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

Human embryonic myosin heavy chain cDNA Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene

AbstractA 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.Myosin heavy chain cDNA; Nucleotide sequence; Amino acid sequence; Myosin rod; Chromosomal mapping; Gene transcription; (Human embryo

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)-γ 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

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

Lung volume reduction surgery restores the normal diaphragmatic length-tension relationship in emphysematous rats

AbstractObjective: Improved respiratory muscle function is a major effect of a lung volume reduction surgery. We studied length adaptation in rat diaphragmatic muscle in an attempt to elucidate the mechanism by which diaphragmatic function improves after this controversial operation. Methods: We developed a model of elastase-induced emphysema and bilateral volume reduction through median sternotomy in rats. Five months after emphysema induction, maximum exchangeable lung volume was determined in intubated and anesthetized control animals and animals with emphysema. Costal diaphragmatic length was measured in vivo, and the length at which maximal twitch force is generated was determined on muscle strips in vitro. Also 5 months after elastase administration, another cohort underwent volume reduction or sham sternotomy. Five months after the operation, these animals were similarly studied. Results: Lung volume was increased in emphysematous rats versus control rats (50.9 ± 1.7 vs 45.4 ± 1.3 mL, P =.001). Lung volume was decreased in emphysematous animals that had undergone volume reduction versus sham sternotomy (44.7 ± 0.60 vs 49.4 ± 1.0 mL, P =.001). In situ diaphragm length (1.99 ± 0.04 vs 2.24 ± 0.07 cm, P =.001) and the length at which maximal twitch force is generated (2.25 ± 0.06 vs 2.48 ± 0.09 cm, P =.038) were shorter in emphysematous than control animals. After volume reduction, in situ diaphragm length (2.13 ± 0.06 vs 1.83 ± 0.02 cm, P <.001) and the length at which maximal twitch force is generated (2.50 ± 0.08 vs 2.27 ± 0.06 cm, P =.013) were longer than in animals undergoing sham sternotomy. Conclusions: In this experimental model of emphysema and lung volume reduction surgery, emphysema shortens the length at which maximal twitch force is generated and shifts the diaphragmatic length-tension curve to lower lengths; volume reduction returns the length at which maximal twitch force is generated toward normal and shifts the diaphragmatic length-tension curve back to longer lengths. This restoration toward normal physiology may enable the improvement in diaphragmatic function seen after lung volume reduction surgery. The mechanism by which these length adaptations occur merits further investigation. (J Thorac Cardiovasc Surg 2001;121:217-24

Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model

In earlier work, we showed that adeno-associated virus–mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy