Approach for in vivo gene delivery into hematopoietic stem cells for hemophilia A therapy

摘要： 我们将测试一种生产体内血细胞分泌的治疗性蛋白的新方法 基因导入造血干细胞(HSCs)。这一方法包括动员来自 骨髓后一次静脉注射整合辅助细胞依赖的HDAd5/35++ 腺病毒载体。在外周血中转导的HSCs返回骨髓，在那里它们持续存在 长期的。转基因整合是通过使用转座酶以随机模式实现的，或者在一个地点- 特定的模式，通过同源引导的DNA修复机制。对于一种秘密的转基因产品，我们 将重点放在体内将人第VIII因子在红细胞中表达后，将第VIII因子基因转移到HSCs。在……里面 与目前使用的rAAV介导的肝脏导向血友病基因治疗相比，我们的技术简单且 成本效益高的方法有可能终身治愈，诱导对第八因子的耐受性。 目的：1.提高基于转座酶的HDAd5/35++体内HSC的有效性和安全性 通过优化动员和载体注射方案以及体内HSC进行转导 扩大或选择机制。2.针对定向集成测试新的HDAd5/35++载体系统， 包括携带CRISPR-Cas9以产生位点特异性DNA断裂和同源性的载体 用于集成的模板。3.在血友病A小鼠模型中测试最好的体内HSC转导系统。 4.在非人类灵长类动物身上进行试点安全性和有效性研究，这是最合适的模型 未来在人类身上的潜在研究。拟议的为期6个月的重复采血和骨髓研究 采样将使我们能够预测对造血的潜在长期副作用，并跟踪 随着时间的推移，基因编辑的造血干细胞。

ABSTRACT: We will test a new approach for the production of therapeutic proteins secreted from blood cells after in vivo gene delivery into hematopoietic stem cells (HSCs). This approach involves the mobilization of HSCs from the bone marrow followed by a single intravenous injection of integrating helper-dependent HDAd5/35++ adenovirus vectors. HSCs transduced in the peripheral blood return to the bone marrow where they persist long-term. Transgene integration is achieved either in a random pattern using a transposase or, in a site- specific pattern, through homology-directed DNA repair mechanisms. For a secreted transgene product, we will focus on human factor VIII expressed in erythrocytes after in vivo factor VIII gene transfer into HSCs. In contrast to currently used rAAV-mediated liver-directed hemophilia gene therapy, our technically simple and cost-efficient approach has the potential for a life-long cure with induction of tolerance to factor VIII. The Specific Aims are 1. Increase the efficacy and safety of transposase-based HDAd5/35++ in vivo HSC transduction through optimization of mobilization and vector injection regimens and through HSC in vivo expansion or selection mechanisms. 2. Test new HDAd5/35++ vector systems for targeted integration, including a vector that carries both a CRISPR-Cas9 to create site-specific DNA breaks and the homology template for integration. 3. Test the best in vivo HSC transduction system in a mouse model for hemophilia A. 4. Perform a pilot safety and efficacy study in non-human primates, which are the most adequate model for potential future studies in humans. The proposed 6-month study with repeated blood and bone marrow sampling will allow us to predict potential long-term side effects on hematopoiesis and follow the expansion of gene-edited HSCs over time.