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404. Intermolecular Concatamerization of Adeno-Associated Viral Genomes Is Not Dependent on Single Strand Interaction
VECTOR GENOME BIOLOGY rAAV targeting in the transgenic model demonstrated substantially reduced efficiencies in primary fibroblasts (~0.01%) and thus far undetectable gene correction in vivo following infection of mouse tibialis muscle, despite high level expression of the mutant EGFP protein target. In conclusion, our studies support the utility of AAV vectors in gene targeting using a non-selectable gene reporter, but suggest that targeting in primary cells and in vivo may be much less efficient at this time. Continued research on the mechanisms of rAAV-mediated gene conversion events may help to enhance the application of this approach for gene correction in the future.
404. Intermolecular Concatamerization of Adeno-Associated Viral Genomes Is Not Dependent on Single Strand Interaction Yongping Yue,1 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO. Intermolecular concatamerization is the foundation for dual vector mediated larger gene transfer by recombinant adeno-associated virus (rAAV). However, the molecular processes underlying the heteroconcatamer formation remain to be defined. Theoretically, AAV intermolecular recombination could occur between single strand (ss) AAV genomes or between double strand (ds) AAV genomes. To distinguish these two possibilities, we have performed a sequential infection experiment with our previously described AAV LacZ transsplicing vectors. In this system, functional LacZ expression is dependent on the head-to-tail recombination between AV.LacZDonor and AV.LacZAcceptor. We administered AV.LacZAcceptor to the mouse anterior tibialis muscles at one month after AV.LacZDonor infection. At this time, a great majority of the ss AAV genomes from previous (AV.LacZDonor) infection were cleared out in muscle. Less than 4.38±1.81 % (BL6 mice) and 12.36±3.54 % (SCID mice) of the inoculated AAV virus remained as ss genomes at one month post-infection. If ss-ss genome interaction is absolutely required for AAV intermolecular concatamerization, LacZ expression from sequential infection will be less than ~5% (BL6) or ~15% (SCID) of that from co-infection. Quantitative analysis of LacZ expression showed that in BL6 muscle, expression from sequential infection reached 54.41±19.98%, 15.87±1.96% and 7.79±11.01% of that from co-infection at 1, 4 and 13 month postAV.LacZAcceptor infection. To exclude the interference of the host immune response in immune competent BL6 mice, similar experiment was performed in immune deficient SCID mice. Consistent with the finding in BL6 mice, LacZ expression from sequential infection reached 56.81±15.71% of that from co-infection at 1m time point. Importantly, the level of LacZ expression from sequential infection was also comparable to that from co-infection at 4- and 13-month time points. Our results strongly suggest that AAV heteroconcatamers are not derived from ss-ss genome interaction. Rather, ds-ds recombination is the predominant pathway for AAV intermolecular concatamer formation.
405. Genome-Wide Amplification for the Detection of Integrated rAAV-2 Vector DNA in Rabbit Tissues Bruce C. Schnepp,1 Michael C. Soult,1 Edward Kelly,2 Keith Munson,2 K. Reed Clark,1 Philip R. Johnson.1 1 Center for Gene Therapy, Columbus Children’s Research Institute, Columbus Children’s Hospital, Columbus, OH; 2 Targeted Genetics Corporation, Seattle, WA. Background. The molecular basis for long-term gene expression mediated by rAAV vectors remains undefined, and this uncertainty could hamper further development of these vectors in situations where vector integration is a safety concern. As part of a preclinical S160
development program for an rAAV-based HIV-1 vaccine, the biodistribution and vector integration status of a rAAV-2 vaccine candidate encoding the HIV-1 gag/protease proteins was assessed in New Zealand White rabbits. Methods. All rabbits received a single intramuscular injection of the rAAV vaccine in 3 dose groups: 3.3e10, 3.3e11, or 3.3e12 DRP. Each group enrolled 36 rabbits (18 male, 18 female). Animals were culled (6/sex/group) at days 5, 90 and 180 and multiple tissues analyzed for the presence of rAAV vector DNA. To determine the status of persisting vector DNA at days 90 and 180, we developed a genome-wide assay that was predicated on the amplification of genomic DNA between interspersed repetitive elements (Alu-like) in rabbits known as “C-repeats.” A single primer representing a consensus C-repeat sequence was used to generate amplicons. A second round of quantitative PCR detected C-repeat amplicons that contained integrated rAAV vector sequences. Significantly, episomal rAAV DNA species did not amplify in the Crpt-PCR assay. Thus, the assay was able to discriminate between integrated and episomal vector persistence. A cell pool containing randomly integrated rAAV vector DNA was used as a standard to define the limit of sensitivity of the assay in each tissue. Results. Vaccine DNA was widely distributed in multiple tissues at 90 and 180 days. Dissemination was dose-dependent and decreased over time. Importantly, gonads were negative at 90 and 180 days. Of 1666 tissue samples tested, 218 contained detectable vector DNA (sensitivity of ≥ 15 vector copies/microgram of cellular DNA). The majority of positive samples were at the site of injection (skin, muscle) or in highly perfused tissues. Of these 218 samples, 37 were determined to have a copy number sufficient to detect integration by the C repeat-PCR assay (≥ 1000 vector copies/microgram). A single sample (lymph node) scored positive at the limit of the assay sensitivity. In fact, this same sample scored negative when the assay was repeated. Most tissues could not be analyzed by Southern blot analysis due to low copy number. Blots from tissues with the highest copy numbers showed that most vector DNA was low molecular weight and single-stranded. Conclusions. Dissemination of vector DNA was dose and time dependent. By 180 days, vector DNA remained only at the injection site and in highly perfused tissues. These data suggest that rAAV vectors given by the intramuscular route demonstrate biologic properties remarkably similar to plasmid DNA.
