Polydactyl Zinc Finger Proteins: Software and Hardware for

Polydactyl Zinc Finger Proteins:

Software and Hardware for Genomes

Prof. Carlos F. Barbas, III

The screen versions of these slides have full details of copyright and acknowledgements 1

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Polydactyl Zinc

Finger Proteins:

Software and Hardware

for Genomes

Carlos F. Barbas, III

Kellogg Professor

Departments of Chemistry

and Molecular Biology

The Scripps Research Institute

La Jolla, CA 92037

USA

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Polydactyl Zinc Finger proteins:

software and hardware for the genome

• Development of a universal system for gene regulation

• Targeted endogenous gene regulation in cells

and organisms

• Transcription factor l ibraries - a powerful new strategy

• The future: cutting and pasting genes and genomes –

zinc finger enzymes and designer epigenetic modifications

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A diversity of recognition domains

EcoRV

Leucine zipper

C4 class

Helix-turn-helix

C2H2 zinc finger protein

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The Zinc Finger domain





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N

C

Zif268

a Cys2-His2 Zinc Finger protein

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-1 3

6

3'

mid

5'

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Life in the major groove

Life in the major groove





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A complete Zinc Finger code

requires 64 defined domains

GNN

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Phage display:

an efficient format for directed molecular evolution

Phage display:

an efficient format for directed molecular evolution

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Results of phage selections of the finger 2 of Zif268

Stabilizing the fold

5’ 3’

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Molecular recognition in the GNN family of Zifs





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Exquisite sequence specificity of Zinc Finger

domains… only half from selections!

Refinement or De Novo design of Fingers is Required for ~50%

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3-finger proteins

are highly specific

A view of 80 proteins

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Three fingers… 9 bp recognition

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The specificity Issue and genome size

Humans

Corn

E. coli

3.5 x 109 bp

5 x 109 bp

4 x 106 bp

Number of zinc

finger domains

3

4

5

6

Recognition

sequence

9 bp

12 bp

15 bp

18 bp

Specificity

49, 2.6 x 105

412, 1.7 x 107

415, 1.1 x 109

418, 6.9 x 1010





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A 6-finger protein can specify a unique site

in the human genome

3 + 3 + 3 + 3 + 3 + 3 = 18 nucleotides

human genome = 3 x 109 nucleotides

418 = 7 x 1010 addressable sequences

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Genomic specificity requires polydactyl proteins

• A 3-finger zinc finger proteins binds ~10,000 genomic sites

• A 6-finger zinc finger proteins binds ~1 genomic site

273Fn16 (-289) 6Fn289 (-269)

Non-additive linkage effects





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Polydactyl proteins:

structure of the first 6-finger protein Aart

Nancy Horton, U. of Arizona; JMB 2006, 363, 405

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Frequency of 6xGNN and 6xRNN sites

in promoter sequences

►Transcription start

►Translation start

● 6xGNN: 11

● 6xRNN: 77

● 6xGNN: 7

● 6xRnn: 94

erbB-2 promoter

erbB-3 promoter

Frequency of 6xGNN and 6xRNN sites

in promoter sequences

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Alignment of erbB-2 target sequence

with erbB-3 5’-UTR

AGCCATGGGGCCGGAGCCGCAGTGAGCACC

GCAATCGGAGCCGGAGCCGGAGTCCGGGGA

-30 -1

-135 -164

erbB-2

erbB-3





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Bicistronic retroviral vector for co-expression

of Zinc Finger proteins and GFP

DNA

mRNA AAAAAA

5’ LTR 3’ LTRGFPZinc finger protein IRES

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Specific regulation of two oncogenes in A431 cells

surface protein levels

ErbB-1 ErbB-2 ErbB-3

KRAB

KRAB

VP16

VP16

ErbB-2 target site:

GGg GCC GGA GCC GcA GTg

Beerli et al., (2000) PNAS 97:1495

ErbB-3 target site: GGa GCC GGA GCC GgA GTc

Cell n

um

ber

Cell n

um

ber“functional

knockouts” !

