Targeted CCR5 Gene Inactivation Using Peptide Nucleic Acids

使用肽核酸进行靶向 CCR5 基因失活

• 批准号: 7494358
• 负责人: Gerald Francis Vovis
• 金额: $ 10万
• 依托单位: HELIX THERAPEUTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7494358
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Acute; Alleles; Binding; CCR5 gene; CD34 gene; Cell Line; Cell Surface Receptors; Cell surface; Cells; Cellular Assay; Chemicals; Chemokine (C-C Motif) Receptor 5; Class; Clone Cells; Condition; DNA; DNA Repair; Development; Disease; Drug resistance; Effectiveness; Electroporation; Face; Frequencies; Gene Silencing; Gene Targeting; Genetic Recombination; Genome; Genotype; HIV; HIV Infections; HIV-1; Helix (Snails); Hematopoietic; Human; Immune; Immune system; Individual; Induced Mutation; Industry; Infection; K562 Cells; Lead; Life; Mammalian Cell; Methods; Modeling; Modification; Mutation; Numbers; Oligonucleotides; Patients; Peptide Nucleic Acids; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Population; Proliferating; Proteins; Public Health; Reagent; Receptor Gene; Reporting; Resistance; Resistance to infection; Sales; Series; Site; Stem cell transplant; T-Lymphocyte; Technology; Testing; Therapeutic; Toxic effect; Transfection; Transplantation; Work; base; chemokine receptor; ear helix; human disease; human stem cells; inhibitor/antagonist; mouse model; mutant; receptor; research study; stem; stem cell therapy; therapeutic gene; triple helix

DESCRIPTION (provided by applicant): The proposed work is to develop a therapeutic gene targeting agent for the treatment of HIV disease. Our approach is to use triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs), which bind to duplex DNA in a sequence-specific manner. Initial work has demonstrated that TFOs can stimulate recombination in mammalian cells by the ability of triple helices to provoke DNA repair and, thus, sensitize the target site to recombination. Using a series of chemical modifications, the intracellular effectiveness of TFOs has been progressively enhanced. Our company, Helix Therapeutics, Inc. (HTI), was formed to commercialize this technology for the treatment of human diseases, such as HIV/AIDS. Entry inhibitors have recently emerged as a new class of HIV therapeutics. These drugs block cell surface receptors required for HIV entry into T-cells, such as the protein encoded by the CCR5 gene. The CCR5 chemokine receptor is a major co-receptor for R5-tropic HIV-1 strains, which are responsible for most cases of initial, acute HIV infection [10]. Individuals, who possess a homozygous inactivating mutation in the CCR5 gene, are almost completely resistant to infection by R5-tropic HIV-1 strains, with no other significant adverse consequences [11]. Pharmaceutical companies are currently trying to develop entry- inhibitor drugs to block the receptor protein, although progress has been hindered by toxicity, efficacy and drug resistance. HTI is optimizing TFO-based technology as an alternative approach by targeting and inactivating the underlying gene for the receptor directly and, thereby, creating cells permanently resistant to infection by the HIV virus. These modified cells, unencumbered by HIV infection, would proliferate and, as a result, restore and maintain a patient's immune system, even in the face of HIV infection. We propose to develop our lead PNA compound to target and inactivate specifically the CCR5 gene in human cells. Experiments will be done to determine the optimal combination of PNA and donor DNA molecules, as well as the most appropriate cellular delivery methods required for maximally enhancing CCR5 gene targeting in several immune and hematopoietic model cell lines. In addition, we will test these conditions for achieving therapeutic gene modification in the intended target cells, human hematopoietic stem (CD34+) cells. Upon isolation of pure populations of cells, containing mutant CCR5 alleles, we will confirm the induced genotype(s) and phenotype(s) and will also test whether the mutant cells are resistant to HIV infection. In addition, we will test whether PNA-modified CD34+ cells engraft and properly differentiate in a mouse model of stem cell transplantation and whether the differentiated, CCR5-mutant immune cells are resistant to HIV infection. The proposed studies are critical for developing a therapeutic gene targeting agent for the treatment of HIV disease because: (1) they will establish a PNA/DNA reagent set and appropriate transfection parameters, which result in high efficiency mutation of the CCR5 gene in both model cell lines and our eventual target cells, CD34+ cells, and (2) they will serve to demonstrate that PNA-induced CCR5 gene inactivation can result in resistance to HIV infection. PUBLIC HEALTH RELEVANCE: Over 40 million people are currently living with AIDS. Helix Therapeutics, Inc. is proposing to create cells permanently resistant to infection by the HIV virus, using a therapeutic gene targeting agent. These modified cells, unencumbered by HIV infection, would proliferate, and, as a result, restore and maintain a patient's immune system, even in the face of HIV infection.

