Compositions and methods for muscle disorders

Abstract

Disclosed herein are methods of correcting DUX-4 expression in cells using novel miRNA molecules.

Description

Technical Field

[0001] This invention relates to miRNA molecules. Background Technology

[0002] Genetic disorders are a major source of disease burden, and many of them have little or no drug treatment or curative treatment. Like other genetically related muscular dystrophy disorders, facioscapulohumeral muscular dystrophy (“FSHD”) is a genetic disorder that causes progressive muscle weakness and atrophy. FSHD is so named because the muscles of the face, shoulder, and upper arm are most affected, but it can also affect other muscles in the body, including those in the legs, eyes, heart, hips, or abdomen. FSHD causes symptoms to progressively worsen, leading to asymmetrical weakness.

[0003] FSHD is a genetic disorder caused by a mutation that leads to inappropriate expression of the DUX4 gene on chromosome 4. DUX4 is the double homologous box protein 4 gene. FSHD can be inherited from only one parent because it is an autosomal dominant disorder. FSHD typically affects individuals under the age of 20, and the estimated prevalence in the United States is 4 per 100,000 people. Currently, there is no drug treatment to stop or reverse the muscle effects of FSHD. Summarize

[0004] This invention provides a novel nucleic acid molecule and method that induces alterations in DUX4 expression, thereby enabling treatment of FSHD. The nucleic acid molecule of this invention comprises a miR scaffold and a miR guidance sequence that targets the dual homeobox 4 (DUX4) transcript. By targeting the DUX4 transcript, the nucleic acid molecule of this invention achieves an appropriate expression profile for the DUX4 gene.

[0005] In aspects of the invention, the nucleic acid molecule may comprise a miRNA guide sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769 or 6687-6770. The nucleic acid molecule may comprise one or more substitutions, such as conserved substitutions, which allow the guide sequence to continue targeting DUX4. Therefore, the miRNA guide sequence may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from SEQ ID NO: 1250-2597 or 6687-6770. In aspects of the invention, the nucleic acid molecule comprises a miRNA guide sequence selected from SEQ ID NO: 1250-2769 or 6687-6770.

[0006] The nucleic acid molecule may contain miRNAs having at least 95% sequence identity with sequences selected from the group consisting of SEQ ID NO: 2770-3597 or 6670-6812, each of which incorporates a guide sequence for targeting DUX4. The nucleic acid molecule may target the DUX4 transcript at the locations shown in Table 6.

[0007] miRNA scaffolds can be derived from any pri-miRNA scaffold. For example, miRNA scaffolds are derived from pri-miRNAs selected from the group consisting of pri-miR-21, pri-miR-22, pri-miR-26a, pri-miR-30a, pri-miR-33, pri-miR-122, pri-miR-375, pri-miR-199, pri-miR-99, pri-miR-194, pri-miR-155, and pri-miR-451. miRNA scaffolds can be derived from pri-miR-33.

[0008] The nucleic acid may contain 5-6 thymidines at its 5' end. The nucleic acid of claim 1, further comprising a promoter sequence. The promoter sequence may be the U6 promoter sequence, MHCK7 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.

[0009] The nucleic acid molecules of the present invention can be delivered into cells by any known method. For example, the nucleic acid molecules of the present invention can be delivered by lipid nanoparticles (LNPs) or viral vectors. The viral vector can be any viral vector, such as an adeno-associated virus (AAV) vector.

[0010] Therefore, various aspects of the present invention provide an AAV vector comprising a promoter sequence, a nucleic acid molecule of the present invention (comprising a miR scaffold and a miR guidance sequence targeting the DUX4 transcript), and a capsid protein.

[0011] The viral vector may contain at least one modification that reduces the hepatic orientation of the AAV vector and / or preferentially targets muscle tissue. The capsid protein may contain at least one modification that is an insertion between any two consecutive amino acids at similar positions in the AAV9 capsid polypeptide, specifically amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof, or between similarly positioned amino acids in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, and AAV rh.10 capsid polypeptides. The capsid protein may be selected from the sequences in Tables 1-5. The vector may further contain a nuclear export sequence capable of enabling intranuclear diffusion.

[0012] Several aspects of the present invention also provide methods for inhibiting the expression of a gene or gene product in cells, the method comprising administering to a subject a composition that causes the nucleic acid molecule of the present invention (comprising a miR scaffold and a miR guide sequence targeting a DUX4 transcript) to be expressed in cells.

[0013] In the method of this invention, the nucleic acid molecules of this invention can be delivered into cells by any known method. In a preferred aspect, the nucleic acid molecules can be delivered via an AAV vector. Advantageously, the AAV vector may contain at least one modification that reduces the hepaticity of the AAV vector and / or preferentially targets muscle tissue. The AAV vector may contain a capsid protein selected from sequences in Tables 1-5.

[0014] The method of this invention can treat muscular dystrophy. Muscular dystrophy is facioscapulohumeral muscular dystrophy. Treatment may include inhibiting the muscle effects of muscular dystrophy. Treatment may include reversing the muscle effects of muscular dystrophy.

[0015] Aspects of nucleic acid molecules (e.g., loadings) and capsid proteins will be described in further detail below.

[0016] For sequences disclosed throughout this application, it should be understood that nucleic acid molecules and peptides may contain one or more substitutions, such as conserved substitutions, to allow the sequence to continue to function. Therefore, sequences may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the disclosed sequences.

[0017] Conservative substitution refers to amino acid substitutions that do not significantly affect or alter the binding characteristics of a specific protein. Generally, a conservative substitution is a substitution in which the substituted amino acid residue is replaced by an amino acid residue with a similar side chain. For example, conservative substitutions can include substitutions found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Alternatively, amino acids can be grouped into conserved substitution groups based on similar functions, chemical structures, or compositions (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, aliphatic groups can include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other conserved substitution groups include sulfur-containing residues: Met and cysteine ​​(Cys or C); acidic residues: Asp, Glu, Asn, and Gln; small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp.

[0018] engineered capsid proteins

[0019] This invention provides capsid protein variants for viral vectors that simultaneously target liver tissue and skeletal muscle and heart tissue. These capsid protein variants can be used to deliver the nucleic acid molecules of this invention, which contain miR scaffolds and miR guidance sequences that target dual homology box 4 (DUX4) transcripts.

[0020] Aspects of the present invention provide an adeno-associated virus (AAV) vector comprising a capsid protein containing amino acid inserts. For example, the capsid protein may comprise inserts selected from Tables 1-5.

[0021] This invention provides an AAV vector comprising a capsid protein containing the amino acid sequence RGDR. In the capsid protein, RGDR may be inserted after amino acid 455, referring to an equivalent position in an AAV9 capsid or another AAV capsid. The AAV vector may comprise the amino acid sequence X1XN2X3X4RGDRX5X6L, wherein X1, X2, X3, X4, X5, and X6 can be any of the amino acids.

[0022] In some aspects of the invention, X1 may be an amino acid selected from the group consisting of: A, I, F, G, H, L, M, Q, S, T, V. In a preferred aspect of the invention, X1 may be an amino acid selected from the group consisting of: A, I, L, M, S, V.

[0023] In this invention, X2 may be an amino acid selected from the group consisting of A, G, S, T, and Y.

[0024] In some aspects of the invention, X3 may be an amino acid selected from the group consisting of S, N, G, and P. In a preferred aspect of the invention, X3 may be S.

[0025] In this invention, X4 may be an amino acid selected from the group consisting of A, G, H, I, M, S, T, and V.

[0026] In this invention, X5 may be an amino acid selected from the group consisting of A, G, and Q.

[0027] In some aspects of the invention, X6 may be an amino acid selected from the group consisting of A, I, L, M, N, and Y. In a preferred aspect of the invention, X6 may be selected from the group consisting of A, S, and Y.

[0028] X1 can be located at amino acid 451, X2 at amino acid 453, X3 at amino acid 454, and X4 at amino acid 455. Referring to the equivalent position in the AAV9 capsid or another AAV capsid, RGDRX5X6L is inserted after amino acid 455.

[0029] In aspects of the invention, two of the amino acids in X1, X2, X3 and X4 are wild-type amino acids at equivalent positions in a reference AAV9 capsid or another AAV capsid, and two of the amino acids in X1, X2, X3 and X4 are not wild-type amino acids.

[0030] For example, the capsid protein variants of the present invention may comprise sequences as listed in Table 5a. It should be noted that the capsid protein variants of the present invention comprise deletions, substitutions, and / or insertions relative to the wild-type viral vector capsid.

[0031] In aspects of the invention, the capsid protein comprises an amino acid sequence selected from Table 5a, and this amino acid sequence is located in the hypervariable region IV (HVR IV) relative to wild-type AAV9. The capsid protein variant may comprise a substitution at amino acids 451-455 relative to the wild-type AAV9 vector capsid. For example, the substitution at amino acids 451-455 may be an amino acid sequence selected from column 1 of Table 5b relative to the wild-type AAV9 vector capsid. In aspects of the invention, the capsid protein variant may further comprise an insert. For example, the capsid protein may comprise a 7-mer insert selected from column 2 of Table 5b. The insert may be located after amino acid 455 relative to the wild-type AAV9 vector.

[0032] Advantageously, viral vectors containing the amino acid sequences of the present invention exhibit muscle-like properties compared to wild-type AAV vectors.

[0033] In aspects of the invention, the capsid protein further includes a deletion of G267 at an equivalent location in the AAV9 capsid or another AAV capsid. Advantageously, the vector can exhibit reduced hepaticity compared to wild-type AAV vectors.

[0034] As mentioned above, for the HVR IV variant, the upstream 5 amino acids are located at positions 451-455 (shown in column 1 of Table 5b). The 7-mer insert of the HVR IV variant begins with "RGD" and is inserted after amino acid 455 (shown in column 2 of Table 5b).

[0035] Exemplary guides and caps

[0036] This invention provides a novel nucleic acid molecule and method that induces alterations in DUX4 expression, thereby enabling treatment of FSHD. The nucleic acid molecule of this invention comprises a miR scaffold and a miR guidance sequence that targets the dual homeobox 4 (DUX4) transcript. By targeting the DUX4 transcript, the nucleic acid molecule of this invention achieves an appropriate expression profile for the DUX4 gene.

[0037] Preferred exemplary guides are described in Table 7 below.

[0038] In an exemplary aspect of the invention, the guiding sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6860-6865. For example, SEQ ID NO: 6860, 6861, 6862, 6863, 6864, or 6865.

[0039] The nucleic acid molecule may contain miRNAs having at least 95% sequence identity with sequences selected from the group consisting of SEQ ID NO: 2770-3597 or 6670-6812, each of which incorporates a guide sequence for targeting DUX4. The nucleic acid molecule may target the DUX4 transcript (with exemplary guide sequences described in Table 7) at the locations shown in Table 6.

