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

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
150kb genome
Double strained DNA virus
~80 genes
nM size
Epichromosome infection
Latent in neuronal cells but lytic in most non- neuronal cells U L Us

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. 4 intratumoural injection all showed some regression of the tumours. One patient had a lasting tumor regression.
2. Overall Survival
G207:median survival 7 m, Mean 6.2 m
1716:median survival 11 m, Mean 10.9 m
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. 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. b
CMV
ICP-4 5’
AAAAAAA-3’ a
miR143-targeted
miR124-targeted
miR143-
ICP-4 mRNA
cleaved mRNA of ICP-4 in normal cells or in neuronal cells
miR124
miR143

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
ARR2PB promoter
ICP27

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
Promoter
ICP4
Survivin
5’UTR
5'UTR of FGF-2
AAAAAAA-3’
miR124T

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
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 is more effective than wt in glioma model
ICP4-
CMV-icp4
SU4124
Icp4 show

22. OV enhancer: Nifuroxazide (NF)
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. 
NF human dose mg/kg
Collected form on 7/6/2016
References#
cited on 28th January, 2016
cited on 28th January, 2016
05/06/2018

23. NF and oHSV-1 combination synergistically enhances oncolysis
Indicated cells were treated with either indicated doses of NF or HrR3 or KOS. Varying concentration of NF was applied first and then indicated viral doses were added alone or in combination for 48 hours. Cytotoxicity was measured by an MTT assay. Error bars represents S.D. and statistically significant difference were indicated by P<0.05 or P<0.01 or P< combination index (CI) values were calculated using Chou-Talalay analysis and plotted against the affected fraction (Fa). CI of < 1, CI = 1 and CI > 1 represents synergistic, additive & antagonistic effect, respectively.
Antagonistic
Synergistic
05/06/2018

24. NF increases oHSV-1 replication
U87 were treated with HrR3 at an MOI of 1 and indicated concentration of NF for 48 hours . Total DNA was extracted and subjected to qPCR to detect HSV-1 using ICP27 primer. NF mediated oHSV-1 % increase is represented in the figure.
05/06/2018

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
U87 cells were treated with indicated concentration of HrR3 or NF or in combination for 24 hours. Extracted protein was then subjected to western blot assay.
05/06/2018

26. NF and oHSV-1 combination augment anti-tumor efficacy in CT26 xenograft in-vivo
HrR3 Injection
Initiation of NF treatment
BALB/C mice subcutaneously bearing the CT26 (colon cancer) tumour were intratumourally injected with a single dose of HrR3 virus (n=4; 2X10^7 PFU/ml) or vehicle with peritoneally injected 50mg/kg NF alone or in combination with HrR3 virus. NF were injected once daily.
05/06/2018

27. NF enhances HSV activity in vivo

28. NF mediated STAT-3 inhibition and enhanced oHSV-1 oncolysis in Glioma xenograft
Mice subcutaneously bearing the U87 tumour were intratumourally injected with a single dose of HrR3 virus (n=2; 1X10^6 PFU/ml) or peritoneally injected indicated concentration of NF alone or in combination with HrR3 virus. Tumours were harvested at day 10 post treatment.
Total genomic DNA was extracted from the harvested tumours .Viral DNA particles (ICP27) were detected by qPCR and normalized to β-actin.
Total protein was extracted from the harvested tumours and subjected to western blot analysis. ICP4 and pSTAT-1 and ICP27 and pSTAT-3 expression were determined by immunohistochemistry assay on harvested tumour tissues. Stained sections were then imaged using a fluroscence microscopy (20X).
Virus load enhancement by NF
NF reduce pSTAT-3 expression
05/06/2018

29. Regulation of STAT pathway

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