Scalable Manufacturing of Gene-Modified Primary Natural Killer Cell Therapies
Outcome/Accomplishment
Researchers at the U.S. National Science Foundation (NSF) Cell Manufacturing Technologies (NSF CMaT) Engineering Research Center (ERC) have made significant progress on the engineering and characterization of natural killer (NK) cells through virus-free payloads. A team, led by Dr. Krishanu Saha, Associate Professor of Biomedical Engineering at the University of Wisconsin-Madison, successfully achieved characterization of nonviral CRISPR CAR (chimeric antigen receptor) NK cells in NSF CMaT’s testbed. No significant off-target modifications were found and cytotoxicity to exogenous DNA was observed. Altogether, this work provides compelling evidence for using the CRISPR-Cas9 gene-spicing system to manufacture virus-free, potent NK cell products. NSF CMaT is headquartered at Georgia Tech.
Impact/Benefits
NK cells are white blood cells that protect the body from harmful invaders by destroying pathogens (viruses, bacteria, and parasites) and cancer cells. NK cells are innate lymphocytes that are human leukocyte antigen (HLA)-agnostic, making them excellent candidates for cell therapies that are both autologous (taken from one individual) and allogeneic (taken from different individuals). However, NK cells are difficult to genetically modify; current methods typically use viral vectors which do not allow for targeted, specific, or multiplex editing. In contrast, NSF CMaT’s virus-free strategy seeks to improve the manufacturing, cryopreservation, and characterization of primary NK cell products that have been gene-modified.
Explanation/Background
Primary NK cells have historically been challenging to modify using standard viral and nonviral techniques. Furthermore, most culture systems use feeder systems (e.g., K562 cells). The virus-free strategy developed by Dr. Saha’s team used the CRISPR-Cas9 system to precisely insert large transgenes into the KLRC1 (Killer Cell Lectin-Like Receptor C1, a protein-coding gene) locus with primary human NK cells.
The team began by developing non-invasive, real-time imaging of NK cell phenotypes. To analyze the genomic outcomes within the KLRC1-CAR product, off-target modifications and integration of transgenes throughout the genome were explored. The genome-wide off-target analysis of KLRC1-CAR cells confirmed high on-target editing with minimal off-target activity, highlighting the specificity of the edit. Moreover, the KLRC1-CAR cells displayed greater cytotoxic function in an in vitro human melanoma model. As a result of the work, a paper is in development, one invention disclosure has been accepted, and intellectual property has been filed.
Location
Atlanta, GeorgiaStart Year
Advanced Manufacturing
Advanced Manufacturing
Lead Institution
Core Partners
Fact Sheet
Outcome/Accomplishment
Researchers at the U.S. National Science Foundation (NSF) Cell Manufacturing Technologies (NSF CMaT) Engineering Research Center (ERC) have made significant progress on the engineering and characterization of natural killer (NK) cells through virus-free payloads. A team, led by Dr. Krishanu Saha, Associate Professor of Biomedical Engineering at the University of Wisconsin-Madison, successfully achieved characterization of nonviral CRISPR CAR (chimeric antigen receptor) NK cells in NSF CMaT’s testbed. No significant off-target modifications were found and cytotoxicity to exogenous DNA was observed. Altogether, this work provides compelling evidence for using the CRISPR-Cas9 gene-spicing system to manufacture virus-free, potent NK cell products. NSF CMaT is headquartered at Georgia Tech.
Location
Atlanta, GeorgiaStart Year
Advanced Manufacturing
Advanced Manufacturing
Lead Institution
Core Partners
Fact Sheet
Impact/benefits
NK cells are white blood cells that protect the body from harmful invaders by destroying pathogens (viruses, bacteria, and parasites) and cancer cells. NK cells are innate lymphocytes that are human leukocyte antigen (HLA)-agnostic, making them excellent candidates for cell therapies that are both autologous (taken from one individual) and allogeneic (taken from different individuals). However, NK cells are difficult to genetically modify; current methods typically use viral vectors which do not allow for targeted, specific, or multiplex editing. In contrast, NSF CMaT’s virus-free strategy seeks to improve the manufacturing, cryopreservation, and characterization of primary NK cell products that have been gene-modified.
Explanation/Background
Primary NK cells have historically been challenging to modify using standard viral and nonviral techniques. Furthermore, most culture systems use feeder systems (e.g., K562 cells). The virus-free strategy developed by Dr. Saha’s team used the CRISPR-Cas9 system to precisely insert large transgenes into the KLRC1 (Killer Cell Lectin-Like Receptor C1, a protein-coding gene) locus with primary human NK cells.
The team began by developing non-invasive, real-time imaging of NK cell phenotypes. To analyze the genomic outcomes within the KLRC1-CAR product, off-target modifications and integration of transgenes throughout the genome were explored. The genome-wide off-target analysis of KLRC1-CAR cells confirmed high on-target editing with minimal off-target activity, highlighting the specificity of the edit. Moreover, the KLRC1-CAR cells displayed greater cytotoxic function in an in vitro human melanoma model. As a result of the work, a paper is in development, one invention disclosure has been accepted, and intellectual property has been filed.