Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma

Donald D Rao, Christopher Jay, Zhaohui Wang, Xiuquan Luo, Padmasini Kumar, Hilary Eysenbach, Maurizio Ghisoli, Neil Senzer, John Nemunaitis, Donald D Rao, Christopher Jay, Zhaohui Wang, Xiuquan Luo, Padmasini Kumar, Hilary Eysenbach, Maurizio Ghisoli, Neil Senzer, John Nemunaitis

Abstract

The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.

Figures

Figure 1
Figure 1
bi-shRNAEWS-FLI1 construct and target sequence. (a) Circular diagram of bi-shRNAEWS-FLI1 construct (pGBI-140). Key vector elements and single cut restriction sites are shown. (b) Target sequence for pGBI-140, -141, and -142 in relation to the translocation break point sequence. (c) Predicted secondary structure of the primary transcript of bi-shRNAEWS-FLI1 (pGBI-140). The predicted stem-loop structure is first processed in the nucleus by the microprocessor complex before transported into the cytoplasm.
Figure 2
Figure 2
Target gene expression knockdown and EWS-FLI1 selective growth inhibition of Ewing's sarcoma cells (SK-N-MC, type I mutation). (a) SK-N-MC cells were transfected with 0.1 µg of each plasmid DNA by electroporation. Two days after transfection, cells were lysed with CelLytic MT solution and process for western immunoblot. The Ewing's type 1 fusion protein was detected via antibody to FLI-1 and percentage reduction in expression was obtained by normalizing fusion protein expression to GAPDH expression. (b) SK-N-MC cells or HEK-293 cells (embryonic kidney cell line without EWS-FLI1 fusion) were transfected with 0.1 or 1 µg of plasmid DNA by electroporation, respectively. After electroporation, cells were plated in 96-well and assayed for growth with CellTiter-Glo Luminescent Cell Viability Assay measuring relative light units (rlu) at 24 hours after. No effect was observed on cells not containing EWS/FLI1 fusion. pUMVC3 is the empty vector control; 140, 141, and 142 are different fusion region targeting constructs.
Figure 3
Figure 3
Tumor growth inhibition characterized with SK-N-MC xenograft model. (a) Study # e203- Tumor volume change demonstrating marked dose related tumor growth control with both higher dose (25 µg, green line) and lower dose (5 µg, blue line) of two bi-shRNAEWS-/FLI1 LPX constructs when compared to no treatment (black line) and vehicle control (gray line). Treatment was twice weekly for four weeks. Graph is average of 10 animals per group. (b) Study # e204- Dose–response experiment assessing percent of animals alive (tumor did not reach the maximum allowed tumor size) with slightly elevated doses and extended treatment time to 6 weeks. Kaplan-Meier survival curve showed survival advantage with high dose groups (25.0 and 18.75 µg dose groups, green line) compared with low-dose groups (12.5 and 6.25 µg dose groups, blue line) and no treatment and vehicle control group (black line). Ten animals were assessed per group. (c) Study # e207- Tumor growth inhibition of bi-shRNAEWS/FLI1 LPX (using improved LP) assessed with further escalated doses; 25 µg (blue line), 50 µg (green line), 75 µg (red line) and control (black line). Treatment was twice weekly for four weeks and tumor measurements over 45 days post the initiation of the treatment. Graph shown is the medium of 10 animals per group. Arrows indicate IV infusion time points.
Figure 4
Figure 4
Ewing's fusion protein expression knockdown demonstrated in vivo. Study # e205-Nude mice carrying SK-N-MC xenograft were treated with 25 µg (lane 3), 18.75 µg (lane 4), 12.5 µg (lane 5), and 6.25 µg (lane 6) of bi-shRNAEWS/FLI1 LPX through slow tail vein infusion at three animal per treatment. The treatment started when tumor was 100–150 mm3 in size, the treatment was twice weekly for 2 weeks. Two days after the final treatment, tumors were taken out and half of the tumors were immersed in Allprotect tissue reagent (Qiagen, Valencia, CA) for molecular analysis. Sections of tumor in Allprotect tissue reagent were processed for western immunoblot with antibody for FLI-1 and for CD99. The fusion protein and CD99 expressions were normalized against β-Actin for comparison. Tumors from untreated (lane 1) and vehicle treated (lane 2) animals were used for comparison. This figure shows a representative result of tumor tissue slices and tumors examined.
Figure 5
Figure 5
Induction of proinflammatory cytokines by bi-shRNAEWS/FLI1LPX. Five male and five female Balb/C mice were administrated with single dose of 100 µg bi-shRNAEWS/FLI1 LPX via slow tail vein infusion. At 1, 3, 6, 24, and 48 hours postinfusion, blood was collected via tail vein bleed and assayed for proinflammaroty cytokines (IFN-γ, TNF-α, IL-12, and IL-6). Graph shown is a plot of the average of serum cytokine value with standard deviations.
Figure 6
Figure 6
Blood GGT level. Comparison of GGT levels representative as assessment of liver function between control LPX (n = 4), pbi-shRNAEWS/FLI1 LPX 0.128 mg/kg dose group (n = 4) and pbi-shRNAEWS/FLI1 LPX 0.384 mg/kg dose group (n = 4) tracked throughout the entire dose schedule (eight doses administered/2× per week). Shaded box indicates treatment dates.

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