Feasibility of Retroviral Vector-Mediated in utero Gene Transfer to the Fetal Rabbit

Abstract

Objectives: 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 β-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 β-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 β-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.

Fetal Liver-Derived Mesenchymal Stem Cell Engraftment After Allogeneic In Utero Transplantation into Rabbits

“…The housing was quiet, at constant room temperature and humidity. Laparotomies were performed under anesthesia at day 17 (E17) or day 21 (E21) of gestation (gestation = 31-32 days) as described previously [20,21]. The fetus was located and fixed by external palpation of the semitransparent uterine wall, and intra-amniotic or intrahepatic injections were performed using 26-gauge needles in view of the common anatomical landmarks of the E15-22 rabbit embryos [26].…”

“…Amplification conditions included an initial 10 min denaturation step at 95°C, followed by 40 cycles (95°C, 45 s; 63°C, 45 s; 72°C, 1 min), and a final 7 min extension at 72°C. Rabbit skeletal muscle myosin heavy chain gene (MIO) primers, RMMHC-F (5¢-AAAGA AGATGGATGTTGAGGC-3¢), and RMMHC-R (5¢-TCACC GTCACTTTCCCTGCT-3¢), were used in parallel to amplify a 429 bp-specific DNA fragment [20], which served as a control for genomic DNA isolation and loading. All samples were evaluated through 2% agarose gel electrophoresis.…”

“…gene transfer studies [20,21]. Indeed, there are several inherited disorders in which a rabbit model is already accessible, such as familial hypercholesterolemia [29], Pelizaeus-Merzbacher disease [30], and osteopetrosis [31].…”

“…According to several reports [17][18][19] and from our own experience [20,21], the pregnant rabbit is a useful and reliable middle-sized animal model to evaluate proof-of-principle strategies based on gene transfer or cell transplantation into the fetus, which allow accurate organ-directed manipulation and delivery. Using fetal instead of adult donor cells for in utero transplantation approaches may also offer some benefit.…”

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

“…Amplification conditions involved an initial 10-min denaturation step at 94°C, followed by 42 cycles (94°C, 1 min; 60°C, 30 s; and 72°C, 45 s), and a final 7-min extension at 72°C, producing a 417-base-pair DNA fragment. Rabbit skeletal muscle myosin heavy-chain gene (MHC) primers, RMMHC-F (5¢-AAAGAAGATGGATGTTGAGGC-3¢), and RMMHC-R (5¢-TCACCGTCACTTTCCCTGCT-3¢), were used to amplify a 429-base-pair-specific DNA fragment as described previously [17], which served as a control for genomic DNA isolation and loading. All samples were evaluated through 2% agarose gel electrophoresis.…”