INDUCTION OF CENTRAL TOLERANCE BY INTRATHYMIC INOCULATION OF ADENOVIRAL ANTIGENS INTO THE HOST THYMUS PERMITS LONG-TERM GENE-THERAPY IN GUNN-RATS

Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral r eadministration. We tested the feasibility of prolonging gene expressi on by induction of central tolerance to adenoviral antigens in bilirub in-UDP-glucuronosyltransferase-1 (BUGT(1))-defficient Gunn rats. Toler ance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recomb inant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (A d-hBUGT(1)) carrying the hBUGT(1) gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic in jections of normal saline, After 12 d all groups were injected intrave nously with 5 x 10(9) pfu of either Ad-hBUGT(1) or adenovirus beta-gal actosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosida se [LacZ] gene). In all three groups of tolerized rats, hBUGT(1) was e xpressed in the liver after administration of Ad-hBUGT(1), with glucur onidation of biliary bilirubin of above 95%. Serum bilirubin levels de creased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. S imilar findings were observed following repeat injections given on day s 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerize d groups, but not in controls, there was a marked inhibition of appear ance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus, Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term co rrection of an inherited metabolic disease following central tolerizat ion with thymic antigen.