ZFN Technology Platform 2018
ZFN Technology Platform
2018
This presentation contains forward-looking statements within the meaning of the "safe harbor" provisions of the Private Securities
Litigation Reform Act of 1995, as amended. These forward-looking statements include, but are not limited to, the design of clinical trials
and expected timing for release of data; the anticipated clinical development milestones and other potential value drivers in the future;
the expected benefits of the collaboration with Pfizer and Kite, the expanded capability of Sangamo’s technologies; the research and
development of novel gene-based therapies and the application of Sangamo’s ZFP technology platform to specific human diseases;
corporate partnerships; and the potential of Sangamo’s genome editing technology to treat genetic diseases. These statements are
based upon our current expectations and speak only as of the date hereof. Our actual results may differ materially and adversely from
those expressed in any forward-looking statements as a result of various factors and uncertainties. Factors that could cause actual
results to differ include, but are not limited to, the dependence on the success of clinical trials of lead programs, the lengthy and
uncertain regulatory approval process, uncertainties related to the timing of initiation and completion of clinical trials, whether clinical trial
results will validate and support the safety and efficacy of Sangamo’s therapeutics, uncertainties related to the initiation and completion
of clinical trials, whether clinical trial results will validate and support the safety and efficacy of Sangamo’s therapeutics, the reliance on
partners and other third-parties to meet their obligations, and the ability to establish strategic partnerships. Further, there can be no
assurance that the necessary regulatory approvals will be obtained or that Sangamo and its partners will be able to develop
commercially viable gene-based therapeutics. Actual results may differ from those projected in forward-looking statements due to risks
and uncertainties that exist in Sangamo’s operations and business environments. These risks and uncertainties are described more fully
in Sangamo’s Annual Report on Form 10-K and Quarterly Reports on Form 10-Q as filed with the Securities and Exchange Commission.
Forward-looking statements contained in this presentation are made as of the date hereof, and Sangamo undertakes no obligation to
update such information except as required under applicable law.
2
3
Sangamo is investing across four technology platforms for genomic medicines
4
Our vision to develop and commercialize our own products, built on Sangamo’s foundational research engine
5
Phase I/II
ClinicalPre-clinicalDiscovery
Phase III /
Commercial
Therapeutic Area Research Preclinical Phase 1/2 Collaborator
Inherited Metabolic Diseases
MPS I (SB-318)
MPS II (SB-913)
Fabry Disease (ST-920)
Hematology
Hemophilia A (SB-525)
Hemophilia B (SB-FIX)
Beta-thalassemia (ST-400)
Sickle Cell Disease (BIVV-003)
CNS Diseases
Tauopathies
ALS/FTLD - C9ORF72
Huntington’s Disease
Oncology
Autologous and Allogeneic CAR/TCR/NKR
Immunology
Undisclosed Autoimmune Disease Targets
Investigator Sponsored Clinical Research
HIV (T cell and Stem Cell)
Current internal and external product portfolios diversified across therapeutic area and technology
6Gene Therapy Genome Editing Cell Therapy Gene Regulation
ZFNs: The platform of choice for therapeutic genome editing
7
Efficiency Level of
modification at the
desired target
nucleotide
Precision Target any desired
nucleotide in the
genome
Specificity Edit the targeted
nucleotide without
editing elsewhere in
the genome
ZFNs
T G C
T G C G C T T A A C G C A T G G G T
A C G C G A A T T G C G T A C C C A
ZFPs
ZFPs
ZFN
ZFN
C C A A C G C G A A T T A T G
G G T G C T T A A T A C
T A C
A T G
