Scalable Electroporation of Adult Keratinocytes with Multiplexed CRISPR Ribonucleoproteins for the Development of Novel Cell Therapeutics
The use of engineered keratinocytes for cell therapy has long been an attractive option to treat various dermatological, oral, and aural disorders. Keratinocytes are easily adapted to in vitro culture and expanded from even small tissue specimens, making them an ideal cell type for a range of autologous and allogeneic cell therapy products. However, engineered keratinocyte-based cell therapies have been limited by the lack of efficient transfection methods for adult keratinocytes. Here we aimed to develop a GMP-compliant, scalable cell engineering process using MaxCyte® electroporation to transfect neonatal and adult primary keratinocytes from four distinct anatomical locations.
Using MaxCyte, we efficiently delivered mRNA or CRISPR-Cas9 ribonucleoproteins (RNPs) to primary keratinocytes from the foreskin, arm, tonsils, and tympanic membrane at two different scales. This was accomplished without compromising cell viability, morphology, or growth capability. Furthermore, delivery of multiple CRISPR RNPs in a single electroporation achieved highly efficient, multiplexed gene editing in a simple, adaptable process. These improvements in transfection efficiency and cell viability reduced keratinocyte engineering times by up to 4 weeks, with a significantly higher success rate than a standard chemical transfection method. Finally, we demonstrate the scalability of the MaxCyte electroporation process, enabling the engineering of millions of primary keratinocytes without any loss of efficacy. In summary, the MaxCyte ExPERTTM platform provides efficient, GMP-compliant, multiplexed transfection for the development and scalable production of engineered keratinocyte cell therapy products.
Summary of Keratinocyte Genome Engineering Process
High Efficiency Delivery of RNA and CRISPR RNPs to Primary Keratinocytes Using MaxCyte Electroporation
Gene Knockout of Multiple Targets in Primary Keratinocytes Derived from Distinct Anatomical Sites
Adaptable Strategies for Development of a Clinical-Scale Keratinocyte Cell Therapy Manufacturing Process
MaxCyte Electroporation Enables Faster Keratinocyte Engineering
- Development of cell therapeutics using engineered keratinocytes has been limited by lack of options for efficient transfection.
- The MaxCyte ATx enables the efficient delivery of mRNA and CRISPR RNPs to hard-to-transfect primary keratinocytes, derived from multiple anatomical locations, in a scalable, GMP-compliant process.
- Electroporation maintains high viability, normal morphology, and low oxidative stress in primary neonatal and adult keratinocytes.
- MaxCyte electroporation enables delivery of up to 6 CRISPR RNPs, over multiple rounds of transfection, with no loss of efficiency.
- Platform flexibility allows for the rapid development of autologous and allogeneic cell therapy processes for specific subsets of keratinocytes and fibroblasts.
Corresponding Author: James Brady; [email protected] MaxCyte, Inc., Tel: (301) 944-1700