Using Zinc Finger Nucleases to Manipulate the Mammalian Genome

Matthew PorteusUT Southwestern Medical Center

Depts. of Pediatrics and BiochemistryDallas, TX

Gene Targeting is a Precise Recombination Event

Definition: Gene targeting is the replacement of genomic DNA with exogenous DNA by homologous

recombination.

Commonly Used For Experimental Purposes in certain cell types (yeast, chicken DT40 cells, murine

embryonic stem cells)

In addition to its usefulness for mammalian somatic cell genetics, it could also be an ideal way to treat genetic

diseases.

GFP Gene Targeting System

CMV/CBA-GFP*-IRES-CD8PGK-Neo

37GFP-IRES-CD8CMV-Sce

Stop-Sce Site

37GFP-IRES-CD8

Stop-Sc e Site

DSB-Induced Gene Targeting

CMV/CBA-GFP-IRES-CD8PGK-Neo

Porteus and Baltimore (2003)

Optimized Rate=3-5% (30-50,000 events per million transfected cells)

Two Components forDSB-Induced Homologous

Recombination

1. Repair Substrate: Fragment of DNA that serves as template for repair of DSB by homologous recombination.

2. Nuclease: Enzyme to create DSB in target gene.

Undamaged DNA (Allele S)

DSB Created (spontaneous or induced, e.g. by ZFN )

Strand Invasion into Undamaged Homologous DNA (Allele A)

In gene targeting exogenous DNA serves as homologous DNA donor.

Repairing of original strands of DNA. Gaps filled by DNA polymerase and nicks sealed

by DNA ligase.

Conversion of Blue Allele(“S”) into Red Allele (“A”) in region of DSB

S

A

A

Schema of DSB-Induced Gene Conversion

Endogenous Genes Do Not have Recognition Sites for

Homing Endonucleases

1. Modify Homing Endonucleases to Recognize new target sites.

2. Use Zinc Finger Nucleases

Zinc Finger Nucleases as Potential Reagents to Create Double-Strand Breaks in Normal Genes

Initially developed by labs of Srinivasan Chandrasegaran (Johns Hopkins) and Dana Carroll (Univ. Utah)

FokI nucleasedomain (Fn)

FokI nucleasedomain (Fn)

CMV/CBA GFP* IRES WRECD8

ZFN Full Site/Sce Site

tGFP

IRES WRECD8

tGFP

CMV/CBA GFP IRES WRECD8

Sce or ZFNExpression

PlasmidGFP Donor

G FPCMV/CBA

14 53

4785 4150

01000200030004000500060007000

Sce QQRL0 Zif QQRL0/Zif

QQR Site 6

Zif Site

Sce Site

Model Zinc Finger Nucleases Stimulate Gene Targeting

Porteus and Baltimore (2003)

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Rel

ativ

e R

ate

of

Gen

e T

arg

etin

g

Time Course of Gene Targeting Using Sce

Time Course of Gene Targeting Using Zinc Finger Nucleases

0100020003000

400050006000

0 5 10 15

QQRLO-CN/Zif-CN withTarget QQR/ZifQQRLO-CN with TargetQQR6

Continuous Expression of ZFNs causes Cytotoxicity

Can we design a pair of zinc finger nucleases to stimulate gene targeting in a real gene

in human somatic cells?

Zinc Fingers Bind Triplets

FokI nucleasedomain (Fn)

FokI nucleasedomain (Fn)

How to assemble a new zinc finger protein?

1. By altering the contact residues one can alter the target triplet.

2. By mixing different fingers one can assemble a zinc finger protein with new target site specificity.

3. Theoretically if one had zinc fingers for all 64 possible triplets one could assemble a zinc finger protein to recognize any sequence.

Zinc fingers have been published that recognize all 16 GNN,ANN, CNN triplets.

But, the GNN fingers are best.

Can we assemble a pair of zinc finger nucleases to stimulate

gene targeting?

