Inherited T cell defects: Diagnosis, Mechanisms and Treatments

在过去的十年中，原发性免疫缺陷的基因发现迅速增长。的到来 新生儿严重联合免疫缺陷（SCID）筛查，加上新的应用， 测序、基因组分析、高通量细胞筛选和CRISPR基因编辑， 这是一个前所未有的机会，可以通过直接测试来确定人类基因序列如何控制免疫细胞。 开发，并最终通过基因组编辑因果突变来治疗SCID。我们的综合方案 将通过以下方式应对利用这一变革机遇必须克服的重大挑战： 将T细胞不足患者的临床数据与基础研究相结合， 免疫学、生物信息学、靶标验证和基因组编辑领域的领导者。SCID新生儿筛查 由Puck博士开创，并在美国所有50个州和越来越多的国家实施， 使用来自婴儿血斑的DNA来计数T细胞受体切除环（TRECs）， 胸腺输出新的T细胞。它对识别T细胞不足的婴儿非常有效，其分子 通常通过免疫表型分析和对一组已知的SCID基因进行测序来揭示病因。重要的是， 然而，这种无偏见的，基于人群的筛查也显示出患有SCID的婴儿缺乏容易辨别的， 有害的致病突变，以及其他婴儿属于以前未被认识的群体， SCID T细胞淋巴细胞减少症（TCL）。我们和其他人已经将全外显子组测序（WES）应用于 SCID和TCL的病例，揭示了意想不到的和令人兴奋的基因变异，直接影响了医疗 护理，同时揭示免疫机制的新见解。然而，重要的是，WES未能识别疾病- 导致60%的神秘病例发生突变这可能是由于不完整的外显子组捕获， 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。这个程序 将通过使用全基因组测序（WGS），T细胞， RNASeq在患者和父母中的应用，以及强大的高通量功能测定来解决这些困难的情况。我们 这项工作将最终迎来一个新的治疗时代， 祖先为了从候选变体中鉴定导致T细胞不足的突变，我们 将在斑马鱼、原代人CD 34+细胞和人多能干细胞中进行功能筛选， 衍生的CD 34+造血干细胞和祖细胞在新型Notch配体微珠上体外分化。 此外，我们将使用Perturb-seq，一种用于单细胞转录组比较和上位性分析的方法， 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 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如何 免疫细胞的发展和功能，同时产生新的治疗方法的重要进展。

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.