Zejing Wang

Original ArticleSuccessful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

Immune Responses to AAV in Canine Muscle Monitored by Cellular Assays and Noninvasive Imaging

We previously demonstrated that direct intramuscular injection of rAAV2 or rAAV6 in wild-type dogs resulted in robust T-cell responses to viral capsid proteins, and others have shown that cellular immunity to adeno-associated virus (AAV) capsid proteins coincided with liver toxicity and elimination of transgene expression in a human trial of hemophilia B. Here, we show that the heparin-binding ability of a given AAV serotype does not determine the induction of T-cell responses following intramuscular injection in dogs, and identify multiple epitopes in the AAV capsid protein that are recognized by T cells elicited by AAV injection. We also demonstrate that noninvasive magnetic resonance imaging (MRI) can accurately detect local inflammatory responses following intramuscular rAAV injection in dogs. These studies suggest that pseudotyping rAAV vectors to remove heparin-binding activity will not be sufficient to abrogate immunogenicity, and validate the utility of enzyme-linked immunosorbent spot (ELISpot) assay and MRI for monitoring immune and inflammatory responses following intramuscular injection of rAAV vectors in preclinical studies in dogs. These assays should be incorporated into future human clinical trials of AAV gene therapy to monitor immune responses.

Expression of human α1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs

We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.We evaluated the potential of lung-directed gene therapy for α1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.

The impact of donor type and ABO incompatibility on transfusion requirements after nonmyeloablative haematopoietic cell transplantation.

We retrospectively analyzed transfusion requirements within the first 100 d among allogeneic haematopoietic cell transplantation (HCT) recipients with haematological malignancies given either myeloablative (n = 1353) or nonmyeloablative conditioning (n = 503). We confirmed that myeloablative recipients required more platelet and red blood cell (RBC) transfusions than nonmyeloablative recipients (P < 0·0001 for both). Myeloablative patients given peripheral blood stem cells required less platelet transfusions (P < 0·0001) than those given marrow while RBC transfusion requirements did not differ significantly. Subsequent analyses were restricted to nonmyeloablative recipients. Platelet and RBC transfusions were less frequent among related compared to unrelated recipients (P < 0·0001 for both), with comparable median numbers of transfused units. Major/bidirectionally ABO‐mismatched recipients required more RBC transfusions than ABO‐matched recipients (P = 0·006). Rates of graft rejection/failure, grades II–IV acute and chronic graft‐versus‐host‐disease (GVHD), 2‐year relapse, 3‐year survivals and non‐relapse mortality were comparable among ABO‐matched, minor‐mismatched, and major/bidirectionally mismatched recipients (P = 0·93, 0·72, 0·57, 0·36, 0·17 and 0·79, respectively). Times to disappearance of anti‐donor IgG and IgM isohemagglutinins among major/bidirectionally ABO‐mismatched recipients were affected by magnitude of pre‐HCT titres (P < 0·001 for both) but not GVHD (P = 0·71 and 0·78, respectively). In conclusion, nonmyeloablative recipients required fewer platelet and RBC transfusions and among them, both unrelated and major/bidirectionally ABO‐mismatched recipients required more RBC transfusions. ABO incompatibility did not affect nonmyeloablative HCT outcomes.

Immunity and AAV-mediated gene therapy for muscular dystrophies in large animal models and human trials

Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery specific to muscles for treating muscular dystrophies and non-muscle diseases in large animal models and human trials, factors that influence the intensity of the immune responses, and immune modulatory strategies to prevent unwanted immune responses and induce tolerance to the vector and therapeutic gene for a successful gene therapy.

Immune responses to rAAV6: the influence of canine parvovirus vaccination and neonatal administration of viral vector

Recombinant adeno-associated viral (rAAV) vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV). rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, 1 month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice.

Local Gene Delivery and Methods to Control Immune Responses in Muscles of Normal and Dystrophic Dogs

Adeno-associated viral vector (AAV)-mediated gene transfer represents a promising gene replacement strategy for treating Duchenne muscular dystrophy (DMD). However, recent studies demonstrated cellular immunity specific to AAV capsid proteins in animal models, which resulted in liver toxicity and elimination of transgene expression in a human trial of hemophilia B. We have recently developed immunosuppressive strategies to prevent such immunity for successful long-term transgene expression in dog muscle. Here, we describe in detail the immunosuppressive regimens employed in both normal and DMD dogs and provide methods for evaluating the efficiency of the regimens following intramuscular injection of AAV in dogs.

Analyzing cellular immunity to AAV in a canine model using ELISPOT assay

Adeno-associated viral (AAV) vector-mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine-specific ELISPOT assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T-cell responses in peripheral blood in dogs following intramuscular injection of AAV. This high-throughput method allows the identification of T-cell epitopes without the need for large cell numbers and the need for major histocompatibility complex molecule-matched cell lines.

791. Immunity to AAV-Mediated Gene Therapy in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

Top of pageAbstract Introduction: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Studies in the mdx mouse model of DMD have shown that muscle membrane integrity and function can be improved by AAV-mediated delivery of a functional dystrophin protein. To assess the potential clinical utility of treating human DMD patients with AAV-mediated gene delivery, we performed a series of direct intra-muscular injections in random-bred normal dogs and in dogs with muscular dystrophy caused by a dystrophin mutation (xmd dogs). Methods: AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for the murine studies and purified either on a heparin column or with a combination of heparin column and cesium chloride gradient. Direct intramuscular injections of virus (1012, 1010, or 108 viral genomes per site) in a total volume of 250 ul were performed. The injection sites were biopsied under anesthesia between 2 and 12 weeks after injection. Results: Injection of either AAV6 or AAV2 expressing CMV-b-galactosidase (b-gal) induced a strong inflammatory response containing both CD8+ and CD4+ T-lymphocytes with peak tissue destruction 4 weeks following viral injection. A similar robust immune response was seen with injection of AAV6-RSV-alkaline-phosphatase, AAV6-CMV-canine-factor-IX (cFIX) and with empty AAV6 capsid alone. Additional purification of the AAV6-CMV-cFIX by centrifugation through a cesium chloride gradient did not diminish the immune response. Continuous immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) largely prevented the immune response to AAV6-CMV-b-gal in a normal dog for up to four weeks and permitted robust transgene expression. The same immunosuppressive regimen did not prevent an immune response to AAV6-CMV-cFIX or AAV6-CMV-human-micro-dystrophin in an xmd dog, suggesting that a more aggressive immunosuppressive regimen might be necessary in the xmd dog. Conclusions: Taken together, our results suggest that AAV capsid proteins, or proteins associated with the capsid, elicit significant immune responses when directly injected into skeletal muscle of normal random-bred dogs. The combination of CSP and MMF effectively prevents the immune responses in a normal dog, but not in an xmd dog, possibly due to the pre-existing inflammatory nature of the DMD muscle disease or due to genetic variation in a random-bred animal population. The robust immune responses to AAV2 and AAV6 in random-bred dogs contrast with the lack of an immune response in studies by others in inbred mice and dogs. Further studies will be necessary to determine the nature of the immune responses and to develop appropriate immunosuppressive regimen for AAV gene delivery to xmd muscle.