Title: Antibodies against gene
products related to Werner's Syndrome
United States Patent: 7,285,641
Issued: October 23, 2007
Inventors: Fu; Ying-Hui
(Seattle, WA), Yu; Chang-En (Seattle, WA), Oshima; Junko (Seattle, WA),
Mulligan; John T (Seattle, WA), Schellenberg; Gerard D (Seattle, WA)
Appl. No.: 10/374,077
Filed: February 25, 2003
Pharm Bus Intell
& Healthcare Studies
The present invention discloses
antibodies that specifically bind to a WRN gene product or a portion
BRIEF SUMMARY OF THE
Briefly stated, the present invention
provides isolated nucleic acid molecules encoding the WRN gene, as well as
portions thereof, representative of which are provided in the Figures (see Original Patent).
The protein which is encoded by the WRN gene is referred to hereinafter as
the "WRN protein". Within other embodiments, nucleic acid molecules are
provided which encode a mutant WRN gene product that increases the
probability of Werner's Syndrome (in a statistically significant manner).
Representative illustrations of such mutants are provided in Example 3.
Within other aspects of the present invention, isolated nucleic acid
molecules are provided, selected from the group consisting of (a) an
isolated nucleic acid molecule as set forth in the Figures (see Original Patent),
or complementary sequence thereof, (b) an isolated nucleic acid molecule
that specifically hybridizes to the nucleic acid molecule of (a) under
conditions of high stringency, and (c) an isolated nucleic acid that
encodes a WRN gene product (WRN protein). As utilized herein, it should be
understood that a nucleic acid molecule hybridizes "specifically" to an
WRN gene (or related sequence) if it hybridizes detectably to such a
sequence, but does not significantly or detectably hybridize to the
Bloom's Syndrome gene (Ellis et al., Cell 83:655-666, 1995).
Within other aspects, expression vectors are provided comprising a
promoter operably linked to one of the nucleic acid molecule described
above. Representative examples of suitable promoters include
tissue-specific promoters, as well as promoters such as the CMV I-E
promoter, SV40 early promoter and MuLV LTR. Within related aspects, viral
vectors are provided that are capable of directing the expression of a
nucleic acid molecule as described above. Representative examples of such
viral vectors include herpes simplex viral vectors, adenoviral vectors,
adenovirus-associated viral vectors and retroviral vectors. Also provided
are host cells (e.g., human, dog, monkey, rat or mouse cells) which carry
the above-described vectors.
Within other aspects of the present invention, isolated proteins or
polypeptides are provided comprising a WRN gene product, as well as
peptides of greater than 12, 13 or 20 amino acids. Within another
embodiment, the protein is a mutant WRN gene product that increases the
probability of Werner's Syndrome.
Within yet another aspect of the present invention, methods of treating or
preventing Werner's Syndrome are provided (as well as for related diseases
which are discussed in more detail below), comprising the step of
administering to a patient a vector containing or expressing a nucleic
acid molecule as described above, thereby reducing the likelihood or
delaying the onset of Werner's Syndrome (or the related disease) in the
patient. Within a related aspect, methods of treating or preventing
Werner's Syndrome (and related diseases) are provided, comprising the step
of administering to a patient a protein as described above, thereby
reducing the likelihood or delaying the onset of Werner's Syndrome (or a
related disease) in the patient. Within certain embodiments, the above
methods may be accomplished by in vivo administration.
Also provided by the present invention are pharmaceutical compositions
comprising a nucleic acid molecule, vector, host cell, protein, or
antibody as described above, along with a pharmaceutically acceptable
carrier or diluent.
Within other aspects of the present invention, antibodies are provided
which specifically bind to an WRN protein or to unique peptides derived
therefrom. As utilized herein, it should be understood that an antibody is
specific for an WRN protein (or peptide) if it binds detectably, and with
a K.sub.d of 10.sup.-7M or less (e.g., 10.sup.-8M, 10.sup.-9M, etc.), but
does not bind detectably (or with an affinity of greater than 10.sup.-7M,
(e.g., 10.sup.-6M, 10.sup.-5M, etc.) to an unrelated helicase (e.g., the
Bloom's Syndrome gene, supra). Also provided are hybridomas which are
capable of producing such antibodies.
Within other aspects of the present invention, nucleic acid probes are
provided which are capable of specifically hybridizing (as defined below)
to an WRN gene under conditions of high stringency. Within one related
aspect, such probes comprise at least a portion of the nucleotide sequence
shown in the Figures (see Original Patent), or its complementary sequence,
the probe being capable of specifically hybridizing to a mutant WRN gene
under conditions of high stringency. Representative probes of the present
invention are generally at least 12 nucleotide bases in length, although
they may be 14, 16, 18 bases or longer. Also provided are primer pairs
capable of specifically amplifying all or a portion of any of the nucleic
acid molecules disclosed herein.
Within other aspects of the invention, methods are provided for diagnosing
a patient having an increased likelihood of contracting Werner's Syndrome
(or a related disease), comprising the steps of (a) obtaining from a
patient a biological sample containing nucleic acid, (b) incubating the
nucleic acid with a probe which is capable of specifically hybridizing to
a mutant WRN gene under conditions and for time sufficient to allow
hybridization to occur, and (c) detecting the presence of hybridized
probe, and thereby determining that said patient has an increased
likelihood of contracting Werner's Syndrome (or a related disease). Within
another aspect, methods are provided comprising the steps of (a) obtaining
from a patient a biological sample containing nucleic acid, (b) amplifying
a selected nucleic acid sequence associated with a mutant WRN gene, and
(c) detecting the presence of an amplified nucleic acid sequence, and
thereby determining that the patient has an increased likelihood of
contracting Werner's Syndrome (or a related disease). Suitable biological
samples include nucleated cells obtained from the peripheral blood, from
buccal swabs, or brain tissue.
Within another aspect, peptide vaccines are provided which comprise a
portion of a mutant WRN gene product containing a mutation, in combination
with a pharmaceutically acceptable carrier or diluent.
Within yet another aspect, transgenic animals are provided whose germ
cells and somatic cells contain a WRN gene (or lack thereof, i.e., a
"knockout") which is operably linked to a promoter effective for the
expression of the gene, the gene being introduced into the animal, or an
ancestor of the animal, at an embryonic stage. Within one embodiment, the
animal is a mouse, rat or dog. Within other embodiments, the WRN gene is
expressed from a vector as described above. Within yet another embodiment,
the WRN gene encodes a mutant WRN gene product.
These and other aspects of the present invention will become evident upon
reference to the following detailed description and attached drawings. In
addition, various references are set forth herein which describe in more
detail certain procedures or compositions (e.g., plasmids, etc.), and are
therefore incorporated by reference in their entirety.
OF THE INVENTION
Genes and Gene Products Related to
The present invention provides isolated nucleic acid molecules comprising
a portion of the gene which is implicated in the onset of WS. Briefly, as
can be seen from FIG. 4 (see Original Patent), this gene encodes a protein
that is similar in amino acid sequence to several known ATP-dependent DNA
helicases (enzymes that unwind the DNA duplex). It is less similar to
known RNA-DNA helicases. Helicases are involved in the replication of DNA,
often binding the replication origin, and/or the replication complex. In
addition, the single stranded DNA that is involved in recombination can be
generated by DNA helicases.
Although various aspects of the WRN gene (or portions thereof) are shown
in the Figures (see Original Patent), it should be understood that within
the context of the present invention, reference to one or more of these
genes includes derivatives of the genes that are substantially similar to
the genes (and, where appropriate, the proteins (including peptides and
polypeptides) that are encoded by the genes and their derivatives). As
used herein, a nucleotide sequence is deemed to be "substantially similar"
if: (a) the nucleotide sequence is derived from the coding region of the
described genes and includes, for example, portions of the sequence or
allelic variations of the sequences discussed above, or alternatively,
encodes a helicase-like activity (Bjornson et al., Biochem.
