WO2026011008A1 - Contrôle d'expression par miarn exprimés par les muscles squelettiques - Google Patents

Contrôle d'expression par miarn exprimés par les muscles squelettiques

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Publication number
WO2026011008A1
WO2026011008A1 PCT/US2025/036161 US2025036161W WO2026011008A1 WO 2026011008 A1 WO2026011008 A1 WO 2026011008A1 US 2025036161 W US2025036161 W US 2025036161W WO 2026011008 A1 WO2026011008 A1 WO 2026011008A1
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Prior art keywords
nucleic acid
aav
mir
acid sequence
vector
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English (en)
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Saurav SESHADRI
Sharif TABEBORDBAR
Shayan TABEBORDBAR
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Kate Therapeutics Inc
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Kate Therapeutics Inc
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    • 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
    • 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
    • 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
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the invention relates to recombinant nucleic acids (e.g., vectors) and methods for differential expression of a transgene.
  • rAAVs Recombinant adeno-associated viruses
  • rAAVs are the most commonly used delivery vehicles for gene therapy and gene editing.
  • rAAVs variants frequently exhibit at least some non-specific transduction of multiple cell types.
  • an rAAV delivering a transgene desired to be expressed in one type of tissue may nevertheless transduce and express that transgene in a different tissue in which that expression is toxic.
  • AAV-delivered gene therapies have been associated with tissuespecific toxicity resulting from expression of a desired gene in an inappropriate tissue. This is especially prominant in muscle-specific therapies, where the overall similarities between skeletal myofibers and cardiomycocytes result in AAV serotypes that typically infect both cell types.
  • compositions and methods that can be used to repress transgene expression in skeletal myofibers but not cardiomycocytes.
  • the present invention provides recombinant nucleic acids (e.g., vectors) that encode a transgene and the binding site(s) for specific microRNAs (miRs) that direct the miRs to selectively inhibit expression of the same transgene.
  • miR-206 and miR-128-3p are highly expressed in skeletal muscle cells and not expressed, or expressed at substantially low levels, in cardiac muscle cells and liver cells.
  • the present invention provides for transgene expression in cardiac cells with limited toxic expression of the same transgene in skeletal muscle, even when the transgene is delivered to both cell types. Advantagouesly, the present invention solves a major hurdle in the selective targeting of cardiac muscle cells when using vectors (e.g., vectors such as AAV vectors) that transduce both cardiac and skeletal muscle tissues.
  • vectors e.g., vectors such as AAV vectors
  • aspects of the invention provide an engineered vector comprising a first nucleic acid sequence encoding a transgene and a second nucleic acid sequence encoding for the binding site(s) of one or more miRs selected from among miR-206 and miR-128-3p.
  • the second nucleic acid sequence directs the miR, when present, to repress expression of the transgene.
  • the selected miRs (miR-206 and miR-128-3p) in cells transduced by the vectors of the invention will be directed to inhibit expression of the transgene.
  • the transgene may provide a therapeutic effect when expressed in cardiac muscle but provide a toxic effect when expressed in skeletal muscle.
  • transduction of both cardiac cells and skeletal muscle cells by vectors of the invention results in transcription of both transgene mRNA and the binding site(s) of the select miR.
  • transgene expression will be substantially repressed.
  • cardiac cells with limited to no expression of miR-206 and miR-128-3p transgene expression will not be substantially repressed.
  • the therapeutic effect of the transgene will continue in cardiac cells and the toxic effect in skeletal cells will be abated.
  • the second nucleic acid sequence may be operably linked to the first nucleic acid sequence, for example, the second nucleic acid sequence may be operably linked to the 3' or 5' end of the first nucleic acid sequence.
  • the engineered vector may be a viral vector.
  • the vector may be an adeno-associated viral (AAV) vector.
  • the engineered vector may comprise a capsid protein with modified tropism for heart muscle in comparison with the wild-type AAV vector.
  • transduction and expression of the transgene may be increased in heart tissue while limited expression of the transgene in off-target tissue types. Advantagouesly, due to the presence of miR-206 and miR-128-3p, even where skeletal muscle tissue is transduced, expression of the transgene is silenced in the skeletal muscle.
  • the first nucleic acid sequence and second nucleic acid sequence may also be operably linked to tissue specific promoters.
  • the first nucleic acid sequence may be operably linked to a cardiac specific promoter.
  • the second nucleic acid is 3' or 5' of the first nucleic acid.
  • the second nucleic acid may be incorporated into a 3' or 5' untranslated region (UTR) of the transgene mRNA when transcribed.
  • UTR untranslated region
  • the cardiac specific promoter promotes transcription of the transgene and the miR binding site has limited to no effect due to the absence of the requisite miR.
  • the cardiac specific promoter limits transcription of the transgene while the miR binding site is still present in the transcript, inhibiting translation of any transgene mRNA still transcribed.
  • aspects of the invention also provide methods and uses of the vectors of the invention, for example, in therapeutic compositions. Accordingly, aspects of the invention provide a method or use of a vector, the method or use comprising providing to a subject an engineered vector comprising a first nucleic acid sequence encoding a transgene and a second nucleic acid sequence expressing the binding site of a miR selected from among miR-206 and miR-128-3p. The second nucleic acid sequence thereby directs the miR, when present (for example endogenously), to repress expression of the transgene.
  • the transgene may be expressed in heart muscle, thereby providing a therapeutic effect.
  • the second nucleic acid sequence may be expressed in skeletal muscle cells and direct endogenous miR-206 and miR-128-3p to repress expression of the transgene in skeletal muscle cells.
  • the transgene may provide a therapeutic effect when expressed in heart muscle but provide atoxic effect when expressed in skeletal muscle cells.
  • a engineered vectorrecombinant nucleic acid comprising a first nucleic acid sequence encoding a transgene; and a second nucleic acid sequence comprising at least one binding site of miR-206 and/or at least one binding site of miR-128-3p; wherein expression of the transgene is repressed in the presence of miR-206 and/or miR-128-3p.
  • the transgene can encode a protein or an inhibitory nucleic acid (e.g., a miRNA, a siRNA).
  • the transgene provides a therapeutic effect when expressed in heart muscle.
  • the transgene provides a toxic effect when expressed in skeletal muscle.
  • the second nucleic acid sequence is operably linked to the first nucleic acid sequence. In some embodiments, the second nucleic acid sequence is operably linked to the 3' or 5' end of the first nucleic acid sequence.
  • the recombinant nucleic acid is comprised in a vector, optionally wherein the vector is a viral vector.
  • the vector is an adeno- associated viral (AAV) vector.
  • the vector comprises a capsid protein with modified tropism for heart muscle in comparison with the a wild-type AAV vector.
  • the first nucleic acid sequence and/or the second nucleic acid sequence are operably linked to a tissue specific promoter.
