[PPT] - Phenotypic HCV-NS3-PI-Resistance Test Arevir Meeting, 11./12.04.2013 PowerPoint Presentation

SLIDE 1

Phenotypic HCV-NS3-PI-Resistance Test

Arevir Meeting, 11./12.04.2013 Daniel Rupp, MD

SLIDE 2

Phenotypic HCV-NS3-PI-Resistance Assay

11./12.04.2013 Arevir Meeting, Köln

Goal: Establishing a phenotypic NS3-PI resistance assay to characterize and monitor mutation development in patient derived samples Principle: Creating replicon-libraries containing patient-derived NS3-protease sequences with subsequent drug titration and IC50/IC90 calculation

SLIDE 3

Characteristics of subgenomic HCV-Replicons

11./12.04.2013 Arevir Meeting, Köln

bicistronic RNA-construct expression of Luciferase and HCV- non-structural-proteins (replicase) Luciferase activity reflects replication

RNA Luciferase

Replication Kinetics

0,01 0,1 1 10 100 1000 24 48 72 Timepoint after Epo RLU (fold 4h) Con1-ET Con1-deltaNS3Prot

SLIDE 4

Characteristics of subgenomic HCV-Replicons

11./12.04.2013 Arevir Meeting, Köln

bicistronic RNA-construct expression of Luciferase and HCV- non-structural-proteins (replicase) Luciferase activity reflects replication

RNA Luciferase

Replication Kinetics

0,01 0,1 1 10 100 1000 24 48 72 Timepoint after Epo RLU (fold 4h) Con1-ET Con1-deltaNS3Prot

Modify Replicons by introducing unique restriction sites for patients‘ sequence shuttling

SLIDE 5

Introducing unique restriction sites enables to shuttle patient derived NS3-protease sequences goal: create library that reflects the quasispecies-pool circulating in patients

Phenotypic NS3-PI-Resistance Assay for gt1b-patients

11./12.04.2013 Arevir Meeting, Köln

AscI ClaI

Replication Fitness Con1-ET-1xAsc

20 40 60 80 100 120 % of Con1-ET

100,00 43,08 Con1-ET Con1-ET-1xAsc

AscI ClaI

Con1-ET: 4000x above background Con1-ET-1xAsc: 2200x above background

SLIDE 6

Phenotypic NS3-PI-Resistance Assay for gt1b-patients

11./12.04.2013 Arevir Meeting, Köln

AscI ClaI

Con1-ET Con1-ET-1xAsc 100,00 43,08

20 40 60 80 100 120 % of Con1-ET

Replication Fitness Con1-ET-1xAsc

AscI ClaI AscI ClaI

Replication Fitness - Con1-ET-1xAsc- Mutants

20 40 60 80 100 120

% of Con1-ET-1xAsc

100,00 37,83 54,92 Con1-ET-1xAsc V36A T54A

Introducing the unique restriction-sites ClaI and AscI and known resistance mutations (V36A, T54A) only slightly reduce the replication fitness of Con1-ET-1xAsc

SLIDE 7

Phenotypic NS3-PI-Resistance Assay for gt1b-patients

11./12.04.2013 Arevir Meeting, Köln

RNA Luciferase 0h 4h 72h

+ DRUG

SLIDE 8

Phenotypic NS3-PI-Resistance Assay for gt1b-patients

11./12.04.2013 Arevir Meeting, Köln Validation Resistence Mutants - Boceprevir

20 40 60 80 100 120 140 0,01 0,1 1 10 Concentration (µM) % compared to untreated

Con1-ET-1xAsc V36A T54A

Validation Resistence Mutants - Telaprevir

20 40 60 80 100 120 140 0,01 0,1 1 10 Concentration (µM) % compared to untreated

Con1-ET-1xAsc V36A T54A

RNA Luciferase 0h 4h 72h

+ DRUG

Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Con1-ET-1xAsc 178 1229 141 748 V36A 1824 9220 383 489 T54A 860 2862 748 2609

shift in IC50!

SLIDE 9

Phenotypic NS3-PI-Resistance Assay for gt1b-patients

11./12.04.2013 Arevir Meeting, Köln

Replication fitness

20 40 60 80 100 120

% Con1-ET-1xAsc

100,00 46,85 33,93 75,48 105,28 Con1-ET- 1xAsc 1-1 1-2 2-1 2-2

Introducing NS3-protease sequences from treatment naive patients (1, 2; sera provided by Sandra Ciesek) can affect replication fitness…

SLIDE 10

Phenotypic NS3-PI-Resistance Assay for gt1b-patients

11./12.04.2013 Arevir Meeting, Köln Replication fitness

20 40 60 80 100 120

% Con1-ET-1xAsc

100,00 46,85 33,93 75,48 105,28 Con1-ET- 1xAsc 1-1 1-2 2-1 2-2

Telaprevir Titration

20 40 60 80 100 120 140 160 0,01 0,1 1 10 Concentration (µM) % of untreated

Con1-ET-1xAsc gt1b 1-1 gt1b 1-2 gt1b 2-1 gt1b 2-2

Boceprevir Titration

20 40 60 80 100 120 140 0,01 0,1 1 10 Concentration (µM) % of untreated

Con1-ET-1xAsc gt1b 1-1 gt1b 1-2 gt1b 2-1 gt1b 2-2

Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Con1-ET-1xAsc 323 411 257 457 1-1 281 476 290 507 1-2 253 532 238 504 2-1 278 399 216 386 2-2 200 396 187 372