3307:14306-14316, 1994); (b) the nucleotide sequence is capable of
hybridization to nucleotide sequences of the present invention under high
or very high stringency (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY,
1989); or (c) the DNA sequences are degenerate as a result of the genetic
code to the DNA sequences defined in (a) or (b). Further, the nucleic acid
molecule disclosed herein includes both complementary and
non-complementary sequences, provided the sequences otherwise meet the
criteria set forth herein. Within the context of the present invention,
high stringency means standard hybridization conditions (e.g.,
5.times.SSPE, 0.5% SDS at 65.degree. C., or the equivalent) while very
high stringency means conditions of hybridization such that the nucleotide
sequence is able to selectively hybridize to a single allele of the
The WRN gene may be isolated from genomic DNA or cDNA. Genomic DNA
libraries constructed in chromosomal vectors, such as YACs (yeast
artificial chromosomes), bacteriophage vectors, such as .lamda.EMBL3,
.lamda.gt10, cosmids, or plasmids are suitable for use. cDNA libraries
constructed in bacteriophage vectors, plasmids, or others, are suitable
for screening. Such libraries may be constructed using methods and
techniques known in the art (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, 1989) or purchased from
commercial sources (e.g., Clontech, Palo Alto, Calif.). Within one
embodiment, the WRN gene is isolated by PCR performed on genomic DNA, cDNA
or DNA from libraries, or is isolated by probe hybridization of genomic
DNA or cDNA libraries. Primers for PCR and probes for hybridization
screening may be designed based on the DNA sequence of WRN presented
herein. The DNA sequence of a portion of the WRN gene and the entire
coding sequence is presented in the Figures. Primers for PCR should be
derived from sequences in the 5' and 3' untranslated region in order to
isolate a full-length cDNA. The primers should not have self-complementary
sequences nor have complementary sequences at their 3' end (to prevent
primer-dimer formation). Preferably, the primers have a GC content of
about 50% and contain restriction sites. The primers are annealed to cDNA
and sufficient cycles of PCR are performed to yield a product readily
visualized by gel electrophoresis and staining. The amplified fragment is
purified and inserted into a vector, such as .lamda.gt10 or pBS (M13+),
and propagated. An oligonucleotide hybridization probe suitable for
screening genomic or cDNA libraries may be designed based on the sequence
provided herein. Preferably, the oligonucleotide is 20-30 bases long. Such
an oligonucleotide may be synthesized by automated synthesis. The
oligonucleotide may be conveniently labeled at the 5' end with a reporter
molecule, such as a radionuclide, (e.g., .sup.32P) or biotin. The library
is plated as colonies or phage, depending upon the vector, and the
recombinant DNA is transferred to nylon or nitrocellulose membranes.
Following denaturation, neutralization, and fixation of the DNA to the
membrane, the membranes are hybridized with the labeled probe. The
membranes are washed and the reporter molecule detected. The hybridizing
colonies or phage are isolated and propagated. Candidate clones or PCR
amplified fragments may be verified as containing WRN DNA by any of
various means. For example, the candidate clones may be hybridized with a
second, nonoverlapping probe or subjected to DNA sequence analysis. In
these ways, clones containing WRN gene, which are suitable for use in the
present invention are isolated.
The structure of the proteins encoded by the nucleic acid molecules
described herein may be predicted from the primary translation products
using the hydrophobicity plot function of, for example, P/C Gene, Lasergen
System, DNA STAR, Madison, Wis., or according to the methods described by
Kyte and Doolittle (J. Mol. Biol. 157:105-132, 1982).
WRN proteins of the present invention may be prepared in the form of
acidic or basic salts, or in neutral form. In addition, individual amino
acid residues may be modified by oxidation or reduction. Furthermore,
various substitutions, deletions, or additions may be made to the amino
acid or nucleic acid sequences, the net effect of which is to retain or
further enhance or decrease the biological activity of the mutant or
wild-type protein. Moreover, due to degeneracy in the genetic code, for
example, there may be considerable variation in nucleotide sequences
encoding the same amino acid sequence.
Other derivatives of the WRN proteins disclosed herein include conjugates
of the proteins along with other proteins or polypeptides. This may be
accomplished, for example, by the synthesis of N-terminal or C-terminal
fusion proteins which may be added to facilitate purification or
identification of WRN proteins (see U.S. Pat. No. 4,851,341; see also,
Hopp et al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteins
such as WRN protein-.beta.-galactosidase or WRN protein-luciferase may be
constructed in order to assist in the identification, expression, and
analysis of WRN proteins.
WRN proteins of the present invention may be constructed using a wide
variety of techniques described herein. Further, mutations may be
introduced at particular loci by synthesizing oligonucleotides containing
a mutant sequence, flanked by restriction sites enabling ligation to
fragments of the native sequence. Following ligation, the resulting
reconstructed sequence encodes a derivative having the desired amino acid
insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific (or segment
specific) mutagenesis procedures may be employed to provide an altered
gene having particular codons altered according to the substitution,
deletion, or insertion required. Exemplary methods of making the
alterations set forth above are disclosed by Walder et al. (Gene 42:133,
1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January
1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods,
Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncation
derivatives of WRN proteins (e.g., a soluble extracellular portion) may
also be constructed by utilizing convenient restriction endonuclease sites
adjacent to the desired deletion. Subsequent to restriction, overhangs may
be filled in, and the DNA religated. Exemplary methods of making the
alterations set forth above are disclosed by Sambrook et al. (Molecular
Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press,
Mutations of the present invention preferably preserve the reading frame
of the coding sequences. Furthermore, the mutations will preferably not
create complementary regions that could hybridize to produce secondary
mRNA structures, such as loops or hairpins, that would adversely affect
translation of the mRNA. Although a mutation site may be predetermined, it
is not necessary that the nature of the mutation per se be predetermined.
For example, in order to select for optimum characteristics of mutants at
a given site, random mutagenesis may be conducted at the target codon and
the expressed mutants screened for indicative biological activity.
Alternatively, mutations may be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked by
restriction sites enabling ligation to fragments of the native sequence.
Following ligation, the resulting reconstructed sequence encodes a
derivative having the desired amino acid insertion, substitution, or
WRN proteins may also be constructed utilizing techniques of PCR
mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS
83:3402-3406, 1986), by forced nucleotide misincorporation (e.g., Liao and
Wise Gene 88:107-111, 1990), or by use of randomly mutagenized
oligonucleotides (Horwitz et al., Genome 3:112-117, 1989).
Proteins can be isolated by, among other methods, culturing suitable host
and vector systems to produce the recombinant translation products of the
present invention. Supernates from such cell lines, or protein inclusions
or whole cells where the protein is not excreted into the supernate, can
then be treated by a variety of purification procedures in order to
isolate the desired proteins. For example, the supernate may be first
concentrated using commercially available protein concentration filters,
such as an Amicon or Millipore Pellicon ultrafiltration unit. Following
concentration, the concentrate may be applied to a suitable purification
matrix such as, for example, an anti-protein antibody bound to a suitable
support. Alternatively, anion or cation exchange resins may be employed in
order to purify the protein. As a further alternative, one or more
reverse-phase high performance liquid chromatography (RP-HPLC) steps may
be employed to further purify the protein. Other methods of isolating the
proteins of the present invention are well known in the skill of the art.
A protein is deemed to be "isolated" within the context of the present
invention if no other (undesired) protein is detected pursuant to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
followed by Coomassie blue staining. Within other embodiments, the desired
protein can be isolated such that no other (undesired) protein is detected
pursuant to SDS-PAGE analysis followed by silver staining.