Specific regulation of two oncogenes in A431 cells





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Prot ein Target Target Sequence Kd, nM

E2C e2c GGg GCC GGA GCC GcA GTg 0.75

E2C e3 GGa GCC GGA GCC GgA GTc 10

E3 e2c GGg GCC GGA GCC GcA GTc 10

E3 e3 GGa GCC GGA GCC GgA GTc 0.35

Affinities of E2C and E3 Six Finger Proteins

Ultra-high affinity will lead to transcriptional control of non-target genes

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Regulation of fetal hemoglobin

for sickle cell therapy

Goal: repress HbS and upregulate HbF

gg1 GTC AAG GCA AGG CTG GCC 0.7 nM

+ transcriptional activator

No regulation

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Efficient and specific γ-globin gene regulation

in retrovirally transduced K562 pools

Intra-cellular staining

FACS analysis

PE-Height

Counts

▬ K562 (unspecific IgG1-PE isotype AB)

▬ K562 (anti-HbF specific AB)

▬ Gg1-VP64 cells (GFP+)

▬ Gg1_KRAB cells (GFP+)

hemoglobin

Visualization

gg1-KRAB K562 gg1-VP64

Efficient and specific γ-globin gene regulation

in retrovirally transduced K562 pools





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Inhibition of HIV replication in primary

T lymphocytes by Zif-Krab

p24 (ng/m

l)

days post infection

Primary blood leukocytes (PBLs)

HIV strain 89.6(dual tropic)

Control

SKD-HLTR3

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Zinc Finger transcription factors in Planta

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Wild Type Flower

sepe

st

ca

AP1::VP64-AP3

Ap3 Activator

AP1::SID-AP3

Ap3 Repressor

Activation of Ap3 in sepals causes

partial sepal → petal transformation

Repression of Ap3 in petals causes

partial petal → sepal transformation

VP64-

SID-





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Creating cell and organism libraries

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Transcription factors libraries

• Activate or repress every gene within a genome

• Create diverse cellular l ibraries

• Modulate multiple pathways to create interesting phenotypes

• Create diverse libraries of organisms with gain of function

and loss of function mutations

Transcription factors libraries

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Transcription factor libraries:

every gene on or up, every gene off

Gene x

Gene y

Gene z

Gene x

Gene y

Gene z

Regulate genes Regulate genes

Assemble

into TF sZF

Assemble

into TF sZF

Repertoire of ZF

DNA-binding domains

3ZF proteins 6ZF proteins





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TFZF selections in vivo: looking for new genes

involved in oncogenesis and/or tumor suppression

and metastasis in mouse models

Infect C8161 cells with pMX-3ZF-VP library

Heterogeneous tumor cell population

(106 cells) Inject in mouse (I.V, sc)

Growth and selection occurs in the host

Remove primary tumor

and organs (metastasis)

PCR ZFs

from genomic

DNA & re-clone

In pMXVP64

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Cellular phenotypes induced by TF-ZF selected

from functional screens

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TFZF 20 promotes cell invasion in vitro and in vivo

Comparison of Cell Invasion in Vitro

0

20

40

60

80

100

120

140

160

Control Hela Hela+#20 ZF

# cells per field 6ZF–20–VP

HeLa

Number of lung metastasis

ControlpMXVP

#20VP0

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15

20

25

30

1

2

3

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Specific regulation of E48 antigen in TF-20VP

expressing cells and tumors

Flow cytometry

FL2-Height

counts

■ HeLa

▬ IRES

▬ 20-VP

▬ 20-SKD

▬ 20-VP-/PE

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100

200

300

400

500

600

700

800

900

DNA Arrays

Specific regulation of E48 antigen in TF-20VP

expressing cells and tumors

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Transcription factors libraries:

opportunities in synthetic biology

• Create and screen complex phenotypes

• 3-finger transcriptions factors might be selected to regulate

multiple genes leading to interesting phenotypes

*3-finger transcription factors hit as many as 10,000 sites in the genome

• Reroute complex biosynthetic pathways: turn some genes

on and others off to create efficient syntheses

• Create novel productive cell types

• Select plants for disease resistance, salt tolerance, increased

yield, and other interesting phenotypes

48Plant J; 2006 Nov; 48 (3):475-83





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Chemical regulation of transgenes

and endogenous genes

• Tetracycline system- tetracycline analogs

• Estrogen receptor system- tamoxifen

• Progesterone receptor system- RU486

• Ecdysone receptor system- ecdysone and analogs

• Designed chemical complementation approach

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Induction of angiogenesis in a mouse model

using engineered transcription factors

Phase I clinical trial in August 2004 for the treatment of intermittent

claudication and a Phase I study in June 2005 for the treatment

of the more severe form of PAD, critical limb ischemia; A Phase Ib

clinical trial of in subjects with diabetic neuropathy in late 2005;

Currently in Phase II studies

Sangamo Biosciences Inc.

