Versatile Lipid Nanoparticle Platform For Efficient CRISPR-Cas9 Gene Editing In Primary T-Cells And CD34+ Hematopoietic Stem Cells

By Reka Geczy, Aruna Balgi, Stella Park, Meenakshi Swaminathan, Mana Novin, Hannah Ly, Rita Zhao, Angela Zhang, Nikita Jain, Anitha Thomas, and Samuel Clarke, Cytiva

Lipid nanoparticles (LNPs) are nano-sized carriers that can deliver gene engineering tools to cells in a more controlled and efficient way. They offer a promising alternative to traditional methods for editing the genes of difficult-to-transfect cells, such as T cells (important for fighting infections and cancer) and blood stem cells (used to treat genetic blood diseases). This study focused on the screening and development of LNPs for both T cells and stem cells, showing the optimization of cell culture conditions for success and the scale-up of both the LNPs and the culture conditions. By fine-tuning different LNP designs, we achieved 90% editing of genes in T cells and blood stem cells. The treated cells stayed healthy, grew well, and, in the case of blood stem cells, retained their ability to develop into different blood cell types. We found that the nutrients and supplements added to the cells during the culture process were important for LNP effectiveness. The developed method was scaled up from small experiments to larger systems approaching clinical production. This work represents a significant step toward using LNP technology to create new therapies that are more robust, more efficient, and accessible for people with cancer and rare diseases.

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