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
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 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
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-TF
ZFN
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 nuclease̶ zinc finger array (binds DNA)̶ FokI domain (catalyzes cleavage)
• Cleaves only when dimerized• Specifies an extended target (36bp)
Zinc finger array
Zinc finger array
7Adapted from Nat Rev Drug Disc 2 361
Alternative functional domains enable diverse possibilities
Targeted integration
8
ZFN
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 nuclease̶ zinc finger array (binds DNA)̶ FokI domain (catalyzes cleavage)
• Cleaves only when dimerized• Specifies an extended target (36bp)
• Each finger specifies 3-4bp
5’ 3’
Zinc finger – DNA recognition
Zinc finger array
9
ZFN
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 nuclease̶ zinc finger array (binds DNA)̶ FokI domain (catalyzes cleavage)
• Cleaves only when dimerized• Specifies an extended target (36bp)
• 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
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
Oneskip
Oneskip
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
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
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 GA T G G G T T G C G C T T A A T A C
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
Amino-terminal attachment
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
NH2COOH
COOH
NH2
NH2
NH2
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
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
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
21
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
Nat Med. 2018 Nov;24(11):1691
Selective clearing of mutant mitochondrial genomes in vivo
22
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
ZFPs: key differentiating features
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
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
ZFPs: key differentiating features
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
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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