新AAV血清型的开发与基因治疗应用

Adeno-associated virus (AAV) is an important vector system for human gene therapy to treat a variety of human diseases. In recent years, AAV-mediated gene replacement, gene silencing and gene editing have been successfully applied to preclinical and clinical research, with two AAV-based gene therapy drugs gaining regulatory approval in Europe or the United States, which helped AAV gain popularity as the ideal therapeutic vector. Although use of AAV serotypes can result in efficient gene transfer, improvements in the transduction efficiency and specificity are still required. Another extrinsic factor that limits the clinical impact of AAV-based gene therapy is the prevalence of pre-existing neutralizing antibodies (NAbs) to AAVs due to natural infection approximately 80% in the human population. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. The project attempt to screen an AAV capsid with high transduction efficiency, specificity in liver and low Nab titer in human population by evolving the AAV using DNA shuffling and in vivo biopanning in a liver hepatocellular cell model. We further apply the bioengineered capsid, harnessing Synthetic biology technology, to construct gene therapy vector for Hemophilia B with high gene expression efficiency and broad application in human population.

腺相关病毒（AAV）是基因治疗研究中重要的载体系统，可以用于治疗多种人类疾病。近些年来，AAV载体平台成功应用于临床前和临床试验中基因替代、基因沉默和基因编辑研究；在欧洲和美国，已有两种基于AAV的药物获批上市，这使得AAV成为基因治疗研究中最普遍使用的基因递送平台。但尽管多种AAV血清型可以实现有效的基因递送，其基因转导效率和组织特异性仍需进一步提高。此外，由于80%人群具有感染过AAV的经历，多种AAV血清型在人体中具有较高水平中和抗体存在，该问题也限制了AAV用于人体基因治疗的适用范围。生物定向进化技术是酶和蛋白质基因工程改造研究中最有效策略之一。本项目旨在通过DNA改组技术并以人源肝癌细胞为生物演变模型，对AAV进行定向进化，筛选高转导效率、组织特异性强和低人体中和抗体比例的新型AAV衣壳，并结合合成生物学技术，开发高基因表达效率、适用人群范围广泛的B型血友病基因治疗载体。

腺相关病毒是基因治疗研究中最具有应用前景的病毒载体，全球已有多个AAV载体药物上市。但天然AAV血清型应用于基因治疗临床试验时，往往存在转导效率不足，预存抗体比例较高等问题，不仅影响疾病治疗效果，还会限制药物受众人群。为解决腺相关病毒载体的缺陷，在本基金支持下，本项目利用DNA 改组技术和定向进化技术成功筛选2种在人源刚脏细胞中高转导效率的新AAV血清型，并且同时具有低人体中和抗体比例的优点。这两种血清型具有广泛的基因治疗应用前景。本项目还通过合成生物学方法设计了全新的凝血因子9（F9）基因表达盒，结合我们筛选的AAV843血清型，可以在血友病B模型小鼠中高效、稳定表达F9蛋白，进而治疗血友病B。本项目开发的血友病B基因治疗药物已经成功用于人体临床试验，显示出良好的安全性和血友病B治疗效果，因此该药物有望造福国内广大血友病B患者。

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