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Highly Efficient and Specific Multiplexed Gene Editing in T cells Using Enhanced Zinc-Finger Nucleases (ZFNs) Enables Strategic Engineering of Allogeneic T Cell Immunotherapies
Sumiti JainMay 19th, 2018
This presentation contains forward-looking statements within the meaning of the "safe harbor" provisions of the Private SecuritiesLitigation Reform Act of 1995, as amended. These forward-looking statements include, but are not limited to, the duration for whichexisting capital resources can provide for planned operations; the design of clinical trials and expected timing for release of data; theanticipated clinical development milestones and other potential value drivers in the future; the expected benefits of the collaboration withPfizer; the expected capability of Sangamo’s technologies; the ability of Sangamo to research and develop novel gene-based therapiesand the anticipated benefits of applying Sangamo’s ZFP technology platform to specific human diseases; anticipated benefits fromcorporate partnerships; and the potential of Sangamo’s genome editing technology to treat genetic diseases. Our actual results may differmaterially and adversely from those expressed in any forward-looking statements as a result of various factors and uncertainties. Factorsthat 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, the ability to establish strategicpartnerships and our ability to control expenses and achieve our milestones that generate revenues under our agreements. Further, therecan be no assurance that the necessary regulatory approvals will be obtained or that Sangamo and its partners will be able to developcommercially viable gene-based therapeutics. Actual results may differ from those projected in forward-looking statements due to risksand uncertainties that exist in Sangamo’s operations and business environments. These risks and uncertainties are described more fullyin Sangamo’s Annual Reports 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 based on our current expectations and are made as of the date hereof.Sangamo undertakes no duty to update such information except as required under applicable law.
2
Forward Looking Statements
3
Vision: Off-the-shelf allogeneic T-cell therapies
HLAs
4
Toolkit to generate healthy donor allogeneic CAR T cells
TCR⍺β
CAR
Healthy Donor
Eliminate HLA Class I
Eliminate Endogenous TCR
CAR Expression
ZFN mRNATRAC Knockout
AAV6 DonorTargeted Insertion
of CD19 CAR into TRAC
ZFN mRNAβ2M Knockout
Prevent rejection
Prevent GvHD
Immune specificity
5
The ZFN platform for genome editing
Efficiency | Ability to edit at the desired target nucleotide
Precision | Ability to target any desired nucleotide
Specificity | Ability to edit the targeted nucleotidewithout editing elsewhere in the genome
6
ZFN Platform: Best-in-class gene editing
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
CGA----------------TTTTGCACTCGTGATAAGGGGGAAAAGAACACCCGCTCACCCGAGTCC--GGGCAATATTTTTGCACTCGTGATAAGGGGGAAACCCGCTCACC
CGAGT---TGGGCAATATTTTTGCACTCGTGATAAGGGAAGAACACCCGCTCACC ACC--------------GCAATATTTTTGCACTCGTGATAAGGGGGAAAACACCCGCTC
7
Single-step multiplexed T cell editing
Activated T Cells Gene Editing
ZFN mRNA Electroporationβ2M and TRAC Knockout
AAV6 TransductionCD19 CAR
Insertion into TRAC
From Healthy Donor
Expand + Analyze
Phenotype(FACS)
Genotype(MiSeq)
ZFN-KO of TRAC achieves 95-99% loss of surface TCR
8
CD3
97% CD3neg
TRAC-ZFN Treated T cells 97% CD3 KO
~1:1 CorrelationGenotype and Phenotype
Untreated control
% T
RAC
Inde
l
% CD3-neg
ZFN-KO of β2M consistently achieves >90% loss of class I HLA
9HLA Class I
91% HLAneg
β2M-ZFN Treated T Cells 91% HLA Class I KO
Untreated control
β2M-KO Achieves Functional Loss of HLA Class I Expression
⍺2 ⍺2
⍺3 β-microgloblin
