The Chimpanzee Model Of HCV: Antiviral...

The Chimpanzee Model Of HCV: Antiviral Therapy

Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA

Antiviral Therapies in Chimpanzees• PEG‐IFN + Ribavirin

• STAT‐C with DAA–Protease Inhibitors–Nucleoside analogues –Polymerase Inhibitors–NS5A Inhibitors

• Immunomodulators‐ TLR7, TLR9, McAb to cytokines and cytokine receptors

• Antisense‐ siRNA targeting viral RNA

• Therapeutic vaccines

• Essential host targets‐Cyclophilin inhibitors‐miR122 antisense – LNA‐Entry Inhibitors – 4 receptors


Phase I, PK in uninfected animals• Dosing, acceptability• Serum to liver ratio

Phase II, Efficacy in HCV chronic animals, n=2‐4• Kinetics of decline of vRNA• Evolution of resistance mutations• Persistence of resistance mutations

Dosing limitations Oral: if accepted by chimps indefinite periods, 3‐6 months.

Tang, Kool‐Aid, Applesauce (micro‐encapsulated)Gavage, IV, SQ , IM all require sedation

IACUC normally limits study to 5‐7 doses with follow‐up bleeds progressively spaced out

Chimps can be trained for oral pills and with some effort for SQand IM injection

Design of Antiviral Studies in Chimpanzees


STAT-C Therapy in Chimps


Chen, C.-M. et al. 2007. Antimicrob. Agents Chemother. 51(12):4290-4296

Abbott A‐837093 NNI (benzothiadiazine) HCV G1a and 1b infected chimpanzees

BID, 14 days

• Very rapid decline, G1b decrease 2.5 logs in 2 days• Rapid emergence of resistance in 1b• Day 5, G1b 13% WT; G1a, 74% WT


Pilot-Matias et al, International Symposium on HCV, Nice France 2009. Abbott

-5.0-4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0-0.50.0

0 2 4 6 8 10 12 14 16 18 20Time (Days)

Vira

l Loa

d C

hang

e Fr

om B

asel

ine

(Log

10IU

/mL)

Last dose

All clones wt

Dosing period (12.8 mg/kg) BID by oral gavage

46%1%1%5%6%

11%10%20%

R155KA156TA156VD168AD168ED168GD168Vwt

Efficacy of Protease Inhibitor EA‐058 in a Genotype 1a‐Infected Chimpanzee

• >4‐log10 reduction in viral load after 2 days of treatment• Only day 4 contained virus with known resistance mutations; • Short‐term treatment (2 days) did not lead to persistence of resistance mutations


Carroll, S. S. et al. 2009. Antimicrob. Agents Chemother. 53(3):926-934

Viral loads in HCV-infected chimps dosed at 1 mg/kg MK-0608 orally once daily for 37 days

282 mutation observed after dosing for single day in X6


HCV Chronic Infection• 10 HCV chronics; viremia vary by 1000‐fold: 104 to 107 ge/ml

• Expression of 971 genes altered at 99% confidence

• ISG and cytokines also elevated during acute infection

• Elevated hepatic IFN response persists for decades with little pathology

Infected Uninfected

Incr

ease

dD

ecre

ased

Bigger et al. J Virol 78: 13779-92,2004.Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA

Hepatic ISG Levels Independent of vRNA Levels

Gene Fold Change Avg fold change vs all 6 baselines

ISG27 50 54 59 56 80 76 71 66 82 70

IP-10 CXCL10 18 12 13 8 14 17 2 13 31 46ITAC CXCL11 11 6 11 6 4 23 4 24 19 51MXA 15 13 9 8 13 12 6 11 11 18OAS2 13 13 13 8 11 11 5 8 11 14

Animals selected to have 1000 fold variation in viremiaVariation in liver ISG response not proportional to viremia; maxed-out

Bigger et al. J Virol 78: 13779-92,2004.Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA

• Previous studies with Ad gene therapy for IFN failed

• How many HCV‐induced genes are ISGs

• Total genome response to IFN in vivo

• Single dose induction in 3 uninfected chimpanzees

human and chimpanzee IFN tested

5x106 IU IFN SQ Liver and PBMC 0, 4, 8, 24 hrs

Chimpanzee Response to IFNαCHIMPANZEE RESPONSE IFN


Hepatic ISG Response Rapidly Down-Regulated

0 4 8 24

ISG 15

25

50

100Fo

ld C

hang

e

0 4 8 24

hr post IFN

Ch IFN

Hu peg-IFN

25

50

100

Fold C

hang

e

>500pg/ml IFN

Rapid Down-Regulation of ISG Response in Presence of High IFN Levels

Contrasts to lack of down-regulation during acute and chronic infection.