Nat Med; 2002 Dec;8(12):1427-32

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Zinc Finger nucleases: scissors for genome surgery

Applications:Applications:

• Gene correction (viaHR using a donor carrying a corrected gene)

• Gene disruption (viaHR using donor with a mutated gene or viamutagenesis NHEJ)

• Targeted integration (viaHR using donor with a inserted gene)

Zinc Finger replaced the DNA

binding domain of FokI

Figures from: Nature 2005 Jun

2;435(7042):646-51

Homology-directed repair

ZFN-driven homology-directed repair

X-ray induced DSB

ZFN-induced DSB

Sister chromatid Donor DNA (plasmid)

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Integration and gene disruptionSeamless gene exchange: recombinase

mediated cassette exchange (RMCE)

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Selected Serine recombinase crystal structures

• Yang W, Steitz TA; Crystal structure of the site-specific recombi nase

gamma delta resolvase complexed with a 34 bp cleavag e site

Cell; 1995 Jul 28;82(2):193- 207

• Li W, Kamtekar S, Xiong Y, Sarkis GJ, Grindley ND, Steitz TA; Structure

of a synaptic gamma delta resolvase tetramer covalentl y linked to two

cleaved DNAs

Science; 2005 Aug 19;309( 5738):1210- 5

• Kamtekar S, Ho RS, Cocco MJ, Li W, Wenwieser SV, Boocock MR,

Grindley ND, Steitz TA; Implicati ons of structures of synaptic tetramer s

of gamma delta resolvase for the mechanism of recombi nati on

Proc Natl Acad Sci U S A; 2006 Jul 11;103(28):10642- 7





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Recombinases: a powerful enzyme class

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4321098765432112345678901234

TGTCTGATAATTTATAATATTTCGAACG

F1

F2

F3

F1 F2 F3 F3 F2 F1

987654321-9bp-11-9bp-123456789

CCCCGCCCC-----AT-----GGGGCGGGG

Short linker

F3 F2 F1

123456789

GGGGCGGGG

γδResolvase Zif268

Zinc Finger - recombinase (RecZF) design

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Zinc Finger - recombinase (RecZF) evolution





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Zinc Finger - recombinase (RecZF) evolution

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Selective recombination

ZFs target catalytic function to corresponding sites

H1 = GGA GGC GTG

P2 = GCA GTG GCG

Inv ersion assay Resolution assay

F1 F2 F3 F3 F2 F1987654321-9bp-11-9bp-123456789CACGCCTCC-----AT-----GGAGGCGTGGTGCGGAGG-----TA-----CCTCCGCAC

F1 F2 F3 F3 F2 F1987654321-9bp-11-9bp-123456789CGCCACTGC-----AT-----GCAGTGGCGGCGGTGACG-----TA-----CGTCACCGC

ZFs

Sub H1 Sub P2

P2P2

H1H1

H1P2

P2H1

ZF:ZF:

BB

P2P2

H1H1

H1P2

P2H1

ZF:ZF:

BB

Selective recombination

ZFs target catalytic function to corresponding sites

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Selective gene excision in human cells

Gordley et al., J Mol Biol (2007)





•• Limitations of gene deliveryLimitations of gene delivery

–– Immunog eni c viral vectorsImmunog eni c viral vectors

–– Inefficient gene targetingInefficient gene targeting

•• Nonviral gene deliveryNonviral gene delivery

–– Phage integrasesPhage integrases

–– TransposasesTransposases

•• Gene targetingGene targeting

–– Homologous recombinati onHomologous recombinati on

–– SiteSite--specific recombinasesspecific recombinases

•• Cre/LoxPCre/LoxP

•• Flp/FRTFlp/FRT

•• Programmable recombinases to edit Programmable recombinases to edit

the endogenous human genomethe endogenous human genome

Gene delivery vehicles

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Targeted integration into ErbB2 locus

