U.S. patent application number 15/499615 was filed with the patent office on 2017-09-07 for regulation of endogenous gene expression in cells using zinc finger proteins.
The applicant listed for this patent is Sangamo Therapeutics, Inc.. Invention is credited to Casey C. Case, George Norbert Cox, III, Stephen P. Eisenberg, Eric Edward Jarvis, Sharon Kaye Spratt.
Application Number | 20170251645 15/499615 |
Document ID | / |
Family ID | 22859587 |
Filed Date | 2017-09-07 |
United States Patent
Application |
20170251645 |
Kind Code |
A1 |
Cox, III; George Norbert ;
et al. |
September 7, 2017 |
REGULATION OF ENDOGENOUS GENE EXPRESSION IN CELLS USING ZINC FINGER
PROTEINS
Abstract
The present invention provides methods for modulating expression
of endogenous cellular genes using recombinant zinc finger
proteins.
Inventors: |
Cox, III; George Norbert;
(Richmond, CA) ; Case; Casey C.; (Richmond,
CA) ; Eisenberg; Stephen P.; (Richmond, CA) ;
Jarvis; Eric Edward; (Richmond, CA) ; Spratt; Sharon
Kaye; (Richmond, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sangamo Therapeutics, Inc. |
Richmond |
CA |
US |
|
|
Family ID: |
22859587 |
Appl. No.: |
15/499615 |
Filed: |
April 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14297197 |
Jun 5, 2014 |
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15499615 |
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13068877 |
May 23, 2011 |
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14297197 |
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10984304 |
Nov 9, 2004 |
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13068877 |
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09897844 |
Jul 2, 2001 |
6979539 |
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10984304 |
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09229037 |
Jan 12, 1999 |
6534261 |
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09897844 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/005 20130101;
A61P 43/00 20180101; A61P 21/04 20180101; A61P 3/06 20180101; C12Q
1/6897 20130101; G01N 33/5091 20130101; C07K 14/47 20130101; C07K
2319/00 20130101; C12N 2830/85 20130101; A61P 9/10 20180101; A61P
17/06 20180101; A61P 25/28 20180101; C12N 2830/008 20130101; A61P
9/00 20180101; A61P 11/00 20180101; C12N 15/8216 20130101; C12N
2830/005 20130101; A01K 67/0275 20130101; A61P 31/00 20180101; A61P
19/02 20180101; A61P 37/00 20180101; A01K 2217/05 20130101; A61P
31/12 20180101; A61P 31/18 20180101; A61P 31/04 20180101; A61P
33/02 20180101; C12N 15/85 20130101; A61P 35/00 20180101; A61P
27/02 20180101; A61P 31/10 20180101; C07K 2319/71 20130101; A61P
21/00 20180101; C07K 14/52 20130101; C12N 15/67 20130101; A61K
48/00 20130101; C12N 15/63 20130101; A61P 7/06 20180101; A61P 7/00
20180101; C12N 2830/002 20130101; C12Q 1/66 20130101; A61P 29/00
20180101; C07K 2319/81 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; A01K 67/027 20060101 A01K067/027; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made with government support under Grant
No. 1 R43 DK52251-01, awarded by the National Institutes of Health.
The government has certain rights in this invention.
Claims
1. A method of modulating expression of an endogenous cellular gene
in a cell, the method comprising the steps of: administering to the
cell a nucleic acid molecule comprising a polynucleotide sequence
which encodes a fusion molecule, the fusion molecule comprising an
engineered DNA-binding domain and a regulatory domain, wherein the
nucleic acid molecule expresses the fusion protein in the cell,
thereby modulating expression of the endogenous cellular gene.
2. The method of claim 1, wherein the fusion protein comprises at
least two regulatory domains.
3. The method of claim 1, wherein the cell is selected from the
group consisting of animal cell, a plant cell, a bacterial cell, a
protozoal cell, or a fungal cell.
4. The method of claim 3, wherein the cell is a mammalian cell.
5. The method of claim 3, wherein the cell is a human cell.
6. The method of claim 1, wherein the regulatory domain is selected
from the group consisting of a transcriptional repressor, a
transcriptional activator, an endonuclease, a methyl transferase,
and a histone deacetylase.
7. The method of claim 1, wherein the step of administering the
nucleic acid molecule to the cell comprises administering the
nucleic acid molecule in a lipid:nucleic acid complex or as naked
nucleic acid.
8. The method of claim 1, wherein the nucleic acid molecule is an
expression vector comprising the fusion protein-encoding nucleic
acid operably linked to a promoter.
9. The method of claim 8, wherein the expression vector is a viral
expression vector.
10. The method of claim 8, wherein the expression vector is a
retroviral expression vector, an adenoviral expression vector, or
an AAV expression vector.
11. The method of claim 8, wherein the promoter is an inducible
promoter.
12. The method of claim 1, wherein the target site is upstream of a
transcription initiation site of the endogenous cellular gene.
13. The method of claim 1, wherein the target site is adjacent to a
transcription initiation site of the endogenous cellular gene.
14. The method of claim 1, wherein the target site is adjacent to
an RNA polymerase pause site, wherein the RNA polymerase pause site
is downstream of a transcription initiation site of the endogenous
cellular gene.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 14/297,197, filed Jun. 5, 2014, which is a
continuation of U.S. patent application Ser. No. 13/068,877, filed
May 23, 2011, now abandoned, which is a continuation of U.S. patent
application Ser. No. 10/984,304, filed Nov. 9, 2004, now abandoned,
which is a continuation of U.S. patent application Ser. No.
09/897,844, filed Jul. 2, 2001, now U.S. Pat. No. 6,979,539, issued
Dec. 27, 2005, which is a continuation of U.S. patent application
Ser. No. 09/229,037, filed Jan. 12, 1999, now U.S. Pat. No.
6,534,261, issued Mar. 18, 2003. The present application claims
priority under 35 U.S.C. .sctn.120 to all of the aforementioned
applications, the disclosures of which are hereby incorporated by
reference in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 27, 2017, is named 8325000215_SL.txt and is 23,207 bytes in
size.
FIELD OF THE INVENTION
[0004] The present invention provides methods for regulating gene
expression of endogenous genes using recombinant zinc finger
proteins.
BACKGROUND OF THE INVENTION
[0005] Many, perhaps most physiological and pathophysiological
processes can be controlled by the selective up or down regulation
of gene expression. If methods existed for gene expression control,
pathologies could be treated. Examples include the inappropriate
expression of proinflamatory cytokines in rheumatoid arthritis,
under expression of the hepatic LDL receptor in hypercholesteremia,
over expression of proangiogenic factors and under expression of
antiangiogenic factors in solid tumor growth, to name just a few.
In addition, pathogenic organisms such as viruses, bacteria, fungi,
and protozoa could be controlled by altering gene expression. There
is a clear unmet need for therapeutic approaches that are simply
able to up-regulate beneficial genes and down-regulate disease
causing genes.
[0006] In addition to the direct therapeutic utility provided by
the ability to manipulate gene expression, this ability can be used
experimentally to determine the function of a gene of interest. One
common existing method for experimentally determining the function
of a newly discovered gene is to clone its cDNA into an expression
vector driven by a strong promoter and measure the physiological
consequence of its over-expression in a transfected cell. This
method is labor intensive and does not address the physiological
consequences of down-regulation of a target gene. Simple methods
allowing the selective over and under-expression of uncharacterized
genes would be of great utility to the scientific community.
Methods that permit the regulation of genes in cell model systems,
transgenic animals and transgenic plants would find widespread use
in academic laboratories, pharmaceutical companies, genomics
companies and in the biotechnology industry.
[0007] An additional use of tools permitting the manipulation of
gene expression is in the production of commercially useful
biological products. Cell lines, transgenic animals and transgenic
plants could be engineered to over-express a useful protein
product. The production of erythropoietin by such an engineered
cell line serves as an example. Likewise, production from metabolic
pathways might be altered or improved by the selective up or
down-regulation of a gene encoding a crucial enzyme. An example of
this is the production of plants with altered levels of fatty acid
saturation.
[0008] Methods currently exist in the art, which allow one to alter
the expression of a given gene, e.g., using ribozymes, antisense
technology, small molecule regulators, over-expression of cDNA
clones, and gene-knockouts. These methods have to date proven to be
generally insufficient for many applications and typically have not
demonstrated either high target efficacy or high specificity in
vivo. For useful experimental results and therapeutic treatments,
these characteristics are desired.
[0009] Gene expression is normally controlled through alterations
in the function of sequence specific DNA binding proteins called
transcription factors. These bind in the general proximity
(although occasionally at great distances) of the point of
transcription initiation of a gene. They act to influence the
efficiency of formation or function of a transcription initiation
complex at the promoter. Transcription factors can act in a
positive fashion (transactivation) or in a negative fashion
(transrepression).
[0010] Transcription factor function can be constitutive (always
"on") or conditional. Conditional function can be imparted on a
transcription factor by a variety of means, but the majority of
these regulatory mechanisms depend of the sequestering of the
factor in the cytoplasm and the inducible release and subsequent
nuclear translocation, DNA binding and transactivation (or
repression). Examples of transcription factors that function this
way include progesterone receptors, sterol response element binding
proteins (SREBPs) and NF-kappa B. There are examples of
transcription factors that respond to phosphorylation or small
molecule ligands by altering their ability to bind their cognate
DNA recognition sequence (Hou et al., Science 256:1701 (1994);
Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene
Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997);
Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat.
Biotechnol. 16:757-761 (1998)). This mechanism is common in
prokaryotes but somewhat less common in eukaryotes.
[0011] Zinc finger proteins ("ZFPs") are proteins that can bind to
DNA in a sequence-specific manner. Zinc fingers were first
identified in the transcription factor TFIIIA from the oocytes of
the African clawed toad, Xenopus laevis. ZFPs are widespread in
eukaryotic cells. An exemplary motif characterizing one class of
these proteins (C.sub.2H.sub.2 class) is
-Cys-(X).sub.2-4-Cys-(X).sub.12-His-(X).sub.3-5-His (where X is any
amino acid). A single finger domain is about 30 amino acids in
length and several structural studies have demonstrated that it
contains an alpha helix containing the two invariant histidine
residues co-ordinated through zinc with the two cysteines of a
single beta turn. To date, over 10,000 zinc finger sequences have
been identified in several thousand known or putative transcription
factors. ZFPs are involved not only in DNA-recognition, but also in
RNA binding and protein-protein binding. Current estimates are that
this class of molecules will constitute about 2% of all human
genes.
[0012] The X-ray crystal structure of Zif268, a three-finger domain
from a murine transcription factor, has been solved in complex with
its cognate DNA-sequence and shows that each finger can be
superimposed on the next by a periodic rotation and translation of
the finger along the main DNA axis. The structure suggests that
each finger interacts independently with DNA over 3 base-pair
intervals, with side-chains at positions -1, 2, 3 and 6 on each
recognition helix making contacts with respective DNA triplet
sub-site. The amino terminus of Zif268 is situated at the 3' end of
its DNA recognition subsite. Recent results have indicated that
some zinc fingers can bind to a fourth base in a target segment
(Isalan et al., PNAS 94:5617-5621 (1997). The fourth base is on the
opposite strand from the other three bases recognized by zinc
finger and complementary to the base immediately 3' of the three
base subsite.
[0013] The structure of the Zif268-DNA complex also suggested that
the DNA sequence specificity of a ZFP might be altered by making
amino acid substitutions at the four helix positions (-1, 2, 3 and
6) on a zinc finger recognition helix. Phage display experiments
using zinc finger combinatorial libraries to test this observation
were published in a series of papers in 1994 (Rebar et al., Science
263:671-673 (1994); Jamieson et al., Biochemistry 33:5689-5695
(1994); Choo et al., PNAS 91:11163-11167 (1994)). Combinatorial
libraries were constructed with randomized side-chains in either
the first or middle finger of Zif268 and then isolated with an
altered Zif268 binding site in which the appropriate DNA sub-site
was replaced by an altered DNA triplet. Correlation between the
nature of introduced mutations and the resulting alteration in
binding specificity gave rise to a partial set of substitution
rules for rational design of ZFPs with altered binding
specificity.
[0014] Greisman & Pabo, Science 275:657-661 (1997) discuss an
elaboration of a phage display method in which each finger of a
zinc finger protein is successively subjected to randomization and
selection. This paper reported selection of ZFPs for a nuclear
hormone response element, a p53 target site and a TATA box
sequence.
[0015] Recombinant ZFPs have been reported to have the ability to
regulate gene expression of transiently expressed reporter genes in
cultured cells (see, e.g., Pomerantz et al., Science 267:93-96
(1995); Liu et al., PNAS 94:5525-5530 1997); and Beerli et al.,
PNAS 95:14628-14633 (1998)).
[0016] For example, Pomerantz et al., Science 267:93-96 (1995)
report an attempt to design a novel DNA binding protein by fusing
two fingers from Zif268 with a homeodomain from Oct-1. The hybrid
protein was then fused with either a transcriptional activator or
repressor domain for expression as a chimeric protein. The chimeric
protein was reported to bind a target site representing a hybrid of
the subsites of its two components. The authors then constructed a
reporter vector containing a luciferase gene operably linked to a
promoter and a hybrid site for the chimeric DNA binding protein in
proximity to the promoter. The authors reported that their chimeric
DNA binding protein could activate or repress expression of the
luciferase gene.
[0017] Liu et al., PNAS 94:5525-5530 (1997) report forming a
composite ZFP by using a peptide spacer to link two component ZFPs,
each having three fingers. The composite protein was then further
linked to transcriptional activation or repression domains. It was
reported that the resulting chimeric protein bound to a target site
formed from the target segments bound by the two component ZFPs. It
was further reported that the chimeric ZFP could activate or
repress transcription of a reporter gene when its target site was
inserted into a reporter plasmid in proximity to a promoter
operably linked to the reporter.
[0018] Beerli et al., PNAS 95:14628-14633 (1998) report
construction of a chimeric six finger ZFP fused to either a KRAB,
ERD, or SID transcriptional repressor domain, or the VP16 or VP64
transcriptional activation domain. This chimeric ZFP was designed
to recognize an 18 bp target site in the 5' untranslated region of
the human erbB-2 gene. Using this construct, the authors of this
study report both activation and repression of a transiently
expressed reporter luciferase construct linked to the erbB-2
promoter.
[0019] In addition, a recombinant ZFP was reported to repress
expression of an integrated plasmid construct encoding a bcr-abl
oncogene (Choo et al., Nature 372:642-645 (1994)). The target
segment to which the ZFPs bound was a nine base sequence GCA GAA
GCC chosen to overlap the junction created by a specific oncogenic
translocation fusing the genes encoding bcr and abl. The intention
was that a ZFP specific to this target site would bind to the
oncogene without binding to abl or bcr component genes. The authors
used phage display to select a variant ZFP that bound to this
target segment. the variant ZFP thus isolated was then reported to
repress expression of a stably transfected bcr-abl construct in a
cell line.
[0020] To date, these methods have focused on regulation of either
transiently expressed genes, or on regulation of exogenous genes
that have been integrated into the genome. The transiently
expressed genes described by Pomerantz et al., Liu et al., and
Beerli et al. are episomal and are not packaged into chromatin in
the same manner as chromosomal genes. Moreover, even the stably
expressed gene described by Choo et al. is randomly integrated into
the genome and is not found in a native chromatin environment as
compared to an endogenous gene. In contrast, specific regulation of
an endogenous cellular gene in its native chromatin environment
using a ZFP has not yet been demonstrated in the art.
SUMMARY OF THE INVENTION
[0021] The present invention thus provides for the first time
methods of regulating endogenous cellular gene expression, where
the endogenous genes are in their native chromatin environment, in
contrast to genes that have been transiently expressed in a cell,
or those that have been exogenously integrated into the genome. In
one preferred embodiment, the methods of regulation use ZFPs with a
K.sub.d of less than about 25 nM to activate or repress gene
transcription. The ZFPs of the invention therefore can be used to
repress transcription of an endogenous cellular gene by 20% or
more, and can be used to activate transcription of an endogenous
cellular gene by about 1.5 fold or more.
[0022] In one aspect, the present invention provides a method of
inhibiting expression of an endogenous cellular gene in a cell, the
method comprising the step of: contacting a first target site in
the endogenous cellular gene with a first zinc finger protein,
wherein the K.sub.d of the zinc finger protein is less than about
25 nM; thereby inhibiting expression of the endogenous cellular
gene by at least about 20%.
[0023] In another aspect, the present invention provides a method
of inhibiting expression of an endogenous cellular gene in a cell,
the method comprising the step of: contacting a target site in the
endogenous cellular gene with a fusion zinc finger protein
comprising six fingers and a regulatory domain, wherein the K.sub.d
of the zinc finger protein is less than about 25 nM; thereby
inhibiting expression of the endogenous cellular gene by at least
about 20%.
[0024] In one embodiment, expression of the endogenous cellular
gene is inhibited by at least about 75%-100%. In another
embodiment, the inhibition of gene expression prevents gene
activation.
[0025] In another aspect, the present invention provides a method
of activating expression of an endogenous cellular gene, the method
comprising the step of: contacting a first target site in the
endogenous cellular gene with a first zinc finger protein, wherein
the K.sub.d of the zinc finger protein is less than about 25 nM;
thereby activating expression of the endogenous cellular gene to at
least about 150%.
[0026] In another aspect, the present invention provides a method
of activating expression of an endogenous cellular gene, the method
comprising the step of: contacting a target site in the endogenous
cellular gene with a fusion zinc finger protein comprising six
fingers and a regulatory domain, wherein the K.sub.d of the zinc
finger protein is less than about 25 nM; thereby activating
expression of the endogenous cellular gene to at least about
150%.
[0027] In one embodiment, expression of the endogenous cellular
gene is activated to at least about 200-500%. In another
embodiment, activation of gene expression prevents repression of
gene expression.
[0028] In another aspect, the present invention provides a method
of modulating expression of an endogenous cellular gene in a cell,
the method comprising the step of: contacting a first target site
in the endogenous cellular gene with a first zinc finger protein;
thereby modulating expression of the endogenous cellular gene.
[0029] In another aspect, the present invention provides a method
of modulating expression of an endogenous cellular gene in a cell,
the method comprising the step of: contacting a target site in the
endogenous cellular gene with a fusion zinc finger protein
comprising six fingers and a regulatory domain; thereby modulating
expression of the endogenous cellular gene.
[0030] In one embodiment, the step of contacting further comprises
contacting a second target site in the endogenous cellular gene
with a second zinc finger protein. In another embodiment, the first
and second target sites are adjacent. In another embodiment, the
first and second zinc finger proteins are covalently linked. In
another embodiment, the first zinc finger protein is a fusion
protein comprising a regulatory domain. In another embodiment, the
first zinc finger protein is a fusion protein comprising at least
two regulatory domains. In another embodiment, the first and second
zinc finger proteins are fusion proteins, each comprising a
regulatory domain. In another embodiment, the first and second zinc
finger protein are fusion proteins, each comprising at least two
regulatory domains.
[0031] In one embodiment, the endogenous cellular gene is a
selected from the group consisting of VEGF, ER.alpha., IGF-I,
c-myc, c-myb, ICAM, Her2/Neu, FAD2-1, EPO, GM-CSF, GDNF, and LDL-R.
In another embodiment, the regulatory domain is selected from the
group consisting of a transcriptional repressor, a transcriptional
activator, an endonuclease, a methyl transferase, a histone
acetyltransferase, and a histone deacetylase.
[0032] In one embodiment, the cell is selected from the group
consisting of animal cell, a plant cell, a bacterial cell, a
protozoal cell, or a fungal cell. In another embodiment, the cell
is a mammalian cell. In another embodiment, the cell is a human
cell.
[0033] In one embodiment, the method further comprises the step of
first administering to the cell a delivery vehicle comprising the
zinc finger protein, wherein the delivery vehicle comprises a
liposome or a membrane translocation polypeptide.
[0034] In one embodiment, the zinc finger protein is encoded by a
zinc finger protein nucleic acid operably linked to a promoter, and
the method further comprises the step of first administering the
nucleic acid to the cell in a lipid:nucleic acid complex or as
naked nucleic acid. In another embodiment, the zinc finger protein
is encoded by an expression vector comprising a zinc finger protein
nucleic acid operably linked to a promoter, and the method further
comprises the step of first administering the expression vector to
the cell. In another embodiment, the expression vector is a viral
expression vector. In another embodiment, the expression vector is
a retroviral expression vector, an adenoviral expression vector, a
DNA plasmid expression vector, or an AAV expression vector.
[0035] In one the zinc finger protein is encoded by a nucleic acid
operably linked to an inducible promoter. In another embodiment,
the zinc finger protein is encoded by a nucleic acid operably
linked to a weak promoter.
[0036] In one embodiment, the cell comprises less than about
1.5.times.10.sup.6 copies of the zinc finger protein.
[0037] In one embodiment, the target site is upstream of a
transcription initiation site of the endogenous cellular gene. In
another embodiment, the target site is adjacent to a transcription
initiation site of the endogenous cellular gene. In another
embodiment, the target site is adjacent to an RNA polymerase pause
site downstream of a transcription initiation site of the
endogenous cellular gene.
[0038] In another embodiment, the zinc finger protein comprises an
SP-1 backbone. In one embodiment, the zinc finger protein comprises
a regulatory domain and is humanized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1: PCR amplification scheme for production of
ZFP-encoding synthetic genes.
[0040] FIGS. 2A-2C. Expression and purification of typical ZFPs.
FIG. 2A: Unfused ZFP before induction (lane 1), after induction
(lane 2), and after purification (lane 3). FIG. 2B: MBP-VEGF
expression before induction (lane 1), after induction (lane 2), and
after French Press lysis (lane 3). FIG. 2C: Purification of
MBP-VEGF by amylose affinity column showing flow-through (FT), and
initial fractions (1-4). Fraction 2 was used for electrophoretic
mobility shift assays ("EMSA"). M, molecular weight markers.
[0041] FIG. 3. Typical EMSA experiment with MBP fused ZFP.
MBP-VEGF1 protein was bound to labeled duplex DNA as described in
the text. A three-fold protein dilution series was carried out;
each point represents the percent shifted at that particular
protein concentration plotted on a semi-log graph. Quantitation was
by phosphorimager. In this case, the protein concentration yielding
50% of maximum shift (the apparent K.sub.d) was 2 nM.
[0042] FIGS. 4A-4B. Off-rate experiment comparing VEGF1 to
VEGF3a/1. FIG. 4A: Protein-DNA complexes were pre-formed and
incubated with a 1000-fold excess of unlabeled oligonucleotide.
Samples were electrophoresed at various times and the amount of
shifted product was measured by phosphorimager. FIG. 4B: Curve
fitting was used to calculate the indicated complex half-lives.
[0043] FIG. 5. Typical expression vector used for transient ZFP
expression in mammalian cells.
[0044] FIG. 6. Co-transfection data showing repression of
luciferase reporter activity via VEGF-KRAB protein expression.
Error bars show the standard deviation of triplicate transfections.
pGL3-C (reporter vector control); pVFR1-4x (VEGF reporter plasmid);
VEGF1 (VEGF1-KRAB); VEGF3a (VEGF3a-KRAB); VEGF3a/1
(VEGF3a/1-KRAB).
