Our scientists have invented a proprietary CRISPR technology that is fueling development of next-generation allogeneic cell therapies with significant potential for the treatment of cancer
CAR-T cell therapies are a class of treatments in which T cells are modified to express chimeric antigen receptors (CARs). CARs are engineered molecules that, when present on the surface of a T cell, enable a T cell to recognize a specific protein that is present on the surface of other cells, including cancer cells. Upon recognition of the target cell via the CAR, the CAR-T cell kills the targeted cell. CAR-T cells have demonstrated great success in the clinic, but currently marketed therapies are derived from a patient’s own cells and are therefore available to a limited patient population. We are developing differentiated, off-the-shelf CAR-T cell therapies, derived from healthy donor T cells, that are genome-edited to improve persistence of antitumor activity and potential therapeutic benefit.
Natural killer (NK) cells, as their name implies, play an important role in ridding the body of cancer and viruses. NK cells are emerging as an increasingly important cell type for therapeutic development. Solid tumors are particularly challenging to treat and CAR-T cells have largely underperformed in the solid tumor setting. NK cells, however, inherently target both primary solid tumors and metastases. We are developing CAR-NK therapies for the treatment of multiple solid tumor types. We are able to differentiate NK cells from induced pluripotent stem cells (iPSCs) that we edit in multiple ways to address targeting, trafficking, proliferation, and overcoming the immunosuppressive tumor microenvironment.
Our next-generation chRDNA genome-editing platform is the foundation for the development of differentiated allogeneic cell therapies. Our chRDNA technology enables multiple genome edits, including multiplex gene insertions, with a high degree of specificity and lower levels of off-target editing than first generation CRISPR-Cas9.
Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.
Donohoue PD, Pacesa M, Lau E, Vidal B, Irby MJ, Nyer DB, Rotstein T, Banh L, Toh MS, Gibson J, Kohrs B, Baek K, Owen ALG, Slorach EM, van Overbeek M, Fuller CK, May AP, Jinek M, Cameron P (2021) Molecular Cell, 81(17):3637-3649.
SUMMARY: We and our collaborators show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity in primary human T cells. The incorporation of 2’-deoxynucleotide into the guide modulates Cas9’s activity and specificity in a target-dependent manner and allows for engineering of chRDNA guides with substantially reduced off-target effects. By solving the structure of target bound Cas9-chRDNA complexes we reveal a distorted guide-target duplex geometry driven by the DNA bases in the chRDNA sequence. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.
Harnessing type I CRISPR–Cas systems for genome engineering in human cells.
Cameron P, Coons MM, Klompe SE, Lied AM, Smith SC, Vidal B, Donohoue PD, Rotstein T, Kohrs BW, Nyer DB, Kennedy R, Banh LM, Williams C, Toh MS, Irby MJ, Edwards LS, Lin CH, Owen ALG, Künne T, van der Oost J, Brouns SJJ, Slorach EM, Fuller CK, Gradia S, Kanner SB, May AP, Sternberg SH (2019) Nature Biotechnology, 37:1471-1477.
Summary: We and our collaborators developed Type I CRISPR-Cas systems for site-specific genome editing applications in eukaryotic cells by fusing the multi-subunit, RNA-guided complex called Cascade to the non-specific FokI nuclease. We show that Cascade can alternatively be deployed with the Cas3 CRISPR enzyme to achieve targeted deletions of up to ~200 kilobases in human cells. Type I CRISPR-Cas systems are highly abundant and are a promising resource for genome editing applications.
Advances in industrial biotechnology using CRISPR-Cas systems.
Donohoue, PD, Barrangou R, May AP (2018) Trends in Biotechnology, 36(2):134-146.
Summary: In this review, we describe the fundamental characteristics of CRISPR-Cas systems and highlight how these features can be used in microbial species important for industrial biotechnology.
Mapping the genomic landscape of CRISPR-Cas9 cleavage.
Cameron P, Fuller CK, Donohoue PD, Jones BN, Thompson MS, Carter MM, Gradia S, Vidal B, Garner E, Slorach EM, Lau E, Banh LM, Lied AM, Edwards LS, Settle AH, Capurso D, Llaca V, Deschamps S, Cigan M, Young JK, May AP (2017) Nature Methods, 14(6):600-606.
Summary: We and our collaborators developed the SITE-Seq® assay to identify potential sites of off-target editing. The SITE-Seq assay is an unbiased, comprehensive, biochemical method that relies on the cutting of unedited, wild type genomic DNA in a test tube and the subsequent analysis of the cut DNA via next-generation sequencing. The SITE-Seq assay produces a list of possible off-target sites that can then be probed in cells to determine whether off-target editing occurred.
DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks.
van Overbeek M, Capurso D, Carter MM, Thompson MS, Frias E, Russ C, Reece-Hoyes JS, Nye C, Gradia S, Vidal B, Zheng J, Hoffman GR, Fuller CK, May AP (2017) Molecular Cell, 63(4):633-646.
Summary: We and our collaborators evaluated how human cells repair a cut made by Cas9, and we made the unexpected finding that the outcome of Cas9-mediated cuts is nonrandom and consistent across experimental replicates, cell lines, and delivery methods. DNA repair outcomes in cell lines are predictive of the DNA repair outcomes in human primary cells.
Guide RNA functional modules direct Cas9 activity and orthogonality.
Briner AE, Donohoue PD, Gomaa AA, Selle K, Slorach EM, Nye CH, Haurwitz RE, Beisel CL, May AP, Barrangou R (2014) Molecular Cell, 56(2):333-339.
Summary: We and our collaborators studied different elements of the Cas9 guide RNA sequence and structure, categorizing them into six distinct features. We determined that certain elements are necessary for Cas9 function while others are not, and we explored ways to engineer the guide RNA and to use Cas9 proteins from multiple bacterial species for genome editing applications.