Targeted Inactivation of the CCR5 HIV Coreceptor with Peptide Nucleic Acids

用肽核酸靶向灭活 CCR5 HIV 辅助受体

• 批准号: 8467486
• 负责人: Elizabeth Peterson-Roth
• 金额: $ 25.03万
• 依托单位: L2 DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8467486
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Acute; Aftercare; Antiviral Therapy; Area; Autologous; Back; Bleomycin; Blood; CCR5 gene; CD34 gene; Camptothecin; Cell Count; Cell Surface Receptors; Cell surface; Cells; Chemokine (C-C Motif) Receptor 5; Clinical; Communication; DNA; DNA Damage; DNA Double Strand Break; DNA Sequence; DNA strand break; Digestion; Disease; Double Strand Break Repair; Frameshift Mutation; Frequencies; Gene Targeting; Gene-Modified; Genes; Genetic Recombination; Goals; HIV; HIV Infections; HIV-1; Hematopoietic Stem Cell Transplantation; Human; Immune; Individual; Induced Mutation; Industry; Infection; Laboratories; Lead; Length; Life; Location; Methods; Modification; Morbidity - disease rate; Mus; Mutation; Nonhomologous DNA End Joining; Null Lymphocytes; Oligonucleotides; Pathway interactions; Patients; Peptide Nucleic Acids; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phase; Procedures; Proteins; Protocols documentation; Publications; Resistance; Sales; Site; Site-Directed Mutagenesis; Stem cells; T-Lymphocyte; Terminator Codon; Testing; Therapeutic; Variant; Viral Load result; Zinc Fingers; adverse outcome; antiretroviral therapy; chemokine receptor; clinically relevant; deep sequencing; design; gene correction; gene therapy; improved; in vivo; inhibitor/antagonist; interest; mortality; mouse model; nanoparticle; novel; novel strategies; nuclease; public health relevance; receptor; repaired; research study; therapeutic gene

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.

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。联合抗病毒疗法显着降低了该病的发病率和死亡率；然而，目前尚无治愈该疾病的方法。发现 CCR5 的天然变异体 delta32 突变可使细胞对 HIV 感染具有抵抗力，这促使一些学术实验室和生物技术公司努力寻找模仿 delta32 突变的基因治疗方法。该项目将证明使用肽核酸 (PNA) 作为治疗剂来基因截断 CD34+ 干细胞中的 CCR5 蛋白并阻止 HIV 细胞进入的可行性。修饰 CCR5 基因的初步实验使用带有供体 DNA 序列的靶向 PNA，在 delta32 突变附近诱导终止密码子。在这个第一阶段项目中，我们将开发一种 PNA 诱导的 CCR5 基因修饰的新策略，以将 CD34+ 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 DNA 链断裂分子直接与 PNA 结合，以诱导 CCR5 基因中的位点特异性断裂。通过容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短的 CCR5 蛋白。这种修复机制预计会导致 CCR5 移码突变显着增加。在特定目标 2 实验中，我们将比较用于修饰人类 CD34+ 干细胞中 CCR5 基因的临床相关 PNA 递送方法。成功的第一阶段项目将确定适合第二阶段项目的 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 结合物 CCR5 基因修饰方案，该方案将在 HIV 患者中产生抗 HIV 免疫细胞，并减少他们对长期抗逆转录病毒治疗的需求。