WO2024226729A2 - Compositions et méthodes de thérapie génique opa1 - Google Patents

Compositions et méthodes de thérapie génique opa1 Download PDF

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WO2024226729A2
WO2024226729A2 PCT/US2024/026180 US2024026180W WO2024226729A2 WO 2024226729 A2 WO2024226729 A2 WO 2024226729A2 US 2024026180 W US2024026180 W US 2024026180W WO 2024226729 A2 WO2024226729 A2 WO 2024226729A2
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nucleic acid
acid sequence
polynucleotide
vector
seq
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WO2024226729A3 (fr
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Kazushi EGUCHI
Masashi KAJIRO
Kasane IMURA
Naoki KOZONO
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Astellas Gene Therapies Inc
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Astellas Gene Therapies Inc
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    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
    • A61L2/16—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using chemical substances
    • A61L2/18—Liquid substances
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2101/00—Chemical composition of materials used in disinfecting, sterilising or deodorising
    • A61L2101/02—Inorganic materials
    • A61L2101/16—Inorganic materials containing phosphorus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
    • A61L2103/05—Living organisms or biological materials
    • 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

Definitions

  • the invention relates to the field of gene therapy of neuro-ocular disorders, such as optic neuropathies, and OPA1 -deficiency disorders, such as Autosomal Dominant Optic Atrophy (ADOA), that are caused by mutations in the Optic Atrophy 1 (OPAT) gene.
  • ADOA Autosomal Dominant Optic Atrophy
  • ADOA Autosomal Dominant Optic Atrophy
  • OPAf Optic Atrophy 1
  • US 11 ,459,584 discloses methods for treating hereditary optic neuropathy, such as ADOA, using human type II mitochondrial dynein-like GTPase enzymes.
  • WO 2019/084050 discloses methods for regulating expression of OPA1 , namely, by way of modulating mRNA splicing.
  • WO 2022/067398 discloses antisense oligomers for modulating mRNA translation of endogenous OPA1 .
  • WO 2022/074396 discloses compositions for upregulating OPA1 gene expression, namely, nucleic acid molecules that contain (1) a sequence reverse complementary to an OPA1 mRNA molecule, and (2) a downstream regulatory sequence.
  • the disclosure features a polynucleotide encoding optic atrophy 1 (OPA1), wherein the polynucleotide has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of any one of SEQ ID NOs: 3-5.
  • the polynucleotide has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the polynucleotide has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the polynucleotide has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide has the nucleic acid sequence of SEQ ID NO: 3.
  • the polynucleotide has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:
  • the polynucleotide has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4.
  • the polynucleotide has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide has the nucleic acid sequence of SEQ ID NO: 4.
  • the polynucleotide has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:
  • the polynucleotide has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 5.
  • the polynucleotide has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide has the nucleic acid sequence of SEQ ID NO: 5.
  • the disclosure features a vector including the polynucleotide of the foregoing aspect.
  • the vector may be, for example, a viral vector or a non-viral vector.
  • the vector is a viral vector.
  • the viral vector is selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, and a synthetic virus.
  • the viral vector is an AAV.
  • the AAV includes capsid proteins from an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhIO, AAVrh74, and AAV2.7m8.
  • the AAV is a pseudotyped AAV.
  • the pseudotyped AAV is AAV2/8 or AAV2/9, optionally wherein the pseudotyped AAV is AAV2/8.
  • the AAV includes a recombinant capsid protein.
  • the polynucleotide is operably linked to a promoter, optionally wherein the promoter is positioned 5’ to the polynucleotide.
  • the promotor is a cytomegalovirus (CMV) promoter, cone-rod homeobox (CRX) promoter, retinal homeobox gene 1 (rx1) promoter, retinal pigment epithelium 65 (RPE65) promoter, CMV early enhancer/chicken beta actin (CAG, CBh, CBA) promoter, gamma-synuclein gene (SNCG) promoter, synapsin I (SYNI) promoter, rhodopsin kinase (GRK1) promoter, or a phosphoglycerate kinase (PGK) promoter.
  • CMV cytomegalovirus
  • CRX cone-rod homeobox
  • rx1 retinal homeobox gene 1
  • RPE65 retinal pigment epithelium 65
  • the promoter is a CMV promoter.
  • the CMV promoter has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of SEQ ID NO: 20.
  • the CMV promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • CMV promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 20, optionally wherein the CMV promoter has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the CMV promoter has the nucleic acid sequence of SEQ ID NO: 20.
  • the polynucleotide is operably linked to a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus
  • the WPRE has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of SEQ ID NO: 10 or 11 .
  • the WPRE has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 10 or 11 .
  • the WPRE has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 10 or 11 , optionally wherein the WPRE has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 10 or 11 . In some embodiments, the WPRE has the nucleic acid sequence of SEQ ID NO: 10 or 11 .
  • the polynucleotide further includes a polyadenylation (PolyA) signal sequence.
  • the PolyA signal sequence includes the simian virus 40 (SV40) late PolyA signal sequence, the SV40 early PolyA signal sequence or human growth hormone PolyA signal sequence (hGH PolyA signal sequence), or a shortened hGH PolyA signal sequence.
  • the PolyA signal sequence includes the SV40 late PolyA signal sequence.
  • the polynucleotide is comprised in a viral vector.
  • the viral vector may be, for example, an AAV.
  • the AAV may further include two inverted terminal repeats (ITRs), wherein the two ITRs include a first ITR (ITR1) and a second ITR (ITR2), wherein ITR1 is positioned 5’ to the polynucleotide and ITR2 is positioned 3’ to the polynucleotide, thereby forming a cassette including the structure ITR1-OPA1-ITR2.
  • the two ITRs are AAV serotype 2 ITRs.
  • the disclosure features a plasmid encoding the viral vector comprising the polynucleotide of the foregoing aspect.
  • the disclosure features a pharmaceutical composition including the polynucleotide of any one of the foregoing aspects or the vector of any one of the foregoing aspects, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the disclosure features a nucleic acid molecule including: a CMV promoter (CMV), a codon-optimized transgene encoding an OPA1 protein (e.g., an OPA1 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 21) (OPA1), a WPRE, and a SV40 PolyA signal sequence (SV40 Poly A), wherein the components are operably linked to each other in the 5’-to-3’ direction as CMV-OPA1-WPRE-SV40 PolyA.
  • CMV CMV promoter
  • OPA1 protein e.g., an OPA1 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 21
  • WPRE e.g., an OPA1 protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 21
  • SV40 PolyA signal sequence SV40 Poly A
  • the CMV promoter has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the CMV promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the CMV promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 20, optionally wherein the CMV promoter has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the CMV promoter has the nucleic acid sequence of SEQ ID NO: 20.
  • the transgene encodes a protein having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the amino acid sequence of SEQ ID NO: 21 .
