Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.

Mechanism of Deletion Removing All Dystrophin Exons in a Canine Model for DMD Implicates Concerted Evolution of X Chromosome Pseudogenes.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder caused by mutations in the large, 2.4-Mb dystrophin gene. The majority of DMD-causing mutations are sporadic, multi-exon, frameshifting deletions, with the potential for variable immunological tolerance to the dystrophin protein from patient to patient. While systemic gene therapy holds promise in the treatment of DMD, immune responses to vectors and transgenes must first be rigorously evaluated in informative preclinical models to ensure patient safety. A widely used canine model for DMD, golden retriever muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to alternative splicing around an intronic point mutation, thereby confounding the interpretation of immune responses to dystrophin-derived gene therapies. Here we characterize a naturally occurring deletion in a dystrophin-null canine, the German shorthaired pointer. The deletion spans 5.6 Mb of the X chromosome and encompasses all coding exons of the DMD and TMEM47 genes. The sequences surrounding the deletion breakpoints are virtually identical, suggesting that the deletion occurred through a homologous recombination event. Interestingly, the deletion breakpoints are within loci that are syntenically conserved among mammals, yet the high homology among this subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific concerted evolution of these atypical sequence elements.

Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse.

Duchenne muscular dystrophy (DMD) is a progressive primary myodegenerative disease caused by a genetic deficiency of the 427-kDa cytoskeletal protein dystrophin. Despite its single-gene etiology, DMD's complex pathogenesis remains poorly understood, complicating the extrapolation from results of preclinical studies in genetic homologs to the design of informative clinical trials. Here we describe novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. By coupling force transduction, high-precision motion tracking, and respiratory measurements, we have achieved a suite of integrative physiological tests that provide novel insights regarding normal and pathological responses to muscular exertion. A common feature of these physiological assays is the precise tracking and analysis of volitional movement, thereby optimizing the relevance to clinical tests. Unexpectedly, the measurable biological distinction between dystrophic and control mice at early time points in the disease process is better resolved with these tests than with the majority of previously used, labor-intensive studies of individual muscle function performed ex vivo. For example, the dramatic loss of volitional movement following a novel, standardized grip test distinguishes control mice from mdx mice by a 17.4-fold difference of the means (3.5 ± 2.2 vs. 60.9 ± 12.1 units of activity, respectively; effect size 1.99). The findings have both mechanistic and translational implications of potential significance to the fields of basic myology and neuromuscular therapeutics.NEW & NOTEWORTHY This study uses novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. A measurable distinction between dystrophic and control mice was seen at early time points in vivo compared with invasive muscle studies performed ex vivo. These assays shed light on normal and pathological responses to muscular exertion and have significant mechanistic and translational implications for the fields of basic myology and neuromuscular therapeutics.

Perspectives on best practices for gene therapy programs.

With recent successes in gene therapy trials for hemophilia and retinal diseases, the promise and prospects for gene therapy are once again garnering significant attention. To build on this momentum, the National Institute of Neurological Disorders and Stroke and the Muscular Dystrophy Association jointly hosted a workshop in April 2014 on "Best Practices for Gene Therapy Programs," with a focus on neuromuscular disorders. Workshop participants included researchers from academia and industry as well as representatives from the regulatory, legal, and patient advocacy sectors to cover the gamut from preclinical optimization to intellectual property concerns and regulatory approval. The workshop focused on three key issues in the field: (1) establishing adequate scientific premise for clinical trials in gene therapy, (2) addressing regulatory process issues, and (3) intellectual property and commercialization issues as they relate to gene therapy. The outcomes from the discussions at this workshop are intended to provide guidance for researchers and funders in the gene therapy field.

Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches.

Cardiac surgical delivery of the sarcoplasmic reticulum calcium ATPase rescues myocytes in ischemic heart failure.