  • the first nucleic acid sequence is operably linked to a cardiac specific promoter, and wherein the second nucleic acid sequence is incorporated at the 3' or 5' untranslated region (UTR) of the transgene mRNA when transcribed.
  • UTR 3' or 5' untranslated region
  • aspects of the present disclosure provide a method of selectively expressing a transgene in a subject, the method comprising administering to a subject a recombinant nucleic acid comprising a first nucleic acid sequence encoding a transgene; and a second nucleic acid sequence encoding the at least one binding site of miR-206 and/or at least one binding site of miR-128-3p; wherein expression of the transgene is repressed in the presence of miR-206 and/or miR-128-3p.
  • the transgene is expressed in heart muscle, thereby providing a therapeutic effect.
  • the second nucleic acid sequence is expressed in skeletal muscle cells and directs endogenous miR-206 and miR-128-3p to repress translation of the transgene in skeletal muscle cells.
  • the second nucleic acid sequence is operably linked to the first nucleic acid sequence. In some embodiments, the second nucleic acid sequence is operably linked to the 3' or 5' end of the first nucleic acid sequence.
  • the recombinant nucleic acid is comprised in a vector, optionally wherein the vector is a viral vector.
  • the vector is an adeno- associated viral (AAV) vector.
  • the vector comprises a capsid protein with modified tropism for heart muscle in comparison with the a wild-type AAV vector.
  • the first nucleic acid sequence and/or the second nucleic acid sequence are operably linked to a tissue specific promoter.
  • the first nucleic acid sequence is operably linked to a cardiac specific promoter, and wherein the second nucleic acid sequence is incorporated at a 3' or 5' untranslated region (UTR) of the transgene mRNA when transcribed.
  • UTR untranslated region
  • FIGs. 1A-1B show expression of miR-206 (FIG. 1 A) and miR-128-39 (FIG. IB) in heart, muscle, and liver tissue from non-human primates (NHPs) or from humans. DETAILED DESCRIPTION
  • the present invention provides recombinant nucleic acids (e.g., vectors such as AAV vectors) that encode a transgene and the binding site of one or more of miR-206 and miR- 128-3p that direct the miRs to selectively inhibit expression of the same transgene.
  • vectors such as AAV vectors
  • aspects of the present disclosure provide a recombinant nucleic acid comprising a nucleic acid sequence encoding a transgene and a nucleic acid sequence comprising at least one binding site of miR-206 and/or at least one binding site of miR-128-3p.
  • the recombinant nucleic acid can further comprise an intron and/or a PolyA signal.
  • the recombinant nucleic acid can further comprise a 5' inverted terminal repeat (ITR) and a 3' ITR.
  • promoters including inducible promoters and constitutive promoters, can be used to drive expression from the recombinant nucleic acids described herein.
  • promoters that can be used in the recombinant nucleic acids disclosed herein include a cytomegalovirus (CMV) promoter, a chicken
  • CMV cytomegalovirus
  • CBA chicken
  • hAAT alpha- 1 antitrypsin
  • the promoter is tissue-specific such that, in a multi-cellular organism, the promoter drives expression only in a subset of specific cells.
  • the promoter is a muscle specific promoter.
  • muscle specific promoters include a CK8 promoter, a desmin promoter, a Mb promoter, a MCK promoter, a MHCK7 promoter, a skeletal muscle alpha actin promoter, or a TTNI2 promoter.
  • Recombinant nucleic acids described herein can comprise a sequence encoding any transgene, e.g., a protein, a peptide, an antibody, an inhibitory nucleic acid (e.g, siRNA, miRNA).
  • transgene e.g., a protein, a peptide, an antibody, an inhibitory nucleic acid (e.g, siRNA, miRNA).
  • inhibitory nucleic acid e.g, siRNA, miRNA
  • Recombinant nucleic acids described herein can comprise at least one binding site of miR-206, e.g., at least one, at least two, at least three, at least four, at least five, or more binding sites of miR-206.
  • Recombinant nucleic acids described herein can comprise at least one binding site of miR-128-3p, e.g., at least one, at least two, at least three, at least four, at least five, or more binding sites of miR-128-3p.
  • Recombinant nucleic acids described herein can comprise at least one binding site of miR-206 and at least one binding site of miR-128- 3p, e.g., at least one, at least two, at least three, at least four, at least five, or more binding sites of miR-206 and at least one, at least two, at least three, at least four, at least five, or more binding sites of miR-128-3p.
  • Recombinant nucleic acids described herein can include the same sequence or a different sequence of binding sites.
  • the recombinant nucleic acid comprises more than one binding site of miR-206
  • the more than one binding site of miR-206 can have the same sequence or a different sequence.
  • the recombinant nucleic acid comprises more than one binding site of miR-128-3p
  • the more than one binding site of miR-128-3p can have the same sequence or a different sequence.
  • the more than one binding site of miR-206 and more than one binding site of miR-128-3p can have the same sequence or a different sequence.
  • the at least one binding site of miR-206 and/or the at least one binding site of of miR- 128-3p can be positioned in the recombinant nucleic acids described herein at any location suitable for binding miR-206 and/or miR-128-3p.
  • the at least one binding site of miR-206 can be positioned upstream or downstream of the sequence encoding the transgene.
  • each of the miR- 206 binding sites can be positioned upstream or downstream of the sequence encoding the transgene.
  • one or more of the miR-206 binding sites can be positioned upstream of the sequence encoding the transgene and one or more of the miR-206 binding sites can be positioned downstream of the sequence encoding the transgene.
  • the at least one binding site of miR-128-3p can be positioned upstream or downstream of the sequence encoding the transgene.
  • each of the miR-128-3p binding sites can be positioned upstream or dow nstream of the sequence encoding the transgene.
  • one or more of the miR-128-3p binding sites can be positioned upstream of the sequence encoding the transgene and one or more of the miR-128- 3p binding sites can be positioned downstream of the sequence encoding the transgene.
  • the at least one binding site of miR-206 and the at least one binding site of miR-128-3p can be positioned upstream or downstream of the sequence encoding the transgene.
  • each of the miR-206 and the miR-128-3p binding sites can be positioned upstream or downstream of the sequence encoding the transgene.
  • one or more of the miR-206 binding sites and one or more of the miR-128-3p binding sites can be positioned upstream of the sequence encoding the transgene and one or more of the miR-206 binding sites and one or more of the miR-128- 3p binding sites can be positioned downstream of the sequence encoding the transgene.
  • Recombinant nucleic acids described herein can further comprise an intron to increase overall efficiency and output of gene expression.
  • the intron is positioned between the promoter and transgene in a 5' to 3' orientation.