Expression of a WRN Gene
The present invention also provides for the manipulation and expression of
the above described genes by culturing host cells containing a vector
capable of expressing the above-described genes. Such vectors or vector
constructs include either synthetic or cDNA-derived nucleic acid molecules
encoding WRN proteins, which are operably linked to suitable
transcriptional or translational regulatory elements. Suitable regulatory
elements may be derived from a variety of sources, including bacterial,
fungal, viral, mammalian, insect, or plant genes. Selection of appropriate
regulatory elements is dependent on the host cell chosen, and may be
readily accomplished by one of ordinary skill in the art. Examples of
regulatory elements include: a transcriptional promoter and enhancer or
RNA polymerase binding sequence, a transcriptional terminator, and a
ribosomal binding sequence, including a translation initiation signal.
Nucleic acid molecules that encode any of the WRN proteins described above
may be readily expressed by a wide variety of prokaryotic and eukaryotic
host cells, including bacterial, mammalian, yeast or other fungi, viral,
insect, or plant cells. Methods for transforming or transfecting such
cells to express foreign DNA are well known in the art (see, e.g., Itakura
et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl. Acad. Sci. USA
75:1929-1933, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshall et
al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950; Axel
et al., U.S. Pat. No. 4,399,216; Goeddel et al., U.S. Pat. No. 4,766,075;
and Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition,
Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and
Marton, Plant Physiol. 104:1067-1071, 1994; and Paszkowski et al.,
Biotech. 24:387-392, 1992).
Bacterial host cells suitable for carrying out the present invention
include E. coli, B. subtilis, Salmonella typhimurium, and various species
within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well
as many other bacterial species well known to one of ordinary skill in the
art. Representative examples of bacterial host cells include DH5.alpha. (Stratagene,
Bacterial expression vectors preferably comprise a promoter which
functions in the host cell, one or more selectable phenotypic markers, and
a bacterial origin of replication. Representative promoters include the
.beta.-lactamase (penicillinase) and lactose promoter system (see Chang et
al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studier et
al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin et al.,
Gene 87:123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. in
Enzymology 101:155, 1983) and the tac promoter (Russell et al., Gene 20:
231, 1982). Representative selectable markers include various antibiotic
resistance markers such as the kanamycin or ampicillin resistance genes.
Many plasmids suitable for transforming host cells are well known in the
art, including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977),
the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth. in
Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268, 1982),
and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla,
Yeast and fungi host cells suitable for carrying out the present invention
include, among others, Saccharomyces pombe, Saccharomyces cerevisiae, the
genera Pichia or Kluyveromyces and various species of the genus
Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349). Suitable
expression vectors for yeast and fungi include, among others, YCp50 (ATCC
No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull,
Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl. Acad. Sci.
USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene 8:121-133, 1979),
pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives
Preferred promoters for use in yeast include promoters from yeast
glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980;
Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982) or alcohol
dehydrogenase genes (Young et al., in Genetic Engineering of
Microorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum,
New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples of
useful promoters for fungi vectors include those derived from Aspergillus
nidulans glycolytic genes, such as the adh3 promoter (McKnight et al.,
EMBO J. 4:2093-2099, 1985). The expression units may also include a
transcriptional terminator. An example of a suitable terminator is the
adh3 terminator (McKnight et al., ibid., 1985).
As with bacterial vectors, the yeast vectors will generally include a
selectable marker, which may be one of any number of genes that exhibit a
dominant phenotype for which a phenotypic assay exists to enable
transformants to be selected. Preferred selectable markers are those that
complement host cell auxotrophy, provide antibiotic resistance or enable a
cell to utilize specific carbon sources, and include leu2 (Broach et al.,
ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3 (Struhl et al.,
ibid.). Another suitable selectable marker is the cat gene, which confers
chloramphenicol resistance on yeast cells.
Techniques for transforming fungi are well known in the literature, and
have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc.
Natl. Acad. Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad.
Sci. USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983). The
genotype of the host cell may contain a genetic defect that is
complemented by the selectable marker present on the expression vector.
Choice of a particular host and selectable marker is well within the level
of ordinary skill in the art.
Protocols for the transformation of yeast are also well known to those of
ordinary skill in the art. For example, transformation may be readily
accomplished either by preparation of spheroplasts of yeast with DNA (see
Hinnen et al., PNAS USA 75:1929, 1978) or by treatment with alkaline salts
such as LiCl (see Itoh et al., J. Bacteriology 153:163, 1983).
Transformation of fungi may also be carried out using polyethylene glycol
as described by Cullen et al. (Bio/Technology 5:369, 1987).
Viral vectors include those which comprise a promoter that directs the
expression of an isolated nucleic acid molecule that encodes an WRN
protein as described above. A wide variety of promoters may be utilized
within the context of the present invention, including for example,
promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviral promoter
(Ohno et al., Science 265: 781-784, 1994), neomycin phosphotransferase
promoter/enhancer, late parvovirus promoter (Koering et al., Hum. Gene
Therap. 5:457-463, 1994), Herpes TK promoter, SV40 promoter,
metallothionein IIa gene enhancer/promoter, cytomegalovirus immediate
early promoter, and the cytomegalovirus immediate late promoter. Within
particularly preferred embodiments of the invention, the promoter is a
tissue-specific promoter (see e.g., WO 91/02805; EP 0,415,731; and WO
90/07936). Representative examples of suitable tissue specific promoters
include neural specific enolase promoter, platelet derived growth factor
beta promoter, bone morpho-genetic protein promoter, human
alpha1-chimaerin promoter, synapsin I promoter and synapsin II promoter.
In addition to the above-noted promoters, other viral-specific promoters
(e.g., retroviral promoters (including those noted above, as well as
others such as HIV promoters), hepatitis, herpes (e.g., EBV), and
bacterial, fungal or parasitic (e.g., malarial)-specific promoters may be
utilized in order to target a specific cell or tissue which is infected
with a virus, bacteria, fungus or parasite.
Thus, WRN proteins of the present invention may be expressed from a
variety of viral vectors, including for example, herpes viral vectors
(e.g., U.S. Pat. No. 5,288,641), adenoviral vectors (e.g., WO 94/26914, WO
93/9191; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS
90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993;
Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell
75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993;
Caillaud et al., Eur. J. Neurosci. 5(10:1287-1291, 1993; Vincent et al.,
Nat. Genet. 5(2):130-134, 1993; Jaffe et al., Nat. Genet. 1(5):372-378,
1992; and Levrero et al, Gene 101(2):195-202, 1991), adeno-associated
viral vectors (WO 95/13365; Flotte et al., PNAS 90(22):10613-10617, 1993),
baculovirus vectors, parvovirus vectors (Koering et al., Hum. Gene Therap.
5:457-463, 1994), pox virus vectors (Panicali and Paoletti, PNAS
79:4927-4931, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm.
193(2):653-660, 1993), and retroviruses (e.g., EP 0,415,731; WO 90/07936;
WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No.
5,219,740; WO 93/11230; WO 93/10218. Viral vectors may likewise be
constructed which contain a mixture of different elements (e.g.,
promoters, envelope sequences and the like) from different viruses, or
non-viral sources. Within various embodiments, either the viral vector
itself, or a viral particle which contains the viral vector may be
utilized in the methods and compositions described below.
Mammalian cells suitable for carrying out the present invention include,
among others: PC12 (ATCC No. CRL1721), NIE-115 neuroblastoma, SK-N-BE(2)C
neuroblastoma, SHSY5 adrenergic neuroblastoma, NS20Y and NG108-15 murine
cholinergic cell lines, or rat F2 dorsal root ganglion line, COS (e.g.,
ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281; BHK 570 cell
line (deposited with the American Type Culture Collection under accession
number CRL 10314), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293
(ATCC No. 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and NS-1
cells. Other mammalian cell lines may be used within the present
invention, including Rat Hep I (ATCC No. CRL 1600), Rat Hep II (ATCC No.
