. No.046192-000118WOPT [0085] FIG.12 shows schematic of ribosome footprint profiling of top selected constructs to identify potentialy translated ARF peptides. Peaks indicates regions along the RNA where the ribosome stals as a proxy indicator of translation initiation.
  • FIG.13 presents a micrograph showing intracellular localization of endogenous mouse LAMP2 protein and LAMP2B protein expressed from constructs in heart.
  • Laminin indicates cardiomyocyte cel membranes
  • DAPI indicates nuclei
  • endogenous mouse LAMP1 a lysosomal marker
  • FIG.14 shows schematic of exemplary vectors for use in treating danon disease.
  • FIG.15A and 15B show schematic and description of pDA023.
  • FIG.15A A plasmid map of pDA023, a 6754 bp plasmid containing the AAV2 ITR-flanked expression cassete packaged in the final vector.
  • the 5’-ITR has a deleted terminal resolution site (trs) which leads to a self- complementary (sc) AAV when packaged into the AAV2i8 capsid.
  • the LAMP2B gene has been codon optimized, and expression is driven by the muscle-specific promoter SPc5-12.
  • FIG.15B Key functional elements of pDA023.
  • FIG.20 presents data showing a summary of LC3-II expression in the indicated animal over time.
  • FIG.21 presents data showing a summary of LC3-II (left panel) and p62 ( right panel) expression in the indicated animal.
  • LC3-II microtubule- associated protein 1A/1B-light chain 3 isoform 2;
  • p62 sequestosome-1;
  • 24h Fast Fasted for 24 hours; 6w Int.
  • Fast Intermitent fasting with one day fed, one day fasted for 6 weeks.
  • FIG.22 presents data showing vector genome copies and transgene expression levels of hLAMP2B.
  • FIG.23 presents data showing a summary of animal body weight from 6-14 weeks of age folowing the indicated treatment.
  • FIG.24 presents data showing animal survival during study period (0-8 weeks) folowing the indicated treatment.
  • FIG.25 presents data showing a summary of LC3-II (left panel) and p62 ( right panel) expression folowing the indicated treatment.
  • LC3-I microtubule-associated protein 1A/1B-light chain 3 isoform 2;
  • p62 sequestosome-1; ns- not significant; **** p ⁇ 0.0001 One-way ANOVA Dunnet’s multiple comparison’s test; **p ⁇ 0.01.
  • FIG.26 presents data showing a summary of heart weight folowing the indicated treatment. Heart weight (left panel) and heart weight normalized to body weight (right panel) is presented.
  • FIG.27 presents data showing a summary of heart weight to tibia length folowing the indicated treatment.
  • FIG.28 presents schematics of the deleted region Lamp2 wild-type alele deleted region in the transgenic mouse.
  • FIG.29 presents schematics of the deletion junction present in the transgenic mouse folowing deletion of exons 2-6 of Lamp2.
  • Danon disease is a rare multisystem lysosomal storage disease caused by mutations in the LAMP2 gene.
  • Lysosome-associated membrane proteins (LAMPs) mediate the acidification of the lumen, the transport of the macromolecules and the fusion of the lysosomes with endosomes, phagosomes and the plasma membrane.
  • the LAMP2 gene undergoes alternative splicing, resulting in 3 diferent spliced isoforms: LAMP2a, LAMP2b and LAMP2c.
  • LAMP2 isoforms in diferent human tissues has shown a variable expression profile across tissue types, with LAMP2a and LAMP2b more widely expressed than LAMP2c (Pérez et al, 2016; Qiao et al, 2023). Most of the mutations in the LAMP2 gene are predicted to result in the deficiency of al 3 LAMP2 isoforms. Isoform-specific mutations have only been found in LAMP2b so far, suggesting that LAMP2b deficiency is enough to cause Danon disease (Cenacchi et al, 2020).
  • Autophagy is a vital process that facilitates the degradation and recycling of damaged organeles, misfolded proteins, and other celular debris.
  • autophagy helps to maintain celular integrity and contraction. Disruption of this process affects the degradation and recycling processes within cels, which is essential for maintaining celular health.
  • LAMP2 results in impaired autophagy and lysosomal function, leading to the accumulation of autophagic vacuoles in cels.
  • aspects of the invention described herein arise from the identification of codon-optimized nucleic acids that encode a human LAMP2B polypeptide.
  • These codon-optimized nucleic acids can be used to produce vectors for gene therapy (e.g., AAV based gene therapy) to treat disorders related to aberant LAMP2B in a subject, e.g., Danon disease.
  • Recombinant vectors e.g., AAV
  • expression cassetes that contain the codon-optimized nucleic acid are used to deliver the LAMP2B coding sequence in expressible form, to the subject.
  • the nucleic acid encoding the LAMP2B polypeptide described herein is codon optimized for enhanced expression in human subjects.
  • the rAAV vectors described herein for delivering a LAMP2B polypeptide to a subject comprise improvements, such as but not limited to, a codon optimized nucleic acid sequence encoding a LAMP2B polypeptide, where the codon optimized nucleic acid sequence encoding the LAMP2B polypeptide is modified to include features for example, to reduce CpG islands or dinucleotides and/or minimize alternative open reading frames, and/or remove alternative splice sites and/or remove bacterial sequences, and/or maximize sequence diversity or other modifications known to those skiled in the art.
  • recombinant AAV (rAAV) vector and constructs described herein for delivering the LAMP2B polypeptide to a subject comprise improvements such as, e.g., incorporation of an intron upstream of the nucleic acid expressing the LAMP2B polypeptide and downstream of the promoter, and use of specific terminator sequences after the 3’ end of nucleic acid expressing the LAMP2B polypeptide, such as, e.g., specific poly A sequences and/or terminator sequences.
  • No.046192-000118WOPT does not comprise Woodchuck Hepatitis Virus Postranscriptional Regulatory Element or WPRE, e.g., as described in Patricio M, et al., Mol. Therapy Nucleic Acids, March 2017, the contenst of which are incorporated herein by reference in their entirety.
  • viral vectors e.g., using rAAV vectors as a non-limiting example, that comprise a nucleotide sequence containing inverted terminal repeats (ITRs), a promoter (e.g., muscle specific promoter), a heterologous gene, a poly-A tail and potentialy other regulator elements (e.g., an intron) for use to treat a disease associated with aberrant LAMP2B expression (e.g., Danon disease), where the heterologous gene is codon-optimized nucleic acid encoding a human LAMP2B polypeptide.
  • ITRs inverted terminal repeats
  • a promoter e.g., muscle specific promoter
  • heterologous gene e.g., a poly-A tail
  • potentialy other regulator elements e.g., an intron
  • the vector e.g., rAAV
  • the vector can be administered to a patient in a therapeuticaly effective dose that is delivered to the appropriate tissue and/ or organ (e.g., cardiac tissue) for expression of the heterologous gene and treatment of the disease, e.g., Danon disease.
  • the appropriate tissue and/ or organ e.g., cardiac tissue
  • the heterologous gene and treatment of the disease e.g., Danon disease.
  • One exemplary rAAV of the invention is a rAAV with self-complementary genome, e.g., as described in U.S. Patent Number 7,790,154, which is incorporated herein by reference in its entirety.
  • One aspect of the invention relates to codon-optimized nucleic acids that encode a human LAMP2B polypeptide.
  • the nucleic acid has the nucleotide sequence set forth in SEQ ID NOs 1, 2 or 3, or a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto. In some embodiments, the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the nucleic acid has the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleic acid having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the codon-optimized nucleic acid described herein is operatively linked to a promoter to thereby generate an expression cassete.
  • the codon-optimized nucleic acid is included in an expression vector in expressible form (e.g., a viral based expression vector).
  • expression vectors include nucleic acid constructs in the form of plasmids that comprise viral sequence elements (e.g., that facilitate integration and expression) and also, e.g., recombinant viral particles, enzymatic DNA (neDNA), and lipid nanoparticles (LNPs).
  • mice model serves as a mice model for Danon disease that shows at least one symptom of the disease symptoms in human including but not limited to autophagy, cardiac hypertrophy, ventricular wal/septum thickening.
  • the mice further comprises a premature stop codon in a deletion alele transcript.
  • the geneticaly altered mice is substantialy free of LAMP2 expression.
  • the mice model serves as a tool to test the eficacy of the Aty. Dkt. No.046192-000118WOPT Danon constructs as described in the invention.
  • Recombinant AAV expressing LAMP2B polypeptide [00123] As disclosed herein, one aspect of the technology relates to the use of the codon-optimized nucleic acid encoding the human LAMP2B polypeptide, described herein, in the treatment of disease (e.g, Danon disease).
  • the native human lysosomal associated membrane protein 2 (LAMP2B) gene has been characterized (Gene ID: 3920; Ensembl:ENSG00000005893 MIM:309060; AlianceGenome:HGNC:6501).
  • GenBank Accession Nos. NM_001122606.1 and NP_001116078.1 provide examples of the nucleotide and amino acid sequences of wild-type native human Lam2B.
  • rAAV vector that contains and expresses the codon- optimized nucleic acid.
  • the rAAV vector comprises a capsid, and within its capsid, is comprised a nucleotide segment (i.e., nucleic acid sequence) refered to as the “rAAV vector genome”.
  • the rAAV vector genome typically includes multiple elements required for expression of a heterologous gene contained therein, including, but not limited to two inverted terminal repeats (ITRs, e.g., the 5’-ITR and the 3’-ITR), and located between the ITRs are additional elements, including an intron, a promoter, the heterologous gene and a poly-A tail; three ITRs present in the self complementary vectors, including two wild type AAV2 ITRs and one mutated ITR; or only one intact ITR, which is the minimal requirement for packaging particles.
  • ITRs inverted terminal repeats
  • the heterologous gene for use in the methods comprises the codon-optimized nucleic acid encoding a human LAMP2B polypeptide, described herein.
  • the invention relates to a rAAV vector comprising in its genome the wild-type nucleic acid encoding a human LAMP2B polypeptide, described herein.
  • the invention relates to a rAAV vector comprising in its genome the codon-optimized nucleic acid encoding a human LAMP2B polypeptide, described herein.
  • the nucleic acid has the nucleotide sequence set forth in one of SEQ ID NOs 1-3, or a nucleic acid having at least 60%, or 70%, or 80%, 85% or 90% or 95%, or 98%, or 99% sequence identity to SEQ ID NO: 1, 2 or 3.
  • the rAAV vector has in its genome: (a) 5’ and 3’ AAV inverted terminal repeats (ITR) sequences, and (b) a heterologous nucleic acid as set forth as the codon-optimized nucleic acid encoding a human LAMP2B polypeptide.
  • the heterologous nucleic acid comprises the nucleotide sequence set forth in one of SEQ ID NOs 1-3, or a nucleic acid having at least 60%, or 70%, or 80%, 85% or 90% or 95%, or 98%, or 99% sequence identity to SEQ ID NO: 1, 2 or 3, and is located between the 5’ and 3’ ITRs in expressible form (e.g., the heterologous nucleic acid is operatively linked to a promoter as disclosed herein).
  • the AAV genome further contains at least one of a 5’ ITR, a promoter sequence, a 5’ UTR sequence, an intron, a poly A sequence, a reverse RNA pol I terminator sequence and a 3’ ITR.
  • the AAV genome comprises in its genome, 5’ and 3’ AAV inverted Aty. Dkt. No.046192-000118WOPT terminal repeats (ITR) sequences; and located between the 5’ and 3’ ITRs, a heterologous nucleic acid encoding codon-optimized LAMP2B transgene.
  • the AAV genome further comprises at least one of a 5’ITR, an intron, a muscle-specific promoter, a polyA sequence, and a 3’ ITR.
  • the rAAV genome is single stranded (ss) genome.
  • the rAAV genome is self-complementary (sc) genome.
  • the rAAV genome comprises, in the 5’ to 3’ direction, a 5’ ITR; a muscle-specific promoter; an intron; heterologous nucleic acid encoding codon-optimized LAMP2B transgene; a poly A sequence; and a 3’ ITR.
  • the muscle specific promoter expresses the LAMP2B polypeptide preferentialy in the muscle. In certain embodiments, the muscle specific promoter expresses the LAMP2B polypeptide preferentialy in the cardiac muscle.
  • the human LAMP2B polypeptide encoded by the codon-optimized nucleic acid has the amino acid sequence shown in SEQ ID NO: 9. In one embodiment of any aspect herein, the human LAMP2B polypeptide, encoded by the codon-optimized nucleic acid, has the amino acid sequence at least 60%, or 70%, or 80%, 85% or 90% or 95%, or 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 9.
  • the human LAMP2B polypeptide is a functional variant of the human LAMP2B polypeptide having the sequence of SEQ ID NO: 9, as defined herein.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide is operably linked with a promoter.
  • the promoter is CMV promoter, CAG promoter, any muscle specific promoter known in the art.
  • the muscle specific promoter is any Spc5-12 promoter known in the art, e.g., as described in as described in Li, X., et al Nat Biotechnol.1999 Mar;17(3), the contents of which are incorporated herein by reference; SEQ ID NO:3 (nucleotides 253 to 586) of US Patent No. 10,647,751; SEQ ID NO: 5 of International Patent Application Publication WO2019154939; and as described in GenBank: MP275696.1.
  • the muscle specific promoter is any Syn100 promoter known in the art, e.g, as described in Qiao, C, et al.
  • the muscle specific promoter is any synthetic muscle specific promoter known in the art, e.g., as described in International Application no: PCT/GB2020/053371, PCT/GB2022/051611, PCT/EP2023/087452, the contents of which are incorporated herein by reference.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence selected from the group consisting of of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and is operably linked to a muscle-specific promoter syn100, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 1 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 4, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence selected from the group consisting of of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and is operably linked to a muscle-specific promoter Spc5-12, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 1 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence selected from the group consisting of of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and is operably linked to a muscle-specific promoter SP0524, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 1 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 6, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 2 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 4, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 2 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 2 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 6, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 3 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 4, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 3 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence of SEQ ID NO: 3 and is operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 6, or a sequence having at least 80% sequence identity thereto.
  • the nucleic acid encoding LAMP2B has a sequence as described in U.S. Patent Number 10,703,797; U. S. Patent Application Number 17/264,275 or 17/430,107; or Aty. Dkt. No.046192-000118WOPT International Patent Application Number WO 2022/12489; the contents of which are incorporated herein in their entireties.
  • the nucleic acid encoding LAMP2B has a sequence of SEQ ID NOs 2-5, 8-10, or 29-32 as described in U.S. Patent Number 10,703,797.
  • the nucleic acid encoding LAMP2B has a sequence of SEQ ID NOs 7-9 as described in U.S. Patent Application Number 17/264,275. In one embodiment, the nucleic acid encoding LAMP2B has a sequence of SEQ ID NOs 6-12 as described in U.S. Patent Application Number 17/430,107. In one embodiment, the nucleic acid encoding LAMP2B has a sequence of SEQ ID NOs 2-5, 8-10, or 29-33 as described in International Patent Application Number WO 2022/125489. In some embodiments of the invention, the nucleic acid encoding Lamp2B as described in U.S. Patent Number 10,703,797; U.S.
  • Patent Application Number 17/264,275 or 17/430,107; or International Patent Application Number WO 2022/125489 further comprises a promoter selected from the group consisting of CMV promoter, Syn100, SP0524, and Spc5-12, or, sequence having at least 80% sequence identity to any of CMV promoter, syn100 promoter, SP0524 promoter or Spc5-12 promoter.
  • the nucleic acid encoding Lamp2B as described in U.S. Patent Number 10,703,797; U. S. Patent Application Number 17/264,275 or 17/430,107; or International Patent Application Number WO 2022/125489 further comprises IVS intron.
  • recombinant AAV vector comprising nucleic acid encoding Lamp2B polypeptide as described herein comprises is a liver detargeting cardiotropic AAV vector.
  • the liver-detargeting cardiotropic AAV vector is AAV2i8 vector.
  • the AAV2i8 vector comprises or consists of the sequence of SEQ ID NO: 116.
  • Patent Number 10,703,797; U. S. Patent Application Number 17/264,275 or 17/430,107; or International Patent Application Number WO 2022/125489 is linked to any of the muscle specific promoters described herein, e.g., SEQ ID NO: 4-6.
  • Patent Application Number 17/264,275 or 17/430,107; or International Patent Application Number WO 2022/125489 is comprised in a vector with any of the regulatory elements as described herein, e.g., an IVS intron, a polyA tail, promoter (e.g., SEQ ID NOs 4-6 herein), etc.
  • the capsid can be AAV2, AAV6, AAV8, AAV2i8, AAVXL-32, AAVXL32.1. Any of these capsids can be used in al embodiments described herein.
  • the AAV capsid protein cannot be an AAV9 serotype. Aty. Dkt.
  • nucleic acid encoding LAMP2B having a sequence of SEQ ID NOs 1-3 is linked to any promoter described in U.S. Patent Number 10,703,797; U. S. Patent Application Number 17/264,275 or 17/430,107; or International Patent Application Number WO 2022/125489, e.g., a CAG promoter.
  • the codon-optimized nucleic acid encoding a human LAMP2B polypeptide has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO: 3 and is operably linked to CMV promoter having a sequence of SEQ ID NO: 117 or a sequence having at least 80% sequence identity thereto.
  • the nucleic acid sequence encoding a human Lamp2B polypeptide has a sequence of SEQ ID NO: 115 and is operably linked to a promoter selected from the group consisting of CMV promoter, Syn100, SP0524, and spc5-12, or, sequence having at least 80% sequence identity thereto.
  • the nucleic acid sequence encoding a human Lamp2B polypeptide has a sequence of SEQ ID NO: 115 and is operably linked to CMV promoter.
  • the rAAV vector comprising nucleic acid sequences selected from the group consisting of SEQ ID NOs: 60, 61, 63, 79, 81 and 82 is administered to a subject who has Danon disease or is in need thereof.
  • the rAAV vector is AAV2i8 as described in US Patent NO: 8,889,641.
  • the administration of the expression vector containing the codon-optimized nucleic acid described herein leads to increased expression of the LAMP2B polypeptide in a subject, as compared to the expression resulting from administration of an otherwise identical expression vector containing a non-codon optimized (native) nucleic acid encoding the same LAMP2B polypeptide.
  • Such expression can be measured by the amount of the expressed polypeptide or by the activity of the polypeptide.
  • increased expression refers to at least 25% greater exogenous or total LAMP2B polypeptide level or activity in a tissue e.g., heart, muscle, diaphragm of a subject (e.g mammal) administered the codon-optimized LAMP2B nucleic acid of the invention, as compared to the level or activity resulting from the native LAMP2B nucleic acid sequence.
  • LAMP2B polypeptide activity is determined by assessing protein levels for the autophagy marker, LC3-II. Loss of Lamp2 results in impaired autophagy, as seen by increased LC3-I levels as compared to wild-type levels of Lamp2.
  • Lamp2b i.e., administration of the expression vector containing the codon-optimized nucleic acid described herein, or expression of Lamp2B in, e.g., a lamp2 knockout mouse
  • increased expression refers to at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 75% greater, at least 100% greater, or at least 1.5 fold greater, at least 2 fold greater, at least 2.5 Aty. Dkt.
  • No.046192-000118WOPT fold greater at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15- fold greater, at least 20-fold greater, at least 25-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 125-fold greater, at least 150-fold greater, at least 175-fold greater, at least 200-fold greater, at least 225-fold greater, or at least 250-fold greater exogenous or total LAMP2B polypeptide or activity in an subject (e.g., a mammal) administered the codon-optimized nucleic acid encoding LAMP2B polypeptide, as compared to the level or activity of exogenous or total LAMP2B
  • Lamp2b gene and protein level are measured by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and western blot analysis respectively or with other methods known in the art.
  • the administration of the expression vector containing the codon-optimized nucleic acid described herein leads to comparable expression of the LAMP2B polypeptide in a subject, as compared to the expression resulting from administration of an otherwise identical expression vector containing a non- codon optimized (native) nucleic acid encoding the same LAMP2B polypeptide, where the codon optimized nucleic acid is modified from the native or wild type sequence in having less or none CG dinucleotide sequences, less or none alternating reading frames (ARF), modified splice donor and/or, acceptor site compared to the native LAMP2B nucleic acid sequence, and thereby is less immunostimulatory.
  • REF Reverse Transcriptase Polymerase Chain Reaction
  • the rAAV comprising the expression construct of the invention is administered localy to the heart or by intracoronary administration to a subject in need thereof.
  • the encoded LAMP2B expression or activity is comparable between codon optimized encoding nucleic acids of the invention and native or non codon optimized encoding nucleic acids
  • the intracoronary administration of the invention alows for the use of love vector doses, and provides greater safety and thus, is an advantage to treating Danon patients using the rAAV vectors of the invention compared to when they are delivered systemicaly.
  • an optimized rAAV vector genome is created from any of the elements disclosed herein and in any combination, including nucleic acid sequences encoding a promoter, an intron, an ITR, a poly-A tail, elements capable of increasing or decreasing expression of a heterologous gene, and in one embodiment, a nucleic acid sequence that is codon optimized for expression of LAMP2B protein in vivo (i.e., coLAMP2B or codon optimized LAMP2B gene) and optionaly, one or more element to reduce immunogenicity.
  • rAAV genome can be used with any AAV capsid that has tropism for the tissue and cels in which the rAAV vector genome is to be transduced and expressed, Aty. Dkt. No.046192-000118WOPT e.g., cardiac tissue.
  • rAAV genome lacks the AAV P5 promoter or a fragment thereof, which is normaly located upstream of the promoter (e.g., muscle-specific promoter) as disclosed herein. Normaly, the P5 promoter controls expression of the AAV rep/cap proteins during AAV replication.
  • this P5 promoter fragment is present in the rAAV vector as disclosed herein which contains predicted transcription factor binding sites, e.g., cyclic AMP- responsive element-binding protein 3 (CREB3), which can be activated by endoplasmic reticulum (ER)/Golgi stress (Sampieri 2019), activating transcription factor 2 (ATF2), which is also involved in stress response (Watson 2017), Nuclear Receptor Subfamily 1 Group I Member 2 (NR1I2) (also known as Pregnane X receptor [PXR]) is known to be enriched in muscle, and is activated by pregnane steroids, rifampin and other molecules including dexamethasone (NR1I2_HGNC) (Xing 2020).
  • CREB3 cyclic AMP- responsive element-binding protein 3
  • ER endoplasmic reticulum
  • ATF2 activating transcription factor 2
  • NR1I2 Nuclear Receptor Subfamily 1 Group I Member 2
  • NR1I2_HGNC
  • the rAAV vector also comprises an RNA polymerase I termination sequence located between the polyA signal and the 3’ ITR.
  • An exemplary terminal sequence is SEQ ID NO: 33, or SEQ ID NO: 34, the later of which introduces two termination codons and one restriction site (e.g., XhoI) replaces TAG, and is located immediately downstream of the last coding amino acids of LAMP2B, and immediately located upstream of the 3’ UTR.
  • the codon-optimized nucleic acid is operatively linked to a muscle specific promoter.
  • a muscle specific promoter enables expression of the operatively linked gene in the muscle, and can in some embodiments, be an inducible muscle specific promoter.
  • a muscle specific promoter is located upstream 5’ and is operatively linked to the heterologous nucleic acid sequence encoding the LAMP2B polypeptide. Exemplary muscle specific promoter are disclosed herein.
  • the promoter is a muscle specific promoter described in US Patent Application Numbers 18/572,668 or 18/024,211, or International Patent Application Number PCT/EP2023/087452, the contents of which are incorporated herein in their entirties.
  • Cis-Regulatory Elements and Functional Variants Thereof Disclosed herein are various cis-regulatory elements (CREs) that can be used in the construction of muscle-specific promoters. These CREs are generaly derived from genomic promoter and enhancer sequences, but they are used herein in contexts quite diferent from their native genomic environment.
  • the CREs constitute smal parts of much larger genomic regulatory domains, which control expression of the genes with which they are normaly associated.
  • Aty. Dkt. No.046192-000118WOPT It has been surprisingly found that these CREs, many of which are very smal, can be isolated form their normal environment and retain muscle-specific regulatory activity when used to construct various synthetic promoters. This is surprising because the removal of a regulatory sequence from the complex and “three dimensional” natural context in the genome often results in a significant loss of activity, so there is no reason to expect a given CRE to retain the levels of activity observed once removed from their natural environment.
  • CREs have been tested and found to be highly effete at enhancing muscle-specific promoter activity when combined with minimal and proximal promoters. It should be noted that the sequences of the CREs of the present invention can be altered without causing a substantial loss of activity. Functional variants of the CREs can be prepared by modifying the sequence of the CREs, provided that modifications which are significantly detrimental to activity of the CRE are avoided. In view of the information provided in the present disclosure, modification of CREs to provide functional variants is straightforward. Moreover, the present disclosure provides methodologies for simply assessing the functionality of any given CRE variant. Functional variant examples for are discussed below.
  • CREs of the present invention comprise certain muscle-specific transcription factor binding sites (TFBS). It is generaly desired that in functional variants of the CREs these muscle-specific TFBS remain functional. The skiled person is wel aware that TFBS sequences can vary yet retain functionality.
  • TFBS sequence for a TFBS is typicaly illustrated by a consensus sequence from which some degree of variation is typicaly present. Further information about the variation that occurs in a TFBS can be illustrated using a positional weight matrix (PWM), which represents the frequency with which a given nucleotide is typicaly found at a given location in the consensus sequence.
  • PWM positional weight matrix
  • Details of TF consensus sequences and associated positional weight matrices can be found in, for example, the Jaspar or Transfac databases htp:/jaspar.genereg.net/ and htp:/gene-regulation.com/pub/databases.html).
  • This information alows the skiled person to modify the sequence in any given TFBS of a CRE in a manner which retains, and in some cases even increases, CRE functionality.
  • the skiled person has ample guidance on how the TFBS for any given TF can be modified, while maintaining ability to bind the desired TF; the Jaspar system wil, for example, score a putative TFBS based on its similarity to a given PWM.
  • CREs can be scanned against al PWM from JASPAR database to identify/analyse al TFBS. The skiled person can of course find additional guidance in the literature, and, moreover, routine experimentation can be used to confirm TF binding to a putative TFBS in any variant CRE.
  • CRMs Synthetic Muscle-Specific Cis-Regulatory Module and Functional Variants Thereof
  • CRMs of the present invention can be used in combination with a wide range of suitable minimal promoters or muscle- specific proximal promoters.
  • Functional variants of a CRM include sequences which vary from the reference CRM element, but which substantialy retain activity as muscle-specific CRMs.
  • a functional variant of a CRM can comprise substitutions, deletions and/or insertions compared to a reference CRM, provided they do not render the CRM substantialy non-functional.
  • a functional variant of a CRM can be viewed as a CRM which, when substituted in place of a reference CRM in a promoter, substantialy retains its activity.
  • a muscle-specific promoter which comprises a functional variant of a given CRM preferably retains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and yet more preferably 100% of its activity (compared to the reference promoter comprising the unmodified CRM).
  • functional variants of a CRM retain a significant level of sequence identity to a reference CRM.
  • functional variants comprise a sequence that is at least 70% identical to the reference CRM, more preferably at least 80%, 90%, 95% or 99% identical to the reference CRM.
  • Functional variants of a given CRM can, in some embodiments, comprise functional variants of one or more of the CREs present in the reference CRM.
  • functional variants of a given CRM can comprise functional variants of 1, 2, 3, 4, 5, or 6 of the CREs present in the reference CRM.
  • Functional variants of a given CRM can, in some embodiments, comprise the same combination CREs as a reference CRM, but the CREs can be present in a diferent order from the reference CRM. It is usualy prefered that the CREs are present in the same order as the reference CRM (thus, the functional variant of a CRM suitably comprises the same permutation of the CREs as set out in a reference CRM).
  • Functional variants of a given CRM can, in some embodiments, comprise one or more additional CREs to those present in a reference CRM. Additional CREs can be provided upstream of the CREs present in the reference CRM, downstream of the CREs present in the reference CRM, and/or between the CREs present in the reference CRM.
  • the additional CREs can be CREs disclosed Aty. Dkt. No.046192-000118WOPT herein, or they can be other CREs.
  • a functional variant of a given CRM comprises the same CREs (or functional variants thereof) and does not comprise additional CREs.
  • Functional variants of a given CRM can comprise one or more additional regulatory elements compared to a reference CRM.
  • ранн ⁇ е как ⁇ ии какл ⁇ ированн ⁇ е какл ⁇ ество may comprise an inducible or repressible element, a boundary control element, an insulator, a locus control region, a response element, a binding site, a segment of a terminal repeat, a responsive site, a stabilizing element, a de-stabilizing element, and a splicing element, etc., provided that they do not render the CRM substantialy non-functional.
  • Functional variants of a given CRM can comprise additional spacers between adjacent CREs or if one or more spacers are present in the reference CRM, said one or more spacers can be longer or shorter than in the reference CRM.
  • the CRMs as disclosed herein, or functional variants thereof can be combined with any suitable promoter elements in order to provide a synthetic muscle-specific promoter according to the present invention.
  • shorter promoter sequences are prefered, particularly for use in situations where a vector (e.g. a viral vector such as AAV) has limited capacity.
  • the synthetic muscle-specific CRM has length of 250 or fewer nucleotides, for example 220, 200, 180, 150, 100, 75, 60, 50 or fewer nucleotides.
  • the synthetic muscle-specific CRM has length of 200 or fewer nucleotides.
  • CREs and CRMs of the present invention can be used in combination with a wide range of suitable minimal promoters or muscle-specific proximal promoters, tenuely caled promoter elements.
  • Functional variants of promoter elements include sequences which vary from the reference promoter element, but which substantialy retain activity as muscle-specific promoter element. It wil be appreciated by the skiled person that it is possible to vary the sequence of a promoter element while retaining its ability to promote expression.
  • a functional variant of a promoter element can comprise substitutions, deletions and/or insertions compared to a reference promoter element, provided they do not render the promoter element substantialy non-functional.
  • a functional variant of a promoter element can be viewed as a promoter element which, when substituted in place of a reference promoter element in a synthetic promoter, substantialy retains its activity.
  • a muscle-specific synthetic promoter which comprises a functional variant of a given promoter element preferably retains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and yet more preferably 100% of its activity (compared to the reference promoter comprising the unmodified promoter element).
  • functional variants of a promoter element retain a significant level of sequence identity to a reference promoter element.
  • functional variants comprise a sequence that is at Aty.
  • Dkt. No.046192-000118WOPT least 70% identical to the reference promoter element, more preferably at least 80%, 90%, 95% or 99% identical to the reference promoter element.
  • Retention of activity can be assessed by comparing expression of a suitable reporter under the control of the reference promoter with an otherwise identical promoter comprising the substituted promoter element under equivalent conditions. Suitable assays for assessing muscle-specific promoter activity are disclosed herein, e.g. in the examples.
  • Synthetic Muscle-Specific Promoters and Functional Variants Thereof are disclosed herein.
  • a functional variant of a reference synthetic muscle-specific promoter is a promoter which comprises a sequence which varies from the reference synthetic muscle-specific promoter, but which substantialy retains muscle-specific promoter activity. It wil be appreciated by the skiled person that it is possible to vary the sequence of a synthetic muscle-specific promoter while retaining its ability to recruit suitable muscle-specific transcription factors (TFs) and to recruit RNA polymerase I to provide muscle-specific expression of an operably linked sequence (e.g. an open reading frame).
