TY - JOUR
T1 - Rapid and efficient gene transfer in human hepatocytes by herpes viral vectors
AU - Fong, Yuman
AU - Federoff, Howard J.
AU - Brownlee, Michael
AU - Blumberg, David
AU - Blumgart, Leslie H.
AU - Brennan, Murray F.
N1 - Funding Information:
Abbreviations:H SV,h erpes simplex viral vectors; HSVlac,l ac-Z/B-galactos- idase; HSVhGH, human growth hormone; EGTA, ethyleneglycodiamine-tetraacetate; FCS, fetal calf serum; MOI, multiplicity of infection. From the 1Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York; and the 2Department of Medicine, and the 3Department of Neuroscienee, Albert Einstein College of Medicine, Bronx, NY. Received August 16, 1994; accepted March 23, 1995. Supported by a grant (HD27116) from the National Institutes of Health, Bethesda, MD, and a grant (192214) from the Juvenile Diabetes Foundation, New York, NY. Address reprint requests to: Yuman Fong, MD, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2203-000553.00/0
PY - 1995/9
Y1 - 1995/9
N2 - Retroviral vectors have been widely studied as vehicles for hepatocyte gene therapy, but they are limited by an inability to infect nondividing cells and the need for prolonged cell culture. Two replication deficient herpes simplex viral vectors (HSV) were constructed with the marker genes lac-Z/β-galactosidase (HSVlac) or human-growth hormone (HSVhGH) to determine the efficiency of HSV gene transfer into adult human hepatocytes. Hepatocytes were isolated by collagenase perfusions and density centrifugation from liver wedge biopsy specimens obtained from six patients. After exposure to HSV (0, 50,000, and 500,000 viral particles/106 hepatocytes) for 20 minutes, 1 hour, or 2 hours, the hepatocytes were washed and placed in culture. Hepatocytes transduced with HSVlac were fixed at 24 hours and histochemically stained with X-gal, and media from HSVhGH-transduced cells were assayed at 48 hours by radioimmunoassay for hGH. After a 20-minute exposure at a multiplicity of infection of 0.5 (1 viral particle per 2 hepatocytes), greater than 35% of the hepatocytes expressed the lac-Z gene (>70% efficiency). hGH was also detected in the media from HSVhGH-transduced cells, showing that proteins coded for by foreign genes are not only expressed by transduced cells but are also secreted. Isolated liver perfusions using HSVlac were also performed in Fischer rats. A 20-minute isolated perfusion using 5 × 106 viral particles resulted in expression of the β-galactosidase gene in the rodent livers 72 hours later without histological signs of tissue injury. HSV vectors are potentially powerful tools for gene therapy of human liver disease, because they are efficient and rapid vehicles for gene transfer. Ex vivo modified hepatocytes theoretically may be ready for reinfusion within 100 minutes of liver resection. Efficient in vivo delivery of foreign gene may also be accomplished using these vectors.
AB - Retroviral vectors have been widely studied as vehicles for hepatocyte gene therapy, but they are limited by an inability to infect nondividing cells and the need for prolonged cell culture. Two replication deficient herpes simplex viral vectors (HSV) were constructed with the marker genes lac-Z/β-galactosidase (HSVlac) or human-growth hormone (HSVhGH) to determine the efficiency of HSV gene transfer into adult human hepatocytes. Hepatocytes were isolated by collagenase perfusions and density centrifugation from liver wedge biopsy specimens obtained from six patients. After exposure to HSV (0, 50,000, and 500,000 viral particles/106 hepatocytes) for 20 minutes, 1 hour, or 2 hours, the hepatocytes were washed and placed in culture. Hepatocytes transduced with HSVlac were fixed at 24 hours and histochemically stained with X-gal, and media from HSVhGH-transduced cells were assayed at 48 hours by radioimmunoassay for hGH. After a 20-minute exposure at a multiplicity of infection of 0.5 (1 viral particle per 2 hepatocytes), greater than 35% of the hepatocytes expressed the lac-Z gene (>70% efficiency). hGH was also detected in the media from HSVhGH-transduced cells, showing that proteins coded for by foreign genes are not only expressed by transduced cells but are also secreted. Isolated liver perfusions using HSVlac were also performed in Fischer rats. A 20-minute isolated perfusion using 5 × 106 viral particles resulted in expression of the β-galactosidase gene in the rodent livers 72 hours later without histological signs of tissue injury. HSV vectors are potentially powerful tools for gene therapy of human liver disease, because they are efficient and rapid vehicles for gene transfer. Ex vivo modified hepatocytes theoretically may be ready for reinfusion within 100 minutes of liver resection. Efficient in vivo delivery of foreign gene may also be accomplished using these vectors.
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U2 - 10.1016/0270-9139(95)90289-9
DO - 10.1016/0270-9139(95)90289-9
M3 - Article
C2 - 7657275
AN - SCOPUS:0029126487
SN - 0270-9139
VL - 22
SP - 723
EP - 729
JO - Hepatology
JF - Hepatology
IS - 3
ER -