CRL 1548), TCMK (ATCC No. CCL 139), Human lung (ATCC No. CCL 75.1), Human
hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065), Mouse liver (ATCC
No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2/0-Ag14 (ATCC No. 1581),
HIT-T15 (ATCC No. CRL 1777), and RINm 5AHT.sub.2B (Orskov and Nielson,
FEBS 229(1):175-178, 1988).
Mammalian expression vectors for use in carrying out the present invention
will include a promoter capable of directing the transcription of a cloned
gene or cDNA. Preferred promoters include viral promoters and cellular
promoters. Viral promoters include the cytomegalovirus immediate early
promoter (Boshart et al., Cell 41:521-530, 1985), cytomegalovirus
immediate late promoter, SV40 promoter (Subramani et al., Mol. Cell. Biol.
1:854-864, 1981), MMTV LTR, RSV LTR, metallothionein-1, adenovirus E1a.
Cellular promoters include the mouse metallothionein-1 promoter (Palmiter
et al., U.S. Pat. No. 4,579,821), a mouse V.sub..kappa. promoter (Bergman
et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983; Grant et al., Nucl.
Acids Res. 15:5496, 1987) and a mouse V.sub.H promoter (Loh et al., Cell
33:85-93, 1983). The choice of promoter will depend, at least in part,
upon the level of expression desired or the recipient cell line to be
Such expression vectors may also contain a set of RNA splice sites located
downstream from the promoter and upstream from the DNA sequence encoding
the peptide or protein of interest. Preferred RNA splice sites may be
obtained from adenovirus and/or immunoglobulin genes. Also contained in
the expression vectors is a polyadenylation signal located downstream of
the coding sequence of interest. Suitable polyadenylation signals include
the early or late polyadenylation signals from SV40 (Kaufman and Sharp,
ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and
the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res.
9:3719-3730, 1981). The expression vectors may include a noncoding viral
leader sequence, such as the Adenovirus 2 tripartite leader, located
between the promoter and the RNA splice sites. Preferred vectors may also
include enhancer sequences, such as the SV40 enhancer. Expression vectors
may also include sequences encoding the adenovirus VA RNAs. Suitable
expression vectors can be obtained from commercial sources (e.g.,
Stratagene, La Jolla, Calif.).
Vector constructs comprising cloned DNA sequences can be introduced into
cultured mammalian cells by, for example, calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson,
Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456,
1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), or
DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987). To
identify cells that have stably integrated the cloned DNA, a selectable
marker is generally introduced into the cells along with the gene or cDNA
of interest. Preferred selectable markers for use in cultured mammalian
cells include genes that confer resistance to drugs, such as neomycin,
hygromycin, and methotrexate. The selectable marker may be an amplifiable
selectable marker. Preferred amplifiable selectable markers are the DHFR
gene and the neomycin resistance gene. Selectable markers are reviewed by
Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham,
Mass., which is incorporated herein by reference).
Mammalian cells containing a suitable vector are allowed to grow for a
period of time, typically 1-2 days, to begin expressing the DNA sequence(s)
of interest. Drug selection is then applied to select for growth of cells
that are expressing the selectable marker in a stable fashion. For cells
that have been transfected with an amplifiable, selectable marker the drug
concentration may be increased in a stepwise manner to select for
increased copy number of the cloned sequences, thereby increasing
expression levels. Cells expressing the introduced sequences are selected
and screened for production of the protein of interest in the desired form
or at the desired level. Cells that satisfy these criteria can then be
cloned and scaled up for production.
Protocols for the transfection of mammalian cells are well known to those
of ordinary skill in the art. Representative methods include calcium
phosphate mediated transfection, electroporation, lipofection, retroviral,
adenoviral and protoplast fusion-mediated transfection (see Sambrook et
al., supra). Naked vector constructs can also be taken up by muscular
cells or other suitable cells subsequent to injection into the muscle of a
mammal (or other animals).
Numerous insect host cells known in the art can also be useful within the
present invention, in light of the subject specification. For example, the
use of baculoviruses as vectors for expressing heterologous DNA sequences
in insect cells has been reviewed by Atkinson et al. (Pestic. Sci.
Numerous plant host cells known in the art can also be useful within the
present invention, in light of the subject specification. For example, the
use of Agrobacterium rhizogenes as vectors for expressing genes in plant
cells has been reviewed by Sinkar et al., (J. Biosci. (Bangalore)
WRN proteins may be prepared by growing (typically by culturing) the
host/vector systems described above, in order to express the recombinant
WRN proteins. Recombinantly produced WRN proteins may be further purified
as described in more detail below.
Within related aspects of the present invention, WRN proteins may be
expressed in a transgenic animal whose germ cells and somatic cells
contain a WRN gene which is operably linked to a promoter effective for
the expression of the gene. Alternatively, in a similar manner transgenic
animals may be prepared that lack the WRN gene (e.g., "knockout" mice).
Such transgenics may be prepared in a variety non-human animals, including
mice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al. Nature
315:680-683, 1985, Palmiter et al. Science 222:809-814, 1983, Brinster et
al. Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985, Palmiter and Brinster
Cell 41:343-345, 1985 and U.S. Pat. Nos. 5,175,383, 5,087,571, 4,736,866,
5,387,742, 5,347,075, 5,221,778, and 5,175,384).
Briefly, an expression vector, including a nucleic acid molecule to be
expressed together with appropriately positioned expression control
sequences, is introduced into pronucleli of fertilized eggs, for example,
by microinjection. Integration of the injected DNA is detected by blot
analysis of DNA from tissue samples. It is preferred that the introduced
DNA be incorporated into the germ line of the animal so that it is passed
on to the animal's progeny. Tissue-specific expression may be achieved
through the use of a tissue-specific promoter, or through the use of an
inducible promoter, such as the metallothionein gene promoter (Palmiter et
al., 1983, ibid), which allows regulated expression of the transgene.
Vectors of the present invention may contain or express a wide variety of
additional nucleic acid molecules in place of or in addition to an WRN
protein as described above, either from one or several separate promoters.
For example, the viral vector may express a lymphokine or lymphokine
receptor, antisense or ribozyme sequence or toxins. Representative
examples of lymphokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF, G-CSF, M-CSF,
alpha-interferon, beta-interferon, gamma-interferon, and tumor necrosis
factors, as well as their respective receptors. Representative examples of
antisense sequences include antisense sequences which block the expression
of WRN protein mutants. Representative examples of toxins include: ricin,
abrin, diphtheria toxin, cholera toxin, saporin, gelonin, pokeweed
antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A.
Within other aspects of the invention, antisense oligonucleotide molecules
are provided which specifically inhibit expression of mutant WRN nucleic
acid sequences (see generally, Hirashima et al. in Molecular Biology of
RNA: New Perspectives (M. Inouye and B. S. Dudock, eds., 1987 Academic
Press, San Diego, p. 401); Oligonucleotides: Antisense Inhibitors of Gene
Expression (J. S. Cohen, ed., 1989 MacMillan Press, London); Stein and
Cheng, Science 261:1004-1012 (1993); WO 95/10607; U.S. Pat. No. 5,359,051;
WO 92/06693; and EP-A2-612844). Briefly, such molecules are constructed
such that they are complementary to, and able to form Watson-Crick base
pairs with, a region of transcribed WRN mutant mRNA sequence containing an
WRN mutation. The resultant double-stranded nucleic acid interferes with
subsequent processing of the mRNA, thereby preventing protein synthesis.
