Use of plasmapheresis to lower anti-AAV antibodies in nonhuman primates with pre-existing immunity to AAVrh74.

Patients with pre-existing immunity to adeno-associated virus (AAV) are currently unable to receive systemic gene transfer therapies. In this nonhuman primate study, we investigated the impact of immunosuppression strategies on gene transfer therapy safety and efficacy and analyzed plasmapheresis as a potential pretreatment for circumvention of pre-existing immunity or redosing. In part 1, animals received delandistrogene moxeparvovec (SRP-9001), an AAVrh74-based gene transfer therapy for Duchenne muscular dystrophy. Cohort 1 (control, n = 2) received no immunosuppression; cohorts 2-4 (n = 3 per cohort) received prednisone at different time points; and cohort 5 (n = 3) received rituximab, sirolimus, and prednisone before and after dosing. In part 2, cohorts 2-4 underwent plasmapheresis before redosing; cohort 5 was redosed without plasmapheresis. We analyzed safety, immune response (humoral and cell-mediated responses and complement activation), and vector genome distribution. After 2 or 3 plasmapheresis exchanges, circulating anti-AAVrh74 antibodies were reduced, and animals were redosed. Plasmapheresis was well tolerated, with no abnormal clinical or immunological observations. Cohort 5 (redosed with high anti-AAVrh74 antibody titers) had hypersensitivity reactions, which were controlled with treatment. These findings suggest that plasmapheresis is a safe and effective method to reduce anti-AAV antibody levels in nonhuman primates prior to gene transfer therapy. The results may inform human studies involving redosing or circumvention of pre-existing immunity.

Gene therapy with bidridistrogene xeboparvovec for limb-girdle muscular dystrophy type 2E/R4: phase 1/2 trial results.

Limb-girdle muscular dystrophy 2E/R4 is caused by mutations in the ß-sarcoglycan (SGCB) gene, leading to SGCB deficiency and consequent muscle loss. We developed a gene therapy approach based on functional replacement of the deficient SCB protein. Here we report interim results from a first-in-human, open-label, nonrandomized, phase 1/2 trial evaluating the safety and efficacy of bidridistrogene xeboparvovec, an adeno-associated virus-based gene therapy containing a codon-optimized, full-length human SGCB transgene. Patients aged 4-15 years with confirmed SGCB mutations at both alleles received one intravenous infusion of either 1.85 × 1013 vector genome copies kg-1 (Cohort 1, n = 3) or 7.41 × 1013 vector gene copies kg-1 (Cohort 2, n = 3). Primary endpoint was safety, and secondary endpoint was change in SGCB expression in skeletal muscle from baseline to Day 60. We report interim Year 2 results (trial ongoing). The most frequent treatment-related adverse events were vomiting (four of six patients) and gamma-glutamyl transferase increase (three of six patients). Serious adverse events resolved with standard therapies. Robust SGCB expression was observed: Day 60 mean (s.d.) percentage of normal expression 36.2% (2.7%) in Cohort 1 and 62.1% (8.7%) in Cohort 2. Post hoc exploratory analysis showed preliminary motor improvements using the North Star Assessment for Limb-girdle Type Muscular Dystrophies maintained through Year 2. The 2-year safety and efficacy of bidridistrogene xeboparvovec support clinical development advancement. Further studies are necessary to confirm the long-term safety and efficacy of this gene therapy. ClinicalTrials.gov registration: NCT03652259 .

Long-term safety and functional outcomes of delandistrogene moxeparvovec gene therapy in patients with Duchenne muscular dystrophy: A phase 1/2a nonrandomized trial.