406. Effect of DNA-PK on rAAV2 Persistence in Mouse Liver Sihong Song, Ge Zhao, Yuanqing Lu, Qiushi Tang, Terence R. Flotte. 1 Departments of Pharmaceutics and Pediatrics, University of Florida, Gainesville, FL, United States. Recombinant adeno-associated virus (rAAV) vectors have been increasingly used for gene therapy because they are relatively nontoxic, and the duration of transgene expression with these vectors is prolonged. As this vector is applied to human clinical trials, understanding the interactions between vector DNA and the cellular genome has become crucial for evaluating safety. We previously demonstrated that in skeletal muscle of SCID (DNA-PK negative) mice, rAAV2 DNA persisted in linear episomes, and gradually integrated into cellular genome, while in the muscle of C57BL/6 (DNA-PK positive) mice, it formed circular episomes. In the present studies, we have tested the effect of DNA-PK on the molecular fate of rAAV2 in liver. The same dose of rAAV2-CB-AT vector (expressing human alpha 1 antitrypsin) was injected via the portal vein into C57BL/6 and SCID mice. Transgene expression in C57BL/ 6 mice was significantly higher (2-fold) than in SCID mice. However, more AAV genomes (4-fold) were detected by real-time PCR in SCID mice than in C57Bl/6 mice. This may suggest concatemer information and additive enhancer effects are more prominent in Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
404. Intermolecular Concatamerization of Adeno-Associated Viral Genomes Is Not Dependent on Single Strand Interaction Yongping Yue,1 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO. Intermolecular concatamerization is the foundation for dual vector mediated larger gene transfer by recombinant adeno-associated virus (rAAV). However, the molecular processes underlying the heteroconcatamer formation remain to be defined. Theoretically, AAV intermolecular recombination could occur between single strand (ss) AAV genomes or between double strand (ds) AAV genomes. To distinguish these two possibilities, we have performed a sequential infection experiment with our previously described AAV LacZ transsplicing vectors. In this system, functional LacZ expression is dependent on the head-to-tail recombination between AV.LacZDonor and AV.LacZAcceptor. We administered AV.LacZAcceptor to the mouse anterior tibialis muscles at one month after AV.LacZDonor infection. At this time, a great majority of the ss AAV genomes from previous (AV.LacZDonor) infection were cleared out in muscle. Less than 4.38±1.81 % (BL6 mice) and 12.36±3.54 % (SCID mice) of the inoculated AAV virus remained as ss genomes at one month post-infection. If ss-ss genome interaction is absolutely required for AAV intermolecular concatamerization, LacZ expression from sequential infection will be less than ~5% (BL6) or ~15% (SCID) of that from co-infection. Quantitative analysis of LacZ expression showed that in BL6 muscle, expression from sequential infection reached 54.41±19.98%, 15.87±1.96% and 7.79±11.01% of that from co-infection at 1, 4 and 13 month postAV.LacZAcceptor infection. To exclude the interference of the host immune response in immune competent BL6 mice, similar experiment was performed in immune deficient SCID mice. Consistent with the finding in BL6 mice, LacZ expression from sequential infection reached 56.81±15.71% of that from co-infection at 1m time point. Importantly, the level of LacZ expression from sequential infection was also comparable to that from co-infection at 4- and 13-month time points. Our results strongly suggest that AAV heteroconcatamers are not derived from ss-ss genome interaction. Rather, ds-ds recombination is the predominant pathway for AAV intermolecular concatamer formation.
