Research Projects

Biotherapeutics are large complex drugs with market value more than US$ 256 billion in 2023 and are produced using live animal/human cells due to its ability to have desired post translational modifications. Usually, a drop in productivity and stability of the transgene is observed during long-term cultivation of the recombinant CHO clones which is attributed to the loss of the transgene copy or due to epigenetic silencing of the transgene cassette. This issue could be addressed at two levels of cell line development - either by inserting the transgene into a chromatin accessible and transcriptionally active genomic site or using an insulating element which ensures that the transgene is not encroached by the nearby heterochromatin. Among these two approaches the use of insulating elements is now increasingly used in cell line development where the insulating elements are chosen in an empirical manner. Although a handful of studies have been reported so far to investigate the effect of various chromatin modifying elements, still several open questions remain regarding their selection: 1) whether the insulating element work in a similar manner irrespective of promoter being used, 2) will the different insulating elements have comparable effect when inserted at exact same genomic locus and 3) what kind of effect the insulating element provide to the transgene. Since epigenetic remodeling is reported to take place in random integration as well as site-specific integration at the insertion site and the activity of insulators is dependent on genomic context, it is ideal to take site-specific integration and genomic loci of choice into consideration while demonstrating the impact of these control elements. This study focuses on investigating the effect of four chromatin modifying elements - insulators (ALOXE3 and cHS4), scaffold/matrix attachment region (S/MAR) (human serine protease B, CSP-B) and UCOE (A2UCOE) and their combinations with three promoters - hCMV, EF1- ɑ and SV40. We will evaluate the expression stability of Dupilumab producing CHO-K1 cell lines in long-term cultivation by multiplexing the different combinations of promoters and insulators that are inserted at the exact same genomic locus. In addition to the stability analyses, the best and poor performing promoter - insulation element configurations will be selected, and the epigenome will be profiled to understand the epigenetic modifications taking place at the insertion site after site-specific integration of transgene with the chromatin modifying elements. While this work will be carried out in Dupilumab producing CHO cell lines, the knowledge gained in this study can be applied across mammalian cell lines producing various recombinant biotherapeutics to improve their yield considerably, thereby reducing the overall manufacturing costs, which will ultimately have a very big social impact.