[0045] FIG. 7. Co-transfection data showing activation of
luciferase reporter activity via VEGF-VP16 protein expression.
Error bars show the standard deviation of triplicate transfections.
pGL3-P (reporter with no VEGF target); pcDNA (empty effector vector
control); pVFR3-4x (VEGF reporter plasmid); VEGF1 (VEGF1-VP16);
VEGF3a (VEGF3a-VP16); VEGF3a/1 (VEGF3a/1-VP16).
[0046] FIG. 8. VEGF ELISA data showing repression of endogenous
VEGF gene expression due to transfection of a VEGF ZFP-KRAB
effector plasmid. DFX treated (control nontransfected Dfx treated
cells; No ZFP (pcDNA-control), VEGF 1 (VEGF1-KRAB), VEGF 3a/1
(VEGF3a/1-KRAB), CCR5 (CCR5-KRAB); Mock uninduced (mock transfected
cells untreated with DFX). Error bars show the standard deviation
of duplicate transfections.
[0047] FIG. 9. VEGF ELISA data showing activation of endogenous
VEGF gene expression due to transfection of a VEGF ZFP-VP16
effector plasmid. Mock (mock transfected cells); No ZFP
(NVF-control), VEGF 1 (VEGF1-VP16), VEGF 3a/1 (VEGF3a/1-VP16).
Error bars show the standard deviation of duplicate
transfections.
[0048] FIGS. 10A-10B. RNase protection assay showing changes in
VEGF specific mRNA by VEGF-specific ZFPs. FIG. 10A: Activation of
VEGF mRNA, NVF-Control (no ZFP), VEGF1-NVF (VEGF1-VP16), CCR5-5-NVF
(CCR5-VP16), CCR5-3-NVF (CCR5-VP16). FIG. 10B: Repression of VEGF
mRNA. NKF-Control (no ZFP), VEGF1-NKF (VEGF1-KRAB), VEGF3a/1-NKF
(VEGF3a/1-KRAB), CCR5-3-NKF (CCR5-KRAB). The size of the 148
nucleotide VEGF specific band is indicated by an arrow. The VEGF
specific probe was synthesized from a human angiogenesis
multi-probe template set (Pharmingen). As a control, signals from
the housekeeping genes L32 and GAPDH are shown (arrrows).
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0049] The present application demonstrates for the first time that
ZFPs can be used to regulate expression of an endogenous cellular
gene that is present in its native chromatin environment. The
present invention thus provides zinc finger DNA binding proteins
that have been engineered to specifically recognize, with high
efficacy, endogenous cellular genes. The experiments described
herein demonstrate that a 3 finger ZFP with a target site affinity
of less than about 10 nM (VEGF1) can be used to effectively
activate or repress activity of an endogenous gene. Furthermore, a
6 finger ZFP (VEGF3a/1) was also shown to effectively repress
activity of an endogenous gene. Preferably, the ZFPs of the
invention exhibit high affinity for their target sites, with
K.sub.ds of less than about 100 nM, preferably less than about 50
nM, most preferably less than about 25 nM or lower.
[0050] As a result, the ZFPs of the invention can be used to
regulate endogenous gene expression, both through activation and
repression of endogenous gene transcription. The ZFPs can also be
linked to regulatory domains, creating chimeric transcription
factors to activate or repress transcription. In one preferred
embodiment, the methods of regulation use ZFPs with a K.sub.d of
less than about 25 nM to activate or repress gene transcription.
The ZFPs of the invention therefore can be used to repress
transcription of an endogenous cellular gene by 20% or more, and
can be used to activate transcription of an endogenous cellular
gene by about 1.5 fold or more.
[0051] Such methods of regulating gene expression allow for novel
human and mammalian therapeutic applications, e.g., treatment of
genetic diseases, cancer, fungal, protozoal, bacterial, and viral
infection, ischemia, vascular disease, arthritis, immunological
disorders, etc., as well as providing means for functional genomics
assays, and means for developing plants with altered phenotypes,
including disease resistance, fruit ripening, sugar and oil
composition, yield, and color.
[0052] As described herein, ZFPs can be designed to recognize any
suitable target site, for regulation of expression of any
endogenous gene of choice. Examples of endogenous genes suitable
for regulation include VEGF, CCR5, ER.alpha., Her2/Neu, Tat, Rev,
HBV C, S, X, and P, LDL-R, PEPCK, CYP7, Fibrinogen, ApoB, Apo E,
Apo(a), renin, NF-.kappa.B, I-.kappa.B, TNF-.alpha., FAS ligand,
amyloid precursor protein, atrial naturetic factor, ob-leptin,
ucp-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, G-CSF, GM-CSF,
Epo, PDGF, PAF, p53, Rb, fetal hemoglobin, dystrophin, eutrophin,
GDNF, NGF, IGF-1, VEGF receptors fit and flk, topoisomerase,
telomerase, bcl-2, cyclins, angiostatin, IGF, ICAM-1, STATS, c-myc,
c-myb, TH, PTI-1, polygalacturonase, EPSP synthase, FAD2-1,
delta-12 desaturase, delta-9 desaturase, delta-15 desaturase,
acetyl-CoA carboxylase, acyl-ACP-thioesterase, ADP-glucose
pyrophosphorylase, starch synthase, cellulose synthase, sucrose
synthase, senescence-associated genes, heavy metal chelators, fatty
acid hydroperoxide lyase, viral genes, protozoal genes, fungal
genes, and bacterial genes. In general, suitable genes to be
regulated include cytokines, lymphokines, growth factors, mitogenic
factors, chemotactic factors, onco-active factors, receptors,
potassium channels, G-proteins, signal transduction molecules, and
other disease-related genes.
[0053] A general theme in transcription factor function is that
simple binding and sufficient proximity to the promoter are all
that is generally needed. Exact positioning relative to the
promoter, orientation, and within limits, distance do not matter
greatly. This feature allows considerable flexibility in choosing
sites for constructing artificial transcription factors. The target
site recognized by the ZFP therefore can be any suitable site in
the target gene that will allow activation or repression of gene
expression by a ZFP, optionally linked to a regulatory domain.
Preferred target sites include regions adjacent to, downstream, or
upstream of the transcription start site. In addition, target sites
that are located in enhancer regions, repressor sites, RNA
polymerase pause sites, and specific regulatory sites (e.g., SP-1
sites, hypoxia response elements, nuclear receptor recognition
elements, p53 binding sites), sites in the cDNA encoding region or
in an expressed sequence tag (EST) coding region. As described
below, typically each finger recognizes 2-4 base pairs, with a two
finger ZFP binding to a 4 to 7 bp target site, a three finger ZFP
binding to a 6 to 10 base pair site, and a six finger ZFP binding
to two adjacent target sites, each target site having from 6-10
base pairs.
[0054] As described herein, two ZFPs can be administered to a cell,
recognizing either the same target endogenous cellular gene, or
different target endogenous cellular gene. The first ZFP optionally
is associated with the second ZFP, either covalently or
non-covalently. Recognition of adjacent target sites by either
associated or individual ZFPs can be used to produce cooperative
binding of the ZFPs, resulting in an affinity that is greater than
the affinity of the ZFPs when individually bound to their target
site.
[0055] In one embodiment, two ZFPs are produced as a fusion protein
linked by an amino acid linker, and the resulting six finger ZFP
recognizes an approximately 18 base pair target site (see, e.g.,
Liu et al., PNAS 94:5525-5530 (1997)). An 18 base pair target site
is expected to provide specificity in the human genome, as a target
site of that size should occur only once in every 3.times.10.sup.10
base pairs, and the size of the human genome is 3.5.times.10.sup.9
base pairs (see, e.g., Liu et al., PNAS 94:5525-5530 (1997)). In
another embodiment, the ZFPs are non-covalently associated, through
a leucine zipper, a STAT protein N-terminal domain, or the FK506
binding protein (see, e.g., O'Shea, Science 254: 539 (1991),
Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211:121-128
(1996); Klemm et al., Annu. Rev. Immunol. 16:569-592 (1998); Ho et
al., Nature 382:822-826 (1996)).
[0056] In another embodiment, the ZFP is linked to at least one or
more regulatory domains, described below. Preferred regulatory
domains include transcription factor repressor or activator domains
such as KRAB and VP16, co-repressor and co-activator domains, DNA
methyl transferases, histone acetyltransferases, histone
deacetylases, and endonucleases such as Fok1. For repression of
gene expression, typically the expression of the gene is reduced by
about 20% (i.e., 80% of non-ZFP modulated expression), more
preferably by about 50% (i.e., 50% of non-ZFP modulated
expression), more preferably by about 75-100% (i.e., 25% to 0% of
non-ZFP modulated expression). For activation of gene expression,
typically expression is activated by about 1.5 fold (i.e., 150% of
non-ZFP modulated expression), preferably 2 fold (i.e., 200% of
non-ZFP modulated expression), more preferably 5-10 fold (i.e.,
500-1000% of non-ZFP modulated expression), up to at least 100 fold
or more.
[0057] The expression of engineered ZFP activators and repressors
can be also controlled by systems typified by the tet-regulated
systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS
89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang
et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood
88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol.
16:757-761 (1998)). These impart small molecule control on the
expression of the ZFP activators and repressors and thus impart
small molecule control on the target gene(s) of interest. This
beneficial feature could be used in cell culture models, in gene
therapy, and in transgenic animals and plants.
Definitions
[0058] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0059] The term "zinc finger protein" or "ZFP" refers to a protein
having DNA binding domains that are stabilized by zinc. The
individual DNA binding domains are typically referred to as
"fingers" A ZFP has least one finger, typically two fingers, three
fingers, or six fingers. Each finger binds from two to four base
pairs of DNA, typically three or four base pairs of DNA. A ZFP
binds to a nucleic acid sequence called a target site or target
segment. Each finger typically comprises an approximately 30 amino
acid, zinc-chelating, DNA-binding subdomain. An exemplary motif
characterizing one class of these proteins (C.sub.2H.sub.2 class)
is -Cys-(X).sub.2-4-Cys-(X).sub.12-His-(X).sub.3-5-His (where X is
any amino acid). Studies have demonstrated that a single zinc
finger of this class consists of an alpha helix containing the two
invariant histidine residues co-ordinated with zinc along with the
two cysteine residues of a single beta turn (see, e.g., Berg &
Shi, Science 271:1081-1085 (1996)).
[0060] A "target site" is the nucleic acid sequence recognized by a
ZFP. A single target site typically has about four to about ten
base pairs. Typically, a two-fingered ZFP recognizes a four to
seven base pair target site, a three-fingered ZFP recognizes a six
to ten base pair target site, and a six fingered ZFP recognizes two
adjacent nine to ten base pair target sites.
[0061] The term "adjacent target sites" refers to non-overlapping
target sites that are separated by zero to about 5 base pairs.
[0062] "K.sub.d" refers to the dissociation constant for the
compound, i.e., the concentration of a compound (e.g., a zinc
finger protein) that gives half maximal binding of the compound to
its target (i.e., half of the compound molecules are bound to the
target) under given conditions (i.e., when
[target]<<K.sub.d), as measured using a given assay system
(see, e.g., U.S. Pat. No. 5,789,538). The assay system used to
measure the K.sub.d should be chosen so that it gives the most
accurate measure of the actual K.sub.d of the ZFP. Any assay system
can be used, as long is it gives an accurate measurement of the
actual K.sub.d of the ZFP. In one embodiment, the K.sub.d for the
ZFPs of the invention is measured using an electrophoretic mobility
shift assay ("EMSA"), as described in Example I and on page 14 of
the present specification. Unless an adjustment is made for ZFP
purity or activity, the K.sub.d calculations made using the method
of Example I may result in an underestimate of the true K.sub.d of
a given ZFP. Preferably, the K.sub.d of a ZFP used to modulate
transcription of an endogenous cellular gene is less than about 100
nM, more preferably less than about 75 nM, more preferably less
than about 50 nM, most preferably less than about 25 nM.
[0063] An "endogenous cellular gene" refers to a gene that is
native to a cell, which is in its normal genomic and chromatin
context, and which is not heterologous to the cell. Such cellular
genes include, e.g., animal genes, plant genes, bacterial genes,
protozoal genes, fungal genes, mitrochondrial genes, and
chloroplastic genes.
[0064] An "endogenous gene" refers to a microbial or viral gene
that is part of a naturally occurring microbial or viral genome in
a microbially or virally infected cell. The microbial or viral
genome can be extrachromosomal or integrated into the host
chromosome. This term also encompasses endogenous cellular genes,
as described above.
[0065] A "native chromatin environment" refers to the naturally
occurring, structural relationship of genomic DNA (e.g., bacterial,
animal, fungal, plant, protozoal, mitochondrial, and chloroplastic)
and DNA-binding proteins (e.g., histones and bacterial DNA binding
protein II), which together form chromosomes. The endogenous
cellular gene can be in a transcriptionally active or inactive
state in the native chromatin environment.
[0066] The phrase "adjacent to a transcription initiation site"
refers to a target site that is within about 50 bases either
upstream or downstream of a transcription initiation site.
"Upstream" of a transcription initiation site refers to a target
site that is more than about 50 bases 5' of the transcription
initiation site (i.e., in the non-transcribed region of the
gene).
[0067] The phrase "RNA polymerase pause site" is described in
Uptain et al., Annu. Rev. Biochem. 66:117-172 (1997).
[0068] "Humanized" refers to a non-human polypeptide sequence that
has been modified to minimize immunoreactivity in humans, typically
by altering the amino acid sequence to mimic existing human
sequences, without substantially altering the function of the
polypeptide sequence (see, e.g., Jones et al., Nature 321:522-525
(1986), and published UK patent application No. 8707252). Backbone
sequences for the ZFPs are preferably be selected from existing
human C.sub.2H.sub.2 ZFPs (e.g., SP-1). Functional domains are
preferably selected from existing human genes, (e.g., the
activation domain from the p65 subunit of NF-.kappa.B). Where
possible, the recognition helix sequences will be selected from the
thousands of existing ZFP DNA recognition domains provided by
sequencing the human genome. As much as possible, domains will be
combined as units from the same existing proteins. All of these
steps will minimize the introduction of new junctional epitopes in
the chimeric ZFPs and render the engineered ZFPs less
immunogenic.
[0069] "Administering" an expression vector, nucleic acid, ZFP, or
a delivery vehicle to a cell comprises transducing, transfecting,
electroporating, translocating, fusing, phagocytosing, shooting or
ballistic methods, etc., i.e., any means by which a protein or
nucleic acid can be transported across a cell membrane and
preferably into the nucleus of a cell.
[0070] A "delivery vehicle" refers to a compound, e.g., a liposome,
toxin, or a membrane translocation polypeptide, which is used to
administer a ZFP. Delivery vehicles can also be used to administer
nucleic acids encoding ZFPs, e.g., a lipid:nucleic acid complex, an
expression vector, a virus, and the like.
[0071] The terms "modulating expression" "inhibiting expression"
and "activating expression" of a gene refer to the ability of a ZFP
to activate or inhibit transcription of a gene. Activation includes
prevention of transcriptional inhibition (i.e., prevention of
repression of gene expression) and inhibition includes prevention
of transcriptional activation (i.e., prevention of gene
activation).
[0072] Modulation can be assayed by determining any parameter that
is indirectly or directly affected by the expression of the target
gene. Such parameters include, e.g., changes in RNA or protein
levels, changes in protein activity, changes in product levels,
changes in downstream gene expression, changes in reporter gene
transcription (luciferase, CAT, .beta.-galactosidase,
.beta.-glucuronidase, GFP (see, e.g., Mistili & Spector, Nature
Biotechnology 15:961-964 (1997)); changes in signal transduction,
phosphorylation and dephosphorylation, receptor-ligand
interactions, second messenger concentrations (e.g., cGMP, cAMP,
IP3, and Ca.sup.2+), cell growth, and neovascularization. These
assays can be in vitro, in vivo, and ex vivo. Such functional
effects can be measured by any means known to those skilled in the
art, e.g., measurement of RNA or protein levels, measurement of RNA
stability, identification of downstream or reporter gene
expression, e.g., via chemiluminescence, fluorescence, colorimetric
reactions, antibody binding, inducible markers, ligand binding
assays; changes in intracellular second messengers such as cGMP and
inositol triphosphate (IP3); changes in intracellular calcium
levels; cytokine release, and the like.
[0073] To determine the level of gene expression modulation by a
ZFP, cells contacted with ZFPs are compared to control cells, e.g.,
without the zinc finger protein or with a non-specific ZFP, to
examine the extent of inhibition or activation. Control samples are
assigned a relative gene expression activity value of 100%.
Modulation/inhibition of gene expression is achieved when the gene
expression activity value relative to the control is about 80%,
preferably 50% (i.e., 0.5.times. the activity of the control), more
preferably 25%, more preferably 5-0%. Modulation/activation of gene
expression is achieved when the gene expression activity value
relative to the control is 110%, more preferably 150% (i.e., 1.5x
the activity of the control), more preferably 200-500%, more
preferably 1000-2000% or more.
[0074] A "transcriptional activator" and a "transcriptional
repressor" refer to proteins or effector domains of proteins that
have the ability to modulate transcription, as described above.
Such proteins include, e.g., transcription factors and co-factors
(e.g., KRAB, MAD, ERD, SID, nuclear factor kappa B subunit p65,
early growth response factor 1, and nuclear hormone receptors,
VP16, VP64), endonucleases, integrases, recombinases,
methyltransferases, histone acetyltransferases, histone
deacetylases etc. Activators and repressors include co-activators
and co-repressors (see, e.g., Utley et al., Nature 394:498-502
(1998)).
[0075] A "regulatory domain" refers to a protein or a protein
domain that has transcriptional modulation activity when tethered
to a DNA binding domain, i.e., a ZFP. Typically, a regulatory
domain is covalently or non-covalently linked to a ZFP to effect
transcription modulation. Alternatively, a ZFP can act alone,
without a regulatory domain, to effect transcription
modulation.
[0076] The term "heterologous" is a relative term, which when used
with reference to portions of a nucleic acid indicates that the
nucleic acid comprises two or more subsequences that are not found
in the same relationship to each other in nature. For instance, a
nucleic acid that is recombinantly produced typically has two or
more sequences from unrelated genes synthetically arranged to make
a new functional nucleic acid, e.g., a promoter from one source and
a coding region from another source. The two nucleic acids are thus
heterologous to each other in this context. When added to a cell,
the recombinant nucleic acids would also be heterologous to the
endogenous genes of the cell. Thus, in a chromosome, a heterologous
nucleic acid would include an non-native (non-naturally occurring)
nucleic acid that has integrated into the chromosome, or a
non-native (non-naturally occurring) extrachromosomal nucleic acid.
In contrast, a naturally translocated piece of chromosome would not
be considered heterologous in the context of this patent
application, as it comprises an endogenous nucleic acid sequence
that is native to the mutated cell.
[0077] Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a "fusion protein,"
where the two subsequences are encoded by a single nucleic acid
sequence). See, e.g., Ausubel, supra, for an introduction to
recombinant techniques.
[0078] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native (naturally
occurring) form of the cell or express a second copy of a native
gene that is otherwise normally or abnormally expressed, under
expressed or not expressed at all.
[0079] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription. As used herein, a promoter
typically includes necessary nucleic acid sequences near the start
site of transcription, such as, in the case of certain RNA
polymerase II type promoters, a TATA element, enhancer, CCAAT box,
SP-1 site, etc. As used herein, a promoter also optionally includes
distal enhancer or repressor elements, which can be located as much
as several thousand base pairs from the start site of
transcription. The promoters often have an element that is
responsive to transactivation by a DNA-binding moiety such as a
polypeptide, e.g., a nuclear receptor, Gal4, the lac repressor and
the like.
[0080] A "constitutive" promoter is a promoter that is active under
most environmental and developmental conditions. An "inducible"
promoter is a promoter that is active under certain environmental
or developmental conditions.
[0081] A "weak promoter" refers to a promoter having about the same
activity as a wild type herpes simplex virus ("HSV") thymidine
kinase ("tk") promoter or a mutated HSV tk promoter, as described
in Eisenberg & McKnight, Mol. Cell. Biol. 5:1940-1947
(1985).
[0082] The term "operably linked" refers to a functional linkage
between a nucleic acid expression control sequence (such as a
promoter, or array of transcription factor binding sites) and a
second nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to
the second sequence.
[0083] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell, and optionally integration
or replication of the expression vector in a host cell. The
expression vector can be part of a plasmid, virus, or nucleic acid
fragment, of viral or non-viral origin. Typically, the expression
vector includes an "expression cassette," which comprises a nucleic
acid to be transcribed operably linked to a promoter. The term
expression vector also encompasses naked DNA operably linked to a
promoter.
[0084] By "host cell" is meant a cell that contains a ZFP or an
expression vector or nucleic acid encoding a ZFP. The host cell
typically supports the replication or expression of the expression
vector. Host cells may be prokaryotic cells such as E. coli, or
eukaryotic cells such as yeast, fungal, protozoal, higher plant,
insect, or amphibian cells, or mammalian cells such as CHO, HeLa,
293, COS-1, and the like, e.g., cultured cells (in vitro), explants
and primary cultures (in vitro and ex vivo), and cells in vivo.
[0085] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0086] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. The term nucleic acid is used interchangeably with gene,
cDNA, mRNA, oligonucleotide, and polynucleotide. The nucleotide
sequences are displayed herein in the conventional 5'-3'
orientation.
[0087] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoproteins. The polypeptide sequences are displayed
herein in the conventional N-terminal to C-terminal
orientation.
[0088] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine, and methyl sulfonium.
Such analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0089] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka
et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol.
Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given protein. For instance, the codons GCA, GCC,
GCG and GCU all encode the amino acid alanine. Thus, at every
position where an alanine is specified by a codon in an amino acid
herein, the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0090] As to amino acid and nucleic acid sequences, individual
substitutions, deletions or additions that alter, add or delete a
single amino acid or nucleotide or a small percentage of amino
acids or nucleotides in the sequence create a "conservatively
modified variant," where the alteration results in the substitution
of an amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants and alleles of
the invention.
[0091] The following groups each contain amino acids that are
conservative substitutions for one another:
[0092] 1) Alanine (A), Glycine (G);
[0093] 2) Serine (S), Threonine (T);
[0094] 3) Aspartic acid (D), Glutamic acid (E);
[0095] 4) Asparagine (N), Glutamine (Q);
[0096] 5) Cysteine (C), Methionine (M);
[0097] 6) Arginine (R), Lysine (K), Histidine (H);
[0098] 7) Isoleucine (I), Leucine (L), Valine (V); and
[0099] 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0100] (see, e.g., Creighton, Proteins (1984) for a discussion of
amino acid properties).
Design of ZFPs
[0101] The ZFPs of the invention are engineered to recognize a
selected target site in the endogenous gene of choice. Typically, a
backbone from any suitable C.sub.2H.sub.2 ZFP, such as SP-1, SP-1C,
or ZIF268, is used as the scaffold for the engineered ZFP (see,
e.g., Jacobs, EMBO J. 11:4507 (1992); Desjarlais & Berg, PNAS
90:2256-2260 (1993)). A number of methods can then be used to
design and select a ZFP with high affinity for its target (e.g.,
preferably with a K.sub.d of less than about 25 nM). As described
above, a ZFP can be designed or selected to bind to any suitable
target site in the target endogenous gene, with high affinity. U.S.
Pat. No. 6,453,242 comprehensively describes methods for design,
construction, and expression of ZFPs for selected target sites.
[0102] Any suitable method known in the art can be used to design
and construct nucleic acids encoding ZFPs, e.g., phage display,
random mutagenesis, combinatorial libraries, computer/rational
design, affinity selection, PCR, cloning from cDNA or genomic
libraries, synthetic construction and the like. (see, e.g., U.S.
Pat. No. 5,786,538; Wu et al., PNAS 92:344-348 (1995); Jamieson et
al., Biochemistry 33:5689-5695 (1994); Rebar & Pabo, Science
263:671-673 (1994); Choo & Klug, PNAS 91:11163-11167 (1994);
Choo & Klug, PNAS 91: 11168-11172 (1994); Desjarlais &
Berg, PNAS 90:2256-2260 (1993); Desjarlais & Berg, PNAS
89:7345-7349 (1992); Pomerantz et al., Science 267:93-96 (1995);
Pomerantz et al., PNAS 92:9752-9756 (1995); and Liu et al., PNAS
94:5525-5530 (1997); Griesman & Pabo, Science 275:657-661
(1997); Desjarlais & Berg, PNAS 91:11-99-11103 (1994)).
[0103] U.S. Pat. No. 6,453,242 provides methods that select a
target gene, and identify a target site within the gene containing
one to six (or more) D-able sites (see definition below). Using
these methods, a ZFP can then be synthesized that binds to the
preselected site. These methods of target site selection are
premised, in part, on the recognition that the presence of one or
more D-able sites in a target segment confers the potential for
higher binding affinity in a ZFP selected or designed to bind to
that site relative to ZFPs that bind to target segments lacking
D-able sites. Experimental evidence supporting this insight is
provided in Examples 2-9 of U.S. Pat. No. 6,453,242.
[0104] A D-able site or subsite is a region of a target site that
allows an appropriately designed single zinc finger to bind to four
bases rather than three of the target site. Such a zinc finger
binds to a triplet of bases on one strand of a double-stranded
target segment (target strand) and a fourth base on the other
strand (see FIG. 2 of U.S. Pat. No. 6,453,242). Binding of a single
zinc finger to a four base target segment imposes constraints both
on the sequence of the target strand and on the amino acid sequence
of the zinc finger. The target site within the target strand should
include the "D-able" site motif 5' NNGK 3', in which N and K are
conventional IUPAC-IUB ambiguity codes. A zinc finger for binding
to such a site should include an arginine residue at position -1
and an aspartic acid, (or less preferably a glutamic acid) at
position +2. The arginine residues at position -1 interacts with
the G residue in the D-able site. The aspartic acid (or glutamic
acid) residue at position +2 of the zinc finger interacts with the
opposite strand base complementary to the K base in the D-able
site. It is the interaction between aspartic acid (symbol D) and
the opposite strand base (fourth base) that confers the name D-able
site. As is apparent from the D-able site formula, there are two
subtypes of D-able sites: 5' NNGG 3' and 5' NNGT 3'. For the former
site, the aspartic acid or glutamic acid at position +2 of a zinc
finger interacts with a C in the opposite strand to the D-able
site. In the latter site, the aspartic acid or glutamic acid at
position +2 of a zinc finger interacts with an A in the opposite
strand to the D-able site. In general, NNGG is preferred over
NNGT.
[0105] In the design of a ZFP with three fingers, a target site
should be selected in which at least one finger of the protein, and
optionally, two or all three fingers have the potential to bind a
D-able site. Such can be achieved by selecting a target site from
within a larger target gene having the formula 5'-NNx aNy bNzc-3',
wherein
[0106] each of the sets (x, a), (y, b) and (z, c) is either (N, N)
or (G, K);
[0107] at least one of (x, a), (y, b) and (z, c) is (G, K). and
[0108] N and K are IUPAC-IUB ambiguity codes
[0109] In other words, at least one of the three sets (x, a), (y,
b) and (z, c) is the set (G, K), meaning that the first position of
the set is G and the second position is G or T. Those of the three
sets (if any) which are not (G, K) are (N, N), meaning that the
first position of the set can be occupied by any nucleotide and the
second position of the set can be occupied by any nucleotide. As an
example, the set (x, a) can be (G, K) and the sets (y, b) and (z,
c) can both be (N, N).
[0110] In the formula 5'-NNx aNy bNzc-3', the triplets of NNx aNy
and bNzc represent the triplets of bases on the target strand bound
by the three fingers in a ZFP. If only one of x, y and z is a G,
and this G is followed by a K, the target site includes a single
D-able subsite. For example, if only x is G, and a is K, the site
reads 5'-NNG KNy bNzc-3' with the D-able subsite highlighted. If
both x and y but not z are G, and a and b are K, then the target
site has two overlapping D-able subsites as follows: 5'-NNG KNG KNz
c-3', with one such site being represented in bold and the other in
italics. If all three of x, y and z are G and a, b, and c are K,
then the target segment includes three D-able subsites, as follows
5'NNG KNG KNG K3', the D-able subsites being represented by bold,
italics and underline.
[0111] These methods thus work by selecting a target gene, and
systematically searching within the possible subsequences of the
gene for target sites conforming to the formula 5'-NNx aNy bNzc-3',
as described above. In some such methods, every possible
subsequence of 10 contiguous bases on either strand of a potential
target gene is evaluated to determine whether it conforms to the
above formula, and, if so, how many D-able sites are present.
Typically, such a comparison is performed by computer, and a list
of target sites conforming to the formula are output. Optionally,
such target sites can be output in different subsets according to
how many D-able sites are present.
[0112] In a variation, the methods of the invention identify first
and second target segments, each independently conforming to the
above formula. The two target segments in such methods are
constrained to be adjacent or proximate (i.e., within about 0-5
bases) of each other in the target gene. The strategy underlying
selection of proximate target segments is to allow the design of a
ZFP formed by linkage of two component ZFPs specific for the first
and second target segments respectively. These principles can be
extended to select target sites to be bound by ZFPs with any number
of component fingers. For example, a suitable target site for a
nine finger protein would have three component segments, each
conforming to the above formula.
[0113] The target sites identified by the above methods can be
subject to further evaluation by other criteria or can be used
directly for design or selection (if needed) and production of a
ZFP specific for such a site. A further criteria for evaluating
potential target sites is their proximity to particular regions
within a gene. If a ZFP is to be used to repress a cellular gene on
its own (i.e., without linking the ZFP to a repressing moiety),
then the optimal location appears to be at, or within 50 bp
upstream or downstream of the site of transcription initiation, to
interfere with the formation of the transcription complex (Kim
& Pabo, J. Biol. Chem. 272:29795-296800 (1997)) or compete for
an essential enhancer binding protein. If, however, a ZFP is fused
to a functional domain such as the KRAB repressor domain or the
VP16 activator domain, the location of the binding site is
considerably more flexible and can be outside known regulatory
regions. For example, a KRAB domain can repress transcription at a
promoter up to at least 3 kbp from where KRAB is bound (Margolin et
al., PNAS 91:4509-4513 (1994)). Thus, target sites can be selected
that do not necessarily include or overlap segments of demonstrable
biological significance with target genes, such as regulatory
sequences. Other criteria for further evaluating target segments
include the prior availability of ZFPs binding to such segments or
related segments, and/or ease of designing new ZFPs to bind a given
target segment.
[0114] After a target segment has been selected, a ZFP that binds
to the segment can be provided by a variety of approaches. The
simplest of approaches is to provide a precharacterized ZFP from an
existing collection that is already known to bind to the target
site. However, in many instances, such ZFPs do not exist. An
alternative approach can also be used to design new ZFPs, which
uses the information in a database of existing ZFPs and their
respective binding affinities. A further approach is to design a
ZFP based on substitution rules as discussed above. A still further
alternative is to select a ZFP with specificity for a given target
by an empirical process such as phage display. In some such
methods, each component finger of a ZFP is designed or selected
independently of other component fingers. For example, each finger
can be obtained from a different preexisting ZFP or each finger can
be subject to separate randomization and selection.
[0115] Once a ZFP has been selected, designed, or otherwise
provided to a given target segment, the ZFP or the DNA encoding it
are synthesized. Exemplary methods for synthesizing and expressing
DNA encoding zinc finger proteins are described below. The ZFP or a
polynucleotide encoding it can then be used for modulation of
expression, or analysis of the target gene containing the target
site to which the ZFP binds.
Expression and Purification of ZFPs
[0116] ZFP polypeptides and nucleic acids can be made using routine
techniques in the field of recombinant genetics. Basic texts
disclosing the general methods of use in this invention include
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.
1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual
(1990); and Current Protocols in Molecular Biology (Ausubel et al.,
eds., 1994)). In addition, essentially any nucleic acid can be
custom ordered from any of a variety of commercial sources.
Similarly, peptides and antibodies can be custom ordered from any
of a variety of commercial sources.
[0117] Two alternative methods are typically used to create the
coding sequences required to express newly designed DNA-binding
peptides. One protocol is a PCR-based assembly procedure that
utilizes six overlapping oligonucleotides (FIG. 1). Three
oligonucleotides (oligos 1, 3, and 5 in FIG. 1) correspond to
"universal" sequences that encode portions of the DNA-binding
domain between the recognition helices. These oligonucleotides
remain constant for all zinc finger constructs. The other three
"specific" oligonucleotides (oligos 2, 4, and 6 in FIG. 1) are
designed to encode the recognition helices. These oligonucleotides
contain substitutions primarily at positions -1, 2, 3 and 6 on the
recognition helices making them specific for each of the different
DNA-binding domains.
[0118] The PCR synthesis is carried out in two steps. First, a
double stranded DNA template is created by combining the six
oligonucleotides (three universal, three specific) in a four cycle
PCR reaction with a low temperature annealing step, thereby
annealing the oligonucleotides to form a DNA "scaffold." The gaps
in the scaffold are filled in by high-fidelity thermostable
polymerase, the combination of Taq and Pfu polymerases also
suffices. In the second phase of construction, the zinc finger
template is amplified by external primers designed to incorporate
restriction sites at either end for cloning into a shuttle vector
or directly into an expression vector.
[0119] An alternative method of cloning the newly designed
DNA-binding proteins relies on annealing complementary
oligonucleotides encoding the specific regions of the desired ZFP.
This particular application requires that the oligonucleotides be
phosphorylated prior to the final ligation step. This is usually
performed before setting up the annealing reactions, but kinasing
can also occur post-annealing. In brief, the "universal"
oligonucleotides encoding the constant regions of the proteins
(oligos 1, 2 and 3 of above) are annealed with their complementary
oligonucleotides. Additionally, the "specific" oligonucleotides
encoding the finger recognition helices are annealed with their
respective complementary oligonucleotides. These complementary
oligos are designed to fill in the region which was previously
filled in by polymerase in the protocol described above. The
complementary oligos to the common oligos 1 and finger 3 are
engineered to leave overhanging sequences specific for the
restriction sites used in cloning into the vector of choice. The
second assembly protocol differs from the initial protocol in the
following aspects: the "scaffold" encoding the newly designed ZFP
is composed entirely of synthetic DNA thereby eliminating the
polymerase fill-in step, additionally the fragment to be cloned
into the vector does not require amplification. Lastly, the design
of leaving sequence-specific overhangs eliminates the need for
restriction enzyme digests of the inserting fragment.
[0120] The resulting fragment encoding the newly designed ZFP is
ligated into an expression vector. Expression vectors that are
commonly utilized include, but are not limited to, a modified
pMAL-c2 bacterial expression vector (New England BioLabs, "NEB") or
a eukaryotic expression vector, pcDNA (Promega).
[0121] Any suitable method of protein purification known to those
of skill in the art can be used to purify ZFPs of the invention
(see Ausubel, supra, Sambrook, supra). In addition, any suitable
host can be used, e.g., bacterial cells, insect cells, yeast cells,
mammalian cells, and the like.
[0122] In one embodiment, expression of the ZFP fused to a maltose
binding protein (MBP-ZFP) in bacterial strain JM109 allows for
straightforward purification through an amylose column (NEB). High
expression levels of the zinc finger chimeric protein can be
obtained by induction with IPTG since the MBP-ZFP fusion in the
pMal-c2 expression plasmid is under the control of the IPTG
inducible tac promoter (NEB). Bacteria containing the MBP-ZFP
fusion plasmids are inoculated in to 2xYT medium containing 10
.mu.M ZnCl.sub.2, 0.02% glucose, plus 50 .mu.g/ml ampicillin and
shaken at 37.degree. C. At mid-exponential growth IPTG is added to
0.3 mM and the cultures are allowed to shake. After 3 hours the
bacteria are harvested by centrifugation, disrupted by sonication,
and then insoluble material is removed by centrifugation. The
MBP-ZFP proteins are captured on an amylose-bound resin, washed
extensively with buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM
NaCl, 5 mM DTT and 50 .mu.M ZnCl.sub.2, then eluted with maltose in
essentially the same buffer (purification is based on a standard
protocol from NEB). Purified proteins are quantitated and stored
for biochemical analysis.
[0123] The biochemical properties of the purified proteins, e.g.,
K.sub.d, can be characterized by any suitable assay. In one
embodiment, K.sub.d is characterized via electrophoretic mobility
shift assays ("EMSA") (Buratowski & Chodosh, in Current
Protocols in Molecular Biology pp. 12.2.1-12.2.7 (Ausubel ed.,
1996); see also U.S. Pat. No. 5,789,538. Affinity is measured by
titrating purified protein against a low fixed amount of labeled
double-stranded oligonucleotide target. The target comprises the
natural binding site sequence (9 or 18 bp) flanked by the 3 bp
found in the natural sequence. External to the binding site plus
flanking sequence is a constant sequence. The annealed
oligonucleotide targets possess a 1 bp 5' overhang which allows for
efficient labeling of the target with T4 phage polynucleotide
kinase. For the assay the target is added at a concentration of 40
nM or lower (the actual concentration is kept at least 10-fold
lower than the lowest protein dilution) and the reaction is allowed
to equilibrate for at least 45 min. In addition the reaction
mixture also contains 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM
MgCl.sub.2, 0.1 mM ZnCl.sub.2, 5 mM DTT, 10% glycerol, 0.02% BSA
(poly (dIdC) or (dAdT) (Pharmacia) can also added at 10-100
.mu.g/.mu.l).
[0124] The equilibrated reactions are loaded onto a 10%
polyacrylamide gel, which has been pre-run for 45 min in
Tris/glycine buffer, then bound and unbound labeled target is
resolved be electrophoresis at 150V (alternatively, 10-20% gradient
Tris-HCl gels, containing a 4% polyacrylamide stacker, can be
used). The dried gels are visualized by autoradiography or
phosphoroimaging and the apparent K.sub.d is determined by
calculating the protein concentration that gives half-maximal
binding.
[0125] Similar assays can also include determining active fractions
in the protein preparations. Active fractions are determined by
stoichiometric gel shifts where proteins are titrated against a
high concentration of target DNA. Titrations are done at 100, 50,
and 25% of target (usually at micromolar levels).
[0126] In another embodiment, phage display libraries can be used
to select ZFPs with high affinity to the selected target site. This
method differs fundamentally from direct design in that it involves
the generation of diverse libraries of mutagenized ZFPs, followed
by the isolation of proteins with desired DNA-binding properties
using affinity selection methods. To use this method, the
experimenter typically proceeds as follows.
[0127] First, a gene for a ZFP is mutagenized to introduce
diversity into regions important for binding specificity and/or
affinity. In a typical application, this is accomplished via
randomization of a single finger at positions -1, +2, +3, and +6,
and perhaps accessory positions such as +1, +5, +8, or +10.
[0128] Next, the mutagenized gene is cloned into a phage or
phagemid vector as a fusion with, e.g., gene III of filamentous
phage, which encodes the coat protein pIII. The zinc finger gene is
inserted between segments of gene III encoding the membrane export
signal peptide and the remainder of pIII, so that the ZFP is
expressed as an amino-terminal fusion with pIII in the mature,
processed protein. When using phagemid vectors, the mutagenized
zinc finger gene may also be fused to a truncated version of gene
III encoding, minimally, the C-terminal region required for
assembly of pIII into the phage particle.
[0129] The resultant vector library is transformed into E. coli and
used to produce filamentous phage which express variant ZFPs on
their surface as fusions with the coat protein pIII (if a phagemid
vector is used, then the this step requires superinfection with
helper phage). The phage library is then incubated with target DNA
site, and affinity selection methods are used to isolate phage
which bind target with high affinity from bulk phage. Typically,
the DNA target is immobilized on a solid support, which is then
washed under conditions sufficient to remove all but the tightest
binding phage. After washing, any phage remaining on the support
are recovered via elution under conditions which totally disrupt
zinc finger-DNA binding.
[0130] Recovered phage are used to infect fresh E. coli, which is
then amplified and used to produce a new batch of phage particles.
The binding and recovery steps are then repeated as many times as
is necessary to sufficiently enrich the phage pool for tight
binders such that these may be identified using sequencing and/or
screening methods.
Regulatory Domains
[0131] The ZFPs of the invention can optionally be associated with
regulatory domains for modulation of gene expression. The ZFP can
be covalently or non-covalently associated with one or more
regulatory domains, alternatively two or more regulatory domains,
with the two or more domains being two copies of the same domain,
or two different domains. The regulatory domains can be covalently
linked to the ZFP, e.g., via an amino acid linker, as part of a
fusion protein. The ZFPs can also be associated with a regulatory
domain via a non-covalent dimerization domain, e.g., a leucine
zipper, a STAT protein N terminal domain, or an FK506 binding
protein (see, e.g., O'Shea, Science 254: 539 (1991), Barahmand-Pour
et al., Curr. Top. Microbiol. Immunol. 211:121-128 (1996); Klemm et
al., Annu. Rev. Immunol. 16:569-592 (1998); Klemm et al., Annu.
Rev. Immunol. 16:569-592 (1998); Ho et al., Nature 382:822-826
(1996); and Pomeranz et al., Biochem. 37:965 (1998)). The
regulatory domain can be associated with the ZFP at any suitable
position, including the C- or N-terminus of the ZFP.
[0132] Common regulatory domains for addition to the ZFP include,
e.g., effector domains from transcription factors (activators,
repressors, co-activators, co-repressors), silencers, nuclear
hormone receptors, oncogene transcription factors (e.g., myc, jun,
fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.);
DNA repair enzymes and their associated factors and modifiers; DNA
rearrangement enzymes and their associated factors and modifiers;
chromatin associated proteins and their modifiers (e.g., kinases,
acetylases and deacetylases); and DNA modifying enzymes (e.g.,
methyltransferases, topoisomerases, helicases, ligases, kinases,
phosphatases, polymerases, endonucleases) and their associated
factors and modifiers.
[0133] Transcription factor polypeptides from which one can obtain
a regulatory domain include those that are involved in regulated
and basal transcription. Such polypeptides include transcription
factors, their effector domains, coactivators, silencers, nuclear
hormone receptors (see, e.g., Goodrich et al., Cell 84:825-30
(1996) for a review of proteins and nucleic acid elements involved
in transcription; transcription factors in general are reviewed in
Barnes & Adcock, Clin. Exp. Allergy 25 Suppl. 2:46-9 (1995) and
Roeder, Methods Enzymol. 273:165-71 (1996)). Databases dedicated to
transcription factors are known (see, e.g., Science 269:630
(1995)). Nuclear hormone receptor transcription factors are
described in, for example, Rosen et al., J. Med. Chem. 38:4855-74
(1995). The C/EBP family of transcription factors are reviewed in
Wedel et al., Immunobiology 193:171-85 (1995). Coactivators and
co-repressors that mediate transcription regulation by nuclear
hormone receptors are reviewed in, for example, Meier, Eur. J.
Endocrinol. 134(2):158-9 (1996); Kaiser et al., Trends Biochem.
Sci. 21:342-5 (1996); and Utley et al., Nature 394:498-502 (1998)).
GATA transcription factors, which are involved in regulation of
hematopoiesis, are described in, for example, Simon, Nat. Genet.
11:9-11 (1995); Weiss et al., Exp. Hematol. 23:99-107. TATA box
binding protein (TBP) and its associated TAF polypeptides (which
include TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250) are
described in Goodrich & Tjian, Curr. Opin. Cell Biol. 6:403-9
(1994) and Hurley, Curr. Opin. Struct. Biol. 6:69-75 (1996). The
STAT family of transcription factors are reviewed in, for example,
Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211:121-8
(1996). Transcription factors involved in disease are reviewed in
Aso et al., J. Clin. Invest. 97:1561-9 (1996).
[0134] In one embodiment, the KRAB repression domain from the human
KOX-1 protein is used as a transcriptional repressor (Thiesen et
al., New Biologist 2:363-374 (1990); Margolin et al., PNAS
91:4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914
(1994); Witzgall et al., PNAS 91:4514-4518 (1994); see also Example
III)). In another embodiment, KAP-1, a KRAB co-repressor, is used
with KRAB (Friedman et al., Genes Dev. 10:2067-2078 (1996)).
Alternatively, KAP-1 can be used alone with a ZFP. Other preferred
transcription factors and transcription factor domains that act as
transcriptional repressors include MAD (see, e.g., Sommer et al.,
J. Biol. Chem. 273:6632-6642 (1998); Gupta et al., Oncogene
16:1149-1159 (1998); Queva et al., Oncogene 16:967-977 (1998);
Larsson et al., Oncogene 15:737-748 (1997); Laherty et al., Cell
89:349-356 (1997); and Cultraro et al., Mol Cell. Biol.
17:2353-2359 (19977)); FKHR (forkhead in rhapdosarcoma gene;
Ginsberg et al., Cancer Res. 15:3542-3546 (1998); Epstein et al.,
Mol. Cell. Biol. 18:4118-4130 (1998)); EGR-1 (early growth response
gene product-1; Yan et al., PNAS 95:8298-8303 (1998); and Liu et
al., Cancer Gene Ther. 5:3-28 (1998)); the ets2 repressor factor
repressor domain (ERD; Sgouras et al., EMBO J. 14:4781-4793
((19095)); and the MAD smSIN3 interaction domain (SID; Ayer et al.,
Mol. Cell. Biol. 16:5772-5781 (1996)).
[0135] In one embodiment, the HSV VP16 activation domain is used as
a transcriptional activator (see, e.g., Hagmann et al., J. Virol.
71:5952-5962 (1997)). Other preferred transcription factors that
could supply activation domains include the VP64 activation domain
(Seipel et al., EMBO J. 11:4961-4968 (1996)); nuclear hormone
receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol.
10:373-383 (1998)); the p65 subunit of nuclear factor kappa B
(Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle &
Hunt, Neuroreport 8:2937-2942 (1997)); and EGR-1 (early growth
response gene product-1; Yan et al., PNAS 95:8298-8303 (1998); and
Liu et al., Cancer Gene Ther. 5:3-28 (1998)).
[0136] Kinases, phosphatases, and other proteins that modify
polypeptides involved in gene regulation are also useful as
regulatory domains for ZFPs. Such modifiers are often involved in
switching on or off transcription mediated by, for example,
hormones. Kinases involved in transcription regulation are reviewed
in Davis, Mol. Reprod. Dev. 42:459-67 (1995), Jackson et al., Adv.
