Inherited T cell defects: Diagnosis, Mechanisms and Treatments

在过去的十年里，发现原发性免疫缺陷的基因迅速增长。随着……的到来 新生儿严重联合免疫缺陷(SCID)筛查与深度免疫治疗的新应用 测序、基因组分析、高通量细胞筛选和CRISPR基因编辑，有一个 通过直接测试确定人类基因序列是如何控制免疫细胞的前所未有的机会 开发，并最终通过对因果突变的基因组编辑来治疗SCID。我们的综合计划 将解决必须克服的主要挑战，以利用这一变革性的机会，通过 将T细胞不足患者的临床数据与基础调查相结合，借鉴 在免疫学、生物信息学、靶标验证和基因组编辑方面处于领先地位。SCID新生儿筛查 由帕克博士首创并在美国所有50个州和越来越多的国家实施的化验方法， 使用婴儿血斑中的DNA来计数T细胞受体切除环(TRECs)，TRECs是 胸腺输出新的T细胞。它对识别婴儿T细胞功能不全是非常有效的，其分子 病因通常是通过免疫表型和一组已知的SCID基因测序来揭示的。重要的是 然而，这种公正的、基于人群的筛查也揭示了患有SCID的婴儿缺乏容易辨别的、 有害的致病突变，以及属于以前未被识别的非 SCID T细胞淋巴细胞减少症(TCL)。我们和其他人已经将完整的外显子组测序(WES)应用于谜题 SCID和TCL病例，揭示了意外和令人兴奋的基因变异，这些基因变异直接影响了医学 护理，同时揭示了对免疫机制的新见解。然而，重要的是，WES未能识别疾病- 导致其中60%的神秘病例发生突变。这可能是由于外显子组捕获不完整、质量不佳造成的 覆盖的外显子，或致病变异可能存在于非编码基因组元件中的事实。本计划 将通过结合使用全基因组测序(WGS)、T细胞 在患者和父母中的RNAseq，以及强大的高通量功能分析来解决这些困难的病例。我们的 这项工作最终将迎来一个使用自体造血细胞基因组编辑的新疗法时代 祖先。为了从候选变异中识别导致T细胞不足的突变(S)，我们 将在斑马鱼、原代人类CD34+细胞和人类多能干细胞中进行功能筛选- CD34+造血干/祖细胞在新型Notch配体微球上的体外分化。 此外，我们还将使用单细胞转录组比较和上位性分析的方法perturb-seq 解开因果变异之间的关系，并构建人类T细胞发育的分子图谱。我们的 综合能力，包括临床专业知识、基因组分析、基因编辑、高通量细胞 筛查、斑马鱼和功能免疫学有能力彻底改变我们对 免疫细胞的发育和功能，同时在新的治疗方法方面取得了重要进展。

The past decade has seen rapid growth of gene discovery for primary immunodeficiencies. With the advent of newborn screening for severe combined immunodeficiency (SCID), coupled with new applications for deep sequencing, genomic analyses, high-throughput cellular screening, and CRISPR gene editing, there is an unprecedented opportunity to establish, by direct testing, how human genetic sequences control immune cell development, and ultimately to treat SCID by genome editing of causal mutations. Our comprehensive program will address the major challenges that must be overcome to capitalize on this transformative opportunity, by integrating clinical data from T cell insufficient patients with basic investigations drawing on the expertise of leaders in immunology, bioinformatics, target validation, and genome editing. The SCID newborn screening assay, pioneered by Dr. Puck and implemented in all 50 states in the USA and an increasing number of countries, employs DNA from infant blood spots to enumerate T cell receptor excision circles (TRECs), a surrogate for thymic output of new T cells. It is highly effective for identifying infants with T cell insufficiency, whose molecular etiologies are often revealed by immune phenotyping and sequencing a panel of known SCID genes. Importantly, however, this unbiased, population-based screening also reveals infants with SCID who lack readily discernable, deleterious causative mutations, as well as other infants belonging to a previously unrecognized group with non- SCID T cell lymphopenia (TCL). We and others have applied whole exome sequencing (WES) to enigmatic cases of SCID and TCL, revealing unanticipated and exciting gene variants that have directly impacted medical care, while revealing new insights into immune mechanisms. Importantly, however, WES fails to identify disease- causing mutations in 60% of these enigmatic cases. This may result from incomplete exome capture, poorly covered exons, or the fact that a disease-causing variant may lie in a non-coding genomic element. This Program will overcome these limitations by combining variant discovery using whole genome sequencing (WGS), T cell RNASeq in patients and parents, and robust high-throughput functional assays to solve these difficult cases. Our work will ultimately usher in an era of novel treatments employing genome editing of autologous hematopoietic progenitors. To identify the mutation(s) responsible for T cell insufficiency from among candidate variants, we will perform functional screening in zebrafish, primary human CD34+ cells and human pluripotent stem cell- derived CD34+ hematopoietic stem and progenitor cells differentiated in vitro on novel Notch-ligand microbeads. Moreover, we will use Perturb-seq, a method for single cell transcriptome comparisons and epistatic analysis to unravel relationships among causal variants and construct a molecular map of human T cell development. Our combined capabilities, encompassing clinical expertise, genomic analysis, gene editing, high-throughput cellular screening, zebrafish and functional immunology, have the power to revolutionize our understanding of how immune cells develop and function while yielding important progress toward new treatment approaches.