John Bell Centre for Innovative Cancer Therapeutics

Transcription

1 Enhancing Oncolytic Virus Activity by Engineering of Artificial micrornas John Bell Centre for Innovative Cancer Therapeutics Affiliated with Affilié à 1

2 Oncolytic Viruses: A Therapy for Metastatic Cancers? Lung Tumours Targeted Infection Tumour Clearance 2

3 Lessons Learned: Ideal Oncolytic Immunotherapeutic Tumor specific and highly lytic Intravenous Delivery: effective against metastatic disease Robust, long lasting tumor specific adaptive immune response Functional, cytolytic TIL and epitope spreading Complements Immune Checkpoint Inhibitors, IO therapies Strong IP and scalable production 3

4 Bio-prospecting for a potent OV Candidate Bioprospecting Rhabdovirus Screening Enhancement/ Augmentation Recombinant system Virus Isolates In Vitro screening assays (cytotoxicity & replication) Candidates (good tumour cell killers) Recombinant Bioselection In vivo efficacy testing Transfect DNA 293T cells Harvest after hours In vivo toxicity/ biodistribution profiling (naïve and tumour bearing animals) Orthotopic models (brain, lung, breast) Plaque Purify & Amplify Candidates (Safe and Effective)

5 Maraba virus: an ideal oncolytic & vaccine candidate Rhabdovirus Structure Rhabdovirus Life Cycle 11 kb single stranded negative sense RNA genome Matrix protein (M) is involved in budding, silencing host gene expression, and apoptosis Little pre-existing immunity; Cytoplasmic life cycle no genotoxicity; Genetically stable; Fully functionalized recombinant system (mutagenesis, payload); Ease of GMP manufacturing; Strong Patent position; Unique immune-stimulating platform.

6 How to increase potency?

7 Manipulate the Infected Host Cell with amirnas Cell Host and Microbe Sept 2013

8 A B Genome-targeting Sindbis virus-based library (16,000 amirna) Library designed using the Hannon and Elledge algorithm In Collaboration with Dr. tenoever (Mt. Sinai, New York)

9 Large-Scale Genome Screening to Identify Virus Replication Factors P0 of SV-LIB ( unique clones) Identification of host factors that enhance virus pathogenicity Varble et al, 2013

10 The challenges Pancreatic Cancer Highest mortality rate of all major cancers (Survival has not improved in the past 30 years) No curative treatment options Fibrotic nature (Cancer-associated fibroblasts are the predominant component of Pancreatic tumor stroma) P013 P019 P023 Pancreatic cancer patient samples- anti-asma

11 Our Strategy Select/engineer oncolytic viruses that have enhanced capacity to replicate in and kill both Pancreatic cancer cells and the CAFs that support their malignant growth/progression In vivo Serial Passaging on CAFS and PCa Deep sequencing In vitro

12 Project Overview MiaPaca-2 Human pancreatic carcinoma In vitro PanC1 Human Pancreatic/ductal carcinoma In vitro PanC02 Mouse Pancreatic carcinoma In vitro PaCF Fetal Pancreatic Fibroblasts (~CAFs) In vitro U125T Patient-derived CAFs In vitro GM38 Normal Lung Fibroblasts In vitro MiaPaca-2 Human pancreatic carcinoma In vivo MiaPaca-2+ PaCF P009 P014 P020 Human pancreatic carcinoma + Pancreatic Fibroblasts Pancreatic cancer Patient-derived grafts In vivo In vivo In vivo In vivo

13 amirna Activity in Maraba Virus 13

14 MG1 virus-mediated Silencing of Luciferase Arbitrary Luciferase Luminescence MG1-amiRLuc 5 N P M G L MOCK MG1 MG1-shLUC/shGFP p< DOX +DOX Induction Condition MiaPaca-Luciferase cells - Tetracycline ON inducible gene expression system

15 Enrichment of amirnas that enhance Oncolytic Virus Growth in PCa and CAFs Fold increase (relative to Library) GM38 PF MP PC2 MP-IV MP+PaCFs-IV amir-1 amir-2&3 amir-4 amir-5 amir-6 amir-7 amir-8 amir-9 amir-10 In Collaboration with Dr. Boutros (Ontario Institute for Cancer Research, Toronto)

16 Cell death (%) % Cell Death % Cell Death % Cell Death % Cell Death % Cell Death Enriched amirnas confer enhanced OV cytotoxicity 60 PanCO2 MiaPaca-2 PanC VSV-miRGFP VSV-amiR VSV-miRGFP VSV-amiR1` VSV-amiR2 VSV-amiR3 VSV-amiR4 VSV-amiR5 VSV-amiR6 VSV-amiR8 VSV-amiR9 VSV-amiR10 MOCK VSV-miRGFP VSV-amiR1` VSV-amiR2 VSV-amiR3 VSV-amiR4 VSV-amiR5 VSV-amiR6 VSV-amiR8 VSV-amiR9 VSV-amiR10 MOCK HPAFII HPAC PanFIB VSV-amiR2 VSV-amiR3 VSV-amiR4 VSV-amiR5 VSV-amiR6 VSV-amiR8 VSV-amiR9 VSV-amiR10 VSV-miRGFP VSV-amiR1` VSV-amiR2 VSV-amiR3 VSV-amiR4 VSV-amiR5 VSV-amiR6 VSV-amiR8 VSV-amiR9 VSV-amiR10 MOCK VSV-miRGFP VSV-amiR1` VSV-amiR2 VSV-amiR3 VSV-amiR4 VSV-amiR5 VSV-amiR6 VSV-amiR8 VSV-amiR9 VSV-amiR10 MOCK VSV-miRGFP VSV-amiR1` VSV-amiR2 VSV-amiR3 VSV-amiR4 VSV-amiR5 VSV-amiR6 VSV-amiR8 VSV-amiR9 VSV-amiR10 MOCK

17 Mock VSVD51-amiRGFP VSVD51-amiR-6 PanC1 PanCO2 HPAF II

18 Objective: To identify synergistic interactions between virotherapy and synthetic lethality for Pancreatic cancers. Synthetic lethal screens can be used to identify genes or small-molecule compounds to specifically target tumour cells while sparing the normal tissue

19 mirna transfer via exosomes may mediate bystander effect mirna are secreted in exosomes (local environment and systemic delivery) Tumor-associated exosomes determine organotropic metastasis (proven in Breast and Pancreatic cancer patients). Integrin-mediated exosomal tropism and cargoes delivery (Hoshino et al. Nature.2015) Sorting of mirna into exosomes is controlled by short motifs (EXOmotifs) (Villarroya-Beltri et al, Nature communications) Epstein-Barr virus-infected cells secrete exosomes that contain EBV-encoded mirnas (Pegtel et al, PNAS. 2010)

20 Infection increases exosome secretion in pancreatic cell lines HPAC Panc1 OVCAR8 SKOV3 HPAFII kda CD63 Secreted exosomes were concentrated by ultracentifugation. Lane 1 = uninfected Lane 2 = infected with VSV-GFP MOI 0.1

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