Efficacy and Safety of AAV Gene Transfer for Hemophilia

描述（由申请人提供） 此职业发展奖申请概述了详细的计划，指导 Dr. Valder R.阿鲁达该研究计划详细介绍了相关实验 用于血友病B的AAV介导的基因转移。在以前的工作中，我们展示了 长期表达的生物活性因子IX在小以及大 肌内注射AAV后血友病B的动物模型 表达人凝血因子IX的载体。这些数据作为 重度血友病B受试者的临床试验基础 通过肌内注射AAV血清型2衍生的载体处理 表达人因子IX。没有证据表明系统或局部 在治疗的受试者中的毒性，但尚未达到 剂量组群中的所有受试者都达到治疗水平的基因表达。 本申请中提出的工作的总体目的是评估 几个假设，可能导致更安全和更有效的基因转移。在 第一个目标，我将测试AAV的替代血清型的假设， 特别是AAV-1和AAV-6，可以指导更高水平的转基因表达 在血友病道夫和人类肌肉细胞中。根据初步数据，我们 已经在小鼠中显示，这些替代血清型可以直接转基因 以比AAV-2的等效剂量高10-20倍的水平表达 导出向量然而，重要的是要确定这种影响是否 仅限于鼠细胞或更广泛地观察到。如果更高水平的 在人肌管中观察到转基因表达，我们将能够使用 这种细胞培养系统，以确定机制（S）负责 更高的表达。在第二个目标中，我们建议使用一种新的载体递送技术来解决困扰肌肉定向基因转移的问题， 即如何实现大量细胞的转导， 肌肉注射。我们假设我们可以实现更多 通过使用一种技术， 该载体沿着血管舒张剂被注入动脉循环 剂.该技术可以允许更有效地递送载体，因此 使肌肉定向基因转移更加可行。最后，我们建议研究 AAV转导骨骼肌的分子和细胞结果 载体使用微阵列技术。这一目标解决了一个最 基因转移中的安全难题，即载体的长期后果 转导这些实验的数据将使我们能够计划更多的 在相关动物模型中进行的安全性研究，以评估 用AAV载体处理。此应用程序的其他组件概述了正式的 课程作业、参加研讨会、负责任地开展研究的培训， 并与一个正式的委员会进行磋商，以提高申请人的能力？S 职业发展。

DESCRIPTION (provided by applicant) This career development award application outlines detailed plans for mentored training of Dr. Valder R. Arruda. The research plan details experiments related to AAV-mediated gene transfer for hemophilia B. In previous work we showed long-term expression of biologically active Factor IX in small as well as large animal models of hemophilia B following intramuscular injection of an AAV vector expressing human blood coagulation Factor IX. These data served as the basis for a clinical trial in which subjects with severe hemophilia B were treated by intramuscular injection with an AAV serotype 2-derived vector expressing human Factor IX. There has been no evidence of systemic or local toxicity in the treated subjects, but a dose has not yet been reached at which all subjects in a dose cohort achieve therapeutic levels of gene expression. The overall foal of the work proposed in this application is to evaluate several hypotheses that may lead to safer and more effective gene transfer. In the first aim, I will test the hypothesis that alternate serotypes of AAV, specifically AAV-1 and AAV-6, can direct higher levels of transgene expression in hemophilic doffs and human muscle cells. As shown in preliminary data, we have already shown in mice that these alternate serotypes can direct transgene expression at levels 10-20 fold higher than equivalent doses of an AAV-2 derived vector. However it is important to determine whether this effect is confined only to murine cells or is more widely observed. If higher levels of transgene expression are observed in human myotubes, we will be able to use this cell culture system to determine the mechanism(s) responsible for the higher expression. In the second aim, we propose to use a novel vector delivery technique to address a problem that plagues muscle-directed gene transfer, namely how to achieve transduction of large numbers of cells without Innumerable intramuscular injections. We hypothesize that we can achieve more extensive delivery to skeletal muscle tissue through the use of a technique in which vector is infused into the arterial circulation along with vasodilatory agents. This technique may allow more efficient delivery of vector and thus render muscle-directed gene transfer more feasible. Finally we propose to study molecular and cellular consequences of transduction of skeletal muscle by AAV vectors using microarray technology. This aim addresses one of the most difficult safety issues in gene transfer, i.e. long-term consequences of vector transduction. The data from these experiments will allow us to plan additional safety studies in relevant animal models to assess the long-term safety of treatment with AAV vectors. Other components of this application outline formal coursework, seminar participation, training in responsible conduct of research, and consultation with a formal committee that will enhance the applicant?s career development.