Gene Therapy For Muscular Dystrophy Research Articles

Systemic delivery of full-length dystrophin in Duchenne muscular dystrophy mice

Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver micro-dystrophin (µDys), which does not provide full protection for striated muscles as it lacks many important functional domains of full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We split FL-dystrophin into three fragments linked to two orthogonal pairs of split intein, allowing efficient assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mdx4cv mice. Dystrophin-glycoprotein complex components are also restored at the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective cavin 4 localization and associated signaling in mdx4cv heart. Therefore, our data support the feasibility of a mutation-independent FL-dystrophin gene therapy for DMD, warranting further clinical development.

Boy Dosed with Pfizer's Duchenne Muscular Dystrophy Gene Therapy Dies a Year After Phase II Trial.

Boy Dosed with Pfizer's Duchenne Muscular Dystrophy Gene Therapy Dies a Year After Phase II Trial.

Prevalence of Adeno-Associated Virus-9-Neutralizing Antibody in Chinese Patients with Duchenne Muscular Dystrophy.

The delivery of a mini-dystrophin gene to skeletal muscles using recombinant adeno-associated virus serotype (AAV) holds great potential as a gene therapy for Duchenne muscular dystrophy (DMD). However, the presence of anti-AAV-neutralizing antibodies (NAbs) may impede the effectiveness of gene transduction. This study aimed to evaluate the prevalence of anti-AAV9 NAbs in Chinese patients with DMD, and to characterize the target population for an AAV gene therapy. A total of one hundred male patients with DMD were included in this study, and demographic and clinical data were collected. A blood specimen was obtained from each participant for the purpose of evaluating the existence of anti-AAV9 NAbs through a cell-based functional assay conducted at a central laboratory. A NAb titer exceeding 1:4 was considered positive. The positivity rates of anti-AAV9 NAb were compared among different subgroups. The median age of this DMD cohort was 8 years old, ranging from 3 to 15 years of age. Forty-two percent of patients tested positive for anti-AAV9 NAb. Notably, all samples from patients under 4 years of age tested negative, and the positivity rates of anti-AAV9 NAb differed significantly across the three age subgroups (<4 years old, ≥4 years old and <12 years old, and ≥12 years old, χ2 = 7.221, p = 0.023). Further investigation into the living environment revealed a higher positivity rate of anti-AAV9 NAb in rural patients compared with urban patients (χ2 = 3.923, p = 0.048). Moreover, the prevalence in patients from different cities/provinces varied greatly (χ2 = 16.550, p = 0.003). There was no statistically significant difference in the positivity rate of NAb among subgroups of patients with different motor functions (ambulatory or nonambulatory) and different treatment strategies (taking or not taking glucocorticoid). In Chinese DMD patients, the prevalence of anti-AAV9 NAb was found to reach 42%. Moreover, the antibody-positive rate in children <4 years of age was low and revealed notable regional discrepancies.

Advances in Dystrophinopathy Diagnosis and Therapy.

Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the Dystrophin gene, including Duchenne and Becker muscular dystrophy, and dilated cardiomyopathy. However, Duchenne muscular dystrophy interconnects with bone loss and osteoporosis, which are exacerbated by glucocorticoids therapy. Procedures for diagnosing dystrophinopathies include creatine kinase assay, haplotype analysis, Southern blot analysis, immunological analysis, multiplex PCR, multiplex ligation-dependent probe amplification, Sanger DNA sequencing, and next generation DNA sequencing. Pharmacological therapy for dystrophinopathies comprises glucocorticoids (prednisone, prednisolone, and deflazacort), vamorolone, and ataluren. However, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and β-blockers are the first-line to prevent dilated cardiomyopathy in dystrophinopathy patients. Duchenne muscular dystrophy gene therapy strategies involve gene transfer, exon skipping, exon reframing, and CRISPR gene editing. Eteplirsen, an antisense-oligonucleotide drug for skipping exon 51 from the Dystrophin gene, is available on the market, which may help up to 14% of Duchenne muscular dystrophy patients. There are various FDA-approved exon skipping drugs including ExonDys-51 for exon 51, VyonDys-53 and Viltolarsen for exon 53 and AmonDys-45 for exon 45 skipping. Other antisense oligonucleotide drugs in the pipeline include casimersen for exon 45, suvodirsen for exon 51, and golodirsen for exon 53 skipping. Advances in the diagnosis and therapy of dystrophinopathies offer new perspectives for their early discovery and care.