[0040] In a preferred aspect of the invention, the miRNA guide sequence comprises a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769, SEQ ID NO: 6855-6963, SEQ ID NO: 6855-6955, SEQ ID NO: 6855-6945, SEQ ID NO: 6855-6935, SEQ ID NO: 6855-6925, SEQ ID NO: 6855-6915, SEQ ID NO: 6855-6905, SEQ ID NO: 6855-6895, SEQ ID NO: 6855-6885, SEQ ID NO: 6855-6875, SEQ ID NO: 6855-6870, SEQ ID NO: 6855-6865, or SEQ ID NO: 6860-6865. For example, SEQ ID NO: 6861, 6862, 6863, 6864 or 6865, for example, SEQ ID NO: 6860, 6861, 6862, 6863, 6864 or 6865.

[0041] In a preferred aspect of the invention, the guide sequence may be capsidated in an AAV vector comprising an engineered AAV vector capsid. The vector capsid may be engineered from an AAV9 capsid protein. The AAV vector capsid may comprise the amino acid sequences described in Tables 1-5 below, wherein Tables 5a / b describe preferred engineered capsid sequences.

[0042] In an exemplary aspect of the invention, the capsid may comprise a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838 or 5036.

[0043] In aspects of the present invention, the capsid protein may comprise sequences selected from sequences 4470-4490, 4500-4520, 4540-4560, 4660-4680, 4830-4850, and 5630-5650.

[0044] The capsid may contain sequences selected from sequences 4475-4480, 4480-4490, 4500-4510, 4540-4550, 4660-4670, 4830-4840, and 5030-5040.

[0045] The capsid may contain sequences selected from sequences 4475-4480, 4485-4490, 4500-4505, 4540-4545, 4665-4670, 4830-4835, 5035-5040, such as sequences SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838, or 5036.

[0046] In summary, exemplary guide miRNAs (e.g., as part of a construct) can be encapsulated using the exemplary capsids described herein. For example:

[0047] In an exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6860, 6861, 6862, 6863, 6864, or 6865. The guide sequence may be capped with an AAV shell comprising a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838, or 5036.

[0048] For example, an exemplary miRNA guide sequence can be encapsulated using an exemplary engineered capsid as shown below:

[0049]

[0050] Therefore, in a preferred aspect of the invention, the miRNA guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769, and the AAV vector capsid of the capsidated miRNA guide may comprise a sequence selected from Tables 1-5.

[0051] For example, in embodiments of the present invention, the miRNA guide sequence may include a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6963, and the capsid protein of the capsidated miRNA guide may include a sequence selected from sequences 4470-4490, 4500-4510, 4540-4550, 4660-4670, 4830-4840, and 5630-5640.

[0052] In a further exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6955, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4470-4490, 4500-4510, 4540-4550, 4660-4670, 4830-4840, and 5630-5640. The guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6945, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6935, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040.

[0053] In a further exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6925, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6915, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6895, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6885, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040.

[0054] In a further exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6875, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040.

[0055] The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6865, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838 or 5036.

[0056] In a preferred aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6860-6865, such as SEQ ID NO: 6861, 6862, 6863, 6864 or 6865, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838 or 5036.

[0057] Table 1: MyoAAV (eMyoAAV) capsid variants

[0058]

[0059] Table 2: Enhanced MyoAAV (eMyoAAV) capsid variants

[0060]

[0062]

[0063]

[0064]

[0065]

[0066] Attached Figure Description

[0067] Figure 1 A schematic diagram of DUX4 mRNA knockdown is shown.

[0068] Figure 2 This is a schematic diagram of a lentiviral construct used for screening miRNAs.

[0069] Figure 3 This is an FACS sorting diagram of positive and negative populations using miRNA sequences.

[0070] Figure 4 This is a graph showing the top-ranked miRNA sequences based on enrichment.

[0071] Figures 5A-5B The expression map of DUX4 and its downstream gene hZSCAN4 in myotubes of FSHD patients transduced with miRNA candidates is shown.

[0072] Figures 6A-6D The expression map of DUX4 and DUX4 target genes in the myotubes of patients after transduction with the lead candidate is shown.

[0073] Figures 7A-7B A volcano plot showing the off-target RNAsq results of candidate miRNAs in human primary myotubes.

[0074] Figures 8A-8B This is a diagram showing the expression of skeletal muscle mRNA and liver capsid DNA after administration of MyoAAV to non-human primates (NHP).

[0075] Figure 9 Immunofluorescence imaging of biceps muscle tissue after administration of MyoAAV to a non-human primate (NHP) is shown.

[0076] Figure 10 The study design for mice after injection of candidate miRNAs is shown.

[0077] Figure 11 The diagram shows tamoxifen-induced DUX4 expression and FSHD complex gene expression after administration of the mediator and lead candidate.

[0078] Figure 12A-12B The expression map of DUX4 and mKif4 in the triceps muscle of mice after administration of candidate miRNA sequences is shown.

[0079] Figure 13 Images show tamoxifen-induced DUX4 expression and muscle degeneration in mice after administration of the mediator and lead candidate. Arrows indicate areas of active degeneration.

[0080] Figures 14A-14BThe figure shows tamoxifen-induced DUX4 expression and the results of treadmill evaluation in mice after administration of the mediator and lead candidate.

[0081] Figure 15 The study design for aged mice after injection of candidate miRNAs is shown.

[0082] Figure 16 The diagram shows tamoxifen-induced DUX4 expression and FSHD complex gene expression after administration of the mediator and lead candidate.

[0083] Figure 17 Images of muscle degeneration in mice after administration of the mediator and lead candidate are shown. Arrows indicate areas of active degeneration. Detailed Implementation

[0084] This invention provides a novel nucleic acid molecule and method that induces alterations in DUX4 expression, thereby enabling treatment of FSHD. The nucleic acid molecule of this invention comprises a miR scaffold and a miR guidance sequence that targets the dual homeobox 4 (DUX4) transcript. By targeting the DUX4 transcript, the nucleic acid molecule of this invention achieves an appropriate expression profile for the DUX4 gene.

[0085] Adeno-associated virus vector

[0086] AAV is a particularly suitable viral vector for delivering genetic material into mammalian cells. It is unclear whether AAV causes disease in mammals, but rather elicits only a very mild immune response. Furthermore, AAV can infect cells at multiple stages, whether quiescent or in the process of cell replication. Advantageously, AAV DNA does not regularly insert into the host genome at random sites, thus reducing the vector's oncogenic properties.

[0087] AAVs have been engineered to deliver a variety of therapies, particularly targeting genetic disorders caused by single nucleotide polymorphisms (“SNPs”). Genetic diseases investigated in conjunction with AAV vectors include cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson's disease, congestive heart failure, and Alzheimer's disease. AAVs can be used as vectors to deliver engineered nucleic acids to a host, which then uses the host's own ribosomes to transcribe the nucleic acid into the desired protein. See, for example, West et al., Virology 160:38-47 (1987); US Patent No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994). AAV has some drawbacks in its replication and / or pathogenicity, and may therefore be safer than adenovirus vectors. In some embodiments, AAV can integrate into a specific site on human chromosome 19 without observable side effects. In some embodiments, the capacity of the AAV vector, its system, and / or AAV particles can be up to about 4.7 kb. The AAV vector or its system may include one or more engineered capsid polynucleotides as described herein.

[0088] AAV is a small, replication-defective, non-enveloped virus that infects humans and other primates and possesses a linear, single-stranded DNA genome. Naturally occurring AAV serotypes exhibit hepatic tropism. Therefore, transfection of non-hepatic tissues with conventional AAV vectors is hindered by the virus's natural hepatic tropism. Furthermore, since the liver's role is to break down substances delivered to the recipient, transfection of non-hepatic tissues with unmodified AAV vectors requires higher doses to provide sufficient viral load to breach the liver and reach non-hepatic tissues. More than 30 naturally occurring AAV serotypes are available. Many natural variants exist within the AAV capsid. AAV serotypes include, but are not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. AAVs can be engineered using conventional molecular biology techniques, allowing for optimization of these particles, such as for cell-specific delivery, minimizing immunogenicity, modulating stability and particle lifespan, efficient degradation, and accurate delivery to the cell nucleus. By selecting appropriate combinations of AAV serotypes, promoters, and delivery methods, AAV vectors can specifically target one or more cell types.

[0089] Previous methods for identifying tropism-related AAV sequences have relied on comparisons of highly correlated existing serotypes with different characteristics, random domain exchanges between uncorrelated serotypes, or consideration of higher-order structures to identify motifs defining hepatic tropism. For example, by comparing highly correlated serotypes, determinants of AAV tropism have been identified. One such example is a single amino acid change (E531K) between AAV1 and AAV6 that improves mouse hepatic transduction in AAV1. See Wu et al. (2006) J. Virol. [Journal of Virology], 80(22):11393-7, which is incorporated herein by reference. Another example is the mutual domain exchange between AAV2 and AAV8, which alters tropism but fails to define any potent tissue-specific targeting motifs. See Raupp et al. (201) J. Virol. [Journal of Virology], 86(17):9396-408, which is incorporated herein by reference. Furthermore, holistic considerations of structure only highlight the overall differences between better or worse liver transducers, differences that are more observational than useful in practice. Nam et al. (2007) J. Virol. [Journal of Virology], 81(22):12260-71.

[0090] AAVs exhibiting altered tissue orientation that can be used with this invention are described in U.S. Patent Nos. 9,695,220, 9,719,070, 10,119,125, 10,526,584, 2018-0369414, 2020-0123504, 2020-0318082, WO 2015 / 054653, WO 2016 / 179496, WO 2017 / 100791, and WO 2019 / 217911 (the entire contents of each of these patents are incorporated herein by reference).

[0091] AAV vectors or systems thereof may include one or more regulatory molecules, such as promoters, enhancers, repressors, etc. In some embodiments, AAV vectors or systems thereof may include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, one or more regulatory proteins may be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. In some embodiments, a muscle-specific promoter can drive the expression of engineered AAV capsid polynucleotides.

[0092] AAV vectors or systems thereof may include one or more polynucleotides that can encode one or more capsid proteins (such as engineered AAV capsid proteins described elsewhere herein). Engineered capsid proteins are capable of assembling into the protein coat (engineered capsid) of AAV viral particles. Engineered capsids may exhibit cell-specific, tissue-specific, and / or organ-specific tropisms.

[0093] AAV vectors or systems thereof can be configured to produce AAV particles having a specific serotype. In some embodiments, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof. The AAV among the AAVs can be selected for the cells to be targeted; for example, AAV serotypes 1, 2, 5, 9, or heterozygous capsids AAV-1, AAV-2, AAV-5, AAV-9, or any combination thereof can be selected for targeting brain and / or neuronal cells; and AAV-4 can be selected for targeting heart tissue; and AAV-8 can be selected for delivery to the liver. Therefore, in some embodiments, AAV vectors or systems thereof capable of producing AAV particles that can target brain and / or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5, or heterozygous capsids AAV-1, AAV-2, AAV-5, or any combination thereof. In some embodiments, an AAV vector or system thereof capable of producing AAV particles that can target cardiac tissue can be configured to generate AAV particles with the AAV-4 serotype. In some embodiments, an AAV vector or system thereof capable of producing AAV particles that can target the liver can be configured to generate AAV particles with the AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. [Contemporary Virology Perspective] 21:75-80.

