Adeno-associated virus (AAV) vector for delivery to muscle cells

Abstract

The present disclosure provides anti-DUX4 shRNA and related AAV vectors for delivering the shRNA to a patient having FSHD (facioscapulohumeral muscular dystrophy). The AAV can further include a transgene encoding MATR3 that synergistically improves the therapeutic effect of the shRNA.

Description

Attorney Docket Ref.: 66CM-405598-WOADENO-ASSOCIATED VIRUS (AAV) VECTOR FOR DELIVERY TO MUSCLE CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the United States Provisional Application Serial No. 63 / 777,313, filed March 25, 2025, the content of which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (405598. xml; Size: 49,287 bytes; and Date of Creation: March 25, 2026) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Diseases associated with the skeletal muscle, in particular degenerative muscle wasting diseases or conditions, are quite challenging to treat. Examples of degenerative muscle wasting diseases and conditions include muscular dystrophy, myopathy, mitochondrial diseases, soft tissue sarcoma, ion channel diseases, cachexia and sarcopenia.

[0004] Muscular dystrophies (MD) are a genetically and clinically heterogeneous group of rare neuromuscular diseases that cause progressive weakness and breakdown of skeletal muscles over time. Over 30 different disorders are classified as muscular dystrophies. Of those, Duchenne muscular dystrophy (DMD) accounts for approximately 50% of cases and affects males beginning around the age of four. Other relatively common muscular dystrophies include Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy, whereas limb-girdle muscular dystrophy and congenital muscular dystrophy are themselves groups of several genetic disorders.

[0005] Muscular dystrophies are caused by mutations in genes, usually those involved in making muscle proteins. The muscle protein, dystrophin, is in most muscle cells and works to strengthen the muscle fibers and protect them from injury as muscles contract and relax. It links the muscle membrane to the thin muscular filaments within the cell. Dystrophin is an integral part of the muscular structure, and an absence of dystrophin can cause impairments such as: healthy muscle tissue can be replaced by fibrous tissue and fat, causing inability toAttorney Docket Ref.: 66CM-405598-WOgenerate force. These mutations are either inherited from parents or may occur spontaneously during early development.

[0006] FSHD is a form of hereditary muscular dystrophy that mainly affects muscles of the face, scapular stabilizers, upper arm, lower leg and hip girdle. FSHD typically presents with weakness of the facial muscles, the stabilizers of the scapula, or the dorsiflexors of the foot. Weakness is slowly progressive and approximately 20% of affected individuals eventually require the aid of a wheelchair.

[0007] There are two classes of FSHD: FSHD1 and FSHD2 which are clinically indistinguishable and share the same pathogenic mechanisms but have different genetics. Both forms are caused by aberrant expression of the transcription factor DUX4. Typically, individuals with FSHD become symptomatic in their teens, but age of onset is variable. More than 50% of individuals with FSHD demonstrate findings by age 20 years.

[0008] With a prevalence 1-9 / 100,000 population, FSHD is a rare disease. There are no disease modifying therapies available for FSHD with current standard of care treatment being solely supportive.

[0009] Gene therapy and antisense drugs need to be delivered to target muscle cells with vectors, such as adeno-associated viral (AAV) vectors. AAV is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV, such as AAV1 to AAV 11 and AAVrh.74. Crv-acling sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, pl 9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pl 9), coupled with the differential splicing of the single AAV intron, result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome.Attorney Docket Ref.: 66CM-405598-WO

[0010] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).

[0011] The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another useful feature of AAV is that it is an quite stable and hearty virus.SUMMARY

[0012] The present disclosure provides anti-DUX4 shRNA and related AAV vectors for delivering the shRNA to a patient having FSHD (facioscapulohumeral muscular dystrophy). The AAV can further include a transgcnc encoding MATR3 that synergistically improves the therapeutic effect of the shRNA.

[0013] One embodiment of the present disclosure provides a plasmid, comprising (a) a first fragment encoding a shRNA targeted at a DUX4 (Double homeobox, 4) mRNA; (b) a U6 promoter operatively linked to the first fragment; (c) a second fragment encoding a MATR3 (Matrin-3) protein; (d) a eukaryotic promoter operatively linked to the second fragment; and (e) a pair of AAV ITR’s flanking the (a), (b), (c) and (d).

[0014] In some embodiments, the plasmid comprises two of (a) and two of (b) each operatively linked to the first fragment of each of the (a).

[0015] In some embodiments, the plasmid comprises three of (a) and three of (b) each operatively linked to the first fragment of each of the (a).Attorney Docket Ref.: 66CM-405598-WO

[0016] In some embodiments, the shRNA comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the shRNA comprises the nucleic acid sequence of SEQ ID NO:7.

[0017] In some embodiments, the eukaryotic promoter is a SkCRM4 promoter. In some embodiments, the eukaryotic promoter further comprises a mini-desmin promoter. In some embodiments, the eukaryotic promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:49.

[0018] In some embodiments, the plasmid further encodes an AAV Rep gene and Cap gene.

[0019] In some embodiments, the plasmid further encodes an AAV capsid protein.

[0020] In some embodiments, the AAV capsid protein is an AAV9 VP1 capsid protein. In some embodiments, the AAV9 VP1 capsid protein comprises a muscle-targeting peptide inserted in the capsid protein. In some embodiments, the insertion is between residues Q588 and A589 of the AAV9 VP1 capsid protein. In some embodiments, the muscle-targeting peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-46. In some embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:47.

[0021] Also provided, in one embodiment, is an isolated shRNA, comprising the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the shRNA comprises the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the shRNA is 80 nucleotides in length or shorter, or 60 nucleotides in length or shorter.

[0022] Also provided, in one embodiment, is a polynucleotide comprising the shRNA and a U6 promoter operatively linked to the shRNA. In some embodiments, the polynucleotide comprises three of the U6 promoters each operatively linked to one of three of the shRNA.

[0023] Also provided, in some embodiments, is a method for treating a FSHD (Facioscapulohumeral Muscular Dystrophy) in a patient in need thereof, comprising administering to the patient a plasmid, shRNA, or polynucleotide of the present disclosure.Attorney Docket Ref.: 66CM-405598-WOBRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows that shRNA molecules reduced the expression of DUX4 and its downstream genes.

[0025] FIG. 2 shows that the triple promoter design (3xU6 and 3xshRNA) resulted in greatly higher shRNA expression and DUX4 inhibition.

[0026] FIG. 3 shows that FZB-400 administration led to significantly higher expression of shRNA301 in gastrocnemius (GAS) and TA (Tibialis Anterior) than a conventional capsid.

[0027] FIG. 4 shows that the single-plasmid design of FZB-400 led to longer lasting and higher in vivo shRNA expression.

[0028] FIG. 5 shows that FZB-400 administration resulted in significantly improved force production and locomotor activity (distance and run time) in tamoxifen-induced FLExDUX4 mice.

[0029] FIG. 6 shows that FZB-400 administration downregulated DUX4 and its downstream genes.

[0030] FIG. 7 shows that FZB-400 administration inhibited mir206 expression, a biomarker for FSHD.

[0031] FIG. 8 presents the results of treadmill assessment on day 22 and 41 in tamoxifen-induced ACTAl-MCM;FLExDUX4 mice after IV injection with AAVMYO-ShRNA301 (FZB-400), AAVMYO-control (FZB-400 control) and MyoAAV2A-ShRNA301 (MyoAAV) compared with untreated ACTA1-MCM / + control mice (WT). Treadmill running to exhaustion test: total running distance (left panels) total running time (right panels). Note that, due to poor health status, animals treated with AAVMYO-control were terminated on day 23, after the first functional assessment. Stat: One-way ANOVA, Holm-Sidak’s test. Mean ± SEM. AAVMYO-ShRNA3()l (n=4), AAVMYO-control (n=6), MyoAAV2A-ShRNA301 (n=4 (day 22), n=3 (day 41)), untreated ACTA1-MCM / + (n=7). *p<0.05, **p<0.01. ns: not significant.Attorney Docket Ref.: 66CM-405598-WO

[0032] FIG. 9 shows changes of animal body weights during the study. Tamoxifen was administered on day 13 (vertical dashed line). Control animals (FZB-400 control) were terminated on day 23. Myo A AV-treated mice were humanely terminated around day 41-48.

