title: A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats.
abstract: OBJECTIVE
  In the quest for a recombinant viral vector for liver-directed gene therapy that would permit both prolonged and efficient transgene 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 were generated at infectious titers of 5 x 10(9) to 1 x 10(10) infectious units (IU)/mL in a Tag-producing packaging cell line (COS-7 cells).
  
  
  RESULTS
  After 1, 3, or 7 daily infusions of 3 x 10(9) IU of SV-hBUGT through an indwelling portal vein catheter in bilirubin-UGT-deficient jaundiced Gunn rats, mean serum bilirubin concentrations decreased by 40%, 60% and 70%, respectively, in 3 weeks and remained at those levels throughout the duration of the study (40 days). Results of liver biopsies from SV-hBUGT-treated Gunn rats, but not from controls, were positive for human BUGT DNA, messenger RNA, and protein. Bilirubin-UGT activity in liver homogenates was 8%-12% of normal, and bilirubin glucuronides were excreted in bile. Immunostaining showed that >50%-60% of hepatocytes stably expressed the transgene. Portal vein infusion of an rSV40 expressing hepatitis B surface antigen (HBsAg) in a naive Gunn rat and a Gunn rat that had received 7 injections of SV-BUGT resulted in approximately equal levels of hepatic expression of HBsAg, indicating that multiple inoculations of SV-BUGT did not elicit neutralizing antibodies. Plasma alanine aminotransferase levels and liver histology remained normal despite repeated injections of rSV40.
  
  
  CONCLUSIONS
  rSV40 vectors may represent a significant advance toward gene therapy for metabolic diseases.
authors.affiliation: Department of Medicine, Seaver Institute of Human Genetics, Albert Einstein College of Medicine, New York, New York, USA.
authors.forename: B V
authors.initials: BV
authors.lastname: Sauter
authors.forename: B
authors.initials: B
authors.lastname: Parashar
authors.forename: N R
authors.initials: NR
authors.lastname: Chowdhury
authors.forename: A
authors.initials: A
authors.lastname: Kadakol
authors.forename: Y
authors.initials: Y
authors.lastname: Ilan
authors.forename: H
authors.initials: H
authors.lastname: Singh
authors.forename: J
authors.initials: J
authors.lastname: Milano
authors.forename: D S
authors.initials: DS
authors.lastname: Strayer
authors.forename: J R
authors.initials: JR
authors.lastname: Chowdhury
mesh_terms: D000818:Animals; D001646:Bile / Q000737:chemistry; D001654:Bile Pigments / Q000032:analysis; D001663:Bilirubin / Q000097:blood / Q000378:metabolism; D019556:COS Cells; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007565:Jaundice / Q000503:physiopathology / Q000628:therapy*; D008099:Liver / Q000503:physiopathology*; D008297:Male; D051381:Rats; D011911:Rats, Gunn; D019233:Retreatment; D013539:Simian virus 40 / Q000235:genetics* / Q000502:physiology; D019076:Transgenes / Q000235:genetics; D019562:Viral Load; D014779:Virus Replication
chemical_list: D001654:Bile Pigments; C019787:bilirubin glucuronoside glucuronosyltransferase; D014453:Glucuronosyltransferase; D001663:Bilirubin