A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in Gunn rats

Background & Aims: In the quest for a recombinant viral vector for liver-di rected gene therapy that would permit both prolonged and efficient transgen e expression in quiescent hepatocytes in vivo and repeated administration, we evaluated a recombinant simian virus 40 (rSV40). Methods: The rSV40 was generated through replacement of the DNA encoding for the T antigens (Tag) by the coding region of human bilirubin-uridine 5'-diphosphate-glucuronosyl -transferase (BUGT) complementary DNA (SV-hBUGT), Helper-free rSV40 units w ere generated at infectious titers of 5 x 10(9) to 1 x 10(10) infectious un its (IU)/mL in a Tag-producing packaging cell line (COS-7 cells). Results: After 1, 3, or 7 daily infusions of 3 x 109 IU of SV-hBUGT through an indwe lling portal vein catheter in bilirubin-UGT-deficient jaundiced Gunn rats, mean serum bilirubin concentrations decreased by 40%, 60% and 70%, respecti vely, in 3 weeks and remained at those levels throughout the duration of th e study (40 days), Results of liver biopsies from SV-hBUGT-treated Gunn rat s, but not from controls, were positive for human BUGT DNA, messenger RNA, and protein. Bilirubin-UGT activity in liver homogenates was 8%-12% of norm al, and bilirubin glucuronides were excreted in bile, Immunostaining showed that >50%-60% of hepatocytes stably expressed the transgene. Portal vein i nfusion of an rSV40 expressing hepatitis B surface antigen (HBsAg) in a nai ve Gunn rat and a Gunn rat that had received 7 injections of SV-BUGT result ed in approximately equal levels of hepatic expression of HBsAg, indicating that multiple inoculations of SV-BUGT did not elicit neutralizing antibodi es, Plasma alanine aminotransferase levels and liver histology remained nor mal despite repeated injections of rSV40, Conclusions: rSV40 vectors may re present a significant advance toward gene therapy for metabolic diseases.