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95. Comprehensive wtAAV Integration Site Analysis Reveals Integration Site Formation Throughout the Whole Viral Genome
AAV Vectors I low integration frequency combined with the AAV inverted terminal repeats (ITR) makes amplification technologically challenging and prone to artifacts that may mis-identify or miss some integration sites. Our lab has utilized a modified Illumina Mate Pair sequencing technique to uncover genomic integration sites and bypass the need to amplify through the AAV ITR. We believe that this technique can accurately establish bona-fide integration sites, uncover novel integration sites, and provide further evidence for existing known hotspots. At its core, mate pair sequencing involves self-circularization of fractured genomic fragments followed by recovery and sequencing of the junction. In essence, it bridges the low throughput shuttle vector systems used in the past with high throughput sequencing. Here we have utilized biotinylated loxP linkers added to the ends of genomic fragments and at unique sites in AAV vectors followed by Cre circularization (Van Nieuwerburgh, 2012). The circularized fragments are enriched by exonuclease treatment to remove linear fragments then fractured to ∼400 bp. The fragments containing the loxP junction are purified using magnetic strepavadin coated beads and Illumina sequencing adapters are added before sequencing. Using this technique, we have successfully recovered control sequences of plasmids mixed with genomic DNA. Importantly, the Cre recombination reaction allows us to accurately distinguish self-circularized fragments from cross-recombined fragments and eliminate apparent false positive integration sites. We are working on applying this technique to DNA samples from mice injected with AAV. Continued study of AAV integration will be extremely important for establishing the safety profile of AAV used in clinical applications.
95. Comprehensive wtAAV Integration Site Analysis Reveals Integration Site Formation Throughout the Whole Viral Genome
Karl Petri,1 Richard Gabriel,1 Leticia Agundez Cortes,2 Raffaele Fronza,1 Saira Afzal,1 Christine Kaeppel,1 Christof von Kalle,1 Els Henckaerts,2 Manfred Schmidt.1 1 Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Department of Infectious Diseases, King’s College, London, United Kingdom. To establish latency, wildtype adeno-associated virus (wtAAV) is able to integrate its DNA into a specific region on chromosome 19 of the human genome, named AAVS1. This targeted integration mechanism relies on the function of the two larger versions (Rep78/68) of the Rep protein family, expressed by wtAAV. Rep is able to bind a 12 bp long sequence, called Rep binding site (RBS). The RBS can be found both in the viral ITRs and in AAVS1. By the formation of a trimeric complex between wtAAV, Rep and the genomic locus, targeted integration can be achieved. However, the exact mechanisms of Rep-mediated integration still remain unknown. To analyze wtAAV IS distribution genome-wide, largescale integration analyses using high-throughput next generation sequencing have been performed. These studies revealed that Repmediated wtAAV integration preferentially occurs in AAVS1, but that additional integration hotspots associated with RBS can be found as well throughout the genome. Up to this point most integration sites (IS) reported for wtAAV were derived from vector-genome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most currently available methods for the analysis of viral IS rely on amplification of S40
the vector-genome junction with primers that bind in the vicinity of the viral ends. Therefore, knowledge about the nature and structure of the internal wtAAV genome remains limited. To address this question, we investigated ~3e6 wtAAV concatemeric structures generated by IS analyses using linear amplification-mediated (LAM) PCR to identify potential alternative breakpoints throughout the wtAAV genome. We discovered that breakpoints occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter, there are a number of other preferred breakpoints throughout the wtAAV genome. Using a novel approach involving targeted enrichment of AAV and AAVS1 sequences, we also were able to consider viral IS at any position of the AAV genome. Targeted HiSeq sequencing of wtAAV and AAVS1 determined several hundreds of exact IS and confirmed that wtAAV genome junctions form at any position of its genome. Those cryptic integration events, not picked up by the majority of methods currently available, shed new light onto wtAAV persistence and may further improve current AAV persistence analyses in gene therapy.
96. Genomic Integration Profile of AAVHSC Vectors in Human CD34+ Long-Term Engrafted Hematopoietic Stem Cell Xenografts in ImmuneDeficient Mice
Manasa Chandra,1 Tahira Ul-Hasan,1 Laura J. Smith,1 K.K. Wong,2 Saswati Chatterjee.1 1 Virology, Beckman Research Institute City of Hope National Medical Center, Duarte, CA; 2Hematology/Stem Cell Transplantation, City of Hope, Duarte, CA. Adeno-associated virus vectors (AAV) are proving to be promising and powerful for gene delivery due to their ability to safely transduce both non-dividing and dividing cells. While AAV mainly persists in episomal copies in non-dividing cells, genomic integration has been reported including in CD34+ hematopoietic stem cells (HSCs). Characterizing the vector integration profile is important to ensure safety since genomic integration has the potential for inducing insertional mutagenesis and genotoxicity. The absence of genotoxicity of AAV has been documented in multiple studies. Our lab has recently described the isolation and gene transfer properties of human HSC derived AAVs (AAVHSCs) (Smith LJ, et al. Mol Therapy 2014). To test the ability of the AAVHSCs to transduce long-term engrafted HSCs, we transduced human CD34+ cells with AAV vectors encoding a luciferase transgene prior to transplantation into irradiated immunodeficient NOD/SCID mice. Through serial bioluminescent imaging, we found that the AAVHSCs supported higher levels of expression than AAV2, 7 and 8. Of the AAVHSC serotypes tested, AAVHSC17 was found to support the highest level of transgene expression in vivo, for at least six months post-transplantation. Since vector genomes were found to persist in both long-term engrafted CD34+ cells as well as their differentiated hematopoietic progeny, we hypothesized that the vector genomes likely persisted as