Sildenafil increases AAV9 transduction after a systemic administration and enhances AAV9-dystrophin therapeutic effect in mdx mice.

Adeno-associated virus (AAV) vectors have been successfully used to deliver genes for treating rare diseases. However, the systemic administration of high AAV vector doses triggers several adverse effects, including immune response, the asymptomatic elevation of liver transaminase levels, and complement activation. Thus, improving AAV transduction and reducing AAV dosage for treatment is necessary. Recently, we found that a phosphodiesterase-5 inhibitor significantly promoted AAV9 transduction in vitro by regulating the caveolae and macropinocytosis pathways. When AAV9-Gaussian luciferase (AAV9-Gluc) and AAV9-green fluorescent protein (AAV9-GFP) were injected intravenously into mice pre-treated with sildenafil, the expressions of Gluc in the plasma and GFP in muscle tissues significantly increased (P < 0.05). Sildenafil also improved Evans blue permeation in tissues. Additionally, we found that sildenafil promoted Treg proliferation, inhibited B-cell activation, and decreased anti-AAV9 IgG levels (P < 0.05). Furthermore, sildenafil significantly promoted Duchenne muscular dystrophy gene therapy efficacy using AAV9 in mdx mice; it increased micro-dystrophin gene expression, forelimb grip strength, and time spent on the rotarod test, decreased serum creatine kinase levels, and ameliorated histopathology by improving muscle cell morphology and reducing fibrosis (P < 0.05). These results show that sildenafil significantly improved AAV transduction, suppressed the levels of anti-AAV9 IgG, and enhanced the efficacy of gene therapy.

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Food and Drug Administration Sets Stage for Approval of First Duchenne Muscular Dystrophy Gene Therapy.

Human Gene TherapyVol. 34, No. 11-12 Gene Therapy BriefsFood and Drug Administration Sets Stage for Approval of First Duchenne Muscular Dystrophy Gene TherapyAlex PhilippidisAlex Philippidis*Correspondence: E-mail Address: aphilippidis@liebertpub.comMary Ann Liebert, Inc., Publishers, New Rochelle, New York, USA.Search for more papers by this authorPublished Online:16 Jun 2023https://doi.org/10.1089/hum.2023.29243.bfsAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 34Issue 11-12Jun 2023 InformationCopyright 2023, by Mary Ann Liebert, Inc., publishersTo cite this article:Alex Philippidis.Food and Drug Administration Sets Stage for Approval of First Duchenne Muscular Dystrophy Gene Therapy.Human Gene Therapy.Jun 2023.477-480.http://doi.org/10.1089/hum.2023.29243.bfsPublished in Volume: 34 Issue 11-12: June 16, 2023PDF download

CFTR and dystrophin encoding plasmids carrying both luciferase reporter gene, nuclear import specific sequences and triple helix sites

Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5–12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine • oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy Gene Therapy in 2023: Status, Perspective, and Beyond.

Duchenne muscular dystrophy (DMD) was named more than 150 years ago. About four decades ago, the DMD gene was discovered, and the reading frame shift was determined as the genetic underpinning. These pivotal findings significantly changed the landscape of DMD therapy development. Restoration of dystrophin expression with gene therapy became a primary focus. Investment in gene therapy has led to the approval of exon skipping by regulatory agencies, multiple clinical trials of systemic microdystrophin therapy using adeno-associated virus vectors, and revolutionary genome editing therapy using the CRISPR technology. However, many important issues surfaced during the clinical translation of DMD gene therapy (such as low efficiency of exon skipping, immune toxicity-induced serious adverse events, and patient death). In this issue of Human Gene Therapy, several research articles highlighted some of the latest developments in DMD gene therapy. Importantly, a collection of articles from experts in the field reviewed the progress, major challenges, and future directions of DMD gene therapy. These insightful discussions have significant implications for gene therapy of other neuromuscular diseases.

Evaluation of rAAVrh74 gene therapy vector seroprevalence by measurement of total binding antibodies in patients with Duchenne muscular dystrophy.

