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，由遗传突变引起的，导致酶-葡萄糖醛酸苷酶（GCase）的缺乏。临床上可分为三种不同亚型：GD I型（伴有内脏症状）和II/III型（症状累及中枢神经系统）。虽然酶替代疗法至少在一定程度上改善了GD I型的症状，但一些患者反应迟钝，缺乏合适的骨髓供体。目前还没有针对GD的神经病变亚型的治疗方案。一种基因治疗方法有可能提供一种独特的可能性，通过解决疾病的实际遗传原因来提供终生的疾病纠正。在本项目中，将评估非病毒睡美人（SB）转座子系统对gcase缺陷造血干细胞（hsc）进行稳定遗传修饰的有效性和生物安全性，用于非神经病变GD小鼠模型（GD1小鼠），该模型具有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传递到造血干细胞，微环技术允许通过删除传统质粒DNA载体中存在的细菌骨干序列来显著减少载体大小。该方法不仅可以提高基因校正的有效性，还可以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，我们还将探讨将酶输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变形式疾病的先决条件。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，在这种载体设计中，一种与GCase偶联的分泌信号有望在移植到骨髓中的基因校正的造血干细胞中表达后实现其全身分布。此外，载脂蛋白B-和载脂蛋白E结构域附着在酶的c端，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。将对GD1小鼠移植经过基因校正的造血干细胞进行植入、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的全面表征。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.