AAV gene therapy methods for treating muscular dystrophy

Abstract

The invention described herein provides a method of treatment for a disease in a human in need thereof, comprising administering to the human an rAAV viral particle comprising a polynucleotide encoding a therapeutic gene-of-interest, as described herein. The method can be used for delivering a gene of interest (GOI), such as a microdystrophin-encoding polynucleotide sequence, for the treatment of a disease, e.g., muscular dystrophy such as Duchenne muscular dystrophy (DMD).

Description

[0001] SLD-023.WO Attorney Docket No.: 129159-04320

[0002] AAV GENE THERAPY METHODS

[0003] FOR TREATING MUSCULAR DYSTROPHY

[0004] REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to and the benefit of the filing dates of U.S. Provisional Patent Application Nos. 63 / 704,824, filed October s, 2024; 63 / 760,057 and 63 / 760,071, both filed February 18, 2025; 63 / 803,412, filed May 9, 2025; and 63 / 805,850, filed May 14, 2025, the entire contents of each of the above referenced applications, including all drawings and sequence listings thereof, are hereby incorporated herein by reference.

[0006] BACKGROUND OF THE INVENTION

[0007] Recombinant muscle-tropic adeno-associated viral (rAAV) vectors demonstrate great promise as the leading platform for in vivo gene delivery. A variety of rAAV vectors enable delivery to multiple tissues, including the muscular system, for the treatment of many genetic and other complex diseases.

[0008] Numerous diseases can be treated with rAAV-mediated gene therapy, including (not limited to): neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and Canavan disease), neuromuscular diseases (such as spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), and X-linked myotubular myopathy (XLMTM)), ocular disorders (such as Leber congenital amaurosis, age-related macular degeneration (AMD), and retinitis pigmentosa), bleeding disorders (such as Hemophilia), lysosomal storage disorders, ornithine transcarbamylase (OTO) deficiency (a congenital metabolic disorder that can lead to central nervous system disorders).

[0009] One exemplary disease treatable by rAAV-mediated gene therapy is muscular dystrophy (MD). MD is a group of diseases that cause progressive weakness and loss of muscle mass. In muscular dystrophy, abnormal genes (mutant genes) produce no functional wild-type proteins needed to form healthy muscle.

[0010] For example, muscular dystrophy (MD) is a group of diseases that cause progressive weakness and loss of muscle mass. In muscular dystrophy, abnormal genes (mutant genes) produce no functional wild-type proteins needed to form healthy muscle.

[0011] Muscular dystrophies have serious debilitating impacts on quality of life of affected patients. Duchenne type muscular dystrophy (DMD) is one of the most devastating muscle diseases affecting 1 in 5,000 newborn males. It is the most well-characterized muscular dystrophy, resulting from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with the loss of sarcolemmal-cytoskeleton tethering by the DAPC.

[0012] Specifically, DMD is caused by mutations in the DMD gene, leading to reductions in DMD mRNA and the absence of dystrophin or functional dystrophin, a 427 kDa sarcolemmal protein associated with the dystrophin- associated protein complex (DAPC) (Hoffman etal., Cell 51 (6):919-928, 1987). The DAPC is composed of multiple proteins at the muscle sarcolemma that form a structural link between the extra-cellular matrix (ECM) and the cytoskeleton via dystrophin, an actin binding protein, and alpha-dystroglycan, a laminin-binding protein. These

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[0014] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 structural links stabilize muscle cell membrane during contraction, and protect against contraction-induced damage.

[0015] Loss of dystrophin as a result of DMD gene mutations disrupts the dystrophin glycoprotein complex, leading to increased muscle membrane fragility. A cascade of events including influx of calcium into the sarcoplasm, activation of proteases and proinflammatory cytokines, and mitochondrial dysfunction results in progressive muscle degeneration. In addition, displacement of neuronal nitric oxide synthase (nNOS) contributes to tissue ischemia, increased oxidative stress, and reparative failure. Disease progression is characterized by increasing muscle necrosis, fibrosis, and fatty tissue replacement and a greater degree of fiber size variation seen in subsequent muscle biopsies.

[0016] Currently there is no cure for DMD. The standard of care includes administering corticosteroids (such as prednisone or deflazacort) to stabilize muscle strength and function, prolonging independent ambulation, and delaying scoliosis and cardiomyopathy; bisphosphonates; and denosumab and recombinant parathyroid hormones.

[0017] With the advent of gene therapy, research and clinical trials for DMD treatment has focused on gene replacement or other genetic therapies aimed to at least partially restore dystrophin function. These include supplying a functional copy of the dystrophin gene, such as a dystrophin minigene, or repairing a defective dystrophin gene product by exon skipping and nonsense mutation suppression.

[0018] Adeno-associated virus (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).

[0019] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. 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). 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 significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0020] Previously, muscular gene delivery was performed by direct injection of rAAV vectors into target tissues, such as skeletal muscle. More recently, intravenous (IV) administration of rAAV has been increasingly employed to facilitate distribution to many types of muscle (including skeletal and cardiac muscles) and is now the preferred administration route for several clinical trials. Some natural serotypes (such as AAV8, AAV9, and AAVrh74) as well as engineered rAAV capsids exhibit enhanced widespread biodistribution to muscle, which could reduce the total

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[0022] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 dose required.

[0023] Multiple studies have demonstrated long-term (> 1 .5 years) recombinant AAV-mediated protein expression in muscle. See, Clark et al., Hum Gene Ther 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. U.S.A. 93:14082- 14087 (1996); and Xiao et al., J Virol 70: 8098-8108 (1996). See also, Chao et al., Mol Ther 2:619-623 (2000) and Chao et al., Mol Ther 4:217-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation following intramuscular injection as described in Herzog et al., Proc Natl Acad Sci U.S.A. 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci U.S.A. 94: 13921-13926 (1997). Moreover, Lewis et al., J Virol 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

[0024] With more than 130 clinical trials and 8 approved gene therapy products as of 2004, rAAV is one of the most popular vehicles to deliver therapeutic DNA in vivo.

[0025] The US FDA has approved several rAAV-based gene therapy products for a variety of diseases, including: ZOLGENSMA (onasemnogene abeparvovec-xioi, which uses AAV9 to deliver a functional copy of the SMN1 gene important for motor neuron survival, and was approved in 2019 to treat spinal muscular atrophy (SMA), a genetic disorder that causes muscle weakness); and ROCTAVIAN® (valoctocogene roxaparvovec-rvox, which consists of an AAV5 capsid containing a DNA sequence encoding the B-domain deleted SQ form of hFVIll-SQ, and was approved in 2023 to treat adults with severe hemophilia A, a rare bleeding disorder caused by a mutation in the blood clot gene factor VIII).

[0026] The first and only FDA approved gene therapy for Duchenne muscular dystrophy is ELEVIDYS® (delandistrogene moxeparvovec-rokl). ELEVIDYS®, an AAV.rh74 gene therapy delivering a unique microdystrophin transgene with a MHCK7 promoter, was first FDA approved in 2023 on an accelerated approval basis to treat DMD, however, only in ambulatory patients who are aged four through five years and who have a confirmed mutation in the DMD gene. FDA review of further clinical data for ELEVIDYS®led FDA to approve ELEVIDYS® in 2024 for (a) the treatment of ambulatory DMD patients who have a confirmed mutation in the DMD gene (traditional approval); and (b) the treatment of non-ambulatory DMD patients who have a confirmed mutation of the DMD gene (accelerated approval).

[0027] Despite this milestone in DMD therapy development, significant challenges have remained. ELEVIDYS® is contraindicated in patients with any deletion in exon 8 and / or exon 9 in the DMD gene because immune-mediated myositis was observed in clinical trials approximately one month following infusion in this patient population. In addition, the package insert for ELEVIDYS® has a warning for immune-mediated myositis for patients with deletions in the DMD gene in exons 1 to 17 and / or exons 59 to 71 . In 2025, it was reported that a first patient died of acute liver failure following a dose of ELEVIDYS®. Shortly thereafter, a report of a second patient death following a dose of ELEVIDYS® emerged (later found not related to treatment). Following these events, there was a halt in further development and a pause in ELEVIDYS® shipments to both ambulatory and non-ambulatory patients, with the FDA since allowing shipments to resume for ambulatory patients. In addition, the CHMP issued a negative opinion for

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[0029] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0030] ELEVIDYS®in the European Opinion.

[0031] Other prominent attempts to deliver a microdystrophin gene therapy were less successful even with the challenges faced by ELEVIDYS®. In 2024, Pfizer discontinued development of fordadistrogene movaparvovec (PF- 06939926), an AAV9 gene therapy delivering its mini-dystrophin, following its Phase 3 CIFFREO trial readout.

[0032] Despite the promise of gene therapy, this technology is not without clinical complications when treating human patients. Many gene therapy products cause acute serious liver injury, with elevated aminotransferases. Acute liver failure with fatal outcomes have been reported. This puts certain patients, such as patients with preexisting liver impairments at higher risk upon receiving such rAAV-mediated gene therapy treatment. To partly mitigate such serious and unfortunate outcomes, certain pre- and post-treatment measures are required. For example, prior to rAAV infusion, patients may be required to go through an array of pre-treatment assessments that may include liver function (e.g., as assessed by clinical examinations and laboratory testing). The patients may also be required to take systemic corticosteroid before and after rAAV infusion. Further, liver function of patients administered rAAV gene therapy products may also require monitoring for a significant period (e.g., for at least 3 months) after rAAV infusion, and at other times as clinically indicated.

[0033] For example, according to the ZOLGENSMA® (onasemnogene abeparvovec-xioi) label, in the clinical trials and in post-marketing experience, asymptomatic aminotransferase elevations were very commonly reported. In addition, cases of acute serious liver injury and acute liver failure, including a few cases with fatal outcomes, have been reported. Some patients have experienced elevations in hepatic alanine transaminase (ALT) and aspartate aminotransferase (AST) > 20 x ULN (upper limit of normal), prolonged prothrombin time and have been symptomatic (e.g., vomiting, jaundice), which required the use of corticosteroids, sometimes with prolonged duration and / or a higher dose. Thus, starting one day prior to rAAV infusion, patients scheduled to receive onasemnogene abeparvovec-xioi treatment are to be administered systemic corticosteroids equivalent to oral prednisolone at 1 mg / kg of body weight per day for a total of 30 days.

[0034] Further, prior to infusion, liver function is required to be assessed by clinical examination and laboratory testing, including hepatic aminotransferases (AST and ALT), total bilirubin level, albumin, prothrombin time, PTT, and INR. Liver function (AST, ALT, total bilirubin, prothrombin time, INR) is required to be monitored for at least 3 months after infusion, and at other times as clinically indicated. This includes monitoring liver function weekly for the first month after onasemnogene abeparvovec-xioi infusion and during the corticosteroid taper period (28 days or longer if needed). If the patient is clinically stable with unremarkable findings at the end of the corticosteroid taper period, continue to monitor liver function every other week for another month. On the other hand, patients with worsening liver function test results and / or signs or symptoms of acute illness (e.g., vomiting, deterioration in health) are required to be promptly assessed and closely monitored. If liver function abnormalities persist, systemic corticosteroids (equivalent to oral prednisolone at 1 mg / kg / day) must continue until findings become unremarkable, before tapering the corticosteroid dose gradually over the next 28 days or longer if needed. In case hepatic injury is suspected, further testing of albumin, PTT, and INR is recommended. If liver function abnormalities continue to persist > 2 x ULN after the 30-day period of systemic corticosteroids, a pediatric gastroenterologist or hepatologist

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[0036] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 needs to be promptly consulted to assess the need for further intervention.

[0037] Transient decreases in platelet counts, some of which met the criteria for thrombocytopenia, were typically observed within the first two weeks after onasemnogene abeparvovec-xioi infusion. This necessitates monitoring platelet counts before onasemnogene abeparvovec-xioi infusion and closely monitoring platelet counts within the first two weeks following infusion and on a regular basis afterwards (at least weekly for the first month; every other week for the second and third months or until platelet counts return to baseline).

[0038] Cases of thrombotic microangiopathy (TMA) were also reported to occur generally within the first two weeks after onasemnogene abeparvovec-xioi infusion in the post-marketing setting. TMA is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury. Concurrent immune system activation (e.g., infections, vaccinations) was identified in some patients receiving onasemnogene abeparvovec-xioi. Since TMA can result in life-threatening or fatal outcomes, patients are advised to pay prompt attention to signs and symptoms of TMA, and monitor platelet counts closely within the first two weeks following infusion and on a regular basis afterwards, as well as signs and symptoms of TMA, such as hypertension, increased bruising, seizures, or decreased urine output. In case these signs and symptoms occur in the presence of thrombocytopenia, further diagnostic evaluation for hemolytic anemia and renal dysfunction must be promptly undertaken. If clinical signs, symptoms and / or laboratory findings consistent with TMA occur, a pediatric hematologist and / or pediatric nephrologist must be immediately consulted to manage TMA as clinically indicated.

[0039] Furthermore, increases in cardiac troponin-l levels (up to 0.176 mcg / L) were observed following onasemnogene abeparvovec-xioi infusion in clinical trials. Though the clinical importance of these findings is not known, cardiac toxicity was observed in animal studies. Therefore, before onasemnogene abeparvovec-xioi infusion, troponin-l is required to be monitored, and monitoring is further required on a regular basis for at least 3 months afterwards (weekly for the first month, and then monthly for the second and third months until troponin-l level returns to baseline). If troponin elevations are accompanied by clinical signs or symptoms (e.g., heart rate changes, cyanosis, tachypnea and respiratory distress), a cardiologist should be consulted.

[0040] Many of these issues are not unique to onasemnogene abeparvovec-xioi. For example, according to the ELEVIDYS® label, liver function, platelet count and troponin-l levels must be assessed before infusion. Further, one day prior to infusion, a corticosteroid regimen is initiated for a minimum period of 60 days, and the corticosteroid dose is modified for patients with liver function abnormalities.

[0041] Similarly, acute serious liver injury has been observed with delandistrogene moxeparvovec-rokl administration, which may result in elevations of liver enzymes (e.g., GGT, ALT) and total bilirubin, typically seen within 8 weeks. Thus, patients with preexisting liver impairment, chronic hepatic condition or acute liver disease (e.g., acute hepatic viral infection) may be at higher risk of acute serious liver injury. Therefore, it is required to monitor liver function before delandistrogene moxeparvovec-rokl infusion, and weekly for the first 3 months after delandistrogene moxeparvovec-rokl infusion. Monitoring is to be continued until results are unremarkable. If acute serious liver injury is suspected, a consultation with a specialist is recommended.

[0042] In clinical studies, liver function test increasing (including increases in GGT, GLDH, ALT, AST, or total

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[0044] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 bilirubin) was commonly reported, typically within 8 weeks following delandistrogene moxeparvovec-rokl infusion, though the majority of cases were asymptomatic and no cases of liver failure were reported. Therefore, post- delandistrogene moxeparvovec-rokl administration, liver function (clinical exam, GGT, and total bilirubin) is to be assessed weekly for the first 3 months. Monitoring is to continue if clinically indicated, until results are unremarkable (normal clinical exam, GGT and total bilirubin levels return to near baseline levels). Platelet counts are also to be obtained weekly for the first two weeks, and continued monitoring is required if clinically indicated.

[0045] Further, acute serious myocarditis and troponin-l elevations have been observed following delandistrogene moxeparvovec-rokl infusion in clinical trials. If a patient experiences myocarditis, those with pre-existing left ventricle ejection fraction (LVEF) impairment may be at higher risk of adverse outcomes. Therefore, troponin-l level must be monitored before delandistrogene moxeparvovec-rokl infusion and weekly for the first month following infusion. Continuous monitoring is required if clinically indicated. More frequent monitoring may be warranted in the presence of cardiac symptoms, such as chest pain or shortness of breath.

[0046] Thus, there is a need to develop further rAAV-mediated gene therapy, such as gene therapy for treating muscular dystrophy, with these common severe and serious side effects associated therewith reduced or mitigated.

[0047] SUMMARY OF THE INVENTION

[0048] One aspect of the invention provides a method of treating Duchenne muscular dystrophy (DMD) in a human subject in need thereof, comprising administering a therapeutically effective dose of a recombinant muscletropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0049] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0050] In certain embodiments, the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0051] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0052] In certain embodiments, the human subject is receiving a stable dose of background glucocorticoids (e.g., at least 0.5 mg / kg / day of oral daily prednisone or 0.75 mg / kg / day deflazacort) for >12 weeks prior to being administered the dose of SGT-003.

[0053] In certain embodiments, the method further comprises administering to the human subject a short-term high dose steroid regimen, the regimen comprising at least one steroid selected from the group consisting of prednisone,

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[0055] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 prednisolone and methylprednisolone.

[0056] In certain embodiments, prednisone or prednisolone is administered daily to the human subject beginning three days prior to being administered the single dose of SGT-003 and continuing for thirty days after the single dose of SGT-003.

[0057] In certain embodiments, methylprednisolone is administered a single IV pulse dose of 20 mg / kg one day after being administered the single dose of SGT-003.

[0058] In certain embodiments, the human subject is male.

[0059] In certain embodiments, the human subject is < 18 years of age.

[0060] In certain embodiments, the human subject is ambulatory.

[0061] In certain embodiments, the human subject is non-ambulatory.

[0062] In certain embodiments, the human subject has not received a dose of a dystrophin modifying drug within three months of being administered the dose of SGT-003.

[0063] In certain embodiments, the dose is 1 E14 (1 x1014) vg / kg.

[0064] In certain embodiments, SGT-003 is administered by a single intravenous (IV) infusion.

[0065] In certain embodiments, the dose is administered over a target duration of 60 minutes at a rate not less than 5 mL / kg / hour and not more than 10 mL / kg / hour.

[0066] Another aspect of the invention provides a method for increasing microdystrophin expression in a human subject in need thereof relative to baseline, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0067] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0068] In certain embodiments, the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0069] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0070] In certain embodiments, the increase in microdystrophin expression is achieved 90 days after the dose of SGT-003 is administered to the human subject.

[0071] In certain embodiments, the increase in microdystrophin expression is measured by Western Blot or mass spectrometer of a muscle biopsy of the human subject.

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[0074] Another aspect of the invention provides a method for delaying progression of Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0075] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0076] In certain embodiments, the the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0077] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0078] In certain embodiments, the delay of progression Duchenne muscular dystrophy is measured relative to baseline by any of: Time to Rise Velocity, stride velocity 95th centile (SV95C), 10-meter walk / run velocity, 4-stair climb velocity, North Star Ambulatory Assessment (NSAA) total score, 6-minute walk test (6MWT) distance, percent predicted forced vital capacity (FVC), in percent predicted peak expiratory flow (PEF), percent predicted forced expiratory volume in 1 second (FEV1), Performance of Upper Limb (PUL) 2.0 score, left ventricular ejection fraction (LVEF) by cardiac MRI, or Bayley Scales of Infant and Toddler Development 4 (Bayley-4) score.

[0079] Another aspect of the invention provides a use of a recombinant muscle-tropic adeno-associated viral vector (rAAV) for the manufacture of a medicament for the treatment of Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of the rAAV to the human subject, wherein the rAAV comprises a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0080] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0081] In certain embodiments, the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0082] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

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[0085] Another aspect of the invention provides a method of improving recombinant muscle-tropic AAV (rAAV)- mediated delivery of a gene of interest (GOI) to muscle in a subject (e.g., a human subject) in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector decreases liver-targeting compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101 .

[0086] Another aspect of the invention provides a method of improving recombinant muscle-tropic AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject (e.g., a human subject) in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector has decreased adverse event or events (such as liver injury or hepatotoxicity) compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101 .

[0087] Another aspect of the invention provides a method of improving recombinant muscle-tropic AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject (e.g., a human subject) in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector has increased GOI copy number per nucleus and / or transduction efficiency compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101 .

[0088] Another aspect of the invention provides a method of improving recombinant muscle-tropic AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject (e.g., a human subject) in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector has reduced minimum dose compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101.

[0089] Another aspect of the invention provides a method of improving recombinant muscle-tropic AAV-mediated delivery of a polynucleotide encoding a microdystrophin to improve cardiac muscle function in a subject (e.g., a human subject) in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and a GOI, wherein said first viral vector improves cardiac muscle function (such as increasing ejection fraction function, reducing cardiac troponin I, and / or reducing titin) compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101.

[0090] In certain embodiments, the subject has muscular dystrophy characterized by a loss-of-function mutation in the dystrophin gene.

[0091] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy.

[0092] In certain embodiments, the first viral vector is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0093] In certain embodiments, the GOI comprises a polynucleotide encoding a microdystrophin (e.g., one that comprises an nNOS binding domain).

[0094] In certain embodiments, the polynucleotide encodes the microdystrophin of SEQ ID NO: 2 ( / .e., the GOI is h-piD5).

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[0096] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0097] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the recombinant muscle-tropic AAV (rAAV) encoding the h-piD5) comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto.

[0098] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5) has a sequence identical to SEQ ID NO: 1 at each capitalized nucleotide, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides.

[0099] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5) substantially lacks CpG islands {e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg plot analysis).

[0100] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5) comprises an operably linked promoter, such as a muscle-specific promoter.

[0101] In certain embodiments, the muscle-specific promoter is CK8 promoter, cardiac troponin T (cTnT) promoter, CK7 promoter, CK9 promoter, truncated MOK (tMCK), myosin heavy chain (MHO) promoter, hybrid o-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a muscle specific creatine kinase (MOK) promoter, human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MOK), truncated MOK (tMCK), myosin heavy chain (MHO), C5-12, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, steroid-inducible element, or glucocorticoid response element (gre).

[0102] In certain embodiments, the muscle-specific promoter is a CK8 promoter of SEQ ID NO: 3; optionally, wherein said CK8 promoter further comprises an enhancer nucleotide sequence or is SEQ ID NO: 4.

[0103] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding h-piD5) further comprises a polyA signal sequence, such as the polyA signal sequence of SEQ ID NO: 8, an SV40 polyadenylation signal sequence {e.g., SEQ ID NO: 9), a bovine growth hormone (bGH) polyadenylation signal sequence {e.g., SEQ ID NO: 10), or a rabbit beta globin (rBG) polyadenylation signal sequence {e.g., SEQ ID NO: 11).

[0104] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5) further comprises a 3' ITR sequence {e.g., an AAV2 3' ITR); and a 5' ITR sequence {e.g., an AAV2 5' ITR).

[0105] In certain embodiments, the 5' ITR sequence, and / or the 3' ITR sequence (1) comprise or are SEQ ID NOs: 12 and 13, respectively; or (2) comprise or are SEQ ID NOs: 24 and 27, respectively.

[0106] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5) further comprises an intron and / or an exon sequence that enhances expression of the microdystrophin.

[0107] In certain embodiments, the intron comprises SEQ ID NO: 14.

[0108] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5) further comprises a 5' UTR sequence, and / or a 3' UTR sequence.

[0109] In certain embodiments, the polynucleotide {e.g., a polynucleotide of the rAAV encoding the h-piD5)

[0110] - 10 -

[0111] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 15 or 20, or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical thereto.

[0112] In certain embodiments, the first viral vector with SLB101 capsid is administered at a dose of about 1 x 1012to about 1 x 1016vector genome (vg) / kg, or about 1 x 1013to about 1 x 1015vector genome (vg) / kg.

[0113] In certain embodiments, the first viral vector is administered as a pharmaceutical composition, at a dose of about 1 x 1014vector genome (vg) / kg.

[0114] In certain embodiments, the first viral vector is administered by a single intravenous (IV) infusion.

[0115] In certain embodiments, the first viral vector is administered over a target duration of 60 minutes at a rate not less than 5 mL / kg / hour and not more than 10 mL / kg / hour.

[0116] In certain embodiments, the human subject is receiving a stable dose of background glucocorticoids (at least 0.5 mg / kg / day of oral daily prednisone or 0.75 mg / kg / day deflazacort) for >12 weeks prior to being administered the first viral vector.

[0117] In certain embodiments, the method further comprises administering to the human subject a short-term high dose steroid regimen, the regimen comprising at least one steroid selected from the group consisting of prednisone, prednisolone and methylprednisolone.

[0118] In certain embodiments, prednisone or prednisolone is administered daily to the human subject beginning three days prior to being administered the first viral vector and continuing for thirty days after the single dose of the first viral vector.

[0119] In certain embodiments, methylprednisolone is administered as a single IV pulse dose of 20 mg / kg one day after being administered the single dose of the first viral vector.

[0120] In certain embodiments, the pharmaceutical composition is suitable or formulated for intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, or intrathecal administration, such as intravenous administration.

[0121] In certain embodiments, the method further comprises administering additional systemic corticosteroid to the subject before (e.g., at least 1 day before, at least 5 days before, at least 10 days before, at least 20 days before, at least 30 days before, at least 40 days before, at least 50 days before, or at least 60 days before) administering the AAV viral vector.

[0122] In certain embodiments, the method does not comprise administering an immunomodulator such as Complement C5 inhibitor (e.g., eculizumab), immunosuppressant (e.g., sirolimus), and anti-CD20 antibody (e.g., rituximab).

[0123] In certain embodiments, the subject in need thereof (e.g., a male human child) is about 2-15 years old, about 4-12 years old, about 4 to 7 years old, or about 7 to 12 years old.

[0124] In certain embodiments, the subject in need thereof has a body weight of no more than 50 kg, 40 kg, 30 kg, 25 kg, 20 kg, 15 kg, or 10 kg.

[0125] In certain embodiments, the subject in need thereof is negative for AAV antibodies prior to administering

[0126] - 11 -

[0127] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 the pharmaceutical composition.

[0128] In certain embodiments, the subject in need thereof has previously been administered an AAV viral vector having a capsid that does not substantially cross-react with SLB101.

[0129] In certain embodiments, the capsid that does not substantially cross-react with SLB101 is AAV8 or AAV- rh74.

[0130] Another aspect of the invention provides a method of assessing muscle integrity and / or resilience in a subject (e.g., a human subject) in need thereof, the method comprising determining the levels of a panel of muscle integrity biomarkers in a sample from the subject, as compared to control or standard, wherein a decrease in at least one of said panel of muscle integrity biomarkers is indicative of improved muscle integrity and / or resilience over the control or standard, wherein said panel of muscle integrity biomarkers comprise: a) serum CK; and b) one or more of: i) serum AST; ii) serum ALT; iii) serum troponin; iv) serum titin; v) serum lactate dehydrogenase (LDH); vi) sarcoglycan; and vii) embryonic myosin heavy chain (eMHC) fibers.

[0131] In certain embodiments, the subject has muscular dystrophy, such as DMD or BMD.

[0132] In certain embodiments, the panel comprises or consists of serum CK, serum AST, serum ALT, serum LDH, serum Titin, and eMHC fibers.

[0133] In certain embodiments, the panel comprises or consists of serum CK, serum Titin, and serum troponin.

[0134] In certain embodiments, the panel comprises or consists of serum CK, serum AST, serum ALT, serum troponin, serum LDH, serum Titin, and eMHC fibers.

[0135] In certain embodiments, the panel comprises or consists of muscle injury and stress biomarkers, such as serum creatine kinase (CK), serum aspartate aminotransferase (AST), serum alanine transaminase (ALT), and / or serum lactate dehydrogenase (LDH).

[0136] In certain embodiments, the panel comprises or consists of muscle breakdown and dystrophic regeneration biomarkers, such as serum titin, and / or embryonic myosin heavy chain (eMHC) positive fibers.

[0137] In certain embodiments, the serum sample is a blood or serum sample.

