Title: Methods and reagents for
diagnosis and treatment of diabetes
United States Patent: 7,144,985
Issued: December 5, 2006
Inventors: Johnson; Jeffrey
D. (Moraga, CA), Blume; John E. (Danville, CA), Palma; John F. (San Ramon,
CA), Zhou; Yun-Ping (San Ramon, CA)
Assignee: Metabolex, Inc.
Appl. No.: 10/308,393
Filed: December 2, 2002
The invention relates to methods of
determining islet cell activity by detecting the level of Archipelin or a
fragment thereof and comparing the level to a baseline level or range
associated with a known islet cell activity. Such methods are useful in
diagnosing and studying the development of diabetes.
OF THE INVENTION
This invention is directed to new polypeptide and polynucleotide
sequences, designated Archipelin sequences, as well as methods of using
the sequences to diagnose and treat diabetes. The present method also
provides methods of identifying modulators of Archipelin expression and
activity. Such modulators are useful for treating type 1 and type 2
diabetes as well as the pathological aspects of such diseases.
II. General Recombinant Nucleic Acids Methods for Use with the Invention
In numerous embodiments of the present invention, nucleic acids encoding a
Archipelin of interest will be isolated and cloned using recombinant
methods. Such embodiments are used, e.g., to isolate Archipelin
polynucleotides (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID
NO:8) for protein expression or during the generation of variants,
derivatives, expression cassettes, or other sequences derived from an
Archipelin polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:7, and SEQ ID NOs:9 14), to monitor Archipelin gene expression, for
the isolation or detection of Archipelin sequences in different species,
for diagnostic purposes in a patient, e.g., to detect mutations in
Archipelin or to detect expression levels of Archipelin nucleic acids or
Archipelin polypeptides. In some embodiments, the sequences encoding the
Archipelin of the invention are operably linked to a heterologous
promoter. In one embodiment, the nucleic acids of the invention are from
any mammal, including, in particular, e.g., a human, a mouse, a rat, etc.
A. General Recombinant Nucleic Acids Methods
This invention relies on routine techniques in the field of recombinant
genetics. Basic texts disclosing the general methods of use in this
invention include Sambrook et al., Molecular Cloning, A Laboratory Manual
(2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory
Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al.,
For nucleic acids, sizes are given in either kilobases (kb) or base pairs
(bp). These are estimates derived from agarose or acrylamide gel
electrophoresis, from sequenced nucleic acids, or from published DNA
sequences. For proteins, sizes are given in kilodaltons (kDa) or amino
acid residue numbers. Proteins sizes are estimated from gel
electrophoresis, from sequenced proteins, from derived amino acid
sequences, or from published protein sequences.
Oligonucleotides that are not commercially available can be chemically
synthesized according to the solid phase phosphoramidite triester method
first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859 1862
(1981), using an automated synthesizer, as described in Van Devanter et.
al., Nucleic Acids Res. 12:6159 6168 (1984). Purification of
oligonucleotides is by either native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137
The sequence of the cloned genes and synthetic oligonucleotides can be
verified after cloning using, e.g., the chain termination method for
sequencing double-stranded templates of Wallace et al., Gene 16:21 26
B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding the
In general, the nucleic acids encoding the subject proteins are cloned
from DNA sequence libraries that are made to encode copy DNA (cDNA) or
genomic DNA. The particular sequences can be located by hybridizing with
an oligonucleotide probe, the sequence of which can be derived from the
sequences provided herein (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
and SEQ ID NO:8), which provides a reference for PCR primers and defines
suitable regions for isolating Archipelin-specific probes. Alternatively,
where the sequence is cloned into an expression library, the expressed
recombinant protein can be detected immunologically with antisera or
purified antibodies made against the Archipelin of interest.
Methods for making and screening genomic and cDNA libraries are well known
to those of skill in the art (see, e.g., Gubler and Hoffman Gene 25:263
269 (1983); Benton and Davis Science, 196:180 182 (1977); and Sambrook,
supra). A islet cells are an example of suitable cells to isolate
Archipelin RNA and cDNA.
Briefly, to make the cDNA library, one should choose a source that is rich
in mRNA. The mRNA can then be made into cDNA, ligated into a recombinant
vector, and transfected into a recombinant host for propagation, screening
and cloning. For a genomic library, the DNA is extracted from a suitable
tissue and either mechanically sheared or enzymatically digested to yield
fragments of preferably about 5 100 kb. The fragments are then separated
by gradient centrifugation from undesired sizes and are constructed in
bacteriophage lambda vectors. These vectors and phage are packaged in
vitro, and the recombinant phages are analyzed by plaque hybridization.
Colony hybridization is carried out as generally described in Grunstein et
al., Proc. Natl. Acad. Sci. USA., 72:3961 3965 (1975).