[0094] It should be understood that while different serotypes can provide a certain level of cell, tissue, and / or organ specificity, each serotype is still multipotent, and therefore, targeting tissues with low transduction efficiency of that serotype with that serotype may result in tissue toxicity. Therefore, in addition to achieving some tissue targeting capability by selecting AAVs of a specific serotype, it should be understood that the tropism of an AAV serotype can be altered by the engineered AAV capsid described herein. As described elsewhere herein, a variant of any serotype of wild-type AAV can be generated via the methods described herein and identified as having a specific cell-specific tropism, which may be the same as or different from the cell-specific tropism of a reference wild-type AAV serotype. In some embodiments, the cell, tissue, and / or specificity of the wild-type serotype can be enhanced (e.g., making it more selective or specific to a particular cell type to which the serotype is already biased). For example, wild-type AAV-9 is biased towards human muscle and brain (see, for example, Srivastava. 2017. Curr. Opin. Virol. [Contemporary Virology Perspective] 21:75-80). By incorporating engineered AAV capsid and / or capsid protein variants, including wild-type AAV-9 as described herein, tropism towards nerve cells and / or muscle specificity can be reduced or eliminated, making nerve specificity appear lower in comparison, thereby enhancing muscle specificity compared to wild-type AAV-9. As previously mentioned, engineered capsid and / or capsid protein variants, including wild-type AAV serotypes, can have different tropisms than wild-type reference AAV serotypes. For example, engineered AAV capsid and / or capsid protein variants of AAV-9 can be specific to tissues other than human muscle or brain.

[0095] In some embodiments, the AAV vector is a hybrid AAV vector or a system thereof. A hybrid AAV is an AAV comprising a genome having elements from one serotype, these elements being packaged into a capsid derived from at least one different serotype. For example, if rAAV2 / 5 is to be generated, and if the generation method is based on the helpless transient transfection method discussed above, the first and third plasmids (adeno helper plasmids) will be the same as those discussed for rAAV2 generation. However, the second plasmid pRepCap will be different. In this plasmid, referred to as pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The generation protocol is the same as the method mentioned above for AAV2 generation. The resulting rAAV is referred to as rAAV2 / 5, wherein the genome is based on recombinant AAV2, and the capsid is based on AAV5. It is presumed that the cellular or tissue tropism exhibited by this AAV2 / 5 hybrid virus should be the same as that of AAV5. It should be understood that wild-type heterozygous AAV particles have the same specificity issues as the previously discussed non-heterozygous wild-type serotypes.

[0096] By generating hybrid AAVs that may include engineered AAV capsids as described elsewhere herein, the advantages achieved by wild-type-based hybrid AAV systems can be combined with the increased and customizable cell specificity achievable with engineered AAV capsids. It should be understood that hybrid AAVs may contain engineered AAV capsids containing genomes with elements from a serotype different from a reference wild-type serotype, and the engineered AAV capsid is a variant of that reference wild-type serotype. For example, hybrid AAVs comprising engineered AAV capsids that are variants of the AAV-9 serotype can be generated for packaging genomes containing components (e.g., rep elements) from the AAV-2 serotype. As with the previously discussed wild-type-based hybrid AAVs, the tropism of the resulting AAV particles will be the tropism of the engineered AAV capsid.

[0097] In some embodiments, the AAV vector or system thereof is configured as a "gutless" vector, similar to the vector described in conjunction with a retroviral vector. In some embodiments, the "gutless" AAV vector or system thereof may have cis-acting viral DNA elements linked to a target heterologous sequence (e.g., one or more engineered AAV capsid polynucleotides) that participate in genome amplification and packaging.

[0098] The vectors described herein can be constructed using any suitable method or technique. In some embodiments, one or more suitable recombination and / or cloning methods or techniques may be used for one or more vectors described herein. Suitable recombination and / or cloning techniques and / or methods may include, but are not limited to, those described in U.S. Application Publication No. US 2004-0171156 A1. Other suitable methods and techniques are described elsewhere herein.

[0099] The construction of recombinant AAV vectors has been described in numerous publications, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. [Molecular Cell Biology] 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. [Molecular Cell Biology] 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS [Proceedings of the National Academy of Sciences] 81:6466-6470 (1984); and Samulski et al., J. Virol. [Journal of Virology] 63:03822-3828 (1989). Any technique and / or method can be used and / or adapted to construct the AAV or other vectors described herein. AAV vectors are discussed elsewhere in this document.

[0100] In some embodiments, the vector may have one or more insertion sites, such as restriction endonuclease recognition sequences (also known as "cloning sites"). In some embodiments, one or more insertion sites (e.g., 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.

[0101] The delivery medium, carrier, particles, nanoparticles, formulations and their components used to express one or more elements of the engineered AAV capping system described herein are as used in the aforementioned literature, such as International Patent Application Publications WO 2021 / 050974 and WO 2021 / 077000 and PCT International Application No. PCT / US2021 / 042812, the contents of which are incorporated herein by reference.

[0102] Additional AAV carriers are described in International Patent Application Publication WO 2019 / 2071632 (the contents of which are incorporated herein by reference).

[0103] Other AAV carriers are described in International Patent Application Publications WO 2020 / 086881 and WO 2020 / 235543 (the contents of each of these patents are incorporated herein by reference).

[0104] Other AAV carriers are described in international patent applications published under WO 2005 / 033321; WO 2006 / 110689; WO2007 / 127264; WO 2008 / 027084; WO 2009 / 073103; WO 2009 / 073104; WO 2009 / 105084; WO2009 / 134681; WO 2009 / 136977; WO 2010 / 051367; WO 2010 / 138675; WO 2001 / 038187; WO2012 / 112832; WO 2015 / 054653; WO 2016 / 179496; WO 2017 / 100791; WO 2017 / 019994; WO2018 / 209154; WO 2019 / 067982; WO 2019 / 195701; WO 2019 / 217911; WO 2020 / 041498; WO2020 / 210839; US Patent No. 7,906,111; US ​​Patent No. 9,737,618; US Patent No. 10,265,417; US Patent No. 10,485,883; US Patent No. 10,695,441; US ​​Patent No. 10,722,598; US Patent No. 8,999,678; US Patent No. 10,301,648; US Patent No. 10,626 ,415; US Patent No. 9,198,984; US Patent No. 10,155,931; US ​​Patent No. 8,524,219; US Patent No. 9,206,238; US Patent No. 8,685,387; US Patent No. 9,359,618; US Patent No. 8,231,880; US Patent No. 8,470,310; US Patent No. 9,597,363; US Patent No. 8,940,290; US Patent No. 9, 593,346; US Patent No. 10,501,757; US Patent No. 10,786,568; US Patent No. 10,973,928; US Patent No. 10,519,198; US Patent No. 8,846,031; US ​​Patent No. 9,617,561; US ​​Patent No. 9,884,071; US ​​Patent No. 10,406,173; US Patent No. 9,596,220; US Patent No. 9,719,010 U.S. Patent Nos. 10,117,125; 10,526,584; 10,881,548; 10,738,087; U.S. Patent Publication No. 2011-023353; 2019-0015527; 2020-155704; 2017-0191079; 2019-0218574;U.S. Patent Publication No. 2020-0208176; U.S. Patent Publication No. 2020-0325491; U.S. Patent Publication No. 2019-0055523; U.S. Patent Publication No. 2020-0385689; U.S. Patent Publication No. 2009-0317417; U.S. Patent Publication No. 2016-0051603; U.S. Patent Publication No. 2016-00244783; U.S. Patent Publication No. 2017-0183636; U.S. Patent Publication No. 2020-0263201; U.S. Patent Publication No. 2020-0101099; U.S. Patent Publication No. 2020-0318082; U.S. Patent Publication No. 2018-0369414; U.S. Patent Publication No. 2019-0330278; U.S. Patent Publication No. 2020-0231986 (the contents of each of these patents are incorporated herein by reference).

[0105] promoter

[0106] This invention may contain a muscle-specific promoter or another type of promoter. The promoter may be linked to a nucleic acid sequence such that transcription preferably occurs within muscle cells. The promoter region enables the host cell to replicate the nucleic acid delivered by AAV only in those cell types and tissues or organs where the desired protein should be produced. Here, a muscle-specific promoter is included because it is primarily desirable for the protein to be translated only in muscle cells. Due to the potentially adverse effects of delivering the nucleic acid and causing it to be translated in cells that do not need that nucleic acid (and therefore the protein), there is a need for specificity for the cell type to which the nucleic acid is delivered and thus the protein is translated.

[0107] Myocyte-specific promoters may be coupled to or otherwise associated with a truncated DUX4 sequence. In some embodiments, the promoter may be directly attached, while in other embodiments, a adapter molecule or another indirect coupling method may be used to attach to the truncated DUX4 sequence. In some embodiments, associated peptides or other particles coupled to the truncated DUX4 sequence may be present.

[0108] In some embodiments, the muscle-specific promoter produces increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof. As used herein, the terms “muscle specificity,” “muscle cell specificity,” “muscle cell potency,” “myotrophic lateral ...

[0109] In some embodiments, the muscle cell-selective promoter utilized is MHCK7. MHCK7 is a 770-base-pair promoter, small enough to be included in an AAV vector. MHCK7 directs expression in fast and slow skeletal muscle and cardiac muscle, and is expressed at low levels in the liver, lung, and spleen. Its activity is lower in smooth muscle. The MHCK7 promoter is associated with high levels of expression in skeletal muscle (including the diaphragm) and includes enhancers to specifically drive expression in the heart, while expression is lowest in off-target tissues.

[0110] In some embodiments, the promoter described herein is inserted into an AAV protein (e.g., an AAV capsid protein) that exhibits reduced specificity (or no detectable, measurable, or clinically relevant interaction) with one or more non-muscle cell types. Exemplary non-muscle cell types include, but are not limited to, liver, kidney, lung, heart, spleen, central or peripheral nervous system cells, bone, immune cells, stomach, intestine, eye, skin cells, etc. In some embodiments, the non-muscle cell is a liver cell.

[0111] The term "operable ligation" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment, such that the function of one is affected by the function of the other.

[0112] Other exemplary tissue-specific promoters include the U6 promoter sequence, MHCK7 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.

[0113] Muscle-specific promoters are described in International Patent Application Publications WO 2020 / 006458 and WO 2021 / 126880, the contents of which are incorporated herein by reference.

[0114] Other muscle-specific promoters are described in U.S. Patent Nos. 9,133,482; 10,105,453; 10,301,367; U.S. Patent Publication No. 2020-0360534; PCT International Patent Publication Nos. WO 2020 / 006458; WO 2021 / 035120; WO 2021 / 053124; and WO 2021 / 077000, the contents of each of which are incorporated herein by reference.