  • TFs muscle-specific transcription factors
  • RNA polymerase I e.g. an open reading frame
  • a functional variant of a synthetic muscle-specific promoter can comprise substitutions, deletions and/or insertions compared to a reference promoter, provided such substitutions, deletions and/or insertions do not render the synthetic muscle- specific promoter substantialy non-functional compared to the reference promoter.
  • a functional variant of a synthetic muscle-specific promoter can be viewed as a variant which substantialy retains the muscle-specific promoter activity of the reference promoter.
  • a functional variant of a synthetic muscle-specific promoter preferably retains at least 70% of the activity of the reference promoter, more preferably at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and yet more preferably 100% of its activity.
  • Functional variants of a synthetic muscle-specific promoter often retain a significant level of sequence similarity to a reference synthetic muscle-specific promoter.
  • functional variants comprise a sequence that is at least 70% identical to the reference synthetic muscle-specific promoter, more preferably at least 80%, 90%, 95% or 99% identical to the reference synthetic muscle-specific promoter.
  • Activity in a functional variant can be assessed by comparing expression of a suitable reporter under the control of the reference synthetic muscle-specific promoter with the expression of a suitable reporter under the control of the putative functional variant under equivalent conditions. Suitable assays for assessing muscle-specific promoter activity are disclosed herein, e.g. in the examples.
  • Functional variants of a given synthetic muscle-specific promoter can comprise functional variants of one or more CREs present in the reference synthetic muscle-specific promoter.
  • functional variant of a given CRM can comprise 1, 2, 3, 4, 5, or 6 of the CREs present in the reference CRM. Functional variants of CREs are discussed above. Aty. Dkt.
  • Functional variants of a given synthetic muscle-specific promoter can comprise functional variants of the promoter element, or a diferent promoter element when compared to the reference synthetic muscle-specific promoter.
  • Functional variants of a given synthetic muscle-specific promoter can comprise the same CREs as a reference synthetic muscle-specific promoter, but the CREs can be present in a diferent order from the reference synthetic muscle-specific promoter.
  • Functional variants of a given synthetic muscle-specific promoter can comprise one or more additional CREs to those present in a reference synthetic muscle-specific promoter.
  • Additional CREs can be provided upstream of the CREs present in the reference CRM, downstream of the CREs present in the reference synthetic muscle-specific promoter, and/or between the CREs present in the reference synthetic muscle-specific promoter.
  • the additional CREs can be CREs disclosed herein, or they can be other CREs.
  • Functional variants of a given synthetic muscle-specific promoter can comprise one or more additional regulatory elements compared to a reference synthetic muscle-specific promoter.
  • a given synthetic muscle-specific promoter can comprise additional spacers between adjacent CREs and promoter elements or if one or more spacer are present in the reference synthetic muscle-specific promoter, said one or more spacers can be longer or shorter than in the reference synthetic muscle-specific promoter. Functional variant examples are provided below.
  • SP0522 is a functional variant of SP0502 and vice versa as SP0522 is a shorter version of SP0502.
  • SP0523 is a functional variant of SP0515 and vice versa as SP0523 is a shorter version of SP0515.
  • SP0524 is a functional variant of SP0521 and vice versa as SP0524 is a shorter version of SP0521.
  • the promoter is SP0527 as described in International Patent Application No. PCT/EP2023/087452, the contents of which are is incorporated herein by reference.
  • synthetic muscle-specific promoters of the present invention can comprise a synthetic muscle-specific promoter of the present invention and additional regulatory sequences.
  • Prefered synthetic muscle-specific promoters of the present invention exhibit muscle- specific promoter activity which is at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, Aty. Dkt.
  • No.046192-000118WOPT 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350% or 400% of the activity exhibited by the CBA or CAG promoter in muscle cels. In many cases higher levels of promoter activity is prefered, but this is not always the case; thus, in some cases more moderate levels of expression may be prefered. In some cases, it is desirable to have available a range of promoters of different activity levels to alow the level of expression to by tailored to requirements; the present disclose provides promoters which are expected to provide such a range of activities.
  • Activity of a given synthetic muscle-specific promoter of the present invention compared to CBA or RSV can be assessed by comparing muscle- specific expression of a reporter gene under control of the synthetic muscle-specific promoter with expression of the same reporter under control of the CBA or RSV promoter, when the two promoters are provided in otherwise equivalent expression constructs and under equivalent conditions.
  • shorter promoter sequences are prefered, particularly for use in situations where a vector (e.g. a viral vector such as AAV) has limited capacity.
  • the synthetic muscle-specific promoter has length of 400 or fewer nucleotides, for example, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 150, 100, 75, 70, 68 or fewer nucleotides.
  • the synthetic muscle- specific promoter has length of 300 or fewer nucleotides, preferably 290 or fewer nucleotides, more preferably 280 or fewer nucleotides, yet more preferably 270 or fewer nucleotides.
  • the synthetic muscle-specific promoter has length of 260 or fewer nucleotides, preferably 250 or fewer nucleotides, more preferably 240 or fewer nucleotides, yet more preferably 230 or fewer nucleotides.
  • Particularly preferred synthetic muscle-specific promoters are those that are both short and which exhibit high levels of activity.
  • the muscle specific promoter is any muscle specific promoter disclosed in International Patent Application Nos WO2022/122733, WO2022/232141, WO2023/141582 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • International Patent Application Nos WO2022/122733, WO2022/232141, WO2023/141582 are incorporated herein by references in their entirties.
  • the promoter is a Syn100 promoter as set forth in SEQ ID NO: 4, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the promoter is a Syn100 promoter as set forth in SEQ ID NO: 114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • Syn100 was previously described in Qiao, C, et al. The American Society fo Gene & Cel Therapy, vol.22 no.11, 1890-1899, Nov.2014, the contents of Aty. Dkt. No.046192-000118WOPT which are incorporated herein.
  • Synthetic muscle promoters activities exceeding naturaly occuring regulatory sequences. Nat Biotechnol 17: 241–245.).
  • the Syn100 promoter is as described in International Publication No. WO2022076556, the contents of which are incorporated herein by reference in its entirety.
  • the syn100 promoter is any syn100 promoter known in the art.
  • the promoter is a SPc5-12 promoter (SEQ ID NO: 5), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the promoter is a SP0524 promoter (SEQ ID NO: 6), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the promoter is selected from the group consisting of SEQ ID NOs 84-114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the promoter is a Syn100 promoter, e.g., as set forth in SEQ ID NO: 4, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease.
  • the promoter is a Syn100 promoter, e.g., as set forth in SEQ ID NO: 114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease.
  • the promoter is a SPc5-12 promoter (SEQ ID NO: 5), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease.
  • the SPc5-12 is any SPc5-12 known in the art.
  • the promoter is a SP0524 promoter (SEQ ID NO: 6), or a variant Aty. Dkt.
  • No.046192-000118WOPT having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease.
  • the promoter is selected from the group consisting of SEQ ID NOs 84-114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease.
  • the promoter is a SP0527 promoter or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease.
  • the promoter is a Syn100 promoter (SEQ ID NO: 4), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is operatively linked to a nucleic acid encoding LAMP2B (e.g., SEQ ID NOs 1-3, or 115).
  • the promoter is a Syn100 promoter (SEQ ID NO: 114), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is operatively linked to a nucleic acid encoding LAMP2B (e.g., SEQ ID NOs 1-3, or 115).
  • the promoter is a SPc5-12 promoter (SEQ ID NO: 5), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is operatively linked to a nucleic acid encoding LAMP2B (e.g., SEQ ID NOs 1-3, or 115).
  • the promoter is a SP0524 promoter (SEQ ID NO: 6), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is operatively linked to a nucleic acid encoding LAMP2B (e.g., SEQ ID NOs 1-3, or 115).
  • the promoter is selected from the group consisting of SEQ ID NOs 84-114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is operatively linked to a nucleic acid encoding LAMP2B (e.g., SEQ ID NOs 1-3, or 115).
  • the promoter is a SP0527 promoter or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is operatively linked to a nucleic acid encoding LAMP2B (e.g., SEQ ID NOs 1-3, or 115).
  • the promoter is a Syn100 promoter (SEQ ID NO: 4), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct that further comprises any of the regulatory elements described herien.
  • the promoter is a SPc5-12 promoter (SEQ ID NO: 5), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct that further comprises any of the regulatory elements described herien.
  • the promoter is a SP0524 promoter (SEQ ID NO: 6), or a variant Aty. Dkt.
  • No.046192-000118WOPT having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct that further comprises any of the regulatory elements described herien.
  • the promoter is selected from the group consisting of SEQ ID NOs 84-114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct that further comprises any of the regulatory elements described herien.
  • the promoter is a SP0527 promoter or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct that further comprises any of the regulatory elements described herien.
  • the promoter is a Syn100 promoter (e.g., SEQ ID NO: 4), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease, the vector or construct further comprising any nucleic acid encoding LAMP2B described herein (e.g., SEQ ID NO: 1-3, or 115) and any of the regulatory elements as described herein.
  • Syn100 promoter e.g., SEQ ID NO: 4
  • a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease, the vector or construct further comprising any nucleic acid encoding LAMP2B described herein (e.g., SEQ ID NO: 1-3, or 115) and any of the regulatory elements as described here
  • the promoter is a SP0524 promoter (SEQ ID NO: 6), or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease, the vector or construct further comprising any nucleic acid encoding LAMP2B described herein (e.g., SEQ ID NO: 1-3, or 115) and any of the regulatory elements as described herein.
  • the promoter is selected from the group consisting of SEQ ID NOs 84-114, or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease, the vector or construct further comprising any nucleic acid encoding LAMP2B described herein (e.g., SEQ ID NO: 1-3, or 115) and any of the regulatory elements as described herein.
  • the promoter is a SP0527 promoter or a variant having a sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto is comprised in a vector or construct for used the treatment of Danon disease, the vector or construct further comprising any nucleic acid encoding LAMP2B described herein (e.g., SEQ ID NO: 1-3, or 115) and any of the regulatory elements as described herein.
  • the promoter is an inducible promoter, or a variant thereof as Aty. Dkt.
  • the synthetic muscle specific promoter has a length of 350 or less nucleotides, preferably 340 or less nucleotides, more preferably 330 or less nucleotides, most preferably 320 or less nucleotides. In some embodiments of the present invention the synthetic muscle specific promoter has a length of 310 or less nucleotides, preferably 300 or less nucleotides, more preferably 290 or less nucleotides, most preferably 280 or less nucleotides.
  • the synthetic muscle specific promoter has a length of 270 or less nucleotides.
  • an expression cassete comprising Syn100 promoter, or a functional variant thereof, operably linked to a sequence encoding an expression product, suitably a gene, e.g., a transgene.
  • an expression cassete comprising SPc5- 12 promoter, or a functional variant thereof, operably linked to a sequence encoding an expression product, suitably a gene, e.g., a transgene.
  • an expression cassete comprising SP0524 promoter, or a functional variant thereof, operably linked to a sequence encoding an expression product, suitably a gene, e.g., a transgene.
  • the muscle specific promoter can be any promoter. In one embodiment, the promoter can be any of the above described promoters.
  • the codon-optimized nucleic acid which can be part of an expression vector, can be operably linked to a muscle specific promoter, wherein the muscle specific promoter is selected from any of: SEQ ID NOS: 4-6 or a functional fragment therein, or a muscle specific promoter having at least 80% sequence identity to SEQ ID NOs: 4-6.
  • the muscle-specific promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114 or a nucleic acid having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • an expression cassete containing any of the codon-optimized nucleic acid described herein, operably linked to a muscle specific promoter, wherein the muscle specific promoter is selected from any of: SEQ ID NOS: 4-6 or a functional fragment thereof, or a muscle specific promoter having at least 80% sequence identity to SEQ ID NOs: 4-6.
  • a vector comprising a synthetic muscle specific promoter Aty. Dkt. No.046192-000118WOPT or an expression cassete according to the present invention.
  • the vector is an expression vector.
  • the vector is a viral vector.
  • the vector is a gene therapy vector, suitably an AAV vector, an adenoviral vector, a retroviral vector, or a lentiviral vector.
  • AAV vectors are of particular interest.
  • a virion viral particle comprising a vector, suitably a viral vector, according to the present invention.
  • a pharmaceutical composition comprising a synthetic muscle specific promoter, expression cassete, vector, or virion according to the present invention.
  • a synthetic muscle specific promoter, expression cassete, vector, virion or pharmaceutical composition according to the present invention for use in therapy, i.e., the prevention or treatment of a medical condition or disease, e.g., Danon disease.
  • a medical condition or disease e.g., Danon disease.
  • the condition or disease is associated with aberrant gene expression, optionaly aberant gene expression in the muscle, e.g., cardiac muscle.
  • the use is for gene therapy, preferably for use in treatment of a disease involving aberant gene expression.
  • the disease is Danon disease.
  • a cel comprising a synthetic muscle specific promoter, expression cassete, vector, or virion as described herein.
  • the cel is a eukaryotic cel, optionaly a mammalian cel, optionaly a human cel.
  • the cel can be a muscle cel, optionaly wherein the cel is a human muscle cel.
  • the synthetic muscle specific promoter or expression cassete can be in a vector or can be in the genome of the cel.
  • a synthetic muscle specific promoter, expression cassete, vector, virion or pharmaceutical composition as described herein for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease as discussed herein, e.g., Danon disease.
  • the disease is Danon disease.
  • the synthetic muscle specific promoter, expression cassete, vector, virion, or pharmaceutical composition as described herein are for use in the manufacture of a pharmaceutical composition for the treatment of Danon disease.
  • a method for producing an expression product comprising providing a synthetic muscle specific expression cassete of the present invention in a muscle cel, e.g., cardiac muscle cel, and expressing the gene present in the synthetic muscle specific expression cassete.
  • the method can be in vitro or ex vivo, or it can be in vivo.
  • the method is a bioprocessing method.
  • the expression product is LAMP2B protein or LAMP2B polypeptide.
  • a method of expressing a therapeutic transgene in a muscle cel comprising introducing into the muscle cel a synthetic muscle specific expression cassete, vector or virion as described herein.
  • the therapeutic transgene is the LAMP2B gene. Aty. Dkt.
  • a method of therapy of a subject comprising: [00250] administering to the subject an expression cassete, vector, virion, or pharmaceutical composition as described herein, which comprises a sequence encoding a therapeutic product operably linked to a promoter according to the present invention; and [00251] expressing a therapeutic amount of the therapeutic product in the muscle, e.g., cardiac muscle, of said subject.
  • the therapeutic product is LAMP2B gene or LAMP2B polypeptide.
  • the method further comprises introducing into the muscle of the subject an expression cassete, vector, virion, or pharmaceutical composition as described herein, which comprises a gene encoding a therapeutic product.
  • the vector is a viral gene therapy vector, preferably an AAV vector.
  • Activity in a functional variant can be assessed by comparing expression of a suitable reporter under the control of the reference synthetic muscle specific promoter with the putative functional variant under equivalent conditions. Suitable assays for assessing muscle specific promoter activity are disclosed herein, e.g., in Examples 2.
  • Functional variants of a given synthetic muscle specific promoter can comprise one or more additional regulatory elements compared to a reference synthetic muscle specific promoter.
  • the muscle promoter is a promoter that has some expression in the muscle.
  • the promoter that has some expression in the muscle is the Syn100 muscle promoter comprising a sequence of SEQ ID NO: 4, or a functional variant have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to SEQ ID NO: 4.
  • the Syn100 promoter is as described in Qiao et al 2014 or International Publication No.
  • the muscle promoter is a promoter that has some expression in the muscle.
  • the promoter that has some expression in the muscle is the SPc5-12 muscle promoter comprising a sequence of SEQ ID NO: 5, or a functional variant have at least 60%, Aty. Dkt.
  • the muscle promoter is a promoter that has some expression in the muscle.
  • the promoter that has some expression in the muscle is the SP0524 muscle promoter comprising a sequence of SEQ ID NO: 6, or a functional variant have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity to SEQ ID NO: 6.
  • a synthetic muscle specific promoter that is at least 50%, 60%, 70%, 80%, 90% or 95% identical to SEQ ID NO: 4-6 comprises a nucleic acid sequence where 2% or 1% or fewer of the nucleotides of SEQ ID NO: 4-6 are altered.
  • a synthetic muscle specific promoter useful in the methods and compositions as disclosed herein is the same length, or not substantialy altered, or 1, 2, 3, 4, 5, or 6 nucleotides longer or 1, 2, 3, 4, 5, or 6 shorter than the length of SEQ ID NO: 4-6.
  • nucleic acid sequences AAV vectors, constructs, and methods to treat Danon disease disclosed herein
  • no nucleotides have been deleted when compared to SEQ ID NO: 4-6.
  • no nucleotides are inserted when compared to SEQ ID NO: 4-6.
  • al modifications made to SEQ ID NO: 4-6 are nucleotide substitutions.
  • a synthetic muscle specific promoter that is at least 50%, 60%, 70%, 80%, 90% or 95% identical to SEQ ID NO: 4-6 comprises a source regulatory nucleic acid sequence which is active in muscle, and the second type of cel or tissue, e.g., muscle, liver, CNS, pancreas, epidermis, etc.
  • the second type of cel or tissue is not a muscle cel.
  • the second type of cel or tissue is a non-cardiac muscle cel.
  • the nucleic acid sequences, AAV vectors (e.g rAAV), constructs and methods to treat Danon disease disclosed herein, the promoter, i.e., the muscle specific promoter, as set out above is operably linked to one or more additional regulatory sequences.
  • An additional regulatory sequence can, for example, enhance expression or cause increased stability of the encoded gene or transcript compared to the encoded transcript where promoter is not operably linked the additional regulatory sequence.
  • the additional regulatory sequence does not substantively reduce the specificity of the muscle specific promoter.
  • the promoter can be operably linked to a sequence encoding a UTR (e.g., a 5’ Aty. Dkt. No.046192-000118WOPT and/or 3’ UTR), an intron, or such.
  • the promoter is operably linked to sequence encoding a UTR, e.g., a 5’ UTR.
  • a 5' UTR can contain various elements that can regulate gene expression. The 5’ UTR in a natural gene begins at the transcription start site and ends one nucleotide before the start codon of the coding region.
  • 5' UTRs as referred to herein may be an entire naturaly occurring 5’ UTR or it may be a portion of a naturaly occurring 5’ UTR.
  • the 5 ’UTR can also be partialy or entirely synthetic. In eukaryotes, 5' UTRs have a median length of approximately 150 nt, but in some cases they can be considerably longer. Regulatory sequences that can be found in 5' UTRs are disclosed in International Application WO2021102107 which is incorporated herein in its entirety by reference. [00264]
  • a 5-UTR sequence is located 3’ of a promoter as disclosed herein, and 5’ of the heterologous nucleic acid sequence (e.g., encoding LAMP2B polypeptide).
  • a muscle specific promoter as set out above is operably linked to a sequence encoding a 5’ UTR derived from the CMV major immediate gene (CMV-IE gene).
  • CMV-IE gene CMV major immediate gene
  • the 5’ UTR from the CMV-IE gene suitably comprises the CMV-IE gene exon 1 and the CMV-IE gene exon 1, or portions thereof.
  • the promoter element may be modified in view of the linkage to the 5 ‘UTR, for example sequences downstream of the transcription start site (TSS) in the promoter element can be removed (e.g., replaced with the 5’ UTR).
  • TSS transcription start site
  • the CMV-IE 5’UTR is described in Simari, et al, Molecular Medicine 4: 700-706, 1998 “Requirements for Enhanced Transgene Expression by Untranslated Sequences from the Human Cytomegalovirus Immediate-Early Gene”, which is incorporated herein by reference. Variants of the CMV-IE 5’ UTR sequences discussed in Simari, et al. are also set out in W02002/031137, incorporated by reference, and the regulatory sequences disclosed therein can also be used. Other UTRs that can be used in combination with a promoter are known in the art, e.g., in Leppek, K., Das, R. & Bama, M.
  • the sequence encoding the 5’ UTR comprises SEQ ID NO: 35-39 as disclosed herein, or a functional variant thereof.
  • functional variants may have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • SEQ ID NO: 35-39 as disclosed herein encodes a CMV-IE 5’ UTR.
  • the 5’ UTR comprises a nucleic acid motif that functions as the protein translation initiation site, e.g., sequences that define a Kozak sequence in the mRNA produced.
  • the sequence encoding the 5’ UTR comprises the sequence motif GCCACC at or near its 3’ end.
  • Kozak sequences or other protein translation initiation sites can be used, as is known in the art (e.g., Marilyn Kozak, “Point Mutations Define a Sequence Flanking the AUG Initiator Codon That Modulates Translation by Eukaryotic Ribosomes” Cel, Vol.44, 283- 292, January 31, 1986; Marilyn Kozak “At Least Six Nucleotides Preceding the AUG Initiator Codon Aty. Dkt. No.046192-000118WOPT Enhance Translation in Mammalian Cels” J. Mol. Rid.
  • a synthetic promoter e.g., a synthetic muscle specific promoter, according to the present invention can be operably linked to a sequence encoding a UTR (e.g., a 5’ and/or 3’ UTR), and/or an intron, or suchlike.
  • a synthetic promoter as set herein is operably linked to a sequence encoding a 5’ UTR and an intron.
  • the 5’ UTR and intron is derived from the CMV major immediate gene (CMV-IE gene).
  • CMV-IE gene CMV major immediate gene
  • the CMV-IE 5’UTR and intron is described in Simari, et al., Molecular Medicine 4: 700-706, 1998 “Requirements for Enhanced Transgene Expression by Untranslated Sequences from the Human Cytomegalovirus Immediate-Early Gene”, which is incorporated herein by reference. Variants of the CMV-IE 5’ UTR and intron sequences discussed in Simari, et al.
  • the 5’ UTR or the 5’ UTR and intron suitably comprises a nucleic acid motif that functions as the protein translation initiation site, e.g., sequences that define a Kozak sequence in the mRNA produced.
  • the sequence encoding the 5’ UTR comprises the sequence motif GCCACC at or near its 3’ end.
  • Kozak sequences or other protein translation initiation sites can be used, as is known in the art (e.g., Marilyn Kozak, “Point Mutations Define a Sequence Flanking the AUG Initiator Codon That Modulates Translation by Eukaryotic Ribosomes” Cel, Vol.44, 283-292, January 31, 1986; Marilyn Kozak “At Least Six Nucleotides Preceding the AUG Initiator Codon Enhance Translation in Mammalian Cels” J. Mol. Rid. (1987) 196, 947-950; Marilyn Kozak “An analysis of 5'’-noncoding sequences from 699 vertebrate messenger RNAs” Nucleic Acids Research.
  • any one of the promoters described herein, or variants thereof, is linked to a sequence encoding a 5’ UTR and/or a 5’UTR and an intron to provide a composite promoter.
  • such composite promoter may be referred to simply as “composite promoters”, or in some cases simply “promoters” for brevity.
  • the rAAV expressing the codon-optimized nucleic acid encoding human LAMP2B polypeptides for use in the methods to treat Danon disease as disclosed herein comprises an intron sequence located between the 3’ end of the promoter sequence and 5’ end of the heterologous nucleic acid (i.e., 5’ of the nucleic acid encoding the LAMP2B polypeptide).
  • Intron sequences serve to increase one or more of: mRNA stability, mRNA transport out of nucleus and/or expression and/or regulation of the expressed LAMP2B polypeptide.
  • a rAAV genome does not comprise an intron sequence. Aty. Dkt.
  • the intron is an IVS intron.
  • the sequence encoding the IVS intron comprises SEQ ID NO: 7 as disclosed herein, or a functional variant thereof.
  • functional variants may have a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • SEQ ID NO: SEQ ID NO: 7 as disclosed herein encodes an IVS intron.
  • the intron sequence is a MVM intron sequence, for example, but not limited to intron sequence of SEQ ID NO: 40, or nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the intron sequence is a HBB2 intron sequence, for example, but not limited to and intron sequence of SEQ ID NO: 41 or SEQ ID NO: 42 or nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the intron sequence is an ubiquitin C (UBC) intron sequence, e.g., intron 1 from the UBC gene, or a portion thereof, e.g., as disclosed in Bianchi et al, 2009, Gene, 448 (1); 88-101, where the intron 1 sequence of the UBC gene is 812bp and starts at chromosomal location 124,914,586, and ends at 124,913,775.
  • UBC ubiquitin C
  • the intron sequence is a UBC intron, for example, but not limited to intron sequence of SEQ ID NO: 43, or nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to SEQ ID NO: 43.
  • the rAAV genotype comprises an intron sequence selected in the group consisting of an IVS intron, a human beta globin b2 (or HBB2) intron, a FIX intron, a chicken beta-globin intron, a CMVIE intron, a UBC intron, a HBB intron sequence, a MVM sequeocne and a SV40 intron.
  • the intron is optionaly a modified intron such as a modified HBB2 intron (see, e.g., SEQ ID NO: 17 in of WO2018046774A1): a modified FIX intron (see., e.g., SEQ ID NO: 19 in WO2018046774A1), or a modified chicken beta-globin intron (e.g., see SEQ ID NO: 21 in WO2018046774A1), or modified HBB2 or FIX introns disclosed in WO2015/162302, which are incorporated herein in their entirety by reference.
  • a modified HBB2 intron see, e.g., SEQ ID NO: 17 in of WO2018046774A1
  • a modified FIX intron see.g., e.g., SEQ ID NO: 19 in WO2018046774A1
  • a modified chicken beta-globin intron e.g., see SEQ ID NO: 21 in WO2018046774A1
  • an rAAV vector genome includes at least one poly A tail that is located 3’ and downstream from the heterologous nucleic acid gene encoding the LAMP2B polypeptide.
  • Any polyA sequence known in the art can be used in the expression constructs of the invention, including but not limited to hGH poly A, bGH poly A, sv40 poly A, synpA polyA and the like.
  • the polyA is a synthetic polyA sequence.
  • the rAAV vector genome comprises two polyA tails, e.g., a hGH poly A sequence and another polyA sequence, where a spacer nucleic acid sequence is located between the two poly A sequences.
  • the polyA signal is 3’ of the heterologous Aty. Dkt. No.046192-000118WOPT nucleic acid sequence encoding the LAMP2B polypeptide.
  • the rAAV genome comprises after the 3’ end of the nucleic acid encoding the LAMP2B polypeptide, a first polyA sequence and a reverse RNA polymerase I terminator sequence (rev RNA PolI terminator sequence), and the 3’ ITR.
  • the rAAV genome comprises 3’ of the nucleic acid encoding the LAMP2B polypeptide, a first polyA sequence, a spacer nucleic acid sequence (e.g., of between 5-10 bp, 5-50 bp, 20-100 bp, 100-400bp, or about 100-250bp, or about 250-400bp), a second poly A sequence, a spacer nucleic acid sequence, and the 3’ ITR.
  • the first and/or second poly A sequence is a hGH poly A sequence, and in some embodiments, the first and second poly A sequences are a synthetic poly A sequence.
  • the first poly A sequence is a hGH poly A sequence and the second poly A sequence is a synthetic poly A sequence, or vice versa – that is, in alternative embodiments, the first poly A sequence is a synthetic poly A sequence and the second poly A sequence is a hGH polyA sequence.
  • first poly A is a 49 bp poly A as described in Levit et al.
  • second poly A is Reverse poly A, or Reverse RNA Pol I terminator sequence. In some embodiments, only one poly A sequence is used.
  • the poly A sequence is selected from any of: SEQ ID NO: 8, or a poly A nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to any of SEQ ID NOS: 8.
  • the poly A sequence is selected from any of: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47, where SEQ ID NO: 44 comprises the signal AATAAA, or a poly A nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to any of SEQ ID NOS: 44-46 or 47.
  • the poly A sequence is selected from any of: SEQ ID NO: 48 or SEQ ID NO: 49, or a poly A nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to any of SEQ ID NOS: 48 or 49.
  • the poly A sequence is, for example, SEQ ID NO: 15 as disclosed in International WO2021102107 (hGH poly A sequence), or a poly A nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to SEQ ID NO: 15 as disclosed in International Application WO2021102107.
  • the hGHpolyA sequence encompassed for use is described in Anderson et al. J. Biol. Chem 264(14); 8222-8229, 1989 (See, e.g., p.8223, 2nd column, first paragraph) which is incorporated herein in its entirety by reference).
  • the recombinant AAV disclosed herein comprises in its genome a transcriptional terminator signal sequence or a transcriptional pause signal sequence in the reverse orientation between polyA, e.g., first poly A and 3’ITR.
  • the recombinant AAV disclosed herein comprises in its genome a reverse RNAPolI transcriptional terminator signal Aty. Dkt.
  • a “reverse RNA Polymerase I terminator sequence” alternatively caled a “dsRNA terminator sequence or termination element,” or “reverse poly A”, is an element that inhibits transcription of double stranded RNA, e.g., from the 3’ ITR.
  • a reverse poly A can be any poly A known in the art in a reverse orientation, i.e., in 3‘-5’ orientation. In one embodiment of the invention, the reverse poly A is same as reverse RNA Polymerase I terminator sequence or dsRNA terminator sequence.
  • the reverse poly A is different than reverse RNA Polymerase I terminator sequence or dsRNA terminator sequence.
  • the termination element or the termination sequence does not alow the transcription from 3’ITR and hence double stranded RNA is not transcribed from 3’ITR.
  • Any termination element can be used including e.g., inverted natural polyA sequences from any species or synthetic polyA signals, or fragments thereof; or other nucleic acid structure terminators known in the art.
  • Exemplary polyA signal and/or, transcription terminators include, but are not limited to polyA signals of BGH, SV40, HGH, Betaglobin, RNA polymerase I transcriptional pause signal from alpha 2 globin gene, transcription termination signal for pol II, fragments thereof and any combination thereof.
  • a “reverse poly A” is a polyA signal sequence placed in a 3’-5’ orientation downstream of the LAMP2B transgene and upstream of 3’ITR. Any natural or synthetic poly A in 3’-5’ orientation can be used as reverse poly A.
  • the reverse poly A is the poly A (pA) as described in International Publication No. WO2019143950 and US Application Publication No.
  • the ‘reverse poly A’ and ‘the double stranded RNA termination element’ and ‘reverse RNA Polymerase I terminator sequence’ are used interchangeably.
  • the poly A signal is a double stranded RNA termination element and/or, a reverse poly A.
  • the reverse poly A or double stranded RNA terminator is located after the homologous or heterologous poly A signal sequence.
  • a transcriptional terminator signal sequence is a reverse RNA polymerase I terminator sequence which is, in a 5’ to 3’ orientation SEQ ID NO: 50, or a rev RNA PolI terminator sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to any of SEQ ID NO: 50, where SEQ ID NO: 50 orientated in a 5’ to 3’ direction is located between the 3’ of the poly A sequence and 5’ of the right ITR sequence (or 3’ ITR).
  • a poly-A tail can be engineered to stabilize the RNA transcript that is transcribed from an rAAV vector genome, including a transcript for a heterologous gene, which in one embodiment is a LAMP2B, and in alternative embodiments, the poly-A tail can be engineered to include elements that are destabilizing.
  • a Aty. Dkt. No.046192-000118WOPT recombinant AAV vector comprises at least one polyA sequence located at, or after the3’ end of the nucleic acid encoding the LAMP2B gene and at or before the 5’ end of the 3’ ITR sequence.
