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

戈谢病（GD）是最常见的溶酶体贮积病，由导致β-葡萄糖苷脂酶（GCase）缺乏的遗传突变引起。在临床上，可以区分三种不同的亚型：GD I型（具有内脏症状）和II/III型（具有影响中枢神经系统的症状）。虽然酶替代疗法至少可以改善GD I型的症状，但有些患者反应迟钝，缺乏合适的骨髓供体。对于GD的神经元病亚型没有可用的治疗选择。基因治疗方法潜在地提供了一种独特的可能性，通过解决其实际遗传原因来提供终身的疾病矫正。在该项目中，将评估非病毒睡美人（SB）转座子系统在非神经元病GD小鼠模型（GD 1小鼠）中对GCase缺陷型造血干细胞（HSC）进行稳定遗传修饰的有效性和生物安全性，该模型表现出正常的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递送到HSC中，所述微环技术通过删除常规质粒DNA载体中存在的细菌骨架序列来显著减小载体大小。这种方法不仅有望提高遗传校正的功效，而且还有助于进一步提高基因治疗应用背景下非病毒基因递送的生物安全性。在这项概念验证研究中，我们还将讨论将酶递送到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形式的先决条件。为此，我们将通过先进的载体设计来挑战我们的临床前基因治疗方法，其中与GCase偶联的分泌信号预期在从移植到骨髓中的遗传校正的HSC表达后能够使其全身分布。此外，载脂蛋白B-和载脂蛋白E结构域连接到酶的C-末端，预计将促进其通过血脑屏障的转移，从而促进其脑特异性递送。移植有遗传校正的HSC的GD 1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方面进行彻底表征。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.