  • introns include a simian virus 40 (SV40) intron sequence, a minute virus of mice (MVM) intron, a RK intron, a P-globin intron, and/or a chicken P-actin (CBA) intron, including functional variants or fragments thereof.
  • Recombinant nucleic acids described herein can further comprise a polyadenylation (Poly A) signal sequence or functional fragment thereof, e.g, a fragment capable of measurably increasing expression as compared to expression in its absence.
  • Poly A signal sequences include a bovine growth hormone poly adenylation (bgh- PolyA) signal, a synthetic PolyA signal, and an SV40 PolyA signal.
  • Recombinant nucleic acids described herein can comprise one or more ITRs, e.g, two ITRs, with one upstream and the other downstream of a sequence encoding a transgene and/or the other elements discussed above (e.g., a promoter, at least one binding site of miR- 206 and/or at least one binding site of miR-128-3p).
  • An ITR sequence may be wild type, or it may comprise one or more mutations, e.g., by the insertion, deletion, or substitution of nucleotides, as long as the sequences retain one or more function of a ild type ITR, such as providing for functional rescue, replication and packaging.
  • a recombinant nucleic acid as described herein can comprise two ITR sequences, each of which is wild type, variant, or modified ITR sequences.
  • a recombinant nucleic acid as described herein can comprise two ITR sequences, which are different (e.g., one wild type ITR sequence and one variant or modified ITR sequence).
  • the “left”’ or 5' ITR is a modified ITR sequence that allows for production of self-complementary genomes, and the ‘Tight” or 3' ITR is a wild type ITR sequence.
  • the “right” or 3' ITR is a modified ITR sequence that allows for the production of self-complementary genomes, and the “left” or 5' ITR is a wild type ITR sequence.
  • a recombinant expression vector e.g.. a viral vector (e.g.. an AAV vector (e.g, a rAAV vector))
  • AAVs are particularly appropriate viral vectors for delivery of genetic material into mammalian cells. AAVs are not known to cause disease in mammals and cause a very mild immune response. Additionally, AAVs are able to infect cells in multiple stages whether at rest or in a phase of the cell replication cycle.
  • AAV DNA is not regularly inserted into the host’s genome at random sites, reducing the oncogenic properties of this vector.
  • AAVs have been engineered to deliver a variety of treatments, especially for genetic disorders caused by single nucleotide polymorphisms (“SNP”). Genetic diseases that have been studied in conjunction with AAV vectors include Cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson’s disease, congestive heart failure, and Alzheimer’s disease.
  • SNP single nucleotide polymorphisms
  • the AAV can be used as a vector to deliver engineered nucleic acid to a host and utilize the host’s own ribosomes to transcribe that nucleic acid into the desired proteins. See, e.g, West et al., Virology 160:38-47 (1987); U.S. Pat. No.
  • AAVs have some deficiency in their replication and/or pathogenicity and thus can be safer that adenoviral vectors.
  • the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects.
  • the capacity' of the AAV vector, system thereof, and/or AAV particles can be up to about 4.7 kb.
  • the AAV vector or system thereof can include one or more engineered capsid polynucleotides described herein.
  • AAVs are small, replication-defective, nonenveloped viruses that infect humans and other primate species and have a linear single-stranded DNA genome.
  • Naturally occurring AAV seroty pes exhibit liver tropism.
  • transduction of non-liver tissue with traditional AAV vectors is impeded by the virus’s natural liver tropism.
  • the liver acts to break down substances delivered to a subject
  • transduction of non-liver tissue with unmodified AAV vectors requires higher dosing to provide sufficient viral load to overcome the liver and reach non-liver tissue. More than 30 naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist.
  • AAV serotypes include, but are not limited to, AAV serotypes AAV1. AAV2. AAV3. AAV3B. AAV4. AAV5. AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13.
  • AAVs may be engineered using conventional molecular biology techniques, making it possible to optimize these particles, for example, for cell specific delivery, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery’ to the nucleus.
  • AAV vectors can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.
  • mapping determinants of AAV tropism have been carried out by comparing highly related serotypes.
  • One such example is the singleamino acid change (E531K) between AAV1 and AAV6 that improves murine liver transduction in AAV1. See, e.g., Wu et al. (2006) J. Virol., 80(22): 11393-7, incorporated by reference herein.
  • Another example is a reciprocal domain swap between AAV2 and AAV8 that alters tropism, but fails to define any robust specific tissue-targeting motifs. See. e.g., Raupp et al. (201) J. Virol., 86(17):9396-408, incorporated by reference herein. Further, global consideration of structure has only highlighted gross differences between better- or worse-liver-transducers that are more observational than useful in practice. Nam et al (2007) J. Virol., 81(22): 12260-71.
  • AAVs exhibiting modified tissue tropism that may be used with the present invention are described in U.S. Patent No. 9,695,220, U.S. Patent No. 9,719,070; U.S. Patent No. 10,119,125; U.S. Patent No. 10,526,584; U.S. Patent Application Publication No. 2018- 0369414; U.S. Patent Application Publication No. 2020-0123504; U.S. Patent Application Publication No. 2020-0318082; PCT International Patent Application Publication No. WO 2015/054653; PCT International Patent Application Publication No. WO 201 /179496; PCT International Patent Application Publication No. WO 2017/100791; and PCT International Patent Application Publication No. WO 2019/217911, the entirety' of the contents of each of which are incorporated by reference herein.
  • the AAV vector or system thereof may include one or more regulatory molecules, such as promoters, enhancers, repressors and the like.
  • the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins.
  • the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof.
  • the muscle specific promoter can drive expression of an engineered AAV capsid polynucleotide.
  • the AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein.
  • the engineered capsid proteins can be capable of assembling into a protein shell (an engineered capsid) of the AAV virus particle.
  • the engineered capsid can have a cell-, tissue-, and/or organ-specific tropism.
  • the AAV vector or system thereof can be configured to produce AAV particles having a specific serotype.
  • the serotype can be AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combinations thereof.
  • the AAV can be AAV1, AAV-2, AAV-5, AAV-9 or any combination thereof.
  • an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and/or neuronal cells can be configured to generate AAV particles having seroty pes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof.
  • an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV-4 serotype.
  • an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 seroty pe. See also Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.
  • each serotype still is multi-tropic and thus can result in tissuetoxicity 7 if using that seroty pe to target a tissue that the serotype is less efficient in transducing.
  • the tropism of the AAV serotype can be modified by an engineered AAV capsid described herein.
  • variants of wild-type AAV of any serotype can be generated via a method described herein and determined to have a particular cell-specific tropism, which can be the same or different as that of the reference wild-type AAV serotype.
  • the cell, tissue, and/or specificity 7 of the wild-type serotype can be enhanced (e.g., made more selective or specific for a particular cell type that the serotype is already biased towards).
  • wild-type AAV-9 is biased towards muscle and brain in humans (see, e.g., Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.)