[0138] In certain embodiments, the control or standard is a sample obtained from the same subject prior to a therapeutic intervention (or a baseline sample).

[0139] In certain embodiments, the method further comprises: a) treating the subject to improve muscle integrity and / or resilience; and / or b) referring the subject to treatment to improve muscle integrity and / or resilience.

[0140] - 12 -

[0141] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0142] In certain embodiments, treating the subject comprises administering to the subject a therapeutically effective amount of AAV viral vector comprising the SLB101 capsid encapsidating a microdystrophin gene.

[0143] In certain embodiments, the microdystrophin gene comprises a sequence encoding an nNOS binding domain of wild-type microdystrophin.

[0144] In certain embodiments, the method further comprises re-assessing muscle integrity and / or resilience based on the levels of the panel of muscle integrity biomarkers following treatment.

[0145] Another aspect of the invention provides a kit for assessing muscle integrity and / or resilience in a subject (e.g., a human subject) in need thereof, the kit comprising reagents for detecting and / or determining the levels of a panel of muscle integrity biomarkers in a sample from the subject, wherein said panel of muscle integrity biomarkers comprise: a) serum CK; and b) one or more of: i) serum AST; ii) serum ALT; iii) serum troponin; iv) serum titin; v) serum lactate dehydrogenase (LDH); vi) sarcoglycan; and vii) embryonic myosin heavy chain (eMHC) fibers.

[0146] In certain embodiments, the reagents comprise: an antibody against a specific biomarker in said panel of muscle integrity biomarkers, an enzyme and / or a substrate that assesses an activity of the specific biomarker in said panel of muscle integrity biomarkers.

[0147] Another aspect of the invention provides a method of treatment for a disease in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide encoding a gene of interest (GOI), an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein: (1) the rAAV viral particle comprises a capsid of the serotype of SLB-101 ; and, (2) wherein the treatment does not lead to and, optionally, does not require assessment and / or monitoring of: (a) serious liver injury; (b) acute liver failure; (c) thrombocytopenia; and / or (d) thrombotic microangiopathies (TMA), e.g., by 45 days after administering the pharmaceutical composition to the human and / or require hospitalization for administration and / or post-administration monitoring of or from the gene therapy.

[0148] In a related aspect, the invention provides a method of treatment for a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide, an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein: (1) the polynucleotide encodes the microdystrophin of SEQ ID NO: 2, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence

[0149] - 13 -

[0150] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto; (2) the rAAV viral particle comprises a capsid of the serotype of SLB-101 ; and, (3) wherein the treatment does not lead to and, optionally, does not require assessment and / or monitoring of: (a) serious liver injury; (b) acute liver failure; (c) thrombocytopenia; and / or (d) thrombotic microangiopathies (TMA), e.g., by 45 days after administering the pharmaceutical composition to the human.

[0151] In another related aspect, the invention provides a method of treatment for a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide, an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein: (1) the polynucleotide encodes the microdystrophin of SEQ ID NO: 2, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 , or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto; (2) the rAAV viral particle comprises a capsid of the serotype of SLB-101 ; and, (3) wherein the treatment is not associated with and, optionally, does not require assessment and / or monitoring of: (a) serious liver injury; (b) acute liver failure; (c) thrombocytopenia; and / or (d) thrombotic microangiopathies (TMA) , e.g., by 45 days after administering the pharmaceutical composition to the human.

[0152] In yet another related aspect, the invention provides a method of treatment for a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide, an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein: (1) the polynucleotide encodes the microdystrophin of SEQ ID NO: 2, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 , or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto; (2) the rAAV viral particle comprises a capsid of the serotype of SLB-101 ; and, (3) wherein the treatment does not require (i) assessing liver function, platelet count, and / or troponin-l level of the human before administering the pharmaceutical composition; (ii) monitoring liver function, platelet count, and / or troponin-l level of the human after administering the pharmaceutical composition; and / or (iii) inhibition of humoral {e.g., depleting B cells with anti- CD20 mAb, inhibiting B cell activation with mTOR inhibitor, and / or cleavage of circulating IgG) and / or cellular {e.g., inhibiting T cell activation with mTOR inhibitor) immune response in the human before and / or after administering the pharmaceutical composition.

[0153] In any of the above aspects of the invention, in certain embodiments, the muscular dystrophy is characterized by a loss-of-function mutation in the dystrophin gene.

[0154] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy.

[0155] In certain embodiments, the rAAV viral particle is administered at a dose of about 1 x 1012to about 1 x 1016vector genome (vg) / kg, or about 1 x 1013to about 1 x 1015vector genome (vg) / kg.

[0156] In certain embodiments, the pharmaceutical composition is administered at a dose of about 1 x 1014vector genome (vg) / kg.

[0157] - 14 -

[0158] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0159] In certain embodiments, the polynucleotide is identical to SEQ ID NO: 1 at each capitalized nucleotide, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides.

[0160] In certain embodiments, the polynucleotide substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg plot analysis).

[0161] In certain embodiments, the polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 1.

[0162] In certain embodiments, the polynucleotide consists of the nucleotide sequence of SEQ ID NO: 1.

[0163] In certain embodiments, the polynucleotide is operably linked to a promoter, such as a muscle-specific promoter.

[0164] In certain embodiments, the muscle-specific promoter is CK8 promoter, cardiac troponin T (cTnT) promoter, CK7 promoter, CK9 promoter, truncated MOK (tMCK), myosin heavy chain (MHO) promoter, hybrid o-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a muscle specific creatine kinase (MCK) promoter, human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), C5-12, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, steroid-inducible element, or glucocorticoid response element (gre).

[0165] In certain embodiments, the muscle-specific promoter is a CK8 promoter; optionally, wherein said CK8 promoter comprises the nucleotide sequence of SEQ ID NO: 3 or 4.

[0166] In certain embodiments, the vector genome further comprises a polyadenylation signal sequence, such as the polyA signal sequence of SEQ ID NO: 8, an SV40 polyadenylation signal sequence (e.g., SEQ ID NO: 9), a bovine growth hormone (bGH) polyadenylation signal sequence (e.g., SEQ ID NO: 10), or a rabbit beta globin (rBG) polyadenylation signal sequence (e.g., SEQ ID NO: 11).

[0167] In certain embodiments, the vector genome further comprises a 3' ITR sequence, such as an AAV2 3' ITR sequence; and a 5' ITR sequence, such as an AAV2 5' ITR sequence.

[0168] In certain embodiments, the 5' ITR sequence, and / or the 3' ITR sequence (1) comprise or are SEQ ID NOs: 12 and 13, respectively; or (2) comprise or are SEQ ID NOs: 24 and 27, respectively.

[0169] In certain embodiments, the vector genome further comprises an intron and / or an exon sequence that enhances expression of the microdystrophin.

[0170] In certain embodiments, the intron comprises SEQ ID NO: 14.

[0171] In certain embodiments, the vector genome further comprises a 5' UTR sequence, and / or a 3' UTR sequence.

[0172] In certain embodiments, the AAV vector genome or the rAAV viral particle comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical thereto.

[0173] - 15 -

[0174] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0175] In certain embodiments, the pharmaceutical composition is suitable or formulated for intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, or intrathecal administration, such as intravenous administration.

[0176] In certain embodiments, the serious liver injury or acute liver failure is characterized by AST or ALT elevations > 2 x ULN, ALT elevations > 3 x ULN, or ALT elevations > 20 x ULN.

[0177] In certain embodiments, thrombocytopenia is characterized by platelet count of less than 140,000, less than 100,000, or less than 50,000 platelets I piL of blood.

[0178] In certain embodiments, thrombotic microangiopathies (TMA) is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury.

[0179] In certain embodiments, assessing liver function comprises performing clinical exam, and / or assessing levels of GGT, GLDH, AST, ALT, total bilirubin, albumin, prothrombin time, partial thromboplastin time (PTT), and / or international normalized ratio (I NR).

[0180] In certain embodiments, monitoring liver function comprises assessing levels of GGT, GLDH, AST, ALT, total bilirubin, prothrombin time, and / or I NR weekly for the first month after administering the pharmaceutical composition.

[0181] In certain embodiments, the method further comprises administering additional systemic corticosteroid to the human before (e.g., at least 1 day before, at least 5 days before, at least 10 days before, at least 20 days before, at least 30 days before, at least 40 days before, at least 50 days before, or at least 60 days before) administering the pharmaceutical composition.

[0182] In certain embodiments, the method further comprises administering maintenance systemic corticosteroid to the human after administering the pharmaceutical composition, e.g., for at least 1 day, at least 5 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, or at least 60 days after administering the pharmaceutical composition.

[0183] In certain embodiments, the additional systemic corticosteroids and the maintenance systemic corticosteroid are independently equivalent to oral prednisolone at about 0.1 - 2 mg / kg of body weight per day, about 0.2 - 1 mg / kg of body weight per day, or about 0.5 mg / kg of body weight per day, for, e.g., > 4 weeks, 8 weeks, or 12 weeks prior to administering the pharmaceutical composition.

[0184] In certain embodiments, the human in need thereof (e.g., a male child) is about 2-15 years old, about 4-12 years old, about 4 to 7 years old, or about 7 to 12 years old.

[0185] In certain embodiments, the human in need thereof has a body weight of no more than 50 kg, 40 kg, 30 kg, 25 kg, 20 kg, 15 kg, or 10 kg.

[0186] In certain embodiments, the human in need thereof is negative for AAV antibodies prior to administering the pharmaceutical composition.

[0187] It should be understood that any one embodiment described herein including those described only in the example or claims, can be combined with one or more additional embodiments of the invention, unless such

[0188] - 16 -

[0189] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 combination is improper or expressly disclaimed.

[0190] BRIEF DESCRIPTION OF THE DRAWINGS

[0191] FIG. 1 represents a study schema for the clinical trial described in Example 1. DSMB: Data and Safety Monitoring Board. Enrolled participants received a single intravenous infusion of SGT-003 at a dose level of 1 E14 (1 xio14) vg / kg.

[0192] FIG. 2A shows a schedule of study visits for the clinical trial described in Example 1 . ET: early termination; IGF: informed consent form.

[0193] FIG. 2B shows a domain map of the h-piD5 microdystrophin protein corresponding to SEQ ID NO: 2 and encoded by SGT-003.

[0194] FIGs. 3A and 3B show the results of microdystrophin expression and average thereof, as assessed by Western Blot (WB) (FIG. 3A) and MS (FIG. 3B), respectively, for Participants 1-3 in the clinical trial described in Example 1 .

[0195] FIG. 30 shows the absolute results, not adjusted for fat and fibrosis, of microdystrophin expression and average thereof, as assessed by percentage of dystrophin-positive muscle fibers, as assessed by immunofluorescence (IF), for Participants 1-3 in the clinical trial described in Example 1.

[0196] FIG. 4 shows representative IF images at baseline and Day 90 following treatment with SGT-003, showing an increase in dystrophin-positive muscle fibers in the clinical trial described in Example 1.

[0197] FIG. 5A shows gamma-glutamyl transferase (GGT) levels during 90 days post dose with AAV-SLB101 at doses up to 3x1014vg / kg in NHPs.

[0198] FIG. 5B shows mean levels of liver injury biomarkers ALT, AST, and GGT with SGT-003 treatment.

[0199] FIG. 6 shows early signs of cardiac benefits (as measured by improved LVEF percentage), for Participants 1-3 in the clinical trial described in Example 1.

[0200] FIG. 7 shows the greater than 5x mean skeletal muscle transduction, using 25% less of SGT-003, as compared with FDA-approved first generation microdystrophin gene therapy, ELEVIDYS® (reported data, crossstudy comparison)

[0201] FIGs. 8A-8C show that for Participants 1-3 in the clinical trial described in Example 1, SGT-003-expressed microdystrophin at Day 90 (FIG. 8A) binds to and restores key elements of the DAPC (Dystrophin-Associated Protein Complex), as evidenced by co-localization of beta-Sarcoglycan (FIG. 8B) and corresponding nNOS activity increase (FIG. 80).

[0202] FIGs. 9A-9C show coordinated decreases in muscle integrity I injury I stress biomarkers in DMD treated patients in the clinical trial described in Example 1, as evidence of comprehensive improvements in muscle integrity and resilience after treatment.

[0203] FIG. 9D shows microdystrophin expression in quadricep tissue and serum creatine kinase, titin, and aspartate aminotransferase (AST) levels at Day 92 in samples collected from mdx mice treated with SGT-003 at

[0204] - 17 -

[0205] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 dose levels of 3E13 vg / kg, 1 E14 vg / kg and 3E14 vg / kg.

[0206] FIG. 9E shows measurement of the same parameters as FIG. 9D at SGT-003 dose levels of 2E12 vg / kg, 6E12 vg / kg and 3E13 vg / kg. *p<0.05, **p<0.01, ****p<0.0001.

[0207] FIGs. 10A-10C show preclinical study results as evidence of low cross-reactivity between AAVSLB101 and AAVrh74 Nabs titers in mice (FIG. 10A), NHPs (FIG. 10B), and human (FIG. 10C) sera samples.

[0208] DETAILED DESCRIPTION OF THE INVENTION

[0209] 1. Overview

[0210] The invention described herein is partly based on the discovery that AAV-SLB101 (SEQ ID NO: 21)- mediated gene therapy is associated with favorable biodistribution of the AAV viral particles comprising the SLB101 capsid, in that the viral particles have enhanced muscle tissue targeting, transduction efficiency, and expression, with reduced liver distribution and the associated liver injury I hepatoxicity, as well as reduced adverse events, as compared to other AAV viral vectors such as AAV9 and AAVrh74. At least partly due to the enhanced muscle targeting I distribution, and the reduced liver toxicity, AAV viral vectors having the SLB101 capsid can be dosed at relatively lower levels with the same or better transduction efficiency and / or expression level compared to the other AAV viral vectors such as AAV91 AAVrh74.

[0211] Advantages of the SLB101 capsid may be further enhanced in AAV-SLB101 -mediated gene therapy for muscular dystrophy (e.g., DMD), such as when the encapsidated gene of interest (GOI) is a mini- or microdystrophin (may generally be referred to as microdystrophin) designed to include an nNOS-binding domain for preventing activity-induced ischemia and associated muscle injury. Such nNOS-binding domain containing microdystrophin, when encoded in a viral particle having the SLB101 capsid, achieved high expression levels in various target muscle tissues, including skeletal muscles and cardiac muscles. Furthermore, the highly expressed microdystrophin proteins bind and restore certain key elements of the Dystrophin-Associated Protein Complex (DAPC), such as beta-Sarcoglycan, resulting in consistent decrease in several biomarkers of muscle injury I stress. This suggests improved and significant muscle resilience and integrity in treated subjects. Furthermore, encouraging early signs of DMD cardiac benefit (as measured by left ventricular ejection fraction (LVEF)) suggest that the treatment is effective to reduce cardiomyopathy - a leading cause of death in DMD.

[0212] The invention described herein is also partly based on the unexpected discovery that certain biomarkers can be used to ascertain and / or establish the status of, and treatment effect on muscle integrity and resilience.

[0213] For example, DMD is a disease of impaired muscle integrity and dysfunction. Muscle integrity is the ability of muscle tissue to remain structurally and functionally whole. Muscle integrity underlies strength and mobility. Preservation of muscle integrity is critical for normal muscle function. Early signals of muscle integrity decline predict negative outcomes in certain organs, such as the heart. In DMD patients, muscle fiber regeneration becomes impaired, leading to deterioration of muscle integrity, resulting in difficulties with mobility (such as due to loss of muscle mass, inflammation, and / or fibrosis), thoracic scoliosis, respiratory failure (such as due to weak diaphragm), and cardiac failure (such as due to decreased heart function, and / or cardiomyopathy). Thus, the impact

[0214] - 18 -

[0215] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 of DMD treatments on muscle integrity for patients with Duchenne is key to determining efficacy.

[0216] The invention described herein is partly based on the unexpected discovery that muscle integrity, resilience, and / or dysfunction can be assessed using a panel of biomarkers that together can be used to establish and / or ascertain the state of muscle integrity I resilience I dysfunction, and the effect of a treatment on muscle integrity and resilience.

[0217] Thus, in one aspect, the invention described herein provides a method of treating Duchenne muscular dystrophy (DMD) in a human subject in need thereof, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0218] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21 , optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0219] In certain embodiments, the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1 , or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0220] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0221] As used herein, "SGT-003” refers to a recombinant adeno-associated virus containing a vector genome that in turn contains a codon-optimized polynucleotide sequence that encodes for a human microdystrophin variant, known as h-piD5, packaged in a novel capsid serotype AAV-SLB101 (SEQ ID NO: 21). The h-piD5 variant expressed by SGT-003 is characterized as having the general structure as shown in FIG. 2A, and is comprised of an actin binding domain, a repeat domain comprising five spectrin-like structural repeats and two hinges (H1 , R1 , R16, R17, R23, R24, H4), including the neuronal nitric oxide synthase (nNOS) binding domain, and a dystroglycan binding domain. The vector genome of SGT-003 is provided herein as SEQ ID NO: 20, and the h-piD5 microdystrophin variant polynucleotide sequence is provided herein as (SEQ ID NO: 1 ). The vector genome of SGT- 003 (SEQ ID NO: 20) encodes a human micro-dystrophin (piD5) protein of SEQ ID NO: 2.

[0222] Thus, in certain embodiments, the rAAV encoding the h-piD5 microdystrophin variant (SEQ ID NO: 2) comprises a coding sequence for h-piD5, wherein the coding sequence comprises, consists essentially of, or consists of (a) the polynucleotide sequence of SEQ ID NO: 1 , or (b) a polynucleotide at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.1 %, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more (but less than 100%) identical to SEQ ID NO: 1 and encoding h-piD5 (SEQ ID NO: 2).

[0223] In certain embodiments, the coding sequence for h-piD5 is operably linked to a promoter, such as a musclespecific promoter.

[0224] - 19 -

[0225] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0226] In certain embodiments, the muscle-specific promoter comprises I is: CK8 promoter, cardiac troponin T (cTnT) promoter, CK7 promoter, CK9 promoter, truncated MCK (tMCK), myosin heavy chain (MHC) promoter, hybrid a-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a muscle specific creatine kinase (MCK) promoter, human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), C5-12, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, steroid-inducible element, or glucocorticoid response element (gre).

[0227] In certain embodiments, the rAAV encoding the h-piD5 microdystrophin variant (SEQ ID NO: 2) has a vector genome comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0228] In certain embodiments, the rAAV encoding the h-piD5 microdystrophin variant (SEQ ID NO: 2) comprises a capsid with muscle-tropism (e.g., a muscle-tropic capsid). In certain embodiments, the muscle-tropic capsid is AAVSLB101 (characterized by a VP1 amino acid sequence of SEQ ID NO: 21). In certain embodiments, the muscle-tropic capsid is AAV1, AAV6, AAV8, AAV9, AAVrh74, or an engineered variant thereof, such as MyoAAV, AAVMYO2, AAVMYO3, MyoAAVIA, LICA1, and AAV-M41.

[0229] In one embodiment, the rAAV encoding the h-piD5 microdystrophin variant (SEQ ID NO: 2) (such as SGT- 003) comprises a capsid and a vector genome. In one embodiment, the VP1 polypeptide of the capsid comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21 . In one embodiment, the vector genome comprises, from 5' to 3', a 5' ITR sequence (e.g., the 5' ITR sequence of AAV2 or a variant thereof, such as SEQ ID NO: 12), a CK-8 promoter (such as a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 3), a coding sequence for a human microdystrophin (such as human piD5), comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 1, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1 and encoding the polypeptide of the amino acid sequence of SEQ ID NO: 2, a polyA signal sequence (e.g., the nucleic acid sequence of SEQ ID NO: 8), and a 3' ITR sequence (e.g., the 3' ITR sequence of AAV2 or a variant thereof, such as SEQ ID NO: 13), optionally, in one embodiment, the vector genome further comprises a consensus Kozak sequence operably linked to the ATG start codon of the human piD5 coding sequence.

[0230] In one embodiment, the rAAV encoding the h-piD5 microdystrophin variant (SEQ ID NO: 2) (such as SGT- 003) comprises, consists essentially of, or consists of: (a) a capsid having a VP1 polypeptide of the amino acid sequence of SEQ ID NO: 21, which encapsidates (b) a vector genome comprising, consisting essentially of, or consisting of a nucleic acid sequence that, from 5' to 3', SEQ ID NOs: 12, 3, 1, 8, and 13, such as SEQ ID NO: 15 or 20 or a nucleic acid at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 15 or 20, and encoding the polypeptide of the amino acid sequence

[0231] - 20 -

[0232] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 of SEQ ID NO: 2.

[0233] While not wishing to be bound by any particular theory, it is believed that human subjects with DMD deletion mutations in exons 1 to 11 or 42 to 45, inclusive, could be at a higher risk for developing an immune response after rAAV (e.g., SGT-003) treatment.

[0234] Any suitable methods in the art can be used to confirm the specific DMD mutation present in a given human subject suspected of having DMD. Exemplary (non-limiting) techniques include genetic testing, protein-based testing, or a combination thereof.

[0235] Typical suitable genetic testing for DMD mutations include MLPA (Multiplex Ligation-Dependent Probe Amplification, which can detect large deletions and duplications that account for up to 70% of all DMD mutations), NGS (Next-Generation Sequencing, which can detect smaller mutations, such as point mutations and small insertions / deletions, and can be used as the initial test or after testing negative using MLPA), and Sanger Sequencing (e.g., when / if NGS is inconclusive or certain small mutations need confirmation, Sanger sequencing can be used to analyze specific regions of the gene).

[0236] Typical protein analysis involves muscle biopsy, which is an invasive procedure that is typically reserved for cases where genetic testing is inconclusive, such as when no mutation is found despite strong clinical signs, or to confirm the significance of a "variant of unknown significance.” Protein analysis may include immunohistochemistry (IHC) (using antibodies to stain for the presence and location of the dystrophin protein in a muscle sample, to reveal a complete absence of dystrophin as a characteristic of DMD), and / or Western Blot (used to measure the amount and size of the dystrophin protein produced by the muscle, which can differentiate DMD from the milder Becker muscular dystrophy and other muscular dystrophin).

[0237] In certain embodiments, the human subject is male.

[0238] In certain embodiments, the human subject is < 18 years of age.

[0239] In certain embodiments, the human subject is ambulatory.

[0240] In certain embodiments, the human subject is non-ambulatory.

[0241] As used herein, "ambulatory” refers to a (human) patient's ability to walk and move about, or to care that doesn't require an overnight hospital stay, such as ambulatory surgery or care in an outpatient clinic. An ambulatory patient I human subject can walk, while ambulatory care is typically provided in settings like physician's offices, clinics, and same-day surgery centers. In certain embodiments, "ambulatory" refers to a human patient's ability to complete a 10-meter walk / run test in <30 seconds.

[0242] In certain embodiments, the human subject has not received a dose of a dystrophin modifying drug within three months of being administered the dose of the rAAV such as SGT-003.

[0243] As used herein, a "dystrophin modifying drug” includes a therapy for a muscular dystrophy disease such as Duchenne Muscular Dystrophy (DMD), which dystrophin-modifying drug aims to at least partially restore or increase functional dystrophin protein in muscles of a patient having such muscular dystrophy disease. Examples of dystrophin-modifying drug includes gene therapy (such as ELEVIDYS®), an exon-skipping drug (such as eteplirsen,

[0244] - 21 -

[0245] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 golodirsen, casimersen, viltolarsen, etc., which helps cells produce shortened but functional dystrophin). Other dystrophin-modifying drug may include ataluren (TRANSLARNA®), which is a read-through therapy that is under FDA evaluation to help cells bypass certain premature stop codons that cause the absence of dystrophin.

[0246] In certain embodiments, the dose for rAAV such as SGT-003 is 1 E14 (1 x1014) vg / kg.

[0247] In certain embodiments, rAAV such as SGT-003 is administered by a single intravenous (IV) infusion.

[0248] In certain embodiments, the dose is administered over a target duration of 60 minutes at a rate not less than 5 mL / kg / hour and not more than 10 mL / kg / hour.

[0249] In certain embodiments, the human subject is receiving a stable dose of background glucocorticoids (at least 0.5 mg / kg / day of oral daily prednisone or 0.75 mg / kg / day deflazacort) for >12 weeks prior to being administered the dose of rAAV such as SGT-003.

[0250] In certain embodiments, the method further comprises administering to the human subject a short-term high dose steroid regimen comprising administering (a) prednisone or prednisolone and (b) methylprednisolone.

[0251] In certain embodiments, the short-term high dose steroid regimen tampers down, or increases coverage against, potential inflammatory / immune responses in the human subject administered the rAAV such as SGT-003.

[0252] In certain embodiments, the short-term high dose steroid regimen comprises about 2 mg / kg / day prednisone or an equivalent thereof, optionally beginning on Day -3 (e.g., 3 days before administering the rAAV I SGT-003) and remaining on the same dose until Day 10, 15, 20, 25, 30, 35, 40 or 45 after administering the rAAV / SGT-003.

[0253] In certain embodiments, the short-term high dose steroid regimen further comprising a single IV pulse dose of about 20 mg / kg methylprednisolone or equivalent thereof, in addition to the 2 mg / kg prednisone or equivalent thereof, starting on Day 2 after administering the rAAV / SGT-003 on Day 1.

[0254] In certain embodiments, the short-term high dose steroid regimen is tapered to the human subject's pretreatment I previous standard of care (SOC) level of steroid, over the following 1 month (e.g., 2 weeks at 1 .5 mg / kg / day, followed by 2 weeks at 1 .0 mg / kg / day, followed by standard of care level).

[0255] Equivalent forms of prednisone (e.g., prednisolone) may be used in place of prednisone in accordance with local clinical practice, and / or as determined to be appropriate by one of skill in the art.

[0256] In certain embodiments, the short-term high dose steroid regimen comprises prednisone or equivalent thereof administered orally, e.g., as a tablet or oral suspension, optionally with a unit dose strength of 1 mg to 50 mg (at a dose level of about 2 mg / kg).

[0257] In certain embodiments, the short-term high dose steroid regimen comprises a dosage formulation of methylprednisolone or equivalent thereof as an injectable suspension, optionally with a unit dose strength of about 20 mg / mL, about 40 mg / mL, or about 80 mg / mL (at a dose level of about 20 mg / kg).

[0258] In certain embodiments, prednisone or prednisolone is administered daily to the human subject beginning three days prior to being administered the single dose of rAAV such as SGT-003, and continuing for thirty days after the single dose of rAAV I SGT-003.

[0259] In certain embodiments, methylprednisolone is administered a single IV pulse dose of 20 mg / kg one day

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[0261] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 after being administered the single dose of rAAV I SGT-003.

[0262] Another aspect of the invention provides a method for increasing microdystrophin expression in a human subject in need thereof relative to baseline, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0263] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0264] In certain embodiments, the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0265] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0266] In certain embodiments, the increase in microdystrophin expression is achieved 90 days, 6 months, 9 months, 1 year, 1 .5 yrs, 2 yrs or more after the dose of SGT-003 is administered to the human subject.

[0267] In certain embodiments, the increase in microdystrophin expression is measured by Western Blot or mass spectrometer of a muscle biopsy of the human subject.

[0268] In certain embodiments, the method further comprises determining, assessing, and / or comparing increases in microdystrophin expression at one or more timepoints at about day 30, 45, 90, 180, 270, 360, 540, year 2, 3, 4, 5, 6 and beyond, postdosing. Optionally, each of the timepoints may vary by ±1, 2, or 3 days.