[0033] FIG. 10, with panels A-H, shows DUX4-downstream gene expression in the Tibialis Anterior (TA) and Gastrocnemius muscles of tamoxifen-induced ACTA1-MCM;FLExDUX4 mice after IV injection with AAVMYO-ShRNA3()l (FZB-400), AAVMYO-control (FZB-400 control) and MyoAAV2A-ShRNA301 (MyoAAV) compared with untreated ACTA1-MCM / + control mice (WT). (A-D) Full-length DUX4, individual DUX4-downstream genes Wfdc3 (B), Trim36 (C) and % composite expression of Wfdc3 and Trim36 (D) in TA muscles of treated ACTAl-MCM;FLExDUX4 and ACTA1-MCM / + healthy controls (WT). (F-H) Full-length DUX4, individual DUX4-downstream genes Wfdc3 (F), Trim36 (G) and % composite expression of Wfdc3 and Trim36 (H) in Gastrocnemius muscles of treated ACTAl-MCM;FLExDUX4 and ACTA1-MCM / + healthy controls (WT). Stat: One-way ANOVA, Holm-Sidak’s test. Mean ± SEM. FZB-400 control (n=6), FZB-400 (n=3), MyoAAV (n=4), WT (ACTA1-MCM / +; n=7). * p<0.05; ** p<0.01.

[0034] FIG. 11, with panels A-D, shows the ShRNA301 and fibrosis gene expression in the TA and GAS of tamoxifen-induced ACTAl-MCM;FLExDUX4 mice after IV injection with AAVMYO-ShRNA301 (FZB-400), AAVMYO-control (FZB-400 control) and MyoAAV2A-ShRNA301 (MyoAAV) compared with untreated ACTA1-MCM / + control mice (WT). (A,C) ShRNA301 in TA (A) and GAS (C) muscles of treated ACTAl-MCM;FLExDUX4 and ACTA1-MCM / + healthy controls. (B,D) Collal in TA (B) and GAS (D) muscles of treated ACTAl-MCM;FLExDUX4 and ACTA1-MCM / + healthy controls. Stat: One-way ANOVA, Holm-Sidak’s test. Mean ± SEM. FZB-400 control(n=6), FZB-400 (n=3), MyoAAV (n=4), WT (ACTA1-MCM / +; n=7). **** p<0.0001.

[0035] FIG. 12 shows the results of treadmill assessment on day 28 in tamoxifen-induced ACTAl-MCM;FLExDUX4 mice after IV injection with AAVMYO-ShRNA301 (FZB-400) administered at three different doses (high, medium and low) or AAVMYO-control (FZB-400 control). Treadmill running to exhaustion test: total running distance (left panel) total running time (right panel). Stat: One-way ANOVA, Holm-Sidak’s test. Mean ± SEM. N=7 for all groups. ***p<0.001, ****p<0.0001.Attorney Docket Ref.: 66CM-405598-WO

[0036] FIG. 13 shows changes of animal body weights during the study. Tamoxifen was administered on day 14 (vertical dashed line).

[0037] FIG. 14, with panels A-H, shows DUX4 and DUX4-downstream gene expression in the TA and GAS in muscles of tamoxifen-induced ACTA1-MCM; FLExDUX4 mice treated with different doses of FZB-400 (AAVMYO-ShRNA301) vs FZB-400 control (AAVMYO-control) at 31 days post treatment. (A-D) Full-length DUX4 (A), individual DUX4-downstream genes Wfdc3 (B), Trim36 (C) and % composite expression of Wfdc3 and Trim36 (D) in TA muscles of treated ACTAl-MCM;FLExDUX4. (E-H) ) Full-length DUX4 (E), individual DUX4-downstream genes Wfdc3 (F), Trim36 (G) and % composite expression of Wfdc3 and Trim36 (H) in GAS muscles of treated ACTAl-MCM;FLExDUX4. Stat: Oneway ANOVA, Holm-Sidak’s test. Mean ± SEM. High dose FZB-400 HD (n=4-6), medium dose FZB-400 MD (n=5-6), low dose FZB-400 LD (n=3-4) and FZB-400 control (n=6) per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0038] FIG. 15, with panels A-D, shows ShRNA301 and fibrosis gene expression in the TA and GAS of tamoxifen-induced ACTAl-MCM;FLExDUX4 mice after IV injection with three doses of AAVMYO-ShRNA301 (FZB-400) or AAVMYO-control (FZB-400 control).(A,C) ShRNA301 in TA (A) and GAS (C) muscles of mice treated with high dose (HD), medium dose (MD) or low dose (LD) FZB-400, compared to FZB-400 controls. (B,D) Coll al in TA (B) and GAS (D) muscles of mice treated with high dose (HD), medium dose (MD) or low dose (LD) FZB-400, compared to FZB-400 controls. Stat: One-way ANOVA, Holm-Sidak's test. Mean ± SEM. FZB-400 control (n=6), FZB-400 HD (n=5-6), FZB-400 MD (n=5-6) and FZB-400 LD (n=3-4). ***p<0.001, **** pcO.OOOLDETAILED DESCRIPTIONDefinitions

[0039] The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.Attorney Docket Ref.: 66CM-405598-WODefinitions

[0040] As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0041] As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a single cell as well as a plurality of cells, including mixtures thereof.

[0042] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. The term “about” also includes the exact value “X” in addition to minor increments of “X” such as “X + 0.1” or “X - 0.1.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0043] The term “muscle cell” as used herein refers to any cell which contributes to muscle tissue. Myoblasts, satellite cells, myotubes, and myofibril tissues are all included in the term “muscle cells” and may all be treated using the methods of the invention. Muscle cell effects may be induced within skeletal, cardiac and smooth muscles. Muscle tissue in adult vertebrates will regenerate from reserve myoblasts called “satellite cells”. Satellite cells are distributed throughout muscle tissue and are mitotically quiescent in the absence of injury or disease. Following muscle injury or during recovery from disease, satellite cells will reenter the cell cycle, proliferate and 1) enter existing muscle fibers or 2) undergo differentiation into multinucleate myotubes which form new muscle fiber. The myoblasts ultimately yield replacement muscle fibers or fuse into existing muscle fibers, thereby increasing fiber girth by the synthesis of contractile apparatus components. This process is illustrated, for example, by the nearly complete regeneration which occurs in mammals following induced muscle fiber degeneration: the muscle progenitor cells proliferate and fuse together regenerating muscle fibers.

[0044] “Myogenic” cells as described herein are those cells that are related to the origin of muscle cells or fibers. Various molecular markers are known to be specific for the middle andAttorney Docket Ref.: 66CM-405598-WOlate stages of myogenic differentiation. For example, in C2C12 cells, myosin and Desmins mark the late stages of myogenesis and are largely restricted to myotubes, whereas myogenin and MRF4 mark the middle stages of myogenesis and are found in all myotubes and in many committed myoblasts.

[0045] As used herein “satellite cells,” or “myosatellite cells,” refers to small multipotent cells with little cytoplasm found in mature muscle. Satellite cells are precursors to skeletal muscle cells, able to give rise to satellite cells or myoblasts, which give rise to skeletal muscle cells. They have the potential to provide additional myonuclei to their parent muscle fiber, or return to a quiescent state. Upon activation, satellite cells can re-enter the cell cycle to proliferate and differentiate into myoblasts. Satellite cells may exhibit one or more features which may be shared with endogenous satellite cells, including, but not limited to, capacity to repopulate the satellite cell niche, ability to drive muscle regeneration, exhibit appropriate expression of gene markers, appropriate expression of glycoproteins, and expandability in culture.

[0046] A “vector,” as used herein, refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.

[0047] The term “polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the nucleic acid can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the nucleic acid can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded nucleic acid can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0048] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers ofAttorney Docket Ref.: 66CM-405598-WOamino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide" refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0049] A “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one, preferably two, inverted terminal repeat sequences (ITRs).

