Non-viral Delivery of CRISPRa Tools for Scalable Directed Differentiation of Induced Pluripotent Stem Cells

Abstract

Background & Aim

Induced pluripotent stem cells (iPSCs) have transformative potential for advanced cell therapies, enabling the development of allogeneic, off-the-shelf products to enhance manufacturing cost-efficiency and accessibility. However, conventional differentiation protocols relying on cocktails of growth factors, cytokines, and media supplements are lengthy, yield suboptimal efficiencies, and produce undesired cell types or residual pluripotent stem cell impurities. These limitations compromise both manufacturing efficiency and product quality, representing key bottlenecks for commercialisation.

Methodology

To address these challenges, we developed an innovative CRISPR-mediated transcriptional activation (CRISPRa) system combined with non-viral delivery to establish a scalable forward programming workflow. Our approach leverages delivery of catalytically dead Cas9-VPR mRNA and four CRISPR guide RNA pools targeting known critical haematopoietic transcription factors (GATA2, TAL1, ETV2, and LMO2), driving differentiation toward haemato-endothelial progenitors.

Results

Optimisation of RNA payload delivery achieved up to 90% transfection efficiency with over 80% cell viability. At 72 hours post-transfection, iPSCs demonstrated robust expression of the four target transcription factors and reductions in pluripotency markers expression. To support scalable biomanufacturing, we then integrated iPSCs aggregates formation with CRISPRa delivery and expression, facilitating the transition from 2D cultures to 3D systems. This adaptation preserved comparable target transcription factors activation and induced the expression of a selection of 14 downstream haematopoietic genes, including CD31, CD34, CD43, and CDH5. Moreover, a 25–75% reduction of pluripotency markers expression was observed, alongside significant morphological changes within 72h (figure below). Finally, we optimised lipid nanoparticle-based delivery for CRISPRa complexes instead of electroporation, achieving high activation efficiency of target genes within a workflow amenable to scalable stir-tank bioreactors.

Conclusion

Integration of these cutting-edge molecular engineering tools into scalable processes has the potential to unlock the next-generation of iPSC-derived manufacturing. Our results demonstrate an innovative approach to enable the generation of a scalable, CRISPRa-mediated differentiation platform, addressing critical inefficiencies in current workflows and supporting enhancements of allogeneic therapies manufacturing capacity.