U.S. patent application number 11/019080 was filed with the patent office on 2005-07-28 for therapeutics and diagnostics for congenital heart disease based on a novel human transcription factor.
This patent application is currently assigned to University of Iowa Research Foundation. Invention is credited to Alward, Wallace L.M., Nishimura, Darryl, Patil, Shiva, Sheffield, Val C., Stone, Edwin M..
Application Number | 20050164256 11/019080 |
Document ID | / |
Family ID | 26766068 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164256 |
Kind Code |
A1 |
Sheffield, Val C. ; et
al. |
July 28, 2005 |
Therapeutics and diagnostics for congenital heart disease based on
a novel human transcription factor
Abstract
Methods and compositions for treating a congenital heart disease
and methods and compositions for prognosing or diagnosing a
congenital heart disease in a subject are disclosed.
Inventors: |
Sheffield, Val C.;
(Coralville, IA) ; Alward, Wallace L.M.; (Iowa
City, IA) ; Stone, Edwin M.; (Iowa City, IA) ;
Nishimura, Darryl; (Coralville, IA) ; Patil,
Shiva; (Iowa City, IA) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
University of Iowa Research
Foundation
|
Family ID: |
26766068 |
Appl. No.: |
11/019080 |
Filed: |
December 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11019080 |
Dec 21, 2004 |
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09612809 |
Jul 10, 2000 |
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6833239 |
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09612809 |
Jul 10, 2000 |
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09083351 |
May 22, 1998 |
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6087107 |
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60081870 |
Apr 15, 1998 |
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Current U.S.
Class: |
435/6.16 ;
435/7.1; 514/16.4 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4702 20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 514/002 |
International
Class: |
C12Q 001/68; G01N
033/53; A61K 038/17 |
Claims
1. A method for treating or preventing the development of a
congenital heart disease in a subject, comprising administering to
the subject an effective amount of an FKHL 7 therapeutic.
2-10. (canceled)
Description
1. BACKGROUND OF THE INVENTION
[0001] "Congenital heart disease" refers to defects in the heart
and major great vessels produced by abnormalities at various stages
of fetal development and present at birth, but which may not be
diagnosed until later. The incidence of such anomalies is 1/120
live births (The Merck Manual of Diagnosis and Therapy, 16.sup.th
Ed. (1992) p. 2051).
[0002] "Atrial septal defect" is form of congenital heart disease
in which there is an opening in the septum that normally separates
the atria. The typical murmur of atrial septal defect is usually
present after age 1 yr., when pulmonary blood flow has increased
significantly.
[0003] Many congenital heart diseases have a genetic basis.
However, surgery offers the only therapeutic option for many of
these disorders. In addition, current identification and diagnosis
of congenital heart disease depends on the recognition of affected
cardiac function, such as heart murmurs representing turbulent
flow, altered systemic and pulmonary blood flow, shunting in either
direction, and evidences of altered work load of the cardiac
chambers. Routine history, physical examination, ECG, and chest
x-ray are usually performed for specific anatomic diagnosis, with
supportive and confirmatory data from echocardiography, cardiac
catheterization, angiocardiography and other laboratory data.
[0004] Improved therapies and diagnostics for genetically based
congenital heart diseases are needed.
2. SUMMARY OF THE INVENTION
[0005] The present invention is based, at least in part, on the
discovery of a novel human gene, which encodes a novel human
protein. These newly identified genes and proteins are referred to
herein as "FKHL7". FKHL7 is a monomeric DNA binding protein that
shares a core binding site (RTAAYA) with four other FKHL7-like
proteins. In addition, the forkhead domain of this protein shows
strong homology to the human gene, FKHL14 and the mouse genes Fkhl
and Fkhl4 by BLASTN analysis.
[0006] A 9.8 kb subclone of BAC471g19 was partially sequenced and
determined to contain the entire coding region of FKHL7 as well as
5' and 3' untranslated sequences (SEQ ID NO. 1). The human FKHL7
coding sequence is 1.7 kb in size and contains no introns. The 1659
bp open reading frame (SEQ ID NO. 3) encodes a 553 amino acid
polypeptide (SEQ ID NO. 2). The COOH-terminal domain contains
several stretches of homopolymeric runs of alanine and glycine. The
FKHL7 coding region contains 5 recognition sites for the
restriction enzyme NotI. A BLASTN screen of the public dbEST
database with the FKHL7 genomic sequence yields only partial human
and mouse cDNA coverage of this gene. Based on the analysis of cDNA
clones identified in the public databases, there is evidence for
the utilization of at least two different polyadenylation signals
within the 3' untranslated region.
[0007] Human FKHL7 is most abundantly expressed during
embryogenesis and of the adult tissue tested, significant
expression was observed in adult eye, heart, kidney and lung, while
relatively little to no expression was observed in adult skeletal
muscle, spleen or liver.
[0008] In one aspect, the invention features isolated FKHL7 nucleic
acid molecules. In one embodiment, the FKHL7 nucleic acid is from a
vertebrate. In a preferred embodiment, the FKHL7 nucleic acid is
from a mammal, e.g. a human. In an even more preferred embodiment,
the nucleic acid has the nucleic acid sequence set forth in SEQ ID
NO. 1 or 3 or a portion thereof. The disclosed molecules can be
non-coding, (e.g. a probe, antisense, or ribozyme molecule) or can
encode a functional FKHL7 polypeptide (e.g. a polypeptide which
functions as either an agonist or antagonist of at least one
bioactivity of the human FKHL7 polypeptide). In one embodiment, the
nucleic acid of the present invention can hybridize to a vertebrate
FKHL7 gene or to the complement of a vertebrate FKHL7 gene. In a
further embodiment, the claimed nucleic acid can hybridize with a
nucleic acid sequence shown in FIG. 1 (SEQ ID NOS. 1 and 3) or a
complement thereof. In a preferred embodiment, the hybridization is
conducted under mildly stringent or stringent conditions.
[0009] In further embodiments, the nucleic acid molecule is an
FKHL7 nucleic acid that is at least about 70%, preferably about
80%, more preferably about 85%, and even more preferably at least
about 90% or 95% homologous to the nucleic acid shown as SEQ ID
NOS: 1 or 3 or to the complement of the nucleic acid shown as SEQ
ID NOS: 1 or 3.
[0010] The invention also provides probes and primers comprising
substantially purified oligonucleotides, which correspond to a
region of nucleotide sequence which hybridizes to at least about 6,
at least about 10, at least about 15, at least about 20, or
preferably at least about 25 consecutive nucleotides of the
sequence set forth as SEQ ID NO. 1 or SEQ ID NO. 3 or complements
of the sequence set forth as SEQ ID NOS. 1 or 3 or naturally
occurring mutants or allelic variants thereof. In preferred
embodiments, the probe/primer further includes a label group
attached thereto, which is capable of being detected.
[0011] For expression, the subject nucleic acids can be operably
linked to a transcriptional regulatory sequence, e.g., at least one
of a transcriptional promoter (e.g., for constitutive expression or
inducible expression) or transcriptional enhancer sequence. Such
regulatory sequences in conjunction with an FKHL7 nucleic acid
molecule can provide a useful vector for gene expression. This
invention also describes host cells transfected with said
expression vector whether prokaryotic or eukaryotic and in vitro
(e.g. cell culture) and in vivo (e.g. transgenic) methods for
producing FKHL7 proteins by employing said expression vectors.
[0012] In another aspect, the invention features isolated FKHL7
polypeptides, preferably substantially pure preparations, e.g. of
plasma purified or recombinantly produced polypeptides. The FKHL7
polypeptide can comprise a full length protein or can comprise
smaller fragments corresponding to one or more particular
motifs/domains, or fragments comprising at least about 5, 10, 25,
50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, 500, 510, 520, 530 or 540 amino acids in
length. In particularly preferred embodiments, the subject
polypeptide is capable of binding to an upstream region of a gene
and/or otherwise regulating expression of a gene.
[0013] In a preferred embodiment, the polypeptide is encoded by a
nucleic acid, which hybridizes with the nucleic acid sequence
represented in SEQ ID NOS. 1 or 3. In a further preferred
embodiment, the FKHL7 polypeptide is comprised of the amino acid
sequence set forth in SEQ ID NO. 2. The subject FKHL7 protein also
includes within its scope modified proteins, e.g. proteins which
are resistant to post-translational modification, for example, due
to mutations which alter modification sites (such as tyrosine,
threonine, serine or aspargine residues), or which prevent
glycosylation of the protein, or which prevent interaction of the
protein with intracellular proteins involved in signal
transduction.
[0014] The FKHL7 polypeptides of the present invention can be
glycosylated, or conversely, by choice of the expression system or
by modification of the protein sequence to preclude glycosylation,
reduced carbohydrate analogs can also be provided. Glycosylated
forms can be obtained, for example, based on derivatization with
glycosaminoglycan chains.
[0015] In yet another preferred embodiment, the invention features
a purified or recombinant polypeptide, which has the ability to
modulate, e.g., mimic or antagonize, an activity of a wild-type
FKHL7 protein. Preferably, the polypeptide comprises an amino acid
sequence identical or homologous to a sequence designated in SEQ ID
NO. 2.
[0016] Another aspect of the invention features chimeric molecules
(e.g., fusion proteins) comprising an FKHL7 protein. For instance,
the FKHL7 protein can be provided as a recombinant fusion protein
which includes a second polypeptide portion, e.g., a second
polypeptide having an amino acid sequence unrelated (heterologous)
to the FKHL7 polypeptide. A preferred FKHL7 fusion protein is an
immunoglobulin-FKHL7 fusion protein, in which an immunoglobulin
constant region is fused to an FKHL7 polypeptide.
[0017] Yet another aspect of the present invention concerns an
immunogen comprising an FKHL7 polypeptide in an immunogenic
preparation, the immunogen being capable of eliciting an immune
response specific for an FKHL7 polypeptide; e.g. a humoral
response, an antibody response and/or cellular response. In a
preferred embodiment, the immunogen comprises an antigenic
determinant, e.g. a unique determinant of a protein encoded by the
nucleic acid set forth in SEQ ID NO. 1 or 3; or as set forth in SEQ
ID NO. 2.
[0018] A still further aspect of the present invention features
antibodies and other binding proteins or peptides that are
specifically reactive with an epitope of an FKHL7 protein.
[0019] The invention also features transgenic non-human animals
which include (and preferably express) a heterologous form of an
FKHL7 gene described herein, or which misexpress an endogenous
FKHL7 gene (e.g., an animal in which expression of one or more of
the subject FKHL7 proteins is disrupted). Such transgenic animals
can serve as animal models for studying cellular and/or tissue
disorders comprising mutated or mis-expressed FKHL7 alleles or for
use in drug screening. Alternatively, such transgenic animals can
be useful for expressing recombinant FKHL7 polypeptides.
[0020] The invention further features assays and kits for
determining whether an individual's FKHL7 genes and/or proteins are
defective or deficient (e.g in activity and/or level), and/or for
determining the identity of FKHL7 alleles. In one embodiment, the
method comprises the step of determining the level of FKHL7
protein, the level of FKHL7 mRNA and/or the transcription rate of
an FKHL7 gene. In another preferred embodiment, the method
comprises detecting, in a tissue of the subject, the presence or
absence of a genetic alteration, which is characterized by at least
one of the following: a deletion of one or more nucleotides from a
gene; an addition of one or more nucleotides to the gene; a
substitution of one or more nucleotides of the gene; a gross
chromosomal rearrangement of the gene; an alteration in the level
of a messenger RNA transcript of the gene; the presence of a
non-wild type splicing pattern of a messenger RNA transcript of the
gene; and/or a non-wild type level of the FKHL7 protein.
[0021] FKHL7 mutations that are particularly likely to cause or
contribute to the development of congential heart disease include
mutations that result in an FKHL7 protein that lacks or contains a
substantially impaired FKHL7 gene.
[0022] FKHL7 mutations can be detected by: i) providing a
probe/primer comprised of an oligonucleotide which hybridizes to a
sense or antisense sequence of an FKHL7 gene or naturally occurring
mutants thereof, or 5' or 3' flanking sequences naturally
associated with the FKHL7 gene; (ii) contacting the probe/primer
with an appropriate nucleic acid containing sample; and (iii)
detecting, by hybridization of the probe/primer to the nucleic
acid, the presence or absence of the genetic alteration.
Particularly preferred embodiments comprise: 1) sequencing at least
a portion of an FKHL7 gene, 2) performing a single strand
conformation polymorphism (SSCP) analysis to detect differences in
electrophoretic mobility between mutant and wild-type nucleic
acids; and 3) detecting or quantitating the level of an FKHL7
protein in an immunoassay using an antibody which is specifically
immunoreactive with a wild-type or mutated FKHL7 protein.
[0023] Information obtained using the diagnostic assays described
herein (alone or in conjunction with information on another genetic
defect, which contributes to the same disease) is useful for
diagnosing or confirming that a symptomatic subject has a genetic
defect (e.g. in an FKHL7 gene or in a gene that regulates the
expression of an FKHL7 gene), which causes or contributes to the
particular disease or disorder. Alternatively, the information
(alone or in conjunction with information on another genetic
defect, which contributes to the same disease) can be used
prognostically for predicting whether a non-symptomatic subject is
likely to develop a disease or condition, which is caused by or
contributed to by an abnormal FKHL7 activity or protein level in a
subject. In particular, the assays permit one to ascertain an
individual's predilection to develop a condition associated with a
mutation in FKHL7, where the mutation is a single nucleotide
polymorphism (SNP). Based on the prognostic information, a doctor
can recommend a regimen (e.g. diet or exercise) or therapeutic
protocol useful for preventing or prolonging onset of a congenital
heart disease in the individual.
[0024] In addition, knowledge of the particular alteration or
alterations, resulting in defective or deficient FKHL7 genes or
proteins in an individual, alone or in conjunction with information
on other genetic defects contributing to the same disease (the
genetic profile of the particular disease) allows customization of
therapy to the individual's genetic profile, the goal of
pharmacogenomics. For example, an individual's FKHL7 genetic
profile or the genetic profile of the congenital heart disease can
enable a doctor to: 1) more effectively prescribe a drug that will
address the molecular basis of glaucoma; and 2) better determine
the appropriate dosage of a particular drug. For example, the
expression level of FKHL7 proteins, alone or in conjunction with
the expression level of other genes known to be involved in
glaucoma, can be measured in many patients at various stages of the
disease to generate a transcriptional or expression profile of the
congenital heart disease. Expression patterns of individual
patients can then be compared to the expression profile of the
congenital heart disease to determine the appropriate drug and dose
to administer to the patient.
[0025] The ability to target populations expected to show the
highest clinical benefit, based on the FKHL7 or congenital heart
disesae genetic profile, can enable: 1) the repositioning of
marketed drugs with disappointing market results; 2) the rescue of
drug candidates whose clinical development has been discontinued as
a result of safety or efficacy limitations, which are patient
subgroup-specific; and 3) an accelerated and less costly
development for drug candidates and more optimal drug labeling
(e.g. since the use of FKHL7 as a marker is useful for optimizing
effective dose).
[0026] In another aspect, the invention provides methods for
identifying a compound which modulates an FKHL7 activity, e.g. the
interaction between an FKHL7 polypeptide and a target peptide In a
preferred embodiment, the method includes the steps of (a) forming
a reaction mixture, which includes: (i) an FKHL7 polypeptide, (ii)
an FKHL7 binding partner and (iii) a test compound; and (b)
detecting interaction of the FKHL7 polypeptide and the FKHL7
binding partner. A statistically significant change (potentiation
or inhibition) in the interaction of the FKHL7 polypeptide and
FKHL7 binding partner in the presence of the test compound,
relative to the interaction in the absence of the test compound,
indicates a potential agonist (mimetic or potentiator) or
antagonist (inhibitor) of FKHL7 bioactivity for the test compound.
The reaction mixture can be a cell-free protein preparation, e.g.,
a reconstituted protein mixture or a cell lysate, or it can be a
recombinant cell including a heterologous nucleic acid
recombinantly expressing the FKHL7 binding partner.
[0027] In preferred embodiments, the step of detecting interaction
of the FKHL7 and FKHL7 binding partner is a competitive binding
assay. In other preferred embodiments, at least one of the FKHL7
polypeptide and the FKHL7 binding partner comprises a detectable
label, and interaction of the FKHL7 and FKHL7 binding partner is
quantified by detecting the label in the complex. The detectable
label can be, e.g., a radioisotope, a fluorescent compound, an
enzyme, or an enzyme co-factor. In other embodiments, the complex
is detected by an immunoassay.
[0028] Yet another exemplary embodiment provides an assay for
screening test compounds to identify agents which modulate the
amount of FKHL7 produced by a cell.
[0029] In one embodiment, the screening assay comprises contacting
a cell transfected with a reporter gene operably linked to an FKHL7
promoter with a test compound and determining the level of
expression of the reporter gene. The reporter gene can encode,
e.g., a gene product that gives rise to a detectable signal such
as: color, fluorescence, luminescence, cell viability, relief of a
cell nutritional requirement, cell growth, and drug resistance. For
example, the reporter gene can encode a gene product selected from
the group consisting of chloramphenicol acetyl transferase,
luciferase, beta-galactosidase and alkaline phosphatase.
[0030] Also within the scope of the invention are methods for
treating a congenital heart disease, comprising administering
(e.g., either locally or systemically) to a subject, a
pharmaceutically effective amount of a composition comprising an
FKHL7 therapeutic.
[0031] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
3. BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 is a DNA sequence of the human FKHL7 gene including
the 5' and 3' untranslated regions (UTRs) (SEQ. ID. No. 1). The
1659 base pair open reading frame is provided herein as SEQ ID NO.
3 and the (SEQ ID NO. 3) 553 amino acid human FKHL7 protein is
provided herein as SEQ ID No. 2. The forkhead region of the protein
is indicated by underline.
[0033] FIG. 2 shows an amino acid comparison of the forkhead
domains of different members of the FKHL-family of genes. The
locations of the three alpha helices and the two wing domains are
shown (Clark, K. L. et al., Nature 364: 412420 (1993)). The
Drosophila forkhead gene sequence is shown above that for FKHL7,
while the positions of the three missense mutations are shown below
FKHL7. Translation of the 11 base pair deletion (bp del) mutation
results in total loss of the forkhead domain. The other FKHL family
members are shown below FKHL7 for comparison. For FKHL10, only
partial sequence is available for the forkhead domain. The last
sequence shown is that for the distantly related FKHR which has
been mapped to 13Q14 near the RIEG2 locus.
[0034] FIG. 3 provides the identity and location of Expressed
Sequence Tags (ESTs) that map to regions of the human FKHL7
gene.
4. DETAILED DESCRIPTION OF THE INVENTION
[0035] 4.1. General
[0036] The present invention is based, at least in part, on the
discovery of a novel human gene, termed "hFKHL7" and the finding
that defects in the gene result in the development of congenital
heart disease. More particularly, a one base pair deletion upstream
of the FKHL7 forkhead domain, resulting in a truncated protein that
lacks the forkhead domain was found in two individuals from a
nuclear family. The proband was found to have Rieger anomaly
similar to other patients as well as an atrial septal defect. His
mother has Rieger anomaly. Both individuals were found to harbor
this mutation. However, the mutation was not found in 128 normal
Caucasian individuals. In addition, as shown in the following FIG.
3, of the 26 human ESTs identified in a BLASTN search, 5 were found
to be derived from a fetal heart library and 2 were from an aorta
cDNA library. Furthermore, an additional 4 ESTs were from a pooled
library of three tissues (melanocytes, uterus and fetal heart). The
fact that such a large proportion of the ESTs are derived from
heart further supports the finding that mutations in the gene can
result in congenital heart defects. Expression of FKHL7 by Northern
blot analysis has been confirmed in human and mouse heart.
[0037] hFKHL7 maps to human chromosome 6p25. The FKHL7 protein is a
monomeric DNA binding protein that shares a core binding site
(RTAAYA) with four other FKHL7-like proteins. The human FKHL7
coding sequence is 1.7 kb in size and contains no introns. The 1659
bp open reading frame (SEQ ID NO. 3) encodes a 553 amino acid
polypeptide (SEQ ID NO. 2). The first in-frame ATG was found to
match well with the Kozak consensus sequence (Kozak, M. Mamm.
Genome 7: 5630574 (1996) and Kozak, M Annu. Rev. Cell. Biol. 8:
197-225 (1992)). The COOH-terminal domain contains several
stretches of homopolymeric runs of alanine and glycine. The FKHL7
coding region contains 5 recognition sites for the restriction
enzyme NotI. A BLASTN screen of the public dbEST database with the
FKHL7 genomic sequence yields only partial human and mouse cDNA
coverage of this gene (SEE FIG. 1). Based on the analysis of cDNA
clones identified in the public databases, there is evidence for
the utilization of at least two different polyadenylation signals
within the 3' untranslated region.
[0038] Human FKHL7 is most abundantly expressed during
embryogenesis and of the adult tissues tested, significant
expression was observed in adult eye, heart, kidney and lung, while
relatively little to no expression was observed in adult skeletal
muscle, spleen or liver.
[0039] 4.2 Definitions
[0040] For convenience, the meaning of certain terms and phrases
employed in the specification, examples, and appended claims are
provided below.
[0041] The term "agonist", as used herein, is meant to refer to an
agent that mimics or upregulates (e.g. potentiates or supplements)
an FKHL7 bioactivity. An FKHL7 agonist can be a wild-type FKHL7
protein or derivative thereof having at least one bioactivity of
the wild-type FKHL7. An FKHL7 therapeutic can also be a compound
that upregulates expression of an FKHL7 gene or which increases at
least one bioactivity of an FKHL7 protein. An agonist can also be a
compound which increases the interaction of an FKHL7 polypeptide
with another molecule, e.g, an upstream region of a gene, which is
regulated by an FKHL7 transcription factor.
[0042] "Antagonist" as used herein is meant to refer to an agent
that down-regulates (e.g. suppresses or inhibits) at least one
FKHL7 bioactivity. An FKHL7 antagonist can be a compound which
inhibits or decreases the interaction between an FKHL7 protein and
another molecule, e.g, an upstream region of a gene, which is
regulated by an FKHL7 transcription factor. Accordingly, a
preferred antagonist is a compound which inhibits or decreases
binding to an upstream region of a gene, which is regulated by an
FKHL7 transcription factor and thereby blocks subsequent activation
of the FKHL7. An antagonist can also be a compound that
downregulates expression of an FKHL7 gene or which reduces the
amount of FKHL7 protein present. The FKHL7 antagonist can be a
dominant negative form of an FKHL7 polypeptide, e.g., a form of an
FKHL7 polypeptide which is capable of interacting with an upstream
region of a gene, which is regulated by an FKHL7 transcription
factor, but which is not capable of regulating transcription. The
FKHL7 antagonist can also be a nucleic acid encoding a dominant
negative form of an FKHL7 polypeptide, an FKHL7 antisense nucleic
acid, or a ribozyme capable of interacting specifically with an
FKHL7 RNA. Yet other FKHL7 antagonists are molecules which bind to
an FKHL7 polypeptide and inhibit its action. Such molecules include
peptides, antibodies and small molecules.
[0043] The term "allele", which is used interchangeably herein with
"allelic variant" refers to alternative forms of a gene or portions
thereof. Alleles occupy the same locus or position on homologous
chromosomes. When a subject has two identical alleles of a gene,
the subject is said to be homozygous for the gene or allele. When a
subject has two different alleles of a gene, the subject is said to
be heterozygous for the gene. Alleles of a specific gene can differ
from each other in a single nucleotide, or several nucleotides, and
can include substitutions, deletions, and insertions of
nucleotides. An allele of a gene can also be a form of a gene
containing a mutation. The term "allelic variant of a polymorphic
region of an FKHL7 gene" refers to a region of an FKHL7 gene having
one or several nucleotide sequences found in that region of the
gene in other individuals.