  • the transgene encodes a protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 21 .
  • the transgene encodes a protein having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21 , optionally wherein the transgene encodes a protein having an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the transgene encodes a protein having the amino acid sequence of SEQ ID NO: 21.
  • the transgene has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of any one of SEQ ID Nos: 2-5. In some embodiments, the transgene has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID Nos: 2-5.
  • the transgene has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID Nos: 2-5, optionally wherein the transgene has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID Nos: 2-5. In some embodiments, the transgene has the nucleic acid sequence of any one of SEQ ID Nos: 2-5.
  • the WPRE has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of SEQ ID NO: 10 or 11 .
  • the WPRE has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 10 or 11.
  • the WPRE has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 10 or 11 , optionally wherein the WPRE has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 10 or 11 . In some embodiments, the WPRE has the nucleic acid sequence of SEQ ID NO: 10 or 11 .
  • the SV40 PolyA signal sequence includes the SV40 late PolyA signal sequence or the SV40 early PolyA signal sequence. In some embodiments, the PolyA signal sequence includes the SV40 late PolyA signal sequence.
  • the disclosure features a vector including the nucleic acid molecule of the foregoing aspect.
  • the vector may be, for example, a viral vector or a non-viral vector.
  • the vector is a viral vector.
  • the viral vector is selected from the group consisting of an AAV, an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, and a synthetic virus.
  • the viral vector is an AAV.
  • the AAV includes capsid proteins from an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhIO, AAVrh74, and AAV2.7m8.
  • the AAV is a pseudotyped AAV.
  • the pseudotyped AAV is AAV2/8 or AAV2/9, optionally wherein the pseudotyped AAV is AAV2/8.
  • the AAV includes a recombinant capsid protein.
  • the vector has a nucleic acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical, or more) to the nucleic acid sequence of any one of SEQ ID NOs: 12-14. In some embodiments, the vector has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 12-14.
  • the vector has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 12- 14, optionally wherein the vector has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 12-14. In some embodiments, the vector has the nucleic acid sequence of any one of SEQ ID NOs: 12-14.
  • the disclosure features a plasmid encoding the viral vector of the foregoing aspect.
  • the plasmid includes a promoter operably linked to a selectable marker gene.
  • the selectable marker gene is an antibiotic resistance gene.
  • the disclosure features a pharmaceutical composition including the nucleic acid molecule of any one of the foregoing aspects or the vector of any one of the foregoing aspects, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the disclosure features a method of treating a human patient diagnosed as having a neuro-ocular disorder or OPA1 -deficiency disorder, the method including administering to the patient a therapeutically effective amount of the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects.
  • the disclosure features a method of improving central and color vision in a human patient diagnosed as having a neuro-ocular disorder or OPA1 -deficiency disorder, the method including administering to the patient a therapeutically effective amount of the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects.
  • the disclosure features a method of improving retinal function in a human patient diagnosed as having a neuro-ocular disorder or OPA1 -deficiency disorder, the method including administering to the patient a therapeutically effective amount of the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects.
  • the disclosure features a method of improving visual acuity in a human patient diagnosed as having a neuro-ocular disorder or OPA1 -deficiency disorder, the method including administering to the patient a therapeutically effective amount of the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects.
  • the disclosure features a method of increasing the expression of OPA1 in a human patient diagnosed as having a neuro-ocular disorder or OPA1 -deficiency disorder, the method including administering to the patient a therapeutically effective amount of the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects.
  • the patient has impaired vision. In some embodiments, the patient has reduced visual acuity. In some embodiments, the patient has blindness. In some embodiments, the patient has retinal damage. In some embodiments, the patient has retinal degeneration.
  • the neuro-ocular disorder is an optic neuropathy. In some embodiments, the neuro-ocular disorder is a hereditary optic neuropathy. In some embodiments, the OPA1 -deficiency disorder is Autosomal Dominant Optic Atrophy (ADOA).
  • ADOA Autosomal Dominant Optic Atrophy
  • the disclosure features a method of ameliorating a symptom associated with a deficiency in OPA1 expression or activity in a human patient, the method including administering to the patient a therapeutically effective amount of the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects.
  • the symptom is selected from peripheral neuropathy, deafness, cerebellar ataxia, spastic paraparesis, and myopathy.
  • the disclosure features a kit including the polynucleotide, the vector, the nucleic acid molecule, or the pharmaceutical composition of any one of the foregoing aspects, and a package insert, wherein the package insert instructs a user of the kit to administer the polynucleotide, nucleic acid, vector, or pharmaceutical composition to a human patient diagnosed as having a neuro- ocular disorder, an OPA1 -deficiency disorder, or a symptom associated with a deficiency in OPA1 expression or activity.
  • the symptom is selected from peripheral neuropathy, deafness, cerebellar ataxia, spastic paraparesis, and myopathy.
  • FIG. 1 is a bar graph measuring expression levels of the OPA1 protein using western blot to quantify the expression ratio of OPA1 protein as expressed by each construct in Example 1 to GAPDH protein. Data were obtained by western blotting performed to visualize OPA1 protein expression levels in HEK293 cells after transfection with the plasmids. The measurement was made as described in Example 4, below.
  • FIG. 1 abbreviations: OPA1 : Optic Atrophy 1 ; pOPA1-A to I: plasmid OPA1-A to I; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
  • the term “about” refers to a value that is within 10% above or below the value being described.
  • the phrase “about 100 nucleic acid residues” refers to a value of from 90 to 110 nucleic acid residues.
  • AAV inverted terminal repeats
  • capsid proteins of any AAV serotype.
  • AAV serotypes include, without limitation, 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 , AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any other AAV now known or later discovered.
  • a “capsid protein” as used herein refers to any of the AAV capsid proteins that are components of AAV viral particles, including AAV8 and AAV9.
  • codon refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule, or coding strand of DNA, that specifies a particular amino acid or a starting or stopping signal for translation.
  • codon also refers to base triplets in a DNA strand.
  • codon optimization refers a process of modifying a nucleic acid sequence in accordance with the principle that the frequency of occurrence of synonymous codons (e.g., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. Sequences modified in this way are referred to herein as “codon-optimized” sequences. This process may be performed on any of the sequences described in this specification to enhance expression or stability. Codon optimization may be performed by any manner known in the art, such as, for example, that described in, e.g., U.S. Patent Nos.
  • Codon-optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or a biologically active fragment of a full-length polypeptide which has substantially the same activity as the full-length polypeptide) encoded by the non-codon-optimized parent polynucleotide.
  • An “inverted terminal repeat” or “ITR” is a palindromic nucleic acid, e.g., of from about 120 nucleotides to about 250 nucleotides in length, that is capable of forming a hairpin.