Two-finger modules used for design
8
• Enables more
specific binding
• Allows one-step
gene assembly
NH2
COOH
T A C C C A 3’5’
Modular platform enables rapid assembly
9
5’ T A C C C A A C G C G A A T T G C G 3’
+ +
six finger ZFP:
two-finger
modules:
6 base pair
subsites:
18 base pair
target site:
5’ T A C C C A 3’ 5’ A C G C G A 3’ 5’ A T T G C G 3’
NH2
COOH
Summary of design components
10
C C A A C G C G A A T T A T G G C G G C G T G C G C T T A A C G C A T G G G T
G G T T G C G C T T A A T A C C G C C G C A C G C G A A T T G C G T A C C C A
T A C
A T G3’
5’
module module module
ZFP-Fok
linker module module module
intermodulelinkers
ZFP-Fok
linkerintermodule
linkers
Modules: 1- and 2-finger units that >8000 hexamer / module combinations
recognize base sequence
Intermodule connect adjacent modules 6 alternatives for skipping 0, 1, or 2 bp
linkers:
ZFP-Fok linker: links the Fok and ZFP domains 5 alternatives for skipping 5-9 bp
4 alternatives for reversing Fok-ZFP polarity
Base-skipping linkers yield a further 80-fold increase
Base-
skipping
linkers
Standard linkers / no skipping:
1 module configuration
Linker variants that skip a base:
4 configurations / ZFN
= 16 configurations / dimer
A T T A A G C A A C G G T A A A T A G G G G G G G GT A A T T C G T T G C C A T T T A T C C C C G G G G
A T T A A G C A A C G G T A A A T A G G G G G G G GT A A T T C G T T G C C A T T T A T C C C C G G G G
A T T A A G C A A C G G T A A A T A G G G G G G G GT A A T T C G T T G C C A T T T A T C C C C G G G G
A T T A A G C A A C G G T A A A T A G G G G G G G GT A A T T C G T T G C C A T T T A T C C C C G G G G
G T G C G A T T A A G A C A T G T G A T AG A C G C T A A T T C T G T A C A C T A T
G T G C G A T T A A G A C A T G T G A T AG A C G C T A A T T C T G T A C A C T A T
G T G C G A T T A A G A C A T G T G A T AG A C G C T A A T T C T G T A C A C T A T
G T G C G A T T A A G A C A T G T G A T AG A C G C T A A T T C T G T A C A C T A T
A T T A A G C A A C G G T A A A T A G G G G G G G G T G C G A T T A A G A C A T G T G A T AT A A T T C G T T G C C A T T T A T C C C C G G G G A C G C T A A T T C T G T A C A C T A T
module module module
module module module
11Single + 2 bp skipping:
81 configurations / dimer (not shown)
One skip
One skip
Two skips
Reversing Fok-ZFP Order Increases Design Options 4-fold
Standard ZFN dimer
Alternative dimers for same target sequence
T G C G C T T A A C G C A T G G G TA C G C G A A T T G C G T A C C C A
T G C G C T T A A C G C A T G G G TA C G C G A A T T G C G T A C C C A
T G C G C T T A A C G C A T G G G TA C G C G A A T T G C G T A C C C A
NH2
COOH
COOH
NH2
NH2
T G C G C T T A A C G C A T G G G T
A C G C G A A T T G C G T A C C C A
T G C G C T T A A C G C A T G G G T
A C G C G A A T T G C G T A C C C A
NH2
NH2
COOH
COOH
Standard Fok attachment point
Amino-terminal attachment
NH2
T G C G C T T A A C G C A T G G G TA C G C G A A T T G C G T A C C C A
COOH
COOH
T A C C C A A C G C G A A T T A T G
A T G G G T T G C G C T T A A T A C
T A C C C A A C G C G A A T T A T GA T G G G T T G C G C T T A A T A C
T A C C C A A C G C G A A T T A T GA T G G G T T G C G C T T A A T A C
T A C C C A A C G C G A A T T A T GA T G G G T T G C G C T T A A T A C
12
Bottom-Top
Top-Top
Bottom-Bottom
Top-Bottom
Elrod-Erickson et al., Structure 1996 – 1AAY.pdb
Conserved
Arg phosphate
contact
DNAzinc finger
Further improvements increase specificity while maintaining high levels of gene modification
13
Removal of conserved, non-specific phosphate contacts from zinc finger proteins increases
targeting specificity
Modified Fok nuclease-DNA contacts significantly reduces off-target cleavage events
Phosphate contacts modulate global specificity
Conserved
Arg phosphate
contact
DNA
zinc finger
14
Fok domain
Fok domain
Lys phosphate
contact
Recent innovations drive exceptional performance
15
Innovation Result
New dimer architectures yield higher
modification activity
Increase DNA editing efficiency to
as high as 99.5%
Phosphate contact tuning via
replacement of key residues
Off-target cleavage undetectable
(>1000 fold reduction)Specificity
Precision
Efficiency
New linkers for configuring
DNA-binding modules
300-fold increase in design options
for targeting any given sequence
Precision
SpecificityEfficiency
Precision
SpecificityEfficiency
16
Notes:
Grey font = No evidence of ZFN cleavage
Test system: ZFNs targeted to BCL11A erythroid enhancer (Nat Methods 12, 927)
CD34+ cells; delivery via RNA transfection (BTX)
(Arg --> Gln) 6 ug 2 ug 0.5ug 6 ug 2 ug 0.5ug 6 ug 2 ug 0.5ug
--- --- 82.7 87.0 76.5 28.00 5.02 .34 4.77 .32 .03
--- R33S 86.4 86.8 77.6 5.07 .58 .10 .75 .07 .03
6 R33S 88.3 86.4 78.0 .55 .08 .01 .04 .01 .02
7 R33S 85.4 86.3 73.4 .17 .03 .03 .02 .00 .01
8 R33S 83.6 86.5 71.7 .03 .01 .02 .02 .03 .00
7 K142S 85.4 83.6 60.6 .01 .02 .02 .01 .00 .01
7 K142S 85.7 84.3 40.1 .02 .00 .01 .00 .00 .00
# modified
fingers
Fok domain
substitution
% Indels
BCL11A Off-target A Off-target C
Parent ZFN
Off-target activity
reduced > 1000xK525S
K525S
R416S
R416S
R416S
R416S
Specificity improved >1000 fold via key substitutions
ZFNs offer significantly higher success rate for cleaving close to an arbitrarily chosen base
17
.59
.96.99 .99 1.0 1.0 1.0 1.0 1.0 1.0
.02
.20
.33
.43
.51
.58.63
.68.72
.76 .79
.0
.25
.5
.75
1.0
0 10 20 30 40 50
Fraction of
bases in globin
test region
Distance (bp) to nearest cleavage site
ZFN platform
G(N20)GG motif
Design density allows Sangamo to select a ZFN optimized for on-target efficiency and maximal therapeutic effect
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G A A G C T a G T C T A G t G C A A G C X T G C T T T T A T C A C A G G C T C C T A G T C t A G T G C A A G C X T G C T T T T A T C A C A G G C T C G A A G C T a G T C T A G t G C A A G C X G C T T T T A T C a C A G G C T C T A G T C T A G T G C a A G C T A A X C T T T T A T C A C A G G C T C T A G T C T A G T G C a A G C T A A X T T T T A T C A C A G G C T C G T C T A G T G C a A G C T A A X T T T T A T C A C A G G C T C G T C T A G t G C A A G C T A A X T T T T A T C A C A G G C T C C T A G T G C A A G C T A A C X T T T A T C A C A G G C T C C C T A G T C t A G T G C A a G C T A A C X T T T A T C A C A G G C T C C C T A G T G C A A G C T A A C X T T T T A T C A C A G G C T C G T C T A G t G C A A G C T A A X T T T A T C A C A G G C T C C T G C A A G C T A A C A X T T T A T C A C A G G C T C C C T A G T C t A G T G C A a G C T A A C X T A T C A C A G G C T C C A G C T A G T G C A A G C T A A C X T A T C A C A G G C T C C A G G T G C A A G C T A A C A G T X T A T C A C A G G C T C C A G G T C T A G t G C A A G C T A A C A G X T A T C A C A G G C T C C A G G T C T A G t G C A A G C T A A C A G X A T C A C A G G C T C C A G G G T G C A A G C T A A C A G T X T A T C A C A G G C T C C A G T A G T G C a A G C T A A C A G X T A T C A C A G G C T C C A G G T C T A G t G C A A G C T A A C A G X T A T C A C A G G C T C C A G G T G C A A G C T A A C A G T X A T C A C A G G C T C C A G G T A G T G C a A G C T A A C A G X T C A C A G g C T C C A G G A A G G G T A G T G C a A G C T A A C A G T T G X T C A C A G g C T C C A G G A A G G G G T G C A A G C T A A C A G T X T C A C A G g C T C C A G G A A G G G T G C A A G C T A A C A G T T X A T C A C A G G C T C C A G G G T G C A A G C T a A C A G T T X T C A C A G g C T C C A G G A A G G G G T G C A A G C T A A C A G T X C A C A G G C T C C A G G A A G G G T G C A A G C T A A C A G T T X T C A C A G g C T C C A G G A A G G G T A G T G C a A G C T A A C A G T T G X T C A C A G G C T C C A G G A G T G C A A G C T A A C A G T X T C A C A G G C T C C A G G A T G C A A G C T A A C A G T T X C A C A G G C T C C A G G A A G G G G T G C A A G C T a A C A G T T X T C A C A G G C T C C A G G A T G C A A G C T A A C A G T T X T C A C A G G C T C C A G G A