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616. rAAV Hairpin Structure Does Not Contribute to Chromosomal Integration
AAV GENOME & HOST INTERACTIONS Differences in AAV transduction in HeLa cells were assessed using a recombinant ssAAV2 vector expressing the firefly Luciferase reporter gene. Analysis of the results obtained from this primary screening identified 1528 genes affecting transduction by AAV vectors by more than 4-fold (184 genes by more than 8-fold). Of these genes, 993 are inhibitors of AAV transduction, whereas 535 are required for efficient transduction by AAV vectors. The effect of these genes on AAV transduction was further confirmed in a secondary screening based on high-content microscopy of cells transduced with a recombinant AAV vector expressing DsRed. Gene ontology analysis of the inhibitor genes revealed a clear overrepresentation of genes related to DNA recombination and repair and cell cycle control, whereas in the subset of genes required for infection an overrepresentation of genes involved in endocytosis, intracellular trafficking and transcription was observed. Consistent with our previous results, among the genes that restrict transduction by AAV vectors, we found members of the MRN complex and other genes involved in cellular DNA damage response, as well as genes involved in the ubiquitin-proteasome system. In addition, a significant number of genes were identified, which have not previously been associated with AAV transduction. Work in progress aims at investigating the mechanisms of action of the identified genes and exploiting this information to develop RNAi or pharmacological strategies to improve AAV transduction in vivo.
615. Enhanced Transgene Expression and SiteSpecific Integration of Adeno-Associated Virus Depends on Methylation Status of the Target Cell Diptiman Chanda,1 Jonathan A. Hensel,1 Jerome T. Higgs,1 Rajat Grover,1 Niroop Kaza,1 Selvarangan Ponnazhagan.1 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL.
Long-term expression of transgenes following vector-mediated delivery is often curtailed due to host defense mechanisms against viral infection. One such event in the eukaryotic cells occurs via DNA methylation and histone modification. Unmethylated CpG regions of microbial DNA are immunogenic and also form targets for DNA methyltransferase activity and recruitment of polycomb and other protein complexes resulting in repression of viral replication as well as transgene expression. Adeno-associated virus (AAV) is a non-pathogenic human parvovirus, which is being tested in many clinical gene therapy applications. Wild-type AAV (wt-AAV) has anti-oncogenic properties mainly in HPV-induced carcinogenesis. There are atleast four CpG islands in the 4.6 kb wtAAV2 genome, which are larger than 400 base-pairs. Moreover, like other gene therapy vectors, recombinant AAV (rAAV) are mostly designed using the cytomegalovirus (CMV) immediate early gene promoter, which also contains a significant number of CpG motifs. Therefore, methylation status of AAV inside the target cell could affect transgene expression. In the present study, we determined the influence of host cell methylation on transgene expression of rAAV and on integration of wt-AAV. When HeLa cells were maintained in a hypomethylating agent, 5-Aza-2’ deoxycytidine (5-Aza) for 72 hours and followed by infection with either recombinant AAV-GFP or wt-AAV showed significantly higher GFP expression and wt-AAV integration respectively when compared to untreated HeLa cells. The enhanced GFP expression was found to be not due to transduction of additional AAV particles following 5-Aza treatment, rather due to the methylation status of the AAV genome. The enhanced GFP expression following 5-Aza treatment was exclusively linked to AAV viral infection and not AAV plasmid transfection, suggesting the role of genomic organization of AAV, flanked by GC-rich terminal repeat hairpin structures. Previous studies have demonstrated that the leading 510 nucleotides at the 5’ end of AAVS1 sequence in the human chromosome 19 are a key to AAV integration and contain homology to the Rep binding site (RBS) and the terminal resolution S236
site (TRS) of AAV. AAVS1 sequence analysis also indicated the 510 nucleotides are a part of a 662 base pairs CpG island starting at 203 nucleotides and ending at 864 nucleotides when the first 1020 base pairs of AAVS1 is analyzed. In order to further characterize the role of methylation on AAV integration, we methylated in vitro a 8.2 kb AAVS1 fragment using CpG methylase and methyl donor, S-adenosyl methionine and transfected this methylated fragment into C18 cells. These cells were infected with wt-AAV after introduction of methylated AAVS1 and when tested failed to show any site-specific integration. Overall, results of the present study showed the influence of host methylation status in AAV-based genetic therapies.
616. rAAV Hairpin Structure Does Not Contribute to Chromosomal Integration
Marcela P. Cataldi,1,3 Douglas M. McCarty.1,2 1 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Dept. of Pediatrics, The Ohio State University, Columbus, OH; 3MCDB Graduate Program, The Ohio State University, Columbus, OH. The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single-stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration by comparing DNA molecules with the TR sequences constrained in the hairpin conformation to molecules with linear duplex ends, with or without TR sequences. In order to measure integration efficiency, 293 cells were transfected with a high dose of each of these AAV genome-like molecules carrying a GFP-expression cassette, and continuously passaged until a stable percentage of GFP-expressing cells was reached, which represented the percentage of cells with DNA molecules integrated into the host genome after loss of episomal DNA by dilution. The results show no significant difference in terms of integration efficiency between the three DNA molecules, suggesting that AAV TR in a hairpin conformation is not more or less likely to integrate than open blunt ends with or without AAV TR sequences. In addition, we found that a previously reported decrease in gene expression mediated by silencing of HP-containing molecules is not observed when molecules are transfected at high doses, suggesting that the pathway that specifically recognizes the hairpin structure and leads to DNA silencing is saturated under these experimental conditions.
