Development of optimized adeno-associated viral capsids for muscle gene therapy

项目摘要。基因疗法为许多罕见肌肉和神经肌肉疾病患者带来了希望 疾病尽管取得了一些早期的成功，但几个严重的脱靶安全问题已经损害了他们的 由于肝脏毒性和腺相关病毒（腺相关病毒）的相关免疫反应而导致的发展 向量。因此，具有上级肌肉特异性的新型衣壳的产生可以彻底改变 肌肉基因治疗的空间，避免脱靶效应，损害药物的疗效和安全性。我们 本发明的目的是工程化具有超过当前“肌肉嗜性”的肌肉嗜性的新AAV衣壳， 血清型（例如AAV 6、AAV 8、AAV 9、AAVrh 74和MyoAAV），因为没有一种实际上是“肌肉特异性的”。事实上， 这些血清型可感染其它组织，尤其是肝脏，其在功能上与所述临床 毒性事实上，肝脏的功能是作为这些载体的接收器，限制肌肉转导并提高 最低有效剂量。我们假设，在增强肌肉的同时， 向性是提高肌肉基因治疗安全性和有效性的关键。其他团体也试图加强 定向进化的肌肉向性。这种高通量方法人工选择具有改善的 肌肉向性，但也不能去靶向肝脏。相比之下，我们将使用合理的设计方法， 同时靶向已知表位用于肝脏去靶向，增强AAV-受体结合和改善 肌肉靶向这些包括用于改善唾液酸/AAV受体结合、受损的肝脏和/或肝细胞的那些。 靶向/硫酸肝素结合、改善的整联蛋白结合和具有组合性质的衣壳。我们还将 使用AAV 6作为肝和肌肉靶向表位对于该血清型是已知的，但对于其它血清型不是。 里程碑1将使用良好建立的体内和免疫组织化学方法开发肝脏去靶向/肌肉靶向AAV 6衣壳。 体外成像方法。里程碑2将通过比较Smad 7肌肉基因来证明功能功效。 在一个实施方案中，本发明涉及一种具有野生型AAV 6（AVGN 7）的治疗剂与具有新的优化的AAV 6衣壳的治疗剂的组合。这些研究 可以理解的高风险，但它们的重要性不成比例地高得多，因为它们会造成肝脏损伤， 具有改进的肌肉嗜性的靶向衣壳，并因此具有非常优越的上级安全性和功效特性。这 将主要通过减少制造负担， 将药物使用限制在更年轻或更少的患者中，从而降低了整体治疗成本，同时扩大了 潜在患者治疗的数量。

PROJECT SUMMARY. Gene therapeutics offer hope to many patients with rare muscle and neuromuscular diseases. Despite some early successes, several serious off-target safety concerns have compromised their development due to hepatic toxicities and related immune responses to the adeno-associated viral (AAV) vectors. The generation of novel capsids with superior muscle specificity could, therefore, revolutionize the muscle gene therapy space by avoiding the off-target effects that compromise drug efficacy and safety. Our objective is to engineer novel AAV capsids with muscle tropism that exceeds the current “muscle tropic” serotypes (e.g. AAV6, AAV8, AAV9, AAVrh74 & MyoAAVs) as none is actually “muscle-specific”. Indeed, all of these serotypes can transduce other tissues, especially the liver, which is functionally linked to the noted clinical toxicities. In fact, the liver functions as a sink for these vectors, limiting muscle transduction and elevating the minimally effective dose. We hypothesize that de-targeting the liver while simultaneously enhancing muscle tropism is key to improving muscle gene therapy safety and efficacy. Other groups have sought to enhance muscle tropism using directed evolution. This high throughput method artificially selects capsids with improved muscle tropism, but cannot also de-target the liver. By contrast, we will use a rational design approach to simultaneously target known epitopes for liver de-targeting, enhanced AAV-receptor binding and improved muscle targeting. These include those for improved sialic acid/AAV-receptor binding, impaired liver targeting/heparin sulfate binding, improved integrin binding and capsids with combined properties. We will also use AAV6 as liver- and muscle-targeting epitopes are known for this serotype, but not for the other serotypes. Milestone 1 will develop liver de-targeted/muscle targeted AAV6 capsids using well-established in vivo and in vitro imaging approaches. Milestone 2 will demonstrate functional efficacy by comparing a Smad7 muscle gene therapeutic featuring a wild-type AAV6 (AVGN7) to one with a novel optimized AAV6 capsid. These studies are understandably high risk yet their significance is disproportionately much higher as they will create a liver de- targeted capsid with improved muscle tropism and as a result, vastly superior safety and efficacy profiles. This would substantially innovate the muscle gene therapy space primarily by reducing a manufacturing burden that limits drug use to younger or fewer patients and thus, reducing the overall treatment costs while expanding the number of potential patient therapies.