INTRODUCTION/AIMS: Delandistrogene moxeparvovec is indicated in the United States for the treatment of ambulatory pediatric patients aged 4 through 5 years with Duchenne muscular dystrophy (DMD) with a confirmed mutation in the DMD gene. Long-term delandistrogene moxeparvovec microdystrophin protein (a shortened dystrophin that retains key functional domains of the wild-type protein) expression may positively alter disease progression in patients with DMD. We evaluated long-term safety and functional outcomes of delandistrogene moxeparvovec in patients with DMD. METHODS: An open-label, phase 1/2a, nonrandomized controlled trial (Study 101; NCT03375164) enrolled ambulatory males, ≥4 to <8 years old, with DMD. Patients received a single intravenous infusion (2.0 × 1014 vg/kg by supercoiled quantitative polymerase chain reaction) of delandistrogene moxeparvovec and prednisone (1 mg/kg/day) 1 day before to 30 days after treatment. The primary endpoint was safety. Functional outcomes were change from baseline in North Star Ambulatory Assessment (NSAA) and timed function tests. RESULTS: Four patients (mean age, 5.1 years) were enrolled. There were 18 treatment-related adverse events; all occurred within 70 days posttreatment and resolved. Mean NSAA total score increased from 20.5 to 27.5, baseline to year 4, with a mean (standard deviation) change of +7.0 (2.9). Post hoc analysis demonstrated a statistically significant and clinically meaningful 9-point difference in NSAA score, relative to a propensity-score-weighted external control cohort (least-squares mean [standard error] = 9.4 [3.4]; P = .0125). DISCUSSION: Gene transfer therapy with delandistrogene moxeparvovec treatment is well tolerated, with a favorable safety profile. Functional improvements are sustained through 4 years, suggesting delandistrogene moxeparvovec may positively alter disease progression.

Expression and function of four AAV-based constructs for dystrophin restoration in the mdx mouse model of Duchenne muscular dystrophy.

Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared the efficacies of four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e. reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.

Delandistrogene Moxeparvovec Gene Therapy in Ambulatory Patients (Aged ≥4 to <8 Years) with Duchenne Muscular Dystrophy: 1-Year Interim Results from Study SRP-9001-103 (ENDEAVOR).

OBJECTIVE: Delandistrogene moxeparvovec is approved in the USA for the treatment of ambulatory patients (4-5 years) with Duchenne muscular dystrophy. ENDEAVOR (SRP-9001-103; NCT04626674) is a single-arm, open-label study to evaluate delandistrogene moxeparvovec micro-dystrophin expression, safety, and functional outcomes following administration of commercial process delandistrogene moxeparvovec. METHODS: In cohort 1 of ENDEAVOR (N = 20), eligible ambulatory males, aged ≥4 to <8 years, received a single intravenous infusion of delandistrogene moxeparvovec (1.33 × 1014 vg/kg). The primary endpoint was change from baseline (CFBL) to week 12 in delandistrogene moxeparvovec micro-dystrophin by western blot. Additional endpoints evaluated included: safety; vector genome copies; CFBL to week 12 in muscle fiber-localized micro-dystrophin by immunofluorescence; and functional assessments, including North Star Ambulatory Assessment, with comparison with a propensity score-weighted external natural history control. RESULTS: The 1-year safety profile of commercial process delandistrogene moxeparvovec in ENDEAVOR was consistent with safety data reported in other delandistrogene moxeparvovec trials (NCT03375164 and NCT03769116). Delandistrogene moxeparvovec micro-dystrophin expression was robust, with sarcolemmal localization at week 12; mean (SD) CFBL in western blot, 54.2% (42.6); p < 0.0001. At 1 year, patients demonstrated stabilized or improved North Star Ambulatory Assessment total scores; mean (SD) CFBL, +4.0 (3.5). Treatment versus a propensity score-weighted external natural history control demonstrated a statistically significant difference in least squares mean (standard error) CFBL in North Star Ambulatory Assessment, +3.2 (0.6) points; p < 0.0001. INTERPRETATION: Results confirm efficient transduction of muscle by delandistrogene moxeparvovec. One-year post-treatment, delandistrogene moxeparvovec was well tolerated, and demonstrated stabilized or improved motor function, suggesting a clinical benefit for patients with Duchenne muscular dystrophy. ANN NEUROL 2023;94:955-968.

Expression of SRP-9001 dystrophin and stabilization of motor function up to 2 years post-treatment with delandistrogene moxeparvovec gene therapy in individuals with Duchenne muscular dystrophy.

