CORE--DEVELOPMENT OF RIBOZYME GENE THERAPY

核心--核酶基因治疗的发展

• 批准号: 3727759
• 负责人: MANG YU
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3727759
• 关键词: AIDS therapy; adeno associated virus group; biomedical facility; cooperative study; gene expression; gene mutation; gene therapy; human immunodeficiency virus; human tissue; molecular cloning; polymerase chain reaction; ribozymes; tissue /cell culture; transfection /expression vector

AIDS, as a result of retroviral infection, represents an acquired genetic disease. Applications of gene therapy to HIV/AIDS hold great theoretic promise. To realize this promise, the expedited transition of this basic discovery from the laboratory bench to clinical application is urgently needed, and the basic strategies themselves must be refined. Hence, the goal of Project 9001 of this SPIRAT is two-fold: supporting a phase 1 trial of an HIV-1 leader sequence ribozyme and improving the ribozyme gene therapy strategy. We have shown that a hairpin ribozyme targeting the 5'- leader sequence of HIV-1 can inhibit the replication of diverse strains of HIV-1 in transient transfection. Stable T-cells conferred long-term resistance to infection of HIV-1 including clinical isolates. Recent demonstrations of the efficacy of ribozyme gene therapy in PBLs for the preclinical studies led to design of a phase I trial that is now RAC approved. To reach the goals of Project 9001, we will: (1) provide GMP vectors to Project 2 for this trial, and perform safety tests on the producer cells and viral supernatant; (2) evaluate the persistence and expression of the ribozyme gene in cells from patients and assist other follow-up studies for phase 1 trial in Project 2, including DNA and RNA PCR to quantitate the ribozyme and control vector in recovered PBLs; (3) evaluate the effects of cellular compartmental targeting strategies to improve ribozyme efficiency. This objective will be approached by cloning the HIV-1 Rev Responsive Elements (RRE) into the ribozyme transcription cassette to create a "fusion RNA" (serving not only as an RRE decoy but more importantly, targeting the ribozyme to the same cellular compartments as the HIV targets), and by cloning the ribozyme into HIV-2-based vectors so that the HIV packaging signal will also co-localize the ribozyme with HIV targets; (4) investigate the interactions between the leader sequence and other ribozymes or other anti-HIV gene products, including the RevM10 transdominant mutant protein (in collaboration with Dr. Gary Nabel) and the interferon inducible cellular gene RBP9-27 (in collaboration with Dr. George Pavlakis).

由于逆转录病毒感染，艾滋病代表获得的遗传 疾病。 基因疗法对艾滋病毒/艾滋病的应用具有良好的理论 承诺。 为了实现这一诺言，这个基本的加快过渡 从实验室长凳到临床应用的发现是迫切的 需要，必须完善基本策略本身。 因此， 该螺旋螺旋的项目9001的目标是两个方面：支持一阶段1 HIV-1领导者序列核酶的试验并改善了核酶基因 治疗策略。 我们已经表明，针对5'-的发夹核酶 HIV-1的领导者序列可以抑制复制的复制 瞬时转染中的HIV-1。 稳定的T细胞赋予了长期 对HIV-1感染的抗性，包括临床分离株。 最近的 核酶基因治疗在PBLS中的功效证明了 临床前研究导致了I期试验的设计，该试验现在是RAC 得到正式认可的。 要达到项目9001的目标，我们将：（1）提供GMP 该试验项目2的向量，并对 生产者细胞和病毒上清液； （2）评估持久性和 核酶基因在患者细胞中的表达并协助其他 项目2中1期试验的后续研究，包括DNA和RNA PCR定量核酶和对照载体在回收的PBL中； （3） 评估细胞隔室靶向策略的影响 提高核酶效率。 克隆将实现此目标 HIV-1 REV响应元素（RRE）进入核酶转录 盒式制作“融合RNA”（不仅用作RRE诱饵，而且使用 更重要的是，将核酶靶向相同的细胞室 作为HIV靶标），并通过将核酶克隆到基于HIV-2的载体中 因此，HIV包装信号还将与核酶共定位 艾滋病毒靶； （4）研究领导者序列之间的相互作用 以及其他核酶或其他抗HIV基因产品，包括Revm10 跨性突变蛋白（与Gary Nabel博士合作）和 干扰素诱导的细胞基因RBP9-27（与博士合作 乔治·帕夫拉基斯（George Pavlakis））。