  • the tropism for nervous cells might be reduced or eliminated and/or the muscle specificity increased such that the nervous specificity appears reduced in comparison, thus enhancing the specificity for muscle as compared to the wild-type AAV-9.
  • an engineered capsid and/or capsid protein variant of a wild-type AAV serotype can have a different tropism than the wild-type reference AAV serotype.
  • an engineered AAV capsid and/or capsid protein variant of AAV-9 can have specificity for a tissue other than muscle or brain in humans.
  • the AAV vector is a hybrid AAV vector or system thereof.
  • Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2/5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the 2nd plasmid, the pRepCap will be different.
  • pRep2/Cap5 In this plasmid, called pRep2/Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5.
  • the production scheme is the same as the above-mentioned approach for AAV2 production.
  • the resulting rAAV is called rAAV2/5, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed by this AAV2/5 hybrid virus should be the same as that of AAV5. It will be appreciated that wild-type hybrid AAV particles suffer the same specificity issues as with the non-hybrid wild-type serotypes previously discussed.
  • hybrid AAVs can contain an engineered AAV capsid containing a genome with elements from a different serotype than the reference wild-type serotype that the engineered AAV capsid is a variant of.
  • a hybrid AAV can be produced that includes an engineered AAV capsid that is a variant of an AAV-9 serotype that is used to package a genome that contains components (e.g., rep elements) from an AAV -2 serotype.
  • the tropism of the resulting AAV particle will be that of the engineered AAV capsid.
  • the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector.
  • the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g, the engineered AAV capsid polynucleotide(s)).
  • the vectors described herein can be constructed using any suitable process or technique.
  • one or more suitable recombination and/or cloning methods or techniques can be used to the vector(s) described herein.
  • Suitable recombination and/or cloning techniques and/or methods can include, but not limited to. those described in U.S. Application publication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein.
  • AAV vectors Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka. PNAS 81 :6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any ofthe techniques and/or methods can be used and/or adapted for constructing an AAV or other vector described herein. AAV vectors are discussed elsewhere herein.
  • the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a '‘cloning site”).
  • one or more insertion sites e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a engineered AAV capsid system described herein are as used in the foregoing documents, such as International Patent Application Publications WO WO 2021/050974 and WO 2021/077000 and PCT International Application No. PCT/US2021/042812. the contents of which are incorporated by reference herein.
  • AAV vectors are described in International Patent Application Publications WO 2005/033321; WO 2006/110689; WO 2007/127264; WO 2008/027084; WO 2009/073103; WO 2009/073104; WO 2009/105084; WO 2009/134681; WO 2009/136977; WO 2010/051367; WO 2010/138675; WO 2001/038187; WO 2012/112832; WO 2015/054653; WO 2016/179496; WO 2017/100791; WO 2017/019994; WO 2018/209154; WO 2019/067982; WO 2019/195701; WO 2019/217911; WO 2020/041498; WO 2020/210839; U.S.
  • the invention may contain a muscle specific promoter or another promoter.
  • the promoter may be linked to the nucleic acid sequence so that the transcription preferably occurs within myocytes. Promoter regions enable the host cells to replicate the AAV delivered nucleic acid only in those cell types and tissues or organs in which the desired protein should be created.
  • the muscle specific promoter is included because it is principally desired that the proteins only be translated in myocytes. Specificity of the cell ty pe into which the nucleic acid is delivered and thus the proteins translated is desired because of the adverse effects that may ensue from delivering the nucleic acid and having it translated in cells in which that nucleic acid and thus protein is not needed.
  • the promoter comprises a cardiac specific promoter. In some embodiments, the promoter is TNNT2. In some embodiments, the promoter is MHCK9. In some embodiments, the promoter is MHCK7. In some embodiments, the promoter is CBA (chicken P-actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is a desmin promoter.
  • the muscle specific promoter yields increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof.
  • muscle-specific refers to the increased specificity, selectivity, or potency, of the muscle-specific targeting moieties and compositions incorporating said muscle-specific targeting moieties of the present invention for myocytes relative to non-muscle cells.
  • the cell specificity', or selectivity, or potency, or a combination thereof of a muscle-specific targeting moiety or composition incorporating a muscle-specific targeting moiety described herein is at least 2 to at least 500 times more specific, selective, and/or potent for/in a muscle cell relative to a non-muscle cell.
  • the promoters described herein are inserted into an AAV protein (e.g., an AAV capsid protein) that has reduced specificity (or no detectable, measurable, or clinically relevant interaction) for one or more non-muscle cell types.
  • AAV protein e.g., an AAV capsid protein
  • non-muscle cell types include, but are not limited to, liver, kidney, lung, heart, spleen, central or peripheral nervous system cells, bone, immune, stomach, intestine, eye, skin cells and the like.
  • the non-muscle cells are liver cells.
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • FIG. 7 promoters include U6 promoter sequence, MHCK.7 promoter sequence, CK6 promoter sequence, tMCK promoter sequence, CK5 promoter sequence, MCK promoter sequence, HAS promoter sequence.
  • MPZ promoter sequence desmin promoter sequence, APOA2 promoter sequence, hAAT promoter sequence, INS promoter sequence, IRS2 promoter sequence, MYH6 promoter sequence, MYL2 promoter sequence, TNNI3 promoter sequence, SYN1 promoter sequence, GFAP promoter sequence, NES promoter sequence, MBP promoter sequence, or TH promoter sequence.
  • RNA polymerase II or III promoter It may be convenient to use an RNA polymerase II or III promoter; these are known to the person skilled in the art and reviewed in, e.g.. Kornberg 1999.
  • transcripts from an RNA II polymerase often have complex transcription terminators and transcripts are polyadenylated; this may hamper with the requirements of the miRNA strand which because both its 5' and 3' ends need to be precisely defined in order to achieve the required secondary structure to produce a functional molecule.
  • the polynucleotide encoding the miRNA strand may also encode self-processing ribozymes and may be operably linked to an RNA polymerase II or III promoter; as such the polynucleotide encodes a pre- miRNA strand comprising the miRNA strand and self-processing ribozymes, wherein, when transcribed, the miRNA strand is released by the self-processing ribozymes from the pre- miRNA strand de transcript.
  • the AAV vector is comprised of an RNA polymerase II promoter or III promoter, and encodes a pre- miRNA strand comprising the miRNA strand and self-processing ribozymes, wherein, when transcribed, the miRNA strand is released by the self-processing ribozymes from the pre- miRNA strand transcript.
  • multiple pre-miRNA strands and multiple selfprocessing ribozymes may be encoded by a single polynucleotide, operably linked to one or more RNA polymerase II promoters.
  • RNA polymerase II or III promoters that are inducible and/or tissue-specific have been previously described.