[0115] Using RNA polymerase II or III promoters may be convenient; these are known to those skilled in the art and have been reviewed, for example, in Kornberg 1999. However, transcripts from RNA II polymerases typically have complex transcriptional terminators and are polyadenylated; this can hinder the requirements of the miRNA chain, as its 5' and 3' ends need to be precisely defined to achieve the secondary structure required to produce a functional molecule. However, these disadvantages can be avoided. In the case of using RNA polymerase II or III promoters, the polynucleotide encoding the miRNA chain can also encode a self-processing ribozyme and can be operatively linked to the RNA polymerase II or III promoter; thus, the polynucleotide encodes a pre-miRNA chain containing the miRNA chain and the self-processing ribozyme, wherein, upon transcription, the miRNA chain is released from the pre-miRNA chain transcript via the self-processing ribozyme.

[0116] Preferably, in the composition according to the invention, the AAV vector comprises an RNA polymerase II or III promoter and encodes a pre-miRNA chain comprising a miRNA chain and a self-processing ribozyme, wherein, upon transcription, the miRNA chain is released from the pre-miRNA chain transcript via the self-processing ribozyme. Conveniently, multiple pre-miRNA chains and multiple self-processing ribozymes can be encoded by a single polynucleotide operably linked to one or more RNA polymerase II promoters.

[0117] Inducible and / or tissue-specific RNA polymerase II or III promoters have been previously described. RNA polymerase promoters are known in the art and are further described in U.S. Patent Publication 11,149,288, the contents of which are incorporated herein by reference.

[0118] Capsid protein

[0119] Capsid proteins are the outer shell or coating of a virus, enabling it to be delivered to the host. Without this protein, the nucleic acid would be destroyed by the host, preventing it from entering the host cell and initiating transcription and translation. Capsid proteins can be in the native conformation of naturally occurring AAVs, or they can be modified.

[0120] In some example embodiments, the AAV capsid protein is an engineered AAV capsid protein with reduced or eliminated uptake in non-muscle cells compared to the corresponding wild-type AAV capsid peptide (e.g., the AAV9 capsid peptide described in SEQ ID NO: 1).

[0121] In some embodiments, the polynucleotide encoding the engineered AAV capsid may be included in a polynucleotide configured as an AAV genome donor in an AAV vector system that can be used to generate engineered AAV particles as described elsewhere herein. In some embodiments, the polynucleotide encoding the engineered AAV capsid may be operatively coupled to a polyadenylated tail. In some embodiments, the polyadenylated tail may be an SV40 polyadenylated tail. In some embodiments, the polynucleotide encoding the AAV capsid may be operatively coupled to a promoter. In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific to: muscle (e.g., cardiac muscle, skeletal muscle, and / or smooth muscle), neurons and supporting cells (e.g., astrocytes, glial cells, Schwann cells, etc.), adipose tissue, spleen, liver, kidney, immune cells, cerebrospinal fluid cells, synovial cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal glands, blood cells, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter may be a constitutive promoter. Suitable tissue-specific and constitutive promoters are discussed elsewhere in this document and are generally known in the art and may be commercially available. Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and its variants, and SPc5-12 synthetic promoter.

[0122] This document describes various embodiments of engineered viral capsids (such as adeno-associated virus (AAV) capsids) that can be engineered to confer cell-specific orientation, such as muscle-specific orientation, to engineered viral particles. The engineered viral capsid can be a lentivirus, retrovirus, adenovirus, or AAV capsid. The engineered capsid can be included in engineered viral particles (e.g., engineered lentiviral particles, retroviral particles, adenovirus particles, or AAV viral particles) and can confer cell-specific orientation, reduced immunogenicity, or both to the engineered viral particles. 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, which may contain a muscle-specific targeting portion containing or constituting an n-mer motif as described elsewhere herein.

[0123] Engineered viral capsids and / or capsid proteins may be encoded by one or more engineered viral capsid polynucleotides. In some embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide. In some embodiments, the engineered viral capsid polynucleotide (e.g., an engineered AAV capsid polynucleotide, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide) may include a 3' polyadenylation signal. The polyadenylation signal may be an SV40 polyadenylation signal.

[0124] Engineered viral capsids can be variants of wild-type viral capsids. For example, in some embodiments, engineered AAV capsids can be variants of wild-type AAV capsids. In some embodiments, the wild-type AAV capsid can be composed of VP1, VP2, VP3 capsid proteins or combinations thereof. In other words, engineered AAV capsids can include one or more variants of wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In some embodiments, the serotype of the reference wild-type AAV capsid can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the serotype of the wild-type AAV capsid can be AAV-9. Engineered AAV capsids can have different tropisms than the reference wild-type AAV capsid.

[0125] The engineered viral capsid may contain 1-60 engineered capsid proteins. In some embodiments, the engineered viral capsid may 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. In some embodiments, the engineered viral capsid may contain 0-59 wild-type viral capsid proteins. In some embodiments, the engineered viral capsid may 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 viral capsid proteins.

[0126] In some embodiments, the engineered AAV capsid may contain 1-60 engineered capsid proteins. In some embodiments, the engineered AAV capsid may 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. In some embodiments, the engineered AAV capsid may contain 0-59 wild-type AAV capsid proteins. In some embodiments, the engineered AAV capsid may 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.

[0127] In some embodiments, the engineered viral capsid protein may have an n-mer amino acid motif, where n may be at least 3 amino acids. In some embodiments, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the engineered AAV capsid may have a 6-mer or 7-mer amino acid motif. In some embodiments, the n-mer amino acid motif may be inserted between two amino acids in the wild-type viral protein (VP) (or capsid protein). In some embodiments, the n-mer motif may be inserted between two amino acids in a variable amino acid region of the viral capsid protein.

[0128] In some embodiments, the n-mer motif may be inserted between two amino acids in the variable amino acid region of the AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-strand β-barrel motif (βB to βI) and an α-helix (αA), which are conserved in autonomous parvovirus capsids (see, for example, DiMattia et al. 2012. J. Virol. [Journal of Virology] 86(12):6947-6958). Structural variable regions (VRs) appear in surface loops connecting the β chains, and these surface loops aggregate to produce local variations on the capsid surface. AAV has 12 variable regions (also known as hypervariable regions) (see, for example, Weitzman and Linden. 2011. “Adeno-Associated Virus Biology. [Adeno-Associated Virus Biology]” in Snyder, RO, Moullier, P. (ed.) Totova, NJ: Humana Press [Humana Press]). In some embodiments, one or more n-mer motifs may be inserted between two amino acids in one or more of the 12 variable regions of the wild-type AVV capsid protein. In some embodiments, one or more n-mer motifs may 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, VR-XI, VR-XII, or combinations thereof. In some embodiments, the n-mer may be inserted between two amino acids in VR-III of the capsid protein. In some embodiments, the engineered capsid may have an n-mer inserted between any two consecutive amino acids between amino acids 262 and 269 of the AAV9 viral protein, between any two consecutive amino acids between amino acids 327 and 332, between any two consecutive amino acids between amino acids 382 and 386, between any two consecutive amino acids between amino acids 452 and 460, between any two consecutive amino acids between amino acids 488 and 505, between any two consecutive amino acids between amino acids 545 and 558, between any two consecutive amino acids between amino acids 581 and 593, and between any two consecutive amino acids between amino acids 704 and 714. In some embodiments, the engineered capsid may have an n-mer inserted between amino acids 588 and 589 of the AAV9 viral protein. In some embodiments, the engineered capsid may have a 7-mer motif inserted between amino acids 588 and 589 of the AAV9 viral protein. In other embodiments, the inserted motif is a 10-mer motif, replacing amino acids 586-88 and inserted before 589. It should be understood that n-mer can insert into similar positions in the proteins of other serotypes of AAV virus.In some embodiments as previously discussed, one or more n-mers may be inserted between any two consecutive amino acids within the AAV viral protein, and in some embodiments, the insertion is performed in a variable region.

[0129] In some embodiments, the first 1, 2, 3, or 4 amino acids of the n-mer motif may replace the first 1, 2, 3, or 4 amino acids preceding the insertion site in the polypeptide into which it is inserted. In some embodiments, the amino acid of the n-mer motif replacing one or more amino acids of the polypeptide into which the n-mer motif is inserted is located before or immediately before “RGD” in the n-mer motif. For example, in one or more of the 10-mer inserts shown in Tables 2-3, the first three amino acids shown may replace 1-3 amino acids in the polypeptide into which they may be inserted. Using AAV as another non-limiting example, one or more of the n-mer motifs may be inserted between amino acids 588 and 589 of, for example, the AAV9 capsid prolyl peptide, and the insert may replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the n-mer motif after insertion is residue 585. It should be understood that this principle can be applied to any other insertion case and is not necessarily limited to the equivalent position between residues 588 and 589 of the AAV9 capsid or in another AAV capsid. It should be further understood that, in some embodiments, no amino acids in the polypeptide with the inserted n-mer motif are replaced by the n-mer motif.

[0130] In some embodiments, the AAV capsid or other viral capsid or composition may be muscle-specific. In some embodiments, the muscle specificity of the engineered AAV or other viral capsid or composition is conferred by a muscle-specific n-mer motif introduced in the engineered AAV or other viral capsid or composition described herein. While not intended to be theoretically constrained, it is thought that the n-mer motif confers a domain or region or a 3D structure within the engineered AAV capsid or other viral capsid or composition such that the interaction of viral particles or other compositions containing the engineered AAV capsid or other viral capsid or composition described herein has an increased or improved interaction with cell surface receptors and / or other molecules on the surface of muscle cells (e.g., increased affinity). In some embodiments, the cell surface receptor is an AAV receptor (AAVR). In some embodiments, the cell surface receptor is a muscle cell-specific AAV receptor. In some embodiments, the cell surface receptor or other molecule is a cell surface receptor or other molecule selectively expressed on the surface of muscle cells. In some embodiments, the cell surface receptor or molecule is an integrin or a dimer thereof. In some embodiments, the cell surface receptor or molecule is a Vb6 integrin heterodimer.

[0131] In some embodiments, muscle-specific engineered viral particles or other compositions containing the muscle-specific capsid, n-mer motif, or muscle-specific targeting portion described herein may have increased uptake, delivery rate, transduction rate, efficiency, quantity, or combinations thereof in muscle cells compared to other cell types and / or other viral particles (including but not limited to AAV) and other compositions that do not contain the muscle-specific n-mer motif of the present invention.

[0132] First- and second-generation muscle-specific AAV capsids were developed using muscle-specific promoters, and the resulting capsid libraries were screened in mice and non-human primates as described elsewhere herein and / or, for example, in U.S. Provisional Application Serials 62 / 899,453, 62 / 916,207, 63 / 018,454, and 63 / 242,008. As previously described, the first- and second-generation myoAAV capsids were further optimized in mice and non-human primates to generate enhanced myoAAV capsids. Capsids that can be used with this invention may be as described in PCT application number PCT / US2024 / 044341.