… but does not cause an shift in IC50/IC90. Introducing NS3-protease sequences from treatment naive patients (1, 2; sera provided by Sandra Ciesek) can affect replication fitness…

SLIDE 11

Phenotypic NS3-PI-Resistance Assay for gt1a-patients

11./12.04.2013 Arevir Meeting, Köln

Modifying the genotype 1a-Replicon H77 by introducing restriction sites for NS3- protease shuttling leads to a complete loss of replication competence. Solution: introduce the gt1a-protease into the gt1b-Replicon backbone to create a hybrid-construct.

AscI ClaI

gt1b gt1a

SLIDE 12

Phenotypic NS3-PI-Resistance Assay for gt1a-patients

11./12.04.2013 Arevir Meeting, Köln

Modifying the genotype 1a-Replicon H77 by introducing restriction sites for NS3- protease shuttling leads to a complete loss of replication competence. Solution: introduce the gt1a-protease into the gt1b-Replicon backbone to create a hybrid-construct.

AscI ClaI

gt1b gt1a

Replication Fitness gt1b/1a Hybrid Vector

20 40 60 80 100 120 140 % of Con1-ET-1xAsc

100,00 7,44 16,29 16,50 Con1-ET-1xAsc Con1/H77 #4 #5

Con1-ET-1xAsc: 5000x above background Con1/H77: 650x above background

SLIDE 13

Phenotypic NS3-PI-Resistance Assay for gt1a-patients

11./12.04.2013 Arevir Meeting, Köln

Modifying the genotype 1a-Replicon H77 by introducing restriction sites for NS3- protease shuttling leads to a complete loss of replication competence. Solution: introduce the gt1a-protease into the gt1b-Replicon backbone to create a hybrid-construct.

AscI ClaI

gt1b gt1a

Replication Fitness gt1b/1a Hybrid Vector

20 40 60 80 100 120 140 % of Con1-ET-1xAsc

100,00 7,44 16,29 16,50 Con1-ET-1xAsc Con1/H77 #4 #5

SLIDE 14

Phenotypic NS3-PI-Resistance Assay

11./12.04.2013 Arevir Meeting, Köln

Patient samples from Cologne (provided by Anna Marie Sikorski, Saleta Sierra-Aragón)

Titration - Boceprevir

20 40 60 80 100 120 140 160 180 0,001 0,01 0,1 1 10 Concentration (µM)

% untreated

Con1-ET-1xAsc #26432 #25724

Titration - Telaprevir

20 40 60 80 100 120 140 0,001 0,01 0,1 1 10 Concentration (µM)

% untreated

Con1-ET-1xAsc #26432 #25724

gt1b

Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Con1-ET-1xAsc 298 703 174 794 #26432 275 6467 332 2490 #25724 317 1354 69 66131

SLIDE 15

Phenotypic NS3-PI-Resistance Assay

11./12.04.2013 Arevir Meeting, Köln

Titration - Boceprevir

20 40 60 80 100 120 140 160 0,001 0,01 0,1 1 10 Concentration (µM)

% untreated

Hybrid #10304 #3524 #10172 #10306 #9719

Titration - Telaprevir

20 40 60 80 100 120 140 160 180 0,001 0,01 0,1 1 10 Concentration (µM)

% untreated

Hybrid #10304 #3524 #10172 #10306 #9719

gt1a Patient samples from Cologne (provided by Anna Marie Sikorski, Saleta Sierra-Aragón)

Telaprevir Boceprevir IC50 (nM) IC90 (nM) IC50 (nM) IC90 (nM) Hybrid 49 756 74 297 #10304 491 4621 2182 20863 #10306 70 695 78 271 #3524 109 2102 156 976 #10172 922 3202 524 1808 #9719 1792 17258 420 1629

SLIDE 16

Summary and Outlook

11./12.04.2013 Arevir Meeting, Köln

drug titration works for gt1b and gt1a/b-Hybrid replicons resistance mutations in paternal replicons cause a shift in IC50/IC90 values sequentially taken samples show no difference in titration curves samples of clinically resistant patients show a shift in titration curves

SLIDE 17

Summary

11./12.04.2013 Arevir Meeting, Köln

drug titration works for gt1b and gt1a/b-Hybrid replicons resistance mutations in paternal replicons cause a shift in IC50/IC90 values sequentially taken samples show no difference in titration curves samples of clinically resistant patients show a shift in titration curves

Tool for phenotypic monitoring of patients under NS3-Protease-Inhibitor treatment

SLIDE 18

Ralf Bartenschlager

Acknowledgements

Rolf Kaiser Saleta Sierra-Aragón Anna Marie Sikorski Ulrike Protzer Thomas von Hahn Sandra Ciesek

11./12.04.2013 Arevir Meeting, Köln