405. Genome-Wide Amplification for the Detection of Integrated rAAV-2 Vector DNA in Rabbit Tissues Bruce C. Schnepp,1 Michael C. Soult,1 Edward Kelly,2 Keith Munson,2 K. Reed Clark,1 Philip R. Johnson.1 1 Center for Gene Therapy, Columbus Children’s Research Institute, Columbus Children’s Hospital, Columbus, OH; 2 Targeted Genetics Corporation, Seattle, WA. Background. The molecular basis for long-term gene expression mediated by rAAV vectors remains undefined, and this uncertainty could hamper further development of these vectors in situations where vector integration is a safety concern. As part of a preclinical S160
development program for an rAAV-based HIV-1 vaccine, the biodistribution and vector integration status of a rAAV-2 vaccine candidate encoding the HIV-1 gag/protease proteins was assessed in New Zealand White rabbits. Methods. All rabbits received a single intramuscular injection of the rAAV vaccine in 3 dose groups: 3.3e10, 3.3e11, or 3.3e12 DRP. Each group enrolled 36 rabbits (18 male, 18 female). Animals were culled (6/sex/group) at days 5, 90 and 180 and multiple tissues analyzed for the presence of rAAV vector DNA. To determine the status of persisting vector DNA at days 90 and 180, we developed a genome-wide assay that was predicated on the amplification of genomic DNA between interspersed repetitive elements (Alu-like) in rabbits known as “C-repeats.” A single primer representing a consensus C-repeat sequence was used to generate amplicons. A second round of quantitative PCR detected C-repeat amplicons that contained integrated rAAV vector sequences. Significantly, episomal rAAV DNA species did not amplify in the Crpt-PCR assay. Thus, the assay was able to discriminate between integrated and episomal vector persistence. A cell pool containing randomly integrated rAAV vector DNA was used as a standard to define the limit of sensitivity of the assay in each tissue. Results. Vaccine DNA was widely distributed in multiple tissues at 90 and 180 days. Dissemination was dose-dependent and decreased over time. Importantly, gonads were negative at 90 and 180 days. Of 1666 tissue samples tested, 218 contained detectable vector DNA (sensitivity of ≥ 15 vector copies/microgram of cellular DNA). The majority of positive samples were at the site of injection (skin, muscle) or in highly perfused tissues. Of these 218 samples, 37 were determined to have a copy number sufficient to detect integration by the C repeat-PCR assay (≥ 1000 vector copies/microgram). A single sample (lymph node) scored positive at the limit of the assay sensitivity. In fact, this same sample scored negative when the assay was repeated. Most tissues could not be analyzed by Southern blot analysis due to low copy number. Blots from tissues with the highest copy numbers showed that most vector DNA was low molecular weight and single-stranded. Conclusions. Dissemination of vector DNA was dose and time dependent. By 180 days, vector DNA remained only at the injection site and in highly perfused tissues. These data suggest that rAAV vectors given by the intramuscular route demonstrate biologic properties remarkably similar to plasmid DNA.
406. Effect of DNA-PK on rAAV2 Persistence in Mouse Liver Sihong Song, Ge Zhao, Yuanqing Lu, Qiushi Tang, Terence R. Flotte. 1 Departments of Pharmaceutics and Pediatrics, University of Florida, Gainesville, FL, United States. Recombinant adeno-associated virus (rAAV) vectors have been increasingly used for gene therapy because they are relatively nontoxic, and the duration of transgene expression with these vectors is prolonged. As this vector is applied to human clinical trials, understanding the interactions between vector DNA and the cellular genome has become crucial for evaluating safety. We previously demonstrated that in skeletal muscle of SCID (DNA-PK negative) mice, rAAV2 DNA persisted in linear episomes, and gradually integrated into cellular genome, while in the muscle of C57BL/6 (DNA-PK positive) mice, it formed circular episomes. In the present studies, we have tested the effect of DNA-PK on the molecular fate of rAAV2 in liver. The same dose of rAAV2-CB-AT vector (expressing human alpha 1 antitrypsin) was injected via the portal vein into C57BL/6 and SCID mice. Transgene expression in C57BL/ 6 mice was significantly higher (2-fold) than in SCID mice. However, more AAV genomes (4-fold) were detected by real-time PCR in SCID mice than in C57Bl/6 mice. This may suggest concatemer information and additive enhancer effects are more prominent in Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy