Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males

Valder R. Arruda, Paul A. Fields, Ross Milner, Luanne Wainwright, Maria P. De Miguel, Peter J. Donovan, Roland W. Herzog, Timothy C. Nichols, Jaclyn A. Biegel, Mahmood Razavi, Mike Dake, Dale Huff, Alan W. Flake, Linda Couto, Mark A. Kay, Katherine A. High

Research output: Contribution to journalArticle

123 Scopus citations

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 × 1013 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 × 1012 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.

Original languageEnglish (US)
Pages (from-to)586-592
Number of pages7
JournalMolecular Therapy
Volume4
Issue number6
DOIs
StatePublished - 2001
Externally publishedYes

Keywords

  • AAV vectors
  • Gene therapy
  • Germline transmission
  • Hemophilia

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery

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  • Cite this

    Arruda, V. R., Fields, P. A., Milner, R., Wainwright, L., De Miguel, M. P., Donovan, P. J., Herzog, R. W., Nichols, T. C., Biegel, J. A., Razavi, M., Dake, M., Huff, D., Flake, A. W., Couto, L., Kay, M. A., & High, K. A. (2001). Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males. Molecular Therapy, 4(6), 586-592. https://doi.org/10.1006/mthe.2001.0491