title: Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.
abstract: A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.
authors.affiliation: The Children's Hospital of Philadelphia, and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: V R
authors.initials: VR
authors.lastname: Arruda
authors.forename: P A
authors.initials: PA
authors.lastname: Fields
authors.forename: R
authors.initials: R
authors.lastname: Milner
authors.forename: L
authors.initials: L
authors.lastname: Wainwright
authors.forename: M P
authors.initials: MP
authors.lastname: De Miguel
authors.forename: P J
authors.initials: PJ
authors.lastname: Donovan
authors.forename: R W
authors.initials: RW
authors.lastname: Herzog
authors.forename: T C
authors.initials: TC
authors.lastname: Nichols
authors.forename: J A
authors.initials: JA
authors.lastname: Biegel
authors.forename: M
authors.initials: M
authors.lastname: Razavi
authors.forename: M
authors.initials: M
authors.lastname: Dake
authors.forename: D
authors.initials: D
authors.lastname: Huff
authors.forename: A W
authors.initials: AW
authors.lastname: Flake
authors.forename: L
authors.initials: L
authors.lastname: Couto
authors.forename: M A
authors.initials: MA
authors.lastname: Kay
authors.forename: K A
authors.initials: KA
authors.lastname: High
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D000229:Dependovirus / Q000235:genetics*; D004285:Dogs; D005164:Factor IX / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D002836:Hemophilia B / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D017404:In Situ Hybridization, Fluorescence; D007273:Injections, Intramuscular; D008297:Male; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D012661:Semen / Q000821:virology; D013094:Spermatozoa / Q000821:virology*; D013737:Testis / Q000821:virology
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D011994:Recombinant Proteins; D005164:Factor IX