Promoter choice: Who should drive the CAR in T cells?

Abstract

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell-impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

“…One recent study compared the lentiviral titer, transduction efficiency, marker and CAR expression levels, cytokine production (IL-2 and IFN-γ), and killing ability of four promoters, including EF-1 (EF-1A in our study), CMV, hPGK, and RPBSA. 14 EF-1 exhibited the best transduction efficiency, killing ability, and cytokine production. Consistent with our observations, these authors observed a reduction in CAR expression driven by the hPGK and RPBSA promoters, which retained acceptable killing ability but reduced cytokine production.…”

“…A recent study considered promoter choice when a long and complicated mRNA was used in gene therapy. 14 Four promoters, EF1A, CMV, hPGK, and RPBSA, were used to compare the packaging and transduction efficiencies, marker and CAR expression, cytokine production levels, and lytic abilities, and EF1A was best of these four promoters. However, MND (myeloproliferative sarcoma virus MPSV enhancer, negative control region NCR deletion, d1587rev primer binding site replacement) had good transcriptional ability in cancer cells in a 1997 study.…”

Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy

“…Another study showed that the MSCV outperformed both EF1α and PGK in terms of expression levels and CAR expression stability [ 35 ]; however, the promotor efficacy may be influenced by the scFv sequence itself [ 36 ] (see section Single-chain fragment variant (scFv) ). Given these and other studies [ 37 ] that report different optimal promoters, it is important to determine the optimal promoter for a given construct (that may also contain additional elements that influence expression levels, such as reporters) and viral vector. The latter is important, since the choice of a promoter also influences the virus titer.…”

Metabolic and Mitochondrial Functioning in Chimeric Antigen Receptor (CAR)—T Cells

“…Recent reports suggest that CD95:CD95L signalling may play a role in the limited long-term persistence of CAR T cells in treating solid tumours [ 49 , 50 ]. Blockade of the signalling pathway has been shown to both increase CAR T cell persistence and number without adverse effects [ 49 , 50 , 51 , 52 , 53 ]. Inhibiting the CD95:CD95L pathway enhanced the CAR T cell therapy in vitro and in vivo.…”

“…The expression of genes involved in a particular metabolic pathway can be modulated using constitutive or inducible systems. The constitutive expression can be achieved using dual-promoter systems [ 132 ], bidirectional promoters [ 133 ], 2A self-cleaving peptides [ 52 ] or adding internal ribosome entry site (IRES) sequences [ 134 ]. For controlled expression over gene-of-interest (GOI), tetracycline (Tet)-On/Off system [ 135 ] or endogenous inducible promoters (e.g., IL-2 minimal promoter) [ 136 ] can be employed.…”