An alternative method combines the use of synthetic oligonucleotide
primers with polymerase extension on an mRNA or DNA template. Suitable
primers can be designed from specific Archipelin sequences, e.g., the
sequences described in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID
NO:8. This polymerase chain reaction (PCR) method amplifies the nucleic
acids encoding the protein of interest directly from mRNA, cDNA, genomic
libraries or cDNA libraries. Restriction endonuclease sites can be
incorporated into the primers. Polymerase chain reaction or other in vitro
amplification methods may also be useful, for example, to clone nucleic
acids encoding specific proteins and express said proteins, to synthesize
nucleic acids that will be used as probes for detecting the presence of
mRNA encoding an Archipelin polypeptide of the invention in physiological
samples, for nucleic acid sequencing, or for other purposes (see, U.S.
Pat. Nos. 4,683,195 and 4,683,202). Genes amplified by a PCR reaction can
be purified from agarose gels and cloned into an appropriate vector.
Appropriate primers and probes for identifying the genes encoding an
Archipelin polypeptide of the invention from mammalian tissues can be
derived from the sequences provided herein, in particular SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5, and SEQ ID NO:8. For a general overview of PCR, see,
Innis et al. PCR Protocols: A Guide to Methods and Applications, Academic
Press, San Diego (1990).
Synthetic oligonucleotides can be used to construct genes. This is done
using a series of overlapping oligonucleotides, usually 40 120 bp in
length, representing both the sense and anti-sense strands of the gene.
These DNA fragments are then annealed, ligated and cloned.
A gene encoding an Archipelin polypeptide of the invention can be cloned
using intermediate vectors before transformation into mammalian cells for
expression. These intermediate vectors are typically prokaryote vectors or
shuttle vectors. The proteins can be expressed in either prokaryotes,
using standard methods well known to those of skill in the art, or
eukaryotes as described infra.
C. Expression in Prokaryotes and Eukaryotes
To obtain high level expression of a cloned gene, such as cDNAs encoding
Archipelin, one typically subclones polynucleotides encoding Archipelin
into an expression vector that contains a strong promoter to direct
transcription, a transcription/translation terminator, and if for a
nucleic acid encoding a protein, a ribosome binding site for translational
initiation. Suitable bacterial promoters are well known in the art and
described, e.g., in Sambrook et al. and Ausubel et al. Bacterial
expression systems for expressing the Archipelin protein are available in,
e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229 235
(1983); Mosbach et al., Nature 302:543 545 (1983). Kits for such
expression systems are commercially available. Eukaryotic expression
systems for mammalian cells, yeast, and insect cells are well known in the
art and are also commercially available.
Selection of the promoter used to direct expression of a heterologous
nucleic acid depends on the particular application. The promoter is
preferably positioned about the same distance from the heterologous
transcription start site as it is from the transcription start site in its
natural setting. As is known in the art, however, some variation in this
distance can be accommodated without loss of promoter function.
In addition to the promoter, the expression vector typically contains a
transcription unit or expression cassette that contains all the additional
elements required for the expression of Archipelin-encoding nucleic acid
in host cells. A typical expression cassette thus contains a promoter
operably linked to the nucleic acid sequence encoding Archipelin and
signals required for efficient polyadenylation of the transcript, ribosome
binding sites, and translation termination. Additional elements of the
cassette may include enhancers and, if genomic DNA is used as the
structural gene, introns with functional splice donor and acceptor sites.
In addition to a promoter sequence, the expression cassette should also
contain a transcription termination region downstream of the structural
gene to provide for efficient termination. The termination region may be
obtained from the same gene as the promoter sequence or may be obtained
from different genes.
The particular expression vector used to transport the genetic information
into the cell is not particularly critical. Any of the conventional
vectors used for expression in eukaryotic or prokaryotic cells may be
used. Standard bacterial expression vectors include plasmids such as
pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as
GST and LacZ. Epitope tags can also be added to recombinant proteins to
provide convenient methods of isolation, e.g., c-myc.
Expression vectors containing regulatory elements from eukaryotic viruses
are typically used in eukaryotic expression vectors, e.g., SV40 vectors,
papilloma virus vectors, and vectors derived from Epstein-Barr virus.
Other exemplary eukaryotic vectors include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+,
pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of
proteins under the direction of the CMV promoter, SV40 early promoter,
SV40 later promoter, metallothionein promoter, murine mammary tumor virus
promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other
promoters shown effective for expression in eukaryotic cells.
Expression of proteins from eukaryotic vectors can be also be regulated
using inducible promoters. With inducible promoters, expression levels are
tied to the concentration of inducing agents, such as tetracycline or
ecdysone, by the incorporation of response elements for these agents into
the promoter. Generally, high level expression is obtained from inducible
promoters only in the presence of the inducing agent; basal expression
levels are minimal. Inducible expression vectors are often chosen if
expression of the protein of interest is detrimental to eukaryotic cells.
