Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In...

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Sustained Tau Reduction via Zinc Finger Protein Transcription Factors as a Potential Next-Generation Therapy for Alzheimer’s Disease and Other Tauopathies Bryan J Zeitler 1 *, Susanne K Wegmann 2 *, Sarah L DeVos 2 *, Kimberly Marlen 1 , Qi Yu 1 , Hoang-Oanh Nguyen 1 , Irina Ankoudinova 1 , Lei Zhang 1 , Jeffrey C Miller 1 , Steven Froelich 1 , Edward J Rebar 1 , Fyodor D Urnov 1 , Philip D Gregory 1 , Brigit E Riley 1 , Michael C Holmes 1 , Bradley T Hyman 2 , and H. Steve Zhang 1 1 Sangamo Therapeutics, Inc 2 Mass General * These authors contributed equally to this work

Transcript of Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In...

Page 1: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

Sustained Tau Reduction via Zinc Finger Protein Transcription Factors as a Potential Next-Generation Therapy for Alzheimer’s Disease and Other TauopathiesBryan J Zeitler1*, Susanne K Wegmann2*, Sarah L DeVos2*, Kimberly Marlen1, Qi Yu1, Hoang-Oanh Nguyen1, Irina Ankoudinova1, Lei Zhang1, Jeffrey C Miller1, Steven Froelich1, Edward J Rebar1, Fyodor D Urnov1, Philip D Gregory1, Brigit E Riley1, Michael C Holmes1, Bradley T Hyman2, and H. Steve Zhang1

1 Sangamo Therapeutics, Inc2 Mass General

* These authors contributed equally to this work

Page 2: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

ZFP-TFs are a potent, specific, and efficacious approach to lowering tau

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1. Lowering tau is a validated therapeutic approach for AD and other tauopathies

2. ZFP-TFs are a potent, single-gene specific solution to lowering tau

3. ZFP-TFs are efficacious in an AD model and well-tolerated for at least 11 months in the brain

4. IV delivery of a ZFP-TF results in global CNS tau reduction of up to 70%

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Clinical symptoms and neuronal loss closely track with tau but not amyloid

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From: Xia C … Dickerson BC. Association of In Vivo [18F]AV-1451 Tau PET Imaging Results With Cortical Atrophy and Symptoms in Typical and Atypical Alzheimer Disease. JAMA Neurol. February 20, 2017

tau PETCorticalAtrophy

Amyloid PET

tau “tangles”Intracellular

Amyloid β “plaques”Extracellular

Aggregates of tau, but not amyloid, are found in many other “tauopathy” diseases

Page 4: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

Neuronal loss in tauopathies can be blocked by reducing tau expression

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Healthy neuronAxonal tau

Diseased neuronPathological, mislocalized tau

Aggregates and tangles

Preventativetau KO or tau Het (mice)H2 haplotype (human)

ReversalTurn off mutant tau (mice)ASOs (mice)

Dying neuronConsumed by NFTs

Atrophied

NFTOligomerPhospho-tau

Multiple tau speciesPathogenic form(s) unclear

Tau reduction has been shown to be safe and neuroprotective

Amyloid betaAlzheimer’s Disease

Mutations in tauFrontal Temporal DementiaProgressive Supranuclear PalsyCorticobasal Degeneration

Brain InjuryChronic Traumatic Encephalopathy

Page 5: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

Differentiated platform for tau targeting: ZFP Transcription Factors

ZFP-TFs block all forms of intracellular tau with a single administration

chaperones

immunotherapyaggregation

inhibitors

smallmolecules

ASO

vectorizedAbsvaccines

ZFP-TF

Page 6: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

ZFP-Transcription Factors can be engineered to regulate any gene

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ZFP-TFs are derived from human proteins and do not edit or cut the DNA

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Therapy: Single AAV-ZFP-TF administration that blocks all tau at the DNA level

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AAV vectorencoding a ZFP-TF

ZFP TF ►Zinc Finger Protein Transcription Factor

AAV vector

+ Option 1Intrathecal (ITC)

Option 2Intravenous (IV)

Option 3Intracranial (IC)

Three delivery options:ITC, IV, or IC

ZFP TF repressor is expressed within neurons, but remains separate from neuron’s DNA

AAV traffics to neurons, ZFP-TF is expressed & represses tau

ZFP TF blocks production of all tau forms at the DNA level

- ZFP TF

+ ZFP TF

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Robust screening capabilities for rapid lead identification