[0269] Another aspect of the invention provides a method for delaying progression of Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0270] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0271] In certain embodiments, the the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

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[0273] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0274] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0275] In certain embodiments, the delay of progression of Duchenne muscular dystrophy (DMD) is measured relative to baseline by any one or more of the clinical measurements selected from: Time to Rise Velocity, stride velocity 95th centile (SV95C), 10-meter walk / run velocity, 4-stair climb velocity, North Star Ambulatory Assessment (NSAA) total score, 6-minute walk test (6MWT) distance, percent predicted forced vital capacity (FVC), in percent predicted peak expiratory flow (PEF), percent predicted forced expiratory volume in 1 second (FEV1), Performance of Upper Limb (PUL) 2.0 score, left ventricular ejection fraction (LVEF) by cardiac MRI, or Bayley Scales of Infant and Toddler Development 4 (Bayley -4) score.

[0276] In some embodiments, the delay of progression of DMD comprises maintaining the disease status, or maintaining or improving a clinical measurement as compared to a baseline value I status.

[0277] In certain embodiments, the delay of progression of DMD is further determined, assessed, and / or compared at one or more timepoints at about day 30, 45, 90, 180, 270, 360, 540, year 2, 3, 4, 5, 6 and beyond, postdosing. Optionally, each of the timepoints may vary by ±1, 2, or 3 days.

[0278] In some embodiments, the time period between two measurements (e.g., between a time point before treatment (such as baseline), and a time point after treatment; or between two time points after treatment), when assessing maintenance and / or improvement, is at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 1 .5 years, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years or more.

[0279] Depending on the clinical measurement, it is within the skill of a person in the field to determine the range within which a particular clinical measurement is "stable”, "maintained” or "not significantly changed” compared to a baseline measurement.

[0280] In some embodiments, the delay of progression of DMD comprises improving the disease status or improving one or more clinical measurement as compared to the baseline. A clinical measurement is considered "improved” compared to baseline if the value of the clinical measurement changes I is improved at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the baseline in the direction correlating with disease improvement.

[0281] For example, if a higher value of a clinical measurement means worse disease status (e.g., the clinical measurement is "time to rise”, where longer time means a subject is less capable of supporting themselves to stand worse disease status), then a decrease in the measurement correlates with improvement.

[0282] In some embodiments, the method described herein delays progression Duchenne muscular dystrophy (DMD) compared to baseline as measured by "time to rise and / or "time to rise velocity.”

[0283] Time to rise, also known as "time to stand from supine” or "TTSTAND” measures the number of seconds taken to rise from a supine position without assistance. A time to rise of less than 5 seconds suggests functional stability; a time to rise of more than 5 seconds indicates some functional impairment, and a time to rise of greater

[0284] - 24 -

[0285] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 than 10 seconds indicate risk of losing ambulation in the ensuing 2 years.

[0286] In some embodiments, the method maintains the "time to rise” measurement of the subject. In some embodiments, the "time to rise” measurement does not increase compared to the baseline, or increases no more than 5 seconds, no more than 4 seconds, no more than 3 seconds, no more than 2 seconds, or no more than 1 second. In some embodiments, the time to rise decreases at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, or at least 10 seconds compared to baseline.

[0287] In some embodiments, the method described herein delays progression of Duchenne muscular dystrophy (DMD) compared to baseline as measured by "time to rise velocity.”

[0288] "Time to rise velocity” is a clinical measurement that converts time to stand (the time it takes to stand up from a supine position) into a measure of speed (rises / second) to provide better statistical properties and highlight meaningful changes in functional ability, especially in patients with Duchenne muscular dystrophy. This method accounts for the fact that a one-second change is more significant in a faster rise than a slower one, giving more clinical emphasis to patients with faster baseline rise times. Time to rise velocity may include a measurement of Baseline Time to Rise (TTSTAND, which measures the time it takes for a patient to rise from a floor position without assistance as measured in seconds), and / or TTSTAND Velocity Calculation (which converts TTSTAND to a velocity by taking its reciprocal (1 / TTSTAND)).

[0289] A time to rise of more than 5 seconds or time to rise velocity of less than 0.2 rises / second indicate some functional impairment, and a time to rise of greater than 10 seconds or time to rise velocity of less than 0.1 rises / second indicate risk of losing ambulation in the ensuing 2 years.

[0290] In some embodiments, the method described herein maintains the "time to rise velocity” measurement of the subject compared to baseline. In some embodiments, the "time to rise velocity” measurement does not decrease compared to the baseline. In some embodiments, the time to rise velocity increases at least 0.05 rises / second, 0.1 rises / second, at least 0.15 rises / second, at least 0.2 rises seconds, at least 0.25 rises / second, at least 0.3 rises / seconds, at least 0.4 rises / second, at least 0.45 rises / second, or at least 0.5 rises / seconds compared to baseline.

[0291] Stride Velocity 95th Gentile (SV95C) is a real-world digital outcome measure that captures a patient's peak ambulation performance using a wearable device (typically a small, passive device such as the Syde® or ActiMyo® system that continuously collects stride data). SV95C represents the velocity of the fastest 5% of strides a person takes over a defined period, providing an objective measure of functional ability that addresses limitations of traditional clinical assessments. The European Medicines Agency (EMA) has qualified SV95C as a primary endpoint for clinical trials in ambulant Duchenne Muscular Dystrophy (DMD) patients, making it the first digitally derived measure to achieve this status.

[0292] In some embodiments, the method described herein maintains the SV95C measurement around, e.g, the baseline measurement, or has a change in the SV95C measurement of no more than 0.07 m / s e.g., no more than 0.1 m / s, no more than 0.15 m / s, or no more than 0.2 m / s. In some embodiments, the SV95C measurement does not

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[0294] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 decrease compared to, e.g., the baseline, or decreases no more than 5% , no more 4%, no more than 3%, no more than 2 %, or no more than 1%. In some embodiments, the SV95C measurement increases at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1 %, at least 1 .1 %, at least 1.2%, at least 1 .3%, at least 1 .4%, at least 1 .5%, at least 1 .6%, at least 1 .7%, at least 1 .8%, at least 1 .9%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5% compared to, e.g he baseline and / or over time. In some embodiments, the SV95C measurement increases at least 0.1 m / s, 0.15 m / s, 0.2 m / s, 0.25 m / s, or 0.3 m / s compared to, e.g. he baseline and / or over time.

[0295] "10-meter walk / run velocity” refers to the speed at which a person walks or runs over a 10-meter distance, typically measured in meters per second (m / s) using the 10-meter walk test (10MWT). This standardized clinical test is used to assess a patient's gait speed, track mobility changes, and evaluate the effectiveness of therapies, especially for individuals with neurological conditions, spinal cord injuries, or older adults. The velocity is calculated by dividing the distance covered (usually 6 or 10 meters) by the time taken to complete it. For example, in a typical test, a 14-meter-long corridor or path is marked with lines at 0m, 2m, 12m, and 14m, etc., before a participant is instructed to walk the 14 meters at a preferred or maximal walking speed, optionally using an assistive device (provided that it is used consistently and is documented). Timing begins when the toes of the leading foot cross the 2-meter mark (start line) and ends when they cross the 12-meter mark (end line). The velocity is calculated by dividing the central 10-meter distance (usually 6 or 10 meters) by the time taken.

[0296] In certain embodiments, the human subject is between 0-18 years old, and is able to complete a 10-meter walk / run test in <30 seconds prior to treatment.

[0297] In certain embodiments, the human subject is between 7-12 years old, and is able to complete a 10-meter walk / run test in <10 seconds prior to treatment.

[0298] In certain embodiments, the human subject is between 12-18 years old, and is able to complete a 10-meter walk / run test in 10-15 seconds prior to treatment.

[0299] In some embodiments, the method described herein maintains the 10-meter walk / run velocity around, e.g., the baseline measurement, or has a change in the 10-meter walk / run velocity of no more than 0.5 m / s, no more than 0.45 m / s, no more than 0.4 m / s, no more than 0.35 m / s, no more than 0.3 m / s, no more than 0.25 m / s, no more than 0.2 m / s, no more than 0.1 m / s, or no more than 0.05 m / s compared to, e.g., the baseline and / or over time. In some embodiments, the 10-meter walk / run measurement does not decrease compared to, e.g., the baseline, or decreases no more than 10%, no more than 9 %, no more than 8%, no more than 7%, no more than 6%, no more than 5% , no more 4%, no more than 3%, no more than 2 %, or no more than 1% compared to the baseline and / or over time. In some embodiments, the 10-meter walk / run velocity measurement increases at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1 %, at least 1 .1 %, at least 1 .2%, at least 1.3%, at least 1 .4%, at least 1 .5%, at least 1 .6%, at least 1 .7%, at least 1 .8%, at least 1 .9%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10 %, at least 15%, at least 20% compared to the baseline and / or over time. In some embodiments, the 10-meter walk / run velocity measurement increases at least 0.1 m / s, 0.15 m / s, 0.2 m / s, 0.25 m / s, 0.3 m / s, 0.35 m / s, 0.4 m / s, 0.45 m / s, or 0.5

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[0301] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 m / s compared to, e.g., the baseline and / or over time.

[0302] A "4-stair climb” test usually refers to a 4-flight stair climb test, which is a quick, easy, and inexpensive way to assess physical fitness and heart health by timing how long it takes to climb four flights of stairs (typically 50-60 total steps) without stopping. A good result is under one minute, while taking longer may indicate a potential health risk. The speed of climbing is measured in stairs per second and is used in clinical settings, particularly for patients with Duchenne muscular dystrophy, to track functional ability and evaluate the efficacy of treatments. The test assesses, among other things, strength, especially in the proximal muscles of the thighs and hip region. A stopwatch is typically used to record the time taken, and ideally the assessment is completed without stopping.

[0303] In some embodiments, the method described herein maintains the 4-stair climb time or velocity around, e.g., the baseline measurement, or has a change in the 4-stair climb velocity of no more than 0.1 stairs / s, no more than 0.05 stairs / s, no more than 0.04 stairs / s, no more than 0.03 stairs / s, no more than 0.02 stairs / s, no more than 0.01 stairs / s compared to, e.g, the baseline and / or over time. In some embodiments, the 4-stair climb velocity measurement does not decrease compared to, e.g, the baseline, or decreases no more than 0.1 stairs / s, no more than 0.05 stairs / s, no more than 0.04 stairs / s, no more than 0.03 stairs / s, no more than 0.02 stairs / s, no more than 0.01 stairs / s compared to, e.g, the baseline and / or over time. In some embodiments, the 4-stair climb velocity measurement increases at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1 .1 %, at least 1 .2%, at least 1 .3%, at least 1 .4%, at least 1 .5%, at least 1 .6%, at least 1 .7%, at least 1 .8%, at least 1 .9%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10 %, at least 15%, at least 20% compared to the baseline and / or over time. In some embodiments, the 4-stair climb velocity measurement increases at least 0.1 stairs / s, 0.15 stairs / s, 0.2 stairs / s, 0.25 stairs / s, 0.3 stairs / s, 0.35 stairs / s, 0.4 stairs / s, 0.45 stairs / s, or 0.5 stairs / s compared to, e.g., the baseline and / or over time.

[0304] The "6-minute walk test (6MWT)” distance refers to the total distance a person walks in six minutes on a flat, hard surface, and it serves as a measure of the person's submaximal aerobic capacity. In healthy adults, the average 6MWT distance is typically between 400 and 700 meters, although this can vary based on factors like age, gender, and height. The primary purpose of the 6MWT is to assess functional exercise capacity, track disease progression in cardiopulmonary conditions, and evaluate the effectiveness of rehabilitation or treatments by measuring performance over time. Typically, participants are instructed to walk as far and as quickly as they can over a 6-minute period on a flat, hard surface, with stopping and resting permitted if needed, but the timer for the 6 minutes continues while stopping I resting. The test is typically performed by a trained clinician walking behind the participant, who monitors the patient and records the total distance walked, but is not allowed to provide assistance or encouragement that could alter the results. This test assesses how much exercise a person can tolerate, and a significant decline in 6MWT distance can indicate disease progression. The test can help to determine if a treatment is effective in improving a patient's functional capacity.

[0305] A baseline of <350 meters often associates with functional decline. Depending on the age of the patient, their 6MWT distance may first increase for a period of time during development. However, unlike healthy subjects

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[0307] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 whose 6MWT distance will reach a plateau, 6WMT distance of DMD patients will start to decline after reaching a peak. For example, a model of DMD population predicts that during development, 6MWT distance improvement is at a rate of about 20 meters / year up until 10 years old, and then a decline at a rate of 85 meters / year (see, e.g., Hamuro et al., "Developing a Natural History Progression Model for Duchenne Muscular Dystrophy Using the Six- Minute Walk Test”, 2017, CPT Pharmacometrics Syst. Pharmacol., 6: 596-603; incorporated herein by reference).

[0308] In some embodiments, the method described herein maintains the 6MWT distance around, e.g., the baseline measurement, or has a change in the 6MWT distance of no more than 20 meters, no more than 15 meters, no more than 10 meters, no more than 10 meters, no more than 9 meters, no more than 8 meters, no more than 7 meters, no more than 6 meters, no more than 5 meters, no more than 4 meters, no more than 3 meters, no more than 2 meters, or no more than 1 meter compared to, e.g., the baseline and / or over time. In some embodiments, the 6MWT measurement does not decrease compared to, e.g., the baseline, or decreases no more than 70 meters, no more than 75 meters, no more than 65 meters , no more than 60 meters, no more than 55 meters, no more than 50 meters, no more than 45 meters, no more than 40 meters, no more than 35 meters, no more than 30 meters, no more than 25 meters, no more than 20 meters, no more than 15 meters, no more than 10 meters, no more than 9 meters, no more than 8 meters, no more than 7 meters, no more than 6 meters, no more than 5 meters compared to, e.g., the baseline and / or over time. In some embodiments, the 6MWT distance increases at least 1 meter, at least 2 meters, at least 3 meters, at least 4 meters, at least 5 meters, at least 6 meters, at least 7 meters, at least 8 meters, at least 9 meters, at least 10 meters, at least 15 meters, at least 20 meters, at least 25 meters, at least 30 meters, or at least 35 meters compared to, e.g., the baseline and / or over time.

[0309] Decline in pulmonary function in DMD contributes to significant morbidity and reduced longevity (see, e.g., Mayer etal., "Characterization of pulmonary function in Duchenne Muscular Dystrophy”, 2015, Pediatr Pulmonol 50 (5): 487-494; incorporated herein by reference). Regular assessment of pulmonary function starting late in the first decade of life is part of the current standard of care in DMD. This monitoring has typically included annual assessments of lung volume (forced vital capacity(FVC)), and measurements of respiratory muscle strength (maximal inspiratory (MIP) and expiratory (MEP) pressures). Assessment of PEF may also be helpful as a measure of disease progression in DMD since it assesses maximal expiratory effort as a surrogate measure for expiratory muscle strength. In an analysis of natural history of DMD patients, up to approximately 10 years of age, subjects showed a nearly linear increase in FVC and PEF, followed by a period of relative stabilization through 18 years, after which there was a rapid decline. However, FVC% and PEF% declined almost linearly from the 6-8 years of age cohort through the 20-22 years of age cohort. The annual rate of change for FVC% was -5.0±0.7% / year and for PEF% was -5.8±0.6% / year (see, e.g., Mayer et al., "Characterization of pulmonary function in Duchenne Muscular Dystrophy”, 2015, Pediatr Pulmonol 50 (5): 487-494; incorporated herein by reference)

[0310] "Percent predicted FVC (forced vital capacity)” (or FVC%) is a measure of lung function that compares an individual's actual FVC to the expected value for someone of their age, height, and gender (Percent predicted FVC = (Measured FVC I Predicted FVC) x 100). A value of 80% or higher is normal, 70-79% represents mild restriction, 60-69% represents moderate restriction, and less than 60% represents severe restriction. A low percent predicted FVC may indicate a restrictive lung disease, capacity, or condition.

[0311] - 28 -

[0312] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0313] In some embodiments, the method described herein maintains the FVC% measurement, e.g., the baseline measurement, or has a change of FVC% of no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the percent FVC% measurement does not decrease compared to, e.g., the baseline, or decreases no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the percent FVC% increases at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least at least 20% compared to, e.g., the baseline and / or over time.

[0314] "Percent predicted peak expiratory flow (PEF)” (or PEF%) measures how a patient's peak expiratory flow (PEF) rate compares to the expected value for their age, height, and sex, and helps to assess lung function and asthma severity. It indicates the percentage of the normal maximum airflow achieved, with values typically categorized into Green (80-100%), Yellow (50-80%), and Red (<50%) zones to guide treatment and alert to potential exacerbations or emergencies. A personal best PEF, if available and reliable, is often preferred over a predicted PEF as it provides a more personalized reference. To measure the PEF, a patient performs a maximal exhalation into a peak flow meter, blowing out as hard and fast as possible. The Predicted PEF is a value calculated based on age, sex, and height using population-based nomograms. The patient's measured PEF is divided by their predicted PEF and multiplied by 100 to arrive at the percent predicted PEF for the patient.

[0315] In some embodiments, the method described herein maintains the PEF% measurement, e.g., the baseline measurement, or has a change of PEF% of no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the PEF% measurement does not decrease compared to, e.g., the baseline, or decreases no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the PEF% measurement increases at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least at least 20% compared to, e.g., the baseline and / or over time.

[0316] "Percent predicted FEV1 (ppFEVI)” is a spirometry measurement that shows how much air a person's lungs can forcefully expel in one second, compared to what's expected for the person's age, sex, height, and ethnicity. A value close to 100% indicates normal lung function, while lower percentages suggest reduced lung capacity. While different guidelines may exist, typically, a ppFEVI above 80% is considered normal or mildly affected, while values below 60% suggest severe lung disease.

[0317] In some embodiments, the method described herein maintains the ppFEVI measurement, e.g., the baseline measurement, or has a change of ppFEVI of no more than 10%, no more than 9%, no more than 8%, no

[0318] - 29 -

[0319] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the ppFEVI measurement does not decrease compared to, e.g., the baseline, or decreases no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the ppFEVI measurement increases at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least at least 20% compared to, e.g., the baseline and / or over time.

[0320] "Performance of the Upper Limb (PUL) 2.0” is a standardized, 22-item functional scale used to assess the upper limb function in individuals, particularly those with DMD. It measures function across three levels - shoulder, middle, and distal - with a maximum total score of 42, where higher scores indicate better function. The scale provides a detailed view of functional ability, offering distinct scores for each level that can be used for clinical trials and to track changes in function over time. The PUL 2.0 measures: Upper Limb Function {e.g., a patient's ability to perform tasks, focusing on the shoulder, elbow, and hand / wrist functions; Functional Levels (specifically, the 22 items are divided into three domains, including Shoulder level (6 items, with a max score of 12), Middle level (9 items, with a max score of 17) and Distal level (7 items, with a max score of 13); and Overall Function. A total score is calculated by summing the scores from the three levels, with a maximum global score of 42. The PUL 2.0 can detect changes in upper limb function over time, making it valuable for monitoring disease progression and evaluating the effectiveness of interventions in clinical trials. The PUL 2.0 is frequently used in clinical trials for DMD to assess treatment efficacy and understand the progression of upper limb decline.

[0321] In certain embodiments, the human subject is between 10-18 years old, and meets performance of Upper Limb (PUL) 2.0 criteria {e.g., has an entry Upper Limb PUL 2.0 score of > 3 and a total score of < 40) prior to treatment.

[0322] In some embodiments, the method described herein maintains the PUL 2.0 score, e.g., the baseline score. In some embodiments, the PUL 2.0 score does not decrease compared to, e.g., the baseline score and / or over time. In some embodiments, the PUL 2.0 score increases at least 1 point, at least 2 points, at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, or at least 10 points compared to, e.g., the baseline and / or over time.

[0323] The "Left Ventricular Ejection Fraction (LVEF)” by Cardiac MRI (CMR) is the gold standard for measuring the percentage of blood pumped out of the left ventricle with each beat, offering high accuracy and reproducibility. CMR, unlike X-rays, provides detailed, non-invasive images of the heart without radiation, and is superior to echocardiography (ECG) because it doesn't depend on geometric models or suffer from poor acoustic windows. The LVEF is a crucial indicator of heart function, with reduced LVEF often forming the basis for heart failure classification and impacting treatment decisions for various heart conditions. LVEF is determined using the Simpson method, a volumetric method that calculates the LVEF as (End-Diastolic Volume - End-Systolic Volume) I End- Diastolic Volume x 100%. Low LVEF is associated with a poorer prognosis, and CMR helps identify patients at

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[0325] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 higher risk for adverse cardiovascular events.

[0326] According to the American Heart Association, LVEF about 50% to 70% is categorized as normal; a mildly reduced LVEF is usually between 41% and 49%. The onset of progressive cardiomyopathy and heart failure currently remains inevitable in DMD, and reduction in LVEF occurs relatively late in the disease process.

[0327] In some embodiments, the method described herein maintains the LVEF measurement, e.g., the baseline measurement, or has a change of LVEF of no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the ppFEVI measurement does not decrease compared to, e.g., the baseline, or decreases no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5 %, no more than 4%, no more than 3%, no more than 2 %, or no more than 1% compared to, e.g., the baseline and / or over time. In some embodiments, the LVEF measurement increases at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least at least 20% compared to, e.g., the baseline and / or over time.

[0328] The "Bayley Scales of Infant and Toddler Development Fourth Edition (Bayley-4)” is an extensive and most comprehensive formal developmental assessment tool for diagnosing developmental delays in early childhood. The Bayley-4 consists of 419 items across 5 scales (subtests), each of which can be administered separately. It includes 81 items in the Cognitive Scale (CG); 79 items in the Language Scale (LANG) including 42 items of Receptive Communication (RC) and 37 items of Expressive Communication (EC); 104 items of Motor Scale (MOT) including 46 items of Fine Motor (FM) and 58 items of Gross Motor (GM); 35 items of Social-Emotional Scale (SOEM) including Social-Emotional (SE) and Social-Emotional Sensory Processing (SP); and 120 items of Adaptive Behavior Scale (ADBE) including 23 items of Receptive Communication (REC), 28 items of Expressive Communication (EXP), 30 items of Daily Living Skills - Personal (PER), 20 items of Socialization - Interpersonal Relationships (IPR), and 19 items of Socialization - Play and Leisure (PLA). The items are scored from 0-2: 0 = not present; 1 = emerging; and 2 = mastery.

[0329] In some embodiments, the method described herein is assessed by The Bayley Scales of Infant and Toddler Development, Fourth Edition (Bayley-4), Gross Motor domain, which measures age-appropriate motor abilities such as crawling, standing, and walking in children aged 1 to 42 months. The Gross Motor Standard Score is derived by converting raw scores to age-normed standard scores (mean = 100). Higher scores indicate better gross motor development.

[0330] In some embodiments, the method described herein maintain the normalized Gross Motor score of the subject, e.g., at baseline score. In some embodiments, the normalized Gross Motor score of the subject does not decrease compared to, e.g., the baseline score, or decreases no more than 10 points, no more than 9 points, no more than 8 points, no more than 7 points, no more than 6 points, no more than 5 points, no more than 4 points, no more than 3 points, no more than 2 points, or no more than 1 point compared to e.g., baseline score and / or over time. In some embodiments, the normalized Gross Motor score of the subject score increases at least 1 point, at

[0331] - 31 -

[0332] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 least 2 points, at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, or at least 10 points compared to, e.g., the baseline score and / or over time.

[0333] The "North Star Ambulatory Assessment (NSAA)” total score ranges from 0 to 34, with a higher score indicating greater ambulatory motor function. This is a 17-item scale that assigns scores from 0 to 2 for each functional skill, with 0 meaning unable to perform, 1 meaning performed with modification, and 2 meaning performed without compensation. The two timed tests, rise from floor and 10-meter walk / run, are included in this 34-point total score. The NSAA assesses patient functional skills {e.g., various gross motor skills relevant to walking and daily activities that become more challenging for individuals with DMD), as well as motor function {e.g., provides a standardized way to measure a patient's ability to perform these functional skills, with higher scores indicating a better level of function). The NSAA is a widely used tool in DMD clinical trials and settings to track disease progression and evaluate the effectiveness of new therapies.

[0334] In some embodiments, the method described herein maintain the NSAA score of the subject, e.g., at baseline score. In some embodiments, the NSAA score of the subject does not decrease compared to, e.g., the baseline score, or decreases no more than 10 points, no more than 9 points, no more than 8 points, no more than 7 points, no more than 6 points, no more than 5 points, no more than 4 points, no more than 3 points, no more than 2 points, or no more than 1 point compared to e.g., baseline score and / or over time. In some embodiments, the NSAA score of the subject score increases at least 1 point, at least 2 points, at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, or at least 10 points compared to, e.g., the baseline score and / or over time.

[0335] Another aspect of the invention provides a use of a recombinant muscle-tropic adeno-associated viral vector (rAAV) for the manufacture of a medicament for the treatment of Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of the rAAV to the human subject, wherein the rAAV comprises a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

[0336] In certain embodiments, the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0337] In certain embodiments, the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

[0338] In certain embodiments, the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

[0339] Another aspect of the invention provides a method of improving AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject in need thereof, or improving an outcome of said method, comprising administering a

[0340] - 32 -

[0341] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector decreases liver-targeting compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV- SLB101.

[0342] Another aspect of the present invention comprises a method of treating a subject with a muscle related condition or disease or symptom wherein the method comprises administering a muscle targeting engineered capsid such as AAV-SLB101 with a muscle related GOI wherein administration of such engineered capsid has reduced adverse events as compared to the method of treating the subject with a naturally occurring capsid.

[0343] Another related aspect of the invention provides a method of improving AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector has decreased adverse event or events (such as liver injury or hepatotoxicity) compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101 .

[0344] Another related aspect of the invention provides a method of improving AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector has increased GOI copy number per nucleus and / or transduction efficiency compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101 .

[0345] Another related aspect of the invention provides a method of improving AAV-mediated delivery of a gene of interest (GOI) to muscle in a subject in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector has reduced minimum dose compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101.

[0346] Another aspect of the invention provides a method of improving AAV-mediated delivery of a polynucleotide encoding a microdystrophin to improve cardiac muscle function in a subject in need thereof, or improving an outcome of said method, comprising administering a first viral vector comprising an AAV-SLB101 capsid (e.g., SEQ ID NO: 21) and GOI, wherein said first viral vector improves cardiac muscle function (such as increasing ejection fraction function, reducing cardiac troponin I, and / or reducing titin) compared to a second viral vector comprising said GOI and an AAV capsid that is not AAV-SLB101 .

[0347] The invention describes herein further provides a method of treatment for a disease in a human in need thereof, the method comprising administering to the subject (e.g., human) a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide encoding a gene of interest (GOI), an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein (1) the rAAV viral particle comprises a capsid of the serotype of SLB-101 (having the VP1 sequence of SEQ ID NO: 21), and (2) wherein the treatment does not lead to, and does not require assessment and / or monitoring of hepatotoxicity, TMA, myocarditis, and / or myositis, including (a) serious liver injury; (b) acute liver failure; (c) thrombocytopenia; and / or (d) thrombotic microangiopathies (TMA), e.g., by 45 days after administering the pharmaceutical composition to the

[0348] - 33 -

[0349] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 human. The invention is partly based on the discovery that administration of rAAV particles having a capsid of the serotype SLB-101 resulted in no or only minor adverse effects that are commonly seen in rAAV-mediated gene therapy, during or from treatment, such as serious liver injury, acute liver failure, thrombocytopenia, and / or thrombotic microangiopathies (TMA).

