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Oncolytic HSV-1 with enhanced oncolysis and safety

Oncolytic HSV-1 with enhanced oncolysis and safety. WILLIAM JIA, PHD University of British Columbia Vancouver, CANADA w.jia@ubc.ca. Disclaimer. I am a founder and CSO of Virogin Biotech Ltd, a company working on oncolytic HSV-1 virus for drug development. Herpes simplex virus type-1. U.

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Oncolytic HSV-1 with enhanced oncolysis and safety

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  1. Oncolytic HSV-1 with enhanced oncolysis and safety WILLIAM JIA, PHD University of British Columbia Vancouver, CANADA w.jia@ubc.ca

  2. Disclaimer I am a founder and CSO of Virogin Biotech Ltd, a company working on oncolytic HSV-1 virus for drug development

  3. Herpes simplex virus type-1 U Us L 150kb genome Double strained DNA virus ~80 genes 150-200nM size Epichromosome infection Latent in neuronal cells but lytic in most non-neuronal cells

  4. The first OV clinical trial in China: VAE-1(2000)

  5. Phase I results of VAE-1 in comparison with other oHSV-1s 1. Image G207:6/20 patients shown tumour regression by image in one month, 1716:2/21 patients shown slightly reduced volume, 2/21 patients were stable VAE-1:16 viral injection after tumour removal, could not evaluate changes in image. 4intratumoural injection all showed some regression of the tumours. One patient had a lasting tumor regression. 2. Overall Survival G207:median survival 7m, Mean 6.2m 1716:median survival 11 m, Mean 10.9m VAE-1:median survival 14 m, Mean 14.3 m

  6. CT scan of a patient treated with VAE-1

  7. Oncolysis vs. tumor specificity • Reduced virulence to normal cells by deletions often causes less oncolytic activity • Can we enhance both oncolysis and safety? • tumor specific controlling of viral essential gene expression • Small molecules to enhance oncolytic activity

  8. TTDR oHSV: Transcription and Translation Dual Regulated oHSV-1

  9. HSV-1 Replication

  10. ICP-4 CMV AAAAAAA-3’ 5’ 3’-miRNA regulated oHSV-1s. • 5 copies of oligonucleotides complementary to the antisense of miR143, miR124 or the combination of 5 copies of each for miR143 and miR124 were integrated in tandem in the 3’ UTR of ICP-4 mRNA. • Theoretically, abundant miR143 in normal tissues or miR124 in neural tissues should induce tissue-specific cleavage of mRNA of ICP-4 and suppress HSV-1 replication. a ICP-4 CMV AAAAAAA-3’ 5’ miR143-targeted miR124-targeted ICP-4 CMV AAAAAAA-3’ 5’ miR143- miR124-targeted ICP-4 ICP-4 CMV CMV AAAAAAA-3’ ICP-4 mRNA AAAAAAA-3’ 5’ 5’ 5’ miR124 b miR143 cleaved mRNA of ICP-4 in normal cells or in neuronal cells

  11. 3’ miRNA regulation completely eliminates oHSV toxicity in animals

  12. 5’ UTR regulated oHSV-1 Molecular Therapy 18(5):929-35 A prostate specific TTDR-oHSV: The viral essential gene ICP27 is regulated by both tumor specific transcription and translation. Transcriptional regulation: a probesin promoter with 2 copies of androgen response elements (ARR2PB) for prostate specificity. Translational regulation: A 5’-UTR from hFGF-2 is inserted in the front of ICP27 ORF and is bound by an eIF-4E complex, which is highly expressed in tumor but not normal cells. A 5’UTR structure of hFGF mRNA ICP27 ARR2PB promoter

  13. eIF4E Expression level • eIF4E is overexpressed in prostate, lung and breast cancer cells (LNCaP, H460, MCF-7) • eIF4E level is low in normal prostate cells (BPH-1, PNT1B)

  14. ARR-UTR-ICP27 is responsive to androgen and replicates in a tumor-specific fashion • A27: oHSV-1 with androgen responsive and prostate specific promoter (ARR2PB) regulated ICP27 viral essential gene (transcriptional control only). • AU27: a TTDR oHSV-1 with the above ARR2PB promoter and 5’UTR to control ICP27 expression. • +/- A: with or without androgen Note: replication of AU27 can be upregulated by androgen but remains 600-fold higher in tumor cells (LNCaP) than non-malignant prostate cells (NT1B)

  15. AU27 is equally effective as A27 or wt HSV by intratumoral injection on LNCaP model

  16. AU27 causes tumor regression by i.v. injection Single injection by tail vein at 105 and 107 pfu virus/mouse on subcutaneous LNCaP model. Even the 105 dose is effective.

  17. SU4124: A triple regulated oHSV-1 ICP4 5’UTR Promoter Survivin 5'UTR of FGF-2 miR124T AAAAAAA-3’

  18. Survivin and eIF4E in glioma Survivin promoter activity and eIF4E level in glioma cells are higher than normal neurons.

  19. SU4124 is 100-fold more selective to glioma than wt HSV-1 Expression of ICP4 in tumour/brain

  20. SU4124 replicates more efficiently than wt in glioma

  21. SU4124 is more effective than wt in glioma model ICP4- CMV-icp4 SU4124 Figure 11. SU4-124 significantly augment antitumor effect without increasing virus spread to other organs. Subcuteneously U87 tumor bearing mice were intratumorally injected with tumor non specific CMV-ICP4 (n=4; 2.4X10^7 PFU/ml helper : 6X10^6 PFU/ml amplicon) or tumor specific SU4-124 (n=5; 2.4X10^7 PFU/ml helper : 6X10^6 PFU/ml amplicon) or ICP4-- 3galΔ3 (n=5; 2.4X10^7 PFU/ml helper only ) at 4:1 helper : amplicon ratio. (A) Tumor volume was measured by using calipers (Height X Length X Wide/2) and represents as fold change difference. Data are presented as mean SD, * P<0.001 vs only helper and ** P<0.05 vs CMV-ICP4 treatment. (B) Total protein from CMV-ICP4 (n=4) & SU4-124 (n=4) virus injected tumors were extracted at the end of the experiment (day 8). ICP27 & β-actin protein expression were determined by western blot analysis. (C) Total genomic DNA was extracted from the different harvested organs of the indicated virus injected subcutaneously U87 bearing mice (n=2). Viral DNA particles (ICP27) were detected by qPCR and normalized to β-actin. SU4124 CMV-icp4 ICP4-

  22. OV enhancer: Nifuroxazide (NF) Collected form https://pubchem.ncbi.nlm.nih.gov/compound/Nifuroxazide#section=2D-Structure on 7/6/2016 • References# • http://pharmacybook.net/nifuroxazide/, cited on 28th January, 2016 • http://theamazingmedicine.blogspot.ca/2013/08/nifuroxazide.html, cited on 28th January, 2016 • Nifuroxazide is an oral nitrofuran antibiotic, and used to treat diarrhea in humans2. • Adults: 4 times a day 200 mg capsules (800 mg daily)1. • Nifuroxazide has no side effects and is well tolerated2. 

  23. NF and oHSV-1 combination synergistically enhances oncolysis Antagonistic Synergistic

  24. NF increases oHSV-1 replication

  25. NF & oHSV-1 combination inhibits STATs activation • oHSV-1 infection increases STAT-1 and STAT-3 phosphorylation • NF & oHSV-1 combination inhibit STATs phosphorylation

  26. NF and oHSV-1 combination augment anti-tumor efficacy in CT26 xenograft in-vivo HrR3 Injection Initiation of NF treatment

  27. NF enhances HSV activity in vivo

  28. NF mediated STAT-3 inhibition and enhanced oHSV-1 oncolysis in Glioma xenograft Virus load enhancement by NF NF reduce pSTAT-3 expression

  29. Regulation of STAT pathway

  30. ACKNOWLEDGEMENT Paul Rennie’s lab • Kevin Zhang • Jun Ding Funding agencies • Canadian Cancer Society • ViroGin Biotech Ltd My lab: • Luke Bu • Guoyu Liu • Cleo Lee • Zahid Delwar

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