Male germ-line cells are at risk following direct-injection retroviral-mediated gene transfer in utero.
The recent observation of vector sequences in the semen of men undergoing clinical gene therapy for hemophilia has highlighted the need to evaluate the risk of inadvertent germ-line transduction in a clinically relevant animal model. In the present study, we used three different approaches to investigate whether the germ line is at risk of inadvertent alteration following in utero retroviral gene transfer in the clinically relevant, random-bred sheep model. First, we conducted breeding studies. All organs from the 10 resultant offspring were devoid of proviral DNA, suggesting that the germ line had not been altered. As a second approach, we performed PCR on gradient-enriched, forensically purified sperm cells from in utero-transduced rams. The purified sperm cells from 6 of 19 of these rams were PCR positive for provirus, providing compelling evidence that the germ line had been transduced. As a third approach, we performed immunohistochemistry on sections of the testis from in utero-transduced sheep. Numerous somatic cells and very low levels of germ cells within the male reproductive tissues were transduced. In conclusion, our analysis on over 3 x 10(9) sperm cells suggests that the direct-injection approach employed in these studies may result in the inadvertent transduction of very low numbers of male germ cells.
Porada
Christopher D
CD
Department of Animal Biotechnology, University of Nevada at Reno, 89557-0104, USA. cporada@cabnr.unr.edu
Park
Paul J
PJ
Tellez
Joe
J
Ozturk
Ferhat
F
Glimp
Hudson A
HA
Almeida-Porada
Graça
G
Zanjani
Esmail D
ED
D000818:Animals; D004247:DNA / Q000302:isolation & purification; D005260:Female; D015316:Genetic Therapy / Q000009:adverse effects*; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D023421:Models, Animal; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000302:isolation & purification*; D012190:Retroviridae / Q000235:genetics* / Q000302:isolation & purification; D012756:Sheep; D013094:Spermatozoa / Q000821:virology*; D013737:Testis / Q000821:virology; D014161:Transduction, Genetic; D014162:Transfection
D004247:DNA