STABLE EXPRESSION OF THERAPEUTIC RNA IN BLOOD CELLS

基因治疗HIV-1感染是一种选择，也许是传统药物治疗的辅助手段。我们已经开发了一种基于RNA的治疗策略，利用锤头核酶靶向HIV-1的tat和rev基因。编码核酶的基因已被表达为起源于LN基逆转录病毒载体LTR的多顺反子转录物的一部分。在临床前试验中，以核酶为基础的载体转导的原发性造血祖细胞产生了单核细胞，能够抵抗HIV-1的攻击。这种载体-核酶组合目前正在HIV-1感染个体的I期临床试验中使用自体核酶和载体转导的CD34细胞。迄今为止获得的结果表明，核酶和载体结构的标记有限。那些早期的试验并没有监测疗效。在拟议的临床试验中，核酶的疗效和长期表达将是一个重要的问题。在这个项目中，我们将开发一种改进的系统来表达核酶和Rev结合适配体，这些适配体来自一种改良的两性逆转录病毒载体MND neo。几种不同的Pol II和Pol III启动子，以及核酶和Rev结合适体的组合将检查表达和抗病毒活性的水平和持续时间。新的核酶靶标将使用一种简单的方法在细胞提取物的天然mrna上进行核酶位点选择。这些新的核酶试验：启动子、核酶和适配体的组合将在长期骨髓培养和SCID-hu小鼠中进行测试。最有前途的盒式磁带将用于项目3。在MND载体中显示出疗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治疗性rna的表达系统，该系统适用于在基因治疗环境中治疗HIV-1。

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.