[0350] The invention describes herein also provides a method of treatment for a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide, an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein (1) the rAAV viral particle comprises a capsid of the serotype SLB-101 {supra), (2) the polynucleotide encodes a microdystrophin {e.g., the microdystrophin of SEQ ID NO: 2), and (3) wherein the treatment is not associated with, does not lead to, and / or does not require assessment and / or monitoring of hepatotoxicity, TMA, myocarditis, and / or myositis, including: (a) serious liver injury; (b) acute liver failure; (c) thrombocytopenia; and / or (d) thrombotic microangiopathies (TMA), e.g., by 45 days after administering the pharmaceutical composition to the human.

[0351] In a related aspect, the invention provides a method of treatment for a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide, an rAAV vector genome comprising the polynucleotide, or an rAAV viral particle comprising the polynucleotide, wherein: (1) the polynucleotide encodes the microdystrophin of SEQ ID NO: 2, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto; (2) the rAAV viral particle comprises a capsid of the serotype of SLB-101; and, (3) wherein the treatment does not require monitoring I assessment of hepatotoxicity, TMA, myocarditis, and / or myositis, including (I) assessing liver function, platelet count, and / or troponin-l level of the human before administering the pharmaceutical composition; (ii) monitoring liver function, platelet count, and / or troponin-l level of the human after administering the pharmaceutical composition; and / or (ill) inhibition of humoral {e.g., depleting B cells with anti-CD20 mAb, inhibiting B cell activation with mTOR inhibitor, and / or cleavage of circulating IgG) and / or cellular {e.g., inhibiting T cell activation with mTOR inhibitor) immune response in the human before and / or after administering the pharmaceutical composition.

[0352] In certain embodiments, the subject has a disease which can be any disease disclosed herein below, e.g., a neuromuscular disease such as: Huntington's disease, X-linked myotubular myopathy (XLMTM), Acid maltase deficiency {e.g., Pompe disease), Spinal Muscular Atrophy (SMA), Myasthenia Gravis (MG), Amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Mitochondrial myopathy, Muscular dystrophies (Duchenne's muscular dystrophy, Myotonic dystrophy, Becker muscular dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Facioscapulohumeral muscular dystrophy (FSH), Congenital muscular dystrophy (CDM), Oculopharyngeal muscular dystrophy (OPMD), Distal muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), Mucopolysaccharidoses (MPS), Metachromatic leukodystrophy (MLD), Rett Syndrome, Krabbe Disease, Canavan disease, Pompe, Fabry's disease, mucopolysaccharide type diseases, e.g., MPS I, II, IIIA, I II B, Gaucher's disease, Danon Disease, Friedreich ataxia, Wilson's Disease, Batten Disease (CLN1, CLN3, CLN6, CLN8), Wolman Disease,

[0353] - 34 -

[0354] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0355] Tay-Sachs, Niemann-Pick Type C, CDKL5 deficiency Disorder, cardiomyopathy, RYR2-Mediated CPVT, CASQ2- Mediated CPVT, BAG3-Mediated DCM, TNNT2 DCM, and RBM20.

[0356] In certain embodiments, the disease is muscular dystrophy characterized by a loss-of-function mutation in the dystrophin gene. In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy

[0357] In some embodiments, the polynucleotide encoding a GOI, the rAAV vector genome comprising the polynucleotide, or the rAAV viral particle for use in the method of the invention comprises a polynucleotide encoding the microdystrophin.

[0358] In certain embodiments, the microdystrophin comprises an nNOS binding domain, such as SEQ ID NO: 2, said polynucleotide encoding the microdystrophin comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto.

[0359] In certain embodiments, the polynucleotide of the invention is identical to SEQ ID NO: 1 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides.

[0360] In certain embodiments, the polynucleotide of the invention substantially lacks CpG islands, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands. The presence or absence of CpG motifs or islands can be predicted based on the polynucleotide sequence using art recognized software, such as the EMBOSS Cpgplot online tool.

[0361] In certain embodiments, the polynucleotide encoding the GOI for use in the method of the invention comprise, consists essentially of, or consists of a nucleotide sequence at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1.

[0362] In certain embodiments, the polynucleotide encoding the GOI for use in the method of the invention comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 .

[0363] Another aspect of the invention provides an adeno associated virus (AAV) vector genome, comprising the polynucleotide of the invention, wherein the AAV vector genome is capable of being packaged inside an AAV capsid, such as SLB-101.

[0364] Another aspect of the invention provides a recombinant adeno associated viral (rAAV) particle, comprising an AAV capsid (such as SLB-101), and an AAV vector genome comprising the polynucleotide of the invention, wherein the AAV vector genome is encapsidated within the AAV capsid.

[0365] In certain embodiments, the polynucleotide encoding the GOI for use in the method of the invention is operably linked to a transcriptional regulatory element. In certain embodiments, the transcriptional regulatory element comprises a promoter, such as a constitutive promoter, or a muscle-specific promoter.

[0366] Numerous muscle-specific promoters can be used to express the polynucleotide encoding the GOI for use in the method of the invention, including, not limited to, CK8 promoter, cardiac troponin T (cTnT) promoter, CK7 promoter, CK9 promoter, truncated MOK (tMCK), myosin heavy chain (MHO) promoter, hybrid o-myosin heavy chain

[0367] - 35 -

[0368] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 enhancer- / MCK enhancer-promoter (MHCK7), a muscle specific creatine kinase (MCK) promoter, human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), C5-12, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, steroid-inducible element, or glucocorticoid response element (gre).

[0369] In certain embodiments, the muscle-specific promoter is a CK8 promoter. In certain embodiments, the CK8 promoter comprises the nucleotide sequence of SEQ ID NO: 3.

[0370] In certain embodiments, the CK8 promoter is a modified CK8 promoter comprising an additional enhancer element. In certain embodiments, the modified CK8 promoter comprises SEQ ID NO: 6 (the basal CK8 promoter, a 269-bp fragment of the CK8 promoter of SEQ ID NO: 3), as well as one additional copy of a 130-bp enhancer (SEQ ID NO: 5) at the 5' end. In certain embodiments, the modified CK8 promoter is CK8e promoter comprising the nucleotide sequence of SEQ ID NO: 4.

[0371] In certain embodiments, the vector genome further comprises a polyadenylation signal sequence.

[0372] In certain embodiments, the polyA signal sequence comprises SEQ ID NO: 8.

[0373] In certain embodiments, the polyA signal sequence comprises an SV40 polyadenylation signal sequence (e.g., SEQ ID NO: 9). In certain embodiments, the polyA signal sequence comprises a bovine growth hormone (bGH) polyadenylation signal sequence (e.g., SEQ ID NO: 10). In certain embodiments, the polyA signal sequence comprises a rabbit beta globin (rBG) polyadenylation signal sequence (e.g., SEQ ID NO: 11).

[0374] In certain embodiments, the vector genome further comprises a 3' ITR sequence. The ITR sequence can be from any AAV, such as an AAV2 3' ITR sequence.

[0375] In certain embodiments, the vector genome further comprises a 5' ITR sequence. The ITR sequence can be from any AAV, such as an AAV2 5' ITR sequence.

[0376] In certain embodiments, the vector genome further comprises a 5' ITR sequence and a 3' ITR sequence. The ITR sequences can be from any AAV, such as an AAV2 5' and 3' ITR sequences.

[0377] Inverted Terminal Repeat (ITR) sequences are important for initiation of viral DNA replication and circularization of adeno-associated virus genomes. Within the ITR sequences, secondary structures (e.g., stems and loops formed by palindromic sequences) are important one or more ITR functions in viral replication and / or packaging. Such sequence elements includes the RBE sequence (Rep binding element), RBE' sequence, and the TRS (terminal resolution sequence).

[0378] In certain embodiments, the 5' and / or 3' ITR sequences are wild-type sequences.

[0379] In certain embodiments, the 5' and / or 3' ITR sequences are modified ITR sequences. For example, the most 5' end or the most 3' end of the wild-type ITR sequences may be deleted. The deletion can be up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides.

[0380] In certain embodiments, up to 15 (such as exactly 15) nucleotides of the most 5' end nucleotides, and / or up

[0381] - 36 -

[0382] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 to 15 (such as exactly 15) nucleotides of the most 3' end nucleotides, of the wild-type AAV2 ITR sequences may be deleted.

[0383] Thus the 5' and / or 3' modified ITR(s) may comprising up to 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, or 127-nt (such as 130 nucleotides) of the 145-nt wild-type AAV ITR sequences.

[0384] In certain embodiments, the modified ITR sequences comprise the RBE sequence, the RBE' sequence, and / or the TRS of the wt ITR sequence.

[0385] In certain embodiments, the modified ITR sequences comprise both the RBE sequence and the RBE' sequence.

[0386] In certain embodiments, the modified ITR sequences confer stability of the plasmids of the invention comprising the AAV vector genome (see below) in bacteria, such as stability during plasmid production.

[0387] In certain embodiments, the modified ITRs do not interfere with sequencing verification of the plasmids of the invention comprising the AAV vector genome.

[0388] In certain embodiments, the modified 5' ITR sequence comprises a 5' heterologous sequence that is not part of wild-type AAV 5' ITR sequence. In certain embodiments, the modified 3' ITR sequence comprises a 3' heterologous sequence that is not part of wild-type AAV 3' ITR sequence.

[0389] In certain embodiments, the modified 5' ITR sequence comprises a 5' heterologous sequence that is not part of wild-type AAV (e.g., wt AAV2) 5' ITR sequence, and the modified 3' ITR sequence comprises a 3' heterologous sequence that is not part of wild-type AAV (e.g., wt AAV2) 3' ITR sequence, wherein the 5' heterologous sequence and the 3' heterologous sequence are complementary to each other.

[0390] In certain embodiments, the 5' heterologous sequence and the 3' heterologous sequence each comprises a type II restriction endonuclease recognition sequence, such as recognition sequence for Sse8387l (CCTGCAGG), or recognition sequence for Pad (TTAATTAA).

[0391] In certain embodiments, the 5' heterologous sequence comprises, consists essentially of, or consists of CCTGCAGGCAG (SEQ ID NO: 19), and the 3' heterologous sequence comprises, consists essentially of, or consists of the reverse complement of SEQ ID NO: 19.

[0392] In certain embodiments, the 5' heterologous sequence comprises, consists essentially of, or consists of TTAATTAAGG (SEQ ID NO: 22), and the 3' heterologous sequence comprises, consists essentially of, or consists of the reverse complement of SEQ ID NO: 22.

[0393] In certain embodiments, the 5' ITR and the 3' ITR are both flip ITR's.

[0394] In certain embodiments, the 5' ITR and the 3' ITR are both flop ITR's.

[0395] In certain embodiments, the 5' ITR and the 3' ITR are independently flip or flop ITR's.

[0396] In certain embodiments, the 5' ITR is a flip ITR, and the 3' ITR is a flop ITR.

[0397] In certain embodiments, the 5' ITR is a flop ITR, and the 3' ITR is a flip ITR.

[0398] In certain embodiments, the 5' ITR is a flip ITR, and the 3' ITR is a flip ITR.

[0399] - 37 -

[0400] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0401] In certain embodiments, the 5' ITR is a flop ITR, and the 3' ITR is a flop ITR.

[0402] As used herein, a 5' flip ITR has the B: B' segment closer to the 5'-terminal than the C:C segment. A 3' flip ITR has the B: B' segment closer to the 3' -terminal than the C:C segment. A 5' flop ITR has the C:C segment closer to the 5' -terminal than the B: B' segment. A 3' flop ITR has the C:C segment closer to the 3'-terminal than the B: B' segment.

[0403] In certain embodiments, the modified 5' ITR and the modified 3' ITR are both flop ITRs, the modified 5' ITR comprises a 5' heterologous sequence that is not part of wild-type AAV2 5' ITR sequence (such as SEQ ID NO: 19 or 22), and the modified 3' ITR sequence comprises a 3' heterologous sequence that is not part of wild-type AAV2 3' ITR sequence, wherein the 5' heterologous sequence and the 3' heterologous sequence are complementary to each other, and each comprises a type II restriction endonuclease recognition sequence, such as recognition sequence for Sse8387l or Pad; optionally, said modified 5' ITR sequence further comprises a deletion in the C:C segment, such as an 11-nts deletion AAAGCCCGGGC (SEQ ID NO: 23).

[0404] In certain embodiments, the 5' ITR comprises, consists essentially of, or consists SEQ ID NO: 12.

[0405] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 12)

[0406] In certain embodiments, the 5' ITR comprises, consists essentially of, or consists SEQ ID NO: 24. CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTT GGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 24)

[0407] In certain embodiments, the 5' ITR comprises, consists essentially of, or consists SEQ ID NO: 25.

[0408] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 25)

[0409] In certain embodiments, the 5' ITR comprises, consists essentially of, or consists SEQ ID NO: 26. TTAATTAAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 26)

[0410] In certain embodiments, the 3' ITR comprises, consists essentially of, or consists SEQ ID NO: 13.

[0411] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA AGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 13)

[0412] In certain embodiments, the 3' ITR comprises, consists essentially of, or consists SEQ ID NO: 27. AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA AGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 27)

[0413] In certain embodiments, the 3' ITR comprises, consists essentially of, or consists SEQ ID NO: 28.

[0414] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA

[0415] - 38 -

[0416] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0417] AGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAA (SEQ ID NO: 28)

[0418] In certain embodiments, the 5' ITR sequence is or comprises SEQ ID NO: 12, and the 3' ITR sequence is or comprises SEQ ID NO: 13.

[0419] In certain embodiments, the 5' ITR sequence is or comprises SEQ ID NO: 24, and the 3' ITR sequence is or comprises SEQ ID NO: 27.

[0420] In certain embodiments, the 5' ITR comprises up to 141 nts of the most 3' nucleotides of the 145-nt wt AAV2 5' ITR (e.g., a deletion of 4 or more most 5' end of the 145-nt wt AAV2 5' ITR).

[0421] In certain embodiments, the 5' ITR comprises up to 130 nts of the most 3' nucleotides of the 145-nt wt AAV2 5' ITR (e.g., a deletion of 15 or more most 5' end of the 145-nt wt AAV2 5' ITR).

[0422] In certain embodiments, the 3' ITR comprises up to 141 nts of the most 5' nucleotides of the 145-nt wt AAV2 3' ITR (e.g., a deletion of 4 or more most 3' end of the 145-nt wt AAV2 3' ITR).

[0423] In certain embodiments, the 3' ITR comprises up to 130 nts of the most 5' nucleotides of the 145-nt wt AAV2 3' ITR (e.g., a deletion of 15 or more most 3' end of the 145-nt wt AAV2 3' ITR).

[0424] In certain embodiments, the 5' and 3' ITR sequences are compatible for AAV production in mammalian-cell based on triple transfection.

[0425] In certain embodiments, the 5' and 3' ITR sequences are compatible for AAV production in insect cell (e.g., Sf9) based on baculovirus vector (see below).

[0426] In certain embodiments, the 5' and 3' ITR sequences are compatible for AAV production in mammalian-cell based on HSV vectors.

[0427] In certain embodiments, the vector genome further comprises an intron and / or an exon sequence that enhances expression of the microdystrophin. In certain embodiments, the intron I exon increases expression of the microdystrophin by up to 2-10 folds.

[0428] In certain embodiments, the intron comprises the sequence of a p-globin splice donor / IgG splice acceptor chimeric intron (see, for example, the chimeric intron in Promega pCMVTnT vector (Cat. No. L5620).

[0429] In certain embodiments, the intron comprises SEQ ID NO: 14. gtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatcc actttgcctttctctccacag (SEQ ID NO: 14)

[0430] In certain embodiments, the promoter is CK8e promoter (infra) that comprises a 48 bp (SEQ ID NO: 7) or 50 bp (SEQ ID NO: 16) MCK UTR exon sequence that enhances expression.

[0431] In certain embodiments, the vector genome does not comprise intron and / or exon sequences that potentially enhances expression of the GOI (e.g., microdystrophin). Eliminating intron I exon sequences may improve packaging efficiency and increase packaging capacity for other sequence elements.

[0432] In certain embodiments, the vector genome further comprises a 5' UTR sequence, and / or a 3' UTR sequence.

[0433] - 39 -

[0434] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0435] In certain embodiments, the AAV vector genome or the rAAV viral particle of the invention comprises, consists essentially of, or consists of, from 5' to 3', the following sequence elements: (1) a 5' ITR (such as a wildtype or modified AAV2 5' ITR, e.g., the 145-nt wild-type AAV2 5' ITR, or the 141 -nt modified AAV2 5' ITR (such as SEQ ID NO: 12)), (2) a muscle-specific promoter (such as a CK8 promoter (e.g., SEQ ID NO: 3) or a modified CK8 protein such as CK8e as described herein (SEQ ID NO: 4)); (3) any one of the CpG reduced I eliminated codon optimized polynucleotide of the invention (such as SEQ ID NO: 1 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto); (4) a polyA signal sequence (such as any one of SEQ ID NOs: 8-11); and (5) a 3' ITR (such as a wild-type or modified AAV2 3' ITR, e.g., the 145-nt wild-type AAV2 3' ITR, or the 141 -nt modified AAV2 3' ITR (Such as SEQ ID NO: 13)); or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical to the AAV vector genome. Optionally, immediately before (3), there is a KOZAK sequence comprising ACC immediately 5' to the ATG start codon.

[0436] In certain embodiments, the AAV vector genome or the rAAV viral particle of the invention comprises, consists essentially of, or consists of, from 5' to 3', the following sequence elements: (1) a 5' ITR (such as SEQ ID NO: 12), (2) a CK8 promoter (e.g., SEQ ID NO: 3); (3) any one of the CpG reduced I eliminated codon optimized polynucleotide of the invention (such as SEQ ID NO: 1); (4) a polyA signal sequence (such as SEQ ID NO: 8); and (5) a 3' ITR (such as SEQ ID NO: 13); or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical to the AAV vector genome. Optionally, immediately before (3), there is a KOZAK sequence comprising ACC immediately 5' to the ATG start codon.

[0437] In certain embodiments, the AAV vector genome or the rAAV viral particle of the invention comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical thereto.

[0438] In certain embodiments, the recombinant adeno-associated virus (rAAV) viral particle comprises an SLB- 101 capsid, and a vector genome encapsidated therein, wherein said vector genome comprises a polynucleotide sequence encoding the MD5 microdystrophin of SEQ ID NO: 2.

[0439] In certain embodiments, the pharmaceutical composition is suitable or formulated for intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, or intrathecal administration, such as intravenous administration.

[0440] In certain embodiments, serious liver injury or acute liver failure is characterized by AST or ALT elevations > 2 x ULN, ALT elevations > 3 x ULN, or ALT elevations > 20 x ULN.

[0441] In certain embodiments, thrombocytopenia is characterized by platelet count of less than 140,000, less than 100,000, or less than 50,000 platelets I piL of blood.

[0442] In certain embodiments, TMA is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury.

[0443] In certain embodiments, assessing liver function comprises performing clinical exam, and / or assessing levels of GGT, GLDH, AST, ALT, total bilirubin, albumin, prothrombin time, partial thromboplastin time (PTT), and / or

[0444] - 40 -

[0445] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 international normalized ratio (I NR).

[0446] In certain embodiments, monitoring liver function comprises assessing levels of GGT, GLDH, AST, ALT, total bilirubin, prothrombin time, and / or I NR weekly for the first month after administering the pharmaceutical composition.

[0447] In certain embodiments, the method further comprises administering additional (e.g., dosing above a baseline standard of care (SOC) dose the patient is already taking (if any) prior to the subject treatment) systemic corticosteroid to the human before (e.g., at least 1 day before, at least 5 days before, at least 10 days before, at least 20 days before, at least 30 days before, at least 40 days before, at least 50 days before, or at least 60 days before) administering the pharmaceutical composition.

[0448] In certain embodiments, the method further comprises administering maintenance systemic corticosteroid to the human after administering the pharmaceutical composition, e.g., for at least 1 day, at least 5 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, or at least 60 days after administering the pharmaceutical composition.

[0449] In certain embodiments, the additional systemic corticosteroids and the maintenance systemic corticosteroid are independently equivalent to oral prednisolone at about 0.1 - 2 mg / kg of body weight per day, about 0.2 - 1 .5 mg / kg of body weight per day, or about 0.5 - 1 mg / kg of body weight per day, for, e.g., > 4 weeks, 8 weeks, or 12 weeks prior to administering the pharmaceutical composition.

[0450] The human in need thereof {e.g., a male child) is about 2-15 years old, about 4-12 years old, about 4 to <7 years old, or about 7 to <12 years old.

[0451] In certain embodiments, the human in need thereof has a body weight of no more than 50 kg, 40 kg, 30 kg, 25 kg, 20 kg, 15 kg, or 10 kg.

[0452] In certain embodiments, the human in need thereof is negative for AAV antibodies prior to administering the pharmaceutical composition.

[0453] In certain embodiments, the subject in need thereof has previously been administered an AAV viral vector having a capsid that does not substantially cross-react with SLB101. In certain embodiments, the capsid that does not substantially cross-react with SLB101 is AAV8 or AAV-rh74.

[0454] Another aspect of the disclosure provides a pharmaceutical composition comprising the polynucleotide of the invention encoding the GOI, the rAAV vector genome or the rAAV viral particle for use in the method of the invention, and a pharmaceutically acceptable carrier.

[0455] In certain embodiments, the pharmaceutical composition is suitable or formulated for intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, or intrathecal administration. In certain embodiments, the pharmaceutical composition is suitable or formulated for intravenous administration.

[0456] Another aspect of the disclosure provides a method of treating a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of the polynucleotide of the invention, the rAAV vector genome or the rAAV viral particle of the invention, or the pharmaceutical

[0457] - 41 -

[0458] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 composition of the invention.

[0459] In certain embodiments, the muscular dystrophy is characterized by a loss-of-function mutation in the dystrophin gene. In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy.

[0460] In certain embodiments, the rAAV viral particle is administered at a dose of about 1 x 1012to about 1 x 1016vector genome (vg) / kg, or about 1 x 1013to about 1 x 1015vector genome (vg) / kg. In certain embodiments, the rAAV viral particle is administered at a dose of about 1 x 1012vg / kg, about 1 x 1013vg / kg, about 1 x 1014vg / kg, about 1 x 1015vg / kg, or about 1 x 1016. In certain embodiments, the rAAV viral particle is administered at a dose of about 1 x 1014(“1 E14”) vg / kg patient body weight.

[0461] Yet another aspect of the invention provides a method of assessing muscle integrity and / or resilience in a subject in need thereof, the method comprising determining the levels of a panel of muscle integrity biomarkers in a sample from the subject, as compared to control or standard, wherein a decrease in at least one of said panel of muscle integrity biomarkers is indicative of improved muscle integrity and / or resilience over the control or standard, wherein said panel of muscle integrity biomarkers comprise: a) serum CK; and b) one or more of:

[0462] I) serum AST; ii) serum ALT; ill) serum troponin; iv) serum titin; v) serum lactate dehydrogenase (LDH); vi) sarcoglycan; and vii) embryonic myosin heavy chain (eMHC) fibers.

[0463] In certain embodiments, the subject has muscular dystrophy, such as DMD or BMD.

[0464] In certain embodiments, the panel comprises or consists of serum CK, serum AST, serum ALT, serum LDH, serum Titin, and eMHC fibers.

[0465] In certain embodiments, the panel comprises or consists of serum CK, serum Titin, and serum troponin.

[0466] In certain embodiments, the panel comprises or consists of serum CK, serum AST, serum ALT, serum troponin, serum LDH, serum Titin, and eMHC fibers.

[0467] In certain embodiments, the panel comprises or consists of muscle injury and stress biomarkers, such as serum creatine kinase (CK), serum aspartate aminotransferase (AST), serum alanine transaminase (ALT), and / or serum lactate dehydrogenase (LDH).

[0468] In certain embodiments, the panel comprises or consists of muscle breakdown and dystrophic regeneration biomarkers, such as serum titin, and / or embryonic myosin heavy chain (eMHC) positive fibers.

[0469] In certain embodiments, the sample is a blood or serum sample.

[0470] - 42 -

[0471] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0472] In certain embodiments, the control or standard is a sample obtained from the same subject prior to a therapeutic intervention (or a baseline sample).

[0473] In certain embodiments, the method further comprises: a) treating the subject to improve muscle integrity and / or resilience; and / or b) referring the subject to treatment to improve muscle integrity and / or resilience.

[0474] In certain embodiments, treating the subject comprises administering to the subject a therapeutically effective amount of AAV viral vector comprising the SLB101 capsid encapsidating a microdystrophin gene.

[0475] In certain embodiments, the microdystrophin gene comprises a sequence encoding an nNOS binding domain of wild-type microdystrophin.

[0476] In certain embodiments, the method further comprises re-assessing muscle integrity and / or resilience based on the levels of the panel of muscle integrity biomarkers following treatment.

[0477] Yet another aspect of the invention provides a kit for assessing muscle integrity and / or resilience in a subject in need thereof, the kit comprising reagents for detecting and / or determining the levels of a panel of muscle integrity biomarkers in a sample from the subject, wherein said panel of muscle integrity biomarkers comprise: a) serum CK; and b) one or more of: i) serum AST; ii) serum ALT; iii) serum troponin; iv) serum titin; v) serum lactate dehydrogenase (LDH); vi) sarcoglycan; and vii) embryonic myosin heavy chain (eMHC) fibers.

[0478] In certain embodiments, the reagents comprise: an antibody against a specific biomarker in said panel of muscle integrity biomarkers, an enzyme and / or a substrate that assesses an activity of the specific biomarker in said panel of muscle integrity biomarkers.

[0479] With the inventions generally described above, the following sections provide more detailed description for specific aspects of the invention. Thus any one embodiment described herein, including those described only in the examples or the claims, can be combined with any one or more additional embodiments of the invention unless expressly disclaimed or improper.

[0480] 2. AAV and Capsid

[0481] As used herein, the term "AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a coinfecting helper virus.

[0482] An "AAV vector” or "(AAV) vector genome” as used herein interchangeably, refers to a vector comprising

[0483] - 43 -

[0484] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious AAV viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0485] An "AAV virion” or "AAV viral particle” or "recombinant AAV (rAAV) viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide ( / .e., a polynucleotide other than a wild-type AAV genome such as the subject CpG reduced codon optimized microdystrophin coding sequence for delivery to a mammalian (muscle) cell), it is typically referred to as an "AAV vector I viral particle.” Thus, production of AAV viral particle necessarily includes production of AAV vector, as such a vector is contained within an AAV viral particle.

[0486] The rAAV viral particle for use in the method of the invention comprises a capsid of the serotype of SLB- 101, which preferentially deliver the GOI to muscle tissues at higher expression level and / or lower dose, thus facilitating more successful gene therapy targeting muscle tissues (e.g., skeletal, cardiac, and / or smooth muscle tissues).

[0487] The SLB-101 capsid, which VP1 capsid comprises the sequence of SEQ ID NO: 21 :

[0488] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALE HDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSS GNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQ RLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQY GYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGS GQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASH KEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQRGDLGLSAQAQTGWV QNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 21).

[0489] 3. Gene of Interest

[0490] The rAAV vector genome or rAAV viral particle for use of the method of the invention may carry any gene of interest (GOI) for treating a disease or condition through gene therapy. The GOI can comprise any gene or coding sequence within the packaging capacity of the rAAV, e.g., about 4-5 kb, or about 4.7 kb including the ITR sequences, or about 4.4 kb without accounting for the ITR sequences.

[0491] In certain embodiments, the rAAV carrying the GOI can be used in gene therapy to treat a disease or condition caused by lacking of function of an endogenous gene in the host, such as a defective version of the GOI.

