14 LONG TERM EXPRESSION OF HUMAN ADENOSINE DEAMINASE IN MURINE HEMATOPOIETIC CELLS

Abstract

Twenty-two defective retrovirus vectors carrying human adenosine (ADA) coding sequences were tested for efficient transduction of human ADA enzyme activity. These vectors were designed to test functional aspects of construction including the use of internal transcriptional units, the importance of the Moloney sequences 563-1038 (gag+), and alterations in the 3′ LTR. Promoters from the human ADA and cFos genes, herpes virus thymidine kinase, and cytomegalovirus immediate early region gene were tested as alternatives to the viral LTR promoter. Several vectors all containing the gag+ region allow high titer virus production. These vectors were then tested in bone marrow transplant experiments. Two vectors, one utilizing the Moloney LTR promoter and one with the HSVTK promoter, gave expression of ADA in CFU-C, CFU-S and in the blood of reconstituted animals. Infection efficiency ranged from 15-85% in CFU-S by Southern analysis. Expression efficiency was also variable. All animals had human ADA in blood 2-4 weeks post-transplant but at 10-12 weeks levels of the enzyme declined. Four of 14 long term recipients however, demonstrated expression for up to 5 months. PCR analysis on nonexpressors indicated a loss of cell with the proviral DNA suggesting that the initial expression resulted from infection of more mature progenitors. Immuno-staining is being used to characterize the lineages and distribution of ADA producing cells in the transplant recipients.