Development of animal models for adeno-associated virus site-specific integration

Abstract

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

FIG. 1

Replication of AAV DNA in AAVS1 transgenic primary cultures. Primary fibroblasts and hepatocytes from AAVS1 transgenic rats, as well as mouse embryonic fibroblasts, were infected with wt AAV and at an MOI of 100. After 1 h for virus adsorption (t = 0), the inoculum was removed and the cells were washed and refed with medium. Two days postinfection (t = 48), low-molecular-weight DNA was isolated from uninfected (mock) and infected cells analyzed on a Southern blot with a ³²P-labeled probe specific for the Rep coding sequence. The positions of the molecular-weight standards (in kilobases) are indicated.

FIG. 2

PCR amplification of ITR-AAVS1 junctions. (A) Schematic representation of the components of the PCR assay. AAV genome integrated at the AAVS1 is represented by dark box. The gray boxes indicate the AAV ITRs. The open box indicates the AAVS1. The primers used in the PCR analysis are indicated by arrows and are labeled with the associated numbers. Primers 16s and 17s are derived from the AAV ITR. Primers 15a and Cr2 are derived from AAVS1 (34). (B) Site-specific integration of wt AAV genome in transgenic rat and mouse fibroblasts. Rat wt (lanes 1 and 4) and transgenic (lanes 3 and 6) fibroblasts were infected with wt AAV virus at an MOI of 100. Mouse transgenic embryonic fibroblasts were infected with wt AAV at MOIs of 100 (lane 8) and 500 (lane 9). Transgenic rat primary hepatocytes were infected with wt AAV at MOIs of 10 (lanes 11 and 14) and 500 (lanes 12 and 15). Two days postinfection, genomic DNA was prepared and subjected to nested set PCR amplification with AAV-derived and AAVS1-derived primers. As a control, high-molecular-weight DNA from mock-infected rat (lanes 2 and 5) and mouse transgenic embryonic fibroblasts (lane 7) and from rat primary hepatocytes (lanes 10 and 13) were also subjected to the PCR amplification protocol. The products were run on an agarose gel, transferred to nylon membrane, and sequentially hybridized to AAVS1 (lanes 1 to 3 and lanes 7 to 12)- and AAV-specific probes (lanes 4 to 6 and lanes 13 to 15) as described earlier (34). The positions of the molecular-weight standards (in kilobases) are indicated.

FIG. 3

Analysis of PCR-amplified AAV-AAVS1 junctions. Amplified junctions from infected primary fibroblasts (F-14, F-44, and F-35) and hepatocytes (H-2 and H-33) of transgenic rats and from embryonic mouse fibroblasts (M-1) were sequenced and compared with the AAVS1 sequence. The ITR sequence is indicated with the nucleotide numbering for the right terminal repeat. Palindromic sequences within the ITR are indicated by the lettering A, C, C′, B, B′, and A, and the two possible orientations Flip and Flop are indicated (39). For each ITR-AAVS1 crossover sequence analyzed, the amount of viral sequence is indicated by the letters representing the ITR palindromic sequences. The numbers above indicate the nucleotide position on the last identifiable viral and AAVS1 sequence. In smaller letters the amplified sequences that cannot be directly associated with either the ITR or the AAVS1 are shown. The number of deleted bases in each ITR analyzed is indicated at the right.

FIG. 4

Southern blot analysis of Neo^r clones derived from the transfection of transgenic primary rat fibroblasts with plasmid pITR(GFP-Neo)P₅Rep. The conditions of transfection and DNA analysis were as described in Materials and Methods. (A) Schematic representation of the predicted head-to-tail configuration of the seven copies of the AAVS1 sequence inserted in rats of line 15. The BamHI restriction sites and the expected restriction fragments are indicated. (B) Hybridization to an AAVS1-specific probe. (C) Same membrane after rehybridization to a neo-specific probe. The bands that are annealed to both probes are considered to be indicative of site-specific integration and are indicated with an arrowhead. Mock-transfected cell DNA sample is indicated with the letter “C”. The number referring to each clone is also shown. The positions of the molecular-weight standards (in kilobases) are indicated.

FIG. 5

In situ hybridization of metaphase chromosomes from selected clone 31. Chromosome preparations were hybridized with a GFP-Neo (in red)- or AAVS1 (in yellow)-specific probes as described earlier (34).

FIG. 6

Analysis of AAV genomic DNA from the injected muscle of transgenic rodents. (A) Southern blot analysis of total muscle DNA from injected transgenic rats. Total cellular DNA was prepared from quadriceps of injected rats 2 or 6 days postinfection. DNA samples (10 μg) were resolved on agarose gel either uncut or after restriction with BamHI or PvuII. The gel was transferred to a nylon membrane and hybridized with a probe specific for the rep gene. Uninjected muscle DNA did not demonstrate any detectable hybridization (data not shown). (B) PCR amplification of the ITR-AAVS1 junction from injected muscle DNA. Total muscle DNA was prepared from injected transgenic rat and mouse tissue 75 and 6 days postinfection, respectively. DNA was subjected to PCR amplification of the viral-chromosomal junction, and amplified DNA was sequentially hybridized to an AAVS1 (lanes 1, 2, 5, and 6)- and an ITR-specific probe (lanes 3, 4, 7, and 8) as described previously (34). The positions of the molecular-weight standards (in kilobases) are indicated. (C) Schematic representation of the sequence of the ITR-AAVS1 junction derived from injected mouse muscle DNA.