GENE-TRANSFER INTO HEPATOCYTES MEDIATED BY HELPER VIRUS-FREE HSV AAV HYBRID VECTORS/

Background: Vectors based on herpes simplex virus type 1 (HSV-1) can e fficiently transduce hepatocytes in the mouse Liver, and vector genome s can persist for at least 2 months. However, 24 hr after gene transfe r, the number of cells that express the transgene decreases rapidly an d no transduced cells are detectable after 7 days. Ln this study, we e xamined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo. Material s and Methods: HSV-1 amplicon or HSV/ AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequ ences were packaged into HSV-1 virions using a helper virus-free packa ging system. To determine relative transduction efficiencies, vector s tocks were titered on four different cell lines, including hamster kid ney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase-/-) and human (NPLC) hepatocytes. After in vivo injection of vector stock s into mouse liver, tissue sections were examined for reporter gene ex pression and cellular inflammatory response. Blood samples were collec ted to measure serum transaminase levels as a biochemical index of liv er toxicity. Results: Expression of a reporter gene from liver-specifi c promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (similar to 2% o f all hepatocytes) remained stable over 7 days after injection of HSV/ AAV hybrid vector, whereas no transduced cells were detected 7 days af ter gene transfer with standard HSV-1 amplicon vector. The rapid decli ne in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occ urred in RAG2-/- mice. Hepatocyte toxicity and cellular inflammatory e ffects associated with HSV/ AAV hybrid vector-mediated gene transfer w ere minimal, and readministration of vector stock proved equally effec tive in naive mice and in animals that received a first vector dose 4 weeks earlier. Conclusions: HSV/AAV hybrid amplicon vectors support ge ne expression in vivo for considerably longer than do traditional HSV- 1 amplicon vectors. Moreover, expression from these vectors does not p rovoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics su ggest that such vectors may hold future promise for hepatic gene repla cement therapy.