[0133] Tables 1 and 2 show the highest hits of the enhanced muscle-specific n-mer motif and its coding sequence in rank order in each table. The enhanced MyoAAV (eMyoAAV) capsid variant transduces mouse muscle more efficiently than the first-generation MyoAAV following systemic delivery. Both first- and second-generation myoAAV capsid variants rely on the aVb6 integrin heterodimer for transduction of human primary myotubes.

[0134] Tables 3 and 4 show the top-ranked capsid variants resulting from multiple rounds of directed evolution targeting skeletal muscle specificity in capsid variants. As shown in the tables above regarding the n-mer inserts of variants containing the P-motif, the first three amino acids of the variant sequences shown are amino acids that replace positions 596, 597, and 598 of the AAV9 capsid polypeptide. Thus, for example, the P-motif is inserted between amino acids 598 and 599 of the AAV9 vector.

[0135] Tables 5a and 5b show the top-ranked capsid variants resulting from multiple rounds of directed evolution of capsid variants specific to skeletal muscle. The capsid protein variants may contain substitutions at amino acid positions 451-455 relative to the wild-type AAV9 vector capsid. For example, the substitutions at amino acid positions 451-455 relative to the wild-type AAV9 vector capsid may be amino acid sequences selected from column 1 of Table 6b. In aspects of the invention, the capsid protein variants may further include an insert. For example, the capsid protein may contain a 7-mer insert selected from column 2 of Table 6b. The insert may be located after amino acid position 455 relative to the wild-type AAV9 vector. For the HVR IV variant, the upstream 5 amino acids are located at positions 451-455 (shown in column 1 of Table 6b). The 7-mer insert of the HVR IV variant begins with "RGD" and is inserted after amino acid position 455 (shown in column 2 of Table 6b).

[0136] microRNA

[0137] MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules. miRNAs pair with complementary base sequences in mRNA molecules, thereby silencing post-transcriptional regulation of gene expression. Typically, miRNA molecules silence mRNA translation by cleaving the mRNA strand into two fragments or by shortening its poly(A) tail to disrupt mRNA stability.

[0138] miRNAs are similar to small interfering RNAs (siRNAs), but miRNAs originate from the backfolding region of the RNA transcript that forms a short hairpin. Animal miRNAs are initially transcribed as part of an arm of an RNA stem-loop, which in turn forms part of a hundreds-of-nucleotides-long miRNA precursor called a primary miRNA (pri-miRNA). A single pri-miRNA can contain one to six miRNA precursors. These hairpin loop structures are typically each about 70 nucleotides long. Each hairpin is flanked by the sequence required for efficient processing.

[0139] Pre-miRNAs are typically cleaved by the RNase Dicer. The RNase interacts with the 5' and 3' ends of the hairpin and cleaves the loop connecting the 3' and 5' arms, resulting in a miRNA:miRNA duplex of approximately 22 nucleotides in length. The overall hairpin length and loop size affect the efficiency of Dicer processing. Although either strand of the duplex may potentially function as a functional miRNA, usually only one strand is introduced into the RNA-induced silencing complex (RISC), in which the miRNA interacts with its mRNA target.

[0140] For example, a pri-miR scaffold, along with its guide and passenger sequences, can form a hairpin loop structure. The length of the hairpin loop structure can be greater than 250 nucleotides (e.g., between 250 and 270 nucleotides). A pri-miR can have the following structure in sequence: a first (“upstream”) scaffold sequence, a guide sequence, a hairpin loop, a sequence complementary or partially complementary to the guide sequence (referred to as the “passenger” sequence), and a second (“downstream”) scaffold sequence. The guide and passenger sequences form a double-stranded RNA (dsRNA), while the first and second scaffold sequences are single-stranded RNAs (sRNAs) (referred to as “arms”) at both ends of the double-stranded molecule. The passenger strand can be completely complementary to the guide sequence, or it can have one or more mismatched nucleotides with the guide strand. For example, mismatches or extra nucleotides may cause a “bump” in the pre-miRNA while overall hybridization is still maintained between the guide and passenger strands. The hairpin loop is located at the end of the dsRNA opposite the first and second scaffold sequences, connecting the 5′ and 3′ ends of the guide and passenger sequences. Examples of pri-miR scaffolds and their guides are shown below:

[0141] Hairpin structures can be processed by cleaving near the junction between the dsRNA and ssRNA arms. This processing can result in substantial cleavage of the ssRNA arms. The loop structure of pri-miRNA is typically 60-100 nucleotides long and is called pre-miRNA.

[0142] Pre-miRNA can be transported to the cytoplasm and further processed by enzymes (e.g., Dicer). Enzymes can process pre-miRNA at the 5' and 3' ends of the hairpin by cutting off the loop connecting the 3' and 5' arms, resulting in a miRNA(guide):miRNA(passenger) double strand of approximately 21 nucleotides in length.

[0143] The double-stranded miRNA can then be processed to form a precursor of the RNA-induced silencing complex (RISC). The complex can then unwind the double strand, and the passenger RNA strand may be discarded, leaving behind a mature RISC carrying the mature single-stranded guide miRNA.

[0144] Advantageously, artificial miRNA molecules can be engineered to include a scaffold of endogenous miRNA and a targeting sequence of the target gene. Aspects of the invention include miRNA molecules targeting the DUX4 transcript. The pri-miRNA scaffold can be selected from any scaffold, such as those based on cell or tissue specificity of mature miRNAs. For example, the pri-miRNA scaffold can be derived from pri-miRNAs selected from the group consisting of pri-miR-21, pri-miR-22, pri-miR-26a, pri-miR-30a, pri-miR-33, pri-miR-122, pri-miR-375, pri-miR-199, pri-miR-99, pri-miR-194, pri-miR-155, and pri-miR-451. In a preferred aspect of the invention, the miRNA scaffold is derived from pri-miR-33.

[0145] miRNA-based therapies (including miRNA inhibition and miRNA replacement) can be used to treat many diseases, such as hepatitis C virus infection, muscular dystrophy, neurodegenerative diseases, peripheral neuropathy, chronic heart failure and post-myocardial infarction remodeling, and cancer. Furthermore, targeted miRNA regulation of gene expression can improve the efficacy of traditional gene therapy methods that utilize vectors to encode protein-coding genes.

[0146] MicroRNA sequences are described in U.S. Patent Publications 2020-0248179, 2019-0300903, 2019-0136235, 2019-0024083, 2017-0029849 and 2014-0322169 (the contents of each of these patents are incorporated herein by reference).

[0147] Pharmaceutical Composition

[0148] Some embodiments of the present invention may include providing an AAV carrier to a subject in any acceptable form. For example, the AAV carrier may be provided to a subject in the form of a composition or formulation comprising an AAV carrier. The expression vectors of the present invention may be formulated and administered by any means of contacting the active ingredient with the site of action of the agent in the subject to treat a variety of disease states. The compositions, polynucleotides, peptides, particles, cells, carrier systems and combinations thereof described herein may be included in formulations (such as pharmaceutical formulations). In some embodiments, formulations may be used to generate peptides and other particles comprising one or more muscle-specific targeting portions described herein. In some embodiments, formulations may be delivered to a subject in need. In some embodiments, one or more components of the engineered AAV capsid system, engineered cells, engineered AAV capsid particles and / or combinations thereof described herein may be included in formulations that may be delivered to a subject or cells. In some embodiments, formulations are pharmaceutical formulations. One or more of the peptides, polynucleotides, carriers, cells and combinations thereof described herein may be provided to a subject or cells in need, individually or as an active ingredient (such as an active ingredient in a pharmaceutical formulation). Therefore, pharmaceutical formulations containing one or more of the peptides, polynucleotides, carriers, cells or combinations thereof described herein are also described herein. In some embodiments, the pharmaceutical formulation may contain an effective amount of one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein. The pharmaceutical formulation described herein may be administered to a subject or cells in need.

[0149] In some embodiments, based on the weight of the subject in need or the average weight of a specific patient group to whom the pharmaceutical formulation can be administered, the amount of one or more of the peptides, polynucleotides, carriers, cells, viral particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in the pharmaceutical formulation may range from about 1 pg / kg to about 10 mg / kg. The amount of one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein in the pharmaceutical formulation may range from about 1 pg to about 10 g, or from about 10 nL to about 10 ml. In embodiments where the pharmaceutical formulation contains one or more cells, the amount may range from about 1 cell to 1 × 10⁻⁶ cells. 2 1 × 10 3 1 × 10 4 1 × 10 5 1 × 10 6 1×10 7 1 × 10 8 1 × 10 9 1 × 10 10The range is from about 1 cell to 1 × 10⁻⁶ cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can be from about 1 cell to 1 × 10⁻⁶ cells. 2 1 × 10 3 1 × 10 4 1 × 10 5 1 × 10 6 1 × 10 7 1 × 10 8 1 × 10 9 1 × 10 10 Within the range of more than one cell / nL, μL, mL or L.

[0150] In embodiments where the formulation contains engineered AAV capping particles, the formulation may contain 1 to 1 × 10⁻⁶ particles. 2 1×10 3 1 × 10 4 1 × 10 5 1 × 10 6 1 × 10 7 1 × 10 8 1 × 10 9 1 × 10 10 1 × 10 11 1 × 10 12 1 × 10 13 1 × 10 14 1 × 10 15 1 × 10 16 1 × 10 17 1 × 10 18 1 × 10 19 Or 1× 10 20 Engineered AAV capsid particles of 1 transduction unit (TU) / mL. In some embodiments, the volume of the formulation may be from 0.1 to 100 mL, and may contain 1 to 1 × 10⁻⁶ TU / mL. 2 1 × 10 3 1 × 10 4 1 × 10 5 1 × 10 6 1 × 10 7 1 × 10 8 1 × 10 9 1 × 10 10 1 × 10 11 1 × 10 12 1 × 10 13 1 × 10 141 × 10 15 1 × 10 16 1 × 10 17 1 × 10 18 1 × 10 19 Or 1 × 10 20 Engineered AAV capsid particles with 1 transduction unit (TU) / mL.

[0151] Pharmaceutically acceptable carriers, excipients and reagents

[0152] In the embodiments, pharmaceutical formulations containing one or more of the peptides, polynucleotides, carriers, cells, viral particles, nanoparticles, other delivery particles, and combinations thereof described herein may further include pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose, amylose, or starch), magnesium stearate, talc, silica, viscous paraffin, fragrance oils, fatty acid esters, hydroxymethyl cellulose, and polyvinylpyrrolidone, which do not adversely react with the active ingredient.

[0153] These pharmaceutical preparations can be sterilized, and if necessary, they can be mixed with adjuvants (such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, etc.) that will not harmfully react with the active composition.

[0154] In some embodiments, the pharmaceutical formulations described herein may be dosage forms. Dosage forms may be suitable for administration via any suitable route. Suitable routes include, but are not limited to, oral (including oral or sublingual), rectal, epidural, intracranial, intraocular, inhalation, intranasal, local (including oral, sublingual, or transdermal), vaginal, urethral, ​​parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavitary, intrathecal, intravitreal, intracerebral, gingival, subgingival, intravenous, and intradermal. Such formulations may be prepared by any method known in the art.

