Bioengineering of phage-derived particles as a discovery platform for muscle gene therapy

项目摘要 基因治疗为患有遗传性疾病的患者提供了功能性治愈，并且特别与遗传性疾病相关。 治疗单基因罕见疾病（由单一基因引起的疾病），如杜氏肌营养不良症 （DMD）。腺相关病毒（AAV）是最流行和临床上最成功的病毒基因治疗。 然而，作为一种递送平台，当涉及免疫原性、货物运输能力和运输能力时，它表现出显著的局限性。 容量、可制造性和细胞类型特异性靶向的容易性。其他平台，如脂质纳米颗粒， cle/mRNA制剂可以大规模生产，但仅诱导非靶向和瞬时基因表达， 锡永。因此，虽然每种平台都有其优点和缺点，但目前还没有一个平台可以 为罕见的遗传性疾病带来了可获得和持久的基因治疗。 M13-Tx的目标是开发下一代体内基因递送平台，以解决当前的基因递送问题。 现有基因治疗技术的主要局限性。该平台基于噬菌体衍生颗粒（PDP） - 一种免疫豁免的、容易工程化的、有效产生的M13噬菌体衍生物-- spe-- 设计用于人类细胞转导的细胞。通过定向进化和理性进化的结合， 标志，这些PDP可以被改造为靶向任何组织，并提供高达20 kb的DNA货物。 在第一阶段SBIR中，M13-Tx建议设计和筛选PDP，以成功部署货物运载系统。 体内肌肉组织中编码肌营养不良蛋白的DMD基因的功能拷贝。相比于现有 AAVs只能包装DMD编码序列的一小部分，而PDP将部署完整的DMD基因 仅在肌肉组织中进行持久和可重复剂量的杜氏肌营养不良症治疗。 该提案的最终产品是铅PDP，准备在DMD小鼠模型中进行测试。主要PDP 肌肉特异性部署可用于治疗由不同基因突变引起的罕见疾病 但表现在相同的组织中（例如，强直性肌营养不良和面肩肱肌营养不良）。 此外，相同的工作流程可以用于找到其他难以靶向的组织（例如，肺和 CNS）。拟议的研究将作为一个概念验证，表明PDP可以选择特定的 基因传递的目的，考虑到他们的工程外套和容易修改的货物。开发这项技术 将通过提供一个多功能和负担得起的基因输送平台来彻底改变基因治疗。

PROJECT SUMMARY Gene therapy provides a functional cure for patients with genetic disorders and is particularly relevant to the treatment of monogenic rare diseases (those caused by a single gene), such as Duchenne muscular dystrophy (DMD). Adeno-associated virus (AAV) is the most prevalent and clinically successful viral gene therapy. However, as a delivery platform, it exhibits significant limitations when it comes to immunogenicity, cargo ca- pacity, manufacturability, and ease of cell type-specific targeting. Other platforms, such as lipid nanoparti- cle/mRNA formulations, can be manufactured at scale but only induce untargeted and transient gene expres- sion. Therefore, while each has its strengths and weaknesses, there is currently no platform available that brings accessible and durable gene therapy to rare genetic disorders. The goal of M13-Tx is to develop a next-generation in vivo gene delivery platform that addresses the current key limitations of existing gene therapy technologies. This platform is based on phage-derived particles (PDPs) – an immune-privileged, easily engineerable, and efficiently produced derivation of M13 bacteriophage – spe- cifically designed for the transduction of human cells. By the combination of directed evolution and rational de- sign, these PDPs can be engineered to target any tissue and deliver up to 20kb DNA cargo. In this Phase I SBIR, M13-Tx proposes to engineer and screen PDPs to successfully deploy a cargo carrying the functional copy of DMD gene coding for dystrophin protein in muscle tissue in vivo. In contrast to existing AAVs, which can only pack a fraction of the DMD coding sequence, the PDPs will deploy the full DMD gene only in muscle tissue for durable and redoseable treatment of Duchenne muscular dystrophy. The final product of this proposal is lead PDPs, ready to be tested in the DMD mouse model. The lead PDPs and muscle-specific deployment can be utilized across rare diseases caused by mutations of different genes but manifested in the same tissue (e.g., Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy). Moreover, the same workflow can be used to find PDPs for other difficult-to-target tissues (e.g., lung and CNS). The proposed study will serve as a proof-of-concept, showing that PDPs can be selected for specific gene delivery purpose, given their engineerable coat and easily modified cargos. Developing this technology will revolutionize gene therapy by presenting a versatile and affordable gene delivery platform.