Oncolytic Activity of Targeted Picornaviruses Formulated as Synthetic Infectious RNA

Figures

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  Figure 1. CVA21-3′miRT Insertion Site Disrupts oriR Structure Resulting in Reduced Specific Infectivity and Oncolytic Activity of iRNA (A) Infectious virus recovery time course in H1-HeLa and Mel624 cells. Cells were transfected with 1 μg in-vitro-derived RNA transcripts encoding CVA21 or CVA21-3′miRT genomes. Samples were collected at various times posttransfection and infectious virus titrated. The experiment was run in triplicate, and data are represented as mean viral titers ± standard deviations. (B and C) CB17 ICR-SCID mice bearing subcutaneous Mel624 xenografts were treated intratumorally with saline (n = 5) or 30 μg iRNA encoding CVA21 (n = 5) or CVA21-3′miRT (n = 5). (B) Viral titers in sera collected from mice on day 7 posttreatment. (C) Tumor volumes (black lines) and weights (gray lines) of all treated mice. (D) Predicted secondary structures of CVA21 and CVA21-3′miRT 3′ NCRs. Stem loops Y and X are labeled, and the predicted pseudoknot interactions are depicted by lines.

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  Figure 2. In Vitro Characterization of MicroRNA-Detargeted CVA21 (A) Schematic representation of the CVA21 genome and location and sequence of the muscle-specific microRNA response elements (REs) analyzed. (B) Infectious virus recovery time course of all iRNAs in H1-HeLa and Mel624 cells. Cells were transfected with 1 μg in-vitro-derived RNA encoding microRNA-detargeted genomes. Samples were collected at various times posttransfection and infectious virus titrated. The experiment was run in triplicate, and data are represented as mean viral titers ± standard deviations. (C) One-step growth curve analysis of microRNA-detargeted CVA21 viruses versus wild-type in H1-HeLa cells. Cells were infected at an MOI of 3. Samples were collected at various times postinfection and total virus titrated. The experiment was run in triplicate, and data are represented as mean viral titers ± standard deviations.

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  Figure 3. In Vitro Characterization of MicroRNA-Detargeting Specificity and Efficacy H1-HeLa cells were transfected with 100 nM microRNA mimics and infected 12 h later with unmodified or miRT-CVA21 at an MOI of 1. (A and B) Cell viability (A) and virus titer in the supernatant (B) were measured 24 h postinfection. This experiment was repeated in duplicate (n = 2; with five distinct technical replicates), and the data are represented as interleaved minimum and maximum box and whisker plots with median marks. ∗p < 0.05 compared with mock infection controls.

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  Figure 4. R-CVA21 iRNA Mounts a Spreading Oncolytic Infection in Mice Bearing Mel624 Xenografts without Causing Toxicity CB17 ICR-SCID mice bearing subcutaneous Mel624 xenografts were treated intratumorally with saline (n = 5) or 30 μg RNA encoding unmodified CVA21 (n = 5), sR-CVA21 (n = 5), R-CVA21 (n = 5), D-CVA21 (n = 5), or CVA21-sTR (n = 4). (A) Tumor volumes (black or colored lines) and weights (gray lines) of all treated mice. (B) Kaplan-Meier survival graphs of the mice in (A). Overall survival comparisons were based on log rank statistics with ∗p ≤ 0.05 and ∗∗p ≤ 0.01 considered significant. Clinical toxicities observed are represented by colored lines or symbols in (A) and (B) as follows: hindlimb paralysis (red), sudden death (orange), weight loss (green), and tumor ulceration (blue). (C) Viral titers in sera collected from mice in (A) on day 7 posttreatment. Horizontal lines represent mean viral titers.

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  Figure 5. Threshold Level of R-CVA21 iRNA Delivery Is Required for Tumor Destruction CB17 ICR-SCID mice bearing subcutaneous Mel624 xenografts were treated intratumorally with saline (n = 4) or 32 μg (n = 5), 16 μg (n = 5), 8 μg (n = 5), 4 μg (n = 5), or 1 μg (n = 5) R-CVA21 iRNA. (A) Tumor volumes (black or colored lines) and weights (gray lines; not taken at end of study) of all treated mice. (B) Viral titers in sera collected from mice in (A) on day 9 posttreatment. (C) Kaplan-Meier survival curves of the mice in (A). Overall survival comparisons were based on log rank statistics with ∗p ≤ 0.05 and ∗∗p ≤ 0.01 considered significant. Clinical toxicities observed are represented by colored lines or symbols in (A) and (B) as follows: hindlimb paralysis (red), sudden death (orange), tumor ulceration (blue), and moribund (gray).