[0050] A “recombinant AAV vector (rAAV vector)’’ refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one, preferably two, AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a “pro- vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. A rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and, in embodiments, encapsidated in a viral particle, particularly an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle)”. AAV helper functions (i.e., functions that allow AAV to be replicated and packaged by a host cell) can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genesAttorney Docket Ref.: 66CM-405598-WOwhich aid in AAV replication and packaging. Other AAV helper functions are known in the art. An AAV may be any one of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, A AV 11 and / or AAV 12.

[0051] An “rAAV virus” or “rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0052] “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a nucleic acid introduced by genetic engineering techniques into a different cell type is a heterologous nucleic acid (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0053] The term “transgene” refers to a nucleic acid that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as siRNA.

[0054] The terms “genome particles (gp),” “genome equivalents,” or “genome copies” as used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as described in the Examples herein, or for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0055] The terms “infection unit (iu),” “infectious particle,” or “replication unit,” as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J.Virol., 62:1963-1973.

[0056] The term “transducing unit (tu)” as used in reference to a viral titer, refers to the number of infectious recombinant AAV vector particles that result in the production of aAttorney Docket Ref.: 66CM-405598-WOfunctional transgene product as measured in functional assays such as described in Examples herein, or for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0057] An “inverted terminal repeat” or “ITR” sequence is a term well understood in the art and refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.

[0058] An “AAV inverted terminal repeat (ITR)” sequence, a term well-understood in the art, is an approximately 145 -nucleotide sequence that is present at both termini of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contains several shorter regions of self-complementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand base-pairing to occur within this portion of the ITR.

[0059] A “terminal resolution sequence” or “trs” is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins.

[0060] A “helper virus” for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. A number of such helper viruses have been identified, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).

[0061] An “isolated” molecule (e.g., nucleic acid or protein) or cell means it has been identified and separated and / or recovered from a component of its natural environment.

[0062] As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizedAttorney Docket Ref.: 66CM-405598-WO(e.g., not worsening) state of disease, preventing spread (e.g., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0063] The terms “subject,” “patient,” “individual,” etc. are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice.

[0064] As used herein, “treating” or “treatment” of a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently.

[0065] As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. In embodiments, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. In embodiments, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. In embodiments, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination. InAttorney Docket Ref.: 66CM-405598-WOembodiments, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.

[0066] The terms “effective amount,” “effective dose,” etc. refer to the amount of an agent that is sufficient to achieve a desired effect, as described herein. In embodiments, the term “effective” when referring to an amount of cells or a therapeutic compound may refer to a quantity of the cells or the compound that is sufficient to yield an improvement or a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of this disclosure. In embodiments, the term “effective” when referring to the generation of a desired cell population may refer to an amount of one or more compounds that is sufficient to result in or promote the production of members of the desired cell population, especially compared to culture conditions that lack the one or more compounds.AAV Vectors and Uses

[0067] The present disclosure provides anti-DUX4 shRNA and related AAV vectors for delivering the shRNA to a patient having FSHD (facioscapulohumeral muscular dystrophy). The AAV can further include a transgene encoding MATR3 that synergistically improves the therapeutic effect of the shRNA.

[0068] As shown in the experimental examples, 5 different shRNA sequences were designed and tested. Only one of them (shRNA 2, also referred to as shRNA301) stood out as a potent inhibitor of DUX4 expression. shRNA301 has a target sequence of SEQ ID NO:2, and a hairpin sequence of SEQ ID NO:7.

[0069] The anti-DUX4 shRNA has been tested with the U6 promoter (SEQ ID NO:48). Interestingly, when three copies of the U6 promoter were used along with three shRNA (the “triple promoter" design), the expression was the most pronounced.

[0070] The instant disclosure designed a single-plasmid AAV system that included all of the AAV elements for transfection and production. Unlike a three -plasmid system which includes the transgene, Rep / Cap and helper functions (e.g., helper virus genes (E2A, E4 andAttorney Docket Ref.: 66CM-405598-WOVA from Adenovirus 2 [Ad2]) separated by insulators (cHS4)) in separate plasmids, the single-plasmid AAV system includes all of them in a single plasmid.

[0071] An AAV capsid with the single -plasmid design, AAVMYO-ShRNA301, or “FZB-400”, was subjected to in vivo testing and compared to a capsid of the conventional design (MyoAAV2A-ShRNA301). While strong myogenic tropism and efficacy was confirmed for both capsids, there was one important advantage of the AAVMYO-ShRNA301 capsid: cardiotoxicity was less severe when ShRNA301 was delivered via AAVMYO vs when it was delivered by MyoAAV2A (Example 5).

[0072] A dose study was then conducted to identify a dose range for AAVMYO-ShRNA301 that could further reduce toxicity without sacrificing efficacy. Three AAVMYO-ShRNA301 (FZB-400) doses were tested in Example 6, including 2xl013vector genomes (vg) / kg, 4xl012vg / kg and 2xl012vg / kg. The lowest lose was chosen as l / 10thof the highest dose. Treatments at all three doses led to significant functional improvements. Also, the expression of all DUX4 target genes was decreased.

[0073] There were no unscheduled deaths or early terminations associated with AAVMYO-ShRNA301 treatment and minimal histopathological changes were noted in the heart tissue of low dose-treated mice. This data shows that at the lowest dose tested, there were no signs of cardiotoxicity. These experimental data, therefore, demonstrate that sufficient treatment efficacy with no observable cardiotoxicity can be achieved by the AAV capsid of the present disclosure.

[0074] In one embodiment, the plasmid includes exogenous sequences between a pair or AAV Inverted Terminal Repeats (ITRs). For instance, the plasmid includes (a) a first fragment encoding a shRNA targeted at a DUX4 (Double homeobox, 4) mRNA; (b) a U6 promoter operatively linked to the first fragment; (c) a second fragment encoding a MATR3 (Matrin-3) protein; (d) a eukaryotic promoter operatively linked to the second fragment; and (e) a pair of AAV ITR’s flanking the (a), (b), (c) and (d).

[0075] In some embodiments, the plasmid includes 2 copies of the U6 promoter and 2 copies of the shRNA, each U6 promoter operatively linked to each of the shRNA. In some embodiments, the plasmid includes 3 copies of the U6 promoter and 3 copies of the shRNA, each U6 promoter operatively linked to each of the shRNA.Attorney Docket Ref.: 66CM-405598-WO

[0076] The shRNA may include any of the shRNA tested herein (see, e.g., Table A). In some embodiments, the shRNA includes the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:6. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:7. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:3. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:8. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:9. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:5. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 10.

[0077] In a particular embodiment, the shRNA includes the nucleic acid sequence of SEQ ID NO:2 . In some embodiments, the shRNA includes the sequence of SEQ ID NO:7.

[0078] In some embodiments, the U6 promoter includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:48. In some embodiments, the U6 promoter includes the sequence of SEQ ID NO:48.

[0079] In some embodiments, the eukaryotic promoter is a SkCRM4 promoter. In some embodiments, the eukaryotic promoter further comprises a mini-desmin promoter (Skopenkova et al., Acta Naturae. 2021 Ian-Mar; 13(1 ):47-58. PMID: 33959386). In someAttorney Docket Ref.: 66CM-405598-WOembodiments, the eukaryotic promoter includes a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:49.

[0080] In some embodiments, the eukaryotic promoter is the synthetic SPc5- 12 promoter (Skopenkova et al., Acta Naturae. 2021 Jan-Mar;13(l):47-58). In some embodiments, the eukaryotic promoter further includes a SkCRM4 promoter. In some embodiments, the eukaryotic promoter is a SkCRM4 / SPc5-12 chimeric promoter.

[0081] In some embodiments, the plasmid further encodes an AAV Rep gene and Cap gene.

[0082] In some embodiments, the plasmid further encodes an AAV capsid protein. A representative AAV capsid protein is the AAV9 VP1 capsid protein (SEQ ID NO: 11). In some embodiments, the capsid protein can include a targeting peptide inserted within. The insertion may be between, e.g., residues Q588 and A589 of SEQ ID NO: 11, or residues Q585 and S586, without limitation.

[0083] The targeting peptide, in some embodiments, is a muscle cell targeting peptide. Examples of such muscle cell targeting peptide include, without limitation, SEQ ID NO: 12-46.

