Jen Hsin-I, Wilkinson Patrick, Lu Xiaohui, Zhang Wei
Global CMC Development, Ultragenyx Pharmaceutical Inc, Woburn, MA, USA.
Gene Therapy Research, Ultragenyx Pharmaceutical Inc, Somerville, MA, USA.
Mol Ther Methods Clin Dev. 2025 Jun 4;33(3):101503. doi: 10.1016/j.omtm.2025.101503. eCollection 2025 Sep 11.
Recombinant adeno-associated viruses (rAAVs) produced by transfecting DNA plasmids into mammalian cells can inadvertently package host cell DNA (hcDNA) and plasmid DNA inside their capsids. Although the percentage of these DNA impurities is low compared to the rAAV genome in vector preparations, it is essential to characterize the DNA impurities in gene therapy products due to the theoretical risks associated with unwanted gene expression and potential immunogenicity and oncogenicity in treated patients. We performed long-read sequencing in rAAV vector, with a focus on analyzing residual, non-transgene DNA within the capsids. Although we detected host cell and residual plasmid DNA impurities, they were predominantly incomplete sequences without coding potential. This indicated that while DNA impurities may be present in rAAV preparations, host cell and residual plasmid genes were unlikely to be expressed. This was supported by RNA sequencing (RNA-seq) analyses that showed minimal plasmid RNA transcripts and host cell RNA transcripts in the livers of mice dosed with rAAV. Overall, the results from these studies enable data-based risk assessment of co-packaged DNA impurities and a better understanding of potential adverse effects associated with rAAV gene therapy.
通过将DNA质粒转染到哺乳动物细胞中产生的重组腺相关病毒（rAAV）可能会在其衣壳内意外地包裹宿主细胞DNA（hcDNA）和质粒DNA。尽管在载体制剂中，与rAAV基因组相比，这些DNA杂质的比例较低，但由于与不必要的基因表达以及治疗患者中潜在的免疫原性和致癌性相关的理论风险，对基因治疗产品中的DNA杂质进行表征至关重要。我们对rAAV载体进行了长读长测序，重点分析衣壳内残留的非转基因DNA。尽管我们检测到了宿主细胞和残留质粒DNA杂质，但它们主要是不完整的序列，没有编码潜力。这表明虽然rAAV制剂中可能存在DNA杂质，但宿主细胞和残留质粒基因不太可能表达。这得到了RNA测序（RNA-seq）分析的支持，该分析显示，在用rAAV给药的小鼠肝脏中，质粒RNA转录本和宿主细胞RNA转录本极少。总体而言，这些研究结果有助于对共包装的DNA杂质进行基于数据的风险评估，并更好地理解与rAAV基因治疗相关的潜在不良反应。
