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
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)
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