Second Messenger Phosphoprotein Res. 28:279-86 (1993), and
Boulikas, Crit. Rev. Eukaryot. Gene Expr. 5:1-77 (1995), while
phosphatases are reviewed in, for example, Schonthal & Semin,
Cancer Biol. 6:239-48 (1995). Nuclear tyrosine kinases are
described in Wang, Trends Biochem. Sci. 19:373-6 (1994).
[0137] As described, useful domains can also be obtained from the
gene products of oncogenes (e.g., myc, jun, fos, myb, max, mad,
rel, ets, bcl, myb, mos family members) and their associated
factors and modifiers. Oncogenes are described in, for example,
Cooper, Oncogenes, 2nd ed., The Jones and Bartlett Series in
Biology, Boston, Mass., Jones and Bartlett Publishers, 1995. The
ets transcription factors are reviewed in Waslylk et al., Eur. J.
Biochem. 211:7-18 (1993) and Crepieux et al., Crit. Rev. Oncog.
5:615-38 (1994). Myc oncogenes are reviewed in, for example, Ryan
et al., Biochem. J. 314:713-21 (1996). The jun and fos
transcription factors are described in, for example, The Fos and
Jun Families of Transcription Factors, Angel & Herrlich, eds.
(1994). The max oncogene is reviewed in Hurlin et al., Cold Spring
Harb. Symp. Quant. Biol. 59:109-16. The myb gene family is reviewed
in Kanei-Ishii et al., Curr. Top. Microbiol. Immunol. 211:89-98
(1996). The mos family is reviewed in Yew et al., Curr. Opin.
Genet. Dev. 3:19-25 (1993).
[0138] ZFPs can include regulatory domains obtained from DNA repair
enzymes and their associated factors and modifiers. DNA repair
systems are reviewed in, for example, Vos, Curr. Opin. Cell Biol.
4:385-95 (1992); Sancar, Ann. Rev. Genet. 29:69-105 (1995);
Lehmann, Genet. Eng. 17:1-19 (1995); and Wood, Ann. Rev. Biochem.
65:135-67 (1996). DNA rearrangement enzymes and their associated
factors and modifiers can also be used as regulatory domains (see,
e.g., Gangloff et al., Experientia 50:261-9 (1994); Sadowski, FASEB
J. 7:760-7 (1993)).
[0139] Similarly, regulatory domains can be derived from DNA
modifying enzymes (e.g., DNA methyltransferases, topoisomerases,
helicases, ligases, kinases, phosphatases, polymerases) and their
associated factors and modifiers. Helicases are reviewed in Matson
et al., Bioessays, 16:13-22 (1994), and methyltransferases are
described in Cheng, Curr. Opin. Struct. Biol. 5:4-10 (1995).
Chromatin associated proteins and their modifiers (e.g., kinases,
acetylases and deacetylases), such as histone deacetylase (Wolffe,
Science 272:371-2 (1996)) are also useful as domains for addition
to the ZFP of choice. In one preferred embodiment, the regulatory
domain is a DNA methyl transferase that acts as a transcriptional
repressor (see, e.g., Van den Wyngaert et al., FEBS Lett.
426:283-289 (1998); Flynn et al., J. Mol. Biol. 279:101-116 (1998);
Okano et al., Nucleic Acids Res. 26:2536-2540 (1998); and Zardo
& Caiafa, J. Biol. Chem. 273:16517-16520 (1998)). In another
preferred embodiment, endonucleases such as Fok1 are used as
transcriptional repressors, which act via gene cleavage (see, e.g.,
WO95/09233; and PCT/US94/01201).
[0140] Factors that control chromatin and DNA structure, movement
and localization and their associated factors and modifiers;
factors derived from microbes (e.g., prokaryotes, eukaryotes and
virus) and factors that associate with or modify them can also be
used to obtain chimeric proteins. In one embodiment, recombinases
and integrases are used as regulatory domains. In one embodiment,
histone acetyltransferase is used as a transcriptional activator
(see, e.g., Jin & Scotto, Mol. Cell. Biol. 18:4377-4384 (1998);
Wolffe, Science 272:371-372 (1996); Taunton et al., Science
272:408-411 (1996); and Hassig et al., PNAS 95:3519-3524 (1998)).
In another embodiment, histone deacetylase is used as a
transcriptional repressor (see, e.g., Jin & Scotto, Mol. Cell.
Biol. 18:4377-4384 (1998); Syntichaki & Thireos, J. Biol. Chem.
273:24414-24419 (1998); Sakaguchi et al., Genes Dev. 12:2831-2841
(1998); and Martinez et al., J. Biol. Chem. 273:23781-23785
(1998)).
[0141] Linker domains between polypeptide domains, e.g., between
two ZFPs or between a ZFP and a regulatory domain, can be included.
Such linkers are typically polypeptide sequences, such as poly gly
sequences of between about 5 and 200 amino acids. Preferred linkers
are typically flexible amino acid subsequences which are
synthesized as part of a recombinant fusion protein. For example,
in one embodiment, the linker DGGGS is used to link two ZFPs. In
another embodiment, the flexible linker linking two ZFPs is an
amino acid subsequence comprising the sequence TGEKP (see, e.g.,
Liu et al., PNAS 5525-5530 (1997)). In another embodiment, the
linker LRQKDGERP is used to link two ZFPs. In another embodiment,
the following linkers are used to link two ZFPs: GGRR (Pomerantz et
al. 1995, supra), (G4S).sub.n (Kim et al., PNAS 93, 1156-1160
(1996.); and GGRRGGGS; LRQRDGERP; LRQKDGGGSERP; LRQKd(G3S).sub.2
ERP. Alternatively, flexible linkers can be rationally designed
using computer program capable of modeling both DNA-binding sites
and the peptides themselves (Desjarlais & Berg, PNAS
90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or by phage display
methods.
[0142] In other embodiments, a chemical linker is used to connect
synthetically or recombinantly produced domain sequences. Such
flexible linkers are known to persons of skill in the art. For
example, poly(ethylene glycol) linkers are available from
Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally
have amide linkages, sulfhydryl linkages, or heterofunctional
linkages. In addition to covalent linkage of ZFPs to regulatory
domains, non-covalent methods can be used to produce molecules with
ZFPs associated with regulatory domains.
[0143] In addition to regulatory domains, often the ZFP is
expressed as a fusion protein such as maltose binding protein
("MBP"), glutathione S transferase (GST), hexahistidine, c-myc, and
the FLAG epitope, for ease of purification, monitoring expression,
or monitoring cellular and subcellular localization.
Expression Vectors for Nucleic Acids Encoding ZFP
[0144] The nucleic acid encoding the ZFP of choice is typically
cloned into intermediate vectors for transformation into
prokaryotic or eukaryotic cells for replication and/or expression,
e.g., for determination of K.sub.d. Intermediate vectors are
typically prokaryote vectors, e.g., plasmids, or shuttle vectors,
or insect vectors, for storage or manipulation of the nucleic acid
encoding ZFP or production of protein. The nucleic acid encoding a
ZFP is also typically cloned into an expression vector, for
administration to a plant cell, animal cell, preferably a mammalian
cell or a human cell, fungal cell, bacterial cell, or protozoal
cell.
[0145] To obtain expression of a cloned gene or nucleic acid, a ZFP
is typically subcloned into an expression vector that contains a
promoter to direct transcription. Suitable bacterial and eukaryotic
promoters are well known in the art and described, e.g., in
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.
1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual
(1990); and Current Protocols in Molecular Biology (Ausubel et al.,
eds., 1994). Bacterial expression systems for expressing the ZFP
are available in, e.g., E. coli, Bacillus sp., and Salmonella
(Palva et al., Gene 22:229-235 (1983)). Kits for such expression
systems are commercially available. Eukaryotic expression systems
for mammalian cells, yeast, and insect cells are well known in the
art and are also commercially available.
[0146] The promoter used to direct expression of a ZFP nucleic acid
depends on the particular application. For example, a strong
constitutive promoter is typically used for expression and
purification of ZFP. In contrast, when a ZFP is administered in
vivo for gene regulation, either a constitutive or an inducible
promoter is used, depending on the particular use of the ZFP. In
addition, a preferred promoter for administration of a ZFP can be a
weak promoter, such as HSV TK or a promoter having similar
activity. The promoter typically can also include elements that are
responsive to transactivation, e.g., hypoxia response elements,
Gal4 response elements, lac repressor response element, and small
molecule control systems such as tet-regulated systems and the
RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992);
Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene
Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996);
and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)).
[0147] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of the
nucleic acid in host cells, either prokaryotic or eukaryotic. A
typical expression cassette thus contains a promoter operably
linked, e.g., to the nucleic acid sequence encoding the ZFP, and
signals required, e.g., for efficient polyadenylation of the
transcript, transcriptional termination, ribosome binding sites, or
translation termination. Additional elements of the cassette may
include, e.g., enhancers, and heterologous spliced intronic
signals.
[0148] The particular expression vector used to transport the
genetic information into the cell is selected with regard to the
intended use of the ZFP, e.g., expression in plants, animals,
bacteria, fungus, protozoa etc. (see expression vectors described
below and in the Example section). Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and commercially available fusion expression systems such
as GST and LacZ. A preferred fusion protein is the maltose binding
protein, "MBP." Such fusion proteins are used for purification of
the ZFP. Epitope tags can also be added to recombinant proteins to
provide convenient methods of isolation, for monitoring expression,
and for monitoring cellular and subcellular localization, e.g.,
c-myc or FLAG.
[0149] Expression vectors containing regulatory elements from
eukaryotic viruses are often used in eukaryotic expression vectors,
e.g., SV40 vectors, papilloma virus vectors, and vectors derived
from Epstein-Barr virus. Other exemplary eukaryotic vectors include
pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any
other vector allowing expression of proteins under the direction of
the SV40 early promoter, SV40 late promoter, metallothionein
promoter, murine mammary tumor virus promoter, Rous sarcoma virus
promoter, polyhedrin promoter, or other promoters shown effective
for expression in eukaryotic cells.
[0150] Some expression systems have markers for selection of stably
transfected cell lines such as thymidine kinase, hygromycin B
phosphotransferase, and dihydrofolate reductase. High yield
expression systems are also suitable, such as using a baculovirus
vector in insect cells, with a ZFP encoding sequence under the
direction of the polyhedrin promoter or other strong baculovirus
promoters.
[0151] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
recombinant sequences.
[0152] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express large quantities
of protein, which are then purified using standard techniques (see,
e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide
to Protein Purification, in Methods in Enzymology, vol. 182
(Deutscher, ed., 1990)). Transformation of eukaryotic and
prokaryotic cells are performed according to standard techniques
(see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss
& Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds,
1983).
[0153] Any of the well known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, liposomes, microinjection, naked DNA,
plasmid vectors, viral vectors, both episomal and integrative, and
any of the other well known methods for introducing cloned genomic
DNA, cDNA, synthetic DNA or other foreign genetic material into a
host cell (see, e.g., Sambrook et al., supra). It is only necessary
that the particular genetic engineering procedure used be capable
of successfully introducing at least one gene into the host cell
capable of expressing the protein of choice.
Assays for Determining Regulation of Gene Expression by ZFPs
[0154] A variety of assays can be used to determine the level of
gene expression regulation by ZFPs. The activity of a particular
ZFP can be assessed using a variety of in vitro and in vivo assays,
by measuring, e.g., protein or mRNA levels, product levels, enzyme
activity, tumor growth; transcriptional activation or repression of
a reporter gene; second messenger levels (e.g., cGMP, cAMP, IP3,
DAG, Ca.sup.2+); cytokine and hormone production levels; and
neovascularization, using, e.g., immunoassays (e.g., ELISA and
immunohistochemical assays with antibodies), hybridization assays
(e.g., RNase protection, northerns, in situ hybridization,
oligonucleotide array studies), colorimetric assays, amplification
assays, enzyme activity assays, tumor growth assays, phenotypic
assays, and the like.
[0155] ZFPs are typically first tested for activity in vitro using
cultured cells, e.g., 293 cells, CHO cells, VERO cells, BHK cells,
HeLa cells, COS cells, and the like. Preferably, human cells are
used. The ZFP is often first tested using a transient expression
system with a reporter gene, and then regulation of the target
endogenous gene is tested in cells and in animals, both in vivo and
ex vivo. The ZFP can be recombinantly expressed in a cell,
recombinantly expressed in cells transplanted into an animal, or
recombinantly expressed in a transgenic animal, as well as
administered as a protein to an animal or cell using delivery
vehicles described below. The cells can be immobilized, be in
solution, be injected into an animal, or be naturally occurring in
a transgenic or non-transgenic animal.
[0156] Modulation of gene expression is tested using one of the in
vitro or in vivo assays described herein. Samples or assays are
treated with a ZFP and compared to control samples without the test
compound, to examine the extent of modulation. As described above,
for regulation of endogenous gene expression, the ZFP typically has
a K.sub.d of 200 nM or less, more preferably 100 nM or less, more
preferably 50 nM, most preferably 25 nM or less.
[0157] The effects of the ZFPs can be measured by examining any of
the parameters described above. Any suitable gene expression,
phenotypic, or physiological change can be used to assess the
influence of a ZFP. When the functional consequences are determined
using intact cells or animals, one can also measure a variety of
effects such as tumor growth, neovascularization, hormone release,
transcriptional changes to both known and uncharacterized genetic
markers (e.g., northern blots or oligonucleotide array studies),
changes in cell metabolism such as cell growth or pH changes, and
changes in intracellular second messengers such as cGMP.
[0158] Preferred assays for ZFP regulation of endogenous gene
expression can be performed in vitro. In one preferred in vitro
assay format, ZFP regulation of endogenous gene expression in
cultured cells is measured by examining protein production using an
ELISA assay (see Examples VI and VII). The test sample is compared
to control cells treated with an empty vector or an unrelated ZFP
that is targeted to another gene.
[0159] In another embodiment, ZFP regulation of endogenous gene
expression is determined in vitro by measuring the level of target
gene mRNA expression. The level of gene expression is measured
using amplification, e.g., using PCR, LCR, or hybridization assays,
e.g., northern hybridization, RNase protection, dot blotting. RNase
protection is used in one embodiment (see Example VIII and FIG.
10). The level of protein or mRNA is detected using directly or
indirectly labeled detection agents, e.g., fluorescently or
radioactively labeled nucleic acids, radioactively or enzymatically
labeled antibodies, and the like, as described herein.
[0160] Alternatively, a reporter gene system can be devised using
the target gene promoter operably linked to a reporter gene such as
luciferase, green fluorescent protein, CAT, or .beta.-gal. The
reporter construct is typically co-transfected into a cultured
cell. After treatment with the ZFP of choice, the amount of
reporter gene transcription, translation, or activity is measured
according to standard techniques known to those of skill in the
art.
[0161] Another example of a preferred assay format useful for
monitoring ZFP regulation of endogenous gene expression is
performed in vivo. This assay is particularly useful for examining
ZFPs that inhibit expression of tumor promoting genes, genes
involved in tumor support, such as neovascularization (e.g., VEGF),
or that activate tumor suppressor genes such as p53. In this assay,
cultured tumor cells expressing the ZFP of choice are injected
subcutaneously into an immune compromised mouse such as an athymic
mouse, an irradiated mouse, or a SCID mouse. After a suitable
length of time, preferably 4-8 weeks, tumor growth is measured,
e.g., by volume or by its two largest dimensions, and compared to
the control. Tumors that have statistically significant reduction
(using, e.g., Student's T test) are said to have inhibited growth.
Alternatively, the extent of tumor neovascularization can also be
measured. Immunoassays using endothelial cell specific antibodies
are used to stain for vascularization of the tumor and the number
of vessels in the tumor. Tumors that have a statistically
significant reduction in the number of vessels (using, e.g.,
Student's T test) are said to have inhibited
neovascularization.
[0162] Transgenic and non-transgenic animals are also used as a
preferred embodiment for examining regulation of endogenous gene
expression in vivo. Transgenic animals typically express the ZFP of
choice. Alternatively, animals that transiently express the ZFP of
choice, or to which the ZFP has been administered in a delivery
vehicle, can be used. Regulation of endogenous gene expression is
tested using any one of the assays described herein.
Nucleic Acids Encoding ZFPs and Gene Therapy
[0163] Conventional viral and non-viral based gene transfer methods
can be used to introduce nucleic acids encoding engineered ZFP in
mammalian cells or target tissues. Such methods can be used to
administer nucleic acids encoding ZFPs to cells in vitro.
Preferably, the nucleic acids encoding ZFPs are administered for in
vivo or ex vivo gene therapy uses. Non-viral vector delivery
systems include DNA plasmids, naked nucleic acid, and nucleic acid
complexed with a delivery vehicle such as a liposome. Viral vector
delivery systems include DNA and RNA viruses, which have either
episomal or integrated genomes after delivery to the cell. For a
review of gene therapy procedures, see Anderson, Science
256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993);
Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH
11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,
Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology
and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British
Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current
Topics in Microbiology and Immunology Doerfler and Bohm (eds)
(1995); and Yu et al., Gene Therapy 1:13-26 (1994).
[0164] Methods of non-viral delivery of nucleic acids encoding
engineered ZFPs include lipofection, microinjection, biolistics,
virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic
acid conjugates, naked DNA, artificial virions, and agent-enhanced
uptake of DNA. Lipofection is described in e.g., U.S. Pat. No.
5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355)
and lipofection reagents are sold commercially (e.g.,
Transfectam.TM. and Lipofectin.TM.). Cationic and neutral lipids
that are suitable for efficient receptor-recognition lipofection of
polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.
Delivery can be to cells (ex vivo administration) or target tissues
(in vivo administration).
[0165] The preparation of lipid:nucleic acid complexes, including
targeted liposomes such as immunolipid complexes, is well known to
one of skill in the art (see, e.g., Crystal, Science 270:404-410
(1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et
al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate
Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995);
Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.
4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728,
4,774,085, 4,837,028, and 4,946,787).
[0166] The use of RNA or DNA viral based systems for the delivery
of nucleic acids encoding engineered ZFP take advantage of highly
evolved processes for targeting a virus to specific cells in the
body and trafficking the viral payload to the nucleus. Viral
vectors can be administered directly to patients (in vivo) or they
can be used to treat cells in vitro and the modified cells are
administered to patients (ex vivo). Conventional viral based
systems for the delivery of ZFPs could include retroviral,
lentivirus, adenoviral, adeno-associated and herpes simplex virus
vectors for gene transfer. Viral vectors are currently the most
efficient and versatile method of gene transfer in target cells and
tissues. Integration in the host genome is possible with the
retrovirus, lentivirus, and adeno-associated virus gene transfer
methods, often resulting in long term expression of the inserted
transgene. Additionally, high transduction efficiencies have been
observed in many different cell types and target tissues.
[0167] The tropism of a retrovirus can be altered by incorporating
foreign envelope proteins, expanding the potential target
population of target cells. Lentiviral vectors are retroviral
vector that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene
transfer system would therefore depend on the target tissue.
Retroviral vectors are comprised of cis-acting long terminal
repeats with packaging capacity for up to 6-10 kb of foreign
sequence. The minimum cis-acting LTRs are sufficient for
replication and packaging of the vectors, which are then used to
integrate the therapeutic gene into the target cell to provide
permanent transgene expression. Widely used retroviral vectors
include those based upon murine leukemia virus (MuLV), gibbon ape
leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human
immuno deficiency virus (HIV), and combinations thereof (see, e.g.,
Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J.
Virol. 66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59
(1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et
al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
[0168] In applications where transient expression of the ZFP is
preferred, adenoviral based systems are typically used. Adenoviral
based vectors are capable of very high transduction efficiency in
many cell types and do not require cell division. With such
vectors, high titer and levels of expression have been obtained.
This vector can be produced in large quantities in a relatively
simple system. Adeno-associated virus ("AAV") vectors are also used
to transduce cells with target nucleic acids, e.g., in the in vitro
production of nucleic acids and peptides, and for in vivo and ex
vivo gene therapy procedures (see, e.g., West et al., Virology
160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin,
Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest.
94:1351 (1994). Construction of recombinant AAV vectors are
described in a number of publications, including U.S. Pat. No.
5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);
Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat
& Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J.
Virol. 63:03822-3828 (1989).
[0169] In particular, at least six viral vector approaches are
currently available for gene transfer in clinical trials, with
retroviral vectors by far the most frequently used system. All of
these viral vectors utilize approaches that involve complementation
of defective vectors by genes inserted into helper cell lines to
generate the transducing agent.
[0170] pLASN and MFG-S are examples are retroviral vectors that
have been used in clinical trials (Dunbar et al., Blood 85:3048-305
(1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al.,
PNAS 94:22 12133-12138 (1997)). PA317/pLASN was the first
therapeutic vector used in a gene therapy trial. (Blaese et al.,
Science 270:475-480 (1995)). Transduction efficiencies of 50% or
greater have been observed for MFG-S packaged vectors. (Ellem et
al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum.
Gene Ther. 1:111-2 (1997).
[0171] Recombinant adeno-associated virus vectors (rAAV) are a
promising alternative gene delivery systems based on the defective
and nonpathogenic parvovirus adeno-associated type 2 virus. All
vectors are derived from a plasmid that retains only the AAV 145 bp
inverted terminal repeats flanking the transgene expression
cassette. Efficient gene transfer and stable transgene delivery due
to integration into the genomes of the transduced cell are key
features for this vector system. (Wagner et al., Lancet 351:9117
1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)).
[0172] Replication-deficient recombinant adenoviral vectors (Ad)
are predominantly used for colon cancer gene therapy, because they
can be produced at high titer and they readily infect a number of
different cell types. Most adenovirus vectors are engineered such
that a transgene replaces the Ad E1a, E1b, and E3 genes;
subsequently the replication defector vector is propagated in human
293 cells that supply deleted gene function in trans. Ad vectors
can transduce multiply types of tissues in vivo, including
nondividing, differentiated cells such as those found in the liver,
kidney and muscle system tissues. Conventional Ad vectors have a
large carrying capacity. An example of the use of an Ad vector in a
clinical trial involved polynucleotide therapy for antitumor
immunization with intramuscular injection (Sterman et al., Hum.
Gene Ther. 7:1083-9 (1998)). Additional examples of the use of
adenovirus vectors for gene transfer in clinical trials include
Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum.
Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther.
2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997);
Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene
Ther. 7:1083-1089 (1998).
[0173] Packaging cells are used to form virus particles that are
capable of infecting a host cell. Such cells include 293 cells,
which package adenovirus, and .psi.2 cells or PA317 cells, which
package retrovirus. Viral vectors used in gene therapy are usually
generated by producer cell line that packages a nucleic acid vector
into a viral particle. The vectors typically contain the minimal
viral sequences required for packaging and subsequent integration
into a host, other viral sequences being replaced by an expression
cassette for the protein to be expressed. The missing viral
functions are supplied in trans by the packaging cell line. For
example, AAV vectors used in gene therapy typically only possess
ITR sequences from the AAV genome which are required for packaging
and integration into the host genome. Viral DNA is packaged in a
cell line, which contains a helper plasmid encoding the other AAV
genes, namely rep and cap, but lacking ITR sequences. The cell line
is also infected with adenovirus as a helper. The helper virus
promotes replication of the AAV vector and expression of AAV genes
from the helper plasmid. The helper plasmid is not packaged in
significant amounts due to a lack of ITR sequences. Contamination
with adenovirus can be reduced by, e.g., heat treatment to which
adenovirus is more sensitive than AAV.
