STABLE EXPRESSION OF THERAPEUTIC RNA IN BLOOD CELLS

HIV-1 感染的基因治疗是一种选择，并且可能是传统药物治疗的辅助手段。我们开发了一种基于 RNA 的治疗策略，该策略利用针对 HIV-1 的 tat 和 rev 基因的锤头核酶。编码核酶的基因已作为源自基于 LN 的逆转录病毒载体的 LTR 的多顺反子转录物的一部分表达。在临床前试验中，用基于核酶的载体转导的原代造血祖细胞产生了对HIV-1攻击具有抵抗力的单核细胞。 这种载体-核酶组合目前正用于感染自体核酶和载体转导的CD34细胞的HIV-1感染个体的I期临床试验。迄今为止获得的结果表明核酶和载体构建体的标记有限。这些早期试验没有监测疗效。在拟议的临床试验中，功效以及核酶的长期表达将是一个重要问题。在这个项目中，我们将开发改进的系统，用于从改良的双嗜性逆转录病毒载体 MND neo 的主链表达核酶和 Rev 结合适体。将检查几种不同的Pol II和Pol III启动子以及核酶和Rev结合适体的组合的表达水平和持续时间以及抗病毒活性。新的核酶靶标将使用一种简单的方法来识别，该方法对细胞提取物中的天然 mRNA 进行核酶位点选择。这些新的核酶试验：启动子、核酶和适体的组合将在长期骨髓培养物和 SCID-hu 小鼠中进行测试。最有前途的表达盒将被引入项目 3。MND 载体中表现出功效和长期表达的构建体将在项目 1 中进行测试，并最终用于项目 4 的人体临床试验。该项目的长期目标是开发适用于基因治疗环境中 HIV-1 治疗的治疗性 RNA 表达系统。

Gene therapy for HIV-1 infection is an option and perhaps an adjuvant to conventional drug therapy. We have developed an RNA based therapeutic strategy that utilizes hammerhead ribozymes targeted to the tat and rev genes of HIV-1. The genes encoding the ribozymes have been expressed as part of a polycistronic transcript originating form the LTR of an LN based retroviral vector. In pre-clinical trials primary hematopoietic progenitor cells transduced with the ribozyme based vectors gave rise to monocytic cells which are resistant to HIV-1 challenge. This vector- ribozyme combination is currently being utilized in a phase I clinical trial of HIV-1 infected individuals infected with autologous ribozyme and vector transduced CD34 cells. The results obtained thus far have demonstrated limited marking with the ribozyme and vector constructs. Those early trials did not monitor efficacy. In the proposed clinical trials, efficacy, and hence long term expression of the ribozymes will be an important issue. In this project we will be developing improved systems for expressing ribozyme and Rev binding aptamers from the backbone of a modified amphotropic retroviral vector, MND neo. Several different Pol II and Pol III promoters, and combinations of ribozymes and Rev binding aptamers will be examined for levels and duration of expression and anti- viral activity. New ribozyme targets will be identified using a facile method for ribozyme site selection on native mRNAs in cell extracts. These new ribozymes trials: Combinations of promoter, ribozyme, and aptamers will be tested in long term bone marrow cultures and a SCID-hu mouse. The most promising cassettes will be channeled into project 3. Constructs in the MND vector which show efficacy and long term expression will be tested in project 1 and ultimately utilized in the human clinical trial of project 4. The long range objectives of this project are the development of expression systems for therapeutic RNAs which are applicable to treatment of HIV-1 in a gene therapy setting.