Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs

Division of Hematology and Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
Molecular Therapy (Impact Factor: 6.23). 07/2010; 18(7):1318-29. DOI: 10.1038/mt.2010.73
Source: PubMed


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.

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Available from: Daniel Hui, Oct 01, 2014
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    • "Staining for FoxP3 revealed the presence of FoxP3+ /CD4+ T cells in muscle tissue after injection of alipogene tiparvovec. Previous work involving intravascular delivery of the canine FIX transgene by AAV2/2 in the muscle in a dog model for hemophilia B (Arruda et al., 2005; Haurigot et al., 2010) showed that stable transgene expression was associated with the presence of Tregs. Our findings support the theory proposed by Mays and Wilson (2011) that in case AAV vector administration does not initiate sufficient innate immune activation, a mechanism of passive tolerance may result through ignorance, anergy, or deletion. "
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    ABSTRACT: Cellular immune responses to adeno-associated viral (AAV) vectors used for gene therapy have been linked to attenuated transgene expression and loss of efficacy. The impact of such cellular immune responses on the clinical efficacy of alipogene tiparvovec (Glybera®, AAV1-LPLS447X, uniQure, Amsterdam, the Netherlands), a gene-therapy consisting of intramuscular administration of a recombinant (r)AAV1 mediating muscle-directed expression of lipoprotein lipase (LPL), was investigated. Five subjects with LPL-deficiency (LPLD) were administered intramuscularly with a dose of 1 x 1012 gc/kg alipogene tiparvovec. All subjects were treated with immune suppression starting shortly before administration of alipogene tiparvovec and maintained until 12 weeks after administration. Systemic antibody and T cell responses against AAV1 and LPLS447X, as well as local cellular immune responses in the injected muscle were investigated in 5 LPLD subjects. Long term transgene expression was demonstrated despite a transient systemic cellular response and a stable humoral immune response against AAV1 capsid protein. Cellular infiltrates were found in 4 of the 5 subjects but were not associated with adverse clinical events or elevation of inflammation markers. Consistent herewith, CD8-positive T cells in the infiltrates lacked cytotoxic potential. Furthermore, FoxP3-positive/CD4-positive T cells were found in the infiltrates suggesting that multiple mechanisms contribute to local tolerance. Systemic and local immune responses induced by intramuscular injection of alipogene tiparvovec have no impact on safety and did not compromise LPL transgene expression. These findings support the use of alipogene tiparvovec in individuals with LPLD and indicate that muscle directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.
    Human gene therapy 12/2013; 25(3). DOI:10.1089/hum.2013.169 · 3.76 Impact Factor
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    • "A number of recent studies have addressed the challenges of establishing safe and effective routes of gene delivery, and reducing immune responses to AAV vectors in adults. Transvenular administration of F.IX in a canine model produced therapeutic levels of F.IX, albeit with anti-AAV capsid responses.50 Addition of transient immunosuppression enhanced F.IX levels and abrogated anti-F.IX antibody responses.51 "
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    ABSTRACT: Neonatal AAV8-mediated Factor IX (F.IX) gene delivery was applied as a model for exploring mechanisms of tolerance induction during immune ontogeny. Intraperitoneal delivery of AAV8/ Factor IX (hF.IX) during weeks 1-4 of life, over a 20-fold dose range, directed stable hF.IX expression, correction of coagulopathy in F.IX-null hemophilia B mice, and induction of tolerance to hF.IX; however, only primary injection at 1-2 days of life enabled increasing AAV8-mediated hF.IX expression after re-administration, due to the absence of anti-viral capsid antibodies. Adoptive splenocyte transfer from tolerized mice demonstrated induction of CD4(+)CD25(+) T regulatory (Treg) populations that specifically suppressed anti-hF.IX antibody responses, but not responses to third party antigen. Induction of hF.IX antibodies was only observed in tolerized mice after in vivo CD4(+)CD25(+) cell depletion and hF.IX challenge. Thus, primary injection of AAV during a critical period in the first week of life does not elicit antiviral responses, enabling re-administration of AAV and augmentation of hF.IX levels. Expansion of hF.IX-specific CD4(+)CD25(+) Tregs has a major role in tolerance induction early in immune ontogeny. Neonatal gene transfer provides a useful approach for defining the ontogeny of immune responses and may suggest approaches for inducing tolerance in the context of genetic therapies.Gene Therapy advance online publication, 13 June 2013; doi:10.1038/gt.2013.22.
    Gene therapy 06/2013; 20(10). DOI:10.1038/gt.2013.22 · 3.10 Impact Factor
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    • "One possible limitation of the results presented here is that the dog model of diabetes used in this study does not fully mimic the immunological state of type 1 diabetic patients. However, although future studies in autoimmune models of diabetes are warranted, studies of mice (36), dogs (37), and humans (38) would suggest that targeting muscle with AAV vectors may at least partially escape immune recognition. This may be the result of lower levels of major histocompatibility complex class I presentation in this tissue, or the result of the induction of apoptosis of reactive T cells (36,38). "
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    ABSTRACT: Diabetes is associated with severe secondary complications, caused largely by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a "glucose sensor" in skeletal muscle through co-expression of glucokinase (Gck) and insulin (Ins), increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adenoassociated viral vectors of serotype 1 (AAV1) encoding for Gck and Ins in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for Ins or Gck alone failed to achieve complete correction of diabetes, indicating that the synergistic action of Ins and Gck are needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.
    Diabetes 02/2013; 62(5). DOI:10.2337/db12-1113 · 8.10 Impact Factor
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