BACKGROUND: The sarcoplasmic reticulum calcium ATPase (SERCA2a) is an important molecular regulator of contractile dysfunction in heart failure. Gene transfer of SERCA2a mediated by molecular cardiac surgery with recirculating delivery (MCARD) is a novel and clinically translatable strategy. METHODS: Ischemic heart failure was induced by ligation of OM1 and OM2 in 14 sheep. Seven sheep underwent MCARD-mediated AAV1-SERCA2a delivery 4 weeks after myocardial infarction, and seven sheep served as untreated controls. Magnetic resonance imaging-based mechanoenergetic studies were performed at baseline, 3 weeks, and 12 weeks after infarction. Myocyte apoptosis was quantified by Tdt-mediated nick-end labeling assay. Myocyte cross-sectional area and caspase-8 and caspase-9 activity was measured with imaging software, specific fluorogenic peptides, and immunohistochemistry. RESULTS: MCARD-mediated AAV1-SERCA2a gene delivery resulted in robust cardiac-specific SERCA2a expression and stable improvements in global and regional contractility. There were significantly higher stroke volume index, left ventricular fractional thickening, and ejection fraction at 12 weeks in the MCARD group than in the control group (30 ± 3 vs 21 ± 2 mL/m(2); 12% ± 5% vs 3% ± 3%; and 43 ± 4 vs 32 ± 4, respectively, all p < 0.05). Apoptotic myocytes were observed more frequently in the control group than in the MCARD-SERCA2a group (0.57.2 ± 0.16 AU vs 0.32.4 ± 0.08 AU, p < 0.05). MCARD-SERCA2a also resulted in decreased caspase-8 and caspase-9 expression and decreased myocyte area in the border zone of transgenic sheep compared with control sheep (14.6% ± 1.2% vs 2.9% ± 0.7%; 18.2% ± 1.9% vs 8.6% ± 1.4%; and 102.1 ± 3.8 µm(2) vs 88.1 ± 3.6 µm(2), all p < 0.05). CONCLUSIONS: MCARD-mediated SERCA2a delivery results in robust cardiac specific gene expression, improved contractility, and a decrease in both myocyte apoptosis and myocyte hypertrophy.

A pharmacokinetic analysis of molecular cardiac surgery with recirculation mediated delivery of ßARKct gene therapy: developing a quantitative definition of the therapeutic window.

BACKGROUND: Two major problems for translating gene therapy for heart failure therapy are: safe and efficient delivery and the inability to establish a relationship between vector exposure and in vivo effects. We present a pharmacokinetics (PK) analysis of molecular cardiac surgery with recirculating delivery (MCARD) of scAAV6-ßARKct. MCARD's stable cardiac specific delivery profile was exploited to determine vector exposure, half-life, and systemic clearance. METHODS AND RESULTS: Five naive sheep underwent MCARD with 10(14) genome copies of scAAV6-ßARKct. Blood samples were collected over the recirculation interval time of 20 minutes and evaluated with quantitative polymerase chain reaction (qPCR). C(t) curves were generated and expressed on a log scale. The exposure, half-life, and clearance curves were generated for analysis. qPCR and Western blots were used to determine biodistribution. Finally, all in vivo transduction data was plotted against MCARD's PK to determine if a relationship existed. Vector concentrations at each time point were (cardiac and systemic, respectively): 5 minutes: 9.16 ± 0.15 and 3.21 ± 0.38; 10 minutes: 8.81 ± 0.19 and 3.62 ± 0.37; 15 minutes: 8.75 ± 0.12 and 3.69 ± 0.31; and 20 minutes: 8.66 ± 0.22 and 3.95 ± 0.26; P < .00001. The half life of the vector was 2.66 ± 0.24 minutes. PK model data revealed that only 0.61 ± 0.43% of the original dose remained in the blood after delivery, and complete clearance from the system was achieved at 1 week. A PK transfer function revealed a positive correlation between exposure and in vivo transduction. Robust ßARKct expression was found in all cardiac regions with none in the liver. CONCLUSION: MCARD may offer a viable method to establish a relationship between vector exposure and in vivo transduction. Using this methodology, it may be possible to address a critical need for establishing an effective therapeutic window.

Gene transfer to muscle from the isolated regional circulation.

Vector transport across the endothelium has long been regarded as one of the central "bottlenecks" in gene therapy research, especially as it pertains to the muscular dystrophies where the target tissue approaches half of the total body mass. Clinical studies of gene therapy for hemophilia B revealed the limitations of the intramuscular route, compelling an aggressive approach to the study of scale-independent circulatory means of vector delivery. The apparent permeability of the microvasculature in small animals suggests that gravitational and/or inertial effects on the circulation require progressive restriction of fluid and solute flow across the capillary wall with increasing body size. To overcome this physiological restriction, we initially used a combined surgical and pharmacological approach to temporarily alter permeability within the isolated pelvic limb. Although this was successful, new information about the cell and molecular biology of histamine-induced changes in microvascular permeability suggested an alternative approach, which substituted pressure-induced transvenular extravasation. Here we outline the details of our surgical approaches in the rat. We also discuss the modifications that are appropriate for the dog.

A translatable, closed recirculation system for AAV6 vector-mediated myocardial gene delivery in the large animal.