  • RNA polymerase promoters are known in the art and further described in U.S. Patent Publication 11,149,288, the contents of which is incorporated by reference herein.
  • any of the recombinant nucleic acids or recombinant expression vectors e.g., recombinant expression vector (e.g., viral vector (e.g., AAV (e.g., rAAV))
  • recombinant expression vector e.g., viral vector (e.g., AAV (e.g., rAAV))
  • viral vector e.g., AAV (e.g., rAAV)
  • viral particle e.g., AAV particle
  • Viral particles for use in compositions and methods described herein can include any viral capsid protein (e.g.. AAV capsid protein or variant thereof) known in the art or described herein.
  • the capsid protein is the shell or coating of the virus that enables its delivery into the host. Without the protein, the nucleic acids would be destroyed by the host without entering into the host cells and beginning transcription and translation.
  • the capsid protein may be in the natural conformation of a naturally occurring AAV, or it may be modified.
  • the AAV capsid protein is an engineered AAV capsid protein having reduced or eliminated uptake in a non-muscle cell as compared to a corresponding wild-type AAV capsid polypeptide.
  • the engineered AAV capsid encoding polynucleotide can be included in a polynucleotide that is configured to be an AAV genome donor in an AAV vector system that can be used to generate engineered AAV particles described elsewhere herein.
  • the engineered AAV capsid encoding polynucleotide can be operably coupled to a poly adenylation tail.
  • the poly adenylation tail can be an SV40 poly adenylation tail.
  • the AAV capsid encoding polynucleotide can be operably coupled to a promoter.
  • the promoter can be a tissue specific promoter.
  • the tissue specific promoter is specific for muscle (e.g., cardiac,), neurons and supporting cells (e.g, astrocytes, glial cells, Schwann cells, etc.), fat, spleen, liver, kidney, immune cells, spinal fluid cells, synovial fluid cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal gland, blood cell, bone marrow cells, placenta, endothelial cells, and combinations thereof.
  • the promoter can be a constitutive promoter. Suitable tissue specific promoters and constitutive promoters are discussed elsewhere herein and are generally known in the art and can be commercially available.
  • engineered viral capsids such as adeno-associated virus (AAV) capsids
  • AAV capsids that can be engineered to confer cell-specific tropism, such as cardiac specific tropism, to an engineered viral particle.
  • Engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids.
  • the engineered capsids can be included in an engineered vims particle (e.g., an engineered lentiviral, retroviral, adenoviral, or AAV virus particle), and can confer cell-specific tropism, reduced immunogenicity, or both to the engineered viral particle.
  • the engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein.
  • the engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein that can contain a muscle-specific targeting moiety containing or composed of an n-mer motif described elsewhere herein.
  • the engineered viral capsid and/or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides.
  • the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide.
  • an engineered viral capsid polynucleotide e.g, an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide
  • the polyadenylation signal can be an SV40 polyadenylation signal.
  • the engineered viral capsids can be variants of wild-ty pe viral capsid.
  • the engineered AAV capsids can be variants of wild-type AAV capsids.
  • the wild-type AAV capsids can be composed of VP1. VP2. VP3 capsid proteins or a combination thereof.
  • the engineered AAV capsids can include one or more variants of a wild-type VP1, wild-type VP2, and/or yvild-type VP3 capsid proteins.
  • the serotype of the reference yvild-type AAV capsid can be AAV-1. AAV-2, AAV-3.
  • the serotype of the wild-type AAV capsid can be AAV-9.
  • the engineered AAV capsids can have a different tropism than that of the reference wild-type AAV capsid.
  • the engineered viral capsid can contain 1-60 engineered capsid proteins.
  • the engineered viral capsids can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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, or 60 engineered capsid proteins.
  • the engineered viral capsid can contain 0- 59 wild-type viral capsid proteins.
  • the engineered viral capsid can contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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, or 59 yvild-type viral capsid proteins.
  • the engineered AAV capsid can contain 1-60 engineered capsid proteins.
  • the engineered AAV capsids can contain 1, 2, 3, 4, 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, or 60 engineered capsid proteins.
  • the engineered AAV capsid can contain 0-59 wild-type AAV capsid proteins.
  • the engineered AAV capsid can contain 0, 1, 2, 3, 4, 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, or 59 wild-type AAV capsid proteins.
  • the engineered viral capsid protein can have an n-mer amino acid motif, where n can be at least 3 amino acids. In some embodiments, n can be 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.
  • an engineered AAV capsid can have a 6-mer or 7-mer amino acid motif.
  • the n-mer amino acid motif can be inserted between two amino acids in the wild-tj pe viral protein (VP) (or capsid protein).
  • the n-mer motif can be inserted between two amino acids in a variable amino acid region in a viral capsid protein.
  • the n-mer motif can be inserted between two amino acids in a variable amino acid region in an AAV capsid protein.
  • the core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958).
  • Structural variable regions (VRs) occur in the surface loops that connect the beta-strands, which cluster to produce local variations in the capsid surface.
  • AAVs have 12 variable regions (also referred to as hypervariable regions) (see, e.g., Weitzman and Linden. 2011. ‘"Adeno-Associated Virus Biology’/’ In Snyder. R.O.. Moullier, P. (eds.) Totowa, NJ: Humana Press).
  • one or more «-mer motifs can be inserted between two amino acids in one or more of the 12 variable regions in the wildtype AVV capsid proteins.
  • the one or more n-mer motifs can be each be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI. VR-VII, VR-III. VR-IX, VR-X.
  • the /7-mer can be inserted between two amino acids in the VR-III of a capsid protein.
  • the engineered capsid can have an n-mer inserted between any two contiguous amino acids betw een amino acids 262 and 269, betw een any two contiguous amino acids between amino acids 327 and 332.
  • the engineered capsid can have an n-mer inserted between amino acids 588 and 589 of an AAV9 viral protein. In some embodiments, the engineered capsid can have a 7- mer motif inserted between amino acids 588 and 589 of an AAV9 viral protein.
  • the motif inserted is a 10-mer motif, with replacement of amino acids 586-88 and an insertion before 589. It will be appreciated that n-mers can be inserted in analogous positions in AAV viral proteins of other serotypes. In some embodiments as previously discussed, the w-mer(s) can be inserted between any two contiguous amino acids within the AAV viral protein and in some embodiments the insertion is made in a variable region.
  • the first 1, 2, 3, or 4 amino acids of an n-mer motif can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted and preceding the insertion site.
  • the amino acids of the n-mer motif that replace 1 or more amino acids of the polypeptide into which the n-mer motif is inserted come before or immediately before an “RGD” in an n-mer motif.
  • the first three amino acids shown can replace 1-3 amino acids into a polypeptide to which they may be inserted.
