MODELING FTDP-17 LINKED TAUOPATHIES AND ALZHEIMER S DISEASE WITH GENETICALLY MODIFIED HUMAN IPSC

Transcription

1 MODELING FTDP-17 LINKED TAUOPATHIES AND ALZHEIMER S DISEASE WITH GENETICALLY MODIFIED HUMAN IPSC An Verheyen, PhD Janssen R&D AVerhey1@its.jnj.com

2 Tauopathies & neurodegeneration Frontotemporal dementia and Alzheimer s disease Most common types of dementia Progressive neurodegenerative disease Loss of synaptic plasticity and connectivity Neuron loss and brain volume loss Cognitive deficits and behavioral changes No cure available Need for translational models Plaque β-amyloid FTDP-17 mutations Neurofibrillary tangles (NFT) hyper-p-tau Spillantini & Goedert. The Lancet Neurology images/alzheimer_brain.jpg

3 Genetically modified ipsc-derived tau neurons 1. Virally-induced mutant tau ipsc-derived neurons for phenotypic screening 2. Zinc finger nuclease engineered mutant tau ipsc-derived neurons for disease mechanisms

4 Genetically modified ipsc-derived tau neurons 1. Virally-induced mutant tau ipsc-derived neurons for phenotypic screening 2. Zinc finger nuclease engineered mutant tau ipsc-derived neurons for disease mechanisms

5 DIV MW6 Plating Axol NPC AAV tau 2N4R-P301L MW 96 K18 fibrils AlphaLISA assay Aggregated tau (htau10/htau10) Total tau (HT7/hTAU10) htau10 TAU aggregation signal

6 HuC/D AT8 DAPI Robust tau aggregation triggered by K18 AT8 staining after 1% triton/pfa fixation Sarkosyl-extractions/WB

7 ipsc - tau screen validation - setup Assay format Source of NPC s (cortical) 384 well plates; 7500 cells/well; independent duplo Axol Biosciences Ltd. (custom differentiation) Assay (tau aggregation) Assay (total tau & toxicity) Compounds >2400 htau10/htau10 alphalisa HT7/hTAU10 alphalisa ( ATP for toxicity)

8 ipsc - tau screen validation compound library Selective kinase inhibitors: 657 compounds L1000 Anti-correlated: 159 Correlated: 154 LOPAC 536 compounds LOPAC, 536 PhenoPrint, 1334 L1000, 313 Selective kinase inhibitors, 657 Other, 909 Targets of interest, 628 Tau compounds, 165 Toxic compounds, 116 PhenoPrint: 1334 compounds Targets of interest (literature based): 628 Tau compounds TAPS (HEK293): false positives Tau PET: 38 compounds Toxic compounds: 116 compounds (historic Janssen data) 3473 unique compounds available (some overlap between groups exists). 8

9 ipsc - tau screen validation - setup Assay format Source of NPC s (cortical) Assay (tau aggregation) Assay (total tau & toxicity) Compound library 384 well plates; 7500 cells/well; independent duplo Axol Biosciences Ltd. (custom differentiation) htau10/htau10 alphalisa HT7/hTAU10 alphalisa ( ATP for toxicity) >2400 cpds Compound treatment (3 M) Quality control criteria Z > 0,5 S/N > 10 spotted in sterile plates diluted in medium 3 hours before addition of K18 fibrils compounds on cells for days

10 2428 cpds 3355 cpds

11 ipsc - tau screen validation - setup Assay format Source of NPC s (cortical) 384 well plates; 7500 cells/well Axol Biosciences Ltd. (custom differentiation) Assay (tau aggregation) Assay (total tau & toxicity) Compound library >2400 Compound treatment (3 M) Quality control criteria Z > 0,5 S/N > 10 htau10/htau10 alphalisa HT7/hTAU10 alphalisa ( ATP for toxicity) spotted in sterile plates diluted in medium 3 hours before addition of K18 fibrils compounds on cells for days Hit aggregated tau (htau10/htau10) total tau (HT7/hTAU10) Amount of active compounds (>40% inhibition) in set 1 + set 2 Further analyses 258 (10%) with toxic cpds included 79 (3,2%) without toxic compounds ongoing

12 Genetically modified ipsc-derived tau neurons 1. Virally-induced mutant tau ipsc-derived neurons for phenotypic screening 2. Zinc finger nuclease engineered mutant tau ipsc-derived neurons for disease mechanisms

13 Zinc finger nuclease engineered tau mutant ipsc MAPT EXON INTRON E1 IVS10+16 C>T E9 E10 E11 E12 E13 p.p301s (C>T) CTRL SM (mono) SM (bi) DM (bi, mono) DM (bi, bi) Gene editing by Sigma = MAPT exon 10 = MAPT intron 10 = IVS10+16 C>T point mutation = p.p301s (C>T) point mutation IVS10+16 C>T intronic mutation destabilization of stem-loop structure that regulates the alternative splicing of exon 10 exon 10 inclusion and 4R tau isoforms p.p301s (C>T) mutation pro-aggregation of tau