[0492] As used herein, "gene of interest” or GOI generally refers to a nucleic acid or polynucleotide sequence, such as a gene, an open reading frame (ORF), or a coding sequence for protein or RNA such as siRNA, miRNA, shRNA, etc. However, in certain circumstances or context, the term GOI also loosely refers to a protein (encoded by the GOI), or a disease or indication that can be remedied by the GOI, or a disease or indication can be (but is not

[0493] - 44 -

[0494] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 necessarily) caused by loss of function of the GOI.

[0495] For example, the gene GALGT2 encodes the protein GalNAc transferase (|3-1,4-N-acetylgalactosamine galactosyltransferase), which is an enzyme that transfers a complex sugar molecule onto a few specific proteins, including dystroglycan. Under normal circumstances, GalNAc transferase is found only at the neuromuscular junction (NMJ), where some components of the dystroglycan-associated protein complex are different than elsewhere in muscle. Importantly, at the NMJ, utrophin is present instead of dystrophin. In the mdx mouse model of muscular dystrophy, viral gene transfer of GALGT2 results in expression of GalNAc transferase across the entire muscle membrane, instead of just at the normal expression domain of the NMJ, as well as upregulation of utrophin across the entire muscle fiber. In the mdx mouse, this expression can correct muscle functional deficits to the same degree as does microdystrophin gene expression. Furthermore, overexpression of GALGT2 corrects muscle pathology in mouse models of other muscular dystrophies, including LGMD2A and congenital muscular dystrophy (MDC1 A). Thus, GALGT2 is a GOI for treating muscular dystrophy such as DMD, BMD, LGMD2A and MDC1 A, even though GALGT2 is not necessarily defective perse in the patient in need of treatment.

[0496] Thus, the GOI can be a gene (or protein) that, when expressed, replaces a mutated, damaged, or inactive gene or protein. The GOI can be a gene (or protein) that, when expressed, assists an already functioning process that requires modification for therapy in a disease, disorder, or dysfunction. The GOI can be a gene (or protein) that, when expressed, assists a dysfunctional process that requires modification for therapy in a disease, disorder, or dysfunction. A GOI nucleic acid sequence can comprise DNA, RNA, or synthetic nucleic acid molecule. The GOI can be a protein, an enzyme, a structural protein, a functional protein, or an adaptable protein based on cell function(s). The GOI can provide therapeutic benefits or a treatment modality for a disease, disorder, or dysfunction.

[0497] In certain embodiments, the GOI may be CRISPR-0as9, Gas 13, TALEN, or other genetic based gene editing protein that are required for intracellular delivery for their intended activity.

[0498] Any and all GOIs as used herein may require codon optimization for enhanced expression and activity via known computer based algorithms.

[0499] The rAAV viral particle for use herein can be produced by using the capsid of serotype of SLB-101 and may encode a gene of interest (GOI) useful for, e.g., gene therapy to treat a disease or condition. Representative (nonlimiting) gene of interest (GOI) may include: a gene responsible for I defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 20), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 2I), or a gene or coding sequence for NAGLU (a-N- acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type I IIB (MPS III B)), sulfamidase or SGSH (for mucopolysaccharidosis type III A or MPS 111 A, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), dystrophin or microdystrophin.

[0500] Diseases or conditions having a potential to benefit from the method of the invention includes: Huntington's disease, X-linked myotubular myopathy (XLMTM), Acid maltase deficiency {e.g., Pompe disease), Spinal Muscular

[0501] - 45 -

[0502] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0503] Atrophy (SMA), Myasthenia Gravis (MG), Amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Mitochondrial myopathy, Muscular dystrophies (Duchenne's muscular dystrophy, Myotonic dystrophy, Becker muscular dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Facioscapulohumeral muscular dystrophy (FSH), Congenital muscular dystrophy (CDM), Oculopharyngeal muscular dystrophy (OPMD), Distal muscular dystrophy, Emery- Dreifuss muscular dystrophy (EDMD), Mucopolysaccharidoses (MPS), Metachromatic leukodystrophy (MLD), Batten Disease, Rett Syndrome, Krabbe Disease, Canavan disease, X-Linked Retinoschisis, Achromatopsia (CNGB3 and CNGA3), Pompe, Fabry's disease, MPS I, II, IIIA, IIIB, Gaucher's disease, Danon Disease, A1 At Deficiency, Friedreich ataxia, Wilson's Disease, Batten Disease (CLN1, CLN3, CLN6, CLN8), Wolman Disease, Niemann-Pick Type C, CDKL5 deficiency Disorder, cardiomyopathies, RYR2-Mediated CPVT, CASQ2-Mediated CPVT, BAG3- Mediated DCM, TNNT2 DCM, and RBM20.

[0504] In certain embodiments, diseases or conditions having a potential to benefit from the method of the invention includes may include: Becker muscular dystrophy (BMD), Congenital muscular dystrophies (CMD), Bethlem CMD, Fukuyama CMD, Muscle-eye-brain diseases (MEBs), Rigid spine syndromes, Ullrich CMD, Walker- Warburg syndromes (WWS), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Motor neuron diseases including ALS (amyotrophic lateral sclerosis), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA).

[0505] In certain embodiments, diseases or conditions having a potential to benefit from the method of the invention may include ion channel diseases, which are typically marked by muscular weakness, absent muscle tone, or episodic muscle paralysis. They include Andersen-Tawil syndrome, Hyperkalemic periodic paralysis, Hypokalemic periodic paralysis, Myotonia congenita, Becker myotonia, Thomsen myotonia, Paramyotonia congenita, Potassium-aggravated myotonia.

[0506] In certain embodiments, diseases or conditions having a potential to benefit from the method of the invention may include mitochondrial diseases, which occur when structures that produce energy for a cell malfunction. Such diseases include: Friedreich's ataxia (FA), Mitochondrial myopathies, Kearns-Sayre syndrome (KSS), Leigh syndrome (subacute necrotizing encephalomyopathy), Mitochondrial DNA depletion syndromes, Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Myoclonus epilepsy with ragged red fibers (MERRF), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Progressive external opthalmoplegia (PEG).

[0507] In certain embodiments, diseases or conditions having a potential to benefit from the method of the invention may include myopathies, which is a disease of muscle in which the muscle fibers do not function properly, resulting in muscular weakness. Myopathies include: Cap myopathies, Centronuclear myopathies, Congenital myopathies with fiber type disproportion, Core myopathies, Central core disease, Multiminicore myopathies, Myosin storage myopathies, Myotubular myopathy, Nemaline myopathies, Distal myopathies, GNE myopathy / Nonaka myopathy / hereditary inclusion-body myopathy (HIBM), Laing distal myopathy, Markesberg-Griggs late-onset distal

[0508] - 46 -

[0509] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 myopathy, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, Vocal cord and pharyngeal distal myopathy, Welander distal myopathy, Endocrine myopathies, Hyperthyroid myopathy, Hypothyroid myopathy, Inflammatory myopathies, Dermatomyositis, Inclusion-body myositis, Polymyositis, Metabolic myopathies, Acid maltase deficiency (AMD, Pompe disease), Carnitine deficiency, Carnitine palmityl transferase deficiency, Debrancher enzyme deficiency (Cori disease, Forbes disease), Lactate dehydrogenase deficiency, Myoadenylate deaminase deficiency, Phosphofructokinase deficiency (Tarui disease), Phosphoglycerate kinase deficiency, Phosphoglycerate mutase deficiency, Phosphorylase deficiency (McArdle disease), Myofibrillar myopathies (MFM), Scapuloperoneal myopathy.

[0510] In certain embodiments, diseases or conditions having a potential to benefit from the method of the invention may include neuromuscular junction diseases, which result from the destruction, malfunction or absence of one or more key proteins involved in the transmission of signals between muscles and nerves. Such diseases include: Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG).

[0511] In certain embodiments, diseases or conditions having a potential to benefit from the method of the invention may include peripheral nerve diseases, in which the motor and sensory nerves that connect the brain and spinal cord to the rest of the body are affected, causing impaired sensations, movement or other functions. Such diseases include: Charcot-Marie-Tooth disease (CMT), Giant axonal neuropathy (GAN), muscle wasting in cachexia and aging.

[0512] In certain embodiments, the GOI is a microdystrophin gene.

[0513] In one aspect, the rAAV vector genome for use herein comprises a polynucleotide sequence encoding a microdystrophin protein, which is also known as "microD5,” "MD5,” or " D5” or "h- D5.” The microdystrophin protein provides stability to the muscle membrane during muscle contraction, e.g., microdystrophin acts as a shock absorber during muscle contraction. MD5 is a specific engineered 5-repeat microdystrophin protein that contains, from N- to C-terminus, the N-terminal actin binding domain, Hinge region 1 (H1), spectrin-like repeats R1, R16, R17, R23, and R24, Hinge region 4 (H4), and the C-terminal dystroglycan binding domain of the human full-length dystrophin protein. The protein sequence of this 5-repeat microdystrophin and the related dystrophin minigene are described in US10,479,821 & WO2016 / 115543 (incorporated herein by reference). A codon-optimized version of this 5-repeat microdystrophin sequence is described in WO2023 / 018854 (incorporated herein by reference).

[0514] In some embodiments, the microdystrophin protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide sequence encoding a microdystrophin protein comprises a codon optimized polynucleotide sequence, such as SEQ ID NO: 1. The polynucleotide sequence of SEQ ID NO: 1, and the protein sequence of SEQ ID NO: 2, are provided below. atgctgtggtgggaggaagtggaagattgctacgagcgcgaggacgtgcagaagaaaaccttcaccaaatgggtcaacgcccagttcagcaagttcggcaagc agcacatcgagaacctgttcagcgacctgcaggacggcagacggctgctggatctgctggaaggcctgaccggacagaagctgcccaaagagaagggcagc accagagtgcacgccctgaacaacgtgaacaaggccctgcgggtgctgcagaacaacaaTgtggacctGgtgaacatTggcagcacAgacatTgtggaTg gcaaccacaagctgaccctgggcctgatctggaacatcatcctgcactggcaagtgaagaacgtgatgaagaacatcatggccggcctgcagcagaccaacag cgagaagatcctgctgagctgggtgcgccagagcaccagaaactacccccaagtgaacgtgatcaacttcaccacctcttggagcgacggcctggccctgaatg ccctgatccacagccacagacccgacctgttTgactggaacagTgtGgtgtgtcagcagagcgccacccagaggctggaacacgccttcaatatcgccagata

[0515] - 47 -

[0516] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 ccagctgggcatTgagaagctgctggaccccgaggatgtggacaccacctaccccgacaagaaatccatcctgatgtatatcaccagcctgttccaggtgctgcct cagcaggtgtccatcgaggccatccaggaagtggaaatgctgcccagaccccccaaagtgaccaaagaggaacacttccagctgcaccaccagatgcactact ctcagcagatcaccgtgtccctggcccagggctacgagagaaccagcagccccaagccccggttcaagagctacgcctatacccaggccgcctacgtgaccac cagcgaccctaccagaagcccattccccagccagcatctggaagcccccgaggacaagagcttcggcagcagcctgatggaaagcgaagtgaacctggatag ataccagaccgccctggaagaggtgctgtcctggctgctgagcgccgaggatacactgcaggctcagggcgagatcagcaaTgaTgtggaagtGgtgaagga ccagttccacacccacgagggctacatgatggacctgacagcccaccagggcagagtgggcaacattctgcagctgggctccaagctgatcggcaccggcaag ctgagcgaggacgaagagacagaggtgcaggaacagatgaacctgctgaacagcagatgggagtgcctgagagtggccagcatggaaaagcagagcaac ctgcacagctacgtgcccagcacctacctgaccgagatcacccatgtgtcccaggccctgctggaagtggaacagctgctgaacgcccccgatctgtgcgccaag gacttcgaggatctgttcaagcaggaagagagcctgaagaatatcaaggactctctgcagcagtccagcggcagaatcgacatcatccacagcaagaaaacag ccgccctgcagtccgccacccccgtggaaagagtgaagctgcaggaagccctgtcccagctggacttccagtgggagaaagtgaacaagatgtacaaggacc ggcagggcagattTgaccgcagTgtggaaaagtggAggAggttccactacgacatcaagatcttcaaccagtggctgacAgaggccgagcagttcctgagaa agacccagatccccgagaactgggagcacgccaagtacaagtggtatctgaaagaactgcaggatggcatTggccagagacagacAgtGgtgcggacactg aatgccaccggcgaggaaatcatccagcagagcagcaagaccgacgccagtattctgcaggaaaagctgggcagcctgaacctgagatggcaggaagtgtg caagcagctgtccgaccggaagaagagactggaagaacagagTgaccagtggaagcggctgcatctgtcactgcaggaactgctGgtgtggctgcagctgaa ggaTgaTgagctgagcagacaggcccctatTggcggcgattttcccgcAgtgcagaaacagaacgaTgtgcaccgggccttcaagagagagctgaaaaca aaagaaccAgtgatcatgagcaccctggaaacAgtgcggatctttctgaccgagcagcccctggaaggactggaaaaactgtaccaggaacccagagagctg ccccctgaagaacgggcccagaacgtgaccagactgctgAggaagcaggccgaggaagtgaacacAgaatgggagaagctgaacctgcactcTgcTgac tggcagAggaagatTgaTgagacactggaacggctgcaggaactgcaggaggccacAgacgagctggacctgaaactgagacaggccgaagtgatcaag ggcagctggcagccagtgggcgacctgctgatcgacagcctgcaggatcacctggaaaaagtgaaagccctgagaggcgagatTgcccccctgaaagaaaa TgtgtcccaTgtgaacgacctggcccggcagctgacaacactgggcatccagctgagcccctacaacctgtccacactggaagatctgaacacccggtggaaa ctgctgcaggtggccgtggaagatagagtgcggcagctgcacgaggcccacagagattttggccctgcctcccagcacttcctgagcacatctgtgcagggcccct gggagagagccatctcccccaacaaggtgccctactacatcaaccacgagacacagaccacctgttgggaccaccccaagatgacAgagctgtaccagagcc tggccgacctgaacaatgtgAggttcagTgcctacAggaccgccatgaagctgcggagactgcagaaagctctgtgcctggacctgctgtccctgtccgccgcttg tgatgccctggaccagcacaacctgaagcagaacgaccagcccatggatatcctgcagatcatcaactgcctgaccaccatctacgaccgcctggaacaggaa cacaacaacctGgtgaatgtgcccctgtgTgtggacatgtgcctgaattggctgctgaatgtgtacgacaccggccggacaggccggatcagagtgctgagcttca agaccggcatcatcagcctgtgcaaggcccacctggaagataagtaccgctacctgttcaaacaggtggccagctccaccggcttttgcgaccagagaaggctgg gcctgctgctgcacgacagcatccagatccctagacagctgggcgaggtggcctctttTggcggcagcaatatTgagcctagTgtgcggagctgcttccagttTg ccaacaacaagcccgagatTgaggccgccctgttcctggactggatgcggctggaaccccagagcatggtgtggctgcctgtgctgcatagagtggccgctgcc gagacagccaagcaccaggccaagtgcaacatctgcaaagagtgccccatcatcggcttccggtacagaagcctgaagcacttcaactacgatatctgccagag ctgctttttcagcggacgggtggccaagggccacaaaatgcactaccccatggtggaatactgcacccccaccacctccggggaggatgtgcgggattttgccaag gtgctgaaaaacaagttccggaccaagcgctacttTgccaaacacccccggatgggctatctgcccgtgcagacagtgctggaaggcgacaacatggaaaccg acaccatgtag (SEQ ID NO: 1)

[0517] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALN NVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQ VNVINFTTSWSDGLALNALIHSHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYI TSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTT SDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGY MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHSYVPSTYLTEITHVSQ ALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKV NKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNA TGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWLQLKDDELSRQAPIG GDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNT EWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKEN VSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPN KVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQP MDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVAS STGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRV AAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKN KFRTKRYFAKHPRMGYLPVQTVLEGDNMETDTM (SEQ ID NO: 2)

[0518] As used herein, "codon-optimized” polynucleotide coding sequence refers to a polynucleotide sequence that has been altered I changed in some respect, such that the resulting codons are optimal for expression in a particular cell, host, or system, such as in a specific mammalian (human) cell type, e.g., muscle cells. Codon

[0519] - 48 -

[0520] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 optimization does not alter the amino acid sequence of the encoded protein, i.e., the codon optimized polynucleotide coding sequence, and the native sequence based on which codon optimization was performed, encode the same amino acid sequence.

[0521] In SEQ ID NO: 1 shown above, certain nucleotides are marked up as capital letters, and these nucleotides are collectively referred to herein as "capitalized nucleotides of SEQ ID NO: 1.” Specifically, the capitalized nucleotides of SEQ ID NO: 1 includes nucleotides 264, 273, 282, 291, 297, 303, 543, 555, 558, 627, 1110, 1113, 1122, 1656, 1665, 1678, 1681, 1722, 1815, 1830, 1833, 1989, 2031, 2052, 2055, 2079, 2097, 2115, 2157, 2181, 2290, 2316, 2343, 2346, 2356, 2364, 2367, 2406, 2532, 2550, 2559, 2844, 2881, 2889, 2896, 3081, 3099, 3339, 3354, 3363, 3384, 3405, and 3735 of SEQ ID NO: 1.

[0522] In certain embodiments, the polynucleotide encoding the GOI, microdystrophin, not only encode the amino acid sequence of SEQ ID NO: 2, but also shares the same set of capitalized nucleotides of SEQ ID NO: 1, yet they differ from SEQ ID NO: 1 at nucleotide positions other than the capitalized nucleotides of SEQ ID NO: 1 .

[0523] In certain embodiments, the polynucleotide encoding microdystrophin not only encode the amino acid sequence of SEQ ID NO: 2, but is also substantially identical to SEQ ID NO: 1 at the capitalized nucleotides of SEQ ID NO: 1, despite additional sequence changes (e.g., to result in 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% overall sequence identity) in positions of SEQ ID NO: 1 other than the capitalized nucleotides. In certain embodiments, the polynucleotide encoding microdystrophin is identical to SEQ ID NO: 1 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

[0524] 18. 19, or 20 capitalized nucleotides.

[0525] In certain embodiments, the polynucleotide encoding microdystrophin comprises, consists essentially of, or consists of a nucleotide sequence at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 1 . That is, the polynucleotide encoding microdystrophin encodes the amino acid of SEQ ID NO: 2, and further, is (1) identical to SEQ ID NO: 1 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

[0526] 15. 16. 17. 18. 19, or 20 capitalized nucleotides; and / or (2) substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg plot analysis).

[0527] Sequence percentage identity between any two or more related or unrelated polynucleotides, or between any two or more related or unrelated protein sequences, can be aligned and the percentage of the matches between the nucleotides or amino acid residues, respectively, can be calculated using any art recognized methods, such as the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403-10, 1990), which is available from online sources, such as the National Center for Biological Information (NCBI) website, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx, depending on the type of query and database. Similar web-based tools can be found at the EMBL-EBI website.

[0528] In certain embodiments, the polynucleotide encoding microdystrophin substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpgplot analysis).

[0529] In certain embodiments, the polynucleotide encoding microdystrophin comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 .

[0530] - 49 -

[0531] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0532] In some embodiments, the polynucleotide encoding the GOI is included in an adeno associated virus (AAV) vector genome, wherein the AAV vector genome is capable of being packaged inside an AAV capsid.

[0533] The packaging capacity of a typical AAV is generally about 4.7 kb, including about 0.2-0.3 kb of 5' and 3' ITR sequences, at least one (maybe both) of which are structural elements required for AAV vector genome packaging into the capsid.

[0534] In certain embodiments, the AAV vector genome comprises certain ITR structural elements, such as the Rep binding element (RBE), the internal hairpin within the TR (RBE'), and the terminal resolution site (TRS).

[0535] In certain embodiments, the polynucleotide encoding the GOI is operably linked to a transcriptional regulatory element. In certain embodiments, the transcriptional regulatory element comprises a promoter, such as a constitutive promoter, or a tissue-specific promoter (e.g., muscle specific promoter) {infra). An exemplary promoter is CK8 or variant thereof {infra).

[0536] In certain embodiments, the vector genome further comprises a polyadenylation signal sequence, such as the polyA signal sequence of any one of SEQ ID NOs: 8-11 {infra).

[0537] In certain embodiments, the vector genome further comprises a 3' ITR sequence, such as an AAV2 3' ITR sequence. In certain embodiments, the vector genome further comprises a 5' ITR sequence, such as an AAV2 5' ITR sequence. In certain embodiments, the 5' ITR sequence, and / or the 3' ITR sequence comprise or are SEQ ID NOs: 12 and 13, respectively.

[0538] In certain embodiments, the vector genome further comprises an intron and / or an exon sequence that enhances expression of the microdystrophin. In certain embodiments, the vector genome does not comprise intron and / or exon sequence that otherwise enhances expression of the microdystrophin.

[0539] In certain embodiments, the vector genome further comprises a 5' UTR sequence, and / or a 3' UTR sequence.

[0540] In certain embodiments, the AAV vector genome or the rAAV viral particle of the invention comprises, consists essentially of, or consists of, from 5' to 3', the following sequence elements: (1) a 5' ITR (such as a wildtype or modified AAV2 5' ITR, e.g., the 145-nt wild-type AAV2 5' ITR, or the 141 -nt modified AAV2 5' ITR), (2) a muscle-specific promoter (such as a CK8 promoter or a modified CK8 protein such as CK8e as described herein); (3) any one of the CpG reduced I eliminated codon optimized polynucleotide of the invention (such as SEQ ID NO: 1 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto); (4) a polyA signal sequence (such as any one of SEQ ID NOs: 8-11); and (5) a 3' ITR (such as a wild-type or modified AAV2 3' ITR, e.g, the 145-nt wild-type AAV2 3' ITR, or the 141 -nt modified AAV2 3’ ITR).

[0541] In certain embodiments, the rAAV vector genome or the rAAV viral particle for use in the invention to deliver a CpG codon optimized sequence encoding microdystrophin comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical thereto.

[0542] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTT

[0543] - 50 -

[0544] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0545] GGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTG

[0546] CGGCCGGCGCGCCACTTTAGACTAGCATGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCC

[0547] TGGTTATAATTAACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCAT

[0548] GCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCA

[0549] AGTCAGCCCTTGGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGC

[0550] ACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGC

[0551] TAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACA

[0552] GCACAGACAGACACTCAGGAGCCAGCCAAAACTAGAACCATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACG

[0553] AGCGCGAGGACGTGCAGAAGAAAACCTTCACCAAATGGGTCAACGCCCAGTTCAGCAAGTTCGGCAAGCAGCA

[0554] CATCGAGAACCTGTTCAGCGACCTGCAGGACGGCAGACGGCTGCTGGATCTGCTGGAAGGCCTGACCGGACA

[0555] GAAGCTGCCCAAAGAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGTGAACAAGGCCCTGCGGGTGCT

[0556] GCAGAACAACAATGTGGACCTGGTGAACATTGGCAGCACAGACATTGTGGATGGCAACCACAAGCTGACCCTG

[0557] GGCCTGATCTGGAACATCATCCTGCACTGGCAAGTGAAGAACGTGATGAAGAACATCATGGCCGGCCTGCAGC

[0558] AGACCAACAGCGAGAAGATCCTGCTGAGCTGGGTGCGCCAGAGCACCAGAAACTACCCCCAAGTGAACGTGAT

[0559] CAACTTCACCACCTCTTGGAGCGACGGCCTGGCCCTGAATGCCCTGATCCACAGCCACAGACCCGACCTGTTT

[0560] GACTGGAACAGTGTGGTGTGTCAGCAGAGCGCCACCCAGAGGCTGGAACACGCCTTCAATATCGCCAGATACC

[0561] AGCTGGGCATTGAGAAGCTGCTGGACCCCGAGGATGTGGACACCACCTACCCCGACAAGAAATCCATCCTGAT

[0562] GTATATCACCAGCCTGTTCCAGGTGCTGCCTCAGCAGGTGTCCATCGAGGCCATCCAGGAAGTGGAAATGCTG

[0563] CCCAGACCCCCCAAAGTGACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACTACTCTCAGCAGATCA

[0564] CCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTATACCC

[0565] AGGCCGCCTACGTGACCACCAGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCCCCGAGG

[0566] ACAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAACCTGGATAGATACCAGACCGCCCTGGAAGAGGT

[0567] GCTGTCCTGGCTGCTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAATGATGTGGAAGTGGT

[0568] GAAGGACCAGTTCCACACCCACGAGGGCTACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACAT

[0569] TCTGCAGCTGGGCTCCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAGAGACAGAGGTGCAGGAACA

[0570] GATGAACCTGCTGAACAGCAGATGGGAGTGCCTGAGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAG

[0571] CTACGTGCCCAGCACCTACCTGACCGAGATCACCCATGTGTCCCAGGCCCTGCTGGAAGTGGAACAGCTGCTG

[0572] AACGCCCCCGATCTGTGCGCCAAGGACTTCGAGGATCTGTTCAAGCAGGAAGAGAGCCTGAAGAATATCAAGG

[0573] ACTCTCTGCAGCAGTCCAGCGGCAGAATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCGCCAC

[0574] CCCCGTGGAAAGAGTGAAGCTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAGAT

[0575] GTACAAGGACCGGCAGGGCAGATTTGACCGCAGTGTGGAAAAGTGGAGGAGGTTCCACTACGACATCAAGATC

[0576] TTCAACCAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCCCCGAGAACTGGGAGCACGCC

[0577] AAGTACAAGTGGTATCTGAAAGAACTGCAGGATGGCATTGGCCAGAGACAGACAGTGGTGCGGACACTGAATG

[0578] CCACCGGCGAGGAAATCATCCAGCAGAGCAGCAAGACCGACGCCAGTATTCTGCAGGAAAAGCTGGGCAGCC

[0579] TGAACCTGAGATGGCAGGAAGTGTGCAAGCAGCTGTCCGACCGGAAGAAGAGACTGGAAGAACAGAGTGACC

[0580] AGTGGAAGCGGCTGCATCTGTCACTGCAGGAACTGCTGGTGTGGCTGCAGCTGAAGGATGATGAGCTGAGCA

[0581] GACAGGCCCCTATTGGCGGCGATTTTCCCGCAGTGCAGAAACAGAACGATGTGCACCGGGCCTTCAAGAGAGA

[0582] - 51 -

[0583] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0584] GCTGAAAACAAAAGAACCAGTGATCATGAGCACCCTGGAAACAGTGCGGATCTTTCTGACCGAGCAGCCCCTG GAAGGACTGGAAAAACTGTACCAGGAACCCAGAGAGCTGCCCCCTGAAGAACGGGCCCAGAACGTGACCAGA CTGCTGAGGAAGCAGGCCGAGGAAGTGAACACAGAATGGGAGAAGCTGAACCTGCACTCTGCTGACTGGCAG AGGAAGATTGATGAGACACTGGAACGGCTGCAGGAACTGCAGGAGGCCACAGACGAGCTGGACCTGAAACTG AGACAGGCCGAAGTGATCAAGGGCAGCTGGCAGCCAGTGGGCGACCTGCTGATCGACAGCCTGCAGGATCAC CTGGAAAAAGTGAAAGCCCTGAGAGGCGAGATTGCCCCCCTGAAAGAAAATGTGTCCCATGTGAACGACCTGG CCCGGCAGCTGACAACACTGGGCATCCAGCTGAGCCCCTACAACCTGTCCACACTGGAAGATCTGAACACCCG GTGGAAACTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGCACGAGGCCCACAGAGATTTTGGCCC TGCCTCCCAGCACTTCCTGAGCACATCTGTGCAGGGCCCCTGGGAGAGAGCCATCTCCCCCAACAAGGTGCCC TACTACATCAACCACGAGACACAGACCACCTGTTGGGACCACCCCAAGATGACAGAGCTGTACCAGAGCCTGG CCGACCTGAACAATGTGAGGTTCAGTGCCTACAGGACCGCCATGAAGCTGCGGAGACTGCAGAAAGCTCTGTG CCTGGACCTGCTGTCCCTGTCCGCCGCTTGTGATGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCC ATGGATATCCTGCAGATCATCAACTGCCTGACCACCATCTACGACCGCCTGGAACAGGAACACAACAACCTGGT GAATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAATGTGTACGACACCGGCCGGACAGGCCG GATCAGAGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCACCTGGAAGATAAGTACCGCTAC CTGTTCAAACAGGTGGCCAGCTCCACCGGCTTTTGCGACCAGAGAAGGCTGGGCCTGCTGCTGCACGACAGC ATCCAGATCCCTAGACAGCTGGGCGAGGTGGCCTCTTTTGGCGGCAGCAATATTGAGCCTAGTGTGCGGAGCT GCTTCCAGTTTGCCAACAACAAGCCCGAGATTGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTGGAACCCCA GAGCATGGTGTGGCTGCCTGTGCTGCATAGAGTGGCCGCTGCCGAGACAGCCAAGCACCAGGCCAAGTGCAA CATCTGCAAAGAGTGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCTGCCAGA GCTGCTTTTTCAGCGGACGGGTGGCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATACTGCACCCCCAC CACCTCCGGGGAGGATGTGCGGGATTTTGCCAAGGTGCTGAAAAACAAGTTCCGGACCAAGCGCTACTTTGCC AAACACCCCCGGATGGGCTATCTGCCCGTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCGACACCATGT AGGAAGTCTTTTAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTGTCAGCGGCCGCAGGAA CCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 15)

[0585] In certain embodiments, the rAAV vector genome or the rAAV viral particle for use in the invention to deliver a CpG codon optimized sequence encoding microdystrophin comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% identical thereto.

