743. Lentiviral Vector Gene Transfer in Monkeys: In Vivo. Detection of Gene Expression Longitudinally Using MicroPET and Optical Imaging

Top of pageAbstract Fetal intraperitoneal (IP) administration of HIV-1-derived lentiviral vectors (HIV/VSV-G/CMV/EGFP; 107 infectious particles/fetus) have consistently resulted in high levels of transduction and gene expression postnatally and for up to three years post-gene transfer in the omentum, peritoneum, and diaphragm when assessed by quantitative PCR and whole tissue fluorescence. Our objective was to develop in vivo imaging techniques that would allow us to monitor gene expression noninvasively and over time. In one series of studies, we explored the potential for imaging gravid long-tailed macaques with fetuses that were administered the VSV-G pseudotyped HIV-1-derived lentiviral vector expressing a mutant herpes simplex virus type 1 thymidine kinase (HSV-1 sr39tk) and firefly luciferase under the control of the CMV promoter IP in the first trimester. Fetuses were monitored sonographically during gestation to assess growth and development. Twice during the second and third trimesters 300-400 |[mu]|Ci F18-FHBG was injected into the fetal circulation under direct ultrasound guidance in preparation for maternal scanning in 3-D mode using a microPET 4 (CTI Concorde Microsystems). Images were reconstructed using an iterative MAP reconstruction algorithm. All newborns were delivered at term by cesarean-section and raised in the nursery for postnatal studies. Blood samples were collected for complete blood counts and chemistry panels monthly thereafter, and no adverse effects have been detected, to date. Beginning at two months postnatal age, animals were administered 650 |[mu]|Ci F18-FHBG and biodistribution assessed by microPET. In vivo optical imaging for firefly luciferase expression was also performed after injection of D-Luciferin using a Xenogen IVIS Imaging System with Living Image Software analysis. Under all imaging conditions gene expression was observed in the abdominal cavity, and closely paralleled findings in our prior studies using whole tissue fluorescence. These investigations have shown that HSV-1 sr39tk and firefly luciferase can be used to detect transgene expression longitudinally in fetal and infant monkeys in vivo, and without evidence of adverse effects.