Full Site Consensus Sequence

5’ nnCnnCnnCnnnnnnGnnGnnGnn 3’

(GNNGNNGNN inverted repeat separated by 6 bp)

Such a sequence occurs in both GFP (twice) and CD8

Lucky, eh?

From Liu et al. (2002)

Empiric Design of Zinc Finger Nucleases

(assembly approach)

Gene Targeting with Zinc Finger Nucleases to GFP

5’ acC atC ttC ttc aag Gac Gac Ggc aac stop-Sce site tac

3’ tgG taG aaG aag ttc Cgc Ctg Ccg ttc

GFPZF1 Fn

GFPZF2Fn

Finger1 Finger2 Finger3

GFPZFN-1 QSSHLTR TRGNLVR QSGNLAR (ggt) (gat) (gaa)

GFPZFN-2 DRSHLTR DRSNLTR DRSNLTR (ggc) (gac) (gac)

CMV/CBA GFP* IRES WRECD8

Sce Site

tGFP

IRES WRECD8

tGFP

CMV/CBA GFP IRES WRECD8

Sce or ZFNExpression

PlasmidGFP Donor

G FPCMV/CBA

GFP ZFN Site

Gene Targeting with Zinc Finger Nucleases to GFP

1071

4543

0

1000

2000

3000

4000

5000

6000

Sce GFP-CN Sce GFPZF1-Fn GFPZF2-Fn

GF

P P

osi

tive

Cel

ls p

er

Mil

lio

n T

ran

sfec

ted

Cel

ls

CD8 Knockout Using Zinc Finger Nucleases

bp 441 5’acc ggCgcCcaC catcgc GtcGcaGcc ctg 3’ bp 471 tgg ccGcgGgtG gtagcg CagCgtCgg gac

CD8ZF1 Fn

CD8ZF2Fn

Knockout of CD8 transgene Using CD8 Zinc Finger Nucleases

M1

M1

16% CD8 Negative

80% CD8 Negative

10/10 clonesCD8+

CD8 Knockout Plasmid

CD8 Knockout Plasmid

+CD8 ZFNs

0/12 clonesCD8+

CMV/CBA GFP* IRES CD885% CD8 PositiveCell Line

Demonstrates in Principle

1. Can make somatic cell knock-outs with ZFNs

2. Can do targeted transgenesis with ZFNs.

i.e. Substitute gene of interest for selectable marker (or both. . .) and insert into pre-selected, “safe” and “permissive” genomic location.

How far from the site of the break can you get targeting?

Co-conversion of Markers by Gene Targeting

CMV/CBA-GFP*-IRES-CD8PGK-Neo

37GFP-IRES-Puro-IRES-CD8CMV-Sce

Stop-Sce Site

37GFP-IRES-Puro-IRES-CD8

Stop-Sc e Site

Frequency of Co-Conversion using DSB Mediated Gene Targeting

CMV/CBA-GFP-IRES-Puro-IRES-CD8PGK-Neo

Day 3 Day17 Fold Change (no puro) (puro selection)

% GFP + Cells 0.041% 58% 1400Total GFP + Cells 1200 66 (-18)

% GFP + Cells 0.013% 17% 1200Total GFP + Cells 405 2 (-200)

Demonstrates:

1. Can get targeting at a distance (up to 400 bp) from site of DSB (at a price).

2. Can do co-conversion

i.e. Correct mutation at one location and insert gene that confers selective advantage nearby.

Can we design zinc finger nucleases to stimulate gene

targeting in a gene that causes human disease?

Collaboration with Sangamo Biosciences (Richmond, CA)

Human Interleukin-2 Receptor Common Gamma Chain Deficiency

(IL2RG)

1. Part of Receptor Complex for IL-2, IL-4, IL-7, IL-9, IL-15, IL-21. . .2. On X-chromosome3. Mutations in which are the most common cause of SCID (severe combined

immunodeficiency)-25% of mutations lie in Exon 5.