[0586] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGGCGCG CCACTTTAGACTAGCATGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTA ACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCATGCCATGTTCCC GGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTT GGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGG

[0587] - 52 -

[0588] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0589] GCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCT

[0590] GTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGA

[0591] CACTCAGGAGCCAGCCAAAACTAGAACCATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGA

[0592] CGTGCAGAAGAAAACCTTCACCAAATGGGTCAACGCCCAGTTCAGCAAGTTCGGCAAGCAGCACATCGAGAAC

[0593] CTGTTCAGCGACCTGCAGGACGGCAGACGGCTGCTGGATCTGCTGGAAGGCCTGACCGGACAGAAGCTGCCC

[0594] AAAGAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGTGAACAAGGCCCTGCGGGTGCTGCAGAACAAC

[0595] AATGTGGACCTGGTGAACATTGGCAGCACAGACATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATCT

[0596] GGAACATCATCCTGCACTGGCAAGTGAAGAACGTGATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAG

[0597] CGAGAAGATCCTGCTGAGCTGGGTGCGCCAGAGCACCAGAAACTACCCCCAAGTGAACGTGATCAACTTCACC

[0598] ACCTCTTGGAGCGACGGCCTGGCCCTGAATGCCCTGATCCACAGCCACAGACCCGACCTGTTTGACTGGAACA

[0599] GTGTGGTGTGTCAGCAGAGCGCCACCCAGAGGCTGGAACACGCCTTCAATATCGCCAGATACCAGCTGGGCAT

[0600] TGAGAAGCTGCTGGACCCCGAGGATGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATATCACC

[0601] AGCCTGTTCCAGGTGCTGCCTCAGCAGGTGTCCATCGAGGCCATCCAGGAAGTGGAAATGCTGCCCAGACCCC

[0602] CCAAAGTGACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACCGTGTCCCT

[0603] GGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTA

[0604] CGTGACCACCAGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCCCCGAGGACAAGAGCTT

[0605] CGGCAGCAGCCTGATGGAAAGCGAAGTGAACCTGGATAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTG

[0606] GCTGCTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAATGATGTGGAAGTGGTGAAGGACCA

[0607] GTTCCACACCCACGAGGGCTACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATTCTGCAGCT

[0608] GGGCTCCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAGAGACAGAGGTGCAGGAACAGATGAACCT

[0609] GCTGAACAGCAGATGGGAGTGCCTGAGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGCTACGTGCC

[0610] CAGCACCTACCTGACCGAGATCACCCATGTGTCCCAGGCCCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCC

[0611] GATCTGTGCGCCAAGGACTTCGAGGATCTGTTCAAGCAGGAAGAGAGCCTGAAGAATATCAAGGACTCTCTGC

[0612] AGCAGTCCAGCGGCAGAATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCGCCACCCCCGTGG

[0613] AAAGAGTGAAGCTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAGATGTACAAGGA

[0614] CCGGCAGGGCAGATTTGACCGCAGTGTGGAAAAGTGGAGGAGGTTCCACTACGACATCAAGATCTTCAACCAG

[0615] TGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAG

[0616] TGGTATCTGAAAGAACTGCAGGATGGCATTGGCCAGAGACAGACAGTGGTGCGGACACTGAATGCCACCGGC

[0617] GAGGAAATCATCCAGCAGAGCAGCAAGACCGACGCCAGTATTCTGCAGGAAAAGCTGGGCAGCCTGAACCTGA

[0618] GATGGCAGGAAGTGTGCAAGCAGCTGTCCGACCGGAAGAAGAGACTGGAAGAACAGAGTGACCAGTGGAAGC

[0619] GGCTGCATCTGTCACTGCAGGAACTGCTGGTGTGGCTGCAGCTGAAGGATGATGAGCTGAGCAGACAGGCCC

[0620] CTATTGGCGGCGATTTTCCCGCAGTGCAGAAACAGAACGATGTGCACCGGGCCTTCAAGAGAGAGCTGAAAAC

[0621] AAAAGAACCAGTGATCATGAGCACCCTGGAAACAGTGCGGATCTTTCTGACCGAGCAGCCCCTGGAAGGACTG

[0622] GAAAAACTGTACCAGGAACCCAGAGAGCTGCCCCCTGAAGAACGGGCCCAGAACGTGACCAGACTGCTGAGG

[0623] AAGCAGGCCGAGGAAGTGAACACAGAATGGGAGAAGCTGAACCTGCACTCTGCTGACTGGCAGAGGAAGATT

[0624] GATGAGACACTGGAACGGCTGCAGGAACTGCAGGAGGCCACAGACGAGCTGGACCTGAAACTGAGACAGGCC

[0625] GAAGTGATCAAGGGCAGCTGGCAGCCAGTGGGCGACCTGCTGATCGACAGCCTGCAGGATCACCTGGAAAAA

[0626] - 53 -

[0627] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0628] GTGAAAGCCCTGAGAGGCGAGATTGCCCCCCTGAAAGAAAATGTGTCCCATGTGAACGACCTGGCCCGGCAG CTGACAACACTGGGCATCCAGCTGAGCCCCTACAACCTGTCCACACTGGAAGATCTGAACACCCGGTGGAAAC TGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGCACGAGGCCCACAGAGATTTTGGCCCTGCCTCCC AGCACTTCCTGAGCACATCTGTGCAGGGCCCCTGGGAGAGAGCCATCTCCCCCAACAAGGTGCCCTACTACAT CAACCACGAGACACAGACCACCTGTTGGGACCACCCCAAGATGACAGAGCTGTACCAGAGCCTGGCCGACCT GAACAATGTGAGGTTCAGTGCCTACAGGACCGCCATGAAGCTGCGGAGACTGCAGAAAGCTCTGTGCCTGGAC CTGCTGTCCCTGTCCGCCGCTTGTGATGCCCTGGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGATA TCCTGCAGATCATCAACTGCCTGACCACCATCTACGACCGCCTGGAACAGGAACACAACAACCTGGTGAATGTG CCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAATGTGTACGACACCGGCCGGACAGGCCGGATCAGA GTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCACCTGGAAGATAAGTACCGCTACCTGTTCA AACAGGTGGCCAGCTCCACCGGCTTTTGCGACCAGAGAAGGCTGGGCCTGCTGCTGCACGACAGCATCCAGA TCCCTAGACAGCTGGGCGAGGTGGCCTCTTTTGGCGGCAGCAATATTGAGCCTAGTGTGCGGAGCTGCTTCCA GTTTGCCAACAACAAGCCCGAGATTGAGGCCGCCCTGTTCCTGGACTGGATGCGGCTGGAACCCCAGAGCATG GTGTGGCTGCCTGTGCTGCATAGAGTGGCCGCTGCCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGC AAAGAGTGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCTGCCAGAGCTGCTT TTTCAGCGGACGGGTGGCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACCACCTCC GGGGAGGATGTGCGGGATTTTGCCAAGGTGCTGAAAAACAAGTTCCGGACCAAGCGCTACTTTGCCAAACACC CCCGGATGGGCTATCTGCCCGTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCGACACCATGTAGGAAG TCTTTTAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTGTCAGCGGCCGCAGGAACCCCTA GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGA CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 20)

[0629] 4. Promoters

[0630] In certain embodiments, the codon optimized microdystrophin coding sequence is operably linked to a transcriptional regulatory element that includes a promoter operably linked to and is capable of driving the transcription of the microdystrophin coding sequence of the invention. The transcriptional regulatory element may further comprise one or more introns or exons that enhance expression of the microdystrophin encoded by the CpG reduced polynucleotide of the invention.

[0631] In certain embodiments, the transcriptional regulatory element comprises a constitutive promoter, such as a CMV promoter, a GAG promoter, an EF-1a promoter, a CB promoter, or a derivative thereof.

[0632] In certain embodiments, the transcriptional regulatory element comprises a muscle-specific control element.

[0633] For example, the muscle-specific control element can be selected from: CK8 promoter, cardiac troponin T (cTnT) promoter, CK7 promoter, CK9 promoter, truncated MCK (tMCK), myosin heavy chain (MHC) promoter, hybrid a-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), a muscle specific creatine kinase (MCK) promoter, human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), C5-12, murine creatine

[0634] - 54 -

[0635] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin I gene element, hypoxia-inducible nuclear factors, steroid-inducible element, or glucocorticoid response element (gre).

[0636] In certain embodiments, muscle-specific control element is 5' to a heterologous intron sequence (that enhanced microdystrophin expression), which is 5' to the microdystrophin coding sequence of the invention, which is 5' to an optional 3'-UTR region including a translation stop codon (such as TAG), a polyA adenylation signal (such as AATAAA), and an mRNA cleavage site (such as CA).

[0637] In certain embodiments, the muscle-specific control element comprises a CK8 promoter, such as one with the following sequence:

[0638] TAGACTAGCATGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCAG ACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCATGCCATGTTCCCGGCGAA GGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCA GCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACG AGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGC TCCTCTATATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCA GGAGCCAGCCA (CK8 PROMOTER, SEQ ID NO: 3)

[0639] In certain embodiments, the CK8 promoter may comprise an additional C at the 5' end and / or an additional dinucleotide GO at the 3' end. The CK8 promoter comprises a 5' end 130-bp enhancer element, followed by a 269- bp basal CK8 promoter, followed by a 48 bp or 50 bp MOK Exon 1 UTR sequence at the 3' end of the CK8 promoter. The 5' end 130-bp enhancer element can be duplicated (e.g., having two tandem copies compared to one copy in CK8) to further enhancer transcription.

[0640] Thus in certain embodiments, the CK8 promoter is modified as a CK8e promoter (SEQ ID NO: 4), which comprises two copies of the 130-bp enhancer of SEQ ID NO: 5, the 269-bp fragment of the basal CK8 promoter of SEQ ID NO: 3 (SEQ ID NO: 6), and the 48-bp or 50-bp MCK exon 1 UTR region sequence (SEQ ID NO: 7 or 16). TAGACTAGCATGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCAG ACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCATGTAGACTAGCATGCTGCC CATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCAGACATGTGGCTGCCCCCC CCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGCATGCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCG CCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGGCCATG GGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTG CTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAG GGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCCAGCCA (SEQ ID NO: 4) tagactagcatgctgcccatgtaaggaggcaaggcctggggacacccgagatgcctggttataattaacccagacatgtggctgcccccccccccccaacacctg ctgcctctaaaaataaccctgcatg (SEQ ID NO: 5, 130-bp enhancer) ccatgttcccggcgaagggccagctgtcccccgccagctagactcagcacttagtttaggaaccagtgagcaagtcagcccttggggcagcccatacaaggccat ggggctgggcaagctgcacgcctgggtccggggtgggcacggtgcccgggcaacgagctgaaagctcatctgctctcaggggcccctccctggggacagcccc tcctggctagtcacaccctgtaggctcctctatataacccaggggcacaggggctgccctc (SEQ ID NO: 6, 269-bp basal CK8 promoter) attctaccaccacctccacagcacagacagacactcaggagccagcca (UTR 48 bp MCK Exon 1, SEQ ID NO: 7). attctaccaccacctccacagcacagacagacactcaggagccagccagc (UTR 50 bp MCK Exon 1, SEQ ID NO: 16)

[0641] - 55 -

[0642] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0643] In certain embodiments, the muscle-specific control element comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 of WO2017 / 181015.

[0644] SEQ ID NO: 10 of WQ2017 / 181015 (SEQ ID NO: 17):

[0645] CAGCCACTAT GGGTCTAGGC TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG 60

[0646] CCTGGTTATA ATTAACCCAG ACATGTGGCT GCTCCCCCCC CCC AACACCT GCTGCCTGAG 120

[0647] CCTCACCCCC ACCCCGGTGC CTGGGTCTTA GGCTCTGTAC ACCATGGAGG AGAAGCTCGC 180

[0648] TCTAAAAATA ACCCTGTCCC TGGTGG 206

[0649] SEQ ID NO: 11 of WQ2017 / 181015 (SEQ ID NO: 18):

[0650] GCTGTGGGGG ACTGAGGGCA GGCTGTAACA GGCTTGGGGG CCAGGGCTTA TACGTGCCTG 60

[0651] GGACTCCCAA AGTATTACTG TTCCATGTTC CCGGCGAAGG GCCAGCTGTC CCCCGCCAGC 120

[0652] TAGACTCAGC ACTTAGTTTA GGAACCAGTG AGCAAGTCAG CCCTTGGGGC AGCCCATACA 180

[0653] AGGCCATGGG GCTGGGCAAG CTGCACGCCT GGGTCCGGGG TGGGCACGGT GCCCGGGCAA 240

[0654] CGAGCTGAAA GCTCATCTGC TCTCAGGGGC CCCTCCCTGG GGACAGCCCC TCCTGGCTAG 300

[0655] TCACACCCTG TAGGCTCCTC TATATAACCC AGGGGCACAG GGGCTGCCCC CGGGTCAC 358

[0656] In certain embodiments, the rAAV vectors of the invention can be operably linked to the muscle-specific control element comprising the MCK enhancer nucleotide sequence (see SEQ ID NO: 10 of WQ2017 / 181015, incorporated herein by reference) and / or the MCK promoter sequence (see SEQ ID NO: 11 of WQ2017 / 181015, incorporated herein by reference).

[0657] 5. PolyA Signal Sequence, Introns, Exons, UTRs

[0658] In certain embodiments, the rAAV further comprises a polyadenylation (polyA) signal sequence for inserting a polyA sequence into a transcribed mRNA.

[0659] In certain embodiments, the polyA signal sequence comprises or is SEQ ID NO: 8, with the AATAAA sequence capitalized and double underlined:

[0660] AATAAAaaatccttattttcattqqatctqtqtqttgqttttttqtqt (SEQ ID NO: 8)

[0661] In certain embodiments, the polyA sequence comprises or is a 197-bp SV40 polyA signal sequence: gatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaacca ttataaactacAATAAAcaaattaacaacaacaattacattcattttatatttcacicittcaciaaaaagqtcitaaaagcitttttta (SEQ ID NO: 9).

[0662] In certain embodiments, the polyA sequence comprises or is a 230-bp bGH polyA signal sequence: gtcgactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtc ctttcctAATAAAatoagoaaattocatcocattotctoaotaogtotcattctattctggggggtggggtggggcaooacaocaagggggaggattgggaaoac aatagcaggcatg (SEQ ID NO: 10).

[0663] In certain embodiments, the polyA sequence comprises or is a 127-bp rBG polyA signal sequence:

[0664] - 56 -

[0665] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 qatctttttccctctaccaaaaattatqaagacatcataaaaccccttqaacatctaacttctqqctAATAAAqqaaatttattttcattacaataqtqtqttgqaattttttq tgtctctcactcg (SEQ ID NO: 11).

[0666] 6. Production of rAA V and Host Cells

[0667] The rAAV viral particles and vector genomes for use in the method of the invention to deliver a GOI (e.g., CpG depleted codon optimized microdystrophin coding sequence) can be produced by any standard rAAV production methods, typically using a producer cell line.

[0668] General principles of rAAV production are reviewed in, for example, Carter, Current Opinions in Biotechnology 1533-1539, 1992; and Muzyczka, Curr. Topics in Microbial, and Immunol. 158:97-129, 1992). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072, 1984; Hermonat et al., Proc. Natl. Acad. Sci. U.S.A. 81 :6466, 1984; Tratschin et al., Mol. Cell. Biol. 5:3251, 1985; McLaughlin et al., J. Virol. 62: 1963, 1988; and Lebkowski et al., Mol. Cell. Biol. 7:349, 1988; Samulski et al., J. Virol. 63:3822-3828, 1989; U.S. Patent No. 5,173,414; WO 95 / 13365, and corresponding U.S. Patent No. 5,658,776; WO95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (POT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin etal., Vaccine 13:1244-1250, 1995; Paul et al., Human Gene Therapy 4:609-615, 1993; Clark etal., Gene Therapy 3:1124-1132, 1996; U.S. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production.

[0669] Overall, a number of strategies differing in principles have been used for rAAV production, all of which can be used to produce the subject rAAV.

[0670] In certain embodiments, the subject rAAV is produced based on the helper-virus-free transient transfection method, with all cis and trans components (vector plasmid and packaging plasmids, along with helper genes isolated from adenovirus) in suitable host cells such as 293 cells. The transient-transfection method is simple in vector plasmid construction and generates high-titer AAV vectors that are free of adenovirus. The VP1 capsid proteins can be encoded by one of the plasmids used in transient transfection of the producer cell line.

[0671] Thus, in certain embodiments, the polynucleotide for use in the method of the invention includes DNA plasmids comprising rAAV vector genomes. Such DNA plasmids can be used in the standard triple transfection method to produce rAAV. Specifically, DNA plasmids of the invention are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, El-deleted adenovirus or herpes virus) for assembly of the rAAV vector genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell, are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from ( / .e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV

[0672] - 57 -

[0673] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 serotypes AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrhIO, AAVrh.32, AAVrh34, or AAVrh.74. In certain embodiments, the capsid is a modified capsid such as SLB- 101.

[0674] Transient Transfection of Packaging Cell Line (HEK293)

[0675] In particular, in certain embodiments, the AAV vector is produced using transient transfection of a packaging cell line such as HEK293 cells. This is the most established AAV production method comprising plasmid transfection of human embryonic HEK293 cells. Typically, HEK293 cells are simultaneously transfected by a vector plasmid (containing the gene of interest, such as the subject polynucleotide encoding both the dystrophin minigene and the one or more additional coding sequences), and one or two helper plasmids, using calcium phosphate or polyethyleneimine (PEI), a cationic polymer.

[0676] The helper plasmid(s) allow the expression of the four Rep proteins, the three AAV structural proteins VP1 , VP2, and VP3, the AAP, and the adenoviral auxiliary functions E2A, E4, and VARNA. The additional adenoviral E1 A / E1 B co-factors necessary for rAAV replication are ex-pressed in HEK293 producer cells. Rep-cap and adenoviral helper sequences are either cloned on two separate plasmids or combined on one plasmid, hence both a triple plasmid system and a two plasmid system for transfection are possible. The triple plasmid protocol lends versatility with a cap gene that can easily be switched from one serotype to another.

[0677] Plasmids can be produced by conventional techniques in E. coli using bacterial origin and anti-biotic- resistance gene or by minicircle technology.

[0678] Transient transfection in adherent HEK293 cells has been used for large-scale manufacturing of rAAV vectors. Recently, HEK293 cells have also been adapted to suspension conditions to be economically viable in the long term.

[0679] HEK293 lines are usually propagated in DMEM completed with L-glutamine, 5%— 10% of fetal bovine serum (FBS), and 1 % penicillin-streptomycin, except for suspension HEK293 cells that are maintained in serum-free suspension F17, Expi293, or other manufacturer-specific media. For adherent cells, the percentage of FBS can be reduced during AAV production to limit contamination by animal-derived components.

[0680] Generally, the rAAV vectors are recovered 48-72 hr after plasmid transfection from the cell pellet and / or supernatant, depending on the serotype.

[0681] Infection of Mammalian Cells with rHSV Vectors

[0682] HSV is a helper virus for replication of AAV in permissive cells. Thus, the HSV can serve both as a helper and as a shuttle to deliver the necessary AAV functions that support AAV genome replication and packaging to the producing cells.

[0683] AAV production based on co-infection with rHSV can efficiently generate a large amount of rAAV. In addition to high overall yields (up to 1.5x105vg / cell), the method is further advantageous in that it creates rAAV stocks with apparently increased quality as measured by an improved viral potency.

[0684] In this method, cells, typically the hamster BHK21 cell line or the HEK293 and derivatives, are infected with

[0685] - 58 -

[0686] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 two rHSVs, one carrying the gene of interest bracketed by AAV ITR (rHSV-AAV), and the second with the AAV rep and cap ORFs of the desired serotype (rHSVrepcap). After 2-3 days, the cells and / or the media are collected, and rAAV is purified over multiple purification steps to remove cellular impurities, HSV-derived contaminants, and unpackaged AAV DNA.

[0687] Thus, in some embodiments, HSV serves as a helper virus for AAV infection. In some embodiments, AAV growth is accomplished using non-replicating mutants of HSV with ICP27 deleted.

[0688] Certain methods for producing recombinant AAV viral particles in a mammalian cell have been known in the art and improved over the past decade. For example, U.S. Application Publication No. 20070202587 describes recombinant AAV production in mammalian cells based on co-infection of the cells with two or more replicationdefective recombinant HSV vectors. U.S. Application Publication No. 20110229971 and Thomas et al. (Hum. Gene Ther. 20(8):861 -870, 2009) describes a scalable recombinant AAV production method using recombinant HSV type 1 coinfection of suspension-adapted mammalian cells. Adamson-Small et al. (Hum. Gene Ther. Methods 28(1): 1- 14, 2017) describes an improved AAV production method in a serum-free suspension manufacturing platform using the HSV system.

[0689] In certain other embodiments, the rAAV for use in the method of the invention is produced using a recombinant herpes simplex virus (rHSV)-based AAV production system, which utilizes rHSV vectors to bring the AAV vector and the Rep and Cap genes ( / .e., the modified VP1 capsid gene of the invention) into the producer cells. The modified cap gene can be present in the rHSV vector that may also hosts the rAAV genome.

[0690] In certain embodiments, the AAV vectors of for use in the method of the invention are produced according to the method described in Adamson-Small etal. (Molecular Therapy - Methods & Clinical Development (2016) 3, 16031; doi:10.1038 / mtm.2016.31 , incorporated herein by reference), a scalable method to produce high-titer and high quality adeno-associated type 9 vectors using the HSV platform. It is a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 x1014rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 xio5vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. This method can also be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

[0691] Infection of Insect Cells with Recombinant Baculovirus

[0692] In certain further embodiments, the rAAV for use in the method of the invention is produced using a baculovirus system that requires simultaneous infection of insect cells with several baculovirus vectors to deliver the AAV vector cassette and the Rep and Cap genes ( / .e., the modified VP1 capsid gene of the invention).

[0693] The baculovirus-Sf9 platform has been established as a GMP-compatible and scalable alternative AAV production method in mammalian cells. It can generate up to 2x105vector genomes (vg) per cell in crude harvests.

[0694] - 59 -

[0695] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0696] Current protocol involves infection of the Sf9 insect cells with two recombinant baculoviruses a baculovirus expression vector (BEV) allowing the synthesis of Rep78 / 52 and Caps, and a recombinant baculovirus carrying the gene of interest flanked by the AAV ITRs. Several serum-free media are adapted for Sf9 cell growth in suspension.

[0697] The dual-baculovirus-Sf9 production system has many advantages over other production platforms regarding these safety issues: (1) the use of serum-free media; (2) despite the discovery of adventitious virus transcripts in Sf cell lines, most viruses infecting insects do not replicate actively in mammalian cells; and (3) no helper virus is required for rAAV production in insect cells besides baculovirus.

[0698] In certain embodiments, stable Sf9 insect cell lines expressing Rep and Cap proteins are used, thus requiring the infection of only one recombinant baculovirus to produce infectious rAAV vectors at high yield. Mammalian Stable Cell Lines.

[0699] The rAAV vectors can also be efficiently and produced in large scale using stable mammalian producer cells stably expressing rep and cap genes. Such cells can be infected by wild-type Ad5 helper virus (which is genetically stable and can be easily produced at high titers) to induce high-level expression of rep and cap. Infectious rAAV vectors can be generated upon infection of these packaging cells lines with wild-type Ad type 5, and providing the rAAV genome by either plasmid transfection or after infection with a recombinant Ad / AAV hybrid virus.

[0700] Alternatively, Ad can be replaced by HSV-1 as the helper virus.

[0701] In certain embodiments, the host cell for producing rAAV particles is a HeLa cell, a Cos7 cell, a HEK293 cell, an A549 cell, a BHK cell, a Vero cell, an RD cell, an HT-1080 cell, an ARPE-19 cell, or a MRC-5 cell. In certain embodiments, the host cell is a HeLa cell or a 293 / 293T cell.

[0702] Suitable stable mammalian producer cells may include HeLa-derived producer cell lines, A549 cells, or HEK293 cells. A preferred HeLa cell line is HeLaS3 cells, a suspension adapted HeLa subclone.

[0703] The AAV vectors for use in the method described herein can be manufactured in animal components-free medium, preferably at 250-L scale, or 2,000-L commercial scale.

[0704] Regardless of how the rAAV viral particle of the invention is produced, the resulting rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther. 10(6): 1031 -1039, 1999; Schenpp and Clark, Methods Mol. Med. 69:427-443, 2002; U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0705] The disclosure provides packaging I producer cells that produce infectious rAAV. In one embodiment, packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

[0706] In certain embodiments, the rAAV for use in the method of the invention is produced based on certain AAV

[0707] - 60 -

[0708] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 producer cell lines derived from, e.g., HeLa or A549 or HEK293 cells, which stably harbored AAV Rep / cap genes. The AAV vector cassette can either be stably integrated in the host genome or be introduced by an adenovirus that contained the cassette.

[0709] In certain embodiments, such producer cell line for rAAV production comprises an rAAV provirus that encodes the microdystrophin flanked by the AAV ITR sequences, wherein the rAAV provirus is integrated into the genome of the producer cell line for rAAV production.

[0710] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV vector genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., Proc. Natl. Acad. Sci. U.S.A. 79:2077-2081, 1982), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., Gene 23:65-73, 1983) or by direct, blunt-end ligation (Senapathy & Carter, J. Biol. Chem. 259:4661-4666, 1984). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV.

