Study of Rag1 hypomorphic mice and their rescue by lentiviral gene transfer

描述（由申请人提供）：Rag 1基因的亚型突变是人类Omenn综合征（OS）的主要原因，这是一种与严重自身免疫表现相关的独特联合免疫缺陷。OS患者遭受压倒性的感染，伴有严重的T细胞浸润和器官损伤。除非通过异基因造血干细胞移植（HSCT）治疗，否则该疾病是迅速致命的，然而当没有匹配的供体时，该程序的结果不太令人满意。基于HLA相同供体的HSCT经验，可以预期基于遗传校正的高度纯化的HSC的骨髓移植的Rag 1亚型突变体的新治疗将是非常有益的。为了设计这种治疗方法，首先至关重要的是：a）在病毒载体的背景下定义用于适当Rag 1基因表达的离散基因组元件，B）在体外和体内表征Rag 1亚型蛋白及其与野生型Rag 1分子竞争的能力，和c）建立用于拯救新产生的Rag 1亚型小鼠的同基因骨髓移植模型，其特征在于T-和B-淋巴细胞发育的严重损伤，与活化T细胞的外周扩增相关，如在具有亚形态Rag缺陷的人中观察到的。该提案提出了初步的数据，显示了新的慢病毒载体的设计Rag 1的表达和它们的使用高度纯化的HSC的转导，除了一个新开发的亚纯型Rag 1小鼠的初步表征。拟议的研究计划将集中在Rag 1亚型的三个方面，这将显着加速Rag相关联合免疫缺陷的慢病毒治疗的临床开发的进展。将在体内研究携带驱动Rag 1表达的各种基因组元件的慢病毒载体诱导特异性和适当基因表达的能力。野生型Rag 1蛋白诱导功能性重排的能力时，竞争与它的亚纯型对应将使用在体外重排试验和前B细胞分离的Rag 1亚纯型小鼠进行研究。最后，本提案的目标1和2的结果将作为临床前研究的基础，旨在使用慢病毒转导HSC挽救Rag 1低形态小鼠。总的来说，这项研究将是重要的，以促进开发新的形式的治疗联合免疫缺陷由于亚型RAG基因缺陷，基因转移的基础上。公共卫生相关性：由于Rag 1突变导致的免疫缺陷导致相当大的死亡率，可用的治疗方法仍然有限。该建议为开发基于通过基因转移校正造血干细胞的Rag相关免疫缺陷的新治疗方法奠定了基础。

DESCRIPTION (provided by applicant): Hypomorphic mutations in the Rag1 gene are the main cause of Omenn syndrome (OS) in humans, a unique combined immunodeficiency associated with severe autoimmune manifestations. OS patients suffer from overwhelming infections accompanied with severe T-cell infiltrate and organ damage. The disease is rapidly fatal unless treated by allogeneic hematopoietic stem cell transplantation (HSCT), however the results of this procedure are less satisfactory when no matched donors are available. Based on the experience of HSCT from HLA-identical donors, it can be anticipated that a new treatment for Rag1 hypomorphic mutants based on bone marrow transplantation of genetically corrected highly purified HSCs would be highly beneficial. In order to design such therapeutic approach it is first crucial to: a) define discrete genomic elements for appropriate Rag1 gene expression in a context of a viral vector, b) characterize the Rag1 hypomorphic protein in vitro and in vivo and its ability to compete with the wild-type Rag1 molecule, and c) establish a syngeneic bone marrow transplantation model for the rescue of newly generated Rag1 hypomorphic mice, that are characterized by a severe impairment in T- and B-lymphocyte development, associated with peripheral expansion of activated T cells, as observed in humans with hypomorphic Rag defects. This proposal presents preliminary data showing the design of new lentiviral vectors for the expression of Rag1 and their use for transduction of highly purified HSCs, in addition to the initial characterization of a newly developed hypomorphic Rag1 mouse. The proposed research program will focus on three aspects of Rag1 hypomorphism, which will significantly accelerate the advancement of the clinical development of a lentiviral based therapy for Rag related combined immunodeficiency. Lentiviral vectors carrying various genomic elements driving Rag1 expression will be studied in vivo for their ability to induce specific and appropriate gene expression. The ability of wild-type Rag1 protein to induce functional rearrangement when competing with its hypomorphic counterpart will be studied using an in vitro rearrangement assay and in pre-B cells isolated from Rag1 hypomorphic mice. Finally, results from aims 1 and 2 of this proposal will serve as the basis for preclinical studies aimed at rescuing Rag1 hypomorphic mice using lentiviral transduction of HSCs. Overall, this study will be important to facilitate development of novel forms of treatment for combined immunodeficiency due to hypomorphic RAG gene defects, based on gene transfer. PUBLIC HEALTH RELEVANCE: Immunodeficiencies due to Rag1 mutations are responsible for considerable mortality and available treatments are still limited. This proposal establishes the basis for the development of a new therapeutic approach for Rag related immunodeficiencies based on correction of hematopoietic stem cells by gene transfer.