[0174] In many gene therapy applications, it is desirable that the
gene therapy vector be delivered with a high degree of specificity
to a particular tissue type. A viral vector is typically modified
to have specificity for a given cell type by expressing a ligand as
a fusion protein with a viral coat protein on the viruses outer
surface. The ligand is chosen to have affinity for a receptor known
to be present on the cell type of interest. For example, Han et
al., PNAS 92:9747-9751 (1995), reported that Moloney murine
leukemia virus can be modified to express human heregulin fused to
gp70, and the recombinant virus infects certain human breast cancer
cells expressing human epidermal growth factor receptor. This
principle can be extended to other pairs of virus expressing a
ligand fusion protein and target cell expressing a receptor. For
example, filamentous phage can be engineered to display antibody
fragments (e.g., FAB or Fv) having specific binding affinity for
virtually any chosen cellular receptor. Although the above
description applies primarily to viral vectors, the same principles
can be applied to nonviral vectors. Such vectors can be engineered
to contain specific uptake sequences thought to favor uptake by
specific target cells.
[0175] Gene therapy vectors can be delivered in vivo by
administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular,
subdermal, or intracranial infusion) or topical application, as
described below. Alternatively, vectors can be delivered to cells
ex vivo, such as cells explanted from an individual patient (e.g.,
lymphocytes, bone marrow aspirates, tissue biopsy) or universal
donor hematopoietic stem cells, followed by reimplantation of the
cells into a patient, usually after selection for cells which have
incorporated the vector.
[0176] Ex vivo cell transfection for diagnostics, research, or for
gene therapy (e.g., via re-infusion of the transfected cells into
the host organism) is well known to those of skill in the art. In a
preferred embodiment, cells are isolated from the subject organism,
transfected with a ZFP nucleic acid (gene or cDNA), and re-infused
back into the subject organism (e.g., patient). Various cell types
suitable for ex vivo transfection are well known to those of skill
in the art (see, e.g., Freshney et al., Culture of Animal Cells, A
Manual of Basic Technique (3rd ed. 1994)) and the references cited
therein for a discussion of how to isolate and culture cells from
patients).
[0177] In one embodiment, stem cells are used in ex vivo procedures
for cell transfection and gene therapy. The advantage to using stem
cells is that they can be differentiated into other cell types in
vitro, or can be introduced into a mammal (such as the donor of the
cells) where they will engraft in the bone marrow. Methods for
differentiating CD34+ cells in vitro into clinically important
immune cell types using cytokines such a GM-CSF, IFN-.gamma. and
TNF-.alpha. are known (see Inaba et al., J. Exp. Med. 176:1693-1702
(1992)).
[0178] Stem cells are isolated for transduction and differentiation
using known methods. For example, stem cells are isolated from bone
marrow cells by panning the bone marrow cells with antibodies which
bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB
cells), GR-1 (granulocytes), and Tad (differentiated antigen
presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702
(1992)).
[0179] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing therapeutic ZFP nucleic acids can be also administered
directly to the organism for transduction of cells in vivo.
Alternatively, naked DNA can be administered. Administration is by
any of the routes normally used for introducing a molecule into
ultimate contact with blood or tissue cells. Suitable methods of
administering such nucleic acids are available and well known to
those of skill in the art, and, although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0180] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention, as described below (see,
e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
Delivery Vehicles for ZFPs
[0181] An important factor in the administration of polypeptide
compounds, such as the ZFPs, is ensuring that the polypeptide has
the ability to traverse the plasma membrane of a cell, or the
membrane of an intra-cellular compartment such as the nucleus.
Cellular membranes are composed of lipid-protein bilayers that are
freely permeable to small, nonionic lipophilic compounds and are
inherently impermeable to polar compounds, macromolecules, and
therapeutic or diagnostic agents. However, proteins and other
compounds such as liposomes have been described, which have the
ability to translocate polypeptides such as ZFPs across a cell
membrane.
[0182] For example, "membrane translocation polypeptides" have
amphiphilic or hydrophobic amino acid subsequences that have the
ability to act as membrane-translocating carriers. In one
embodiment, homeodomain proteins have the ability to translocate
across cell membranes. The shortest internalizable peptide of a
homeodomain protein, Antennapedia, was found to be the third helix
of the protein, from amino acid position 43 to 58 (see, e.g.,
Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)).
Another subsequence, the h (hydrophobic) domain of signal peptides,
was found to have similar cell membrane translocation
characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1
4255-14258 (1995)).
[0183] Examples of peptide sequences which can be linked to a ZFP
of the invention, for facilitating uptake of ZFP into cells,
include, but are not limited to: an 11 amino acid peptide of the
tat protein of HIV; a 20 residue peptide sequence which corresponds
to amino acids 84-103 of the p16 protein (see Fahraeus et al.,
Current Biology 6:84 (1996)); the third helix of the 60-amino acid
long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem.
269:10444 (1994)); the h region of a signal peptide such as the
Kaposi fibroblast growth factor (K-FGF) h region (Lin et al.,
supra); or the VP22 translocation domain from HSV (Elliot &
O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties
that provide enhanced cellular uptake may also be chemically linked
to ZFPs.
[0184] Toxin molecules also have the ability to transport
polypeptides across cell membranes. Often, such molecules are
composed of at least two parts (called "binary toxins"): a
translocation or binding domain or polypeptide and a separate toxin
domain or polypeptide. Typically, the translocation domain or
polypeptide binds to a cellular receptor, and then the toxin is
transported into the cell. Several bacterial toxins, including
Clostridium perfringens iota toxin, diphtheria toxin (DT),
Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus
anthracis toxin, and pertussis adenylate cyclase (CYA), have been
used in attempts to deliver peptides to the cell cytosol as
internal or amino-terminal fusions (Arora et al., J. Biol. Chem.,
268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156
(1993); Stenmark et al., J. Cell Biol. 113:1025-1032 (1991);
Donnelly et al., PNAS 90:3530-3534 (1993); Carbonetti et al.,
Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al.,
Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNAS U.S.A.
89:10277-10281 (1992); and Novak et al., J. Biol. Chem.
267:17186-17193 1992)).
[0185] Such subsequences can be used to translocate ZFPs across a
cell membrane. ZFPs can be conveniently fused to or derivatized
with such sequences. Typically, the translocation sequence is
provided as part of a fusion protein. Optionally, a linker can be
used to link the ZFP and the translocation sequence. Any suitable
linker can be used, e.g., a peptide linker.
[0186] The ZFP can also be introduced into an animal cell,
preferably a mammalian cell, via a liposomes and liposome
derivatives such as immunoliposomes. The term "liposome" refers to
vesicles comprised of one or more concentrically ordered lipid
bilayers, which encapsulate an aqueous phase. The aqueous phase
typically contains the compound to be delivered to the cell, i.e.,
a ZFP.
[0187] The liposome fuses with the plasma membrane, thereby
releasing the drug into the cytosol. Alternatively, the liposome is
phagocytosed or taken up by the cell in a transport vesicle. Once
in the endosome or phagosome, the liposome either degrades or fuses
with the membrane of the transport vesicle and releases its
contents.
[0188] In current methods of drug delivery via liposomes, the
liposome ultimately becomes permeable and releases the encapsulated
compound (in this case, a ZFP) at the target tissue or cell. For
systemic or tissue specific delivery, this can be accomplished, for
example, in a passive manner wherein the liposome bilayer degrades
over time through the action of various agents in the body.
Alternatively, active drug release involves using an agent to
induce a permeability change in the liposome vesicle. Liposome
membranes can be constructed so that they become destabilized when
the environment becomes acidic near the liposome membrane (see,
e.g., PNAS 84:7851 (1987); Biochemistry 28:908 (1989)). When
liposomes are endocytosed by a target cell, for example, they
become destabilized and release their contents. This
destabilization is termed fusogenesis.
Dioleoylphosphatidylethanolamine (DOPE) is the basis of many
"fusogenic" systems.
[0189] Such liposomes typically comprise a ZFP and a lipid
component, e.g., a neutral and/or cationic lipid, optionally
including a receptor-recognition molecule such as an antibody that
binds to a predetermined cell surface receptor or ligand (e.g., an
antigen). A variety of methods are available for preparing
liposomes as described in, e.g., Szoka et al., Ann. Rev. Biophys.
Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344,
4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,
4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,
4,946,787, PCT Publication No. WO 91\17424, Deamer & Bangham,
Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., PNAS
76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65
(1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986);
Williams et al., PNAS 85:242-246 (1988); Liposomes (Ostro (ed.),
1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986);
Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from
Physics to Applications (1993)). Suitable methods include, for
example, sonication, extrusion, high pressure/homogenization,
microfluidization, detergent dialysis, calcium-induced fusion of
small liposome vesicles and ether-fusion methods, all of which are
well known in the art.
[0190] In certain embodiments of the present invention, it is
desirable to target the liposomes of the invention using targeting
moieties that are specific to a particular cell type, tissue, and
the like. Targeting of liposomes using a variety of targeting
moieties (e.g., ligands, receptors, and monoclonal antibodies) has
been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and
4,603,044).
[0191] Examples of targeting moieties include monoclonal antibodies
specific to antigens associated with neoplasms, such as prostate
cancer specific antigen and MAGE. Tumors can also be diagnosed by
detecting gene products resulting from the activation or
over-expression of oncogenes, such as ras or c-erbB2. In addition,
many tumors express antigens normally expressed by fetal tissue,
such as the alphafetoprotein (AFP) and carcinoembryonic antigen
(CEA). Sites of viral infection can be diagnosed using various
viral antigens such as hepatitis B core and surface antigens (HBVc,
HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human
immunodeficiency type-1 virus (HIV1) and papilloma virus antigens.
Inflammation can be detected using molecules specifically
recognized by surface molecules which are expressed at sites of
inflammation such as integrins (e.g., VCAM-1), selectin receptors
(e.g., ELAM-1) and the like.
[0192] Standard methods for coupling targeting agents to liposomes
can be used. These methods generally involve incorporation into
liposomes lipid components, e.g., phosphatidylethanolamine, which
can be activated for attachment of targeting agents, or derivatized
lipophilic compounds, such as lipid derivatized bleomycin. Antibody
targeted liposomes can be constructed using, for instance,
liposomes which incorporate protein A (see Renneisen et al., J.
Biol. Chem., 265:16337-16342 (1990) and Leonetti et al., PNAS
87:2448-2451 (1990).
Doses of ZFPs
[0193] For therapeutic applications of ZFPs, the dose administered
to a patient, in the context of the present invention should be
sufficient to effect a beneficial therapeutic response in the
patient over time. In addition, particular dosage regimens can be
useful for determining phenotypic changes in an experimental
setting, e.g., in functional genomics studies, and in cell or
animal models. The dose will be determined by the efficacy and
K.sub.d of the particular ZFP employed, the nuclear volume of the
target cell, and the condition of the patient, as well as the body
weight or surface area of the patient to be treated. The size of
the dose also will be determined by the existence, nature, and
extent of any adverse side-effects that accompany the
administration of a particular compound or vector in a particular
patient.
[0194] The maximum therapeutically effective dosage of ZFP for
approximately 99% binding to target sites is calculated to be in
the range of less than about 1.5.times.10.sup.5 to
1.5.times.10.sup.6 copies of the specific ZFP molecule per cell.
The number of ZFPs per cell for this level of binding is calculated
as follows, using the volume of a HeLa cell nucleus (approximately
1000 .mu.m.sup.3 or 10.sup.-12 L; Cell Biology, (Altman & Katz,
eds. (1976)). As the HeLa nucleus is relatively large, this dosage
number is recalculated as needed using the volume of the target
cell nucleus. This calculation also does not take into account
competition for ZFP binding by other sites. This calculation also
assumes that essentially all of the ZFP is localized to the
nucleus. A value of 100.times.K.sub.d is used to calculate
approximately 99% binding of to the target site, and a value of
10.times.K.sub.d is used to calculate approximately 90% binding of
to the target site. For this example, K.sub.d=25 nM
ZFP + target site complex ##EQU00001## i . e . , DNA + protein DNA
: protein complex ##EQU00001.2## K d = [ DNA ] [ protein ] [ DNA :
protein complex ] ##EQU00001.3## When 50 % of ZFP is bound , K d =
[ protein ] ##EQU00001.4## So when [ protein ] = 25 nM and the
nucleus volume is 10 - 12 L ##EQU00001.5## [ protein ] = ( 25
.times. 10 - 9 moles / L ) ( 10 - 12 L / nucleus ) ( 6 .times. 10
23 molecules / mole ) = 15 , 000 molecules / nucleus for 50 %
binding ##EQU00001.6## When 99 % target is bound ; 100 .times. K d
= [ protein ] ##EQU00001.7## 100 .times. K d = [ protein ] = 2.5 M
( 2.5 .times. 10 - 6 moles / L ) ( 10 - 12 L / nucleus ) ( 6
.times. 10 23 molecules / mole ) = about 1 , 500 , 000 molecules
per nucleus for 99 % binding of target site . ##EQU00001.8##
[0195] The appropriate dose of an expression vector encoding a ZFP
can also be calculated by taking into account the average rate of
ZFP expression from the promoter and the average rate of ZFP
degradation in the cell. Preferably, a weak promoter such as a
wild-type or mutant HSV TK is used, as described above. The dose of
ZFP in micrograms is calculated by taking into account the
molecular weight of the particular ZFP being employed.
[0196] In determining the effective amount of the ZFP to be
administered in the treatment or prophylaxis of disease, the
physician evaluates circulating plasma levels of the ZFP or nucleic
acid encoding the ZFP, potential ZFP toxicities, progression of the
disease, and the production of anti-ZFP antibodies. Administration
can be accomplished via single or divided doses.
Pharmaceutical Compositions and Administration
[0197] ZFPs and expression vectors encoding ZFPs can be
administered directly to the patient for modulation of gene
expression and for therapeutic or prophylactic applications, for
example, cancer, ischemia, diabetic retinopathy, macular
degeneration, rheumatoid arthritis, psoriasis, HIV infection,
sickle cell anemia, Alzheimer's disease, muscular dystrophy,
neurodegenerative diseases, vascular disease, cystic fibrosis,
stroke, and the like. Examples of microorganisms that can be
inhibited by ZFP gene therapy include pathogenic bacteria, e.g.,
chlamydia, rickettsial bacteria, mycobacteria, staphylococci,
streptococci, pneumococci, meningococci and conococci, klebsiella,
proteus, serratia, pseudomonas, legionella, diphtheria, salmonella,
bacilli, cholera, tetanus, botulism, anthrax, plague,
leptospirosis, and Lyme disease bacteria; infectious fungus, e.g.,
Aspergillus, Candida species; protozoa such as sporozoa (e.g.,
Plasmodia), rhizopods (e.g., Entamoeba) and flagellates
(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viral
diseases, e.g., hepatitis (A, B, or C), herpes virus (e.g., VZV,
HSV-1, HSV-6, HSV-II, CMV, and EBV), HIV, Ebola, adenovirus,
influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie
virus, cornovirus, respiratory syncytial virus, mumps virus,
rotavirus, measles virus, rubella virus, parvovirus, vaccinia
virus, HTLV virus, dengue virus, papillomavirus, poliovirus, rabies
virus, and arboviral encephalitis virus, etc.
[0198] Administration of therapeutically effective amounts is by
any of the routes normally used for introducing ZFP into ultimate
contact with the tissue to be treated. The ZFPs are administered in
any suitable manner, preferably with pharmaceutically acceptable
carriers. Suitable methods of administering such modulators are
available and well known to those of skill in the art, and,
although more than one route can be used to administer a particular
composition, a particular route can often provide a more immediate
and more effective reaction than another route.
[0199] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention (see, e.g., Remington's
Pharmaceutical Sciences, 17.sup.th ed. 1985)).
[0200] The ZFPs, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can
be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0201] Formulations suitable for parenteral administration, such
as, for example, by intravenous, intramuscular, intradermal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically or intrathecally. The
formulations of compounds can be presented in unit-dose or
multi-dose sealed containers, such as ampules and vials. Injection
solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described.
Regulation of Gene Expression in Plants
[0202] ZFPs can be used to engineer plants for traits such as
increased disease resistance, modification of structural and
storage polysaccharides, flavors, proteins, and fatty acids, fruit
ripening, yield, color, nutritional characteristics, improved
storage capability, and the like. In particular, the engineering of
crop species for enhanced oil production, e.g., the modification of
the fatty acids produced in oilseeds, is of interest.
[0203] Seed oils are composed primarily of triacylglycerols (TAGs),
which are glycerol esters of fatty acids. Commercial production of
these vegetable oils is accounted for primarily by six major oil
crops (soybean, oil palm, rapeseed, sunflower, cotton seed, and
peanut.) Vegetable oils are used predominantly (90%) for human
consumption as margarine, shortening, salad oils, and frying oil.
The remaining 10% is used for non-food applications such as
lubricants, oleochemicals, biofuels, detergents, and other
industrial applications.
[0204] The desired characteristics of the oil used in each of these
applications varies widely, particularly in terms of the chain
length and number of double bonds present in the fatty acids making
up the TAGs. These properties are manipulated by the plant in order
to control membrane fluidity and temperature sensitivity. The same
properties can be controlled using ZFPs to produce oils with
improved characteristics for food and industrial uses.
[0205] The primary fatty acids in the TAGs of oilseed crops are 16
to 18 carbons in length and contain 0 to 3 double bonds. Palmitic
acid (16:0 [16 carbons:0 double bonds]), oleic acid (18:1),
linoleic acid (18:2), and linolenic acid (18:3) predominate. The
number of double bonds, or degree of saturation, determines the
melting temperature, reactivity, cooking performance, and health
attributes of the resulting oil.
[0206] The enzyme responsible for the conversion of oleic acid
(18:1) into linoleic acid (18:2) (which is then the precursor for
18:3 formation) is .DELTA.12-oleate desaturase, also referred to as
omega-6 desaturase. A block at this step in the fatty acid
desaturation pathway should result in the accumulation of oleic
acid at the expense of polyunsaturates.
[0207] In one embodiment ZFPs are used to regulate expression of
the FAD2-1 gene in soybeans. Two genes encoding microsomal .DELTA.6
desaturases have been cloned recently from soybean, and are
referred to as FAD2-1 and FAD2-2 (Heppard et al., Plant Physiol.
110:311-319 (1996)). FAD2-1 (delta 12 desaturase) appears to
control the bulk of oleic acid desaturation in the soybean seed.
ZFPs can thus be used to modulate gene expression of FAD2-1 in
plants. Specifically, ZFPs can be used to inhibit expression of the
FAD2-1 gene in soybean in order to increase the accumulation of
oleic acid (18:1) in the oil seed. Moreover, ZFPs can be used to
modulate expression of any other plant gene, such as delta-9
desaturase, delta-12 desaturases from other plants, delta-15
desaturase, acetyl-CoA carboxylase, acyl-ACP-thioesterase,
ADP-glucose pyrophosphorylase, starch synthase, cellulose synthase,
sucrose synthase, senescence-associated genes, heavy metal
chelators, fatty acid hydroperoxide lyase, polygalacturonase, EPSP
synthase, plant viral genes, plant fungal pathogen genes, and plant
bacterial pathogen genes.
[0208] Recombinant DNA vectors suitable for transformation of plant
cells are also used to deliver the ZFP of the invention to plant
cells. Techniques for transforming a wide variety of higher plant
species are well known and described in the technical and
scientific literature (see, e.g., Weising et al. Ann. Rev. Genet.
22:421-477 (1988)). A DNA sequence coding for the desired ZFP is
combined with transcriptional and translational initiation
regulatory sequences which will direct the transcription of the ZFP
in the intended tissues of the transformed plant.
[0209] For example, a plant promoter fragment may be employed which
will direct expression of the ZFP in all tissues of a regenerated
plant. Such promoters are referred to herein as "constitutive"
promoters and are active under most environmental conditions and
states of development or cell differentiation. Examples of
constitutive promoters include the cauliflower mosaic virus (CaMV)
35S transcription initiation region, the 1'- or 2'-promoter derived
from T-DNA of Agrobacterium tumafaciens, and other transcription
initiation regions from various plant genes known to those of
skill.
[0210] Alternatively, the plant promoter may direct expression of
the ZFP in a specific tissue or may be otherwise under more precise
environmental or developmental control. Such promoters are referred
to here as "inducible" promoters. Examples of environmental
conditions that may effect transcription by inducible promoters
include anaerobic conditions or the presence of light.
[0211] Examples of promoters under developmental control include
promoters that initiate transcription only in certain tissues, such
as fruit, seeds, or flowers. For example, the use of a
polygalacturonase promoter can direct expression of the ZFP in the
fruit, a CHS-A (chalcone synthase A from petunia) promoter can
direct expression of the ZFP in flower of a plant.
[0212] The vector comprising the ZFP sequences will typically
comprise a marker gene which confers a selectable phenotype on
plant cells. For example, the marker may encode biocide resistance,
particularly antibiotic resistance, such as resistance to
kanamycin, G418, bleomycin, hygromycin, or herbicide resistance,
such as resistance to chlorosluforon or Basta.
[0213] Such DNA constructs may be introduced into the genome of the
desired plant host by a variety of conventional techniques. For
example, the DNA construct may be introduced directly into the
genomic DNA of the plant cell using techniques such as
electroporation and microinjection of plant cell protoplasts, or
the DNA constructs can be introduced directly to plant tissue using
biolistic methods, such as DNA particle bombardment. Alternatively,
the DNA constructs may be combined with suitable T-DNA flanking
regions and introduced into a conventional Agrobacterium
tumefaciens host vector. The virulence functions of the
Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria.
[0214] Microinjection techniques are known in the art and well
described in the scientific and patent literature. The introduction
of DNA constructs using polyethylene glycol precipitation is
described in Paszkowski et al. EMBO J. 3:2717-2722 (1984).
Electroporation techniques are described in Fromm et al. PNAS
82:5824 (1985). Biolistic transformation techniques are described
in Klein et al. Nature 327:70-73 (1987).
[0215] Agrobacterium tumefaciens-meditated transformation
techniques are well described in the scientific literature (see,
e.g., Horsch et al. Science 233:496-498 (1984)); and Fraley et al.
PNAS 80:4803 (1983)).
[0216] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype and thus the
desired ZFP-controlled phenotype. Such regeneration techniques rely
on manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker
which has been introduced together with the ZFP nucleotide
sequences. Plant regeneration from cultured protoplasts is
described in Evans et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, pp. 124-176 (1983); and Binding,
Regeneration of Plants, Plant Protoplasts, pp. 21-73 (1985).
Regeneration can also be obtained from plant callus, explants,
organs, or parts thereof. Such regeneration techniques are
described generally in Klee et al. Ann. Rev. of Plant Phys.
38:467-486 (1987).
