Bidirectional promoters for the use of CAR T cell therapy

Abstract

Cancer is currently a leading cause of death in the world, with 9.6 million deaths in 2018 alone according to the Global Cancer Observatory (GLOBOCAN). Conventional methods such as surgery, chemotherapy, and radiotherapy, carry a risk of adverse effects, but without the guarantee of remission. To treat B cell acute lymphoblastic leukaemia (B-ALL), single antigen directed therapies such as CD19 targeted chimeric antigen receptor (CAR) T cell therapy have been developed. Unfortunately, the loss of the CD19 antigen on malignant cells occurs in a subset of patients, rendering the tumour cell variants refractory to CAR T cell attack. Targeting two antigens in one treatment could counter CD19 antigen loss. We propose using a bidirectional promoter to express two CAR constructs on a T cell. A reporter vector using the Sleeping Beauty transposon system was developed to determine the bidirectional activity of promoters using RFP and GFP. The EF1α, LMP2/TAP1 and synthetic RPBSA promoters, as well as RPBSA deletion variants, all showed bidirectional activity in transiently transfected HEK293T cells, furthermore bidirectional activity was maintained for over 60 days in stably transfected Jurkat cells. Intron deletion from RPBSA led to reduced bidirectional activity, while exon deletion resulted in increased activity. Interestingly, at 60 days, the WT RPBSA showed the least reduction in bidirectional activity when compared to its deletion variants and was therefore selected for further experimentation. Promoters were next compared with other constitutive promoters to determine their ability to express a dual CAR. RPBSA effectively drove CAR in the sense orientation; however, activity was reduced for CAR expression in the antisense direction in HEK293T cells. Introduction of the dual CAR construct into lentiviral vectors resulted in low viral titres and compromised transduction into primary human T cells, as compared with the single CAR controls. Although the bidirectional promoters could not drive CAR expression, they offer the potential for dual expression of shorter mRNA (e.g. reporter genes) or miRNA transcripts.