Adeno-associated virus (AAV) vectors are a promising platform for in vivo transfer of transgenes designed to treat diseases. Pre-existing humoral immunity to these vectors can potentially impact the safety and efficacy of gene therapies. Consequently, individuals with pre-existing antibodies to the specific AAV serotypes used may be excluded from clinical trials and treatments. Recombinant AAV serotype rh74 (rAAVrh74), a vector originally isolated from rhesus monkeys and potentially less immunogenic than other serotypes isolated from humans (e.g. AAV2, AAV5, and AAV9), efficiently transduces muscle and is being investigated for use in gene therapy for Duchenne muscular dystrophy (DMD). To evaluate prevalence of total binding antibodies (neutralizing and non-neutralizing) against rAAVrh74 in patients with DMD. Eligible individuals (N = 107) were ⩾ 4 to < 18 years old with genetically confirmed DMD and were excluded from the study if they lived with a person who had known exposure to rAAVrh74 or other gene transfer therapy, or if they received prior treatment with gene transfer therapy. A single blood sample was obtained from each participant, and anti-rAAVrh74 total binding antibodies were measured by enzyme-linked immunosorbent assay. Total binding antibody level < 1:400 was defined as not elevated or seronegative. Primary endpoint was the percentage of subjects with elevated total antibody titers to rAAVrh74. A large preponderance (86.1%) of patients with DMD in this data set was seronegative for anti-rAAVrh74 total binding antibodies. These patients would potentially meet the antibody status eligibility criterion for entry into rAAVrh74-based gene therapy clinical trials. Measuring total binding antibodies is a more comprehensive approach to assess pre-existing immune response versus measuring neutralizing antibodies alone. The low seroprevalence of total binding antibodies against rAAVrh74 shown here supports the broad applicability of rAAVrh74-based gene transfer therapy for patients with DMD and potentially other neuromuscular diseases.

PCR51 Reassessing the Acceptability of Risk of Death Associated With Gene Therapy for Duchenne Muscular Dystrophy: A Qualitative Study to Inform Updating a Quantitative Instrument

In 2017/2018, we concluded a study on risk tolerance for gene therapy (GT) for Duchenne muscular dystrophy (Duchenne). Duchenne is a progressive, fatal condition. GTs under trial could be disease modifying, not curative. GT has serious risks, including risk of death. In our prior study we assessed maximum acceptable risk (MAR) of death in U.S.-based adults with Duchenne and parents. In GT trials over the past 5 years there were serious adverse events, clinical holds, and one death in late 2021. We initiated a second study to assess current MAR in the U.S., United Kingdom, and to explore experiences and attitudes of clinicians.

Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full- length dystrophin. Thus increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain1 (ABD1). We engineered a novel fluorescence resonance energy transfer (FRET) biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C- terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, high-throughput screening (HTS) fluorescence lifetime assay, identifying several compounds from a FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.

P.142 Improving FSHD RNAi gene therapy using myotropic MyoAAVs

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder affecting more than 870,000 people worldwide. FSHD is caused by de-repression of the toxic <i>DUX4</i> gene in skeletal muscle. There are currently no approved treatments for FSHD, but several promising <i>DUX4</i>-inhibition therapies are under development. Previously, our lab published pre-clinical efficacy and safety studies for a <i>DUX4</i>-targeted RNAi-based gene therapy using an artificial microRNA called mi405. In unpublished data, we performed dose-finding studies in FSHD animal models, demonstrated inhibition of human <i>DUX4</i>-associated biomarkers in xenografts, confirmed long-term expression in vivo, and characterized the biodistribution of two lead AAV vector serotypes (AAV6 & AAV9). In line with muscular dystrophy gene therapy studies now in clinical trials (e.g., micro-dystrophin), our dose-finding work demonstrated that sufficient muscle transduction required high dose systemic delivery of AAV9 (1e14vg/kg). Though high doses were non-pathogenic in our toxicology studies [WL1] and several systemic gene therapy studies used doses that exceeded our target range, serious adverse events and deaths in other programs raised concerns about the safety of first generation AAV vector doses exceeding 10^14 vg/kg. Here we seek to reduce potential perceived or real safety issues. We aim to boost muscle transduction and reduce vector doses required to provide therapeutic benefit using new, engineered MyoAAV serotypes. Here, we compare our first generation AAV9 system with MyoAAVs carrying the same mi405 genome and a GFP reporter, using intramuscular and systemic delivery. Consistent with published data, systemically delivered MyoAAVs provided drastically superior muscle transduction compared to AAV9. Specifically, we observed widespread muscle transduction following 3e13 vg/kg doses in mice. We are now performing dose finding efficacy and safety studies in FSHD mice to support a future clinical trial (9e12 vg/kg, 3e13 vg/kg, and 6e13 vg/kg). We expect that new muscle-enhanced vectors combined with the highly efficacious mi405 will enable lower AAV doses while preserving the therapeutic efficacy of high dose first generation vectors.