Functional Genomics Assays
[0217] ZFPs also have use for assays to determine the phenotypic
consequences and function of gene expression. The recent advances
in analytical techniques, coupled with focussed mass sequencing
efforts have created the opportunity to identify and characterize
many more molecular targets than were previously available. This
new information about genes and their functions will speed along
basic biological understanding and present many new targets for
therapeutic intervention. In some cases analytical tools have not
kept pace with the generation of new data. An example is provided
by recent advances in the measurement of global differential gene
expression. These methods, typified by gene expression microarrays,
differential cDNA cloning frequencies, subtractive hybridization
and differential display methods, can very rapidly identify genes
that are up or down-regulated in different tissues or in response
to specific stimuli. Increasingly, such methods are being used to
explore biological processes such as, transformation, tumor
progression, the inflammatory response, neurological disorders etc.
One can now very easily generate long lists of differentially
expressed genes that correlate with a given physiological
phenomenon, but demonstrating a causative relationship between an
individual differentially expressed gene and the phenomenon is
difficult. Until now, simple methods for assigning function to
differentially expressed genes have not kept pace with the ability
to monitor differential gene expression.
[0218] Using conventional molecular approaches, over expression of
a candidate gene can be accomplished by cloning a full-length cDNA,
subcloning it into a mammalian expression vector and transfecting
the recombinant vector into an appropriate host cell. This approach
is straightforward but labor intensive, particularly when the
initial candidate gene is represented by a simple expressed
sequence tag (EST). Under expression of a candidate gene by
"conventional" methods is yet more problematic. Antisense methods
and methods that rely on targeted ribozymes are unreliable,
succeeding for only a small fraction of the targets selected. Gene
knockout by homologous recombination works fairly well in
recombinogenic stem cells but very inefficiently in somatically
derived cell lines. In either case large clones of syngeneic
genomic DNA (on the order of 10 kb) should be isolated for
recombination to work efficiently.
[0219] The ZFP technology can be used to rapidly analyze
differential gene expression studies. Engineered ZFPs can be
readily used to up or down-regulate any endogenous target gene.
Very little sequence information is required to create a
gene-specific DNA binding domain. This makes the ZFP technology
ideal for analysis of long lists of poorly characterized
differentially expressed genes. One can simply build a zinc
finger-based DNA binding domain for each candidate gene, create
chimeric up and down-regulating artificial transcription factors
and test the consequence of up or down-regulation on the phenotype
under study (transformation, response to a cytokine etc.) by
switching the candidate genes on or off one at a time in a model
system.
[0220] This specific example of using engineered ZFPs to add
functional information to genomic data is merely illustrative. Any
experimental situation that could benefit from the specific up or
down-regulation of a gene or genes could benefit from the
reliability and ease of use of engineered ZFPs.
[0221] Additionally, greater experimental control can be imparted
by ZFPs than can be achieved by more conventional methods. This is
because the production and/or function of an engineered ZFP can be
placed under small molecule control. Examples of this approach are
provided by the Tet-On system, the ecdysone-regulated system and a
system incorporating a chimeric factor including a mutant
progesterone receptor. These systems are all capable of indirectly
imparting small molecule control on any endogenous gene of interest
or any transgene by placing the function and/or expression of a ZFP
regulator under small molecule control.
Transgenic Mice
[0222] A further application of the ZFP technology is manipulating
gene expression in transgenic animals. As with cell lines,
over-expression of an endogenous gene or the introduction of a
heterologous gene to a transgenic animal, such as a transgenic
mouse, is a fairly straightforward process. The ZFP technology is
an improvement in these types of methods because one can circumvent
the need for generating full-length cDNA clones of the gene under
study.
[0223] Likewise, as with cell-based systems, conventional
down-regulation of gene expression in transgenic animals is plagued
by technical difficulties. Gene knockout by homologous
recombination is the method most commonly applied currently. This
method requires a relatively long genomic clone of the gene to be
knocked out (ca. 10 kb). Typically, a selectable marker is inserted
into an exon of the gene of interest to effect the gene disruption,
and a second counter-selectable marker provided outside of the
region of homology to select homologous versus non-homologous
recombinants. This construct is transfected into embryonic stem
cells and recombinants selected in culture. Recombinant stem cells
are combined with very early stage embryos generating chimeric
animals. If the chimerism extends to the germline homozygous
knockout animals can be isolated by back-crossing. When the
technology is successfully applied, knockout animals can be
generated in approximately one year. Unfortunately two common
issues often prevent the successful application of the knockout
technology; embryonic lethality and developmental compensation.
Embryonic lethality results when the gene to be knocked out plays
an essential role in development. This can manifest itself as a
lack of chimerism, lack of germline transmission or the inability
to generate homozygous back crosses. Genes can play significantly
different physiological roles during development versus in adult
animals. Therefore, embryonic lethality is not considered a
rationale for dismissing a gene target as a useful target for
therapeutic intervention in adults. Embryonic lethality most often
simply means that the gene of interest can not be easily studied in
mouse models, using conventional methods.
[0224] Developmental compensation is the substitution of a related
gene product for the gene product being knocked out. Genes often
exist in extensive families. Selection or induction during the
course of development can in some cases trigger the substitution of
one family member for another mutant member. This type of
functional substitution may not be possible in the adult animal. A
typical result of developmental compensation would be the lack of a
phenotype in a knockout mouse when the ablation of that gene's
function in an adult would otherwise cause a physiological change.
This is a kind of false negative result that often confounds the
interpretation of conventional knockout mouse models.
[0225] A few new methods have been developed to avoid embryonic
lethality. These methods are typified by an approach using the cre
recombinase and lox DNA recognition elements. The recognition
elements are inserted into a gene of interest using homologous
recombination (as described above) and the expression of the
recombinase induced in adult mice post-development. This causes the
deletion of a portion of the target gene and avoids developmental
complications. The method is labor intensive and suffers form
chimerism due to non-uniform induction of the recombinase.
[0226] The use of engineered ZFPs to manipulate gene expression can
be restricted to adult animals using the small molecule regulated
systems described in the previous section. Expression and/or
function of a zinc finger-based repressor can be switched off
during development and switched on at will in the adult animals.
This approach relies on the addition of the ZFP expressing module
only; homologous recombination is not required. Because the ZFP
repressors are trans dominant, there is no concern about germline
transmission or homozygosity. These issues dramatically affect the
time and labor required to go from a poorly characterized gene
candidate (a cDNA or EST clone) to a mouse model. This ability can
be used to rapidly identify and/or validate gene targets for
therapeutic intervention, generate novel model systems and permit
the analysis of complex physiological phenomena (development,
hematopoiesis, transformation, neural function etc.). Chimeric
targeted mice can be derived according to Hogan et al.,
Manipulating the Mouse Embryo: A Laboratory Manual, (1988);
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,
Robertson, ed., (1987); and Capecchi et al., Science 244:1288
(1989.
[0227] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0228] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Examples
[0229] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of noncritical parameters that could be
changed or modified to yield essentially similar results.
Example I: Design and Testing of ZFPs Targeted to the Human VEGF
Gene
[0230] This first Example demonstrates the construction of ZFPs
designed to recognize DNA sequences contained in the promoter of
the human vascular endothelial growth factor (VEGF) gene. VEGF is
an approximately 46 kDa glycoprotein that is an endothelial
cell-specific mitogen induced by hypoxia. VEGF has been implicated
in angiogenesis associated with cancer, various retinopathies, and
other serious diseases. The DNA target site chosen was a region
surrounding the transcription initiation site of the gene. The two
9 base pair (bp) sites chosen are found within the sequence
agcGGGGAGGATcGCGGAGGCTtgg, where the upper-case letters represent
actual 9-bp targets. The protein targeting the upstream 9-bp target
was denoted VEGF1, and the protein targeting the downstream 9-bp
target was denoted VEGF3a. The major start site of transcription
for VEGF is at the T at the 3' end of the first 9-bp target, which
is underlined in the sequence above.
[0231] The human SP-1 transcription factor was used as a progenitor
molecule for the construction of designed ZFPs. SP-1 has a three
finger DNA-binding domain related to the well-studied murine Zif268
(Christy et al., PNAS 85:7857-7861 (1988)). Site-directed
mutagenesis experiments using this domain have shown that the
proposed "recognition rules" that operate in Zif268 can be used to
adapt SP-1 to other target DNA sequences (Desjarlais & Berg,
PNAS 91:11099-11103 (1994)). The SP-1 sequence used for
construction of zinc finger clones corresponds to amino acids 533
to 624 in the SP-1 transcription factor.
[0232] The selection of amino acids in the recognition helices of
the two designed ZFPs, VEGF1 and VEGF3a, is summarized in Table
1.
TABLE-US-00001 TABLE 1 Amino acids chosen for recognition helices
of VEGF-recognizing ZFPs Position: Finger 1 Finger 2 Finger 3
Protein -1 2 3 6 -1 2 3 6 -1 2 3 6 VEGF1 T S N R R S N R R D H R
VEGF3A Q S D R R S N R R D E R
[0233] Coding sequences were constructed to express these peptides
using a PCR-based assembly procedure that utilizes six overlapping
oligonucleotides (FIG. 1). Three oligonucleotides (oligos 1, 3, and
5 in FIG. 1) corresponding to "universal" sequences that encode
portions of the DNA-binding domain between the recognition helices.
These oligonucleotides remain constant for any zinc finger
construct. The other three "specific" oligonucleotides (oligos 2,
4, and 6 in FIG. 1) were designed to encode the recognition
helices. These oligonucleotides contained substitutions at
positions -1, 2, 3 and 6 on the recognition helices to make them
specific for each of the different DNA-binding domains. Codon bias
was chosen to allow expression in both mammalian cells and E.
coli.
[0234] The PCR synthesis was carried out in two steps. First, the
double stranded DNA template was created by combining the six
oligonucleotides (three universal, three specific) and using a four
cycle PCR reaction with a low temperature (25.degree.) annealing
step. At this temperature, the six oligonucleotides join to form a
DNA "scaffold." The gaps in the scaffold were filled in by a
combination of Taq and Pfu polymerases. In the second phase of
construction, the zinc finger template was amplified in thirty
cycles by external primers that were designed to incorporate
restriction sites for cloning into pUC19. Accuracy of clones for
the VEGF ZFPs were verified by DNA sequencing. The DNA sequences of
each of the two constructs are listed below.
[0235] VEGF1:
TABLE-US-00002 GGTACCCATACCTGGCAAGAAGAAGCAGCACATCTGCCACATCCAGGGCT
GTGGTAAAGTTTACGGCACAACCTCAAATCTGCGTCGTCACCTGCGCTG
GCACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAAC
GCTTCACCCGTTCGTCAAACCTGCAGCGTCACAAGCGTACCCACACCGGT
GAGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGTAGTGA
CCACCTGTCCCGTCACATCAAGACCCACCAGAATAAGAAGGGTGGATCC
[0236] VEGF1 translation:
TABLE-US-00003 VPIPGKKKQHICHIQGCGKVYGTTSNLRRHLRWHTGERPFMCTWSYCGKR
FTRSSNLQRHKRTHTGEKKFACPECPKRFMRSDHLSRHIKTHQNKKGGS
[0237] VEGF3a:
TABLE-US-00004 GGTACCCATACCTGGCAAGAAGAAGCAGCACATCTGCCACATCCAGGGCT
GTGGTAAAGTTTACGGCCAGTCCTCCGACCTGCAGCGTCACCTGCGCTGG
CACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACG
CTTCACCCGTTCGTCAAACCTACAGAGGCACAAGCGTACACACACCGGTG
AGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGAAGTGAC
GAGCTGTCACGACATATCAAGACCCACCAGAACAAGAAGGGTGGATCC
[0238] VEGF3a translation:
TABLE-US-00005 VPIPGKKKQHICHIQGCGKVYGQSSDLQRHLRWHTGERPFMCTWSYCGKR
FTRSSNLQRHKRTHTGEKKFACPECPKRFMRSDELSRHIKTHQNKKGGS
[0239] The ability of the designed ZFPs to bind their target sites
was verified by expressing and purifying recombinant protein from
E. coli and performing electrophoretic mobility shift assays
(EMSAs). The expression of ZFPs was carried out in two different
systems. In the first, the DNA-binding peptides were expressed in
E. coli by inserting them into the commercially available pET15b
vector (Novagen). This vector contains a T7 promoter sequence to
drive expression of the recombinant protein. The constructs were
introduced into E. coli BL21/DE3 (lacI.sup.q) cells, which contain
an IPTG-inducible T7 polymerase. Cultures were supplemented with 50
.mu.M ZnCl.sub.2, were grown at 37.degree. C. to an OD at 600 nm of
0.5-0.6, and protein production was induced with IPTG for 2 hrs.
ZFP expression was seen at very high levels, approximately 30% of
total cellular protein (FIG. 2). These proteins are referred to as
"unfused" ZFPs.
[0240] Partially pure unfused ZFPs were produced as follows
(adapted from Desjarlais & Berg, Proteins: Structure, Function
and Genetics 12:101-104 (1992)). A frozen cell pellet was
resuspended in 1/50th volume of 1 M NaCl, 25 mM Tris HCl (pH 8.0),
100 .mu.M ZnCl.sub.2, 5 mM DTT. The samples were boiled for 10 min.
and centrifuged for 10 min. at 3,000.times.g. At this point the ZFP
protein in the supernatant was >50% pure as estimated by
staining of SDS polyacrylamide gels with Coomassie blue, and the
product migrated at the predicted molecular weight of around 11 kDa
(FIG. 2).
[0241] The second method of producing ZFPs was to express them as
fusions to the E. coli Maltose Binding Protein (MBP). N-terminal
MBP fusions to the ZFPs were constructed by PCR amplification of
the pET15b clones and insertion into the vector pMal-c2 under the
control of the Tac promoter (New England Biolabs). The fusion
allows simple purification and detection of the recombinant
protein. It had been reported previously that zinc finger
DNA-binding proteins can be expressed from this vector in soluble
form to high levels in E. coli and can bind efficiently to the
appropriate DNA target without refolding (Liu et al. PNAS
94:5525-5530 (1997)). Production of MBP-fused proteins was as
described by the manufacturer (New England Biolabs). Transformants
were grown in LB medium supplemented with glucose and ampicillin,
and were induced with IPTG for 3 hrs at 37.degree. C. The cells
were lysed by French press, then exposed to an agarose-based
amylose resin, which specifically binds to the MBP moiety, thus
acting as an affinity resin for this protein. The MBP fusion
protein was eluted with 10 mM maltose (FIG. 2C) to release ZFP of
>50% purity. In some cases, the proteins were further
concentrated using a Centricon 30 filter unit (Amicon).
[0242] Partially purified unfused and MBP fusion ZFPs were tested
by EMSA to assess binding to their target DNA sequences. The
protein concentrations in the preparations were measured by
Bradford assay (BioRad). Since SDS polyacrylamide gels demonstrated
>50% homogeneity by either purification method, no adjustment
was made for ZFP purity in the calculations. In addition, there
could be significant amounts of inactive protein in the
preparations. Therefore, the data generated by EMSAs below
represent an underestimate of the true affinity of the proteins for
their targets (i.e., overestimate of K.sub.ds). Two separate
preparations were made for each protein to help control for
differences in ZFP activity.
[0243] The VEGF DNA target sites for the EMSA experiments were
generated by embedding the 9-bp binding sites in 29-bp duplex
oligonucleotides. The sequences of the recognition ("top") strand
and their complements ("bottom") used in the assays are as
follows:
TABLE-US-00006 VEGF site 1, top: 5'-CATGCATAGCGGGGAGGATCGCCATCGAT
VEGF site 1, bottom: 5'-ATCGATGGCGATCCTCCCCGCTATGCATG VEGF site 3,
top: 5'-CATGCATATCGCGGAGGCTTGGCATCGAT VEGF site 3, bottom:
5'-ATCGATGCCAAGCCTCCGCGATATGCATG
[0244] The VEGF DNA target sites are underlined. The 3 bp on either
side of the 9 bp binding site was also derived from the actual VEGF
DNA sequence. The top strand of each target site was labeled with
polynucleotide kinase and .gamma.-.sup.32P dATP. Top and bottom
strands were annealed in a reaction containing each oligonucleotide
at 0.5 .mu.M, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 50 mM NaCl.
The mix was heated to 95.degree. C. for 5 min. and slow cooled to
30.degree. C. over 60 min. Duplex formation was confirmed by
polyacrylamide gel electrophoresis. Free label and ssDNA remaining
in the target preparations did not appear to interfere with the
binding reactions.
[0245] Binding of the ZFPs to target oligonucleotides was performed
by titrating protein against a fixed amount of duplex substrate.
Twenty microliter binding reactions contained 10 fmole (0.5 nM)
5'-.sup.32P-labeled double-stranded target DNA, 35 mM Tris HCl (pH
7.8), 100 mM KCl, 1 mM MgCl.sub.2, 1 mM dithiothreitol, 10%
glycerol, 20 .mu.g/ml poly dI-dC (optionally), 200 .mu.g/ml bovine
serum albumin, and 25 .mu.M ZnCl.sub.2. Protein was added as one
fifth volume from a dilution series made in 200 mM NaCl, 20 mM Tris
(pH 7.5), 1 mM DTT. Binding was allowed to proceed for 30 min. at
room temperature. Polyacrylamide gel electrophoresis was carried
out at 4.degree. C. using precast 10% or 10-20% Tris-HCl gels
(BioRad) and standard Tris-Glycine running buffer containing 0.1 mM
ZnCl.sub.2.
[0246] The results of a typical EMSA using an MBP fused ZFP are
shown in FIG. 3. In this case, a 3-fold dilution series of the
MBP-VEGF1 protein was used. The shifted product was quantitated on
a phosphorimager (Molecular Dynamics) and the relative signal
(percent of plateau value) vs. the log.sub.10 of nM protein
concentration was plotted. An apparent K.sub.d was found by
determining the protein concentration that gave half maximal
binding of MBP-VEGF1 to its target site, which in this experiment
was approximately 2 nM.
[0247] The binding affinities determined for the VEGF proteins can
be summarized as follows. VEGF1 showed the stronger DNA-binding
affinity; in multiple EMSA analyses, the average apparent K.sub.d
was determined to be approximately 10 nM when bound to VEGF site 1.
VEGF3a bound well to its target site but with a higher apparent
K.sub.d than VEGF1; the average K.sub.d for VEGF3a was about 200
nM. In both cases the MBP-fused and unfused versions of the
proteins bound with similar affinities. K.sub.ds were also
determined under these conditions for MBP fusions of the wild-type
Zif268 and SP-1 ZFPs, which yielded K.sub.ds of 60 and 65 nM,
respectively. These results are similar to binding constants
reported in the literature for Zif268 of approximately 2-30 nM
(see, e.g., Jamieson et al., Biochemistry 33:5689-5695 (1994)). The
K.sub.ds for the synthetic VEGF ZFPs therefore compare very
favorably with those determined for these naturally-occurring
DNA-binding proteins.
[0248] In summary, this Example demonstrates the generation of two
novel DNA-binding proteins directed to specific targets near the
transcriptional start of the VEGF gene. These proteins bind with
affinities similar to those of naturally-occurring transcription
factors binding to their targets.
Example II: Linking ZFPs to Bind an 18-Bp Target in the Human VEGF
Gene
[0249] An important consideration in ZFP design is DNA target
length. For random DNA, a sequence of n nucleotides would be
expected to occur once every 0.5.times.4.sup.n base-pairs. Thus,
DNA-binding domains designed to recognize only 9 bp of DNA would
find sites every 130,000 bp and could therefore bind to multiple
locations in a complex genome (on the order of 20,000 sites in the
human genome). 9-bp putative repressor-binding sequences have been
chosen for VEGF in the 5' UTR where they might directly interfere
with transcription. However, in case zinc finger domains that
recognize 9-bp sites lack the necessary affinity or specificity
when expressed inside cells, a larger domain was constructed to
recognize 18 base-pairs by joining separate three-finger domains
with a linker sequence to form a six-finger protein. This should
ensure that the repressor specifically targets the appropriate
sequence, particularly under conditions where only small amounts of
the repressor are being produced. The 9-bp target sites in VEGF
were chosen to be adjacent to one another so that the zinc fingers
could be linked to recognize an 18-bp sequence. The linker DGGGS
was chosen because it permits binding of ZFPs to two 9-bp sites
that are separated by a one nucleotide gap, as is the case for the
VEGF1 and VEGF3a sites (see also Liu et al., PNAS 5525-5530
(1997)).
[0250] The 6-finger VEGF3a/1 protein encoding sequence was
generated as follows. VEGF3a was PCR amplified using the primers
SPE7 (5'-GAGCAGAATTCGGCAAGAAGAAGCAGCAC) and SPEamp12
(5'-GTGGTCTAGACAGCTCGTCACTTCGC) to generate EcoRI and XbaI
restriction sites at the ends (restriction sites underlined). VEGF1
was PCR amplified using the primers SPEamp13
(5'-GGAGCCAAGGCTGTGGTAAAGTTTACGG) and SPEamp11
(5'-GGAGAAGCTTGGATCCTCATTATCCC) to generate StyI and HindIII
restriction sites at the ends (restriction sites underlined). Using
synthetic oligonucleotides, the following sequence was ligated
between the XbaI and StyI sites, where XbaI and StyI are
underlined: TCT AGA CAC ATC AAA ACC CAC CAG AAC AAG AAA GAC GGC GGT
GGC AGC GGC AAA AAG AAA CAG CAC ATA TGT CAC ATC CAA GG. This
introduced the linker sequence DGGGS between the two SP-1 domains.
The ligation product was reamplified with primers SPE7 and SPEamp11
and cloned into pUC19 using the EcoRI and HindIII sites. The linked
ZFP sequences were then amplified with primers
[0251] (1) GB19 GCCATGCCGGTACCCATACCTGGCAAGAAGAAGCAGCAC)
[0252] (2) GB10 CAGATCGGATCCACCCTTCTTATTCTGGTGGGT to introduce KpnI
and BamHI sites for cloning into the modified pMAL-c2 expression
vector as described above.
[0253] The nucleotide sequence of the designed, 6-finger ZFP
VEGF3a/1 from KpnI to BamHI is:
TABLE-US-00007 GGTACCCATACCTGGCAAGAAGAAGCAGCACATCTGCCACATCCAGGGCT
GTGGTAAAGTTTACGGCCAGTCCTCCGACCTGCAGCGTCACCTGCGCTGG
CACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACG
CTTCACACGTTCGTCAAACCTACAGAGGCACAAGCGTACACACACAGGTG
AGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGAAGTGAC
GAGCTGTCTAGACACATCAAAACCCACCAGAACAAGAAAGACGGCGGTGG
CAGCGGCAAAAAGAAACAGCACATATGTCACATCCAAGGCTGTGGTAAAG
TTTACGGCACAACCTCAAATCTGCGTCGTCACCTGCGCTGGCACACCGGC
GAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACGCTTCACCCG
TTCGTCAAACCTGCAGCGTCACAAGCGTACCCACACCGGTGAGAAGAAAT
TTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGTAGTGACCACCTGTCC
CGTCACATCAAGACCCACCAGAATAAGAAGGGTGGATCC
[0254] The VEGF3a/1 amino acid translation (using single letter
code) is:
TABLE-US-00008 VPIPGKKKQHICHIQGCGKVYGQSSDLQRHLRWHTGERPFMCTWSYCGKR
FTRSSNLQRHKRTHTGEKKFACPECPKRFMRSDELSRHIKTHQNKKDGGG
SGKKKQHICHIQGCGKVYGTTSNLRRHLRWHTGERPFMCTWSYCGKRFTR
SSNLQRHKRTHTGEKKFACPECPKRFMRSDHLSRHIKTHQNKKGGS
[0255] The 18-bp binding protein VEGF3a/1 was expressed in E. coli
as an MBP fusion, purified by affinity chromatography, and tested
in EMSA experiments as described in Example I. The target
oligonucleotides were prepared as described and comprised the
following complementary sequences:
TABLE-US-00009 (1) JVF9 AGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAG,
and (2) JVF 10 CGCTCTACCCGGCTGCCCCAAGCCTCCGCGATCCTCCCCGCT.
