engineering zinc finger proteins for therapeutic applications · 2020-06-18 · engineering zinc...

Engineering zinc finger proteins for therapeutic applications

This presentation contains forward-looking statements regarding Sangamo’s current expectations. These forward-looking statements include, without limitation, statements relating to the potential to develop, obtain regulatory approvals for and commercialize safe and effective therapies to treat certain diseases and the timing, availability and costs of such therapies, the potential to use ZFP, ZFP-TF, ZFN and other technologies to develop safe and effective therapies, the potential for Sangamo to benefit from its collaborations and the timing of such benefits, plans and timelines for Sangamo and our collaborators to conduct clinical trials and share clinical data and other statements that are not historical fact. These statements are not guarantees of future performance and are subject to certain risks and uncertainties that are difficult to predict. Sangamo’s actual results may differ materially and adversely from those expressed. Factors that could cause actual results to differ include, without limitation, risks and uncertainties related to the research and development process; the uncertain timing and unpredictable results of clinical trials, including whether initial clinical trial data will be representative of final clinical trial data and whether final clinical trial data will validate the safety and efficacy of product candidates; the unpredictable regulatory approval process for product candidates across multiple regulatory authorities; the manufacturing of products and product candidates; the commercialization of approved products; the potential for technological developments that obviate technologies used by Sangamo; the potential for collaborators to breach or terminate collaboration agreements; the potential for Sangamo to fail to realize its expected benefits of its collaborations; and the uncertainty of Sangamo’s future capital requirements, financial performance and results. There can be no assurance that Sangamo and its collaborators will be able to develop commercially viable products. These risks and uncertainties are described more fully in Sangamo’s Quarterly Report on Form 10-Q for the quarter ended March 31, 2020 and Annual Report on Form 10-K. Forward-looking statements contained in this presentation speak only as of the date hereof, and Sangamo undertakes no duty to update such information except as required under applicable law. This presentation concerns investigational product candidates that are under preclinical and/or clinical investigation and which have not yet been approved for marketing by any regulatory agency. They are currently limited to investigational use, and no representations are made as to their safety or efficacy for the purposes for which they are being investigated. Any discussions of safety or efficacy are only in reference to the specific results presented here and may not be indicative of an ultimate finding of safety or efficacy by regulatory agencies.

Forward-Looking Statements

2

Genomic medicines offer the prospect of true precision therapies

3

• Highly characterized operating environment (the genome)• Digitally defined targets• Molecularly sophisticated outcomes (not just binding/blocking)• Requirement for fine-scale discrimination for many applications

- e.g. single base alternatives / methylation status / repeat element count

• Million-fold dynamic range for qualifying performance

4

Number of proteins

Genome-wide comparison of transcriptional activator families in eukaryotes

Tupler R, Perini G, Green MR, Nature 409: 832.

The C2H2 zinc finger: Mediator of metazoan evolution

Zinc Finger Nuclease (ZFN) and Transcription Factor (ZFP-TF)

5

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 TG 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 CA T G3’

5’

• Synthetic, chimeric transcription factor̶ zinc finger array (binds DNA)̶ transcription activation or repression domain

• Binds 18 bp

p65 orKRAB

ZFP-TFZinc finger array

ZFN


T A CA T G3’

5’

• Synthetic, chimeric nuclease̶ zinc finger array (binds DNA)̶ FokI domain (catalyzes cleavage)

• Cleaves only when dimerized• Specifies an extended target (36bp)

Zinc finger array

Zinc Finger Nuclease (ZFN) and Transcription Factor (ZFP-TF)

6


T A CA T G3’

5’

• Synthetic, chimeric transcription factor̶ zinc finger array (binds DNA)̶ transcription activation or repression domain

• Binds 18 bp

p65 orKRAB

ZFP-TF

ZFN


T A CA T G3’

5’



Zinc finger array

Zinc finger array

7Adapted from Nat Rev Drug Disc 2 361

Alternative functional domains enable diverse possibilities

Targeted integration

8

ZFN


T A CA T G3’

5’



• Each finger specifies 3-4bp

5’ 3’

Zinc finger – DNA recognition

Zinc finger array

9

ZFN


T A CA T G3’

5’



• Each finger specifies 3-4bp

• Helix residues determine base preferences

5’ 3’ Helix sequence Binding-1 +2+3 +6 preference

LKQNLCM CATAQCCLFH AGCDQSNLRA AACRSDELTR GCGG… etc …

Zinc finger – DNA recognition

Zinc finger array

10

CRISPR-CasTALENsMeganucleasesMegaTALsZinc Finger Proteins (ZFPs)

Diverse platforms available for site-specific genome targeting

11

ZFPs: key differentiating features

High targeting precision

Compactness (deliverability / accessibility)

Nice balance of modularity vs customizability

- enables rapid development of reagents with therapeutically beneficial properties

Capacity for very high activity & specificity

12

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 pairsubsites:

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

Zinc finger protein design and assembly

• Thousands of two-finger units for hexamer recognition- Pre-assembled, pre-characterized

• Derived from selected, designed and natural fingers

• Highly specific for diverse sequence compositions

13

Sangamo’s archive of two-finger modules

Base skipping architectureFour module configurations available on each side of targeted base

14

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 moduleChosen base Canonical ZFN architectureOnly one module configuration is compatible with cleavage at any chosen base

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



Oneskip

Oneskip


Twoskips

Base-skipping linkers

16 module configurations enable cleavage

Targeted cleavage requires six precisely positioned modulesBase-skipping linkers increase configurational options