Food and Drug Administration Lifts Clinical Hold on Pfizer Duchenne Muscular Dystrophy Gene Therapy Linked to Patient Death.

Food and Drug Administration Lifts Clinical Hold on Pfizer Duchenne Muscular Dystrophy Gene Therapy Linked to Patient Death.

Gene Therapy for the Limb-Girdle Muscular Dystrophy

Gene Therapy for the Limb-Girdle Muscular Dystrophy

First person – Chady Hakim

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models &amp; Mechanisms, helping early-career researchers promote themselves alongside their papers. Chady Hakim is first author on ‘ Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber-type switch in Duchenne muscular dystrophy dogs’, published in DMM. Chady is a Research Assistant Professor in the lab of Dongsheng Duan at the University of Missouri, Colombia, MO, USA, investigating the preclinical development of gene therapy for Duchenne muscular dystrophy (DMD), with a particular interest in using the canine DMD model.

Changes in Myonuclear Number During Postnatal Growth - Implications for AAV Gene Therapy for Muscular Dystrophy.

Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration.AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for microdystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies.

DMD – ANIMAL MODELS: EP.90 A novel AAV8-based gene therapy for Duchenne muscular dystrophy: preclinical studies in the mdx mouse

RGX-202, a recombinant adeno-associated virus of serotype 8 (AAV8) with an optimized human microdystrophin transgene under the control of a muscle specific promoter (spc5-12), is a potential gene therapy candidate for the treatment of DMD. The microdystrophin includes 4 rods and 3 hinges and an extended coding region of the carboxyl terminal (CT) domain, known to bind α-dystrobrevin and α-syntrophin. Enhanced association of α-syntrophin may help recruiting neuronal nitric oxide synthase (nNOS) at the sarcolemma, facilitating vasodilation during exercise and protecting muscle against ischemic stress. The protein coding sequence was codon-optimized and depleted of CpG dinucleotides. RGX-202 was administered intravenously to male mdx mice and evaluated over 26 weeks. RGX-202-treated animals exhibited significant increases in muscle strength and normalization of gait as shown by fine kinematic analysis. Using T2 magnetic resonance imaging (MRI) to probe muscle damage characteristic of mdx, dose-dependent reductions of the hyper intense lesions were measured, consistent with an improvement in dystrophic pathology. High levels of RGX-202 vector DNA and microdystrophin protein were detected in the skeletal and cardiac muscles, and membrane assembly of the Dystrophin Associated Protein Complex (DAPC) was observed by immunostaining of muscle sections. Alongside staining for α-syntrophin, dystrobrevin, and ß-dystroglycan, there was also evidence for nNOS recruitment at the sarcolemma. RGX-202 treatment resulted in a reduction of the number of activated Pax7 positive satellite cells, further suggesting an improvement in abnormal muscle regeneration. Our data showed that treatment with RGX-202 resulted in the expression of an active microdystrophin which was able to improve muscle function and reduce dystrophic pathology in mdx mice. We are currently developing RGX-202 as a candidate for microdystrophin gene therapy in DMD patients.

Incorporating baseline covariates to validate surrogate endpoints with a constant biomarker under control arm.

A surrogate endpoint S in a clinical trial is an outcome that may be measured earlier or more easily than the true outcome of interest T. In this work, we extend causal inference approaches to validate such a surrogate using potential outcomes. The causal association paradigm assesses the relationship of the treatment effect on the surrogate with the treatment effect on the true endpoint. Using the principal surrogacy criteria, we utilize the joint conditional distribution of the potential outcomes T, given the potential outcomes S. In particular, our setting of interest allows us to assume the surrogate under the placebo, , is zero-valued, and we incorporate baseline covariates in the setting of normally distributed endpoints. We develop Bayesian methods to incorporate conditional independence and other modeling assumptions and explore their impact on the assessment of surrogacy. We demonstrate our approach via simulation and data that mimics an ongoing study of a muscular dystrophy gene therapy.

Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin.

ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of μDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.