Targeted Inactivation of the CCR5 HIV Coreceptor with Peptide Nucleic Acids

描述（由申请人提供）：目前有超过3400万人感染艾滋病毒/艾滋病。联合抗病毒治疗显著降低了该病的发病率和死亡率;然而，目前尚无治愈该病的方法。发现CCR 5的一种天然变体，即delta 32突变，使细胞对HIV感染具有抵抗力，这使得几个学术实验室和生物技术公司努力寻找模拟delta 32突变的基因治疗方法。该项目将证明使用肽核酸（PNAs）作为治疗剂的可行性，以遗传方式截短CD 34+干细胞中的CCR 5蛋白并阻断HIV细胞进入。修饰CCR 5基因的初步实验使用具有供体DNA序列的靶向PNA来诱导靠近delta 32突变的终止密码子。在这个I期项目中，我们将开发一种新的策略，用于PNA诱导的CCR 5基因修饰，以将CD 34+突变频率提高到临床有用的水平。具体目标1实验将测试PNA靶向策略的可行性，其中我们将使DNA链断裂分子直接缀合至PNA以诱导CCR 5基因中的位点特异性断裂。通过易错的非同源末端连接途径对这些断裂进行诱变修复将导致移码突变和截短的CCR 5蛋白。这种修复机制预计会导致CCR 5移码突变的显著增加。在特定目标2实验中，我们将比较用于修饰人CD 34+干细胞中CCR 5基因的临床相关PNA递送方法。一个成功的I期项目将确定适合II期项目的PNA缀合物和递送方法，该项目将使用我们的新策略在HIV感染的人源化小鼠模型中建立HIV抗性T细胞。我们的长期目标是开发一种高度特异性的临床PNA缀合物CCR 5基因修饰方案，该方案将在HIV患者中产生HIV抗性免疫细胞，并减少他们对长期抗逆转录病毒治疗的需求。

DESCRIPTION (provided by applicant): Over 34 million people are currently living with HIV/AIDS. Combination antiviral therapies have significantly reduced the morbidity and mortality of the disease; however, there is currently no cure for the disease. Discovery that a naturally occurring variant of CCR5, the delta32 mutation, renders cells resistant to HIV infection has led to efforts by several academic laboratories and biotech companies to find gene therapy approaches to mimic the delta32 mutation. This project will demonstrate the feasibility of using peptide nucleic acids (PNAs) as a therapeutic to genetically truncate the CCR5 protein in CD34+ stem cells and block HIV cell entry. Preliminary experiments that modified the CCR5 gene used a targeting PNA with a donor DNA sequence to induce a stop codon near the delta32 mutation. In this Phase I project, we will develop a novel strategy for PNA-induced CCR5 gene modification to increase the CD34+ mutation frequency to clinically useful levels. Specific Aim 1 experiments will test the feasibility of a PNA targeting strategy where we will conjugate DNA strand breaking molecules directly to the PNA to induce site specific breaks in the CCR5 gene. Mutagenic repair of these breaks via the error-prone non-homologous end joining pathway will lead to frameshift mutations and a truncated CCR5 protein. This repair mechanism is expected to generate a significant increase in CCR5 frameshift mutations. In Specific Aim 2 experiments, we will compare clinically relevant PNA delivery methods for modification of the CCR5 gene in human CD34+ stem cells. A successful Phase I project will identify PNA conjugates and delivery methods suitable for a Phase II project that will use our novel strategy to establish HIV-resistant T cells in the humanized mouse model of HIV infection. Our long- term goal is to develop a highly specific clinical PNA conjugate CCR5 gene modification protocol that will create HIV-resistant immune cells in HIV patients and reduce their need for long-term antiretroviral therapy.