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.

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.

Myosin gene mutation correlates with anatomical changes in the human lineage.

Powerful masticatory muscles are found in most primates, including chimpanzees and gorillas, and were part of a prominent adaptation of Australopithecus and Paranthropus, extinct genera of the family Hominidae. In contrast, masticatory muscles are considerably smaller in both modern and fossil members of Homo. The evolving hominid masticatory apparatus--traceable to a Late Miocene, chimpanzee-like morphology--shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. Here, we show that the gene encoding the predominant myosin heavy chain (MYH) expressed in these muscles was inactivated by a frameshifting mutation after the lineages leading to humans and chimpanzees diverged. Loss of this protein isoform is associated with marked size reductions in individual muscle fibres and entire masticatory muscles. Using the coding sequence for the myosin rod domains as a molecular clock, we estimate that this mutation appeared approximately 2.4 million years ago, predating the appearance of modern human body size and emigration of Homo from Africa. This represents the first proteomic distinction between humans and chimpanzees that can be correlated with a traceable anatomic imprint in the fossil record.

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.

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.

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.

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.

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.

Uniform scale-independent gene transfer to striated muscle after transvenular extravasation of vector.

BACKGROUND: The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for treatment of the underlying genetic disorder. Somatic gene therapy will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large-animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions to achieve this. METHODS AND RESULTS: Rats and dogs underwent limb or hemibody isolation via atraumatic tourniquet placement or myocardial isolation via heterotopic transplantation. Recombinant adenovirus (10(13) particles per kilogram) or recombinant adeno-associated virus (10(14) genome copies/kg) encoding the lacZ transgene was delivered through pressurized venous infusion without pharmacological mediators. Muscle exhibited almost 100% myofiber transduction in rats and dogs by X-galactosidase staining and significantly higher beta-galactosidase levels compared with nonpressurized delivery. No significant difference was seen in beta-galactosidase levels between 100- or 400-mm Hg groups. The <50-mm Hg group yielded inhomogeneous and significantly lower transgene expression. CONCLUSIONS: Uniform scale- and vector-independent skeletal and cardiac myofiber transduction is facilitated by pressurized venous infusion in anatomic domains isolated from the central circulation without pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to muscle fibers of an entire extremity in the dog, providing a firm foundation for further translational studies of efficacy in canine models for human diseases.

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.

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.

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.

Sarcomeres are added in series to emphysematous rat diaphragm after lung volume reduction surgery.

STUDY OBJECTIVES: The diaphragm adapts to its shortened state in experimental emphysema primarily by losing sarcomeres in series, thus reducing its optimal operating length. One would expect improved diaphragmatic function after lung volume reduction surgery (LVRS) only if the muscle can readapt to its elevated, lengthened postoperative position by either adding back sarcomeres or lengthening sarcomeres. We used a model of elastase-induced emphysema in rats to test the hypothesis that sarcomere addition occurs following LVRS. DESIGN: A cohort of emphysematous rats was created by the intratracheal instillation of elastase. Five months after the instillation, one group of rats underwent measurement of in situ costal diaphragm length via laparotomy, the determination of optimal muscle fiber operating length (Lo) on stimulated diaphragm strips in vitro, and the measurement of sarcomere length by electron microscopy on strips fixed at Lo. Another group of rats underwent LVRS or sham sternotomy 5 months after the instillation, and 5 months following the operation these animals underwent the same series of diaphragmatic studies. RESULTS: Lo was significantly greater in rats that underwent LVRS than those that underwent sternotomy (mean [+/- SE] Lo after LVRS, 2.50 +/- 0.08 cm; mean Lo after sternotomy, 2.27 +/- 0.06 cm; p = 0.013). There was no significant difference in sarcomere lengths between the two groups (2.95 +/- 0.04 vs 3.04 +/- 0.04 microm, respectively; p = 0.10). Using Lo as the length basis, the mean sarcomere number was calculated to be 8,712 +/- 192 in animals that had undergone LVRS and 7,144 +/- 249 in animals that had undergone sternotomy (p < 0.001). CONCLUSION: Sarcomere length is not significantly altered but sarcomeres are added in series following LVRS in this experimental model of emphysema/LVRS. It is likely that this sarcomere addition is a prerequisite to the improvement in inspiratory muscle function that has been observed following LVRS in humans.

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema.

BACKGROUND: Several physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema. METHODS: We sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro. RESULTS: In addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 +/- 2.7 ms versus 53.9 +/- 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema. CONCLUSION: This is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans.

Inspiratory loading does not accelerate dystrophy in mdx mouse diaphragm: implications for regenerative therapy.

Since the finding that the mdx mouse diaphragm, in contrast to limb muscles, undergoes progressive degeneration analogous to that seen in Duchenne muscular dystrophy, the relationship between the workload on a muscle and the pathogenesis of dystrophy has remained controversial. We increased the work performed by the mdx mouse diaphragm in vivo by tracheal banding and evaluated the progression of dystrophic changes in that muscle. Despite the establishment of dramatically increased respiratory workload and accelerated myofiber damage documented by Evans blue dye, no change in the pace of progression of dystrophy was seen in banded animals vs. unbanded, sham-operated controls. At the completion of the study, more centrally nucleated fibers were evident in the diaphragms of banded mdx mice than in sham-operated mdx controls, indicating that myofiber regeneration increases to meet the demands of the work-induced damage. These data suggest that there is untapped regenerative capacity in dystrophin-deficient muscle and validates experimental efforts aimed at augmenting regeneration within skeletal muscle as a therapeutic strategy in the treatment of dystrophinopathies.

Global cardiac-specific transgene expression using cardiopulmonary bypass with cardiac isolation.

BACKGROUND: The available techniques for intravascular gene delivery to the heart are inefficient and not organ-specific. Yet, effective treatment of heart failure will likely require transgene expression by the majority of cardiac myocytes. To address this problem, we developed a novel cannulation technique that achieves efficient isolation of the heart in situ using separate cardiopulmonary bypass (CPB) circuits for the heart and body in dogs. METHODS: The arterial inflow and venous effluent from the two circuits were physically isolated. The efficiency of separation was 98% to 99% in three preliminary experiments using Evans Blue dye-labeled albumin. In 6 dogs, the cardiac circuit was perfused with oxygenated crystalloid cardioplegia at 37 degrees C containing approximately 4 x 10(11) particles of an adenovirus encoding LacZ (AdCMVLacZ) with a perfusion pressure of 170 to 200 mm Hg for 15 minutes allowing virus to recirculate through the heart approximately 15 times. Cross-clamp time was 26 +/- 2 minutes and CPB time was 90 +/- 3 minutes. RESULTS: Five animals survived and were euthanized at 7 days. Beta-galactosidase activities measured using a chemiluminescent assay were three orders of magnitude higher in all areas of the heart than in the liver. Histological analyses revealed heterogeneous X-Gal staining of myocytes in all areas of the myocardium. CONCLUSIONS: Despite using a constitutive promoter, this technique yields relatively cardiac-specific transgene expression and is potentially translatable to clinical applications. Future studies will allow for further optimization of the conditions necessary for vector-mediated gene delivery to the heart.

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.