  • the poly A is a ful length poly A (fl-polyA) sequence.
  • the polyA is a truncated polyA sequence as disclosed in International WO2021102107, which is incorporated herein in its entirety.
  • a poly-A tail can be engineered to become a destabilizing element by altering the length of the poly-A tail.
  • the poly-A tail can be lengthened or shortened.
  • the rAAV genome may also comprise a Stuffer DNA nucleic sequence.
  • An exemplary stuffer DNA sequence is SEQ ID NO: 71 as disclosed in International Application WO2021102107, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity thereto.
  • the 91r sequence is located 3’ of the poly A tail, for example, and is located 5’ of the ‘3 ITR sequence.
  • the stuffer DNA sequence comprises a synthetic polyadenylation signal in the reverse orientation.
  • a stuffer nucleic acid sequence (also referred to as a “spacer” nucleic acid fragment) can be located between the poly A sequence and the 3’ ITR (i.e., a holder nucleic acid sequence is located 3’ of the polyA sequence and 5’ of the 3’ ITR).
  • a kerr nucleic acid sequence can be about 30bp, 50pb, 75bp, 100bp, 150bp, 200bp, 250bp, 300bp or longer than 300bp.
  • a breaker nucleic acid fragment is between 20-50bp, 50-100bp, 100-200bp, 200-300bp, 300-500bp, or any integer between 20-500bp.
  • Exemplary telomere (or spacer) nucleic acid sequence can be selected from any of: SEQ ID NO: 16, SEQ ID NO: 71 or SEQ ID NO: 78 as disclosed in International Application WO2021102107, the contents of which are incorporated herein in its entirety, or a nucleic acid sequence at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical to SEQ ID NO: 16 or SEQ ID NO: 71 or SEQ ID NO: 78 as disclosed in International Application WO2021102107, the contents of which are incorporated herein in its entirety.
  • the stuffer sequence is used outside of ITRs, e.g., outside of 5’ITR and/or, outside of 3‘ITR.
  • AAV ITRs [00297]
  • the rAAV vector or genome, or constructs thereof, as disclosed herein for use in the methods to treat Danon disease can comprise AAV ITRs that have desirable characteristics and can be designed to modulate the activities of, and celular responses to vectors that incorporate the ITRs.
  • the AAV ITRs are synthetic AAV ITRs that has desirable characteristics and can be designed to manipulate the activities of and celular responses to vectors comprising one or two synthetic ITRs, including, as set forth in U.S.
  • an ITR exhibits modified transcription activity relative to a naturaly occuring ITR, e.g., ITR2 from AAV2. It is known that the ITR2 sequence inherently has promoter activity. It also inherently has termination activity, similar to a poly(A) sequence.
  • the minimal functional ITR of the present invention exhibits transcription activity as shown in the examples, although at a diminished level relative to ITR2. Thus, in some embodiments, the ITR is functional for transcription. In other embodiments, the ITR is defective for transcription.
  • the ITR can act as a transcription insulator, e.g., preventing transcription of a transgenic cassete present in the vector when the vector is integrated into a host chromosome.
  • a transcription insulator e.g., preventing transcription of a transgenic cassete present in the vector when the vector is integrated into a host chromosome.
  • One aspect of the invention relates to an rAAV vector genome, or construct thereof, comprising at least one synthetic AAV ITR, wherein the nucleotide sequence of one or more transcription factor binding sites in the ITR is deleted and/or substituted, relative to the sequence of a naturaly occurring AAV ITR such as ITR2. In some embodiments, it is the minimal functional ITR in which one or more transcription factor binding sites are deleted and/or substituted.
  • At least 1 transcription factor binding site is deleted and/or substituted, e.g., at least 5 or more or 10 or more transcription factor binding sites, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 transcription factor binding sites.
  • a rAAV vector or construct thereof, including an rAAV vector genome as described herein comprises a polynucleotide comprising at least one synthetic AAV ITR, wherein one or more CpG islands (a cytosine base folowed immediately by a guanine base (a CpG) in which the cytosines in such arrangement tend to be methylated) that typicaly occur at, or near the transcription start site in an ITR are deleted and/or substituted.
  • deletion, or reduction in the number of CpG islands can reduce the immunogenicity of the rAAV vector. This results from a reduction or complete inhibition in TLR-9 binding to the rAAV vector DNA sequence, which occurs at CpG islands.
  • methylation of CpG motifs results in transcriptional silencing. Removal of CpG motifs in the ITR is expected to result in decreased TLR-9 recognition and/or decreased methylation and therefore decreased transgene silencing. In some embodiments, it is the minimal functional ITR in which one or more CpG islands are deleted and/or substituted. In an embodiment, AAV ITR2 is known to contain 16 CpG islands of which one or more, or al 16 can be deleted.
  • the synthetic ITR comprises, consists essentialy of, or consists of Aty. Dkt. No.046192-000118WOPT one of the nucleotide sequences listed in Table 1.
  • the synthetic ITR comprises, consist essentialy of, or consist of a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the nucleotide sequences listed in Table 1.
  • the ITR is a sequence is disclosed in FIG.1 of Samulski et al., 1983, Cel, 33; 135-143 (refered to “Samulski et al, 1983” as which is incorporated herein in its entirety by reference), which discloses modified ITR sequences in FIG.1.
  • the ITR sequence comprises, or consists of a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of the ITR sequences in FIG.1 as disclosed in Samulski et al, 1993.
  • the ITR comprises, or consists of a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5% identical to the ITR sequence of pSM 609 right disclosed in the middle panel of FIG.1 (that lacks the 9bp) disclosed in Samulski et al, 1983.
  • the ITR comprises a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5% identical to the ITR sequence of any of SEQ ID NOs: 51-58.
  • the ITR sequence e.g., Right ITR (or 3’ ITR) is SEQ ID NO: 58 or SEQ ID NO: 54 or a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5% identical to SEQ ID NO: 58 or SEQ ID NO: 54.
  • the ITR sequence e.g., left ITR (or 5’ ITR) is SEQ ID NO: 51 or SEQ ID NO: 53 or a nucleotide sequence that is at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 99.5% identical to SEQ ID NO: 51 or SEQ ID NO: 53.
  • the ITR is a mutant ITR that is at least 106 bp in length.
  • the mutant ITR has a sequence of SEQ ID NO: 16.
  • one or both of the ITR sequences is a wt ITR or an ITR sequence disclosed herein, having an insertion, deletion or substitution.
  • any one or both of 5’ITR and 3’ITR is 145 bp long or smaler than 145 bp in length e.g, 142 bp, 141 bp, 140 bp, 135 bp, 130 bp, 128 bp, 120 bp, 117 bp, 115 bp, or smaler than 115 bp in length.
  • the 5’ITR or 3’ITR is 130 bp long.
  • both 5’ITR and 3’ITR are 130 bp long.
  • the ITR is at least 145 bp in length.
  • the ITR has a sequence of SEQ ID NO: 17. aggaaccc ctagtgatgg agtggccac tccctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggcttgcc cgggcggcct cagtgagcga gcgagcgcgc agagagggag tggccaa (SEQ ID NO: 17).
  • Table 1 Exemplary synthetic ITR sequences Aty. Dkt.
  • the AAV capsid used in the treatment of Danon disease can be a modified AAV capsid that is derived in whole or in part from the AAV capsid set forth in SEQ ID NO: 44.
  • the amino acids from an AAV3b capsid as set forth in SEQ ID NO: 44 can be, or are substituted with amino Aty. Dkt. No.046192-000118WOPT acids from another capsid of a diferent AAV serotype, wherein the substituted and/or inserted amino acids can be from any AAV serotype, and can include either naturaly occuring or partialy or completely synthetic amino acids.
  • an AAV capsid used in the treatment of Danon disease is an AAV3b265D capsid.
  • an AAV3b265D capsid comprises a modification in the amino acid sequence of the two-fold axis loop of an AAV3b capsid via replacement of amino acid G265 of the AAV3b capsid with D265.
  • an AAV3b265D capsid comprises SEQ ID NO: 46 as set forth in International Patent Applications WO 2020/102645 and WO2021102107.
  • the modified virus capsids of the invention are not limited to AAV capsids set forth in SEQ ID NO: 46 as set forth in International Patent Applications WO 2020/102645 and WO2021102107.
  • the amino acids from AAV3b265D as set forth in SEQ ID NO.46 can be, or are substituted with amino acids from a capsid from an AAV of a diferent serotype, wherein the substituted and/or inserted amino acids can be from any AAV serotype, and can include either naturaly occuring or partialy or completely synthetic amino acids.
  • an rAAV vector useful in the treatment of Danon disease as disclosed herein is an AAV3b265D549A capsid.
  • an AAV3b265D549A capsid comprises a modification in the amino acid sequence of the two-fold axis loop of an AAV3b capsid via replacement of amino acid G265 of the AAV3b capsid with D265 and replacement of amino acid T549 of the AAV3b capsid with A549.
  • an AAV3b265D549A capsid comprises SEQ ID NO: 50 as disclosed herein International Patent Applications WO 2020/102645 and WO2021102107.
  • the modified virus capsids of the invention are not limited to AAV capsids set forth in SEQ ID NO: 50.
  • the amino acids from AAV3b265D549A as set forth in SEQ ID NO: 50 can be, or are substituted with amino acids from a capsid from an AAV of a diferent serotype, wherein the substituted and/or inserted amino acids can be from any AAV serotype, and can include either naturaly occuring or partialy or completely synthetic amino acids.
  • the amino acids from AAV3bSASTG can be, or are substituted with amino acids from a capsid from an AAV of a diferent serotype, wherein the substituted and/or inserted amino acids can be from any AAV serotype, and can include either naturaly occuring or partialy or completely synthetic amino acids.
  • an rAAV vector useful in the treatment of Danon disease as disclosed herein is an AAV3b549A capsid.
  • an AAV3b549A capsid comprises a modification in the amino acid sequence of the two-fold axis loop of an AAV3b capsid via replacement of amino acid T549 of the AAV3b capsid with A549.
  • an AAV3b549A capsid comprises SEQ ID NO: 52 as disclosed herein International Patent Applications WO 2020/102645 and WO2021102107.
  • the modified virus capsids of the invention are not limited to AAV capsids set forth in SEQ ID NO: 52.
  • the amino acids from AAV3b549A as set forth in SEQ ID NO: 52 can be, or are substituted with amino acids from a capsid Aty. Dkt.
  • an rAAV vector useful in the treatment of Danon disease as disclosed herein is an AAV3bQ263Y capsid.
  • an AAV3bQ263Y capsid comprises a modification in the amino acid sequence of the two-fold axis loop of an AAV3b capsid via replacement of amino acid Q263 of the AAV3b capsid with Y263.
  • an AAV3b549A capsid comprises SEQ ID NO: 54 as disclosed herein International Patent Applications WO 2020/102645 and WO2021102107.
  • the modified virus capsids of the invention are not limited to AAV capsids set forth in SEQ ID NO: 54.
  • the amino acids from AAV3bQ263Y as set forth in SEQ ID NO: 54 can be, or are substituted with amino acids from a capsid from an AAV of a different serotype, wherein the substituted and/or inserted amino acids can be from any AAV serotype, and can include either naturaly occurring or partialy or completely synthetic amino acids.
  • an rAAV vector useful in the treatment of Danon disease as disclosed herein is AAV3bSASTG serotype or comprises a AAV3bSASTG capsid.
  • an AAV3bSASTG capsid comprises a modification in the amino acid sequence to comprise a SASTG mutation, in particular, the AAV3b capsid was modified to resemble AAV2 Q263A/T265 subvariant by introducing these modifications at similar positions in the AAV3b capsid (as disclosed in Messina EL, et al., Adeno-associated viral vectors based on serotype 3b use components of the fibroblast growth factor receptor signaling complex for transduction. Hum.
  • an rAAV vector useful in the treatment of Danon disease as disclosed herein is AAV3bSASTG serotype or comprises a AAV3bSASTG capsid comprising a AAV3b Q263A/T265 capsid.
  • the amino acids from AAV3bSASTG can be, or are substituted with amino acids from a capsid from an AAV of a different serotype, wherein the substituted and/or inserted amino acids can be from any AAV serotype, and can include either naturaly occurring or partialy or completely synthetic amino acids.
  • an rAAV vector genome useful in the invention are recombinant nucleic acid constructs that include (1) a heterologous sequence to be expressed (in one embodiment, a polynucleotide encoding a LAMP2B polypeptide) and (2) viral sequence elements that facilitate integration and expression of the heterologous genes.
  • the viral sequence elements may include those sequences of an AAV vector genome that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into an AAV capsid.
  • the heterologous gene encodes LAMP2B, which is useful for correcting a LAMP2B-deficiency in a Aty. Dkt. No.046192-000118WOPT patient suffering from Danon disease.
  • such an rAAV vector genome may also contain marker or reporter genes.
  • an rAAV vector genome can have one or more of the AAV3b wild-type (WT) cis genes replaced or deleted in whole or in part, but retain functional flanking ITR sequences.
  • WT wild-type
  • nanoparticles e.g., a lipid nanoparticule (LNP)
  • LNP lipid nanoparticule
  • nanoparticles also encompasses liposomes and lipid particles having the size of a nanoparticle.
  • Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.
  • the carier is, comprises, or consists of a lipid nanoparticle (LNP).
  • Lipid nanoparticles can comprise multiple components, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.
  • ionizable lipids such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102
  • pegylated lipids such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159
  • phospholipids such as DSPC
  • the lipid nanoparticle has a mean diameter from about 30 nm to about 150 nm, more typicaly from about 50 nm to about 150 nm, more typicaly about 60 nm to about 130 nm, more typicaly about 70 nm to about 110 nm, most typicaly about 85 nm to about 105nm, and preferably about 100 nm.
  • the disclosure provides for lipid particles that are larger in relative size to common nanoparticles and about 150 to 250 nm in size. Lipid nanoparticle particle size can be determined by quasi-elastic light scatering using, for example, a Malvern Zetasizer Nano ZS (Malvern, UK) system.
  • the nucleic acid can be complexed with the lipid portion of the particle or encapsulated in the lipid position of the lipid nanoparticle. In some embodiments, the nucleic acid can be fuly encapsulated in the lipid position of the lipid nanoparticle, thereby protecting it from degradation by a nuclease, e.g., in an aqueous solution.
  • the nucleic acid in the lipid nanoparticle is not substantialy degraded after exposure of the lipid nanoparticle to a nuclease at 37°C. for at least about 20, 30, 45, or 60 minutes. In some embodiments, the nucleic acid in the lipid nanoparticle is not substantialy degraded after incubation of the particle in serum at 37oC. for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. [00329] In certain embodiments, the lipid nanoparticles are substantialy non-toxic to mammals such as humans. Aty. Dkt.
  • lipid nanoparticles are solid core particles that possess at least one lipid bilayer.
  • the lipid nanoparticles have a non-bilayer structure, i.e., a non- lamelar (i.e., non-bilayer) morphology.
  • the non-bilayer morphology can include, for example, three dimensional tubes, rods, cubic symmetries, etc.
  • the non-lamelar morphology (i.e., non-bilayer structure) of the lipid particles can be determined using analytical techniques known to and used by those of skil in the art.
  • lipid nanoparticles lamelar vs. non- lamelar
  • Cryo-TEM Cryo- Transmission Electron Microscopy
  • DSC Differential Scanning calorimetry
  • X- Ray Difraction X- Ray Difraction
  • the morphology of the lipid nanoparticles lamelar vs. non- lamelar
  • the lipid nanoparticles having a non-lamelar morphology are electron dense.
  • the lipid nanoparticle is either unilamelar or multilamelar in structure.
  • rAAVs can be administered with vasoactive agents or vasculature permeability agents along with vasodilators. In some embodiments, rAAVs can be administered with only vasoactive agents or vasculature permeability agents. In some embodiments, vasoactive agents and vasodilators are co administered at different times.
  • treatment of a subject with Danon disease may comprise multiple administrations of a pharmaceutical composition comprising a AAV vector comprising the heterologous codon-optimized nucleic acid encoding a human LAMP2B polypeptide, described herein, wherein the multiple administrations can be carried out over a range of time periods, such as, e.g., once yearly, or every 6-months, or about every 2-years, or about every 3-years, or about every 4 years, or about every 5-years or longer than 5-year intervals.
  • the timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms.
  • an efective dose of a AAV vector as disclosed herein can be administered to an individual once every year, or once every two years, or every six months for an indefinite period of time, or until the individual no longer requires any additional Danon disease therapy.
  • a person of ordinary skil in the art wil recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of a AAV vector as disclosed herein that is administered can be adjusted accordingly.
  • the AAV vector can be administered to the lungs of a subject by any suitable means, optionaly by administering an aerosol suspension of respirable particles comprised of the virus vectors and/or virus capsids, which the subject inhales.
  • the respirable particles can be liquid or solid. Aerosols of liquid Aty. Dkt. No.046192-000118WOPT particles comprising the virus vectors and/or virus capsids may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skil in the art. See, e.g., U.S. Patent No.4,501,729.
  • Aerosols of solid particles comprising the virus vectors and/or capsids may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • a AAV vector comprising the heterologous codon-optimized nucleic acid encoding a human LAMP2B polypeptide, as disclosed herein can be formulated in a solvent, emulsion or other diluent in an amount sufficient to dissolve an rAAV vector.
  • the rAAV vectors and/or rAAV genome encoding LAMP2B polypeptide as disclosed herein can herein may be formulated in a solvent, emulsion or a diluent in an amount of, e.g., less than about 90% (v/v), less than about 80% (v/v), less than about 70% (v/v), less than about 65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than about 50% (v/v), less than about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less than about 30% (v/v), less than about 25% (v/v), less than about 20% (v/v), less than about 15% (v/v), less than about 10% (v/v), less than about 5% (v/v), or less than about 1% (v/v).
  • a solvent, emulsion or a diluent in an amount of, e.g.,
  • the rAAV vectors and/or rAAV genome encoding a LAMP2B polypeptide as disclosed herein can disclosed herein may comprise a solvent, emulsion or other diluent in an amount in a range of, e.g., about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v
  • a AAV vector comprising the heterologous codon-optimized nucleic acid encoding a human LAMP2B polypeptide can be an AAV of any serotype, including but not limited to encapsulated by any AAV capsid descibred herein, e.g., AAV2i8.
  • AAV2i8 e.g., AAV2i8.
  • it can be mixed with a carrier or excipient.
  • Carriers and excipients that might be used include saline (especialy sterilized, pyrogen- free saline) saline bufers (for example, citrate bufer, phosphate buffer, acetate bufer, and bicarbonate bufer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade cariers and excipients are particularly useful for delivery of virions to human subjects. Aty. Dkt.
  • a AAV vector comprising the heterologous codon-optimized nucleic acid encoding a human LAMP2B polypeptide, as disclosed herein can also be formulated as a depot preparation.
  • Such long-acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by IM injection.
  • a rAAV vector and/or rAAV genome as disclosed herein may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives.
  • the method is directed to treating a disease or disorder, e.g., Danon disase, that results from a deficiency of LAMP2B in a subject, wherein a AAV vector comprising the heterologous codon-optimized nucleic acid encoding a human LAMP2B polypeptide, as disclosed herein is administered to a patient suffering from Danon disease, and folowing administration, LAMP2B polypeptide is transduced, e.g., in cardiac muscle.
  • a disease or disorder e.g., Danon disase
  • the AAV vector is encapsulated in a capsid, e.g., encapsulated by any AAV capsid descibred herein, e.g., AAV2i8.
  • a capsid e.g., encapsulated by any AAV capsid descibred herein, e.g., AAV2i8.
  • at least about 1.0x1009 to about 1.0x1013 vg/kg wil be administered per dose in a pharmaceuticaly acceptable carrier.
  • dosages of the virus vector and/or capsid to be administered to a subject depend upon the mode of administration, the individual subject's condition, age and gender, and the particular virus vector or capsid, the nucleic acid encoding LAMP2B polypeptide to be delivered, and the like, and can be determined in a routine manner.
  • Exemplary doses for achieving therapeutic effects are AAV doses of at least about 1.5X1010 vg/kg, at least about 1.5 x 1011 vg/kg, or at least about 1.5x1012 vg/kg, at least about 4.0 x1012 vg/kg, at east 1.0 x 1013 vg/kg, at least about 3 x 1014 vg/kg; 1.0 x 1013 vg/kg, at least about 2 x 1014 vg/kg; 3.0 x 1013 vg/kg, at least about 3 x 1014 vg/kg; 1.0 x 1013 vg/kg, at least about 3 x 1013 vg/kg; 1.0 x 1013 vg/kg, at least about 2 x 1013 vg/kg; 3.0 x 1013 vg/kg, at least about 3 x 1013 vg/kg; 1.0 x 1014 vg/kg, at least about 3 x 1014 vg/kg
  • the dose for achieving therapeutic effects as disclosed herein may also be determined by the strength of the promoter operatively linked to the nucleic acid encoding the LAMP2B polypeptide.
  • the dose of the AAV herein can be lower than about 1.6x1012 when the promoter, for example, is stronger than the muscle specific promoter, however, the dose of AAV should be titrated and determined based on the level of LAMP2B polypeptide expressed in the cel, as determined by transduction efficiency of the AAV capsid and the promoter, and the ability of the cel to expressed LAMP2B polypeptide in order to avoid LAMP2B polypeptide accumulation in the transfected cel and any associated cel toxicity.
  • a method of treating Danon disease by administering a codon-optimized nucleic acid encoding a human LAMP2B polypeptide in expressible form to a cel, of a patient, comprising contacting the cel with a rAAV vector and/or rAAV genome as disclosed Aty. Dkt. No.046192-000118WOPT herein, under conditions for the nucleic acid to be introduced into the cel and expressed to produce the LAMP2B polypeptide.
  • the cel is a cel in vivo.
  • the cel is a mammalian cel in vivo.
  • a AAV vector encoding a LAMP2B polypeptide as disclosed herein is useful in methods to decrease symptoms a mammal caused by Danon disease and/or insuficient LAMP2B levels.
  • a rAAV capsid of the rAAV virion used to treat Danon disease is any of those listed in Table 1 as disclosed in International Applications WO2020/102645, and WO2020/102667, each of which are incorporated herein in their entirety.
  • treatment with the rAAV virion comprising the codon-optimized nucleic acid encoding the human LAMP2B, as disclosed herein is capable of reducing any one or more of symptoms of in a patient suffering from Danon disease (i.e., cardiomyopathy, myopathy, propos disability, palpitations, or arrhymias) by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment.
  • Danon disease i.e., cardiomyopathy, myopathy, propos disability, palpitations, or arrhymias
  • an AAV containing encoding LAMP2B of any serotype is capable of reducing any one or more of the symptoms in a patient suffering from Danon disease (i.e., cardiomyopathy, myopathy, propos disability, palpitations, or arrhymias) by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not
  • At least one symptom associated with Danon disease, or at least one adverse side effect associated with Danon disease are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, and the severity of at least one symptom associated with Danon disease, or at least one adverse side effect is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%
  • At least one symptom associated with Danon disease is reduced by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.
  • the technology relates to a method of treating Danon disease in a subject, comprising administering to the subject a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector comprising in its genome, a heterologous codon- optimized nucleic acid sequence encoding an LAMP2B polypeptide in expressible form wherein the heterologous nucleic acid is operatively linked to a promoter (e.g., a muscle specific promoter), in the absence or presence of administration of an additional anti-Danon disease therapy.
  • AAV recombinant adeno-associated virus
  • the dosage of the recombinant AAV ranges from 1.0E8 vg/kg to 5.0E14 vg/kg, 1.0E9 vg/kg to 5.0E12vg/kg, and al ranges in between, and in some embodiments, the LAMP2B is expressed to a level that the subject has a level of LAMP2B expressed by the AAV at a pharmaceutical activity range from at least 25% to about 150% of normal, or at least 50% to about 150% of normal, e.g., at least within two weeks of administration.
  • the LAMP2B is expressed to a level that the subject has a level of LAMP2B expressed by the AAV at a pharmaceutical activity range from at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 7
  • the LAMP2B is expressed to a level that the subject has a level of LAMP2B expressed by the AAV at a pharmaceutical activity no more than 150%, e.g., at least within two weeks of administration. [00360] In some embodiments, the LAMP2B is expressed to a level that the subject has a level of Aty. Dkt.
  • No.046192-000118WOPT LAMP2B expressed by the AAV at a pharmaceutical activity range from at least 15-140%, 55-150%, 60-150%, 65-150%, 70-150%, 75-150%, 80-150%, 85-150%, 90-150%, 95-150%, 100-150%, 105- 150%, 110-150%, 115-150%, 120-150%, 125-150%, 130-150%, 135-150%, 140-150%, 145-150%, 50-145%, 50-140%, 50-135%, 50-130%, 50-125%, 50-120%, 50-115%, 50-110%, 50-105%, 50- 100%, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-140%, 70-130%, 80-120%, 90-110%, 100-110% of normal activity, e.g., at least within two weeks of administration.
  • Pharamceutial activity can be assessed, e.g., by measuring protein, DNA or RNA levels, or assessing for the localization of lysosomes in biopsy samples.
  • the dosage of the AAV ranges from 1.0E9 vg/kg to 5.0E12vg/kg. In some embodiments, the dosage of the AAV is no more than 4.0E13 vg/kg. In some embodiments, the dosage ranges from 1.0E8 vg/kg to 1.0E12vg/kg.
  • the dosage of AAV containing the codon-optimized nucleic acid encoding the LAMP2B polypeptide is no more than 5.0E12vg/kg.
  • the dosages range from 1.0E9 vg/kg to 5.0E12vg/kg.
  • the total dosage of AAV containing the codon- optimized nucleic acid encoding the LAMP2B polypeptide to treat a subject in need thereof is about e12vg, about 5e12 vg, about e13vg, about 3e13 vg, about 3.25e13 vg, about 5e13 vg, about 6e13vg, about 6.5e13vg, about e14vg, about 1.08e14vg, about 2e14vg, about 3e14vg, about 3.5e14vg, or about 4e14vg.
  • the technology described herein relates to the discovery that a single infusion of a rAAV vector containing the codon-optimized nucleic acid encoding the LAMP2B polypeptide can be a stand-alone therapeutic.
  • a one-time administration of the AAV leads to long-term transduction of the LAMP2B polypeptide into muscle cels, e.g., skeletal or cardiac cels and continuous constitutive expression of LAMP2B polypeptide in the systemic circulation or in the tissue of interest, e.g., heart.
  • a method of treating Danon disase in a subject in need thereof by administering the subject a composition comprising a AAV vector containing the codon-optimized nucleic acid encoding the LAMP2B polypeptide, where the subject is not being concurrently administered any additional Danon disease therapies.
  • the technology relates to a method of administering the AAV where the subject has not been administrered any additional -Danon disease therapies for an extended period of time, e.g., at least 3 months, at least 4 months, at least 5 months, at least 1 year, at least 11 ⁇ 2 years and points in between 6 months or longer.
  • the subject has not been administered an additional Danon disease therapy on the day of, or shortly before administration of the AAV.
  • the subject receiving the Danon treatment of the invention is concurrently undergoing or had previously been under standard of care therapies available, e.g., to stabilize muscle and or heart function.
  • described herein is a method of treating Danon disease in a subject in need thereof by administering the subject a composition comprising a AAV vector comprising the Aty. Dkt. No.046192-000118WOPT codon-optimized nucleic acid encoding the LAMP2B polypeptide, where the subject is concurently administered at least one additional Danon disease therapies.
  • the technology relates to a method of administering the AAV where the subject has been administrered at least one additional -Danon disease therapies for an extended period of time, e.g., at least 3 months, at least 4 months, at least 5 months, at least 1 year, at least 11 ⁇ 2 years and points in between 6 months or longer.
  • the subject has been administered at least one additional Danon disease therapy on the day of, or shortly before administration of the AAV.
  • Subjects administered a AAV encoding the codon-optimized nucleic acid encoding the LAMP2B polypeptide according to the methods and dose ranges as disclosed herein, can exhibit a minimal immune response to the LAMP2B protein expressed by the AAV.
  • immune modulation or administration of immune suppressants at the time of, or before, or after the administration of the AAV to the subject, and therefore normal immune suppressants protocols which are typicaly administered when a subject is administered a viral vector, or undergoing gene therapy are not required.
  • the AAV that comprise a nucleotide sequence containing inverted terminal repeats (ITRs), an intron, a promoter, a heterologous gene, a poly-A tail and potentialy other regulator elements for use to treat a at least one
  • the heterologous gene is the codon- optimized nucleic acid encoding the LAMP2B polypeptide
  • the vector, e.g., rAAV can be administered to a patient in a therapeuticaly effective dose that is delivered to the appropriate tissue and/ or organ for expression of the LAMP2B polypeptide and treatment of the disease, e.g., at least one.
  • the method to treat at least one with rAAV comprising the heterologous codon-optimized nucleic acid encoding a human LAMP2B polypeptide, as disclosed herein comprises administration of a therapeuticaly effete amount of a rAAV disclosed herein to result in a level of the expressed LAMP2B polypeptide e.g., in a tissue sample.
  • eficacy of any of the therapeutics of methods of treatment described herein is assessed by at least one endpoint.
  • Endpoints are assessments used to determine if the therapeutic or method of treatment described herein were effecious in treating the disease (e.g., Danon disease), preventing onset of the disease, or preventing or slowing progression of the disease.
  • Examplary endpoints include, but are not limited to, change in cardiac wal thickness (e.g., as assessed by either echocardiography or cardiac MRI), change in left ventricle mass, change in left ventricle wal thickness, LVEF, ECG findings (e.g., Atrial fibrilation/fluter, AV block, or Wolf- Parkinson-White), Holter recording results (if available, including heart rate, occurrence of atrial fibrilation/fluter, occurence of ventricular or supraventricular ectopy, or AV block), ICD findings, occurance of Heart transplantation, occurance of cardiac device implantation (e.g., ICD, CRT-D, pacemaker), NYHA class determination, time since Danon Disease diagnosis, changes in blood tests (e.g., BNP, NT-
  • an endpoint is assessed at least once post-administration of the therapeutic, e.g, rAAV comprising Lamp2B nucleic acid of the invention.
  • an endpoint is assessed at least once per day, at least once per week, at least per month, at least once in months, at least once in 6 months, and/ or at least one year post-administration. Endpoints can be assessed at any given interval folowing adminstration, e.g., at 3 months, 6 months, 9 months, and 12 months post-administration, or at 2 months, 4 months, 6 months, 8 months, and 12 months post- administration, or at 6 months and 12 months post-administration, or at 4 months, 8 months, and 12 months post-administration. In one embodiment, an endpoint is assessed at at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months post-administration.
  • cardiac wal thickness is stabilized or improved post-administration of the rAAV comprising Lamp2B of the invention.
  • stabilized cardiac wal thickness is indicative of a cardic wal thinkness that has remained unchanged as compared to the cardiac wal thickness at or prior to administration.
  • cardiac wal thickness is improved post- administration by at least 5% as compared to the cardiac wal thickness at or prior to administration.
  • cardiac wal thickness is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 30
  • left ventricle mass is stabilized or improved post-administration of the therapeutic, e.g., rAAV comprising Lamp2B nucleic acid of the invention.
  • “stabilized” left ventricle mass is indicative of a left ventricle mass that has remained unchanged as compared to the left ventricle mass at or prior to administration.
