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

摘要： 我们将测试一种新的方法，用于生产治疗性蛋白质分泌的血细胞后，在体内 基因递送到造血干细胞（HSC）中。这种方法涉及从造血干细胞中动员造血干细胞。 骨髓，然后单次静脉注射整合辅助因子依赖性HDAd 5/35++ 腺病毒载体在外周血中转导的HSC返回骨髓，在那里它们持续存在。 长期的转基因整合可以通过转座酶以随机模式实现，也可以通过转座酶以定点模式实现。 特定的模式，通过同源定向的DNA修复机制。对于分泌的转基因产物，我们 将集中在体内因子VIII基因转移到HSC后在红细胞中表达的人因子VIII。在 与目前使用的rAAV介导的肝定向血友病基因治疗相反，我们的技术简单， 具有成本效益的方法具有终身治愈的潜力，并诱导对因子VIII的耐受性。的 具体目标是1。提高基于转座酶的HDAd 5/35++在体内HSC的有效性和安全性 通过优化动员和载体注射方案以及通过体内HSC进行转导 扩展或选择机制。2.测试新的HDAd 5/35++载体系统的靶向整合， 包括携带CRISPR-Cas9以产生位点特异性DNA断裂和同源性的载体， 集成的模板。3.在血友病A小鼠模型中测试最佳体内HSC转导系统。 4.在非人灵长类动物中进行初步安全性和有效性研究，这是最适当的模型， 未来在人类身上的潜在研究。拟定的6个月重复血液和骨髓研究 采样将使我们能够预测对造血的潜在长期副作用，并跟踪 基因编辑的HSC。

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.