GenScript’s first Gene&Cell Engineering Summit will showcase leaders in the field advancing cell immunotherapeutic development. The keynote speaker, Dr. Alexander Marson, has developed innovative CRISPR-based tools to engineer T cells and understand the molecular mechanisms regulating T cell function.
T cell immunotherapeutics, CAR/TCR-T cells, have significantly changed the landscape of cancer treatment. For T cell engineering, viral approaches represent the standard method of choice for transgene delivery. However, viral transduction leads to random insertions and potential mutagenesis. Therefore, recognizing the need for a more specific approach to T cell engineering in 2018 the Marson lab provided the first proof-of-principle for the effective use of CRISPR-based non-viral approaches to T cell engineering (Roth et al. 2018).
This new strategy relied on the electroporation of CRISPR/Cas9 ribonucleoprotein (RNP) complex and single (ssDNA) or double (dsDNA) stranded DNA payloads for specific T cell genome targeting. To demonstrate the potential of this CRISPR-based approach in clinically relevant T cell engineering, the Marson group targeted long dsDNA and short ssDNA to repair autoimmune associated mutations in the IL2RA gene. Additionally, this approach enabled them to replace the TCR locus with a new sequence encoding specificity for the tumor antigen NY-ESO-1. Thus, besides improving the safety of edited cells for therapeutic applications, their approach meant that T cells could be modified with long dsDNA sequences, a previously thought impossibility marred by cellular toxicity.
More recently, the Marson lab implemented two new approaches to improve the efficiency and safety of their CRISPR/Cas9 non-viral knockin method. First, the use of modified DNA payloads containing Cas9 target sequences helped them enhance template nuclear shuttling. Second, the use of anionic polymers (i.e., poly(glutamic acid)) or PGA, supported RNP particle stabilization and improved delivery through electroporation (Nguyen et al. 2020). Together, these approaches improved NY-ESO-1 TCR knockin efficiency and reduced T cell toxicity.
Lastly, in an effort to expedite the screening of edited immune cells for improved functionality following CRISPR/Cas9 non-viral gene knockin, the Marson lab implemented the use of barcoded homology-directed repair (HDR) templates (Roth et al. 2020). In this approach, T cells are electroporated with a library of non-viral barcoded HDR templates and Cas9 RNP, targeting the TRAC locus for insertion. The resulting edited T cells are then screened for improved functionality both in vitro (i.e., T cell expansion, cytokine expression) and in vivo (i.e., T cell tumor infiltration, tumor cell killing). This new platform already allowed the Marson lab to identify TGFβR2–41BB as a modification that imparts improved T cell function against solid tumors. Therefore, this strategy has the potential to fast-track the discovery of valuable candidates for TCR engineering to support future T cell therapeutics.