[0155] Dosage forms suitable for oral administration may be discrete dosage units, such as capsules, pills or tablets, powders or granules, solutions, or suspensions in aqueous or non-aqueous liquids; edible foams or whisks; or oil-in-water or water-in-oil liquid emulsions. In some embodiments, pharmaceutical formulations suitable for oral administration may also include one or more agents for flavoring, preserving, coloring, or aiding in the dispersion of the pharmaceutical formulation. Dosage forms for oral administration may also be prepared in the form of liquid solutions that can be delivered as foams, sprays, or liquid solutions. In some embodiments, oral dosage forms may contain from about 1 ng to 1000 g of a pharmaceutical formulation containing a therapeutically effective amount or an appropriate fraction of a targeted effector fusion protein and / or its complex, or a composition containing one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein. Oral dosage forms may be administered to subjects in need.

[0156] Where appropriate, the dosage forms described herein can be microencapsulated.

[0157] Dosage forms can also be formulated to prolong or maintain the release of any component. In some embodiments, one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein may be delayed-release components. In other embodiments, the release of optionally included auxiliary components is delayed. Suitable methods for delaying component release include, but are not limited to, coating or embedding the component with materials such as polymers, waxes, and gels. Delayed-release dosage forms can be prepared according to descriptions in, for example, the following standard references: "Pharmaceutical dosage form tablets," Liberman et al. (New York, Marcel Dekker, Inc., 1989); "Remington - The science and practice of pharmacy," 20th edition, Lippincott Williams and Wilkins Publishing House, Baltimore, MD, 2000; and "Pharmaceutical dosage forms and drug delivery systems," 6th edition, Ansel et al. (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and methods for preparing tablets and capsules, as well as delayed-release dosage forms of tablets, pills, capsules, and granules. Delayed release can be any time from about one hour to about three months or longer.

[0158] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate; polyvinyl acetate phthalates; acrylic polymers and copolymers; and methacrylic resins, zein, shellac, and polysaccharides commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany).

[0159] Coating can be formed with varying ratios of water-soluble polymers, water-insoluble polymers, and / or pH-dependent polymers, with or without water-insoluble / water-soluble nonpolymeric excipients, to produce a desired release profile. Coating is performed on dosage forms (matrix-based or simple dosage forms), including but not limited to tablets (compressed, with or without coating), capsules (with or without coating), beads, granule compositions, and "ingredient as is" formulated as, but not limited to, suspensions or spray formulations.

[0160] Dosage forms suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treating the eyes or other external tissues (e.g., the mouth or skin), the pharmaceutical formulation is applied as a topical ointment or cream. When formulated into an ointment, one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein can be formulated with a paraffin or water-miscible ointment base. In some embodiments, the active ingredient can be formulated as an oil-in-water or water-in-oil cream base. Dosage forms suitable for topical oral application include lozenges, tablets, and mouthwashes.

[0161] Dosage forms suitable for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein are included in a dosage form suitable for inhalation, which is in a form with reduced particle size obtained or available through micronization. In some embodiments, the particle size of the size-reduced (e.g., micronized) compound or its salt or solvate is defined by a D50 value of about 0.5 to about 10 micrometers, as measured by suitable methods known in the art. Dosage forms suitable for inhalation administration also include particulate dust or aerosols. Carriers or excipients are suitable dosage forms for liquids administered in the form of nasal sprays or drops, comprising aqueous or oil solutions / suspensions of active ingredients (e.g., one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein and / or co-active agents), which can be generated by various types of metered-dose pressurized aerosols, nebulizers, or blowpipes.

[0162] In some embodiments, the dosage form may be an aerosol formulation suitable for inhalation administration. In some embodiments of these embodiments, the aerosol formulation may contain a solution or fine suspension of one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein, and a pharmaceutically acceptable aqueous or non-aqueous solvent. The aerosol formulation may be presented in a sterile form in a single-dose or multi-dose quantity in a sealed container. In some embodiments of these embodiments, the sealed container is a single-dose or multi-dose nasal container or an aerosol dispenser equipped with a metering valve (e.g., a metered-dose inhaler) designed to be discarded once the contents of the container are used up.

[0163] When an aerosol formulation is contained in an aerosol dispenser, the dispenser contains a suitable pressurized propellant, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to hydrofluorocarbons. In other embodiments, the aerosol formulation is contained in a pump-type nebulizer. Pressurized aerosol formulations may also contain a solution or suspension of one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation may also contain co-solvents and / or modifiers introduced to improve, for example, the stability and / or taste and / or fine particulate quality characteristics (quantity and / or characteristic profile) of the formulation. The aerosol formulation may be administered once daily or several times daily, for example, 2, 3, 4, or 8 times daily, wherein each administration delivers 1, 2, or 3 doses.

[0164] For some dosage forms suitable and / or adapted for inhalation, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein, co-active ingredients, and / or pharmaceutically acceptable salts thereof, such dosage forms may contain a powder matrix such as lactose, glucose, trehalose, mannitol, and / or starch. In some embodiments of these examples, one or more of the peptides, polynucleotides, carriers, cells, and combinations thereof described herein are in a reduced particle size form. In other embodiments, performance modifiers such as L-leucine or another amino acid, cellobiose octaacetate, and / or metal salts of stearic acid, such as magnesium stearate or calcium stearate, are used.

[0165] In some embodiments, an aerosol dosage form may be arranged such that each dose of the aerosol contains a predetermined amount of an active ingredient, such as one or more of peptides, polynucleotides, carriers, cells, and combinations thereof as described herein.

[0166] Dosage forms suitable for vaginal administration may be available as vaginal suppositories, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms suitable for rectal administration include suppositories or enemas.

[0167] Dosage forms suitable for parenteral administration and / or suitable for any type of injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intra-articular, cavernous sinus, gingival, subgingival, intrathecal, intravitreal, intracerebral, and intraventricular) may include aqueous and / or non-aqueous sterile injectable solutions (which may contain antioxidants, buffers, antibacterial agents, and solutes that make the composition isotonic with the blood of the subject) and aqueous and non-aqueous sterile suspensions (which may include suspending agents and thickeners). Dosage forms suitable for parenteral administration may be available in single-dose or multi-dose containers, including but not limited to sealed ampoules or vials. The dose may be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. In some embodiments, injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0168] Dosage forms suitable for ocular application may include aqueous and / or non-aqueous sterile solutions (which may optionally be suitable for injection and may optionally contain antioxidants, buffers, antibacterial agents, or solutes that make the composition isotonic with the subject’s eye or fluids contained in or around the eye), and aqueous and non-aqueous sterile suspensions (which may include suspending agents and thickeners).

[0169] In some embodiments, the dosage form contains a predetermined amount of one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein per unit dose. In some embodiments, this predetermined amount of such unit dose can therefore be administered once or more daily. Such pharmaceutical formulations can be prepared by any method well known in the art.

[0170] DUX4 and facioscapulohumeral muscular dystrophy

[0171] Muscular dystrophy (MD) is a group of inherited disorders. This group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement or breathing. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. These disorders vary in the distribution and severity of muscle weakness (some forms of MD also affect the cardiac muscle), age of onset, rate of progression, and genetic patterns.

[0172] Facial-scapular muscular dystrophy (FSHD) is a complex autosomal dominant genetic disorder characterized by progressive and asymmetrical weakness of the muscles in the face, shoulders, and limbs. Symptoms typically occur in adulthood, with most patients showing clinical features before age 30. Approximately 5% of patients develop symptoms in infancy or adolescence, and these patients are usually more severely affected. Clinical presentation can range from mild (some limited muscle weakness) to severe (wheelchair dependence). Historically, FSHD was classified as the third most common MD, affecting one in 20,000 people worldwide. However, recent data suggest that FSHD is the most common MD in Europe, indicating a global prevalence that may be as high as 1 in 8,333. Typical cases of FSHD (FSHD1A, formerly known as FSHD) are associated with heterozygous chromosome deletions that reduce the copy number of the 3.3 kb D4Z4 repeat sequence on human chromosome 4q35. In short, normal individuals have 11-100 copies of tandemly repeated D4Z4 on both 4q35 alleles, while FSHD patients have one normal allele and one contracted allele containing 1-10 repetitive sequences. Furthermore, FSHD-associated D4Z4 contraction must occur on a specific disease-admissible chromosomal 4q35 background (called 4qA). Importantly, no gene is completely lost or structurally mutated due to FSHD-associated deletions. Instead, FSHD-associated genetic changes result in the expression of the toxic DUX4 gene, which is detrimental to muscles. FSHD2 (also known as FSHD1B) is phenotypically identical to FSHD1, is associated with DUX4 expression, and requires a 4qA chromosomal background. FSHD2 is not associated with D4Z4 repetitive sequence contraction but is caused by mutations in the SMCHD1 gene, a chromatin regulator that is normally involved in repressing the DUX4 locus at 4qA. The mutated SMCHD1 protein is unable to participate in the addition of heterochromatin to the 4qA DUX4 allele, thus allowing DUX4 gene expression.

[0173] In the dominant pathogenesis model of FSHD, D4Z4 contraction is thought to induce epigenetic changes that allow for the expression of the DUX4 gene. Therefore, the aberrant overexpression of the normally silenced or near-silenced DUX4 gene and the genes it regulates may ultimately lead to FSHD. Consistent with data showing that normal 4q35 D4Z4 repeat sequences exhibit heterochromatin characteristics, while FSHD-associated D4Z4 repeat sequences contain markers of euchromatin that are more indicative of active transcription. These transcriptionally permissive epigenetic changes, combined with the observation that complete monosomy of D4Z4 (i.e., zero repeat sequences) does not cause FSHD, support the hypothesis that D4Z4 repeat sequences possess potential myopathyal open reading frames (ORFs) that are aberrantly expressed in FSHD muscles. This idea was first proposed in 1994 when an ORF located at D4Z4 was first identified and named DUX4. However, this locus possessed some characteristics of unexpressed pseudogenes, thus DUX4 was hastily dismissed as a candidate gene for FSHD. For many years afterward, research into FSHD-related genes primarily focused on sequences other than the D4Z4 repeat. Although some interesting candidate genes emerged in these studies, none were clearly associated with the development of FSHD. This slow progress led to DUX4 re-emerging as a candidate gene for FSHD in 2007.

