74. Cellular Immunity to Adeno-Associated Virus Capsid Attenuates Transgene Expression in the Liver
Authors: Samuel L. Murphy, Denise Sabatino, Federico Mingozzi, Shyrie Edmonson, Katherine High
Journal: Molecular Therapy (2006)

Adeno-associated virus (AAV) vectors hold the potential to treat a variety of inherited and acquired human diseases. One of the confounding issues regarding the use of AAV in humans is the cellular immune response to the virus vector. While the ability of humoral immunity to interfere with AAV transduction is well-established, the ability of cellular immunity targeting the vector to ablate AAV transduction has only recently been identified. Our laboratory previously described the use of adenovirus expressing AAV capsid as a means to invoke a cellular immune response towards AAV in a mouse model system. These studies identified the immunodominant CD8 T-cell epitopes in Balb/C and C57Bl/6 mice immunized against AAV capsid of serotypes 2 and 8 (Mol Ther. 2005 Dec;12(6):1023–33). Cytolytic potential of these AAV capsid specific T-cells, however, was not investigated. In the current study, we seek to determine whether cellular immune responses reduce the efficiency of AAV transduction. We first evaluated the cytolytic activity of AAV capsid specific T-cells using a sensitive in vivo killing assay. In this assay, naïve murine splenocytes were loaded with either an immunodominant AAV peptide or an irrelevant AAV peptide. To discriminate between the two populations, each was labeled with a different concentration of CFSE dye. The two cell populations were then mixed together in equal numbers and injected into the tail vein of naïve and AAV- immune mice. Nearly equal proportions of both labeled subsets were recovered from naïve mice. In contrast, AAV-immune mice efficiently destroyed splenocytes loaded with the immunodominant AAV peptide. Percent specific lysis was greater than 70% for three out of four immunodominant peptides tested. These results show that AAV immunized mice harbor potent cytotoxic activity directed towards AAV capsid. We subsequently tested the ability of splenocytes from AAV-immune mice to reduce long-term AAV-mediated transgene expression in the liver. Naïve mice were first injected via the portal vein with 5×1010 vector genomes encapsidated by AAV-2 or AAV-8 capsid. These vectors encoded the human factor IX (hFIX) gene expressed under the control of a liver-specific promoter. Following a delay of 1–2 hours, these mice then received an adoptive transfer of splenocytes harvested from capsid matched AAV-immune mice. Control mice received adoptive transfer of splenocytes from naïve mice. Two months post-infusion, mice that received adoptive transfer from AAV-2 immune mice expressed significantly lower levels of hFIX than mice receiving adoptive transfer of naïve splenocytes (775ng/mL vs. 1771ng/mL, p=.03). Recipients of adoptive transfer from AAV-8 immune mice similarly exhibited reduced levels of hFIX two months post-infusion compared to control mice (606ng/mL vs. 1637ng/mL, p=.04). We conclude from these studies that a cellular immune response specific for AAV capsid can reduce AAV-mediated gene transfer in the liver.