[0044] "Biological activity" or "bioactivity" or "activity" or
"biological function", which are used interchangeably, for the
purposes herein means an effector or antigenic function that is
directly or indirectly performed by an FKHL7 polypeptide (whether
in its native or denatured conformation), or by any subsequence
thereof. Biological activities include binding to a target nucleic
acid e.g, an upstream region of a gene, which is regulated by an
FKHL7 transcription factor. An FKHL7 bioactivity can be modulated
by directly affecting an FKHL7 polypeptide. Alternatively, an FKHL7
bioactivity can be modulated by modulating the level of an FKHL7
polypeptide, such as by modulating expression of an FKHL7 gene.
[0045] As used herein the term "bioactive fragment of an FKHL7
polypeptide" refers to a fragment of a full-length FKHL7
polypeptide, wherein the fragment specifically mimics or
antagonizes the activity of a wild-type FKHL7 polypeptide. The
bioactive fragment preferably is a fragment capable of interacting
with e.g, an upstream region of a gene, which is regulated by an
FKHL7 transcription factor.
[0046] The term "an aberrant activity", as applied to an activity
of a polypeptide such as FKHL7, refers to an activity which differs
from the activity of the wild-type or native polypeptide or which
differs from the activity of the polypeptide in a healthy subject.
An activity of a polypeptide can be aberrant because it is stronger
than the activity of its native counterpart. Alternatively, an
activity can be aberrant because it is weaker or absent relative to
the activity of its native counterpart. An aberrant activity can
also be a change in an activity. For example an aberrant
polypeptide can interact with a different target peptide. A cell
can have an aberrant FKHL7 activity due to overexpression or
underexpression of the gene encoding FKHL7.
[0047] "Cells", "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0048] A "chimeric polypeptide" or "fusion polypeptide" is a fusion
of a first amino acid sequence encoding one of the subject FKHL7
polypeptides with a second amino acid sequence defining a domain
(e.g. polypeptide portion) foreign to and not substantially
homologous with any domain of an FKHL7 polypeptide. A chimeric
polypeptide may present a foreign domain which is found (albeit in
a different polypeptide) in an organism which also expresses the
first polypeptide, or it may be an "interspecies", "intergenic",
etc. fusion of polypeptide structures expressed by different kinds
of organisms. In general, a fusion polypeptide can be represented
by the general formula X-FKHL7-Y, wherein FKHL7 represents a
portion of the polypeptide which is derived from an FKHL7
polypeptide, and X and Y are independently absent or represent
amino acid sequences which are not related to an FKHL7 sequence in
an organism, including naturally occurring mutants.
[0049] "Congenital heart disease" refers to defects in the heart
and major great vessels produced by abnormalities at various stages
of fetal development and present at birth, but which may not be
diagnosed until later. Examples include: ventricular septal defect,
atrial septal defect, patent ductus arteriosus, atrioventricular
canal defects, congential aortic valve stenosis, pulmonic valve
stenosis, peripheral pulmonic stenosis, coarctation of the aorta,
tetralogy of fallot, transposition of the great arteries, complex
cyanotic congenital heart disease and underdeveloped left ventricle
syndrome.
[0050] The term "nucleotide sequence complementary to the
nucleotide sequence set forth in SEQ ID NO. x" refers to the
nucleotide sequence of the complementary strand of a nucleic acid
strand having SEQ ID NO. x. The term "complementary strand" is used
herein interchangeably with the term "complement". The complement
of a nucleic acid strand can be the complement of a coding strand
or the complement of a non-coding strand. When referring to double
stranded nucleic acids, the complement of a nucleic acid having SEQ
ID NO. x refers to the complementary strand of the strand having
SEQ ID NO. x or to any nucleic acid having the nucleotide sequence
of the complementary strand of SEQ ID NO. x. When referring to a
single stranded nucleic acid having the nucleotide sequence SEQ ID
NO. x, the complement of this nucleic acid is a nucleic acid having
a nucleotide sequence which is complementary to that of SEQ ID NO.
x. The nucleotide sequences and complementary sequences thereof are
always given in the 5' to 3' direction.
[0051] A "delivery complex" shall mean a targeting means (e.g. a
molecule that results in higher affinity binding of a gene,
protein, polypeptide or peptide to a target cell surface and/or
increased cellular or nuclear uptake by a target cell). Examples of
targeting means include: sterols (e.g. cholesterol), lipids (e.g. a
cationic lipid, virosome or liposome), viruses (e.g. adenovirus,
adeno-associated virus, and retrovirus) or target cell specific
binding agents (e.g. ligands recognized by target cell specific
receptors). Preferred complexes are sufficiently stable in vivo to
prevent significant uncoupling prior to internalization by the
target cell. However, the complex is cleavable under appropriate
conditions within the cell so that the gene, protein, polypeptide
or peptide is released in a functional form.
[0052] As is well known, genes may exist in single or multiple
copies within the genome of an individual. Such duplicate genes may
be identical or may have certain modifications, including
nucleotide substitutions, additions or deletions, which all still
code for polypeptides having substantially the same activity. The
term "DNA sequence encoding an FKHL7 polypeptide" may thus refer to
one or more genes within a particular individual. Moreover, certain
differences in nucleotide sequences may exist between individual
organisms, which are called alleles. Such allelic differences may
or may not result in differences in amino acid sequence of the
encoded polypeptide, yet still encode a polypeptide with the same
biological activity.
[0053] The term "FKHL7 nucleic acid" refers to a nucleic acid
encoding an FKHL7 protein, such as nucleic acids having SEQ ID NOs.
1 or 3, as well as fragments thereof, complements thereof, and
derivatives thereof.
[0054] The terms "FKHL7 polypeptide" and "FKHL7 protein" are
intended to encompass polypeptides comprising the amino acid
sequence shown as SEQ ID NO. 2 or fragments thereof, and homologs
thereof and include agonist and antagonist polypeptides.
[0055] The term "FKHL7 therapeutic" refers to various forms of
FKHL7 polypeptides, as well as peptidomimetics, nucleic acids, or
small molecules, which can modulate at least one activity of an
FKHL7 polypeptide, e.g., binding to and/or otherwise regulating
expression of a gene, by mimicking or potentiating (agonizing) or
inhibiting (antagonizing) the effects of a naturally-occurring
FKHL7 polypeptide. An FKHL7 therapeutic which mimics or potentiates
the activity of a wild-type FKHL7 polypeptide is a "FKHL7 agonist".
Conversely, an FKHL7 therapeutic which inhibits the activity of a
wild-type FKHL7 polypeptide is a "FKHL7 antagonist".
[0056] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are identical at that position. A
degree of homology or similarity or identity between nucleic acid
sequences is a function of the number of identical or matching
nucleotides at positions shared by the nucleic acid sequences. An
"unrelated" or "non-homologous" sequence shares less than about 40%
identity, though preferably less than about 25% identity, with one
of the FKHL7 sequences of the present invention.
[0057] The term "interact" as used herein is meant to include
detectable relationships or associations (e.g. biochemical
interactions) between molecules, such as interaction between
protein-protein, protein-nucleic acid, nucleic acid-nucleic acid,
and protein-small molecule or nucleic acid-small molecule in
nature.
[0058] The term "isolated" as used herein with respect to nucleic
acids, such as DNA or RNA, refers to molecules separated from other
DNAs, or RNAs, respectively, that are present in the natural source
of the macromolecule. For example, an isolated nucleic acid
encoding one of the subject FKHL7 polypeptides preferably includes
no more than about 10 kilobases (kb) of nucleic acid sequence which
naturally immediately flanks the FKHL7 gene in genomic DNA, more
preferably no more than about 5 kb of such naturally occurring
flanking sequences, and most preferably less than about 1.5 kb of
such naturally occurring flanking sequence. The term isolated as
used herein also refers to a nucleic acid or peptide that is
substantially free of cellular material, viral material, or culture
medium when produced by recombinant DNA techniques, or chemical
precursors or other chemicals when chemically synthesized.
Moreover, an "isolated nucleic acid" is meant to include nucleic
acid fragments which are not naturally occurring as fragments and
would not be found in the natural state.
[0059] The term "isolated" is also used herein to refer to
polypeptides which are isolated from other cellular proteins and is
meant to encompass both purified and recombinant polypeptides. The
term "modulation" as used herein refers to both upregulation (i.e.,
activation or stimulation (e.g., by agonizing or potentiating)) and
downregulation (i.e. inhibition or suppression (e.g., by
antagonizing, decreasing or inhibiting)).
[0060] The term "mutated gene" refers to an allelic form of a gene,
which is capable of altering the phenotype of a subject having the
mutated gene relative to a subject which does not have the mutated
gene. If a subject must be homozygous for this mutation to have an
altered phenotype, the mutation is said to be recessive. If one
copy of the mutated gene is sufficient to alter the genotype of the
subject, the mutation is said to be dominant. If a subject has one
copy of the mutated gene and has a phenotype that is intermediate
between that of a homozygous and that of a heterozygous subject
(for that gene), the mutation is said to be co-dominant.
[0061] The "non-human animals" of the invention include mammals
such as rodents, non-human primates, sheep, dog, cow, chickens,
amphibians, reptiles, etc. Preferred non-human animals are selected
from the rodent family including rat and mouse, most preferably
mouse, though transgenic amphibians, such as members of the Xenopus
genus, and transgenic chickens can also provide important tools for
understanding and identifying agents which can affect, for example,
embryogenesis and tissue formation. The term "chimeric animal" is
used herein to refer to animals in which the recombinant gene is
found, or in which the recombinant gene is expressed in some but
not all cells of the animal. The term "tissue-specific chimeric
animal" indicates that one of the recombinant FKHL7 genes is
present and/or expressed or disrupted in some tissues but not
others.
[0062] As used herein, the term "nucleic acid" refers to
polynucleotides or oligonucleotides such as deoxyribonucleic acid
(DNA), and, where appropriate, ribonucleic acid (RNA). The term
should also be understood to include, as equivalents, analogs of
either RNA or DNA made from nucleotide analogs and as applicable to
the embodiment being described, single (sense or antisense) and
double-stranded polynucleotides.
[0063] The term "polymorphism" refers to the coexistence of more
than one form of a gene or portion (e.g., allelic variant) thereof.
A portion of a gene of which there are at least two different
forms, i.e., two different nucleotide sequences, is referred to as
a "polymorphic region of a gene". A polymorphic region can be a
single nucleotide, the identity of which differs in different
alleles. A polymorphic region can also be several nucleotides
long.
[0064] A "polymorphic gene" refers to a gene having at least one
polymorphic region.
[0065] As used herein, the term "promoter" refers to a DNA sequence
that regulates expression of a selected DNA sequence operably
linked to the promoter, and which effects expression of the
selected DNA sequence in cells. The term encompasses "tissue
specific" promoters, i.e. promoters, which effect expression of the
selected DNA sequence only in specific cells (e.g. cells of a
specific tissue). The term also covers so-called "leaky" promoters,
which regulate expression of a selected DNA primarily in one
tissue, but cause expression in other tissues as well. The term
also encompasses non-tissue specific promoters and promoters that
constitutively express or that are inducible (i.e. expression
levels can be controlled).
[0066] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0067] The term "recombinant protein" refers to a polypeptide of
the present invention which is produced by recombinant DNA
techniques, wherein generally, DNA encoding an FKHL7 polypeptide is
inserted into a suitable expression vector which is in turn used to
transform a host cell to produce the heterologous protein.
Moreover, the phrase "derived from", with respect to a recombinant
FKHL7 gene, is meant to include within the meaning of "recombinant
protein" those proteins having an amino acid sequence of a native
FKHL7 polypeptide, or an amino acid sequence similar thereto which
is generated by mutations including substitutions and deletions
(including truncation) of a naturally occurring form of the
polypeptide.
[0068] "Small molecule" as used herein, is meant to refer to a
composition, which has a molecular weight of less than about 5 kD
and most preferably less than about 4 kD. Small molecules can be
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic (carbon containing) or
inorganic molecules. Many pharmaceutical companies have extensive
libraries of chemical and/or biological mixtures, often fungal,
bacterial, or algal extracts, which can be screened with any of the
assays of the invention to identify compounds that modulate an
FKHL7 bioactivity.
[0069] As used herein, the term "specifically hybridizes" or
"specifically detects" refers to the ability of a nucleic acid
molecule of the invention to hybridize to at least approximately 6,
12, 20, 30, 50, 100, 150, 200, 300, 350, 400, 425, 450, 475 or 500
consecutive nucleotides of a vertebrate gene, preferably an FKHL7
gene.
[0070] "Transcriptional regulatory sequence" is a generic term used
throughout the specification to refer to DNA sequences, such as
initiation signals, enhancers, and promoters, which induce or
control transcription of protein coding sequences with which they
are operably linked. In preferred embodiments, transcription of one
of the FKHL7 genes is under the control of a promoter sequence (or
other transcriptional regulatory sequence) which controls the
expression of the recombinant gene in a cell-type in which
expression is intended. It will also be understood that the
recombinant gene can be under the control of transcriptional
regulatory sequences which are the same or which are different from
those sequences which control transcription of the
naturally-occurring forms of a FKHL7 polypeptide.
[0071] As used herein, the term "transfection" means the
introduction of a nucleic acid, e.g., via an expression vector,
into a recipient cell by nucleic acid-mediated gene transfer.
"Transformation", as used herein, refers to a process in which a
cell's genotype is changed as a result of the cellular uptake of
exogenous DNA or RNA, and, for example, the transformed cell
expresses a recombinant form of an FKHL7 polypeptide or, in the
case of anti-sense expression from the transferred gene, the
expression of a naturally-occurring form of the FKHL7 polypeptide
is disrupted.
[0072] As used herein, the term. "transgene" means a nucleic acid
sequence (encoding, e.g., one of the FKHL7 polypeptides, or an
antisense transcript thereto) which has been introduced into a
cell. A transgene could be partly or entirely heterologous, i.e.,
foreign, to the transgenic animal or cell into which it is
introduced, or, can be homologous to an endogenous gene of the
transgenic animal or cell into which it is introduced, but which is
designed to be inserted, or is inserted, into the animal's genome
in such a way as to alter the genome of the cell into which it is
inserted (e.g., it is inserted at a location which differs from
that of the natural gene or its insertion results in a knockout). A
transgene can also be present in a cell in the form of an episome.
A transgene can include one or more transcriptional regulatory
sequences and any other nucleic acid, such as introns, that may be
necessary for optimal expression of a selected nucleic acid.
[0073] A "transgenic animal" refers to any animal, preferably a
non-human mammal, bird or an amphibian, in which one or more of the
cells of the animal contain heterologous nucleic acid introduced by
way of human intervention, such as by transgenic techniques well
known in the art. The nucleic acid is introduced into the cell,
directly or indirectly by introduction into a precursor of the
cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with a recombinant virus. The term
genetic manipulation does not include classical cross-breeding, or
in vitro fertilization, but rather is directed to the introduction
of a recombinant DNA molecule. This molecule may be integrated
within a chromosome, or it may be extrachromosomally replicating
DNA. In the typical transgenic animals described herein, the
transgene causes cells to express a recombinant form of one of the
FKHL7 polypeptides, e.g. either agonistic or antagonistic forms.
However, transgenic animals in which the recombinant FKHL7 gene is
silent are also contemplated, as for example, the FLP or CRE
recombinase dependent constructs described below. Moreover,
"transgenic animal" also includes those recombinant animals in
which gene disruption of one or more FKHL7 genes is caused by human
intervention, including both recombination and antisense
techniques.
[0074] The term "treating" as used herein is intended to encompass
curing as well as ameliorating at least one symptom of the
condition or disease.
[0075] The term "vector" refers to a nucleic acid molecule, which
is capable of transporting another nucleic acid to which it has
been linked. One type of preferred vector is an episome, i.e., a
nucleic acid capable of extra-chromosomal replication. Preferred
vectors are those capable of autonomous replication and/or
expression of nucleic acids to which they are linked. Vectors
capable of directing the expression of genes to which they are
operatively linked are referred to herein as "expression vectors".
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of "plasmids" which refer
generally to circular double stranded DNA loops which, in their
vector form are not bound to the chromosome. In the present
specification, "plasmid" and "vector" are used interchangeably as
the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of expression
vectors which serve equivalent functions and which become known in
the art subsequently hereto.
[0076] The term "wild-type allele" refers to an allele of a gene
which, when present in two copies in a subject results in a
wild-type phenotype. There can be several different wild-type
alleles of a specific gene, since certain nucleotide changes in a
gene may not affect the phenotype of a subject having two copies of
the gene with the nucleotide changes.
[0077] 4.3. Nucleic Acids of the Present Invention
[0078] The invention provides FKHL7 nucleic acids, homologs
thereof, and portions thereof. Preferred nucleic acids have a
sequence, which is at least about 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, and more preferably 85% homologous with a nucleotide
sequence of an FKHL7 gene, e.g., such as a sequence shown in one of
SEQ ID NOS: 1 or 3 or complements thereof. Nucleic acids at least
90%, more preferably 95%, and most preferably at least about 98-99%
homologous with a nucleic sequence represented in one of SEQ ID
NOS. 1 or 3 or a complement thereof are of course also within the
scope of the invention.
[0079] The invention also pertains to isolated nucleic acids
comprising a nucleotide sequence encoding FKHL7 polypeptides,
variants and/or equivalents of such nucleic acids. The term
equivalent is understood to include nucleotide sequences encoding
functionally equivalent FKHL7 polypeptides or functionally
equivalent peptides having an activity of an FKHL7 protein such as
described herein. Equivalent nucleotide sequences will include
sequences that differ by one or more nucleotide substitutions,
additions or deletions, such as allelic variants; and therefore
includes sequences that differ from the nucleotide sequence of the
FKHL7 gene shown in SEQ ID NOS. 1 or 3, due to the degeneracy of
the genetic code.
[0080] Preferred nucleic acids are vertebrate FKHL7 nucleic acids.
Particularly preferred vertebrate FKHL7 nucleic acids are
mammalian. Regardless of species, particularly preferred FKHL7
nucleic acids encode polypeptides that are at least about 60%, 65%,
70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to
an amino acid sequence of a vertebrate FKHL7 protein. In one
embodiment, the nucleic acid is a cDNA encoding a polypeptide
having at least one bio-activity of the subject FKHL7 polypeptide.
Preferably, the nucleic acid includes all or a portion of the
nucleotide sequence corresponding to the nucleic acid of SEQ ID
NOS. 1 or 3.
[0081] Still other preferred nucleic acids of the present invention
encode an FKHL7 polypeptide which is comprised of at least 50, 100,
150, 200, 250, 300, 350, 400, 450 or 500 amino acid residues. For
example, such nucleic acids can comprise about 150, 300, 450, 600,
750, 900, 1050, 1200, 1350 or 1500 base pairs. Also within the
scope of the invention are nucleic acid molecules for use as
probes/primer or antisense molecules (i.e. noncoding nucleic acid
molecules), which can comprise at least about 6, 12, 20, 30, 50,
60, 70, 80, 90 or 100 base pairs in length.
[0082] Another aspect of the invention provides a nucleic acid
which hybridizes under stringent conditions to a nucleic acid
represented by SEQ ID NOS. 1 or 3 or a complement thereof.
Appropriate stringency conditions which promote DNA hybridization,
for example, 6.0.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C., are known to those skilled in the art or can be
found in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration
in the wash step can be selected from a low stringency of about
2.0.times.SSC at 50.degree. C. to a high stringency of about
0.2.times.SSC at 50.degree. C. In addition, the temperature in the
wash step can be increased from low stringency conditions at room
temperature, about 22.degree. C., to high stringency conditions at
about 65.degree. C. Both temperature and salt may be varied, or
temperature and salt concentration may be held constant while the
other variable is changed. In a preferred embodiment, an FKHL7
nucleic acid of the present invention will bind to one of SEQ ID
NOS. 1 or 3 or complement thereof under moderately stringent
conditions, for example at about 2.0.times.SSC and about 40.degree.
C. In a particularly preferred embodiment, an FKHL7 nucleic acid of
the present invention will bind to one of SEQ ID NOS. 1 or 3 or a
complement thereof under high stringency conditions.
[0083] Nucleic acids having a sequence that differs from the
nucleotide sequences shown in one of SEQ ID NOS. 1 or 3 or a
complement thereof due to degeneracy in the genetic code are also
within the scope of the invention. Such nucleic acids encode
functionally equivalent peptides (i.e., peptides having a
biological activity of an FKHL7 polypeptide) but differ in sequence
from the sequence shown in the sequence listing due to degeneracy
in the genetic code. For example, a number of amino acids are
designated by more than one triplet. Codons that specify the same
amino acid, or synonyms (for example, CAU and CAC each encode
histidine) may result in "silent" mutations which do not affect the
amino acid sequence of an FKHL7 polypeptide. However, it is
expected that DNA sequence polymorphisms that do lead to changes in
the amino acid sequences of the subject FKHL7 polypeptides will
exist among mammals. One skilled in the art will appreciate that
these variations in one or more nucleotides (e.g., up to about 3-5%
of the nucleotides) of the nucleic acids encoding polypeptides
having an activity of an FKHL7 polypeptide may exist among
individuals of a given species due to natural allelic
variation.
[0084] The polynucleotide of the present invention may also be
fused in frame to a marker sequence, also referred to herein as
"Tag sequence" encoding a "Tag peptide", which allows for marking
and/or purification of the polypeptide of the present invention. In
a preferred embodiment, the marker sequence is a hexahistidine tag,
e.g., supplied by a PQE-9 vector. Numerous other Tag peptides are
available commercially. Other frequently used Tags include
myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:
21150-21157) which includes a 10-residue sequence from c-myc, the
pFLAG system (International Biotechnologies, Inc.), the
pEZZ-protein A system (Pharmacia, NJ), and a 16 amino acid portion
of the Haemophilus influenza hemagglutinin protein. Furthermore,
any polypeptide can be used as a Tag so long as a reagent, e.g., an
antibody interacting specifically with the Tag polypeptide is
available or can be prepared or identified.
[0085] In another embodiment, a fusion gene coding for a
purification leader sequence, such as a poly-(His)/enterokinase
cleavage site sequence at the N-terminus of the desired portion of
the recombinant protein, can allow purification of the expressed
fusion protein by affinity chromatography using a Ni.sup.2+ metal
resin. The purification leader sequence can then be subsequently
removed by treatment with enterokinase to provide the purified
protein (e.g., see Hochuli et al. (1987) J. Chromatography 411:
177; and Janknecht et al. PNAS 88: 8972).
[0086] Techniques for making fusion genes are known to those
skilled in the art. Essentially, the joining of various DNA
fragments coding for different polypeptide sequences is performed
in accordance with conventional techniques, employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed to generate a chimeric
gene sequence (see, for example, Current Protocols in Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
[0087] Other preferred FKHL7 fusion proteins include
FKHL7-immunoglobulin (FKHL7-Ig) polypeptides. An FKHL7-Ig
polypeptide can comprise the entire extracellular domain of FKHL7,
e.g, human FKHL7, or a variant thereof. For example, an FKHL7-Ig
fusion proteins can be prepared as described e.g., in U.S. Pat. No.
5,434,131.
[0088] As indicated by the examples set out below, FKHL7
protein-encoding nucleic acids can be obtained from mRNA present in
any of a number of eukaryotic cells, e.g., from cardiac tissue. It
should also be possible to obtain nucleic acids encoding FKHL7
polypeptides of the present invention from genomic DNA from both
adults and embryos. For example, a gene encoding an FKHL7 protein
can be cloned from either a cDNA or a genomic library in accordance
with protocols described herein, as well as those generally known
to persons skilled in the art. cDNA encoding an FKHL7 protein can
be obtained by isolating total mRNA from a cell, e.g., a vertebrate
cell, a mammalian cell, or a human cell, including embryonic cells.
Double stranded cDNAs can then be prepared from the total mRNA, and
subsequently inserted into a suitable plasmid or bacteriophage
vector using any one of a number of known techniques. The gene
encoding an FKHL7 protein can also be cloned using established
polymerase chain reaction techniques in accordance with the
nucleotide sequence information provided by the invention. The
nucleic acid of the invention can be DNA or RNA or analogs thereof.
A preferred nucleic acid is a cDNA represented by a sequence
selected from the group consisting of SEQ ID NOS. 1 or 3.
