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: 6.43). 07/2010; 18(7):1318-29. DOI: 10.1038/mt.2010.73
Source: PubMed

ABSTRACT 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 · 3.62 Impact Factor
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    • "Transient immunosuppression with cyclophosphamide prevented anti-cFIX antibody formation and resulted in sustained transgene expression via either an intramuscular or intravascular delivery route (Herzog et al., 2001; Arruda et al., 2005, 2010). In another study by Haurigot et al. (2010), 6 weeks of immunosuppression with cyclophosphamide did not prevent the development of anti-AAV antibody and T cell infiltrates in muscle, and the T cells were transgene specific. "
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    ABSTRACT: Hemophilia is an inherited bleeding disorder caused by a deficiency of functional clotting factors VIII or IX in the blood plasma. The drawbacks of the classical protein substitution therapy fueled interest in alternative treatments by gene therapy. Hemophilia has been recognized as an ideal target disease for gene therapy because a relatively modest increase in clotting factor levels can result in a significant therapeutic benefit. Consequently, introducing a functional FVIII or FIX gene copy into the appropriate target cells could ultimately provide a cure for hemophilic patients. Several cell types have been explored for hemophilia gene therapy, including hepatocytes, muscle, endothelial and hematopoietic cells. Both nonviral and viral vectors have been considered for the development of hemophilia gene therapy, including transposons, γ-retroviral, lentiviral, adenoviral and adeno-associated viral vectors. Several of these strategies have resulted in stable correction of the bleeding diathesis in hemophilia A and B murine as well as canine models, paving the way towards clinical trials. Although clotting factor expression has been detected in hemophilic patients treated by gene therapy, the challenge now lies in obtaining prolonged therapeutic FVIII or FIX levels in these patients. This review highlights the benefits and potential risks of the different gene therapy strategies for hemophilia that have been developed.
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