描述（由申请人提供）：拟开展的工作是开发用于治疗HIV疾病的治疗性基因靶向剂。我们的方法是使用三链体形成寡核苷酸（TFO）和肽核酸（PNA），它们以序列特异性的方式与双链体DNA结合。最初的工作已经证明，TFO可以通过三螺旋的能力刺激哺乳动物细胞中的重组，以引起DNA修复，从而使靶位点对重组敏感。通过一系列化学修饰，TFO的细胞内有效性已逐步增强。我们的公司，Hynthia Therapeutics，Inc. (HTI)该公司成立的目的是将这项技术商业化，用于治疗人类疾病，如艾滋病毒/艾滋病。进入抑制剂最近作为一类新的HIV治疗剂出现。这些药物阻断HIV进入T细胞所需的细胞表面受体，如CCR 5基因编码的蛋白质。CCR 5趋化因子受体是R5嗜性HIV-1毒株的主要共受体，其是大多数初始急性HIV感染病例的原因[10]。在CCR 5基因中具有纯合失活突变的个体几乎完全抵抗R5嗜性HIV-1毒株的感染，没有其他显著的不良后果[11]。制药公司目前正试图开发进入抑制剂药物来阻断受体蛋白，尽管进展受到毒性，疗效和耐药性的阻碍。HTI正在优化基于TFO的技术，作为一种替代方法，通过直接靶向和灭活受体的潜在基因，从而创造出永久抵抗HIV病毒感染的细胞。这些经过修饰的细胞不受艾滋病毒感染的影响，可以增殖，从而恢复和维持患者的免疫系统，即使面对艾滋病毒感染。我们建议开发我们的前导PNA化合物，以靶向并特异性地抑制人类细胞中的CCR 5基因。将进行实验以确定PNA和供体DNA分子的最佳组合，以及在几种免疫和造血模型细胞系中最大限度地增强CCR 5基因靶向所需的最合适的细胞递送方法。此外，我们将测试这些条件，以在预期的靶细胞（人造血干细胞（CD 34+）细胞）中实现治疗性基因修饰。在分离含有突变型CCR 5等位基因的纯细胞群体后，我们将确认诱导的基因型和表型，并将测试突变型细胞是否对HIV感染具有抗性。此外，我们将测试PNA修饰的CD 34+细胞是否在干细胞移植的小鼠模型中移植并正确分化，以及分化的CCR 5突变免疫细胞是否对HIV感染具有抗性。拟议的研究对于开发用于治疗HIV疾病的治疗性基因靶向剂至关重要，因为：（1）他们将建立PNA/DNA试剂组和适当的转染参数，这导致模型细胞系和我们最终的靶细胞CD 34+细胞中CCR 5基因的高效突变，和（2）它们将用于证明PNA诱导的CCR 5基因失活可导致对HIV感染的抗性。 公共卫生相关性：目前有4 000多万人感染艾滋病。Hefei Therapeutics，Inc.正在计划使用一种治疗性的基因靶向剂来制造永久抵抗HIV病毒感染的细胞。这些经过修饰的细胞不受HIV感染的影响，可以增殖，因此，即使面对HIV感染，也可以恢复和维持患者的免疫系统。