Targeted gene repair of primary immune deficiencies

LAGLIDADG归巢结构/功能关系的最新进展 核酸内切酶（LHE）的使用已经允许产生具有新的识别和切割位点的人工LHE，这表明人工LHE可以被工程化以在人类基因组中的特定靶位点处切割作为增强这些位点处的同源重组的手段的可能性。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，用于原发性免疫缺陷的靶向基因修复。我们选择IL 2 R γ基因作为我们的初始靶点，该基因参与X连锁严重联合免疫缺陷综合征（XSCID），因为它是最常见的形式。 严重联合免疫缺陷（SCID），并有证据表明，存在一个选择性优势的细胞表达的功能版本的蛋白质编码的XSCID基因座。后一种性质非常重要，因为它允许将来在体内尝试基因修复，这些基因修复仅在靶细胞群体的百分之几中成功，以扩增到足以对免疫功能产生临床相关影响的水平。 在具体目标1中，我们将构建用于基准LHE诱导的IL 2 R γ基因靶向修复的模型。Subaim a将涉及工程化培养的淋巴细胞系，以在IL 2 R γ基因的外显子6附近或外显子6内携带I-Scel和I-Anil LHE的切割位点。然后，我们将在子目标B中使用这些细胞系来评估用于引入I-Scel或I-Ani LHE和DNA修复模板的各种方法，作为在造血/淋巴细胞环境中诱导IL 2 R γ基因的靶向修复的手段。具体目标2。我们将修改IAni LHE，以便在我们确定的特定部位进行切割，根据LHE的修改原则， 我们的合作者，在人或鼠IL 2 R γ基因的外显子6处或附近。在子目标a中，我们将使用遗传筛选、生化评价和基本原理设计的组合来鉴定在所鉴定的序列处切割的I-Anil变体。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定每种修饰的LHE诱导靶向基因修复的能力及其对基因组完整性的影响，其中I-Scel诱导的靶向修复用作比较基准。总的来说，这里提出的工作将建立一个基础的材料和经验的发展，LHE的一般工具， 人类原发性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.