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体组织培养模型系统，可用于鉴定和表征导致PID（原发性免疫缺陷）的基因突变。在本申请中，我们证明了这种方法可以用于提供调节V（D）J {可变（多样性）连接}重组、CSR（类别转换重组）和SHM（体细胞超突变）的DNA修复基因的结构：功能理解。V（D）J重组和CSR是人类免疫系统发育绝对需要的位点特异性DNA重排过程。SHM利用非必需的DNA诱变过程，进化以积极的方式招募该过程以增加免疫系统的功效。在过去的十年中，已经证明，在大约十几个基因中的任何一个基因突变的患者，其中大多数是DNA DSB（双链断裂）修复基因，这是一个或所有这些过程所必需的，突变时会导致PID。重要的是，这些基因中的大多数具有免疫系统之外的功能（例如，信号传导、基因组稳定性和端粒维持），并且并不总是清楚哪些人类症状/病理与基因的哪些活性相关，使得治疗干预困难，甚至潜在危险。在过去的几年里，我们已经开发了一种使用rAAV（重组腺相关病毒）的系统，其中可以在几乎任何人类基因和几乎任何人类细胞系中进行靶向突变。我们已经在原理验证实验中使用了这个系统，使人类细胞系中的许多PID致病基因无效。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领域获得试剂、检测和专业知识。通过将野生型cDNA或含有人突变的cDNA引入这些遗传修饰的细胞系中，可以准确评估患者突变对每种代谢途径的影响。这些实验将增强我们对这些基因的基本结构域和活动的理解。此外，可以合理预期，本研究产生的信息（即，患者突变与活性的相关性）可以有效地用于临床治疗和/或诊断PID。我们认为，我们的提案具有若干重要的一般优势：1）原则上它可以用于已知在突变时引起PID的120多个基因中的任何一个，2）它可以用于研究人员选择的任何人类体细胞系，3）它允许严格评估天然存在的序列变体是无关紧要的多态性还是使人衰弱的突变，4）它允许对所研究的基因进行详细的结构和功能分析，以及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：项目叙述：原发性免疫缺陷目前影响约50万美国人，新感染的个体以每1,200例活产1例的速度增加。这种疾病在遗传上是非常异质的，有超过120种致病基因与超过200种临床上不同类型的免疫缺陷相关。在这里，我们建议开发一个人类体细胞培养系统，我们可以在其中描述这些基因的大部分及其相关活动。这样一个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.)