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Non-viral delivery of CRISPR activation tools for directed differentiation of induced pluripotent stem cells.

O Awonusi(1) S Perez(1) R Esse(1) S Krishnan(1) M Braam(1) M Jayawardena(1) M Lopes Tiburcio(1) E Zucchelli(1) V Di Cerbo(1)

1:Cell Therapy Catapult

Induced pluripotent stem cells (iPSCs) are a promising solution for allogeneic cell therapy production. To enhance their potential, cell engineering methods have been applied to modify their genome and/or promote their differentiation. This is made possible by the delivery of cell engineering tools to iPSCs, which has been demonstrated through viral and non-viral delivery methods.

To support the development of the next generation of allogeneic cell therapy, we are establishing an iPSC directed differentiation workflow based on non-viral delivery of CRISPR-mediated transcriptional activation (CRISPRa) tools, aiming at the transcriptional activation of four key factors leading to the differentiation into haematopoietic progenitors.

We initially optimised the non-viral delivery process by electroporation, achieving up to 95% transfection of a dead Cas9 (dCas9)/CRISPRa complex multiplexed with four guide RNAs for the selected transcriptional factors. Additionally, we established flow cytometry and droplet digital PCR analytics to evaluate the levels of gene activation. Next, we have implemented the workflow into a haemato-endothelium differentiation protocol to investigate the benefits of a directed differentiation strategy to improve allogeneic therapy manufacturing.

Our current work is focusing on benchmarking alternative non-viral delivery technologies including lipid nanoparticles, which we aim to implement in a full directed differentiation process in stirred tanked bioreactor. We anticipate that building the bioprocessing and analytical capabilities with innovative technologies has the potential to accelerate the development of the next generation of allogeneic therapies as well as establish a testbed for technology developers to evaluate the performance of their solutions against industry standards.

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