In vivo evaluation of anti-hbv CRISPR/Cas9 therapy in the FRG mouse

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1 In vivo evaluation of anti-hbv CRISPR/Cas9 therapy in the FRG mouse Keith R. Jerome, MD PhD Fred Hutchinson Cancer Research Center University of Washington December 5, 2017

2 Gene editing as an approach to cure HBV Entry Uncoating Exocytosis Approved antiviral drugs only inhibit replication cccdna persists in hepatocyte nucleus throughout its life time cccdna elimination or inactivation could prevent HBV persistence Nuclear import Recycling Budding into ER Reverse Transcription HBsAg HBcAg/Polymerase Targeted Endonuclease rcdna Repair Linearization and degradation RTi pgrna Encapsidation Viral proteins NUCLEUS Transcription cccdna Mutation/inactivation Viral RNAs Translation CYTOPLASM

3 Gene editing Cas9 CRISPR/Cas Nishimasu et al, Cell ORF + guide RNA >3.3 kb coding sequence Leaves blunt ends Trivial retargeting Specificity controversial Difficult vectorization modified from Schiffer et al, J Virol 2012

4 Gene editing as a genetic therapy If cleavage and mutation were to occur within the coding sequence for an essential viral protein, viral production and pathogenesis would be prevented

5 Potential Viral Targets for Gene Editing HIV HBV HSV HPV +

6 HBV-specific ZFNs inhibit HBV replication Weber et al, PLoS ONE 2014

7 Humanized FRG mouse as a model for HBV de Jong et al 2010

8 Control mouse scaav-lk03 transduces human hepatocytes in FRG mice αhuman albumin 20X αmouse albumin 20X merge 2 X genomes EVOS 10X αgfp 4X αgfp 20X 2 X genomes αhuman albumin 4X αgfp 4X merge scaav-lk03-smcba-egfp 14 days post intravenous delivery

9 HBV replication in the FRG mouse (Genotype C) 11 Treatment 10 HBV Genome Copies (Log 10/mL) Vehicle Entecavir Compound A Compound B 4 3

10 S.aureus CRISPR/Cas9 for HBV S.pyogenes Cas9 has shown good activity against HBV in vitro spcas9 is too large to be effectively used with AAV vectors S.aureus Cas9 is smaller and can be readily used with AAV vectors Target 1 Target 2 ITR EFS-MVM NLS-saCas9-NLS-HA SV40pA sgrna hu6 sgrna hh1 ITR Genotype A Genotype C A12 A4 A1 C3 A10 C7 3.2 Kb 3 C Kb A5 A8 C16 C6 C1

11 Reporter constructs (4 total) HBV genotype C sacas9 sgrna test GFP6-20 GFP7-20 MND egfp SV40pA Target 1-Target 2-Target 3 Untreated Reporter GFP6-20 GFP7-20

12 Timeline for HBV-C + FRG mice Group A1 # 1M Group A1 # 1M Group B1 # 2M Group B1 # 2M Anti-GFP Anti-HBV Group A2 # 3M001 Group A2 # 3M002 Group A2 # 3M Anti-GFP Group A2 # 3M Group A2 # 3M005 Group B2 # 4M001 Group B2 # 4M002 Group B2 # 4M003 Group B2 # 4M Group B2 # 4M Group B2 # 4M006 28# Anti-HBV Entecavir No Entecavir Weeks IV AAV delivery 5 x vg/mouse scheduled sacrifice found dead (No terminal blood sample) sacrificed due to health (Terminal blood sample) # sacrificed early as part of group B1

13 In vivo tolerability of anti-hbv gene editing No weight loss that differed from animals receiving control anti-gfp therapy No morbidity or behavioral findings that differed from control animals No histopathologic changes attributable to AAV/Cas9 therapy 35.0 Study Day versus Body Weight (Weekly) 30.0 Body Weight (grams) Group 1 (GFP-AAV) Group 2 (HBV-AAV) Group 3 (GFP-AAV) Group 4 (HBV-AAV) Day 1 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 62

14 in vivo gene editing of HBV

15 Viral loads during CRISPR/Cas9 treatment AAV day Anti-GFP Anti-HBV 10 8 HBV IU/ml Anti-GFP Anti-HBV 10 4 Entecavir 10 3 Premature death Planned sacrifice day Days SLU qpcr UW qpcr

16 Viral loads during CRISPR/Cas9 treatment AAV day Anti-GFP Anti-HBV Anti-GFP Anti-HBV Entecavir 10 8 HBV IU/ml Days SLU qpcr UW qpcr

17 Conclusions from initial in vivo trial in vivo therapy with AAV/Cas9 is well tolerated Gene editing of HBV in liver can be achieved No gene-edited HBV was observed in plasma, consistent with loss of replicative capacity Low-level gene editing does not result in decreased plasma viremia Next steps What was the cause of low editing frequency? poor viral suppression with entecavir Is Cas9 the optimal enzyme? confirmation of efficient hepatocyte transduction and Cas9 expression quantitation of gene editing in rcdna vs. cccdna

18 Acknowledgments Daniel Stone Martine Aubert Tom Andrus Chung Dang Harshana De Silva Feelixge Meei-Li Huang Shiu Liang Michelle Loprieno Nixon Niyonzima Harlan Pietz Ruth Hall Sedlak Larry Stensland Nick Weber Mary Lamery Alexander Astrakhan Paula Cannon Jordan Jarjour Hans-Peter Kiem David Rawlings Pavitra Roychoudhury Andrew Scharenberg Joshua Schiffer Barry Stoddard 7 th Wave Labs Yecuris Cellectis Pregenen/Bluebird Sangamo Caladan Foundation