Rhesus monkey model for fetal gene transfer: Studies with retroviral-basedvector systems

Many life-threatening conditions that can be diagnosed early in gestation m ay be treatable in utero using gene therapy. In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhe sus monkeys as a model for the human. Included in these studies were Molone y murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stoma titis virus-G (VSV-G) pseudotyped MLV, and a VSV-C pseudotyped HIV-l-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a r eporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16). Rhesus monkey fetuses were administered viral vector supernatant prepa rations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) r outes via ultrasound guidance at 55 +/- 5 days gestation (late first trimes ter; term 165 +/- 10 days). Fetuses were monitored sonographically, specime ns were collected prenatally and postnatally, and tissue harvests were perf ormed at birth or 3 or 6 months postnatal age (3-10 months post-gene transf er). PCR analyses demonstrated that transduced cells were present at simila r to1.2% in peripheral blood mononuclear cells from fetuses administered am photropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and <similar to>4.2% f or the lentiviral vector, which decreased to 2% at birth. Hematopoietic pro genitors showed that overall (mean of all time points assessed), similar to 25% of the collected colonies were positive for the EGFP transgene with th e lentiviral vector, which war; significantly greater than results achieved with the MLV-based vector systems (4-9%; P less than or equal to 0.001-0.0 16). At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups. EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%). Overall, the results of these studies have shown : (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results i n gene transfer to multiple organ systems, (3) the highest level of gene tr ansfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams. The rhesus monkey is an important preclinical pri mate model system for exploring gene transfer approaches for future applica tions in humans.