[0089] Preferred nucleic acids encode a vertebrate FKHL7
polypeptide comprising an amino acid sequence that is at least
about 60% homologous, more preferably at least about 70% homologous
and most preferably at least about 80% homologous with an amino
acid sequence contained in SEQ ID NO. 2. Nucleic acids which encode
polypeptides with at least about 90%, more preferably at least
about 95%, and most preferably at least about 98-99% homology with
an amino acid sequence represented in SEQ ID NO. 2 are also within
the scope of the invention. In one embodiment, the nucleic acid is
a cDNA encoding a peptide having at least one activity of the
subject vertebrate FKHL7 polypeptide. Preferably, the nucleic acid
includes all or a portion of the nucleotide sequence corresponding
to the coding region of SEQ ID NOS. 1 or 3.
[0090] Preferred nucleic acids encode a bioactive fragment of a
vertebrate FKHL7 polypeptide comprising an amino acid sequence,
which is at least about 60% homologous or identical, more
preferably at least about 70% homologous or identical, still more
preferably at least about 75% homologous or identical and most
preferably at least about 80% homologous or identical with an amino
acid sequence of SEQ ID NO. 2. Nucleic acids which encode
polypeptides which are at least about 90%, more preferably at least
about 95%, and most preferably at least about 98-99% homologous or
identical, with an amino acid sequence represented in SEQ ID NO. 2
are also within the scope of the invention.
[0091] Bioactive fragments of FKHL7 polypeptides can be
polypeptides, which bind upstream of and/or regulate the expression
of a gene. Assays for determining whether an FKHL7 polypeptide has
any of these or other biological activities are known in the art
and are further described herein.
[0092] Nucleic acids encoding modified forms or mutant forms of
FKHL7 also include those encoding FKHL7 proteins having mutated
glycosylation sites, such that either the encoded FKHL7 protein is
not glycosylated, partially glycosylated and/or has a modified
glycosylation pattern.
[0093] Other preferred nucleic acids of the invention include
nucleic acids encoding derivatives of FKHL7 polypeptides which lack
one or more biological activities of FKHL7 polypeptides. Such
nucleic acids can be obtained, e.g., by a first round of screening
of libraries for the presence or absence of a first activity and a
second round of screening for the presence or absence of another
activity.
[0094] Also within the scope of the invention are nucleic acids
encoding splice variants or nucleic acids representing transcripts
synthesized from an alternative transcriptional initiation site,
such as those whose transcription was initiated from a site in an
intron.
[0095] In preferred embodiments, the FKHL7 nucleic acids can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids
(see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4
(1): 5-23). As used herein, the terms "peptide nucleic acids" or
"PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. PNAS 93:
14670-675.
[0096] PNAs of FKHL7 can be used in therapeutic and diagnostic
applications and are further described herein. Such modified
nucleic acids can be used as antisense or antigene agents for
sequence-specific modulation of gene expression or in the analysis
of single base pair mutations in a gene by, e.g., PNA directed PCR
clamping or as probes or primers for DNA sequence and hybridization
(Hyrup B. et al (1996) supra; Perry-O'Keefe supra).
[0097] PNAs of FKHL7 can further be modified, e.g., to enhance
their stability or cellular uptake, e.g., by attaching lipophilic
or other helper groups to the FKHL7 PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. FKHL7 PNAs can also be linked to
DNA as described, e.g., in Hyrup B. (1996) supra and Finn P. J. et
al. (1996) Nucleic Acids Research 24 (17): 3357-63. For example, a
DNA chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry and modified nucleoside analogs,
e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite,
can be used between the PNA and the 5' end of DNA (Mag, M. et al.
(1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn P. J. et al. (1996)
supra). Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment (Peterser, K. H. et al. (1975)
Bioorganic Med Chem. Lett. 5: 1119-11124).
[0098] In other embodiments, FKHL7 nucleic acids may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents that facilitate transport across the
cell membrane as described herein.
[0099] 4.3.1 Probes and Primers
[0100] The nucleotide sequences determined from the cloning of
FKHL7 genes from mammalian organisms will further allow for the
generation of probes and primers designed for use in identifying
and/or cloning FKHL7 homologs in other cell types, e.g., from other
tissues, as well as FKHL7 homologs from other mammalian organisms.
For instance, the present invention also provides a probe/primer
comprising a substantially purified oligonucleotide, which
oligonucleotide comprises a region of nucleotide sequence that
hybridizes under stringent conditions to at least approximately 12,
preferably 25, more preferably 40, 50 or 75 consecutive nucleotides
of sense or anti-sense sequence selected from the group consisting
of SEQ ID NOS. 1 and 3 or naturally occurring mutants thereof. For
instance, primers based on the nucleic acid represented in SEQ ID
NOS. 1 or 3 can be used in PCR reactions to clone FKHL7
homologs.
[0101] Likewise, probes based on the subject FKHL7 sequences can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins, for use, e.g, in prognostic or diagnostic
assays (further described below). In preferred embodiments, the
probe further comprises a label group attached thereto and able to
be detected, e.g., the label group is selected from amongst
radioisotopes, fluorescent compounds, enzymes, and enzyme
co-factors.
[0102] Probes and primers can be prepared and modified, e.g., as
previously described herein for other types of nucleic acids.
[0103] 4.3.2 Antisense, Ribozyme and Triplex Techniques
[0104] Another aspect of the invention relates to the use of the
isolated nucleic acid in "antisense" therapy. As used herein,
"antisense" therapy refers to administration or in situ generation
of oligonucleotide molecules or their derivatives which
specifically hybridize (e.g., bind) under cellular conditions, with
the cellular mRNA and/or genomic DNA encoding one or more of the
subject FKHL7 proteins so as to inhibit expression of that protein,
e.g., by inhibiting transcription and/or translation. The binding
may be by conventional base pair complementarity, or, for example,
in the case of binding to DNA duplexes, through specific
interactions in the major groove of the double helix. In general,
"antisense" therapy refers to the range of techniques generally
employed in the art, and includes any therapy which relies on
specific binding to oligonucleotide sequences.
[0105] An antisense construct of the present invention can be
delivered, for example, as an expression plasmid which, when
transcribed in the cell, produces RNA which is complementary to at
least a unique portion of the cellular mRNA which encodes an FKHL7
protein. Alternatively, the antisense construct is an
oligonucleotide probe which is generated ex vivo and which, when
introduced into the cell causes inhibition of expression by
hybridizing with the mRNA and/or genomic sequences of an FKHL7
gene. Such oligonucleotide probes are preferably modified
oligonucleotides which are resistant to endogenous nucleases, e.g.,
exonucleases and/or endonucleases, and are therefore stable in
vivo. Exemplary nucleic acid molecules for use as antisense
oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in antisense therapy
have been reviewed, for example, by Van der Krol et al. (1988)
BioTechniques 6: 958-976; and Stein et al. (1988) Cancer Res 48:
2659-2668. With respect to antisense DNA, oligodeoxyribonucleotides
derived from the translation initiation site, e.g., between the -10
and +10 regions of the FKHL7 nucleotide sequence of interest, are
preferred.
[0106] Antisense approaches involve the design of oligonucleotides
(either DNA or RNA) that are complementary to FKHL7 mRNA. The
antisense oligonucleotides will bind to the FKHL7 mRNA transcripts
and prevent translation. Absolute complementarity, although
preferred, is not required. In the case of double-stranded
antisense nucleic acids, a single strand of the duplex DNA may thus
be tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with an RNA it
may contain and still form a stable duplex (or triplex, as the case
may be). One skilled in the art can ascertain a tolerable degree of
mismatch by use of standard procedures to determine the melting
point of the hybridized complex.
[0107] Oligonucleotides that are complementary to the 5' end of the
mRNA, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have recently been shown to be
effective at inhibiting translation of mRNAs as well. (Wagner, R.
1994. Nature 372: 333). Therefore, oligonucleotides complementary
to either the 5' or 3' untranslated, non-coding regions of an FKHL7
gene could be used in an antisense approach to inhibit translation
of endogenous FKHL7 mRNA. Oligonucleotides complementary to the 5'
untranslated region of the mRNA should include the complement of
the AUG start codon. Antisense oligonucleotides complementary to
mRNA coding regions are less efficient inhibitors of translation
but could also be used in accordance with the invention. Whether
designed to hybridize to the 5', 3' or coding region of FKHL7 mRNA,
antisense nucleic acids should be at least six nucleotides in
length, and are preferably less than about 100 and more preferably
less than about 50, 25, 17 or 10 nucleotides in length.
[0108] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense oligonucleotide are
compared with those obtained using a control oligonucleotide. It is
preferred that the control oligonucleotide is of approximately the
same length as the test oligonucleotide and that the nucleotide
sequence of the oligonucleotide differs from the antisense sequence
no more than is necessary to prevent specific hybridization to the
target sequence.
[0109] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors), or agents facilitating
transport across the cell membrane (see, e.g., Letsinger et al.,
1989, Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre et al.,
1987, Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No.
WO88/09810, published Dec. 15, 1988) or the blood-brain barrier
(see, e.g., PCT Publication No. WO89/10134, published Apr. 25,
1988), hybridization-triggered cleavage agents. (See, e.g., Krol et
al., 1988, BioTechniques 6: 958-976) or intercalating agents. (See,
e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0110] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxytiethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-
hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0111] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0112] The antisense oligonucleotide can also contain a neutral
peptide-like backbone. Such molecules are termed peptide nucleic
acid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et
al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93: 14670 and in Eglom et
al. (1993) Nature 365: 566. One advantage of PNA oligomers is their
ability to bind to complementary DNA essentially independently from
the ionic strength of the medium due to the neutral backbone of the
DNA. In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0113] In yet a further embodiment, the antisense oligonucleotide
is an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15: 6625-6641). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:
6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215: 327-330).
[0114] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16: 3209), methylphosphonate
olgonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85: 7448-7451), etc.
[0115] While antisense nucleotides complementary to the FKHL7
coding region sequence can be used, those complementary to the
transcribed untranslated region and to the region comprising the
initiating methionine are most preferred.
[0116] The antisense molecules can be delivered to cells which
express FKHL7 in vivo. A number of methods have been developed for
delivering antisense DNA or RNA to cells; e.g., antisense molecules
can be injected directly into the tissue site, or modified
antisense molecules, designed to target the desired cells (e.g.,
antisense linked to peptides or antibodies that specifically bind
receptors or antigens expressed on the target cell surface) can be
administered systematically.
[0117] However, it may be difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation
on endogenous mRNAs in certain instances. Therefore a preferred
approach utilizes a recombinant DNA construct in which the
antisense oligonucleotide is placed under the control of a strong
pol III or pol II promoter. The use of such a construct to
transfect target cells in the patient will result in the
transcription of sufficient amounts of single stranded RNAs that
will form complementary base pairs with the endogenous FKHL7
transcripts and thereby prevent translation of the FKHL7 mRNA. For
example, a vector can be introduced in vivo such that it is taken
up by a cell and directs the transcription of an antisense RNA.
Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired
antisense RNA. Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid,
viral, or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence encoding
the antisense RNA can be by any promoter known in the art to act in
mammalian, preferably human cells. Such promoters can be inducible
or constitutive and can include but not be limited to: the SV40
early promoter region (Bernoist and Chambon, 1981, Nature 290:
304-310), the promoter contained in the 3' long terminal repeat of
Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), the
herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.
Acad. Sci. U.S.A. 78: 1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al, 1982, Nature 296: 39-42),
etc. Any type of plasmid, cosmid, YAC or viral vector can be used
to prepare the recombinant DNA construct which can be introduced
directly into the tissue site. Alternatively, viral vectors can be
used which selectively infect the desired tissue, in which case
administration may be accomplished by another route (e.g.,
systematically).
[0118] Ribozyme molecules designed to catalytically cleave FKHL7
mRNA transcripts can also be used to prevent translation of FKHL7
mRNA and expression of FKHL7 (See, e.g., PCT International
Publication WO90/11364, published Oct. 4, 1990; Sarver et al.,
1990, Science 247: 1222-1225 and U.S. Pat. No. 5,093,246). While
ribozymes that cleave mRNA at site specific recognition sequences
can be used to destroy FKHL7 mRNAs, the use of hammerhead ribozymes
is preferred. Hammerhead ribozymes cleave mRNAs at locations
dictated by flanking regions that form complementary base pairs
with the target mRNA. The sole requirement is that the target mRNA
have the following sequence of two bases: 5'-UG-3'. The
construction and production of hammerhead ribozymes is well known
in the art and is described more fully in Haseloff and Gerlach,
1988, Nature, 334: 585-591. There are a number of potential
hammerhead ribozyme cleavage sites within the nucleotide sequence
of human FKHL7 cDNA. Preferably the ribozyme is engineered so that
the cleavage recognition site is located near the 5' end of the
FKHL7 mRNA; i.e., to increase efficiency and minimize the
intracellular accumulation of non-functional mRNA transcripts.
[0119] The ribozymes of the present invention can also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and which has been extensively described by
Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:
574-578; Zaug and Cech, 1986, Science, 231: 470-475; Zaug, et al.,
1986, Nature, 324: 429-433; published International patent
application No. WO88/04300 by University Patents Inc.; Been and
Cech, 1986, Cell, 47: 207-216). The Cech-type ribozymes have an
eight base pair active site which hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes which target eight
base-pair active site sequences that are present in an FKHL7
gene.
[0120] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.) and should be delivered to cells which express the
FKHL7 gene in vivo. A preferred method of delivery involves using a
DNA construct "encoding" the ribozyme under the control of a strong
constitutive pol III or pol II promoter, so that transfected cells
will produce sufficient quantities of the ribozyme to destroy
endogenous FKHL7 messages and inhibit translation. Because
ribozymes unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
[0121] Endogenous FKHL7 gene expression can also be reduced by
inactivating or "knocking out" the FKHL7 gene or its promoter using
targeted homologous recombination. (e.g., see Smithies et al.,
1985, Nature 317: 230-234; Thomas & Capecchi, 1987, Cell 51:
503-512; Thompson et al., 1989 Cell 5: 313-321; each of which is
incorporated by reference herein in its entirety). For example, a
mutant, non-functional FKHL7 (or a completely unrelated DNA
sequence) flanked by DNA homologous to the endogenous FKHL7 gene
(either the coding regions or regulatory regions of the FKHL7 gene)
can be used, with or without a selectable marker and/or a negative
selectable marker, to transfect cells that express FKHL7 in vivo.
Insertion of the DNA construct, via targeted homologous
recombination, results in inactivation of the FKHL7 gene. Such
approaches are particularly suited in the agricultural field where
modifications to ES (embryonic stem) cells can be used to generate
animal offspring with an inactive FKHL7 (e.g., see Thomas &
Capecchi 1987 and Thompson 1989, supra). However this approach can
be adapted for use in humans provided the recombinant DNA
constructs are directly administered or targeted to the required
site in vivo using appropriate viral vectors.
[0122] Alternatively, endogenous FKHL7 gene expression can be
reduced by targeting deoxyribonucleotide sequences complementary to
the regulatory region of the FKHL7 gene (i.e., the FKHL7 promoter
and/or enhancers) to form triple helical structures that prevent
transcription of the FKHL7 gene in target cells in the body. (See
generally, Helene, C. 1991, Anticancer Drug Des., 6(6): 569-84;
Helene, C., et al., 1992, Ann. N.Y. Acad. Sci., 660: 27-36; and
Maher, L. J., 1992, Bioassays 14(12): 807-15).
[0123] Nucleic acid molecules to be used in triple helix formation
for the inhibition of transcription are preferably single stranded
and composed of deoxyribonucleotides. The base composition of these
oligonucleotides should promote triple helix formation via
Hoogsteen base pairing rules, which generally require sizable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleotide sequences may be pyrimidine-based,
which will result in TAT and CGC triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen that are purine-rich, for example, containing a stretch
of G residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC pairs, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in CGC triplets across the three strands in the
triplex.
[0124] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3',3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0125] Antisense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules. These include techniques
for chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides well known in the art such as for example
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors which
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0126] Moreover, various well-known modifications to nucleic acid
molecules may be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule or
the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the oligodeoxyribonucleotide
backbone.
[0127] 4.3.3. Vectors Encoding FKHL7 Proteins and FKHL7 Expressing
Cells
[0128] The invention further provides plasmids and vectors encoding
an FKHL7 protein, which can be used to express an FKHL7 protein in
a host cell. The host cell may be any prokaryotic or eukaryotic
cell. Thus, a nucleotide sequence derived from the cloning of
mammalian FKHL7 proteins, encoding all or a selected portion of the
full-length protein, can be used to produce a recombinant form of
an FKHL7 polypeptide via microbial or eukaryotic cellular
processes. Ligating the polynucleotide sequence into a gene
construct, such as an expression vector, and transforming or
transfecting into hosts, either eukaryotic (yeast, avian, insect or
mammalian) or prokaryotic (bacterial) cells, are standard
procedures well known in the art.
[0129] Vectors that allow expression of a nucleic acid in a cell
are referred to as expression vectors. Typically, expression
vectors used for expressing an FKHL7 protein contain a nucleic acid
encoding an FKHL7 polypeptide, operably linked to at least one
transcriptional regulatory sequence. Regulatory sequences are
art-recognized and are selected to direct expression of the subject
FKHL7 proteins. Transcriptional regulatory sequences are described
in Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). In one embodiment, the
expression vector includes a recombinant gene encoding a peptide
having an agonistic activity of a subject FKHL7 polypeptide, or
alternatively, encoding a peptide which is an antagonistic form of
an FKHL7 protein.
[0130] Suitable vectors for the expression of an FKHL7 polypeptide
include plasmids of the types: pBR322-derived plasmids,
pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived
plasmids and pUC-derived plasmids for expression in prokaryotic
cells, such as E. coli.
[0131] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2,
and YRP17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used. In an
illustrative embodiment, an FKHL7 polypeptide is produced
recombinantly utilizing an expression vector generated by
subcloning the coding sequence of one of the FKHL7 genes
represented in SEQ ID NOS. 1 or 3.
[0132] The preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2.sup.nd
Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989) Chapters 16 and 17.
[0133] In some instances, it may be desirable to express the
recombinant FKHL7 polypeptide by the use of a baculovirus
expression system. Examples of such baculovirus expression systems
include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the .beta.-gal containing pBlueBac III).
[0134] When it is desirable to express only a portion of an FKHL7
protein, such as a form lacking a portion of the N-terminus, i.e. a
truncation mutant which lacks the signal peptide, it may be
necessary to add a start codon (ATG) to the oligonucleotide
fragment containing the desired sequence to be expressed. It is
well known in the art that a methionine at the N-terminal position
can be enzymatically cleaved by the use of the enzyme methionine
aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat
et al. (1987) J. Bacteriol. 169: 751-757) and Salmonella
typhimurium and its in vitro activity has been demonstrated on
recombinant proteins (Miller et al. (1987) PNAS 84: 2718-1722).
Therefore, removal of an N-terminal methionine, if desired, can be
achieved either in vivo by expressing FKHL7 derived polypeptides in
a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae),
or in vitro by use of purified MAP (e.g., procedure of Miller et
al., supra).
[0135] Moreover, the gene constructs of the present invention can
also be used as part of a gene therapy protocol to deliver nucleic
acids encoding either an agonistic or antagonistic form of one of
the subject FKHL7 proteins. Thus, another aspect of the invention
features expression vectors for in vivo or in vitro transfection
and expression of an FKHL7 polypeptide in particular cell types so
as to reconstitute the function of, or alternatively, abrogate the
function of FKHL7 in a tissue. This could be desirable, for
example, when the naturally-occurring form of the protein is
misexpressed or the natural protein is mutated and less active.
[0136] In addition to viral transfer methods, non-viral methods can
also be employed to cause expression of a subject FKHL7 polypeptide
in the tissue of an animal. Most nonviral methods of gene transfer
rely on normal mechanisms used by mammalian cells for the uptake
and intracellular transport of macromolecules. In preferred
embodiments, non-viral targeting means of the present invention
rely on endocytic pathways for the uptake of the subject FKHL7
polypeptide gene by the targeted cell. Exemplary targeting means of
this type include liposomal derived systems, poly-lysine
conjugates, and artificial viral envelopes.
[0137] In other embodiments, transgenic animals, described in more
detail below could be used to produce recombinant proteins.
[0138] 4.4. Polypeptides of the Present Invention
[0139] The present invention makes available FKHL7 polypeptides
which are isolated from, or otherwise substantially free of other
cellular proteins. The term "substantially free of other cellular
proteins" (also referred to herein as "contaminating proteins") or
"substantially pure or purified preparations" are defined as
encompassing preparations of FKHL7 polypeptides having less than
about 20% (by dry weight) contaminating protein, and preferably
having less than about 5% contaminating protein. Functional forms
of the subject polypeptides can be prepared, for the first time, as
purified preparations by using a cloned gene as described
herein.
[0140] Preferred FKHL7 proteins of the invention have an amino acid
sequence which is at least about 60%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, or 95%
identical or homologous to an amino acid sequence of SEQ ID NO. 2.
Even more preferred FKHL7 proteins comprise an amino acid sequence
which is at least about 97, 98, or 99% homologous or identical to
an amino acid sequence of SEQ ID NO. 2. Such proteins can be
recombinant proteins, and can be, e.g., produced in vitro from
nucleic acids comprising a nucleotide sequence set forth in SEQ ID
NOS. 1 or 3 or homologs thereof. For example, recombinant
polypeptides preferred by the present invention can be encoded by a
nucleic acid, which is at least about 85% homologous and more
preferably at least about 90% homologous and most preferably at
least about 95% homologous with a nucleotide sequence set forth in
SEQ ID NOS. 1 or 3. Polypeptides which are encoded by a nucleic
acid that is at least about 98-99% homologous with the sequence of
SEQ ID NOS. 1 or 3 are also within the scope of the invention.
[0141] In a preferred embodiment, an FKHL7 protein of the present
invention is a mammalian FKHL7 protein. In a particularly preferred
embodiment an FKHL7 protein is set forth as SEQ ID NO. 2. In
particularly preferred embodiments, an FKHL7 protein has an FKHL7
bioactivity. It will be understood that certain post-translational
modifications, e.g., phosphorylation and the like, can increase the
apparent molecular weight of the FKHL7 protein relative to the
unmodified polypeptide chain.
[0142] The invention also features protein isoforms encoded by
splice variants of the present invention. Such isoforms may have
biological activities identical to or different from those
possessed by the FKHL7 proteins specified by SEQ ID NO. 2.
[0143] FKHL7 polypeptides preferably are capable of functioning as
either an agonist or antagonist of at least one biological activity
of a wild-type ("authentic") FKHL7 protein of the appended sequence
listing. The term "evolutionarily related to", with respect to
amino acid sequences of FKHL7 proteins, refers to both polypeptides
having amino acid sequences which have arisen naturally, and also
to mutational variants of human FKHL7 polypeptides which are
derived, for example, by combinatorial mutagenesis.
[0144] Full length proteins or fragments corresponding to one or
more particular motifs and/or domains or to arbitrary sizes, for
example, at least 5, 10, 25, 50, 75 and 100, amino acids in length
are within the scope of the present invention.
[0145] For example, isolated FKHL7 polypeptides can be encoded by
all or a portion of a nucleic acid sequence shown in any of SEQ ID
NOS. 1 or 3. Isolated peptidyl portions of FKHL7 proteins can be
obtained by screening peptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding such peptides.
In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, an FKHL7 polypeptide
of the present invention may be arbitrarily divided into fragments
of desired length with no overlap of the fragments, or preferably
divided into overlapping fragments of a desired length. The
fragments can be produced (recombinantly or by chemical synthesis)
and tested to identify those peptidyl fragments which can function
as either agonists or antagonists of a wild-type (e.g.,
"authentic") FKHL7 protein.
[0146] Preferred FKHL7 polypeptides contain the forkhead domain
located from about amino acid 73 to about 178 of SEQ ID NO. 2 (i.e.
the underlined region of the protein shown in FIG. 1). Other
preferred FKHL7 polypeptides bind to an RTAAYA target region of a
nucleic acid.
