1. Volume 20, Issue 6, Pages 1177-1186 (June 2012)
Integration Frequency and Intermolecular Recombination of rAAV Vectors in Non- human Primate Skeletal Muscle and Liver 
Ali Nowrouzi, Magalie Penaud-Budloo, Christine Kaeppel, Uwe Appelt, Caroline Le Guiner, Philippe Moullier, Christof von Kalle, Richard O Snyder, Manfred Schmidt 
Molecular Therapy 
Volume 20, Issue 6, Pages (June 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Recombinant AAV insertional standards (rAIS) for LAM-PCR coupled with 454 sequencing. (a) Cell clones harboring integrated rAAV genomes were generated after transduction of a Cos cell line with the rAAV-RSV-tGFP-IRES-Puro-wpre-ISceI vector and puromycin selection. Two clones were selected that each harbor a single rAAV integration site (IS) (dark gray box) with a partial ITR sequence (gray arrow); these truncated ITRs mimic the majority of the rAAV forms found following transduction in vivo. (b) LAM-PCR was performed from 100 ng of DNA and the amplicon size was 272 bp and 186 bp for Cos 1 and Cos 7 clone, respectively. To the sizes of the COS 1 and COS 7 amplicons depicted in the agarose gel, ∼30 bp resulting from the linker ligation required for subsequent amplification by LAM-PCR (see methods) have to be added. (c) The efficacy of amplifying and sequencing the vector–host genome junction through an ITR with varying length was measured by the relative retrieval frequency of the IS following 454 pyrosequencing. The primer used for linear amplification is located in the RSV promoter sequence (white arrow on a). AAV, adeno-associated virus; ITR, inverted terminal repeat; LAM-PCR, linear amplification-mediated PCR.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
High resolution mapping of ITR breakpoints in rAAV concatemeric fragments and proviruses in vivo reveals hotspots of recombination. (a) Percentage of rAAV proviral insertions with ITR breakpoints at particular regions within the ITR in skeletal muscle and liver. (b) Depth sequence analysis was used to detect ITR breakpoints of rAAV concatemers in skeletal muscle and liver of nine NHP. Therefore, the sequence coverage was calculated for each basepair starting from the primer that was used to prepare the samples for sequencing, through the 5′ITR until reaching the first break. Only sequences that continue with a rAAV-specific fragment after the break were taken into account. Preferentially, breakpoints were detected in the B region of the ITR indicated by a significant decrease in sequence coverage. (c) H-H or H-T concatmers were distinguished by analyzing the orientation of each side of the concatemeric amplicons. Red arrows indicate location of recombination hotspots which result in up to 3 kb deletions of single concatemerized vector units (see e). (d) The relative proportion of concatemers was calculated by measuring the total sequence counts of retrieved H-H and H-T concatemers obtained both in liver and skeletal muscle of all analyzed NHP. In cases where only ITR-ITR fragments were sequenced the H-T or H-H configuration cannot be determined. Designated as u.d. (e) To identify the configuration of the rAAV concatemers, 454-sequence reads were identified that discontinuously align to exactly two parts to the vector sequence. The resulting concatemeric sequences can be divided by a junction resembling the recombination spot in the 5′ITR (x-axis) and the whole vector sequence (y-axis). The x-axis resembles the coordinates of the 5′ITR starting with the position of the sequencing primer (462 bp) and coordinates on the y-axis indicates the exact position of breakpoints on the whole vector sequence (1–4,173 bp). The two vector coordinates describing the transitions of the two-part-alignments were drawn as red (head-to-tail) and blue (head-to-head) spots. Each dot in the graphs represents the exact coordinates of breakpoints according to the 5′ITR junction (x-axis) and the whole vector (y-axis) which can be in the ITR itself or on other locations of the vector. ITR, inverted terminal repeat; NHP, non-human primates; rAAV, recombinant adeno-associated virus; u.d, undetermined.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Retrieval of rAAV insertion sites by LAM-PCR and deep sequencing is copy number dependent. (a) For analyzing the sensitivity of capturing rAAV insertion sites mimicking the in vivo context, genomic DNA obtained from transduced primate muscle (harboring predominantly episomes) was mixed with DNA derived from the Cos 7 rAAV insertion standard and LAM-PCR was performed followed by deep sequencing. To calculate the relative retrieval frequency of the rAIS, we measured the sequence count of the rAIS in each dilution. The retrieval frequency of all integration sites was calculated from the total amount of break-evident reads revealing a copy number dependent retrieval of rAAV insertion sites. (b) The retrieval frequency of rAIS (Cos7) spiked with genomic DNA was compared in samples obtained from muscle of NHP containing high (Mac1; 31.4 vector copies/diploid genome) and low vector copy numbers (Mac7; 1.3 vector copies/diploid genome). (c) The quantitative relation of rAAV insertion sites compared to concatemeric vector configurations predominantly derived from episomal vector genomes was calculated for each individual animal analyzed for muscle and liver respectively. Therefore, the relative sequence count of all LAM-PCR amplicons which passed our validation criteria for being concatemeric or potential insertion sites were divided into groups resembling either rAAV insertion sites and concatemers. (d) Quantitative measurement of captured rAAV proviruses in NHP liver and muscle following IM and RI vector administration. IM, intramuscular; IS, integration sites; LAM-PCR, linear amplification-mediated PCR; NHP, non-human primates; rAAV, recombinant adeno-associated virus; rAIS, rAAV insertional standards; RI, regional intravenous.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions