DNA palindromes with a modest arm length of greater, similar 20 base pairs are a significant target for recombinant adeno-associated virus vector integration in the liver, muscles, and heart in mice

Abstract

Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of greater, similar 20 bp (total length, greater, similar 40 bp) are a significant target for rAAV integration. Up to approximately 30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of greater, similar 20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

FIG. 1.

AAV-ISce I.AO3 vector map. The vector genome consists (left to right) of an AAV2-ITR sequence, a stuffer sequence, an ISceI-BamHI combination site, a shortened Tn3 prokaryotic promoter (Pr), the β-lactamase gene, the pUC plasmid origin of replication, a portion of MLV long terminal repeat (MLV-LTR), and an ITR. Diagrammed below is a schema of the preparative vector-cellular DNA junction fragments obtained by BamHI and BglII double digestion. Below that are fragments predicted for a diagnostic BstYI digestion used to analyze plasmid clones.

FIG. 2.

Histogram of in silico BamHI-BglII-digested mouse genomic DNA fragments. In silico-digested DNA fragments were grouped based upon size in 500-bp increments. •, BamHI-BglII-digested mouse genomic DNA; □, BamHI-BglII-digested mouse genomic DNA flanked with computer-simulated random integrants (see the text).

FIG. 3.

Frequency of palindrome labeling by rAAV integration compared to that of labeling by random integration. Palindromes at or near rAAV and random integration sites are categorized based on their arm length. (A to L) Frequency of palindrome labeling as a percentage of total integrations is plotted for each size category. Experimental variables are given above each graph: rAAV vector serotype (AAV2 or AAV8)/mouse strain (B6, C57BL/6J; Sc, SCID; HTI, hereditary tyrosinemia type I mouse)/tissue type (Lv, liver; M, skeletal muscle; H, heart)/vector dose (hi, high dose; lo, low dose; see Tables 1 and 2). The number in parentheses in each panel indicates the total number of rAAV integration sites analyzed in each group. For panels A to J, 3′ rAAV integration sites were isolated without selection, while integration sites were isolated after in vivo selection for panels K and L, in which 5′ and 3′ rAAV integration sites are separately displayed. (M) Palindrome labeling frequency data from panels A to J are com- bined and plotted as a function of palindrome arm length. For this analysis, labeling frequency of palindromes with the same arm length is compared between rAAV and random integrations. For palindromes with an arm length of ≥11 bp, points represent palindrome arm lengths in increments of 2 bp (i.e., 11 to 12 bp, 13 to 14 bp, and so on). Asterisks and solid triangles indicate statistical significance compared to results for random integrations (P < 0.001 [two asterisks] and 0.001 ≤ P < 0.01 [one asterisk] by χ² test; P < 0.001 [closed triangle] by Fisher's exact test).

FIG. 4.

Positional relationship between rAAV integration sites and DNA palindromes (pal.). The positions of rAAV-labeled DNA palindromes are displayed relative to their associated rAAV integration sites. A 1-kb sequence window represents ±500 bp centromeric (minus) and telomeric (plus) to each rAAV integration site (centered at the 0-bp position). The histogram gives the number of palindromes located within each 50-bp increment relative to the rAAV integration sites (i.e., positions −500 to −451, −450 to −401, and so on). An exception is the 51-bp center window from positions −50 to 0, which includes an rAAV integration site. (A) rAAV-labeled DNA palindromes with an arm length of ≥6 bp (369 palindromes in all). (B) rAAV-labeled DNA palindromes with an arm length of ≥20 bp (134 in all). (C) rAAV-labeled DNA palindromes with an arm length between 6 and 19 bp (235 in all).

FIG. 5.

Patterns of palindrome (Pal.) center retention/deletion associated with rAAV integration. (A) Definition of center-deleted and center-retained rAAV integration. (B) Percentage of center-deleted rAAV integrations of total rAAV integrations is plotted as a function of palindrome arm length and compared to that for random integration. To increase the power of analysis, palindromes with different arm lengths are combined in the following manner: 9 to 10 bp (shown as 10 in the figure), 11 to 14 bp (14), 15 to 18 bp (18), 19 to 22 bp (22), 23 to 26 bp (26), 27 to 28 bp (28), 29 to 30 bp (30), and 31 bp or more (≥31). We compared the observed frequency to that of 20 independently generated random integration data sets (see Materials and Methods) by the χ² test or Fisher's exact test. Solid triangles indicate statistical significance (P < 0.05; Fisher's exact test) for all 20 random data set comparisons. Three representative random integration data sets are included. (C and D) Positional relationship between center-deleted or center-retained rAAV integrations and the palindrome symmetry center. rAAV integration sites are shown as histograms, giving their locations in 50-bp increments across a 1-kb region centered on the palindrome symmetry center. An exception is the 51-bp center window from positions −50 to 0, which includes the palindrome symmetry center. Center-deleted or center-retained rAAV integrations are displayed in the left (clear background) or right (gray background) portion of the 1-kb window as labeled. (C) rAAV-labeled DNA palindromes with an arm length of ≥20 bp (134 palindromes in all). (D) rAAV-labeled DNA palindromes with an arm length between 6 and 19 bp (235 in all).

FIG. 6.

Significance of (AT)n and (AC)n (GT)n palindromes in rAAV-labeled palindromes susceptible to breakage. (A) Percentage of rAAV-labeled (AT)n palindromes among the 134 rAAV-labeled palindromes with an arm length of ≥20 bp is plotted as a function of n. Random integration-labeled palindromes are likewise plotted. Asterisks and solid triangles indicate statistical significance between rAAV and random integrations (*, P < 0.001 by χ² test; closed triangle, P < 0.001 by Fisher's exact test). (B) DNA sequences of six (AC)n (GT)n palindromes (numbered 1 to 6) labeled by rAAV integration. Black arrowheads indicate the positions at which joining to the rAAV vector genome occurred. Uppercase letters denote the portion of the palindrome sequence that was retained in each junction, while lowercase letters denote the nonretained portion. Underlined nucleotides are central spacer sequences. All six (AC)n (GT)n palindromes show a center-deleted pattern. Two representative (AT)n palindromes are shown as 7 and 8 for comparison. Coordinates of the palindromes numbered 1 to 8 (UCSC mm8) are chr 10: 38176562, chr 5: 118212140, chr 6: 7083048, chr 15: 29735040, chr 8: 120556025, chr 16: 69258562, chr X: 150903099, and chr 15: 3434443, respectively.

FIG. 7.

Analyses of dinucleotide labeling by rAAV integration. Relative frequency of rAAV-labeling of the four possible types of dinucleotide repeats among total integration events is plotted as a function of n (n is the number of dinucleotide repeats). For comparison, the frequency of random integration labeling is also plotted. The four types of dinucleotide repeats are (AT)n (A), (AC or GT)n (B), (AG or CT)n (C), and (CG)n (D).

FIG. 8.

Distribution of break-prone palindromes in the mouse genome. A total of 134 rAAV-labeled palindromes with an arm length of ≥20 bp are mapped on a normal mouse karyotype. Symbols indicate center-deleted integration in (white circles) or near (black circles) palindromes and center-retained integration in (white squares) or near (gray squares) palindromes, respectively. There was no rAAV-labeled break-prone palindrome on the Y chromosome in the present study.

FIG. 9.

Proposed model for rAAV integration at palindromes in either a cruciform or hairpin structure. In this model, hairpin loops of palindromic AAV-ITR and DNA palindromes on the genome are nicked, trimmed, and joined by cellular DNA repair mechanisms. Various degrees of host chromosomal DNA deletions occur at rAAV integration, resulting in exclusively center-deleted integrations.