PDF(Pubmed)
Abstract：
Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.
摘要：
腺相关病毒载体（AAV）是动物模型和人类基因治疗的主要递送系统。市场上有几种食品和药物管理局批准的AAV基因疗法，与载体制造相关的问题变得越来越重要。在这项研究中,我们专注于AAV前病毒质粒可能产生的潜在毒性DNA污染物，在真核细胞中制造重组AAV所需的原材料。典型的AAV前病毒质粒是含有侧翼为天然AAV反向末端重复(ITR)序列的治疗基因盒的环状DNA。和携带细菌中质粒复制和选择所需的原核序列的质粒骨架。虽然大多数AAV颗粒包装预期的治疗有效载荷，一些衣壳代替包装位于前病毒质粒骨架上的细菌序列。由于ITR序列也具有启动子活性，潜在有毒的细菌开放阅读框架可以在体内产生，从而代表安全风险。在这项研究中,我们描述了一种用于载体制造的新的AAV前病毒质粒，该质粒（1）显着减少交叉包装的细菌序列，（2）增加正确包装的AAV有效载荷，和(3)钝化ITR驱动的交叉包装材料的转录以避免表达潜在的毒性细菌序列。该系统可以帮助提高AAV载体产物的安全性。
