Sleeping Beauty-mediated Gene Therapy of X-linked SCID

描述（由申请人提供）：原发性免疫缺陷包括一组由正常淋巴细胞发育中断引起的遗传性遗传病。这些疾病被认为是基因治疗的主要候选者，通过将缺失的基因引入造血干细胞，然后可以分化成淋巴样细胞，从而恢复免疫力。由编码IL-2、IL-4、IL-7、IL-9、IL-15和IL-213受体的共同链基因(c)的基因突变引起的x -连锁严重联合免疫缺陷（X-SCID）是一种较常见的原发性免疫缺陷之一。X-SCID的临床基因治疗试验侧重于使用逆转录病毒作为整合病毒载体引入c基因，从而使移植了转导的自体造血干细胞的患者恢复免疫力。然而，在这些临床试验中出现了严重的不良事件，即21例接受治疗的患者中有5例出现t细胞白血病样综合征。因此，需要更安全的替代疗法。

DESCRIPTION (provided by applicant): Primary immune deficiencies comprise a group of inherited genetic disorders caused by interruption of normal lymphoid development. These diseases are considered prime candidates for gene therapy by introduction of the missing gene into hematopoietic stem cells that can then differentiate into lymphoid cells, and thus restore immunity. X-linked severe combined immunodeficiency (X-SCID), caused by a mutation in the gene encoding the common ( chain gene ((c) of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-213, is one of the more common of the primary immunodeficiencies. Clinical gene therapy trials for X-SCID have focused on the use of retroviruses as an integrating viral vector for introduction of the (c gene, resulting in restoration of immunity in patients that have engrafted with transduced, autologous hematopoietic stem cells. However, serious adverse events have emerged in these clinical trials, namely a T-cell leukemia-like syndrome developing in 5 of 21 treated patients. Therefore, safer, alterative therapies are needed. At Discovery Genomics, Inc. (DGI), we are working on developing the Sleeping Beauty (SB) transposon system for gene therapy, including the targeting of hematopoietic stem cells for treatment of primary immunodeficiencies. The two-component SB system consists of a transposon (inverted repeats (IR's) flanking a therapeutic gene of interest) and a transposase that catalyzes excision of the transposon at the ends of the IRs and then integration into host cell chromosomal sequence. As a lead immunodeficiency, here we propose that X-SCID may be treatable without the use of a virus by combining the power of electroporation for introduction of DNA into cells, along with use of DGI's Sleeping Beauty transposon system to achieve integration and long-term (c gene expression. In this Phase I application, the overall goal is to establish conditions for SB-mediated gene therapy for X-SCID. In this regard, there are two key questions that will need to be addressed: (i) Will SB-mediated transposition of the (c gene allow functional correction of lymphoid cells? (ii) What is the effectiveness of SB-mediated (c gene insertion in the treatment of an animal model of X-SCID? There are two Specific Aims: Aim 1. Evaluate the effectiveness of Sleeping Beauty transposons for correction of (c chain deficiency in a lymphoblastoid cell line derived from an X-linked SCID patient. Aim 2. Evaluate Sleeping Beauty-mediated transposition and long term expression of (c gene in hematopoietic stem cells derived of X-SCID mice, as a model for SB-mediated gene therapy of X-SCID. Successful accomplishment of these goals will provide evidence for the effectiveness of the SB system to mediate non-viral gene transfer in murine hematopoietic stem cells (previously undemonstrated), as well as provide key preclinical data for the development of SB transposons for X-SCID as a lead primary immunodeficiency. PUBLIC HEALTH RELEVANCE: Results from these experiments will provide the technical basis for achieving transposon-mediated integration and long-term expression in hematopoietic stem cells, the cellular target inr gene therapy of primary immunodeficiencies. In addition, it will provide the basis for the development of the SB transposon system for treatment of other hematologic diseases.