  • left ventricle mass is improved post-administration by at least 5% as compared to the left ventricle mass at or prior to administration.
  • left ventricle mass is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %,
  • LVMI is decreased post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • LVMI is stabilized post-administration of the rAAV of the invention.
  • stabilized LVMI is indicative of a LVMI that has remained unchanged as compared to the LVMI at or prior to administration.
  • LVMI is decreased post-administration by at least 10% as compared to the LVMI at or prior to administration.
  • LVMI is decreased post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least
  • myocardial Lamp2B expression is stabilized or improved post- administration of the rAAV of the invention comprisin Lamp2B nucleic acids as discussed herein.
  • “stabilized” myocardial Lamp2B expression is indicative of a myocardial Lamp2B expression that has remained unchanged as compared to the left myocardial Lamp2B expression at or prior to administration.
  • myocardial Lamp2B expression is improved post- administration by at least 5% as compared to the myocardial Lamp2B expression at or prior to administration.
  • myocardial Lamp2B expression is improved post-administration by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40
  • left ventricle wal thickness is stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized left ventricle wal thickness is indicative of a left ventricle wal thickness that has remained unchanged as compared to the left ventricle wal thickness at or prior to administration.
  • left ventricle wal thickness is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 5% as compared to the left ventricle wal thickness at or prior to administration.
  • left ventricle wal thickness is improved post-administration by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at Aty. Dkt.
  • No.046192-000118WOPT least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %
  • LVEF is stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized is indicative of a LVEF that has remained unchanged as compared to the LVEF at or prior to administration.
  • LVEF is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 5% as compared to the LVEF at or prior to administration.
  • LVEF is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least
  • ECG findings are stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized ECG findings are indicative of a ECG findings are that has remained unchanged as compared to the ECG findings at or prior to administration.
  • ECG findings are improved post-administration by at least 5% as compared to the ECG findings at or prior to administration.
  • ECG findings are improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least
  • Holter recording results are stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized Holter recording results are indicative of a Holter recording results that has remained unchanged as compared to the Holter recording results at or prior to administration.
  • Holter recording results are improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 5% as compared to the Holter recording results at or prior to administration.
  • Holter recording results are improved post-administration by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least
  • Dkt. No.046192-000118WOPT least 60 %, at least 61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %
  • ICD findings are stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized ICD findings are indicative of a ICD findings that has remained unchanged as compared to the ICD findings at or prior to administration.
  • ICD findings are improved post-administration by at least 5% as compared to the ICD findings at or prior to administration.
  • ICD findings are improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least
  • NYHA class determination is stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized NYHA class determination is indicative of a NYHA class determination that has remained unchanged as compared to the NYHA class determination at or prior to administration.
  • NYHA class determination is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 5% as compared to the NYHA class determination at or prior to administration.
  • NYHA class determination is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, Aty. Dkt.
  • No.046192-000118WOPT at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52
  • NYHA class is improved by at least 1 class, e.g, by 2, or, 3 class.
  • Non limiting examples include the treatment as described herein causes improvement from NYHA class IV to class II, or, to class I or, to classI.
  • the treatment causes improvement from NYHA class II to class I, or to class I.
  • treatment causes improvement from NYHA class I to class I.
  • blood test readouts e.g., BNP, NT-proBNP, Troponin, CK-MB, LFTs (e.g., ALT, AST, bilirubin), or LDH
  • BNP BNP
  • NT-proBNP Troponin
  • CK-MB e.g., LFTs (e.g., ALT, AST, bilirubin), or LDH) are stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • LFTs e.g., ALT, AST, bilirubin
  • stabilized blood test readouts are indicative of a blood test readouts that has remained unchanged as compared to the blood test readouts at or prior to administration.
  • blood test readouts are improved post- administration by at least 5% as compared to the blood test readouts at or prior to administration.
  • blood test readouts are improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %,
  • Dkt. No.046192-000118WOPT least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or more, or at least 1x, 2x, 3x, 4x, 5x or more as compared to the blood test readouts at or prior to administration.
  • occurrence and severity of extracardiac manifestations of Danon Disease is stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • “stabilized” occurrence and severity of extracardiac manifestations of Danon Disease is indicative of a occurrence and severity of extracardiac manifestations of Danon Disease that has remained unchanged as compared to the occurrence and severity of extracardiac manifestations of Danon Disease at or prior to administration.
  • occurence and severity of extracardiac manifestations of Danon Disease is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 5% as compared to the occurence and severity of extracardiac manifestations of Danon Disease at or prior to administration.
  • occurrence and severity of extracardiac manifestations of Danon Disease is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least
  • retinal involvement is stabilized or improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized retinal involvement is indicative of a retinal involvement that has remained unchanged as compared to the retinal involvement at or prior to administration.
  • retinal involvement is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 5% as compared to the retinal involvement at or prior to administration.
  • No.046192-000118WOPT involvement is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37
  • the subjects’ required treatment is stabilized or improved post- administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • stabilized sujbects’ required treatment is indicative of a subjects’ required treatment that has remained unchanged as compared to the sujbects’ required treatment at or prior to administration.
  • subjects’ required treatment is improved post-administration by at least 5% as compared to the sujbects’ required treatment at or prior to administration.
  • subjects’ required treatment is improved post-administration of the rAAV comprising Lamp2B nucleic acids of the invention by at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at
  • Dkt. No.046192-000118WOPT least 98 %, at least 99 % or more, or at least 1x, 2x, 3x, 4x, 5x or more as compared to the subjects’ required treatment at or prior to administration.
  • the subject does not undergo a heart transplant post-administration of the rAAV comprising Lamp2B nucleic acids of the invention.
  • the subject does not undergo a heart transplant at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer post-administration.
  • the subject is not hospitalized for device implantation or revision at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer post-administration.
  • improvement is seen in at least two of these endpoints. In one embodiment, improvement is seen in at least three of these endpoints, in at least four of these endpoints, in at least five of these endpoints, in at least six of these endpoints, in at least seven of these endpoints, or more.
  • the methods disclosed herein relate to human subjects can be administered a rAAV containing the codon-optimized nucleic acid encoding the LAMP2B polypeptide as disclosed herein at a dose in the range of 1.0E8 vg/kg to 5.0E14 vg/kg, 1.0E9 vg/kg to 5.0E12vg/kg.
  • the methods disclosed herein relate to human subjects can be administered a rAAV containing the codon-optimized nucleic acid encoding the LAMP2B polypeptide as disclosed herein at a dose in the range of 3.0E13 vg/kg to 3.0E14vg/kg.
  • the dosage is lower than 1.0E8 vg/kg to 3.0E13vg/kg.
  • the dose of the a rAAV vector or rAAV genome to be administered to the subject according to the method to treat Danon disase as disclosed herein depends upon the mode of administration, the promoter used, the severity of the disease or other condition to be treated and/or prevented, the individual subject's condition, the particular virus vector or capsid, the promoter being used and the nucleic acid to be delivered, including but not limited to, nucleic acid encoding the signal peptide atached to the 5’ of the nucleic acid encoding expressible LAMP2B polypeptide, and the like, and can be determined in a routine manner.
  • the dose of the rAAV vector comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide is a therapeuticaly effete amount to increase the level of LAMP2B polypeptide levels in the subject to therapeutic levels.
  • the dose of the rAAV vector is a therapeuticaly effective amount to increase LAMP2B polypeptide in the subject to within 40%, or within 30%, or within 20%, or within 10%, or within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the native active form of LAMP2B content.
  • Female subjects produce a native form of LAMP2B.
  • the native active form of LAMP2B content is zero.
  • the dose of the rAAV vector is a therapeuticaly effete amount to increase LAMP2B polypeptide content in the subject more than 2-fold, or 3-fold, or 4-fold, or 5-fold, or 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold, or more than 10-fold of the level of LAMP2B content in the subject with Danon disease.
  • the dose of the rAAV vector is a therapeuticaly effective amount to increase LAMP2B polypeptide content in the subject to about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% of the level of LAMP2B in a healthy subject.
  • the LAMP2B polypeptide activity is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 8 fold, or at least 10 fold than the level prior to AAV administration.
  • the dose of the rAAV vector comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide is a therapeuticaly effective amount of rAAV vector to exhibit an improvement in the therapeutic index of 3- to 5-fold.
  • the dose of the rAAV vector is a therapeuticaly effete amount to result in the subject having clinicaly stable levels of LAMP2B polypeptide at 10-weeks, or at least 20 weeks, or 30 weeks post rAAV administration.
  • the dose of the rAAV vector comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide wil be therapeuticaly effective in at least 5% of tissue penetrance.
  • the dose of the rAAV vector comprising the codon-optimized Aty. Dkt. No.046192-000118WOPT nucleic acid encoding a human LAMP2B polypeptide wil be therapeuticaly effete in at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of tissue penetrance.
  • the term “effective amount” is synonymous with “therapeuticaly effective amount”, “efective dose”, or “therapeuticaly effete dose.”
  • the efectiveness of a therapeutic compound disclosed herein to treat Danon disease can be determined, without limitation, by observing an improvement in an individual based upon one or more clinical symptoms, and/or physiological indicators associated with Danon disease.
  • exemplary doses for achieving therapeutic effects of a rAAV comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein is within the range of 1.0E8 vg/kg to 5.0E12vg/kg.
  • the dose administered to a subject is at least about 1.0E8 vg/kg, at least about 1.0E9 vg/kg, at least about 1.0E10 vg/kg, at least about 1.0E11 vg/kg, at least about 1.0E12 vg/kg, about 1.1E12 vg/kg, about 1.2E12 vg/kg, about 1.3E12 vg/kg, about 1.4E12 vg/kg, about 1.5E12 vg/kg, about 1.6E12 vg/kg, about 1.7E12 vg/kg, about 1.8E12 vg/kg, about 1.9E12 vg/kg, about 2.0E12 vg/kg, about 3.0E12 vg/kg, about 4.0E12 vg/kg, about 5.0E12 vg/kg, about 6.0E12 vg/kg, about 7.0E12 vg/kg, about 8.0E12 vg/kg, about 9.0
  • exemplary doses for achieving therapeutic effects according to the methods as disclosed herein are titers of at between 1.2E12 and 4.0E12 vg/kg, for example, least about 1.0E12 vg/kg, about 1.1E12 vg/kg, about 1.2E12 vg/kg, about 1.3E12 vg/kg, about 1.4E12 vg/kg, about 1.5E12 vg/kg, about 1.6E12 vg/kg, about 1.7E12 vg/kg, about 1.8E12 vg/kg, about 1.9E12 vg/kg, about 2.0E12 vg/kg, about 2.1E12 vg/kg, about 2.2E12 vg/kg, about 2.3E12 vg/kg, about 2.4E12 vg/kg, about 2.5E12 vg/kg, about 2.6E12 vg/kg, about 2.7E12 vg/kg, about 2.8E12 vg/kg,
  • a rAAV vector comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein useful for the methods to treat Danon disease
  • exemplary doses for achieving therapeutic effects are titers of at least about 1.0E12 to 4.0E12 vg/kg, or about 1.2E12 to 3.0E12 vg/kg, or about 1.2E12 to 2.5E12 vg/kg, or about 2.5E12 to 4.0E12 vg/kg.
  • the dosage may be modified by a person of ordinary skil in the art, e.g., the dose administered can be lower than 1.0E12 vg/kg, or lower than about 5.0E12 vg/kg where a stronger promoter is operatively linked to the nucleic acid encoding LAMP2B polypeptide.
  • the dosage may be modified by a person of ordinary skil in the Aty. Dkt.
  • the dose of the rAAV vector administered can be higher than about 1.6E12 vg/kg, or higher than about 5.0E12 vg/kg when a weaker promoter used in the vector is operatively linked to the nucleic acid encoding the LAMP2B polypeptide.
  • Exemplary doses for achieving therapeutic effects are titers of at least about 1.0E51.0E61.0E71.0E81.0E91.0E10, 1.0E111.0E12 vg/kg, optionaly a 10 , , , , , , bout 1.0E to about 1.0E12 transducing units (vg/kg), and optionaly does not exceed about 4.0E12 vg or optionaly is about 3.0E12 transducing units (vg).
  • the total dosage of AAV containing the codon-optimized nucleic acid encoding the LAMP2B polypeptide to treat a subject in need thereof is about e12vg, about 5e12 vg, about e13vg, about 3e13 vg, about 3.25e13 vg, about 5e13 vg, about 6e13vg, about 6.5e13vg, about e14vg, about 1.08e14vg, about 2e14vg, about 3e14vg, about 3.5e14vg, or about4e14vg.
  • administration of rAAV vector or rAAV genome according to the methods as disclosed herein to treat a subject with Danon disease can result in production of a LAMP2B polypeptide with a circulatory half-life of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months or more.
  • the methods for treatment of Danon disease as disclosed herein relate to a single dose of a rAAV comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide is used to treat a subject in a single administration.
  • the dose of rAAV to be administered can be given to the subject in multiple administrations, e.g., a dose of rAAV can be divided into sub-doses and administered in multiple administrations.
  • the methods for treatment of Danon disease as disclosed herein can comprise multiple administrations of a single dose of a rAAV comprising the codon-optimized nucleic acid encoding a human LAMP2B polypeptide, that is, the subject can be treated with a booster administration (i.e., a second, third, fourth, etc.) of the rAAV after a defined period of time after the initial or first administration.
  • a booster administration i.e., a second, third, fourth, etc.
  • the dose of a booster administration can be the same dose (amount) of rAAV administered in the first administration, or can be a higher dose, or a lower dose, depending on the factors above, including, but not limited to, a therapeuticaly effete dose to achieve any one or more of (i) LAMP2B polypeptide levels indicating steady state of LAMP2B polypeptide expression and (i) substantial reduction in one or more Danon disease symptoms, including, without limitation weakening of heart muscle or skeletal muscle, inteluctual disability, heart palpitations, or arrhymia.
  • the total dose of rAAV comprising the Lamp2b nucleic acids of the invention is subdivided in two or more subdoses and are administered to the subject at the same time or subsequently one after another over a period of time, e.g., over a period of about 2 minutes, about 5 Aty. Dkt. No.046192-000118WOPT minutes, about 8 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or over a longer period of time.
  • a steady state of LAMP2B polypeptide expression by the rAAV as disclosed herein is a level of LAMP2B polypeptide thatprovides a therapeutic effect.
  • Stability of one or more symptoms of Danon disease can be determined by the clinical stability of one or more parameters or endpoints as disclosed herein.
  • the time period of between administration of a first dose, and a subsequent dose (i.e., a booster dose) of a rAAV vector according to the methods for treatment of Danon disease as disclosed herein is selected from any of the folowing: about 4 months, about 6 months, about 7 months, about 8 months, about 9 months, about 12 months, about 18 months, about 24 months, or about 3 years, about 4 years, about 5 years, or more than 5 years.
  • the technology relates to methods to treat Danon disease by administering a rAAV vector containing a codon-optimised nucleic acid encoding a human LAMP2B polypeptide as disclosed herein, where the administration of a composition comprising a AAV vector is administered to the subject without ongoing immune suppression. That is, in some embodiments, immune suppression is not administered to the subject long term.
  • an immune modulator is administered starting at about 24hrs before the rAAV administration and is administered for at least 1 day, or at least 2 days, or at least 3 days or at least 4 days, or at least 5 days, or at least 6 days, or for about 1 week, or for longer than 1 week after administration of the rAAV vector. In some embodiments, an immune modulator is administered starting at, or about 24 hrs before rAAV administration and is administered for no more than 1 day, or 2 days, 3 days, or 4 days, or 5 days, or 6 days, or for 1 week, or for 2 weeks, or for 3 weeks or for 1 month after administration of the rAAV.
  • the first dose of an immune modulator is started at, or about 24hrs before rAAV administration and is administered for at least 1 day, or at least 2 days, or at least 3 days or at least 4 days, or at least 5 days, or at least 6 days, or for about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, after which the immune modulator is reduced to a third dose (which is lower than the second dose) for a third period of time (e.g., for at least 1 day, or at least 2 days, or at least 3 days or at least 4 days, or at least 5 days, or at least 6 days, or for about 1 week).
  • a third dose which is lower than the second dose
  • a third period of time e.g., for at least 1 day, or at least 2 days, or at least 3 days or at least 4 days, or at least 5 days, or at least 6 days, or for about 1 week.
  • the methods to treat Danon disease comprises administering prednisone as an immune suppressant, i.e., immune prophylaxis, at a first dose of 60 miligrams (given oraly) starting 24 hours prior to rAAV vector administration.
  • prednisone is continued at 60 mg/day po through the completion of week four after vector administration, after which, at the beginning of week 5 the prednisone dose is tapered to a second dose level of 55 mg/day po and maintained for 7 days.
  • the dose is tapered to a third dose level of 50 mg/day po and maintained for 7 days etc., so that the dose of the immune suppressant (i.e., prednisone) is tapered on a weekly basis by 5 mg/day, after an initial immune suppressant dose for 4 weeks.
  • the immune suppressant i.e., prednisone
  • prednisone is exemplified herein as an immune suppressant for immune prophylaxis according to the methods as disclosed herein.
  • prednisone can be readily substituted with a different immune modulator and administration regimen known by a person of ordinary skil in the art.
  • normal immune prophylaxis for preventing immune reactivity to the rAAV or the expressed LAMP2B polypeptide is stopped, or withdrawn on day 1, or shortly before or after administration of the rAAV according to the methods as disclosed herein.
  • Immune Modulation and Immunosuppression [00415] As disclosed herein, in some embodiments, the methods to treat Danon disease by administering a rAAV containing a codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein, to the subject without ongoing immune suppression.
  • immune suppression is not administered to the subject long term and is only administered for a short and pre-defined period, including an initial period (with an initial dose) and a tapering period (with incremental tapering doses) after the administration of the AAV vector to the subject.
  • the immune suppression is administered for between 4 weeks to up to about 15 weeks after the administration of the AAV vector to the subject and can be administered in an initial and tapering doses as disclosed herein.
  • the methods and compositions using the AAV vectors and AAV genomes as described herein, for treating Danon disease further comprises administering an immune modulator for an initial period folowed by a tapering period.
  • the Aty is administered to the subject long term and is only administered for a short and pre-defined period, including an initial period (with an initial dose) and a tapering period (with incremental tapering doses) after the administration of the AAV vector to the subject.
  • the immune suppression is administered for between 4 weeks to up to about 15 weeks after the administration of the AAV vector to the subject and can be administered in an initial and tapering
  • Dkt. No.046192-000118WOPT immune modulator can be administered at the time of rAAV vector administration, before rAAV vector administration or after the rAAV vector administration.
  • a subject being administered a rAAV vector or rAAV genome as disclosed herein is also administered an immunosuppressive agent.
  • an immunosuppressive agent such as a proteasome inhibitor.
  • proteasome inhibitor known in the art, for instance as disclosed in U.S. Patent No.9,169,492 and U.S.
  • an immunosuppressive agent can be an antibody, including polyclonal, monoclonal, scfv or other antibody derived molecule that is capable of suppressing the immune response, for instance, through the elimination or suppression of antibody producing cels.
  • the immunosuppressive element can be a short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • the coding region of the shRNA is included in the rAAV cassete and is generaly located downstream, 3’ of the poly-A tail.
  • the shRNA can be targeted to reduce or eliminate expression of immunostimulatory agents, such as cytokines, growth factors (including transforming growth factors ⁇ 1 and ⁇ 2, TNF and others that are publicly known).
  • immunostimulatory agents such as cytokines, growth factors (including transforming growth factors ⁇ 1 and ⁇ 2, TNF and others that are publicly known).
  • the immune modulator is an immunoglobulin degrading enzyme such as IdeS, IdeZ, IdeS/Z, Endo S, or their functional variant.
  • the immune modulator or immunosuppressive agent is a proteasome inhibitor.
  • the proteasome inhibitor is Bortezomib.
  • the immune modulator comprises bortezomib and anti CD20 antibody, Rituximab. In other aspects of the embodiment, the immune modulator comprises bortezomib, Rituximab, methotrexate, and intravenous gamma globulin.
  • Non-limiting examples of such references disclosing proteasome inhibitors and their combination with Rituximab, methotrexate and intravenous gamma globulin, as described in US 10,028,993, US 9,592,247, and US 8,809,282, each of which are incorporated in their entirety by reference.
  • One such proteasome inhibitor known in the art for instance as disclosed in U.S. Patent No.9,169,492 and U.S.
  • an immunosuppressive agent can be an antibody, including polyclonal, monoclonal, scfv or other antibody derived molecule that is capable of suppressing the immune response, for instance, through the elimination or suppression of antibody producing cels.
  • the immunosuppressive element can be a short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • the coding region of the shRNA is included in the rAAV cassete and is generaly located downstream, 3’ of the poly-A tail.
  • the immune modulator can be targeted to reduce or eliminate expression of immunostimulatory agents, such as cytokines, growth factors (including transforming growth factors ⁇ 1 and ⁇ 2, TNF and others that are publicly known).
  • immunostimulatory agents such as cytokines, growth factors (including transforming growth factors ⁇ 1 and ⁇ 2, TNF and others that are publicly known).
  • the immune modulator is an inhibitor of the NF-kB pathway.
  • the immune modulator is Rapamycin, or a functional variant. Non- limiting examples of references disclosing rapamycin and its use described in US 10,071,114, US 20160067228, US 20160074531, US 20160074532, US 20190076458, US 10,046,064, are incorporated in their entirety.
  • the immune modulator is synthetic nanocarriers comprising an immunosuppressant.
  • the immune modulator is synthetic nanocariers comprising rapamycin (ImmTORTM nanoparticles) (Kishimoto, et al., 2016, Nat Nanotechnol, 11(10): 890-899; Maldonado, et al., 2015, PNAS, 112(2): E156-165), as disclosed in US20200038463, US Patent 9,006,254 each of which is incorporated herein in its entirety.
  • the immune modulator is an engineered cel, e.g., an immune cel that has been modified using SQZ technology as disclosed in WO2017192786, which is incorporated herein in its entirety by reference.
  • the immune modulator is selected from the group consisting of poly- ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21
  • the immunomodulator or adjuvant is poly-ICLC.
  • the immune modulator is a smal molecule that inhibit the innate immune response in cels, such as chloroquine (a TLR signaling inhibitor) and 2-aminopurine (a PKR inhibitor), can also be administered in combination with the composition comprising at least one rAAV as disclosed herein.
  • TLR signaling inhibitor chloroquine
  • PKR inhibitor 2-aminopurine
  • TLR-signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (al Aty. Dkt. No.046192-000118WOPT available for purchase from INVIVOGENTM).
  • inhibitors of patern recognition receptors such as 2-aminopurine, BX795, chloroquine, and H-89
  • PRR patern recognition receptors
  • a rAAV vector can also encode a negative regulator of innate immunity such as NLRX1.
  • NLRX1 negative regulator of innate immunity
  • a rAAV vector can also optionaly encode one or more, or any combination of NLRX1, NS1, NS3/4A, or A46R.
  • a composition comprising at least one rAAV vector as disclosed herein can also comprise a synthetic, modified RNA encoding inhibitors of the innate immune system to avoid the innate immune response generated by the tissue or the subject.
  • an immune modulator for use in the administration methods as disclosed herein is an immunosuppressive agent.
  • immunosuppressive agent is intended to include pharmaceutical agents which inhibit or interfere with normal immune function.
  • immunosuppressive agents suitable with the methods disclosed herein include agents that inhibit T-cel/B- cel costimulation pathways, such as agents that interfere with the coupling of T-cels and B-cels via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No 2002/0182211.
  • an immunosuppressive agent is cyclosporine A.
  • Other examples include myophenylate mofetil, rapamicin, and anti- thymocyte globulin.
  • the immunosuppressive drug is administered in a composition comprising at least one rAAV vector as disclosed herein or can be administered in a separate composition but simultaneously with, or before or after administration of a composition comprising at least one rAAV vector according to the methods of administration as disclosed herein.
  • An immunosuppressive drug is administered in a formulation which is compatible with the route of administration and is administered to a subject at a dosage sufficient to achieve the desired therapeutic effect.
  • the immunosuppressive drug is administered transiently for a sufficient time to induce tolerance to the rAAV vector as disclosed herein.
  • the immunosuppressant is a monoclonal antibody which specificaly inhibits FcRn-IgG binding without interfering with FcRn-albumin binding and that can be used to treat patients having neutralizing antibodies to viral vectors, e.g, AAV.
  • the monoclonal antibody is nipocalimab (M281), rozanolixizumab (UCB7665); IMVT-1401, RVT-1401, HL161, HBM916, ARGX-113 (efgartigimod), SYNT001, SYNT002, ABY-039, or DX-2507, derivatives or combinations thereof as described in published US application US20230220069, which is incorporated by reference in its entirety.
  • the monoclonal antibody is delivered one to seven days prior to administration of the viral vector. In some embodiments, the monoclonal antibody is delivered daily. In some other embodiments, the monoclonal antibody is dosed or administered on the same day the viral vector is Aty.
  • the monoclonal antibody is dosed for one day to four weeks post-vector administration.
  • Various methods are known to result in the immunosuppression of an immune response of a patient being administered rAAV. Methods known in the art include administering to the patient an immunosuppressive agent, such as a proteasome inhibitor.
  • an immunosuppressive agent such as a proteasome inhibitor.
  • proteasome inhibitor known in the art, for instance as disclosed in U.S. Patent No.9,169,492 and U.S. Patent Application No. 15/796,137, both of which are incorporated herein by reference, is bortezomib.
  • an immunosuppressive agent can be an antibody, including polyclonal, monoclonal, scfv or other antibody derived molecule that is capable of suppressing the immune response, for instance, through the elimination or suppression of antibody producing cels.
  • the immunosuppressive element can be a short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • the coding region of the shRNA is included in the rAAV cassete and is generaly located downstream, 3’ of the poly-A tail.
  • the shRNA can be targeted to reduce or eliminate expression of immunostimulatory agents, such as cytokines, growth factors (including transforming growth factors ⁇ 1 and ⁇ 2, TNF and others that are publicly known).
  • the use of such immune modulating agents facilitates the ability to for one to use multiple dosing (e.g., multiple administration) over numerous months and/or years. This permits for using multiple agents as discussed below, e.g., a rAAV vector encoding multiple genes, or multiple administrations to the subject.
  • Manufacturing of the rAAV of the invention [00430]
  • the recombinant AAV comprising a nucleic acid encoding LAMP2B is produced by the triple transfection method that uses close ended linear duplexed DNA molecules that lack bacterial backbone sequences, for example, as described in International Patent Application No.
  • the rAAV of the invention is manufactured where one or more, or al of nucleic acids, e.g., AAV rep-cap, Adenovirus helper, and transgene, used as starting material are plasmid. In some embodiments, the rAAV of the invention is manufactured where one or more, or al of nucleic acids, e.g., AAV rep-cap, Adenovirus helper, and transgene, used as starting materials are no end DNA (neDNA) or close ended linear duplexed DNA.
  • neDNA no end DNA
  • close ended linear duplexed DNA is dumbbel shaped DNA.
  • Another example of close ended linear duplexed DNA is doggy bone DNA.
  • Non-limiting examples of methods describing cel free in vitro synthesis of dumbbel-shaped DNA and doggy bone DNA are described in U.S. Patent No.6,451,563; Efficient production of superior dumbbel-shaped DNA minimal vectors for smal hairpin RNA expression-Nucleic Acids Res.2015 Oct 15; 43(18): e120; High-Purity Preparation of a Large DNA Dumbbel-Antisense & nucleic acid drug development 11:149–153 (2001);US 9,109,250; U.S. Patent No.9,499,847; U.S.
  • rAAV is manufactured using a adherent HEK293 cel or a HEK293 cel in suspension.
  • the rAAV of the invention is manufactured using Pro10 cel as described in US Patent No: 9,441,206 which is incorporated herein by reference in its entirety.
  • the recombinant AAV comprising a nucleic acid encoding LAMP2B is produced by the method as described in PCT/US2021/013689, published as WO/2021/146591, or as described in PCT/US2022/013279, published as WO/2022/159679, which is incorporated herein by reference in its entirety.
  • compositions [00432]
  • the rAAV vectors containing a codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein, for use in the methods of administration as disclosed herein can be formulated in a pharmaceutical composition with a pharmaceuticaly acceptable excipient, i.e., one or more pharmaceuticaly acceptable carier substances and/or additives, e.g., bufers, carriers, excipients, stabilizers, etc.
  • the pharmaceutical composition may be provided in the form of a kit.
  • Pharmaceutical compositions comprising the rAAV vectors as disclosed herein for use in the methods of administration as disclosed herein and uses thereof are known in the art.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a rAAV vector containing a codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein, for use in the methods of administration as disclosed herein.
  • Relative amounts of the active ingredient e.g., a rAAV vectors as disclosed herein
  • a pharmaceuticaly acceptable excipient e.g., any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1 percent and 99 percent (w/w) of the active ingredient.
  • the composition may comprise between 0.1 percent and 100 percent, e.g., between.5 and 50 percent, between 1-30 percent, between 5- 80 percent, at least 80 percent (w/w) active ingredient.
  • the pharmaceutical compositions can be formulated using one or more excipients or diluents to (1) increase stability; (2) increase cel transfection or transduction; (3) permit the sustained or delayed release of the payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cel types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein and/or (7) alow for regulatable expression of the payload of the invention.
  • a pharmaceuticaly acceptable excipient may be at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or 100 percent pure.
  • an excipient is approved for use for humans and for veterinary use.
  • an excipient may be approved by United States Food and Drug Administration.
  • an excipient may be of pharmaceutical grade.
  • an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • Excipients include, but are not limited to, any and al solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincot, Wiliams and Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
  • compositions/formulations [00435]
  • the rAAV vectors containing a codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein, can be formulated in a composition.
  • the rAAV vectors as disclosed herein can be formulated in a pharmaceutical composition with a pharmaceuticaly acceptable excipient, i.e., one or more pharmaceuticaly acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilisers, etc.
  • a pharmaceuticaly acceptable excipient i.e., one or more pharmaceuticaly acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilisers, etc.
  • the composition e.g., the pharmaceutical composition may be provided in the form of a kit.
  • composition and “formulation” are used interchangeably here.
  • the composition comprises the recombinant AAV vector particles described herein at a concentration from about 1e9 vg/ml to about 1e15vg/ml. In some embodiments, the composition comprises the recombinant AAV vector particles described herein at a concentration from about 1e10vg/ml to about 1e14 vg/ml. In some embodiments, the composition comprises the recombinant AAV vector particles described herein at a concentration from about 1e12vg/ml to about 1e14 vg/ml. In some embodiments, the composition comprises the recombinant AAV vector particles described herein at a concentration from about 1e12vg/ml to about 1e15 vg/ml.
  • the composition comprises the recombinant AAV vector particles described herein at a concentration from about 3e12vg/ml to about 3e13 vg/ml, from about 2.5e12vg/ml to about 1e14 vg/ml, from about 3e13vg/ml to about 1e14 vg/ml, or from 1e13vg/ml to about 1e14 vg/ml.
  • the composition comprises the recombinant AAV vector particles described herein at a concentration of about about 1e10vg/ml, or about 1.5e10 vg/ml, or about 2e10 Aty. Dkt.
  • No.046192-000118WOPT vg/ml or about 2.5e10 vg/ml, or about 3e10 vg/ml, or about 3.5e10 vg/ml, or about 4e10 vg/ml, or about 4.5e10 vg/ml, or about 5e10 vg/ml, or about 5.5e10 vg/ml, or about 6e10 vg/ml, or about 6.5e10 vg/ml, or about 7e10 vg/ml, or about 7.5e10 vg/ml, or about 8e10 vg/ml, or about 8.5e10 vg/ml, or about 9e10 vg/ml, or about 1e11vg/ml, or about 1.5e11 vg/ml, or about 2e11 vg/ml, or about 2.5e11 vg/ml, or about 3e11 vg/ml, or about 3.5e
  • the composition comprises the recombinant AAV vector particles described herein at a concentration of 6.7e13vg/ml.
  • the composition comprises the recombinant AAV vector particles described herein at a concentration of 6.7e13vg/ml when administered to a pedicatric subject.
  • the pharmaceutical composition comprises the population of purified recombinant adeno- associated virus (rAAV) described herein.
  • the pharmaceutical composition comprising the rAAV comprises a buffer of pH about 6.5 to about 8.0. In some embodiments, the pH is about 6.5 to about 7.5.
  • the pH is from about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4 or about 7.5. In some preferred embodiments, the pH is less than about 7.5. For example, the pH is less than about 7.4, less than about 7.3, less than about 7.2, less than about 7.1, less than about 7.0, less than about 6.9, less than about 6.8, less than about 6.7, or less than about 6.6.
  • the pharmaceutical composition comprises one or more excipients, comprising one or more multivalent ions and/or, salts thereof.
  • the multivalent ions can be selected or optionaly selected from the group consisting of citrate, sulfate, magnesium and phosphate.
  • the pharmaceutical composition comprises one or more excipients, comprising one or more ions selected or optionaly selected from the group consisting of, sodium, potassium, chroride, ammonium, carbonate, nitrate, chlorate, chlorite, and calcium.
  • the pharmaceutical composition comprising the rAAV further comprises a non-ionic surfactant.
  • the non-ionic surfactant is selected from the group consisting of polyoxyethylene faty alcohol ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkylglucosides, alkyl phenol ethoxylates, preferably polysorbates, polyoxyethylene alkyl phenyl ethers, and any combinations thereof. In some Aty. Dkt.
  • non-ionic surfactant is selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, Polyoxyethylene (18) tridecyl ether, Polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, Poloxamer P188, Poloxamer P407, Poloxamer P338 IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, Brij S20, BrijSl0, Brij 010, Brij Cl0, BRIJ 020, ECOSURF EH-9 ,ECOSURF EH-14, TERGITOL 15-S-7, PF-68, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOLNP-7, TERGITIT
  • the pharmaceutical composition further comprises polyol, or sugar, or similar. See, e.g., International Patent No. WO2022/159679, which is incorporated herein by reference in its entirety.
  • the composition comprises a buffer. It is noted that any physiological buffer can be used. Non-limiting examples of bufers include, but are not limited to, PBS, Tris.HCl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, ⁇ -ketoglutaric acid, carbonate (bicarbonate-carbonic acid buffer), and protein buffers.
  • the buffer is PBS.
  • the buffer comprises Tris.
  • buffer is Tris.HCl.
  • the bufer is histidine bufer.
  • the buffer has a salt concentration of from about 50 mM to about 750 mM.
  • the buffer has a salt concentration from about 75 mM to about 700 mM, from about 100 mM to about 650 mM, from about 120 mM to about 600 mM, or from about 140 mM to about 550 mM.
  • the buffer has a salt concentration from about 150mM to about 400mM.
  • the bufer has a salt concentration of about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, or about 475 mM.
  • the bufer has a salt concentration of about 150 mM, about 200 mM or about 365 mM.
  • the ionic strength of the composition is at least about 100 mM.
  • the ionic strength of the composition is from about 125 mM to about 750 mM, or from about 150 mM to about 500 mM, or from about 175 mM to about 700 mM, from about 200mM to about 600 mM, or from about 225 mM to about 550 mM, or from about 250 mM to about 500 mM, or from about 275 mM to about 450 mM, or from about 300 mM to about 400 mM.
  • the ionic strength of the composition is at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 275 mM, at least about 300 mM, at least about 325 mM, at least about 350 mM, at least about 375 mM, at least about 400 mM, at least about 425 mM, at least about 450 mM, at least about 475 mM or at least about 500 mM.
  • the ionic strength of the composition is less than 100mM, for example about 95mM, about 90mM, about 85mM, about 80mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, about 50mM, or even less.
  • the osmolarity of the composition is maintained at near isotonic levels.
  • the osmolarity of the composition can be from about 100 mOsm to about 600 mOsm, such as from about 125 mOsm to about 500 mOsm, or from about 130 mOsm to about 350 mOsm, or from about 140 mOsm to about 400 mOsm, or from about 140 mOsm to about 350 mOsm, or from about 200 mOsm to about 400 mOsm, or from about 500 mOsm to about 600 mOsm, or from about 200 mOsm to about 600 mOsm, or from about 300 mOsm to about 600 mOsm, or from about 200 mOsm to about 500 mOsm, or from about 300 mOsm to about 400 mOsm, or from about 150 mOsm to about 350 mOsm, or from about 175 mOsm to about 300 mOsm, or from about 300 m
  • the composition comprises an isotonic solution.
  • the composition has a pH of about 6.5 to about 8.0.
  • the composition has a pH of about 6.5 to about 7.5.
  • the composition has a pH of from about 7 to about 8.
  • the composition has a pH of from about 7.3 to about 7.9.
  • the composition has a pH of from about 7.4 to about 7.8 or from about 7.4 to about 7.7.
  • the composition has a pH of from about 7.3 to about 7.6, e.g., from about 7.3 to about 7.55.
  • the composition has a pH less than about 7.5.
  • the composition has a pH about 7.4 or lower, about 7.3 or lower, about 7.2 or lower, about 7.1 or lower, about 7.0 or lower, about 6.9 or lower, about 6.8 or lower, about 6.7 or lower, about 6.6 or lower, or about 6.5 or lower.
  • the composition has a pH of about 6.5 to about 8.0.
  • the composition has a pH of about 6.5 to about 7.5.
  • the composition has a pH of from about 7 to about 8.
  • the composition has a pH of from about 7.3 to about 7.9.
  • the composition has a pH of from about 7.4 to about 7.8 or from about 7.4 to about 7.7.
  • the composition has a pH of from about 7.3 to about 7.6, e.g., from about 7.3 to about 7.55. In some prefered embodiments, the composition has a pH less than about 7.5.
  • the composition has a pH about 7.4 or lower, about 7.3 or lower, about 7.2 or lower, about 7.1 or lower, about 7.0 or lower, about 6.9 or lower, about 6.8 or lower, about 6.7 or lower, about 6.6 or lower, or about 6.5 or lower.
  • the composition can comprise one or more ions and/or salts thereof. Exemplary ions include, but are not limited to sodium, potassium, chloride, magnesium ammonium, carbonate, nitrate, chlorate, chlorite, and calcium.
  • the ions can be provided as a salt, such as a halide (F, Cl, Br, I) salt of sodium, potassium, magnesium, and/or calcium, non-limiting examples of which include NaCl, KCl, MgCl2, CaCl2, and combinations thereof.
  • a halide (F, Cl, Br, I) salt of sodium, potassium, magnesium, and/or calcium non-limiting examples of which include NaCl, KCl, MgCl2, CaCl2, and combinations thereof.
  • Additional exemplary salts that can be used include, but are not limited to, carboxylic acid salts, such as acetates, propionates, pyrol idonecarboxylates (or pidolates) or sorbates; poly hydroxylated carboxylic acid salts, such as gluconates, heptagluconates, ketogluconates, lactate gluconates, ascorbates or pantothenates; mono- or polycarboxyl hydroxy acid Aty. Dkt. No.046192-000118WOPT salts, such as citrates or lactates; amino acid salts, such as aspartates or glutamates; and fulvate salts.
  • carboxylic acid salts such as acetates, propionates, pyrol idonecarboxylates (or pidolates) or sorbates
  • poly hydroxylated carboxylic acid salts such as gluconates, heptagluconates, ketogluconates,
  • the composition comprises one or more multivalent ions and/or salts thereof.
  • Exemplary multivalent ions include, but are not limited to, calcium, citrate, sulfate, magnesium, and phosphate.
  • Multivalent ions and/or salts thereof can be individualy included in the composition at a concentration of from about 500 ⁇ M to about 500 mM, for example, at a concentration of about 500 ⁇ M, about 750 ⁇ M, about 1 mM, about 1.3 mM, about 1.5 mM, about 1.7 mM, about 2.3 mM, about 2.5 mM, about 2.7 mM, about 3.3 mM, about 3.5 mM, about 3.7 mM, about 4.3 mM, about 4.5 mM, about 4.7 mM, about 5 mM, about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about
  • Non limiting examples of salts are NaCl, KCl, CaCl2, CaSO4, MgSO4, Na3PO4, CaCO3, NaNO3, Al2(SO4)3.
  • the composition comprises NaCl.
  • NaCl can be at a concentration from about 100 mM to about 500 mM, or from about 125 mM to about 450 mM, or from about 100 mM to about 200 mM, or from about 150 mM to about 200 mM.
  • the composition can comprise NaCl at a concentration from about 150 mM to about 425 mM, from about 175 mM to about 400 mM, or from about 175 mM to about 375 mM, or from about 200 mM to about 375 mM.
  • the composition comprises KCl.
  • KCl can be at a concentration from about 1 mM to about 10 mM.
  • the composition can comprise KCl at a concentration from about 1.5 mM to about 7.5 mM.
  • the composition comprises CaCl2.
  • CaCl2 can be at a concentration from about 0.1 mM to about 2 mM.
  • the composition can comprise CaCl2 at a concentration from about 0.5 mM to about 1.5 mM. In some embodiments, the composition comprises CaCl2 at a concentration from about 0.75 mM to about 1.25 mM. [00452] In some embodiments, the composition comprises MgCl2. When present, MgCl2 can be at a concentration from about 0.1 mM to about 1.5 mM. For example, the composition can comprise MgCl2 at a concentration from about 0.25 mM to about 1 mM or from about 0.25 mM to about 0.75 mM. [00453] In some embodiments, the composition comprises MgSO4.
  • MgSO4 can be at a concentration from about 5 mM to about 150 mM.
  • the composition can comprise MgSO4 at a concentration from about 10 mM to about 120 mM, or from about 10 mM to about 50 mM, or from about 15 mM to about 45 mM, or about 75 mM to about 125 mM, or from about 80 mM to about 100 mM, or from about 85 mM to about 95 mM, or from about 15 mM to about 100 mM.
  • Dkt Aty. Dkt.
  • the composition comprises phosphate, e.g., mono basic or dibasic phosphate or a salt thereof.
  • the phosphate e.g., mono basic or dibasic phosphate or a salt thereof can be at a concentration from about 5 mM to about 30 mM.
  • the composition can comprise phosphate, e.g., mono basic or dibasic phosphate or a salt thereof at a concentration from about 7.5 mM to about 25 mM.
  • the composition comprises phosphate, e.g., mono basic or dibasic phosphate or a salt thereof at a concentration from about 10 mM to about 20 mM.
  • the composition comprises a mono basic phosphate or a salt thereof at a concentration from about 0.25 mM to about 3 mM.
  • the composition comprises a mono basic phosphate or a salt thereof at a concentration from about 0.5 mM to about 2.75 mM, or from about 0.75 mM to about 2.5 mM or from about 1 mM to about 2.25 mM.
  • the mono basic phosphate or salt thereof is potassium phosphate monobasic.
  • the composition comprises a dibasic phosphate or a salt thereof at a concentration from about 5 mM to about 15 mM.
  • the composition comprises a dibasic phosphate or a salt thereof at a concentration from about 7.5 mM to about 12.5 mM or from about 8 mM to about 10 mM.
  • the dibasic phosphate or a salt thereof is sodium phosphate dibasic.
  • the composition is substantialy free of dibasic phosphate, e.g., sodium phosphate dibasic.
  • the composition comprises Tris (e.g., Tris.HCl) or a salt thereof at a concentration from about 1 mM to about 50 mM.
  • the composition comprises Tris (e.g., Tris.HCl) or a salt thereof at a concentration of from about 5 mM to about 40 mM, or from about 7.5 mM to about 35 mM, or from about 10 mM to about 30 mM or from about 15 mM to about 25 mM.
  • the composition comprises histidine or a salt thereof at a concentration from about 1 mM to about 50 mM.
  • the composition comprises histidine or a salt thereof at a concentration of from about 5 mM to about 40 mM, or from about 7.5 mM to about 35 mM, or from about 10 mM to about 30 mM or from about 15 mM to about 25 mM.
  • the composition can also comprise a bulking agent.
  • Exemplary bulking agents include, but are not limited to sugars, polyols and (PVP K24).
  • Exemplary polyols include, but are not limited to, polyhydroxy hydrocarbons, monosaccharides, disaccharides, and trisaccharides.
  • polyols include but are not limited to, sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, dulcitol, sucrose, lactose, maltose, trehalose and dextran.
  • polyol is sorbitol, sucrose or mannitol.
  • the bulking agent is sorbitol.
  • the bulking agent is sucrose.
  • the bulking agent is mannitol.
  • the bulking agent is trehalose, e.g., trehalose dehydrate.
  • the bulking agent is a dextran, e.g., Dextran T40 and/or Dextran T10.
  • the bulking agent can be present at a concentration of from about 0.5 % (w/v) to about 10% (w/v).
  • the composition can comprise a bulking agent, e.g., a polyol or Aty. Dkt. No.046192-000118WOPT providone (PVP K24) at a concentration from about from about 1 % (w/v) to about 7.5% (w/v), e.g., from about 1%(w/v) to about 4% (w/v) or from about 4%(w/v) to about 6% (w/v).
  • PVP K24 polyol or Aty. Dkt. No.046192-000118WOPT providone
  • the composition comprises glycerol, sorbitol, sucrose, or mannitol at a concentration from about 1% (w/v) to about 10% (w/v). In some embodiments, the composition comprises glycerol, sorbitol, sucrose, or mannitol at a concentration from about 1%(w/v) to about 10%(w/v). In some embodiments, the composition comprises sorbitol at concentration from about 3%(w/v) to about 6% (w/v).
  • the composition comprises sorbitol at concentration of about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v).
  • the composition comprises sucrose at concentration from about 3%(w/v) to about 6% (w/v).
  • the composition comprises sucrose at concentration of about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v).
  • the composition comprises mannitol at concentration from about 3%(w/v) to about 6% (w/v).
  • the composition comprises mannitol at concentration of about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v).
  • the composition can also comprise a non-ionic surfactant.
  • the non-ionic surfactant can be selected from the group consisting of polyoxyethylene faty alcohol ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkylglucosides, alkyl phenol ethoxylates, preferably polysorbates, polyoxyethylene alkyl phenyl ethers, and any combinations thereof.
  • Non-limiting examples of suitable non-ionic surfactants include polyoxyethylene (12) isooctylphenyl ether (e.g., IGEPAL® CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylenesorbitan monooleate (e.g., TWEEN® 80 polyoxyethylenesorbitan monooleate), polyethylene glycol octadecyl ether (e.g., Brij® S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., EcosurfTM SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., TergitolTM NP-10 nonylphenol ethoxylate), and combinaitons thereof.
  • polyoxyethylene (12) isooctylphenyl ether e.g., IGEPAL® CA-270
  • the non-ionic surfactant is selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, Pluronic F-68 (PF 68), Polyoxyethylene (18) tridecyl ether, Polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, Poloxamer P188, Poloxamer P407, Poloxamer P 338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, Brij S20, BrijSl0, Brij 010, Brij Cl0, BRIJ 020, ECOSURF EH-9 ,ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOLNP-7, TERGITIT
  • the non-ionic surfactant is Poloxamer P 188, Poloxamer P407, Pluronic F-68, Ecosurf Aty. Dkt. No.046192-000118WOPT SA-15, Brij S20, Tergitol NP-10, IGEPAL CA 720 or Tween 80.
  • the composition is substantialy free of a non-ionic surfactant.
  • the non-ionic surfactant is not a polysorbate, e.g., Tween 80 (also referred to as polysorbate 80 or PS80).
  • the non-ionic surfactant can be present at a concentration from about 0.0001% (w/v) to about 0.01% (w/v).
  • the composition can comprise a non-ionic surfactant at a concentration from about 0.0005% (w/v) to about 0.0015% (w/v).
  • the composition can comprise a non-ionic surfactant at a concentration of about 0.0001% (w/v), about 0.0002% (w/v), about 0.0003% (w/v), about 0.0004% (w/v), about 0.0005% (w/v), about 0.0006% (w/v), about 0.0007% (w/v), about 0.0008% (w/v), about 0.0009% (w/v), about 0.001% (w/v), about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), about 0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), or about 0.01%.
  • the composition comprises a non-ionic surfactant at a concentration of about 0.0005% (w/v) or about 0.001% (w/v).
  • the composition comprises, in addition to the rAAV, a bufer (e.g., PBS, Tris.HCl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, ⁇ - ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, dulcitol, sucrose, lactose, maltose, trehalose and dextran) and a non-ionic surfactant (e.g., Poloxamer P 188, Poloxamer P407, Pluronic F-68, Ecosurf SA-15, Brij S20,
  • a bufer e.g., PBS, Tris.HCl
  • the composition comprises, in addition to the rAAV, a bufer (e.g., PBS, Tris.HCl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, ⁇ - ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, dulcitol, sucrose, lactose, maltose, trehalose and dextran), a non-ionic surfactant (e.g., Poloxamer P 188, Poloxamer P407, Pluronic F-68, Ecosurf SA- 15, Brij S20, Tergitol NP-10, IGEPAL CA 720 or Tween 80), and a multivalent ion (e.g., a multivalent ion selected from the group consisting of calcium,
  • a bufer e.
  • the composition comprises, in addition to the rAAV, a bufer (e.g., PBS, Tris.HCl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, ⁇ - ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, dulcitol, sucrose, lactose, maltose, trehalose and dextran), and a multivalent ion (e.g., a multivalent ion selected from the group consisting of calcium, citrate, sulfate, and magnesium).
  • a bufer e.g., PBS, Tris.HCl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, ⁇ - ketoglutaric acid, carbonate buffer
  • any one of the specific bufers or group of buffers listed in the description of the compositions can be used with any one of the specific bulking agents or group of bulking agents listed in the description of the compositions and with any of the specific non-ionic surfactants or group of surfactants listed in the description of the compositions and with any of the specific multivalent ions and multivalent ion group listed in the description of the compositions.
  • any one of the specific bufers or group of buffers listed in the description of the compositions can be used with any one of the specific bulking agents or group of bulking agents listed in the description of the compositions and with any of the specific non-ionic surfactants or group of surfactants listed in the description of the compositions and with any of the specific multivalent ions and multivalent ion group listed in the description of the compositions.
  • any one of the specific bulking agents or group of bulking agents listed in the description of the compositions can be used with any one of the specific buffers or group of buffers listed in the description of the compositions and with any of the specific non-ionic surfactants or group of surfactants listed in the description of the compositions and with any of the specific multivalent ions and multivalent ion group listed in the description of the compositions.
  • any of the specific non-ionic surfactants or group of surfactants listed in the description of the compositions can be used with any one of the specific buffers or group of buffers listed in the description of the compositions and with any one of the specific bulking agents or group of bulking agents listed in the description of the compositions and with any of the specific multivalent ions and multivalent ion group listed in the description of the compositions.
  • any of the specific multivalent ions and multivalent ion group listed in the description of the compositions can be used with any one of the specific buffers or group of buffers listed in the description of the compositions and with any one of the specific bulking agents or group of bulking agents listed in the description of the compositions and with any of the specific non-ionic surfactants or group of surfactants listed in the description of the compositions.
  • al individual specific combinations of buffers, buffer group, bulking agents, bulking agent groups, non-ionic surfactants, non-ionic surfactant groups, multivalent ions and multivalent ion groups listed in the description of the compositions are specificaly contemplated and claimed.
  • the formulation comprises sodium phosphate, dibasic at a concentration of from about 0.1 mg/ml to about 3 mg/ml, sodium phosphate monobasic monohydrate at a concentration of from about 0.1 mg/ml to about 3 mg/ml, sodium chloride at a concentration of from about 1 mg/ml to about 20 mg/ml, mannitol at a concentration of from about 5 mg/ml to about 40 mg/ml, and poloxamer 188 at a concentration of from about 0.1 mg/ml to about 4 mg/ml.
  • the formulation of the present invention comprises sodium phosphate, dibasic at a concentration of about 1.42 mg/ml, sodium phosphate monobasic monohydrate at a concentration of about 1.38 mg/ml, sodium chloride at a concentration of about 8.18 mg/ml, mannitol at a concentration of about 20 mg/ml, and poloxamer 188 at a concentration of about 2 mg/ml.
  • the formulations of the present invention may be in liquid form and may comprise the AAV LAMP2B virus particle at a concentration of from about 1E12 vg/ml to about 2E14 vg/ml, or at a concentration of about 2E13 vg/ml.
  • the AAV LAMP2B formulation of the invention comprises one or more pharmaceuticaly acceptable excipients to provide the formulation with advantageous properties for storage and/or administration to subjects for the treatment of Danon disease.
  • the formulations of the present invention are capable of being stored at ⁇ 65° C for a period of at least 2 weeks, at least 4 weeks, at least 6 weeks and at least about 8 weeks, without detectable change in stability.
  • stable means that the recombinant AAV LAMP2B virus present in the formulation essentialy retains its physical stability, chemical stability and/or biological activity during storage.
  • the recombinant AAV LAMP2B Aty. Dkt.
  • No.046192-000118WOPT virus present in the formulation retains at least about 80% of its biological activity in a human patient during storage for a determined period of time at ⁇ 65° C., or at least about 85%, 90%, 95%, 98% or 99% of its biological activity in a human patient.
  • the formulation comprising recombinant AAV LAMP2B virions further comprises one or more bufering agents.
  • the formulation of the present invention comprises sodium phosphate dibasic at a concentration of about 0.1 mg/ml to about 3 mg/ml, about 0.5 mg/ml to about 2.5 mg/ml, about 1 mg/ml to about 2 mg/ml, or about 1.4 mg/ml to about 1.6 mg/ml.
  • the AAV LAMP2B formulation of the present invention comprises about 1.42 mg/ml of sodium phosphate, dibasic (dried).
  • Another bufering agent that may find use in the recombinant AAV LAMP2B formulations of the present invention is sodium phosphate, monobasic monohydrate which, in some embodiments, finds use at a concentration of from about 0.1 mg/ml to about 3 mg/ml, about 0.5 mg/ml to about 2.5 mg/ml, about 1 mg/ml to about 2 mg/ml, or about 1.3 mg/ml to about 1.5 mg/ml.
  • the AAV LAMP2B formulation of the present invention comprises about 1.38 mg/ml of sodium phosphate, monobasic monohydrate.
  • the recombinant AAV LAMP2B formulation of the present invention comprises about 1.42 mg/ml of sodium phosphate, dibasic and about 1.38 mg/ml of sodium phosphate, monobasic monohydrate.
  • the recombinant AAV LAMP2B formulation of the present invention may comprise one or more isotonicity agents, such as sodium chloride, at a concentration of about 1 mg/ml to about 20 mg/ml, for example, about 1 mg/ml to about 10 mg/ml, about 5 mg/ml to about 15 mg/ml, or about 8 mg/ml to about 20 mg/ml.
  • the formulation of the present invention comprises about 8.18 mg/ml sodium chloride.
  • Other buffering agents and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations of the present disclosure.
  • the recombinant AAV LAMP2B formulations of the present invention may comprise one or more bulking agents.
  • Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24).
  • the formulations of the present invention comprise mannitol, which may be present in an amount from about 5 mg/ml to about 40 mg/ml, or from about 10 mg/ml to about 30 mg/ml, or from about 15 mg/ml to about 25 mg/ml. In one embodiment, mannitol is present at a concentration of about 20 mg/ml.
  • the recombinant AAV LAMP2B formulations of the present invention may comprise one or more surfactants, which may be non-ionic surfactants. Exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof.
  • the surfactant can be, without limitation, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecylsulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188 and the like, and combinations thereof.
  • the formulation of the present invention comprises Aty. Dkt.
  • No.046192-000118WOPT poloxamer 188 which may be present at a concentration of from about 0.1 mg/ml to about 4 mg/ml, or from about 0.5 mg/ml to about 3 mg/ml, from about 1 mg/ml to about 3 mg/ml, about 1.5 mg/ml to about 2.5 mg/ml, or from about 1.8 mg/ml to about 2.2 mg/ml.
  • poloxamer 188 is present at a concentration of about 2.0 mg/ml.
  • the formulation is stable at a temperature of about 5° C. (e.g., 2° C. to 8° C.) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more.
  • the formulation is stable at a temperature of less than or equal to about ⁇ 20° C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.
  • the formulation is stable at a temperature of less than or equal to about ⁇ 40° C.
  • the formulation is stable at a temperature of less than or equal to about ⁇ 60° C. for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10 mM Phosphate pH 7.4, 350 mM NaCl, about 2.7 mM KCl, 5 % (w/v) sorbitol, and 0.001% (w/v) poloxamer 188.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10 mM Phosphate pH 7.4, about 200 mM NaCl, about 5 mM KCl, about 1% (w/v) mannitol, and about 0.0005% (w/v) IGEPAL CA 720.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 20 mM Phosphate pH 7.4, about 300 mM NaCl, about 3 mM KCl, about 3 % (w/v) mannitol, and about 0.001% (w/v) Brij S20.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 20 mM Phosphate pH 7.4, about 300 mM NaCl, about 3 mM KCl, about 3 % (w/v) sorbitol, and about 0.001% (w/v) Ecosurf SA-15.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10 mM Phosphate pH 7.4, about 350 mM NaCl, about 2.7 mM KCl, about 5 % (w/v) sorbitol, and about 0.001% (w/v) poloxamer 188.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 6.95-7.2, about 137mM NaCl, about 2.7mM KCl, about 0.9mM CaCl2, about 0.5mM MgCl2, and about 0.001% (w/v) Pluronic F-68. Aty. Dkt.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.3, about 180 mM NaCl, about 2.7 mM KCl, about 5 % (w/v) sorbitol, and about 0.001% (w/v) Poloxamer 188.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 15 mM Phosphate pH 7.4, about 375 mM NaCl, about 3.5 mM KCl, about 5 % (w/v) sorbitol, and about 0.0005% (w/v) Tergitol NP-10.
  • the c composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 15 mM Phosphate pH 7.4, about 375 mM NaCl, about 3.5 mM KCl, about 3 % (w/v) glycerol, and about 0.0005% (w/v) Tween 80.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 9.0 mM Na2HPO4.7H2O, about 1.0 mM KH2PO4 pH 7.4, about 350 mM NaCl, about 2.7 mM KCl, about 5 % (w/v) sorbitol, and about 0.001% (w/v) poloxamer 188.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.6, about 137 mM NaCl, about 2.7 mM KCl, about 5% (w/v) sorbitol, and about 0.01% Pluronic F-68.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.4, about 137 mM NaCl, about 2.7 mM KCl, about 5% (w/v) sorbitol, about 0.01% Pluronic F-68, and about 20 mM MgSO4.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.6, about 137 mM NaCl, about 2.7 mM KCl, about 5% (w/v) mannitol, and about 0.01% Pluronic F-68.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.3, about 137 mM NaCl, about 2.7 mM KCl, about 5% (w/v) mannitol, about 0.01% Pluronic F-68, and about 20 mM MgSO4.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.4, about 137 mM NaCl, about 2.7 mM KCl, about 5% (w/v) sorbitol, and about 20 mM MgSO4.
  • the composition e.g., the pharmaceutical composition comprises, in addition to the rAAV, about 10mM Phosphate pH 7.4, about 137 mM NaCl, about 2.7 mM KCl, about 5% (w/v) mannitol, and about 20 mM MgSO4.
  • the composition e.g., the pharmaceutical composition comprises recombinant AAV vector (rAAV), in 10 mM Phosphate pH 7.4, 200 mM NaCl, 5 mM KCl, 1% (w/v) mannitol, 0.0005% (w/v) IGEPAL CA 720 to a fil volume of 5ml.
  • the fil volume is 1ml, 2ml, 3ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml.
  • the composition e.g., the pharmaceutical composition comprises recombinant AAV vector (rAAV), in 20 mM Phosphate pH 7.4, 300 mM NaCl, 3 mM KCl, 3 % (w/v) Aty. Dkt. No.046192-000118WOPT mannitol, 0.001% (w/v) Brij S20 to a fil volume of 5ml.
  • the fil volume is 1ml, 2ml, 3ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml.
  • the composition e.g., the pharmaceutical composition comprises recombinant AAV vector (rAAV), in 20 mM Phosphate pH 7.4, 300 mM NaCl, 3 mM KCl, 3 % (w/v) sorbitol, 0.001% (w/v) Ecosurf SA-15 to a fil volume of 5ml.
  • the fil volume is 1ml, 2ml, 3ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml.
  • the composition e.g., the pharmaceutical composition comprises recombinant AAV vector (rAAV), in 10 mM Phosphate pH 7.4, 350 mM NaCl, 2.7 mM KCl, 5 % (w/v) sorbitol, 0.001% (w/v) poloxamer 188 to a fil volume of 5ml.
  • the fil volume is 1ml, 2ml, 3ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml.
  • the composition e.g., the pharmaceutical composition comprises recombinant AAV vector (rAAV), in 15 mM Phosphate pH 7.4, 375 mM NaCl, 3.5 mM KCl, 5 % (w/v) sorbitol, 0.0005% (w/v) Tergitol NP-10 to a fil volume of 5ml.
  • the fil volume is 1ml, 2ml, 3ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml.
  • the composition e.g., the pharmaceutical composition comprises recombinant AAV vector (rAAV), in 15 mM Phosphate pH 7.4, 375 mM NaCl, 3.5 mM KCl, 3 % (w/v) glycerol, 0.0005% (w/v) Tween 80 to a fil volume of 5ml.
  • the fil volume is 1ml, 2ml, 3ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml.
  • the AAV vector described herein is formulated to a concentration of ⁇ 5.0 ⁇ 1012 genome copies (GC) AAV/mL in a solution of 20 mM Tris, 1 mM magnesium chloride (MgCl2).6 H2O, 200 mM sodium chloride (NaCl), containing 0.01% (Weight to volume) Pluronic® F- 68 poloxamer, pH 8.0+/ ⁇ 0.2.
  • the formulation is stored as a frozen liquid in a 2 mL 13 mm Type I clear glass vial at ⁇ 60° C.
  • the AAV is administered with a diluent, if necessary to obtain the desired therapeutic dose.
  • an AAV described herein is formulated to a concentration of 2.0 ⁇ 1013 vg/mL in 20 mM Tris pH 8.0, 1 mM MgCl2, 200 mM NaCl, and 0.005% poloxamer 188.
  • the AAV vector is formulated at a concentration from about 1 ⁇ 1012 vg/ml to 1 ⁇ 1013 vg/ml; from about 10 mM to 30 mM Tris; from about 150 mM to 300 mM NaCl; from about 0.5 mM to 3.0 mM MgCl2.6H2O; and from about 0.002% (w/v) to 0.02% (w/v) poloxamer 188, such as Pluronic® F-68, wherein the formulation has a pH of from 7.8 to 8.2.
