661. Immune System Regulation of Transgene Expression in the Brain 3: Effects of the Immune Response on Transgene Expression from HIV-Derived Lentiviral Vectors Injected into the Rat Striatum

Abstract

HIV-derived pseudotyped lentiviral vectors are potent vectors for the transfer of potentially therapeutic genes into the brain. However, the efficiency of LV-mediated gene transfer into the brain has not been examined in detail. Further, nothing is known on the effects of the systemic priming of an anti-lentiviral immune response on transgene expression and inflammation in the brain. The self-inactivating pRRLsin.cPPT.CMV.Wpre, and pRRLsin.cPPT.ALB.Wpre constructs were used to generate VSV-pseudotyped LV stocks expressing either GFP (LV-GFP) or human FIX (LV-hFIX). Titers for LV-GFP were determined on HeLa and mouse liver (MLP29) cells and were approx. 5 × 109 to 2 × 1010 transducing units (TU)/ml with 100–400 ug HIV-1 p24/ml. To study the innate inflammatory responses to LV vectors in the CNS, LV-GFP was injected into the striatum at doses of 1 × 102−1 × 107 TU. Transduced cells could be found at all doses, and transgene expression increased up to the highest dose. Even at the highest dose, there was no statistically significant increase in the influx of either activated macrophages or CD8+-T cells. To analyze the effects of the systemic anti-LV immunization on gene expression in the CNS, LV-GFP was injected into the striatum, followed 30 days later, by the systemic immunization with either LV-GFP or LV-FIX, or the injection of saline. The effectiveness of the immune responses were determined by the presence of anti-envelope and anti-transgene antibodies by ELISA. In animals immunized against LV-FIX or injected with saline, there was no changes in the number of immunoreactive cells in the brain. However, in animals immunized against LV-GFP, there was a 30–50% reduction in the number of immunoreactive neurons. Concomitantly, there was a highly significant increase in the number of CD8+ T-cells in the brains of animals immunized with LV-GFP, but not those immunized with LV-FIX. This indicates that LV-injected into the brain cause minimal activation of the CNS innate inflammatory response. However, in the presence of a systemic anti-transgene immune response, activated lymphocytes can enter the brain and contribute to a down-regulation of LV-encoded transgene expression. Since LV-derived vectors do not express any vector proteins, immunization against a LV vector expressing a different transgene did not cause brain inflammation or down-regulation of CNS transgene expression. In conclusion, a systemic anti-transgene immune response can down-regulate gene expression from a LV vector in the CNS.