Gene edited lymphoid progenitors for adoptive transfer as a treatment of primary immunodeficiency

我们的项目提供了一种新的基于基因和细胞疗法的策略来治疗严重联合免疫缺陷(RAG-SCID)和高IgM综合征1(HIGM1)。这两种罕见的原发免疫缺陷(PID)都出现在生命的第一年，都是由RAG1和CD40LG的突变引起的。这两种基因的表达都受到严格的调控。因此，遗传缺陷不能用经典的基因转移方法来治疗。我们的策略是通过基因编辑方法纠正RAG1和CD40LG突变，然后过继转移基因编辑的淋巴祖细胞(GE-LP)。这两种基因缺陷都会影响B和T淋巴细胞的生成。之所以选择它们，是因为它们可以通过移植少量的淋巴祖细胞来治愈。为了纠正RAG1和CD40LG的原位突变，我们将应用核酸酶介导的同源重组，因为这恢复了基因功能，同时保留了对基因表达的内源控制。基因治疗的选择也受到来自RAG-SCID和HIGM1患者的造血祖细胞(HSC)可用性的限制。为了克服这一限制，我们将把我们的人类基因编辑策略应用于骨髓来源的HSC和来自诱导多能干细胞(IPSCs)的多潜能造血祖细胞，IPSCs可以从许多类型的人类细胞中产生。我们将通过测量GE-LP的功能和分子特征来验证RAG1和CD40LG突变在体外对人类HSC的基因编辑。然后，我们将通过将细胞移植到新的小鼠品系中来评估GE-LPs在体内的发展潜力，这些品系优化了人类HSC的植入和多系分化。我们的项目分为五个工作包(WPS；图1)：1)在特定情况下通过基因编辑技术纠正致病突变2)从患者来源的IPSC产生GE-LP到从患者来源的造血干细胞产生GE-LP 4)在体外验证从GE-LP到GE-LP的B和T细胞发育5)评估GE-LP在体内的植入能力和功能我们的联合专业知识将使基于GE-LP的治疗在体外和体内的功能验证成为可能。我们方法的几个方面已经适应了GMP的条件。再加上我们在将基因疗法转化为临床方面的成熟经验，这就保证了该项目开发的方法学能够持续和无缝地转化为临床应用。我们期待，我们的研究将提供一种创新的、量身定做的、精确的基于基因的治疗方法。

Our project provides a new gene and cell therapy-based strategy to treat severe combined immunodeficiency (RAG-SCID) and hyper-IgM syndrome 1 (HIGM1). Both rare primary immunodeficiencies (PIDs) manifest during the first year of life and are caused by mutations in RAG1 and CD40LG. The expression of both genes is tightly regulated. Therefore, the genetic defects cannot be treated by classical gene transfer methodology. Our strategy is based on the correction of RAG1 and CD40LG mutations by a gene-editing approach, which is followed by the adoptive transfer of gene-edited lymphoid progenitor cells (GE-LP). Both genetic defects affect B and T lymphopoiesis. They were chosen, because they can be cured by transplanting even small numbers of lymphoid progenitor cells. To correct RAG1 and CD40LG mutations in situ we will apply nuclease-mediated homologous recombination as this restores gene function while retaining the endogenous control of gene expression. Gene therapy options are also limited by the availability of hematopoietic precursor cells (HSC) from RAG-SCID and HIGM1 patients. To overcome this limitation, we will apply our gene-editing strategy human to bone marrow-derived HSC and to multipotent hematopoietic progenitors derived from induced pluripotent stem cells (iPSCs), which can be generated from many human cell types. We will validate gene-editing of RAG1 and CD40LG mutations in human HSC in vitro by measuring both functionality and molecular signatures of GE-LPs. We will then evaluate the developmental potential of GE-LPs in vivo by transplanting the cells into novel mouse strains optimized for the engraftment and multilineage differentiation of human HSC. Our project is structured into five Work Packages (WPs; Fig. 1):1) To correct disease-causing mutations by gene editing technology in situ.2) To generate GE-LPs from patient-derived iPSCs3) To generation GE-LPs from patient-derived hematopoietic stem cells4) To validate B and T cell development from GE-LPs in vitro5) To evaluate the engraftment capacity and function of GE-LPs in vivoOur combined expertise will allow the functional validation of a GE-LPs based therapy both in vitro and in vivo. Several aspects of our approach have been already adapted to GMP conditions. Together with our proven experience in translating gene therapy to the clinic, this warrants the sustained and seamless transfer of the methodology developed in this project into clinical application. We expect, that our study will provide an innovative tailored and precise gene-based treatment of PIDs.