Within other related aspects of the invention, ribozyme molecules are
provided wherein an antisense oligonucleotide sequence is incorporated
into a ribozyme which can specifically cleave mRNA molecules transcribed
from a mutant WRN gene (see generally, Kim et al. Proc. Nat. Acad. Sci.
USA 84:8788 (1987); Haseloff, et al. Nature 234:585 (1988), Cech, JAMA
260:3030 (1988); Jeffries, et al. Nucleic Acids Res. 17:1371 (1989); U.S.
Pat. Nos. 5,093,246; 5,354,855; 5,144,019; 5,272,262; 5,254,678; and
4,987,071). According to this aspect of the invention, the antisense
sequence which is incorporated into a ribozyme includes a sequence
complementary to, and able to form Watson-Crick base pairs with, a region
of the transcribed mutant WRN mRNA containing an WRN mutation. The
antisense sequence thus becomes a targeting agent for delivery of
catalytic ribozyme activity specifically to mutant WRN mRNA, where such
catalytic activity cleaves the mRNA to render it incapable of being
subsequently processed for WRN protein translation.
As discussed above, nucleic acid molecules which encode the WRN proteins
of the present invention (or the vectors which contain and/or express
related mutants) may readily be introduced into a wide variety of host
cells. Representative examples of such host cells include plant cells,
eukaryotic cells, and prokaryotic cells. Within preferred embodiments, the
nucleic acid molecules are introduced into cells from a vertebrate or
warm-blooded animal, such as a human, macaque, dog, cow, horse, pig,
sheep, rat, hamster, mouse or fish cell, or any hybrid thereof.
Preferred prokaryotic host cells for use within the present invention
include E. coli, Salmonella, Bacillus, Shigella, Pseudomonas, Streptomyces
and other genera. Techniques for transforming these hosts and expressing
foreign DNA sequences cloned therein are well known in the art (see, e.g.,
Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, 1982, which is incorporated herein by reference; or
Sambrook et al., supra). Vectors used for expressing cloned DNA sequences
in bacterial hosts will generally contain a selectable marker, such as a
gene for antibiotic resistance, and a promoter that functions in the host
cell. Appropriate promoters include the trp (Nichols and Yanofsky, Meth.
Enzymol. 101:155-164, 1983), lac (Casadaban et al., J. Bacteriol.
143:971-980, 1980), and phage .lamda. (Queen, J. Mol. Appl. Genet. 2:1-10,
1983) promoter systems. Plasmids useful for transforming bacteria include
the pUC plasmids (Messing, Meth. Enzymol. 101:20-78, 1983; Vieira and
Messing, Gene 19:259-268, 1982), pBR322 (Bolivar et al., Gene 2:95-113,
1977), pCQV2 (Queen, ibid.), and derivatives thereof. Plasmids may contain
both viral and bacterial elements.
Preferred eukaryotic cells include cultured mammalian cell lines (e.g.,
rodent or human cell lines) and fungal cells, including species of yeast
(e.g., Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces
spp., or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp.,
Neurospora spp.). Strains of the yeast Saccharomyces cerevisiae are
particularly preferred. Methods for producing recombinant proteins in a
variety of prokaryotic and eukaryotic host cells are generally known in
the art (see, "Gene Expression Technology," Methods in Enzymology, Vol.
185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990; see also,
"Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology,
Guthrie and Fink (eds.), Academic Press, San Diego, Calif., 1991). In
general, a host cell will be selected on the basis of its ability to
produce the protein of interest at a high level or its ability to carry
out at least some of the processing steps necessary for the biological
activity of the protein. In this way, the number of cloned DNA sequences
that must be introduced into the host cell can be minimized and overall
yield of biologically active protein can be maximized.
The nucleic acid molecules (or vectors) may be introduced into host cells
by a wide variety of mechanisms, including for example calcium
phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978),
lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen. 7:603, 1981;
Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et
al., EMBO J. 1:841-845, 1982), retroviral, adenoviral, protoplast
fusion-mediated transfection or DEAE-dextran mediated transfection (Ausubel
et al., (eds.), Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, N.Y., 1987).
Host cells containing vector constructs of the present invention are then
cultured to express a DNA molecule as described above. The cells are
cultured according to standard methods in a culture medium containing
nutrients required for growth of the chosen host cells. A variety of
suitable media are known in the art and generally include a carbon source,
a nitrogen source, essential amino acids, vitamins and minerals, as well
as other components, e.g., growth factors or serum, that may be required
by the particular host cells. The growth medium will generally select for
cells containing the DNA construct(s) by, for example, drug selection or
deficiency in an essential nutrient which is complemented by the
selectable marker on the DNA construct or co-transfected with the DNA
Suitable growth conditions for yeast cells, for example, include culturing
in a chemically defined medium, comprising a nitrogen source, which may be
a non-amino acid nitrogen source or a yeast extract, inorganic salts,
vitamins and essential amino acid supplements at a temperature between
4.degree. C. and 37.degree. C., with 30.degree. C. being particularly
preferred. The pH of the medium is preferably maintained at a pH greater
than 2 and less than 8, more preferably pH 5-6. Methods for maintaining a
stable pH include buffering and constant pH control. Preferred agents for
pH control include sodium hydroxide. Preferred buffering agents include
succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Due to
the tendency of yeast host cells to hyperglycosylate heterologous
proteins, it may be preferable to express the nucleic acid molecules of
the present invention in yeast cells having a defect in a gene required
for asparagine-linked glycosylation. Such cells are preferably grown in a
medium containing an osmotic stabilizer. A preferred osmotic stabilizer is
sorbitol supplemented into the medium at a concentration between 0.1 M and
1.5 M, preferably at 0.5 M or 1.0 M.
Cultured mammalian cells are generally cultured in commercially available
serum-containing or serum-free media. Selection of a medium and growth
conditions appropriate for the particular cell line used is well within
the level of ordinary skill in the art.
Antibodies to the WRN proteins discussed above may readily be prepared
given the disclosure provided herein. Such antibodies may, within certain
embodiments, specifically recognize wild type WRN protein rather than a
mutant WRN protein, mutant WRN protein rather than wild type WRN protein,
or equally recognize both the mutant and wild-type forms of WRN protein.
Antibodies may be used for isolation of the protein, establishing
intracellular localization of the WRN protein, inhibiting activity of the
protein (antagonist), or enhancing activity of the protein (agonist).
Knowledge of the intracellular location of the WRN gene product may be
abnormal in patients with WRN mutations, thus allowing the development of
a rapid screening assay. As well, assays for small molecules that interact
with the WRN gene product will be facilitated by the development of
antibodies and localization studies.
Within the context of the present invention, antibodies are understood to
include monoclonal antibodies, polyclonal antibodies, anti-idiotypic
antibodies, antibody fragments (e.g., Fab, and F(ab').sub.2, F.sub.v
variable regions, or complementarity determining regions). As discussed
above, antibodies are understood to be specific against an WRN protein if
it binds with a K.sub.d of greater than or equal to 10.sup.-7M, preferably
greater than of equal to 10.sup.-8M. The affinity of a monoclonal antibody
or binding partner can be readily determined by one of ordinary skill in
the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949).
