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: