A Self-Complementary AAV Proviral Plasmid System to Reduce Aberrant Cross-Packaging and ITR Promoter Activity in AAV Vector Preparations (Short-read)

Adeno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-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. Overall design: Novel AAV plasmids were packaged into 1st-generation AAV serotypes. Cross-packaging of AAV backbone and other contaminants was assessed by short-read Illumina sequencing.

腺相关病毒载体（Adeno-associated viral vectors, AAV）是用于动物模型及人类基因治疗的主流递送系统。目前已有多款获美国食品药品监督管理局（FDA）批准的AAV基因疗法上市，载体生产相关的问题也愈发受到重视。本研究聚焦于源自AAV前病毒质粒的潜在毒性DNA污染物——这类质粒是在真核细胞中生产重组腺相关病毒载体（recombinant AAV）所需的原材料。典型的AAV前病毒质粒为环状DNA，包含一段由天然AAV反向末端重复序列（inverted terminal repeat, ITR）侧翼包裹的治疗基因盒，以及一段携带原核序列的质粒骨架，该原核序列用于质粒在细菌内的复制与筛选。尽管大多数AAV颗粒会包装预期的治疗有效载荷，但部分衣壳却会包装前病毒质粒骨架上的细菌序列。由于ITR序列同样具备启动子活性，体内可能会表达具有潜在毒性的细菌开放阅读框，进而带来安全风险。本研究介绍了一款用于载体生产的新型AAV前病毒质粒，其具备三大优势：(1) 显著减少交叉包装的细菌序列；(2) 提升正确包装的AAV治疗有效载荷比例；(3) 抑制ITR驱动的交叉包装物质转录，从而避免表达具有潜在毒性的细菌序列。该系统有助于提升AAV载体产品的安全性。整体实验设计：将新型AAV质粒包装至第一代AAV血清型中，通过短读长Illumina测序评估AAV骨架及其他污染物的交叉包装情况。