chromosomal integrants since episomal copies would have been lost during mitosis. Here, we evaluated the vector genome integration profile in the long-term engrafted CD34+ HSCs transduced with AAVHSC17. High molecular weight DNA was extracted from flow sorted human CD34+ cells isolated from engrafted mouse marrow and sonicated to fragment the DNA before ligation of double stranded DNA adaptors. Using LM-PCR we amplified AAV-chromosomal sequences using AAV-specific and adaptor-specific primers, and further enriched vector-chromosome junctions with nested PCR. The PCR generated library was then used for paired-end Illumina sequencing. The sequence reads were paired using CLC Genomics Workbench. High-throughput analysis resources including PLAN, BLAST (NCBI), and ENSEMBL were used to determine the locations of the AAV-chromosomal junctions. AAV integration was found to occur randomly throughout the genome, including the X and Y Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
95. Comprehensive wtAAV Integration Site Analysis Reveals Integration Site Formation Throughout the Whole Viral Genome
Karl Petri,1 Richard Gabriel,1 Leticia Agundez Cortes,2 Raffaele Fronza,1 Saira Afzal,1 Christine Kaeppel,1 Christof von Kalle,1 Els Henckaerts,2 Manfred Schmidt.1 1 Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Department of Infectious Diseases, King’s College, London, United Kingdom. To establish latency, wildtype adeno-associated virus (wtAAV) is able to integrate its DNA into a specific region on chromosome 19 of the human genome, named AAVS1. This targeted integration mechanism relies on the function of the two larger versions (Rep78/68) of the Rep protein family, expressed by wtAAV. Rep is able to bind a 12 bp long sequence, called Rep binding site (RBS). The RBS can be found both in the viral ITRs and in AAVS1. By the formation of a trimeric complex between wtAAV, Rep and the genomic locus, targeted integration can be achieved. However, the exact mechanisms of Rep-mediated integration still remain unknown. To analyze wtAAV IS distribution genome-wide, largescale integration analyses using high-throughput next generation sequencing have been performed. These studies revealed that Repmediated wtAAV integration preferentially occurs in AAVS1, but that additional integration hotspots associated with RBS can be found as well throughout the genome. Up to this point most integration sites (IS) reported for wtAAV were derived from vector-genome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most currently available methods for the analysis of viral IS rely on amplification of S40
the vector-genome junction with primers that bind in the vicinity of the viral ends. Therefore, knowledge about the nature and structure of the internal wtAAV genome remains limited. To address this question, we investigated ~3e6 wtAAV concatemeric structures generated by IS analyses using linear amplification-mediated (LAM) PCR to identify potential alternative breakpoints throughout the wtAAV genome. We discovered that breakpoints occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter, there are a number of other preferred breakpoints throughout the wtAAV genome. Using a novel approach involving targeted enrichment of AAV and AAVS1 sequences, we also were able to consider viral IS at any position of the AAV genome. Targeted HiSeq sequencing of wtAAV and AAVS1 determined several hundreds of exact IS and confirmed that wtAAV genome junctions form at any position of its genome. Those cryptic integration events, not picked up by the majority of methods currently available, shed new light onto wtAAV persistence and may further improve current AAV persistence analyses in gene therapy.
96. Genomic Integration Profile of AAVHSC Vectors in Human CD34+ Long-Term Engrafted Hematopoietic Stem Cell Xenografts in ImmuneDeficient Mice
Manasa Chandra,1 Tahira Ul-Hasan,1 Laura J. Smith,1 K.K. Wong,2 Saswati Chatterjee.1 1 Virology, Beckman Research Institute City of Hope National Medical Center, Duarte, CA; 2Hematology/Stem Cell Transplantation, City of Hope, Duarte, CA. Adeno-associated virus vectors (AAV) are proving to be promising and powerful for gene delivery due to their ability to safely transduce both non-dividing and dividing cells. While AAV mainly persists in episomal copies in non-dividing cells, genomic integration has been reported including in CD34+ hematopoietic stem cells (HSCs). Characterizing the vector integration profile is important to ensure safety since genomic integration has the potential for inducing insertional mutagenesis and genotoxicity. The absence of genotoxicity of AAV has been documented in multiple studies. Our lab has recently described the isolation and gene transfer properties of human HSC derived AAVs (AAVHSCs) (Smith LJ, et al. Mol Therapy 2014). To test the ability of the AAVHSCs to transduce long-term engrafted HSCs, we transduced human CD34+ cells with AAV vectors encoding a luciferase transgene prior to transplantation into irradiated immunodeficient NOD/SCID mice. Through serial bioluminescent imaging, we found that the AAVHSCs supported higher levels of expression than AAV2, 7 and 8. Of the AAVHSC serotypes tested, AAVHSC17 was found to support the highest level of transgene expression in vivo, for at least six months post-transplantation. Since vector genomes were found to persist in both long-term engrafted CD34+ cells as well as their differentiated hematopoietic progeny, we hypothesized that the vector genomes likely persisted as chromosomal integrants since episomal copies would have been lost during mitosis. Here, we evaluated the vector genome integration profile in the long-term engrafted CD34+ HSCs transduced with AAVHSC17. High molecular weight DNA was extracted from flow sorted human CD34+ cells isolated from engrafted mouse marrow and sonicated to fragment the DNA before ligation of double stranded DNA adaptors. Using LM-PCR we amplified AAV-chromosomal sequences using AAV-specific and adaptor-specific primers, and further enriched vector-chromosome junctions with nested PCR. The PCR generated library was then used for paired-end Illumina sequencing. The sequence reads were paired using CLC Genomics Workbench. High-throughput analysis resources including PLAN, BLAST (NCBI), and ENSEMBL were used to determine the locations of the AAV-chromosomal junctions. AAV integration was found to occur randomly throughout the genome, including the X and Y Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy