Gene Therapy For Muscular Dystrophy Research Articles

Development of A2G80 peptide-gene complex for targeted delivery to muscle cells

Therapeutic strategies based on antisense oligonucleotides and therapeutic genes are being extensively investigated for the treatment of hereditary muscle diseases and hold great promise. However, the cellular uptake of these polyanions to the muscle cells is inefficient. Therefore, it is necessary to develop more effective methods of gene delivery into the muscle tissue. The A2G80 peptide (VQLRNGFPYFSY) from the laminin α2 chain has high affinity for α-dystroglycan (α-DG) which is expressed on the membrane of muscle cells. In this study, we designed a peptide-modified A2G80 with oligoarginine and oligohistidine (A2G80-R9-H8), and prepared peptide/plasmid DNA (pDNA) complex, to develop an efficient gene delivery system for the muscle tissue. The peptide/pDNA complex showed α-DG-dependent cellular uptake of the A2G80 sequence and significantly improved gene transfection efficiency mediated by the oligohistidine sequence in C2C12 myoblast cells. Further, the peptide/pDNA complex promoted efficient and sustained gene expression in the Duchenne muscular dystrophy mouse models. The A2G80-R9-H8 peptide has the potential for use as a specific carrier for targeting muscle in gene therapy in muscular dystrophy.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.206 Evaluation of the lipid-binding and stability properties of recombinant dystrophin spectrin-like repeat constructs

Gene therapy for Duchenne muscular dystrophy (DMD) is being evaluated as a therapeutic strategy to restore production of functional dystrophin. Replacement of full-length dystrophin gene is not feasible due to its large size relative to the cloning capacity of adeno-associated virus (AAV)-derived vectors (∼5kb). Strategies based on delivery of shortened, but functional forms of dystrophin have emerged as a viable option. Identification of the optimal shortened micro-dystrophin construct that most normalizes muscle function is a key translation question.

Overview of gene therapy in spinal muscular atrophy and Duchenne muscular dystrophy.

Both 5q-linked spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD) are fatal monogenic neuromuscular disorders caused by loss-of-function mutations. SMA is an autosomal recessive disorder affecting motor neurons that is typically caused by homozygous whole-gene deletions of SMN1. DMD is an X-linked recessive muscle disease most often due to exon deletions, but also duplications and smaller sized variants within the DMD gene. Gene replacement therapy offers the opportunity to correct the underlying genetic defect by the introduction of a functional gene. We review the transformative work from clinical trials to United States Food and Drug Administration approval of onasemnogene abeparvovec-xioi in SMA and its application in clinical practice and the early results of microdystrophin delivery in DMD. We also review the introduction of antisense oligonucleotides to alter pre-messenger RNA splicing to promote exon inclusion (as in nusinersen in SMA) or exclusion (as in eteplirsen in DMD) into neuromuscular therapeutics. There are multiple promising novel genetically mediated therapies on the horizon, which in aggregate point towards a hopeful future for individuals with SMA and DMD.

RAAV8 and rAAV9-Mediated Long-Term Muscle Transduction with Tacrolimus (FK506) in Non-Human Primates

To establish an efficient, safe immunosuppressive regimen of adeno-associated vector (AAV)-mediated gene therapy for Duchenne muscular dystrophy (DMD), we evaluated the effect of tacrolimus (FK506) on skeletal muscle transduction with AAV8 and AAV9 vectors expressing the LacZ and microdystrophin (M3) genes labeled by FLAG. We utilized 3- to 4-year-old Macaca fascicularis, screened for neutralizing antibodies against AAV. 3 days before AAV injection and throughout the experiment, 0.06 mg/kg tacrolimus was intravenously administered. A viral suspension of 1 × 1013 viral genomes/muscle was intramuscularly injected bilaterally at the tibialis anterior and biceps brachii muscles, which were biopsied at 8, 16, 24, and 42 weeks after injection. Without tacrolimus, AAV8- and AAV9-mediated LacZ expression disappeared 8 and 16 weeks after transduction, respectively. With tacrolimus, AAV8/9-mediated LacZ expression persisted for at least 42 weeks after injection. At 42 weeks after AAV8CMVLacZ and AAV9CMVLacZ injection, nearly 50% and 17% of muscle fibers were positive for β-galactosidase, respectively. AAV8/9-mediated M3-FLAG expression lasted for up to 42 weeks using tacrolimus. No significant generalized toxicity was observed in any monkey. These results indicate that tacrolimus administration regulated the immune response to transgenes and truncated microdystrophin in normal primates and may enhance the benefits of AAV-mediated gene therapy for DMD.

