TARGETING OF AAV REP MEDIATED SPECIFIC DNA INTEGRATION

靶向 AAV REP 介导的特异性 DNA 整合

• 批准号: 6177447
• 负责人: RALPH MICHAEL LINDEN
• 金额: $ 16.76万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177447
• 关键词: DNA binding protein; adeno associated virus group; gene targeting; nucleic acid sequence; protein binding; recombinant proteins; tissue /cell culture

The primary focus of this project will be the targeting of DNA integration as mediated by the Rep proteins of adeno-associated virus (AAV). As shown previously, AAV integrates its genome site-specifically into human chromosome 19. Using an episome-based assay, we were able to demonstrate that a 33 nucleotide signal, present within the target sequence, is necessary and sufficient to mediate this highly specific integration/recombination event. The DNA targeting signal contains two sequence motifs which are also present in the viral origin of replication and which are bound by the viral Rep proteins. The hypothesis is that the viral Rep proteins determine site-specific integration through recognition and binding of these origin signals present on chromosome 19. This project will test the feasibility of retargeting Rep-mediated site-specific DNA integration by means of exchanging the DNA binding domain of the AAV Rep proteins. In order to establish a model system, we first propose to exchange the DNA binding domain of AAV Rep78 with its counterpart from goose parvovirus (GPV). Interestingly, the similarity between the two proteins exceeds 90 percent within the active domain of Rep. The DNA-binding domains share much lower similarity consistent with the observation that GPV Rep1 recognizes a different DNA motif. Following biochemical characterization of the hybrid protein a recombinant AAV will be generated containing a rep gene that encodes the hybrid protein. This virus will be used in an episome based integration assay to test for altered site-specific DNA integration into the sequence motif that is recognized by the GPV Rep1 protein. The second goal will be to redirect integration to sites within the human genome other than AAVS1. A genetic screen will be used to identify mutants of the Rep protein capable of binding defined cellular target sequences with high affinity. Once these mutant proteins are purified and biochemically characterized, the episomal integration assay will be used to test for integration into the new target sequences. The experiments proposed are designed to provide further insights into the mechanism of targeted DNA integration with regard to functions of AAV Rep as well as to provide us with a tool to target selected sites within the human genome.

这个项目的主要焦点将是DNA的靶向

项目成果

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system.

在重构系统中，单纯疱疹病毒 1 型复制复合物依赖 Rep 启动腺相关病毒 2 型 DNA 复制。

• DOI: 10.1128/jvi.75.21.10250-10258.2001
• 发表时间: 2001
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Ward,P;Falkenberg,M;Elias,P;Weitzman,M;Linden,RM
• 通讯作者: Linden,RM

A role for single-stranded templates in cell-free adeno-associated virus DNA replication.

单链模板在无细胞腺相关病毒 DNA 复制中的作用。

• DOI: 10.1128/jvi.74.2.744-754.2000
• 发表时间: 2000
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Ward,P;Linden,RM
• 通讯作者: Linden,RM

Adeno-associated virus site-specifically integrates into a muscle-specific DNA region.

腺相关病毒位点特异性地整合到肌肉特异性 DNA 区域中。

• DOI: 10.1073/pnas.080079397
• 发表时间: 2000
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Dutheil,N;Shi,F;Dupressoir,T;Linden,RM
• 通讯作者: Linden,RM