Highly efficient double KO and GFP TI into TRAC
10GFP
GFP TI into TRAC91% GFP expression
91% GFPpos
TRAC + β2M double KO91% double neg
CD3
HLA
-AB
C
Unedited T cells Double KO T cells(TRAC/B2M)
91%
AAV TI of CD19 CAR into TRAC yields functional CAR-T cells
11
Protein-L/ CARE:T Ratio
% C
D19
+ K
562 Untreated
CD19 CARTRAC KO
CD19 CAR Double KO
0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5
0
2 0
4 0
6 0
8 0
1 0 077%CARpos
Specific Lysis of CD19+ K562 Cells4hrs; co-culture of CAR-T cells with K562wt and CD19+ K562
CAR TI into Double KO77% CARpos
12
Improve specificity of ZFNs by varying phosphate contact
Arg phosphatecontact variant
DNA
zinc finger Fok domain
Fok domain
Lys phosphatecontact variant
13
% Indels
ZFN Pair B2M (ON-Target) OFF-Target A # Architecture 4ug 1ug 0.5ug 4ug 1ug 0.5ug 1 parent 100.0 96.6 89.1 4.89 1.32 0.12 2 optimized 99.3 89.9 73.9 0.02 0.00 0.00 3 optimized 100.0 95.1 81.6 0.02 0.01 0.00 4 optimized 100.0 96.9 83.3 0.01 0.00 0.00 5 optimized 100.0 93.4 74.2 0.02 0.00 0.01
Optimized ZFN pairs exhibit highly efficient editing and improved specificity
Loss of off-target activity vs. parent
Optimized ZFNs targeting B2M exhibit no off-target activity
Determine if the optimized ZFN pairs exhibit off-target activity at new sites by performing an unbiased integration site analysisNext
Optimized ZFNs targeting B2M exhibit no off-target activity
Genome-wide unbiased oligonucleotide duplex integration analysis revealed NO off-target activity for the optimized ZFN pairs
14
Verify off-target activity in T cells in an at-scale process which achieves clinically relevant editing efficienciesNext
Off-Target Site?
DSB
Integrated Donor
Donor
Sequence Reveals Candidate Off-Target Site
Adaptor
Genome
• Treat cells with ZFNs + donor oligo duplex
• Sequence genome segments adjacent to integrated donors
• Treat K562 or HepG2 cells with ZFNs• PCR-amplify candidate off-target sites• Assess for indels
Identify Candidate OTsOligo-Duplex End-Capture Assay
Confirm OTsDeep Sequencing forActual ZFN Activity
2
1
15
Locus Integrant count
% Indels p-value if < 0.05ZFN-treated Control
On Target 3386 95.23 0.14
Highly efficient genome editing is critical for multiple gene modifications
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1 KO + 1 KO + 1 TI Compounded Efficiency
99% x 99% x 99% = 97%
90% x 90% x 90% = 73%
70% x 70% x 70% = 34%
50% x 50% x 50% = 13%
Very High
Medium
Low
High
Simultaneous multiplex editing efficiency: 3x ZFN KO + 1x TI
17
76% of cells have all 4 edits
Potential ApplicationUniversal T cells with checkpoint gene knock-out
Single Step Editing
ZFN Knock-out1. TRAC (TCR)2. β2M (HLA-class I)3. CISH (checkpoint gene)
Targeted Insertion4. GFP (into TRAC)
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 %
TRAC -
93% 96% 93% 91%
β2M - CISH - GFP +
% E
ditin
g
Acknowledgements
Genome EditingGary LeeLynn TruongNimisha GandhiAnthony Conway and TeamAndreas Reik and Team
TechnologyEdward RebarJeff Miller and TeamLei Zhang and TeamDeepak PatilBioinformatics
LeadershipSandy MacraeMichael Holmes
AAV Production Richard Surosky and Team
Slide Number 1Forward Looking StatementsVision: Off-the-shelf allogeneic T-cell therapiesToolkit to generate healthy donor allogeneic CAR T cellsThe ZFN platform for genome editingZFN Platform: Best-in-class gene editingSingle-step multiplexed T cell editingZFN-KO of TRAC achieves 95-99% loss of surface TCRZFN-KO of β2M consistently achieves >90% loss of class I HLAHighly efficient double KO and GFP TI into TRACAAV TI of CD19 CAR into TRAC yields functional CAR-T cellsSlide Number 12Slide Number 13Slide Number 14Slide Number 15Highly efficient genome editing is critical for multiple gene modificationsSimultaneous multiplex editing efficiency: 3x ZFN KO + 1x TISlide Number 18