Targeted gene repair of primary immune deficiencies

LAGLIDG寻的结构/功能关系的研究进展 核酸内切酶(LHE‘s)使人工LHE具有新的识别和切割位点成为可能，这表明人工LHE可以被设计成在人类基因组中特定的靶点切割，作为一种增强这些位置的同源重组的手段。在这项资助中，我们建议开发和评估将LHE和DNA修复模板引入造血细胞的方法，目的是有针对性地修复原发免疫缺陷的基因。我们选择IL2RGamma基因作为我们的初始目标，它与X连锁严重联合免疫缺陷综合征(XSCID)有关，因为它是最常见的形式 严重的联合免疫缺陷(SCID)，有证据表明，细胞表达由XSCID基因编码的蛋白的功能版本存在选择性优势。后一种特性非常重要，因为它允许将来在体内尝试的基因修复，这些尝试只在少数目标细胞群体中成功地被放大到足以对免疫功能产生临床相关影响的水平。 在特定的目标1中，我们将构建LHE诱导的IL2RGamma基因靶向修复的标杆模型。Subaim a将涉及对培养的淋巴细胞系进行工程改造，使其携带IL2RGamma基因外显子6附近或内的I-SCEL和I-ANIL LHEs的切割位点。然后，我们将在亚目的b中使用这些细胞系来评估引入I-SCEL或I-ANI LHE‘s和DNA修复模板的各种方法，作为一种在造血细胞/淋巴细胞环境中诱导IL2Rγ基因的靶向修复的手段。在具体目标2中，我们将根据以下定义的修改LHE的原则，修改Iani LHE以在我们确定的特定位置切割 我们的合作者，在人类或小鼠IL2RGamma基因的外显子6或附近。在子目标a中，我们将使用遗传筛选、生化评估和基本原理设计的组合来鉴定切割已鉴定序列的I-ANIL变异体。在子目标b中，我们将使用在特定目标1中开发的模型和方法来测试每个修饰的LHE诱导靶向基因修复的能力以及它对基因组完整性的影响，并以I-SCEL诱导的靶向修复作为比较基准。总体而言，这里提出的工作将为LHE作为通用工具的开发奠定材料和经验基础 人类原发免疫缺陷的靶向基因治疗。

Recent advances in knowledge of the structure/function relationships of LAGLIDADG homing endonucleases (LHE's) has allowed the creation of artificial LHE's with novel recognition and cut sites, suggesting the possibility that artificial LHE's can be engineered to cut at specific target sites in the human genome as a means to enhance homologous recombination at those sites. In this grant, we propose to develop and evaluate methods for introducing LHE's and DNA repair templates into hematopoietic cells for the purpose of targeted gene repair of primary immune deficiencies. We have chosen as our initial target the IL2Rgamma gene, involved in X-linked severe combined immune deficiency syndrome (XSCID), as it is the most common form of severe combined immune deficiency (SCID), and there is evidence for the existence of a selective advantage for cells expressing a functional version of the protein encoded by XSCID locus. The latter property is of great importance, as it allows future in-vivo attempts at gene repair which are successful in only a few percent of a target cell population to be amplified to a level sufficient to make a clinically relevant impact on immune function. In Specific Aim 1, we will construct models for benchmarking LHE-induced targeted repair of the IL2Rgamma gene. Subaim a will involve engineering cultured lymphocyte cell lines to carry cut sites for I-Scel and I-Anil LHEs near or within exon 6 of the IL2Rgamma gene. We will then use these cell lines in subaim b to evaluate various methods for introducing the I-Scel or I-Ani LHE's and a DNA repair template as a means to induce targeted repair of the IL2Rgamma gene in a hematopoietic/lymphocytic cell context. In Specific Aim 2. we will modify the IAni LHE to cut at specific sites we have identified, based on principles for modification of LHE's defined by our collaborators, at or near exon 6 of the human or murine IL2Rgamma genes. In subaim a, we will use a combination of genetic screens, biochemical evaluation, and rationale design to identify I-Anil variants which cut at the identified sequences. In subaim b, we will use the models and methods developed in specific Aim 1 to assay each modified LHE for its capacity to induce targeted gene repair as well as its effects on genomic integrity, with I-Scel-induced targeted repair serving as a comparison benchmark. Overall, the work proposed here will build a foundation of materials and experience for the development of LHE's as general tools for targeted gene therapy of human primary immune deficiencies.