14 ZFN engineered tau mutant ipsc are pluripotent and have a normal karyotype IVS10+16 C>T p.p301s (C>T) IVS10+16 SM mono P301S IVS10+16 IVS10+16 SM bi IVS10+16/P301S DM bi/bi IVS10+16/P301S DM bi/mono

15 n p c : is o g e n ic c o n tro l n p c : S M m o n o n p c : S M b i n p c : D M m o n o n p c : D M b i w 5 : is o g e n ic c tr w 5 : S M m o n o w 5 : S M b i w 5 : D M m o n o w 5 : D M b i N o rm a liz e d e x p r e s s io n IVS10+16 promotes the inclusion of exon 10 and increases the expression of 4R tau RTPCR (NPC and week 5) Western blot week 5 (total tau) 4 3 R 4 R Tau ladder mono bi P301S - mono bi bi bi IVS N4R 0N3R 0

16 ra tio E d U /D A P I E d U c o u n t n o rm a liz e d to N P C s ta g e DAPI count normalized to NPC Reduced proliferation in IVS10+16 neurons 8 6 CTRL 1F05-D12 (SM, bi) 7H6A1 (DM, bi) ISOGENIC CONTROL IVS10+16 (SM) IVS10+16/P301S DM NPC N P C Preliminary N P C w e e k s in c u ltu re DAPI+ and EdU+ cells in IVS10+16 and IVS10+16/P301S neurons compared to isogenic control P<0.05, P<0.01 and P<0.001

17 IVS10+16/P301S (DM) IVS10+16 (SM) ISOGENIC CTR IVS10+16 neurons are different from controls DAPI TAU MAP2 TUBB3 DAPI TBR1 CTIP2 DAPI vglut2 TUBB3 DAPI vgat TUBB3 week 5

18 IVS10+16 neurons are different from isogenic controls microarray analyses Limma adjusted P<0, genes = 9.61% 878 genes and 900 genes

19 IVS10+16 neurons are different from isogenic controls microarray analyses Gene ontology biological process Gene ontology molecular function

20 Neuron fate commitment significant genes

21 Differentially expressed genes (IVS10+16 vs ctr)

22 Differentially expressed genes (IVS10+16 vs ctr)

23 Functional differences in ZFN engineered Tau ipsc-derived neurons calcium imaging 4 S M (b i) D M (b i/m o n o ) D M (b i/b i) B u r s ts p e r m in u te S M (b i) D M (m o n o ) D M (b i) Live cell calcium imaging (Fluo4), 5 weeks after final plating on human astrocytes SM = IVS10+16 (bi) DM = IVS10+16 (bi) / P301S Increased calcium bursting frequency due to P301S mutation Preliminary n=2 experiments; P<0.05 and P<0.01

24 n o rm a liz e d to is o g e n ic c tr w ith o u t K 1 8 s e e d in g Tau aggregation in bi-allelic IVS10+16/P301S ipsc-derived neurons after K18 seeding is o g e n ic c tr S M (m o n o ) S M (b i) D M (m o n o ) D M (b i) 4 3 n o K 1 8 s e e d in g K 1 8 s e e d in g 25nM K18 (P301L) 2 weeks after plating AlphaLISA 5-7 weeks after K18 seeding htau10/htau10 AlphaLISA Labeled K18 Studying uptake of K18 tau fibrils P<0.0001; n=3

25 Summary & next steps AAV-P301L-tau ipsc-derived neurons currently tested in phenotypic screen of tau aggregation inhibitors/modulators analyses and identification of hits ongoing & confirmation of hits in dose response planned Successful generation and differentiation of ZFN engineered IVS10+16 and IVS+16/P301S tau ipsc lines Accelerated expression of 4R tau in IVS10+16 neurons ± P301S Early phenotypes IVS10+16 neurons are a different subtype of neurons compared to isogenic controls Reduced proliferation of IVS+16 neurons ± P301S Increased calcium activity and bursting frequency Tau aggregation after addition of K18 fibrils in bi-allelic P301S mutant ipsc neurons Further characterization of ZFN gene edited neurons ongoing Single cell transcriptomics analyses brain area s 4R tau expression in different neuronal subtypes Compare with IVS10+16 ipsc-derived neurons (Selena Wray) from FTD patients

26 Acknowledgements Janssen R&D Annick Diels Ines Royaux An De Bondt Vladimir Chupakhin Peter Roevens Pieter Peeters Janssen R&D Neuroscience TA Jacobine Kuijlaars Louis De Muynck Alfredo Cabrera-Socorro Rony Nuydens Andreas Ebneth