Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit model.
BACKGROUND
Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors.


METHODS
Rabbit fetuses received 8.5 x 10(6) HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post-in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein.


RESULTS
Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP.


CONCLUSIONS
At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer.
Moreno
Rafael
R
Medical and Molecular Genetics Centre, Institut d'Investigació Biomèdica de Bellvitge, Hospital Duran i Reynals, Barcelona, Spain.
Rosal
Marta
M
Martinez
Itziar
I
Vilardell
Felip
F
Gonzalez
Juan Ramón
JR
Petriz
Jordi
J
Hernandez-Andrade
Edgard
E
Gratacós
Eduard
E
Aran
Josep M
JM
D000818:Animals; D005260:Female; D005333:Fetus / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D023421:Models, Animal; D011247:Pregnancy; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D019076:Transgenes / Q000276:immunology / Q000502:physiology
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins