Salerno Paola, Leckenby Matthew W, Humphrey Bruce, Cranenburgh Rocky M
London Bioscience Innovation Centre, Prokarium Ltd, London, UK.
Cobra Biologics Ltd, Keele, Staffordshire, UK.
Synth Biol (Oxf). 2022 May 13;7(1):ysac005. doi: 10.1093/synbio/ysac005. eCollection 2022.
Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark™, a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in cytosol aminopeptidase ( mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both and bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins. Graphical Abstract.
抗生素抗性基因被广泛用于筛选用质粒转化的细菌，并防止培养物中质粒的丢失，然而抗生素在生物制药生产过程中属于污染物，监管机构不鼓励在疫苗和生物疗法中保留抗生素抗性基因。为克服这些限制，我们开发了X-mark™，这是一种利用Xer重组来产生用于下游治疗应用的无选择标记基因质粒的新技术。使用该技术，在胞质溶胶氨基肽酶（突变体中产生带有位于XerC/D靶位点两侧的抗生素抗性基因的X-mark质粒，这些突变体在质粒上缺乏Xer重组，随后将其转化到具有功能性Xer系统的肠道细菌中。这导致抗性基因以高分辨率（100%）快速缺失，并且在没有抗生素选择压力的情况下，已解析的质粒稳定复制超过40代。由于包括已开发用作临床口服疫苗的菌株在内的辅助序列之间具有高度同源性，该技术在革兰氏阴性菌和革兰氏阳性菌中均有效。X-mark有效地消除了生物制药产品（如疫苗和治疗性蛋白质）中抗生素抗性残留带来的任何监管和安全问题。图形摘要。
