SUSTAINED/TARGETED TRANSGENE EXPRESSION--DEVELOPMENT OF NEW VIRAL VECTOR SYSTEMS

尽管最近在基因领域取得了许多技术进步 转会方面，一些技术问题继续阻碍着转会的成功 实施以基因转移为基础的疾病临床治疗 影响心脏、血液和肺部。因此，它正在成为 日益重要的是考虑开发新的治疗方法 基因传递，克服了现有系统的局限性。这个 这项研究计划的总体目标是开发新的逆转录病毒 能够有效地将基因直接传递给 体内心血管系统中增殖和静止的细胞， 并为转移基因的持续表达提供了条件。 推动这项研究的主要假设是，一种逆转录病毒 静止时耦合积分能力的矢量系统 能够产生极高滴度病毒库的细胞 将在体外和体内基因治疗中有广泛的应用 旨在治疗心脏、血液和血液疾病的战略 肺部。在第一系列研究中，我们建议生成安全的、 可靠、稳定的HIV来源包装细胞株适用于 大量生产高滴度无辅助慢病毒- 可以高度集中的派生向量。要最小化 涉及同源包装的重组事件的机会 和载体序列，这些序列可能导致出现 复制能力强的病毒或特定病毒基因的传播 产品，我们将尝试精确地工程艾滋病毒的表达 通过使用特定的非逆转录病毒的Gag-Poll编码序列 表达式元素。与稳定包装的产生并行 细胞系，我们建议开发混合HLV/MLV载体 可被HIV有效包装的MFG载体的设计 衍生成分和高效表达插入基因。一秒钟内 系列研究中，我们将考虑以下几种策略 主要是基于MLV的包装细胞系，这将有可能 支持生产能够转导的鼠基载体 增殖和静止的细胞。不同载体的能力 高效转导增殖和静止细胞的系统 心血管系统将在体外和体内进行评估， 由向量评估和模型系统核心进行的研究。

Despite many recent technological advances in the field of gene transfer, a number of technical issues continue to thwart the successful implementation of gene transfer-based clinical therapies for diseases affecting the heart, blood, and lungs. Accordingly, it is becoming increasingly important to consider the development of new methods for gene delivery which overcome the limitations of existing systems. The overall goal of this research program is to develop new retroviral vectors which are able to efficiently deliver genes directly to both proliferating and quiescent cells of the cardiovascular system in vivo, and which provide for the sustained expression of the transferred genes. The major hypothesis driving the proposed studies is that a retroviral vector system which couples the capacity for integration in quiescent cells with the ability to generate extremely high titer virus stocks will have broad application in both ex vivo and in vivo gene therapy strategies aimed at the treatment of diseases of the heart, blood, and lungs. In a first series of studies, we propose to generate safe, reliable, stable HIV-derived packaging cell lines useful for the production of large quantities of high titer helper-free lentivirus- derived vectors which can be highly concentrated. To minimize the opportunity for recombinational events involving homologous packaging and vector sequences that could lead to the emergence of either replication competent virus or the transmission of specific viral gene products, we will attempt to precisely engineer the expression of HIV gag-pol coding sequences through the use of specific non-retroviral expression elements. In parallel with the generation of stable packaging cell lines, we propose to develop hybrid HlV/MLV vectors modeled after the design of the MFG vector which can be efficiently packaged by HIV- derived components and efficiently express inserted genes. In a second series of studies, we will consider several strategies for generating predominantly MLV-based packaging cell lines which will potentially support the production of murine-based vectors able to transduce proliferating and quiescent cells. The ability of the different vectors systems to efficiently transduced proliferating and quiescent cells of the cardiovascular system will be assessed both in vitro and in vivo in studies conducted by the Vector Evaluation and Model Systems Core.