[0147] In general, polypeptides referred to herein as having an
FKHL7 activity (e.g., are "bioactive") are defined as polypeptides
which include an amino acid sequence encoded by all or a portion of
the nucleic acid sequences shown in one of SEQ ID NOS. 1 or 3 and
which mimic or antagonize all or a portion of the
biological/biochemical activities of a naturally occurring FKHL7
protein. Examples of such biological activity include: regulation
of gene expression. Furthermore these fragments can either promote
or inhibit these processes or agonize or antagonize the activity of
another agent which itself promotes or inhibits these processes.
Other biological activities of the subject FKHL7 proteins will be
reasonably apparent to one of skill in the art. According to the
present invention, a polypeptide has biological activity if it is a
specific agonist or antagonist of a naturally-occurring form of an
FKHL7 protein. Assays for determining whether a compound, e.g, a
protein, such as an FKHL7 protein or variant thereof, has one or
more of the above biological activities are well known in the
art.
[0148] Other preferred proteins of the invention are those encoded
by the nucleic acids set forth in the section pertaining to nucleic
acids of the invention. In particular, the invention provides
fusion proteins, e.g., FKHL7-immunoglobulin fusion proteins. Such
fusion proteins can provide, e.g., enhanced stability and
solubility of FKHL7 proteins and may thus be useful in therapy.
Fusion proteins can also be used to produce an immunogenic fragment
of an FKHL7 protein. For example, the VP6 capsid protein of
rotavirus can be used as an immunologic carrier protein for
portions of the FKHL7 polypeptide, either in the monomeric form or
in the form of a viral particle. The nucleic acid sequences
corresponding to the portion of a subject FKHL7 protein to which
antibodies are to be raised can be incorporated into a fusion gene
construct which includes coding sequences for a late vaccinia virus
structural protein to produce a set of recombinant viruses
expressing fusion proteins comprising FKHL7 epitopes as part of the
virion. It has been demonstrated with the use of immunogenic fusion
proteins utilizing the Hepatitis B surface antigen fusion proteins
that recombinant Hepatitis B virions can be utilized in this role
as well. Similarly, chimeric constructs coding for fusion proteins
containing a portion of an FKHL7 protein and the poliovirus capsid
protein can be created to enhance immunogenicity of the set of
polypeptide antigens (see, for example, EP Publication No: 0259149;
and Evans et al. (1989) Nature 339: 385; Huang et al. (1988) J.
Virol. 62: 3855; and Schlienger et al. (1992) J. Virol. 66: 2).
[0149] The Multiple antigen peptide system for peptide-based
immunization can also be utilized to generate an immunogen, wherein
a desired portion of an FKHL7 polypeptide is obtained directly from
organo-chemical synthesis of the peptide onto an oligomeric
branching lysine core (see, for example, Posnett et al. (1988) JBC
263: 1719 and Nardelli et al. (1992) J. Immunol. 148: 914).
Antigenic determinants of FKHL7 proteins can also be expressed and
presented by bacterial cells.
[0150] In addition to utilizing fusion proteins to enhance
immunogenicity, it is widely appreciated that fusion proteins can
also facilitate the expression of proteins, and accordingly, can be
used in the expression of the FKHL7 polypeptides of the present
invention. For example, FKHL7 polypeptides can be generated as
glutathione-S-transferase (GST-fusion) proteins. Such GST-fusion
proteins can enable easy purification of the FKHL7 polypeptide, as
for example by the use of glutathione-derivatized matrices (see,
for example, Current Protocols in Molecular Biology, eds. Ausubel
et al. (N.Y.: John Wiley & Sons, 1991)).
[0151] The present invention further pertains to methods of
producing the subject FKHL7 polypeptides. For example, a host cell
transfected with a nucleic acid vector directing expression of a
nucleotide sequence encoding the subject polypeptides can be
cultured under appropriate conditions to allow expression of the
peptide to occur. Suitable media for cell culture are well known in
the art. The recombinant FKHL7 polypeptide can be isolated from
cell culture medium, host cells, or both using techniques known in
the art for purifying proteins including ion-exchange
chromatography, gel filtration chromatography, ultrafiltration,
electrophoresis, and immunoaffinity purification with antibodies
specific for such peptides. In a preferred embodiment, the
recombinant FKHL7 polypeptide is a fusion protein containing a
domain which facilitates its purification, such as GST fusion
protein.
[0152] Moreover, it will be generally appreciated that, under
certain circumstances, it may be advantageous to provide homologs
of one of the subject FKHL7 polypeptides, which function in a
limited capacity as one of either an FKHL7 agonist (mimetic) or an
FKHL7 antagonist, in order to promote or inhibit only a subset of
the biological activities of the naturally-occurring form of the
protein. Thus, specific biological effects can be elicited by
treatment with a homolog of limited function, and with fewer side
effects relative to treatment with agonists or antagonists which
are directed to all of the biological activities of naturally
occurring forms of FKHL7 proteins.
[0153] Homologs of each of the subject FKHL7 proteins can be
generated by mutagenesis, such as by discrete point mutation(s), or
by truncation. For instance, mutation can give rise to homologs
which retain substantially the same, or merely a subset, of the
biological activity of the FKHL7 polypeptide from which it was
derived. Alternatively, antagonistic forms of the protein can be
generated which are able to inhibit the function of the naturally
occurring form of the protein, such as by competitively binding to
an FKHL7 receptor.
[0154] The recombinant FKHL7 polypeptides of the present invention
also include homologs of the wildtype FKHL7 proteins, such as
versions of those protein which are resistant to proteolytic
cleavage, as for example, due to mutations which alter
ubiquitination or other enzymatic targeting associated with the
protein.
[0155] FKHL7 polypeptides may also be chemically modified to create
FKHL7 derivatives by forming covalent or aggregate conjugates with
other chemical moieties, such as glycosyl groups, lipids,
phosphate, acetyl groups and the like. Covalent derivatives of
FKHL7 proteins can be prepared by linking the chemical moieties to
functional groups on amino acid sidechains of the protein or at the
N-terminus or at the C-terminus of the polypeptide.
[0156] Modification of the structure of the subject FKHL7
polypeptides can be for such purposes as enhancing therapeutic or
prophylactic efficacy, stability (e.g., ex vivo shelf life and
resistance to proteolytic degradation), or post-translational
modifications (e.g., to alter phosphorylation pattern of protein).
Such modified peptides, when designed to retain at least one
activity of the naturally-occurring form of the protein, or to
produce specific antagonists thereof, are considered functional
equivalents of the FKHL7 polypeptides described in more detail
herein. Such modified peptides can be produced, for instance, by
amino acid substitution, deletion, or addition. The substitutional
variant may be a substituted conserved amino acid or a substituted
non-conserved amino acid.
[0157] For example, it is reasonable to expect that an isolated
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
(i.e. isosteric and/or isoelectric mutations) will not have a major
effect on the biological activity of the resulting molecule.
Conservative replacements are those that take place within a family
of amino acids that are related in their side chains. Genetically
encoded amino acids can be divided into four families: (1)
acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. In similar fashion, the amino acid repertoire can be
grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine,
arginine histidine, (3) aliphatic=glycine, alanine, valine,
leucine, isoleucine, serine, threonine, with serine and threonine
optionally be grouped separately as aliphatic-hydroxyl; (4)
aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine,
glutamine; and (6) sulfur-containing=cysteine and methionine. (see,
for example, Biochemistry, 2.sup.nd ed., Ed. by L. Stryer, WH
Freeman and Co.: 1981). Whether a change in the amino acid sequence
of a peptide results in a functional FKHL7 homolog (e.g.,
functional in the sense that the resulting polypeptide mimics or
antagonizes the wild-type form) can be readily determined by
assessing the ability of the variant peptide to produce a response
in cells in a fashion similar to the wild-type protein, or
competitively inhibit such a response. Polypeptides in which more
than one replacement has taken place can readily be tested in the
same manner.
[0158] This invention further contemplates a method for generating
sets of combinatorial mutants of the subject FKHL7 proteins as well
as truncation mutants, and is especially useful for identifying
potential variant sequences (e.g., homologs). The purpose of
screening such combinatorial libraries is to generate, for example,
novel FKHL7 homologs which can act as either agonists or
antagonist, or alternatively, possess novel activities all
together. Thus, combinatorially-derived homologs can be generated
to have an increased potency relative to a naturally occurring form
of the protein.
[0159] In one embodiment, the variegated library of FKHL7 variants
is generated by combinatorial mutagenesis at the nucleic acid
level, and is encoded by a variegated gene library. For instance, a
mixture of synthetic oligonucleotides can be enzymatically ligated
into gene sequences such that the degenerate set of potential FKHL7
sequences are expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display) containing the set of FKHL7 sequences therein.
[0160] There are many ways by which such libraries of potential
FKHL7 homologs can be generated from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
carried out in an automatic DNA synthesizer, and the synthetic
genes then ligated into an appropriate expression vector. The
purpose of a degenerate set of genes is to provide, in one mixture,
all of the sequences encoding the desired set of potential FKHL7
sequences. The synthesis of degenerate oligonucleotides is well
known in the art (see for example, Narang, S A (1983) Tetrahedron
39: 3; Itakura et al. (1981) Recombinant DNA, Proc 3.sup.rd
Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:
Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:
323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983)
Nucleic Acid Res. 11: 477. Such techniques have been employed in
the directed evolution of other proteins (see, for example, Scott
et al. (1990) Science 249: 386-390; Roberts et al. (1992) PNAS 89:
2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al.
(1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0161] Likewise, a library of coding sequence fragments can be
provided for an FKHL7 clone in order to generate a variegated
population of FKHL7 fragments for screening and subsequent
selection of bioactive fragments. A variety of techniques are known
in the art for generating such libraries, including chemical
synthesis. In one embodiment, a library of coding sequence
fragments can be generated by (i) treating a double stranded PCR
fragment of an FKHL7 coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule;
(ii) denaturing the double stranded DNA; (iii) renaturing the DNA
to form double stranded DNA which can include sense/antisense pairs
from different nicked products; (iv) removing single stranded
portions from reformed duplexes by treatment with S1 nuclease; and
(v) ligating the resulting fragment library into an expression
vector. By this exemplary method, an expression library can be
derived which codes for N-terminal, C-terminal and internal
fragments of various sizes.
[0162] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations or truncation, and for screening cDNA libraries for gene
products having a certain property. Such techniques will be
generally adaptable for rapid screening of the gene libraries
generated by the combinatorial mutagenesis of FKHL7 homologs. The
most widely used techniques for screening large gene libraries
typically comprises cloning the gene library into replicable
expression vectors, transforming appropriate cells with the
resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates relatively easy isolation of the vector encoding the
gene whose product was detected. Each of the illustrative assays
described below are amenable to high through-put analysis as
necessary to screen large numbers of degenerate FKHL7 sequences
created by combinatorial mutagenesis techniques. Combinatorial
mutagenesis has a potential to generate very large libraries of
mutant proteins, e.g., in the order of 10.sup.26 molecules.
Combinatorial libraries of this size may be technically challenging
to screen even with high throughput screening assays. To overcome
this problem, a new technique has been developed recently,
recrusive ensemble mutagenesis (REM), which allows one to avoid the
very high proportion of non-functional proteins in a random library
and simply enhances the frequency of functional proteins, thus
decreasing the complexity required to achieve a useful sampling of
sequence space. REM is an algorithm which enhances the frequency of
functional mutants in a library when an appropriate selection or
screening method is employed (Arkin and Yourvan, 1992, PNAS USA 89:
7811-7815; Yourvan et al., 1992, Parallel Problem Solving from
Nature, 2, In Maenner and Manderick, eds., Elsevir Publishing Co.,
Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering
6(3): 327-331).
[0163] The invention also provides for reduction of the FKHL7
proteins to generate mimetics, e.g., peptide or non-peptide agents,
such as small molecules, which are able to disrupt binding of an
FKHL7 polypeptide of the present invention with a molecule, e.g.
target peptide. Thus, such mutagenic techniques as described above
are also useful to map the determinants of the FKHL7 proteins which
participate in protein-protein interactions involved in, for
example, binding of the subject FKHL7 polypeptide to a target
peptide. To illustrate, the critical residues of a subject FKHL7
polypeptide which are involved in molecular recognition of its
receptor can be determined and used to generate FKHL7 derived
peptidomimetics or small molecules which competitively inhibit
binding of the authentic FKHL7 protein with that moiety. By
employing, for example, scanning mutagenesis to map the amino acid
residues of the subject FKHL7 proteins which are involved in
binding other proteins, peptidomimetic compounds can be generated
which mimic those residues of the FKHL7 protein which facilitate
the interaction. Such mimetics may then be used to interfere with
the normal function of an FKHL7 protein. For instance,
non-hydrolyzable peptide analogs of such residues can be generated
using benzodiazepine (e.g., see Freidinger et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), substituted gamma lactam rings (Garvey et al.
in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), keto-methylene
pseudopeptides (Ewenson et al. (1986) J Med Chem 29: 295; and
Ewenson et al. in Peptides: Structure and Function (Proceedings of
the 9.sup.th American Peptide Symposium) Pierce Chemical Co.
Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al. (1985)
Tetrahedron Lett 26: 647; and Sato et al. (1986) J Chem Soc Perkin
Trans 1: 1231), and .beta.-aminoalcohols (Gordon et al. (1985)
Biochem Biophys Res Commun 126: 419; and Dann et al. (1986) Biochem
Biophys Res Commun 134: 71).
[0164] 4.5. Anti-FKHL7 Antibodies and Uses Therefor
[0165] Another aspect of the invention pertains to an antibody
specifically reactive with a mammalian FKHL7 protein, e.g., a
wild-type or mutated FKHL7 protein. For example, by using
immunogens derived from an FKHL7 protein, e.g., based on the cDNA
sequences, anti-protein/anti-peptide antisera or monoclonal
antibodies can be made by standard protocols (See, for example,
Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring
Harbor Press: 1988)). A mammal, such as a mouse, a hamster or
rabbit can be immunized with an immunogenic form of the peptide
(e.g., a mammalian FKHL7 polypeptide or an antigenic fragment which
is capable of eliciting an antibody response, or a fusion protein
as described above). Techniques for conferring immunogenicity on a
protein or peptide include conjugation to carriers or other
techniques well known in the art. An immunogenic portion of an
FKHL7 protein can be administered in the presence of adjuvant. The
progress of immunization can be monitored by detection of antibody
titers in plasma or serum. Standard ELISA or other immunoassays can
be used with the immunogen as antigen to assess the levels of
antibodies. In a preferred embodiment, the subject antibodies are
immunospecific for antigenic determinants of an FKHL7 protein of a
mammal, e.g., antigenic determinants of a protein set forth in SEQ
ID No: 2 or closely related homologs (e.g., at least 90%
homologous, and more preferably at least 94% homologous).
[0166] Following immunization of an animal with an antigenic
preparation of an FKHL7 polypeptide, anti-FKHL7 antisera can be
obtained and, if desired, polyclonal anti-FKHL7 antibodies isolated
from the serum. To produce monoclonal antibodies,
antibody-producing cells (lymphocytes) can be harvested from an
immunized animal and fused by standard somatic cell fusion
procedures with immortalizing cells such as myeloma cells to yield
hybridoma cells. Such techniques are well known in the art, and
include, for example, the hybridoma technique originally developed
by Kohler and Milstein ((1975) Nature, 256: 495-497), the human B
cell hybridoma technique (Kozbar et al., (1983) Immunology Today,
4: 72), and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., (1985 Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be
screened immunochemically for production of antibodies specifically
reactive with a mammalian FKHL7 polypeptide of the present
invention and monoclonal antibodies isolated from a culture
comprising such hybridoma cells. In one embodiment anti-human FKHL7
antibodies specifically react with the protein encoded by a nucleic
acid having SEQ ID NO. 1 or 3.
[0167] The term antibody as used herein is intended to include
fragments thereof which are also specifically reactive with one of
the subject mammalian FKHL7 polypeptides. Antibodies can be
fragmented using conventional techniques and the fragments screened
for utility in the same manner as described above for whole
antibodies. For example, F(ab).sub.2 fragments can be generated by
treating antibody with pepsin. The resulting F(ab).sub.2 fragment
can be treated to reduce disulfide bridges to produce Fab
fragments. The antibody of the present invention is further
intended to include bispecific, single-chain, and chimeric and
humanized molecules having affinity for an FKHL7 protein conferred
by at least one CDR region of the antibody. In preferred
embodiments, the antibody further comprises a label attached
thereto and able to be detected, (e.g., the label can be a
radioisotope, fluorescent compound, enzyme or enzyme
co-factor).
[0168] Anti-FKHL7 antibodies can be used, e.g., to monitor FKHL7
protein levels in an individual for determining, e.g., whether a
subject has a disease or condition associated with an aberrant
FKHL7 protein level, or allowing determination of the efficacy of a
given treatment regimen for an individual afflicted with such a
disorder. The level of FKHL7 polypeptides may be measured from
cells in bodily fluid, such as in blood samples.
[0169] Another application of anti-FKHL7 antibodies of the present
invention is in the immunological screening of cDNA libraries
constructed in expression vectors such as .lambda.gt11,
.lambda.gt18-23, .lambda.ZAP, and .lambda.ORF8. Messenger libraries
of this type, having coding sequences inserted in the correct
reading frame and orientation, can produce fusion proteins. For
instance, .lambda.gt11 will produce fusion proteins whose amino
termini consist of .beta.-galactosidase amino acid sequences and
whose carboxy termini consist of a foreign polypeptide. Antigenic
epitopes of an FKHL7 protein, e.g., other orthologs of a particular
FKHL7 protein or other paralogs from the same species, can then be
detected with antibodies, as, for example, reacting nitrocellulose
filters lifted from infected plates with anti-FKHL7 antibodies.
Positive phage detected by this assay can then be isolated from the
infected plate. Thus, the presence of FKHL7 homologs can be
detected and cloned from other animals, as can alternate isoforms
(including splice variants) from humans.
[0170] 4.6. Transgenic Animals
[0171] The invention further provides for transgenic animals, which
can be used for a variety of purposes, e.g., to identify FKHL7
therapeutics. Transgenic animals of the invention include non-human
animals containing a heterologous FKHL7 gene or fragment thereof
under the control of an FKHL7 promoter or under the control of a
heterologous promoter. Accordingly, the transgenic animals of the
invention can be animals expressing a transgene encoding a
wild-type FKHL7 protein or fragment thereof or variants thereof,
including mutants and polymorphic variants thereof. Such animals
can be used, e.g., to determine the effect of a difference in amino
acid sequence of an FKHL7 protein from the sequence set forth in
SEQ ID NO. 2, such as a polymorphic difference. These animals can
also be used to determine the effect of expression of an FKHL7
protein in a specific site or for identifying FKHL7 therapeutics or
confirming their activity in vivo.
[0172] The transgenic animals can also be animals containing a
transgene, such as reporter gene, under the control of an FKHL7
promoter or fragment thereof. These animals are useful, e.g., for
identifying compound that modulate production of FKHL7, such as by
modulating FKHL7 gene expression. An FKHL7 gene promoter can be
isolated, e.g., by screening of a genomic library with an FKHL7
cDNA fragment and characterized according to methods known in the
art. In a preferred embodiment of the present invention, the
transgenic animal containing said FKHL7 reporter gene is used to
screen a class of bioactive molecules known as steroid hormones for
their ability to modulate FKHL7 expression. In a more preferred
embodiment of the invention, the steroid hormones screened for
FKHL7 expression modulating activity belong to the group known as
androgens. In a still more preferred embodiment of the invention,
the steroid hormone is testosterone or a testosterone analog. Yet
other non-human animals within the scope of the invention include
those in which the expression of the endogenous FKHL7 gene has been
mutated or "knocked out". A "knock out" animal is one carrying a
homozygous or heterozygous deletion of a particular gene or genes.
These animals could be useful to determine whether the absence of
FKHL7 will result in a specific phenotype, in particular whether
these mice have or are likely to develop a specific disease, such
as high susceptibility to heart disease or cancer. Furthermore
these animals are useful in screens for drugs which alleviate or
attenuate the disease condition resulting from the mutation of the
FKHL7 gene as outlined below. These animals are also useful for
determining the effect of a specific amino acid difference, or
allelic variation, in an FKHL7 gene. That is, the FKHL7 knock out
animals can be crossed with transgenic animals expressing, e.g., a
mutated form or allelic variant of FKHL7, thus resulting in an
animal which expresses only the mutated protein and not the
wild-type FKHL7 protein.
[0173] In a preferred embodiment of this aspect of the invention, a
transgenic FKHL7 knock-out mouse, carrying the mutated FKHL7 locus
on one or both of its chromosomes, is used as a model system for
transgenic or drug treatment of the condition resulting from loss
of FKHL7 expression.
[0174] Methods for obtaining transgenic and knockout non-human
animals are well known in the art. Knock out mice are generated by
homologous integration of a "knock out" construct into a mouse
embryonic stem cell chromosome which encodes the gene to be knocked
out. In one embodiment, gene targeting, which is a method of using
homologous recombination to modify an animal's genome, can be used
to introduce changes into cultured embryonic stem cells. By
targeting a FKHL7 gene of interest in ES cells, these changes can
be introduced into the germlines of animals to generate chimeras.
The gene targeting procedure is accomplished by introducing into
tissue culture cells a DNA targeting construct that includes a
segment homologous to a target FKHL7 locus, and which also includes
an intended sequence modification to the FKHL7 genomic sequence
(e.g., insertion, deletion, point mutation). The treated cells are
then screened for accurate targeting to identify and isolate those
which have been properly targeted.
[0175] Gene targeting in embryonic stem cells is in fact a scheme
contemplated by the present invention as a means for disrupting a
FKHL7 gene function through the use of a targeting transgene
construct designed to undergo homologous recombination with one or
more FKHL7 genomic sequences. The targeting construct can be
arranged so that, upon recombination with an element of a FKHL7
gene, a positive selection marker is inserted into (or replaces)
coding sequences of the gene. The inserted sequence functionally
disrupts the FKHL7 gene, while also providing a positive selection
trait. Exemplary FKHL7 targeting constructs are described in more
detail below.
[0176] Generally, the embryonic stem cells (ES cells) used to
produce the knockout animals will be of the same species as the
knockout animal to be generated. Thus for example, mouse embryonic
stem cells will usually be used for generation of knockout
mice.
[0177] Embryonic stem cells are generated and maintained using
methods well known to the skilled artisan such as those described
by Doetschman et al. (1985) J. Embryol. Exp. MoFKHL7hol. 87:
27-45). Any line of ES cells can be used, however, the line chosen
is typically selected for the ability of the cells to integrate
into and become part of the germ line of a developing embryo so as
to create germ line transmission of the knockout construct. Thus,
any ES cell line that is believed to have this capability is
suitable for use herein. One mouse strain that is typically used
for production of ES cells, is the 129J strain. Another ES cell
line is murine cell line D3 (American Type Culture Collection,
catalog no. CKL 1934) Still another preferred ES cell line is the
WW6 cell line (Ioffe et al. (1995) PNAS 92: 7357-7361). The cells
are cultured and prepared for knockout construct insertion using
methods well known to the skilled artisan, such as those set forth
by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. IRL Press, Washington,
D.C. [1987]); by Bradley et al. (1986) Current Topics in Devel.
Biol. 20: 357-371); and by Hogan et al. (Manipulating the Mouse
Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. [1986]).
[0178] A knock out construct refers to a uniquely configured
fragment of nucleic acid which is introduced into a stem cell line
and allowed to recombine with the genome at the chromosomal locus
of the gene of interest to be mutated. Thus a given knock out
construct is specific for a given gene to be targeted for
disruption. Nonetheless, many common elements exist among these
constructs and these elements are well known in the art. A typical
knock out construct contains nucleic acid fragments of not less
than about 0.5 kb nor more than about 10.0 kb from both the 5' and
the 3' ends of the genomic locus which encodes the gene to be
mutated. These two fragments are separated by an intervening
fragment of nucleic acid which encodes a positive selectable
marker, such as the neomycin resistance gene (neo.sup.R). The
resulting nucleic acid fragment, consisting of a nucleic acid from
the extreme 5' end of the genomic locus linked to a nucleic acid
encoding a positive selectable marker which is in turn linked to a
nucleic acid from the extreme 3' end of the genomic locus of
interest, omits most of the coding sequence for FKHL7 or other gene
of interest to be knocked out. When the resulting construct
recombines homologously with the chromosome at this locus, it
results in the loss of the omitted coding sequence, otherwise known
as the structural gene, from the genomic locus. A stem cell in
which such a rare homologous recombination event has taken place
can be selected for by virtue of the stable integration into the
genome of the nucleic acid of the gene encoding the positive
selectable marker and subsequent selection for cells expressing
this marker gene in the presence of an appropriate drug (neomycin
in this example).