  • the terms “inverted terminal repeat” and “ITR” include the site of the viral genome replication that can be recognized and bound by a parvoviral protein (e.g., Rep78/68).
  • An ITR may be from any adeno- associated virus (AAV), for example, from AAV serotype 2, among others described herein.
  • An ITR includes a replication protein binding element (RBE) and a terminal resolution sequence (TRS).
  • ITR does not require a wild-type parvoviral ITR (e.g., a wild-type nucleic acid sequence may be altered by insertion, deletion, truncation, or missense mutations), as long as the ITR functions to mediate virus packaging, replication, integration, and/or provirus rescue, and the like.
  • the term “5’ ITR” is intended to mean the parvoviral ITR located at the 5’ boundary of the nucleic acid molecule; similarly, the term “3’ ITR” is intended to mean the parvoviral ITR located at the 3’ boundary of the nucleic acid molecule.
  • the “length” of a nucleic acid refers to the linear size of the nucleic acid as assessed by measuring the quantity of nucleotides from the 5’ to the 3’ end of the nucleic acid. Exemplary molecular biology techniques that may be used to determine the length of a nucleic acid of interest are known in the art.
  • the term “operably linked” refers to a first molecule (e.g., a first nucleic acid) joined to a second molecule (e.g., a second nucleic acid), wherein the molecules are so arranged that the first molecule affects the function of the second molecule.
  • the two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent to one another.
  • a promoter is “operably linked” to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in target cells.
  • two portions of a transcription regulatory element are “operably linked” to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion.
  • Two transcription regulatory elements may be “operably linked” to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be “operably linked” to one another with no intervening nucleotides present.
  • a linker nucleic acid e.g., an intervening non-coding nucleic acid
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, such as a polynucleotide, a nucleic acid molecule or vector comprising the polynucleotide or the nucleic acid molecule described herein, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and/or carriers, to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.
  • a therapeutic agent such as a polynucleotide, a nucleic acid molecule or vector comprising the polynucleotide or the nucleic acid molecule described herein, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and/or carriers, to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit/risk ratio.
  • wild-type and non-mutant form of a gene refer to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation that results in higher risk of developing, onset, or progression of a neurodegenerative disease).
  • mutant refers to any change in the gene sequence, which may be passed onto subsequent generations (hereditary mutation) or not (somatic mutation) or any change in the amino acid sequence, for example, the substitution, insertion, or deletion of one or more amino acids.
  • Gene mutations include the substitution, insertion, or deletion of a single base in a polynucleotide or the substitution, insertion, deletion, or rearrangement of multiple bases or larger sections of genes or chromosomes, including repeat expansions.
  • expression construct refers to any type of genetic construct containing a nucleic acid (e.g., transgene) in which part or all the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically active polypeptide product.
  • promoter refers to a recognition site on DNA that is bound by an RNA polymerase.
  • the polymerase drives transcription of the transgene.
  • exemplary promoters suitable for use with the compositions and methods described herein include ubiquitous promoters (e.g., a cytomegalovirus (CMV) promoter), among others described herein.
  • CMV cytomegalovirus
  • terapéutica protein refers to (i) a protein whose deficiency or lack of activity is associated with a disorder (e.g., Optic Atrophy 1 (OPA1)), as well as (ii) a protein that is not necessarily deficient in a patient, but whose supplementation would nonetheless have a beneficial effect on the patient.
  • OPA1 Optic Atrophy 1
  • transgene refers to an exogenous nucleic acid that has been transferred by genetic engineering means into another cell and is capable of being transcribed, and optionally translated.
  • nucleic acid and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally- occurring nucleotides.
  • a polynucleotide encoding OPA1 may comprise DNA sequence encoding OPA1 and/or RNA equivalent thereof.
  • RNA equivalent refers to an RNA polynucleotide (i.e., a polynucleotide comprised of ribonucleotides or variants thereof) that encodes the same polypeptide sequence as is encoded by the corresponding DNA polynucleotide counterpart.
  • RNA polynucleotides of the disclosure may include naturally occurring nucleosides and internucleoside linkages. Additionally or alternatively, RNA polynucleotides of the disclosure may include one or more chemically modified nucleosides or internucleoside linkages.
  • RNA polynucleotides of the disclosure may include a chemically modified nucleobase, such as one or more chemically modified forms of adenine, uracil, cytosine, and guanine.
  • a chemically modified nucleobase such as one or more chemically modified forms of adenine, uracil, cytosine, and guanine.
  • endogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • the term “gene expression” refers to the process by which a nucleic acid is transcribed from a nucleic acid molecule, and often, translated into a peptide or protein.
  • the process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof.
  • Reference to a measurement of “gene expression” may refer to measurement of the product of transcription (e.g., RNA or mRNA), the product of translation (e.g., peptides or proteins).
  • the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as a genetic disease described herein).
  • the subject may be, for example, a patient suffering from a genetic disease, for example, an OPA1- deficiency disorder such as Autosomal Dominant Optic Atrophy (ADOA).
  • OPA1-deficiency disorder means a disorder or condition which is caused by mutations in the 0PA1 gene and shows at least one symptom selected from optic neuropathy, peripheral neuropathy, deafness, cerebellar ataxia, spastic paraparesis, and myopathy.
  • transcription regulatory element refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., PolyA signal sequences) that control or help to control gene transcription. Examples of transcription regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).
  • treat and “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a genetic disease described herein.
  • the term “vector” refers to a vehicle for the delivery of a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), tissue, organ, or organism, such as a patient undergoing treatment for a disease or condition described herein, for purposes of expressing an encoded transgene.
  • a vector can be non-viral or viral vector.
  • non-viral vectors are plasmids, closed-ended DNA (ceDNA), chromosomes, artificial chromosomes, naked nucleic acid (DNA or RNA) and lipid nanoparticles, such as lipid nanoparticles comprising a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable cationic lipid, or any combination thereof.
  • exemplary viral vectors are an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, and a synthetic virus.
  • AAV adeno-associated virus
  • administration refers to providing or giving a subject a therapeutic agent (e.g., a pharmaceutical composition containing a vector containing a polynucleotide that encodes OPA1), by any effective route. Exemplary routes of administration are described herein below.
  • a therapeutic agent e.g., a pharmaceutical composition containing a vector containing a polynucleotide that encodes OPA1
  • the terms “effective amount”, “therapeutically effective amount”, and a “sufficient amount” of a pharmaceutical composition described herein refer to a quantity sufficient to, when administered to the subject in need thereof, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied.
  • the amount of a given pharmaceutical composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • a “therapeutically effective amount” of a pharmaceutical composition of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. As defined herein, a therapeutically effective amount of a pharmaceutical composition of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regime may be adjusted to provide the optimum therapeutic response.
  • the terms “cytomegalovirus promoter” and “CMV promoter” refer to the cytomegalovirus immediate early enhancer/promoter.