Introduction: Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy designed for targeted expression of SRP-9001 dystrophin protein, a shortened dystrophin retaining key functional domains of the wild-type protein. Methods: This Phase 2, double-blind, two-part (48 weeks per part) crossover study (SRP-9001-102 [Study 102]; NCT03769116) evaluated delandistrogene moxeparvovec in patients, aged ≥4 to <8 years with Duchenne muscular dystrophy. Primary endpoints (Part 1) were change from baseline (CFBL) in SRP-9001 dystrophin expression (Week 12), by Western blot, and in North Star Ambulatory Assessment (NSAA) score (Week 48). Safety assessments included treatment-related adverse events (TRAEs). Patients were randomized and stratified by age to placebo (n = 21) or delandistrogene moxeparvovec (n = 20) and crossed over for Part 2. Results: SRP-9001 dystrophin expression was achieved in all patients: mean CFBL to Week 12 was 23.82% and 39.64% normal in Parts 1 and 2, respectively. In Part 1, CFBL to Week 48 in NSAA score (least-squares mean, LSM [standard error]) was +1.7 (0.6) with treatment versus +0.9 (0.6) for placebo; p = 0.37. Disparity in baseline motor function between groups likely confounded these results. In 4- to 5-year-olds with matched baseline motor function, CFBL to Week 48 in NSAA scores was significantly different (+2.5 points; p = 0.0172), but not significantly different in 6-to-7-year-olds with imbalanced baseline motor function (-0.7 points; p = 0.5384). For patients treated with delandistrogene moxeparvovec in Part 2, CFBL to Week 48 in NSAA score was +1.3 (2.7), whereas for those treated in Part 1, NSAA scores were maintained. As all patients in Part 2 were exposed to treatment, results were compared with a propensity-score-weighted external control (EC) cohort. The LSM difference in NSAA score between the Part 2 treated group and EC cohort was statistically significant (+2.0 points; p = 0.0009). The most common TRAEs were vomiting, decreased appetite, and nausea. Most occurred within the first 90 days and all resolved. Discussion: Results indicate robust expression of SRP-9001 dystrophin and overall stabilization in NSAA up to 2 years post-treatment. Differences in NSAA between groups in Part 1 were not significant for the overall population, likely because cohorts were stratified only by age, and other critical prognostic factors were not well matched at baseline.

Assessing the value of delandistrogene moxeparvovec (SRP-9001) gene therapy in patients with Duchenne muscular dystrophy in the United States.

Background: Delandistrogene moxeparvovec (SRP-9001) is an investigational gene therapy that may delay progression of Duchenne muscular dystrophy (DMD), a severe, rare neuromuscular disease caused by DMD gene mutations. Early cost-effectiveness analyses are important to help contextualize the value of gene therapies for reimbursement decision making. Objective: To determine the potential value of delandistrogene moxeparvovec using a cost-effectiveness analysis. Study design: A simulation calculated lifetime costs and equal value of life years gained (evLYG). Inputs included extrapolated clinical trial results and published utilities/costs. As a market price for delandistrogene moxeparvovec has not been established, threshold analyses established maximum treatment costs as they align with value, including varying willingness-to-pay up to $500,000, accounting for severity/rarity. Setting: USA, healthcare system perspective Patients: Boys with DMD Intervention: Delandistrogene moxeparvovec plus standard of care (SoC; corticosteroids) versus SoC alone Main outcome measure: Maximum treatment costs at a given willingness-to-pay threshold Results: Delandistrogene moxeparvovec added 10.30 discounted (26.40 undiscounted) evLYs. The maximum treatment cost was approximately $5 M, assuming $500,000/evLYG. Varying the benefit discount rate to account for the single administration increased the estimated value to #$5M, assuming $500,000/evLYG. Conclusion: In this early economic model, delandistrogene moxeparvovec increases evLYs versus SoC and begins to inform its potential value from a healthcare perspective.

Systemic γ-sarcoglycan AAV gene transfer results in dose-dependent correction of muscle deficits in the LGMD 2C/R5 mouse model.