G T G C A A G C T A A C A G T X C A C A G G C T C C A G G A A T G C A A G C T A A C A G T T X C A C A G G C T C C A G G A A G T G C A A G C T A A C A G T T G C X T C A C A G g C T C C A G G A A G G G G T G C A A G C T A A C A G T T G C X T C A C A G G C T C C A G G A T G C A A G C T A A C A G T T X A C A G G C T C C A G G A A G G G T T G C A A G C T A A C A G T T X A C A G G C T C C A G G A A G T A G T G C a A G C T A A C A G T T G X C A C A G G C T C C A G G A A G G G T A G T G C a A G C T A A C A G T T G X C A C A G G C T C C A G G A A G T G C A A G C T A A C A G T T G C X C A G G C T C C A G G A A G G G T T G T G C A A G C T A A C A G T T G C X C A C A G G C T C C A G G A A G G G G T G C A A G C T A A C A G T T G C X A C A G G C T C C A G G A A G G G T G T G C A A G C T A A C a G T T G C T X A C A G G C T C C A G G A A G G G T G T G C A A G C T A A C A G T T G C X A C A G G C T C C A G G A A G G T G C A A G C T A A C a G T T G C T X A C A G G C T C C A G G A A G G T G C A A G C T A A C A G T T G C X C A G G C T C C A G G A A G G G T G C A A G C T A A C a G T T G C T X A C A G G C T C C A G G A A G G G T G T G C A A G C T A A C a G T T G C T X A C A G G C T C C A G G A A G G T G C A A G C T A A C A G T T G C X C A C A G G C T C C A G G A A T A G T G C a A G C T A A C A G T T G X C A G G C T C C A G G A A G G G T T G T G C A A G C T A A C a G T T G C T X C A C A G G C T C C A G G A A G G G T A G T G C a A G C T A A C A G T T G X A C A G G C T C C A G G A A G G G T T A G T G C a A G C T A A C A G T T G X A C A G G C T C C A G G A A G T A G T G C a A G C T A A C A G T T G X C A G G C T C C A G G A A G G G T G C A A G C T A A C a G T T G C T X C A C A G G C T C C A G G A A G T G C A A G C T A A C a G T T G C T X C A G G C T C C A G G A A G G G T T G T G C A A G C T A A C a G T T G C T X C A G G C T C C A G G A A G G G T T G T G C A A G C T A A C a G T T G C T X C A G G C T C C A G G A A G G G T G C A A G C T A A C a G T T G C T X C A G G C T C C A G G A A G G G T G C A A G C T A A C A G T T G C X A G G C T C C A G G A A G G G G C T A A C A G T T G C T T T X C A G G C T C C A G G A A G G G T T A G C T A A C A G T T G C T T X C A G G C T C C A G G A A G G G T T G T G C A A G C T A A C a G T T G C T X G G C T C C A G G A A G G G T G T G C A A G C T A A C a G T T G C T X A G G C T C C A G G A A G G G G C T A A C A G T T G C T T T X A G G C T C C A G G A A G G G G C T A A C A G T T G C T T T X C A G G C T C C A G G A A G G A G C T A A C A G T T G C T T X C A G G C T C C A G G A A G G G C T A A C A G T T G C T T T X G G C T C C A G G A A G G G T G C T A A C A G T T G C T T T X C T C C A G G A A G G G t T T G G C C G C T A A C A G T T G C T T T X G C T C C A G G A A G G G T T A G C T A A C A G T T G C T T T T A X G C T C C A G G A A G G G T T G C T A A C A G T T G C T T T T A T X G C T C C A G G A A G G G T T A G C T A A C A G T T G C T T T T A X C T C C A G G A A G G G t T T G G C C G C T A A C A G T T G C T T T T A T X C T C C A G G A A G G G t T T G G C C G C T A A C a G T T G C T T T T A T C X T C C A G G A A G G G T T T G G C C G C T A A C A G T T G C T T T T A T X T C C A G G A A G G G T T T G G C C G C T A A C A G T T G C T T T T A T X C C A G G A A G G G T T T G G G C T A A C a G T T G C T T T T A T C X C T C C A G G A A G G G t T T G G C C A G C T A A C A G T T G C T T T T A X T C C A G G A A G G G T T T G G C C A G C T A A C A G T T G C T T T T A X C C A G G A A G G G T T T G G G C T A A C a G T T G C T T T T A T C X C C A G G A A G G G T T T G G G C T A A C A G T T G C T T T T A T X C A G G A A G G G t T T G G C C G C T A A C a G T T G C T T T T A T C X A G G A A G G G T T T G G C C A A C A G T T G C T T T T A T C A C X C A G G A A G G G t T T G G C C G C T A A C a G T T G C T T T T A T C X C A G G A A G G G t T T G G C C A A C A G T T G C T T T T A T C A C X C C A G G A A G G G T T T G G C A G T T G C T T T T A