Improving delivery of therapeutic material to skeletal muscle

项目摘要/摘要 尽管基因治疗取得了进展，但将治疗材料输送到特定组织仍然是一个挑战。 这项建议通过设计新的车辆来解决基因治疗领域中长期存在的递送问题。 肌肉组织。我们应对这一挑战的方法是利用直接控制蛋白质的活性 成肌细胞融合，是多核骨骼肌纤维发育、修复和修复所必需的过程 重生。成肌细胞膜与肌纤维膜的融合允许祖细胞进入 合胞体，这是一个过程，如果对分子的理解和适当的工程，可以使交付 传递肌肉的交通工具。我们将利用我们对Myomaker和Mymer的发现，这两个发现代表了 成肌细胞融合的最小和必要的机制，将被包裹的病毒和外切体工程到 将治疗材料输送到肌肉细胞的特定和高效的载体。挑战的证据 与肌肉基因治疗相关的是缺乏对肌肉等遗传性肌肉疾病的治疗 营养不良。腺相关病毒(AAV)是目前有效的骨骼肌基因治疗标准， 但该领域不得不调和一个明显无法瞄准肌肉干细胞的问题，这一目标可能需要 要实现可持续的纠正结果。这项建议的主要理由是 随着肌肉特异性融合原的发现，是时候重新审视非AAV载体的潜力了，例如 慢病毒载体和非病毒颗粒(外切体)作为递送载体，可用于AAV或 独立的。因为Myomaker和Mymer在成肌细胞的细胞表面起作用，以驱动 融合所必需的膜重塑过程，我们认为它们在病毒被膜和 外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Mymer，或这些蛋白质的区域，在病毒的包膜和表面 外显体。我们还将评估和优化这些工程车辆进入肌肉的能力 和非肌肉组织的体外和体内实验。在R33阶段，我们将验证送货车辆的使用 在R61阶段进行优化，以测试他们提供临床相关水平治疗材料的能力。 具体地说，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和改善肌肉 在dystrophin缺陷的mdx小鼠模型中发挥作用。总的来说，这项工作有望开辟一个新的领域 通过创新可用于多种用途的新型递送工具来研究再生医学 肌肉骨骼状况。

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.