Adenoviruses encoding HPRT correct biochemical abnormalities of HPRT-deficient cells and allow their survival in negative selection medium

The Lesch-Nyhan syndrome is an X-linked disorder caused by a virtually comp lete absence of the key enzyme of purine recycling, hypoxanthine-guanine ph osphoribosyltransferase (HPRT). It is characterized by uric acid overproduc tion and severe neurological dysfunction. No treatment is yet available for the latter symptoms. A possible long-term solution is gene therapy, and re combinant adenoviruses have been proposed as vectors for gene transfer into postmitotic neuronal cells. We have constructed an adenoviral vector expre ssing the human HPRT cDNA under the transcriptional control of a short huma n cytomegalovirus major immediate early promoter (RAd-HPRT). Here we show t hat infection of human 1306, HPRT-negative cells with RAd-HPRT, expressed h igh enough levels of HPRT enzyme activity, as to reverse their abnormal bio chemical phenotype, thus enhancing hypoxanthine incorporation and restoring purine recycling, increasing GTP levels, decreasing adenine incorporation, and allowing cell survival in HAT medium in which only cells expressing hi gh levels of HPRT can survive, infection of murine STO cells, increased hyp oxanthine incorporation and restored purine recycling, thus allowing cell s urvival in HAT medium, and reduced de novo purine synthesis. Although both cells were able to survive in HAT medium post infection with RAd-HPRT, some of the biochemical consequences differed. In summary, even though adenovir al vectors do not integrate into the genome of target HPRT-deficient human or murine cells, RAd-HPRT mediated enzyme replacement corrects abnormal pur ine metabolism, increases intracellular GTP levels, and allows cells to sur vive in a negative selection medium.