Patent WO-2019039962-A1 (WO)
Title: GENE THERAPY DNA VECTOR VTvaf17, METHOD OF PRODUCTION; ESCHERICHIA COLI STRAIN SCS110-AF, METHOD OF PRODUCTION; ESCHERICHIA COLI STRAIN SCS110-AF/VTvaf17 BEARING GENE THERAPY DNA VECTOR VTvaf17, METHOD OF PRODUCTION
Inventor: Savelieva Natalia
IPC: A61K48/00
Filed: 2018-03-26
Abstract: ﻿The invention refers to genetic engineering and can be used in biotechnology, medicine and agriculture. This invention requires construction of 3165-bp gene therapy DNA vector VTvaf17 for genetic modification of animal and human cells containing nucleotide sequence SEQ ID No. 1. The method of constructing 3165-bp gene therapy DNA vector VTvaf17 involves, first of all, constructing a 4182-bp vector that contains a 1188-bp promoter region of human elongation factor EF1A with an intrinsic enhancer, a 35-bp polylinker with sites for restriction endonucleases BamHI, EcoRV, Sail, Hindlll, Kpnl, EcoRI, a 466-bp transcription terminator and a polyadenylation sequence of the human growth factor, a 136-bp regulatory element RNA-OUT of transposon Tn10 allowing for antibiotic-free positive selection, a 1299-bp origin of replication for autonomous replication with a single nucleotide substitution to increase vector production in the cells of most Escherichia coli strains, a 1010-bp kanamycin resistance gene, and then it is cleaved by Spel restriction sites, and the remaining fragment is ligated to itself. The specified purpose is achieved by obtaining Escherichia coli strain SCS110-AF for the production of gene therapy DNA vector VTvaf17 or gene therapy DNA vectors based on it allowing for antibiotic-free positive selection. The method of obtaining Escherichia coli strain SCS110-AF for the production of gene therapy DNA vector VTvaf17 or gene therapy DNA vectors based on it involves constructing a 64-bp linear DNA fragment which contains regulatory element RNA-IN of transposon Tn10 allowing for antibiotic-free positive selection, 1422-bp levansucrase gene sacB the product of which ensures selection within a sucrose-containing medium, 763-bp chloramphenicol resistance gene catR required for the picking of strain clones in which homologous recombination occurs, and two homologous sequences, 329-bp and 233-bp, ensuring homologous recombination in the region of gene recA concurrent with gene inactivation, and then the Escherichia coli cells are transformed by electroporation, and clones surviving in a medium containing 10 µg/ml of chloramphenicol are picked. Escherichia coli strain SCS110-AF/VTvaf17 (registered at the Russian National Collection of Industrial Microorganisms under number B-12990, INTERNATIONAL DEPOSITARY AUTHORITY No. NCIMB 42801) carrying gene therapy DNA vector VTvaf17 is also constructed for its further development allowing for antibiotic-free selection. The method of obtaining Escherichia coli strain SCS110-AFA/Tvaf17 (registered at the Russian National Collection of Industrial Microorganisms under number B-12990, INTERNATIONAL DEPOSITARY AUTHORITY No. NCIMB 42801) carrying gene therapy DNA vector VTvaf17 involves making electrocompetent cells of Escherichia coli strain SCS110-AF and subjecting these cells to electroporation with gene therapy DNA vector VTvaf17. After that, the cells are poured into agar plates (Petri dishes) with a selective medium containing yeastrel, peptone, 6% sucrose, and 10 µg/ml of chloramphenicol.