Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks - PubMed (original) (raw)

Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks

Daniel G Miller et al. Mol Cell Biol. 2003 May.

Abstract

The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.

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Figures

FIG. 1.

FIG. 1.

Vectors used in the study. Maps of foamy retrovirus target site vector CnZ1450+22PNO, AAV targeting vector AAV2-nZ3113, and MLV vectors LXSHD (control) and I-Sce_I-expressing LSceISHD are shown with the viral long terminal repeats (LTR), CMV, PGK, Tn_5, and simian virus 40 (SV40) promoters, lacZ, hisD, and neo genes, nuclear localization signals (nls), and the p15A replication origin. Arrows indicate transcription start sites. The probe used in Southern blot analysis as well as relevant restriction enzyme sites and the I-_Sce_I site in CnZ1450+22PNO are shown.

FIG. 2.

FIG. 2.

Experimental design. A schematic view of the gene-targeting experimental protocol is shown with the relevant vectors (Fig. 1) and selection (G418 or

l

-histidinol) used.

FIG. 3.

FIG. 3.

DSBs increase AAV-mediated gene-targeting frequencies in HT-1080 cells. (A) Southern blot of genomic DNAs from HT-1080/CnZ1450+22PNO cells containing a single copy of the CnZ1450+22PNO provirus, which were digested with _Bgl_II and incubated with I-_Sce_I in vitro or exposed to I-_Sce_I in vivo by infection with MLV vector LSceISHD and selection of transduced cells. DNAs were probed for 3′ lacZ sequences, and the positions of size standards (in kilobases) are shown on the left. (B) HT-1080/CnZ1450+22PNO cells transduced by MLV vector LSceISHD (+) or LXSHD (−) were infected with AAV2-nZ3113 at the indicated MOIs (vector particles/cell) and assayed for AAV-mediated gene targeting by staining infected cell cultures for β-Gal expression (see Materials and Methods). Gene-targeting frequencies are shown as β-Gal+ foci/105 cells. The black and gray columns represent results from two independent experiments. (C) Southern blot analysis of HT-1080 clones targeted with AAV2-nZ3113. Genomic DNAs were purified from parental, untargeted HT-1080/CnZ1450+22PNO cells and five targeted clones isolated by fluorescence-activated sorting of cells that contained lacZ target sites corrected by AAV-mediated gene targeting in the presence of I-_Sce_I. These DNAs were digested in vitro with _Bgl_II and I-_Sce_I or _Bgl_II alone (far right lane) and probed for 3′ lacZ sequences. The positions of size standards (in kilobases) are shown on the left.

FIG. 4.

FIG. 4.

DSBs increase AAV-mediated gene-targeting frequencies in normal human fibroblasts. (A) Polyclonal human fibroblasts containing CnZ1450+22PNO lacZ target sites were transduced by MLV vector LSceISHD (+) or LXSHD (−), infected with AAV2-nZ3113 at the indicated MOIs (vector particles/cell), and assayed for AAV-mediated gene targeting by staining infected cell cultures for β-Gal expression (see Materials and Methods). Gene-targeting frequencies are shown as β-Gal+ foci/105 cells. Values are the means and standard errors of three experiments. (B) Southern blot analysis of DSB levels in normal human fibroblasts expressing I-_Sce_I. Genomic DNAs were purified from the normal human fibroblasts used for the experiment shown in panel A after transduction with LSceISHD (in vivo I-_Sce_I +) or LXSHD (in vivo I-_Sce_I −). Samples were digested in vitro with _Spe_I with (+) or without (−) in vitro I-_Sce_I as indicated and probed for 3′ lacZ sequences. The positions of size standards (in kilobases) are shown on the left.

FIG. 5.

FIG. 5.

Nonhomologous integration of AAV vectors. (A) Diagram of AAV vector concatemer forms and restriction enzyme sites used for Southern blot analysis as described for panel B. Vector orientations are indicated by the arrows. Inverted terminal repeats (ITRs) are indicated as black or white boxes. The vector-vector junction can contain 1 to 2 ITRs (9), so predicted sizes are accurate to within about 200 bp. The probe binds to sites indicated by the black bars underneath the diagrams. Note that the probe does not hybridize to the tail-tail junction fragment but detects flanking head-head or head-tail junctions from the same concatemer. (B) Southern blot of genomic DNAs from polyclonal human fibroblasts containing CnZ1450+22PNO target sites that had been transduced by LSceISHD and infected with AAV2-nZ3113 at the indicated MOIs (using the same samples as described in Table 1). DNA samples were digested with _Bss_SI (lanes 1 to 4) or _Msc_I (lanes 5 to 8) and hybridized to an 800-bp _Cla_I fragment from the 5′ end of the lacZ gene. The positions of size standards (in kilobases) are shown on the left. ss, single stranded.

FIG. 6.

FIG. 6.

Possible mechanisms of DSB repair by AAV vectors. A chromosome containing a DSB that was processed to leave single-stranded 3′ tails and the AAV targeting vector genome with ITRs are shown pairing and undergoing gene targeting by two different pathways.

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