Improving AAV-transduction efficiencies for skeletal muscle delivery

项目总结 腺相关病毒(AAV)是最常用的基因治疗载体。目前，FDA已经 批准了两种AAV介导的疗法，还有更多的正在筹备中。然而，仍然有 对有效利用AAV媒介的挑战。例如，高剂量(~1014个矢量粒子)是 这是大多数人类应用程序所必需的。如此高的媒介剂量会导致组织毒性并引发高 抗转基因抗体水平，限制了治疗效果。高剂量还需要大量注射 体积和/或多次注射以达到所需的转基因表达水平。此外，高 制造成本阻碍了这种方法的广泛使用。这些困难中的每一个都可能是 通过提高AAV转导的效率从而减少AAV的数量来解决 达到预期治疗效果所需的颗粒。 肌肉内传播媒介的途径具有关键优势。它有助于绕过前的问题 现有的甲型肝炎病毒衣壳抗体，否则会阻碍有效的转导。A的表达式 由于肌肉组织的长寿，来自肌肉的转基因可以持续数年或数十年。最后，不同于 其他靶向表达AAV转基因的组织，如肝脏，组织的后果 局部炎症造成的损害并不严重，也是可控的。然而，相对的 AAV转导人类和非人类灵长类肌肉组织的效率低下 高媒介剂量。因此，基于AAV的疗法面临的许多挑战可以通过 提高肌肉特异性转导的效率。 在初步研究中，我们发现AAV9在可变区VIII用胰岛素修饰 受体结合肽比野生型AAV9更有效地转导人 体内分化的肌肉细胞和小鼠肌肉。我们在目标1中建议进一步改进这一点 通过定向进化和选择，并评估该多肽在 其他AAV血清型。我们还在目标2中提出了通过增强向量来验证我们的假设 高效，我们可以减少组织毒性、炎症和抗转基因抗体的产生，从而 提高了这些载体的安全性和治疗效果。总之，这些研究将确定一种 更有效地转导肌肉组织的AAV载体，在这样做的过程中，他们还将解决 与AAV介导的基因治疗相关的几个挑战。

PROJECT SUMMARY Adeno-associated virus (AAV) is the most commonly used gene therapy vector. Currently, FDA has approved two AAV-mediated therapies, and many more are in pipeline. However, there still are challenges to the effective use of AAV vectors. For example, high doses (~1014 vector particles) are necessary for most human applications. Such high vector doses cause tissue toxicity and elicit high levels of anti-transgene antibodies, limiting therapeutic efficacy. High doses also require large injection volumes and/or multiple injections to achieved desired levels of transgene expression. In addition, high manufacturing costs preclude widespread use of this approach. Each of these difficulties can be addressed by increasing the efficiency of AAV transduction and thereby reducing the number of AAV particles necessary to achieve a desired therapeutic outcome. The intramuscular route of vector delivery has key advantages. It helps circumvent the problem of pre- existing antibodies to the AAV capsid, which otherwise prevents efficient transduction. Expression of a transgene from muscle persists for years or decades due the longevity of this tissue. Finally, unlike other tissues targeted to express AAV transgenes such as the liver, the consequences of tissue damage through local inflammation are modest and manageable to address. However, the relative inefficiency with which AAV transduces human and non-human primate muscle tissues necessitates high vector doses. Therefore many challenges to the AAV-based therapies can be addressed by improving the efficiency of muscle-specific transduction. In the Preliminary Studies, we show that AAV9 modified in the variable region VIII with an insulin receptor-binding peptide is dramatically more efficient than wild-type AAV9 in transducing human differentiated muscle cells and mouse muscles in vivo. We propose in Aim 1 to further improve this vector through directed evolution and selection, and to assess the utility of this peptide in the context of other AAV serotypes. We also propose in Aim 2 to verify our hypotheses that by enhancing vector efficiency, we can reduce tissue toxicity, inflammation, and anti-transgene antibody production, thereby increasing the safety and therapeutic efficacy of these vectors. Together, these studies will identify an AAV vector that much more efficiently transduce muscle tissue, and in doing so, they will also address several challenges associated with AAV-mediated gene therapy.