Evolving High Potency AAV Vectors for Neuromuscular Genome Editing

摘要 重组腺相关病毒（AAV）已成为临床基因治疗的安全有效的载体 治疗应用，包括神经肌肉疾病的全身治疗，如脊髓性肌萎缩症 (SMA)、杜氏肌营养不良症（DMD）和巨轴突神经病（GAN）等。然而，在这方面， 使基因编辑疗法能够治疗人类疾病需要改进的系统， 在多种动物模型中以低全身性AAV载体剂量进行基因组编辑。此外，理想的靶细胞 基因编辑的类型，包括祖细胞，与常规基因递送的靶细胞不同。 vector载体has been optimized优化for.特别是神经肌肉组织中的基因组编辑具有挑战性。到目前为止， 尚未确定有效的非病毒递送载体用于肌肉骨骼中广泛的基因组编辑。 和神经组织。此外，在AAV载体的情况下，几个挑战 与基因组编辑器的递送相关的问题仍然存在。我们目前提议的理由是， 通过（i）在AAV嗜性中观察到的种属相关差异，（ii）存在预先存在的中和抗体 （iii）需要高的全身性AAV剂量以实现神经肌肉基因转移，（iv）载体 剂量相关毒性，如通过检测患者的肝转氨酶所示，和（v）独特的机会 以原位纠正祖细胞。为了解决这些问题，我们组建了一个跨部门的合作团队， 削减专业知识，并开发了全面和创新的方法，以发展高效能的AAV变体 用于系统性神经肌肉基因组编辑。具体来说，我们将集中精力发展高效能的AAV 使用适用于不同物种、组织的三层方法进行神经肌肉基因组编辑的载体 和细胞类型。

ABSTRACT Recombinant adeno-associated viruses (AAV) have emerged as safe and effective vectors for clinical gene therapy applications including systemic treatment of neuromuscular diseases such as Spinal Muscular Atrophy (SMA), Duchenne Muscular Dystrophy (DMD), and Giant Axonal Neuropathy (GAN) amongst others. However, enabling gene editing therapeutics to treat human disease requires improved systems that achieve effective genome editing at low systemic AAV vector doses in multiple animal models. Additionally, the ideal target cell types for gene editing, including progenitor cells, are distinct from the target cells that conventional gene delivery vectors have been optimized for. Genome editing in neuromuscular tissue, in particular, is challenging. To date, no effective non-viral delivery vehicles have been identified for widespread genome editing in musculoskeletal and neurological tissue following systemic administration. Further, in case of AAV vectors, several challenges pertinent to delivery of genome editors remain. The rationale for our current proposal stems from barriers posed by (i) species-related differences observed in AAV tropism, (ii) presence of pre-existing neutralizing antibodies to natural AAV, (iii) the need for high systemic AAV doses to achieve neuromuscular gene transfer, (iv) vector dose-related toxicity as indicated by detection of liver transaminases in patients, and (v) the unique opportunity to correct progenitor cells in situ. To address these aspects, we have assembled a collaborative team with cross- cutting expertise and developed a comprehensive and innovative approach to evolve high potency AAV variants for systemic neuromuscular genome editing. Specifically, we will focus our efforts on evolving high potency AAV vectors for neuromuscular genome editing using a three-tiered approach applied across different species, tissues and cell types.