  • one or more of the n-mer motifs can be inserted into, e.g., and AAV9 capsid prolylpeptide between amino acids 588 and 589 and the insert can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the n-mer motif after insertion is residue 585.
  • this principle can apply in any other insertion context and is not necessarily limited to insertion between residues 588 and 589 of an AAV9 capsid or equivalent position in another AAV capsid.
  • no amino acids in the polypeptide into which the n-mer motif is inserted are replaced by the n-mer motif.
  • the AAV capsids or other viral capsids or compositions can be muscle-specific.
  • muscle-specificity of the engineered AAV or other viral capsid or other composition is conferred by a cardiac specific n-mer motif incorporated in the engineered AAV or other viral capsid or other composition described herein.
  • the n-mer motif confers a 3D structure to or within a domain or region of the engineered AAV capsid or other viral capsid or other composition such that the interaction of the viral particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein has increased or improved interactions (e.g, increased affinity) with a cell surface receptor and/or other molecule on the surface of a muscle cell.
  • the cell surface receptor is AAV receptor (AAVR).
  • the cell surface receptor is a muscle cell specific AAV receptor.
  • the cell surface receptor or other molecule is a cell surface receptor or other molecule selectively expressed on the surface of a muscle cell.
  • the cell surface receptor or molecule is an integrin or dimer thereof. In some embodiments, the cell surface receptor or molecule is an Vb6 integrin heterodimer.
  • a cardiac specific engineered viral particle or other composition described herein containing the muscle-specific capsid, n-mer motif, or musclespecific targeting moiety described herein can have an increased uptake, del i ⁇ ery rate, transduction rate, efficiency, amount, or a combination thereof in a muscle cell as compared to other cells types and/or other virus particles (including but not limited to AAVs) and other compositions that do not contain the muscle-specific n-mer motif of the present invention.
  • First-and second-generation muscle specific AAV capsids were developed using a muscle specific promoter and the resulting capsid libraries were screened in mice and nonhuman primates as described elsewhere herein and/or in, e.g, WO 2021/050974 and WO 2021/077000.
  • First and second generation myoAAV capsids were further optimized in mice and non-human primates as previously described to generate enhanced myoAAV capsids with cardiac specificity.
  • the present invention provides miR-206 and miR-128-3p binding sites that inhibit expression of a transgene.
  • 'miRNA binding site 7 refers to a nucleic acid sequence that has sufficient complementarity to all or a portion of a nucleic acid sequence of a miRNA to allow hybridization or binding of the miRNA binding site to the miRNA.
  • miRNA binding site can be used interchangeably with the term “binding site of miRNA”.
  • a binding site of miR-206 refers to a nucleic acid sequence that has sufficient complementarity’ to all or a portion of a nucleic acid sequence of miR-206 to allow hybridization or binding of the binding site of miR-206 to the miR-206; and “a binding site of miR-128-3p” refers to a nucleic acid sequence that has sufficient complementarity to all or a portion of a nucleic acid sequence of miR-128-3p to allow hybridization or binding of the binding site of miR-128-3p to the miR-128-3p.
  • Nucleic acid sequences of miR-206 and miR- 128-3p are known in the art and can be found in, e.g., NCBI Reference Sequence: NR_029713.1 and NR_029672.1, respectively.
  • MicroRNAs are small, single-stranded, non-coding RNA molecules. miRNAs basepair to complementary sequences in mRNA molecules, thereby producing post- transcriptional regulation of gene expression. Typically, miRNA molecules silence messenger RNA (mRNA) translation by facilitating a process that leads to cleavage of mRNA strand into two pieces or destabilization of the mRNA by shortening its poly(A) tail. miRNAs are typically ⁇ 22 nt and are expressed in a cell and tissue type specific manner. miRNAs resemble small interfering RNAs (siRNAs), however miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins.
  • siRNAs small interfering RNAs
  • RNA stem-loop that in turn forms part of a several hundred nucleotide-long miRNA precursor termed a pri-miRNA.
  • a single pri-miRNA may contain from one to six miRNA precursors.
  • These hairpin loop structures are typically composed of about 70 nucleotides each. Each hairpin is flanked by sequences necessary for efficient processing.
  • the pre-miRNA hairpin is typically cleaved by the RNase enzyme Dicer.
  • the RNase interacts with 5' and 3' ends of the hairpin and cuts away the loop joining the 3' and 5' arms, resulting in a miRNA:miRNA duplex about 22 nucleotides in length.
  • Overall hairpin length and loop size influence the efficiency of Dicer processing.
  • either strand of the duplex may potentially act as a functional miRNA, only one strand is generally incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact.
  • RISC RNA-induced silencing complex
  • compositions e.g.. recombinant nucleic acid, rAAV particle, AAV vector, pharmaceutical composition
  • the composition comprises a recombinant nucleic acid.
  • the composition is a rAAV capsid protein described herein.
  • the composition is an isolated and purified rAAV capsid protein described herein.
  • the rAAV particle encapsidates an AAV vector comprising a transgene (e.g., therapeutic nucleic acid).
  • the composition is a rAAV capsid protein described herein conjugated with a therapeutic agent disclosed herein.
  • the composition is a pharmaceutical composition comprising the rAAV particle and a pharmaceutically acceptable carrier.
  • the one or more compositions are administered to the subject alone (e.g., stand-alone therapy).
  • the composition is a first-line therapy for the disease or condition.
  • the composition is a second-line, third-line, or fourth-line therapy, for the disease or condition.
  • Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g.. a transgene, therapeutic nucleic acid).
  • an episomal heterologous nucleic acid e.g.. a transgene, therapeutic nucleic acid.
  • a rAAV Upon delivery to a host in vivo environment, a rAAV will (1) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid , (5) convert of the heterologous nucleic acid from single-stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell (“transduction’”).
  • aspects disclosed herein provide methods of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the recombinant nucleic acid of the present disclosure, the rAAV of the present disclosure, or the pharmaceutical formulation of the present disclosure, wherein the gene product is a therapeutic gene product.
  • the administering is by intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracistemal, or subcutaneous.
  • a disease or a condition associated with an aberrant expression or activity of a target gene or gene expression product thereof comprising modulating the expression or the activity of a target gene or gene expression product in a subject by administering a rAAV encapsidating a heterologous nucleic acid of the present disclosure.
  • the expression or the activity of the target gene or gene expression product is decreased, relative to that in a normal (nondiseased) individual; and administering the rAAV to the subject is sufficient to increase the expression of the activity of the target gene or gene expression product.
  • the expression or the activity of the gene or gene expression product is increased, relative to that in a normal individual; and administering the rAAV to the subject is sufficient to decrease the expression or the activity of the target gene or gene expression product.
  • Also provided are methods of preventing a disease or condition disclosed herein in a subject comprising administering to the subject a therapeutically effective amount of an rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein.
