Zfn Technology Overview

New Zinc Finger Nuclease Updates - Production Cell Line Uses

1. Zinc Finger Nuclease (ZFN) Technology Overview
Sigma-Aldrich Corporation

3. x
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.
x
Single cell cloneSingle cell clone
Targeted Genome Editing in CHO Using
Engineered Zinc Finger Nucleases (ZFN’s)

4. Cell Line Engineering Using ZFNs
Knockouts in CHO
•dhfr-
•Fut8-
•GS-
•Bax/Bak
•Neu3
4

5. Cell Line Engineering Using ZFNs
Purpose: 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-
5

6. CHO Dihydrofolate Reductase (dhfr-)
Clones
- 83%
- 73%
-
- 5%
Not tested
7%
1%
33%
Transfection
Efficiency.
Compared to
CHOK1
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)
DE7
3.0E+06 18 23.95 D13 Not depleted 1.8 7.71
8E7
2.6E+06 18 27.70 D13 Not depleted 1.67 6.35
GE6
2.8E+06 18 26.64 Not depletedNot depleted 1.96 6.67
FN18
2.1E+06 11 31.03 Not depletedNot depleted 1.9 7.03
6G7
2.5E+06 15 27.25 Not depletedNot depleted 2.81 7.97
CHO K1
6.8E+06 9 20.11 D6 D4 1.57 5.39
Chasin DG44
2.4E+06 18+ 25.23 D6 D4 1.76 5.61
Commercially
purchased
DG44
2.5E+06 7 26.55 Not depletedNot depleted 2.35 9.78
6

7. 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
VCD (cells/ml *1e6)
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
Percent Viability
CD CHO Fusion + HT CD CHO Fusion
7

8. 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 glutamine
8

9. 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
9

10. 10
Wild
type
cells
GS
-/-
GS
ZFNs
WTGS
-/-
GS
GS = glutamine synthetase
GS
-/-
DHFR
-/-
DHFR
ZFNs
DHFR
GS
b-Tubulin
2KO
1F1.6 2B12.8 DG44 GS
-/-
WT
DHFR = dihydrofolate reductase
GS
-/-
DHFR
-/-
Fut8
-/-
Fut8
ZFNs
……...
WT, no F-LCA
_____
WT, + F-LCA
_____
14C1, + F-LCA (+1/+4)
_____
35F2, + F-LCA (D4/D5)
FUT8 = a1,6-fucosyltransferase
Multiple Gene Knockout in Mammalian
Cells

11. 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: