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.
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This work was supported by the Canadian Institutes of Health Research [#PJT-156317] and the Natural Science and Engineering Research Council NSERC [#RGPIN-2019-04183].
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Supplementary Figure 1
HEK293 transiently transfected with plasmids encoding SRE promoters. (DOCX 89 kb)
Supplementary Video 1 Fluorescence microscopy time lapse of HEK293 cells stably transfected with RCaMP after stimulation with ATP. (AVI 163 kb)
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Lam, C.K.C., Truong, K. Engineering a synthesis-friendly serum responsive promoter for antibiotic selection of genomic integration in cell-based therapy. Biotechnol Lett 44, 605–611 (2022). https://doi.org/10.1007/s10529-022-03244-z
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DOI: https://doi.org/10.1007/s10529-022-03244-z