[0174] The role of DUX4 in the pathogenesis of FSHD can be explained as follows. First, the D4Z4 repeat sequence contains the same DUX4 coding region, and the D4Z4 repeat sequence also has small sense and antisense transcripts, including some similar microRNAs. Overexpressed DUX4 transcripts and a full-length DUX4 protein of approximately 50 kDa are present in biopsies and cell lines from FSHD patients. These data are consistent with the transcriptional derepression model of FSHD pathogenesis. Furthermore, unlike pseudogenes, the D4Z4 repeat sequence and DUX4 may have functional importance because the tandemly arranged D4Z4 repeat sequence is conserved in at least 11 different placental mammal species (non-placental animals lack the D4Z4 repeat sequence), with the highest sequence conservation occurring within the DUX4 ORF. Second, overexpressed DUX4 is toxic to tissue culture cells and embryonic progenitor cells of lower-developing organisms in vivo. This toxicity occurs at least in part through a pro-apoptotic mechanism, indicated by caspase-3 activation in DUX4-transfected cells and the presence of TUNEL-positive nuclei in Xenopus embryos that experienced developmental arrest after injection of DUX4 mRNA at the two-cell stage. These results are consistent with studies showing the presence of several pro-apoptotic proteins, including caspase-3, in the muscle of FSHD patients. In addition to stimulating apoptosis, DUX4 may negatively regulate myogenesis. Human DUX4 potentially inhibits the differentiation of mouse C2C12 myoblasts in vitro by interfering with PAX3 and / or PAX7, and leads to developmental arrest and reduced staining of several muscle markers upon delivery to progenitor cells of zebrafish or Xenopus embryos. Finally, aberrant DUX4 function is directly associated with potentially important molecular changes observed in the muscle of FSHD patients. In particular, full-length human DUX4 encodes a 50 kDa dual-homogeneous-domain transcription factor, and elevated levels of DUX4 targets are found in the muscle of FSHD patients. These data support the idea that DUX4 catalyzes many downstream molecular changes that are incompatible with maintaining normal muscle integrity.

[0175] Exemplary guides and caps

[0176] This invention provides a novel nucleic acid molecule and method that induces alterations in DUX4 expression, thereby enabling treatment of FSHD. The nucleic acid molecule of this invention comprises a miR scaffold and a miR guidance sequence that targets the dual homeobox 4 (DUX4) transcript. By targeting the DUX4 transcript, the nucleic acid molecule of this invention achieves an appropriate expression profile for the DUX4 gene.

[0177] Preferred exemplary guides are described in Table 7 below.

[0178] In an exemplary aspect of the invention, the guiding sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6860-6865. For example, SEQ ID NO: 6860, 6861, 6862, 6863, 6864, or 6865.

[0179] The nucleic acid molecule may contain miRNAs having at least 95% sequence identity with sequences selected from the group consisting of SEQ ID NO: 2770-3597 or 6670-6812, each of which incorporates a guide sequence for targeting DUX4. The nucleic acid molecule may target the DUX4 transcript (with exemplary guide sequences described in Table 7) at the locations shown in Table 6.

[0180] In a preferred aspect of the invention, the miRNA guide sequence comprises a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769, SEQ ID NO: 6855-6963, SEQ ID NO: 6855-6955, SEQ ID NO: 6855-6945, SEQ ID NO: 6855-6935, SEQ ID NO: 6855-6925, SEQ ID NO: 6855-6915, SEQ ID NO: 6855-6905, SEQ ID NO: 6855-6895, SEQ ID NO: 6855-6885, SEQ ID NO: 6855-6875, SEQ ID NO: 6855-6870, SEQ ID NO: 6855-6865, or SEQ ID NO: 6860-6865. For example, SEQ ID NO: 6861, 6862, 6863, 6864 or 6865, for example, SEQ ID NO: 6860, 6861, 6862, 6863, 6864 or 6865.

[0181] In a preferred aspect of the invention, the guide sequence may be capsidated in an AAV vector comprising an engineered AAV vector capsid. The vector capsid may be engineered from an AAV9 capsid protein. The AAV vector capsid may comprise the amino acid sequences described in Tables 1-5 below, wherein Tables 5a / b describe preferred engineered capsid sequences.

[0182] In an exemplary aspect of the invention, the capsid may comprise a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838 or 5036.

[0183] In aspects of the present invention, the capsid protein may comprise sequences selected from sequences 4470-4490, 4500-4520, 4540-4560, 4660-4680, 4830-4850, and 5630-5650.

[0184] The capsid may contain sequences selected from sequences 4475-4480, 4480-4490, 4500-4510, 4540-4550, 4660-4670, 4830-4840, and 5030-5040.

[0185] The capsid may contain sequences selected from sequences 4475-4480, 4485-4490, 4500-4505, 4540-4545, 4665-4670, 4830-4835, 5035-5040, such as sequences SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838, or 5036.

[0186] In summary, exemplary guide miRNAs (e.g., as part of a construct) can be encapsulated using the exemplary capsids described herein. For example:

[0187] In an exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6860, 6861, 6862, 6863, 6864, or 6865. The guide sequence may be capped with an AAV shell comprising a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838, or 5036.

[0188] For example, an exemplary miRNA guide sequence can be encapsulated using an exemplary engineered capsid as shown below:

[0189]

[0190] Therefore, in a preferred aspect of the invention, the miRNA guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769, and the AAV vector capsid of the capsidated miRNA guide may comprise a sequence selected from Tables 1-5.

[0191] For example, in embodiments of the present invention, the miRNA guide sequence may include a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6963, and the capsid protein of the capsidated miRNA guide may include a sequence selected from sequences 4470-4490, 4500-4510, 4540-4550, 4660-4670, 4830-4840, and 5630-5640.

[0192] In a further exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6955, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4470-4490, 4500-4510, 4540-4550, 4660-4670, 4830-4840, and 5630-5640. The guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6945, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6935, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040.

[0193] In a further exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6925, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6915, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6895, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040. The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6885, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040.

[0194] In a further exemplary aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6875, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from sequences 4475-4490, 4500-4505, 4540-4505, 4665-4670, 4830-4840, and 5035-5040.

[0195] The guide sequence may contain a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6855-6865, and the capsid protein of the capsidated miRNA guide may contain a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838 or 5036.

[0196] In a preferred aspect of the invention, the guide sequence may comprise a sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 6860-6865, such as SEQ ID NO: 6861, 6862, 6863, 6864 or 6865, and the capsid protein of the capsidated miRNA guide may comprise a sequence selected from SEQ ID NO: 4479, 4486, 4487, 4503, 4544, 4667, 4832, 4838 or 5036. Experimental Examples

[0197] Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant inherited disorder, affecting an estimated 1 in 8,000 people. FSHD patients typically exhibit progressive atrophy of the muscles in the face, shoulder, upper arm, leg, and abdomen; approximately 20% of patients will eventually require a wheelchair, and most experience debilitating pain and fatigue. De-repression of the transcription factor DUX4 has been identified as a pathogenesis of FSHD: insufficient epigenetic silencing of the DUX4 gene in the muscles of affected individuals leads to aberrant DUX4 expression, which is toxic to mature muscle fibers.

[0198] We analyzed the knockdown of DUX4 transcripts when gene therapy is used as a treatment strategy for FSHD.

[0199] Figure 1 A schematic diagram of DUX4 mRNA knockdown is shown.

[0200] High-throughput flat-panel screening of artificial miRNAs targeting DUX4 identified the most potent miRNAs targeting the transcript. Rigorous in vitro and in vivo screening was used to identify the most potent and selective miRNA knockdown sequences targeting DUX4, thereby enabling the identification of lead and backup miRNA knockdown sequences. More than 14,000 candidate miRNA sequences were screened in a cell-based DUX4 knockdown assay. Candidate sequences were computer-selected to exclude sequences with perfect 21-mer matches to off-target RNAs. The top 13 lead sequences were identified based on potency (DUX4 kD) and selectivity (RNAseq). The top 5 lead sequences were tested in ACTA1-MCC; in vivo potency was confirmed in FLExDUX4 mice. The lead miRNA candidates of this invention are being advanced to NHP biodistribution / safety studies.

[0201] In vitro screening identified the most effective artificial miRNA sequence targeting DUX4.

[0202] HEK293 cell lines that stably express the mCherry marker DUX4 (Hox1 mutant) were generated.

[0203] Figure 2This is a schematic diagram of a lentiviral construct used for screening miRNAs. A stable cell line was transduced with low MOI. The miRNA sequences were then subjected to fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS).

[0204] Lentiviral / FACS-based screening was conducted to identify the most effective artificial miRNA sequences targeting DUX4.

[0205] Figure 3 This is an FACS sorting diagram of positive and negative populations using miRNA sequences.

[0206] Several effective sequences were selected for individual testing in myotubes derived from FSHD patients.

[0207] Figure 4 This is a graph showing the top-ranked miRNA sequences based on enrichment.

[0208] Primary human myotube expression

[0209] When tested alone in myotubes of FSHD1 patients, the top-ranked miRNAs from the screening hit DUX4 knockdown.

[0210] Figures 5A-5B The expression profile of DUX4 and its downstream gene hZSCAN4 in myotubes of FSHD patients transduced with a miRNA candidate is shown. The selected FSHD lead candidate effectively knocked down DUX4 and reduced the expression of its downstream genes, regardless of transduction time.

[0211] Figures 6A-6D The graph shows the expression of DUX4 and its target genes in myotubes of patients after transduction with the lead candidate. When transducing cells during induced differentiation, the FSHD lead candidate knocked down DUX4 in myotubes of FSHD patients in a dose-dependent manner, as shown in the FSHD complex gene expression calculated by averaging the normalized expression of several downstream DUX4 genes (CCNA1, MBD3L2, ZSCAN4, TRIM43, KHDC1L). When transducing myotubes after differentiation, the FSHD lead candidate knocked down DUX4 in myotubes of FSHD patients in a dose-dependent manner.

[0212] The FSHD lead candidate effectively knocked down DUX4 and reduced the expression of downstream genes of DUX in cells of FSHD patients.

[0213] RNAseq and small RNAseq of control cells were used as measures of relative selectivity based on off-target expression changes in human myotubes to assess differential gene and endogenous miRNA expression between myotubes transduced with the lead candidate and scrambled controls.

[0214] Figures 7A-7B A volcano plot showing RNAseq results for differentially expressed genes and differentially expressed endogenous miRNAs in human myotubes transduced with FSHD-guided miRNA candidates relative to scrambled controls.

[0215] The lead miRNA was expressed at therapeutic levels. Based on RNA-seq and small RNA-seq, the lead candidate did not cause off-target effects, and no endogenous mRNA or miRNA met the criteria of adjusted p < 0.001, 33% decrease, or 50% increase.

[0216] Candidate Capsule

[0217] The muscle-targeted, liver-detargeted capsid of the capsidated FSHD lead candidate described herein was engineered for transduction of all muscle fibers after systemic administration in the NHP. A new class of muscle-targeted and liver-detargeted MyoAAV-LD capsid variants evolved, with the top-ranked MyoAAV-LD from directed evolution based on a naturally occurring and engineered AAV capsid in the NHP. MyoAAV-LD 6.1 was identified as the lead capsid.

[0218] The transgenic mRNA expression of MyoAAV-LD-6.1 was compared with different naturally occurring and engineered capsids in non-human primate (NHP) skeletal muscle. Barcoded hFXN was packaged in different capsids and injected as a whole into NHP. The abundance of transgenic mRNA was measured by NGS. The results showed that transgenic expression in MyoAAV-LD-6.1 was approximately 60-fold higher than that in AAVrh74.

