zfn technology overview
DESCRIPTION
New Zinc Finger Nuclease Updates - Production Cell Line UsesTRANSCRIPT
Zinc Finger Nuclease (ZFN) Technology Overview
Sigma-Aldrich Corporation
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Wild type cells
ZFNs introduces a double stranded DNA break in the gene (transient exposure).
Break repaired imperfectly by non-homologous end-joining (NHEJ).
Gene ORF disrupted.
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Single cell cloneSingle cell clone
Targeted Genome Editing in CHO Using Engineered Zinc Finger Nucleases (ZFN’s)
Cell Line Engineering Using ZFNsKnockouts in CHO
•dhfr-
•Fut8-
•GS-
•Bax/Bak
•Neu3
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Cell Line Engineering Using ZFNsPurpose: Create a dhfr- genotype in CHO K1 parental cell line
Superior transfection efficiency
Shorter doubling time
Higher cell densities
Does not clump in suspension
DHFR selection and gene amplification
Easy to adapt to CD formulations
Cells mutagenized
DG44CHO K1 CHO K1/ DHFR-
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CHO Dihydrofolate Reductase (dhfr-) Clones
Clone Peak VCD
Days in culture
(> 60% viable)
Doubling time
(hours)
Glucose depletion
(< 1.0 mmol/L)
Glutamine depletion
(< 0.5 mmol/L)
Max lactate production
(g/L)
Max NH4+ production(mmol/L)
Transfection Efficiency.
Compared to CHOK1
DE7 3.0E+06 18 23.95 D13 Not depleted 1.8 7.71 33%
8E72.6E+06 18 27.70 D13 Not depleted 1.67 6.35 1%
GE62.8E+06 18 26.64 Not depleted Not depleted 1.96 6.67 7%
FN182.1E+06 11 31.03 Not depleted Not depleted 1.9 7.03 Not tested
6G72.5E+06 15 27.25 Not depleted Not depleted 2.81 7.97 - 5%
CHO K16.8E+06 9 20.11 D6 D4 1.57 5.39 -
Chasin DG44 2.4E+06 18+ 25.23 D6 D4 1.76 5.61 - 73%
Commercially purchased DG44 2.5E+06 7 26.55 Not depleted Not depleted 2.35 9.78 - 83%
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CHO K1 Dihydrofolate Reductase (dhfr-) Clone DE7
DE7 Viable Cell Density-HT Growth Characterization
0
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10 12 14 16 18 20 22 24
Days in Culture
VC
D (
cell
s/m
l *1
e6)
CD CHO Fusion + HT CD CHO Fusion
DE7 Percent Viability-HT Growth Characterization
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18 20 22 24
Days in Culture
Per
cen
t V
iab
ilit
yCD CHO Fusion + HT CD CHO Fusion
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Cell Line Engineering Using ZFNs: Glutamine Synthetase (GS-) Cells
Purpose: create a GS genotype in CHO K1 parental cell line
Benefit: Better growth characteristics than CHOK1SV in your parental cell line
Arrow = withdrawal of glutamine8
Cell Line Engineering Using ZFNs
• Fut8- Cells• Purpose: Create a Fut8- genotype in CHO parental cell line
• Benefit: Better characteristics in your parental cell line than the current fut8- CHO line
• Neu3- Cells• Purpose: Prevent the removal of terminal sialic acid from r-
protein in CHO parental cells• Benefit: Improve protein activity and circulating half-life
(clearance rate)
• Bax/Bak- Cells• Purpose: Create a bak/bax - genotype in CHO parental cell line• Benefit: Better longevity in bioreactor
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Wildtypecells
GS -/-
GSZFNs
WT GS -/-
GS
GS = glutamine synthetase
GS -/-
DHFR -/-
DHFRZFNs
DHFR
GS
-Tubulin
2KO
1F1.
62B
12.8
DG
44G
S-/
-
WT
DHFR = dihydrofolate reductase
GS -/-
DHFR -/-
Fut8 -/-
Fut8ZFNs
……... WT, no F-LCA_____ WT, + F-LCA_____ 14C1, + F-LCA (+1/+4)_____ 35F2, + F-LCA (D4/D5)
FUT8 = 1,6-fucosyltransferase
Multiple Gene Knockout in Mammalian Cells
Applications in Bioproduction
• Create new CHO Parental Lines• Extend culture life• Increasing productivity• Create selection marker
• Change the efficacy of a therapeutic through cell engineering• Altering glycosylation patterns-protein quality
• Eliminate endogenous CHO proteins that co-purify with therapeutic product
• Eliminate viral elements in CHO genome
• Eliminate waste byproducts (e.g lactate)
Customers have purchased ZFN’s in BioPharmaceutical companies to: