zfn technology platform€¦ · therapeutic area and technology 6 gene therapy genome editing cell...

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

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




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



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



NH2

COOH

COOH

NH2

NH2





NH2

NH2

COOH

COOH

Standard Fok attachment point

Amino-terminal attachment

NH2


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



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

18

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.

zfn technology platform€¦ · therapeutic area and technology 6 gene therapy genome editing cell...

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