Novel Human Cell Lines for the Study of Primary Immunodeficiencies

描述(由申请人提供)：我们建议开发一种人类体细胞组织培养模型系统，该系统可用于识别和表征导致PID(初级免疫缺陷)的基因突变。在这个应用中，我们证明了这种方法可以用来提供一种结构：调节V(D)J{变量(多样性)连接}重组、CSR(类开关重组)和SHM(体细胞高突变)的DNA修复基因的功能。V(D)J重组和CSR是人类免疫系统发育所必需的位点特异性DNA重排过程。SHM利用进化以积极方式征召的非必要的DNA突变过程来增加免疫系统的效率。在过去的十年里，已经证明，大约12个基因中的任何一个发生突变的患者，当突变时会导致PID，其中大多数是DNA DSB(双链断裂)修复基因，这些基因是这些过程中的一个或所有过程所必需的。重要的是，这些基因中的大多数都具有免疫系统以外的功能(例如，信号、基因组稳定性和端粒维持)，而且并不总是清楚哪些人类症状/病理与基因的哪些活性有关，这使得治疗干预变得困难，甚至是潜在的危险。在过去的几年里，我们开发了一种使用重组腺相关病毒(RAAV)的系统，其中几乎可以在任何人类基因和几乎任何人类细胞系中进行靶向突变。我们已经使用这个系统--在原理验证实验中--使人类细胞系中许多导致产后应激障碍的基因缺失。此外，我们花了几十年的时间在DNA修复、V(D)J、CSR、SHM和端粒维护等一系列名副其实的领域获得试剂、分析和专业知识。通过将野生型cDNA或含有人类突变的cDNA引入这些转基因细胞系中，可以准确地评估患者突变对每条代谢途径的影响。这些实验应该会加深我们对这些基因的基本结构域和活性的理解。此外，有理由预计，这项研究产生的信息(即患者突变与活动的相关性)可以在临床上有效地用于治疗和/或诊断PID。我们相信，我们的建议有许多重要的一般优势：1)原则上它可以用于任何120+个基因-当突变时-已知导致PIDS，2)它可以用于研究人员选择的任何人类体细胞系，3)它允许严格评估自然发生的序列变体是无关紧要的多态还是衰弱的突变，4)它允许详细的结构：正在研究的基因的功能分析和5)它提供了可用于临床前治疗研究的新细胞系。 公共卫生相关性：项目描述：亨德里克森初级免疫缺陷目前影响到约50万美国人，新感染的人以每1200名活产中有1人的速度增加。这种疾病在遗传上是非常不同的，有120多个致病基因与200多种临床上不同类型的免疫缺陷相关。在这里，我们建议开发一种人类体细胞培养系统，在该系统中，我们可以表征大部分这些基因及其相关的活性。这种系统的开发有能力产生更好的治疗方法，因此显然与NIH的使命有关。)

DESCRIPTION (provided by applicant): We propose to develop a human somatic tissue culture model system that can be used to identify and characterize mutations in genes that cause PIDs (primary immune deficiencies). In this application we demonstrate that this approach can be used to provide a structure:function understanding of the DNA repair genes that regulate V(D)J {variable(diversity)joining} recombination, CSR (class switch recombination) and SHM (somatic hypermutation). V(D)J recombination and CSR are site-specific DNA rearrangement processes absolutely required for the development of the human immune system. SHM utilizes a non-essential DNA mutagenesis process that evolution has conscripted in a positive fashion to increase the efficacy of the immune system. Over the past decade it has been demonstrated that patients with mutations in any of about a dozen genes, most of them DNA DSB (double-strand break) repair genes, which are required for either one or all of these processes, result in PIDs when mutated. Importantly, most of these genes have functions outside of the immune system (e.g., signaling, genomic stability, and telomere maintenance) and it is not always clear which human symptoms/pathologies are associated with which activity of the gene, rendering therapeutic intervention difficult and even potentially dangerous. We have developed over the past several years a system using rAAV (recombinant adeno-associated virus) where targeted mutations can be made in virtually any human gene and in virtually any human cell line. We have used this system - in proof-of-principle experiments - to make human cell lines null for a number of PID-causative genes. In addition, we have spent several decades acquiring reagents, assays and expertise in a veritable bevy of areas relevant to DNA repair, V(D)J, CSR, SHM and telomere maintenance. By introducing either wild-type cDNAs or cDNAs containing human mutations into these genetically modified cell lines the impact of patient mutations on each metabolic pathway can be accurately assessed. These experiments should enhance our understanding of the basic domains and activities of these genes. In addition, it is reasonable to anticipate that the information generated from this study (i.e., a correlation of patient mutations to activity) can be productively used in the clinic to treat and/or diagnose PIDs. We believe that our proposal has a number of important general strengths: 1) it can in principle be used for any of the 120+ genes that - when mutated - are known to cause PIDs, 2) it can be used with any human somatic cell line of the researcher's choosing, 3) it allows for a rigorous assessment of whether a naturally occurring sequence variant is an inconsequential polymorphism or a debilitating mutation, 4) it permits a detailed structure:function analysis of the gene under study and 5) it provides novel cell lines that can be used for pre-clinical therapeutic studies. PUBLIC HEALTH RELEVANCE: Project Narrative: Hendrickson Primary immune deficiencies currently affect ~500,000 Americans and newly affected individuals are accruing at the rate of 1 per 1,200 live births. This disease is genetically very heterogeneous with over 120 disease-causing genes correlated with over 200 clinically distinct types of immune deficiencies. Here we propose to develop a human somatic cell culture system in which we can characterize the great bulk of these genes and their relevant activities. The development of such a system has the ability to yield better therapies and is thus clearly relevant to the mission of NIH.)