Standard ZFN dimer

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

NH2COOH

Standard Fok attachment point


COOH

COOH

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


NH2COOH

Amino-terminal attachment

Alternative dimers for same target sequence




NH2COOH

COOH

NH2

NH2

NH2




15

Reversing ZFP-nuclease order further increases design options

Amino-terminal attachment enables 3 more options

Design options increased 64-fold using both approaches

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 TTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTATCACAGGCTCCAGGAAGGGTTTGGCCTCTGATTAGGGAACTTCGATCAGATCACGTTCGATTGTCAACGAAAATAGTGTCCGAGGTCCTTCCCAAACCGGAGACTAATCCC

16

SANGAMO CONFIDENTIAL

Erythroid-specific enhancerof BCL11A

104 ZFN architectures available for GATAA ± 10bp

Platform enables dense tiling of targeted regions

Net result: high targeting density

17

Target region: 28 bp window in the HBG promoter spanning key HPFH mutations

Screened via transient RNA delivery in K562 cells

0

20

40

60

80

100

-199 -198 -197 -196 -195 -194 -193 -192 -191 -190 -189 -188 -187 -186 -185 -184 -183 -182 -181 -180 -179 -178 -177 -176 -175 -174 -173 -172

Base number within the HBG promoter

% Indels

Targeting capabilities featured in recent Sangamo manuscript

18

Nat Commun. 2019 Mar 8;10(1):1133

Targeting example: mouse mitochondria mutation m.5024C>T

19

Goal: selective clearance of mutant mitochondrial genomes in heteroplasmic cells

Resolution of pathological phenotypes due to mitochondrial dysfunction

Studies performed in vivo in mouse heart. ZFNs delivered via AAV

Selective clearing of mutant mitochondrial genomes in vivo

20




21




Nat Med. 2018 Nov;24(11):1691

Selective clearing of mutant mitochondrial genomes in vivo

22







23

ZFPs offer a sleeker recognition unit

ZFP

TALE

CRISPR-Cas9

174 amino acids

781 amino acids

1,368 amino acids

24

ZFPs: modular and optimizable

Finger: triplet concordance enables rapid design & assembly

Interface complexity (>107 options)enables fine tuning of properties,

e.g.• allelic discrimination• base modifications• specificity vs affinity

25







26

ZFP

ZFN

G C G G C GC G C C G C

C C A A C G C G A A T T A T GG G T T G C G C T T A A T A C

T A CA T G3’

5’

FokT 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

zinc finger

1) Removing Arg-phosphate contacts

Chimeric structure enables two strategies for optimizing specificity

Fok domain

Fok domain

2) Substituting key FokI residues

27

zinc finger

1) Removing Arg-phosphate contacts to tune dissociation rate (k-1)

Independent tuning of dissociation and catalysis

Fok domain

Fok domain

2) Substituting key FokI residues to modulate rate of catalysis (k2)

28

Fok domain

2) Substituting key FokI residues to modulate rate of catalysis (k2)

Residues identified (e.g. Q481) that enable catalytic tuning

Arginine substitutions enable affinity tuning over 50x range

29

G T G C A A G C T A A C G T T G C TC A C G T T C G A T T G C A A C G A

C A G G C T C C A G G A A G GG T C C G A G G T C C T T C C

AT

ZFN-L 1x

2x

3x

4x

5x

Half life analysis

none(parent)

ZFN structure

# of fingerssubstituted

Bindinghalf life

(minutes)

740

410

120

50

25

1200

1x

2x

3x

4x5x

parent

ZFNs targeted to TCRα

Affinity-reducing mutations in 6 fingers

Fok variant that slows catalysis

G T G A T T G G G T T C C G A A T CC A C T A A C C C A A G G C T T A G

C C T G A A A G T G G C C G GG G A C T T T C A C C G G C C

Q481E

New approaches used to develop TCRα ZFNsComplete gene modification achieved with no off-targets

1.2% CD3 neg

98.5% CD3-neg

On-target activity assessment:

% DNA modification(via sequencing)

ZFN-treated Control

% functionalknockout(via FACS)

98.2% indels 0% indels Guide-Seq, followed by sequencing of candidate off-target loci

High nuclease dose used for Guide-Seq & follow-up studies (90% & 98% on-target indels)

No evidence off-target cleavage seen with median background levels of 0.01%

Specificity assessment:

Q481E

30

31

Study published July 2019

First point of application is T-cell programs

32

Highly efficient multiplexed edits

33

76% of cells have all four modifications

T C R-

β 2 M-

C I S H-

G F P+

0

5 0

1 0 0

9 3 % 9 6 % 9 3 % 9 1 %

% ofcells

ZFN Knock-out1. TRAC (TCR)2. β2M (HLA-class I)3. CISH (checkpoint gene)

Targeted Insertion4. GFP (into TRAC)

*Data generated prior to Kite collaboration

Take home points

34

Designed zinc finger proteins provide the platform of choice for fully realizing the prospect of precision genomic medicines.

Design versatility enables genome targeting with base pair resolution.

Use of precharacterized, modular components enables rapid lead development with option for fine scale tuning of properties

New strategies for specificity optimization enable complete (>98%) gene editing with no detectable off-targets

Modular nature of ZFPs enables continued extensions to capabilities

engineering zinc finger proteins for therapeutic applications · 2020-06-18 · engineering zinc...

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