  • the CMV promoter comprises the nucleic acid set forth in SEQ ID NO: 20, as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of SEQ ID NO: 20 and that promote the expression of a transgene in target cells when the transgene is operably linked to the promoter.
  • the CMV promoter has the nucleic acid sequence of SEQ ID NO: 20.
  • OPA1 mitochondrial dynamin like GTPase
  • OPA1 mitochondrial dynamin like GTPase
  • OPA1 or “OPA1 gene” refers to a polynucleotide encoding the OPA1 protein.
  • the OPA1 gene includes a wild-type OPA1 gene (e.g., NCBI Reference Sequence: NC_000003.12:193593208-193697811 and SEQ ID NO: 2) or a variant thereof.
  • Exemplary variants of wild-type OPA1 are polynucleotides that have at least 85% nucleic acid sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% nucleic acid sequence identity, or more) to the nucleic acid sequence of a wild-type OPA1 gene (e.g., SEQ ID NO: 2) and/or that retain the therapeutic function of a wild-type OPA1 protein when translated in target cells.
  • nucleic acid sequence identity e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% nucleic acid sequence identity, or more
  • an OPA1 gene comprises a codon-optimized polynucleotide encoding an OPA1 protein, such as a codon-optimized polynucleotide having at least 85% nucleic acid sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% nucleic acid sequence identity, or more) to the nucleic acid sequence of any one of SEQ ID NOs: 3-5.
  • the codon-optimized polynucleotide encoding an OPA1 protein has the nucleic acid sequence of any one of SEQ ID NOs: 3-5.
  • 5’ UTR refers to the 5' untranslated region (also referred to as leader sequence, transcript leader, or leader RNA) in mRNA. It is placed right before the initiation codon and plays an important role in the regulation of translation.
  • exemplary 5’ UTRs may include an internal ribosomal entry site (IRES), central polypurine tract (cPPT), RNA localization signal, small RNA binding site, and RNA/RNA polymerase recognition site.
  • IRS internal ribosomal entry site
  • cPPT central polypurine tract
  • RNA localization signal RNA localization signal
  • small RNA binding site small RNA binding site
  • RNA/RNA polymerase recognition site examples of 5’ UTRs that may be used in conjunction with the pharmaceutical compositions and methods of the disclosure are those having the nucleic acid sequence of SEQ ID NO: 6 or 8.
  • 3’ UTR refers to the 3' untranslated region in mRNA and is placed right after the translation termination codon.
  • the 3' UTR may contain regulatory regions that affect gene expression in a post-transcriptional manner.
  • Exemplary 3’ UTRs may include a PolyA signal sequence and a WPRE.
  • Examples of 3’ UTRs that may be used in conjunction with the pharmaceutical compositions and methods of the disclosure are those having the nucleic acid sequence of any one of SEQ ID NOs: 7, 9, 10, and 11.
  • the terms “woodchuck hepatitis virus posttranscriptional regulatory element” and “WPRE” refer to a DNA sequence, which when transcribed, creates a tertiary structure enhancing expression.
  • the WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells.
  • the addition of the WPRE may result in a substantial improvement in the level of transgene expression, both in vitro and in vivo.
  • WPRE elements that may be used herein include those having the nucleic acid sequence of SEQ ID NO: 10 or 11 , as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequences of SEQ ID NO: 10 or 11 and that promote the expression of a transgene in target cells.
  • the WPRE has the nucleic acid sequence of SEQ ID NO: 10 or 11.
  • the term “ocular” refers to the eye and may designate a composition as being derived from, or related to, the eye.
  • neuro-ocular disorder and “neuro-visual disorder” refer to a disorder in which the neurons in the eye are affected, for example, a disorder caused by injury or damage to the retinal ganglion cells in the eye.
  • neuro-ocular disorders include an optic neuropathy (for example, a hereditary optic neuropathy, such as ADOA) caused by mutations in the OPA1 gene.
  • a neuro-ocular disorder may be associated with genetic defects (e.g., a mutation in the OPA1 gene), protein misfolding, defects in protein degradation, programmed cell death, membrane damage, or other processes.
  • neuro-ocular disorders include optic neuropathy, ADOA, retinal degeneration, blindness, blindness-related disorders, retinopathy, optic neuropathies, optic neuritis, retinitis pigmentosa, chiasm disorders, giant cell (temporal) arteritis, eye movement disorders, double vision, nystagmus, oscillopsia, disorders of the pupils (for example, anisocoria), and temporary vision problems related to auras, among others.
  • central vision refers to a subject’s ability to see what is directly in front of the subject.
  • Central vision primarily relies upon the activity of cone cells.
  • color vision refers to the ability of a subject to perceive color (i.e., the ability to perceive and distinguish between different wavelengths of light). Color vision primarily relies upon the activity of cone photoreceptors in the retina. As used herein, the term “visual acuity” is a measure of the spatial resolution achieved by the eye (i.e., a measure of one’s sharpness and clarity of vision).
  • retina degeneration refers to the progressive loss of cells in the retina, for example, rod-cone degeneration leads to loss of rods and cones. Retinal degeneration can cause blindness.
  • optical neuropathy refers to damage caused to the optic nerve, which carries visual information from the eye to the brain.
  • Optic neuropathy can be hereditary in nature, for example, ADOA is a common hereditary optic neuropathy which can be caused by mutations in the 0PA1 gene.
  • Optic neuropathy can arise due to a variety of reasons such as infections (e.g., toxoplasmosis, herpes simplex), inflammatory diseases, neurological disorders, certain medication, dietary deficiencies, and toxins (e.g., methanol, alcohol, tobacco).
  • retina damage refers to any damage to the retina that can be caused by, for example, old age, light, trauma, and retinal disorders, among others. Retinal damage is a common problem of the eye and can cause vision loss or blindness.
  • retina function refers to the ability of the retina to convert the light that enters the eye into electrical signals that can be sent to the brain using the optic nerve so that a visual image can be formed.
  • compositions and methods for the treatment of optic neuropathy for example, Autosomal Dominant Optic Atrophy (ADOA)
  • ADOA Autosomal Dominant Optic Atrophy
  • OPAT Optic Atrophy 1
  • a subject such as a mammalian subject, for example, a human
  • ADOA Autosomal Dominant Optic Atrophy
  • OPAT Optic Atrophy 1
  • the pharmaceutical compositions described herein may include nucleic acid molecules that include a transgene encoding OPA1 , and methods for the delivery of this transgene into target cells to express the resulting protein OPA1 .
  • the target cells are eukaryotic cells, such as mammalian cells.
  • the mammalian cells are human cells, non-human primate cells or mouse cells.
  • the human cells are neurons, retinal cells (e.g., retinal ganglion cells, retinal pigment epithelium, or photoreceptor cells), cells in the inner ear, cells in muscle tissues.
