Gene therapy using homologous recombination in mouse spermatogonial stem cells

描述（申请人提供）：通过基因治疗治愈遗传疾病的道路已经取得了一些成功，但也有相当大的失望。特别地，通过转基因的不受控制的插入的基因添加已经与插入肿瘤发生和沉默相关。基因靶向，内源基因序列的校正，是优选的，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非常低，但最近已显示锌指核酸酶（ZFN）是通过在感兴趣的基因座中引入双链断裂来刺激外源修复（供体）DNA模板与内源基因座之间的同源重组的极好工具。ZFN是由工程化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已被用于刺激各种体细胞类型和生物体中的基因靶向;然而，要实现其治疗益处，需要将其成功应用于干细胞。为了建立临床相关的ZFN介导的基因靶向，必须首先获得非常高的基因靶向绝对率，或者必须开发用于在体外选择和扩增稀有靶细胞的方法。本建议涉及这两种方法。先前验证的ZFN将用于刺激精原干细胞（SSC）中的基因靶向，所述精原干细胞（SSC）来源于敲入Rosa 26基因座的突变GFP基因的纯合小鼠。这种小鼠是常染色体隐性遗传病的模型。目的1通过对基因导入方法、细胞周期和细胞分化状态等参数的检测，优化ZFN介导的基因打靶在精原干细胞中的应用。这一目标的完成将有助于确定生殖系和体干细胞基因治疗的要求。在目标2中，培养的SSC将用于模拟离体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的分离和扩增方法外，这一目标的实现还为小鼠染色体位点的跨代基因治疗提供了原理证明;即生殖细胞将校正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这样的建议，即睾丸可能是用于男性自体移植的组织相容性细胞的极好来源。有一天，通过从睾丸活检组织中获得患者特异性多能细胞，在体外纠正突变基因，将细胞分化为所需的细胞类型，然后进行移植以取代患病细胞，可能治愈遗传疾病。实现这一雄心勃勃的目标的重要的第一步是开发本提案中所述的在SSCs中进行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纠正后的干细胞移植到患者体内可能是治疗某些遗传疾病的有效方法。一种有希望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，这一过程然后刺激突变基因序列与外源提供的正常基因序列的交换。我们建议在培养的精原干细胞中测试和优化这种基因校正过程，精原干细胞具有很大的治疗潜力，并且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.