Improving delivery of therapeutic material to skeletal muscle

项目总结/摘要 尽管基因治疗取得了进展，但将治疗材料递送至特定组织仍然是一个挑战。 该提案通过设计新颖的载体来解决基因治疗领域长期存在的递送问题 用于肌肉组织。我们应对这一挑战的方法是利用蛋白质的活动， 成肌细胞融合是多核骨骼肌纤维发育、修复和 再生成肌细胞膜与肌纤维膜的融合允许祖细胞进入成肌细胞膜。 合胞体，这是一个过程，如果理解分子和适当的工程， 车辆到肌肉。我们将利用我们对Myomaker和Myomerger的发现， 成肌细胞融合的最小和必要的机制，工程包膜病毒和外来体， 特异性和有效的载体，将治疗材料递送到肌肉细胞。挑战的证据 与肌肉基因治疗相关的是缺乏对遗传性肌肉疾病的治疗， 营养不良腺相关病毒（AAV）是有效的骨骼肌基因治疗的当前标准， 但该领域不得不调和一个明显无法靶向肌肉干细胞的问题，这一目标可能需要 以获得持续的矫正效果。这项建议的主要理由是， 随着肌肉特异性融合基因的发现，现在是时候重新审视非AAV载体的潜力了， 慢病毒载体和非病毒颗粒（外来体）作为递送媒介物，其可以与AAV或 独立地。由于Myomaker和Myomerger在成肌细胞的细胞表面起作用， 融合所必需的膜重塑过程，我们提出，它们在病毒包膜上的存在， 外泌体将增加进入肌肉的机会。在该项目的R61阶段，我们将设计和优化 Myomaker和Myomerger，或这些蛋白质的区域，在病毒的包膜上和病毒的表面， 外来体我们还将评估和优化这些工程车辆的能力，以驱动进入肌肉 和非肌肉组织的体外和体内研究。在R33阶段，我们将验证交付工具的使用 在R61阶段进行优化，以测试其递送临床相关水平的治疗材料的能力。 具体来说，我们将确定我们优化的运输工具是否可以恢复肌营养不良蛋白并改善肌肉 在肌营养不良蛋白缺陷型mdx小鼠模型中的功能。总的来说，这项工作有望开辟一个新的领域， 通过创新可用于无数目的的新型运载工具来研究再生医学。 肌肉骨骼疾病

Project Summary/Abstract Despite advances in gene therapy, delivery of therapeutic material to a specific tissue remains a challenge. This proposal tackles the long-standing delivery issue in the gene therapy field by engineering novel vehicles for muscle tissue. Our approach to this challenge is to harness the activities of the proteins that directly control myoblast fusion, a process essential for multinucleated skeletal muscle fibers to develop, repair, and regenerate. Fusion of a myoblast membrane with a myofiber membrane allows entry of the progenitor into the syncytium, which is a process that, if understood molecularly and properly engineered, could empower delivery vehicles to transduce muscle. We will leverage our discoveries of Myomaker and Myomerger, which represent the minimal and essential machinery for myoblast fusion, to engineer enveloped viruses and exosomes into specific and efficient vehicles that deliver therapeutic material to muscle cells. Evidence for the challenges associated with muscle gene therapy is the lack of a treatment for genetic muscle diseases such as muscular dystrophy. Adeno-associated virus (AAV) is the current standard for efficacious skeletal muscle gene therapy, but the field has had to reconcile an apparent inability to target muscle stem cells, a goal that would likely need to be achieved for sustained corrective outcomes. The principal rationale of this proposal is that with the discovery of muscle-specific fusogens, it is time to re-examine the potential of non-AAV vectors such as lentiviral vectors and non-viral particles (exosomes) as delivery vehicles that could be used with AAV or independently. Because Myomaker and Myomerger function at the cell surface of myoblasts to drive the membrane remodeling processes necessary for fusion, we propose that their presence on viral envelopes and exosomes will increase entry into muscle. In the R61 phase of this project, we will engineer and optimize Myomaker and Myomerger, or regions of these proteins, on the envelope of viruses and the surface of exosomes. We will also assess and optimize the ability of these engineered vehicles to drive entry into muscle and non-muscle tissues in vitro and in vivo. In the R33 phase, we will validate the use of the delivery vehicles optimized in the R61 phase to test their ability to deliver clinically relevant levels of therapeutic material. Specifically, we will determine if our optimized delivery vehicles can restore dystrophin and improve muscle function in the dystrophin-deficient mdx mouse model. Overall, this work promises to open up a new area of investigation into regenerative medicine by innovating novel delivery vehicles that could be utilized for a myriad of musculoskeletal conditions.