[0179] Variations on this basic technique also exist and are well
known in the art. For example, a "knock-in" construct refers to the
same basic arrangement of a nucleic acid encoding a 5' genomic
locus fragment linked to nucleic acid encoding a positive
selectable marker which in turn is linked to a nucleic acid
encoding a 3' genomic locus fragment, but which differs in that
none of the coding sequence is omitted and thus the 5' and the 3'
genomic fragments used were initially contiguous before being
disrupted by the introduction of the nucleic acid encoding the
positive selectable marker gene. This "knock-in" type of construct
is thus very useful for the construction of mutant transgenic
animals when only a limited region of the genomic locus of the gene
to be mutated, such as a single exon, is available for cloning and
genetic manipulation. Alternatively, the "knock-in" construct can
be used to specifically eliminate a single functional domain of the
targetted gene, resulting in a transgenic animal which expresses a
polypeptide of the targetted gene which is defective in one
function, while retaining the function of other domains of the
encoded polypeptide. This type of "knock-in" mutant frequently has
the characteristic of a so-called "dominant negative" mutant
because, especially in the case of proteins which homomultimerize,
it can specifically block the action of (or "poison") the
polypeptide product of the wild-type gene from which it was
derived. In a variation of the knock-in technique, a marker gene is
integrated at the genomic locus of interest such that expression of
the marker gene comes under the control of the transcriptional
regulatory elements of the targeted gene. A marker gene is one that
encodes an enzyme whose activity can be detected (e.g.,
.beta.-galactosidase), the enzyme substrate can be added to the
cells under suitable conditions, and the enzymatic activity can be
analyzed. One skilled in the art will be familiar with other useful
markers and the means for detecting their presence in a given cell.
All such markers are contemplated as being included within the
scope of the teaching of this invention.
[0180] As mentioned above, the homologous recombination of the
above described "knock out" and "knock in" constructs is very rare
and frequently such a construct inserts nonhomologously into a
random region of the genome where it has no effect on the gene
which has been targeted for deletion, and where it can potentially
recombine so as to disrupt another gene which was otherwise not
intended to be altered. Such nonhomologous recombination events can
be selected against by modifying the abovementioned knock out and
knock in constructs so that they are flanked by negative selectable
markers at either end (particularly through the use of two allelic
variants of the thymidine kinase gene, the polypeptide product of
which can be selected against in expressing cell lines in an
appropriate tissue culture medium well known in the art--i.e. one
containing a drug such as 5-bromodeoxyuridine). Thus a preferred
embodiment of such a knock out or knock in construct of the
invention consist of a nucleic acid encoding a negative selectable
marker linked to a nucleic acid encoding a 5' end of a genomic
locus linked to a nucleic acid of a positive selectable marker
which in turn is linked to a nucleic acid encoding a 3' end of the
same genomic locus which in turn is linked to a second nucleic acid
encoding a negative selectable marker Nonhomologous recombination
between the resulting knock out construct and the genome will
usually result in the stable integration of one or both of these
negative selectable marker genes and hence cells which have
undergone nonhomologous recombination can be selected against by
growth in the appropriate selective media (e.g. media containing a
drug such as 5-bromodeoxyuridine for example). Simultaneous
selection for the positive selectable marker and against the
negative selectable marker will result in a vast enrichment for
clones in which the knock out construct has recombined homologously
at the locus of the gene intended to be mutated. The presence of
the predicted chromosomal alteration at the targeted gene locus in
the resulting knock out stem cell line can be confirmed by means of
Southern blot analytical techniques which are well known to those
familiar in the art. Alternatively, PCR can be used.
[0181] Each knockout construct to be inserted into the cell must
first be in the linear form. Therefore, if the knockout construct
has been inserted into a vector (described infra), linearization is
accomplished by digesting the DNA with a suitable restriction
endonuclease selected to cut only within the vector sequence and
not within the knockout construct sequence.
[0182] For insertion, the knockout construct is added to the ES
cells under appropriate conditions for the insertion method chosen,
as is known to the skilled artisan. For example, if the ES cells
are to be electroporated, the ES cells and knockout construct DNA
are exposed to an electric pulse using an electroporation machine
and following the manufacturer's guidelines for use. After
electroporation, the ES cells are typically allowed to recover
under suitable incubation conditions. The cells are then screened
for the presence of the knock out construct as explained above.
Where more than one construct is to be introduced into the ES cell,
each knockout construct can be introduced simultaneously or one at
a time.
[0183] After suitable ES cells containing the knockout construct in
the proper location have been identified by the selection
techniques outlined above, the cells can be inserted into an
embryo. Insertion may be accomplished in a variety of ways known to
the skilled artisan, however a preferred method is by
microinjection. For microinjection, about 10-30 cells are collected
into a micropipet and injected into embryos that are at the proper
stage of development to permit integration of the foreign ES cell
containing the knockout construct into the developing embryo. For
instance, the transformed ES cells can be microinjected into
blastocytes. The suitable stage of development for the embryo used
for insertion of ES cells is very species dependent, however for
mice it is about 3.5 days. The embryos are obtained by perfusing
the uterus of pregnant females. Suitable methods for accomplishing
this are known to the skilled artisan, and are set forth by, e.g.,
Bradley et al. (supra).
[0184] While any embryo of the right stage of development is
suitable for use, preferred embryos are male. In mice, the
preferred embryos also have genes coding for a coat color that is
different from the coat color encoded by the ES cell genes. In this
way, the offspring can be screened easily for the presence of the
knockout construct by looking for mosaic coat color (indicating
that the ES cell was incorporated into the developing embryo).
Thus, for example, if the ES cell line carries the genes for white
fur, the embryo selected will carry genes for black or brown
fur.
[0185] After the ES cell has been introduced into the embryo, the
embryo may be implanted into the uterus of a pseudopregnant foster
mother for gestation. While any foster mother may be used, the
foster mother is typically selected for her ability to breed and
reproduce well, and for her ability to care for the young. Such
foster mothers are typically prepared by mating with vasectomized
males of the same species. The stage of the pseudopregnant foster
mother is important for successful implantation, and it is species
dependent. For mice, this stage is about 2-3 days
pseudopregnant.
[0186] Offspring that are born to the foster mother may be screened
initially for mosaic coat color where the coat color selection
strategy (as described above, and in the appended examples) has
been employed. In addition, or as an alternative, DNA from tail
tissue of the offspring may be screened for the presence of the
knockout construct using Southern blots and/or PCR as described
above. Offspring that appear to be mosaics may then be crossed to
each other, if they are believed to carry the knockout construct in
their germ line, in order to generate homozygous knockout animals.
Homozygotes may be identified by Southern blotting of equivalent
amounts of genomic DNA from mice that are the product of this
cross, as well as mice that are known heterozygotes and wild type
mice.
[0187] Other means of identifying and characterizing the knockout
offspring are available. For example, Northern blots can be used to
probe the mRNA for the presence or absence of transcripts encoding
either the gene knocked out, the marker gene, or both. In addition,
Western blots can be used to assess the level of expression of the
FKHL7 gene knocked out in various tissues of the offspring by
probing the Western blot with an antibody against the particular
FKHL7 protein, or an antibody against the marker gene product,
where this gene is expressed. Finally, in situ analysis (such as
fixing the cells and labeling with antibody) and/or FACS
(fluorescence activated cell sorting) analysis of various cells
from the offspring can be conducted using suitable antibodies to
look for the presence or absence of the knockout construct gene
product.
[0188] Yet other methods of making knock-out or disruption
transgenic animals are also generally known. See, for example,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent
knockouts can also be generated, e.g. by homologous recombination
to insert target sequences, such that tissue specific and/or
temporal control of inactivation of an FKHL7-gene can be controlled
by recombinase sequences (described infra).
[0189] Animals containing more than one knockout construct and/or
more than one transgene expression construct are prepared in any of
several ways. The preferred manner of preparation is to generate a
series of mammals, each containing one of the desired transgenic
phenotypes. Such animals are bred together through a series of
crosses, backcrosses and selections, to ultimately generate a
single animal containing all desired knockout constructs and/or
expression constructs, where the animal is otherwise congenic
(genetically identical) to the wild type except for the presence of
the knockout construct(s) and/or transgene(s).
[0190] A FKHL7 transgene can encode the wild-type form of the
protein, or can encode homologs thereof, including both agonists
and antagonists, as well as antisense constructs. In preferred
embodiments, the expression of the transgene is restricted to
specific subsets of cells, tissues or developmental stages
utilizing, for example, cis-acting sequences that control
expression in the desired pattern. In the present invention, such
mosaic expression of a FKHL7 protein can be essential for many
forms of lineage analysis and can additionally provide a means to
assess the effects of, for example, lack of FKHL7 expression which
might grossly alter development in small patches of tissue within
an otherwise normal embryo. Toward this and, tissue-specific
regulatory sequences and conditional regulatory sequences can be
used to control expression of the transgene in certain spatial
patterns. Moreover, temporal patterns of expression can be provided
by, for example, conditional recombination systems or prokaryotic
transcriptional regulatory sequences.
[0191] Genetic techniques, which allow for the expression of
transgenes can be regulated via site-specific genetic manipulation
in vivo, are known to those skilled in the art. For instance,
genetic systems are available which allow for the regulated
expression of a recombinase that catalyzes the genetic
recombination of a target sequence. As used herein, the phrase
"target sequence" refers to a nucleotide sequence that is
genetically recombined by a recombinase. The target sequence is
flanked by recombinase recognition sequences and is generally
either excised or inverted in cells expressing recombinase
activity. Recombinase catalyzed recombination events can be
designed such that recombination of the target sequence results in
either the activation or repression of expression of one of the
subject FKHL7 proteins. For example, excision of a target sequence
which interferes with the expression of a recombinant FKHL7 gene,
such as one which encodes an antagonistic homolog or an antisense
transcript, can be designed to activate expression of that gene.
This interference with expression of the protein can result from a
variety of mechanisms, such as spatial separation of the FKHL7 gene
from the promoter element or an internal stop codon. Moreover, the
transgene can be made wherein the coding sequence of the gene is
flanked by recombinase recognition sequences and is initially
transfected into cells in a 3' to 5' orientation with respect to
the promoter element. In such an instance, inversion of the target
sequence will reorient the subject gene by placing the 5' end of
the coding sequence in an orientation with respect to the promoter
element which allow for promoter driven transcriptional
activation.
[0192] The transgenic animals of the present invention all include
within a plurality of their cells a transgene of the present
invention, which transgene alters the phenotype of the "host cell"
with respect to regulation of cell growth, death and/or
differentiation. Since it is possible to produce transgenic
organisms of the invention utilizing one or more of the transgene
constructs described herein, a general description will be given of
the production of transgenic organisms by referring generally to
exogenous genetic material. This general description can be adapted
by those skilled in the art in order to incorporate specific
transgene sequences into organisms utilizing the methods and
materials described below.
[0193] In an illustrative embodiment, either the cre/loxP
recombinase system of bacteriophage P1 (Lakso et al. (1992) PNAS
89: 6232-6236; Orban et al. (1992) PNAS 89: 6861-6865) or the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251: 1351-1355; PCT publication WO 92/15694) can be
used to generate in vivo site-specific genetic recombination
systems. Cre recombinase catalyzes the site-specific recombination
of an intervening target sequence located between loxP sequences.
loxP sequences are 34 base pair nucleotide repeat sequences to
which the Cre recombinase binds and are required for Cre
recombinase mediated genetic recombination. The orientation of loxP
sequences determines whether the intervening target sequence is
excised or inverted when Cre recombinase is present (Abremski et
al. (1984) J. Biol. Chem. 259: 1509-1514); catalyzing the excision
of the target sequence when the loxP sequences are oriented as
direct repeats and catalyzes inversion of the target sequence when
loxP sequences are oriented as inverted repeats.
[0194] Accordingly, genetic recombination of the target sequence is
dependent on expression of the Cre recombinase. Expression of the
recombinase can be regulated by promoter elements which are subject
to regulatory control, e.g., tissue-specific, developmental
stage-specific, inducible or repressible by externally added
agents. This regulated control will result in genetic recombination
of the target sequence only in cells where recombinase expression
is mediated by the promoter element. Thus, the activation
expression of a recombinant FKHL7 protein can be regulated via
control of recombinase expression.
[0195] Use of the cre/loxP recombinase system to regulate
expression of a recombinant FKHL7 protein requires the construction
of a transgenic animal containing transgenes encoding both the Cre
recombinase and the subject protein. Animals containing both the
Cre recombinase and a recombinant FKHL7 gene can be provided
through the construction of "double" transgenic animals. A
convenient method for providing such animals is to mate two
transgenic animals each containing a transgene, e.g., an FKHL7 gene
and recombinase gene.
[0196] One advantage derived from initially constructing transgenic
animals containing a FKHL7 transgene in a recombinase-mediated
expressible format derives from the likelihood that the subject
protein, whether agonistic or antagonistic, can be deleterious upon
expression in the transgenic animal. In such an instance, a founder
population, in which the subject transgene is silent in all
tissues, can be propagated and maintained. Individuals of this
founder population can be crossed with animals expressing the
recombinase in, for example, one or more tissues and/or a desired
temporal pattern. Thus, the creation of a founder population in
which, for example, an antagonistic FKHL7 transgene is silent will
allow the study of progeny from that founder in which disruption of
FKHL7 mediated induction in a particular tissue or at certain
developmental stages would result in, for example, a lethal
phenotype.
[0197] Similar conditional transgenes can be provided using
prokaryotic promoter sequences which require prokaryotic proteins
to be simultaneous expressed in order to facilitate expression of
the FKHL7 transgene. Exemplary promoters and the corresponding
trans-activating prokaryotic proteins are given in U.S. Pat. No.
4,833,080.
[0198] Moreover, expression of the conditional transgenes can be
induced by gene therapy-like methods wherein a gene encoding the
trans-activating protein, e.g. a recombinase or a prokaryotic
protein, is delivered to the tissue and caused to be expressed,
such as in a cell-type specific manner. By this method, a FKHL7
transgene could remain silent into adulthood until "turned on" by
the introduction of the trans-activator.
[0199] In an exemplary embodiment, the "transgenic non-human
animals" of the invention are produced by introducing transgenes
into the germline of the non-human animal. Embryonal target cells
at various developmental stages can be used to introduce
transgenes. Different methods are used depending on the stage of
development of the embryonal target cell. The specific line(s) of
any animal used to practice this invention are selected for general
good health, good embryo yields, good pronuclear visibility in the
embryo, and good reproductive fitness. In addition, the haplotype
is a significant factor. For example, when transgenic mice are to
be produced, strains such as C57BL/6 or FVB lines are often used
(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those
with H-2.sup.b, H-2.sup.d or H-2.sup.q haplotypes such as C57BL/6
or DBA/1. The line(s) used to practice this invention may
themselves be transgenics, and/or may be knockouts (i.e., obtained
from animals which have one or more genes partially or completely
suppressed).
[0200] In one embodiment, the transgene construct is introduced
into a single stage embryo. The zygote is the best target for
micro-injection. In the mouse, the male pronucleus reaches the size
of approximately 20 micrometers in diameter which allows
reproducible injection of 1-2 pl of DNA solution. The use of
zygotes as a target for gene transfer has a major advantage in that
in most cases the injected DNA will be incorporated into the host
gene before the first cleavage (Brinster et al. (1985) PNAS 82:
4438-4442). As a consequence, all cells of the transgenic animal
will carry the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene.
[0201] Normally, fertilized embryos are incubated in suitable media
until the pronuclei appear. At about this time, the nucleotide
sequence comprising the transgene is introduced into the female or
male pronucleus as described below. In some species such as mice,
the male pronucleus is preferred. It is most preferred that the
exogenous genetic material be added to the male DNA complement of
the zygote prior to its being processed by the ovum nucleus or the
zygote female pronucleus. It is thought that the ovum nucleus or
female pronucleus release molecules which affect the male DNA
complement, perhaps by replacing the protamines of the male DNA
with histones, thereby facilitating the combination of the female
and male DNA complements to form the diploid zygote.
[0202] Thus, it is preferred that the exogenous genetic material be
added to the male complement of DNA or any other complement of DNA
prior to its being affected by the female pronucleus. For example,
the exogenous genetic material is added to the early male
pronucleus, as soon as possible after the formation of the male
pronucleus, which is when the male and female pronuclei are well
separated and both are located close to the cell membrane.
Alternatively, the exogenous genetic material could be added to the
nucleus of the sperm after it has been induced to undergo
decondensation. Sperm containing the exogenous genetic material can
then be added to the ovum or the decondensed sperm could be added
to the ovum with the transgene constructs being added as soon as
possible thereafter.
[0203] Introduction of the transgene nucleotide sequence into the
embryo may be accomplished by any means known in the art such as,
for example, microinjection, electroporation, or lipofection.
Following introduction of the transgene nucleotide sequence into
the embryo, the embryo may be incubated in vitro for varying
amounts of time, or reimplanted into the surrogate host, or both.
In vitro incubation to maturity is within the scope of this
invention. One common method in to incubate the embryos in vitro
for about 1-7 days, depending on the species, and then reimplant
them into the surrogate host.
[0204] For the purposes of this invention a zygote is essentially
the formation of a diploid cell which is capable of developing into
a complete organism. Generally, the zygote will be comprised of an
egg containing a nucleus formed, either naturally or artificially,
by the fusion of two haploid nuclei from a gamete or gametes. Thus,
the gamete nuclei must be ones which are naturally compatible,
i.e., ones which result in a viable zygote capable of undergoing
differentiation and developing into a functioning organism.
Generally, a euploid zygote is preferred. If an aneuploid zygote is
obtained, then the number of chromosomes should not vary by more
than one with respect to the euploid number of the organism from
which either gamete originated.
[0205] In addition to similar biological considerations, physical
ones also govern the amount (e.g., volume) of exogenous genetic
material which can be added to the nucleus of the zygote or to the
genetic material which forms a part of the zygote nucleus. If no
genetic material is removed, then the amount of exogenous genetic
material which can be added is limited by the amount which will be
absorbed without being physically disruptive. Generally, the volume
of exogenous genetic material inserted will not exceed about 10
picoliters. The physical effects of addition must not be so great
as to physically destroy the viability of the zygote. The
biological limit of the number and variety of DNA sequences will
vary depending upon the particular zygote and functions of the
exogenous genetic material and will be readily apparent to one
skilled in the art, because the genetic material, including the
exogenous genetic material, of the resulting zygote must be
biologically capable of initiating and maintaining the
differentiation and development of the zygote into a functional
organism.
[0206] The number of copies of the transgene constructs which are
added to the zygote is dependent upon the total amount of exogenous
genetic material added and will be the amount which enables the
genetic transformation to occur. Theoretically only one copy is
required; however, generally, numerous copies are utilized, for
example, 1,000-20,000 copies of the transgene construct, in order
to insure that one copy is functional. As regards the present
invention, there will often be an advantage to having more than one
functioning copy of each of the inserted exogenous DNA sequences to
enhance the phenotypic expression of the exogenous DNA
sequences.
[0207] Any technique which allows for the addition of the exogenous
genetic material into nucleic genetic material can be utilized so
long as it is not destructive to the cell, nuclear membrane or
other existing cellular or genetic structures. The exogenous
genetic material is preferentially inserted into the nucleic
genetic material by microinjection. Microinjection of cells and
cellular structures is known and is used in the art.
[0208] Reimplantation is accomplished using standard methods.
Usually, the surrogate host is anesthetized, and the embryos are
inserted into the oviduct. The number of embryos implanted into a
particular host will vary by species, but will usually be
comparable to the number of off spring the species naturally
produces.
[0209] Transgenic offspring of the surrogate host may be screened
for the presence and/or expression of the transgene by any suitable
method. Screening is often accomplished by Southern blot or
Northern blot analysis, using a probe that is complementary to at
least a portion of the transgene. Western blot analysis using an
antibody against the protein encoded by the transgene may be
employed as an alternative or additional method for screening for
the presence of the transgene product. Typically, DNA is prepared
from tail tissue and analyzed by Southern analysis or PCR for the
transgene. Alternatively, the tissues or cells believed to express
the transgene at the highest levels are tested for the presence and
expression of the transgene using Southern analysis or PCR,
although any tissues or cell types may be used for this
analysis.
[0210] Alternative or additional methods for evaluating the
presence of the transgene include, without limitation, suitable
biochemical assays such as enzyme and/or immunological assays,
histological stains for particular marker or enzyme activities,
flow cytometric analysis, and the like. Analysis of the blood may
also be useful to detect the presence of the transgene product in
the blood, as well as to evaluate the effect of the transgene on
the levels of various types of blood cells and other blood
constituents.
[0211] Progeny of the transgenic animals may be obtained by mating
the transgenic animal with a suitable partner, or by in vitro
fertilization of eggs and/or sperm obtained from the transgenic
animal. Where mating with a partner is to be performed, the partner
may or may not be transgenic and/or a knockout; where it is
transgenic, it may contain the same or a different transgene, or
both. Alternatively, the partner may be a parental line. Where in
vitro fertilization is used, the fertilized embryo may be implanted
into a surrogate host or incubated in vitro, or both. Using either
method, the progeny may be evaluated for the presence of the
transgene using methods described above, or other appropriate
methods.
[0212] The transgenic animals produced in accordance with the
present invention will include exogenous genetic material. As set
out above, the exogenous genetic material will, in certain
embodiments, be a DNA sequence which results in the production of a
FKHL7 protein (either agonistic or antagonistic), and antisense
transcript, or a FKHL7 mutant. Further, in such embodiments the
sequence will be attached to a transcriptional control element,
e.g., a promoter, which preferably allows the expression of the
transgene product in a specific type of cell.
[0213] Retroviral infection can also be used to introduce transgene
into a non-human animal. The developing non-human embryo can be
cultured in vitro to the blastocyst stage. During this time, the
blastomeres can be targets for retroviral infection (Jaenich, R.
(1976) PNAS 73: 1260-1264). Efficient infection of the blastomeres
is obtained by enzymatic treatment to remove the zona pellucida
(Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1986). The viral vector
system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al. (1985) PNAS 82: 6927-6931; Van der Putten et al. (1985) PNAS
82: 6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6: 383-388).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298: 623-628). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic non-human animal.
Further, the founder may contain various retroviral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0214] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984)
Nature 309: 255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and
Robertson et al. (1986) Nature 322: 445-448). Transgenes can be
efficiently introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a non-human animal.
The ES cells thereafter colonize the embryo and contribute to the
germ line of the resulting chimeric animal. For review see
Jaenisch, R. (1988) Science 240: 1468-1474.
[0215] 4.7. Screening Assays for FKHL7 Therapeutics
[0216] The invention further provides screening methods for
identifying FKHL7 therapeutics, e.g., for treating and/or
preventing the development of a congenital heart disease.
[0217] An FKHL7 therapeutic can be any type of compound, including
a protein, a peptide, peptidomimetic, small molecule, and nucleic
acid. A nucleic acid can be, e.g., an FKHL7 gene, an antisense
nucleic acid, a ribozyme, or a triplex molecule. An FKHL7
therapeutic of the invention can be an agonist or an antagonist.
