Targeted CCR5 Gene Inactivation Using Peptide Nucleic Acids

描述(申请人提供)：建议的工作是开发一种用于治疗HIV疾病的治疗性基因靶向试剂。我们的方法是使用三链形成寡核苷酸(TFOS)和肽核酸(PNA)，它们以序列特异性的方式与双链DNA结合。最初的工作表明，TFOS可以通过三螺旋刺激DNA修复，从而使靶部位对重组敏感，从而刺激哺乳动物细胞中的重组。通过一系列的化学修饰，TFOS的细胞内效应已逐步增强。我们的公司Helix Treateutics，Inc.(HTI)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。进入抑制药最近已成为一类新的艾滋病毒治疗药物。这些药物阻断了HIV进入T细胞所需的细胞表面受体，例如CCR5基因编码的蛋白质。CCR5趋化因子受体是R5嗜性HIV-1毒株的主要共同受体，这些毒株是大多数初始、急性HIV感染病例的罪魁祸首[10]。在CCR5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药物来阻断受体蛋白，尽管进展受到毒性、有效性和耐药性的阻碍。HTI正在优化基于TFO的技术，作为一种替代方法，通过直接针对受体的潜在基因并使其失活，从而创建永久抵抗艾滋病毒感染的细胞。这些经过修饰的细胞不受艾滋病毒感染的阻碍，会增殖，结果是恢复和维持患者的免疫系统，即使面对艾滋病毒感染也是如此。我们建议开发我们的先导PNA化合物来靶向并特异性地灭活人类细胞中的CCR5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CCR5基因靶向性所需的最合适的细胞递送方法。此外，我们将测试这些条件，以便在预期的目标细胞--人造血干细胞(CD34+)中实现治疗性基因修改。在分离出含有突变的CCR5等位基因的纯细胞群体后，我们将确认诱导的基因型(S)和表型(S)，并将测试突变细胞是否对艾滋病毒感染具有抵抗力。此外，我们将在干细胞移植的小鼠模型中测试PNA修饰的CD34+细胞是否移植并正确分化，以及分化后的CCR5突变免疫细胞是否对HIV感染具有抵抗力。这些研究对于开发治疗HIV疾病的基因靶向剂至关重要，因为：(1)他们将建立一套PNA/DNA试剂集和合适的转染参数，从而在两个模型细胞系和我们的最终靶细胞CD34+细胞中导致CCR5基因的高效突变，以及(2)它们将有助于证明PNA诱导的CCR5基因失活可以导致对HIV感染的抵抗力。 公共卫生相关性：目前有4000多万人患有艾滋病。Helix治疗公司正在提议使用一种治疗性基因靶向剂来创造永久抵抗艾滋病毒感染的细胞。这些经过修饰的细胞不受艾滋病毒感染的阻碍，会增殖，结果是恢复和维持患者的免疫系统，即使面对艾滋病毒感染也是如此。

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.