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications, hurdles and possible optimizations.

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well-tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non-viral gene transfer following hydrodynamic limb vein injection.

Roche will acquire a Sarepta gene therapy

Roche is expanding its reach in gene therapy with a $1.15 billion deal to commercialize outside the US a Sarepta Therapeutics gene therapy for Duchenne muscular dystrophy. In December, Roche completed its delayed $4.8 billion acquisition of Spark Therapeutics, a gene therapy firm focused on eye and blood diseases. Sarepta’s therapy, which uses a virus to deliver a microdystrophin gene, originally came from Nationwide Children’s Hospital. The firm is recruiting for a Phase II clinical trial.

Surrogate gene therapy for muscular dystrophy.

An engineered truncated gene derived from the dystrophin-related protein (utrophin), prevents pathology without an immune response in an animal model of Duchenne muscular dystrophy (DMD) gene therapy.

DMD TREATMENT: P.334PPMO pre-treatment is beneficial for AAV-based gene therapy in Duchenne muscular dystrophy

DMD TREATMENT: P.334PPMO pre-treatment is beneficial for AAV-based gene therapy in Duchenne muscular dystrophy

Catalent will enter gene-therapy service market

Seeking to enter the business of gene-therapy manufacturing, the drug service firm Catalent Pharma Solutions has agreed to pay $1.2 billion for the custom manufacturer Paragon Bioservices. Paragon will put Catalent in the business of making adeno-associated virus vectors, plasmids, and lentivirus vectors used to deliver gene and cell therapies. Earlier this month, Paragon opened a gene-therapy manufacturing facility in Maryland’s Anne Arundel County, complementing the production capacity at its headquarters at University of Maryland’s BioPark. The new capacity will support customers’ clinical- and commercial-stage programs. After the Catalent deal was unveiled, Paragon announced it will expand its relationship with a key customer, Sarepta Therapeutics. In October, Sarepta enlisted Paragon to help make its experimental Duchenne muscular dystrophy gene therapy; now, Paragon and Sarepta are considering a joint venture to manufacture gene therapies at a new site. The deal for Paragon, which expects to have sa...

Gene and Cell Therapy for Muscular Dystrophies: Are We Getting There?

In the last few years, significant advances have occurred in the preclinical and clinical work toward gene and cell therapy for muscular dystrophy. At the time of this writing, several trials are ongoing and more are expected to start. It is thus a time of expectation, even though many hurdles remain and it is unclear whether they will be overcome with current strategies or if further improvements will be necessary.

Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.

D11 - Combining iPS cell-derived myogenic progenitors and human artificial chromosomes as a potential genomic integration-free cell and gene therapy for Duchenne muscular dystrophy

D11 - Combining iPS cell-derived myogenic progenitors and human artificial chromosomes as a potential genomic integration-free cell and gene therapy for Duchenne muscular dystrophy

Gene Therapy for Hemophilia and Duchenne Muscular Dystrophy in China.

Gene therapy is a new technology that provides potential for curing monogenic diseases caused by mutations in a single gene. Hemophilia and Duchenne muscular dystrophy (DMD) are ideal target diseases of gene therapy. Important advances have been made in clinical trials, including studies of adeno-associated virus vectors in hemophilia and antisense in DMD. However, issues regarding the high doses of viral vectors required and limited delivery efficiency of antisense oligonucleotides have not yet been fully addressed. As an alternative strategy to classic gene addition, genome editing based on programmable nucleases has also shown promise to correct mutations in situ. This review describes the recent progress made by Chinese researchers in gene therapy for hemophilia and DMD.

Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD

RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy.

P.305 - Molecular therapy in a novel translational large animal model for Duchenne muscular dystrophy

The development of efficient and safe strategies in gene therapy for Duchenne muscular dystrophy (DMD) remains a huge challenge. Primary myoblast culture models derived from our recently developed large animal model, the exon 52-deficient DMD pig, facilitate to assess various approaches towards dystrophin restoration. The DMD pig carries the most frequent exon deletion occurring in DMD patients and is amenable to targeted exon 51 or exon 53 antisense oligonucleotide (AON)-mediated therapies. Differentiating porcine myoblasts represent a preclinical in vitro model to examine advanced splice modulation approaches including novel oligonucleotide chemistries. Dystrophin mRNA and protein expression are primary endpoints that will be qualitatively and quantitatively examined to identify the most promising candidates for in vivo applications. Dystrophin expression is only present in differentiating myogenic cells. As compared to other species, pig myoblasts differentiate very poorly in vitro. However, by systematically improving coating of cell culture dishes and differentiation medium, we even observed spontaneous twitching of pig myotubes in vitro suggesting high-level differentiation. Besides the translational potential to DMD therapy in affected patients, differentiating myogenic cell culture models are an essential prerequisite to screen novel substances in terms of efficacy and toxicity, prior to their application in vivo.

