TABLE 3.
Summary of palindromes recurrently labeled by rAAV integration
| Features of palindromes | Features of rAAV integration breakpoints
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Chromosome no. | Band | Coordinate | Arm length (bp) | Spacer (bp) | Mismatch in the arm (bp) | AT/TA repeat no. | Sample | Mouse genome (coordinate) | rAAV nucleotide positiona | Insertion size (bp)b | Orientation of rAAVc | In or near palindromed |
| 1 | E2.3 | 117802950 | 34 | 0 | 0 | 34 | AAV8/Sc/H/hi | 117802899 | 2951 | 0 | Plus | Near |
| AAV8/Sc/H/hi | 117803000 | 2891 | 0 | Minus | Near | |||||||
| 3 | H1 | 139340183 | 45 | 0 | 1 | 27 | AAV8/B6/H/hi | 139340211 | 3011 | 0 | Minus | In |
| AAV8/B6/H/hi | 139340237 | 3024 | 0 | Minus | Near | |||||||
| 4 | B1 | 47412002 | 72 | 0 | 3 | 20 | AAV8/B6/H/hi | 47412045 | 3034 | 0 | Minus | In |
| AAV8/B6/H/hi | 47412059 | 3034 | 0 | Minus | In | |||||||
| 4 | C6 | 97561203 | 8 | 0 | 0 | 2 | AAV8/Sc/H/hi | 97561059 | 2939 | 0 | Plus | In |
| AAV8/Sc/H/hi | 97561027 | 2970 | 0 | Plus | In | |||||||
| 10 | D1 | 98632057 | 28 | 0 | 0 | 28 | AAV8/B6/Lv/lo | 98632020 | 2999 | 0 | Plus | Near |
| AAV8/Sc/Lv/lo | 98632105 | 2965 | 0 | Minus | Near | |||||||
| 11 | B1.1 | 44726916 | 33 | 0 | 0 | 32 | AAV8/Sc/H/hi | 44726958 | 2947 | 0 | Minus | Near |
| AAV8/Sc/M/hi | 44726988 | 2971 | 0 | Minus | Near | |||||||
| 14e | A2 | 14983628 | 25 | 0 | 0 | 25 | AAV8/Sc/M/hi | 14983597 | 2654 | 0 | Minus | Near |
| AAV8/B6/Lv/lo | 14983597 | 2654 | 0 | Minus | Near | |||||||
| 15 | D1 | 63699599 | 47 | 0 | 4 | 32 | AAV8/B6/H/hi | 63699609 | 3015 | 2 | Minus | In |
| AAV8/B6/M/hi | 63699610 | 3020 | 1 | Minus | In | |||||||
| 15 | F1 | 99702254 | 47 | 0 | 0 | 46 | AAV8/Sc/M/hi | 99702203 | 2968 | 5 | Plus | Near |
| AAV8/B6/H/hi | 99702257 | 2989 | 0 | Minus | In | |||||||
| 19 | A | 11606158 | 28 | 0 | 0 | 27 | AAV8/Sc/M/hi | 11606437 | 2831 | 0 | Minus | Near |
| AAV8/Sc/M/hi | 11606302 | 2842 | 0 | Minus | Near | |||||||
The total length of the rAAV vector is 3,069 bases. The nucleotide positions are numbered from 1 to 3069 from the left (5′) to the right (3′).
Insertion indicates the length of nucleotide insertions at rAAV-cellular DNA junctions.
Plus and minus indicate that rAAV vectors at the junctions were found in a plus orientation (centromere:5′ side of rAAV:3′ side of rAAV:telomere) and in a minus orientation (centromere:3′ side of rAAV:5′ side of rAAV:telomere), respectively.
In and near indicate that rAAV integration occurred in or near palindromes, respectively.
Independence of the two integration events was confirmed by the different structures of the isolated rAAV integration plasmids. Plasmid AAV8/Sc/M/hi contained a longer vector genome-flanking mouse genomic sequence than that in plasmid AAV8/B6/Lv/lo. A BglII site in the mouse genome was retained in plasmid AAV8/Sc/M/hi, while it was digested and ligated with the BamHI vector end in plasmid AAV8/B6/Lv/lo. Although such an event caused by incomplete digestion with restriction enzymes was rarely found, this finding has provided strong evidence that they were independent integration events.