  • the AAV vector is formulated at a concentration of from 5.0 ⁇ 1012 to 1 ⁇ 1013 GC/ml; about 20 mM Tris; about 200 mM NaCl; about 1.0 mM MgCl2.6H2O; and about 0.01% (w/v) poloxamer 188, such as Pluronic® F-68; wherein the formulation has a pH of about 8.0.
  • the AAV vector described herein is formulated to comprise a recombinant AAV LAMP2B -encoding virus, a bufering agent, an isotonicity agent, a bulking agent and a surfactant.
  • the formulations of the present invention comprises any of the Aty. Dkt. No.046192-000118WOPT AAV- LAMP2B viruses described herein, p-100 ATGB or any of the other herein described vectors and/or are stable during storage at ⁇ 65° C. for at least 2 weeks.
  • the formulation of the present invention comprises any of the AAV- LAMP2B described herein formulated in a liquid solution that comprises about 1.42 mg/ml of sodium phosphate, dibasic, about 1.38 mg/ml of sodium phosphate, monobasic monohydrate, about 8.18 mg/ml sodium chloride, about 20 mg/ml mannitol and about 2 mg/ml poloxamer 188.
  • the pH of the formulation is 7.4.
  • the concentration of recombinant AAV virus in the above described formulation was 2E13 vg/ml.
  • the concentration of recombinant AAV virus in the above described formulation was 2E13 vg/ml.
  • Additional exemplary compositions/compositions comprising rAAV are described in International Patent Application No. PCT/US2022/0137279, US Patent Application No.17/725,086, and US Patent No.10,512,675 the content of which is incorporated herein by reference in its entirety.
  • the rAAV vectors containing a codon-optimized nucleic acid encoding a human LAMP2B polypeptide as disclosed herein, for use in the methods of administration as disclosed herein may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents.
  • compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • the delivery of one treatment e.g., gene therapy vectors
  • the delivery of the second (e.g., one or more therapeutic) is stil occurring when the delivery of the second (e.g., one or more therapeutic) begins, so that there is overlap in terms of administration. This is sometimes refered to herein as “simultaneous" or “concurrent delivery.”
  • the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration.
  • the second treatment is more effete, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partialy additive, wholy additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is stil detectable when the second is delivered.
  • the composition described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentialy.
  • the gene therapy vectors described herein can be administered first, and the one or more therapeutic can be administered second, or the order of administration can be reversed.
  • the gene therapy vectors and the one or more therapeutic can be Aty. Dkt. No.046192-000118WOPT administered during periods of active disorder, or during a period of remission or less active disease.
  • the gene therapy vectors can be administered before another treatment, concurently with the treatment, post-treatment, or during remission of the disorder.
  • the rAAV vectors as disclosed herein for use in the methods of administration as disclosed herein and the one or more therapeutic (e.g., second or third therapeutic), or al can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of each used individualy, e.g., as a monotherapy.
  • the administered amount or dosage of a rAAV vector as disclosed herein for use in the methods of administration as disclosed herein and the one or more therapeutic (e.g., second or third agent), or al is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each used individualy.
  • the amount or dosage of the rAAV vector as disclosed herein for use in the methods of administration as disclosed herein and the one or more therapeutic (e.g., second or third agent), or al, that results in a desired effect (e.g., treatment of a cardiovascular disease or heart disease) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each individualy required to achieve the same therapeutic effect.
  • the methods of administration of a rAAV vector as disclosed herein can deliver a rAAV vector disclosed herein alone, or in combination with an additional agent, for example, an immune modulator and/or a vasodilator as disclosed herein.
  • One aspect herein described is a transgenic mouse model that mimics Danon disease; i.e., a transgenic mouse having a genome that cannot express Lamp2 gene and/or protein. Accordingly, one aspect provides a Lamp2 gene deficient transgenic mouse, the transgenic mouse’s cels comprising deletions of exons 2-6 of the Lamp2 gene and a premature stop codon upstream of exon 7 of the Lamp2 gene. [00509] In one embodiment, the premature stop codon results in an out-of-frame shift of exons 7-9 of the Lamp2 gene. In one embodiment, the premature stop codon results in nonsense-mediated mRNA decay of the Lamp2 gene.
  • the transgenic mouse lacking Lamp2 expression exhibits a complete loss of the Lamp2 gene and/or protein expression.
  • the transgenic mouse lacking Lamp2 expression does not produce any known isoform of Lamp2.
  • the transgenic mouse lacking Lamp2 expression can be used to assess any combination of pathologies observed related to Danon disease, e.g., weakening of heart muscle or skeletal muscle, inteluctual disability, heart palpitations, or arrhymia.
  • the transgenic mouse having a genome that cannot express Lamp2 gene and/or protein is a mouse model for hypertrophic cardiomyopathy. Lamp2 gene variants can develop Aty. Dkt.
  • the mouse model disclosed herein is a hypertrophic cardiomyopathy mouse model.
  • the transgenic mouse having a genome that cannot express Lamp2 gene and/or protein is a mouse model any disease or disorder resulting from loss of Lamp2.
  • the mouse model is used to assess any disease or disorder resulting from loss of Lamp2 in cardiac tissue, e.g., ischemic cardiomyopathy.
  • the mouse model disclosed herein is a ischemic cardiomyopathy mouse model.
  • the mouse model is used to assess any disease or disorder resulting from loss of Lamp2 in muscle tissue, e.g., skeletal myopathy.
  • the mouse model disclosed herein is a skeletal myopathy mouse model.
  • the mouse model is used to assess any disease or disorder resulting from loss of Lamp2 in central nervous tissue, e.g., hippocampal dysfunction (see, e.g., Rothaug, Acta Neuropathologica Communications volume 3, Article number: 6 (2015).
  • the mouse model disclosed herein is a hippocampal dysfunction mouse model.
  • the mouse model is used to assess any disease or disorder resulting from loss of Lamp2 in retinal tissue, e.g., age-related macular degeneration (see, e.g., Notomi, PNAS, 116 (47) 23724-23734 (2019). Accordingly, in one embodiment, the mouse model disclosed herein is a age-related macular degeneration mouse model. [00518] In one embodiment, the mouse model is used to assess any disease or disorder resulting from loss of Lamp2, e.g., chaperone mediated autophagy (see, e.g., Robert, Autophagy, Volume 15, Issue 5 (2019)). Accordingly, in one embodiment, the mouse model disclosed herein is a chaperone mediated autophagy mouse model.
  • the mouse model is used to assess any disease or disorder resulting from loss of Lamp2, e.g., lysosomal storage diseases or disorders. Accordingly, in one embodiment, the mouse model disclosed herein is a lysosomal storage disease or disorder mouse model. [00520] In one embodiment, the mouse model is used to assess any disease or disorder resulting from loss of Lamp2, e.g., autophagy diseases or disorders. Accordingly, in one embodiment, the mouse model disclosed herein is an autophagy disease or disorder mouse model. [00521] In one embodiment, the mouse model is used to assess any disease or disorder resulting from loss of Lamp2, e.g., selective disabilities. Accordingly, in one embodiment, the mouse model disclosed herein is an an intellectual disability mouse model.
  • the Lamp2 gene knockout strategy entailed deletion of exons 2-6 of the 9 exon gene in the transgenic mouse’s cels. The remaining exons wil be out-of-frame with the presence of a premature stop codon in the deletion alele transcript that should trigger nonsense-mediated mRNA decay.
  • Cas guide RNAs targeting introns 1 and 6 were designed using Benchling software. Three guide RNAs Aty. Dkt. No.046192-000118WOPT each were selected for activity testing. Guide RNAs were cloned into a T7 promoter vector folowed by in vitro transcription and silica spin column purification. Functional testing was performed by transfecting a mouse embryonic fibroblast cel line with each guide RNA and Cas9 protein.
  • the target regions were amplified from transfected cel lysates using genotyping assays B and C listed below. Amplified PCR products were analyzed by Sanger sequencing folowed by ICE Analysis (Synthego) to detect cleavage events.
  • One active guide RNA at each site was selected for genome editing in embryos: i1g90B (5’- TAGTCGATCCTTGATGCGGA -3”) (SEQ ID NO: 119) and i6g75T (5’- AACAGTGGTAGGTGTATGCG -3’) (SEQ ID NO: 120).
  • Embryo Electroporation, Founder Generation and Mating Another aspect provided herein is a method of making a transgenic mouse having a genome that cannot express functional Lamp2 expression comprising contacting a mouse embryo or mouse embryonic cels with at least one sgRNAsthat target exons 1 and 6 of the Lamp2 gene and a Cas nuclease. [00525] In one embodiment, the contacting is performed by electroporation. [00526] In one embodiment, the mouse embryo is a C57BL/6J mouse embryo.
  • the at least one sgRNA is selected from TAGTCGATCCTTGATGCGGA (SEQ ID NO: 119) and AACAGTGGTAGGTGTATGCG (SEQ ID NO: 120).
  • the contacted mouse embryos are implanted in pseudopregnant recipient female mice.
  • C57BL/6J zygotes were electroporated with a CRISPR reagent mix comprised of 1200 nM eSpCas9 protein and 1800 nM each guide RNA.
  • Electroporated embryos were implanted in pseudopregnant recipient females and resultant pups were screened by PCR as described below for the presence of the approximately 11.5 kb deletion between the two guide RNA cut sites. Multiple founders were identified containing the desired deletion event. Sequencing was performed on a subset of founders to determine the deletion endpoints. Two male founders were mated to wild- type C57BL/6J females for germline transmission of the deletion alele. Both founders transmited the deletion alele to offspring. Since Lamp2 is an X-linked gene, only female N1 offspring contained the deletion alele. The N1 females from founder line #15 were sequenced to confirm the presence of a 11,496 bp deletion in this founder line.
  • the N1 heterozygous females were backcrossed to C57BL/6J males and to founder #15 to generate hemizygous males and homozygous females.
  • Genotyping Summary [00531] The Lamp2 knockout was detected by PCR Assay A which produces a 539 bp deletion alele band in animals from founder line #15. The sequence of this PCR product detailing the deletion junctions is shown in the Appendix. Assay A primers span a region of 12,035 bp between guide RNA target sites in the wild-type Ppp1r1a locus, such that a band is not obtained with the wild-type alele. Aty. Dkt.
  • Intron 1 gRNA Cut Site Spanning Primers Lamp2 i1ScF1: TGACTGGGTTGAGAGAAGCAG (SEQ ID NO: 123) Lamp2 i1ScR1: GGCAGATATGTTTACTTACATTGGTTTGG (SEQ ID NO: 124) Expected product from knockout alele: none Expected product from WT alele: 579 bp [00536] Assay C.
  • ordinal indicators – such as “first,” “second,” “third,” etc. – for identified elements are used to distinguish between the elements, and do not indicate or imply a required or limited number of such elements, and do not indicate a particular position or order of such elements unless otherwise specificaly stated.
  • Al methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
  • the use of any and al examples, or exemplary language (e.g., “such as”) provided herein is intended merely to beter illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.
  • the term "about,” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • “and/or” refers to and encompasses any and al possible combinations of one or more of the associated listed items, as wel as the lack of combinations when interpreted in the alternative (“or").
  • the transitional phrase "consisting essentialy of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materialy afect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03.
  • amino acid can be selected from any subset of these amino acid(s) for example A, G, I or L; A, G, I or V; A or G; only L; etc. as if each such subcombination is expressly set forth herein.
  • amino acid can be disclaimed (e.g., by negative proviso).
  • the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer is expressly set forth herein.
  • parvovirus encompasses the family Parvoviridae, including autonomously replicating parvoviruses and dependoviruses.
  • the autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus.
  • Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, B19 virus, and any other autonomous parvovirus now known or later discovered.
  • Other autonomous parvoviruses are known to those skiled in the art.
  • AAV adeno-associated virus
  • AAV includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, any AAV disclosed in Table 2 herein, and any other AAV now known or later discovered.
  • capsid structures of autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincot-Raven Publishers). See also, description of the crystal structure of AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci.99:10405-10), AAV4 (Padron et al., (2005) J. Viral.79: 5047-58), AAV5 (Walters et al., (2004) J. Viral.78: 3361- 71) and CPV (Xie et al., (1996) J. Mal.
  • tropism refers to preferential entry of the virus into certain cels or tissues, optionaly folowed by expression (e.g., transcription and, optionaly, translation) of a sequence(s) caried by the viral genome in the cel, e.g., for a recombinant virus, expression of a heterologous nucleic acid(s) of interest.
  • systemic tropism and “systemic transduction” (and equivalent terms) indicate that the virus capsid or virus vector of the invention exhibits tropism for and/or transduces tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney and/or pancreas).
  • selective tropism or “specific tropism” means delivery of virus vectors to and/or specific transduction of certain target cels and/or certain tissues.
  • “eficient transduction” or “eficient tropism,” or similar terms can be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or tropism, respectively, of the control).
  • the virus vector efficiently transduces or has tropism for muscle cels.
  • Suitable controls wil depend on a variety of factors including the desired tropism and/or transduction profile.
  • a virus “does not efficiently transduce” or “does not have efficient tropism” for a target tissue, or similar terms by reference to a suitable control.
  • the virus vector does not efficiently transduce (i.e., has does not have tropism) for kidney, gonads and/or germ cels.
  • transduction e.g., undesirable transduction
  • tissue(s) e.g., kidney
  • tissue(s) e.g., kidney
  • the level of transduction of the desired target tissue(s) e.g., liver, skeletal muscle, diaphragm muscle, cardiac muscle and/or cels of the central nervous system.
  • desired target tissue(s) e.g., liver, skeletal muscle, diaphragm muscle, cardiac muscle and/or cels of the central nervous system.
  • a "polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturaly occurring and non-naturaly occuring nucleotides), but in representative embodiments are either single or double stranded DNA sequences.
  • the terms “heterologous nucleotide sequence” and “heterologous nucleic acid molecule” are used interchangeably herein and refer to a nucleic acid sequence that is not naturaly occuring in the virus.
  • the heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide and/or nontranslated RNA of interest (e.g., for delivery to a cel and/or subject).
  • a “chimeric nucleic acid” comprises two or more nucleic acid sequences covalently linked together to encode a fusion polypeptide.
  • the nucleic acids may be DNA, RNA, or a hybrid thereof.
  • the term “fusion polypeptide” comprises two or more polypeptides covalently linked together, typicaly by peptide bonding.
  • an "isolated" polynucleotide e.g., an "isolated DNA” or an “isolated RNA" means a polynucleotide at least partialy separated from at least some of the other components of the naturaly occurring organism or virus, for example; the cel or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • an "isolated" nucleotide is enriched by at least about 10-fold, 100'-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • an "isolated" polypeptide means a polypeptide that is at least partialy separated from at least some of the other components of the naturaly occurring organism or virus, for example, the cel or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • An "isolated cel” refers to a cel that is separated from other components with which it is normaly associated in its natural state.
  • an isolated cel can be a cel in culture medium and/or a cel in a pharmaceuticaly acceptable carrier of this invention.
  • an isolated cel can be delivered to and/or introduced into a subject.
  • an isolated cel can be a cel that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.
  • a population of virions can be generated by any of the methods described herein. In one embodiment, the population is at least 101 virions.
  • the population is at least 102 virions, at least 103, virions, at least 104 virions, at least 105 virions, at least 106 virions, at least 107 virions, at least 108 virions, at least 109 virions, at least 1010 virions, at least 1011 virions, at least 1012 virions, at least 1013 virions, at least 1014 virions, at least 1015 virions, at least 1016 virions, or at least 1017 virions.
  • a population of virions can be heterogeneous or can be homogeneous (e.g., Aty. Dkt. No.046192-000118WOPT substantialy homogeneous or completely homogeneous).
  • a substantialy homogeneous population is at least 90% of identical virions (e.g., the desired virion), and can be at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% of identical virions.
  • a population of virions that is completely homogeneous contains only identical virions.
  • virus vector or virus particle or population of virus particles it is meant that the virus vector or virus particle or population of virus particles is at least partialy separated from at least some of the other components in the starting material.
  • an "isolated” or “purified” virus vector or virus particle or population of virus particles is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • eficient transduction or “eficient tropism,” or similar terms, can be determined by reference to a suitable control (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or tropism, respectively, of the control).
  • the virus vector ly transduces or has tropism for neuronal cels and cardiomyocytes.
  • a "therapeutic polypeptide” is a polypeptide that can aleviate, reduce, prevent, delay and/or stabilize symptoms that result from an absence or defect in a protein in a cel or subject and/or is a polypeptide that otherwise confers a benefit to a subject, e.g., enzyme replacement to reduce or eliminate symptoms of a disease, or improvement in transplant survivability or induction of an immune response.
  • heterologous nucleotide sequence and “heterologous nucleic acid molecule” are used interchangeably herein and refer to a nucleic acid sequence that is not naturaly occuring in the virus.
  • the heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide and/or nontranslated RNA of interest (e.g., for delivery to a cel and/or subject), for example the LAMP2B polypeptide.
  • virus vector e.g., AAV
  • vector genome e.g., viral DNA [vDNA]
  • vector may be used to refer to the vector genome/vDNA alone.
  • An "rAAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) that comprises one or more heterologous nucleic acid sequences. rAAV vectors generaly require only the inverted terminal repeat(s) (TR(s)) in cis to generate virus.
  • Al other viral sequences are dispensable Aty. Dkt. No.046192-000118WOPT and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbial. Immunol.158:97). Typicaly, the rAAV vector genome wil only retain the one or more TR sequence so as to maximize the size of the transgene that can be efficiently packaged by the vector.
  • the structural and non- structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cel).
  • the rAAV vector genome comprises at least one ITR sequence (e.g., AAV TR sequence), optionaly two ITRs (e.g., two AAV TRs), which typicaly wil be at the 5' and 3' ends of the vector genome and flank the heterologous nucleic acid, but need not be contiguous thereto.
  • the TRs can be the same or diferent from each other.
  • terminal repeat or "TR” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., an ITR that mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like).
  • the TR can be an AAV TR or a non-AAV TR.
  • a non-AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • the TR can be partialy or completely synthetic, such as the "double-D sequence" as described in United States Patent No.5,478,745 to Samulski et al.
  • An "AAV terminal repeat” or “AAV TR,” including an “AAV inverted terminal repeat” or “AAV ITR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any other AAV now known or later discovered.
  • An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV TR or AAV ITR sequence may be altered by insertion, deletion, truncation and/or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like.
  • AAV proteins VP1, VP2 and VP3 are capsid proteins that interact together to form an AAV capsid of an icosahedral symmetry.
  • VP1.5 is an AAV capsid protein described in US Publication No. 2014/0037585.
  • the virus vectors of the invention can further be "targeted" virus vectors (e.g., having a directed tropism) and/or a "hybrid" parvovirus (i.e., in which the viral TRs and viral capsid are from diferent parvoviruses) as described in international patent publication WO 00/28004 and Chao et al., (2000) Molecular Therapy 2:619.
  • the virus vectors of the invention can further be duplexed parvovirus particles as described in international patent publication WO O1/92551 (the disclosure of which is incorporated herein by reference in its entirety).
  • double stranded (duplex) genomes can be packaged into the virus capsids of the invention.
  • the viral capsid or genomic elements can contain other modifications, including insertions, deletions and/or substitutions. Aty. Dkt.
  • a "chimeric' capsid protein as used herein means an AAV capsid protein (e.g., any one or more of VP1, VP2 or VP3) that has been modified by substitutions in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to wild type, as wel as insertions and/or deletions of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to wild type.
  • complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wild type domain, functional region, epitope, etc. of a different AAV serotype, in any combination, to produce a chimeric capsid protein of this invention.
  • Production of a chimeric capsid protein can be carried out according to protocols wel known in the art and a significant number of chimeric capsid proteins are described in the literature as wel as herein that can be included in the capsid of this invention.
  • haploid AAV shal mean that AAV as described in International Application WO2018/170310, or US Application US2018/037149, which are incorporated herein in their entirety by reference.
  • a population of virions is a haploid AAV population where a virion particle can be constructed wherein at least one viral protein from the group consisting of AAV capsid proteins, VP1, VP2 and VP3, is diferent from at least one of the other viral proteins, required to form the virion particle capable of encapsulating an AAV genome.
  • VP1, VP2, and/or VP3 For each viral protein present (VP1, VP2, and/or VP3), that protein is the same type (e.g., al AAV2 VP1).
  • At least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not a chimeric.
  • VP1 and VP2 are chimeric and only VP3 is non- chimeric.
  • only the viral particle composed of VP1/VP2 from the chimeric AAV2/8 (the N-terminus of AAV2 and the C-terminus of AAV8) paired with only VP3 from AAV2; or only the chimeric VP1/VP228m-2P3 (the N-terminal from AAV8 and the C-terminal from AAV2 without mutation of VP3 start codon) paired with only VP3 from AAV2.
  • VP3 is chimeric and VP1 and VP2 are non-chimeric.
  • at least one of the viral proteins is from a completely diferent serotype.
  • a "hybrid" AAV vector or parvovirus refers to a rAAV vector where the viral TRs or ITRs and viral capsid are from diferent parvoviruses. Hybrid vectors are described in international patent publication WO 00/28004 and Chao et al., (2000) Molecular Therapy 2:619.
  • a hybrid AAV vector typicaly comprises the adenovirus 5' and 3' cis ITR sequences sufficient for adenovirus replication and packaging (i.e., the adenovirus terminal repeats and PAC sequence).
  • polyploid AAV refers to a AAV vector which is composed of capsids from two or more AAV serotypes, e.g., and can take advantages from individual serotypes for higher transduction but not in certain embodiments eliminate the tropism from the parents.
  • amino acid encompasses any naturaly occurring amino acid, modified forms thereof, and synthetic amino acids. Naturaly occuring, levorotatory (L-) amino acids Aty.
  • Dkt. No.046192-000118WOPT are disclosed in Table 2 of US Publication 2018/0371496, which is incorporated herein in its entirety.
  • the amino acid can be a modified amino acid residue (nonlimiting examples are shown in Table 4 of US Publication of US Publication 2018/0371496) and/or can be an amino acid that is modified by post-translation modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation).
  • the non-naturaly occuring amino acid can be an “unnatural” amino acid as described by Wang et al., Annu Rev Biophys Biomol Struct.35:225-49 (2006).
  • amino acids can advantageously be used to chemicaly link molecules of interest to the AAV capsid protein.
  • this language also indicates that the amino acid can be selected from any subset of these amino acid(s) for example A, G, I or L; A, G, I or V; A or G; only L; etc. as if each such subcombination is expressly set forth herein.
  • such language also indicates that one or more of the specified amino acids can be disclaimed (e.g., by negative proviso).
  • the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer is expressly set forth herein.
  • promoter refers to a region of DNA that generaly is located upstream of a nucleic acid sequence to be transcribed that is needed for transcription to occur, i.e., which initiates transcription. Promoters permit the proper activation or repression of transcription of a coding sequence under their control. A promoter typicaly contains specific sequences that are recognized and bound by plurality of TFs.
  • TFs bind to the promoter sequences and result in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene.
  • a great many promoters are known in the art.
  • the term “synthetic promoter” as used herein relates to a promoter that does not occur in nature. Parts of the synthetic promoter may be naturaly occurring (e.g., the minimal promoter), but the synthetic promoter as a complete entity is not naturaly occuring.
  • “minimal promoter” also known as the “core promoter” refers to a short DNA segment which is inactive or largely inactive by itself, but can mediate transcription when combined with other transcription regulatory elements.
  • Minimum promoter sequence can be derived from various diferent sources, including prokaryotic and eukaryotic genes. Examples of minimal promoters are discussed above, and include the dopamine beta-hydroxylase gene minimum promoter, cytomegalovirus (CMV) immediate early gene minimum promoter (CMV-MP), and the herpes thymidine kinase minimal promoter (MinTK).
  • a minimal promoter typicaly comprises the transcription start site (TSS) and elements directly upstream, a binding site for RNA polymerase I, and general transcription factor binding sites (often a TATA box).
  • TSS transcription start site
  • proximal promoter relates to the minimal promoter plus the proximal sequence upstream of the gene that tends to contain primary regulatory elements.
  • the proximal Aty. Dkt. No.046192-000118WOPT promoter can be a naturaly occurring muscle -specific proximal promoter. However, the proximal promoter can be synthetic.
  • a “functional variant” of a promoter or other nucleic acid sequence in the context of the present invention is a variant of a reference sequence that retains the ability to function in the same way as the reference sequence, e.g., as a muscle -specific promoter.
  • Alternative terms for such functional variants include “biological equivalents” or “equivalents”.
  • polypeptide refers to a polypeptide resulting from one or more amino acid substitution, deletion or insertions, which retains a substantial amount of one or more biological activities (e.g., activity involved in treating Danon disease) of the reference polypeptide, e.g., by at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more, as determined by various available in vitro and/or in vivo assays.
  • biological activities e.g., activity involved in treating Danon disease
  • the activity of the Factor VII polypeptide described herein is its activity as a Factor IXa cofactor.
  • CpG island refers to a region within a polynucleotide having a statisticaly elevated density of CpG dinucleotides.
  • An "exogenous” molecule is a molecule that is introduced into a subject (e.g., by introducing into cels of the subject) by one or more genetic, biochemical or other methods.
  • An exogenous molecule can comprise, for example, a functioning version of an absent or malfunctioning endogenous molecule.
  • an "endogenous" molecule is one that is present naturaly in subject or a cel.
  • expression cassete refers minimaly to a nucleic acid that encodes a polypeptide operatively linked to a promoter.
  • the coding region may further be operatively linked to other elements such as a polyA sequence and other regulatory elements such as 5’ UTR, enhancers, etc.
  • muscle-specific or “muscle-specific expression” when in reference to a promoter refers to the ability of promoter to enhance or drive expression of a gene in the muscle (or in muscle- derived cels) in a preferential or predominant manner as compared to other tissues (e.g., spleen, muscle, heart, lung, and brain). Expression of the gene can be in the form of mRNA or protein. In some embodiments, muscle-specific expression is such that there is negligible expression in other (i.e., non- muscle) tissues or cels, i.e., expression is highly muscle-specific.
  • a muscle-specific promoter drives expression preferentialy in the muscle, it can also drive expression of the gene in another tissue of interest at a lower level, e.g., liver.
  • the skiled person can thus easily determine whether any variant of the muscle specific promoter recited above remains functional (i.e., it is a functional variant as defined above).
  • any given promoter to be assessed can be operably linked to a minimal promoter (e.g., positioned upstream of CMV-MP) and the ability of the promoter to drive muscle specific expression Aty. Dkt. No.046192-000118WOPT of a gene (typicaly a reporter gene) is measured.
  • a promoter to drive muscle specific expression can be readily assessed by the skiled person (e.g., as described in the examples below).
  • Expression levels of a gene driven by a variant of a reference promoter can be compared to the expression levels driven by the reference sequence.
  • muscle specific expression levels driven by a variant promoter are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the expression levels driven by the reference promoter, it can be said that the variant remains functional.
  • Suitable nucleic acid constructs and reporter assays to assess muscle-specific expression enhancement can easily be constructed, and the examples set out below give suitable methodologies.
  • Muscle specificity can be identified wherein the expression of a gene (e.g., a therapeutic or reporter gene) occurs preferentialy or predominantly in muscle derived cels.
  • a gene e.g., a therapeutic or reporter gene
  • Preferential or predominant expression can be defined, for example, where the level of expression is significantly greater in muscle derived cels than in other types of cels (i.e., non- muscle derived cels).
  • expression in muscle derived cels is suitably at least 5-fold higher than non- muscle cels, preferably at least 10-fold higher than non-liver cels, and it may be 50-fold higher or more in some cases.
  • muscle specific expression can suitably be demonstrated via a comparison of expression levels in a cardiac or skeltal cel line or muscle primary cels, compared with expression levels in a kidney-derived cel line (e.g., HEK-293), a cervical tissue-derived cel line (e.g., HeLa) and/or a lung-derived cel line (e.g., A549).
  • the synthetic muscle specific promoters of the present invention are preferably suitable for promoting expression in the muscle of a subject, e.g., driving muscle specific expression of a transgene, preferably a therapeutic transgene.
  • Prefered synthetic muscle specific promoters of the present invention are suitable for promoting muscle specific transgene expression and have an activity in muscle cels which is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350% or 400% of the activity of the non-muscle specific promoter.
  • the terms "identity” and “identical” and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules.
  • Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originaly described by Altschul et al.1990 (J Mol Biol 215: 403- 10), such as the "Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Let 174: 247-250).
  • BLAST Basic Local Alignment Search Tool
  • Methods for aligning sequences for comparison are wel-known in the art.
  • Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math.2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
  • BLASTTM Basic Local Alignment Search Tool
  • Bethesda, MD National Center for Biotechnology Information
  • Blastn Align Sequence Nucleotide BLAST
  • Nucleic acid sequences with even greater similarity to the reference sequences show increasing percentage identity when assessed by this method. Typicaly, the percentage sequence identity is calculated over the entire length of the sequence.
  • a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the folowing scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2.
  • the percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.
  • the term “synthetic” as used herein means a nucleic acid molecule that does not occur in nature.
  • Synthetic nucleic acid expression constructs of the present invention are produced artificialy, typicaly by recombinant technologies.
  • Such synthetic nucleic acids may contain naturaly occurring sequences (e.g., promoter, enhancer, intron, and other such regulatory sequences), but these are present in a non-naturaly occuring context.
  • a synthetic gene or portion of a gene
  • typicaly contains one or more nucleic acid sequences that are not contiguous in nature (chimeric sequences), and/or may encompass substitutions, insertions, and deletions and combinations thereof.
  • a “spacer sequence” or “spacer” as used herein is a nucleic acid sequence that separates two functional nucleic acid sequences.
  • treat By the terms “treat,” “treating” or “treatment of' (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partialy improved or stabilized and/or that some aleviation, mitigation, decrease or stabilization in at least one clinical symptom is Aty. Dkt. No.046192-000118WOPT achieved and/or there is a delay in the progression of the disease or disorder.
  • prevent refers to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
  • the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is substantialy less than what would occur in the absence of the present invention.
  • a “treatment effete” amount as used herein is an amount that is adequate to provide some improvement or benefit to the subject.
  • a “treatment effete” amount is an amount that wil provide some aleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject.
  • a "prevention effete" amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention.
  • Those skiled in the art wil appreciate that the level of prevention need not be complete, as long as some preventative benefit is provided to the subject.
  • the phrase a “therapeuticaly effective amount” and like phrases mean a dose or plasma concentration in a subject that provides the desired specific pharmacological effect, e.g., to express a therapeutic gene in the muscle, and secretion into the plasma.
  • a therapeuticaly effective amount may not always be effective in treating the conditions described herein, even though such dosage is deemed to be a therapeuticaly effective amount by those of skil in the art.
  • the therapeuticaly effete amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the disease or condition being treated.
  • the terms “individual,” “subject,” and “patient” are used interchangeably, and refer to any individual subject with a disease or condition in need of treatment.
  • the subject may be a primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or bovine, and the like.
  • a codon-optimized nucleic acid encoding a LAMP2B polypeptide wherein the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NOs 1-3, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • the codon-optimized nucleic acid of paragraph 1 wherein the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NO: 1, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NO: 2, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto. 4.
  • nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NO: 3, or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto. 5.
  • the codon-optimized nucleic acid of any preceding paragraph wherein the LAMP2B polypeptide is a functional variant of the LAMP2B polypeptide having the amino acid sequence shown in SEQ ID NO: 9, or at least 60%, or 70%, or 80%, 85% or 90% or 95%, or 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 9.
  • the codon-optimized nucleic acid of any preceding paragraph that is comprised within a nucleic acid construct that further comprises viral sequence elements that facilitate integration and expression.
  • the muscle-specific promoter comprises a nucleic acid sequence selected from the group consisting of SP0497, SP0498, SP0499, SP0500, SP0508, SP0509, SP0510, SP0511, SP0512, SP0513, SP0522, SP0524, Syn100, and Spc5-12 or a nucleic acid having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto. 12.
  • the expression cassete of any preceding paragraph further comprising one or more additional regulatory elements and/or a poly A sequence.
  • the one or more additional regulatory elements is selected from the group consisting of an enhancer, a 5’ untranslated region (5’UTR), an intron, a reverse RNA pol I terminator sequence, and combinations thereof.
  • the intron is an IVS intron.
  • the expression cassete of any preceding paragraph, wherein the intron is an IVS intron comprises a nucleic acid sequence of SEQ ID NO: 7, a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or 99% sequence identity thereto.
  • a recombinant adeno-associated virus (rAAV) vector comprising in its genome the expression cassete of any preceding paragraph. 17.
  • a recombinant adeno-associated virus (rAAV) vector comprising in its genome: a) 5’ and 3’ AAV inverted terminal repeats (ITR) sequences; and b) located between the 5’ and 3’ ITRs, the expression cassete specified in any preceding paragraphs.
  • the AAV genome further comprises at least one of: a) a 5’ ITR; b) an intron; c) a poly A sequence; and d) a 3’ ITR.
  • No.046192-000118WOPT b) a muscle-specific promoter; c) an intron d) a codon-optimized nucleic acid specified in any preceding paragraphs; e) a poly A sequence; f) a 3’ ITR.
  • the rAAV vector of any preceding paragraph wherein one or more CpG islands in the ITR are removed.
  • the rAAV vector of any preceding paragraph, wherein the poly A sequence is a ful length SV40 polyA sequence or HGF poly A sequence.
  • poly A sequence is selected from SEQ ID NO: 8, or a nucleic acid sequence at least 80% sequence identity thereto.
  • the rAAV vector of any preceding paragraph, wherein the rAAV vector is a chimeric AAV vector, haploid AAV vector, a hybrid AAV vector or polyploid AAV vector.
  • rAAV vector of any preceding paragraph wherein the rAAV vector is a rational haploid capsid, a mosaic AAV capsid, a chemicaly modified AAV capsid, or a AAV capsid from any AAV serotypes.
  • rAAV capsid is a capsid from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6i, AAV1P5i, AAV4A1i, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2i, AAVS10P1i, AAV JEA, AAV
  • a pharmaceutical composition comprising the rAAV vector of any preceding paragraph in a pharmaceuticaly acceptable carrier.
  • a method for treating a subject in need of LAMP2B the method comprising administering the rAAV vectors of any preceding paragraph or the pharmaceutical composition of any preceding paragraph, or the expression cassete of any preceding paragraph or the codon-optimized nucleic acid of any preceding paragraph, to the subject.
  • a method for treating danon disease the method comprising administering the rAAV vectors of any preceding paragraph or the pharmaceutical composition of any preceding Aty. Dkt. No.046192-000118WOPT paragraph, or the expression cassete of any preceding paragraph or the codon-optimized nucleic acid of any preceding paragraph, to the subject.
  • administering to the subject is by systemic administration.
  • systemic administration is by intravenous administration.
  • administering to the subject is by local administration.
  • local administration is by injection to the heart.
  • rAAV vector is administered at a dosage range of between 1.0E8 vg to 5.0E14vg.
  • a rAAV vector in the preparation of a medicament for treating danon disease, the medicament comprising the rAAV vector specified in any preceding paragraph.
  • An expression cassete containing the codon-optimized nucleic acid sequence of SEQ ID NO: 2 operably linked to a muscle-specific promoter having a sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity thereto.
  • a recombinant adeno-associated virus (rAAV) vector comprising in its genome the expression cassete of any preceding paragraph.
  • An expression cassete comprising SP0524 promoter sequence, wherein the expression cassete is used to treat Danon disease.
  • An expression cassete of any preceding paragraph wherein the expression cassete further comprises nucleic acid sequence that encodes LAMP2B polypeptide sequence as set forth by SEQ ID NO: 1-3.
  • An expression cassete of any preceding paragraph further comprising an intron sequence, wherein the intron is selected from the group consisting of an IVS sequence, a MVM sequence, a HBB2 sequence, an CMVIE intron sequence, a UBC intron sequence, and a SV40 sequence.
  • An expression cassete of any preceding paragraph further comprising a poly A sequence.
  • An expression cassete of any preceding paragraphs comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:s 60, 61, 63, 79, 81 and 82, or a sequence having at least 80% sequence identity thereto.
  • An expression cassete of any preceding paragraphs is delivered to a subject in need thereof by using a non-viral vector.
  • An expression cassete of any preceding paragraphs is delivered to a subject in need thereof by using a viral vector.
  • the viral vector of any preceding paragraphs is recombinant adeno associated virus (rAAV) vector.
  • the viral vector of any preceding paragraphs is a AAV2i8 vector.
  • a transgenic mouse having a genome that cannot express functional Lamp2 expression the transgenic mouse’s cels comprising: deletions of exons 2-6 of the Lamp2 gene, and Aty. Dkt. No.046192-000118WOPT a premature stop codon upstream of exon 7 of the Lamp2 gene.
  • the premature stop codon results in an out-of-frame shift of exons 7-9 of the Lamp2 gene.
  • the premature stop codon results in nonsense-mediated mRNA decay of the Lamp2 gene.
  • the mouse exhibits a complete loss of the Lamp2 expression.
  • a method of making a transgenic mouse having a genome that cannot express functional Lamp2 expression comprising contacting a mouse embryo with at least one sgRNAsthat target exons 1 and 6 of the Lamp2 gene, and a Cas nuclease.
  • the contacting is electroporation.
  • the mouse embryo is a C57BL/6J mouse embryo.
  • the at least one sgRNA is selected from TAGTCGATCCTTGATGCGGA (SEQ ID NO: 119) and AACAGTGGTAGGTGTATGCG (SEQ ID NO: 120).
  • MCB Master Cel Bank
  • FBS fetal bovine serum
  • the MCB cels were cultured and passaged over a 4 week period while the amount of FBS in the tissue culture media was gradualy reduced from 10% to 2.5%.
  • the cels were then transfered from DMEM 2.5% FBS into serum free suspension media and grown in shaker flasks.
  • the cels were then cultured in the serum-free media for another 3 weeks while their growth rate and viability is monitored.
  • the adapted cels were then expanded and frozen down.
  • the cels were counted using a ViCel XR Viability Analyzer (Beckman Coulter) and diluted for transfection.
  • the folowing reagents were added to a conical tube in this order: plasmid DNA, OPTIMEM® I (Gibco) or OptiPro SFM (Gibco), or other serum free compatible transfection media, and then the transfection reagent at a specific ratio to plasmid DNA or close ended linear duplexed DNA.
  • the plasmid DNA or close ended linear duplexed DNA of the transgene construct had a sequence comprising a heterologous nucleic acid sequence of any of the codon-optimized LAMP2B transgene as described herein (e.g., any of SEQ ID NOs 1-3) operatively linked to any of the muscle specific promoters described herein (e.g., any of SEQ ID NOs 4-6).
  • the cocktail further comprised a Packaging plasmid encoding Rep2 and serotype- specific CapAAV2i8: AAV-Rep/Cap, and the Ad-Helper plasmid (XX680: or XX85 encoding adenoviral helper sequences as disclosed in WO2023250416 which is incorporated by reference).
  • the cocktail was inverted to mix prior to being incubated at room temperature.
  • the transfection cocktail was then pipeted into the flasks and placed back in the shaker/incubator. Al optimization studies were caried out at 30 mL culture volumes folowed by validation at larger culture volumes. Cels were harvested 48 hours post-transfection.
  • rAAV comprising the expression constructs of the invention was manufactured using Pro10 cels as described in US Patent No: 9,441,206, which is incorporated by reference in its entirety.
  • Production of rAAV using wave bioreactor systems Wave bags were seeded 2 days prior to transfection. Two days post-seeding the wave bag, cel culture counts were taken and the cel culture was then expanded/diluted before transfection. The wave bioreactor cel culture was then transfected. Cel culture was harvested from the wave bioreactor bag at least 48 hours post-induction.
  • the pelet could either be stored in NLT- 60°C or continued through purification.
  • the cel pelet was then resuspended in 5 mL of DNase bufer (5 mM CaC12, 5 mM MgC12, 50 mM Tris-HC1 pH 8.0) folowed by sonication to lyse the cels efficiently.300 ul was then removed and placed into a 1.5 mL microfuge tube.140 units of DNase I was then added to each sample and incubated at 37°C for 1 hour. To determine the Aty. Dkt.
  • DNase bufer 5 mM CaC12, 5 mM MgC12, 50 mM Tris-HC1 pH 8.0
  • the nylon membrane was soaked in 0.4 M Tris-HC1, pH 7.5 and then cross linked using UV strata linker 1800 (Stratagene) at 600 ujou1s x 100.
  • the membrane was then pre-hybridized in CHURCH buffer (1% BSA, 7% SDS, 1 mM EDTA, 0.5 M Na3PO4, pH 7.5). After pre-hybridization, the membrane was hybridized overnight with a 32P-CTP labeled transgene probe (Roche Random Prime DNA labeling kit). The folowing day, the membrane was washed with low stringency SSC buffer (1xSSC, 0.1% SDS) and high stringency (0.1xSSC, 0.1% SDS).
  • HeLaRC-32 cels (Chadeuf et al., J Gene Med.2:260 (2000) were plated at 2x105 cels/wel of a 24 wel plate and incubated at 37°C overnight. The cels were observed for 90-100% confluence.50 mL of DMEM with 2% FBS, 1% Pen/Strep is pre-warmed, and adenovirus (d1309)was added at a MOI of 10. The d1309 containing media was aliquoted in 900 ul fractions and used to dilute the rAAV in a series of ten-fold dilutions. The rAAV was then plated at 400 ⁇ l and alowed to incubate for 48 hours at 37°C.
  • Purified dialyzed rAAV vectors were placed on a 400-mesh glow-discharged carbon grid by inversion of the grid on a 20 ul drop of virus. The grid was then washed 2 times by inversion on a 20 ul drop of ddH2O folowed by inversion of the grid onto a 20 ul drop of 2% uranyl acetate for 30 seconds. The grids were bloted dry by gently touching Whatman paper to the edges of the grids. Each vector was visualized using a Zeiss EM 910 electron microscope.
  • EXAMPLE 2 Three promoters (SEQ ID NO: 4-6) and a benchmark (e.g., pDA013 having a CAG promter) were placed upstream of a coding sequence encoding a codon optimized LAMP2B sequence (SEQ ID NO: 1-3) and packaged within an AAV2i8 capsid; their potency was analyzed folowing intravenous injection of the AAV vectors to C57BL/6J mice over the course of 4 weeks.
  • the Syn100 promoter is as described in Qiao et al 2014 or International Publication No. WO2022076556, the contents of which are incorporated herein by reference in its entirety.
  • AAV production [00626] AAV vectors described in this Example were produced by triple transfection in high density Pro10 cels. Helper plasmid xx680 and RepCap plasmid 2/2i8 were used. Cels were harvested on day three post-transfection and lysed via sonication. Lysates were purified by iodixanol gradient and concentrated via Amicon filtration units. The produced AAVs were quantified by PCR-based methods as described herein below and purity of each was confirmed by silver stain. Aty. Dkt.
  • test article were diluted in saline to achieve the desired concentrations of vg/mouse which were administered at a fixed dosing volume of 150 ⁇ L.
  • Al dosing solutions were formulated according to independently verified dilution sheets. Each dilution was witnessed by a verifier and was signed off as such within the dilution sheets.
  • the test article was pipeted ten times to ensure uniformity and was administered within one hour of preparation. Al stock solutions, and remaining test articles were stored at -80°C for potential further analysis.
  • Clinical Observations Any clinical observations were documented while observing the animals, and could include, but wil not be limited to, changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior. Al animals in poor health were identified for further monitoring folowed by potential unscheduled euthanasia, and tissue harvest.
  • Body Weights [00633] Each animal was weighted individualy prior to dosing, and once a week until end of study. Body weight loss in excess of 20% was noted and flagged for further clinical observation. Loss in excess of 30% is grounds for unscheduled euthanasia.
  • RNA Later Tissue Colection Samples stored in RNA Later were to be placed into a cryovial filed with 1 mL of RNA later solution. Samples sat at 4C for at least 24 hours, not to exceed 30 days. After this period, the RNA later solution were separated from the sample.
  • Total protein stain and hLAMP2B immunobloting [00657] Total protein was stained using the RevertTM 700 Total Protein Stain kit (Li-Cor 926-11021) and imaged on a Li-Cor Odyssey® M system. Immunobloting was caried out with primary mouse monoclonal (H4B4) anti-hLAMP2 primary antibody (Abcam #25631) at a concentration of 1:100. Detection was achieved using IRDye® 800CW conjugated Donkey anti-Mouse secondary antibody (Li- cor 926-32212) at a concentration of 1:10,000 imaged on a Li-Cor Odyssey® M system.
  • H4B4B4 anti-hLAMP2 primary antibody Abcam #25631
  • RNA isolation For each sample ⁇ 10-30 mg of tissue were homogenized with the TissueLyser I system (Qiagen 85300) QIAzol lysis reagent (Qiagen 79306). Folowing addition of chloroform, the aqueous phase was separated and DNA-free RNA isolation was then carried out using the RNeasy 96 QIAcube HT kit (Qiagen 74171) and QIAcube HT instrument for automated nucleic acid purification (Qiagen 9001896). RNA concentrations were quantified using the QuibitTM Flex Fluorometer (Invitrogen Q33327) and the Qubit RNA Broad Range (BR) Assay Kit (Thermo Fisher Q10211).
  • PCR reactions were setup as duplex assays targeting transgene specific sequences (DA01 or WPRE) and the mouse Transferrin receptor gene, mTFRC as an internal control (VIC; Thermo Fisher 4458367).
  • the folowing primers were designed at AskBio and synthesized by Integrated DNA Technologies (IDT): [00665]
  • DA01 DA01 Forward: ctgactgacacgtgtaccaca (SEQ ID NO: 63) DA01 Reverse: caacaatgcatcattatgttca (SEQ ID NO: 64) DA01 Probe (FAM): acctcccacacctcccctgaacc (SEQ ID NO: 65) [00667] PCR reactions with DNA inputs were assembled as folows: 5 ⁇ L sample DNA (4 ng/ ⁇ L), 3 ⁇ L 4x QIAcuity Probe Mastermix (Qiagen 250103), 0.6 ⁇ L of 20x primer/probe mix (10 ⁇ M/ 5 ⁇ M; DA01 or WPRE), 0.6 ⁇ L of 20x mTFRC primer/probe mix, 0.15 ⁇ L XhoI (20 U/ ⁇ L; NEB R0146), and nuclease-free water were mixed to reach a final reaction volume
  • PCR reactions were manualy loaded into QIAcuity 8.5K partition 96-wel nanoplate (Qiagen 250021) then dPCR was caried out on a QIAcuity digital PCR system (Qiagen 911042) according to the folowing protocol: 1) Samples were partitioned by standard priming 2) The PCR protocol comprised an initial denaturation/enzyme activation step at 95°C for 2 minutes, folowed by 40 thermocycles of denaturation at 95 °C for 15s and annealing/elongation at 52°C (DA01) or 60°C (WPRE) for 30s.3) Partitions were imaged using a 500ms exposure duration with the gain set to 6.
  • RT-PCR reactions with RNA input were assembled as folows: 5 ⁇ L sample RNA (10 ng/ ⁇ L), 3 ⁇ L 4x OneStep Advanced Probe Master Mix (Qiagen 250132), 0.12 ⁇ L 100x OneStep RT Mix (Qiagen 250132), 1.5 ⁇ L 8x Enhancer GC (Qiagen 250132), 0.6 ⁇ L of 20x primer/probe mix (10 ⁇ M/ 5 ⁇ M; DA01 or WPRE), 0.6 ⁇ L of 20x mTFRC primer/probe mix, and nuclease-free water were mixed to reach a final reaction volume of 12 ⁇ L.
  • RT-PCR reactions were manualy loaded into QIAcuity 8.5K partition 96-wel nanoplate (Qiagen 250021) then dPCR was carried out on a QIAcuity digital PCR system (Qiagen 911042) according to the folowing protocol: 1) Samples were partitioned by standard priming.2) The RT-PCR protocol comprised an initial one-step reverse-transcription stage at 50°C for 40 minutes folowed by denaturation/enzyme activation step at 95°C for 2 minutes, then by 40 thermocycles of denaturation at 95 °C for 5s and annealing/elongation at 52°C (DA01) or 60°C (WPRE) for 30s.3) Partitions were imaged using a 500ms exposure duration with the gain set to 6.
  • Ribosome footprint analysis [00670] Ribo-seq analysis of LAMP2B expression cassetes was performed in C2C12 cels transiently transfected with pDA plasmids. The day before transfection, C2C12 cels were seeded at 2e10 cel per 10 cm plate density.
  • Cels were transfected with plasmid DNA using Lipofectamine 3000 (Invitrogen) transfection reagent according to the manufacturer instructions.48 h post transfection, Haringtonine was added to the culture medium at a final concentration of 2 ⁇ g/mL and cels were incubated for 120 s at 37°C before cycloheximide was added at a concentration of 100 ⁇ g/ml. Cels were washed twice in 5 mL cold PBS, transfered to a 1.5 mL tube on ice, and subsequently peleted for 2 min at 6000 rpm and 4°C.
  • Cel pelets were then lysed in 300 ⁇ l ice cold lysis buffer (1 % NP40 alternative (Milipore), 200 mM KOAc (Sigma-Aldrich), 25 mM K-Hepes pH 7.2 (Sigma-Aldrich), 10 mM Aty. Dkt. No.046192-000118WOPT MgCl2 (Sigma-Aldrich), 4 mM CaCl2 (Sigma-Aldrich) for 5 min on ice. Cel debris was removed by centrifugation at 9000 rpm and 4°C.
  • RNA pelet was resuspended in 10 ⁇ l PNK solution (1x PNK buffer, 1 mM ATP, 1 U/ ⁇ l PNK (NEB) and incubated at 37°C for 30 min.
  • PNK reaction was stopped by addition of 10 ⁇ l RNA loading dye and samples were loaded onto a 15% urea-acrylamide gel in TBE buffer. Gels were stained with SybrGreen diluted 1:10,000 in TBE buffer.
  • Ribosomal footprints at ⁇ 30-35 nt were cut out under UV light with a clean razor blade and transfered to 400 ⁇ l of 400 mM NaOAc (pH 5.2) solution.
  • Gel slices were frozen for at least 10 min at - 80°C, thawed at 95°C, crushed using a pestle, and RNA was extracted by 3x incubating for 5 min at 95°C folowed by vortexing for 20 min. Gel pieces were removed by spinning samples through a SpinX columns (Sigma-Aldrich), and RNA was precipitated after addition of 1 mL EtOH and 2 ⁇ l GlycoBlue for 1 h at -20°C.
  • Immunohistochemistry [00672] Whole hearts were harvested from C57Bl/6J mice on day 29 folowing saline or test article injection. Samples were immersion fixed in 4% paraformaldehyde (PFA) at 4°C for 24 hours, then infiltrated with a 30% sucrose solution.
  • PFA paraformaldehyde
  • Samples were embedded and frozen in optimal cuting temperature compound (Tissue-Tek OCT; Sakura 4583) in an isopentane and liquid nitrogen bath, then sectioned transversaly with a Leica cryostat (CM1950) at a 5 ⁇ m thickness and stored at -80°C.
  • CM1950 Leica cryostat
  • Immunohistochemistry assays were carried out on the Leica BOND RX automated staining system (21.2821). Tissue sections were prepared for automated staining by washing in PBS (Fisher Scientific BP3991) for 5 minutes, permeabilizing with 0.3% Triton X-100 (Sigma-Aldrich X100) for 20 minutes, then washing in PBS for 2 minutes.
  • the folowing BOND-RX staining protocol was as used: Tissue sections were blocked in 5% BSA in PBS (Thermo Scientific J61089AP) for 30 minutes at room temperature, then washed thrice for 30 seconds, with BOND Wash Solution (BWS, Leica AR9590). Tissue sections were then incubated for 1 hour at room temperature in primary antibody solution, then washed thrice for 3 minutes with BWS. Tissue sections were incubated for 1 hour at room temperature in secondary antibody solution containing Alexa Fluor 488-conjugated Wheat Germ Agglutinin (Invitrogen W11261, 1:250), then washed thrice for 30 seconds with BWS.
  • Tissue sections were counterstained with DAPI (Thermo Scientific 62248, 1:5000), then washed with BWS thrice for Aty. Dkt. No.046192-000118WOPT 3 minutes.
  • Tissue sections were mounted in ProLongTM Gold antifade mounting medium (Invitrogen P36930) and imaged using a Leica THUNDER imaging system (Leica DM6B upright microscope equipped with a Scientific CMOS camera K8).
  • the folowing antibodies were used: mouse anti- LAMP2 (Abcam ab25631, 1:250, reactive to human LAMP2), rat anti-LAMP2 (Sigma-Aldrich MABC40, 1:250, reactive to mouse LAMP2), goat anti-mouse Alexa Fluor 594 (Invitrogen A11005, 1:500) and goat anti-rat Alexa Fluor Plus 647 (Invitrogen A21247, 1:500).
  • mouse anti- LAMP2 Abcam ab25631, 1:250, reactive to human LAMP2
  • rat anti-LAMP2 Sigma-Aldrich MABC40, 1:250, reactive to mouse LAMP2
  • goat anti-mouse Alexa Fluor 594 Invitrogen A11005, 1:500
  • goat anti-rat Alexa Fluor Plus 647 Invitrogen A21247, 1:500.
  • Muscle specific promoter for example, Syn100 (SEQ ID NO: 4, SPc5-12 (SEQ ID NO: 5) or SP524 (SEQ ID NO: 6) are utilized herein to promote strong transgene expression in the target tissue (heart and skeletal muscles) as compared to a ubiquitous promoter, and decreased expression in non-muscle tissues.
  • the LAMP2B coding sequence is codon optimized and deprived of immunogenic motifs (e.g., CGs, bacterial sequences) and non-self proteins (e.g., ARF and alternative splice sites).
  • the oncogenic WPRE element has been removed.
  • a short, bidirectional terminator, SV40 poly A is incorporated to terminate transcription in both directions and reduce the formation of double-stranded RNA.
  • a screen was prepared. Promoters with different atributes were included in the cassete design (Fig.1A). Expression data from representative promoters shows luciferase protein expression in heart, skeletal muscles and liver in comparison to CK7 promoter, a control promoter (Fig.1B). [00676] A total of 31 constructs were developed (Fig.2) to identify the optimal configuration that promotes the best muscle-specific expression of the LAMP2B transgene. Each of the contructs were packaged into a AAV2i8 capsids, e.g., as described in Example 1. The packaged AAV are injected into mice via tail vein injection.
  • LAMP2B protein expression was normalized to total protein in heart (Fig.4). Blue bars indicate control prior art coding sequence in the context of Syn100 promoter (Syn100 RS5) and ITR- ITR construct (RS10). A new construct was designed by combining SP524 with the 7prob coding sequence variant. Data show that LAMP2B protein expression was high in some constructs (Syn100 Aty. Dkt. No.046192-000118WOPT 2prob, Syn1007pref, Syn1007prob, SP524 cDNA) and low to medium in others including control constructs (Syn100 RS5 and RS10) (Fig.5).
  • RNA expression in heart was additionaly measured as copies/ug RNA (Fig.5).
  • Asterisks indicate selected constructs. Blue bars indicate control coding sequence in the context of Syn100 promoter (Syn100 RS5) and control ITR-ITR construct (RS10). RNA expression was uniform among the diferent groups.
  • constructs e.g., driven by Syn100 promoter
  • RNA expression e.g., LAMP2B protein and RNA expression were observed in the heart, with lower expression in skeletal muscles and very low expression in other organs.
  • Top selected constructs DA004, DA022 and DA023 display similar tissue biodistribution. Folowing assessment of expression of constructs, seven constructs were chosen in up-selection and study design (Fig.6). See methods for study protocols (Studies #6-8).
  • the control A vector was control ITR-ITR sequence packaged in AAV9 and the control vector was the same ITR- ITR sequence packaged in AAV2i8.
  • AAV9 used herein in the Examples was used specificaly as a comparator.
  • the AAV capsid protein cannot be an AAV9 serotype.
  • Each vector was manufactured at two scales to test for vector lot reproducibility and mice were injected at two doses to validate dose dependent protein expression.
  • LAMP2B protein expression and vector genome copies were measured from al 4 chambers of the heart.
  • RNA expression was measured from both ventricles due to insufficient tissue availability from atria. Dose dependent increase in LAMP2B protein expression is observed in al groups across the diferent chambers of the heart (Fig.7). Most of the down selected constructs have similar protein expression levels.
  • the 7prob coding sequence for LAMP2B (i.e., SEQ ID NO: 3) was selected in combination with the IVS intron and SV40 poly A sequences. The top candidates difered from each other in the promoter used. Promoter characteristics are indicated in Fig.14. EXAMPLE 3 – Study and sampling design [00683] Methods for administration and sampling were described herein above in Example 2. [00684] STUDY #1 [00685] A set of 19 codon optimized, CG free, ARF depleted LAMP2B constructs with diferent promoters were designed for Danon disease and screened in wild type mice to select the highest expressers (see Table 3).
  • Liver were 3 smals parts from left lobe (2 frozen and 1 RNAlater) with the rest of the liver frozen. Brain were 3 smal parts cortex (2 frozen and 1 RNAlater) with the rest of the brain frozen. [00691] STUDY #2 [00692] A set of 21 codon optimized, CG free, ARF depleted LAMP2B constructs with diferent promoters were designed for Danon disease and screened in wild type mice to select the highest expressers. The top performers from this screen were up selected in the LAMP2 knockout mouse model with efficacy studies thereby selecting the therapeutic candidate Characterization of four of the optimized down-selected codons and immunogenic sequence depleted constructs, comparing them to a benchmark construct. Aty. Dkt.
  • Group 1 was injected with saline as a negative control, while groups 2 – 9 are injected with one of 4 test constructs at a dose level of either 6.00e10 or 3.00e11.
  • Weekly body weights were taken through study endpoint on day 29. At endpoint, animals were anesthetized and ful volume terminal cardiac blood colection for serum processing is Aty. Dkt. No.046192-000118WOPT performed. Animals were euthanized for subsequent brain, spinal column, heart, lung, diaphragm, liver, spleen, testes, quadriceps, gastrocnemius, tibialis anterior, and soleus harvest. [00698] Male C57BL/6J mice were used for this study.
  • C57BL/6J is the most common, and most used wild type mouse strain. Mice were 6 – 8 weeks old and weigh approximately 20 grams at study initiation. [00699] Table 8 shows study design from Study #3. [00700] Table 9 shows sampling design from Study #3. Day 28; Groups 1-9 Animals 1-5 Aty. Dkt. No.046192-000118WOPT [00701] Table 10 shows sampling design from Study #3. Day 29; Groups 1-9 Animals 6-8 [00702] STUDY #4 [00703] Five groups containing eight male C56Bl/6J mice aged 9 weeks were injected intravenously on day 1 with 150uL of test article.
  • Group 1 were injected with saline as a negative control, while groups 2 – 5 were injected with one of 2 test constructs at a dose level of either 6.00e10 or 3.00e11. Weekly body weights were taken through study endpoint on day 28 and 29. At endpoint, animals were anesthetized and ful volume terminal cardiac blood colection (D28) and terminal mandibular bleeds (D29) for serum processing is performed. Animals were euthanized for subsequent brain, spinal column, heart, lung, diaphragm, liver, spleen, testes, quadriceps, gastrocnemius, tibialis anterior, and soleus harvest (D28), with heart and liver samples for 4% PFA fixation (D29).
  • D28 cardiac blood colection
  • D29 terminal mandibular bleeds
  • Table 11 shows study design from Study #4. Aty. Dkt. No.046192-000118WOPT
  • Table 12 shows sampling design from Study #4. Day 28; Groups 1-5 Animals 1-5.
  • Table 13 shows sampling design from Study #4. Day 29; Groups 1-5 Animals 6-8
  • STUDY #5 [00708] Characterization of two of the downselected codon optimized and immunogenic sequence depleted constructs and compared to benchmark construct.
  • Table 14 shows study design from Study #5. Aty. Dkt. No.046192-000118WOPT
  • Table 15 shows study design from Study #5.
  • Table 15 shows second sampling design from Study #5. Day 29; Group 1 Animals 3-4, Groups 2-5 Animals 6-8, Groups 6-7 Animal 3 Aty. Dkt. No.046192-000118WOPT [00712] STUDY #6 [00713] Characterization of three optimized down-selected codons and immunogenic sequence depleted constructs, at 20L scale prep, compared them to a benchmark construct. [00714] Table 17 shows study design from Study #6. [00715] Table 18 shows sampling design from Study #6.
  • Table 19 shows second sampling design from Study #6. Day 29; Group 1 Animals 4-5, Groups 2-5 Animals 6-8, Groups 8-9 Animal 3. [00717] STUDY #7 [00718] Characterization of three optimized down-selected codons and immunogenic sequence depleted constructs, at 20L scale prep, compared to a benchmark construct. [00719] Table 20 shows study design from Study #7. Table 20 Aty. Dkt. No.046192-000118WOPT [00720] Table 21 shows sampling design from Study #.
  • Table 21 shows sampling design from Study #7. Day 29; Group 1 Animals 4-5, Groups 2-5 Animals 6-8, Groups 8-9 Animal 3. Aty. Dkt. No.046192-000118WOPT [00722] STUDY #8 [00723] Characterization of three of the optimized down-selected codons and immunogenic sequence depleted constructs, at 20L scale prep, compared to a benchmark construct. [00724] Table 23 shows study design from Study #8. Table 23 [00725] Table 24 shows sampling design from Study #8.
  • AAV2i8-LAMP2B the study drug, comprised of an AAV serotype 2i8 vector containing a DNA expression cassete encoding the codon-optimized sequence for human LAMP2B under the control of a muscle specific promoter (e.g., Syn100, Spc5-12 or SP0524).
  • a muscle specific promoter e.g., Syn100, Spc5-12 or SP0524.
  • This example herein describes administrating a rAAV viral vector expressing the codon optimized LAMP2B gene to human patients for the treatment of Danon disease, according to the methods disclosed herein.
  • the rAAV vector AAV2i8.sc-LAMP2B constructs discussed herein are exemplary rAAV, and any rAAV disclosed herein can be used in the methods to treat Danon disease, as disclosed herein.
  • one of ordinary skil in the art can readily substitute the muscle specific promoter in the AAV2i8.sc-LAMP2B constructs for any of Syn100, SPc5-12, or SP0524, or Aty. Dkt. No.046192-000118WOPT any muscle specific promoter as described herein.