[0219] Figures 8A-8B This is a map showing the expression of skeletal muscle mRNA and liver capsid DNA after administration of MyoAAV to non-human primates (NHP). It indicates that the vector genome level of MyoAAV-LD-6.1 in the liver is approximately one-tenth that of AAVrh74.

[0220] Figure 9 Immunofluorescence imaging of biceps muscle tissue after administration of MyoAAV to cynomolgus monkeys that received a systemic injection of MyoAAV-LD-6.1-KEP1.1-microDystrophin-FLAG at a dose of 4E+13 vg / kg is shown.

[0221] When administered systemically in NHP, MyoAAV-LD-6.1 transduces all muscle fibers.

[0222] mouse expression

[0223] The lead candidate was tested in ACTA1-MCM;FLExDUX4 mice. Double transgenic ACTA1-MCM;FLExDUX4 mice expressed DUX4 and DUX4 downstream genes after tamoxifen induction.

[0224] Mice approximately 3 months old

[0225] Figure 10 The study design for mice after injection of candidate miRNAs is shown.

[0226] ACTA1-MCM;FLExDUX4 mice (approximately 3 months old) were injected with MyoAAV containing different top-ranked DUX4 miRNAs. Three weeks later, the mice were injected with tamoxifen to induce DUX4 expression. The expression of DUX4 and DUX4 target genes was quantified 10 days after tamoxifen induction.

[0227] Figure 11 The diagram shows tamoxifen-induced DUX4 expression and FSHD complex gene expression after administration of the mediator and lead candidate. DUX4 downstream gene expression was reduced in induced mice treated with the lead candidate. FSHD complex gene expression is the mean of DUX4 downstream genes SLN, KIF4, WFDC3, and ENTPD3.

[0228] Figure 12A-12B The expression map of DUX4 and mKif4 in the triceps muscle of mice after administration of candidate miRNA sequences is shown.

[0229] Figure 13 Images show tamoxifen-induced DUX4 expression and muscle degeneration in mice after administration of the mediator and lead candidate. Arrows indicate areas of active degeneration. Muscle histology was improved in induced mice treated with the lead candidate compared to induced animals injected with the mediator.

[0230] Figures 14A-14B The figure shows tamoxifen-induced DUX4 expression and treadmill assessment results in mice after administration of the mediator and lead candidate. ACTA1-MCM;FLExDUX4 mice treated with the lead candidate ran longer on the treadmill compared to animals induced by the mediator.

[0231] aged mice

[0232] Figure 15 This study design demonstrates the administration of candidate miRNAs to aged mice. ACTA1-MCM;FLExDUX4 mice (approximately 14 months old) were injected with MyoAAV containing different top-ranked DUX4 miRNAs.

[0233] Figure 16The diagram shows tamoxifen-induced DUX4 expression and FSHD complex gene expression after administration of the mediator and lead candidate. DUX4 downstream gene expression was reduced in aged ACTA1-MCM;FLExDUX4 mice treated with the lead candidate.

[0234] Figure 17 Images of muscle degeneration in mice after administration of the vector and the lead candidate are shown. Arrows indicate areas of active degeneration. Muscle histology was improved in aged ACTA1-MCM;FLExDUX4 mice treated with the lead candidate compared to animals injected with the vector.

[0235] The FSHD lead candidate resulted in decreased expression of downstream DUX4 genes and improved muscle histology in aged ACTA1-MCM; FLExDUX4 mice.

[0236] in conclusion

[0237] MyoAAV engineered AAV significantly outperforms competing capsids in transducing NHP into muscle and detargets from the liver after systemic administration.

[0238] The lead candidate knocked down DUX4 and its downstream genes in the myotubes of FSHD patients, regardless of transduction time.

[0239] The lead candidate knocked down downstream genes of DUX4 in a severe FSHD mouse model and rescued functional phenotype and histopathology.

[0240] The FSHD lead candidate does not produce off-target effects on the expression of endogenous mRNA and miRNA in human myotubes. By incorporating references

[0241] This disclosure references and cites other literature, such as patents, patent applications, patent publications, journals, books, papers, and online content. For all purposes, all such literature is incorporated herein by reference in its entirety. equivalent

[0242] Based on the entire contents of this document (including references to scientific and patent literature cited herein), various modifications to the invention and its many further embodiments will become apparent to those skilled in the art, in addition to those shown and described herein. The subject matter contains important information, examples, and guidance suitable for practicing the invention in its various embodiments and equivalents.

Claims

1. A nucleic acid molecule comprising a miR scaffold and a miR guidance sequence targeting dual homology box 4 (DUX4) transcripts.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a miRNA guide sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769 or 6687-6770.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises a miRNA guide sequence selected from SEQ ID NO: 1250-2769 or 6687-6770.

4. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a miRNA having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2770-3597 or 6671-6812.

5. The nucleic acid molecule of claim 4, wherein the miRNA targets the dual homology box 4 (DUX4) transcript at the location shown in Table 6.

6. The nucleic acid molecule of claim 1, wherein the miRNA scaffold is derived from pri-miRNAs selected from the group consisting of pri-miR-21, pri-miR-22, pri-miR-26a, pri-miR-30a, pri-miR-33, pri-miR-122, pri-miR-375, pri-miR-199, pri-miR-99, pri-miR-194, pri-miR-155, and pri-miR-451.

7. The nucleic acid molecule of claim 6, wherein the miRNA scaffold is derived from pri-miR-33.

8. The nucleic acid of claim 1, wherein the miRNA contains 5-6 thymidines at its 5' end.

9. The nucleic acid of claim 1, wherein the nucleic acid further comprises a promoter sequence.

10. The nucleic acid of claim 9, wherein the promoter sequence is a U6 promoter sequence, MHCK7 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.

11. An adeno-associated virus (AAV) vector, said adeno-associated virus vector comprising: Starter sequence, Nucleic acid molecules, the nucleic acid molecules comprising a miR scaffold and a miR-guided sequence targeting dual homeobox 4 (DUX4) transcripts; and Capsid protein.

12. The AAV vector of claim 11, wherein the nucleic acid molecule comprises a miRNA guide sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 2010-2769.

13. The AAV vector of claim 12, wherein the nucleic acid molecule comprises a miRNA guide sequence selected from SEQ ID NO: 1250-2769 or 6687-6770.

14. The AAV vector of claim 13, wherein the nucleic acid molecule comprises a miRNA having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2770-3597 or 6670-6812.

15. The AAV vector of claim 14, wherein the nucleic acid molecule comprises a miRNA guide sequence selected from SEQ ID NO: 2770-3597 or 6670-6812.

16. The AAV vector of claim 11, wherein the miRNA targets the dual homology box 4 (DUX4) transcript at the location shown in Table 6.

17. The AAV vector of claim 11, wherein the miRNA scaffold is derived from pri-miRNAs selected from the group consisting of pri-miR-21, pri-miR-22, pri-miR-26a, pri-miR-30a, pri-miR-33, pri-miR-122, pri-miR-375, pri-miR-199, pri-miR-99, pri-miR-194, pri-miR-155, and pri-miR-451.

18. The AAV vector of claim 17, wherein the miRNA scaffold is derived from pri-miR-33.

19. The AAV vector of claim 11, wherein the miRNA contains 5-6 thymidines at its 5' end.

20. The AAV vector of claim 1, wherein the AAV vector further comprises a promoter sequence.

21. The AAV vector of claim 20, wherein the promoter sequence is the U6 promoter sequence, the MHCK7 promoter sequence, the CK6 promoter sequence, the tMCK promoter sequence, or the CK5 promoter.

22. The AAV vector of claim 11, wherein the capsid protein comprises at least one modification that reduces the hepatic orientation of the AAV vector and / or the AAV vector preferentially targets muscle tissue.

23. The AAV vector of claim 11, wherein the vector further comprises a nuclear output sequence capable of achieving intranuclear diffusion.

24. The AAV vector of claim 11, wherein the capsid protein comprises at least one modification, said modification being an insertion between any two consecutive amino acids at similar positions in the AAV9 capsid polypeptide, namely amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714 or any combination thereof, or in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptides.

25. The AAV vector of claim 24, wherein the capsid protein may be selected from the sequences in Tables 1-5.

26. A method for inhibiting the expression of a gene or gene product in a cell, the method comprising administering to a subject a composition that causes a nucleic acid molecule comprising a pri-miR scaffold and a miR directing sequence targeting a dual homeobox 4 (DUX4) transcript to be expressed in the cell.

27. The method of claim 26, wherein the nucleic acid molecule comprises a miRNA guide sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1250-2769.

28. The method of claim 27, wherein the nucleic acid molecule comprises a miRNA guide sequence selected from SEQ ID NO: 2010-2769 or 2770-3597.

29. The method of claim 28, wherein the nucleic acid molecule comprises a miRNA having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2770-3529 or 6670-6812.

30. The method of claim 29, wherein the miRNA targets the dual homology box 4 (DUX4) transcript at the location shown in Table 6.

31. The method of claim 1, wherein the miRNA scaffold is derived from pri-miRNAs selected from the group consisting of pri-miR-21, pri-miR-22, pri-miR-26a, pri-miR-30a, pri-miR-33, pri-miR-122, pri-miR-375, pri-miR-199, pri-miR-99, pri-miR-194, pri-miR-155, and pri-miR-451.

32. The method of claim 31, wherein the miRNA scaffold is derived from pri-miR-33.

33. The method of claim 1, wherein the miRNA contains 5-6 thymidines at its 5' end.

34. The method of claim 1, further comprising a promoter sequence.

35. The method of claim 34, wherein the promoter sequence is a U6 promoter sequence, MHCK7 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.

36. The method of claim 26, wherein the composition comprises an adeno-associated virus (AAV) vector, the AAV vector comprising a promoter sequence, the nucleic acid molecule comprising a pri-miR scaffold and a miR guiding sequence, and a capsid protein.

37. The method of claim 26, wherein the capsid protein comprises at least one modification that reduces the hepatic orientation of the AAV vector and / or the AAV vector preferentially targets muscle tissue.

38. The method of claim 36, wherein the AAV vector comprises at least one modification, said modification being an insertion between any two consecutive amino acids at similar positions in the AAV9 capsid polypeptide, namely amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714 or any combination thereof, or in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptides.

39. The method of claim 38, wherein the capsid protein may be selected from the sequences in Tables 1-5.

40. The method of claim 36, wherein the carrier further comprises a nuclear output sequence capable of achieving intranuclear diffusion.

41. The method of claim 26, wherein the inhibition of the gene or gene product results in treatment of muscular dystrophy.

42. The method of claim 41, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy 1.

43. The method of claim 42, wherein the treatment may include inhibiting the muscle effects of muscular dystrophy.

44. The method of claim 43, wherein the treatment may include reversing the muscle effects of the muscular dystrophy.

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