Research progress on disease models and gene therapy of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked, recessive and lethal genetic disease, which usually caused by gene mutations and the underlying mechanisms are complicated and diverse. The causal gene of DMD is the largest one in human that locates in the region of Xp21.2, encoding dystrophin. Currently there is no effective treatment for DMD patients. The treatment of DMD depends on gene mutation and molecular mechanism study of the disease, which requires reliable disease models such as mdx mouse model. Recently, researchers have increasingly discovered gene therapy strategies for DMD, and the efficacy has been demonstrated in DMD animal models. In addition, induced pluripotent stem cell technology can provide patient-specific cell source, offering a new platform for mechanism and therapy study of DMD.

Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

The Action Duchenne 13th International Annual Conference, November 6–7, 2015, London, UK

Article history: Received 3 December 2015 Accepted 3 December 2015 Available online 3 December 2015 trophin protein. Two groups recently published data, a single-dose intravascular delivery of rAAV2/8-Spc512-μDys (Le Guiner et al., 2015) and AAV-9 reporter or micro-dystrophin (μDys) vector at doses of 1.92–6.24 × 10 viral genome particles/kg (Yue et al., 2015) in Golden Retriever Muscular Dystrophy Dogs, the canine model of DMD. Overall, the improvement of multiple muscle parameters was observed with no

Gene Therapy for LMNA-related Congenital Muscular Dystrophy (L-CMD) by Trans-Splicing

LMNA-related Congenital Muscular Dystrophy (L-CMD) is a rare genetic disorder characterized by the onset of selective axial weakness and wasting in the first year of life with limited motor achievements, associated with multiple severe contractures and frequent respiratory failure requiring early ventilatory support. We identified heterozygous de novo mutations in LMNA, encoding lamins A/C, as responsible for this sub-group of CMD in which no therapeutic treatment is available[1]. Lamins A/C are nuclear envelope proteins, ubiquitously expressed in all post mitotic cells, which play essential roles in the nucleus structure and in the regulation of gene expression. We generated the first Knock-In mouse model of L-CMD (KI-LmnadelK32) reproducing a LMNA mutation identified in L-CMD patients. Homozygous mice die within the first 3 weeks of life from striated muscles maturation delay and severe metabolic defects[2]. Heterozygous mice develop an isolated dilated cardiomyopathy and die by one year of age[3]. We aim to assess the possibility of LMNA-mRNA repair by spliceosome-mediated RNA trans-splicing (SMarT) as a potential therapeutic approach for L-CMD. This gene therapy strategy will allow inhibition of mutated LMNA transcript expression for the benefit of corresponding wild type transcripts. We developed 5'-RNA pre-trans-splicing molecules (PTM) capable of repairing the murine LMNA transcripts. Efficiency of these PTM was assessed in vitro in C2C12 cells and in vivo using Adeno-Associated Virus (AAV) transduction in tibialis anterior of WT mice. We will now determine the ability of the best PTM to restore normal muscular phenotype, in vitro in KI myoblasts/myotubes and in vivo after injection of AAV-PTM vectors in new born homozygous and adult heterozygous mice. Histological and metabolic parameters will be monitored to evaluate the degree of phenotype rescue.

Viral vector-mediated gene therapies.

Gene therapy as a treatment for neuromuscular disease has significantly advanced over the past decade. In the present review, the progress of adeno-associated viruses (AAV) vector-mediated gene therapy for Duchenne muscular dystrophy (DMD) during the past year is highlighted. Modulating the immune response to AAV vector capsid or the transgene has helped to increase stable transduction efficiency. Full-length dystrophin expression via gene editing with targeted nucleases may ultimately be an ideal treatment option. Also genes with homologues function may ameliorate many aspects of the DMD pathophysiology. The work during the past year has increased our understanding of AAV vector-mediated therapy and has also validated new approaches to treat DMD. The results will aid in the design of both preclinical and clinical trials.