4. Selective Advantage for corrected cells.

5. Treatment-Bone Marrow Transplantation

: Allogeneic (sibling): Haploidentical (parent)

-Gene Therapy: Alain Fischer trial in France: Ooops, leukemia.

ZFN Gene Correctionat the IL2RG gene

IL2RG ZFN-R5’CTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTGTGGAAGTGCTC 3’3’GATGTGCAAAGCACAAGCCTCGGCGAAATTGGGTGAGACACCTTCACGAG 5’

IL2RG ZFN-L

GFP Gene Targeting Reporter for IL2RG ZFNs

5’ GFP Sce site 3’ GFP

Target site of GFP ZFNs

IL2RG site

Stimulation of Gene Targeting Using ZFNs for the IL2RG Gene

GF

P P

osi

tive

Cel

ls p

er

Mil

lio

n T

ran

sfec

ted

Cel

ls 1968

715

0

500

1000

1500

2000

2500

5-8L0/5-9L0 M16/M17IL2RG ZFN-LIL2RG ZFN-R

GFPZFNs

Optimization of IL2RG ZFN-LG

FP

Po

siti

ve C

ells

per

M

illi

on

Tra

nsf

ecte

d C

ells

1276

2897

3892

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

IL2RG ZFN-R IL2RG ZFN-R IL2RG ZFN-RIL2RG ZFN-L IL2RG ZFN-L(D) IL2RG ZFN-L(G)

Optimization of cc ZFN-RG

FP

Po

siti

ve C

ells

per

M

illi

on

Tra

nsf

ecte

d C

ells

2943 29402656

4420

1937

2689

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1 2 3 4 5 6cc ZFN-LG cc ZFN-LG cc ZFN-LG cc ZFN-LG cc ZFN-LG cc ZFN-LG cc ZFN-R cc ZFN-R cc ZFN-R cc ZFN-R cc ZFN-R cc ZFN-R (A) (B) (C) (D) (E)

4-Finger Zinc Finger Nucleases Seem to Have Less Cytotoxicity

0

1000

2000

3000

4000

5000

6000

Day3 Day5 Day7

GF

P P

osi

tive

Cel

ls p

er

Mil

lio

n T

ran

sfec

ted

Cel

ls

Experimental Design to Detect Targeting at

Endogenous IL2RG Locus

1. Transfect K562 cells with IL2RG ZFNs with repair substrate that contains BsrBI polymorphism.

2. Isolate individual clones (no selection).

3. Expand individual clones (no selection).

4. Harvest genomic DNA from individual clones.

5. Analyze genomic DNA for BsrBI polymorphism.

bB bB bB bB

bBbBbB

bBbB

BB

BBBB

BB

BB

Total clones: 76 Corrected: 14 (18%) bB: 9 (11.5%) BB: 5 (6.5%)

Bi-Allelic Targeting in Human Somatic Cells (K562)

Future Directions

1. Design ZFNs to other target genes.

2. Develop efficient method to make specific ZFNs that recognize a broad range of sequences.

3. Refine ZFNs for use in primary cells, including stem cells.

4. Assess possible induction of genomic rearrangements by ZFNs.

I. Eliminate

II. Use as a tool to study sequence specific DSBs in genetic instability.

5. Develop as a therapeutic tool.

Potential Applications to Aging Research

1. Audience will be more clever than I.

2. Use as an experimental tool to study genetics of aging in mammalian cells.

3. Create allele specific gene variants in stem cells that are associated with slower “aging.”

Thank You

UT Southwestern

Patrick ConnellyRuth EbangitBrian EllisShondra PruettKimberly Wilson

Funding

Burroughs-Wellcome Fund Career Development AwardNIH Career Development AwardUT Southwestern Medical Center

Sangamo Biosciences

Fyodor UrnovMichael Holmes

Jeff MillerPhilip Gregory

Casey Case