[0084] In some embodiments, the AAV capsid protein, with a targeting peptide includes, is an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:47.Table A. SequencesName Sequence SEQ ID NO:AAV9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGY 11 capsid KYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFprotein VP1 QERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQP IGEPPAAPSGVGS LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALP TYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYF PSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKT INGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSE FAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEE IKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQG ILPGMVWQDRDVYLQGP IWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIK NTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQ YTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNLTargeting RGDLGLS 12 peptideTargeting RGDMSRE 13peptideAttorney Docket Ref.: 66CM-405598-WOTargeting GEARISA 14 peptideTargeting ESGLSQS 15 peptideTargeting EYRDSSG 16 peptideTargeting DLGSARA 17 peptideTargeting SGNSGAA 18 peptideTargeting CDCRGDCFC 19 peptideTargeting NDVRSAN 20 peptideTargeting NDVRAVS 21 peptideTargeting GGGRGDLGLSGGG 22 peptideTargeting GGSRGDLGLSGGS 23 peptideTargeting RGDLTTP 24 peptideTargeting RGDQTTL 25 peptideTargeting GPGRGDQTTL 26 peptideTargeting AEGRGDQYTR 27 peptideTargeting ATGRGDLGQA 28 peptideTargeting AVARGDQGLI 29 peptideTargeting NISRGDQGYQ 30 peptideTargeting APARGDQGSQ 31 peptideTargeting CGGPQVTRGDVFTnP (n is norleucine) 32 peptideTargeting CGGRGDVF 33 peptideTargeting CGGNGEPRGDNYRAY 34 peptideTargeting CGGRGDNY 35 peptideTargeting CGGRGDAA 36 peptideTargeting CGGRGD 37 peptideTargeting RIPRGDMPDDR 38 peptideTargeting PEILDVPSTV 39 peptideTargeting CRIPRGDMPDDRC 40 peptideTargeting CGPEILDVPSTVC 41 peptideTargeting CRIPILDVPDDRC 42peptideAttorney Docket Ref.: 66CM-405598-WOTargeting CRIPALDVPDDRC 43 peptideTargeting CRIPIADVPDDRC 44 peptideTargeting CRIPILAVPDDRC 45 peptideTargeting CRIPAAAVPDDRC 46 peptideAAVMyo MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYCapsid KYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNP YLKYNHADAEF 47QERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQP IGEPPAAPSGVGS LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALP TYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYF PSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKT INGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSE FAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGR DNVDADKVMITNEEE IKTTNPVATESYGQVATNHQGQSGRGDLGLSAQAA QTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK HPPPQIL IKNTPVPADPPTAFNKDKLNSF ITQYSTGQVSVE IEWELQKEN SKRWNPE IQYTSNYYKSNNVEFAVNTEGVYSEPRP IGTRYLTRNLU6 gagggcctatttcccatgattcctt catatttgcatatacgat acaaggc 48tgttagagagataattggaattaatttgactgt aaacacaaagat attag tacaaaat aegtgaegt agaaagtaat aatttcttgggt agtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaa gt atttcgatttcttggcttt at at atcttgtggaaaggacgaaacaccSKCM4- tt ctgagtcct ctaaggtccctcactcccaact cagccccatgtcctgt c 49 miniDes aattcccactcagtgtctgatctccttctcctcacctttcccatctcccg tttgacccagctt cctgagct ct cctcccattcccctttttggagtcct c ctcctctcccagaacccagtaataagtgggctcctccctggcctggaccc ccgtggtaaccctat aaggcgaggcagctgctgtctgaggcagggagggg ctggt gtgggaggct aagggcagctgctaagtttagggtggct cctt ct c tettett agagacaacaggtggctggggcct cagtgcccagaaaagaaaa tgt ettagaggt at eggcat gggcctggaggaggggggacagggcagggg gaggcatcttcctcaggacat cgggtcct agaggt gt acaacgcgtt acg cct caggtaccccctgccccccacagctcct ct cctgtgccttgttt ccc agccatgcgttctcctctataaatacccgctctggtatttggggttggca gctgttgctgccagggagatggttgggttgacatgcggctcctgacaaaa cacaaacccctggtgtgtgtgggcgtgggtggtgtgagt agggggatgaa tcagggagggggcgggggacccagggggcaggagccacacaaagt ctgtg cgggggtgggagcgcacat ageaattggaaaetgaaagett at cagaccc ttt ct ggaaat cagcccactgtttataaacttgaggccccaccct cgaca gt accggggaggaagagggcctgcact agt ccagagggaaactgaggct c agggctagctcgcccat agacat acatggcaggcaggctttggccaggat ccctccgcctgccaggcgt ctccct gccctc cctt cct gcctagaga ccc ccaccct caagcctggctggt ettt gcct gaga cccaaa cct cttcgact tcaagagaatatttaggaacaaggtggttt agggccttt cctgggaacag gccttgaccctttaagaaatgacccaaagtctctccttgaccaaaaaggg gaccctcaaactaaagggaagcctctctt ctgctgtctcccctgacccca ct ccccccCaccccaggacgaggagataaccagggctgaaagaggcccgc ct gggggctgc agacat get t get gcct gee ct ggcgaaggattggcagg cttgcccgt cacaggacccccgctggctgactcaggggcgcaggcct ett gcgggggagctggcctccccgcccccacggccacgggccgccctttcctg gcaggacagcgggat cttgcagctgtcaggggaggggaggcgggggctga tgtcaggagggatacaaatagtgccgacggctgggggccctgtctcccct cgccgcatccactctccggccggccgcctgcccgccgcctcctccgtgcgcc ege cage ct egee cgcgccgtcaccAttorney Docket Ref.: 66CM-405598-WOL-ITR ggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccggg 50cgt cgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagt ggccaact ccatcact aggggttcctR-ITR aggaacccctagtgatggagttggccact ccct ct ctgcgcgctcgctcg 51ct cactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcc

[0085] Also provided, in one embodiment, is an isolated shRNA. In some embodiments, the shRNA includes the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:6. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:2. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:7. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:3. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:8. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:9. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:5. In some embodiments, the shRNA includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 10.

[0086] In a particular embodiment, the shRNA includes the nucleic acid sequence of SEQ ID NO:2 . In some embodiments, the shRNA includes the sequence of SEQ ID NO:7.

[0087] In some embodiments, the shRNA is 100 nucleotides in length or shorter, or 90, 80, 70, 60, 50, 40, 30, 25, 24, 23, 22 or 21 nucleotides in length or shorter. In some embodiments, the shRNA is at least 20 nucleotides in length or longer, or 20, 21, 22, 23, 24, 25, 30, 35, or 40 nucleotides in length or longer.Attorney Docket Ref.: 66CM-405598-WO

[0088] Also provided, in one embodiment, is a polynucleotide comprising the shRNA and a U6 promoter operatively linked to the shRNA. In some embodiments, the polynucleotide comprises two of the U6 promoters each operatively linked to one of two of the shRNA. In some embodiments, the polynucleotide comprises three of the U6 promoters each operatively linked to one of three of the shRNA.

[0089] In some embodiments, the U6 promoter includes a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:48. In some embodiments, the U6 promoter includes the sequence of SEQ ID NO:48.

[0090] Also provided, in some embodiments, is a method for treating a FSHD (Facioscapulohumeral Muscular Dystrophy) in a patient in need thereof, comprising administering to the patient a plasmid, shRNA, or polynucleotide of the present disclosure.

[0091] An AAV gene therapy vector for use in the present technology may be produced either in mammalian cells or in insect cells. Both methods are described in the art. For example Grimm et al. (2003 Molecular Therapy 7(6):839-850) disclose a strategy to produce AAV vectors in a helper virus free and optically controllable manner, which is based on transfection of only two plasmids into 293T cells. They disclose a method for production of a hybrid AAV vector comprising AAV2 ITRs and AAV5 capsid proteins. Further information can also be found in Blits ct al. (2010) (Journal of Neuroscience methods 185(2):257-263). The terms “hybrid” and “pseudotyped” are used interchangeably herein and are used to indicate vectors of which the Rep proteins, ITRs and / or capsid proteins are of different serotypes. For example, the ITRs and the Rep proteins are of AAV2 and the capsid proteins are of AAV5. The term “chimeric” is used herein to describe that a single gene, such as for example the capsid, is composed of at least two sequences derived from different serotypes.