[0711] Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0712] Thus, any of the packaging cells are within the scope of the host cell of the invention that comprise a polynucleotide, an AAV vector genome, or an AAV viral particle of the invention.

[0713] 7. Treatment of Muscular Dystrophy using rAA V Gene Delivery

[0714] Another aspect of the invention provides a method of treating a muscular dystrophy in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of the polynucleotide of the invention, the rAAV vector genome or the rAAV viral particle of the invention, or the pharmaceutical composition of the invention.

[0715] In certain embodiments, the muscular dystrophy is characterized by loss-of-function mutation in the dystrophin gene.

[0716] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy.

[0717] Thus a related aspect of the invention provides a method of treating muscular dystrophy (such as DMD and BMD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a polynucleotide encoding a gene of interest (GOI) encoding a functional version of the gene defective in the muscular dystrophy, such as a microdystrophin gene, wherein the rAAV vector genome comprises any of the CpG reduced codon optimized polynucleotide of the invention (such as SEQ ID NO: 1) and wherein the rAAV viral particle comprises a capsid of the serotype of SLB-101 .

[0718] - 61 -

[0719] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0720] In certain embodiments, the microdystrophin gene comprises a coding sequence for the R1, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as one described in PCT / US2016 / 013733).

[0721] In certain embodiments, the microdystrophin gene comprises a coding sequence for the microdystrophin protein of SEQ ID NO: 2, and the coding sequence comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% identical thereto. Optionally, the coding sequence is identical to SEQ ID NO: 1 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides, further optionally, the coding sequence substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands).

[0722] In certain embodiments, the method further comprises producing the rAAV prior to administering to the subject the rAAV so produced.

[0723] In any of the methods of the invention, the rAAV vector can be administered by intramuscular injection or intravenous injection.

[0724] In any of the methods of the invention, the rAAV vector or composition is administered systemically. For example, the rAAV vector or composition is parentally administration by injection, infusion or implantation.

[0725] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is between 4 to <7 years of age, 7 to <12 years of age, 0 to < 4 years of age, 12 to < 18 years of age, or 10 to < 18 years of age.

[0726] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) has, prior to treatment by the subject rAAV (e.g., SGT-003), an ambulatory status characterized by an ability to complete a 10-meter walk / run test in < 30 seconds, optionally the human subject is between 0 to <18 years of age.

[0727] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) has, prior to treatment by the subject rAAV (e.g., SGT-003), an ambulatory status characterized by an ability to complete a 10-meter walk / run test in < 10 seconds, wherein the human subject is between 7 to <12 years of age.

[0728] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) has, prior to treatment by the subject rAAV (e.g., SGT-003), an ambulatory status characterized by an ability to complete a 10-meter walk / run test in 10-15 seconds, wherein the human subject is between 12 to <18 years of age.

[0729] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is, prior to treatment by the subject rAAV (e.g., SGT-003), non-ambulatory, but has been previously ambulatory by history.

[0730] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) has established clinical diagnosis of DMD and documented dystrophin gene mutation predictive of DMD phenotype (e.g., as confirmed by a suitable genetic testing).

[0731] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is, prior to treatment by the subject rAAV (e.g., SGT-003), negative for anti-AAV antibodies.

[0732] - 62 -

[0733] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0734] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is or has been, prior to treatment by the subject rAAV (e.g., SGT-003), administered a stable daily oral steroid regimen comprising, consisting essentially of, or consisting of: at least 0.5 mg / kg / day of prednisone, or 0.75 mg / kg / day of deflazacort, for >12 weeks prior to treatment. Optionally, the dose of the stable daily oral steroid regimen is adjusted according to weight-based modifications consistent with clinical practice. Further optionally, the human patient is between 4 to <18 years of age.

[0735] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is, prior to treatment by the subject rAAV (e.g., SGT-003), characterized by an ability to rise from supine in 3 to <7 seconds, optionally the human subject is between 7 to <12 years of age.

[0736] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is, prior to treatment by the subject rAAV (e.g., SGT-003), characterized by an ability to meet Performance of Upper Limb (PUL) 2.0 criteria (e.g., having an entry item score > 3 and total score of < 40), optionally the human subject is between 10 to <18 years of age.

[0737] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) has, prior to treatment by the subject rAAV (e.g., SGT-003), a body weight of <90 kg.

[0738] In certain embodiments, the human subject that can be treated by the subject rAAV (e.g., SGT-003) is, prior to treatment by the subject rAAV (e.g., SGT-003), associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive, in the human DMD gene (e.g., as documented by a genetic report and / or confirmed by a suitable genetic testing such as MLPA, NGS, and / or Sanger Sequencing).

[0739] 8. Pharmaceutical Composition and Uses Thereof

[0740] The disclosure also provides a composition, such as a pharmaceutical composition, comprising any of the rAAV vectors, viral particles and vector genome comprising the GOI coding sequence for use in the method of the invention.

[0741] In certain embodiments, the active ingredient for use in the pharmaceutical composition is SGT-003.

[0742] In certain embodiments, the composition is a pharmaceutical composition, which may further comprise a therapeutically compatible carrier, excipient, diluents and / or adjuvants, in addition to the SGT-003 drug substance as the active ingredient. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and / or nonionic surfactants such as Tween, poloxamers, or polyethylene glycol (PEG).

[0743] In another embodiment, the composition comprises any of the rAAV vectors, viral particles and vector

[0744] - 63 -

[0745] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 genome comprising the GOI coding sequence described herein (e.g., CpG reduced codon optimized microdystrophin coding sequence) for use in treating a subject suffering from a disease or condition, such as dystrophinopathy or a muscular dystrophy, such as DMD or Becker Muscular dystrophy.

[0746] The compositions (e.g., pharmaceutical compositions) for use in the method of the invention can be formulated for intramuscular injection or intravenous injection. The composition of the invention can also be formulated for systemic administration, such as parental administration by injection, infusion or implantation. In addition, any of the compositions are formulated for administration to a subject suffering from dystrophinopathy or a muscular dystrophy, such as DMD, Becker muscular dystrophy or any other dystrophin associated muscular dystrophy.

[0747] In a further embodiment, the disclosure provides for use of any of the rAAV vectors, viral particle and vector genome comprising the GOI encoding sequence described herein (e.g, CpG reduced codon optimized microdystrophin coding sequence) for preparation of a medicament for reducing the subject suffering from dystrophinopathy or muscular dystrophy, such as DMD, Becker muscular dystrophy or any other dystrophin associated muscular dystrophy.

[0748] In any of the uses of the invention, the medicament can be formulated for intramuscular injection. In addition, any of the medicaments may be prepared for administration to a subject suffering from muscular dystrophy such as DMD or any other dystrophin associated muscular dystrophy.

[0749] 9. Dosing, Administration, and Kit

[0750] Titers of rAAV (such as SGT-003) to be administered in methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV (such as SGT-003) may range from about 1 x106, about 1 x107, about 1 x108, about 1 xio9, about 1 xio10, about 1 xio11, about 1 xio12, about 1 x1013, about 1 xio14, about 2x1014, about 3x1014, about 5x1014, about 1 xio15, about 2x1015, about 3x1015, about 5x1015, about 1 xi o16or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) or viral genomes per kg of patient body weight (vg / kg).

[0751] In certain embodiments, the titer or dose of the rAAV (e.g., SGT-003) administered is 1 E14 (1 x1014) vg / kg.

[0752] The method of the invention comprises the step of administering an effective dose, or effective single dose or multiple doses, of a composition comprising the rAAV described herein (such as SGT-003) to a subject in need thereof. If the dose is administered prior to development of a disorder / disease, the administration is prophylactic. If the dose is administered after the development of a disorder / disease, the administration is therapeutic. In embodiments of the invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, that slows or prevents progression to a disorder / disease state, that slows or prevents progression of a disorder / disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and / or that prolongs survival.

[0753] Administration of an effective dose of the compositions may be by routes standard in the art including, but

[0754] - 64 -

[0755] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration a may be chosen and / or matched by those skilled in the art taking into account the infection and / or disease state being treated and the target cells / tissue(s) that are to express the one or more GOI coding sequences and / or microdystrophin.

[0756] Specifically, the formulations described herein may be administered by, without limitation, injection, infusion, perfusion, inhalation, lavage, and / or ingestion. Routes of administration may include, but are not limited to, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topically, intratumoral, intramuscular, intravesicular, intrapericardial, intraumbilical, intraocularal, mucosal, oral, subcutaneous, and / or subconjunctival. In certain embodiments, the formulations described herein may be administered by infusion or intravenous infusion.

[0757] In certain embodiments, the formulations described herein (e.g., SGT-003) is administered by a single intravenous (IV) infusion.

[0758] In certain embodiments, the formulations described herein (e.g., SGT-003) is administered over a target duration of 60 minutes, at a rate not less than 5 mL / kg / hour and not more than 10 mL / kg / hour.

[0759] In particular, actual administration of rAAV (such as SGT-003) may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal, such as the skeletal muscles. Administration according to the invention includes, but is not limited to, injection into muscle, the bloodstream and / or directly into the liver. Simply re-suspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV).

[0760] Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0761] The dose of rAAV (such as SGT-003) to be administered in methods disclosed herein will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art.

[0762] The actual dose amount administered to a particular subject may also be determined by a physician, a veterinarian, or a researcher, taking into account parameters such as, but not limited to, physical and physiological factors including body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject, and / or route of administration.

[0763] Titers of each rAAV (such as SGT-003) administered may range from about 1 x106, about 1 x107, about 1 xio8, about 1 xio9, about 1 x1O10, about 1 xio11, about 1 xio12, about 1 xio13, about 1 xio14, about 5x1014, about 1 x1015, or about 5x1015or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) ( / .e., 1 x107vg, 1 x108vg, 1 x109vg, 1 xio10vg, 1 x1011vg, 1 x1012vg, 1 x1013vg, 1 xio14vg, 1 x1015

[0764] - 65 -

[0765] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 vg, respectively). Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of bodyweight ( / .e., 1 xio10vg / kg, 1 *1011vg / kg, 1 x1012vg / kg, 1 x1013vg / kg, 1 x1014vg / kg, 1 x1015vg / kg respectively). Methods for tittering AAV are described in Clark et al., Hum. Gene Ther. 10:1031 -1039, 1999.

[0766] Exemplary doses may range from about 1 x101° to about 1 x1015vector genomes (vg)Zkilogram of body weight. In some embodiments, doses may comprise 1 xio10vg / kg of body weight, 1 xio11vg / kg of body weight, 1 xio12vg / kg of body weight, 1 xio13vg / kg of body weight, 1 x1014vg / kg of body weight, or 1 xio15vg / kg of body weight. Doses may comprise 1 x101° vg / kg / day, 1 x1011vg / kg / day, 1 x1012vg / kg / day, 1 x1013vg / kg / day, 1 x1014vg / kg / day, or 1 x1015vg / kg / day. Doses may range from 0.1 mg / kg / day to 5 mg / kg / day or from 0.5 mg / kg / day to 1 mg / kg / day or from 0.1 mg / kg / day to 5 pig / kg / day or from 0.5 mg / kg / day to 1 pig / kg / day. In other non-limiting examples, a dose may comprise 1 pig / kg / day, 5 pig / kg / day, 10 pig / kg / day, 50 pig / kg / day, 100 pig / kg / day, 200 pig / kg / day, 350 pig / kg / day, 500 pig / kg / day, 1 mg / kg / day, 5 mg / kg / day, 10 mg / kg / day, 50 mg / kg / day, 100 mg / kg / day, 200 mg / kg / day, 350 mg / kg / day, 500 mg / kg / day, or 1000 mg / kg / day. Therapeutically effective amounts may be achieved by administering single or multiple doses during the course of a treatment regimen ( / .e., days, weeks, months, etc.).

[0767] In some embodiments, the pharmaceutical composition is in a dosage form of 10 mL of aqueous solution having at least 1 .6x1013or 1 .6x1014vector genomes. In some embodiments, the dosage has a potency of at least 2x1012or 2x1013vector genomes per milliliter. In some embodiments, the dosage comprises a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride. In some embodiments, the pharmaceutical composition is in a dosage form of 10 mL of a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride; and having at least 1 .6x1013or 1.6x1014vector genomes.

[0768] In some embodiments, the pharmaceutical composition may be a dosage comprising between 1 x101° and 1 x1015vector genomes in 10 mL aqueous solution; between 1 x1011and 1 x1014vector genomes in 10 mL aqueous solution; between 1 x 1012and 2x1013vector genomes in 10 mL aqueous solution; or greater than or equal to about 1 .6x1013vector genomes in 10 mL aqueous solution. In some embodiments the aqueous solution is a sterile aqueous solution comprises about 10 mM L histidine pH 6.0, with 150 mM sodium chloride, and 1 mM magnesium chloride. In some embodiments, the dosage has a potency of greater than about 1 xio12vector genomes per milliliter (vg / mL), greater than about 1 x1013vg / mL, greater than about 2x1013vg / mL, greater than about 3x1013vg / mL, or greater than about 4x1013vg / mL.

[0769] In some embodiments, at least one AAV vector is provided as part of a pharmaceutical composition. The pharmaceutical composition may comprise, for example, at least 0.1% w / v of the AAV vector. In some other embodiments, the pharmaceutical composition may comprise between 2% to 75% of compound per weight of the pharmaceutical composition, or between 25% to 60% of compound per weight of the pharmaceutical composition.

[0770] In some embodiments, the dosage is in a kit. The kit may further include directions for use of the dosage.

[0771] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be

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[0773] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropy Icell ulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0774] In some embodiments, for injection, formulations may be made as aqueous solutions, such as in buffers including, but not limited to, Hanks' solution, Ringer's solution, and / or physiological saline. The solutions may contain formulator agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the formulation may be in lyophilized and / or powder form for constitution with a suitable vehicle control (e.g., sterile pyrogen-free water) before use.

[0775] Any formulation disclosed herein may advantageously comprise any other pharmaceutically acceptable carrier or carriers which comprise those that do not produce significantly adverse, allergic, or other untoward reactions that may outweigh the benefit of administration, whether for research, prophylactic, and / or therapeutic treatments. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, which is incorporated by reference herein for its teachings regarding the same. Moreover, formulations may be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the United States FDA's Division of Biological Standards and Quality Control and / or other relevant U.S. and foreign regulatory agencies.

[0776] Exemplary, generally used pharmaceutically acceptable carriers may comprise, but are not limited to, bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, and vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and / or lubricants.

[0777] Exemplary buffering agents may comprise, but are not limited to, citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0778] Exemplary preservatives may comprise, but are not limited to, phenol, benzyl alcohol, meta-cresol, methylparaben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, and / or 3-pentanol.

[0779] Exemplary isotonic agents may comprise polyhydric sugar alcohols comprising, but not limited to, trihydric or higher sugar alcohols, (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and / or mannitol).

[0780] Exemplary stabilizers may comprise, but are not limited to, organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, and / or polysaccharides.

[0781] Formulations may also be depot preparations. In some embodiments, such long-acting formulations may

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[0783] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 be administered by, without limitation, implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, compounds may be formulated with suitable polymeric and / or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0784] Additionally, in various embodiments, the AAV vectors may be delivered using sustained-release systems, such as semipermeable matrices of solid polymers comprising the AAV vector. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release capsules may, depending on their chemical nature, release the vector following administration for a few weeks up to over 100 days.

[0785] The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0786] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0787] Muscle tissue is an attractive target for in vivo DNA delivery, because it is not a vital organ and is easy to access. As used herein, "muscle cell” or "muscle tissue” is meant a cell or group of cells derived from muscle of any kind (for example, skeletal muscle and smooth muscle, e.g., from the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts.

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[0789] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0790] EXAMPLE

[0791] Example 1 A Study of SGT-003 Gene Therapy in Duchenne Muscular Dystrophy (INSPIRE DUCHENNE) (NCT06138639)

[0792] This is a first-in-human Phase 1 / 2 ongoing multicenter, open-label, non-randomized study to investigate the safety, tolerability, and efficacy of a single intravenous (IV) infusion of SGT-003 in pediatric male participants (study subjects) with Duchenne muscular dystrophy. There currently are 5 cohorts planned in this study. Cohort 1 includes participants 4 to < 7 years of age. Cohort 2 includes participants 7 to < 12 years of age. Cohort 3 includes participants 0 to < 4 years of age. Cohort 4 includes participants 12 to < 18 years of age. Cohort 5 includes participants 10 to < 18 years of age. Initiation of participant enrollment in Cohorts 4 and 5 will be subject to the accrual of safety and efficacy data from Cohorts 1-3. All participants will receive SGT-003 and will be enrolled in the study for 5 total years for long-term follow up.

[0793] The estimated enrollment for this clinical trial is 40 patients. To date, at least 15 patients have been dosed. The data disclosed herein concerning this clinical trial, unless stated otherwise, result from the first three patients who have reached the 90-day follow up milestone with two of these patients having reached the 180-day follow up milestone.

[0794] Recruitment and eligibility for this Inspire Duchenne clinical trial is dependent on participants meeting the following inclusion criteria and exclusion criteria as well as any additional criteria set forth in the clinical trial protocol.

[0795] Inclusion Criteria:

[0796] • Cohort 1 : 4 to <7 years of age

[0797] • Cohort 2: 7 to <12 years of age

[0798] • Cohort 3: 0 to < 4 years of age

[0799] • Cohort 4: 12 to < 18 years of age

[0800] • Cohort 5: 10 to < 18 years of age

[0801] • Participant ambulatory status at the time of Screening Part A or Rescreening, with reference to FIG. 2A, as defined by the ability to complete a 10-meter walk / run test in < 30 seconds: o Cohorts 1, 2, and 4: Ambulatory o Cohort 3: Either ambulatory or non-ambulatory o Cohort 5: Non-ambulatory, but having been previously ambulatory by history

[0802] • Established clinical diagnosis of DMD and documented dystrophin gene mutation predictive of DMD phenotype confirmed by Sponsor genetic testing. In cases where a genotype may be predictive of residual dystrophin production and / or a clear clinical diagnosis of DMD cannot be made (e.g., due to age), evaluation of dystrophin levels in baseline muscle biopsies may be required to determine eligibility under this criterion.

[0803] • Negative for AAV antibodies, including AAV9.

[0804] • Steroid regimen: o Cohorts 1 , 2, 4, and 5: A stable daily oral steroid regimen of at least 0.5 mg / kg / day of prednisone

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[0806] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 or 0.75 mg / kg / day of deflazacort for >12 weeks prior to Screening Part A or Rescreening, allowing for weight-based modifications consistent with clinical practice. o Cohort 3: N / A

[0807] • Meet 10-meter walk / run time criteria (e.g., ability to complete a 10-meter walk / run test in <30 seconds for Cohorts 1-4, ability to complete the 10-meter walk / run test in <10 seconds for Cohort 2, and ability to complete the 10-meter walk / run test in 10-15 seconds for Cohort 4)

[0808] • Meet time to rise from supine criteria (e.g., ability to rise from supine in 3 to <7 seconds for Cohort 2)

[0809] • Meet Performance of Upper Limb (PUL) 2.0 criteria (e.g., entry item score > 3 and total score of < 40 for Cohort 5)

[0810] • Participant has body weight: < 90 kg

[0811] • Biological sex: Male

[0812] Exclusion Criteria:

[0813] • Treatment with dystrophin modifying drugs within 3 months prior to screening.

[0814] • Current or prior treatment with an approved or investigational gene transfer drug.

[0815] • Exposure to certain approved or investigational drugs within 3 months prior to screening or 5 half-lives since last administration, whichever is longer.

[0816] • Established clinical diagnosis of DMD that is associated with any deletion mutation in exons 1 to 11 or 42 to 45, inclusive, in the DMD gene as documented by a genetic report and confirmed by Sponsor genetic testing.

[0817] No participants were allowed to have abnormal liver or renal function, abnormal or impaired cardiac or pulmonary function, significant abnormalities of coagulation (e.g., Activated partial thromboplastin time, D-dimer, Fibrinogen, INR / prothrombin time) or hypersensitivity reactions.

[0818] Other inclusion or exclusion criteria apply.

[0819] Arms and Interventions:

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[0821] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0822] Primary Outcome Measures:

[0823] Secondary and Exploratory Outcome Measures:

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[0825] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

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[0827] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0828] Secondary and exploratory outcomes in addition to those recited in the foregoing table were measured. Baseline assessments were determined prior to administering the single dose of SGT-003.

[0829] Study participants admitted into the INSPIRE Duchenne clinical trial were administered a single intravenous dose of SGT-003 at a dose level of 1 E14 (1 x1014) vg / kg as a single IV infusion over a target duration of 60 minutes on study Day 1 in a hospital setting. Infusion durations were not less than 5 mL / kg / hour and did not exceed 10 mL / kg / hour.

[0830] All study participants were required to be on a stable dose of background glucocorticoids (at least 0.5 mg / kg / day of oral daily prednisone or 0.75 mg / kg / day deflazacort) for >12 weeks prior to screening, allowing for weight-based modifications consistent with clinical practice.

[0831] To increase coverage against potential inflammatory / immune responses, participants were initiated on a short-term high dose steroid regimen of 2 mg / kg / day prednisone beginning on Day -3 and remained on that dose until Day 30. On Day 2, participants received a single IV pulse dose of 20 mg / kg methylprednisolone in addition to the 2 mg / kg prednisone. If deemed appropriate, participants' steroid doses are tapered to their previous standard of care level over the following 1 month (e.g., 2 weeks at 1.5 mg / kg / day followed by 2 weeks at 1.0 mg / kg / day followed by standard of care level). Equivalent forms of prednisone (e.g., prednisolone) may be used in place of prednisone in accordance with local clinical practice. In accordance with this high dose steroid regimen, prednisone was administered orally as a tablet or oral suspension with a unit dose strength of 1 mg to 50 mg at a dose level of 2 mg / kg. The dosage formulation of methylprednisolone was an injectable suspension with a unit dose strength of 20 mg / mL, 40 mg / mL, or 80 mg / mL at a dose level of 20 mg / kg.

[0832] SGT-003 targets integrin receptors that are upregulated in dystrophic muscles. The transgene of SGT-003, h-piDys5, encodes for a functional dystrophin protein surrogate that localizes to the muscle membrane and stabilizes essential associated proteins, including neuronal nitric oxide synthase (nNOS), to rescue muscle integrity and function. In muscle, membrane localized nNOS has been associated with the ability to effectively generate the signaling molecule nitric oxide in response to exercise-induced energy demand to regulate local blood flow by antagonizing sympathetic vasoconstriction. We discovered that h-piDys5 and this next-generation capsid design in

[0833] - 73 -

[0834] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 the SGT-003 construct, SLB-101 , effectively de-targeted the liver, while delivering therapeutic benefit to skeletal muscles, and improved cardiac and pulmonary functions.

[0835] Here, SGT-003 was systemically administered via a single IV infusion to reach target skeletal and cardiac muscle tissues, and produced a functional microdystrophin protein that stabilized the muscle membrane, by restoring the dystrophin-associated protein complex (DAPC), including members such as beta-sarcoglycan and nNOS, to rescue muscle integrity and function.

[0836] Microdystrophin expression and membrane localization across skeletal and cardiac muscle tissues, the restoration of dystrophin-associated protein complex members to the muscle membrane, and improved muscle function were observed. As a result, potential benefits following administration, include slowing the progression of the disease by preserving motor abilities, were realized in treated patients.

[0837] Primary and secondary outcomes to determine safety and efficacy of SGT-003 in this FIH trial of the studied patient population comprise a variety of biopsy-based biomarker measures as well as functional outcome tests that are described in the foregoing tables entitled, "Primary Outcome Measures" and "Secondary Outcome Measures." These measures and tests were performed according to the time schedule specified in these tables with reference to the date in which SGT-003 was administered, Day 1 .

[0838] In addition to the frequent monitoring that will occur in the first 45 days following dosing, participants will be monitored for safety, tolerability, and efficacy for 5 years post-administration of SGT-003. Muscle biopsies were and will be performed at baseline, Day 90, and Day 360 timepoints to evaluate microdystrophin expression. Functional efficacy assessments were and will be performed at baseline, Days 90, 180, 360, and 540, and annual visits at Years 2, 3, 4, and 5 postdosing. Based on the visit and the functional status of each participant, these assessments will include evaluations of ambulatory function (North Star Ambulatory Assessment [NSAA], 6-minute walk test [6MWT], 10-meter walk / run, 4-stair climb, time to rise from supine), pulmonary function (% predicted forced vital capacity [FVC], peak expiratory flow [PEF], forced expiratory volume in 1 second [FEV1 ]), activity monitoring by wearable device (stride velocity 95th centile [SV95C]), development (Bayley Scales of Infant and Toddler Development 4 [Bayley-4]), upper limb function (Performance of Upper Limb [PUL] 2.0), and patient-reported outcomes (Pediatric Outcomes Data Collection Instrument [PODCI]). Individual outcome assessments will be evaluated at each timepoint and change from baseline will be calculated for interpretation of differences over time.

[0839] Microdystrophin expression was evaluated in muscle biopsies collected at baseline and day 90 to assess the relationship between the expression of this dystrophin surrogate as a direct result of treatment and overall functional capacity. Muscle biopsies (needle biopsies) were obtained, under general anesthesia using imaging guidance, to evaluate microdystrophin protein levels and distribution.

[0840] Safety Assessments

[0841] Safety assessments included vector kinetics and biodistribution; immunogenicity (assessed by AAV antibody titers and microdystrophin T-cell reactivity by enzyme-linked immunospot (ELISpot)); etc.

[0842] Additional safety assessments included:

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[0844] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0845] Electrocardiograms

[0846] Three 12-lead ECGs were conducted at least 1 minute apart after the subject was positioned supine (or semi recumbent position within 45 degrees of supine), resting, and quiet for a minimum of 10 minutes. The results of the ECG report include the findings as normal or abnormal (clinically significant [CS] or not clinically significant [NCS]).

[0847] Echocardiography (ECHO)

[0848] ECHO, performed to measure cardiovascular function, aims to include left ventricular ejection fraction, shortening fraction, EF, end-systolic diameter, end diastolic diameter, left ventricular dimensions, left atrial volume, posterior wall thickness, septum wall thickness, left ventricular mass, left ventricular Ecc, and diastolic function. Whenever possible, z scores were included.

[0849] Clinical laboratory assessments

[0850] Safety laboratory parameters were analyzed prior to dosing on Day 1 and post-administration up to Day 30.

[0851] Laboratory parameters included

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[0853] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0854] ALP=alkaline phosphatase; ALT=alanine aminotransferase; AST=aspartate aminotransferase; CK=creatine kinase; CRP=C-reactive protein; DNA=deoxyribonucleic acid; GGT=gamma-glutamyl transferase; GLDH=glutamate dehydrogenase; I NR=international normalized ratio; LDH=lactate dehydrogenase; RBC=red blood cell.

[0855] Cytokines and Complement

[0856] Complement panel included C3, C4, CH50, and C5b-9. The following cytokines and analytes may also be included if permittable: C-X-C motif chemokine ligand 10 (CXCL10), IL-1, IL-12, IL-18, TNFo, VEGF, Von Willebrand factor antigen, and procalcitonin. Prioritization is given to a cytokine panel containing IL-18 and VEGF followed by all remaining cytokine panels.

[0857] Adverse Events (AE) and Serious Adverse Events (SAE)

[0858] An AE means any untoward medical occurrence in a subject temporally associated with the use of an intervention, whether or not considered related to the intervention.

