STABLE EXPRESSION OF THERAPEUTIC RNA IN BLOOD CELLS

针对HIV-1感染的基因治疗是传统药物治疗的一种选择，也可能是一种辅助治疗。我们开发了一种基于RNA的治疗策略，利用针对HIV-1的TAT和REV基因的锤头状核酶。编码核酶的基因已经作为多顺反子转录本的一部分表达，该转录本源于基于LN的逆转录病毒载体的LTR。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-核酶组合目前正被用于感染自体核酶和载体转导的CD34细胞的HIV-1感染者的I期临床试验。到目前为止所获得的结果表明，用核酶和载体构建的标记有限。这些早期试验没有监测疗效。在拟议的临床试验中，核酶的有效性和长期表达将是一个重要的问题。在这个项目中，我们将开发更好的系统来表达核酶和REV结合适体，这些适体来自修改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结合适配子的组合将被检测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便的方法在细胞提取液中的天然mRNAs上进行核酶位点选择。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和SCID-HU小鼠中进行测试。最有希望的盒式磁带将被引入项目3。MND载体中显示出有效性和长期表达的构建物将在项目1中进行测试，并最终用于项目4的人类临床试验。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.