RAPID AND EFFICIENT GENE-TRANSFER IN HUMAN HEPATOCYTES BY HERPES VIRAL VECTORS

Retroviral vectors have been widely studied as vehicles for hepatocyte gene therapy, but they are limited by an inability to infect nondivid ing cells and the need for prolonged cell culture, Two replication def icient herpes simplex viral vectors (HSV) were constructed with the ma rker genes lac-Z/beta-galactosidase (HSVlac) or human-growth hormone ( HSVhGH) to determine the efficiency of HSV gene transfer into adult hu man hepatocytes. Hepatocytes were isolated by collagenase perfusions a nd density centrifugation from liver wedge biopsy specimens obtained f rom six patients. After exposure to HSV (0, 50,000, and 500,000 viral particles/10(6) hepatocytes) for 20 minutes, 1 hour, or 2 hours, the h epatocytes were washed and placed in culture, Hepatocytes transduced w ith HSVlac were fixed at 24 hours and histochemically stained with X-g al, and media from HSVhGH-transduced cells were assayed at 48 hours by radioimmunoassay for hGH. After a 20-minute exposure at a multiplicit y of infection of 0.5 (1 viral particle per 2 hepatocytes), greater th an 35% of the hepatocytes expressed the lac-Z gene (>70% efficiency). hGH was also detected in the media from HSVhGH-transduced cells, showi ng that proteins coded for by foreign genes are not only expressed by transduced cells but are also secreted. Isolated liver perfusions usin g HSVlac were also performed in Fischer rats. A 20-minute isolated per fusion using 5 x 10(6) viral particles resulted in expression of the b eta-galactosidase gene in the rodent livers 72 hours later without his tological signs of tissue injury. HSV vectors are potentially powerful tools for gene therapy of human liver disease, because they are effic ient and rapid vehicles for gene transfer. Ex vivo modified hepatocyte s theoretically may be ready for reinfusion within 100 minutes of live r resection. Efficient in vivo delivery of foreign gene may also be ac complished using these vectors.