[0859] Certain abnormal laboratory test or other safety assessments (e.g., ECG, radiological scans, vital signs measurements), considered as an AE include those that worsen from baseline, and considered clinically significant by medical and scientific judgment (e.g., not related to progression of underlying disease, or more severe than expected for the condition).

[0860] AE also includes exacerbation of a chronic or intermittent pre-existing condition (including either an increase in frequency and / or severity of the condition), and new condition detected or diagnosed after administration, even though it may have been present before the start of the study. AE further includes signs, symptoms, or the clinical sequelae of a suspected intervention-intervention interaction; and signs, symptoms, or the clinical sequelae of a suspected overdose of either study intervention or a concomitant medication.

[0861] Meanwhile, AE does not include any abnormal laboratory findings or other abnormal safety assessments associated with the underlying disease, unless more severe than expected for the condition. AE also does not include the disease / disorder being studied or expected progression, signs, or symptoms of the disease / disorder being studied, unless more severe than expected for the condition. AE does not include medical or surgical

[0862] - 76 -

[0863] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 procedure (e.g., endoscopy, appendectomy). AE does not include situations in which an untoward medical occurrence did not occur or anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected prior to administration that do not worsen.

[0864] An SAE is defined as an AE that: a) results in death, b) is life-threatening, c) requires inpatient hospitalization or prolongation of existing hospitalization, d) results in persistent or significant disability / incapacity, e) is a congenital anomaly / birth defect; f) is an important medical event; or g) is a malignancy.

[0865] An assessment of the severity for each AE and SAE was made. The severity, or intensity, refers to the extent to which the AE affects the subject's daily activities. Severity is graded according to the CTCAE version 5.0.

[0866] Preliminary Results

[0867] To date, at least 15 patients (study participants) of the estimated 40 total have been dosed in the INSPIRE Duchenne clinical trial. A total of 6 participants have been dosed, and the data disclosed herein from this clinical trial result, unless stated otherwise, are from the first three patients who have reached the 90-day follow up milestone with two of these patients having reached the 180-day follow up milestone. For the three reported participants, dosing has been well tolerated by all. There were no observed serious adverse events (SAEs) and no SUSARs (Suspected Unexpected Serious Adverse Reactions). In addition, no TMA / aHUS was observed in the dosed participants.

[0868] The ages at dosing were 5, 6, 7, 6, 7, and 7 years for the 6 participants, respectively. Body weight at dosing (in kg) were 19.6, 26.4, 27.8, 22.0, 23.2 and 18.9 for Participants 1-6, respectively. Approximate time lapsed since dosing was 8, 7, and 5 months for Participants 1-3, and was <3 months or the other 3 participants.

[0869] Hematology results include: Basophils (Count and %), Eosinophils (Count and %), Hematocrit, Hemoglobin, Lymphocytes (Count and %), MCH, MCHC, MCV, Monocytes (Count and %), Neutrophils (Count and %), Platelets, RBC, Reticulocytes (Count and %), and WBC.

[0870] Chemistry results include: Albumin, Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT), Amylase, Aspartate Aminotransferase (AST), Bilirubin (Direct and Total), Calcium, Chloride, Creatine Kinase (CK), Creatinine, C-Reactive Protein (CRP), Cystatin C, Gamma-Glutamyl Transferase (GGT), Glutamate Dehydrogenase (GLDH), Globulin, Glucose, Lactate Dehydrogenase (LDH), Potassium, Sodium, Total Protein, Troponin I, and Urea.

[0871] Complement results include: C3, C4, CH50, and sC5b-9.

[0872] Coagulation results include: Activated Partial Thromboplastin Time (aPTT), D-Dimer, Fibrinogen, International Normalized Ratio (INR), and Prothrombin Time (PT).

[0873] Urinalysis results include: pH, Specific Gravity, and Urine Creatinine.

[0874] Muscle biopsy results include: Dystrophin % positive fibers, Dystrophin mean fiber mean stain density (MSD), % Muscle, % Fibrosis, % Fat, Western blot % of normal dystrophin, Western blot muscle-corrected % of normal dystrophin (calculated by dividing the Western blot % of normal dystrophin by the % Muscle result (as a decimal) from the same participant and same visit), MANEX44A Dystrophin % positive fibers, DYS2 Dystrophin % positive fibers, p-sarcoglycan (b-Sarc) % positive fibers, neuronal nitric oxide synthase (nNOS) % positive fibers,

[0875] - 77 -

[0876] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 nNOS activity % positive fibers, embryonic myosin heavy chain (eMHC) % positive fibers, Dystrophic pathology score, Mass spectrometry VGN concentration, Mass spectrometry LEE concentration, Mass spectrometry % of normal dystrophin VGN (calculated by dividing concentration results by a constant of 3672 pM and multiplying by 10 - the constant represents the measured pM concentration of normal human dystrophin), Mass spectrometry % of normal dystrophin LEE (also calculated by dividing concentration results by a constant of 3672 pM and multiplying by 10), Mass spectrometry muscle-corrected % of normal dystrophin VGN (calculated by dividing % of normal dystrophin results by the % Muscle result (as a decimal) from the same participant and same visit), Mass spectrometry muscle-corrected % of normal dystrophin LEE (calculated by dividing % of normal dystrophin results by the % Muscle result (as a decimal) from the same participant and same visit). Outputs include results for each participant and cumulative results for the 3 participants. Absolute values per visit and change from baseline to each visit are shown. Results that are below the lower limit of quantification of a test are imputed as 0. Results that are above the upper limit of quantification of a test are imputed as the upper limit of quantification of that test. Results from multiple aliquots tested from the same participant from the same visit are presented individually and as an average.

[0877] Data concerning serums biomarkers associated with muscle membrane stability obtained include CK, ALT, AST, LDH, and Titin. Outputs include results for each participant and cumulative results for the 3 participants. Absolute values per visit and change from baseline to each visit are shown. Results that are below the lower limit of quantification of a test are imputed as 0. Results that are above the upper limit of quantification of a test are imputed as the upper limit of quantification of that test. The mean of replicates from each sample is considered the result for titin outputs.

[0878] Vector genome copies results include: vector genome copies / pig and vector genome copies / diploid genome for muscle, blood, urine, saliva, and feces (when available). Outputs include results for each participant and cumulative results for the 3 participants. Results that are below the lower limit of quantification of a test are imputed as 0. • Results that are above the upper limit of quantification of a test are imputed as the upper limit of quantification of that test. Results from multiple aliquots tested from the same participant from the same visit are presented individually and as an average.

[0879] Adverse events are summarized in the section below entitled "Safety Summary.”

[0880] The preliminary findings in the first few dosed patients, based on these assays, are described in further details below.

[0881] Microdystrophin Expression

[0882] Among Participants 1-3 who reached Day 90, significant microdystrophin expression was observed based on comprehensive orthogonal measurements.

[0883] Specifically, mean microdystrophin expression, as measured by Western Blot (WB), was observed to be 110% for SGT-003 at Day 90 (n=3), >2x greater than that of FDA-approved first-generation Duchenne gene therapy (at Weeks 12 & 64). See FIG. 3A. Similar observations were made when microdystrophin expression was

[0884] - 78 -

[0885] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 measured by MS (mass spectrometer) - 108% on average for SGT-003 at Day 90 (n=3). See FIG. 3B. In FIG. 3C, an average of 78% of the dystrophin fibers were positive, as measured by immunofluorescence (IF). Also see a representative IF staining image in FIG. 4. In these experiments, the baseline Western Blot and MS were both 0% mean normal dystrophin, and the baseline dystrophin positive fibers were 1 .5% as measured by IF.

[0886] Muscle Integrity Biomarkers

[0887] Maintenance of muscle integrity for patients with DMD is vital for preserving normal muscle function and supporting strength and mobility.

[0888] As part of the treatment evaluation, several selected biomarkers were chosen to have a fuller I better representation of muscle integrity, resilience, and breakdown. Improved muscle integrity, as indicated by a coordinated biomarker profile, may support a slowing of disease progression and better long-term clinical outcomes. Meanwhile, monitoring of multiple Duchenne biomarkers offers a powerful approach for assessing disease trajectory, with long-term assessment to establish treatment effectiveness.

[0889] These biomarkers include one or more of: CK (released from muscle fibers as a result of muscle damage), AST and ALT (leakage of either is caused by skeletal muscle injury), LDH (released into the bloodstream from cells upon tissue damage), Titin (fragments are released into serum and urine when muscle is damaged), eMHC (expressed in newly generated dystrophic muscle), and Cardiac troponin I (released during myocardial cell injury). Together, these biomarkers constitute a comprehensive assessment of muscle integrity, resilience, & preservation.

[0890] Reductions in these biomarkers of muscle breakdown in Participants 1-3 showed comprehensive improvements in muscle health upon SGT-003 treatment. These biomarkers included: serum AST (-45%, see FIG. 9A), serum ALT (-54%, see FIG. 9A), serum CK (-57%, see FIG. 9A), serum LDH (-60%, see FIG. 9A), serum Titin (- 42%, see FIG. 9B), histologic eMHC (-59%, see FIG. 9B), and serum Troponin (-36%, in Participant 3 only, see FIG. 9C). Reduction in serum cardiac hs-troponin I (hs-cTnl) of -36% was observed at Day 90 in Participant 3, who entered the trial with elevated hs-cTnl levels. Participants 1 and 2 entered the study with normal baseline cTnl levels. Preliminary data also showed that two participants of the first six participants measured had elevated troponin at baseline reduced below initial baseline values post-dose. Consistent decrease in these biomarkers of muscle injury I stress at Day 90 (mean: N=3 patients) suggested improved and significant muscle resilience and integrity in SGT-003 treated DMD patients. High sensitivity-troponin I (Hs-cTnl) is an important marker that can be predictive of severe cardiac disease in neuromuscular diagnoses. Indeed, higher mean reductions from baseline in serum cardiac high sensitivity-troponin I (hs-cTnl) of 85% at Day 90 (N=14) and 70% at Day 360 (N=3) were seen in participants who entered the study with elevated baseline levels.

[0891] In children with DMD, eMHC is elevated primarily due to muscle breakdown and regeneration. As muscle fibers deteriorate, muscle stem cells are activated to replace damaged fibers. During this regenerative process, newly formed muscle fibers express embryonic myosin heavy chain (eMHC). High eMHC protein levels in muscle biopsies indicate active muscle regeneration. In DMD, muscle regeneration cannot keep up with muscle breakdown, leading to progressive weakness despite ongoing repair attempts. During maturation phase, muscle fibers often remain in an immature state, leading to persistent eMHC expression, due to constant muscle injury and incomplete - 79 -

[0892] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 repair in DMD. eMHC expression is significantly upregulated in dystrophic muscle fibers, making it a biomarker for disease progression and response to therapies.

[0893] SGT-003 was observed to reduce muscle breakdown and increase muscle preservation in treated DMD patients. Robust microdystrophin expression and nNOS co-localization led to reduced markers of muscle loss and dystrophic regeneration at Day 90 (mean: N=3 patients). See FIG. 9B. Reduced titin combined with reduced eMHC suggests that SGT-003 has interrupted muscle breakdown and preserved muscle fibers in treated patients.

[0894] Overall, 7 simultaneous biomarker reductions observed at Day 90 (mean: N=3 patients) suggest SGT-003 enhanced muscle integrity and resilience in treated patients. nNOS Co-Localization and Activity nNOS plays an important role in muscle health, adaptation, and performance. It prevents muscle wasting, protects against fibrosis and oxidative stress, supports muscle repair and recovery, promotes anti-inflammatory effects, supports cellular repair and regeneration, improves blood flow and stamina, and regulates muscle contraction. Loss of nNOS at the sarcolemma leads to impaired NO-mediated vasodilation, functional ischemia, and muscle fatigue and breakdown. Thus, restoring properly localized nNOS activity is essential to more fully protect cardiac and skeletal muscle.

[0895] Preliminary data demonstrated that SGT-003-mediated microdystrophin gene therapy achieved nNOS colocalization for the expressed microdystrophin, as well as activity in the treated DMD patients. More specifically, about 42% of the muscle fibers showed appropriately localized nNOS expression I activity at Day 90 (mean: N=3 patients, see FIG. 8C).

[0896] Early Signs of DMD Cardiac Benefit

[0897] Loss of dystrophin in DMD leads to progressive degeneration of cardiac muscle. Cardiac disease is underway long before overt cardiac dysfunction appears. About 25% of the patients showed evidence of cardiomyopathy by age 6. This proportion increased to 59% by age 10, and 98% by age 18. About 40% of these patients eventually progress to heart failure. This is further compounded by the fact that cardiac tissue has limited regenerative capacity - by age 25, only ~1% of cardiomyocytes will turn over annually. Not surprisingly, cardiomyopathy is a leading cause of death in DMD.

[0898] Early troponin elevation is predictive of severe cardiac disease in neuromuscular diseases. A hs-cTnl level >7.6 ng / L is correlated with a 3-fold increased risk of cardiac disease. Early detection of changes in the heart using troponin inform interventions to slow disease progression, improve quality of life, and lower the risk of severe cardiomyopathy.

[0899] Gamma-glutamyl transferase (GGT) levels remained stable during 90 days post dosing with AAV-SLB101 at doses up to 3x1014vg / kg in NHPs. No biomarkers (ALT, AST, and GGT) or clinical evidence of liver injury was observed in mdx mice treated with SGT-003 (FIG. 5B). These findings suggest that the decrease in biodistribution to the liver could be driving an increased safety profile of AAV-SLB-101.

[0900] - 80 -

[0901] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0902] In mdx mice treated with SGT-003 at dose levels ranging from 3E13 vg / kg to 3E14 vg / kg, microdystrophin expression resulted in decreased levels of muscle damage biomarkers. Serum creatine kinase (CK), titin, and aspartate aminotransferase (AST) levels at Day 92 were significantly decreased in samples collected from mdx mice treated with SGT-003. Immunofluorescence evaluation of microdystrophin expression in quadriceps tissue from treated mice showed greater than 95% positive fibers across all groups. At these doses where robust piDys expression (>95%) was observed, a statistically significant reduction in all serum biomarkers tested was observed (see FIG. 9D). At a lower dosage of SGT-003 ranging from 2E12 vg / kg to 3E13 vg / kg, microdystrophin expression also resulted in decreased levels of muscle damage biomarkers; declines in all three biomarkers were observed in a dose dependent manner in mice treated with ascending doses of SGT-003 (see FIG. 9E).

[0903] Consistent with these findings, the SGT-003 clinical study showed early signs of cardiac benefit in DMD Participants 1-3, based on assessing LVEF (left ventricular ejection fraction - the central measure of left ventricular systolic function). As shown in FIG. 6, in the first 2 participants at Day 180, mean baseline cardiac function (as assessed by LVEF(%)) increased by 8% from a Baseline LVEF of 56%. Participant #3 has 90 days of LVEF follow up, and also showed promising sign of LVEF increase over the baseline (FIG. 6).

[0904] Since lower LVEF correlates with a higher 5-year probability of death, the observed mean increase in LVEF is a promising early sign of cardiac benefit in treated DMD patients.

[0905] A concurrent 36% reduction in Cardiac troponin I in Participant 3 (FIG. 9C), as well as 85% reduction in 14 enrolled Participants who entered the study with elevated baseline levels and who have reached Day 90 post injection, and 70% reduction in 3 such enrolled Participants who have reached Day 360 post injection, are consistent with early signs of cardiac benefit in DMD treatment using SGT-003.

[0906] Lower Dose Gene Therapy

[0907] Partly due to the observed higher muscle transduction efficiency I rate, the SLB101 capsid-mediated gene therapy is able to be administered at a comparatively lower dose than other gene therapy capsids with lower transduction efficiency, such as AAV-rh74.

[0908] More specifically, in the SGT-003 clinical study, the SLB101 capsid (e.g., in SGT-003) was administered at a 25% lower dose (1 E14 vg / kg) when compared to approved first generation microdystrophin gene therapy (1 .33E14 vg / kg), but was observed to have greater than 5-times mean skeletal muscle transduction (N=3). See FIG. 7. The mean copy number for Participants 1-3 was 18.7 per nucleus using SLB101, while the same was 2.91-3.44 copies per nucleus using AAV-rh74.

[0909] Thus, SLB101 has achieved greater than 5-times of transduction rate compared to the FDA-approved first generation microdystrophin gene therapy, with 25% lower dose, and with no use of eculizumab or other intensive immunomodulation co-therapy, such as immunosuppressant (e.g., sirolimus) and anti-CD20 antibody (e.g, rituximab) (cross-trial comparison).

[0910] Functional Microdystrophin Binds and Restores Key Elements of DAPC (Dystrophin-Associated Protein Complex)

[0911] - 81 -

[0912] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320

[0913] Microdystrophin expressed from the SGT-003 construct exhibited not only enhanced expression, but also functional binding to key elements of the Dystrophin-Associated Protein Complex (DAPC), including beta- Sarcoglycan, as well as restored nNOS activity.

[0914] As shown in FIG. 8A, based on IF analysis, there was on average a 54-fold increase in microdystrophin expression among Participants 1-3, when comparing Day 90 microdystrophin expression (mean = 78%, n=3) to baseline expression (mean = 1 .5%, n=3).

[0915] Meanwhile, as shown in FIG. 8B, based there was on average a 54-fold increase in co-localized beta- Sarcoglycan among Participants 1-3, when comparing Day 90 values (mean = 70%, n=3) to baseline expression (mean = 1 .3%, n=3). This suggests that the expressed microdystrophin correctly localized to cell membrane and restored ket elements of the DPAC such as beta-Sarcoglycan.

[0916] This is further supported by the nNOS assessment shown in FIG. 8C. At Day 90, there was on average about 42% positive fibers having nNOS activity in Participants 1-3, which represented a 60-fold increase from the baseline value of 0.7% (mean, n=3).

[0917] Safety Summary

[0918] SGT-003 was well-tolerated in all 6 participants dosed as of Day 90 for reported Participants 1-6. No hospitalizations were reported. No evidence of TMA or aHUS was observed. The treatment was accompanied by prophylactic glucocorticoids alone, but there was no need for intensive immunomodulation therapy.

[0919] The most common AEs included nausea / vomiting (all have resolved); transient thrombocytopenia (one CTCAE Grade 3 episode that resolved within days without intervention, all returned to within normal laboratory range with no clinical consequence (no bleeding), no evidence of hemolysis (hemoglobin stable and no schistocytes on smears), and no eculizumab, steroids or other agents used (such as immunosuppressant (e.g., sirolimus) and anti- CD20 antibody (e.g., rituximab)); infusion related hypersensitivity reaction (one CTCAE Grade 3 episode of prolonged fever that resolved within days without intervention); and fever (all have resolved). These Adverse Events were expected, and were consistent with AEs in AAV gene therapy.

[0920] All treatment related AEs were resolved with no sequelae. Such AEs began in days following treatment, and were resolved within weeks of onset.

[0921] There were no observed Serious Adverse Events (SAEs), and there were no SUSARs. There were also no AEs of hepatic transaminitis (including no elevated gamma-glutamyl transferase (GGT) levels), a result consistent with the consistent declines in AST and ALT values in treated patients.

[0922] As to Adverse Events of Special Interest (AESI), there was 1 episode of mild transient hs-troponin I elevation (CTCAE Grade 1) in the first week post dosing ( / .e., Participant Troponin I was elevated at baseline, which increased during the first week of therapy but returned to baseline without intervention), but there was no clinical evidence of myocarditis, and was no EKG or Echocardiographic changes.

[0923] Overall, the data show that SGT-003-mediated AAV therapy using the SLB-101 capsid is associated with reduced adverse events and increased functional results (in terms of biodistribution, gene expression level, and

[0924] - 82 -

[0925] MEl\58298049.vl SLD-023.WO Attorney Docket No.: 129159-04320 better functional outcomes, etc.).

[0926] On the other hand, according to the ELEVIDYS® (delandistrogene moxeparvovec-rokl) label, during its clinical trial, liver function tests showed increased liver enzyme and total bilirubin levels, including increases in GGT, GLDH, ALT, AST, or total bilirubin, typically within 8 weeks following drug infusion, though the majority of the cases were asymptomatic. Cases resolved spontaneously or with systemic corticosteroids and resolved without clinical sequelae within 2 months. No cases of liver failure were reported. Liver function monitoring (including clinical exam, GGT, and total bilirubin) was required weekly for the first 3 months following infusion. Continued monitoring is required if clinically indicated, until results are unremarkable (normal clinical exam, GGT and total bilirubin levels return to near baseline levels).

[0927] Similarly, according to the ZOLGENSMA (onasemnogene abeparvovec-xioi) label, liver enzyme increase in "Study 3” occurred at a higher frequency compared with the previous 4 studies. AST or ALT elevations > 2 x ULN were observed in the majority of patients (23 out of 24 patients), including 21 patients with ALT elevations > 3 x ULN and 5 patients with ALT elevations > 20 x ULN, though these patients were clinically asymptomatic and there were no elevations of bilirubin. The AST and ALT elevations were managed with the use of corticosteroids, typically with prolonged duration and / or given at a higher dose.

[0928] Example 2 Low Cross-Reactivity Between AAVSLB101 & AAVrh74

[0929] AAV vectors are promising gene therapy candidates, but pre-existing anti-AAV neutralizing antibodies (NAbs) pose a significant challenge to successful gene delivery.

[0930] Ongoing preclinical studies showed low cross-reactivity between AAVSLB101 and AAVrh74 titers in mice, NHPs (non-human primates), and human sera samples, thus supporting re-dosing with AAV-SLB101 -based gene therapy capsids after prior treatment with AAV-rh74 capsid, such as the one used in the FDA-approved first- generation microdystrophin gene therapy.

[0931] FIG. 10A shows the result of a longitudinal assessment of anti-AAV-SLB101 Nab titers in mice (n=5) previously dosed with AAV-rh74. As is evident, anti-AAV-rh74 Nabs titer increased steadily over the 60-day or so follow up period, while anti-AAV-SLB101 remained undetectable.

[0932] Similar findings were observed in NHP (monkey). FIG. 10B shows the result of Day 30 assessment of AAV-SLB101 Nabs titer in monkeys (n=6) previously dosed with AAV-rh74. Nabs titer against AAV-rh74 was very high, compared to that against AAV-SLB101, which is negligible.

[0933] Likewise, in serum samples from human (n=3) previously dosed with AAV-rh74, Nabs against AAV-rh74 was very high. Since the AAV rhesus serotype 74 (rh74) is a Clade E member closely related to AAV8, another clade E member, sharing 93% homology, Nabs against AAV8 was also comparably very high, likely due to crossreactivity. In contrast, Nabs titer against AAV-SLB101 was very low / negligible.

[0934] The low cross-reactivity observed between AAVrh74 and AAV-SLB101 NAb titers, combined rapid transduction and expression observed with AAV-SLB101, provide an opportunity for re-dosing DMD patients previously treated with AAVrh74, such as DMD patients treated with the FDA-approved first-generation microdystrophin gene therapy using AAV-rh74 as capsid.

[0935] - 83 -

[0936] MEl\58298049.vl

Claims

SLD-023.WO Attorney Docket No.: 129159-04320CLAIMS1 . A method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

2. The method according to claim 1, wherein the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

3. The method according to claim 1, wherein the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

4. The method according to any one of claims 1-3, wherein the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

5. The method according to any one of claims 1-4, wherein the human subject is receiving a stable dose of background glucocorticoids (at least 0.5 mg / kg / day of oral daily prednisone or 0.75 mg / kg / day deflazacort) for >12 weeks prior to being administered the dose of SGT-003.

6. The method according to any one of claims 1-5, further comprising administering to the human subject a short-term high dose steroid regimen, the regimen comprising at least one steroid selected from the group consisting of prednisone, prednisolone and methylprednisolone.

7. The method according to claim 6, wherein prednisone or prednisolone is administered daily to the human subject beginning three days prior to being administered the single dose of SGT-003 and continuing for thirty days after the single dose of SGT-003.

8. The method according to claim 7, wherein methylprednisolone is administered a single IV pulse dose of 20 mg / kg one day after being administered the single dose of SGT-003.

9. The method according to any one of claims 1-8, wherein the human subject is male.

10. The method according to any one of claims 1-9, wherein the human subject is < 18 years of age.

11. The method according to any one of claims 1-10, wherein the human subject is ambulatory.

12. The method according to any one of claims 1-10, wherein the human subject is non-ambulatory.

13. The method according to any one of claims 1-12, wherein the human subject has not received a dose of a dystrophin modifying drug within three months of being administered the dose of SGT-003.- 84 -MEl\58298049.vlSLD-023.WO Attorney Docket No.: 129159-0432014. The method according to any one of claims 1-13, wherein the dose is 1 E14 (1 xio14) vg / kg.

15. The method according to any one of claims 1-14, wherein SGT-003 is administered by a single intravenous (IV) infusion.

16. The method according to any one of claims 1-15, wherein the dose is administered over a target duration of 60 minutes at a rate not less than 5 mL / kg / hour and not more than 10 mL / kg / hour.

17. A method for increasing microdystrophin expression in a human subject in need thereof relative to baseline, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

18. The method according to claim 17, wherein the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

19. The method according to claim 17, wherein the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

20. The method according to any one of claims 17-19, wherein the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

21. The method according to any one of claims 17-20, wherein the increase in microdystrophin expression is achieved 90 days after the dose of SGT-003 is administered to the human subject.

22. The method according to any one of claims 17-21, wherein the increase in microdystrophin expression is measured by Western Blot or mass spectrometer of a muscle biopsy of the human subject.

23. A method for delaying progression of Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of a recombinant muscle-tropic adeno-associated viral vector (rAAV) to the human subject, the rAAV comprising a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

24. The method according to claim 23, wherein the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.- 85 -MEl\58298049.vlSLD-023.WO Attorney Docket No.: 129159-0432025. The method according to claim 23, wherein the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

26. The method according to any one of claims 23-25, wherein the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.

27. The method of any one of claims 23-26, wherein the delay of progression Duchenne muscular dystrophy is measured relative to baseline by any of: Time to Rise Velocity, stride velocity 95th centile (SV95C), IOmeter walk / run velocity, 4-stair climb velocity, North Star Ambulatory Assessment (NSAA) total score, 6- minute walk test (6MWT) distance, percent predicted forced vital capacity (FVC), percent predicted peak expiratory flow (PEF), percent predicted forced expiratory volume in 1 second (FEV1), Performance of Upper Limb (PUL) 2.0 score, left ventricular ejection fraction (LVEF) by cardiac MRI, or Bayley Scales of Infant and Toddler Development 4 (Bayley-4) score.

28. Use of a recombinant muscle-tropic adeno-associated viral vector (rAAV) for the manufacture of a medicament for the treatment of Duchenne muscular dystrophy in a human subject in need thereof, comprising administering a therapeutically effective dose of the rAAV to the human subject, wherein the rAAV comprises a polynucleotide sequence encoding a human microdystrophin protein having the amino acid sequence of SEQ ID NO: 2 ("h-piD5”), and wherein the human subject has a confirmed mutation of the DMD gene that is not associated with a deletion mutation in exons 1 to 11 or 42 to 45, inclusive.

29. The use according to claim 28, wherein the rAAV has a capsid of AAV-SLB101 characterized by a VP1 amino acid sequence of SEQ ID NO: 21, optionally, the rAAV comprises a vector genome having the polynucleotide sequence of SEQ ID NO: 20, or a polynucleotide at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.

30. The use according to claim 28, wherein the polynucleotide sequence has the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical thereto.31 . The use according to any one of claims 28-30, wherein the rAAV is SGT-003 comprising a vector genome having the polynucleotide sequence of SEQ ID NO: 20.- 86 -MEl\58298049.vl

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