Integrase-defective Lentiviral Vectors as a Delivery Platform for Targeted Modification of Adenosine Deaminase Locus

被引：56

作者：

Joglekar, Alok V. ^{[1
,2
]}

Hollis, Roger P. ^{[1
,2
]}

Kuftinec, Gabriela ^{[1
,2
]}

Senadheera, Shantha ^{[1
,2
]}

Chan, Rebecca ^{[2
,3
]}

Kohn, Donald B. ^{[1
,2
]}

机构：

[1] Univ Calif Los Angeles, Dept Microbiol Immunol & Mol Genet, Los Angeles, CA 90095 USA

[2] Univ Calif Los Angeles, Eli & Edythe Broad Ctr Regenerat Med & Stem Cell, Los Angeles, CA 90095 USA

[3] Univ Calif Los Angeles, Dept Pathol & Lab Med, Los Angeles, CA 90095 USA

来源：

MOLECULAR THERAPY | 2013年 / 21卷 / 09期

关键词：

ZINC-FINGER NUCLEASES; GENE-THERAPY; RETROVIRAL VECTORS; NONDIVIDING CELLS; STEM-CELLS; IN-VIVO; EXPRESSION; TRANSGENE; TRANSDUCTION; INHIBITION;

D O I：

10.1038/mt.2013.106

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

页码：1705 / 1717

页数：13

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