Assessment of therapeutic potential and risks of retrovirus mediated in utero gene transfer

In utero gene therapy (IUGT), while in its infancy, offers numerous potential advantages that promise to bypass the causes of previous postnatal gene therapy clinical trial failures. Using the fetal sheep model of IUGT, we investigated in detail several aspects of IUGT pertinent to its efficacy and safety. Forty-eight sheep fetuses were injected at approximately 40-102 days of gestation (term: 148 days) with retroviral containing supernatants. None of the fetuses injected during the first trimester (<50 days) survived to term, but fetal mortality declined to 33% and 40% for second trimester and third trimester injections, respectively. Analysis of transduction and transgene expression levels within fetal tissues revealed an inverse correlation between gestational recipient age and transduction efficiency of the liver. In contrast, the transgene expression levels within the lung and thymus showed a direct correlation with recipient age. The brain showed the lowest transgene expression indicating that the intraperitoneal route is not efficient for targeting the fetal brain. The types of cells that were transduced were diverse and appeared to correlate with their cycling and proliferation states within fetal tissues, particularly within the lung. Furthermore, amphotropic receptor mRNA expression levels within the liver did not vary significantly between getational days 58-103 indicating that amphotropic receptor expression levels may not be an important determinant of retroviral transduction efficiency. Examination of adult tissues 2.5 years after in utero gene transfer revealed transgene expression within the lung, pancreas, heart, and lymph nodes. Immunohistochemical analysis of fetal and adult testes as well as real-time PCR analysis of purified mature spermatocytes provided strong evidence that male germ cells were transduced following in utero gene transfer. Detailed analysis of primordial germ cells (PGC) within the fetal testes revealed that PGC compartmentalization continues from the fetal to neonatal period and involves the sequential expression of stage-specific embryonic antigens. Migrating PGCs remained quiescent until they came into close proximity to circumferentially developing Sertoli cells, providing a potential mechanistic model for retroviral transduction of PGCs within the fetal testes. Collectively, these studies provide a greater understanding of the therapeutic potential of IUGT, which may outweigh its associated risks.