Enhanced genomic stability of new miRNA-regulated oncolytic coxsackievirus B3

Figures

• 

  Figure 1. Construction of the second-generation miR-CVB3s (A) Relative expression of miR-1, miR-133, miR-216, and miR-375 in various NOS-SCID mouse tissues and in H526-derived xenograft tumors was measured by qRT-PCR and presented as mean ± SD (n = 3). (B) New miRNA-modified CVB3s (miR-CVB3s), namely miR-CVB3-2.1 and miR-CVB3-2.2, were generated by substituting viral genomic RNA within 5′UTR with target sequences (TS) of organ-specific miRs (miR-CVB3-2.1) or inserting these organ-specific miR-TS into P1 region of viral genome (miR-CVB3-2.2) as indicated. miR-CVB3-1.1 was previously generated by inserting tumor-suppressive and organ-selective miR-TS into the 5′UTR of CVB3 as indicated and used as a control.

• 

  Figure 2. Measurement of genomic stability of newly generated miR-CVB3s in vitro (A) Illustration of the primer binding sites (blue lines) on TS of indicated miR-CVB3s. Primers binding to P1 region or 2A within P2 region were used as references (internal controls). Note that two primer pairs (denoted as “site 1” and “site 2”) were designed to assess the integrity of miR-CVB3-2.2 by monitoring the existence of two separate miR-TS inserts. (B, C) miR-CVB3s were serially passaged in HL-1 cardiomyocytes for up to 20 passages. Cell supernatants were harvested every five passages for viral genome quantification by qRT-PCR using primers as described above targeting miR-TS (B) or P1 region (C) relative to the copy numbers of 2A (mean ± SD, n = 3). (D) Absolute quantification of viral genome copy numbers in the supernatants was determined using primers targeting 2A (mean ± SD, n = 3 for each group). ∗, p < 0.05; #, p < 0.01; &, p < 0.005 compared with miR-CVB3-1.1. ns, not significant.

• 

  Figure 3. Evaluation of lytic and replicative ability of new miR-CVB3s in vitro (A) KRASmut lung adenocarcinoma H2030 and TP53mut/RB1mut SCLC H526 cell lines were sham-infected or infected with indicated WT- or miR-CVB3s at MOI = 0.01, 0.1, 1 for 72 h. Cell viability was assessed by alamarBlue assay (mean ± SD, n = 3). (B) HL-1 cardiomyocytes, H2030, and H526 lung cancer cells were inoculated with WT- or miR-CVB3s at MOI = 0.01 for indicated hours. Cell lysates were harvested for RNA extraction and quantification of viral genome by qRT-PCR using 2A primer pairs. The viral copy number at day 1 is set arbitrarily to 1 and fold changes are presented throughout the time course (mean ± SD, n = 3). Unpaired Student’s t test was conducted. ∗, p < 0.05; #, p < 0.01; &, p < 0.005; $, p < 0.001 compared with WT-CVB3. PI, post-infection.

• 

  Figure 4. Safety evaluation of new miR-CVB3s in immunocompetent mice A/J mice were inoculated intraperitoneally with PBS (sham infection), WT- or miR-CVB3s at a dose of 1 × 106 PFU for up to 14 days. (A) Body weight was measured every other day (mean ± SD, n = 3 for each group) and normalized to that on day 0, which was arbitrarily set a value of 1.0. (B) Kaplan-Meier survival rate (n = 3 for each group) was recorded and plotted daily until experimental endpoint (day 14 following treatment). p < 0.05 comparing survivals in WT-CVB3 group with those in PBS, miR-CVB3-2.1, or miR-CVB3-2.2 group by log rank test. (C, D) H&E staining was performed on the heart and pancreas collected at day 4 post-treatment from a different cohort of mice (C), and pathological scores (mean ± SD, n = 3 mice) were evaluated based on the H&E staining (D). Arrows indicate tissue damage and inflammatory infiltration. (E) Blood was collected at day 4 post-infection (mean ± SD, n = 4 mice) for biochemical analysis of serum lipase levels using Advia 1800 autoanalyzer. (F, G) Immunostaining was conducted to assess viral protein VP1 expression in mouse heart and pancreas harvested on day 4 and 10/14 (WT-CVB3 and miR-CVB3,…

• 

  Figure 5. Assessment of safety and oncolytic efficacy of new miR-CVB3s in a xenograft SCLC mouse model SCID mice bearing H526-derived xenograft tumors were treated intraperitoneally once a week with PBS (n = 8), miR-CVB3-1.1 (n = 4), miR-CVB3-2.1 (n = 8), or miR-CVB3-2.2 (n = 8) at a dose of 1 × 106 PFU. (A) Kaplan-Meier survival rate was monitored until experimental endpoint (day 150 post-treatment). (B) Body weight was measured twice every week and normalized to those on day 0 (mean ± SD). (C) Tumor volume was measured twice weekly until experimental endpoint (mean ± SD). (D) Average number of viral injections until tumor growth was firmly suppressed. (E) Peak tumor volumes in each group. (F) Days taken to stably suppress tumor growth for 2 weeks. ∗, p < 0.05; #, p < 0.01; &, p < 0.005; $, p < 0.001. ns, not significant. PI, post-infection.