  • the pharmaceutical compositions described herein may ameliorate the negative effects of ADOA by increasing the expression of OPA1 in human patients diagnosed with or displaying one or more symptoms of ADOA.
  • the pharmaceutical compositions and methods described herein may be used to treat neuro-ocular disorders, such as ADOA, or symptoms of such disorders, such as blindness or reduced visual acuity.
  • the nucleic acids described herein may be carried in a vector, such as a viral vector.
  • a vector such as a viral vector.
  • AAV vectors such as pseudotyped AAV vectors (e.g., an AAV2/8 vector) containing a transgene encoding OPA1 operably linked to a promoter, e.g., a CMV promoter.
  • the present invention is based, at least in part, on the discovery that a codon-optimized polynucleotide encoding the OPA1 protein leads to a highly efficient and robust expression of OPA1.
  • the expression of OPA1 can be increased in patients diagnosed with or displaying one or more symptoms of ADOA, such as blindness or reduced visual acuity. This change in gene expression and subsequent shift in protein expression can mitigate symptoms of ADOA and may ameliorate the disease.
  • ADOA Autosomal Dominant Optic Atrophy
  • optic neuropathy which can be hereditary in nature, for example, because of a mutation in the 0PA 1 gene. It is one of the most common forms of hereditary optic neuropathy.
  • the symptoms of this disease include impaired vision, impaired color vision (color blindness), impaired central vision, central scotoma, reduced visual acuity, vision loss, and/or blindness and phenotypes such as retinal damage, retinal degeneration, retinal ganglion cell loss, optic nerve damage, among others.
  • ADOA can cause blindness, vision loss or vision impairment, starting in childhood, thus, resulting in childhood blindness.
  • ADOA causes bilateral optic neuropathy, but other neurological phenotypes include peripheral neuropathy, deafness, cerebellar ataxia, spastic paraparesis, and myopathy. Some of these phenotypes are extraocular in nature. DOA generally affects both eyes in a roughly symmetrical manner and causes a slowly progressive pattern of vision loss starting in childhood. Vision examinations in ADOA patients reveal scotomas (areas of impaired visual acuity) in the central visual fields with sparing of peripheral vision. Visual acuity loss tends to vary from mild to severe. Symptoms of ADOA are typically observable between four to six years of age but ADOA has been diagnosed even in patients as young as a one-year-old. In some cases, DOA is not readily observable until early adulthood. The severity of ADOA by adolescence reflects the overall level of visual function that the patient can expect to have for the rest of the adult life.
  • OPA1 is a mitochondrial dynamin-like GTPase protein and is ubiquitously expressed. It plays an important role in mitochondrial fusion dynamics. It is required for forming protein complexes that provide structural support to the cristae junctions in mitochondria and for keeping cytochrome C within the lumen of the cristae. OPA1 also functionally interacts with the oxidative phosphorylation complexes. Since OPA1 is required for normal mitochondrial fusion, mutations in this protein cause inherited neuropathies because the nervous system has a high metabolic rate and therefore, the proper functioning of the mitochondria is important for its optimal activity.
  • OPA1 is located on the inner mitochondrial membrane and protects cells from apoptosis by binding cytochrome C. Mutations in OPA1 cause mitochondrial respiratory dysfunction and lead to the loss of retinal ganglion cells, the axons of which form the optic nerve, which is in turn responsible for carrying visual information from the eye to the brain.
  • the pharmaceutical compositions and methods described herein target this dysfunction by providing codon-optimized transgenes encoding the OPA1 protein, as well as nucleic acid molecules, expression cassettes, and vectors containing the same.
  • Nucleic acid molecules described herein include codon-optimized polynucleotides encoding OPA1 .
  • the nucleic acid molecules described herein my further contain a promoter, as operably linking a codon-optimized version of the OPA 1 gene to a promoter (e.g., CMV promoter) advantageously and robustly increases the OPA1 protein in target cells.
  • the nucleic acid molecule may be carried in a vector to optimize delivery into target cells.
  • the polynucleotide encoding OPA1 comprises a wild-type OPA 1 (e.g., SEQ ID NO: 2) or a variant thereof, such as a polynucleotide having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of the wild-type OPA 1 gene and retaining the therapeutic function of the wildtype OPA1 protein when translated in target cells.
  • the OPA1 gene has the nucleic acid sequence of SEQ ID NO: 2.
  • SEQ ID NO: 2 is shown below:
  • the OPA1 gene comprises codon-optimized polynucleotides encoding the OPA1 protein.
  • Exemplary codon-optimized polynucleotides encoding OPA1 are shown in Table 1 , below.
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 3-5 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 3-5).
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding OPA1 has the nucleic acid sequence of SEQ ID NO: 3.
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4.
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding OPA1 has the nucleic acid sequence of SEQ ID NO: 4.
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 5.
  • the polynucleotide encoding OPA1 has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding OPA1 has the nucleic acid sequence of SEQ ID NO: 5.
  • the OPA1 protein may be wild-type OPA1 protein (e.g., NCBI Reference Sequence: NP_056375.2 and SEQ ID NO: 21) or a variant of the wild-type OPA1 protein, e.g., a variant protein having at least 85% amino acid sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% amino acid sequence identity, or more) to the amino acid sequence of a wild-type OPA1 protein (e.g., SEQ ID NO: 21), provided that the OPA1 variant retains the therapeutic function of wild-type OPA1 protein when translated in target cells.
  • the OPA1 protein has the amino acid sequence of SEQ ID NO: 21 .
  • SEQ ID NO: 21 is shown below:
  • Polynucleotides encoding an OPA1 protein disclosed herein may include a regulatory element capable of modulating (i.e., increasing or attenuating) gene expression.
  • the polynucleotide or nucleic acid molecule of the disclosure may further include a transcription regulatory element operably linked to the polynucleotide encoding OPA1 so as to regulate gene transcription.
  • the polynucleotide or nucleic acid molecule of the disclosure can comprise promoters, enhancers, and other nucleic acids (e.g., PolyA signal sequences and WPRE), and so on.
  • Promoters that may be used in conjunction with the polynucleotides of the disclosure include, without limitation, a cytomegalovirus (CMV) promoter, cone-rod homeobox (CRX) promoter, retinal homeobox gene 1 (rx1) promoter, retinal pigment epithelium 65 (RPE65) promoter, CMV early enhancer/chicken beta actin (CAG, CBh) promoter, gamma-synuclein gene (SNCG) promoter, human rhodopsin kinase (GRK1) promoter, or a phosphoglycerate kinase (PGK) promoter, a synapsin (Syn) promoter, synapsin I (SYNI) promoter, a chicken p-actin promoter with cytomegalovirus enhancer elements (CB7), a tetracycline-controlled transactivator protein (tTA) promoter, a reverse tetracycline- controlled transactivator
  • CX3CR1 C-X3-C motif chemokine receptor 1
  • IGAM integrin subunit alpha M
  • AIF1 allograft inflammatory factor 1
  • P2Y12 purinergic receptor P2Y12
  • TMEM119 transmembrane protein 119
  • CSF1 R colony stimulating factor 1 receptor
  • the CMV promoter has the nucleic acid sequence set forth in SEQ ID NO: 20, or a nucleic acid sequence having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of SEQ ID NO: 20 and that promotes the expression of a transgene in target cells when the transgene is operably linked to the promoter.
