Polycytidine tract deletion from microRNA-detargeted oncolytic Mengovirus optimizes the therapeutic index in a murine multiple myeloma model

Figures

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  Figure 1. PolyC tract length variably affects Mengovirus propagation in different cell types (A) Schematic representation of Mengovirus genome: 5′ and 3′ NCRs flanking the coding region (P1-3) with individual genes are shown. Detailed 5′ NCR RNA secondary structures in MC0 (polyC-deleted), MC24 (polyC truncated), and wild type (Mwt) are represented. The polyC positioning between pseudoknot (pk) and stem-loop D is highlighted in red. Based on Carocci and Bakkali-Kassimi,22 Martin et al.,45 and Duque and Palmenberg.51 (B) Propagation of Mengovirus wild-type (Mwt) or the polyC-deleted variant (MC0) in primary cells isolated/differentiated from BALB/c mice. Bone marrow-derived macrophages (BM-Mφ), bone marrow-derived dendritic cells (BM-DCs), splenocytes, and adult fibroblasts were infected at MOI 1 and cell viability (left) was determined 48 h post-infection through MTT test. Multi-step growth curves (MOI, 0.01) (right) of Mwt and MC0 in splenocytes and in adult fibroblasts were used to evaluate replication. (C) Viral propagation of Mwt, MC24, or MC0 in immune (A20 and MPC-11) and non-immune murine tumor cell lines. Cytotoxicity (left) was assessed through MTT assay performed 48 h…

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  Figure 2. MicroRNA-detargeted variants with expanded polyC tracts exhibit miRT instability and reduced replicative fitness but maintain targeting specificity (A) Schematic representation of miRNA-detargeted Mengovirus polyC variant viruses. Length of the polyC tract, and length and location of the miRT inserts are shown. RNA structural elements highlighted: pseudoknot (pk), stem-loop D (D), and stem-loop I–III. Tables (right) summarize miRT sequences of target tissues and features of the vectors compared with wild-type (Mwt). (B) Rescue protocol and stability of miRT inserts at 32°C versus 37°C. Viruses were rescued in triplicate by RNA transfection and single passage in H1-Hela cells. A summary of the number of rescue attempts that successfully retained the miRT sequences at each temperature is shown on the right, sequencing data shown in Figure S2. (C) Functionality and specificity of microRNA detargeting. H1-Hela cells reverse transfected with cognate miRNA mimics (miR-124, miR-133, miR-208) or negative controls (neg ctrl, miR-125, mock transfection) were infected with miRNA-detargeted viruses or MC24 at an MOI of 0.2. Cell viability (left) determined by MTT assay and viral titers…

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  Figure 3. miRNA-detargeted polyC-deleted MC0-NC is associated with more rapid intratumoral replication and superior in vivo oncolytic efficacy BALB/c mice bearing s.c. MPC-11 tumors were treated with a single intratumoral injection of PBS or 1 × 107 TCID50 of MC0-NC, MC24-NC or MC37-NC, according to the protocol outlined in (A). A second cohort of mice (Exp2) with identical protocol set-up were killed at day 2 and day 6 post-injection to assess viral load in tumors and relevant organs. (B) Overall survival of control PBS-treated (n = 10), MC0-NC (n = 10), MC24-NC (n = 10), or MC37-NC (n = 10) mice was assessed using Kaplan-Meier survival curves, and statistical significance established through log rank (Mantel-Cox) test (∗p < 0.5; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). Responses to miRNA-detargeted virus therapies are summarized in the table. (C) Disease burden monitored by repeated caliper measurements of tumor volume over time. (D) Tumor regression post miRNA-detargeted virus treatment measured by tumor volume assessed by caliper measurements of tumor volume on day 2 and day 6 post-injection (n = 5 per group, per time point). (E) Viral loads in the tumors of miRNA-detargeted…

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  Figure 4. Systemically administered MC0-NC is also associated with more rapid intratumoral replication and superior in vivo oncolytic efficacy BALB/c mice bearing s.c. MPC-11 tumors were treated with a single intravenous injection of PBS or 1 × 107 TCID50 of MC0-NC, MC24-NC, or MC37-NC, according to the protocol outlined in (A). (B) Overall survival of control PBS-treated (n = 10), MC0-NC (n = 10), MC24-NC (n = 10), or MC37-NC (n = 10) mice assessed using Kaplan-Meier survival curves, and statistical significance established through log rank (Mantel-Cox) test (∗p < 0.5; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). Responses to miRNA-detargeted viruses therapies are summarized in the table. (C) Disease burden monitored by repeated caliper measurements of tumor volume over time. (D) Plasma viral loads on day 2 post-injection in miRNA-detargeted virus-treated mice (n = 5 per group) measured as TCID50/mL of plasma. Statistical significance was established through one-way ANOVA with Tukey multiple comparison (∗p < 0.5; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). Cartoons created with BioRender.com.

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  Figure 5. The MC0-NC genome shows greater miRT insert stability compared with MC24-NC and MC37-NC genomes Sequence alignment of the 3′ NCR insertion sites (A) and the RNA polymerase gene (3D) (B) from viral RNA isolated from the plasma of mice treated intratumorally (i.t.) and intravenously (i.v.) with MC0-NC (mice 11–15), MC24-NC (mice 21–25) and MC37-NC (mice 31–35). A 150-nt-long sub-region of the 3D gene sequence is shown for comparison with the ∼150 miRT insert region (B). Bold red letters represent point mutations and dashed lines represent deletions. Numbering on the reference sequences refers to the nucleotide position with respect to the beginning of the 3′ NCR and 3D gene, to account for the difference in length among the three viruses. Results for miRT sequence retention on 3′ NCR are summarized in the graph (right).