Preferred FKHL7 agonists include FKHL7 genes or proteins or
derivatives thereof which mimic at least one FKHL7 activity. Other
preferred agonists include compounds which are capable of
increasing the production of an FKHL7 protein in a cell, e.g.,
compounds capable of upregulating the expression of an FKHL7 gene,
and compounds which are capable of enhancing an FKHL7 activity
and/or the interaction of an FKHL7 protein with another molecule,
such as a target peptide. Preferred FKHL7 antagonists include FKHL7
proteins which are dominant negative proteins. Other preferred
antagonists include compounds which decrease or inhibit the
production of an FKHL7 protein in a cell and compounds which are
capable of downregulating expression of an FKHL7 gene, and
compounds which are capable of downregulating an FKHL7 activity
and/or interaction of an FKHL7 protein with another molecule. In
another preferred embodiment, an FKHL7 antagonist is a modified
form of a target peptide, which is capable of binding to a gene,
but which does not regulate expression of the gene.
[0218] The invention also provides screening methods for
identifying FKHL7 agonist and antagonist compounds, comprising
selecting compounds which are capable of interacting with an FKHL7
protein or with a molecule capable of interacting with an FKHL7
protein. In general, a molecule which is capable of interacting
with an FKHL7 protein is referred to herein as "FKHL7 binding
partner".
[0219] The compounds of the invention can be identified using
various assays depending on the type of compound and activity of
the compound that is desired. In addition, as described herein, the
test compounds can be further tested in animal models. Set forth
below are at least some assays that can be used for identifying
FKHL7 therapeutics. However, based on the instant disclosure, one
of skill in the art could use additional assays for identifying
FKHL7 therapeutics without requiring undue experimentation.
[0220] 4.7.1. Cell-Free Assays
[0221] Cell-free assays can be used to identify compounds which are
capable of interacting with an FKHL7 protein or binding partner, to
thereby modify the activity of the FKHL7 protein or binding
partner. Such a compound can, e.g., modify the structure of an
FKHL7 protein or binding partner and thereby effect its activity.
Cell-free assays can also be used to identify compounds which
modulate the interaction between an FKHL7 protein and an FKHL7
binding partner, such as a target peptide. In a preferred
embodiment, cell-free assays for identifying such compounds consist
essentially in a reaction mixture containing an FKHL7 protein and a
test compound or a library of test compounds in the presence or
absence of a binding partner. A test compound can be, e.g., a
derivative of an FKHL7 binding partner, e.g., a biologically
inactive target peptide, or a small molecule.
[0222] Accordingly, one exemplary screening assay of the present
invention includes the steps of contacting an FKHL7 protein or
functional fragment thereof or an FKHL7 binding partner with a test
compound or library of test compounds and detecting the formation
of complexes. For detection purposes, the molecule can be labeled
with a specific marker and the test compound or library of test
compounds labeled with a different marker. Interaction of a test
compound with an FKHL7 protein or fragment thereof or FKHL7 binding
partner can then be detected by determining the level of the two
labels after an incubation step and a washing step. The presence of
two labels after the washing step is indicative of an
interaction.
[0223] An interaction between molecules can also be identified by
using real-time BIA (Biomolecular Interaction Analysis, Pharmacia
Biosensor AB) which detects surface plasmon resonance (SPR), an
optical phenomenon. Detection depends on changes in the mass
concentration of macromolecules at the biospecific interface, and
does not require any labeling of interactants. In one embodiment, a
library of test compounds can be immobilized on a sensor surface,
e.g., which forms one wall of a micro-flow cell. A solution
containing the FKHL7 protein, functional fragment thereof, FKHL7
analog or FKHL7 binding partner is then flown continuously over the
sensor surface. A change in the resonance angle as shown on a
signal recording, indicates that an interaction has occurred. This
technique is further described, e.g., in BIAtechnology Handbook by
Pharmacia.
[0224] Another exemplary screening assay of the present invention
includes the steps of (a) forming a reaction mixture including: (i)
an FKHL7 polypeptide, (ii) an FKHL7 binding partner, and (iii) a
test compound; and (b) detecting interaction of the FKHL7 and the
FKHL7 binding protein. The FKHL7 polypeptide and FKHL7 binding
partner can be produced recombinantly, purified from a source,
e.g., plasma, or chemically synthesized, as described herein. A
statistically significant change (potentiation or inhibition) in
the interaction of the FKHL7 and FKHL7 binding protein in the
presence of the test compound, relative to the interaction in the
absence of the test compound, indicates a potential agonist
(mimetic or potentiator) or antagonist (inhibitor) of FKHL7
bioactivity for the test compound. The compounds of this assay can
be contacted simultaneously. Alternatively, an FKHL7 protein can
first be contacted with a test compound for an appropriate amount
of time, following which the FKHL7 binding partner is added to the
reaction mixture. The efficacy of the compound can be assessed by
generating dose response curves from data obtained using various
concentrations of the test compound. Moreover, a control assay can
also be performed to provide a baseline for comparison. In the
control assay, isolated and purified FKHL7 polypeptide or binding
partner is added to a composition containing the FKHL7 binding
partner or FKHL7 polypeptide, and the formation of a complex is
quantitated in the absence of the test compound.
[0225] Complex formation between an FKHL7 protein and an FKHL7
binding partner may be detected by a variety of techniques.
Modulation of the formation of complexes can be quantitated using,
for example, detectably labeled proteins such as radiolabeled,
fluorescently labeled, or enzymatically labeled FKHL7 proteins or
FKHL7 binding partners, by immunoassay, or by chromatographic
detection.
[0226] Typically, it will be desirable to immobilize either FKHL7
or its binding partner to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of FKHL7 to an FKHL7
binding partner, can be accomplished in any vessel suitable for
containing the reactants. Examples include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows the protein
to be bound to a matrix. For example,
glutathione-5-transferase/FKHL7 (GST/FKHL7) fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtitre plates, which are
then combined with the FKHL7 binding partner, e.g. an
.sup.35S-labeled FKHL7 binding partner, and the test compound, and
the mixture incubated under conditions conducive to complex
formation, e.g. at physiological conditions for salt and pH, though
slightly more stringent conditions may be desired. Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly (e.g.
beads placed in scintilant), or in the supernatant after the
complexes are subsequently dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of FKHL7 protein or FKHL7 binding partner
found in the bead fraction quantitated from the gel using standard
electrophoretic techniques such as described in the appended
examples.
[0227] Other techniques for immobilizing proteins on matrices are
also available for use in the subject assay. For instance, either
FKHL7 or its cognate binding partner can be immobilized utilizing
conjugation of biotin and streptavidin. For instance, biotinylated
FKHL7 molecules can be prepared from biotin-NHS
(N-hydroxysuccinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with can be
derivatized to the wells of the plate, and FKHL7 trapped in the
wells by antibody conjugation. As above, preparations of an FKHL7
binding protein and a test compound are incubated in the FKHL7
presenting wells of the plate, and the amount of complex trapped in
the well can be quantitated. Exemplary methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the FKHL7 binding partner, or which
are reactive with FKHL7 protein and compete with the binding
partner; as well as enzyme-linked assays which rely on detecting an
enzymatic activity associated with the binding partner, either
intrinsic or extrinsic activity. In the instance of the latter, the
enzyme can be chemically conjugated or provided as a fusion protein
with the FKHL7 binding partner. To illustrate, the FKHL7 binding
partner can be chemically cross-linked or genetically fused with
horseradish peroxidase, and the amount of polypeptide trapped in
the complex can be assessed with a chromogenic substrate of the
enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or
4-chloro-1-napthol. Likewise, a fusion protein comprising the
polypeptide and glutathione-5-transferase can be provided, and
complex formation quantitated by detecting the GST activity using
1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:
7130).
[0228] For processes which rely on immunodetection for quantitating
one of the proteins trapped in the complex, antibodies against the
protein, such as anti-FKHL7 antibodies, can be used. Alternatively,
the protein to be detected in the complex can be "epitope tagged"
in the form of a fusion protein which includes, in addition to the
FKHL7 sequence, a second polypeptide for which antibodies are
readily available (e.g. from commercial sources). For instance, the
GST fusion proteins described above can also be used for
quantification of binding using antibodies against the GST moiety.
Other useful epitope tags include myc-epitopes (e.g., see Ellison
et al. (1991) J Biol Chem 266: 21150-21157) which includes a
10-residue sequence from c-myc, as well as the pFLAG system
(International Biotechnologies, Inc.) or the pEZZ-protein A system
(Pharmacia, NJ).
[0229] Cell-free assays can also be used to identify compounds
which interact with an FKHL7 protein and modulate an activity of an
FKHL7 protein. Accordingly, in one embodiment, an FKHL7 protein is
contacted with a test compound and the catalytic activity of FKHL7
is monitored. In one embodiment, the abililty of FKHL7 to bind a
target molecule is determined. The binding affinity of FKHL7 to a
target molecule can be determined according to methods known in the
art. Determination of the enzymatic activity of FKHL7 can be
performed with the aid of the substrate
furanacryloyl-L-phenylalanyl-glycyl-glycine (FAPGG) under
conditions described in Holmquist et al. (1979) Anal. Biochem. 95:
540 and in U.S. Pat. No. 5,259,045.
[0230] 4.7.2. Cell Based Assays
[0231] In addition to cell-free assays, such as described above,
FKHL7 proteins as provided by the present invention, facilitate the
generation of cell-based assays, e.g., for identifying small
molecule agonists or antagonists. Cell based assays can be used,
for example, to identify compounds which modulate expression of an
FKHL7 gene, modulate translation of an FKHL7 mRNA, which modulate
the stability of an FKHL7 mRNA or protein or which otherwise
interfere with an interaction between an FKHL7 gene or protein and
an FKHL7 binding partner. Accordingly, in one embodiment, a cell
which is capable of producing FKHL7 is incubated with a test
compound and the amount of FKHL7 produced in the cell medium is
measured and compared to that produced from a cell which has not
been contacted with the test compound. The specificity of the
compound vis a vis FKHL7 can be confirmed by various control
analysis, e.g., measuring the expression of one or more control
genes. Compounds which can be tested include small molecules,
proteins, and nucleic acids. In particular, this assay can be used
to determine the efficacy of FKHL7 antisense molecules or
ribozymes.
[0232] In another embodiment, the effect of a test compound on
transcription of an FKHL7 gene is determined by transfection
experiments using a reporter gene operatively linked to at least a
portion of the promoter of an FKHL7 gene. A promoter region of a
gene can be isolated, e.g., from a genomic library according to
methods known in the art. The reporter gene can be any gene
encoding a protein which is readily quantifiable, e.g, the
luciferase or CAT gene. Such reporter gene are well known in the
art.
[0233] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0234] 4.8. Predictive Medicine
[0235] The invention further features predictive medicines, which
are based, at least in part, on the identity of the novel FKHL7
gene and alterations in the genes and related pathway genes, which
affect the expression level and/or function of the encoded FKHL7
protein in a subject.
[0236] For example, as described herein, FKHL7 mutations that are
particularly likely to cause or contribute to the development of a
congenital heart disease are those mutations that negatively impact
normal (wildtype) functioning of the forkhead domain that is
involved with the DNA binding properties of FKHL7. Examples of such
mutations include: i) upstream mutations that encode truncated
transcripts that lack the DNA-binding, forkhead domain (e.g. an 11
base pair deletion encoding an FKHL7 transcript that is missing 477
amino acids); and ii) missense mutations occurring within the
forkhead domain (e.g. a cytosine to thymine transition that causes
an amino acid change at position 131 from serine to leucine
(Ser131Leu); a cytosine to guanine transition that causes an amino
acid change at position 126 from isoleucine to methionine
(Ile126Met); and a thymine to cytosine transition, which results in
a replacement of phenylalanine with serine at position 112
(Phe112Ser). In addition, mutations or translocations that result
in expression of only one copy of FHKL7 (e.g. monosomy of 6p25),
can result in a congenital heart disease phenotype.
[0237] Information obtained using the diagnostic assays described
herein (alone or in conjunction with information on another genetic
defect, which contributes to the same disease) is useful for
prognosing, diagnosing or confirming that a subject has a genetic
defect (e.g. in an FKHL7 gene or in a gene that regulates the
expression of an FKHL7 gene), which causes or contributes to the
development of glaucoma. Based on prognostic information, a doctor
can recommend a regimen (e.g. diet or exercise) or therapeutic
protocol, which is useful for preventing or prolonging onset of
congenital heart disease in the individual.
[0238] In addition, knowledge of the particular alteration or
alterations, resulting in defective or deficient FKHL7 genes or
proteins in an individual (the FKHL7 genetic profile), alone or in
conjunction with information on other genetic defects contributing
to a congenital heart disease (the congenital heart disease genetic
profile) allows customization of therapy to the individual's
genetic profile, the goal of "pharmacogenomics". For example, an
individual's FKHL7 genetic profile or the congenital heart disease
genetic profile, can enable a doctor to: 1) more effectively
prescribe a drug that will address the molecular basis of the
glaucoma; and 2) better determine the appropriate dosage of a
particular drug for the particular individual. For example, the
expression level of FKHL7 proteins, alone or in conjunction with
the expression level of other genes, known to contribute to the
same disease, can be measured in many patients at various stages of
the disease to generate a transcriptional or expression profile of
the disease. Expression patterns of individual patients can then be
compared to the expression profile of the disease to determine the
appropriate drug and dose to administer to the patient.
[0239] The ability to target populations expected to show the
highest clinical benefit, based on the FKHL7 or disease genetic
profile, can enable: 1) the repositioning of marketed drugs with
disappointing market results; 2) the rescue of drug candidates
whose clinical development has been discontinued as a result of
safety or efficacy limitations, which are patient
subgroup-specific; and 3) an accelerated and less costly
development for drug candidates and more optimal drug labeling
(e.g. since the use of FKHL7 as a marker is useful for optimizing
effective dose).
[0240] These and other methods are described in further detail in
the following sections.
[0241] 4.8.1. Prognostic and Diagnostic Assays
[0242] The present methods provide means for determining if a
subject has (diagnostic) or is at risk of developing (prognostic) a
disease, condition or disorder that is associated with an aberrant
FKHL7 activity, e.g., an aberrant level of FKHL7 protein or an
aberrant bioactivity, such as results in the development of a
congenital heart disease.
[0243] Accordingly, the invention provides methods for determining
whether a subject has or is likely to develop a congenital heart
disease, comprising determining the level of an FKHL7 gene or
protein, an FKHL7 bioactivity and/or the presence of a mutation or
particular polymorphic variant in the FKHL7 gene.
[0244] In one embodiment, the method comprises determining whether
a subject has an abnormal mRNA and/or protein level of FKHL7, such
as by Northern blot analysis, reverse transcription-polymerase
chain reaction (RT-PCR), in situ hybridization,
immunoprecipitation, Western blot hybridization, or
immunohistochemistry. According to the method, cells are obtained
from a subject and the FKHL7 protein or mRNA level is determined
and compared to the level of FKHL7 protein or mRNA level in a
healthy subject. An abnormal level of FKHL7 polypeptide or mRNA
level is likely to be indicative of an aberrant FKHL7 activity.
[0245] In another embodiment, the method comprises measuring at
least one activity of FKHL7. For example, regulation of the
expression of a gene by an FKHL7 can be determined, e.g., as
described herein. Comparison of the results obtained with results
from similar analysis performed on FKHL7 proteins from healthy
subjects is indicative of whether a subject has an abnormal FKHL7
activity.
[0246] In preferred embodiments, the methods for determining
whether a subject has or is at risk for developing a disease, which
is caused by or contributed to by an aberrant FKHL7 activity is
characterized as comprising detecting, in a sample of cells from
the subject, the presence or absence of a genetic alteration
characterized by at least one of: (i) an alteration affecting the
integrity of a gene encoding an FKHL7 polypeptide, or (ii) the
mis-expression of the FKHL7 gene. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of: (i) a deletion of one or more nucleotides from an
FKHL7 gene, (ii) an addition of one or more nucleotides to an FKHL7
gene, (iii) a substitution of one or more nucleotides of an FKHL7
gene, (iv) a gross chromosomal rearrangement of an FKHL7 gene, (v)
a gross alteration in the level of a messenger RNA transcript of an
FKHL7 gene, (vi) aberrant modification of an FKHL7 gene, such as of
the methylation pattern of the genomic DNA, (vii) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of an
FKHL7 gene, (viii) a non-wild type level of an FKHL7 polypeptide,
(ix) allelic loss of an FKHL7 gene, and/or (x) inappropriate
post-translational modification of an FKHL7 polypeptide. As set out
below, the present invention provides a large number of assay
techniques for detecting alterations in an FKHL7 gene. These
methods include, but are not limited to, methods involving sequence
analysis, Southern blot hybridization, restriction enzyme site
mapping, and methods involving detection of the absence of
nucleotide pairing between the nucleic acid to be analyzed and a
probe. These and other methods are further described infra.
[0247] Specific diseases or disorders, e.g., genetic diseases or
disorders, are associated with specific allelic variants of
polymorphic regions of certain genes, which do not necessarily
encode a mutated protein. Thus, the presence of a specific allelic
variant of a polymorphic region of a gene, such as a single
nucleotide polymorphism ("SNP"), in a subject can render the
subject susceptible to developing a specific disease or disorder.
Polymorphic regions in genes, e.g, FKHL7 genes, can be identified,
by determining the nucleotide sequence of genes in populations of
individuals. If a polymorphic region, e.g., SNP is identified, then
the link with a specific disease can be determined by studying
specific populations of individuals, e.g, individuals which
developed a specific disease, such as glaucoma. A polymorphic
region can be located in any region of a gene, e.g., exons, in
coding or non coding regions of exons, introns, and promoter
region.
[0248] It is likely that FKHL7 genes comprise polymorphic regions,
specific alleles of which may be associated with specific diseases
or conditions or with an increased likelihood of developing such
diseases or conditions. Thus, the invention provides methods for
determining the identity of the allele or allelic variant of a
polymorphic region of an FKHL7 gene in a subject, to thereby
determine whether the subject has or is at risk of developing a
disease or disorder that is associated with a specific allelic
variant of a polymorphic region.
[0249] In an exemplary embodiment, there is provided a nucleic acid
composition comprising a nucleic acid probe including a region of
nucleotide sequence which is capable of hybridizing to a sense or
antisense sequence of an FKHL7 gene or naturally occurring mutants
thereof, or 5' or 3' flanking sequences naturally associated with
the subject FKHL7 genes or naturally occurring mutants thereof. The
nucleic acid of a cell is rendered accessible for hybridization,
the probe is contacted with the nucleic acid of the sample, and the
hybridization of the probe to the sample nucleic acid is detected.
Such techniques can be used to detect alterations or allelic
variants at either the genomic or mRNA level, including deletions,
substitutions, etc., as well as to determine mRNA transcript
levels.
[0250] A preferred detection method is allele specific
hybridization using probes overlapping the mutation or polymorphic
site and having about 5, 10, 20, 25, or 30 nucleotides around the
mutation or polymorphic region. In a preferred embodiment of the
invention, several probes capable of hybridizing specifically to
allelic variants, such as single nucleotide polymorphisms, are
attached to a solid phase support, e.g., a "chip". Oligonucleotides
can be bound to a solid support by a variety of processes,
including lithography. For example a chip can hold up to about
250,000 oligonucleotides. Mutation detection analysis using these
chips comprising oligonucleotides, also termed "DNA probe arrays"
is described e.g., in Cronin et al. (1996) Human Mutation 7: 244.
In one embodiment, a chip comprises all the allelic variants of at
least one polymorphic region of a gene. The solid phase support is
then contacted with a test nucleic acid and hybridization to the
specific probes is detected. Accordingly, the identity of numerous
allelic variants of one or more genes can be identified in a simple
hybridization experiment.
[0251] In certain embodiments, detection of the alteration
comprises utilizing the probe/primer in a polymerase chain reaction
(PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as
anchor PCR or RACE PCR, or, alternatively, in a ligase chain
reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:
1077-1080; and Nakazawa et al. (1994) PNAS 91: 360-364), the latter
of which can be particularly useful for detecting point mutations
in the FKHL7 gene (see Abravaya et al. (1995) Nuc Acid Res 23:
675-682). In a merely illustrative embodiment, the method includes
the steps of (i) collecting a sample of cells from a patient, (ii)
isolating nucleic acid (e.g., genomic, mRNA or both) from the cells
of the sample, (iii) contacting the nucleic acid sample with one or
more primers which specifically hybridize to an FKHL7 gene under
conditions such that hybridization and amplification of the FKHL7
gene (if present) occurs, and (iv) detecting the presence or
absence of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
It is anticipated that PCR, LCR or any other amplification
procedure (e.g. self sustained sequence replication (Guatelli, J.
C. et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878),
transcriptional amplification system (Kwoh, D. Y. et al., 1989,
Proc. Natl. Acad. Sci. USA 86: 1173-1177), or Q-Beta Replicase
(Lizardi, P. M. et al., 1988, Bio/Technology 6: 1197)), may be used
as a preliminary step to increase the amount of sample on which can
be performed, any of the techniques for detecting mutations
described herein.
[0252] In a preferred embodiment of the subject assay, mutations
in, or allelic variants, of an FKHL7 gene from a sample cell are
identified by alterations in restriction enzyme cleavage patterns.
For example, sample and control DNA is isolated, amplified
(optionally), digested with one or more restriction endonucleases,
and fragment length sizes are determined by gel electrophoresis.
Moreover, the use of sequence specific ribozymes (see, for example,
U.S. Pat. No. 5,498,531) can be used to score for the presence of
specific mutations by development or loss of a ribozyme cleavage
site.
[0253] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
FKHL7 gene and detect mutations by comparing the sequence of the
sample FKHL7 with the corresponding wild-type (control) sequence.
Exemplary sequencing reactions include those based on techniques
developed by Maxim and Gilbert (Proc. Natl Acad Sci USA (1977) 74:
560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74: 5463).
It is also contemplated that any of a variety of automated
sequencing procedures may be utilized when performing the subject
assays (Biotechniques (1995) 19: 448), including sequencing by mass
spectrometry (see, for example PCT publication WO 94/16101; Cohen
et al. (1996) Adv Chromatogr 36: 127-162; and Griffin et al.
(1993)Appl Biochem Biotechnol 38: 147-159). It will be evident to
one skilled in the art that, for certain embodiments, the
occurrence of only one, two or three of the nucleic acid bases need
be determined in the sequencing reaction. For instance, A-track or
the like, e.g., where only one nucleic acid is detected, can be
carried out.
[0254] In a further embodiment, protection from cleavage agents
(such as a nuclease, hydroxylamine or osmium tetroxide and with
piperidine) can be used to detect mismatched bases in RNA/RNA or
RNA/DNA or DNA/DNA heteroduplexes (Myers, et al. (1985) Science
230: 1242). In general, the art technique of "mismatch cleavage"
starts by providing heteroduplexes formed by hybridizing (labelled)
RNA or DNA containing the wild-type FKHL7 sequence with potentially
mutant RNA or DNA obtained from a tissue sample. The
double-stranded duplexes are treated with an agent which cleaves
single-stranded regions of the duplex such as which will exist due
to base pair mismatches between the control and sample strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA
hybrids treated with S1 nuclease to enzymatically digest the
mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA
duplexes can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions. After
digestion of the mismatched regions, the resulting material is then
separated by size on denaturing polyacrylamide gels to determine
the site of mutation. See, for example, Cotton et al (1988) Proc.
Natl Acad Sci USA 85: 4397; Saleeba et al (1992) Methods Enzymol.
217: 286-295. In a preferred embodiment, the control DNA or RNA can
be labeled for detection.
[0255] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in FKHL7
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15: 1657-1662). According to an exemplary
embodiment, a probe based on an FKHL7 sequence, e.g., a wild-type
FKHL7 sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[0256] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations or the identity of the
allelic variant of a polymorphic region in FKHL7 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA 86: 2766, see also Cotton (1993) Mutat Res 285: 125-144;
and Hayashi (1992) Genet Anal Tech Appl 9: 73-79). Single-stranded
DNA fragments of sample and control FKHL7 nucleic acids are
denatured and allowed to renature. The secondary structure of
single-stranded nucleic acids varies according to sequence, the
resulting alteration in electrophoretic mobility enables the
detection of even a single base change. The DNA fragments may be
labelled or detected with labelled probes. The sensitivity of the
assay may be enhanced by using RNA (rather than DNA), in which the
secondary structure is more sensitive to a change in sequence. In a
preferred embodiment, the subject method utilizes heteroduplex
analysis to separate double stranded heteroduplex molecules on the
basis of changes in electrophoretic mobility (Keen et al. (1991)
Trends Genet 7: 5).
