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治疗的策略来治疗严重联合免疫缺陷（RAG-SCID）和高igm综合征1 （HIGM1）。这两种罕见的原发性免疫缺陷（PIDs）都在出生后的第一年表现出来，由RAG1和CD40LG突变引起。这两种基因的表达都受到严格调控。因此，传统的基因转移方法无法治疗遗传缺陷。我们的策略是基于通过基因编辑方法纠正RAG1和CD40LG突变，随后是基因编辑淋巴祖细胞（GE-LP）的过继转移。这两种遗传缺陷都会影响B淋巴和T淋巴的生成。之所以选择它们，是因为即使移植少量的淋巴祖细胞也能治愈它们。为了原位纠正RAG1和CD40LG突变，我们将应用核酸酶介导的同源重组，因为这可以恢复基因功能，同时保留基因表达的内源性控制。基因治疗的选择也受到来自ragg - scid和HIGM1患者的造血前体细胞（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）的多能造血祖细胞，iPSCs可以从许多人类细胞类型中产生。我们将通过测量GE-LPs的功能和分子特征来验证体外人HSC中RAG1和CD40LG突变的基因编辑。然后，我们将通过将GE-LPs细胞移植到适合人类HSC移植和多系分化的新型小鼠品系中，来评估GE-LPs在体内的发育潜力。我们的项目分为五个工作包（WPs，图1）：1)通过基因编辑技术原位纠正致病突变。2)从患者来源的ipscs中生成GE-LPs 3)从患者来源的造血干细胞中生成GE-LPs 4)验证GE-LPs在体外的B细胞和T细胞发育5)评估GE-LPs在体内的植入能力和功能我们的综合专业知识将允许GE-LPs为基础的治疗在体外和体内的功能验证。我们方法的几个方面已经适应了GMP条件。再加上我们在将基因治疗转化为临床方面的成熟经验，这保证了该项目开发的方法持续无缝地转移到临床应用中。我们期望，我们的研究将提供一种创新的定制和精确的基于基因的pid治疗。

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.