  • the CMV promoter has the nucleic acid sequence of SEQ ID NO: 20. SEQ ID NO: 20 is shown below:
  • Enhancers represent another class of regulatory elements that induce a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site.
  • Many enhancer sequences are now known from mammalian genes or derived from the genetic material of a virus capable of infecting a eukaryotic cell. Exemplary enhancers from mammalian genes are those from the genes that encode mammalian globin, elastase, albumin, a-fetoprotein, and insulin.
  • Exemplary enhancers derived from the genetic material of a virus are the SV40 enhancer on the late side of the replication origin (bp 100- 270), the CMV early promoter enhancer, the cytomegalovirus immediate early enhancer, the cytomegalovirus early enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription are disclosed in Yaniv et al., Nature 297:17 (1982).
  • the polynucleotide or nucleic acid molecule of the disclosure may include a WPRE.
  • the WPRE has the nucleic acid of SEQ ID NO: 10 or 11 , or a nucleic acid having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity, or more) to the nucleic acid sequence of SEQ ID NO: 10 or 11 and that promotes the expression of a transgene in target cells when the transgene is operably linked to the WPRE.
  • the WPRE has the nucleic acid sequence of SEQ ID NO: 10 or 11 .
  • SEQ ID Nos: 10 mutant WPRE (mtWPRE)
  • WPREs short WPRE
  • the polynucleotide or nucleic acid molecule of the disclosure may further include a PolyA signal sequence.
  • Exemplary PolyA signal sequence include those known in the art and described in the working examples, below.
  • the PolyA signal sequence includes the simian virus 40 (SV40) late PolyA signal sequence, the SV40 early PolyA signal sequence, human growth hormone PolyA signal sequence (hGH PolyA signal sequence), or shortened hGH PolyA signal sequence.
  • the PolyA signal sequence includes the SV40 late PolyA signal sequence.
  • Untranslated regions (UTRs) other than WPRE and PolyA signal sequence also occur on either side of a coding sequence in an mRNA.
  • the polynucleotide or nucleic acid molecule of the disclosure may comprise 5’ UTR and/or 3’ UTR.
  • Exemplary 5’ UTRs include the nucleic acids set forth in any one of SEQ ID NO: 6 or 8, as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of any one of SEQ ID NO: 6 or 8 and that regulate the translation in target cells.
  • the 5’ UTR has the nucleic acid sequence of any one of SEQ ID NO: 6 or 8.
  • Exemplary 3’ UTRs include the nucleic acids set forth in any one of SEQ ID NO: 7 or 9, as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of any one of SEQ ID NO: 7 or 9 and that regulate the translation in target cells.
  • the 3’ UTR has the nucleic acid sequence of any one of SEQ ID NO: 7 or 9.
  • SEQ ID Nos: 6-9 are shown in Table 3, below:
  • the polynucleotide or nucleic acid molecule of the disclosure may be carried in a vector.
  • a vector can be non-viral or viral vector. Examples of non-viral vectors are plasmids, closed-ended DNA (ceDNA), chromosomes, artificial chromosomes, naked nucleic acid (DNA or RNA) and lipid nanoparticles.
  • viral vectors are a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • retrovirus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example.
  • retroviruses are avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996)).
  • murine leukemia viruses murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.
  • vectors are described, for example, in McVey et al., (US 5,801 ,030), the teachings of which are incorporated herein by reference.
  • the viral vector is selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, and a synthetic virus.
  • AAV adeno-associated virus
  • the viral vector is an AAV.
  • AAV vectors that may be used in conjunction with the pharmaceutical compositions and methods of the disclosure include those having one or more of the naturally occurring AAV genes deleted in whole or in part but retain functional flanking ITR sequences.
  • the AAV ITRs may be of any serotype (e.g., derived from serotype 2) suitable for a particular application. Methods for using AAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.
  • the polynucleotide or nucleic acid molecule of the disclosure can be incorporated into a AAV virion to facilitate introduction of the polynucleotide or nucleic acid molecule into a cell.
  • the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
  • the cap gene encodes three viral coat proteins, VP1 , VP2, and VP3, which are required for virion assembly.
  • the construction of AAV virions has been described, for example, in US Patent Nos.
  • AAV virions useful in conjunction with the pharmaceutical compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1 , 2, 3, 4, 5, 6, 7, 8 and 9. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2 :619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97 :3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000); Halbert et al., J. Virol.
  • AAV AAV2.7m8
  • WO 2012/145601 WO 2012/145601
  • US Patent No. 11 ,236,402 the disclosures of each of which are incorporated herein by reference.
  • Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1 , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, among others).
  • AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9.
  • pseudotyped AAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075- 3081 (2001).
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635-45 (2000).
  • Other AAV virions that can be used in methods of the disclosure include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).
  • a patient diagnosed with or displaying one or more symptoms of optic neuropathy or OPA1 -deficiency disorder may be administered a pseudotyped AAV2/8 vector including a polynucleotide encoding, from 5’ to 3’, a CMV promoter, a transgene encoding OPA1 , a WPRE, and a simian virus 40 late PolyA signal sequence.
  • ADOA Autosomal Dominant Optic Atrophy
  • the CMV promoter has a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 20 and that promotes the expression of the transgene in target cells.
  • the transgene encoding OPA1 has a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NOs: 3-5.
  • the OPA1 protein has an amino acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 21 and retains the therapeutic function of wild-type OPA1 protein.
  • the WPRE has a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NO: 10 or 1 1 and that promote the expression of a transgene in target cells.
  • the AAV vector comprises a nucleic acid sequence that is at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NOs: 12-14 to express the OPA1 protein in target cells.
  • Exemplary AAV vectors of the disclosure include those having the nucleic acid sequence of any one of SEQ ID NOs: 1 and 12-19.
  • SEQ ID NOs: 1 and 12-19 are shown in Table 4, below.
  • AAV vector of the disclosure comprises the nucleic acid set forth in any one of SEQ ID NOs: 12-14, as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to the nucleic acid sequence of any one of SEQ ID NOs: 12-14 and that achieve expression of the OPA1 gene in target cells.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more
  • the vector including the nucleic acid molecule may be delivered to desired tissues, such as the eye (e.g., to the retina), of a patient diagnosed with or displaying one or more symptoms of optic neuropathy and OPA1 -deficiency disorder, for example ADOA.
