Therapeutic gene delivery with Sleeping Beauty transposon vectors: Assessment of preclinical efficacy and safety in a mouse model of Gaucher disease

高谢病(GD)是一种最常见的溶酶体储存性疾病，由遗传突变导致酶β-葡萄糖苷酶(GCase)缺陷引起。临床上可以区分三种不同的亚型：GDI型(有内脏症状)和II/III型(有中枢神经系统症状)。虽然酶替代疗法至少在一定程度上改善了GDI型的症状，但一些患者反应迟钝，缺乏合适的骨髓捐赠者。目前尚无治疗GD神经病亚型的方法。基因治疗方法潜在地提供了一种独特的可能性，通过解决疾病的实际遗传原因来提供终身纠正疾病的方法。在这个项目中，非病毒睡美人(SB)转座子系统的有效性和生物安全性将在一个非神经病理性GD小鼠模型(GD1小鼠)中进行稳定的GCase缺陷造血干细胞(HSCs)遗传修饰评估，该模型显示出正常寿命和相对缓慢的疾病进展。我们的目标是通过将SB转座子系统与微环技术相结合来改善我们的非病毒治疗性基因输送到HSCs中，该技术允许通过删除传统质粒DNA载体中存在的细菌骨架序列来显著减少载体大小。这种方法不仅有望提高基因矫正的效率，而且还有助于在基因治疗应用的背景下进一步提高非病毒基因传递的生物安全性。在这项概念验证研究中，我们还将探讨酶进入中枢神经系统的可行性，这是成功应用该策略治疗严重神经病变形式的疾病的先决条件。为此，我们将通过一种先进的载体设计来挑战我们的临床前基因治疗方法，在这种设计中，与GCase耦合的分泌信号有望在从植入骨髓的经过基因修正的HSCs中表达后，使其能够在全身分布。此外，连接到该酶C-末端的载脂蛋白B-和载脂蛋白E结构域有望促进其通过血脑屏障的转移，从而促进其脑特异性递送。移植了转基因HSCs的GD1小鼠将在植入、基因标记和载体插入位点分析、转基因表达和生物分布以及内脏器官表型等方面得到彻底的表征。该项目有望显著加快GD基因治疗安全临床前方案的开发和临床转化。

Gaucher disease (GD) is the most common lysosomal storage disease caused by inherited mutations that result in a deficiency of the enzyme beta-glucorebrosidase (GCase). Clinically, three different subtypes can be distinguished: GD type I (with visceral symptoms), and II/III (with symptoms affecting the central nervous system). While enzyme replacement therapy offers at least some improvement of symptoms of GD type I, some patients are unresponsive and lack a suitable bone marrow donor. There are no treatment options available for the neuronopathic subforms of GD. A gene therapy approach potentially offers a unique possibility to provide life-long correction of disease by tackling their actual genetic cause. In this project, efficacy and biosafety of the non-viral Sleeping Beauty (SB) transposon system will be assessed for stable genetic modification of GCase-deficient hematopoietic stem cells (HSCs) in a non-neuronopathic GD mouse model (GD1 mice), which exhibits normal lifespan and relatively slow disease progression. We aim at improving our non-viral therapeutic gene delivery into HSCs by combining the SB transposon system with minicircle technology, which allows for a significant reduction in vector size by deleting bacterial backbone sequences present in conventional plasmid DNA vectors. This approach is expected not only to enhance the efficacy of genetic correction, but it also serves to further improve the biosafety of non-viral gene delivery in the context of a gene therapy application. In this proof-of-concept study, we will also address the feasibility of enzyme delivery into the central nervous system, which is a prerequisite for a successful application of the strategy for the severe neuronopathic forms of the disease. For that purpose, we are going to challenge our preclinical gene therapy approach by an advanced vector design, in which a secretion signal coupled to GCase is expected to enable its systemic distribution after being expressed from genetically corrected HSCs engrafted in the bone marrow. In addition, apolipoprotein B- and apolipoprotein E domains attached to the C-terminus of the enzyme are expected to promote its transfer through the blood-brain-barrier and hence its brain-specific delivery. GD1 mice transplanted with genetically corrected HSCs will be thoroughly characterized with respect to engraftment, gene marking and vector insertion site analysis, transgene expression and biodistribution and visceral organ phenotyping. This project is expected to significantly accelerate the development and clinical translation of safe preclinical protocols for GD gene therapy.