Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection

Abstract

Lentiviral vectors derived from human immunodeficiency virus type I are widely used to deliver functional gene copies to mammalian cells for research and gene therapies. Post-transcriptional splicing of lentiviral vector transgene in transduced host and transfected producer cells presents barriers to widespread application of lentiviral vector-based therapies. The present study examined effects of indole derivative compound IDC16 on splicing of lentiviral vector transcripts in producer cells and corresponding yield of infectious lentiviral vectors. Indole IDC16 was shown previously to modify alternative splicing in human immunodeficiency virus type I. Human embryonic kidney 293T cells were transiently transfected by 3rd generation backbone and packaging plasmids using polyethyleneimine. Reverse transcription-quantitative polymerase chain reaction of the fraction of unspliced genomes in human embryonic kidney 293T cells increased up to 31% upon the indole's treatment at 2.5 uM. Corresponding yield of infectious lentiviral vectors decreased up to 4.5-fold in a cell transduction assay. Adjusting timing and duration of IDC16 treatment indicated that the indole's disruption of early stages of transfection and cell cycle had a greater effect on exponential time course of lentiviral vector production than its reduction of post-transcriptional splicing. Decrease in transfected human embryonic kidney 293T proliferation by IDC16 became significant at 10 uM. These findings indicated contributions by early-stage transfection, cell proliferation, and post-transcriptional splicing in transient transfection of human embryonic kidney 293T cells for lentiviral vector production.

Fig 1

Green fluorescent protein (GFP) expression identified transfected cells in (a) and transduced cells in (b). (a) GFP expression and cell confluency in a well section at 6 and 24 h post-transfection. (b) GFP expression by cells transduced via supernatant from transfected cells without or with DMSO imaged 24 h post-transfection in a well (below) and section (above). Untreated and DMSO-treated transfected cells expressed GFP; non-transfected cells did not. (c) GFP expression per 2.33 mm² of well measured at 6 h post-transfection (n = 6). (d) Total genome RNA viral copies per uL of supernatant 24 h post-transfection (n = 5 to 6). (e) Transduction units (TUs) or infectious virus per uL of supernatant (n = 6). (f) Ratio of total viral genomes to TU (n = 5 to 6). Bars and error bars show mean and SD. * indicates significant differences at p ≤ 0.05, ** at p ≤ 0.01, *** at p ≤ 0.001, **** at p ≤ 0.0001, and ns are ‘not significant’ differences.

Fig 2. Infectious virus and viral genome copies remained consistent across three untreated cell transfections.

(a) Cell transduction assay of TUs (n = 4 to 6), (b) total viral genome copies in cell extract (n = 4 to 5), and (c) unspliced viral genome copies in cell extract (n = 4 to 5). Symbols and error bars show mean and SD. ns are ‘not significant’ differences.

Fig 3. IDC16 increased the proportion of unspliced viral genomes in cells and reduced infectious LV production.

(a) Ratio of unspliced to total genome RNA viral copies (n = 6). (b) and (c) Infectious LV measured by cell transduction assay (TU/uL) for IDC16 treatment from 1 to 2.5 uM (n = 4) and from 0.5 to 2.5 uM (n = 6), respectively. Bars and error bars show mean and SD. * indicates significant differences at p ≤ 0.05, ** at p ≤ 0.01, *** at p ≤ 0.001, and **** at p ≤ 0.0001.

Fig 4. IDC16 was detrimental to cell proliferation and morphology in transfected cells.

(a) Cell proliferation at 0.5–2.5 uM IDC16 (n = 3). b) Cell proliferation at 2.5–10 uM IDC16 (n = 4). (c) Images at 24 h post-transfection for untreated cells, IDC16 treated cells, and DMSO-only treated controls at different dilutions. Bars and error bars show mean and SD. * indicates significant differences at p ≤ 0.05, ** at p ≤ 0.01, and *** at p ≤ 0.001.

Fig 5. Time trajectories of LV production post-transfection.

(a) Cell transduction assay and (b) production trajectory of LV in TU/uL at 10, 16, and 24 h post-transfection for untreated cells, 2.5 uM IDC16 + 35 mM DMSO treatment, and 35 mM DMSO control (n = 5 to 6). At 6 h post-transfection, treatments (none, IDC16 + DMSO or DMSO) were removed and washed out with DPBS from the transfected cell and only the enhancer solution (NaBu + KSR) was added. Points and error bars show mean and SD. * indicates significant differences at p ≤ 0.05, ** at p ≤ 0.01, and **** at p ≤ 0.0001.

Fig 6. Effect of timing and duration of IDC16 treatment on infectious LV production, and GFP expression 6 h post-transfection.

(a) Timing and duration of IDC16 treatment modified impact on infectious LV. Left: TU/uL after exposing cells from 0 to 24 h post-transfection to 2.5 uM IDC16 + 35 mM DMSO, 35 mM DMSO alone, or untreated control (n = 6). Middle: TU/ul after exposing cells from 0 to 6 h post-transfection to the same treatments (n = 5 to 6). Right: TU/ul after exposing cells from 6 to 24 h post-transfection to the same treatments (n = 5 to 6). (b) Number of fluorescing HEK293T cells per 2.33 mm² of well was measured by automatic fluorescent cell count after treatment by IDC16 across different dilutions (n = 5 to 6). Bars and error bars show mean and SD. ** indicates significant differences at p ≤ 0.01, **** at p ≤ 0.0001, and. ns are ‘not significant’ differences.