Facilitate by-stander effects by EV-mRNA cargo in AAV gene therapy for MPS IIIC

在 MPS IIIC 的 AAV 基因治疗中通过 EV-mRNA 货物促进旁观者效应

• 批准号: 10040153
• 负责人: HAIYAN FU
• 金额: $ 42.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040153
• 关键词: Address; Binding Proteins; Cell Culture Techniques; Cells; Code; Data; Development; Disease; Dose; Enzymes; Family; Fibroblasts; Funding Mechanisms; GAG Gene; Gene Delivery; Gene Transduction Agent; Goals; Human; In Situ; In Vitro; Lysosomal Storage Diseases; Mediating; Membrane Proteins; Messenger RNA; Nervous system structure; Patients; Plant Roots; Plasmid Cloning Vector; Plasmids; Production; Property; Proteins; Recombinant adeno-associated virus (rAAV); Ribosomes; Signal Transduction; Signaling Protein; Skin; Testing; Therapeutic; Transgenes; Translations; Vectorial capacity; Viral Vector; adeno-associated viral vector; cellular transduction; clinical application; experience; extracellular vesicles; gene therapy; high risk; human disease; in vitro testing; intercellular communication; neurogenetics; neuropathology; novel; novel strategies; protein expression; scale up; success; tool; vector

Project Summary MPS IIIC is a devastating lysosomal storage disease (LSD), for which there is no definitive treatment. To address this unmet need, this proposed project is to develop a novel effective gene therapy product for treating MPS IIIC using AAV vector to target the root cause. Notably, the neuropathologies in MPS IIIC are global, involving the entire nervous system, and HGSNAT is a trans-lysosomal-membrane protein, which, unlike the majority of lysosomal enzymes, cannot be secreted and taken-up by neighboring cells. Therefore, optimal therapeutic benefits requires targeting as many cells as possible, which can be achieved by high-dose vector delivery, such as recently approved Zolgensma for SMA.25 In comparison, studies have demonstrated that gene therapy for MPS disorders involving a secretable protein required significantly lower vector doses. The high vector doses has been a major challenge in translation of AAV gene therapy due to the scale-up capacity of vector production for human application. All cells are known to continuously release extracellular vesicles (EVs) and communicate by exchanging large molecules via EV traffic. The demonstrated properties of EV intercellular interaction offer a potential tool to ease this challenge to translation of rAAV gene therapy. We hypothesize that incorporation of EV-mRNA packaging signals in gene therapy vectors will engender by-stander effects for normally non-secreted protein via the mRNA-containing EVs. To test this hypothesis, we will construct rAAV vectors that express hHGSNAT and contain different putative EV packaging signals, and identify effective signals for EV-mRNA loading. The goal is to develop an effective rAAV-hHGSNAT gene delivery approach for treating MPS IIIC. Data generated may also provide powerful tools for developing gene therapy to treat other diseases involving non- secreted transgene products. We believe this project is ideally suited to the R21 high risk/high impact funding mechanism.

项目摘要 MPS IIIC是一种毁灭性的溶酶体贮积病（LSD），目前尚无明确的治疗方法。解决 针对这一未满足的需求，本拟议项目旨在开发一种新型有效的基因治疗产品，用于治疗MPS IIIC 使用AAV载体来针对根本原因。值得注意的是，MPS IIIC中的神经病理学是全球性的，涉及 HGSNAT是一种跨溶酶体膜蛋白，与大多数神经系统不同， 溶酶体酶不能被分泌并被邻近细胞吸收。因此，最佳治疗 益处需要靶向尽可能多的细胞，这可以通过高剂量载体递送来实现，例如 25相比之下，研究表明， 涉及分泌性蛋白质的MPS疾病需要显著较低的载体剂量。高载体剂量 由于载体生产的规模化能力， 用于人类应用。已知所有细胞都持续释放细胞外囊泡（EV）并进行通信。 通过EV交通交换大分子。EV细胞间相互作用的特性提供了一个 这是一个潜在的工具，可以缓解rAAV基因治疗翻译的挑战。我们假设， 基因治疗载体中的EV-mRNA包装信号将对正常非分泌的EV-mRNA产生旁观者效应。 蛋白质通过含有mRNA的EV。为了验证这一假设，我们将构建表达 hHGSNAT和含有不同推定的EV包装信号，并鉴定EV-mRNA的有效信号 加载中目标是开发用于治疗MPS IIIC的有效rAAV-hHGSNAT基因递送方法。数据 产生的基因也可能为开发基因疗法提供强有力的工具，以治疗其他涉及非肿瘤的疾病。 分泌的转基因产物。我们相信这个项目非常适合R21高风险/高影响力资金 机制