title: Integrase-defective lentiviral vectors as a delivery platform for targeted modification of adenosine deaminase locus.
abstract: We investigated the use of integrase-defective lentiviral vectors (IDLVs) for transient delivery of zinc finger nucleases (ZFNs) and donor templates for site-specific modification of the human adenosine deaminase (hADA) gene. Initially, we constructed IDLVs carrying ZFN monomers (Single-IDLVs) and found them to be able to deliver their gene-editing payload to K562 cells successfully upon cotransduction, with minimal cytotoxicity. To simplify delivery, we designed an IDLV construct to deliver both ZFN monomers from the same vector (Double-IDLV). However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem. We modified the Double-IDLV constructs to reduce recombination and restored simultaneous delivery of both ZFNs. We also tested an IDLV construct for delivery of donor templates and demonstrated its efficacy for gene modification. In summary, we highlighted the importance of modifying vector design for co-delivery of highly similar sequences inherent to genome-editing nucleases, and demonstrated significant improvement in the use of IDLVs for delivery of ZFNs and donor templates for genome modification.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
authors.forename: Alok V
authors.initials: AV
authors.lastname: Joglekar
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hollis
authors.forename: Gabriela
authors.initials: G
authors.lastname: Kuftinec
authors.forename: Shantha
authors.initials: S
authors.lastname: Senadheera
authors.forename: Rebecca
authors.initials: R
authors.lastname: Chan
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D000243:Adenosine Deaminase / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D056426:Genetic Loci; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics*; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic; D016662:Virus Integration; D016335:Zinc Fingers
chemical_list: D019426:Integrases; D004720:Endonucleases; C548482:ADA protein, human; D000243:Adenosine Deaminase