  • desired tissues such as the eye (e.g., to the retina)
  • the patient may exhibit an increase in the expression of OPA1 , an increase in visual acuity, improvement of retinal function, improvement of central vision, improvement of color vision and/or overall improvement in other symptoms within sometime after treatment.
  • the patient may receive additional treatments.
  • compositions and methods of the present disclosure may be used to treat a variety of subjects having a disease or condition associated with a mutation in one or more 0PA1 alleles.
  • subjects that may be treated as described herein include those subjects having an optic neuropathy, such as a hereditary optic neuropathy (e.g., ADOA).
  • ADOA is hereditary in nature and can be caused, for example, because of a mutation in the 0PA1 gene.
  • Additional examples of subjects that may be treated using the pharmaceutical compositions and methods of the disclosure are those having diseases or conditions caused by OPA1 protein deficiency.
  • OPA1 deficiency is associated not only with optic neuropathy, but also with systemic pathologies, including peripheral neuropathy, deafness, cerebellar ataxia, spastic paraparesis, and myopathy. Accordingly, the pharmaceutical compositions and methods disclosed herein can be used to ameliorate these systemic conditions.
  • compositions and methods described herein may also be used as a preventative treatment for patients having a deleterious mutation in an 0PA1 gene, patients with reduced OPA1 activity, and patients exhibiting symptoms of ADOA.
  • exemplary symptoms of ADOA include impaired vision, reduced visual acuity, vision loss, color blindness, central vision loss, blindness, retinal damage, retinal degeneration, retinal ganglion cell loss, and optic nerve damage, among others.
  • a subject may be administered a vector comprising the polynucleotide or nucleic acid molecule of the disclosure (e.g., an AAV vector of the disclosure) to deliver the OPA1 protein that supplements the expression of the OPA1 protein that is already expressed in the subject, but whose supplementation gives rise to a therapeutic effect.
  • a vector comprising the polynucleotide or nucleic acid molecule of the disclosure (e.g., an AAV vector of the disclosure) to deliver the OPA1 protein that supplements the expression of the OPA1 protein that is already expressed in the subject, but whose supplementation gives rise to a therapeutic effect.
  • Vectors such as AAV vectors and others described herein, containing an 0PA1 gene operably linked to a promoter described herein may be administered to a patient (e.g., a human patient) by a variety of routes of administration.
  • Administration of an effective dose of the pharmaceutical composition comprising the vectors may be by exemplary routes of administration standard in the art, including, but not limited to, systemic (e.g., by intravenous administration), local (e.g., into the eye), and direct injection (e.g., subretinal).
  • the route of administration may vary, for example, with the onset and severity of disease, and may include, e.g., subretinal, intraocular, intravitreal, intravenous, intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and administration.
  • Treatment regimens may vary, and often depend on disease severity and the age, weight, and sex of the patient.
  • Treatment may include administration of vectors (e.g., viral vectors) or other agents described herein as useful for the introduction of a transgene into a target cell in various unit doses.
  • Each unit dose will ordinarily contain a predetermined quantity of the pharmaceutical composition.
  • the AAV vector comprising the transgene encoding OPA1 described herein can be administered in an amount sufficient to improve one or more pathological features of ADOA.
  • Administration of the pharmaceutical compositions described herein may improve visual acuity in the subject, reduce the amount of vision loss in the subject, improve central vision of the subject, improve color vision of the subject, increase the expression of OPA1 in the subject, and/or improve retinal function in the subject.
  • the polynucleotide, the nucleic acid molecule, or the vector comprising the polynucleotide or the nucleic acid molecule (e.g., the AAV vector) described herein can be formulated into a pharmaceutical composition for administration to a patient, such as a human patient having a disease or condition associated with a mutation in one or more OPA1 alleles.
  • the pharmaceutical compositions of the disclosure may be administered to a subject having an optic neuropathy, such as a subject diagnosed with or displaying one or more symptoms of a hereditary optic neuropathy (e.g., ADOA).
  • the pharmaceutical compositions of the disclosure may also be administered to a subject having a systemic disease or condition caused by OPA1 protein deficiency, such as peripheral neuropathy, deafness, cerebellar ataxia, spastic paraparesis, and myopathy.
  • compositions of the disclosure may be provided in a biologically compatible form suitable for administration in vivo.
  • a pharmaceutical composition containing a polynucleotide encoding OPA1 or a nucleic acid molecule including a transgene encoding OPA1 for example, the AAV vector comprising the polynucleotide or the nucleic acid molecule, typically includes a pharmaceutically acceptable diluent or carrier.
  • a sterile saline solution typically a pharmaceutical grade saline
  • sterile water typically a pharmaceutical grade water
  • PBS phosphate-buffered saline
  • compositions of the disclosure can be formulated using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
  • Treatment regimens may vary, and often depend on disease severity and the age, weight, and sex of the patient.
  • the disclosure provides the polynucleotide, the nucleic acid molecule, or the vector comprising the polynucleotide or the nucleic acid molecule (e.g., the AAV vector) for use in the treatment or prevention of an optic neuropathy or OPA1 -deficiency disorder, such as ADOA.
  • an optic neuropathy or OPA1 -deficiency disorder such as ADOA.
  • the disclosure provides use of the polynucleotide, the nucleic acid molecule, or the vector comprising the polynucleotide or the nucleic acid molecule (e.g., the AAV vector) in the manufacture of a pharmaceutical composition for the treatment or prevention of an optic neuropathy or OPA1 -deficiency disorder, such as ADOA. Kits
  • the polynucleotide, nucleic acid molecule, vector, or pharmaceutical composition that achieves the expression of OPA1 protein in a subject described herein can be provided in a kit for use in treating OPA1 -deficiency disorder, such as ADOA.
  • the kit can include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein.
  • the kit may optionally include a syringe or other device for administering the composition.
  • the plasmid tested in this Example has a CMV promoter sequence, a nucleotide sequence encoding OPA1 , and a PolyA signal sequence.
  • the sequence from CMV promoter to PolyA signal was inserted between the Nrul site and F1 ori start site of pcDNA3.1 (+) (ThermoFisher Scientific V79020) from the 5' end to the 3' end, as shown in SEQ ID NO: 1 .
  • This plasmid is referred to as pOPA1-A.
  • pOPA1-A includes the native codon nucleotide sequence of OPA1 (SEQ ID NO: 2).
  • pOPA1-A Based on pOPA1-A, two codon-optimized sequences encoding OPA1 (SEQ ID NO: 3 and SEQ ID NO: 4) were inserted between the Agel site and BamHI site.
  • the plasmid containing the sequence of SEQ ID NO: 3 is referred to as pOPA1-B and the plasmid containing the sequence of SEQ ID NO: 4 is referred to as pOPA1-C.
  • Each codon-optimized sequence is inserted to replace SEQ ID NO: 2 in pOPA1-A.
  • pOPA1-A Based on pOPA1-A, a fragment was prepared by PCR from a plasmid containing a codon- optimized sequence encoding OPA1 (SEQ ID NO: 5) and inserted between the Agel site and BamHI site.
  • the plasmid containing the sequence of SEQ ID NO: 5 is referred to as pOPA1-D.
  • the codon- optimized sequence was inserted to replace SEQ ID NO: 2 in pOPA1-A.
  • pOPA1-A human alpha-1-globin derived 5’ UTR (SEQ ID NO: 6) was inserted between the Sacl site and PfIMI site.
  • the plasmid containing the sequence of SEQ ID NO: 6 is referred to as pOPA1-E.
  • pOPA1-F Based on pOPA1-A, a fragment was prepared by PCR from a plasmid containing human alpha-1 -globin derived 3’ UTR (SEQ ID NO: 7) and inserted between the BamHI site and Xbal site.
  • SEQ ID NO: 7 The plasmid containing the sequence of SEQ ID NO: 7 is referred to as pOPA1-F.
  • a virus derived sequence (SEQ ID NO: 8) was inserted between the Sacl site and PfIMI site. After this step, linked sequence of a virus derived sequence (SEQ ID NO: 9) was inserted between the BamHI site and Xbal site.
  • pOPA1-G 8 and 9 is referred to as pOPA1-G.
  • pOPA1-A a fragment was prepared by PCR from a plasmid containing Woodchuck hepatitis virus (WHV) derived mutant WHV Posttranscriptional Regulatory Element (WPRE) sequence (SEQ ID NO: 10) and inserted between the BamHI site and Xbal site.
  • WPRE Woodchuck hepatitis virus
  • SEQ ID NO: 10 The plasmid containing the sequence of SEQ ID NO: 10 is referred to as pOPA1-H.
  • pOPA1-A Based on pOPA1-A, a fragment was prepared by PCR from a plasmid containing Woodchuck hepatitis virus derived short WPRE sequence (SEQ ID NO: 11) and inserted between the BamHI site and Xbal site.
  • the plasmid containing the sequence of SEQ ID NO: 11 is referred to as pOPA1-L pOPA1-A to I were transformed using stbl3 (ThermoFisher Scientific C7373-03), and each colony was collected and incubated at 37°C for 1 day. Plasmid purification was performed for pOPA1- A using Nucleo Bond Xtra Maxi Plus EF (TaKaRa bio, 740426.50).
  • pOPA1-B to I were purified using NucleoBond Xtra Midi Plus EF (TaKaRa bio, 740422.50).
  • each plasmid The sequences contained by each plasmid are shown in Table 5 below.
  • the full-length sequences from the promoter to the PolyA signal are SEQ ID NOs: 12- 19, respectively.
  • HEK293 cells (ATCC CRL-1573) were seeded into a collagen l-coated 24 well plate (IWAKI 4820-010) at a density of 100,000 cells per well and cultured with Dulbecco Modified Eagle Medium (Sigma D6429-500ML) containing 10% fetal bovine serum (FBS, Hyclone SH30084.03) and 1% Penicillin-Streptomycin (ThermoFisher Scientific 15070063) at 37°C with 5% CO2 for 1 day.
  • Dulbecco Modified Eagle Medium Sigma D6429-500ML
  • FBS fetal bovine serum
  • Hyclone SH30084.03 fetal bovine serum
  • Penicillin-Streptomycin ThermoFisher Scientific 15070063
  • Cells were transfected with 500 ng of each plasmid by using the ViaFectTM Transfection Reagent (Promega E4981). The cells were incubated for 2 days after transfection. Cells were lysed with 100 uL of RIPA buffer (ThermoFisher Scientific 89901) containing 1 % Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (ThermoFisher scientific 78441) and 0.1% Benzonase® Nuclease, Purity > 99% (Millipore, 70664-10KUN). Cell lysates were centrifuged at 12,000 g for 10 mins at 4°C and the supernatants were collected in new 1 .5 mL tubes.
  • the amount of total protein was calculated using the BCA protein assay kit (ThermoFisher Scientific 23227) on 96 well assay plates (IWAKI 3881-096). Absorbance was measured at 562 nm by Infinite M200PRC (TECAN). The concentrations of protein lysates were normalized with RIPA buffer (ThermoFisher Scientific 89901) containing 1 % Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (ThermoFisher scientific 78441) and 0.1% Benzonase® Nuclease, Purity > 99% (Millipore, 70664-10KUN).
  • Protein lysates were mixed with NuPAGETM LDS Sample Buffer (ThermoFisher scientific NP0007) and NuPAGETM Sample Reducing Agent (ThermoFisher scientific NP0009). The samples were heated at 95°C for 10 mins.
  • Total protein was separated by SDS-PAGE on NuPAGETM 4 to 12%, Bis-Tris, 1.0-1 .5 mm, Mini Protein Gels (ThermoFisher scientific NP0329BOX) and then transferred onto PVDF membranes using Trans-Blot Turbo Mini 0.2 pm PVDF Transfer Packs (Bio-Rad 1704156). Non-specific protein binding was blocked using Blocking One (NACALAI TESQUE, INC. 03953-95). The proteins on the membranes were immunoblotted against anti-OPA1 (D6U6N) Rabbit mAb (Cell Signaling 80471 S) and anti-GAPDH (14C10) Rabbit mAb (Cell Signaling 2118S).
  • Immunoreactive bands were visualized using Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling 7074S) and the ECL detection system (GE Healthcare RPN2236). Chemiluminescence signals were captured using a ChemiDoc Touch (BioRad) and the protein expression levels were quantified using Image Lab (Bio-Rad). Results
  • OPA1 protein was normalized by GAPDH protein.
  • the relative OPA1 protein expression level from each construct compared to the not treated sample was calculated (Fig. 1).
  • the OPA1 expression levels of pOPA1-B, pOPA1-C, and pOPA1-D were higher than that of pOPA1-A.

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Abstract

L'invention concerne des compositions pharmaceutiques et des méthodes de traitement d'un sujet diagnostiqué comme atteint, par exemple, d'un trouble neuro-oculaire ou d'un trouble de déficience en OPA1, notamment une atrophie optique autosomique dominante (ADOA) provoquée par des mutations dans le gène de l'atrophie optique (OPA1). Les compositions pharmaceutiques et les méthodes de l'invention comprennent des polynucléotides, des vecteurs viraux (par exemple, des vecteurs viraux adéno-associés), et des vecteurs non viraux, qui contiennent un transgène à codons optimisés codant pour la protéine OPA1.
PCT/US2024/026180 2023-04-26 2024-04-25 Compositions et méthodes de thérapie génique opa1 Ceased WO2024226729A2 (fr)

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