Episomal lenti vector for a humanized hemophilia mouse

描述（由申请人提供）：可以通过使用慢病毒载体治疗性解决的遗传疾病谱受到其致癌潜力的限制，这是其整合性质所固有的。在这项研究中，我们建议开发和利用新的非整合慢病毒载体，以解决上述障碍。我们的方法是基于我们实验室最近的发现，证明HIV-1 Rev/RRE系统能够作为二级包装系统发挥作用，介导非HIV-1 mRNA包装成HIV-1颗粒。我们表明，HIV-1 RRE含有EIAV载体有效地包装成HIV-1颗粒。新的嵌合载体缺乏整合到宿主细胞基因组中的能力，其表现出的转基因表达水平显著高于其他非整合载体，例如包装有HIV-1整合酶突变体E152 A的HIV-1载体。在此，我们建议：i）表征涉及嵌合EIAV/HIV-1载体的Rev/RRE依赖性包装的机制; ii）表征HIV-1加工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嵌合体载体的转基因表达的影响; iv）表征嵌合体EIAV/HIV-1载体的生物分布;和v）确定嵌合体载体在人源化血友病小鼠模型中递送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开发有效的非整合慢病毒载体，其将更适合于人基因治疗。

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.