IntegrationIntegration--specificspecific

PCR of genomic DNAPCR of genomic DNA

• Targeted integration into a natural site

in the human genome

• Ongoing work

– Quantificati on of ErbB2 protein levels

– Phenotypi c assays

– Cancer model & therapy

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Designer recombinase applications

• Gene Therapy

• Knockout of harmful genes (by excision or integration)

• Regulation of endogenous proteins (promoter modification)

• Allele replacement

• Enable Personalized Stem Cell Therapies

• Biotechnology

• Facilitated generation of transgenic organisms (ES cells)

• Marker excision (from plants)

• Genome modification without exogenous DNA

• Research

• Experiment al genetics in model organisms

• Synthetic biology

• Tools for molecular biology





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Epigenetic control: heritable repression of gene

expression by CpG methylation

Razin A, (1998) EMBO J. 17, 4905-8

Transient repression Stable repression

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Reassembly of M.HhaI

Split C-domain Split N-domain

Intact

Choe, W. et al., Biochem. Biophys. Res. Commun. (2005)

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In vivo site-specific DNA methylation

with a designed sequence-enabled DNA

methylase





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Targeted methylation at only 1 of 19 HhaI sites by the sequence assembled methylase

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Sequence-enabled methylase provides

for exquisite site-specificity as compared

to simple M.HhaI-ZF fusions

HS2 zf HS1 zf

HS2 zfIntact

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N

N NH2

O

H

Site-specific DNA modification by DNA methyltransferase:

sequence specific organic chemistry for pattern

generation and Nano-construction

MTase

cytosine

Lukinavicius, G. et al., J.Am. Chem. Soc. (2007)

N

N NH2

O

HN

O

H2N

N

NN

N

NH 2

O

HOHHH

S

CO OHH2 N

NH

O

H 2 N

H H





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Opportunities in Nano-construction

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Expression Vectors encoding DNA binding proteins

and transcription factors in under 2 w eeks…

as fast as you can make a synthetic gene

you can make a synthetic TF or enzyme

w ww.zincfingertools.org

Transcription factor and enzyme design

now automated

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Zinc Finger technology

• Zinc finger proteins binding 9- and 18-bp DNA target sites with high

affinity and specificity can be rapidly prepared using predefined zinc

finger “modules”

• Domains binding 5’-GNN-3’ 5’-ANN-3’ and 5’-CNN-3’ sites prov ide

ready access to 466 or 9.5 billion nov el proteins that bind DNA

sequences with the structure 5’-VNN VNN VNN VNN VNN VNN-3’

• One 5’-([A/C/G]NN)6-3’ site should be present ev ery 3 nucleotides

therefore this technology can target any gene

• Any laboratory can build their own custom DNA-binding protein

today; endogenous genes can also be chemically regulated

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Zinc Finger technology… or RNAi?

Complementary approaches

Zifs provide:

• Activation - graded if desired

• Repression - graded if desired

• Wide variety of compatible chemistries

for regulated expression

• Linked transcription factors provide for the regulation

of multiple genes

• Opportunities to design networks of regulated genes

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• Roger R. Beerli

• Russell Gordley

• David Segal

• Pilar Blancafort

• Birgit Dreier

• Scott Eberhardy

• Karin Effertz

• Torbjorn Graslund

• Charles Gersbach

• Xuelin Li

• Caren V. Lund

• Laurent Magnenat

• Jeff Mandell

• Beatriz Gonzalez

• Justin T. Stege

• Beate Koksch

• Ulrich Schopfer

• Roberta Fuller

Acknowledgments

The Scripps

Research Institutecarlos@scripps. edu





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Anti-HIV Strategies

Joao Goncal ves- URIA-Centro de Patogenese Molecular

Bruce Torbett- Scripps

Christina Swan- Scripps

Regulation of Plant Genes

Roger Beachy and Isabel Ordiz - Donald Danforth Center

Steve Briggs and Xuen Guan-Torr ey Mesa Research Institute, Syngenta

Directing HIV-1 Integrase

Samson Chow- UCLA School of Medicine

Structures of 6-finger Proteins

Nancy Horton- Arizona

David Segal- UC Davis

Our collaborators

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Sangamo Biosciences

Johnson & Johnson

Novartis

Syngenta/Diversa

Licensees of our Zif technology

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Documents

Polydactyl Zinc Finger Proteins: Software and Hardware for