Lanford et al, Hepatology 43:961-972, 2006.Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA

HCV Chronic Chimps Null IFNα Response

Time After First Dose of IFN

-4 wk

-2 wk

4 hr8 hr24 hr

1 wk

2 wk

4 wk

6 wk

8 wk

9 wk

10 wk

11 wk

12 wk

13 wk

14 wk

18 wk

GE/

ml

10 3

10 4

10 5

10 6

10 7

10 8

10 9

Genotype 1b

Genotype 3a Genotype 1a

Peg IFN

Lanford et al, Hepatology 46:999-1008, 2007.


Uninfected

0 4 8 24

Fold

Inc

reas

e

0

200

400

600

800

Liver

Hours Post-IFN0 4 8 24

Fold

Inc

reas

e

0

100

200

300

400

500

HCV Chronic

0 4 8 240

20406080

100120


020406080

100120

Fold Change IP-10 Transcription


PBMC


Uninfected

0 4 8 24

Fold

Inc

reas

e

0

200

400

600

800

Liver


Fold

Inc

reas

e

0

100

200

300

400

500

HCV Chronic

0 4 8 240

20406080

100120


020406080

100120

Fold Change IP-10 Transcription


PBMC

FC vs uninfected

FC vs HCV baseline


Conclusions from IFN Studies• ISG response highly elevated in chronic chimpanzee liver

• May be maximally induced

• Exogenous IFN does not further induce liver ISG response

• Chimps maybe be representative of human null responders

• Humans: two phenotypes high or low ISG prior to therapylow ISG correlates with protective IL28B alleles

• Potential of antivirals to convert null response phenotypereduced vRNA reduce ISGs = responder phenotype?

• Only 35% of chimps progress to chronic infection, protective allele• All chimps examined have high ISG phenotype and lack response to IFN,

non‐protective allele• Chimps are not polymorphic at same sites in IL28B as humans


miR122 liver specific miRNA

70% of all miRNA in liver - 50,000 copies per cell

miR122 regulates cholesterol and fatty acid synthesis

Jopling et al demonstrated HCV requires miR122Two miR122 binding sites in 5’ NCR,

Santaris developed LNA technologymiR122 knockdown in vivo in AGMreduced TSC ~40%

Potential for HCV therapy

Targeting Host Factors for Antiviral Therapy


• SPC3649: 15-mer high-affinity LNA-antimiR122

•(8 LNAs, Tm=80 oC, PS backbone)

UGUUUGUGGUAACAGUGUGAGGU 5’ miR122CcAttGTcAcaCtCC 3’ SPC3649

CGACACUCCACCAUGAAUCACUCCC HCVGUGAGGU miR‐122GUGAGGU5’

CGACACUCCACCAUGAAUCACUCCC HCV

SPC3649 treatment


Safety–Efficacy Trial in HCV Infected Chimpanzees

4 HCV genotype 1 infected chimpanzees: 2 high dose - 5 mg/kg2 low dose - 1 mg/kg

4 weeks pre-study12 weeks IV dosing in saline14 weeks follow up

Proof of Concept Study


• 2.6-log reduction in HCV RNA• No viral rebound during dosing• Maximum HCV suppression extended 11 weeks after dosing• miR122-LNA duplex still present in liver at 14 weeks after dosing• Total serum cholesterol reduced by 45%.

SPC3649-Prolonged Antiviral Activity

Lanford et al, Science 327:198-201, 2010.

HCV VIRAL RNA

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

-4 -2 0 2 4 6 8 10 12 14 17 21 25

week

GE/

ml

placebo dosing

-100

-80

-60

-40

-20

-10 1 2 3 4 5 6 7 8 910111213141517192123

Chn

age

in T

SC

Total Serum Cholesterol


No Selection of Adaptive Changesin miR-122 Seed Matches

• Clonal and Deep sequencing of high dose animals• No increase in nt changes in 5’ NCR during dosing


Normalization of Hepatic ISG Expression

• Affymetrix microarray data – Supervised analysis of ISGs• ISGs decline during treatment or normalize• HCV infected chimpanzees are Null responders to IFN • Conversion of Null Phenotype to Responder Phenotype


CONCLUSIONS

•SPC3649 provides long term sequestration of miR122.

•No rebound of virus and no selection of adaptive variants.

•Very long half life in liver

•SPC3649 provides a high barrier to resistance.

•TSC was reduced by up to 44% in chimpanzees and 30% in humans.

•The ISG response was normalized suggesting the opportunity to more effectively treat IFN null responders.


Collaborators

Southwest FoundationDeborah ChavezBernadette GuerraHelen LeeLena NotvallYunmi ChungSNPRCKathleen Brasky DVM

Stan LemonChris WalkerDave Thomas

Santaris PharmaLisa Hildebrandt-EriksenAndreas PetriSakari KauppinenHenrik Oerum


The Chimpanzee Model Of HCV: Antiviral...

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