[0092] AAV ITR and Rep sequences that may be used in the present invention for the production of rAAV vectors in insect cells can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels. This provides an identical set of genetic functions to produce virions which are essentially physically and functionally equivalent. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities sec c.g. GcnBank Accession number U89790; GcnBank Accession number J01901 ; GcnBankAttorney Docket Ref.: 66CM-405598-WOAccession number AF043303; GenBank Accession number AF085716; Chiorini et al. (1997, J. Vir. 71: 6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chiorini et al. (1999, J. Vir.73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74: 8635-47). rAAV serotypes 1, 2, 3, 4 and 5 are preferred source of AAV nucleotide sequences for use in the context of the present invention. Preferably the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, and / or AAV5. More preferably, the ITR sequences for use in the present invention are AAV2 ITR. Likewise, the Rep (Rep78 / 68 and Rep52 / 40) coding sequences are preferably derived from AAV1, AAV2, and / or AAV5, more preferably AAV2.

[0093] AAV Rep and ITR sequences are particularly conserved among most serotypes. The Rep78 proteins of various AAV serotypes are e.g., more than 89% identical and the total nucleotide sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82% (Bantel-Schaal et al., 1999, J. Virol., 73(2):939-947). Moreover, the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells. US2003148506 reports that AAV Rep and ITR sequences also efficiently cross-complement other AAV Rep and ITR sequences in insect cells.

[0094] The AAV VP proteins are known to determine the cellular tropicity of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The sequences coding for the viral proteins (VP) VP1, VP2, and VP3 capsid proteins for use in the context of the present invention are derived from AAV5. Most preferably, VP1, VP2 and VP3 are AAV5 VP1, VP2 and VP3.Alternatively, VP1, VP2 and VP3 are wild-type AAV5 sequences. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of one serotype and the ITR sequences of another AAV serotype. Such pseudotyped rAAV particles are a part of the present invention.

[0095] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.Attorney Docket Ref.: 66CM-405598-WO

[0096] “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. Examples of pharmaceutically acceptable excipients include, without limitation, sterile liquids, water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), oils, detergents, suspending agents, carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or suitable mixtures thereof.

[0097] A pharmaceutical composition can be provided in bulk or in dosage unit form. It is especially advantageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. The term “dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved. A dosage unit form can be an ampoule, a vial, a suppository, a dragee, a tablet, a capsule, an IV bag, or a single pump on an aerosol inhaler.

[0098] In therapeutic applications, the dosages vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be a therapeutically effective amount. Dosages can be provided in mg / kg / day units of measurement (which dose may be adjusted for the patient’s weight in kg, body surface area in m2, and age in years). Exemplary doses and dosages regimens for the compositions in methods of treating muscle diseases or disorders are described herein.

[0099] Dosage of the AAV can be determined experimentally to ensure efficacy with minimized adverse effects. In some embodiments, a suitable dose for an AAV of the present disclosure is not higher than lOxlO13vg (vector genomes ) / kg body weight, per administration. In some embodiments, a suitable dose for an AAV of the present disclosure is not higher than 9xl013vg / kg, or not higher than 9xl013vg / kg, 8xl013vg / kg, 7xl013vg / kg,Attorney Docket Ref.: 66CM-405598-WO6x10° vg / kg, 5x10° vg / kg, 4xl013vg / kg, 3xlO13vg / kg, 2x10° vg / kg, 1x10° vg / kg, 0.9x10° vg / kg, 0.8x10° vg / kg, 0.7x10° vg / kg, O.6xlO13vg / kg, 0.5xl013vg / kg, 0.4xl013vg / kg, 0.3x10° vg / kg, 0.2x10° vg / kg, 0.1x10° vg / kg, 0.09x10° vg / kg, 0.08xl013vg / kg, 0.07x10° vg / kg, 0.06x10° vg / kg, O.O5xlO13vg / kg, 0.04xl013vg / kg, O.O3xlO13vg / kg, 0.02xl013vg / kg, 0.01x10° vg / kg, 0.009x10° vg / kg, O.OO8xlO13vg / kg, 0.007xl013vg / kg, O.OO6xlO13vg / kg, O.OO5xlO13vg / kg, 0.004x10° vg / kg, 0.003x10° vg / kg, 0.002x10° vg / kg, or 0.001x10° vg / kg.

[0100] In some embodiments, a suitable dose for an AAV of the present disclosure is at least 0.0005x10° vg (vector genomes) / kg body weight, per administration. In some embodiments, a suitable dose for an AAV of the present disclosure is at least 0.001x10° vg / kg, or at least 0.002x10° vg / kg, 0.003x10° vg / kg, 0.004x10° vg / kg, 0.005x10° vg / kg, 0.006x10° vg / kg, 0.007x10° vg / kg, 0.008x10° vg / kg, 0.009x10° vg / kg, 0.01x10° vg / kg, or at least 0.02x10° vg / kg, 0.03x10° vg / kg, 0.04x10° vg / kg, 0.05x10° vg / kg, 0.06x10° vg / kg, 0.07x10° vg / kg, 0.08x10° vg / kg, 0.09x10° vg / kg, O.lxlO13vg / kg, or at least 0.2x10° vg / kg, 0.3x10° vg / kg, 0.4x10° vg / kg, 0.5x10° vg / kg, 0.6x10° vg / kg, 0.7x10° vg / kg, 0.8x10° vg / kg, 0.9x10° vg / kg, 1x10° vg / kg, or at least 2x10° vg / kg, 3x10° vg / kg, 4x10° vg / kg, or 5x10° vg / kg.

[0101] In some embodiments, the AAV is administered with any method known in the art. Example administration methods include, without limitation, intravenous injection, subcutaneous injection, and intramuscular injection.EXPERIMENTAL EXAMPLESExample 1. Preparation and Testing of shRNA

[0102] This example designed and prepared 5 shRNA against the mRNA of the DUX4 (Double homeobox, 4) gene. The sequences of these shRNA sequences are shown in Table 1.Table 1. shRNA SequencesName Sequence (in DNA sequence) SEQ ID NO: shRNA 1 target GCCCTTGTTCTTCCGTGAAAT 1 shRNA 1 insert GCCCTTGTTCTTCCGTGAAATCTCGAGATTTCACGGAAGAACAAGGGC 6 shRNA 2 target GGCAAACCTGGATTAGAGTTA 2 (shRNA301)shRNA 2 insert GGCAAACCTGGATTAGAGTTACTCGAGTAACTCTAATCCAGGTTTGCC 7 shRNA 3 target GGATGATTAGTTCAGAGATAT 3shRNA 3 insert GGATGATTAGTTCAGAGATATCTCGAGATATCTCTGAACTAATCATCC 8Attorney Docket Ref.: 66CM-405598-WOshRNA 4 target CCCTCCCTGTGGATCCTATAG 4 shRNA 4 insert CCCTCCCTGTGGATCCTATAGCTCGAGCTATAGGATCCACAGGGAGGG 9 shRNA 5 target GTTACATCTCCTGGATGATTA 5shRNA 5 insert GTTACATCTCCTGGATGATTACTCGAGTAATCATCCAGGAGATGTAAC 10

[0103] Each of the shRNA insert was included in an AAV plasmid and transfected into MFC (muscle progenitor cell) formed FSHD myotubes. As shown in FIG. 1, at least shRNA 1-3 and 5 were able to reduce the expression of DUX4 and its downstream genes MBD3L2 and LEUTX. Strikingly, shRNA 2 outperformed all the other shRNA by a significant margin. shRNA 2 was therefore selected for further studies, and was referred to as shRNA301.Example 2. Testing of shRNA301 in 3D Muscle Constructs

[0104] The effect of shRNA301 was then tested in a 3D muscle construct, through AAV delivery. Two AAV constructs were prepared, one of which contained a shRNA301 driven by a U6 promoter (AAV9-lXshRNA) and the other three shRNA301 each preceded by a U6 promoter (AAV9-3XshRNA).

