Engineering a synthesis-friendly serum responsive promoter for antibiotic selection of genomic integration in cell-based therapy.

Abstract

For clinical cell-based therapies (e.g. CAR-T cells), the genomic integration of therapeutic genes into cells are selected using inefficient resistance genes of host origin to avoid potential immune response from using more efficient resistance genes of foreign origin. In principle, a serum-responsive promoter could express efficient resistance genes during the cell manufacturing stage that could then diminish during in vivo administration. To avoid genomic instability, we designed a synthesis-friendly serum-responsive promoter (SFSp) with no extreme GC ratios or repeats greater than 9 base pairs. SFSp was used to express a fluorescent reporter, whose expression was diminished after serum starvation. Furthermore, SFSp could be used in replacement of weak promoters (e.g. SV40p) for expressing efficient resistance genes (e.g. blasticidin resistance) from genomic integration via lentiviral infection. Thus, the regulation of resistance genes using SFSp could be a valuable tool in cell-based therapeutics to increase selection efficiency and reduce immunogenicity.