617. Assessment of Oncogenic Insertional Activation Potential from rAAV Vector
Lucia E. Rosas,1 Jessica L. Grieves,1,2 Douglas M. McCarty.1,3 Center for Gene Therapy, The Reseach Institute at Nationwide Children’s Hospital, Columbus, OH; 2Department of Veterinary Biosciences, Ohio State University College of Veterinary Medicine, Columbus, OH; 3Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH. 1
Although recombinant adeno-associated virus (rAAV) gene delivery vectors have generally proven safe in animal models of human disease, their potential for low frequency integration into host chromosomal DNA raises concerns about genotoxicity and oncogenesis. Interactions between a rAAV vector expressing β-glucuronidase and a host common integration site have been associated with hepatocellular carcinoma (HCC) in MPS VII mice. We Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
615. Enhanced Transgene Expression and SiteSpecific Integration of Adeno-Associated Virus Depends on Methylation Status of the Target Cell Diptiman Chanda,1 Jonathan A. Hensel,1 Jerome T. Higgs,1 Rajat Grover,1 Niroop Kaza,1 Selvarangan Ponnazhagan.1 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL.
Long-term expression of transgenes following vector-mediated delivery is often curtailed due to host defense mechanisms against viral infection. One such event in the eukaryotic cells occurs via DNA methylation and histone modification. Unmethylated CpG regions of microbial DNA are immunogenic and also form targets for DNA methyltransferase activity and recruitment of polycomb and other protein complexes resulting in repression of viral replication as well as transgene expression. Adeno-associated virus (AAV) is a non-pathogenic human parvovirus, which is being tested in many clinical gene therapy applications. Wild-type AAV (wt-AAV) has anti-oncogenic properties mainly in HPV-induced carcinogenesis. There are atleast four CpG islands in the 4.6 kb wtAAV2 genome, which are larger than 400 base-pairs. Moreover, like other gene therapy vectors, recombinant AAV (rAAV) are mostly designed using the cytomegalovirus (CMV) immediate early gene promoter, which also contains a significant number of CpG motifs. Therefore, methylation status of AAV inside the target cell could affect transgene expression. In the present study, we determined the influence of host cell methylation on transgene expression of rAAV and on integration of wt-AAV. When HeLa cells were maintained in a hypomethylating agent, 5-Aza-2’ deoxycytidine (5-Aza) for 72 hours and followed by infection with either recombinant AAV-GFP or wt-AAV showed significantly higher GFP expression and wt-AAV integration respectively when compared to untreated HeLa cells. The enhanced GFP expression was found to be not due to transduction of additional AAV particles following 5-Aza treatment, rather due to the methylation status of the AAV genome. The enhanced GFP expression following 5-Aza treatment was exclusively linked to AAV viral infection and not AAV plasmid transfection, suggesting the role of genomic organization of AAV, flanked by GC-rich terminal repeat hairpin structures. Previous studies have demonstrated that the leading 510 nucleotides at the 5’ end of AAVS1 sequence in the human chromosome 19 are a key to AAV integration and contain homology to the Rep binding site (RBS) and the terminal resolution S236
site (TRS) of AAV. AAVS1 sequence analysis also indicated the 510 nucleotides are a part of a 662 base pairs CpG island starting at 203 nucleotides and ending at 864 nucleotides when the first 1020 base pairs of AAVS1 is analyzed. In order to further characterize the role of methylation on AAV integration, we methylated in vitro a 8.2 kb AAVS1 fragment using CpG methylase and methyl donor, S-adenosyl methionine and transfected this methylated fragment into C18 cells. These cells were infected with wt-AAV after introduction of methylated AAVS1 and when tested failed to show any site-specific integration. Overall, results of the present study showed the influence of host methylation status in AAV-based genetic therapies.
616. rAAV Hairpin Structure Does Not Contribute to Chromosomal Integration
Marcela P. Cataldi,1,3 Douglas M. McCarty.1,2 1 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Dept. of Pediatrics, The Ohio State University, Columbus, OH; 3MCDB Graduate Program, The Ohio State University, Columbus, OH. The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single-stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration by comparing DNA molecules with the TR sequences constrained in the hairpin conformation to molecules with linear duplex ends, with or without TR sequences. In order to measure integration efficiency, 293 cells were transfected with a high dose of each of these AAV genome-like molecules carrying a GFP-expression cassette, and continuously passaged until a stable percentage of GFP-expressing cells was reached, which represented the percentage of cells with DNA molecules integrated into the host genome after loss of episomal DNA by dilution. The results show no significant difference in terms of integration efficiency between the three DNA molecules, suggesting that AAV TR in a hairpin conformation is not more or less likely to integrate than open blunt ends with or without AAV TR sequences. In addition, we found that a previously reported decrease in gene expression mediated by silencing of HP-containing molecules is not observed when molecules are transfected at high doses, suggesting that the pathway that specifically recognizes the hairpin structure and leads to DNA silencing is saturated under these experimental conditions.
617. Assessment of Oncogenic Insertional Activation Potential from rAAV Vector
Lucia E. Rosas,1 Jessica L. Grieves,1,2 Douglas M. McCarty.1,3 Center for Gene Therapy, The Reseach Institute at Nationwide Children’s Hospital, Columbus, OH; 2Department of Veterinary Biosciences, Ohio State University College of Veterinary Medicine, Columbus, OH; 3Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH. 1
Although recombinant adeno-associated virus (rAAV) gene delivery vectors have generally proven safe in animal models of human disease, their potential for low frequency integration into host chromosomal DNA raises concerns about genotoxicity and oncogenesis. Interactions between a rAAV vector expressing β-glucuronidase and a host common integration site have been associated with hepatocellular carcinoma (HCC) in MPS VII mice. We Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy