Gene therapy using homologous recombination in mouse spermatogonial stem cells

描述（由申请人提供）：通过基因疗法治疗遗传疾病的道路取得了一些成功，但也有很大的失望。特别是，通过不受控制的转基因插入的基因添加与插入性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。虽然在大多数细胞类型中基因靶向的自发率很低，但锌指核酸酶（ZFNs）最近被证明是一种极好的工具，可以通过在感兴趣位点引入双链断裂来刺激外源修复（供体）DNA模板和内源位点之间的同源重组。ZFNs是由工程DNA结合结构域和核酸酶结构域组成的人工蛋白质。迄今为止，ZFNs已被用于刺激多种体细胞类型和生物体的基因靶向；然而，要使它们的治疗效果成为现实，需要它们在干细胞中的成功应用。为了建立与临床相关的zfn介导的基因靶向，首先必须达到很高的绝对基因靶向率，或者必须开发出在体外选择和扩增罕见靶向细胞的方法。本提案涉及这两种方法。先前验证的ZFNs将用于刺激来自小鼠纯合子的精原干细胞（SSCs）中的基因靶向，该细胞具有敲入Rosa26位点的突变GFP基因。这只小鼠模拟了常染色体隐性遗传病。在Aim 1中，我们将通过测试基因传递方式、细胞周期和细胞分化状态等参数来优化zfn介导的SSCs基因靶向。这一目标的完成将有助于确定对生殖系和体细胞干细胞进行基因治疗的要求。在Aim 2中，培养的SSCs将用于模拟体外zfn介导的基因校正、选择、扩增、分子表征和移植的治疗过程。除了分离和扩增罕见基因校正干细胞的测试方法外，这一目标的实现为小鼠染色体位点的跨代基因治疗提供了原理证明；也就是说，生殖细胞将纠正后的基因传递给下一代的能力。小鼠和人精原干细胞（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使人们提出，睾丸可能是用于男性自体移植的组织相容性细胞的极佳来源。有一天，通过从睾丸活检中提取患者特异性多能细胞，在体外纠正突变基因，将细胞分化成所需的细胞类型，然后进行移植以取代患病细胞，可能治愈遗传疾病。实现这一宏伟目标的重要第一步是开发高效的SSCs基因靶向技术，如本提案所述。

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.