Biology of Subgenomic AAV Vector Particles

项目摘要/摘要 血友病A是一种X连锁出血性疾病，由F8基因(编码凝血)的遗传缺陷引起 第八因子)，全世界大约每5000名男婴中就有一名受到影响。临床上，血友病A是 表现为严重出血表型，通常需要昂贵的蛋白质替代疗法。最近的 基因疗法的发展有望治愈血友病A，尽管临床试验的结果 使用AAV载体治疗血友病A一直是令人鼓舞的，腺相关病毒(AAV)介导的 FVIII尚未获得监管部门的批准。主要障碍依然存在，因为人们对 犬循环中FVIII水平的降低和肝细胞克隆性扩张的长期研究 模型，这引起了人们对使用AAV载体时的遗传毒性甚至癌症形成的担忧 基因疗法。目前的AAV-FVIII基因递送策略(AAV-F8)遵循标准设计，在该设计中，迷你 启动子用于驱动F8的表达，侧翼有两个ITR。然而，临床级别的载体产生了 对载体基因组进行Southern杂交分析，结果表明它们具有较好的同源性。然而，我们的新研究， 使用单分子测序(SMRT)，已经发现了载体种群的广泛复杂性 用传统的分析方法漏掉了。一个值得注意的亚基因组载体颗粒包含 Snapback基因组(SBG)，这可能导致宿主细胞产生dsRNA。对于其他仅具有 启动子序列，它们存在促进致癌基因通读的风险，这可能是一个实质性的 安全问题。该项目的中心假设是，宿主细胞等反式因子的组合 蛋白质、病毒辅助功能和载体核苷酸组成等顺式元件导致SGP的形成。 因此，对这些因素如何调节SGP形成的全面机制的理解将是 对开发具有减少/消除的SGP颗粒的新的媒介生产平台和 控制和管理SGP潜在危害的策略的设计。为了实现这些目标，我们将 追求以下目标：1)确定反式因子在亚基因组颗粒形成中的作用；2)确定 载体基因组组成在亚基因组颗粒形成中的作用；以及3)制定策略以 减少/消除亚基因组颗粒的形成。

PROJECT SUMMARY/ABSTRACT Hemophilia A is an X-linked bleeding disorder caused by hereditary defects in the F8 gene (encoding coagulation factor VIII, FVIII), which affects approximately 1 in 5000 male births worldwide. Clinically, hemophilia A is manifested as a severe bleeding phenotype that typically requires costly protein replacement therapy. The recent development of a gene therapy holds the promise of curing hemophilia A. Although the outcome of clinical trials using AAV vectors for hemophilia A has been encouraging, Adeno-associated virus (AAV)-mediated delivery of FVIII has not yet received regulatory approval. Major hurdles persist since there are concerns on gradually decreasing levels of FVIII in circulation as well as clonal hepatocellular expansion in long-term studies in a canine model, which raises concerns over genotoxicity or even cancer formation when using AAV vectors for human gene therapy. Current AAV-FVIII gene delivery strategies (AAV-F8) follow a standard design, in which a mini- promoter is used to drive F8 expression with two flanking ITRs. Nevertheless, clinical-grade vectors produced were reasonably homogenous based on Southern blot analysis of vector genomes. Our new studies however, using single molecule sequencing (SMRT), have uncovered a broad complexity of vector population that has been missed using conventional methods of analysis. One notable subgenomic vector particle contains the snapback genome (SBG), which may lead to dsRNA production in the host cells. For other SBGs having only the promoter sequences, they run the risk of promoting oncogenic readthrough, which may be a substantial safety concern. The central hypothesis of this project is that the combination of trans factors such as host cellular proteins, viral helper functions and cis elements such as vector nucleotide compositions lead to SGP formation. Therefore, a comprehensive mechanistic understanding of how these factors mediate SGP formation will be essential for the development of new a vector production platform with reduced/eliminated SGP particles and for the design of strategies to control and manage the potential hazards of SGP. To achieve these goals, we will pursue the following aims: 1): To define the roles of trans factors in subgenomic particle formation; 2) to define the role of vector genome composition in subgenomic particle formation; and 3) to develop strategies to reduce/eliminate subgenomic particle formation.