Optimization of a CRISPR/Cas9-based platform to produce allogeneic chimeric antigen receptor (CAR) T cells

Author(s)

Sarah Caroline Gomes de Lima

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-02-20

Examining board members

Lucas Eduardo Botelho de Souza; Thiago Aparecido da Silva; Eloah Rabello Suarez

• Advisor: Lucas Eduardo Botelho de Souza

Abstract

Allogeneic chimeric antigen receptor (CAR) T cell therapy represents a promising strategy to increase treatment accessibility. This approach has numerous advantages, such as the availability of an off-the-shelf, standardized product at a reduced cost due to production scaling. However, the large-scale implementation of this approach raises concerns for the graft-versus-host disease development, a potentially fatal complication caused by antigen recognition by the T cell receptor (TCR) on CAR T cells. A solution to overcome this challenge is the TCR deletion from CAR T cell surface using gene editing tools. This work described the establishment process of a platform for allogeneic anti-CD19 CAR T-cell production based on TCR depletion by gene knockout or targeted insertion using CRISPR/Cas9. First, the efficiency of two guide RNAs (gRNAs) in depleting the TCR was evaluated by targeting the TCR alpha-chain constant region (TRAC) gene. A T cell model was electroporated with the Cas9-encoding plasmid and a gRNA capable of generating up to 80% of CD3- cells was selected for the subsequent steps. For the gene knockout strategy, T cells were transduced with lentiviral particles for CAR expression and electroporated for delivering Cas9-gRNA ribonucleoprotein complexes (RNPs). Higher cell editing efficiency and recovery were observed when cell activation preceded the electroporation. Knockout efficiency was greater than 86%, with ~39% of the cells showing the phenotype of interest (CAR+CD3-). Cell viability reached 75% two days after transduction and remained above 60% after electroporation. Compared to conventional CAR T-cells, the edited ones maintained selective cytotoxicity to the CD19 antigen and showed increased levels of interferon-γ secretion. We then repurposed the CRISPR/Cas9 tool for targeted CAR knock-in and TCR knockout in a single-step approach, eliminating the need for viral vectors. Knock-in at the TRAC locus was performed by providing a double-stranded DNA template, co-delivered with the RNPs via electroporation, to direct the homology repair. We observed high percentages of TCR knockout (~91.5%) and CAR knock-in (~83.7%) 5 days post-electroporation. The moderate cell growth reflected the viability drop, reaching 27% of viable cells on day 3 and ~60% on day 7 of manufacturing. Although we did confirm the integration of the CAR transgene into the TRAC locus using an \"in-out\" PCR assay, we observed a drop in the frequency of CAR+ cells at prolonged culture times. Nevertheless, at this stage, edited CAR T cells exhibited activity against target tumor cells. Despite its potential, the knock-in approach requires refinements. On the other hand, the production of edited CAR T cells through gene knockout has proven to be a robust methodology, allowing for high editing efficiency and satisfactory cell yield. (AU)

• FAPESP's process: 20/02043-2 - Development of CRISPR/Cas9-based genome editing for the generation of allogeneic CAR-T cells
• Grantee: Sarah Caroline Gomes de Lima
• Support Opportunities: Scholarships in Brazil - Master