L. Rodino-Klapac

Gentamicin-induced readthrough of stop codons in duchenne muscular dystrophy

The objective of this study was to establish the feasibility of long‐term gentamicin dosing to achieve stop codon readthrough and produce full‐length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD).

Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates

A vector delivered into muscles of monkeys generates a natural regulatory molecule, which increases muscle size and strength and may be useful therapeutically. Beyond Mighty Mouse: Building Muscle Mass Strength in Monkeys Patients with progressive neuromuscular disorders all experience the foreboding of the severe disability that awaits them and from which there is little to no relief. Although this class of disorders has multiple genetic and physiological origins, a therapy that directly addresses the debilitating muscle weakness that is the hallmark of these maladies would enhance the lives of millions. Now, in an extension of their previous work in dystrophic mice, Kota et al. describe such a therapeutic approach in preclinical studies performed in nonhuman primates. This treatment mode is applicable to several progressive neuromuscular disorders whether or not scientists have defined their precise genetic defects. The authors used a gene therapy approach to introduce a version of the human gene encoding follistatin into the muscles of the femurs of healthy cynomolgus macaques. Follistatin is a potent inhibitor of myostatin, a signaling molecule that regulates skeletal muscle mass. Follistatin blocks myostatin signaling and augments muscle size and strength safely in mice but, until now, has not been tested in primates. Kota et al. injected a follistatin-producing gene therapy vector into the leg muscles of the monkeys and measured increases in muscle mass and strength. Sustained follistatin expression caused no aberrations in the structures or functions of a variety of organs. This promising progress comes with some caveats. Because healthy monkeys served as subjects for this therapeutic protocol, these findings are not predictive of the outcome in a clinical setting with patients suffering from muscle disorders. In certain genetic neuromuscular diseases, the muscles undergo a repeated cycle of degeneration and regeneration. The vector used in this study does not integrate into the muscle cell genome and thus can be lost from the cells during the degeneration-regeneration cycles. However, the authors point out that the enhancement of muscle size and strength observed in similarly treated dystrophic mice persisted for more than a year even though there was appreciable muscle turnover. More study is needed before follistatin enters the clinic, such as a molecular assessment of gene and vector sequences in multiple tissues. Nonetheless, the work of Kota et al. constitutes proof of principle for the use of myostatin inhibitors to build muscle in primates. Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.

Gene Therapy for Muscular Dystrophy: Lessons Learned and Path Forward

Our Translational Gene Therapy Center has used small molecules for exon skipping and mutation suppression and gene transfer to replace or provide surrogate genes as tools for molecular-based approaches for the treatment of muscular dystrophies. Exon skipping is targeted at the pre-mRNA level allowing one or more exons to be omitted to restore the reading frame. In Duchenne Muscular Dystrophy (DMD), clinical trials have been performed with two different oligomers, a 2O-methyl-ribo-oligonucleoside-phosphorothioate (2OMe) and a phosphorodiamidate morpholino (PMO). Both have demonstrated early evidence of efficacy. A second molecular approach involves suppression of stop codons to promote readthrough of the DMD gene. We have been able to establish proof of principle for mutation suppression using the aminoglycoside, gentamicin. A safer, orally administered, alternative agent referred to as Ataluren (PTC124) has been used in clinical trials and is currently under consideration for approval by the FDA. Using a gene therapy approach, we have completed two trials and have initiated a third. For DMD, we used a mini-dystrophin transferred in adeno-associated virus (AAV). In this trial an immune response was seen directed against transgene product, a quite unexpected outcome that will help guide further studies. For limb girdle muscular dystrophy 2D (alpha-sarcoglycan deficiency), the transgene was again transferred using AAV but in this study, a muscle specific creatine kinase promoter controlled gene expression that persisted for six months. A third gene therapy trial has been initiated with transfer of the follistatin gene in AAV directly to the quadriceps muscle. Two diseases with selective quadriceps muscle weakness are undergoing gene transfer including sporadic inclusion body myositis (sIBM) and Becker muscular dystrophy (BMD). Increasing the size and strength of the muscle is the goal of this study. Most importantly, no adverse events have been encountered in any of these clinical trials.

Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

Plasmapheresis Eliminates the Negative Impact of AAV Antibodies on Microdystrophin Gene Expression Following Vascular Delivery

Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0u2009±u200920.8%) that was attenuated in sero-positive animals (19.6u2009±u200918.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8u2009±u200918.0%), which is comparable with that of AAV sero-negative animals (53.7u2009±u20097.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1u2009±u20096.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

Update on the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is the most severe childhood form of muscular dystrophy caused by mutations in the gene responsible for dystrophin production. There is no cure, and treatment is limited to glucocorticoids that prolong ambulation and drugs to treat the cardiomyopathy. Multiple treatment strategies are under investigation and have shown promise for Duchenne muscular dystrophy. Use of molecular-based therapies that replace or correct the missing or nonfunctional dystrophin protein has gained momentum. These strategies include gene replacement with adeno-associated virus, exon skipping with antisense oligonucleotides, and mutation suppression with compounds that “read through” stop codon mutations. Other strategies include cell therapy and surrogate gene products to compensate for the loss of dystrophin. All of these approaches are discussed in this review, with particular emphasis on the most recent advances made in each therapeutic discipline. The advantages of each approach and challenges in translation are outlined in detail. Individually or in combination, all of these therapeutic strategies hold great promise for treatment of this devastating childhood disease.

Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D

Background: Alpha-sarcoglycan (α-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of α-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. Methods: The human α-SG gene (hα-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old α-SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 × 109 vg) and high (3 × 1010 vg) doses of AAV1.hα-SG were administered. Results: Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of α-SG gene expression by fiber counts yielded similar levels of myofiber transduction for both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed α-SG overexpression using CMV and both the MCK promoters. Conclusion: Our data demonstrate robust and sustained adeno-associated virus type 1 alpha-sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.

AAV1.NT-3 Gene Therapy for Charcot–Marie–Tooth Neuropathy

Charcot–Marie–Tooth (CMT) neuropathies represent a heterogeneous group of peripheral nerve disorders affecting 1 in 2,500 persons. One variant, CMT1A, is a primary Schwann cell (SC) disorder, and represents the single most common variant. In previous studies, we showed that neurotrophin-3 (NT-3) improved the tremblerJ (TrJ) mouse and also showed efficacy in CMT1A patients. Long-term treatment with NT-3 was not possible related to its short half-life and lack of availability. This led to considerations of NT-3 gene therapy via adenoassociated virus (AAV) delivery to muscle, acting as secretory organ for widespread distribution of this neurotrophic agent. In the TrJ model of demyelinating CMT, rAAV1.NT-3 therapy resulted in measurable NT-3 secretion levels in blood sufficient to provide improvement in motor function, histopathology, and electrophysiology of peripheral nerves. Furthermore, we showed that the compound muscle action potential amplitude can be used as surrogate for functional improvement and established the therapeutic dose and a preferential muscle-specific promoter to achieve sustained NT-3 levels. These studies of intramuscular (i.m.) delivery of rAAV1.NT-3 serve as a template for future CMT1A clinical trials with a potential to extend treatment to other nerve diseases with impaired nerve regeneration.

Vascular Delivery of rAAVrh74.MCK.GALGT2 to the Gastrocnemius Muscle of the Rhesus Macaque Stimulates the Expression of Dystrophin and Laminin α2 Surrogates

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44u2009±u20094% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9u2009±u20094% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.

Gene Therapy for Muscular Dystrophy: Moving the Field Forward

Gene therapy for the muscular dystrophies has evolved as a promising treatment for this progressive group of disorders. Although corticosteroids and/or supportive treatments remain the standard of care for Duchenne muscular dystrophy, loss of ambulation, respiratory failure, and compromised cardiac function is the inevitable outcome. Recent developments in genetically mediated therapies have allowed for personalized treatments that strategically target individual muscular dystrophy subtypes based on disease pathomechanism and phenotype. In this review, we highlight the therapeutic progress with emphasis on evolving preclinical data and our own experience in completed clinical trials and others currently underway. We also discuss the lessons we have learned along the way and the strategies developed to overcome limitations and obstacles in this field.