Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR

Abstract

Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.

FIG. 1.

Real-time PCR assay for the quantification of site-specific integration of AAV-2 into AAVS1 on chr-19. Sequence elements of integrated AAV-2 are represented as boxes differentially shaded in gray. Amplification, detection, and quantification of site-specific integration were performed with the real-time PCR LightCycler system. The primers used for the amplification of AAV ITR/AAVS1 junctions are indicated by arrows. Primer PITR hybridizes to AAV-2 sequences at positions 4526 to 4545 (23), and primer PAAVS1 hybridizes to AAVS1 on chr-19 19q13.3-qter at positions 1609 to 1593 (9). Sequence elements within the ITR are as outlined in Fig. 3B. Further sequence specificity is guaranteed by the hybridization probe assay format used for detection of the PCR product. Fluorescent dye-labeled probes hybridize to the amplified DNA fragment (donor probe, 1541 to 1560; acceptor probe, 1562 to 1583), thereby bringing the attached dyes into close proximity, thus eliciting fluorescence resonance energy transfer (FRET). Fluorescence emission intensity is directly proportional to the amount of PCR product.

FIG. 2.

Kinetics of AAV site-specific integration. HeLa cells were infected with AAV-2 at an MOI of 500. Total genomic DNA was isolated at different times p.i. Purified DNA (1 μg) was preamplified in 50 μl for 13 cycles by conventional PCR. Samples of 2 μl were subjected to LightCycler PCR as outlined in Materials and Methods. (A) Raw data of the LightCycler analysis of HeLa cell DNA (1 μg) spiked with known copy numbers (10², 10³, 10⁴, and 10⁵) of standard plasmid pAAVS1-TR. (B) The copy numbers of AAV ITR/chr-19 integration site junctions per microgram of AAV-infected HeLa DNA were quantified by comparison to values of a standard curve run in parallel as outlined for panel A. Each value represents the mean ± the standard deviation of three independent cultures. (C) LightCycler PCR products were analyzed on agarose gels. Standards were 10¹, 10², 10³, 10⁴, and 10⁵ copies of pAAVS1-TR added to 1 μg of uninfected HeLa cell DNA. The bands in the range below 100 bp present in all lanes represent input primers. M, molecular size marker.

FIG. 3.

Analysis of PCR-amplified AAV ITR/AAVS1 site junctions. PCR fragments of the sample at 96 h p.i. (Fig. 2) were cloned into pCR4-TOPO. Colonies were picked at random. (A) DNAs were digested with EcoRI to release the PCR fragments. Agarose gel electrophoresis visualizes the variability of fragment lengths. (B) The hairpin structure of the AAV ITR is represented in the “flop” orientation. Small letters (d′, a, and b) indicate palindromic sequence elements of the right AAV ITR. (C) Structural maps deduced from DNA sequence analysis of cloned PCR fragments. With the exception of clones 12 and 13, clone numbers refer to the lane numbers in panel A. The black arrow indicates the hybridization site of primer PAAVS1 on chr-19. The gray arrow indicates the binding site of primer PITR on the AAV ITR. Positions of the last unambiguous cellular and/or viral nucleotide are indicated in accordance with the published sequence information (9, 23). Overlapping sequences between the AAV ITR and the chr-19 integration site are underlined.

FIG. 4.

Compilation of published AAVS1-AAV ITR crossover sequences. Schematic representation of published integration sites within AAVS1 in different cell systems with respect to PCR primers used for their detection. The bottom line represents the nucleotide sequence and numbering as determined by Kotin et al. (9). The trs and RBS homology sequences, as determined by Linden et al. (13), are indicated as boxes, as is the recently described DNase-hypersensitive site (DHS-S1) (11). The primers used for detection of junction fragments are indicated below with the designations used in the respective publications. Nucleotide positions, in the 5′ to 3′ direction, are as follows: Cr2, 1222 to 1201; C, 1345 to 1326; PAAVS1, 1609 to 1593; AAVS1-2R, 2299 to 2280. Integration sites determined in the indicated studies are given as characteristic symbols above. Each symbol represents one integration site. The cell system, the primers used for determination of junction sites, and the respective references are given to the right of the symbols.