At this year’s ASGCT annual meeting, one of the first series of talks was dedicated to advances in cell therapies, in a session titled “Off the Shelf Cell Therapies - Beyond T Cells.” This session featured leaders in the field leveraging CRISPR/Cas9 tools to develop cell therapies, including Alexander Marson, Director at Gladstone, and Charles Gersbach, Duke University.
Dr. Marson opened the session by talking about his team’s vision for developing cell therapies based on the use of precise gene editing. His group has successfully developed new CRISPR/Cas9-based methods that leverage synthetic DNA payloads, stirring away from conventional delivery tools such as retrovirus and lentivirus vectors. In addition, his lab has continued to advance strategies for precise engineering of T cells by targeting the TRAC locus, thus changing the antigen specificity of these cells through fully non-viral approaches. Currently, non-viral CRISPR approaches enable his team to edit T cell specificity with high efficiency, opening the door to developing GMP grade, clinically relevant cell products for cancer therapies (Shy et al. 2021).
Despite these advances, Dr. Marson acknowledges that several challenges must be overcome to successfully use CAR T cells in treating some cancer types, such as solid tumors. For example, downregulation of tumor target antigens or antigen-escape and the immunosuppressive tumor microenvironment are two critical obstacles limiting CAR T cell therapy efficacy. Therefore, the Marson lab has leveraged various forward genome-wide CRISPR screens, including knock-out and knock-in approaches, to identify T cell properties that may provide an edge in tackling the many obstacles imposed by solid tumors (Shifrut et al. 2018).
Enabled by these unbiased genome-wide CRISPR screen strategies, the Marson team has identified new factors that may be targeted to improve cancer immunotherapies. For example, the gene RASA2 was identified as an important factor for T cell activation through CRISPR knock-out approaches. Loss of RASA2 expression improves T cell antigen sensitivity and persistence, thereby providing new opportunities to enhance T cell efficacy against solid tumors. Ultimately, through CRISPR knock-out, knock-in, and CRISPRa/CRISPRi screens, the Marson group is gaining insight into the molecular factors and pathways critical for T cells to maintain their fitness within the immunosuppressive tumor microenvironment (Schmidt et al. 2022).
CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi). “Catalytically inactive “dead” Cas9 (dCas9), in which point mutations abrogate DNA cleavage by the HNH and RuvC domains, can be repurposed as a transcription activator (CRISPRa) or repressor (CRISPRi). For CRISPRa (top), dCas9 is fused to a transcription activator. For CRISPRi (bottom), dCas9 is fused to a transcription repressor.” Retrieved and modified from Gebre et al. 2018, only part of the legend for Figure 5 is shown. (https://creativecommons.org/licenses/by/4.0/)
Continuing to illustrate how CRISPR/Cas9 tools are charting the way forward to develop cell therapies, Dr. Gersbach shared his team’s work leveraging CRISPR/Cas9 based screens, CRISPRa/CRISPRi, to elucidate programs controlling neuronal differentiation and T cell fates.
Gersbach’s team has leveraged CRISPR/Cas9 (CRISPRa) screens to uncover new transcription programs directing neuronal fate by introducing guide RNA libraries targeting the promoters of ~1,500 transcription factor genes into induced pluripotent stem cells (iPSCs). This approach has enabled his team to identify new transcriptional programs involved in neuronal subtype specification and maturation (Black et al. 2020).
Beyond factors involved in neuronal differentiation, the Gersbach group has more recently applied similar CRISPRa and CRISPRi screens to understand transcriptional programs regulating T cell fate. More specifically, factors that play a role in the transition of CD8+ T cells between naïve, central memory, effector memory, and effector state. Among the factors identified, BATF3 appears to be relevant in enacting programs controlling CD8+ T cell fate and influencing properties relevant for T cells as therapeutics, such as exhaustion and activation.
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