Limb-girdle muscular dystrophy (LGMD) type 2C/R5 results from mutations in the γ-sarcoglycan (SGCG) gene and is characterized by muscle weakness and progressive wasting. Loss of functional γ-sarcoglycan protein in the dystrophin-associated protein complex destabilizes the sarcolemma, leading to eventual myofiber death. The SGCG knockout mouse (SGCG -/-) has clinical-pathological features that replicate the human disease, making it an ideal model for translational studies. We designed a self-complementary rAAVrh74 vector containing a codon-optimized human SGCG transgene driven by the muscle-specific MHCK7 promoter (SRP-9005) to investigate adeno-associated virus (AAV)-mediated SGCG gene transfer in SGCG -/- mice as proof of principle for LGMD 2C/R5. Gene transfer therapy resulted in widespread transgene expression in skeletal muscle and heart, improvements in muscle histopathology characterized by decreased central nuclei and fibrosis, and normalized fiber size. Histopathologic improvements were accompanied by functional improvements, including increased ambulation and force production and resistance to injury of the tibialis anterior and diaphragm muscles. This study demonstrates successful systemic delivery of the hSGCG transgene in SGCG -/- mice, with functional protein expression, reconstitution of the sarcoglycan complex, and corresponding physiological and functional improvements, which will help establish a minimal effective dose for translation of SRP-9005 gene transfer therapy in patients with LGMD 2C/R5.

Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations.

Given the increasing number of gene transfer therapy studies either completed or underway, there is growing attention to the importance of preexisting adaptive immunity to the viral vectors used. The recombinant viral vectors developed for gene transfer therapy share structural features with naturally occurring wild-type virus. Antibodies generated against viral vectors obtained through a previous exposure to wild-type virus can potentially compromise transgene expression by blocking transduction, thereby limiting the therapeutic efficacy of the gene transfer therapy; they may also pose potential safety concerns. Therefore, systemic gene transfer delivery requires testing patients for preexisting antibodies. Two different assays have been used: (1) binding assays that focus on total antibodies (both neutralizing and non-neutralizing) and (2) neutralizing assays that detect neutralizing antibodies. In this review we focus on adeno-associated virus-based gene therapies, describing the immune response that occurs to naturally occurring adeno-associated viruses, the implications for patients with this exposure, the assays used to detect preexisting immune responses, and strategies to circumvent preexisting adaptive immunity to expand the patient base that could benefit from such therapies.

Unmet needs and evolving treatment for limb girdle muscular dystrophies.

Limb-girdle muscular dystrophies (LGMDs) represent a major group of muscle disorders. Treatment is sorely needed and currently expanding based on safety and efficacy adopting principles of single-dosing gene therapy for monogenic autosomal recessive disorders. Gene therapy has made in-roads for LGMD and this review describes progress that has been achieved for these conditions. This review first provides a background on the definition and classification of LGMDs. The major effort focuses on progress in LGMD gene therapy, from experimental studies to clinical trials. The disorders discussed include the LGMDs where the most work has been done including calpainopathies (LGMD2A/R1), dysferlinopathies (LGMD2B/R2) and sarcoglycanopathies (LGMD2C/R5, LGMD2D/R3, LGMD2E/R4). Early success in clinical trials provides a template to move the field forward and potentially apply emerging technology like CRISPR/Cas9 that may enhance the scope and efficacy of gene therapy applied to patient care.

Lay abstract Limb-girdle muscular dystrophy is a term that is applied to a group of relatively rare forms of muscular dystrophy. The term 'LGMD' was introduced in the 1950's, but there were no strict rules for defining the condition. This changed as a result of the 229th European Neuromuscular Center International Workshop in 2017 providing a clear definition and classification discussed in this article. Limb-girdle muscular dystrophy is now recognized as a genetic muscle disease with an elevated serum creatine kinase and dystrophic changes on muscle histology. Most treatments up to now rely on supportive measures for heart and lungs and assisting the physical limitations. Medications have not proven to be beneficial to stop progression of disease. This article focuses on new innovations of treatment that target the effected gene and the use special methods to replace the abnormal gene.

Evaluation of the Lipid-binding Properties of Recombinant Dystrophin Spectrin-like Repeat Domains R1-3.

Recombinant micro-dystrophin genes are designed to treat Duchenne muscular dystrophy (DMD) by retaining dystrophin domains believed to play key functional roles while fitting the packaging capacity of adeno-associated virus vectors. Domains R1-3 are important for muscle force generation and for association with the sarcolemma, but the nature of this interaction is not fully understood. We measured lipid-binding affinity of 3 peptides containing different spectrin-like repeat modules (R1-3; R1-2; and R1, 2, 22). Lipid-binding affinity was highest with R1-3, suggesting that the complete R1-R3 region could be beneficial and should be considered for inclusion in micro-dystrophin constructs.

