Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London , London, UK.
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus , Cambridge, UK.
Mol Ther Methods Clin Dev. 2015 Sep 16;2:15035. doi: 10.1038/mtm.2015.35. eCollection 2015.
The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.
目前，痘苗病毒（VACV）载体的构建方法涉及使用选择和纯化标记物，然而，在临床应用中，将没有治疗价值的基因包含在最终载体中是不可取的。Cre-LoxP 系统已被用于制造无标记痘病毒，但效率非常低。为了获得无标记 VACV 载体，我们开发了标记基因切除系统，分别使用 Cre-Loxp 和 Flp-FRET 系统修饰胸苷激酶（TK）区和 N1L 区。CRISPR-Cas9 系统显著提高了生成标记基因阳性 TK 突变 VACV 载体的效率（约 90%）。可以使用 Cre 重组酶从重组病毒中切除标记基因（RFP）。为了构建针对 TK 和 N1L 基因的双基因缺失的无标记 VV 载体，我们构建了一个针对 N1L 基因的供体修复载体，该载体可以携带治疗基因和标记基因（RFP），可以使用 Flp 重组酶从重组病毒中切除。这里开发的无标记系统可用于在 TK 区或 N1L 区有效构建携带任何治疗基因的无标记 VACV 载体，而无需标记基因。我们的无标记系统平台具有为临床应用开发新的无标记 VACV 载体的巨大潜力。