[0256] For the EMSA studies, 20 .mu.l binding reactions contained
10 fmole (0.5 nM) 5'-.sup.32P-labeled double-stranded target DNA,
35 mM Tris HCl (pH 7.8), 100 mM KCl, 1 mM MgCl.sub.2, 5 mM
dithiothreitol, 10% glycerol, 20 .mu.g/ml poly dI-dC, 200 .mu.g/ml
bovine serum albumin, and 25 .mu.M ZnCl.sub.2. Protein was added as
one fifth volume from a 3-fold dilution series. Binding was allowed
to proceed for 60 min at either room temperature or 37.degree. C.
Polyacrylamide gel electrophoresis was carried out at room
temperature or 37.degree. C. using precast 10% or 10-20% Tris-HCl
gels (BioRad) and standard Tris-Glycine running buffer. The room
temperature assays yielded an apparent K.sub.d for this VEGF3a/1
protein of approximately 1.5 nM. Thus, the 18-bp binding ZFP bound
with high affinity to its target site. In a parallel experiment,
VEGF1 protein was tested against its target using the
oligonucleotides described in Example I, yielding an apparent
K.sub.d of approximately 2.5 nM. When binding and electrophoresis
were performed at 37.degree. C., the apparent K.sub.d of VEGF3a/1
was approximately 9 nM when tested against the 18-bp target,
compared to a K.sub.d of 40 nM for VEGF1 tested against its target.
This indicates that the difference in binding affinities is
accentuated at the higher temperature.
[0257] The apparent K.sub.d is a useful measure of the affinity of
a protein for its DNA target. However, for a DNA binding site
either in vitro or in vivo, its occupancy is determined to a large
extent by the off-rate of the DNA-binding protein. This parameter
can be measured by competition experiments as shown in FIG. 4. The
conditions for EMSA were as described above; binding and
electrophoresis were performed at 37.degree. C. These data indicate
that the half-life of the protein-DNA complex is more than ten
times longer for VEGF3a/1 than for VEGF1. Thus, under these in
vitro conditions, the occupancy of the target site is much higher
for the 18-bp binding protein than for the 9-bp binding
protein.
Example III: Fusing Designed ZFP Sequences to Functional Domains in
Mammalian Expression Vectors
[0258] This Example describes the development of expression vectors
for producing ZFPs within mammalian cells, translocating them to
the nucleus, and providing functional domains that are localized to
the target DNA sequence by the ZFP. The functional domains employed
are the Kruppel-Associated Box (KRAB) repression domain and the
Herpes Simplex Virus (HSV-1) VP16 activation domain.
[0259] Certain DNA-binding proteins contain separable domains that
function as transcriptional repressors. Approximately 20% of ZFPs
contain a non-DNA-binding domain of about 90 amino acids that
functions as a transcriptional repressor (Thiesen, The New
Biologist 2:363-374 (1990); Margolin et al., PNAS 91:4509-4513
(1994); Pengue et al., (1994), supra; Witzgall et al., (1994),
supra). This domain, termed the KRAB domain, is modular and can be
joined to other DNA-binding proteins to block expression of genes
containing the target DNA sequence (Margolin et al., (1994); Pengue
et al., (1994); Witzgall et al., (1994), supra). The KRAB domain
has no effect by itself; it needs to be tethered to a DNA sequence
via a DNA-binding protein to function as a repressor. The KRAB
domain has been shown to block transcription initiation and can
function at a distance of up to at least 3 kb from the
transcription start site. The KRAB domain from the human KOX-1
protein (Thiesen, The New Biologist 2:363-37 (1990)) was used for
the studies described here. This 64 amino acid domain can be fused
to ZFPs and has been shown to confer repression in cell culture
(Liu et al., supra).
[0260] The VP16 protein of HSV-1 has been studied extensively, and
it has been shown that the C-terminal 78 amino acids can act as a
trans-activation domain when fused to a DNA-binding domain (Hagmann
et al., J. Virology 71:5952-5962 (1997)). VP16 has also been shown
to function at a distance and in an orientation-independent manner.
For these studies, amino acids 413 to 490 in the VP16 protein
sequence were used. DNA encoding this domain was PCR amplified from
plasmid pMSVP16.DELTA.C+119 using primers with the following
sequences:
TABLE-US-00010 (1) JVF24 CGCGGATCCGCCCCCCCGACCGATG, and (2) JVF25
CCGCAAGCTTACTTGTCATCGTCGTCCTTGTAGTCGCTGCCCCCACCGTAC
TCGTCAATTCC.
[0261] The downstream primer, JVF25, was designed to include a
downstream FLAG epitope-encoding sequence.
[0262] Three expression vectors were constructed for these studies.
The general design is summarized in FIG. 5. The vectors are derived
from pcDNA3.1(+) (Invitrogen), and place the ZFP constructs under
the control of the cytomegalovirus (CMV) promoter. The vector
carries ampicillin and neomycin markers for selection in bacteria
and mammalian cell culture, respectively. A Kozak sequence for
proper translation initiation (Kozak, J. Biol. Chem.
266:19867-19870 (1991)) was incorporated. To achieve nuclear
localization of the products, the nuclear localization sequence
(NLS) from the SV40 large T antigen (Pro-Lys-Lys-Lys-Arg-Lys-Val)
(Kalderon et al., Cell 39:499-509 (1984)) was added. The insertion
site for the ZFP-encoding sequence is followed by the functional
domain sequence. The three versions of this vector differ in the
functional domain; "pcDNA-NKF" carries the KRAB repression domain
sequence, "pcDNA-NVF" carries the VP16 activation domain, and
"NF-control" carries no functional domain. Following the functional
domain is the FLAG epitope sequence (Kodak) to allow specific
detection of the ZFPs.
[0263] The vectors were constructed as follows. Plasmid
pcDNA-.DELTA.HB was constructed by digesting plasmid pcDNA3.1(+)
(Invitrogen) with HindIII and BamHI, filling in the sticky ends
with Klenow, and religating. This eliminated the HindIII, KpnI, and
BamHI sites in the polylinker. The vector pcDNA3.1(+) is described
in the Invitrogen catalog. Plasmid pcDNA-NKF was generated by
inserting a fragment into the EcoRI/XhoI sites of pcDNA-.DELTA.HB
that contained the following: 1) a segment from EcoRI to KpnI
containing the Kozak sequence including the initiation codon and
the SV40 NLS sequence, altogether comprising the DNA sequence
GAATTCGCTAGCGCCACCATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCCAT GGGGTAC,
where the EcoRI and KpnI sites are underlined; and 2) a segment
from KpnI to XhoI containing a BamHI site, the KRAB-A box from KOX1
(amino acid coordinates 11-53 in Thiesen, 1990, supra), the FLAG
epitope (from Kodak/IBI catalog), and a HindIII site, altogether
comprising the sequence
GGTACCCGGGGATCCCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCA
CCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATG
TGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGCAGCGACTACAAGGACG
ACGATGACAAGTAAGCTTCTCGAG where the KpnI, BamHI and XhoI sites are
underlined.
[0264] The VEGF3a/1-KRAB effector plasmid was generated by
inserting a KpnI-BamHI cassette containing the ZFP sequences into
pcDNA-NKF digested with KpnI and BamHI. The VEGF1-KRAB and
VEGF3a-KRAB effector plasmids were constructed in a similar way
except that the ZFP sequences were first cloned into the
NLS-KRAB-FLAG sequences in the context of plasmid pLitmus 28 (New
England Biolabs) and subsequently moved to the BamHI-XhoI sites of
pcDNA3.1(+) as a BglII-XhoI cassette, where the BglII site was
placed immediately upstream of the EcoRI site (see Example IV for
expression of these vectors).
[0265] The effector plasmids used in Example V were constructed as
follows. Plasmid pcDNA-NVF was constructed by PCR amplifying the
VP16 transactivation domain, as described above, and inserting the
product into the BamHI/HindIII sites of pcDNA-NKF, replacing the
KRAB sequence. The sequence of the inserted fragment, from BamHI to
HindIII, was:
TABLE-US-00011 GGATCCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGA
CGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATC
TGGACATGTTGGGGGACGGGGATTCCCCGGGGCCGGGATTTACCCCCCAC
GACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCA
GATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGGGCAGCGACT
ACAAGGACGACGATGACAAGTAAGCTT.
[0266] VEGF1-VP16 and VEGF3a/1-VP16 vectors were constructed by
inserting a KpnI-BamHI cassette containing the ZFP sequences into
pcDNA-NVF digested with KpnI and BamHI.
[0267] The effector plasmids used in Example VI were constructed as
follows.
[0268] Plasmid NF-control was generated by inserting the sequence
GAATTCGCTAGCGCCACCATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCCAT
GGGGTACCCGGGGATGGATCCGGCAGCGACTACAAGGACGACGATGACAAGTA
AGCTTCTCGAG
into the EcoRI-XhoI sites of pcDNA-NKF, thereby replacing the
NLS-KRAB-FLAG sequences with NLS-FLAG only.
[0269] VEGF1-NF and VEGF3a/1-NF were constructed by inserting a
KpnI-BamHI cassette containing the ZFP sequences into NF-control
digested with KpnI and BamHI. CCR5-KRAB was constructed in the same
way as the VEGF KRAB vectors, except that the ZFP sequences were
designed to be specific for a DNA target site that is unrelated to
the VEGF targets.
[0270] Finally, control versions of both the KRAB and VP16
expression plasmids were constructed. Plasmid NKF-control was
designed to express NLS-KRAB-FLAG without zinc finger protein
sequences; plasmid NVF-control was designed to express
NLS-VP16-FLAG without ZFP sequences. These plasmids were made by
digesting pcDNA-NKF and -NVF, respectively, with BamHI, filling in
the ends with Klenow, and religating in order to place the
downstream domains into the proper reading frame. These plasmids
serve as rigorous controls for cell culture studies.
[0271] Mammalian cell expression and nuclear localization of the
VEGF engineered ZFPs was demonstrated through immunofluorescence
studies. 293 (human embryonic kidney) cells were transfected with
the expression plasmid encoding the NLS-VEGF1-KRAB-FLAG chimera.
Lipofectamine was used as described below. After 24-48 hours, cells
were fixed and exposed to a primary antibody against the FLAG
epitope. A secondary antibody labeled with Texas Red was applied,
and the cells were counter stained with DAPI. Texas Red staining
was observed to consistently co-localize with the DAPI staining,
indicating that the ZFP being expressed from this plasmid was
nuclear localized.
Example IV: Repression of VEGF Reporters in Co-Transfection
Experiments
[0272] This Example demonstrates the use of transient
co-transfection studies to measure the activity of the ZFP
repressor proteins in cells. Such experiments involve
co-transfection of ZFP-KRAB expression ("effector") plasmids with
reporter plasmids carrying the VEGF target sites. Efficacy is
assessed by the repression of reporter gene expression in the
presence of the effector plasmid relative to empty vector
controls.
[0273] The reporter plasmid system was based on the pGL3 firefly
luciferase vectors (Promega). Four copies of the VEGF target sites
were inserted upstream of the SV40 promoter, which is driving the
firefly luciferase gene, in the plasmid pGL3-Control to create
pVFR1-4x. This plasmid contains the SV40 enhancer and expresses
firefly luciferase to high levels in many cell types. Insertions
were made by ligating together tandem copies of the two
complementary 42-bp oligonucleotides, JVF9 and JVF10, described in
Example II. Adaptor sequences were ligated on, and the assembly was
inserted into the MluI/BglII sites of pGL3-Control. This resulted
in the insertion of the following sequence between those sites:
TABLE-US-00012 ACGCGTaagcttGCTAGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGC
CGGGTAGAGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGA
GCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGAGCGAGCGG
GGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGAGCGCTCAGaagcttAG ATCT.
[0274] The first six and last six nucleotides shown are the MluI
and BglII sites; the lowercase letters indicate HindIII sites. The
binding sites for VEGF1 and VEGF3a are underlined.
[0275] The effector plasmid construction is described above. The
VEGF1-KRAB, VEGF3a-KRAB, and VEGF3a/1-KRAB expression vectors were
designed to produce a fusion of the SV40 nuclear localization
sequence, the VEGF ZFP, the KRAB repression domain, and a FLAG
epitope marker all under the control of the CMV promoter. The empty
pcDNA3.1 expression vector was used as a control (pcDNA).
[0276] All vectors were prepared using Qiagen DNA purification
kits. FIG. 6 shows a typical set of transfections using COS-1
(African green monkey kidney) cells. Approximately 40,000 cells
were seeded into each well of a 24-well plate and allowed to grow
overnight in Dulbecco's Modified Eagle Medium (D-MEM) medium
containing 10% fetal bovine serum at 37.degree. C. with 5%
CO.sub.2. Cells were washed with PBS and overlayed with 200 .mu.l
of serum-free D-MEM. Plasmids were introduced using lipofectamine
(Gibco-BRL). Each well was transfected with about 0.3 .mu.g of
effector plasmid, 0.3 .mu.g of reporter plasmid, and 0.01 .mu.g of
plasmid pRL-SV40 (Promega) that had been complexed with 6 .mu.l of
lipofectamine and 25 .mu.l of D-MEM for 30 min at 37.degree. C.
Transfections were done in triplicate. After 3 hrs, 1 ml of medium
containing 10% serum was added to each well. Cells were harvested
40-48 hours after transfection. Luciferase assays were done using
the Dual Luciferase.TM. System (Promega). The third plasmid
transfected, pRL-SV40, carries the Renilla luciferase gene and was
co-transfected as a standard for transfection efficiency. The data
shown in FIG. 6 are the averages of triplicate assays normalized
against the Renilla activity.
[0277] For the control reporter plasmid pGL3-Control (pGL3-C), the
presence or absence of the ZFP-KRAB expression plasmid does not
influence the luciferase expression level. However, for pVFR1-4x,
the reporter containing four copies of the VEGF target site,
presence of the VEGF1 (9-bp-binding ZFP) or VEGF3a/1 (18-bp-binding
ZFP) expression plasmid reduces luciferase expression by a factor
of 2-3 relative to the empty pcDNA vector control. The VEGF3a
(9-bp-binding ZFP) expression plasmid appears to exhibit little or
no effect. These experiments clearly demonstrate that a designed
ZFP is capable of functioning in a cell to repress transcription of
a gene when its target site is present. Furthermore, it appears
that a certain level of affinity is required for function; i.e.,
VEGF1 and VEGF3a/1, with K.sub.ds of 10 nM or less, are functional,
whereas VEGF3a, with a K.sub.d of 200 nM, is not.
[0278] A second reporter plasmid, pVFR2-4x, was constructed by
removing the four copies of the VEGF target sites using HindIII and
inserted them into the HindIII site of pGL3-Control (in the forward
orientation). This places the target sites between the start site
of transcription for the SV40 promoter and the translational start
codon of the luciferase gene. In similar co-transfection
experiments to those described, approximately 3-4 fold repression
of the luciferase signal was observed with the VEGF1-KRAB or
VEGF3a/1-KRAB repressors relative to the pcDNA controls (data not
shown). This indicates that the repressors are active when bound
either upstream or downstream of the start of transcription.
Example V: Activation of VEGF Reporters in Co-Transfection
Experiments
[0279] This Example demonstrates the use of transient
co-transfection studies to measure the activity of the ZFP
transcriptional activators in cells. The experimental setup is
similar to that of Example IV except that a different transfection
method, a different cell line, and a different set of reporter and
effector plasmids was used.
[0280] For activation experiments, a reporter was constructed
labeled pVFR3-4x. This reporter contains the four copies of the
VEGF targets, with the sequence shown above, at the MluI/BglII
sites of plasmid pGL3-Promoter (Promega). This vector has been
deleted for the SV40 enhancer sequence and therefore has a lower
basal level of firefly luciferase expression. pVFR3-4x was
constructed by swapping the KpnI/NcoI fragment of pVFR1-4x into the
KpnI/NcoI sites of pGL3-Promoter.
[0281] The effector plasmid construction is described above. The
VEGF1-VP16, VEGF3a-VP16, and VEGF3a/1-VP16 expression vectors were
designed to produce a fusion of the SV40 nuclear localization
sequence, the VEGF ZFP, the VP16 trans-activation domain, and a
FLAG epitope tag all under the control of the CMV promoter. The
empty pcDNA3 expression vector was used as a control.
[0282] All vectors were prepared using Qiagen DNA purification
kits. FIG. 7 shows a typical set of transfections using 293 (human
embryonic kidney) cells. Approximately 40,000 cells were seeded
into each well of a 24-well plate and allowed to grow overnight in
D-MEM medium containing 10% fetal bovine serum at 37.degree. C.
with 5% CO.sub.2. Cells were washed with serum-free D-MEM and
overlayed with 200 .mu.l of the same. Plasmids were introduced
using a calcium phosphate transfection kit (Gibco-BRL) according to
the manufacturer's instructions. Cells in each well were
transfected with 1.5 .mu.g of reporter plasmid, 1.5 .mu.g of
effector plasmid, and 0.5 .mu.g of an actin/.beta.-gal plasmid.
Plasmids were combined with 15 .mu.l of CaCl.sub.2 and brought to
100 .mu.l with dH.sub.2O. 100 .mu.l of HEPES solution was added
dropwise while vortexing. The mix was incubated for 30 min at room
temperature. The 200 .mu.l of calcium phosphate-treated DNA was
then added to the medium in each well. Transfections were done in
triplicate. After 5 hours, the medium was removed and 1 ml of
medium containing 10% serum was added. Cells were harvested 40-48
hours after transfection. Luciferase assays were done using the
Dual-Light.TM. system (Tropix). The third plasmid transfected,
actin/.beta.-gal, carries the .beta.-galactosidase gene under the
control of the actin promoter and was co-transfected as a standard
for transfection efficiency. The .beta.-galactosidase assays were
also done according to the manufacturer's protocol (Tropix). The
data shown in FIG. 7 are the average of triplicate assays
normalized against the .beta.-galactosidase activity.
[0283] For the control reporter plasmid, pGL3-Promoter (pGL3-P),
the presence or absence of the ZFP-VP16 expression plasmid does not
significantly influence the luciferase expression level. For
pVFR3-4x, the reporter containing four copies of the VEGF target
site, presence of VEGF1 (the 9-bp-binding ZFP) shows a very slight
activation relative to the empty pcDNA vector control. VEGF3a/1
(the 18-bp-binding ZFP) expression plasmid activates luciferase
expression very substantially, showing about a 14-fold increase
relative to pcDNA. These experiments clearly demonstrate that a
designed ZFP, when fused to the VP16 activation domain, is capable
of functioning in a cell to activate transcription of a gene when
its target site is present. Furthermore, these results clearly
demonstrate that an 18-bp binding protein, VEGF3a/1, is a much
better activator in this assay than a 9-bp binding VEGF1 protein.
This could be a result of the improved affinity or decreased
off-rate of the VEGF3a/1 protein.
[0284] A fourth VEGF reporter plasmid was constructed by cloning
the KpnI/NcoI fragment of pVFR2-4x into pGL3-Promoter to create
plasmid pVFR4-4x. Activation was observed in co-transfections using
this reporter in combination with effector plasmids expressing the
VEGF1-VP16 and VEGF3a/1-VP16 fusions (data not shown). This
indicates that these artificial trans-activators are functional
when bound either upstream or downstream of the start of
transcription.
[0285] These co-transfection data demonstrate that ZFPs can be used
to regulate expression of reporter genes. Such experiments serve as
a useful tool for identifying ZFPs for further use as modulators of
expression of endogenous cellular genes. As is shown below,
modulation results can vary between co-transfection experiments and
endogenous gene experiments, while using the same ZFP
construct.
Example VI: Repression of an Endogenous VEGF Gene in Human
Cells
[0286] This Example demonstrates that a designed ZFP can repress
expression of an endogenous cellular gene that is in its natural
context and chromatin structure. Specifically, effector plasmids
expressing VEGF ZFPs fused to the KRAB repression domain were
introduced into cells and were shown to down-regulate the VEGF
gene.
[0287] Eucaryotic expression vectors were constructed that fuse the
VEGF3a/1 and the VEGF1 ZFPs to the SV40 NLS and KRAB, as described
above in Example III. Transfections were done using Lipofectamine,
a commercially available liposome preparation from GIBCO-BRL. All
plasmid DNAs were prepared using Qiagen Midi DNA purification
system. 10 .mu.g of the effector plasmid was mixed with 100 .mu.g
of Lipofectamine (50 .mu.l) in a total volume of 1600 .mu.l of
Opti-MEM. A pCMV.beta.-gal plasmid (Promega) was also included in
the DNA mixture as an internal control for transfection efficiency.
Following a 30 minute incubation, 6.4 ml of DMEM was added and the
mixture was layered on 3.times.10.sup.6 293 cells. After five
hours, the DNA-Lipofectamine mixture was removed, and fresh culture
medium containing 10% fetal bovine serum was layered on the
cells.
[0288] Eighteen hours post transfection, the 293 cells were induced
by treatment with 100 .mu.M DFX (desferrioxamine), resulting in a
rapid and lasting transcriptional activation of the VEGF gene and
also in a gradual increase in VEGF mRNA stability (Ikeda et al., J.
Biol. Chem. 270:19761-19766 (1995)). Under routine culture
conditions, 293 cells secrete a low level of VEGF in the culture
media. The cells were allowed to incubate an additional 24 hours
before the supernatants were collected for determination of VEGF
levels by an ELISA assay.
[0289] In parallel experiments that demonstrated a similar level of
repression, cell viability was monitored using the Promega
Celltiter 96.RTM. Aqueous One Solution cell proliferation assay
(Promega). After Dfx treatment for 18 hours, 500 .mu.L of the
original 2 ml of media was removed and analyzed for VEGF
expression, as described above. To evaluation cell viability, 300
.mu.L of Promega Celltiter 96.RTM. Aqueous One Solution Reagent was
added to the remaining 1.5 ml. The cells were then incubated at
37.degree. C. for approximately 2 hours. 100 .mu.L from each well
was transferred to a 96-well plate and read on an ELISA plate
reader at OD 490 nm. There was no significant reduction in
viability of cells expressing the VEGF3a/1-KRAB construct relative
to those transfected with empty vector controls, indicating that
the VEGF repression observed was not due to generalized cell
death.
