Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome

Figures

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  FIG 1. Reverse genetics system for CVA21. (a) Schematic of reverse genetics system of CVA21 cDNA plasmid to yield rCVA21. The CVA21 cDNA is flanked by a T7 promoter at the 5′ end and a 25A sequence at the 3′ end. The plasmid was linearized and transcribed in vitro to generate vRNA. rCVA21 was generated following transfection of SK-Mel-28 cells with transcribed vRNA. (b) Growth titers of original WT CVA21 versus rCVA21 at 12 and 24 h after infection of SK-Mel-28 cells at an MOI of 1. The experiments were performed in triplicate, and titers were determined using the TCID50 method (mean with SD; n = 3). Error bars represent standard deviations. ns, P ≥ 0.05, not significant.

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  FIG 2. Random transposon insertion mutagenesis system identifies potential transgene locations in the CVA21 genome. (a) Schematic of Mutation Generation System (Thermo Scientific) applied to CVA21 cDNA followed by recombinant CVA21 virus library generation. The vRNA was purified from the resulting virus library and sent for standard RNA next-generation sequencing (NGS). (b) Relative frequency of cDNA plasmids with 15-bp insert in plasmid library measured by NGS indicates approximately random, equal insertion across CVA21 cDNA. Only a random small portion of the insertions (5%) was plotted in the graph here due to limited space. Right below the x axis, the CVA21 genome diagram was drawn to scale to show the positions of viral genes. (c) Relative frequency of rCVA21 with 15-nt insert in rescued virus library measured by NGS indicates tolerable insert locations identified using the procedures listed in panel a. Two independent experiments were performed, and the insertion positions and frequencies are shown for each experiment.

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  FIG 3. The 5′ UTR region next to the ORF region of the CVA21 genome is amenable to engineering to carry small miRNA sponge sequences. (a) Schematic showing insertion of 1 or 3 copies of miRNA21 (miR21) sponge sequences between nucleotides 641 and 698. (b) Growth titers of rCVA21 versus two rCVA21s carrying miRNA21 (miR21) sponge sequences at 12 and 24 h after infection of SK-Mel-28 cells at an MOI of 1. The experiments were performed in triplicate, and titers were determined using the TCID50 method (mean with SD; n = 3). Error bars represent standard deviations. ns, P ≥ 0.05, not significant; *, 0.01 < P < 0.05, significant.

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  FIG 4. Tolerability of up to 141 amino acids at the 5′ UTR-VP4 junction. (a) Schematic showing insertion of various-sized GFP inserts placed upstream of the VP4 gene. Each truncated (102- to 197-aa) or full-length GFP (239-aa) ORF followed by a tetracysteine (TC) tag sequence was inserted and connected to the VP4 gene through an in-frame 3C cleavage site. (b) RT-PCR results showed the stability of GFP and TC inserts encoding up to 141 aa in rescued recombinant viruses. The primer pair NA41 and NA42, whose positions are shown in panel a, is used for PCR to amplify the region spanning the 5′ UTR-VP4 junction. The top gel shows the PCR from input cDNA plasmids; the bottom is the RT-PCR result from purified vRNAs derived from rescued viruses. The solid arrows designate intact inserts, and hollow ones are shortened inserts compared with cDNA inputs. (c) Growth titers of rCVA21 versus two rCVA21s carrying GFP sequences at 12 and 24 h after infection of SK-Mel-28 cells at an MOI of 1. The experiments were performed in triplicate, and titers were determined using the TCID50 method (mean with SD; n = 3). Error bars represent standard deviations. **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001;…

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  FIG 5. Tumor cell-killing activity of rCVA21-UnaG compared to rCVA21. (a) Cell viability of SK-Mel-28 cells postinfection with rCVA21 or rCVA21-UnaG at an MOI range of 10 to 0.0001 as assessed using CellTiter-Glo 2.0 assay in 96 wells (mean with SD; n = 3). For a continuous 3 days (1 to 3 days postinfection [dpi]), the infected cells were subjected to the assay. Data were normalized to uninfected conditions with blank subtracted. (b) Comparison of the apoptosis caused by rCVA21 versus rCVA21-UnaG using annexin V staining monitored by IncuCyte. Cells were seeded in 96-well assay plates, and the viruses and annexin V reagents were added the following day under MOIs of 10 to 0.0001. The data were taken at 2-h intervals for 3 days. (b, Left) Degree of apoptosis measured by red fluorescent protein (RFP) channel in a time-lapsed manner. (b, Right) Cell confluence of SK-Mel-28 cells monitored by IncuCyte. Duplicate experiments are shown here.

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  FIG 6. miniCMV promoter-driven CVA21 cDNA integration into the cell genome and subsequent de novo generation of recombinant virus from stable cell pool. (a) Schematic of design of CVA21 cDNA cassettes driven by either a miniCMV or normal CMV promoter. A 25A stretch and SV40 poly(A) sequence were placed at the 3′ ends. (b) Diagram of experimental procedures used to generate stable cell pools by integrating miniCMV-CVA21 or CMV-CVA21 into the cell genome followed by cell passaging and virus titer determination in growth media. (c) Titer of supernatants from 293T cells electroporated (P0) and passaged 6 times (P6) in the presence of puromycin selection. The P0 samples were harvested from cells 1 day postelectroporation in triplicate wells for each plasmid. The P6 samples were harvested from triplicate wells on day 3 of passage 6 for each group. Error bars represent standard deviations. **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001. (d) qPCR quantification of CVA21 cDNA levels at passages 3 (P3) and 7 (P7) of miniCMV-CVA21 293T cell pool. The experiment was done in 12 repeats for each sample. Error bars represent standard deviations. ns, P ≥ 0.05, not significant.