[0105] The muscle construct was prepared on day 0, AAV transfected on day 4 and the tissue was harvested on day 14 for examination. As shown in FIG.2, the triple promoter design (3xU6 and 3xshRNA) resulted in greatly higher shRNA expression and DUX4 (and its downstream gene TRIMM43 and LEUTX) inhibition.Example 3. In vivo Testing of shRNA301

[0106] An FHSD mouse model was used in this example. FHSD was induced with tamoxifen (TMX).

[0107] The expression of shRNA301 was examined after AAV administration of a singleplasmid capsid design (FZB-400). As shown in FIG. 3, shRNA301 levels following FZB-400 administration were 30 to 10000 higher in gastrocnemius (GAS) and TA (Tibialis Anterior), respectively, compared to a conventional AAV capsid.Example 4. Single Plasmid Design of shRNA301

[0108] In this example, a single-plasmid design (AAVMYO-ShRNA3()l, or “FZB-400”) was implemented that contained all required elements for AAV transfection and production and a modified capsid protein with a muscle-targeting RGD peptide inserted. A conventional AAV design that included three separate plasmids (“MYO AAV”, transgene plasmid, AAV helper plasmid, and plasmid with Rep / Cap) was used as control.Attorney Docket Ref.: 66CM-405598-WO

[0109] As shown in FIG. 4, the single-plasmid design led to longer lasting and higher in vivo shRNA expression.

[0110] Delivery of the single -plasmid AAV (FZB-400) also resulted in significantly improved muscle functions. In this animal study, FLExDUX4 mice injected with 2.5xl013pg / kg of the AAV. Mice were either uninduced or induced with 2.5mg / Kg of Tamoxifen (day 14). Significantly improved force production in tamoxifen-induced FLExDUX4 mice compared to control AAV, and significantly improved locomotor activity (distance and run time) on day 28 was also observed (FIG. 5).

[0111] Molecular signals were also evaluated in the animal model, including the expression of DUX4, DUX4 downstream genes, and fibrosis genes on day 28. As shown in FIG. 6, all of these genes were down-regulated by FZB-400.

[0112] The expression of mir206, an exploratory biomarker for FSHD, was also measured. As shown in FIG. 7, mir206 expression in the blood was significantly inhibited by FZB-400.Example 5. Comparative efficacy of a single systemic injection of AAVMYO-shRNA301 vs MyoAAV2A-ShRNA301 in tamoxifen-induced ACTA1-MCM; FLExDUX4 mice.

[0113] This study was conducted to characterize the comparative efficacy of a single IV injection of AAVMYO-ShRNA301 (FZB-400) or a conventional design with a different AAV capsid, MyoAAV2A-ShRNA301, via functional and molecular outcomes upon administration into tamoxifen-induced adult ACTA1-MCM; FLExDUX4 mice.Methods

[0114] Twenty one (21) double transgenic ACTAl-MCM;FLExDUX4 female mice (10-12 weeks of age at enrollment) and seven (7) age-matched ACTA1-MCM / + control female mice were used in this study, according to the design of Table 2.Table 2. Experimental DesignGroup Mouse strain Tamoxifen # ROA Treatment Imaging &Induction Functional 2xl013vg / kg IVIS*:1 7 AAVMYO-S11RNA301 day 14*, 28 and organ ACTA1- Yes <200pl at terminationIV MCM;FLExDUX4 2.5mg / Kg 2xl013vg / kg2 7 AAVMYO-control Treadmill:<20 day 22, 410plAttorney Docket Ref.: 66CM-405598-WO2xl013vg / kg3 7 MyoAAV2A- ShRNA301<200j.il4 ACTA1-MCM / + No 7 n / a NTvg: vector genome* day 14: IVIS prior to tamoxifen induction.# group 4 was not IVIS imaged at any time point.NT: no treatment

[0115] Briefly, ACTAl-MCM;FLExDUX4 mice and ACTA1-MCM / + control mice were enrolled in the study at about 10-12 weeks of age. On day 0, groups 1-3 received AAV (IV) injections as per study design (Table 2). On day 13 (one day prior to that defined by study protocol, to accommodate staff assignment), animals from groups 1-3 were TMX-induced (2.5mg / kg); the day after (day 14), AAV-treated animals were imaged via whole body IVIS. On day 22, treadmill running-to-exhaustion performance was measured in animals from groups 1-4 and on day 28, animals from groups 1-3 were imaged via whole body IVIS. On day 41, animals from groups 1, 3 and 4 were tested again on the treadmill. Termination was on day 80 for surviving animals. Group 1 mice had whole body and organ IVIS on day 80. Terminal blood was collected with serum separated and frozen. Both TA and both GAS muscles were collected and frozen for molecular analysis. Tissues were also collected for histopathology, as indicated below.ResultsBiodistribution via luciferase expression upon IV injection of AAVMYO and MyoAAV2A capsids expressing ShRNA301 or luciferase control

[0116] Biodistribution and qualitative gene expression kinetics were examined via bioluminescence imaging at 14, 28 and 80 days after treatment. The luminescence signal, representing transgene expression, was detected in AAVMYO-ShRNA3()l (group 1, Gl), AAVMYO-control (group 2, G2) and MyoAAV2A-ShRNA301 (group 3, G3) treated mice and was compatible with skeletal muscle targeting. On day 14, luminescence signal appeared weaker for group 3 compared to groups 1 and 2. On day 28 post treatment, the luminescence signal was relatively similar between groups 1 and 3. The biodistribution pattern persisted throughout the examination period.Attorney Docket Ref.: 66CM-405598-WO

[0117] On the scheduled termination day 80, three animals from group 1 had whole body as well as organ luminescence imaging. Two mice exhibited strong luminescence signal, similar to day 28 while one exhibited low luminescence; this animal died right after IVIS and therefore, its health status may have contributed to this. Organ imaging confirmed skeletal muscle tropism (quads and diaphragm) with variable signal in the heart and, importantly, detargeting of the liver.Treadmill assessment

[0118] TMX induction led to a significant reduction of muscle function as demonstrated by a decrease in treadmill running distance and time in control treated ACTA1-MCM;FLExDUX4 mice on day 22 post treatment (8 days post TMX induction). Treatment of TMX-induced mice with AAVMYO-ShRNA301 (FZB-400) or MyoAAV2A-ShRNA301 (MyoAAV) rescued this functional decline to levels similar to that of ACTA1-MCM / + untreated mice (WT), which served as healthy controls (FIG.8A-B).

[0119] Upon re-testing on day 41, the mice treated with ShRNA301 (via AAVMYO or MyoAAV2A) performed as ACTA1-MCM / + untreated mice. Interestingly, AAVMYO-ShRNA301 mice improved their treadmill performance compared to day 22; furthermore, on day 41, although not statistically significantly, AAVMYO-ShRNA3()l treated mice run longer distances and for a longer time than MyoAAV2A-ShRNA301 treated or untreated ACTA1-MCM / + mice (FIG. 8C-D).In life observations and histopathology assessment

[0120] Animal weights assessed throughout the study are presented in FIG. 9. Unscheduled deaths were reported in groups 1, 2 and 3, at different time points. On the day after and up to four days after tamoxifen induction, i.e., day 14-17 post treatment, three animals on group 1, one animal on group 2 and three animals on group 3. One group 1 animal was found dead on day 72 post treatment and one died after IVIS on scheduled termination day 80. Early terminations were also reported.

[0121] The hearts from four group 1 animals were submitted to a pathology CRO: one animal found dead at day 72 post treatment, one that died after IVIS on day 80 and two animals terminated on day 80.Attorney Docket Ref.: 66CM-405598-WO

[0122] Hearts from all examined animals dosed with AAVMYO-ShRNA301 exhibited microscopic findings of similar character. Ventricular wall thinning with luminal dilation (resulting in cardiomegaly) was mild or moderate in animals with no macroscopic findings and marked in two animals which were found dead or died prior to the scheduled sacrifice.

