Improving AAV-transduction efficiencies for skeletal muscle delivery

项目摘要 腺相关病毒（AAV）是最常用的基因治疗载体。目前，FDA已 批准了两种AAV介导的疗法，还有更多的疗法正在酝酿中。然而， 有效利用AAV载体的挑战。例如，高剂量（~1014个载体颗粒） 这是大多数人类应用所必需的。这样高的载体剂量引起组织毒性并引起高的 抗转基因抗体的水平，限制了治疗效果。高剂量也需要大量注射 体积和/或多次注射以实现所需的转基因表达水平。此外，高 制造成本排除了这种方法的广泛使用。这些困难中的每一个都可以 通过增加AAV转导的效率并由此减少AAV转导的数量来解决。 实现所需治疗结果所需的颗粒。 载体递送的肌内途径具有关键优势。它有助于避免预- 现有的抗体的AAV衣壳，否则阻止有效的转导。表达 来自肌肉的转基因由于该组织的寿命而持续数年或数十年。最后，不像 靶向表达AAV转基因的其他组织，如肝脏， 通过局部炎症造成的损害是适度的，并且可以处理。然而，相对 AAV转导人和非人灵长类动物肌肉组织的效率低， 更高的载体剂量。因此，基于AAV的疗法的许多挑战可以通过以下方式解决： 提高肌肉特异性转导的效率。 在初步研究中，我们表明在可变区VIII中用胰岛素修饰的AAV 9 受体结合肽在转导人类细胞中比野生型AAV 9显著更有效。 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 载体通过定向进化和选择，并评估这种肽在 其他AAV血清型。我们还在目标2中提出通过增强向量来验证我们的假设， 效率，我们可以减少组织毒性，炎症和抗转基因抗体的产生，从而 增加这些载体的安全性和治疗功效。总之，这些研究将确定一个 AAV载体，更有效地促进肌肉组织，在这样做的时候，他们也将解决 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.