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. 2019 Dec 24;17:209–219. doi: 10.1016/j.omtm.2019.11.021

Figure 1.

Figure 1

Production of Gene Therapy LVs in HEK293T Triggers NF-κB Activation but Does Not Impact LV Yield

(A) HEK293T cells were transfected with the indicated firefly luciferase reporter constructs, pRL-TK Renilla luciferase, and empty pcDNA3 as a control or LV constructs including VSV-G envelope encoding pMD2.G, pCMV-dR8.74 Gag-Pol expression plasmid, and a genome plasmid bearing IRES-GFP as follows: LV-EV is LV genome bearing empty vector, LV-T5C encodes full-length humanized TRIM5-CypA chimera, LV-T5C* encodes full-length humanized TRIM5-CypA chimera with mutated start methionines at positions 1 and 47. pLV-T5C and pLV-T5C* denote genome plasmids alone without pMD2.G or pCMV-dR8.74 co-transfection. Firefly luciferase values were normalized to Renilla luciferase values. Data represent mean fold change in reporter activity ± SD (n = 3) 48 h post transfection presented relative to cells transfected with an equivalent amount of pcDNA3. (B) Culture supernatants from (A) were harvested at 48 h and mean viral titers ± SD of biological replicates (n = 2) were determined in duplicate in HEK293T and THP-1 cells by enumerating GFP-positive cells. TU, transducing units. (C) HEK293T cells were transfected with the indicated firefly luciferase reporter constructs, pRL-TK Renilla luciferase, and pcDNA-based expression plasmids encoding cGAS and/or STING as shown and/or a LV encoding a chimeric antigen receptor (CAR), as well as pCMV-dR8.74 and pMD2.G (LV-CAR). Mean fold change in reporter activity ± SD (n = 3) was assessed 48 h later using a dual-luciferase reporter assay and is presented relative to cells transfected with an equivalent amount of pcDNA. (D) Cell extracts from transfected HEK293T cells were subjected to immunoblot detecting cGAS or STING or vinculin as loading control. Molecular mass markers are shown. (E) Culture supernatants from (C) containing infectious LV CAR were harvested at 48 h and viral titers ± SD of biological replicates (n = 2) were determined in duplicate in HEK293T cells by qPCR with CAR encoding LV-specific probes.