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Targeted lentiviral vector integration at "safe harbor" genomic loci

Friday, November 08, 2013 — Poster Session III

10:00 a.m. – 12:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • PJ Li
  • M Marino
  • J Reiser


Lentiviral vectors provide powerful tools for therapeutic gene delivery in vitro, ex vivo and in vivo. Our goal is to engineer lentiviral vectors so that their integration preference is shifted to genomic “safe harbor” sites. The AAVS1 locus on chromosome 19 constitutes such a genomic “safe harbor”. We generated integrase-defective lentiviral vectors (IDLVs) capable of integrating at the AAVS1 locus through homologous recombination (HR). We are now in the process of improving the efficiency of this approach using site-specific nucleases including zinc finger nucleases (ZFNs). To assess the efficiency of HR at the endogenous AAVS1 locus in HEK 293 cells, we co-delivered the coding regions for the AAVS1 site-specific ZFN pair and a donor plasmid bearing a puromycin resistance gene, flanked by homology arms corresponding to the AAVS1 site. Puromycin-resistant cell clones were characterized by PCR. Our data indicate that AAVS1-specific ZFNs can significantly increase the efficiency of HR at the AAVS1 site. We expect this gene editing approach to be useful for incorporating other transgene sequences at the AAVS1 locus. In an attempt to further improve the efficiency of this approach, we are currently testing IDLVs to co-deliver ZFN encoding sequences plus donor sequences into target cells.

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