PhD Student Technical University of Denmark, Denmark
Abstract: Induced pluripotent stem cells (iPSC) are a promising avenue for producing next-generation adoptive cell therapies (ACT). They serve as a potentially unlimited source of cells to differentiate any immune cell of interest. Specifically in T cell-based ACTs, this approach allows the customization and personalization of the final T cell product and the generation of an off-the-shelf, allogeneic product. The differentiation of iPSCs to T cells poses great challenges, mainly associated with the low efficiency of the process and protocol variability. Therefore, decoding the molecular signature of this process is of significant importance for advancing this type of therapy. In this regard, CRISPR activation (a) or interference (i) screens offer a comprehensive and systematic approach to unravel the complexities of gene function and regulate the differentiation process.
We have generated iPS cell lines that constitutively express dead Cas9 (dCas9) fused to either the VP64 transactivating domain or the transinhibiting domain KRAB. We have further optimized the constructs to avoid transgene silencing by incorporating an antisilencing element (UCOE). We have validated the CRISPRa technology in two iPS cell lines, where a robust gene upregulation of CD4, CD8 and CD14 has been achieved.
For CRISPRa screens, these iPS cell lines will be transduced with gRNA libraries specifically targeting drugable targets, phosphatases, and signaling pathways. We will then follow the enrichment of gRNAs in differentiated populations to evaluate which genes regulate the differentiation at different stages.
We will use our in-house scaffold technology to improve the presentation of the current differentiation factors, as well as implementing the drugable hits from the screen, which allows for a more controlled and localized delivery and improvement of the T-cell yield.
We anticipate that the knowledge obtained from the screens will not only enhance the understanding of T cell development but will also provide opportunities for more precise manipulation of gene expression, which will lead to the generation of robust protocols for iPSC-derived T cell products. Moreover, hits from early stages of the differentiation (at HPSC for instance) can also be used to improve the yields of other immune populations of interest such as NK cells.