Dose-Escalation Study of Systemically Delivered rAAVrh74.MHCK7.micro-dystrophin in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by mutations in the DMD gene. More than 2,000 mutations of the DMD gene are responsible for progressive loss of muscle strength, loss of ambulation, and generally respiratory and cardiac failure by age 30. Recently, gene transfer therapy has received widespread interest as a disease-modifying treatment for all patients with DMD. We designed an adeno-associated virus vector (rAAVrh74) containing a codon-optimized human micro-dystrophin transgene driven by a skeletal and cardiac muscle-specific promoter, MHCK7. To test the efficacy of rAAVrh74.MHCK7.micro-dystrophin, we evaluated systemic injections in mdx (dystrophin-null) mice at low (2 × 1012 vector genome [vg] total dose, 8 × 1013 vg/kg), intermediate (6 × 1012 vg total dose, 2 × 1014 vg/kg), and high doses (1.2 × 1013 vg total dose, 6 × 1014 vg/kg). Three months posttreatment, specific force increased in the diaphragm (DIA) and tibialis anterior muscle, with intermediate and high doses eliciting force outputs at wild-type (WT) levels. Histological improvement included reductions in fibrosis and normalization of myofiber size, specifically in the DIA, where results for low and intermediate doses were not significantly different from the WT. Significant reduction in central nucleation was also observed, although complete normalization to WT was not seen. No vector-associated toxicity was reported either by clinical or organ-specific laboratory assessments or following formal histopathology. The findings in this preclinical study provided proof of principle for safety and efficacy of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin at high vector titers, supporting initiation of a Phase I/II safety study in boys with DMD.

Preclinical Systemic Delivery of Adeno-Associated α-Sarcoglycan Gene Transfer for Limb-Girdle Muscular Dystrophy.

Limb-girdle muscular dystrophy type 2D/R3 (LGMD2D/R3) is a progressive muscular dystrophy that manifests with muscle weakness, respiratory abnormalities, and in rare cases cardiomyopathy. LGMD2D/R3 is caused by mutations in the SGCA gene resulting in loss of protein and concomitant loss of some or all components of the dystrophin-associated glycoprotein complex. The sgca-null (sgca-/-) mouse recapitulates the clinical phenotype of patients with LGMD2D/R3, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduction in the generation of absolute muscle force and locomotor activity. Thus, sgca-/- mice provide a relevant model to test the safety and efficacy of gene transfer. We designed a self-complementary AAVrh74 vector containing a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter, shortened muscle creatine kinase (tMCK). In this report, we test the efficacy and safety of scAAVrh74.tMCK.hSGCA in sgca-/- mice using a dose-escalation design to evaluate a single systemic injection of 1.0 × 1012, 3.0 × 1012, and 6.0 × 1012 vg total dose compared with vehicle-treatment and wild-type mice. In sgca-/- mice, treatment with scAAVrh74.tMCK.hSGCA resulted in robust expression of α-sarcoglycan protein at the sarcolemma membrane in skeletal muscle at all doses tested. In addition, scAAVrh74.tMCK.hSGCA was effective in improving the histopathology of limb and diaphragm muscle of sgca-/- mice, as indicated by reductions in fibrosis, central nucleation, and normalization of myofiber size. These molecular changes were concomitant with significant increases in specific force generation in the diaphragm and tibialis anterior muscle, protection against eccentric force loss, and reduction in serum CK. Locomotor activity was improved at all doses of vector-treated compared with vehicle-treated sgca-/- mice. Lastly, vector toxicity was not detected in a serum chemistry panel and by gross necropsy. Collectively, these findings provide support for a systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D/R3.

Current Clinical Applications of In Vivo Gene Therapy with AAVs.

Hereditary diseases are caused by mutations in genes, and more than 7,000 rare diseases affect over 30 million Americans. For more than 30 years, hundreds of researchers have maintained that genetic modifications would provide effective treatments for many inherited human diseases, offering durable and possibly curative clinical benefit with a single treatment. This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome and has a low immunogenicity. There are now five treatments approved for commercialization and currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials. The review article presents a broad overview of the field of therapy by in vivo gene transfer. We review gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system, including Alzheimer's disease, Parkinson's disease, Canavan disease, aromatic l-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy), ocular disorders (Leber congenital amaurosis, age-related macular degeneration [AMD], choroideremia, achromatopsia, retinitis pigmentosa, and X-linked retinoschisis), the bleeding disorder hemophilia, and lysosomal storage disorders.

