Human embryonic kidney 293T (ATCC CRL-1573) and MCF-7 (ATCC HTB-22) cell lines were cultured in high glucose Dulbeccos Modified Essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Pan-Biotech GmbH), penicillin (100 U/mL) and streptomycin (100 g/mL) (Gibco). 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. Even though four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of main T cells. Major differences were found in the ability 6-Thioinosine of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important concern for gene therapy applications requiring the expression of long and complex mRNA. Introduction Promoters are of crucial importance 6-Thioinosine for expressing optimal levels of the transgene in CAR T cells for the Rabbit Polyclonal to MRPL14 production of functional proteins or non-coding RNA [1C5]. It is also obvious that high expression of the CAR can result in antigen-independent CAR signaling, resulting in T cell exhaustion and sub-optimal anti-tumor responses, or lead to the inappropriate acknowledgement of tumor antigen on self-tissue [1, 2]. In addition, controlling CAR T cell signaling is critical for proper memory cell formation [6]. Because surface expression of the CAR may be limited by mRNA levels, the choice of promoter is critical [1, 2]. There have been limited studies that directly compare the efficiency of different promoters for driving long mRNA comprising multiple genes within CAR T cells [1, 2, 7]. Recent studies investigating promoter overall performance in mouse or human T cells were usually limited to either the CAR, a single gene of interest alone, or single fluorescent reporter genes of limited size [1, 2, 7C9]. For the generation of lentiviral particles for transduction, using multiple internal promoters or internal ribosome access sites (IRES) for multiple genes may interfere with transcription or reverse transcription of viral genomic RNA (vgRNA), impacting upon lentiviral particle titre, and/or around the efficiency of integration into the target cell [8, 10]. Therefore, strategies that employ single promoters to drive multiple genes may be favored for CAR T cell engineering [9]. Although all current, clinically-approved second and third generation CAR T cells rely on the expression of a single gene encoding a single polypeptide, it may be advantageous to express longer RNA made up of the CAR, together with one or more genes of interest. For example, endogenous growth factors or membrane bound or secreted cytokines could improve T cell growth and survival [6, 11]. Alternatively, markers of transduction efficiency or death switches could be incorporated into the CAR element [4, 12C14]. Promoter choice for such applications is crucial to obtain optimised gene expression of multiple, linked genes. Because requirements for driving short versus long RNA might be unique in CAR T cell genetic elements, we investigated the ability of several promoters to drive an extended downstream genetic sequence comprised of GFP, anti-Her2-CAR and an additional cell survival gene Myeloid leukemia cell differentiation protein (Mcl-1), an anti-apoptotic Bcl2 family member. Mcl-1 aids in T cell development, mitochondrial function and lifespan and appears to a suitable candidate for enhancing CAR T cell overall performance [15, 16]. Mcl-1 inhibits the action of pro-apoptotic BIM / BAK / BAX at the mitochondrial membrane and is expressed throughout T cell differentiation and is essential for memory T cell formation [16C20]. The individual elements were 6-Thioinosine tested at 6-Thioinosine protein level and for functional activity. The results demonstrated clear differences in the ability of these internal promoters to drive expression of multiple CAR-cassette associated transgenes. Material and methods Plasmid construction The third-generation lentiviral vector pCCLsin.cPPT.hPGK.GFP.WPRE (pCCLsin) and VSV-G-based packaging plasmids were a kind gift from Prof. Dr. Naldini and have been explained elsewhere [21]. The anti-Her-2 CAR FRP5, anti-CD19 CAR FMC63 (withCEQKLISEEDLCc-myc tag between scFv and CD8 hinge) and codon-optimized human Mcl-1 (cop-Mcl-1) were synthesized as gene blocks (IDT Technologies). Both CAR constructs are second generation CAR with CD28 costimulatory domains (Fig 1A). Sap I Type IIs restriction enzyme cloning was utilized for scarless assembly of the eGFP-P2A-CAR-P2A-Mcl-1. This cassette was then cloned into the BamHI and SalI sites of the pCCLsin (Fig 1A). Promoters were amplified with 5′ EcoRV and 3′ BamHI sites from respective plasmids: CMV from pcDNA3.1(-), EF-1 from Sleeping Beauty (pSBbiRP) and RPBSA from Sleeping Beauty (pSBtet-GP) and ligated upstream of the GFP-CAR-mcl1 cassette. Codon optimized Leucine Zipper CD95 (LZ-CD95L) gene 6-Thioinosine was synthesized by IDT with EcoRI and BamHI sites and cloned into pcDNA3.1(-) (Addgene #104349). Open in a separate windows Fig 1 The effect of internal promoters in generating functional lentiviral particles.(a) Schematic illustration of the pCCLsin backbone bearing four.