Briefly, polyclonal antibodies may be readily generated by one of ordinary
skill in the art from a variety of warm-blooded animals such as horses,
cows, various fowl, rabbits, mice, or rats. Typically, an WRN protein or
unique peptide thereof of 13-20 amino acids (preferably conjugated to
keyhole limpet hemocyanin by cross-linking with glutaraldehyde) is
utilized to immunize the animal through intraperitoneal, intramuscular,
intraocular, or subcutaneous injections, an adjuvant such as Freund's
complete or incomplete adjuvant. Merely as an example, a peptide
corresponding to residues 1375 through 1387 of the WRN polypeptide
sequence is used to raise a rabbit polyclonal antiserum. Following several
booster immunizations, samples of serum are collected and tested for
reactivity to the WRN protein or peptide. Particularly preferred
polyclonal antisera will give a signal on one of these assays that is at
least three times greater than background. Once the titer of the animal
has reached a plateau in terms of its reactivity to the protein, larger
quantities of antisera may be readily obtained either by weekly bleedings,
or by exsanguinating the animal.
Monoclonal antibodies may also be readily generated using conventional
techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and
4,411,993 which are incorporated herein by reference; see also Monoclonal
Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum
Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A
Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory
Press, 1988, which are also incorporated herein by reference).
Briefly, within one embodiment a subject animal such as a rat or mouse is
injected with an WRN protein or portion thereof as described above. The
protein may be admixed with an adjuvant such as Freund's complete or
incomplete adjuvant in order to increase the resultant immune response.
Between one and three weeks after the initial immunization the animal may
be reimmunized with another booster immunization, and tested for
reactivity to the protein utilizing assays described above. Once the
animal has reached a plateau in its reactivity to the injected protein, it
is sacrificed, and organs which contain large numbers of B cells such as
the spleen and lymph nodes are harvested.
Cells which are obtained from the immunized animal may be immortalized by
transfection with a virus such as the Epstein-Barr virus (EBV) (see Glasky
and Reading, Hybridoma 8(4):377-389, 1989). Alternatively, within a
preferred embodiment, the harvested spleen and/or lymph node cell
suspensions are fused with a suitable myeloma cell in order to create a "hybridoma"
which secretes monoclonal antibody. Suitable myeloma lines include, for
example, NS-1 (ATCC No. TIB 18), and P3.times.63-Ag 8.653 (ATCC No. CRL
Following the fusion, the cells may be placed into culture plates
containing a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's
Modified Eagles Medium) (JRH Biosciences, Lenexa, Kans.), as well as
additional ingredients, such as fetal bovine serum (FBS, i.e., from
Hyclone, Logan, Utah, or JRH Biosciences). Additionally, the medium should
contain a reagent which selectively allows for the growth of fused spleen
and myeloma cells such as HAT (hypoxanthine, aminopterin, and thymidine)
(Sigma Chemical Co., St. Louis, Mo.). After about seven days, the
resulting fused cells or hybridomas may be screened in order to determine
the presence of antibodies which are reactive against an WRN protein. A
wide variety of assays may be utilized to determine the presence of
antibodies which are reactive against the proteins of the present
invention, including for example countercurrent immuno-electrophoresis,
radioimmunoassays, radioimmunoprecipitations, enzyme-linked immuno-sorbent
assays (ELISA), dot blot assays, western blots, immunoprecipitation,
Inhibition or Competition Assays, and sandwich assays (see U.S. Pat. Nos.
4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, Harlow
and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Following
several clonal dilutions and reassays, a hybridoma producing antibodies
reactive against the WRN protein may be isolated.
Other techniques may also be utilized to construct monoclonal antibodies
(see William D. Huse et al., "Generation of a Large Combinational Library
of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281,
December 1989; see also L. Sastry et al., "Cloning of the Immunological
Repertoire in Escherichia coli for Generation of Monoclonal Catalytic
Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA
Library," Proc. Natl. Acad. Sci. USA 86:5728-5732, August 1989; see also
Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A
Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9,
January 1990; these references describe a commercial system available from
Stratacyte, La Jolla, Calif., which enables the production of antibodies
through recombinant techniques). Briefly, mRNA is isolated from a B cell
population, and utilized to create heavy and light chain immunoglobulin
cDNA expression libraries in the .lamda.ImmunoZap(H) and .lamda.ImmunoZap(L)
vectors. These vectors may be screened individually or co-expressed to
form Fab fragments or antibodies (see Huse et al., supra; see also Sastry
et al., supra). Positive plaques may subsequently be converted to a non-lytic
plasmid which allows high level expression of monoclonal antibody
fragments from E. coli.
Similarly, portions or fragments, such as Fab and Fv fragments, of
antibodies may also be constructed utilizing conventional enzymatic
digestion or recombinant DNA techniques to incorporate the variable
regions of a gene which encodes a specifically binding antibody. Within
one embodiment, the genes which encode the variable region from a
hybridoma producing a monoclonal antibody of interest are amplified using
nucleotide primers for the variable region. These primers may be
synthesized by one of ordinary skill in the art, or may be purchased from
commercially available sources. Stratacyte (La Jolla, Calif.) sells
primers for mouse and human variable regions including, among others,
primers for V.sub.Ha, V.sub.Hb, V.sub.Hc, V.sub.Hd, C.sub.H1, V.sub.L and
C.sub.Lregions. These primers may be utilized to amplify heavy or light
chain variable regions, which may then be inserted into vectors such as
ImmunoZAP.TM. H or ImmunoZAP.TM. L (Stratacyte), respectively. These
vectors may then be introduced into E. coli, yeast, or mammalian-based
systems for expression. Utilizing these techniques, large amounts of a
single-chain protein containing a fusion of the V.sub.H and V.sub.L
domains may be produced (see Bird et al., Science 242:423-426, 1988). In
addition, such techniques may be utilized to change a "murine" antibody to
a "human" antibody, without altering the binding specificity of the
Once suitable antibodies have been obtained, they may be isolated or
purified by many techniques well known to those of ordinary skill in the
art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold
Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide
or protein affinity columns, HPLC or RP-HPLC, purification on protein A or
protein G columns, or any combination of these techniques.
Assays useful within the context of the present invention include those
assays for detecting agonists or antagonists of WRN protein activity.
Other assays are useful for the screening of peptide or organic molecule
libraries. Still other assays are useful for the identification and/or
isolation of nucleic acid molecules and/or peptides within the present
invention, the identification of proteins that interact or bind the WRN
protein, for diagnosis of a patient with an increased likelihood of
contracting Werner's Syndrome, or for diagnosis of a patient with
susceptibility to or manifestation of a WRN-related disease.
Nucleic Acid Based Diagnostic Tests
Briefly, another aspect of the present invention provides probes and
primers for detecting the WRN genes and/or mutants thereof. In one
embodiment of this aspect, probes are provided that are capable of
specifically hybridizing to DNA or RNA of the WRN genes. For purposes of
the present invention, probes are "capable of hybridizing" to DNA or RNA
of the WRN gene if they hybridize to an WRN gene under conditions of
either high or moderate stringency (see Sambrook et al., supra) but not
significantly or detectably to the an unrelated helicase gene such as the
Bloom's Syndrome gene (Ellis et al., Cell 83:655-666, 1995). Preferably,
the probe hybridizes to suitable nucleotide sequences under high
stringency conditions, such as hybridization in 5.times.SSPE, 1.times.
Denhardt's solution, 0.1% SDS at 65.degree. C., and at least one wash to
remove unhybridized probe in the presence of 0.2.times.SSC, 1.times.
Denhardt's solution, 0.1% SDS at 65.degree. C. Except as otherwise
provided herein, probe sequences are designed to allow hybridization to
WRN genes, but not to DNA or RNA sequences from other genes. The probes
are used, for example, to hybridize to nucleic acid that is present in a
biological sample isolated from a patient. The hybridized probe is then
detected, thereby indicating the presence of the desired cellular nucleic
acid. Preferably, the cellular nucleic acid is subjected to an
amplification procedure, such as PCR, prior to hybridization.