[0257] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al (1985) Nature 313: 495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing agent gradient to identify differences in the mobility
of control and sample DNA (Rosenbaum and Reissner (1987) Biophys
Chem 265: 12753).
[0258] Examples of other techniques for detecting point mutations
or the identity of the allelic variant of a polymorphic region
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation or nucleotide difference (e.g., in allelic
variants) is placed centrally and then hybridized to target DNA
under conditions which permit hybridization only if a perfect match
is found (Saiki et al. (1986) Nature 324: 163); Saiki et al (1989)
Proc. Natl. Acad. Sci USA 86: 6230). Such allele specific
oligonucleotide hybridization techniques may be used to test one
mutation or polymorphic region per reaction when oligonucleotides
are hybridized to PCR amplified target DNA or a number of different
mutations or polymorphic regions when the oligonucleotides are
attached to the hybridizing membrane and hybridized with labelled
target DNA.
[0259] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation or
polymorphic region of interest in the center of the molecule (so
that amplification depends on differential hybridization) (Gibbs et
al (1989) Nucleic Acids Res. 17: 2437-2448) or at the extreme 3'
end of one primer where, under appropriate conditions, mismatch can
prevent, or reduce polymerase extension (Prossner (1993) Tibtech
11: 238. In addition it may be desirable to introduce a novel
restriction site in the region of the mutation to create
cleavage-based detection (Gasparini et al (1992) Mol. Cell Probes
6: 1). It is anticipated that in certain embodiments amplification
may also be performed using Taq ligase for amplification (Barany
(1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation
will occur only if there is a perfect match at the 3' end of the 5'
sequence making it possible to detect the presence of a known
mutation at a specific site by looking for the presence or absence
of amplification.
[0260] In another embodiment, identification of the allelic variant
is carried out using an oligonucleotide ligation assay (OLA), as
described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et
al., Science 241: 1077-1080 (1988). The OLA protocol uses two
oligonucleotides which are designed to be capable of hybridizing to
abutting sequences of a single strand of a target. One of the
oligonucleotides is linked to a separation marker, e.g,
biotinylated, and the other is detectably labeled. If the precise
complementary sequence is found in a target molecule, the
oligonucleotides will hybridize such that their termini abut, and
create a ligation substrate. Ligation then permits the labeled
oligonucleotide to be recovered using avidin, or another biotin
ligand. Nickerson, D. A. et al. have described a nucleic acid
detection assay that combines attributes of PCR and OLA (Nickerson,
D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87: 8923-8927 (1990).
In this method, PCR is used to achieve the exponential
amplification of target DNA, which is then detected using OLA.
[0261] Several techniques based on this OLA method have been
developed and can be used to detect specific allelic variants of a
polymorphic region of an FKHL7 gene. For example, U.S. Pat. No.
5,593,826 discloses an OLA using an oligonucleotide having 3'-amino
group and a 5'-phosphorylated oligonucleotide to form a conjugate
having a phosphoramidate linkage. In another variation of OLA
described in To be et al. ((1996) Nucleic Acids Res 24: 3728), OLA
combined with PCR permits typing of two alleles in a single
microtiter well. By marking each of the allele-specific primers
with a unique hapten, i.e. digoxigenin and fluorescein, each OLA
reaction can be detected by using hapten specific antibodies that
are labeled with different enzyme reporters, alkaline phosphatase
or horseradish peroxidase. This system permits the detection of the
two alleles using a high throughput format that leads to the
production of two different colors.
[0262] The invention further provides methods for detecting single
nucleotide polymorphisms in an FKHL7 gene. Because single
nucleotide polymorphisms constitute sites of variation flanked by
regions of invariant sequence, their analysis requires no more than
the determination of the identity of the single nucleotide present
at the site of variation and it is unnecessary to determine a
complete gene sequence for each patient. Several methods have been
developed to facilitate the analysis of such single nucleotide
polymorphisms.
[0263] In one embodiment, the single base polymorphism can be
detected by using a specialized exonuclease-resistant nucleotide,
as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127).
According to the method, a primer complementary to the allelic
sequence immediately 3' to the polymorphic site is permitted to
hybridize to a target molecule obtained from a particular animal or
human. If the polymorphic site on the target molecule contains a
nucleotide that is complementary to the particular
exonuclease-resistant nucleotide derivative present, then that
derivative will be incorporated onto the end of the hybridized
primer. Such incorporation renders the primer resistant to
exonuclease, and thereby permits its detection. Since the identity
of the exonuclease-resistant derivative of the sample is known, a
finding that the primer has become resistant to exonucleases
reveals that the nucleotide present in the polymorphic site of the
target molecule was complementary to that of the nucleotide
derivative used in the reaction. This method has the advantage that
it does not require the determination of large amounts of
extraneous sequence data.
[0264] In another embodiment of the invention, a solution-based
method is used for determining the identity of the nucleotide of a
polymorphic site. Cohen, D. et al. (French Patent 2,650,840; PCT
Appln. No. WO91/02087). As in the Mundy method of U.S. Pat. No.
4,656,127, a primer is employed that is complementary to allelic
sequences immediately 3' to a polymorphic site. The method
determines the identity of the nucleotide of that site using
labeled dideoxynucleotide derivatives, which, if complementary to
the nucleotide of the polymorphic site will become incorporated
onto the terminus of the primer.
[0265] An alternative method, known as Genetic Bit Analysis or
GBA.TM. is described by Goelet, P. et al. (PCT Appln. No.
92/15712). The method of Goelet, P. et al. uses mixtures of labeled
terminators and a primer that is complementary to the sequence 3'
to a polymorphic site. The labeled terminator that is incorporated
is thus determined by, and complementary to, the nucleotide present
in the polymorphic site of the target molecule being evaluated. In
contrast to the method of Cohen et al. (French Patent 2,650,840;
PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is
preferably a heterogeneous phase assay, in which the primer or the
target molecule is immobilized to a solid phase.
[0266] Recently, several primer-guided nucleotide incorporation
procedures for assaying polymorphic sites in DNA have been
described (Komher, J. S. et al., Nucl. Acids. Res. 17: 7779-7784
(1989); Sokolov, B. P., Nucl. Acids Res. 18: 3671 (1990); Syvanen,
A.-C., et al., Genomics 8: 684-692 (1990); Kuppuswamy, M. N. et
al., Proc. Natl. Acad. Sci. (U.S.A.) 88: 1143-1147 (1991); Prezant,
T. R. et al., Hum. Mutat. 1: 159-164 (1992); Ugozzoli, L. et al.,
GATA 9: 107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:
171-175 (1993)). These methods differ from GBA.TM. in that they all
rely on the incorporation of labeled deoxynucleotides to
discriminate between bases at a polymorphic site. In such a format,
since the signal is proportional to the number of deoxynucleotides
incorporated, polymorphisms that occur in runs of the same
nucleotide can result in signals that are proportional to the
length of the run (Syvanen, A.-C., et al., Amer. J. Hum. Genet. 52:
46-59 (1993)).
[0267] For mutations that produce premature termination of protein
translation, the protein truncation test (PTT) offers an efficient
diagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet. 2:
1719-21; van der Luijt, et. al., (1994) Genomics 20: 1-4). For PTT,
RNA is initially isolated from available tissue and
reverse-transcribed, and the segment of interest is amplified by
PCR. The products of reverse transcription PCR are then used as a
template for nested PCR amplification with a primer that contains
an RNA polymerase promoter and a sequence for initiating eukaryotic
translation. After amplification of the region of interest, the
unique motifs incorporated into the primer permit sequential in
vitro transcription and translation of the PCR products. Upon
sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
translation products, the appearance of truncated polypeptides
signals the presence of a mutation that causes premature
termination of translation. In a variation of this technique, DNA
(as opposed to RNA) is used as a PCR template when the target
region of interest is derived from a single exon.
[0268] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid, primer set; and/or antibody reagent described
herein, which may be conveniently used, e.g., in clinical settings
to diagnose patients exhibiting symptoms or family history of a
disease or illness involving an FKHL7 polypeptide.
[0269] Any cell type or tissue may be utilized in the diagnostics
described below. In a preferred embodiment a bodily fluid, e.g.,
blood, is obtained from the subject to determine the presence of a
mutation or the identity of the allelic variant of a polymorphic
region of an FKHL7 gene. A bodily fluid, e.g, blood, can be
obtained by known techniques (e.g. venipuncture). Alternatively,
nucleic acid tests can be performed on dry samples (e.g. hair or
skin). For prenatal diagnosis, fetal nucleic acid samples can be
obtained from maternal blood as described in International Patent
Application No. WO91/07660 to Bianchi. Alternatively, amniocytes or
chorionic villi may be obtained for performing prenatal
testing.
[0270] When using RNA or protein to determine the presence of a
mutation or of a specific allelic variant of a polymorphic region
of an FKHL7 gene, the cells or tissues that may be utilized must
express the FKHL7 gene. Preferred cells for use in these methods
include cardiac cells (see Examples). Alternative cells or tissues
that can be used, can be identified by determining the expression
pattern of the specific FKHL7 gene in a subject, such as by
Northern blot analysis.
[0271] Diagnostic procedures may also be performed in situ directly
upon tissue sections (fixed and/or frozen) of patient tissue
obtained from biopsies or resections, such that no nucleic acid
purification is necessary. Nucleic acid reagents may be used as
probes and/or primers for such in situ procedures (see, for
example, Nuovo, G. J., 1992, PCR in situ hybridization: protocols
and applications, Raven Press, NY).
[0272] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, profiles may also be
assessed in such detection schemes. Fingerprint profiles may be
generated, for example, by utilizing a differential display
procedure, Northern analysis and/or RT-PCR.
[0273] Antibodies directed against wild type or mutant FKHL7
polypeptides or allelic variants thereof, which are discussed
above, may also be used in disease diagnostics and prognostics.
Such diagnostic methods, may be used to detect abnormalities in the
level of FKHL7 polypeptide expression, or abnormalities in the
structure and/or tissue, cellular, or subcellular location of an
FKHL7 polypeptide. Structural differences may include, for example,
differences in the size, electronegativity, or antigenicity of the
mutant FKHL7 polypeptide relative to the normal FKHL7 polypeptide.
Protein from the tissue or cell type to be analyzed may easily be
detected or isolated using techniques which are well known to one
of skill in the art, including but not limited to western blot
analysis. For a detailed explanation of methods for carrying out
Western blot analysis, see Sambrook et al, 1989, supra, at Chapter
18. The protein detection and isolation methods employed herein may
also be such as those described in Harlow and Lane, for example,
(Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual",
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),
which is incorporated herein by reference in its entirety.
[0274] This can be accomplished, for example, by immunofluorescence
techniques employing a fluorescently labeled antibody (see below)
coupled with light microscopic, flow cytometric, or fluorimetric
detection. The antibodies (or fragments thereof) useful in the
present invention may, additionally, be employed histologically, as
in immunofluorescence or immunoelectron microscopy, for in situ
detection of FKHL7 polypeptides. In situ detection may be
accomplished by removing a histological specimen from a patient,
and applying thereto a labeled antibody of the present invention.
The antibody (or fragment) is preferably applied by overlaying the
labeled antibody (or fragment) onto a biological sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the FKHL7 polypeptide, but also its distribution in
the examined tissue. Using the present invention, one of ordinary
skill will readily perceive that any of a wide variety of
histological methods (such as staining procedures) can be modified
in order to achieve such in situ detection.
[0275] Often a solid phase support or carrier is used as a support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0276] One means for labeling an anti-FKHL7 polypeptide specific
antibody is via linkage to an enzyme and use in an enzyme
immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay
(ELISA)", Diagnostic Horizons 2: 1-7, 1978, Microbiological
Associates Quarterly Publication, Walkersville, Md.; Voller, et
al., J. Clin. Pathol. 31: 507-520 (1978); Butler, Meth. Enzymol.
73: 482-523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press,
Boca Raton, Fla., 1980; Ishikawa, et al., (eds.) Enzyme
Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound
to the antibody will react with an appropriate substrate,
preferably a chromogenic substrate, in such a manner as to produce
a chemical moiety which can be detected, for example, by
spectrophotometric, fluorimetric or by visual means. Enzymes which
can be used to detectably label the antibody include, but are not
limited to, malate dehydrogenase, staphylococcal nuclease,
delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alphaglycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0277] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect
fingerprint gene wild type or mutant peptides through the use of a
radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles
of Radioimmunoassays, Seventh Training Course on Radioligand Assay
Techniques, The Endocrine Society, March, 1986, which is
incorporated by reference herein). The radioactive isotope can be
detected by such means as the use of a gamma counter or a
scintillation counter or by autoradiography.
[0278] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, Q-phthaldehyde and
fluorescamine.
[0279] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0280] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0281] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0282] Moreover, it will be understood that any of the above
methods for detecting alterations in a gene or gene product or
polymorphic variants can be used to monitor the course of treatment
or therapy.
[0283] 4.8.2. Pharmacogenomics
[0284] Knowledge of the particular alteration or alterations,
resulting in defective or deficient FKHL7 genes or proteins in an
individual (the FKHL7 genetic profile), alone or in conjunction
with information on other genetic defects contributing to the same
disease (the genetic profile of the particular disease) allows a
customization of the therapy for a particular disease to the
individual's genetic profile, the goal of "pharmacogenomics". For
example, subjects having a specific allele of an FKHL7 gene may or
may not exhibit symptoms of a particular disease or be predisposed
of developing symptoms of a particular disease. Further, if those
subjects are symptomatic, they may or may not respond to a certain
drug, e.g., a specific FKHL7 therapeutic, but may respond to
another. Thus, generation of an FKHL7 genetic profile, (e.g.,
categorization of alterations in FKHL7 genes which are associated
with the development of glaucoma), from a population of subjects,
who are symptomatic for a disease or condition that is caused by or
contributed to by a defective and/or deficient FKHL7 gene and/or
protein (an FKHL7 genetic population profile) and comparison of an
individual's FKHL7 profile to the population profile, permits the
selection or design of drugs that are expected to be safe and
efficacious for a particular patient or patient population (i.e., a
group of patients having the same genetic alteration).
[0285] For example, an FKHL7 population profile can be performed,
by determining the FKHL7 profile, e.g., the identity of FKHL7
genes, in a patient population having a disease, which is caused by
or contributed to by a defective or deficient FKHL7 gene.
Optionally, the FKHL7 population profile can further include
information relating to the response of the population to an FKHL7
therapeutic, using any of a variety of methods, including,
monitoring: 1) the severity of symptoms associated with the FKHL7
related disease, 2) FKHL7 gene expression level, 3) FKHL7 mRNA
level, and/or 4) FKHL7 protein level and (iii) dividing or
categorizing the population based on the particular genetic
alteration or alterations present in its FKHL7 gene or an FKHL7
pathway gene. The FKHL7 genetic population profile can also,
optionally, indicate those particular alterations in which the
patient was either responsive or non-responsive to a particular
therapeutic. This information or population profile, is then useful
for predicting which individuals should respond to particular
drugs, based on their individual FKHL7 profile.
[0286] In a preferred embodiment, the FKHL7 profile is a
transcriptional or expression level profile and step (i) is
comprised of determining the expression level of FKHL7 proteins,
alone or in conjunction with the expression level of other genes,
known to contribute to the same disease. The FKHL7 profile can be
measured in many patients at various stages of the disease.
[0287] Pharmacogenomic studies can also be performed using
transgenic animals. For example, one can produce transgenic mice,
e.g., as described herein, which contain a specific allelic variant
of an FKHL7 gene. These mice can be created, e.g, by replacing
their wild-type FKHL7 gene with an allele of the human FKHL7 gene.
The response of these mice to specific FKHL7 therapeutics can then
be determined.
[0288] 4.8.3. Monitoring of Effects of FKHL7 Therapeutics During
Clinical Trials
[0289] The ability to target populations expected to show the
highest clinical benefit, based on the FKHL7 or disease genetic
profile, can enable: 1) the repositioning of marketed drugs with
disappointing market results; 2) the rescue of drug candidates
whose clinical development has been discontinued as a result of
safety or efficacy limitations, which are patient
subgroup-specific; and 3) an accelerated and less costly
development for drug candidates and more optimal drug labeling
(e.g. since the use of FKHL7 as a marker is useful for optimizing
effective dose).
[0290] The treatment of an individual with an FKHL7 therapeutic can
be monitored by determining FKHL7 characteristics, such as FKHL7
protein level or activity, FKHL7 mRNA level, and/or FKHL7
transcriptional level. This measurements will indicate whether the
treatment is effective or whether it should be adjusted or
optimized. Thus, FKHL7 can be used as a marker for the efficacy of
a drug during clinical trials.
[0291] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a preadministration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of an FKHL7 protein, mRNA, or genomic DNA
in the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the FKHL7 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the FKHL7 protein, mRNA, or
genomic DNA in the preadministration sample with the FKHL7 protein,
mRNA, or genomic DNA in the post administration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly. For example, increased administration of the agent may
be desirable to increase the expression or activity of FKHL7 to
higher levels than detected, i.e., to increase the effectiveness of
the agent. Alternatively, decreased administration of the agent may
be desirable to decrease expression or activity of FKHL7 to lower
levels than detected, i.e., to decrease the effectiveness of the
agent.
[0292] Cells of a subject may also be obtained before and after
administration of an FKHL7 therapeutic to detect the level of
expression of genes other than FKHL7, to verify that the FKHL7
therapeutic does not increase or decrease the expression of genes
which could be deleterious. This can be done, e.g., by using the
method of transcriptional profiling. Thus, mRNA from cells exposed
in vivo to an FKHL7 therapeutic and mRNA from the same type of
cells that were not exposed to the FKHL7 therapeutic could be
reverse transcribed and hybridized to a chip containing DNA from
numerous genes, to thereby compare the expression of genes in cells
treated and not treated with an FKHL7-therapeutic. If, for example
an FKHL7 therapeutic turns on the expression of a proto-oncogene in
an individual, use of this particular FKHL7 therapeutic may be
undesirable.
[0293] 4.8.4 Kits
[0294] The invention further provides kits for use in diagnostics
or prognostic methods for glaucoma or for determining which FKHL7
therapeutic should be administered to a subject, for example, by
detecting the presence of FKHL7 mRNA or protein in a biological
sample. For example, the kit can comprise a labeled compound or
agent capable of detecting FKHL7 protein or mRNA in a biological
sample; means for determining the amount of FKHL7 in the sample;
and means for comparing the amount of FKHL7 in the sample with a
standard. The compound or agent can be packaged in a suitable
container. The kit can further comprise instructions for using the
kit to detect FKHL7 mRNA or protein. Such a kit can comprise, e.g.,
one or more nucleic acid probes capable of hybridizing specifically
to at least a portion of an FKHL7 gene or allelic variant thereof,
or mutated form thereof.
[0295] 4.9. Methods of Treatment
[0296] The present invention provides for both prophylactic and
therapeutic methods of treating a subject having a congenital heart
disease. Subjects at risk for such a disease can be identified by a
diagnostic or prognostic assay, e.g., as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the FKHL7 aberrancy,
such that development of the congenital heart disease is prevented
or, alternatively, delayed in its progression. In general, the
prophylactic or therapeutic methods comprise administering to the
subject an effective amount of a compound which is capable of
agonizing a wildtype FKHL7 activity or antagonizing a mutant
(defective) FKHL7 activity. Examples of suitable compounds include
the antagonists, agonists or homologues described in detail
herein.
[0297] 4.9.1. Effective Dose
[0298] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining The Ld.sub.50 (The
Dose Lethal To 50% Of The Population) And The Ed.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic induces are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0299] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0300] 4.9.2. Formulation and Use
[0301] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates may be formulated for administration by, for example,
injection, inhalation or insufflation (either through the mouth or
the nose) or oral, buccal, parenteral or rectal administration.
[0302] For such therapy, the compounds of the invention can be
formulated for a variety of loads of administration, including
systemic and topical or localized administration. Techniques and
formulations generally may be found in Remmington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa. For systemic
administration, injection is preferred, including intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the
compounds of the invention can be formulated in liquid solutions,
preferably in physiologically compatible buffers such as Hank's
solution or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included.
[0303] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0304] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0305] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0306] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0307] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. Other suitable delivery systems include microspheres which
offer the possiblity of local noninvasive delivery of drugs over an
extended period of time. This technology utilizes microspheres of
precapillary size which can be injected via a coronary chatheter
into any selected part of the e.g. heart or other organs without
causing inflammation or ischemia. The administered therapeutic is
slowly released from these microspheres and taken up by surrounding
tissue cells (e.g. endothelial cells).
[0308] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives in addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the oligomers of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0309] In clinical settings, a gene delivery system for the
therapeutic FKHL7 gene can be introduced into a patient by any of a
number of methods, each of which is familiar in the art. For
instance, a pharmaceutical preparation of the gene delivery system
can be introduced systemically, e.g., by intravenous injection, and
specific transduction of the protein in the target cells occurs
predominantly from specificity of transfection provided by the gene
delivery vehicle, cell-type or tissue-type expression due to the
transcriptional regulatory sequences controlling expression of the
receptor gene, or a combination thereof. In other embodiments,
initial delivery of the recombinant gene is more limited with
introduction into the animal being quite localized. For example,
the gene delivery vehicle can be introduced by catheter (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.
(1994) PNAS 91: 3054-3057). An FKHL7 gene, such as any one of the
sequences represented in the group consisting of SEQ ID NOS 1 and 3
or a sequence homologous thereto can be delivered in a gene therapy
construct by electroporation using techniques described, for
example, by Dev et al. ((1994) Cancer Treat Rev 20: 105-115).
[0310] The pharmaceutical preparation of the gene therapy construct
or compound of the invention can consist essentially of the gene
delivery system in an acceptable diluent, or can comprise a slow
release matrix in which the gene delivery vehicle or compound is
imbedded. Alternatively, where the complete gene delivery system
can be produced intact from recombinant cells, e.g., retroviral
vectors, the pharmaceutical preparation can comprise one or more
cells which produce the gene delivery system.
[0311] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0312] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references (including literature
references, issued patents, published patent applications as cited
throughout this application are hereby expressly incorporated by
reference. The practice of the present invention will employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2.sup.nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold
Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and
II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait
ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986).
5. EXAMPLES
[0313] 5.1. Cloning and Analysis of Human FKHL7
[0314] Methods
[0315] Construction of Somatic Cell Hybrids. Lymphoblastoid cell
lines (LCLs) were established whole blood from the two
translocations patients. Somatic cell hybrids were created from the
LCLs of patient with the balanced translocation using a
modification of previously published protocols (Puck, J. M. et al.,
J. Clin. Invest 79: 1395-1400 (1987); Nussbaum, R. L. et al., Hum.
Genet. 64: 148-150 (1983)). Briefly, LCLs were expanded to roughly
2-5.times.10.sup.7 cells in RPMI 1640 media with 10% inactivated
fetal calf serum. The were pelleted at 1200 g in a table top
centrifuge and resuspended in 2 m. of Dulbecco's Modified Eagles
Medium with 10% uninactivated fetal calf serum (DMEM/UFCS). The
plate was then incubated overnight in 4 ml of DMEM/UFCS.
[0316] The following day, the cells were trypsinized and split 1:5
into 100 mm plates. The cells grown in 5 ml of DMEM/UFC
supplemented with 10.sup.-4M hypoxanthine and 4.times.10.sup.-5M
azaserine. This supplemented media was placed as needed until the
colonies started to appear (2 to 4 weeks post-fusion). The
individual colonies were allowed to grow until they were clearly
visible without magnification. They were then removed from the
plate using cloning rings to avoid contamination of the hybrid from
others on the plate and put in 12-well tissue culture plates.