Current strategies for managing congestive heart failure are limited, validating the search for an alternative treatment modality. Gene therapy holds tremendous promise as both a practical and translatable technology platform. Its effectiveness is evidenced by the improvements in cardiac function observed in vector-mediated therapeutic transgene delivery to the murine myocardium. A large animal model validating these results is the likely segue into clinical application. However, controversy still exists regarding a suitable method of vector-mediated cardiac gene delivery that provides for efficient, global gene transfer to the large animal myocardium that is also clinically translatable and practical. Intramyocardial injection and catheter-based coronary delivery techniques are attractive alternatives with respect to their clinical applicability; yet, they are fraught with numerous challenges, including concerns regarding collateral gene expression in other organs, low efficiency of vector delivery to the myocardium, inhomogeneous expression, and untoward immune response secondary to gene delivery. Cardiopulmonary bypass (CPB) delivery with dual systemic and isolated cardiac circuitry precludes these drawbacks and has the added advantage of allowing for control of the pharmacological milieu, multiple pass recirculation through the coronary circulation, the selective addition of endothelial permeabilizing agents, and an increase in vector residence time. Collectively, these mechanics significantly improve the efficiency of global, vector-mediated cardiac gene delivery to the large animal myocardium, highlighting a potential therapeutic strategy to be extended to some heart failure patients.

Safety of AAV factor IX peripheral transvenular gene delivery to muscle in hemophilia B dogs.

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector-mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4(+)FoxP3(+)IL-10(+) T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Cardiac gene therapy: optimization of gene delivery techniques in vivo.

Vector-mediated cardiac gene therapy holds tremendous promise as a translatable platform technology for treating many cardiovascular diseases. The ideal technique is one that is efficient and practical, allowing for global cardiac gene expression, while minimizing collateral expression in other organs. Here we survey the available in vivo vector-mediated cardiac gene delivery methods--including transcutaneous, intravascular, intramuscular, and cardiopulmonary bypass techniques--with consideration of the relative merits and deficiencies of each. Review of available techniques suggests that an optimal method for vector-mediated gene delivery to the large animal myocardium would ideally employ retrograde and/or anterograde transcoronary gene delivery,extended vector residence time in the coronary circulation, an increased myocardial transcapillary gradient using physical methods, increased endothelial permeability with pharmacological agents, minimal collateral gene expression by isolation of the cardiac circulation from the systemic, and have low immunogenicity.

Safety and efficacy of regional intravenous (r.i.) versus intramuscular (i.m.) delivery of rAAV1 and rAAV8 to nonhuman primate skeletal muscle.

We developed a drug-free regional intravenous (r.i.) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (i.m.) delivery of the same dose of vector. We show that r.i. delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After i.m., muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although r.i. delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that r.i. is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.

Safety and Efficacy of Regional Intravenous (RI) Versus Intramuscular (IM) Delivery of rAAV1 and rAAV8 to Nonhuman Primate Skeletal Muscle.

We developed a drug-free regional intravenous (RI) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (IM) delivery of the same dose of vector. We show that RI delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After IM, muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although RI delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that RI is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.

Limb-girdle muscular dystrophy in the United States.

Limb-girdle muscular dystrophy (LGMD) has been linked to 15 chromosomal loci, 7 autosomal-dominant (LGMD1A to E) and 10 autosomal-recessive (LGMD2A to J). To determine the distribution of subtypes among patients in the United States, 6 medical centers evaluated patients with a referral diagnosis of LGMD. Muscle biopsies provided histopathology and immunodiagnostic testing, and their protein abnormalities along with clinical parameters directed mutation screening. The diagnosis in 23 patients was a disorder other than LGMD. Of the remaining 289 unrelated patients, 266 had muscle biopsies sufficient for complete microscopic evaluation; 121 also underwent Western blotting. From this combined evaluation, the distribution of immunophenotypes is 12% calpainopathy, 18% dysferlinopathy, 15% sarcoglycanopathy, 15% dystroglycanopathy, and 1.5% caveolinopathy. Genotypes distributed among 2 dominant and 7 recessive subtypes have been determined for 83 patients. This study of a large racially and ethnically diverse population of patients with LGMD indicates that establishing a putative subtype is possible more than half the time using available diagnostic testing. An efficient approach to genotypic diagnosis is muscle biopsy immunophenotyping followed by directed mutational analysis. The most common LGMDs in the United States are calpainopathies, dysferlinopathies, sarcoglycanopathies, and dystroglycanopathies.