Alternatively, the WRN gene may be amplified and the amplified product
subjected to DNA sequencing. Mutants of WRN may be detected by DNA
sequence analysis or hybridization with allele-specific oligonucleotide
probes under conditions and for time sufficient to allow hybridization to
the specific allele. Typically, the hybridization buffer and wash will
contain tetramethyl ammonium chloride or the like (see Sambrook et al.,
Nucleic acid probes of the present invention may be composed of either
deoxyribonucleic acids (DNA), ribonucleic acids (RNA), nucleic acid
analogues (e.g., peptide nucleic acids), or any combination thereof, and
may be as few as about 12 nucleotides in length, usually about 14 to 18
nucleotides in length, and possibly as large as the entire sequence of a
WRN gene. Selection of probe size is somewhat dependent upon the use of
the probe, and is within the skill of the art.
Suitable probes can be constructed and labeled using techniques that are
well known in the art. Shorter probes of, for example, 12 bases can be
generated synthetically and labeled with .sup.32P using T.sub.4
polynucleotide kinase. Longer probes of about 75 bases to less than 1.5 kb
are preferably generated by, for example, PCR amplification in the
presence of labeled precursors such as [.alpha.-.sup.32P]dCTP,
digoxigenin-dUTP, or biotin-dATP. Probes of more than 1.5 kb are generally
most easily amplified by transfecting a cell with a plasmid containing the
relevant probe, growing the transfected cell into large quantities, and
purifying the relevant sequence from the transfected cells. (See Sambrook
et al., supra.)
Probes can be labeled by a variety of markers, including for example,
radioactive markers, fluorescent markers, enzymatic markers, and
chromogenic markers. The use of .sup.32P is particularly preferred for
marking or labeling a particular probe.
It is a feature of this aspect of the invention that the probes can be
utilized to detect the presence of WRN mRNA or DNA within a sample.
However, if the relevant sample is present in only a limited number, then
it may be beneficial to amplify the relevant sequence so that it may be
more readily detected or obtained.
A variety of methods may be utilized in order to amplify a selected
sequence, including, for example, RNA amplification (see Lizardi et al.,
Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989;
Lomeli et al., Clinical Chem. 35(9):1826-1831, 1989; U.S. Pat. No.
4,786,600), and DNA amplification utilizing LCR or polymerase chain
reaction ("PCR") (see, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159)
(see also U.S. Pat. Nos. 4,876,187 and 5,011,769, which describe an
alternative detection/amplification system comprising the use of scissile
linkages), or other nucleic acid amplification procedures that are well
within the level of ordinary skill in the art. With respect to PCR, for
example, the method may be modified as known in the art. Transcriptional
enhancement of PCR may be accomplished by incorporation of bacteriophage
T7 RNA polymerase promoter sequences in one of the primary
oligonucleotides, and immunoenzymatic detection of the products from the
enhanced emitter may be effected using anti-RNA:DNA antibodies (Blais,
Appl. Environ. Microbiol. 60:348-352, 1994). PCR may also be used in
combination with reverse dot-blot hybridization (Iida et al., FEMS
Microbiol. Lett. 114:167-172, 1993). PCR products may be quantitatively
analyzed by incorporation of dUTP (Duplaa et al., Anal. Biochem.
212:229-236, 1993), and samples may be filter sampled for PCR-gene probe
detection (Bej et al., Appl. Environ. Microbiol. 57:3529-3534, 1991).
Within a particularly preferred embodiment, PCR amplification is utilized
to detect the WRN DNA. Briefly, as described in greater detail below, a
DNA sample is denatured at 95.degree. C. in order to generate
single-stranded DNA. The DNA sample may be a cDNA generated from RNA.
Specific primers are then annealed to the single-stranded DNA at
37.degree. C. to 70.degree. C., depending on the proportion of AT/GC in
the primers. The primers are extended at 72.degree. C. with Taq DNA
polymerase or other thermostable DNA polymerase in order to generate the
opposite strand to the template. These steps constitute one cycle, which
may be repeated in order to amplify the selected sequence. For greater
specificity, nested PCR may be performed. In nested PCR, a second
amplification is performed using a second set of primers derived from
sequences within the first amplified product. The entire coding region of
WRN may be amplified from cDNA using three sets of primers to generate
fragment lengths that are a convenient size for determining their
sequence. In a preferred embodiment, nested PCR is performed.
Within an alternative preferred embodiment, LCR amplification is utilized
for amplification. LCR primers are synthesized such that the 5' base of
the upstream primer is capable of hybridizing to a unique base pair in a
desired gene to specifically detect an WRN gene.
Within another preferred embodiment, the probes are used in an automated,
non-isotopic strategy wherein target nucleic acid sequences are amplified
by PCR, and then desired products are determined by a calorimetric
oligonucleotide ligation assay (OLA) (Nickerson et al., Proc. Natl. Acad.
Sci. USA 81:8923-8927, 1990).
Primers for the amplification of a selected sequence should be selected
from sequences that are highly specific to WRN (and not, e.g., the Bloom's
Syndrome gene, supra) and form stable duplexes with the target sequence.
The primers should also be non-complementary, especially at the 3' end,
should not form dimers with themselves or other primers, and should not
form secondary structures or duplexes with other regions of DNA. In
general, primers of about 18 to 20 nucleotides are preferred, and can be
easily synthesized using techniques well known in the art. PCR products,
and other nucleic acid amplification products, may be quantitated using
techniques known in the art (Duplaa et al., Anal. Biochem. 212:229-236,
1993; Higuchi et al., Bio/Technology 11:1026-1030).
Within one embodiment of the invention, nucleic acid diagnostics may be
developed which are capable of detecting the presence of Werner's
Syndrome, or of various related diseases that may be caused by Werner's
Syndrome. Briefly, severe mutations in the WRN gene may lead to Werner's
Syndrome, as well as a host of related diseases, including for example,
increased frequency of some benign and malignant neoplasms (especially
sarcomas), cataracts, cardiovascular disease, osteoporosis, type I or type
II diabetes, cataracts, sclerodoma-like skin changes and hyperkeratosis.
Less severe mutations of the gene may lead to the onset of the same set of
diseases, but at an older age. In addition, many of the related diseases
may be associated with mutations in the WRN gene. For example, diabetes
and osteoporosis are often associated with aging. Aging population and
individuals with these (or other) diseases are screened for mutations in
WRN. Any of the assays described herein may be used. RT-PCR is especially
preferred in conjunction with DNA sequence determination. To correlate a
mutation or polymorphism with disease, sibling pairs in which one sibling
has disease are preferred subjects. Once a mutation is identified, other
convenient screening assays may be used to assay particular nucleotide
Since the sequences of the two copies of the gene from non-Werner's
affected individuals can be correlated with the medical histories of these
patients to define these correspondences, these alleles can therefore be
used as diagnostics for susceptibilities to these diseases, once the
relationship is defined. Certain non-null forms of the gene, for example,
in either the homozygous or heterozygous state may significantly affect
the propensity for the carriers to develop, for example, cancer. These
propensities can be ascertained by examining the sequences of the gene
(both copies) in a statistically significant sample of cancer patients.
Other diseases (see above) can be similarly examined for significant
correlations with certain alleles. To detect such a causal relationship
one can use a chi-squared test, or other statistical test, to examine the
significance of any correlation between the appropriate genotypes and the
disease state as recorded in the medical records, using standard good
practices of medical epidemiology. The sequences that define each of the
alleles are then valuable diagnostic indicators for an increased
susceptibility to the disease. Thus, from the nucleic acid sequences
provided herein, a wide variety of Werner's Syndrome-related diseases may
be readily detected.
