IN-UTERO GENE-THERAPY - TRANSFER AND LONG-TERM EXPRESSION OF THE BACTERIAL NEO(GAMMA) GENE IN SHEEP AFTER DIRECT-INJECTION OF RETROVIRAL VECTORS INTO PREIMMUNE FETUSES

We investigated whether directly injecting retroviral vectors into pre immune fetuses could result in the transfer and long-term expression o f exogenous genes. Twenty-nine preimmune sheep fetuses were injected w ith helper-free retroviral vector preparations. Twenty-two fetuses sur vived to term, 4 of which were sacrificed at birth. Of the remaining 1 8 animals, 3 were controls and 15 had received vector preparations. Tw elve of these 15 animals demonstrated transduction of hematopoietic ce lls when blood and marrow were analyzed by neo(r)-specific PCR, Eight experimental sheep have been followed for 5 years, during which time w e have consistently observed proviral DNA and G418-resistant hematopoe tic progenitors. The G418-resistant colonies were positive when analyz ed by neo(r)-specific PCR. neo(r) gene expression was also demonstrate d using several immunological and biochemical methods, The transductio n of hematopoietic stem cells was confirmed when lambs transplanted wi th bone marrow from in utero-transduced sheep exhibited neo(r) activit y in marrow and blood. Vector distribution was widespread in primary a nimals without pathology. PCR analysis indicates that the germ line wa s not altered. These studies demonstrate that direct injection of an e ngineered retrovirus is a feasible means of safely delivering a foreig n gene to a developing fetus and achieving long-term expression withou t modifying the germ line of the recipient.