  • the AAV2i8.sc-LAMP2B can be readily substituted for a rAAV comprising a codon-optimized nucleic acid sequence encoding LAMP2B, such as, but not limited to the nucleic acid sequences of SEQ ID NO: 1-3, or any of the sequences as described herein.
  • a single intracoronary infusion of AAV2i8.sc-LAMP2B wil be administered to human patients with Danon disease, where administration can occur as single administration of a total dose selected from any of: 1 x 1010vg, 3 x 1010vg/kg, 1 x 1011vg/kg, 3 x 1011vg/kg, 1 x 1012vg/kg, 3 x 1012vg/kg, 1 x 1013vg/kg, 3 x 1013vg/kg, 1 x 1014vg/kg, 3 x 1014vg/kg, 1 x 1015vg/kg, or 1 x 1015vg/kg
  • the total dose wil be administered in a series of 3 or 5 sub-doses, each sub-dose administered from a separate syringe or catheter, and where the sub-
  • the total rAAV dose is diluted in 50 ml saline and administered as 5 sub-doses in 5 syringes, with 10ml volume in each syringe.
  • the administration wil be performed over a time period of about 5 minutes for each of the syringes, one syringe after another, with frequent stops checking the position of catheters in each coronary artery using contrast injections.
  • the patients wil not receive any prophylactic immunomodulator administration before, at the time, or soon after the rAAV administration.
  • Al patients wil be folowed until 12 months post treatment intervention, and then undergo long-term folow-up via semi-structured telephone questionnaires every 6 months for an additional 24 months (+/- 30 days).
  • coronary angiography wil be performed in the usual manner.
  • three infusion areas wil be used to subserve the largest portion of LV blood flow
  • the selected, appropriate catheter for the first infusion wil be engaged in the first coronary artery (subtending the largest teritory) after back-bleeding/flushing in the usual manner.
  • two locations (infusions) wil folowing the first location
  • the selected appropriate catheter for the second infusion wil be engaged in the second coronary artery (subtending the lesser territory) after back-bleeding/flushing in the usual fashion.
  • Similar exchanges wil be performed for a third catheter exchange should a three infusion (location) sequence be required.
  • rAAV of invention wil be delivered by manual or by pump intracoronary push at a rate of 1-10mL/minute. The infusion may be slowed or interupted for subjects who experience infusion- associated symptoms or if a catheter needs to be repositioned. [00731] Before administration, and after 4-weeks (+/- 3 days), 6 months and 12-months post- administration, the patients wil be assessed using a variety of physiological assessments.
  • echocardiographic assessments of LVEF, LVEVD, LVEDVI, VLESV, LVEVI, SpI and GLS and degree of mitral regurgitation can be assessed, as wel as serum NT-proBNP or BNP levels, as wel as secondary effects assessed to determine efficacy of the administration of the AAV2i8.sc- Aty. Dkt. No.046192-000118WOPT LAMP2B vector.
  • Secondary effects include, e.g., but are not limited to (i) Peak VO2 assessed by cardiopulmonary exercise testing, (i) 6-minute walk test (6MWT), (ii) New York Heart Association (NYHA) Classification, (iv), total number of days alive out-of-hospital (as wel as total days out-of- hospital as a % of total days alive post study intervention) (v), Quality of Life at 6 and 12 months compared to baseline, (vi) Health related quality of life as assessed by Minnesota Living with Heart Failure Questionnaire (MLWHFQ). Patients can also be assessed at 12-months and long-term folow- up period (until month 36 post-intervention), or assessed for a need for cardiac transplantation, or left ventricular assist device (LVAD) implantation.
  • 6MWT 6-minute walk test
  • NYHA New York Heart Association
  • iv total number of days alive out-of-hospital (as wel as total days out-of- hospital as a % of total days alive post study intervention)
  • v Quality of Life at 6
  • Peak VO2 wil Primary outcome measures wil be assessed, which include a change in Peak VO2 and LVEF levels.
  • the Peak VO2 wil be measured at 6- and 12-months post- administration as compared to the Peak VO2 before, or at the time of administration (i.e., as compared to baseline), as determined by Cardiopulmonary exercise testing using a modified Bruce protocol.
  • LVEF Left Ventricular Ejection Fraction
  • Secondary outcome measures can also be assessed, which include a change in results from the 6MWT (6-minute walk test), measured at 6- and 12-months post administration as compared to the results from the 6MWT measured at, or before administration (i.e., as compared to the baseline). Secondary outcome measures of the analysis of % predicted in heart failure subjects as compared to normal subjects was also determined.
  • AAV2i8sc-LAMP2B as described herein wil be administered to pig by single intracoronary administration whereas AAV2i8 (not self complementary genome) -LAMP2B wil be administered to pig by single intravenous administration. It is expected that AAV2i8sc-LAMP2B wil be administered intracoronary at a log lower dose compared to AAV2i8 (not sc)-LAMP2B by intravenous administration to achieve comparable level cardiomyocyte transduction and comparable LAMP2B expression in heart. Aty. Dkt.
  • the open-ended transitional term “comprising” (along with equivalent open-ended transitional phrases thereof such as “including,” “containing” and “having”) encompasses al the expressly recited elements, limitations, steps and/or features alone or in combination with un-recited subject mater; the named elements, limitations and/or features are essential, but other unnamed elements, limitations and/or features may be added and stil form a construct within the scope of the claim.
  • the open-ended transitional phrase “comprising” (along with equivalent open-ended transitional phrases thereof) includes within its meaning, as a limiting case, claimed subject mater specified by the closed-ended transitional phrases “consisting of” or “consisting essentialy of.”
  • embodiments described herein or so claimed with the phrase “comprising” are expressly or inherently unambiguously described, enabled and supported herein for the phrases “consisting essentialy of” and “consisting of.”
  • LAMP-2 lysosome-associated membrane protein 2
  • LAMP-2 is known to be involved in autophagy, and a characteristic accumulation of autophagic vacuoles in the affected tissues further supports the idea that autophagy is disrupted in this disease (Rowland et al, 2016).
  • the X chromosome caries LAMP-2 which means that boys (who have only one X chromosome) often have a more severe version of the disease than girls (who have two X chromosomes, which may dilute the effect of a single mutated gene).
  • LAMP2B therapeutic is an experimental adeno-associated virus (AAV)-based gene therapy, which replaces the missing or nonworking LAMP-2 gene with a normal LAMP-2 gene. Patients also receive immunosuppressants for several months. This prevents their immune systems from reacting negatively to the genetic infusion.
  • AAV adeno-associated virus
  • Inventors are conducting a Phase 1 dose escalation gene therapy trial to evaluate the safety and efficacy of a single antegrade intracoronary artery infusion of 3 dose levels of AAV2i8 capsid expressing the LAMP2B transgene (AAV2i8-LAMP2B) in subjects with non-ischemic cardiomyopathy and NYHA Class II symptoms of HF.
  • AAV2i8-LAMP2B LAMP2B transgene
  • OBJECTIVES AND ENDPOINTS [00749] Primary Objective: [00750] To evaluate the efficacy and safety of a single antegrade intracoronary artery infusion of a LAMP2B therapeutic (e.g., as described herein) compared to compared to an external control arm in increasing myocardial LAMP2 protein expression and reducing LVMI. [00751] Secondary Objectives: [00752] To evaluate the impact of LAMP2B therapeutic on LAMP2 protein expression, cardiac function, quality of life and the cardiac biomarkers (NT-proBNP level, Troponin I).
  • Efficacy Endpoints are assessed from baseline to 52-weeks folowing administration of the LAMP2B therapeutic including: • Primary Eficacy Endpoint ⁇ Co-primary endpoint of myocardial expression of LAMP2 and decrease in LVMI (Left Ventricular Mass Index) • Secondary Efficacy Endpoints o LAMP2 protein expression o LVMI o hs-TnI Aty. Dkt.
  • Subjects in the Phase 1 single ascending dose study receive either a high dose or low dose of a LAMP2B therapeutic. Subjects are observed and folowed for the 52-week observation period and Long-Term Folow-Up period of 4 years for a total study duration of 5 years. Subjects in the [00757] Phase 2 study receive a single dose of a LAMP2B therapeutic and are observed for an initial 52 week period folowed by a long-term-folow-up period of 4 years. [00758] Number of Subjects [00759] Approximately 24 subjects are assigned to the study intervention. [00760] Intervention Groups and Duration [00761] Table 1 shows Intervention Groups and Duration Aty. Dkt.
  • the clinical trial wil also have an external control arm.
  • Inclusion Criteria [00770] Main Criteria for Inclusion: [00771] The study wil enrol adult and pediatric males with a confirmed diagnosis of DD. Patients may be of any race or ethnicity. Patients and/or competent custodial parents must provide informed writen consent and meet al of the enrolment criteria as detailed subsequently to be eligible to participate. 1. Documentation of a pathogenic or likely pathogenic variant of the LAMP2 gene. 2. Male gender. 3. Age ⁇ 15 years for cohorts 1 and 2; 8-14 years for cohorts 1A. 4. Evidence of left ventricular hypertrophy with preserved systolic function phenotype as defined by each of the folowing: 1.
  • left ventricular posterior wal or interventricular septum at end diastole >13 mm (>12 mm if family history of clinicaly significant Danon disease), 2.
  • Left ventricular ejection fraction (LVEF) ⁇ 50%. 5.
  • NYHA New York Heart Association
  • Aty. Dkt. No.046192-000118WOPT 6.
  • hsTnI ⁇ 20% above the ULN 7.
  • LVAD Left Ventricular Assisted Device
  • Active hepatitis B or C infection including patients with positive hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), hepatitis B core antibody (HBcAb), or detectable hepatitis B virus (HBV) or hepatitis C virus (HCV) viral load). Patients with previous, adequately resolved HBV or HCV are eligible. 13. Significant medical conditions including documented human immunodeficiency virus (HIV) infection, active viral or other hepatitis, poorly-controled hypertension or diabetes, poorly controled cardiac arrhythmia, or uncontroled viral, bacterial, or fungal infection. 14.
  • HBV human immunodeficiency virus
  • Active hematologic or solid organ malignancy not including non-melanoma skin cancer or other carcinoma in situ. Patients with previously resected solid organ malignancies or definitively treated hematologic malignancies may be eligible if there has been no evidence of active malignancy during the prior 3 years.
  • Aty. Dkt. No.046192-000118WOPT The study shows stabilized or improved myocardial Lamp2B expression and at least 10% reduction in LVMI among other clinicaly meaningful improvement in endpoints.
  • EXAMPLE 6 Objectives, Endpoints, and/or Estimands OVERALL DESIGN
  • This study assesses the baseline characteristics, cardiac effects, extracardiac effectss and drug utilization amongst patients with Danon disease. The study period extends from 2 years prior to the index date (date of Danon Disease diagnosis) of the earliest diagnosed patient to 2 years after the index date of the most recently diagnosed patient.
  • Extracardiac manifestations of disease record diagnoses of pulmonary, liver and skeletal muscle disease including severity and duration.
  • Use of medications before and after the index date are recorded, including dose, posology, duration of use.
  • Data sources [00787] Danon Disease is a rare disease and there are few centralized colections of data. Data are identified from the participating center health care records and registry data where available.
  • Enrolment [00789] The study wil provide a 2:1 ratio of patients with respect to the planned interventional trial. Based on an effect size of 0.4 and 80% power with respect to LV mass reduction the interventional study would require an estimated 52 patients.
  • the cohort study aim to colect data on a larger number of cohort e.g., 104 patients overal. Aty. Dkt. No.046192-000118WOPT [00790] Data Analysis [00791] The individual cohort of patients are described by descriptive statistics of the folowing variables [00792] Detailed descriptive variables including baseline characteristics, demographics, co- medications and comorbidities are captured. [00793] Cardiac structure and function data are captured using records of echocardiograms and/or cardiac MRI, NYHA Class, and ECG findings. [00794] Extracardiac manifestations of disease record diagnoses of pulmonary, liver and skeletal muscle disease including severity and duration.
  • EXAMPLE 7 Nonclinical data demonstrating eficacy of DA023 in a Danon mouse model
  • Danon disease is a severe form of hypertrophic cardiomyopathy caused by loss-of-expression of Lamp2. This leads to lysosome dysfunction and vacuolization of heart and skeletal muscle, impaired autophagy, reduced cardiac output, and rapidly progressing heart failure.
  • Current treatments are focused on preserving cardiac function, or in late-stage disease, heart transplantation.
  • the therapeutic strategy of DA023 is to restore Lamp2B expression in heart, addressing the most critical manifestation of the disease.
  • This example summarizes the development and characterization of a Lamp2 KO mouse model that recapitulates features of Danon disease in humans, including the key phenotypes of cardiac hypertrophy and impaired autophagy.
  • the inventor developed DA023, a scAAV using the cardiotropic capsid AAV2i8, and a muscle-specific promoter, to restore Lamp2B expression in the critical target organ of Danon disease, the heart (FIGs 15A and 15B).
  • the inventor conducted four studies in the Lamp2 KO mouse, using DA023, to demonstrate restoration of the key phenotypes of Danon disease, cardiac hypertrophy and autophagy, summarized herein this example.
  • LAMP2 KO MOUSE MODEL CHARACTERIZATION (DAN-PD4-011) Generation of Lamp2 KO mouse model
  • DAN-PD4-011 LAMP2 KO MOUSE MODEL CHARACTERIZATION
  • Lamp2 KO mouse model To generate a mouse model that mimics the molecular cause of disease and manifests Danon disease pathophysiology, and should be predicative of therapeutic efficacy, the inventor developed a Aty. Dkt. No.046192-000118WOPT model that is based on complete knockout of Lamp2 expression.
  • the Lamp2 gene knockout strategy entailed deletion of exons 2-6 of the 9 exon gene (Fig.28). The remaining exons were out-of-frame with the presence of a premature stop codon in the deletion alele transcript that should trigger nonsense-mediated mRNA decay (Fig.29).
  • Lamp2 KO embryos were generated by Crispr/Cas deletion in the C57BL/6J strain. Founders were sequence-verified to confirm deletion endpoints. Complete loss of Lamp2 expression was confirmed by western blot analysis of hemizygous males (Fig.16).
  • Characterization of Lamp2 KO model has been conducted via an ongoing study (Study DAN-PD4-011) focused on 1 year of baseline model characterization, summarized in Table 27. Characterization is focused on hemizygous males. The study includes assessments of survival and in-life observation, cardiac hypertrophy and function, autophagy in cardiac tissue, and serum biomarker analysis.
  • Lamp2 KO mice were obtained via IVF at 5 weeks of age and in-life observations were taken (up to 44 weeks currently). For time points earlier than 5 weeks of age, survival information was obtained from the IVF vendor. KO mice displayed some early lethality, with ⁇ 20% of animals dead by 10 weeks (Fig.17). Thereafter, survival was maintained. As with other published Danon mouse models, the KO does not progress to end-stage heart failure in young adult animals, as occurs with human patients. The cause of early mortality was not determined. KO animals gained weight over the duration of the study, however at a slower rate than WT controls (Fig.18).
  • cardiac hypertrophy as measured to heart weight, was consistently observed in the KO mouse model, it is moderate in nature compared to models such as thoracic aorta banding.
  • the inventor also employed echocardiography (long axis B mode, short axis M mode) to measure numerous cardiac parameters, including left ventricular wal and intraventricular septum thickness, for measurement of hypertrophy. These methods, however, were not able to yield statisticaly significant and/or reproducible measures of hypertrophy, due to moderate levels of hypertrophy and variability across samples.
  • Another key feature of Danon pathology is the alteration of autophagy pathways, measured by increased expression of LC3-II.
  • LC3 is proteolyticaly cleaved to LC3-I and then conjugated to lipid to form LC3-I.
  • This diferentialy migrating protein can be detected by western blot. Elevated LC3-II, normalized to total protein, is an indication of aberant autophagy, and is observed in Danon patient biopsies.
  • the Lamp2 KO model displays increased LC3-II as early as 8 weeks and maintains this increase (Fig.20). Twenty-four hour starvation or 6-week intermitent fasting further increase LC3-I and a second autophagy marker, p62 (Fig.21).
  • Lamp2 KO Model Summary [00808] Lamp2 KO Model Summary [00809] The Lamp2 KO model that the inventor developed exhibits key phenotypes of Danon disease including cardiac hypertrophy and autophagy defects. These phenotypes are similar to published mouse models of Danon. The inventor’s model has some phenotypic differences from the models described by Tanaka 2000 and Manso 2020. Homozygous females were infertile and matings with heterozygous females led to high neonatal fatality, requiring the use of IVF to generate animals to perform the experiments. Mortality was observed mostly during the first week and then from week 3 to week 10, up to a total of roughly 20% males. The Tanaka model, however, had higher mortality between days 20 and 40 (50%).
  • Lamp2 KO mouse model mimics the molecular pathology of Danon disease by eliminating expression of Lamp2.
  • the model presents some of the most prominent symptoms of the disease in humans: heart hypertrophy (as assessed by heart weight), increased autophagy markers, and increased serum liver enzymes.
  • the Lamp2 KO murine model of Danon disease does not fuly replicate al of the cardiac features of the clinical disorder.
  • the mouse model does not exhibit cardiac fibrosis or elevated serum cardiac Troponin-T. These mice do not develop reduced ejection fraction, even with intermitent starvation.
  • Vector DNA was measured by dPCR, transgene mRNA was measured by dRT-PCR, and transgene protein was measured by western blot.
  • Systemic delivery of DA023 resulted in transduction of the heart (left ventricle measured) as measured by vector genome recovery (0.17 vgs/diploid genome at a dose of 1e12 vg/mouse) and dose dependent expression of hLAMP2b mRNA and protein expression (Fig. 22).
  • Densitometry quantitation for transgene protein was not statisticaly significant at low dose, but protein bands were clearly visible on the blot. Data shown is for study DAN-PD4-015, but results from al 4 studies were similar (DAN-PD4-017 DNA, results stil pending).
  • DA023 prevented anesthesia-induced mortality in Lamp2 KO animals
  • Lamp2 KO mice showed anesthsia-induced mortality, with around 50% mortality over the duration of the study. WT mice were unaffected.
  • treatment with DA023 appeared to rescue anesthesia- induced mortality, with al treated KO mice surviving to the study endpoint in both high and low dose groups (Fig.24).
  • the Lamp2-deficient mouse model that the inventor developed has key features of Danon disease in humans, similar to published models.
  • the Lamp2 KO model disclosed herein is distinct from the model in the prior art.
  • the published model of Lamp2 knockout claims to Aty. Dkt. No.046192-000118WOPT have a complete knockout of the Lamp2 gene by homologous recombination (see, e.g., Tanaka et al, Nature.2000 Aug 24;406(6798):902-6. doi: 10.1038/35022595).
  • the rAAV comprising a codon optimized Lamp2b nucleic acid of the invention has demonstrated efficacy in 2 key measures of Danon disease: cardiac hypertrophy (as measured by heart weight) and impaired autophagy (as measured by LC3-II), across multiple smal interventional studies. These studies support further development of the rAAV comprising codon optimized Lamp2b expression constructs of the invention for their use in treating subjects with Danon disease and or having LAMP2B deficiency.
  • EXAMPLE 8 – GLP Toxicology Study in Yorkshire Pigs [00833] A summary of study design for GLP toxicology study in yorkshire pigs is presented herein in Table 29.
  • the porcine model is a desirable biologicaly relevant large animal species that has a close resemblance to the anatomy of the human heart.
  • a Danon disease porcine model does not exist, using healthy Yorkshire farm pigs wil alow the inventors to design a pivotal toxicology and biodistribution study to test the relevant clinical procedure in a similar cardiovascular system.
  • the target clinical population for DA023 is an adult and pediatric population ⁇ 6 years of age.
  • Yorkshire pigs that are 3 months old are dosed with DA023.
  • Yorkshire pigs that are 3 months old weigh ⁇ 25-30kgs and are developmentaly similar to human children that are ⁇ 2-11 years old.
  • Organ systems, such as the cardiovascular, nervous, and skeletal muscle systems are at similar developmental stages, or maturation levels, and thus the use of a 3 month old pig is used to support the safety profile in the dosing of human subjects ⁇ 2 years old.
  • GLP pig toxicology study pigs are dosed to atain LAMP2b protein levels in cardiac tissue that are equivalent or greater to the LAMP2b protein expression levels that showed eficacy in our mouse model.
  • Duration of GLP Toxicity Study [00841] The GLP toxicology study is a 3 month study with interim time points of 7 days and 30 days post DA023 administration. Table 30 presents a summary of the summary of study design for GLP toxicology study in yorkshire pigs.
  • Table 30 presented a summary of dosing for the GLP toxicology study in yorkshire pigs
  • Pre-Screening for Total Antibodies to AAV2i8 Prior to selection, al animals are screened for total antibodies (TAbs) to the AAV2i8 capsid. Animals negative for AAV2i8 TAbs are included in the study.
  • TAbs total antibodies
  • Animals negative for AAV2i8 TAbs are included in the study.
  • Prior to dosing 2 mL of serum is colected from prospective study animals to screen for serological status to AAV2i8 by TAb assay. Only AAV2i8 seronegative animals are included in the study.
  • Echocardiography Aty. Dkt. No.046192-000118WOPT On Day 1 prior to dosing and on the day before terminal necropsy echocardiography examinations occur. Echocardiography evaluations are conducted by a board certified veterinary cardiologist on echocardiograms performed by the Testing Facility. Animals are anesthetized according to Testing Facility SOP for the exams. For the echocardiogram examination, each animal has hair clipped from the thoracic region, if necessary. Coupling gel is applied to the animal and transducer, 70% isopropyl alcohol may be applied prior to gel application.
  • a right parasternal short-axis view, right parasternal long-axis view, right parasternal short-axis aortic view, and a short axis m-mode view is recorded. Additional views may be recorded.
  • the folowing parameters are evaluated: Left ventricular internal dimension diastole (LVIDd), left ventricular internal dimension systole (LVIDs), systolic and diastolic left ventricular posterior wal thickness, systolic and diastolic interventricular septum thickness, E point to septal separation, and aorta diameter. Fractional shortening is calculated based on LVIDd and LVIDs measurements.
  • Electrocardiogram On Day 1 prior to dosing and on the day before terminal necropsy Electrocardiograms are performed. Animals are anesthetized according to Testing Facility SOP for the exams. Insofar as possible, care is taken to avoid causing undue excitement of the animals before the recording of electrocardiograms to minimize extreme fluctuations or artifacts in these measurements. Standard ECGs (6 Lead) are recorded at 50 mm/sec. Using Lead II (or another appropriate lead) the RR, PR, and QT intervals, and QRS duration is measured and recorded. The QT interval is corrected (QTc).
  • Hematology parameters include: leukocyte count (total and absolute diferential), erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration (calculated), absolute reticulocytes, platelet count, RDW, blood smear preserve and stain.
  • Coagulation parameters include; prothrombin time, activated partial thromboplastin time, and fibrinogen.
  • Clinical chemistry parameters include; alkaline phosphatase (ALP), total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), sorbitol dehydrogenase, urea nitrogen, creatinine, total protein, albumin, globulin and A/G (albumin/globulin) ratio (calculated), glucose, total cholesterol, triglycerides, electrolytes (sodium, potassium, chloride), calcium, phosphorus, Creatine Kinase (CK).
  • Biomarker parameters include troponin I and CK-MB. Additional serum may be colected for exploratory analysis.
  • the AAV2i8 total antibody (TAb) assay is an Electrochemiluminescence Immunoassay to detect total antibody Aty. Dkt. No.046192-000118WOPT against AAV2i8 in pig serum.
  • the LAMP2b total antibody (TAb) assay is an Electrochemiluminescence Immunoassay to detect total antibody against LAMP2b in pig serum.
  • DA023 elicits a T cel response that is measured at 31 and 91 days post dosing.
  • the T cels response is either the AAV2i8 capsid or the human LAMP2b protein or both.
  • the human LAMP2B protein is an intracellular protein the potential stil exists that it could elicit a T cels response.
  • ELISpot assays to both the AAV2i8 capsid and the human LAMP2B protein is used to investigate the potential of a T cel response.
  • 40 mL of EDTA whole blood is colected, then PBMCs isolated and cryopreserved.
  • cels are seeded into pig IFN- ⁇ ELISpot plates (CTL pIFNg-1M) and stimulated with peptide pools encompassing the protein sequence of either the AAV 2i8 capsid or LAMP2b transgene product (15mers overlapping by 10 or 11 amino acids) for 48 hours.
  • DMSO and Concanavalin A (ConA) are included as negative and positive controls respectively.
  • Pig IFN- ⁇ ELISpots are developed according to the manufacturer’s recommended protocol, then imaged and quantified using an ImmunoSpot S6 plate reader and expressed as IFN- ⁇ spot forming units per milion cels (SFU/106 cels).
  • Viral Shedding Urine, blood, feces, and saliva are colected to analyze viral shedding. Samples are colected prior to dosing, Day 3, Day 8, Day 15, Day 30, Day 60, and Day 90. Viral shedding samples are analyzed by measuring DNA levels of the LAMP2b transgene via ddPCR. [00860] POSTMORTEM EVALUATIONS [00861] Necropsy [00862] Al animals are examined carefuly for external abnormalities including palpable masses. Macroscopic evaluations are performed by reflecting the skin from a ventral midline incision, and any subcutaneous masses are identified and correlated with antemortem findings.
  • the abdominal, thoracic, and cranial cavities are examined for abnormalities and the organs removed, examined, and, where required, placed in the appropriate fixative.
  • al tissues are placed in a neutral buffered formalin, except that the eyes, including the optic nerves, which are fixed using a modified Davidson’s fixative. Eyes are placed into formalin after fixation. Formalin is infused into the lung via the trachea. Body weight and the organ weights are recorded for al animals at the scheduled necropsies, and appropriate organ weight ratios are calculated (relative to body and brain weights). Paired organs are weighed together. Organs are weighed folowing qPCR tissue colection.
  • No.046192-000118WOPT gastrocnemius, biceps femoris and sartorius
  • lung liver, diaphragm, spleen, lymph node (mesenteric), ovaries (2), kidney (2) pulmonary artery, aorta, left circumflex coronary artery, left anterior descending coronary artery, right atrium, left atrium, right ventricle free wal (basal and apical layers), interventricular septum (basal and middle layers), left ventricle anterior wal (basal, middle and apical layers), left ventricle posterior wal (basal, middle and apical layers), left ventricle free wal (basal and middle layers), and brain.
  • Tissues are colected in individualy labeled tubes for specified organs from al biodistribution animals for analysis. Tissue samples are colected using strict aseptic techniques. Care is given to ensure that cross contamination between tissues does not occur. Biodistribution analysis is done by quantifying levels of LAMP2b transgene DNA, mRNA LAMP2 protein is measured in heart, skeletal muscle (quadriceps femoris), liver and brain.
  • ddPCR Droplet digital PCR assays are used to measure DA023 DNA copy number and Lamp2B transgene mRNA expression in whole blood and tissue samples. Samples are homogenized as needed, and nucleic acid extracted using magnetic bead-based processes to obtain purified DNA and RNA.
  • DNA is quantified using a duplexed ddPCR assay with primers and probes targeting the DA023 vector and a single-copy gene as an internal control. The quantity of DNA loaded in each PCR reaction is determined by measurement of the single-copy internal control gene. Copies of DA023 per mass of genomic DNA for tissues, or per mililiter of whole blood, is reported to describe DA023 DNA biodistribution.
  • Lamp2B transgene mRNA is quantified using a singleplex ddPCR assay with primers and probe targeting the Lamp2B transgene. Total RNA is quantified using the commercialy available QuantIT RNA fluorometric assay.
  • LAMP2b transgene protein is quantified in heart tissue samples using established methods and normalized to an internal control. Specificity of the assays for DA023 DNA and Lamp2B transgene mRNA and protein is confirmed.
  • LAMP2 Immunohistochemistry in Cardiac Tissue [00867] Detection of LAMP2 via immunohistochemistry (IHC) is performed at al necropsy timepoints. Porcine cardiac tissues samples are colected from animals treated with DA023 or vehicle control.
  • the tissue is cut longitudinaly, and a cross section of the left ventricular wal is dissected, avoiding regions with trabeculations and papilary muscle, to alow for assessment of LAMP2 localization in cardiomyocytes in the three layers of the left ventricular wal: endocardium, myocardium, and epicardium.
  • the samples are fixed in 10% neutral-buffered formalin for 24 hours, dehydrated, and processed in ethanol and xylene for parafin embedding on a 12-hour schedule using the Leica HistoCore Peloris 3.
  • the sections are then be embedded in parafilm to create formalin-fixed parafilm-embedded (FFPE) blocks.
  • FFPE formalin-fixed parafilm-embedded
  • DAB IHC is performed using the Ventana Ultra automated staining system, which includes deparafinization, rehydration, and antigen retrieval. Staining is then be conducted using the rabbit anti- Aty. Dkt. No.046192-000118WOPT human LAMP2 antibody (Invitrogen, clone 5C1N2), folowed by addition of an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Omni-MAP HRP), and detection using the Discovery ChoromoMap DAB Detection Kit, which produces a brown chromogenic signal.
  • Ventana Ultra automated staining system which includes deparafinization, rehydration, and antigen retrieval. Staining is then be conducted using the rabbit anti- Aty. Dkt. No.046192-000118WOPT human LAMP2 antibody (Invitrogen, clone 5C1N2), folowed by addition of an anti-rabbit secondary antibody conjugated to horseradish peroxidase (

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Abstract

La présente invention concerne des acides nucléiques à codon optimisé codant pour un polypeptide LAMP2B. La présente invention concerne également des cassettes d'expression et des vecteurs d'expression (par exemple, des vecteurs AAV recombinés (rAAV)) qui contiennent les acides nucléiques à codon optimisé sous une forme exprimable. La présente invention concerne également des méthodes de traitement de la maladie de Danon. La présente invention concerne également un vecteur d'expression contenant les acides nucléiques à codon optimisé (par exemple, un vecteur AAV recombiné (rAAV)).
PCT/US2025/024314 2024-04-12 2025-04-11 Virus adéno-associé thérapeutique utilisant un acide nucléique à codon optimisé codant lamp2b Pending WO2025217543A1 (fr)

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US202463633410P 2024-04-12 2024-04-12
US63/633,410 2024-04-12
US202463666567P 2024-07-01 2024-07-01
US63/666,567 2024-07-01
US202563746601P 2025-01-17 2025-01-17
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066334A1 (en) * 2010-12-24 2014-03-06 Geneart Ag Method for the production of reading-frame-correct fragment libraries
US20210381003A1 (en) * 2018-07-12 2021-12-09 Spacecraft Seven, Llc Gene therapy vectors for treatment of danon disease
US20230133924A1 (en) * 2020-01-08 2023-05-04 Tenaya Therapeutics, Inc. Optimized expression cassettes for gene therapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066334A1 (en) * 2010-12-24 2014-03-06 Geneart Ag Method for the production of reading-frame-correct fragment libraries
US20210381003A1 (en) * 2018-07-12 2021-12-09 Spacecraft Seven, Llc Gene therapy vectors for treatment of danon disease
US20230133924A1 (en) * 2020-01-08 2023-05-04 Tenaya Therapeutics, Inc. Optimized expression cassettes for gene therapy

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