Another cellular phenotype of the cells from Werner's patients is the
increased frequency of deletion mutation in these cells. Clearly, the
defective helicase in these cells leads to a specific mutator phenotype,
while not rendering the cells hypersensitive to a variety of chemical or
physical mutagens that damage DNA, like ionizing radiation. Disease
states, or sensitivities that result from an elevated deletion frequency
can therefore be controlled, in part, by alterations of the Werner's gene,
and some alleles may therefore be diagnostic of this class of medical
Assays for Agonists and Antagonists
An agonist or antagonist of the WRN gene product comprising a protein,
peptide, chemical, or peptidomimetic that binds to the WRN gene product or
interacts with a protein that binds to the WRN gene product such that the
binding of the agonist or antagonist affects the activity of the WRN gene
product. An agonist will activate or increase the activity of the WRN gene
product. An antagonist will inhibit or decrease the activity of the WRN
gene product. The activity of the WRN gene product may be measured in an
assay, such as a helicase assay or other assay that measures an activity
of the WRN gene product. Other assays measure the binding of protein that
interacts with WRN and is necessary for its activity.
Agonists and antagonists of the WRN gene product may be used to enhance
activity or inhibit activity of the gene product. Such agonists and
antagonists may be identified in a variety of methods. For example,
proteins that bind and activate WRN may be identified using a yeast
2-hybrid detection system. In this system, the WRN gene is fused to either
a DNA-binding domain or an activating domain of a yeast gene such as GAL4.
A cDNA library is constructed in a vector such that the inserts are fused
to one of the domains. The vectors are co-transfected into yeast and
selected for transcriptional activation of a reporter gene (Fields and
Song, Nature 340: 245, 1989). The protein(s) that bind to WRN are
candidate agonists. Three different proteins that bind WRN have been
identified in an initial screen using the 2-hybrid system.
When the binding site on WRN gene product is determined, molecules that
bind and activate WRN protein may be designed and evaluated. For example,
computer modeling of the binding site can be generated and mimetics that
bind can be designed. Antibodies to the binding site may be generated and
analogues of native binding proteins generated as well. Any of these
molecules is tested for agonist or antagonist activity by a functional
assay of the WRN gene product. For example, to test for antagonist
activity, yeast are co-transfected with the WRN and binding protein each
fused to a DNA binding domain or an activation domain. The test molecule
is administered and activation is monitored. An antagonist will inhibit
the activation of the reporter gene by at least 50%. Similarly, agonist
activity may be measured by either enhancing WRN activity in a yeast
2-hybrid system or by coupling the test compound to a DNA binding or
activation domain and monitoring activity of the reporter gene.
WRN proteins, nucleic acid molecules which encodes such proteins, anti-WRN
protein antibodies and agonists or antagonists, as described above and
below, may be labeled with a variety of molecules, including for example,
fluorescent molecules, toxins, and radionuclides. Representative examples
of fluorescent molecules include fluorescein, Phycobili proteins, such as
phycoerythrin, rhodamine, Texas red and luciferase. Representative
examples of toxins include ricin, abrin diphtheria toxin, cholera toxin,
gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and
Pseudomonas exotoxin A. Representative examples of radionuclides include
Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-123,
I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and
Bi-212. In addition, the antibodies described above may also be labeled or
conjugated to one partner of a ligand binding pair. Representative
examples include avidin-biotin, and riboflavin-riboflavin binding protein.
Methods for conjugating or labeling the WRN proteins, nucleic acid
molecules which encode such proteins, anti-WRN protein antibodies and
agonists or antagonists, as discussed above, with the representative
labels set forth above may be readily accomplished by one of ordinary
skill in the art (see Trichothecene Antibody Conjugate, U.S. Pat. No.
4,744,981,; Antibody Conjugate, U.S. Pat. No. 5,106,951; Fluorogenic
Materials and Labeling Techniques, U.S. Pat. No. 4,018,884; Metal
Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat. No.
4,897,255; and Metal Radionuclide Chelating Compounds for Improved
Chelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Methods In
Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B,
Jakoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; see also
Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical
Applications," Anal. Biochem. 171:1-32, 1988).
As noted above, the present invention also provides a variety of
pharmaceutical compositions, comprising one of the above-described WRN
proteins, nucleic acid molecules, vectors, antibodies, host cells,
agonists or antagonists, along with a pharmaceutically or physiologically
acceptable carrier, excipients or diluents. Generally, such carriers
should be nontoxic to recipients at the dosages and concentrations
employed. Ordinarily, the preparation of such compositions entails
combining the therapeutic agent with buffers, antioxidants such as
ascorbic acid, low molecular weight (less than about 10 residues)
polypeptides, proteins, amino acids, carbohydrates including glucose,
sucrose or dextrins, chelating agents such as EDTA, glutathione and other
stabilizers and excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary appropriate diluents.
In addition, the pharmaceutical compositions of the present invention may
be prepared for administration by a variety of different routes. In
addition, pharmaceutical compositions of the present invention may be
placed within containers, along with packaging material which provides
instructions regarding the use of such pharmaceutical compositions.
Generally, such instructions will include a tangible expression describing
the reagent concentration, as well as within certain embodiments, relative
amounts of excipient ingredients or diluents (e.g., water, saline or PBS)
which may be necessary to reconstitute the pharmaceutical composition.
Methods of Treating or Preventing Werner's Syndrome
The present invention also provides methods for treating or preventing
Werner's Syndrome (or related diseases), comprising the step of
administering to a patient a vector (e.g., expression vector, viral
vector, or viral particle containing a vector) or nucleic acid molecules
alone, as described above, thereby reducing the likelihood or delaying the
onset of Werner's Syndrome (or the related disease).
Similarly, therapeutic peptides, peptidomimetics, or small molecules may
be used to delay onset of Werner's Syndrome, lessen symptoms, or halt or
delay progression of the disease. Such therapeutics may be tested in a
transgenic animal model that expresses mutant protein, wild-type and
mutant protein, or in an in vitro assay system (e.g., a helicase assay
such as that described by Bjornson et al., Biochem. 3307:14306-14316,
As noted above, the present invention provides methods for treating or
preventing Werner's Syndrome through the administration to a patient of a
therapeutically effective amount of an antagonist or pharmaceutical
composition as described herein. Such patients may be identified through
clinical diagnosis based on the classical symptoms of Werner's Syndrome.
As will be evident to one of skill in the art, the amount and frequency of
administration will depend, of course, on such factors as the nature and
severity of the indication being treated, the desired response, the
condition of the patient, and so forth. Typically, the compositions may be
administered by a variety of techniques, as noted above.
Within other embodiments of the invention, the vectors which contain or
express the nucleic acid molecules which encode the WRN proteins described
above, or even the nucleic acid molecules themselves may be administered
by a variety of alternative techniques, including for example
administration of asialoosomucoid (ASOR) conjugated with poly-L-lysine DNA
complexes (Cristano et al., PNAS 92122-92126, 1993), DNA linked to killed
adenovirus (Curiel et al., Hum. Gene Ther. 3(2):147-154, 1992), cytofectin-mediated
introduction (DMRIE-DOPE, Vical, Calif.), direct DNA injection (Acsadi et
al., Nature 352:815-818, 1991); DNA ligand (Wu et al., J. of Biol. Chem.
264:16985-16987, 1989); lipofection (Felgner et al., Proc. Natl. Acad. Sci.
USA 84:7413-7417, 1989); liposomes (Pickering et al., Circ. 89(1):13-21,
1994; and Wang et al., PNAS 84:7851-7855, 1987); microprojectile
bombardment (Williams et al., PNAS 88:2726-2730, 1991); and direct
delivery of nucleic acids which encode the WRN protein itself either alone
(Vile and Hart, Cancer Res. 53: 3860-3864, 1993), or utilizing PEG-nucleic
Claim 1 of 6 Claims
1. A purified antibody which specifically
binds to a WRN gene product comprising the amino acid sequence set forth
in SEQ ID NO:71.
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