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，目前还没有治愈这种疾病的方法。发现CCR5的一种自然变异，即delta32突变，可以使细胞抵抗HIV感染，这促使一些学术实验室和生物技术公司努力寻找模拟delta32突变的基因治疗方法。该项目将证明使用肽核酸（PNAs）作为一种治疗方法在CD34+干细胞中基因截断CCR5蛋白并阻断HIV细胞进入的可行性。修改CCR5基因的初步实验使用带有供体DNA序列的靶向PNA在delta32突变附近诱导一个停止密码子。在这个I期项目中，我们将开发一种新的pna诱导CCR5基因修饰策略，以将CD34+突变频率提高到临床有用的水平。特异性Aim 1实验将测试PNA靶向策略的可行性，我们将把DNA链断裂分子直接结合到PNA上，以诱导CCR5基因的位点特异性断裂。通过易出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断CCR5蛋白。这种修复机制有望显著增加CCR5移码突变。在Specific Aim 2实验中，我们将比较临床相关的PNA递送方法在人类CD34+干细胞中修饰CCR5基因。一个成功的一期项目将确定适合二期项目的PNA偶联物和递送方法，二期项目将使用我们的新策略在人源化小鼠HIV感染模型中建立抗HIV T细胞。我们的长期目标是开发一种高度特异性的临床PNA偶联CCR5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.