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Designed Zinc Finger Protein Transcription Factors for Single-Gene Regulation Throughout the Central Nervous System
Bryan J Zeitler1*, Sarah L DeVos2*, Susanne K Wegmann2*, Kimberly Marlen1, Qi Yu1, Hoang-Oanh Nguyen1, Annemarie Ledeboer1, David S. Ojala1, Lei Zhang1, David A. Shivak1, Jeffrey C Miller1, Edward J Rebar1, Brigit E Riley1, Bradley T Hyman2, Michael C Holmes1
1 Sangamo Therapeutics
2 Mass General
* These authors contributed equally to this work.
Disclosure
2
I am an employee of Sangamo Therapeutics
Single-administration AAV ZFP to lower all tau forms at the DNA level
3
Administration options:
Intracranial, Intrathecal
or Intravenous
ZFP-TFs reduce
tau at the DNA levelPackaging into AAV
AAV Vector
Zinc Finger Protein Transcription
Factor (ZFP-TF)
Stable repression of
Tau/MAPT gene
Non-integrating
CNS Disease
Gene Regulation
1. ZFP-TF technology
2. Targeting tau for the treatment of tauopathies
3. Repression of mouse and human tau in vitro
Outline
CNS Disease
Gene Regulation
1. ZFP-TF technology
2. Targeting tau for the treatment of tauopathies
3. Repression of mouse and human tau in vitro
Outline
Zinc Fingers can be engineered to recognize specific DNA sequences
6
• Most abundant DNA
binding domain in the
human genome
• 28 amino acid peptide
• Binds 3-4 bp of DNA
• Specificity and affinity
can be engineered
5’ 3’
ZFP-TFs can be engineered to regulate any gene
7
ZFP-TFs can be engineered to regulate any gene
8
• Fully human-derived sequence
• 3 ZFPs can fit in 1 rAAV genome
• High potency – 2 targets per cell
CNS Disease
Gene Regulation
1. ZFP-TF technology
2. Targeting tau for the treatment of tauopathies
3. Repression of mouse and human tau in vitro
Outline
10Xia 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 PETCortical loss Amyloid PET
tau “tangles”Intracellular
Amyloid b “plaques”Extracellular
Tau defects causes many other “tauopathy” diseases:
• Frontal Temporal Dementia• Progressive Supranuclear Palsy• Corticobasal Degeneration• Chronic Traumatic Encephalopathy
Tau tracks closely with clinical symptoms and neuronal loss
Neuronal loss in tauopathies is blocked by tau reduction
11
Healthy neuronAxonal tau
NFTOligomerPhospho-tau
Multiple tau speciesPathogenic form(s) unclear
Pathogenic tau
Diseased neuronPathological, mislocalized tau
Aggregates and tangles
Dying neuronConsumed by tau pathology
Atrophied
Lowering tau
Prevent Reverse
Disease
Single-administration AAV-ZFP-TF to lower all tau forms at DNA level
12
Routes of Administration:
Intracranial, Intrathecal
or Intravenous
Packaging into AAV ZFP-TFs repress tau
at the DNA level
Reduced tau
prevents
neurodegeneration
+ ZFP-TF
- ZFP-TF
Zinc Finger Protein Transcription
Factor (ZFP-TF)
AAV Vector
Non-integrating
CNS Disease
Gene Regulation
1. ZFP-TF technology
2. Targeting tau for the treatment of tauopathies
3. Repression of mouse and human tau in vitro
Outline
High ZFP density design for rapid lead identification
14
=185 ZFP-TFs
Neuro2A cells were harvested after 24 hours and analyzed by RT-qPCR.
High ZFP density design for rapid lead identification
15
=185 ZFP-TFs
~30% of ZFP-TFs from the initial screen reduced tau by ≥ 50%
ZFP CZFP BZFP A
1000 / 300 / 100 / 30 / 10 / 3ng
Ta
u m
RN
A
ZFP-TF mRNA / txn
Neuro2A cells were harvested after 24 hours and analyzed by RT-qPCR.
tau mRNA
1000
Highly specific ZFPs can be identified at the initial design stage
16
ZFP CZFP BZFP A
Neuro2A + 300 ng ZFP mRNA txn / Genechip @ 24 hrs / n=4-6 biological replicates
12717
00
40
Down > 2x
Up >2x
tau
tautau
• Most zinc fingers make a
conserved DNA phosphate
contact from their β-sheet
• Alteration of this contact may
improve ZFP specificity
Elrod-Erickson et al., Structure 1996 – 1AAY.pdb
Conserved
Arg PO4
contact
DNA
Zinc Finger
Altering Zinc Finger-Phosphate Contacts to Improve Specificity
Phosphate contacts modulate global specificity
183E5 / 1E5 / 3E4 / 1E4AAV9 VG per cell
ZFP-D
R Q
ZFP-D
Microarray dose: 1E5 VG per cell, n=4-6 biological replicates; primary mouse cortical neurons
microarray
tau mRNA
microarray
11075
tau
P V
alu
e (
-lo
g10)
5
10
15
20
25
0-32 -16 -8 -4 -2 2 4 8 16 32
43
tau
P V
alu
e (
-lo
g10)
5
10
15
20
25
0
-32 -16 -8 -4 -2 2 4 8 16 32
+
AAV9ZFP-D
Neurons
qRT-PCRmicroarray
7d
tau
Potent, single-gene specific repression of tau in primary neurons
19
Mouse
tau mRNA
1 % 2 %
Mouse
tau protein
Mock ZFP E Mock ZFP E
+
AAV9ZFP-E
Neurons
qRT-PCRELISA
microarray
7d
Microarray dose: 1E5 VG per cell, n = 4-6 biological replicates
primary mouse cortical neurons; Mouse Clariom D Genechip
P V
alu
e (
-lo
g1
0)
Fold change
Global specificityN=26,491 transcripts evaluated
20
+
AAV6ZFP-E
iPS Neurons
qRT-PCRmicroarray
19d
Single-gene specific tau repression in human iPS neurons
**** P < 0.0001, n = 5-7 biological replicates. Dose: 1E5 VG per cell. Human Clariom S Genechip.
tau
Global specificityN=19,959 transcripts evaluated
P V
alu
e (
-lo
g1
0)
Fold change
47%****
Human Tau
mRNA
% N
orm
alized
Exp
ressio
n
Mo ACCCACCAGCTCCAGCAC
Hu ACCCACCAGCTCCGGCAC
Target site
alignment
ZFP-E
ZFP-TFs are a potent and specific tau-targeted therapay
21
Activity
Specificity
Efficacy
Potent tau reduction in mouse and human neurons
ZFPs with no off-targets can be efficiently engineered
ZFP-treatment reduced APP/PS1 neuritic dystrophies by 50% across the cortex
Tau repression persists for at least 11 months in the hippocampus
ZFP-TFs can reduce tau by up to 80% across the brain
Single-gene specific tau reduction in the hippocampus following IV delivery
Sangamo Research
Kim MarlenQi YuOanh NguyenAnnemarie LedeboerDavid OjalaMohammad SamieRainier AmoraTanja MeyerKathy MeyerBrigit RileyMichael Holmes
Lei ZhangDavid ShivakJeff MillerSarah HinkleyStephen LamYuri BendanaDave PaschonEd Rebar
Mass General
Sarah DeVosSusanne WegmannDanny MackenzieCaitlin ComminsBrad Hyman
Alicia GoodwinHung TranTim GabrieleAndrea KangRichard Surosky