[0123] Increased interstitial cellularity, which included mononuclear inflammatory cells (lymphocytes and macrophages), fibroblasts, and / or capillary endothelial cells; one animal also exhibited a focus of interstitial fibrosis (minimal severity). Cellular infiltrates appeared to be more severe in samples with less significant degeneration / atrophy. Overt necrosis was not a significant feature.DUX4 target genes, ShRNA30I and fibrosis marker expression

[0124] DUX4 and DUX4-target genes

[0125] Using qPCR, DUX4 expression was evaluated in the TA and gastrocnemius muscles of all available animals. TMX induction increased DUX4 expression in both TA and GAS (FIG. 10A,E). Treatment of TMX-induced mice with AAVMYO-ShRNA301 (FZB-400) and MyoAAV-ShRNA301 reduced the DUX4 expression to -half the amount detected in respective control mice (i.e., FZB-400 control treated, TMX-induced mice). FZB-400 treatment was slightly better than MyoAAV-ShRNA301 at reducing DUX4.

[0126] DUX4-target gene expression (Triin36 and Wfdc3) was assessed in the TA and GAS muscles and, in general, ShRNA301 expression led to a decrease in their expression. In TA muscles, treatment with AAVMYO-ShRNA301 (FZB-400) as well as MyoAAV2A-ShRNA301 decreased Wfdc3 and Trim36 expression to ACTA1-MCM / + levels (FIG.10B,C). In GAS muscles, treatment with AAVMYO-ShRNA30 (FZB-400) as well as MyoAAV2A-ShRNA301 decreased Wfdc3 and Trim36 expression to ACTA1-MCM / + levels (FIG. 10F,G). Interestingly, in the gastrocnemius muscle, this decrease in Trim36 expression was more evident than in the TA.

[0127] 1'he % composite expression of Wfdc3 and Trim36 is shown in FIG. 10D,H, showing a substantial decrease in the combined expression of those two DUX4 target genes in both muscles of animals treated with ShRNA301, independently of capsid.

[0128] ShRNA301 and fibrosis gene expressionAttorney Docket Ref.: 66CM-405598-WO

[0129] ShRNA301 was expressed in the TA and GAS of mice treated with MyoAAV2A-ShRNA301 or AAVMYO-ShRNA301 (FZB-400) but not in AAVMYO control (FZB-400 control) treated or WT animals (FIG. 11A,C). Expression was higher in animals treated with AAVMYO-ShRNA301 (FZB-400) compared to animals treated with MyoAAV2A-ShRNA301, reaching high statistical significance in the TA (but not in the GAS).

[0130] Collagen expression was decreased by ShRNA301 treatment in the TA (in both AAVMYO / FZB-400 and MyoAAV2A-treated animals) compared to WT and FZB-control treated animals (FIG. 11B). In the GAS, the collagen decreasing effect of ShRNA301 was not evident in FZB-400-treated animals when compared to MyoAAV-treated animals (FIG.11D)

[0131] In this example, whole body bioluminescence imaging showed that systemic AAVMYO-ShRNA301 (FZB-400) treatment resulted in robust and sustained expression of luciferase (the reporter gene) in skeletal muscle up to 80 days post treatment, the longest time point tested. Liver and brain de-targeting was also noted in animals treated with FZB-400.

[0132] Functional evaluation via treadmill demonstrated a significant functional improvement of FZB-400 treated, TMX -induced ACTAl-MCM;FLExDUX4 mice at day 22 and 41 post treatment, indicating that the DUX4-mediated muscle toxicity was rescued by the treatment. Importantly, muscle function in the FZB-400-treated group was improved to normal (WT) levels at both timepoints. At day 22 post treatment, treadmill performance was more robust in FZB-400-treated mice at day 41, compared to that of mice treated with MyoAAV2A-ShRNA310.

[0133] Unscheduled deaths shortly after tamoxifen induction across study groups were likely due to tamoxifen toxicity in younger female mice.

[0134] Expression of DUX4 and DUX4 downstream genes was reduced in TA and GAS muscles of MyoAAV2A-shRNA301 and FZB-400-treated mice, indicating a consistent, capsid-independent effect of the ShRNA301 cargo. Collagen expression (a fibrosis marker) was reduced in the TA of FZB-400 treated mice in the TA and GAS of MyoAAV2A-ShRNA301 treated mice.

[0135] Expression of ShRNA301 was confirmed in muscles of MyoAAV2A-ShRNA301 and FZB-400 treated ACTAl-MCM;FLExDUX4 mice and not in FZB-400-control orAttorney Docket Ref.: 66CM-405598-WOuntreated WT mice. The ShRNA301 expression levels were higher in FZB-400 treated mice, reaching statistical significance in the TA.

[0136] Taken together, the results of this study demonstrated functional and molecular efficacy of systemic administration of a DUX4-targeted myogenic AAV-delivered ShRNA301 in induced ACTAl-MCM;FLExDUX.Example 6. Dose study of systemic injection of AAVMYO-ShRNA301 in tamoxifen- induced ACTA1-MCM; FLExDUX4 mice.

[0137] This study was conducted characterize the initial dose response of a single IV injection of FZB-400 (AAVMYO-ShRNA301), administered to tamoxifen-induced adult ACTA1-MCM; FLExDUX4 mice.Methods

[0138] Twenty one (21) double transgenic ACTAl-MCM;FLExDUX4 female mice (17 weeks of age at enrollment) were used in this study. Details regarding the rAAVs tested are depicted in Table 3.Table 3. Experimental DesignGroup Mouse strain Tamoxifen # ROA Treatment* Imaging &Induction Functional 2xl013vg / kg1 7 AAVMYO- ShRNA301 IVIS:4xl012vg / kg day 14*, 28 and 2 7 AAVMYO- organ at ACTA1- Yes ShRNA301 termination MCM;FLExDUX4 2.5mg / Kg IV2xl012vg / kg3 7 AAVMYO- Treadmill:ShRNA301 day 28 2xl013vg / kg4 7AAVMYO-controlvg: vector genome#IV injections were all <200pl total volume.* day 14: IVIS prior to tamoxifen induction.

[0139] Briefly, ACTAl-MCM;FLExDUX4 mice were enrolled in the study at about 17 weeks of age. On day 0, groups 1-4 received one rAAV (IV) injection as per study design (Table 3). On day 14, all animals were TMX-induced (2.5mg / kg) and subsequently imaged via whole body IVIS. On day 28, treadmill running-to-exhaustion performance was measured in all animals, followed by via whole body IVIS and termination on day 31. OrganAttorney Docket Ref.: 66CM-405598-WOIVIS was obtained, terminal blood was collected with serum separated and frozen. Both TA and both GAS muscles were collected and frozen for molecular analysis. Tissues were also collected for histopathology, as indicated below.ResultsBiodistribution via luciferase expression following IV injection of AAVMYO-ShRNA301 (at three doses) or AAVMYO control

[0140] Biodistribution and qualitative gene expression kinetics were examined via bioluminescence imaging at 14 and ~31 days after treatment. The luminescence signal, representing transgene expression, was detected in all treated groups: AAVMYO-ShRNA301 high dose (group 1, Gl), mid dose (group 2, G2), low dose (group 3, G3) and AAVMYO-control (group 4, G4). Transgene expression was compatible with skeletal muscle targeting. On day 14, the luminescence signal was strongest in group 4 (control) and weaker in groups 1 , 2 and 3. An expected dose response was detected, with group 3 appearing to have the lowest luminescence signal. On day 31 post treatment, the luminescence signal of group 4 had increased compared to day 14; groups 1-3 exhibited dose-dependent signal decrease, similar to that of day 14.

[0141] On the scheduled termination day, all animals had target organs extracted and evaluated for luminescence. Organ imaging confirmed skeletal muscle tropism (quads and diaphragm) with mid- low signal in the heart (G4 only) and, importantly, de-targeting of the liver and brain. In the quadricep muscles, the luminescence signal in the low dose animals (group 3) appeared to be as strong as in the higher dose groups (Gl and G2).Treadmill assessment

[0142] As in previous studies, TMX induction led to a significant reduction of muscle function as demonstrated by a decrease in treadmill running distance and time in AAVMYO AAV-control (FZB-400 control) treated ACTAl-MCM;FLExDUX4 mice on day 28 post treatment (14 days post TMX induction). Treatment of TMX-induced mice with AAVMYO-ShRNA301 (FZB-400) rescued this functional decline (FIG. 12A,B).Attorney Docket Ref.: 66CM-405598-WO

[0143] All AAVMYO-ShRNA301 (FZB-400) doses resulted in similar and highly significant efficacy in both assessments, indicating that a minimally effective dose is yet to be defined (FIG. 12).In life observations and histopathology assessment

[0144] Animal weights throughout the study are presented in FIG. 13.

