Evolving High Potency AAV Vectors for Neuromuscular Genome Editing

摘要 重组腺相关病毒(AAV)是一种安全有效的临床基因表达载体 治疗应用包括系统治疗神经肌肉疾病，如脊髓性肌萎缩 Duchenne肌营养不良症(DMD)和巨大轴索神经病(GAN)等。然而， 使基因编辑疗法能够治疗人类疾病需要改进的系统，以实现有效 在多种动物模型中的低系统AAV载体剂量下的基因组编辑。此外，理想的目标细胞 基因编辑的类型，包括祖细胞，与传统基因传递的靶细胞不同 矢量已针对以下方面进行了优化。神经肌肉组织的基因组编辑尤其具有挑战性。到目前为止， 还没有确定在肌肉骨骼中广泛进行基因组编辑的有效的非病毒传递工具 以及全身给药后的神经组织。此外，在AAV载体的情况下，几个挑战 与基因组编辑的交付有关的问题仍然存在。我们目前提议的理由来自于所设置的障碍 通过(I)在AAV趋向性上观察到的物种相关差异，(Ii)先前存在的中和抗体的存在 对于自然AAV，(Iii)需要高系统AAV剂量以实现神经肌肉基因转移，(Iv)载体 通过检测患者的肝脏转氨酶表明的剂量相关毒性，以及(V)独特的机会 以原位纠正祖细胞。为了解决这些方面的问题，我们组建了一个协作团队，与 削减专业知识，并开发了一种全面而创新的方法来进化高效AAV变体 进行全身神经肌肉基因组编辑。具体地说，我们将把我们的努力集中在进化高效AAV上 用于神经肌肉基因组编辑的载体使用三层方法应用于不同物种、组织 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.