Gene therapy using homologous recombination in mouse spermatogonial stem cells

描述(申请人提供)：通过基因疗法治疗遗传病的道路已经取得了一些成功，但也有很大的失望。特别是，通过不受控制的转基因插入而增加的基因与插入致癌和沉默有关。基因打靶，即纠正内源基因序列，是更可取的，因为它避免了这两个问题。虽然在大多数细胞类型中基因打靶的自发比率很低，但锌指核酸酶(ZFN)最近被证明是一种很好的工具，通过在感兴趣的位置引入双链断裂来刺激外源修复(供体)DNA模板和内源基因座之间的同源重组。ZFN是由DNA结合结构域和核酸酶结构域组成的人工蛋白质。到目前为止，ZFN已经被用来在各种体细胞类型和生物体中刺激基因靶向；然而，要使它们的治疗效果发挥作用，还需要成功地将它们应用于干细胞。为了建立临床相关的ZFN介导的基因打靶，首先必须获得非常高的基因打靶绝对率，或者必须开发出在体外选择和扩增稀有靶向细胞的方法。这项提案涉及这两种方法。此前验证过的ZFN将用于刺激精原干细胞(SSCs)中的基因靶向，该干细胞来自一只敲入rosa26基因的突变GFP基因的纯合子小鼠。这只小鼠是一种常染色体隐性遗传病的模型。目的1通过检测基因传递方式、细胞周期和细胞分化状态等参数，优化ZFN介导的基因打靶在SSCs中的应用。这一目标的完成应该有助于确定生殖系和体细胞干细胞基因治疗的要求。在AIM中，2个培养的SSCs将被用于模拟ZFN介导的体外基因纠正、选择、扩增、分子鉴定和移植的治疗过程。除了用于分离和扩增稀有基因校正的干细胞的测试方法外，这一目标的实现还为小鼠染色体上的跨代基因治疗提供了原理证明；即生殖细胞将校正的基因传递给下一代的能力。小鼠和人类精原干细胞(SSCs)最近被证明能够去分化为多能胚胎干细胞样细胞。这一令人兴奋的发现导致了一项建议，即睾丸可能是组织相容细胞的极好来源，用于男性自体移植。有朝一日，有可能通过从睾丸活检中获得针对患者的多能细胞，在体外纠正突变基因，将细胞分化为所需的细胞类型，然后进行移植来取代患病的细胞，从而治愈遗传性疾病。朝着这一雄心勃勃的目标迈出的重要的第一步是开发这项提案中描述的在SSCs中有效的基因靶向技术。 与公共卫生相关：纠正干细胞中的突变基因并将纠正后的干细胞移植到患者体内可能是治愈某些遗传性疾病的有效方法。一种有前景的纠正突变基因的新方法是使用分子剪刀(锌指核酸酶)切割基因，这一过程随后刺激突变的基因序列与外源提供的正常基因序列交换。我们建议在培养的精原干细胞中测试和优化这一基因校正过程，精原干细胞是一种具有巨大治疗潜力的细胞，与多能胚胎干细胞高度相关。

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.