[0317] Marker typing. PCR amplification for the analysis of short
tandem repeat polymorphism's (STRPs) was performed using 20 ng of
genomic DNA in 5-pl reactions contain 0.5 .mu.l of 10.times.PCR
buffer [100 mM Tris-HCl (pH 8.8), 500 mM MgCl.sub.2 0.01% gelatin
(w/v)], 200 .mu.m each of dATP, dCTP, dGTP and dTTP, 2.5 pmol of
each primer and 0.2 unit of Taq polymerase (BMB, ISC). Samples were
subjected to 35 cycles of 94.degree. C. as required) for 30 s and
72.degree. C. for 30 s. Amplication products were electrophoresed
on 6% polyacrylamide gels contain 7.7 M urea at 60 W for
approximately 2 h. The bands are detected by silver staining
(Bassam, B. J., et al., Anal. Biochem. 196: 80-83 (1991)).
[0318] Marker typing for physical mapping performed on 2% agarose
gels using a PCR reaction size 10 .mu.l. Reaction conditions were
as described above with the following exception. For markers which
proved difficult to amplify using the standard Taq polymerase, we
substituted an equal amount of AmpliTaq (ABI) along with an initial
incubation of the PCR mixture at 94.degree. C. for 10 m. For the
amplification of the FKHL7 fragments, 10% DMSO was also added to
the reaction mixture. For PCR reactions involving YAC, BAC or
plasmid DNA, 1 to 2 ng of DNA was utilized as template. For colony
PCR, a small number of cells were inoculated into 20 .mu.l of
ddH.sub.2O. 1 .mu.l of this suspension was used as template for the
PCR reaction.
[0319] Oligonucleotide primers for the STRPs were obtained as
MapPairs (Research Genetics). The custom primers required for this
study were designed using the PRIMER 0.5 program and synthesized
commercially (Research Genetics). Primer sequences for the
screening assay and expected amplification sizes are available on
request. Size standards for the 2% agarose gels were 100 bp ladder
(Gibco/BRL) and for the denaturing acrylamide gels a 50 bp ladder
(Gibco/BRL). For the 0.8% agarose gels, lambda DNA digested with
StyT was used as a size marker.
[0320] YAC, BAC and cDNA Identification. Initial YACs were
identified by searching a database at the Whitehead Institute/MIT
Genome Center (http://www-genome.wi.mit.edu) (Hudson, T. J. et al.,
Science 270: 1945-1954 (1995) with STSs known to be in the 6p25 and
13q22 region. Subsequently, YACs and BACs were identified by a
PCR-based screening assay of pooled libraries (Research Genetics)
using various STSs within each region. A few of the chimeric YACs
that were in critical areas were also obtained from a second source
(Genome Systems). cDNA clones were identified by a BLASTN search of
the public dbEST database available through a web interface
(http://www.ncbi.nlm.nih.gov).
[0321] DNA Isolation. DNA was prepared from the somatic cell hybrid
cell lines using a rapid salt isolation procedure (Laitinen, J. et
al., Biotechniques 17: 316, 318, 320-322). The initial screening of
the cell lines utilized a 500 ol volume of cells, while for the
second stage of the DNA preparation the entire contents of a T75
flask was used. YAC DNA was isolated using the DNA-Pure yeast
genomic kit (CPG Inc.). BAC DNA was prepared via an alkaline lysis
protocol as implemented in the Wizard Plus Miniprep Kit (Promega)
with the following modification to the protocol. Instead of loading
the supernatant onto a vacuum column, it as precipitated with a
2.times. volume of absolute EtOH. In additin, 150 .mu.l volumes
were used for the commercial solutions in place of the 200 .mu.l
volumes suggested in the protocol. The precipitated DNA was then
washed with 70% EtOH and dried. The DNA pellet was then resuspended
in 501 .mu.l of ddH.sub.2O. Finally, plasmid DNA was prepared using
a Wizard Plus Miniprep kit (Promega) following the recommended
protocol. Culture sizes for DNA preparation from YACs, BACs and
plasmids were 1.5 ml of the appropriate media and antibiotics for
each construct.
[0322] Subcloning of BACs. BAC DNA was digested with either EcoRI
or HindIII for 8 h at 37.degree. C. in a 50 .mu.l reaction volume.
Vector DNA (pUC19) was also digested with either EcoRI or HindIII
under similar conditions. All restriction digests were purified by
drop dialysis against ddH.sub.2O using VS filters with a pore size
of 0.025 .mu.M (Millipore) for 15 minutes. Integrity of the digest
was verified by gel electrophoresis of a portion of the reaction on
0.8% agarose gels. Equal amounts of digested BAC DNA and pUC19
vector were mixed and ligated overnight at 14.degree. C. 1 to 3
.mu.l of ligation mix was transformed into DH5.alpha. competent
cells (Gibco/BRL). Recombinant clones were selected and the inserts
were characterized by restriction enzyme digestion.
[0323] Sequencing plasmids and PCR products. PCR products for
sequencing were amplified in a 50 .mu.l reaction size and purified
using the Quiaquick PCR Clean-up kit (Promega). 500 ng of plasmid
DNA (in 4.5 .mu.l) or 4.5 .mu.l of purified PCR product was used as
template for a sequencing reation. 1 .mu.l of primer (20 pmoles)
and 4.5 .mu.l of terminator sequencing mix (Amersham) was added for
a final reaction size of 10 .mu.l. Cycling conditions were
performed as specified by the manufacturer. The sequencing
reactions were precipitated in the presence of linear acrylamide
and resuspended in 2 ol of loading buffer. The reactions were
analyzed on an ABI 377 using a run time of 3 h.
[0324] Gene Identification and Characterization. Raw SCF files from
ABI 373A and 377 sequences were imported directly into the
Sequencher v3.0 program (GeneCodes). Contigs were generated by
comparing all fragments in a project with the parameters of at
least a 50 bp overlap in sequence with a 75% level of homology.
Genomic sequence of both the 6p25 and 13q22 regions were submitted
to the BLAST server at NCBI for a BLASTN analysis on both the NR
and dbEST databases. Any region which gave a significant score
(p<10.sup.-5) was also submitted for a BLASTX screen of the
SWISS-PROT database. EST sequence was obtained from GENBANK and SCF
files from the WashU-Merck ftp site (ftp://genome.wusd.edu).
[0325] RNA Isolation and Blot Analysis. Freshly dissected embryos
and adult tissues from NIH Swiss mice were rapidly frozen in liquid
nitrogen and stored at -70.degree. C. until use. Total cellular RNA
was prepared using RNA STAT-60 (Tel-Test "B", Inc.) according to
the manufacturer's specifications. Poly (A) mRNA was isolated using
a Poly (A) Quick mRNA Isolation Kit (Stratagene). Two .mu.g of poly
(A) mRNA were electrophoresed through a 0.8% agarose gel containing
formaldehyde. RNA length standards (0.4-9.5 kb) were obtained from
Gibco-BRL. The gel was stained with ethidium bromide, destained
overnight in 0.1 M ammonium acetate and the RNA was transferred to
Gene Screen Plus (NEN) following the manufacturer's
specifications.
[0326] Hybridization probes were gel-purified inserts of the
following plasmids: human FKHL7 cDNA corresponding to the 3' UTR
(I.M.A.G.E. Consortium Clone ID 864392, Research Genetics), murine
Fkhl7 cDNA corresponding to the same region (I.M.A.G.E. Consortium
Clone ID 864300, Research Genetics), the murine cDNA homologue of
mannose deyhdratase (I.M.A.G.E. Consortium Clone ID 717347,
Research Genetics) and murine B-actin (Clontech). Hybridization
probes were labeled with .sup.32P-(dCTP) and hybridized for 16 h at
42.degree. C. in 50% formamide, 5.times.SSC (SCC is a standard
saline citrate: 0.15 M NaCl, 0.015 M Na citrate), 1.times.
Denhardt's solution, 20 mM phosphate buffer (pH 7.6), 1% sodium
dodecyl sulfate (SDS), 100 .mu.g/ml salmon sperm DNA and 10%
dextran sulfate. The filter was then washed twice at room
temperature in 1.times.SSC followed by 2 rinses at 65.degree. C. in
1.times.SSC-1% SDS and a final room temperature wash in
0.1.times.SSC. Kodac XAR-5 film was exposed at -70.degree. C. with
Dupont Cronex Lightning Plus intensifying screens (Dupont).
Following autoradiography, the filter was stripped of radioactivity
and subsequently rehybridized.
[0327] Mutation Detection and Confirmation. Mutation detection was
performed using single strand conformation polymorphism (SSCP)
analysis and direct sequencing of PCR products, PCR products were
electrophoresed on SSCP gels (5 ml glycerol, 5 ml 5.times.TBE, 12.5
ml 37.5:1 acrylamide/bis and 77.5 ml ddH.sub.2O) for 3 to 4 hr in
0.25.times.TBE at room temperature. Gels were silver stained as
described above. Abnormal variants were sequenced and compared to a
control sample to detect any changes from that of the normal
sequence. Mutations were confirmed by amplification-refractory
mutation system (ARMS) analysis (Newton, C. R. et al., Nucleic
Acids Res. 17: 2503-2516 (1989).
[0328] Results
[0329] Clinical features of translocation patients An infant female
was delivered at 38 weeks gestation with an apparent de novo
balanced translocation: 46,XX,t(6;13)(p25.3;q22.3). She was noted
to have a number of congenital anomalies including a small
mandible, cleft palate, hypoplastic lungs, segmental abnormalities
of the cervical spine, and agenesis of the corpus callosum. Eye
findings included nasolacrimal duct obstruction, persistent tunica
vasculosa lentis, lower lid epiblepharon, ectropion, fistula to the
nasolacrimal system, fat prolapse in the left eye and
hypertelorism. She was diagnosed with PCG at the age of 6 months.
Her parents and siblings are phenotypically normal and her parents
have normal karyotypes.
[0330] Cytogenetic evaluation of a second infant female presenting
with multiple congenital anomalies (cardiac defects, poor muscle
tone, craniofacial abnormalities and hydronephrosis) revealed an
unbalanced translocation: 46,XX,der(6).sub.t(2;6)(q35;p25) with the
loss of the region 6p25->pter and gain of 2q35->qter. At 5
days of age, she was found to have PCG based on diffuse corneal
haze, presence of posterior embryotoxon, increased axial eye
lengths, barely visible irides and elevated intraocular
pressures.
[0331] Since the rearrangements in the above two patients appeared
to occur in the same region of chromosome 6, we hypothesized that a
gene causing PCG was present in this region, and that
identification of the 6p25 breakpoint from the balanced
translocation patient would allow for the identification of the
gene responsible for PCG.
[0332] Mapping of the balanced translocation breakpoints. To
facilitate the identification and cloning of the t(6; 13)
breakpoints, somatic cell hybrids were constructed from cell lines
derived from the balanced translocation patient. Such hybrids are a
useful tool in the mapping of chromosomal rearrangements as they
allow for the molecular analysis of the derivative chromosomes
apart from their normal homologues. Two somatic cell hybrids (H14
and H17) that each contained a single derivative chromosome were
identified by genotyping with highly polymorphic markers. H17 was
found to contain the derivative 13 chromosome and the normal human
chromosome 6. H14 was found to contain the derivative 6 chromosome
in the absence of the normal 6 and 13 chromosomes.
[0333] To map the t(6;13) breakpoints, DNA from hybrids H14 and H17
along with DNA from controls (CEPH individuals 1331-01 and 1331-02,
the balanced translocation patient and the hamster cell line,
RJK88) were used as PCR templates to screen genetic markers to
identify those markers flanking the chromosome 6 and 13
breakpoints. Markers within the genetic map of 6p25 were selected
for the screen. The 6p25 breakpoint was found to be located in a 5
cM region flanked by the markers D6S344 and D6S477. Similarly
markers within the genetic map of 13q22 (Murray J. C. et al.,
(1994) Science 265: 2049-2054) were evaluated. The chromosome 13q22
breakpoint was found to be contained in a 3 cM region flanked by
the markers D13S160 and D13S170.
[0334] A high resolution physical map of the 6p25 region was
constructed to aid in the cloning of the 6p breakpoint. This map,
along with the development of STSs from YACs, allowed mapping of
the breakpoint to a small region near D6S344. BACs were then
isolated from the region surrounding D6S344. Two BACs (185d15 and
471g19) were selected for subcloning based on their ability to
cover the region as determined by STS content analysis. Primers
derived from these BAC subclones were screened by PCR using hybrid
H14 as template. This allowed identification of a clone that
contained the 6p25 breakpoint. STS content mapping within the clone
as compared to hybrid H14 allowed precise localization of the 6p25
breakpoint and obtainment of the surrounding sequence. The junction
fragment from the H14 hybrid DNA was isolated using a primer
flanking the 6p25 breakpoint in combination with a set of Alu-based
primers (Dorin, J. R. et al., Hum. Mol. Genet. 1: 53-59 (1992)).
Sequence analysis of this fragment confirmed that it was the
junction fragment from hybrid H14. Since this junction fragment
contained chromosome 13 sequence adjacent to the breakpoint, an STS
was developed from this sequence and mapped onto the YAC/BAC contig
of 13q22. This STS mapped distal to the 13q22 breakpoint and its
location within the physical map of 13q22 was consistent with it
being in close proximity to the breakpoint. This marker also mapped
to the BAC 163n9 which had been isolated with markers that were
proximal to the breakpoint. This result indicated that BAC 163n9
contained the 13q22 breakpoint of the balanced translocation
patient.
[0335] Subclones from the 163n9 BAC were screened using the STS
developed from the hybrid H14 junction fragment. A 3.5 kb subclone
was identified that contained the 13q22 breakpoint. Sequence
comparison with the normal chromosome 6 sequence and that from the
hybrid H14 junction fragment revealed the location of the 13q22
breakpoint within the normal 13q22 sequence. Finally, the junction
fragment from the hnybrid H17 was evaluated to determine if there
had been any gain or loss of material at the site of the
translocation. Using hybrid H17 DNA, a single fragment was
generated by PCR using a primer proximal to the 13q22 breakpoint
and one distal to the 6p25 breakpoint. Sequence analysis confirmed
that this fragment was the junction fragment from hybrid H17.
Comparison of normal chromosome 6p25 sequence and normal chromosome
13q22 sequence along with that from the two junction fragments
revealed that there had been a loss of 11 bp.
[0336] Identification of candidate genes within 6p25. Sequence
generated from both sides of the 6p25 breakpoint (total of 10 kb)
was analyzed for the presence of gene sequences by using both
BLASTN (Alstchul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990)
and BLASTX (Gish, W. et al., Nat. Genet. 3: 266-272 (1993)) to
search public databases for homology to known genes. This sequence
analysis resulted in the identification of a novel human gene
showing strong homology to the GDP-Mannose 4,6-Dehydratase gene
(E.C.4.2.1.47) that has been identified in a number of other
organisms (Currie, H. L. et al., Clin. Diagn. Lab. Immunol 2:
554-562 (1995); Li. Y. et al., Virology 212: 134-150 (1995);
Stevenson et al., Bacteriol. 178: 4885-4893 (1996); Bonin, C. P. et
al. Proc. Natl. Acad. Sci. USA 94: 2085-2090 (1997)). By comparing
the genomic sequence to the human cDNA sequence, the 6p25
breakpoint was localized to an intron upstream of the penultimate
exon of this gene. Sequence analysis of BACs containing this gene
has been used to determine the partial intron/exon boundaries for
this gene. Human mannose dehydratase appears to be 1.1 kb in size
and has at least 7 exons. The genomic structure of two areas of
coding sequence (345 and 253 bp) remain to be determined.
[0337] Physical mapping of the 6p25 region indicated that a human
forkhead transcription factor gene, FKHL7, is within 25 kb of the
6p25 breakpoint and is translocated to the derivative 13
chromosome. Sequence of the forkhead domain of FKHL7 has been
published (Pierrou, S. et al., EMBO J. 13: 5002-5012 (1994)), along
with FISH and somatic cell mapping data that confirm the
localization of this gene to 6p25.
[0338] To determine if a gene on chromosome 13 could be considered
a candidate gene for the glaucoma in the balanced translocation
patient, 2 kb of DNA surrounding the 13q22 breakpoint was
sequenced. GRAIL (Xu, Y. et al., Gen. Engin. 16: (241-253 (1994);
and Uberbacher, E. C. and R. J. Mural Proc. Natl. Acad. Sci. USA
88: 11261-11265 (1991)) analysis of this sequence failed to find
evidence for the presence of any predicted exons in close proximity
to the breakpoint. BLAST (Alstchul, S. F. et al., J. Mol. Biol.
215: 403-410 (1990); Gish, W. et al., Nat. Genet. 3: 266-272
(1993)) analysis also failed to identify any homologies to known
genes or ESTs. The failure to detect a gene within the 13q22 region
sequenced does not rule out the presence of a transcript as the
possibility exists that the 13q22 breakpoint has occurred within a
large intron.
[0339] Analysis of the t(2;6) unbalanced translocation patient. The
patient with the t(2;6) unbalanced translocation is monosomic for a
portion of distal 6p. In order to determine if this patient is
monosomic for the t(6;13) breakpoint region of the balanced
translocation patient, highly polymorphic genetic markers were
amplified using genomic DNA from the unbalanced translocation
patient as template. Markers proximal to D6S2652 were found to be
heterozygous and markers distal to D6S2652 were found to be
homozygous. This indicates that mannose dehydratase and FKHL7 which
are distal to D6S2652 are present in only a .backslash.single copy
in the t(2;6) patient. Co-amplification of STSs in this region
using quantitative PCR confirms the loss of chromosomal material
containing mannose dehydratase and FKHL7 in this patient.
[0340] Expression studies of FKHL7 and mannose dehydratase. In
order to prioritize FKHL7 and mannose dehydratase as candidates for
congenital glaucoma, the expression pattern of each gene was
evaluated by Northern blot analysis. Previous expression studies of
FKHL7 demonstrated that a 3.9 kb transcript was widely expressed in
a variety of human adult and fetal tissues, while the expression of
a second 3.4 kb transcript was limited to fetal kidney (Pierrou, S
et al., EMBO J. 13: 5002-5012 (1994)). Northern blot analysis of a
variety of human adult tissues (brain, heart, kidney, spleen, liver
and colon) confirmed the broad expression pattern of the 3.9 kb
transcript and showed the co-expression of a 3.0 kb transcript.
These multiple FKHL7 transcripts may arise by differential
polyadenylation, consistent with the presence of several
polyadenylation signals in the FKHL7 3' UTR. Using a murine
orthologue of the FKHL7 3' UTR, expression was analyzed in staged
mouse embryos and in various adult tissues including the eye. A 3.7
and 3.0 kb doublet was most abundantly expressed during
embryogenesis, and of the adult tissues tested, expression in the
eye and kidney were significantly higher than that seen in other
adult tissues.
[0341] The expression pattern of mouse mannose dehydratase was also
analyzed on the identical Northern blot used for the FKHL7
experiments. A basal level of expression was found during
embryogenesis as well as in most adult tissues, including the eye.
The transcript size of mouse mannose dehydratase appears to be
approximately 1.9 kb in size which is in agreement with the size
predicted from the human mannose dehydratase coding sequence.
[0342] Based on expression, both FKHL7 and mannose dehydratase are
viable candidate genes for causing glaucoma phenotypes. However,
based on the higher level of expression in the eye, the
developmental regulation and putative function (Semina, E. V. et
al., Nat. Genet 14: 392-399 (1966); Alward, W. L. M. et al., Am. J.
Ophthalmol. 125: 98-100 (1998); Glaser, T. et al., Nat. Genet. 2:
232-239 (1992); Jordan, T. et al., Nat. Genet. 1: 328-332 (1992)
FKHL7 was favored as the better candidate.
[0343] Characterization of FKHL7 gene. FKHL7 is a monomeric DNA
binding protein that shares a core binding site (RTAAAYA) with four
other FKHL-like proteins (Pierrou, S. et al., EMBO J. 13: 5002-5012
(1994). The forkhead domain shows strong homology to the human
gene, FKHL14, and the mouse genes Fkh1 and FKH14 by BLASTN
(Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990)
analysis. A 9.8 kb subclone of BAC 471g19 was partially sequenced
and determined to contain the entire coding region of FKHL7 as well
as 5' and 3' untranslated sequences. The human FKHL7 coding
sequence is 1.7 kb in size (553 amino acids) and contains no
introns. The 1659 bp open reading frame was found to contain the
previously published DNA binding forkhead domain of FKHL7 (Pierrou,
S. et al., EMBO J. 13: 5002-5012 (1994). The first in-frame ATG was
found to match well to the Kozak consensus sequence (Kozak, M.
Annu. Rev. Cell. Biol. 8: 197-225 (1992); Kozak, M. Annu. Rev. Cell
Biol. 8: 197-225 (1992)). The COOH-terminal domain contains several
stretches of homopolymeric runs of alanine and glycine. The FKHL7
coding region contains 5 recognition sites for the restriction
enzyme NotI. The large number of NotI sites within the coding
region of FKHL7 has adversely affected the identification of a
full-length cDNA since many cDNA libraries are constructed with
this restriction enzyme to prevent cloning at an internal site. A
BLASTN (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990)
screen of the public dbEST database with the FKHL7 genomic sequence
yields only partial human and mouse cDNA coverage of this gene.
Based on the analysis of cDNA clones identified in the public
databases, there is evidence for the utilization of at least two
different polyadenylation signals within the 3' untranslated
region.
[0344] Mutation screen. Although molecular analysis of the two
translocation patients was extremely useful for identifying FKHL7
and mannose dehydratase as candidates for causing glaucoma, neither
gene was conclusively demonstrated to be disease causing.
Therefore, these two genes were screened for mutations in a cohort
of unrelated probands with either PCG or anterior segment defects
(RA and/or IH). Twenty-nine Caucasian probands were initially
identified. Of these, 10 proved to have SSCP evidence of a mutation
in another glaucoma related gene (either CYP1B1 or PITX2), and were
therefore eliminated from the screen. The remaining 19 probands (6
PCG and 13 anterior chamber defect patients) were screened by SSCP
for mutations in mannose dehydratase and FKHL7. No mannose
dehydratase mutations were identified in a screen of 60% of the
coding sequence of this gene. FKHL7 mutations were found in four
probands and subsequently in related affected family members.
[0345] An 11 bp deletion upstream of the FKHL7 forkhead domain was
identified in two brothers diagnosed with different anterior
segment defects (RA and IH). Both brothers had glaucoma, and
neither had the extra-ocular manifestations of Rieger syndrome
(RS). Their father was found to have isolated posterior embryotoxon
(PE), suggesting that the disease was inherited through him as an
autosomal dominant. He was also found to carry the deletion. A
second mutation was found in a proband and her mother who both were
diagnosed with classic RA and glaucoma. This mutation, a C to T
transition within the forkhead domain causes a change from a serine
to a leucine (SER131Leu). A third mutation, a C to G transversion
within the forkhead domain, was found in a proband with severe
Axenfeld anomaly and glaucoma. This change results in the
replacement of isoleucine with methionine (Ile126MET) and is also
found in the father who was diagnosed with AA. Finally, a T to C
transition was found in a proband of an extended family with a
spectrum of anterior segment defects. This change results in the
replacement of phenylalanine with serine. Phe112Ser) within the
forkhead domain. The pedigrees of each family (FIG. 6) and the
associated mutations (FIG. 7) are shown. Three of the mutations
were not found in 128 unrelated normal individuals from an
ethnically similar control population (Caucasian). The fourth
mutation (Phe112Ser) was only detected by direct sequencing of PCR
products from patient genomic DNA. This mutation was found to
segregate with the disease in an extended pedigree and was not
present in an additional 12 Caucasian individuals by sequence
analysis.
[0346] The 11 bp deletion upstream of the FKHL7 forkhead domain is
predicted to cause a truncated transcript (missing 477 aa) lacking
the DNA-binding forkhead domain. All three missense mutations occur
within highly conserved regions of the forkhead domain that has
been implicated in the DNA binding properties of the molecule
(Pierrou, S. et al., EMBO J. 13: 5002-5012). The alteration of
amino acids at these sites would be expected to have an effect on
the DNA binding specificity of FKHL7. Finally, screening of FKHL7
in the translocation patients failed to identify mutations,
suggesting that the presence of one abnormally expressed copy of
the gene results in a disease phenotype.
Sequence CWU 1
1
* * * * *
References