T C A X C C A G G A A G G G T T T G G C A G T T G C T T T T A T C A X A G G A A G G G T T T G G C C A A C A G T T G C T T T T A T C A C X A G G A A G G G T T T G G C C A A C A G T T G C T T T t A T C A C A X C A G G A A G G G t T T G G C C A A C A G T T G C T T T T A T C A C X G A A G G G t T T G G C C T C T G A T A A C A G T T G C T T T t A T C A C A X A G G A A G G G T T T G G C C A A C A G T T G C T T T t A T C A C A X G A A G G G t T T G G C C T C T G A T A A C A G T T G C T T T T A T C A C X G G A A G G g t T T G G C C T C T G A T A A C A G T T G C T T T t A T C A C A X G G A A G G g t T T G G C C T C T G A T A A C A G T T G C T T T t A T C A C A X A A G G G T T T G G C C T C T G A T A G T T G C T T T T A T C A C A G G X G G A A G G g t T T G G C C T C T G A T A G T T G C T T T T A T C A C A G G X G A A G G G t T T G G C C T C T G A T A G T T G C T T T T A T C A C A G G X A A G G G T T T G G C C T C T G A T T G C T T T t A T C A C A G G C X A A G G G T T T G G C C T C T G A T T G C T T T t A T C A C A G G C X G A A G G G t T T G G C C T C T G A T G T T G C T T T T A T C a C A G G C T X A A G G G T T T G G C C T C T G A T
TTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATCACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGG C T A A C A G T T G C T T T T A T X A C A G G T T G C T T T T A T C A C A G G C X C C A G G
Structural flexibility provides diverse architectures for targeting any DNA segment
19
1
10
100
1000
-300 -200 -100 0 100 2000
Base location in
β-globin gene
70-130 270
ZFN
Distinct architectures
available for design
(20 bp window)
01
10
100
1000
Sickle
mutation
ZFN average (455)
HiFi Cas9
HiFi Cas9 average (1.07)
Reactivation of fetal hemoglobin (HbF) to treat beta-thalassemia and sickle cell disease
20
β-globin defects (SCD) and
scarcity (β-thal) caused by
hundreds of different
mutations
γ-globin in HbF raises oxygen
affinity, confers anti-sickling
effects
HbF protects infants from β-
thal, SCD manifestations until
birth, when γ-globin is
downregulated by BCL11A
Adult Hemoglobin (HbA)Fetal Hemoglobin (HbF)
g
g
⍺
⍺
⍺
⍺
β
β
Reactivation of HbF in erythroid lineage achieved by targeted KO in CD34+ stem cells at key nucleotide within BCL11A enhancer
211 Platt et al. Blood (1994)
2 Musallam et al. Blood (2012)
3 Vierstra, J. et al. Nat. Methods (2015)
Natural genetic variants in BCL11A
reactivate HbF, which protects against
disease symptoms and increases
lifespan in β-thal1 and SCD patients2
Erythroid-specific enhancer in
BCL11A selectively reactivates HbF
in erythroid lineage while preserving
normal immune cell functions
Precise disruption of key
nucleotide in BCL11A erythroid-
specific enhancer, based on
naturally protective variations
Non-viral delivery of ZFN
mRNA for possibly superior
long-term safety
Fetal hemoglobin has natural
features (stronger oxygen
affinity, anti-sickling properties)
found to protect against disease
symptoms in patients1,2
Strategic Advantages for Treating β-thalassemia and SCD
Precise disruption at key nucleotide maximizes γ-globin expression3
BCL11A geneenhancer sequence
Deep understanding of protein DNA interaction allows us to engineer ZFNs with unparalleled specificity
22
Strategic Advantages
Defined engineering
refinements to protein
structures rapidly generate
ZFP constructs with high
on-target activity
No detectable off-target
activity using state-of-the-
art, unbiased oligo capture
assay and deep
sequencing
Deep sequencing results for 55 loci identified by state-of-art unbiased oligo capture methods (assay background generally
<0.1%) for potential off-target activity. Performed in CD34 hematopoietic stem cells treated with ZFNs at clinical scale.
0
10
20
30
40
50
60
70
80
90
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
High on-target
modification (82%)
No significant off-target modification
seen compared to control
% inde
ls
Locus rank
Thank you.