[0145] There were no unscheduled deaths, early terminations or abnormal clinical signs reported. All animals reached their intended temrination timepoint.

[0146] The hearts from all mice (n=7) treated with the lowest dose of AAVMYO-ShRNA301 (FZB-400 LD) (group 3) and from two randomly selected mice treated with control vims (group 4) were submitted to Dallas Tissue Research (DTR), a pathology CRO, for histopathology assessment.

[0147] Hearts from all examined animals dosed with either 2xl013vg / Kg control AAVMYO (FZB-400 control) or 2xl012vg / Kg AAVMYO-ShRNA301 (FZB-400 LD) exhibited minimal findings, as follows. Slight increase was observed in the extent of cardiomyocyte vacuolation in animals dosed with AAVMYO-ShRNA301, compared to those dosed with control AAVMYO. Sporadic, minimal severity mononuclear cell infiltrates were observed with no evidence of atrophy or ventricular wall thinning in all animals.Gene expression assessment

[0148] DUX4 and DUX4-target genes

[0149] Using qPCR, DUX4 expression was evaluated in the TA and GAS muscles of treated mice (FIG. 14A,E). Treatment of TMX-induced mice with FZB-400 (AAVMYO-ShRNA301) resulted in no obvious reduction of DUX4 at any dose tested, when compared to FZB-400 control-treated mice.

[0150] DUX4-target gene expression (Trim36 and W / dc3) was assessed in the TA and GAS muscles. In TA muscles of TMX-induced mice, there was a dose-independent reduction in Wfdc3 expression in AAVMYO-ShRNA301 (FZB-400) treated mice compared to FZB-400 control mice (FIG. 14B). Expression of Trim36 decreased with dose in AAVMYO-ShRNA301 (FZB-400) treated mice compared to FZB-400 controls (FIG. 14C).Attorney Docket Ref.: 66CM-405598-WO

[0151] In GAS muscles of TMX-induced mice, there was a dose-independent reduction in Wfdc3 expression in AAVMYO-ShRNA301 (FZB-400) treated mice compared to FZB-400 control mice (FIG. 14F). Expression of Trim36 decreased with dose in AAVMYO-ShRNA301 (FZB-400) treated mice compared to FZB-400 controls (FIG. 14G).

[0152] The % composite expression showed a substantial decrease in the combined expression of the two DUX4-target genes Wfdc3 and Trim36 in both muscles of animals treated with FZB-400, when compared to animals treated with FZB-400 control (FIG.14D,H).

[0153] ShRNA301 and fibrosis gene expression

[0154] ShRNA301 was expressed in the TA and GAS of TMX-induced ACTA1-MCM;FLExDUX4 mice treated with AAVMYO-ShRNA301 (FZB-400) in a dose-dependent matter. At the lowest dose of AAVMYO-ShRNA301 tested (FZB-400 LD), the ShRNA301 expression was negligible in both muscles. No ShRNA301 signal was detected in FZB-400 control treated mice (FIG. 15A,C).

[0155] Collagen expression was not affected by ShRNA301 treatment in the TA and GAS muscles of AAVMYO / FZB-400-treated animals compared to FZB-400 control treated animals (FIG. 15B,D).

[0156] Whole body bioluminescence imaging showed that systemic treatment with decreasing doses of AAVMYO-ShRNA301 (FZB-400) resulted in a dose-dependent expression of luciferase. At termination, organ luminescence assessment confirmed skeletal muscle tropism but with less evident dose-dependent signal. Importantly, liver, brain and heart de-targeting was observed.

[0157] Functional evaluation via treadmill demonstrated a significant functional improvement of all AAVMYO-ShRNA301 (FZB-400) treated, TMX-induced ACTA1-MCM;FLExDUX4 mice on day 28 post treatment, regardless of dose, compared to FZB-400 control treated mice.

[0158] There was no mortality detected in any animal treated with AAVMYO-ShRNA301 (FZB-400) at any dose. Histopathology assessment reported slight changes in cardiomyocyteAttorney Docket Ref.: 66CM-405598-WOappearance of AAVMYO-ShRNA301 -treated mice (FZB-400, low dose group) but no evidence of atrophy or ventricular wall thinning.

[0159] Expression of DUX4 was not affected by AAVMYO-ShRNA301 (FZB-400) treatment at any dose, compared to AAVMYO-control (FZB-400 control). Despite the above, expression of DUX4-target genes was reduced in TA and GAS muscles from AAVMYO-ShRNA301 (FZB-400) treated mice in a dose-independent way.

[0160] Expression of ShRNA301 was confirmed in muscles of AAVMYO-ShRNA301 (FZB-400) treated ACTAl-MCM;FLExDUX4 mice in a dose dependent manner.

[0161] Taken together, the results of this study demonstrated functional and molecular efficacy of systemic administration of FZB-400 in induced ACTAl-MCM;FLExDUX4 mice in a mostly dose-independent manner, suggesting that a minimally effective dose can be lower than 2xlO12vg / Kg.* * *

[0162] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0163] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0164] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods,Attorney Docket Ref.: 66CM-405598-WOand examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0165] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0166] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0167] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0168] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Claims

Attorney Docket Ref.: 66CM-405598-WOCLAIMS:

1. A plasmid, comprising(a) a first fragment encoding a shRNA targeted at a DUX4 (Double homeobox, 4) mRNA;(b) a U6 promoter operatively linked to the first fragment;(c) a second fragment encoding a MATR3 (Matrin-3) protein;(d) a eukaryotic promoter operatively linked to the second fragment; and(e) a pair of AAV ITR’s flanking the (a), (b), (c) and (d).

2. The plasmid of claim 1, which comprises two of (a) and two of (b) each operatively linked to the first fragment of each of the (a).

3. The plasmid of claim 1, which comprises three of (a) and three of (b) each operatively linked to the first fragment of each of the (a).

4. The plasmid of any one of claims 1-3, wherein the shRNA comprises the nucleic acid sequence of SEQ ID NO:2.

5. The plasmid of any one of claims 1-3, wherein the shRNA comprises the nucleic acid sequence of SEQ ID NO:7.

6. The plasmid of any preceding claim, wherein the eukaryotic promoter is a SkCRM4 promoter.

7. The plasmid of claim 6, wherein the eukaryotic promoter further comprises a minidesmin promoter or a SPc5- 12 promoter.

8. The plasmid of claim 7, wherein the eukaryotic promoter comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:49.

9. The plasmid of any preceding claim, further encoding an AAV Rep gene and Cap gene.Attorney Docket Ref.: 66CM-405598-WO10. The plasmid of any preceding claim, further encoding an AAV capsid protein.

11. The plasmid of claim 10, wherein the AAV capsid protein is an AAV9 VP1 capsid protein.

12. The plasmid of claim 11, wherein the AAV9 VP1 capsid protein comprises a muscletargeting peptide inserted in the capsid protein.

13. The plasmid of claim 12, wherein the insertion is between residues Q588 and A589 of the AAV9 VP1 capsid protein.

14. The plasmid of claim 12, wherein the muscle-targeting peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-46.

15. The plasmid of claim 10, wherein the AAV capsid protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:47.

16. An isolated shRNA, comprising the nucleic acid sequence of SEQ ID NO:2.

17. The shRNA of claim 16, comprising the nucleic acid sequence of SEQ ID NO:7.

18. The shRNA of claim 16 or 17, which is 80 nucleotides in length or shorter, or 60, 50, 40, 30, 25, 24, 23, 22 or 21 nucleotides in length or shorter.

19. A polynucleotide comprising the shRNA of any one of claims 16-18 and a U6 promoter operatively linked to the shRNA.

20. The polynucleotide of claim 19, which comprises three of the U6 promoters each operatively linked to one of three of the shRNA.

21. A method for treating a FSHD (Facioscapulohumeral Muscular Dystrophy) in a patient in need thereof, comprising administering to the patient a plasmid of any one of claims 1-15, the shRNA of any one of claims 16-18, or the polynucleotide of claim 19 or 20.

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