1086. T Cell Responses to Canine Factor IX and AAV Capsid Antigens in Hemophilia B Dogs after Intravascular Gene Delivery to Skeletal Muscle
Authors: Daniel J. Hui, Federico Mingozzi, Aaron Dillow, Stephanie McCorquodale, Timothy C. Nichols, Valder R. Arruda, Katherine A. High
Journal: Molecular Therapy (2006)

Previous studies have shown successful correction of hemophilia B in dogs by muscle-directed gene delivery of canine F.IX (cF.IX) using an adeno-associated virus (AAV) serotype 2. Intravascular delivery of the cF.IX gene to skeletal muscle allowed long-term expression of cF.IX of 4-20%, thereby correcting the disease phenotype. However, concerns over the risk of immune responses against the transgene and viral vector antigens have focused recent work on further characterizing the immunogenicity of both AAV capsid and F.IX in a gene therapy setting. Six HB dogs from the Chapel Hill colony received three different doses of an AAV2-cF.IX vector (1x1012 vg/kg, n=3; 3 × 1012 vg/kg, n=2; 8 × 1012 vg/kg, n=1) by intravascular delivery to skeletal muscle, in addition to an immunosuppression (IS) regimen (6 weekly infusions of cyclophosphamide beginning at the time of vector administration). T cell responses were measured on PBMCs isolated from whole blood drawn prior to vector infusion, during IS and after discontinuing IS. PBMCs were used for ELISpot assays to measure IL-10 and IFN-γ secretion in response to both cF.IX and AAV-2 antigens. This approach utilized a peptide library consisting of 15- mers overlapping by 10 amino acids, spanning the entire antigen sequence, arranged in a matrix such that each peptide was contained in two orthogonal pools. All HB dogs in this study exhibited a unique Th2 response consisting of IL-10 secretion in response to a common epitope corresponding to peptide 68 in the cF.IX library. This epitope was also found in normal dogs, which have the natural form of the protein, suggesting a role in tolerance to the cF.IX transgene rather than immunity. Interestingly, the dog that received the highest vector dose (1.5 μg cF.IX/ml, 30%) at day 15 post- injection, developed a non-neutralizing antibody response to cF.IX, as well as a T cell response to an epitope corresponding to peptide 84. This peptide maps to the region containing the missense mutation responsible for the disease phenotype (Chapel Hill mutation, Glu379→Gly). No IFN-γ secretion was detected following AAV2-cF.IX gene transfer to muscle, contrasting the Th1 response previously seen in intramuscularly delivered cF.IX using an AAV-1 vector. While all animals that received an AAV2-cF.IX vector developed B cell responses against the viral capsid antigen, T cell responses to the capsid were undetectable in PBMCs even after in vitro expansion with the antigen. In conclusion, intravascular delivery of AAV2-cF.IX to skeletal muscle elicited a Th2 T cell response characterized by the secretion of IL-10 in response to a specific cF.IX peptide. An additional immunodominant peptide was detected in the presence of a non- neutralizing immune response to the cF.IX transgene at the highest dose. These results suggest that while route of administration and/ or AAV serotype influence the type of immune response elicited, transgene level may impose additional safety limitations in terms of T cell response.