CORE--DEVELOPMENT OF RIBOZYME GENE THERAPY

艾滋病，作为逆转录病毒感染的结果，代表了一种获得性基因 疾病。基因治疗在艾滋病毒/艾滋病中的应用具有重要的理论意义 我保证。为了实现这一承诺，加快这一基本转型 从实验室到临床的发现迫在眉睫 这是必要的，基本战略本身必须完善。因此， 该SPIRAT的9001项目有两个目标：支持第一阶段 HIV-1前导序列核酶的试验及核酶基因的改良 治疗策略。我们已经证明针对5‘-的发夹核酶- HIV-1的前导序列可抑制不同毒株的复制 瞬时转染法中的HIV-1。稳定的T细胞可长期存活 对HIV-1感染的抵抗力，包括临床分离株。近期 核酶基因治疗外周血淋巴细胞的疗效论证 临床前研究导致了一项I期试验的设计，该试验现在是RAC 批准了。为了达到9001项目的目标，我们将：(1)提供GMP 定向到此试验的项目2，并对 产生细胞和病毒上清液；(2)评估持久性和 核酶基因在患者细胞中的表达及辅助治疗 项目2中第一阶段试验的后续研究，包括DNA和RNA 回收的外周血淋巴细胞中核酶和对照载体的定量 评估细胞间隔区靶向策略对 提高核酶效率。这一目标将通过克隆来实现 HIV-1 Rev反应元件(RRE)进入核酶转录 用于制造“融合RNA”的盒式磁带(不仅用作RRE诱饵，而且 更重要的是，将核酶定位于相同的细胞隔间 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包装信号也将与核酶共同定位 HIV靶点；(4)研究前导序列之间的相互作用 和其他核酶或其他抗艾滋病毒基因产品，包括RevM10 跨显性突变蛋白(与Gary Nabel博士合作)和 干扰素诱导细胞基因RBP9-27(与Dr。 乔治·帕夫拉基斯)。

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).