Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks - PubMed (original) (raw)

Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks

Matthew H Porteus et al. Mol Cell Biol. 2003 May.

Abstract

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

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Figures

FIG. 1.

FIG. 1.

Schematics of the GFP gene targeting system components and rAAVs used. (A) Schematic depiction of artificial gene target (A658). A 35-bp insertion consisting of an in-frame stop codon followed by the recognition site for the Sce endonuclease was inserted at bp 327 of the GFP coding region. The entire insertion sequence is as follows: 5′ TAAGCTCTCGAGATTACCCTGTTATCCCTAAGCTT 3′. (B) Schematic representations of the rAAVs used in this paper. The viruses consist of a single-stranded DNA core with hairpin ends. Repair substrate viruses rAAV.Subs and rAAV.Subs-Puro are missing the first 36 nucleotides of the GFP coding region. Abbreviations: CMV/CBA, cytomegalovirus enhancer/chicken β-actin promoter; EGFP, enhanced GFP; IRES, internal ribosomal entry site; CD8, coding region for the human CD8α coding region; WPRE, woodchuck posttranscriptional regulatory element (36); PGK-Neo, neomycin phosphotransferase gene driven by the phosphoglycerate kinase promoter; TruncGFP, GFP coding region that begins at bp 37 of the coding region; Sce, coding region for the I-_Sce_I endonuclease; Puro, puromycin acetyltransferase gene driven by the SV40 promoter and containing a polyadenylation signal sequence; CMV, cytomegalovirus promoter and enhancer; pA, polyadenylation signal sequence; LacZ, coding region for the β-galactosidase gene.

FIG. 2.

FIG. 2.

Random integration with rAAV. (A) The time course of rAAV transduction is illustrated. This graph shows representative examples of the time course of transduction of 293 cells with rAAV.GFP at different MOIs. (B) The random integration frequency of rAAV.GFP at various MOIs is shown. The random integration frequency at each MOI was determined by measuring the percentage of GFP-positive cells by flow cytometry at day 14 after infection from at least four different infections. (C) The random integration frequency of rAAV.GFP is not affected by the presence of a DSB. 293/A658 cells were infected with rAAV.GFP ± rAAV.Sce at the MOIs indicated, and the percentage of stably transduced cells was measured by flow cytometry. (D) The random integration frequency with rAAV.Subs-Puro is shown. 293/A658 cells were infected with rAAV.Subs-Puro at an MOI of either 300 or 1,000, and the numbers of puromycin-resistant colonies were counted at day 21 after infection.

FIG. 3.

FIG. 3.

Gene targeting with rAAV. (A) The spontaneous frequency of gene targeting with rAAV-Subs is dependent on the MOI. (B) Transfection of the Sce expression plasmid stimulates rAAV-mediated gene targeting. 293/A658 cells were either cotransfected with an Sce expression plasmid (Sce driven by the PGK promoter) and repair substrate plasmid (RS2100) or simultaneously transfected with the Sce expression plasmid and infected with rAAV.Subs. Plasmid RS2100 contains the 2,100-bp repair substrate in rAAV.Subs-Puro, but in the pBS SK(+) (Stratagene) backbone (22). The targeting frequencies in this figure are not adjusted for transfection or infection efficiency.

FIG. 4.

FIG. 4.

DSB-mediated gene targeting can be stimulated by coinfection of rAAV.Sce and rAAV.Subs. (A) Gene targeting frequencies were obtained after coinfecting different MOIs of rAAV.Subs (along the x axis) and rAAV.Sce (in the figure key). The data for MOIs between 300 and 3,000 are the averages of four to six different samples from two or three different experiments. The data when the MOI was 10,000 for either virus are the result of one or two samples from a single experiment. (B) rAAV DSB-mediated gene targeting frequency was the same for rAAV.Subs and rAAV.Subs-Puro. (C) rAAV DSB-mediated gene targeting frequency was not changed by coinfection with a nonspecific rAAV.

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