Transposon-Mediated Gene Therapy for Fanconi Anemia

描述（由申请人提供）：范可尼贫血（FA）是一种罕见的常染色体隐性遗传疾病，由缺乏调节DNA修复的八种不同蛋白质中的任何一种引起。受影响的人在生命早期遭受骨髓衰竭。异体造血干细胞移植可有效治疗FA。然而，大多数患者缺乏匹配的相关供体，并且使用非相关来源的干细胞移植后发病率和死亡率显着增加。Discovery Genomics, Inc. （DGI）正在开发其睡美人（SB）转座子系统，作为FA基因治疗的非病毒载体。双组分SB系统由转座子（治疗基因两侧的反向重复序列）和转座酶组成，该转座酶催化IR末端转座子的切除，然后整合到宿主细胞染色体序列中。在I期研究中，DGI成功地将转座子DNA和RNA装载到培养的造血细胞中，然后通过sb介导的转座子对Fanconi贫血补体C组（Fanconi anemia complementation group C, fanc）缺陷的人淋巴母细胞样细胞进行了纠正。在这个II期申请中，总体目标是提供关键的临床前数据，这些数据将是支持sb介导的基因治疗FA的预期疗效所必需的。在这方面，有两个关键问题需要解决；(i) sb介导的FANC基因插入治疗FA动物模型的有效性如何？（ii）对于人体试验，将编码fnc的转座子DNA引入人造血干细胞（HSC）的临床适用方法是什么？有两个具体目标：目标1。评价睡美人转座子介导的fnc - c基因在fnc - c缺陷小鼠造血干细胞中的整合和长期表达，作为sb介导的基因治疗范可尼贫血的模型。目标2。评估睡美人转座子介导的人类CD34+造血干细胞整合和长期表达。为了实现这两个目标，我们将使用Cyto Pulse电穿孔技术将SB转座子DNA和转座子酶编码序列引入HSC。这些实验结果将为在FA基因治疗的目标细胞群造血干细胞中实现转座子介导的整合和长期表达提供技术基础，也将为利用SB转座子系统开发FA基因治疗中的DGI生长提供商业基础。该项目描述了由小型生物技术初创公司Discovery Genomics， Inc. （DGI）开发的一种非病毒基因转移方法，用于治疗范可尼贫血（一种罕见的血液疾病）。使用DGI的转座子系统成功治疗范可尼贫血，旨在为该技术在治疗其他血液疾病方面的潜在应用提供原理证明。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a rare, autosomal recessive inherited disease caused by the absence of any of eight different proteins that regulate DNA repair. Affected individuals suffer from bone marrow failure early in life. FA can be effectively treated by allogeneic hematopoietic stem cell transplant. However, most patients lack a matched related donor, and there is a marked increase in morbidity and mortality following transplant using stem cells from an unrelated source. Discovery Genomics, Inc. (DGI) is developing its Sleeping Beauty (SB) transposon system as a non- viral vector for gene therapy of FA. The two-component SB system consists of a transposon (inverted repeats flanking a therapeutic gene of interest) and a transposase that catalyzes excision of the transposon at the ends of the IR's and then integration into host cell chromosomal sequence. In Phase I studies, DGI demonstrated the successful loading of transposon DNA and RNA into cultured hematopoietic cells, and then correction of human lymphoblastoid cells deficient in Fanconi anemia complementation group C (FANC-C) by SB-mediated transposition. In this Phase II application, the overall goal is to provide key preclinical data that will be necessary to support the anticipated efficacy of SB-mediated gene therapy for FA. In this regard, there are two key questions that will need to be addressed; (i) What is the effectiveness of SB-mediated FANC gene insertion in the treatment of an animal model of FA? (ii) For the human trial, what is the clinically applicable methodology that will be used for introduction of FANC-encoding transposon DNA into human hematopoietic stem cells (HSC)? There are two Specific Aims: Aim 1. Evaluate Sleeping Beauty transposon-mediated integration and long-term expression of the FANC-C gene in hematopoietic stem cells of FANC-C deficient mice as a model for SB-mediated gene therapy for Fanconi anemia. Aim 2. Evaluate Sleeping Beauty transposon-mediated integration and long-term expression in human CD34+ hematopoietic stem cells. For both of these Aims, SB transposon DNA and transposase-encoding sequence will be introduced into HSC using Cyto Pulse electroporation technology. Results from these experiments will provide the technical basis for achieving transposon-mediated integration and long-term expression in hematopoietic stem cells, the cell population that will be targeted for FA gene therapy, and will also provide the commercial basis for growth of DGI in the development of FA gene therapy using the SB transposon system. This project describes the development of a non-viral gene transfer method for treatment of Fanconi anemia, a rare disorder of the blood, by Discovery Genomics, Inc. (DGI), a small biotech startup company. Successful treatment of Fanconi anemia using DGI's transposon system is intended to provide proof of principle for the potential application of this technology to the treatment of other disorders of the blood as well.