Feasibility of retroviral vector-mediated in utero gene transfer to the fetal rabbit.
OBJECTIVE
Successful treatment or prevention of severe hereditary diseases could conceivably be achieved by genetic intervention early in development. Viral vector-mediated fetal gene transfer is proving a valuable tool to test the above concept in relevant animal models. Although the pregnant rabbit is a well-recognized model for fetal therapy, few preclinical assays have used it to validate fetal gene transfer approaches. In this preliminary study we assessed for the first time the feasibility of retroviral vector-mediated in utero gene transfer in the fetal rabbit.


METHODS
Different amounts of the vesicular stomatitis virus G pseudotyped MFG(nls)LacZ retroviral vector, expressing a nuclear-localized beta-galactosidase reporter protein were injected intraperitoneally and -hepatically into 20- to 22-day-old fetuses. At 8-9 days post-treatment, the pups were sacrificed and the tissues harvested for analysis. Evidence of gene transfer was obtained by PCR amplification of proviral sequences within genomic DNA isolated from the treated samples. Transgenic beta-galactosidase expression was assessed by X-gal histochemical staining.


RESULTS
By intraperitoneal injection 43% of the viable fetuses treated (3/7) showed evidence of successful LacZ gene transfer and low-level beta-galactosidase expression into liver and heart, whereas by intrahepatic injection roughly 38% (3/8) of the livers were positive for LacZ gene transfer and expression. The success rate for the viable fetuses rose to 67% positive livers (4/6) when a near double amount of recombinant virus was injected using a 10-fold concentrated virus stock. In terms of short-term safety, fetal and maternal survival rates approached 80% of treated fetuses, and 100% of treated does.


CONCLUSIONS
The pregnant rabbit is a useful and reliable model allowing the design of further studies to optimize the conditions for effective, safer, and persistent retroviral vector-mediated fetal gene transfer.
Moreno
Rafael
R
Medical and Molecular Genetics Center, Institut de Recerca Oncològica, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Spain.
Rosal
Marta
M
Cabero
Lluis
L
Gratacós
Eduard
E
Aran
Josep M
JM
D000818:Animals; D005240:Feasibility Studies; D005260:Female; D005333:Fetus / Q000601:surgery*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008099:Liver / Q000601:surgery; D023421:Models, Animal; D010529:Peritoneal Cavity / Q000601:surgery; D011247:Pregnancy; D011817:Rabbits; D012190:Retroviridae