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tau mRNA1000

=185 ZFPs

Activity1000-3 ng ZFP mRNAqRT-PCR @ 24 hrs

ZFP A ZFP B ZFP C

40

01

12425Specificity

300 ng ZFP mRNAGenechip @ 24 hrs

tauG

~30% of tau ZFP-TFs from the initial screen reduced tau by ≥ 50%

Page 9: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

Fold Repression Fold Activation

N=5 replicates / tmt

Potent, single-gene specific tau repression in primary neurons

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tau mRNA

1 % 2 %

tau protein

+AAV9

CMV-ZFPNeurons

mouse cortical

tau mRNAtau proteinmicroarray7 days

~100-fold reduction in tau levels with no detectable off-targets

Tau is the only generepressed by >2 fold out of 26,491 coding transcripts assayed

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ZFP-TFs stably repress endogenous tau for 11 months in the mouse brain

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AAV9 hippocampal coverageusing dual stereotaxic injections

AAV9CMV-ZFP

+

N=3-4. Mean +/- SD. * P <0.05; **** P < 0.0001. PBS from 6 wk shown. Normalized to mean of ATP5b, EIF4a2 & GAPDH.

6 wkqRT-PCR 48 wk

qRT-PCR

Wild-type3 months old

tauneurons

NeuNastrocytes

GFAPmicroglia

IBA1

Sustained >75% tau reduction with no neuronal loss and minimal glial elevation

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tau mRNA tau protein

Strong tau reduction in vivo driven by neuronal promoters

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Wild-type3 months old

AAV9hSYN1-ZFP

+ 8 weeks

qRT-PCRtau ELISA

Neuronal specific expression confirmed by histologyN=3-6 hemispheres. Mean +/- SD. **** P < 0.0001. Normalized to mean of ATP5b, EIF4a2 & GAPDH.

AAV9CamKIIα-ZFP

AAV9MeCP2-ZFP

OR OR

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Can a tau ZFP protect mice against amyloid neurotoxicity found in AD?

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4.5 months

+ ZFP-TF - ZFP-TF

APP/PS1WT

Plaque

DystrophicNeurites AD Human

APP/PS1 Mouse

• Dystrophic neurites are a pathological hallmark of AD• Hyperphosphorylated tau surrounds plaques in AD and APP/PS1• Lowering tau in vitro protects cultured neurons against amyloid• No tau lowering agent has previously translated this result in vivo

7 months

10 weeks

ZFPT2A

Venus

RFP

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ZFP-TFs reduce neuritic dystrophy in AD mice with established disease

13Quantitation: Dystrophies were counted for 121-256 plaques per mouse. Shown are average number of dystrophies per plaque per by mouse (n=4); mean +/- SEM, Two-tailed paired T-test.

Left CortexZFP-T2A-Venus

Right CortexRFP P = 0.0346

66%

ZFP ControlHemisphere:

Dystrophies per Aβ plaque

First in vivo demonstration of a tau lowering agent reducing neuritic dystrophy

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Broad CNS coverage may provide greater therapeutic benefit for tauopathies and other neurodegenerative disorders

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Global CNS tau reduction following a single IV administration

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Wild-type8 wk old

IV delivery

AAV-SangamoZFP-TF

or

AAV9ZFP-TF

+After 4 wks,

micro-dissectbrain regions,

qRT-PCR

~40-70% tau reduction throughout the CNS after one ZFP-TF treatmentN=3-4. Mean +/- SD. * P <0.05; **** P < 0.0001. Normalized to mean of ATP5b, EIF4a2 & GAPDH.

Motor Cortex Cortex (other) Cervical SC

Hippocampus Midbrain Cerebellum

Thalamus Hypothalamus Striatum

Page 16: Sustained Tau Reduction via Zinc Finger Protein ... · PDF fileDickerson BC. Association of In Vivo ... Dystrophies per A. β . plaque First . in vivo. ... Lei Zhang. Jeff Miller.

ZFP-TFs are a potent, specific, and efficacious approach to lowering tau

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Activity Potent, single-gene specific tau reduction in primary neurons

Tolerability Sustained tau lowering for 11 mo. in the brain; minimal inflammatory marker elevation

Efficacy First demonstration of a tau lowering agent reducing neuritic dystrophies

Delivery AAV-ZFPs can drive global CNS tau reduction via IV delivery

These results support the further development of atau-targeted ZFP-TF for the treatment of AD and tauopathies

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ResearchBryan Zeitler*Kim MarlenQi YuOanh NguyenRainier AmoraSteve ZhangSteven FroelichJulie QiaoBrigit RileyMichael Holmes

AAV TeamRichard SuroskyAlicia GoodwinHung TranTim GabrieleAndrea KangTim

TechnologySarah HinkleyIrina AnkoudinovaLei ZhangJeff MillerEd RebarPatrick LiDavid ShivakDave PaschonStephen Lam

Mass GeneralSarah DeVos*Suzanne Wegmann*Danny MackenzieCaitlin ComminsBrad HymanTim

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