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

• Approved antiviral drugs only inhibit replication

Gene editing as an approach to cure HBV

Uncoating

Nuclear import

Repair

cccDNA

rcDNA

TranscriptionTranslation

Encapsidation

Viral proteins

Budding into ER

Exocytosis

Entry

CYTOPLASMNUCLEUS

Viral RNAs

RTi

Reverse Transcription

Targeted Endonuclease

HBcAg/Polymerase

HBsAg

Recycling

pgRNA

• cccDNA elimination or inactivation could prevent HBV persistence

Linearization anddegradation

• cccDNA persists in hepatocyte nucleus throughout its life time

Mutation/inactivation

Gene editing ≠ Cas9

modified from Schiffer et al, J Virol 2012

CRISPR/Cas1 ORF + guide RNA

>3.3 kb coding sequenceLeaves blunt endsTrivial retargeting

Specificity controversialDifficult vectorization

Nishimasu et al, Cell 2014

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

www.ltk.uzh.ch/de/dyn_output.html

Potential Viral Targets for Gene Editing

HBV HSVHIV HPV

+

HBV-specific ZFNs inhibit HBV replication

Weber et al, PLoS ONE 2014

Humanized FRG mouse as a model for HBV

de Jong et al 2010

scAAV-LK03-smCBA-eGFP14 days post intravenous delivery

2 X

1011

geno

mes

EVOS 10X αGFP 4X αGFP 20X

αhuman albumin 20X αmouse albumin 20X mergeC

ontr

ol m

ouse

αhuman albumin 4X αGFP 4X merge

2 X

1011

geno

mes

scAAV-LK03 transduces human hepatocytes in FRG mice

HBV replication in the FRG mouse(Genotype C)

3

4

5

6

7

8

9

10

11

HBV

Gen

ome

Copi

es (L

og 1

0/m

L)

Vehicle

Entecavir

Compound A

Compound B

Treatment

S.aureus CRISPR/Cas9 for HBV

Target 1

ITR ITREFS-MVM NLS-saCas9-NLS-HA SV40pA sgRNA hU6 sgRNAhH1

Target 2

• 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

5’5’3’

3’3.2 Kb

5’5’3’

3’3.2 Kb

A1

A10 C7

A12A4 C3

C6C1

C16A5A8

C14

Genotype A Genotype C

HBV genotype C saCas9 sgRNA test

eGFP

Target 1-Target 2-Target 3

SV40pAMND

GFP6-20 GFP7-20

Reporter constructs (4 total)

Untreated Reporter

GFP6-20 GFP7-20

Timeline for HBV-C+ FRG mice Group A1 # 1M001 14**Group A1 # 1M002 28*

Group B1 # 2M001 21***

Group B1 # 2M002 11***

Group A2 # 3M00162*Group A2 # 3M002

56***Group A2 # 3M003 26***

Group A2 # 3M004 19**

Group A2 # 3M00562*

Group B2 # 4M00162*Group B2 # 4M00262*Group B2 # 4M00362*

Group B2 # 4M00445***Group B2 # 4M005

55**Group B2 # 4M006 28*#

Anti-GFP

Anti-HBV

Anti-GFP

Anti-HBV

IV AAV delivery 5 x 1011 vg/mouse

0 84

* scheduled sacrifice** found dead (No terminal blood sample)*** sacrificed due to health (Terminal blood sample)# sacrificed early as part of group B1

Weeks

No EntecavirEntecavir

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

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

Day 1 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 62

Body

Wei

ght (

gram

s)

Study Day versus Body Weight (Weekly)

Group 1 (GFP-AAV)

Group 2 (HBV-AAV)

Group 3 (GFP-AAV)

Group 4 (HBV-AAV)

in vivo gene editing of HBV

Viral loads during CRISPR/Cas9 treatmentH

BV

IU/m

l

-24 -3 7 21 28 35 42 49-10-17 14 56102

103

104

105

108

109

1010

AAV day 0

Entecavir

Anti-GFP

Anti-HBV

Anti-GFP

Anti-HBV

*

**

**

**

**

Premature death

Planned sacrifice day 28

*

Days

106

107

SLU qPCR UW qPCR

Viral loads during CRISPR/Cas9 treatment

-24 -3 7 21 28 35 42 49-10-17 14

AAV day 0

Anti-GFPAnti-HBVAnti-GFPAnti-HBV

Days

Entecavir

HB

V IU

/ml

103

104

105

108

109

1010

106

107

SLU qPCR UW qPCR

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

AcknowledgmentsAlexander AstrakhanPaula CannonJordan JarjourHans-Peter KiemDavid RawlingsPavitra RoychoudhuryAndrew ScharenbergJoshua SchifferBarry Stoddard

Daniel StoneMartine Aubert

Tom AndrusChung DangHarshana De Silva FeelixgeMeei-Li HuangShiu LiangMichelle LoprienoNixon NiyonzimaHarlan PietzRuth Hall SedlakLarry StenslandNick WeberMary Lamery

7th Wave LabsYecurisCellectisPregenen/BluebirdSangamo

Caladan Foundation