Gene therapy using homologous recombination in mouse spermatogonial stem cells

在小鼠精原干细胞中使用同源重组进行基因治疗

• 批准号: 8019497
• 负责人: Christina Tenenhaus Dann
• 金额: $ 18.48万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019497
• 关键词: Address; Antibiotic Resistance; Autologous Transplantation; Biological Assay; Biopsy; Breeding; Cell Cycle; Cell Cycle Stage; Cell Differentiation process; Cell Line; Cell Transplants; Cells; DNA; DNA Binding Domain; Dependovirus; Development; Disease; Embryo; Engineering; Event; Fertilization; Fluorescence; Gene Delivery; Gene Targeting; Genes; Genetic; Germ Cells; Goals; Green Fluorescent Proteins; Hereditary Disease; Human; In Vitro; Knock-in Mouse; Lentivirus Vector; Mediating; Methods; Modeling; Modification; Molecular; Mus; Mutate; Mutation; Oncogene Activation; Organism; Patients; Process; Processed Genes; Proteins; Route; Site; Somatic Cell; Source; Stem cell transplant; Stem cells; Testing; Testis; Therapeutic; Transgenes; Transgenic Organisms; Transplantation; Validation; Zinc Fingers; base; cDNA Expression; cell type; clinically relevant; embryonic stem cell; gene correction; gene therapy; homologous recombination; interest; men; mouse model; mutant; next generation; nuclease; protein expression; public health relevance; repaired; self-renewal; stem; stem cell differentiation; success; technology development; theories; tool; transmission process; tumorigenesis

DESCRIPTION (provided by applicant): The road toward curing genetic diseases by gene therapy has seen some success but also significant disappointment. In particular, gene addition via uncontrolled insertion of transgenes has been associated with insertional oncogenesis and silencing. Gene targeting, correction of the endogenous gene sequence, is preferable as it avoids both of these issues. While the spontaneous rate of gene targeting in most cell types is very low, zinc finger nucleases (ZFNs) have recently been shown to be an excellent tool for stimulating homologous recombination between an exogenous repair (donor) DNA template and the endogenous locus by introducing a double strand break in the locus of interest. ZFNs are artificial proteins consisting of an engineered DNA binding domain and nuclease domain. So far ZFNs have been used to stimulate gene targeting in a variety of somatic cell types and organisms; however, to bring their therapeutic benefit to fruition will require their successful application in stem cells. To establish clinically relevant ZFN-mediated gene targeting, very high absolute rates of gene targeting must first be attained, or methods for selecting and expanding rare targeted cells in vitro must be developed. This proposal addresses both of these approaches. Previously validated ZFNs will be used to stimulate gene targeting in spermatogonial stem cells (SSCs) derived from a mouse homozygous for a mutant GFP gene knocked-in to the Rosa26 locus. This mouse models an autosomal recessive disease. In Aim 1 ZFN-mediated gene targeting will be optimized in SSCs by testing parameters including gene delivery method, cell cycle and cell differentiation state. The completion of this aim should help to define the requirements for gene therapy in germline and somatic stem cells. In Aim 2 cultured SSCs will be used to model the therapeutic process of ex vivo ZFN-mediated gene correction, selection, amplification, molecular characterization and transplantation. In addition to testing methods for isolation and expansion of rare gene-corrected stem cells, the accomplishment of this aim provides proof of principle for transgenerational gene therapy at a chromosomal locus in mice; that is, the ability of germ cells to transmit a corrected gene to the next generation. Mouse and human spermatogonial stem cells (SSCs) have recently been shown to be capable of dedifferentiation into pluripotent embryonic stem - like cells. This exciting discovery has led to the proposal that the testis could be an excellent source of histocompatible cells for autologous transplantation in men. One day it may be possible to cure genetic diseases by deriving patient-specific pluripotent cells from a testicular biopsy, correcting a mutated gene in vitro, differentiating the cells into the desired cell type and then performing transplantation to replace the diseased cells. An important first step toward this ambitious goal is the development of technology for efficient gene targeting in SSCs as described in this proposal. PUBLIC HEALTH RELEVANCE: Correcting a mutated gene in stem cells and transplanting the corrected stem cells into a patient could be an effective way to cure certain genetic diseases. A promising new method to correct mutant genes is to cut the gene using molecular scissors (zinc finger nucleases), a process that then stimulates the exchange of the mutated gene sequence with an exogenously provided normal gene sequence. We propose to test and optimize this process of gene correction in cultured spermatogonial stem cells, cells with great therapeutic potential and that are highly related to pluripotent embryonic stem cells.

描述（由申请人提供）：通过基因疗法治愈遗传疾病的道路已经成功，但也令人失望。特别是，通过不受控制的转基因插入基因与插入肿瘤发生和沉默有关。基因靶向，内源基因序列的校正是可取的，因为它避免了这两个问题。虽然大多数细胞类型中的基因靶向的自发速率非常低，但最近已证明锌指核酸酶（ZFN）是刺激外源修复（供体）DNA模板与内源性位点之间同源重组的绝佳工具，该工具通过引入了感兴趣的轨迹中的双链破裂。 ZFN是由工程DNA结合结构域和核酸酶结构域组成的人造蛋白。到目前为止，ZFN已用于刺激各种体细胞类型和生物的基因靶向。但是，为了实现其治疗益处，将需要成功地在干细胞中应用。为了建立临床相关的ZFN介导的基因靶向，必须首先达到非常高的基因靶向率，或者必须在体外选择和扩展稀有靶向细胞的方法。该提案解决了这两种方法。先前经过验证的ZFN将用于刺激源自纯合子的小鼠的精子干细胞（SSC），用于将突变的GFP基因撞到Rosa26基因座。该小鼠对常染色体隐性疾病进行了建模。在AIM 1中，将通过测试包括基因递送方法，细胞周期和细胞分化状态（细胞周期和细胞分化状态）来优化SSC中ZFN介导的基因靶向。该目标的完成应有助于定义种系和体干细胞中基因治疗的要求。在AIM 2中，培养的SSC将用于模拟体内ZFN介导的基因校正，选择，扩增，分子表征和移植的治疗过程。除了测试稀有基因校正干细胞分离和扩展的测试方法外，该目标的完成还提供了小鼠染色体基因座的转世基因治疗原理证明。也就是说，生殖细胞将校正基因传递到下一代的能力。小鼠和人类精子干细胞（SSC）最近被证明能够将其去分化为多能胚胎干 - 像细胞一样。这一令人兴奋的发现导致了这样的提议，即睾丸可能是男性自体移植的组织兼容细胞的极好来源。有一天，有可能通过从睾丸活检中得出患者特异性多能细胞来治愈遗传疾病，从而在体外纠正突变的基因，从而将细胞区分为所需的细胞类型，然后进行移植以替代患病细胞。朝着这个雄心勃勃的目标迈出的重要第一步是，如本提案中所述，开发了在SSC中有效基因靶向的技术。 公共卫生相关性：纠正干细胞中的突变基因并将校正的干细胞移植到患者中可能是治愈某些遗传疾病的有效方法。纠正突变基因的一种有希望的新方法是使用分子剪刀（锌指核酸酶）切割基因，然后该过程刺激了与外源提供的正常基因序列的突变基因序列的交换。我们建议在培养的精子干细胞，具有巨大治疗潜力的细胞中测试和优化基因校正过程，并且与多能胚胎干细胞高度相关。