title: Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit model.
abstract: 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.
authors.affiliation: Medical and Molecular Genetics Centre, Institut d'Investigació Biomèdica de Bellvitge, Hospital Duran i Reynals, Barcelona, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Moreno
authors.forename: Marta
authors.initials: M
authors.lastname: Rosal
authors.forename: Itziar
authors.initials: I
authors.lastname: Martinez
authors.forename: Felip
authors.initials: F
authors.lastname: Vilardell
authors.forename: Juan Ramón
authors.initials: JR
authors.lastname: Gonzalez
authors.forename: Jordi
authors.initials: J
authors.lastname: Petriz
authors.forename: Edgard
authors.initials: E
authors.lastname: Hernandez-Andrade
authors.forename: Eduard
authors.initials: E
authors.lastname: Gratacós
authors.forename: Josep M
authors.initials: JM
authors.lastname: Aran
mesh_terms: 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
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins