Episomal lenti vector for a humanized hemophilia mouse

描述（由申请人提供）：使用慢病毒载体进行治疗的遗传疾病的范围受到其致癌潜力的限制，这是其固有的整合性质。在这项研究中，我们建议开发和利用新的非积分慢病毒载体来解决上述障碍。我们的方法是基于我们实验室最近的发现，证明了HIV-1 Rev/RRE系统作为二次包装系统的能力，该系统介导非HIV-1 mrna包装成HIV-1颗粒。我们发现含有EIAV载体的HIV-1 RRE有效地包装到HIV-1颗粒中。这种新型嵌合载体缺乏整合宿主细胞基因组的能力，其转基因表达水平明显高于其他非整合载体，如包装有HIV-1整合酶突变体E152A的HIV-1载体。在此，我们提出：i)表征EIAV/HIV-1嵌合载体Rev/RRE依赖性包装的机制；ii)描述HIV-1处理EIAV att位点的能力及其对嵌合体载体整合的影响；iii)研究组蛋白修饰和染色质结构对EIAV/HIV-1嵌合体载体转基因表达的影响；iv)描述嵌合体EIAV/HIV-1载体的生物分布特征；v)确定嵌合体载体在人源化血友病小鼠模型中传递和维持高水平人因子IX表达的功效。我们相信，所提出的研究将导致有效的非整合慢病毒载体的发展，这将更适合于人类基因治疗。

DESCRIPTION (provided by applicant): The spectrum of genetic diseases that can be therapeutically addressed by using lentiviral vectors is restricted by their oncogenic potential, which is inherent to their integrative nature. In this study we propose to develop and utilize novel non-integrating lentiviral vectors to address the above obstacle. Our approach is based on recent findings in our laboratory demonstrating the ability of the HIV-1 Rev/RRE system to function as a secondary packaging system, which mediates packaging of non-HIV-1 mRNAs into HIV-1 particles. We showed that HIV-1 RRE containing EIAV vectors efficiently packaged into HIV-1 particles. The novel chimeric vectors, which lacked the ability to integrate into a host cells' genome, exhibited transgene expression levels significantly higher than other non-integrating vectors, such as HIV-1 vectors packaged with the HIV-1 integrase mutant E152A. Here we propose to: i) characterize the mechanism involved in the Rev/RRE dependent packaging of chimeric EIAV/HIV-1 vectors; ii) characterize the ability of the HIV-1 to process the EIAV att sites and its effects on chimera vector integration; iii) investigate the effects of histone modifications and chromatin structure on transgene expression from EIAV/HIV-1 chimera vectors; iv) to characterize the biodistribution of the chimera EIAV/HIV-1 vectors; and v) to determine the efficacy of the chimera vectors at delivering and maintaining high levels of human factor IX expression in a humanized hemophilia mouse model We believe that the proposed studies will result in the development of efficacious non-integrating lentiviral vectors, which will be better suited for human gene therapy.