  • the rAAV vector may be encapsidated in the modified capsid protein or rAAV viral particle described herein.
  • the therapeutic gene expression product is effective to modulate the activity or expression of a target gene or gene expression product.
  • compositions of the present disclosure are particularly useful for the treatment of the diseases or conditions described herein because they specifically or more efficiently target the in vivo environment and deliver a therapeutic nucleic acid engineered to modulate the activity or the expression of a target gene expression product involved with the pathogenesis or pathology of the disease or condition.
  • a disease or a condition, or a symptom of the disease or condition in a subject, comprising: (a) diagnosing a subject with a disease or a condition affecting a target in vivo environment; and (b) treating the disease or the condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., recombinant nucleic acid, rAAV particle. AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased specificity for the target in vivo environment.
  • a composition disclosed herein e.g., recombinant nucleic acid, rAAV particle. AAV vector, pharmaceutical composition
  • compositions e.g., recombinant nucleic acid. rAAV particle. AAV vector, pharmaceutical composition
  • expressing the therapeutic nucleic acid into a target in vivo environment in the subject with an increased transduction enrichment comprising: (a) administering to the subject a composition (e.g., recombinant nucleic acid. rAAV particle. AAV vector, pharmaceutical composition); and (b) expressing the therapeutic nucleic acid into a target in vivo environment in the subject with an increased transduction enrichment.
  • a composition e.g., recombinant nucleic acid. rAAV particle. AAV vector, pharmaceutical composition
  • methods of modulating a target gene expression product comprising administering to a subject in need thereof a composition (e.g., recombinant nucleic acid, rAAV particle, AAV vector, pharmaceutical composition) disclosed herein.
  • a composition e.g., recombinant nucleic acid, rAAV particle, AAV vector, pharmaceutical composition
  • methods provided herein comprise administering to a subject a rAAV with a rAAV capsid protein encapsidating a viral vector comprising a heterologous nucleic acid that modulates the expression or the activity' of the target gene expression product.
  • any of the recombinant nucleic acids, the expression vectors (e.g., AAV vectors), or the AAV particles described herein can be included in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • Some embodiments of the invention may include any acceptable form of providing the AAV vector to a subject.
  • the AAV vector may be provided to the subject in the form of a composition or formulation comprising the AAV vector.
  • the expression vector (e.g, AAV vector) of this invention can be formulated and administered to treat a variety’ of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the subject.
  • the compositions, polynucleotides, polypeptides, particles, cells, vector systems and combinations thereof described herein can be contained in a formulation, such as a pharmaceutical formulation.
  • the formulations can be used to generate polypeptides and other particles that include one or more musclespecific targeting moieties described herein.
  • the formulations can be delivered to a subject in need thereof.
  • component(s) of the engineered AAV capsid system, engineered cells, engineered AAV capsid particles, and/or combinations thereof described herein can be included in a formulation that can be delivered to a subject or a cell.
  • the formulation is a pharmaceutical formulation.
  • One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided to a subject in need thereof or a cell alone or as an active ingredient, such as in a pharmaceutical formulation.
  • compositions containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein.
  • the pharmaceutical formulation can contain an effective amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.
  • the pharmaceutical formulations described herein can be administered to a subject in need thereof or a cell.
  • the amount of the one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in the pharmaceutical formulation can range from about 1 pg/kg to about 10 mg/kg based upon the body weight of the subject in need thereof or average body weight of the specific patient population to which the pharmaceutical formulation can be administered.
  • the amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in the pharmaceutical formulation can range from about 1 pg to about 10 g, from about 10 nL to about 10 ml.
  • the amount can range from about 1 cell to 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 . 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 . 1 x IO 10 or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x IO 10 or more cells per nL, pL, mL, or L.
  • the formulation can contain 1 to 1 x 10 2 , 1 x 10 3 , 1 x IO 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 X IO 8 , 1 X 10 9 , 1 X IO 10 , 1 X IO 11 , 1 X 10 12 . 1 X IO 13 , 1 X 10 14 , 1 x IO 15 , 1 x 10 16 , 1 x 10 17 , 1 x IO 18 , 1 x 10 19 , or 1 x IO 20 transducing units (TU)/mL of the engineered AAV capsid particles.
  • TU transducing units
  • the formulation can be 0.1 to 100 mL in volume and can contain 1 to 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x IO 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 , 1 x 10 18 , 1 x 10 19 , or 1 x IO 20 transducing units (TU)/mL of the engineered AAV capsid particles.
  • TU transducing units
  • the pharmaceutical formulation containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein can further include a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil. fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.
  • the pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition.
  • the pharmaceutical formulations described herein may be in a dosage form.
  • the dosage forms can be adapted for administration by any appropriate route.
  • Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavemous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intracerebroventricular, and intradermal.
  • Such formulations may be prepared by any method known in the art.
  • Dosage forms adapted for oral administration can be discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or nonaqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation.
  • Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as foam, spray, or liquid solution.
  • the oral dosage form can contain about 1 ng to 1000 g of a pharmaceutical formulation containing a therapeutically effective amount or an appropriate fraction thereof of the targeted effector fusion protein and/or complex thereof or composition containing the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.
  • the oral dosage form can be administered to a subject in need thereof.
  • the dosage forms described herein can be microencapsulated.
  • the dosage form can also be prepared to prolong or sustain the release of any ingredient.
  • the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the ingredient whose release is delayed.
  • the release of an optionally included auxiliary ingredient is delayed.
  • Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
  • cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate
  • polyvinyl acetate phthalate acrylic acid polymers and copolymers
  • methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany),
  • Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non-polymeric excipient, to produce the desired release profile.
  • the coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle compositions, “ingredient as is” formulated as, but not limited to, suspension form or as a sprinkle dosage form.
  • Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • the pharmaceutical formulations are applied as a topical ointment or cream.
  • the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated with a paraffinic or water- miscible ointment base.
  • the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
  • Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders.
  • the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is contained in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization.
  • the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art.
  • Dosage forms adapted for administration by inhalation also include particle dusts or mists.
  • Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active ingredient (e.g., the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and/or auxiliary’ active agent), which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.
  • an active ingredient e.g., the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and/or auxiliary’ active agent
  • the dosage forms can be aerosol formulations suitable for administration by inhalation.
  • the aerosol formulation can contain a solution or fine suspension of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multidose quantities in sterile form in a sealed container.
  • the sealed container is a single dose or multi-dose nasal, or an aerosol dispenser fitted with a metering valve (e.g.. metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.
  • the dispenser contains a suitable propellant under pressure, such as compressed air. carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon.
  • a suitable propellant under pressure such as compressed air. carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon.
  • the aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer.
  • the pressurized aerosol formulation can also contain a solution or a suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.
  • the aerosol formulation can also contain co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation.
  • Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2. or 3 doses are delivered each time.
  • the pharmaceutical formulation is a dry powder inhalable formulation.
  • an auxiliary’ active ingredient, and/or pharmaceutically acceptable salt thereof such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and/or starch.
  • the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is in a particle-size reduced form.
  • a performance modifier such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate.
  • the aerosol dosage forms can be arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.
  • Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.
  • Dosage forms adapted for rectal administration include suppositories or enemas.
  • Dosage forms adapted for parenteral administration and/or adapted for any type of injection can include aqueous and/or non-aqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • the dosage forms adapted for parenteral administration can be presented in a single- unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials.
  • the doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration.
  • Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets.
  • Dosage forms adapted for ocular administration can include aqueous and/or nonaqueous sterile solutions that can optionally be adapted for injection, and which can optionally contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the eye or fluid contained therein or around the eye of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents.
  • the dosage form contains a predetermined amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose.
  • the predetermined amount of the Such unit doses may therefore be administered once or more than once a day.
  • Such pharmaceutical formulations may be prepared by any of the methods well know n in the art.
  • the nucleic acid sequences contemplated can be DNA, RNA, or modified versions thereof.
  • Modified nucleic acids can be distinguished from naturally occurring nucleic acids by modifications to the backbone of the polynucleotide chain, for example, peptide nucleic acids (PNA), morpholinos, locked nucleic acids (LNA). glycol nucleic acids (GNA) and threose nucleic acid (TNA). Modified nucleic acids can also include analogs with modifications to the four nucleobases.
  • the nucleic acids are PNAs.
  • the nucleic acids are LNAs.
  • the nucleic acids are morpholines.
  • the nucleic acids are in a single-stranded form. In some embodiments, the nucleic acids are in doublestranded form. In some embodiments, the nucleic acids are linear. In some embodiments, the nucleic acids are circular. In some embodiments, the nucleic acids are plasmids.
  • Embodiment I is an engineered vector comprising a first nucleic acid sequence encoding a transgene; and a second nucleic acid sequence encoding the binding site(s) of one or more microRNAs (miRs) selected from among miR-206 and miR-128-3p, wherein the second nucleic acid sequence directs the miR to repress expression of the transgene.
  • miRs microRNAs
  • Embodiment 2 is the engineered vector of Embodiment 1. wherein the transgene provides therapeutic effect when expressed in heart muscle.
  • Embodiment 3 is the engineered vector of Embodiment 2, wherein the transgene provides a toxic effect when expressed in skeletal muscle.
  • Embodiment 4 is the engineered vector of Embodiment 1. wherein the second nucleic acid sequence is operably linked to the first nucleic acid sequence.
  • Embodiment 5 is the engineered vector of Embodiment 4, wherein the second nucleic acid sequence is operably linked to the 3' or 5' end of the first nucleic acid sequence.
  • Embodiment 6 is the engineered vector of Embodiment 1. wherein the vector is a viral vector.
  • Embodiment 7 is the engineered vector of Embodiment 6. wherein the vector is an adeno-associated viral (AAV) vector.
  • Embodiment 8 is the engineered vector of Embodiment 7, wherein the vector comprises a capsid protein with modified tropism for heart muscle in comparison with the wild-type AAV vector.
  • Embodiment 9 is the engineered vector of Embodiment 8. wherein the first nucleic acid sequence and second nucleic acid sequence are operably linked to tissue specific promoters.
  • Embodiment 10 is the engineered vector of Embodiment 9, wherein the first nucleic acid sequence is operably linked to a cardiac specific promoter and wherein the second nucleic acid sequence is incorporated at the 3' or 5' untranslated region (UTR) of the transgene mRNA when transcribed.
  • UTR untranslated region
  • Embodiment 11 is a method of selectively expressing a transgene in a subject, the method comprising administering to a subject an engineered vector comprising a first nucleic acid sequence encoding a transgene; and a second nucleic acid sequence encoding the binding site(s) of one or more microRNAs (miRs) selected from among miR-206 and miR- 128-3p, wherein the second nucleic acid sequence directs the miR to repress expression of the transgene.
  • miRs microRNAs
  • Embodiment 12 is the method of Embodiment 11, wherein the transgene is expressed in heart muscle, thereby providing a therapeutic effect.
  • Embodiment 13 is the method of Embodiment 12, wherein the second nucleic acid sequence is expressed in skeletal muscle cells and directs endogenous miR-206 and miR-128-3p to repress translation of the transgene in skeletal muscle.
  • Embodiment 14 is the method of Embodiment 11, wherein the second nucleic acid sequence is operably linked to the first nucleic acid sequence.
  • Embodiment 15 is the method of Embodiment 14, wherein the second nucleic acid sequence is operably linked to the 3' or 5' end of the first nucleic acid sequence.
  • Embodiment 16 is the method of Embodiment 11 , wherein the vector is a viral vector.
  • Embodiment 17 is the method of Embodiment 16, wherein the vector is an adeno-associated viral (AAV) vector.
  • Embodiment 18 is the method of Embodiment 17, wherein the vector comprises a capsid protein with modified tropism for heart muscle in comparison with the wild-type AAV vector.
  • Embodiment 19 is the method of Embodiment 18, wherein the first nucleic acid sequence and second nucleic acid sequence are operably linked to tissue specific promoters.
  • Embodiment 20 is the method of Embodiment 19. wherein the first nucleic acid sequence is operably linked to a cardiac specific promoter and wherein the second nucleic acid sequence is incorporated at a 3' or 5' untranslated region (UTR) of the transgene mRNA when transcribed.
  • UTR untranslated region
  • Unbiased small RNAseq was used to identify miRNAs highly expressed in nonhuman primate (NHP) and human skeletal muscle and lowly expressed in NHP and human heart and liver.
  • FIGs. 1A-1B show expression of miR-206 and miR-128-3p, respectively, in heart, muscle, and liver from NHPs and humans.
  • miR-206 and miR-128-3p were identified as the most differentially expressed miRNAs between skeletal muscle versus heart and liver in humans and NHPs.
  • miR-206 and miR-128-3p binding sites were identified as being able to be incorporated into the 3’ or 5' UTR of a transgene to promote repression of transgene expression in skeletal muscle but not heart and liver.

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Abstract

La présente invention concerne des acides nucléiques recombinants qui expriment un transgène et au moins un site de liaison de miR-206 et/ou au moins un site de liaison de miR-128-3 p de sorte que l'expression du transgène est inhibée par la présence de miR-206 endogène et/ou de miR-128-3 p dans les muscles squelettiques.
PCT/US2025/036161 2024-07-02 2025-07-01 Contrôle d'expression par miarn exprimés par les muscles squelettiques Pending WO2026011008A1 (fr)

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