[0290] A 40-50-fold decrease in VEGF expression was noted in the
DFX treated cells transfected with VEGF3a/1-KRAB, an expression
vector encoding the 18 bp binding VEGF high affinity ZFP. A
two-fold decrease in expression was observed when cells were
transfected with VEGF1-KRAB, an expression vector encoding the 9 bp
binding VEGF high affinity ZFP. No significant decrease in VEGF
expression was observed in cells that were transfected with a
non-VEGF ZFP (CCR5-KRAB) or NKF-control (FIG. 8). Similar results
have been obtained in three independent transfection
experiments.
[0291] In a separate experiment, the following results were
obtained (data not shown). VEGF1-NF, which expresses the
9-bp-binding VEGF1 ZFP without a functional domain, showed no
effect on VEGF gene expression. A significant reduction in VEGF
expression was observed with VEGF3a/1-NF, which expresses the 18-bp
binding protein without a functional domain. This result suggests
that binding to the start site of transcription, even without a
repression domain, interferes with transcription. Even when fused
to the KRAB domain, the VEGF3a ZFP is unable to affect expression
levels (plasmid VEGF3a-KRAB). However, VEGF1 fused to KRAB
(VEGF1-KRAB) results in a dramatic decrease in expression. VEGF3a/1
fused to KRAB (VEGF3a/1-KRAB) prevents expression of VEGF
altogether.
[0292] These data indicate that a designed ZFP is capable of
locating and binding to its target site on the chromosome and
preventing expression of an endogenous cellular target gene. In
particular, the results indicate that ZFPs with a K.sub.d of less
than about 25 nM (e.g., VEGF1 has an average apparent K.sub.d of
about 10 nM) provide dramatic decreases in expression. In addition,
the data demonstrate that the KRAB functional domain enhances gene
silencing. Because in this experiment the introduction of the
repressor occurs before the inducer of VEGF is added (DFX), the
data demonstrate the ability of a designed repressor to prevent
activation of an already quiescent gene. In addition, these results
demonstrate that a six-finger engineered ZFP (VEGF3a/1) with
nanomolar affinity for its target is able to inhibit the hypoxic
response of the VEGF gene when it binds a target that overlaps the
transcriptional start site.
Example VII: Activation of Endogenous VEGF Gene in Human Cells
[0293] This Example demonstrates that a designed ZFP can activate
the expression of a gene that is in its natural context and
chromatin structure. Specifically, effector plasmids expressing
VEGF ZFPs fused to the VP16 activation domain were introduced into
cells and were shown to up-regulate the VEGF gene.
[0294] Eucaryotic expression vectors were constructed that fuse the
VEGF3a/1 and the VEGF1 ZFPs to the SV40 NLS and VP16, as described
in Example III. Transfections were done using Lipofectamine, a
commercially available liposome preparation from GIBCO-BRL. All
plasmid DNAs were prepared using the Qiagen Midi DNA purification
system. 10 .mu.g of the effector plasmid (containing the engineered
ZFP) was mixed with 100 .mu.g of Lipofectamine (50 .mu.l) in a
total volume of 1600 .mu.l of Opti-MEM. A pCMV.beta.-gal plasmid
(Promega) was also included in the DNA mixture as an internal
control for transfection efficiency. Following a 30 minute
incubation, 6.4 ml of DMEM was added and the mixture was layered on
3.times.10.sup.6 293 cells. After five hours, the DNA-Lipofectamine
mixture was removed, and fresh culture medium containing 10% fetal
bovine serum was layered on the cells. One day later, fresh media
was added and the supernatant was collected 24 hours later for
determination of VEGF levels using a commercially available ELISA
kit (R and D Systems).
[0295] For the three-fingered VEGF1-specific ZFP (VEGF1-VP16), a
7-10 fold increase in VEGF expression was observed when compared to
control plasmid (NVF-control) and mock transfected cells (FIG. 9).
Similar results have been obtained in 5 independent experiments. It
is important to note that the level of VEGF secretion in VEGF1-VP16
transfected cells was equivalent or greater than the level in cells
that have been treated with DFX (FIG. 9). Introduction of
VEGF3a/1-VP16 stimulated a more modest induction of VEGF. This
result is consistent with the finding in Example VI, in which
expression of the 18-bp binding protein without a functional domain
prevented activation to a certain degree. This result suggested
that the tight binding of this protein to the start site of
transcription interferes with activation.
[0296] These data indicate that a designed ZFP is capable of
locating and binding to its target site on the chromosome,
presenting a transcriptional activation domain, and dramatically
enhancing the expression level of that gene. In particular, the
results indicate that ZFPs with a K.sub.d of less than about 25 nM
(e.g., VEGF1 has an average apparent K.sub.d of about 10 nM)
provide dramatic increases in expression.
Example VIII: RNase Protection Assay
[0297] To further substantiate the results in Examples VI and VII,
a ribonuclease protection assay (RPA) was performed to correlate
the increased level of VEGF protein with an increase in VEGF mRNA
levels (Example VII), and to correlate the decreased level of VEGF
protein with a decrease in VEGF mRNA levels (Example VI).
[0298] RNA was isolated from the transfected cells using an RNA
isolation kit (Pharmingen). Radiolabeled multi template probes,
which included a VEGF specific probe, were prepared by in vitro
transcription and hybridized overnight at 56.degree. C. to 5 .mu.g
of each of the RNAs from the experimental and control transfected
cells. The hybridization mixture was treated with RNase and the
protected probes were purified and subjected to 5% denaturing
polyacrylamide gel electrophoresis and the radioactivity was
evaluated by autoradiography. 293 cells transfected with the
VEGF1-VP16 had a 2-4 fold increase in the level of VEGF mRNA when
compared to cells transfected with NVF-control (FIG. 10A; see
Example VII for experimental details). The size of the protected
probe was identical to the size of the probe generated from the
control human RNA provided as a control for RNA integrity. (FIG.
10A).
[0299] In a separate experiment, the level of VEGF specific mRNA
was also quantitated in cells that had been transfected with a
VEGF-KRAB effector plasmid (FIG. 10B; see Example VI for
experimental details). The details of the transfection are
described in Example VI. A dramatic decrease in the level of VEGF
mRNA was observed when cells were transfected with the
VEGF3a/1-KRAB effector plasmid. No significant decrease in VEGF
mRNA was observed when cells were transfected with NKF-control or a
non-VEGF specific ZFP (CCR5-5-KRAB and CCR5-3-KRAB, which recognize
different CCR5 target sites).
[0300] This experiment demonstrates that the increase in VEGF
protein observed upon transfection with the VEGF1-VP16 chimeric
transcription factor is mediated by an increase in the level of
VEGF mRNA. Similarly, the decrease in VEGF protein observed upon
transfection with the VEGF3a/1-KRAB chimeric transcription factor
is mediated by a decrease in the level of VEGF mRNA.
Sequence CWU 1
1
40125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic exemplary motif peptide of C2H2 class of zinc finger
proteins (ZFP)MOD_RES(2)..(3)Any amino acidMOD_RES(4)..(5)Any amino
acid, may be present or absentMOD_RES(7)..(18)Any amino
acidMOD_RES(20)..(22)Any amino acidMOD_RES(23)..(24)Any amino acid,
may be present or absent 1Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa His Xaa Xaa Xaa Xaa Xaa
His 20 25 210DNAArtificial SequenceDescription of Artificial
Sequence Synthetic ZFP target site oligonucleotide with two
overlapping D-able subsitesmodified_base(1)..(2)a, c, t, or
gmodified_base(5)..(5)a, c, t, or gmodified_base(8)..(8)a, c, t, or
gmodified_base(9)..(9)a, c, t, or g; if g, then position 10 cannot
be g or tmodified_base(10)..(10)a, c, t, or g; if g or t, then
position 9 cannot be g 2nngkngknnn 10310DNAArtificial
SequenceDescription of Artificial Sequence Synthetic ZFP target
site oligonucleotide with three overlapping D-able
subsitesmodified_base(1)..(2)a, c, t, or gmodified_base(5)..(5)a,
c, t, or gmodified_base(8)..(8)a, c, t, or g 3nngkngkngk
1045PRTArtificial SequenceDescription of Artificial Sequence
Synthetic linker peptide 4Asp Gly Gly Gly Ser 1 5 55PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
5Thr Gly Glu Lys Pro 1 5 69PRTArtificial SequenceDescription of
Artificial Sequence Synthetic linker peptide 6Leu Arg Gln Lys Asp
Gly Glu Arg Pro 1 5 74PRTArtificial SequenceDescription of
Artificial Sequence Synthetic linker peptide 7Gly Gly Arg Arg 1
85PRTArtificial SequenceDescription of Artificial Sequence
Synthetic linker peptide 8Gly Gly Gly Gly Ser 1 5 98PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
9Gly Gly Arg Arg Gly Gly Gly Ser 1 5 109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
10Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 1112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
11Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10
1216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic linker peptide 12Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly
Gly Gly Ser Glu Arg Pro 1 5 10 15 1325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic ZFP target
site oligonucleotide region surrounding initiation site of vascular
endothelial growth factor (VEGF) gene containing two 9-base pair
target sitesmisc_feature(4)..(12)Upstream 9-base pair ZFP VEGF1
target sitemisc_feature(14)..(22)Downstream 9-base pair ZFP VEGF3a
target site 13agcggggagg atcgcggagg cttgg 2514298DNAArtificial
SequenceDescription of Artificial Sequence Synthetic VEGF1 ZFP
construct polynucleotide targeting upstream 9-base pair target site
in VEGF promoterCDS(2)..(298)VEGF1 14g gta ccc ata cct ggc aag aag
aag cag cac atc tgc cac atc cag ggc 49 Val Pro Ile Pro Gly Lys Lys
Lys Gln His Ile Cys His Ile Gln Gly 1 5 10 15 tgt ggt aaa gtt tac
ggc aca acc tca aat ctg cgt cgt cac ctg cgc 97Cys Gly Lys Val Tyr
Gly Thr Thr Ser Asn Leu Arg Arg His Leu Arg 20 25 30 tgg cac acc
ggc gag agg cct ttc atg tgt acc tgg tcc tac tgt ggt 145Trp His Thr
Gly Glu Arg Pro Phe Met Cys Thr Trp Ser Tyr Cys Gly 35 40 45 aaa
cgc ttc acc cgt tcg tca aac ctg cag cgt cac aag cgt acc cac 193Lys
Arg Phe Thr Arg Ser Ser Asn Leu Gln Arg His Lys Arg Thr His 50 55
60 acc ggt gag aag aaa ttt gct tgc ccg gag tgt ccg aag cgc ttc atg
241Thr Gly Glu Lys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met
65 70 75 80 cgt agt gac cac ctg tcc cgt cac atc aag acc cac cag aat
aag aag 289Arg Ser Asp His Leu Ser Arg His Ile Lys Thr His Gln Asn
Lys Lys 85 90 95 ggt gga tcc 298Gly Gly Ser 1599PRTArtificial
SequenceDescription of Artificial Sequence Synthetic VEGF1 ZFP
construct polypeptide targeting upstream 9-base pair target site in
VEGF promoter 15Val Pro Ile Pro Gly Lys Lys Lys Gln His Ile Cys His
Ile Gln Gly 1 5 10 15 Cys Gly Lys Val Tyr Gly Thr Thr Ser Asn Leu
Arg Arg His Leu Arg 20 25 30 Trp His Thr Gly Glu Arg Pro Phe Met
Cys Thr Trp Ser Tyr Cys Gly 35 40 45 Lys Arg Phe Thr Arg Ser Ser
Asn Leu Gln Arg His Lys Arg Thr His 50 55 60 Thr Gly Glu Lys Lys
Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met 65 70 75 80 Arg Ser Asp
His Leu Ser Arg His Ile Lys Thr His Gln Asn Lys Lys 85 90 95 Gly
Gly Ser 16298DNAArtificial SequenceDescription of Artificial
Sequence Synthetic VEGF3a ZFP construct polynucleotide targeting
downstream 9-base pair target site in VEGF
promoterCDS(2)..(298)VEGF3a 16g gta ccc ata cct ggc aag aag aag cag
cac atc tgc cac atc cag ggc 49 Val Pro Ile Pro Gly Lys Lys Lys Gln
His Ile Cys His Ile Gln Gly 1 5 10 15 tgt ggt aaa gtt tac ggc cag
tcc tcc gac ctg cag cgt cac ctg cgc 97Cys Gly Lys Val Tyr Gly Gln
Ser Ser Asp Leu Gln Arg His Leu Arg 20 25 30 tgg cac acc ggc gag
agg cct ttc atg tgt acc tgg tcc tac tgt ggt 145Trp His Thr Gly Glu
Arg Pro Phe Met Cys Thr Trp Ser Tyr Cys Gly 35 40 45 aaa cgc ttc
acc cgt tcg tca aac cta cag agg cac aag cgt aca cac 193Lys Arg Phe
Thr Arg Ser Ser Asn Leu Gln Arg His Lys Arg Thr His 50 55 60 acc
ggt gag aag aaa ttt gct tgc ccg gag tgt ccg aag cgc ttc atg 241Thr
Gly Glu Lys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met 65 70
75 80 cga agt gac gag ctg tca cga cat atc aag acc cac cag aac aag
aag 289Arg Ser Asp Glu Leu Ser Arg His Ile Lys Thr His Gln Asn Lys
Lys 85 90 95 ggt gga tcc 298Gly Gly Ser 1799PRTArtificial
SequenceDescription of Artificial Sequence Synthetic VEGF3a ZFP
construct targeting polypeptide downstream 9-base pair target site
in VEGF promoter 17Val Pro Ile Pro Gly Lys Lys Lys Gln His Ile Cys
His Ile Gln Gly 1 5 10 15 Cys Gly Lys Val Tyr Gly Gln Ser Ser Asp
Leu Gln Arg His Leu Arg 20 25 30 Trp His Thr Gly Glu Arg Pro Phe
Met Cys Thr Trp Ser Tyr Cys Gly 35 40 45 Lys Arg Phe Thr Arg Ser
Ser Asn Leu Gln Arg His Lys Arg Thr His 50 55 60 Thr Gly Glu Lys
Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met 65 70 75 80 Arg Ser
Asp Glu Leu Ser Arg His Ile Lys Thr His Gln Asn Lys Lys 85 90 95
Gly Gly Ser 1829DNAArtificial SequenceDescription of Artificial
Sequence Synthetic VEGF DNA target site 1 recognition (top) strand.
oligonucleotidemisc_feature(11)..(19)VEGF DNA ZFP target site 1
18catgcatagc ggggaggatc gccatcgat 291929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic VEGF DNA site
1 complementary (bottom) strand oligonucleotide 19atcgatggcg
atcctccccg ctatgcatg 292029DNAArtificial SequenceDescription of
Artificial Sequence Synthetic VEGF DNA target site 3 recognition
(top) strand oligonucleotidemisc_feature(11)..(19)VEGF DNA ZFP
target site 3 20catgcatatc gcggaggctt ggcatcgat 292129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic VEGF DNA
target site 3 complementary (bottom) strand oligonucleotide
21atcgatgcca agcctccgcg atatgcatg 292229DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer SPE7
22gagcagaatt cggcaagaag aagcagcac 292326DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
SPEamp12 23gtggtctaga cagctcgtca cttcgc 262428DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer SPE
amp13 24ggagccaagg ctgtggtaaa gtttacgg 282526DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
SPEamp11 25ggagaagctt ggatcctcat tatccc 262683DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide sequence ligated between XbaI and StyI sites
26tctagacaca tcaaaaccca ccagaacaag aaagacggcg gtggcagcgg caaaaagaaa
60cagcacatat gtcacatcca agg 832739DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer GB19 27gccatgccgg
tacccatacc tggcaagaag aagcagcac 392833DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer GB10
28cagatcggat ccacccttct tattctggtg ggt 3329589DNAArtificial
SequenceDescription of Artificial Sequence Synthetic designed
polynucleotide 6-finger ZFP VEGF3a/1 from KpnI to
BamHICDS(2)..(589)VEGF3a/1 29g gta ccc ata cct ggc aag aag aag cag
cac atc tgc cac atc cag ggc 49 Val Pro Ile Pro Gly Lys Lys Lys Gln
His Ile Cys His Ile Gln Gly 1 5 10 15 tgt ggt aaa gtt tac ggc cag
tcc tcc gac ctg cag cgt cac ctg cgc 97Cys Gly Lys Val Tyr Gly Gln
Ser Ser Asp Leu Gln Arg His Leu Arg 20 25 30 tgg cac acc ggc gag
agg cct ttc atg tgt acc tgg tcc tac tgt ggt 145Trp His Thr Gly Glu
Arg Pro Phe Met Cys Thr Trp Ser Tyr Cys Gly 35 40 45 aaa cgc ttc
aca cgt tcg tca aac cta cag agg cac aag cgt aca cac 193Lys Arg Phe
Thr Arg Ser Ser Asn Leu Gln Arg His Lys Arg Thr His 50 55 60 aca
ggt gag aag aaa ttt gct tgc ccg gag tgt ccg aag cgc ttc atg 241Thr
Gly Glu Lys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met 65 70
75 80 cga agt gac gag ctg tct aga cac atc aaa acc cac cag aac aag
aaa 289Arg Ser Asp Glu Leu Ser Arg His Ile Lys Thr His Gln Asn Lys
Lys 85 90 95 gac ggc ggt ggc agc ggc aaa aag aaa cag cac ata tgt
cac atc caa 337Asp Gly Gly Gly Ser Gly Lys Lys Lys Gln His Ile Cys
His Ile Gln 100 105 110 ggc tgt ggt aaa gtt tac ggc aca acc tca aat
ctg cgt cgt cac ctg 385Gly Cys Gly Lys Val Tyr Gly Thr Thr Ser Asn
Leu Arg Arg His Leu 115 120 125 cgc tgg cac acc ggc gag agg cct ttc
atg tgt acc tgg tcc tac tgt 433Arg Trp His Thr Gly Glu Arg Pro Phe
Met Cys Thr Trp Ser Tyr Cys 130 135 140 ggt aaa cgc ttc acc cgt tcg
tca aac ctg cag cgt cac aag cgt acc 481Gly Lys Arg Phe Thr Arg Ser
Ser Asn Leu Gln Arg His Lys Arg Thr 145 150 155 160 cac acc ggt gag
aag aaa ttt gct tgc ccg gag tgt ccg aag cgc ttc 529His Thr Gly Glu
Lys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe 165 170 175 atg cgt
agt gac cac ctg tcc cgt cac atc aag acc cac cag aat aag 577Met Arg
Ser Asp His Leu Ser Arg His Ile Lys Thr His Gln Asn Lys 180 185 190
aag ggt gga tcc 589Lys Gly Gly Ser 195 30196PRTArtificial
SequenceDescription of Artificial Sequence Synthetic designed
polypeptide 6-finger ZFP VEGF3a/1 from KpnI to BamHI 30Val Pro Ile
Pro Gly Lys Lys Lys Gln His Ile Cys His Ile Gln Gly 1 5 10 15 Cys
Gly Lys Val Tyr Gly Gln Ser Ser Asp Leu Gln Arg His Leu Arg 20 25
30 Trp His Thr Gly Glu Arg Pro Phe Met Cys Thr Trp Ser Tyr Cys Gly
35 40 45 Lys Arg Phe Thr Arg Ser Ser Asn Leu Gln Arg His Lys Arg
Thr His 50 55 60 Thr Gly Glu Lys Lys Phe Ala Cys Pro Glu Cys Pro
Lys Arg Phe Met 65 70 75 80 Arg Ser Asp Glu Leu Ser Arg His Ile Lys
Thr His Gln Asn Lys Lys 85 90 95 Asp Gly Gly Gly Ser Gly Lys Lys
Lys Gln His Ile Cys His Ile Gln 100 105 110 Gly Cys Gly Lys Val Tyr
Gly Thr Thr Ser Asn Leu Arg Arg His Leu 115 120 125 Arg Trp His Thr
Gly Glu Arg Pro Phe Met Cys Thr Trp Ser Tyr Cys 130 135 140 Gly Lys
Arg Phe Thr Arg Ser Ser Asn Leu Gln Arg His Lys Arg Thr 145 150 155
160 His Thr Gly Glu Lys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe
165 170 175 Met Arg Ser Asp His Leu Ser Arg His Ile Lys Thr His Gln
Asn Lys 180 185 190 Lys Gly Gly Ser 195 3142DNAArtificial
SequenceDescription of Artificial Sequence Synthetic JVF9 VEGF3a/1
target oligonucleotide 31agcgagcggg gaggatcgcg gaggcttggg
gcagccgggt ag 423242DNAArtificial SequenceDescription of Artificial
Sequence Synthetic JVF10 VEGF3a/1 target oligonucleotide
complementary sequence 32cgctctaccc ggctgcccca agcctccgcg
atcctccccg ct 423325DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer JVF24 33cgcggatccg cccccccgac cgatg
253462DNAArtificial SequenceDescription of Artificial Sequence
Synthetic downstream primer JVF25 34ccgcaagctt acttgtcatc
gtcgtccttg tagtcgctgc ccccaccgta ctcgtcaatt 60cc 62357PRTSimian
virus 40misc_feature(1)..(7)SV40 large T antigen nuclear
localization sequence (NLS) 35Pro Lys Lys Lys Arg Lys Val 1 5
3661DNAArtificial SequenceDescription of Artificial Sequence
Synthetic segment oligonucleotide from EcoRI to KpnI containing
Kozak sequence including initiation codon and SV40 NLS 36gaattcgcta
gcgccaccat ggcccccaag aagaagagga aggtgggaat ccatggggta 60c
6137187DNAArtificial SequenceDescription of Artificial Sequence
Synthetic segment polynucleotide from KpnI to XhoI containing BamHI
site, KRAB-A box from KOX1, FLAG epitope and HindIII site
37ggtacccggg gatcccggac actggtgacc ttcaaggatg tatttgtgga cttcaccagg
60gaggagtgga agctgctgga cactgctcag cagatcgtgt acagaaatgt gatgctggag
120aactataaga acctggtttc cttgggcagc gactacaagg acgacgatga
caagtaagct 180tctcgag 18738277DNAArtificial SequenceDescription of
Artificial Sequence Synthetic inserted fragment polynucleotide from
BamHI to HindIII sites 38ggatccgccc ccccgaccga tgtcagcctg
ggggacgagc tccacttaga cggcgaggac 60gtggcgatgg cgcatgccga cgcgctagac
gatttcgatc tggacatgtt gggggacggg 120gattccccgg ggccgggatt
taccccccac gactccgccc cctacggcgc tctggatatg 180gccgacttcg
agtttgagca gatgtttacc gatgcccttg gaattgacga gtacggtggg
240ggcagcgact acaaggacga cgatgacaag taagctt 27739118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
sequence replacing NLS-KRAB-FLAG with NLS-FLAG only 39gaattcgcta
gcgccaccat ggcccccaag aagaagagga aggtgggaat ccatggggta 60cccggggatg
gatccggcag cgactacaag gacgacgatg acaagtaagc ttctcgag
11840204DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
insert into MluI/BglII sites of pGL3-Control to create pVFR1-4x
40acgcgtaagc ttgctagcga gcggggagga tcgcggaggc ttggggcagc cgggtagagc
60gagcggggag gatcgcggag gcttggggca gccgggtaga gcgagcgggg aggatcgcgg
120aggcttgggg cagccgggta gagcgagcgg ggaggatcgc ggaggcttgg
ggcagccggg 180tagagcgctc agaagcttag atct 204
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