Assessment of Systemic Delivery of rAAVrh74.MHCK7.micro-dystrophin in Children With Duchenne Muscular Dystrophy: A Nonrandomized Controlled Trial.

Importance: Micro-dystrophin gene transfer shows promise for treating patients with Duchenne muscular dystrophy (DMD) using recombinant adeno-associated virus serotype rh74 (rAAVrh74) and codon-optimized human micro-dystrophin driven by a skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7). Objective: To identify the 1-year safety and tolerability of intravenous rAAVrh74.MHCK7.micro-dystrophin in patients with DMD. Design, Setting, and Participants: This open-label, phase 1/2a nonrandomized controlled trial was conducted at the Nationwide Children's Hospital in Columbus, Ohio. It began on November 2, 2017, with a planned duration of follow-up of 3 years, ending in March 2021. The first 4 patients who met eligibility criteria were enrolled, consisting of ambulatory male children with DMD without preexisting AAVrh74 antibodies and a stable corticosteroid dose (≥12 weeks). Interventions: A single dose of 2.0 × 1014 vg/kg rAAVrh74.MHCK7.micro-dystrophin was infused through a peripheral limb vein. Daily prednisolone, 1 mg/kg, started 1 day before gene delivery (30-day taper after infusion). Main Outcomes and Measures: Safety was the primary outcome. Secondary outcomes included micro-dystrophin expression by Western blot and immunohistochemistry. Functional outcomes measured by North Star Ambulatory Assessment (NSAA) and serum creatine kinase were exploratory outcomes. Results: Four patients were included (mean [SD] age at enrollment, 4.8 [1.0] years). All adverse events (n = 53) were considered mild (33 [62%]) or moderate (20 [38%]), and no serious adverse events occurred. Eighteen adverse events were considered treatment related, the most common of which was vomiting (9 of 18 events [50%]). Three patients had transiently elevated γ-glutamyltransferase, which resolved with corticosteroids. At 12 weeks, immunohistochemistry of gastrocnemius muscle biopsy specimens revealed robust transgene expression in all patients, with a mean of 81.2% of muscle fibers expressing micro-dystrophin with a mean intensity of 96% at the sarcolemma. Western blot showed a mean expression of 74.3% without fat or fibrosis adjustment and 95.8% with adjustment. All patients had confirmed vector transduction and showed functional improvement of NSAA scores and reduced creatine kinase levels (posttreatment vs baseline) that were maintained for 1 year. Conclusions and Relevance: This trial showed rAAVrh74.MHCK7.micro-dystrophin to be well tolerated and have minimal adverse events; the safe delivery of micro-dystrophin transgene; the robust expression and correct localization of micro-dystrophin protein; and improvements in creatine kinase levels and NSAA scores. These findings suggest that rAAVrh74.MHCK7.micro-dystrophin can provide functional improvement that is greater than that observed under standard of care. Trial Registration: ClinicalTrials.gov Identifier: NCT03375164.

Clinical development on the frontier: gene therapy for duchenne muscular dystrophy.

Introduction: The development of adeno-associated virus (AAV) vectors as safe vehicles for in vivo delivery of therapeutic genes has been a major milestone in the advancement of gene therapy, enabling a promising strategy for ameliorating a wide range of diseases, including Duchenne muscular dystrophy (DMD).Areas covered: Based on experience with the development of a gene transfer therapy agent for DMD, we discuss ways in which gene therapy for rare disease challenges traditional clinical development paradigms, and recommend a step-wise approach for design and evaluation to support broader applicability of gene therapy.Expert opinion: The gene therapy development approach should intentionally design the therapeutic construct and the clinical study to systematically evaluate agent delivery, safety, and efficacy. Rigorous preclinical work is essential for establishing an effective gene delivery platform and determining the efficacious dose. Clinical studies should thoroughly evaluate transduction, on-target transgene expression at the tissue and cellular level, and functional efficacy.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

Gene Delivery for Limb-Girdle Muscular Dystrophy Type 2D by Isolated Limb Infusion.

In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/µg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model - the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.