Some expression systems have markers that provide gene amplification such
as thymidine kinase and dihydrofolate reductase. Alternatively, high yield
expression systems not involving gene amplification are also suitable,
such as using a baculovirus vector in insect cells, with an Archipelin-encoding
sequence under the direction of the polyhedrin promoter or other strong
The elements that are typically included in expression vectors also
include a replicon that functions in E. coli, a gene encoding antibiotic
resistance to permit selection of bacteria that harbor recombinant
plasmids, and unique restriction sites in nonessential regions of the
plasmid to allow insertion of eukaryotic sequences. The particular
antibiotic resistance gene chosen is not critical, any of the many
resistance genes known in the art are suitable. The prokaryotic sequences
are preferably chosen such that they do not interfere with the replication
of the DNA in eukaryotic cells, if necessary.
Standard transfection methods are used to produce bacterial, mammalian,
yeast or insect cell lines that express large quantities of Archipelin
protein, which are then purified using standard techniques (see, e.g.,
Colley et al., J. Biol. Chem. 264:17619 17622 (1989); Guide to Protein
Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)).
Transformation of eukaryotic and prokaryotic cells are performed according
to standard techniques (see, e.g., Morrison, J. Bact. 132:349 351 (1977);
Clark-Curtiss & Curtiss, Methods in Enzymology 101:347 362 (Wu et al., eds,
Any of the well-known procedures for introducing foreign nucleotide
sequences into host cells may be used. These include the use of calcium
phosphate transfection, polybrene, protoplast fusion, electroporation,
liposomes, microinjection, plasma vectors, viral vectors and any of the
other well known methods for introducing cloned genomic DNA, cDNA,
synthetic DNA or other foreign genetic material into a host cell (see,
e.g., Sambrook et al., supra). It is only necessary that the particular
genetic engineering procedure used be capable of successfully introducing
at least one gene into the host cell capable of expressing Archipelin.
After the expression vector is introduced into the cells, the transfected
cells are cultured under conditions favoring expression of Archipelin,
which is recovered from the culture using standard techniques identified
III. Purification of Proteins of the Invention
Either naturally occurring or recombinant Archipelin can be purified for
use in functional assays. Naturally occurring Archipelin can be purified,
e.g., from mouse or human tissue such as islet cells or any other source
of an Archipelin ortholog. Recombinant Archipelin can be purified from any
suitable expression system.
The Archipelin may be purified to substantial purity by standard
techniques, including selective precipitation with such substances as
ammonium sulfate; column chromatography, immunopurification methods, and
others (see, e.g., Scopes, Protein Purification: Principles and Practice
(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et
A number of procedures can be employed when recombinant Archipelin are
being purified. For example, proteins having established molecular
adhesion properties can be reversible fused to Archipelin. With the
appropriate ligand, Archipelin can be selectively adsorbed to a
purification column and then freed from the column in a relatively pure
form. The fused protein is then removed by enzymatic activity. Finally
Archipelin can be purified using immunoaffinity columns.
A. Purification of Proteins from Recombinant Bacteria
When recombinant proteins are expressed by the transformed bacteria in
large amounts, typically after promoter induction, although expression can
be constitutive, the proteins may form insoluble aggregates. There are
several protocols that are suitable for purification of protein inclusion
bodies. For example, purification of aggregate proteins (hereinafter
referred to as inclusion bodies) typically involves the extraction,
separation and/or purification of inclusion bodies by disruption of
bacterial cells typically, but not limited to, by incubation in a buffer
of about 100 150 .mu.g/ml lysozyme and 0.1% Nonidet P40, a non-ionic
detergent. The cell suspension can be ground using a Polytron grinder
(Brinkman Instruments, Westbury, N.Y.). Alternatively, the cells can be
sonicated on ice. Alternate methods of lysing bacteria are described in
Ausubel et al. and Sambrook et al., both supra, and will be apparent to
those of skill in the art.
The cell suspension is generally centrifuged and the pellet containing the
inclusion bodies resuspended in buffer which does not dissolve but washes
the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM
NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to
repeat the wash step to remove as much cellular debris as possible. The
remaining pellet of inclusion bodies may be resuspended in an appropriate
buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other
appropriate buffers will be apparent to those of skill in the art.
Following the washing step, the inclusion bodies are solubilized by the
addition of a solvent that is both a strong hydrogen acceptor and a strong
hydrogen donor (or a combination of solvents each having one of these
properties). The proteins that formed the inclusion bodies may then be
renatured by dilution or dialysis with a compatible buffer. Suitable
solvents include, but are not limited to, urea (from about 4 M to about 8
M), formamide (at least about 80%, volume/volume basis), and guanidine
hydrochloride (from about 4 M to about 8 M). Some solvents that are
capable of solubilizing aggregate-forming proteins, such as SDS (sodium
dodecyl sulfate) and 70% formic acid, are inappropriate for use in this
procedure due to the possibility of irreversible denaturation of the
proteins, accompanied by a lack of immunogenicity and/or activity.
Although guanidine hydrochloride and similar agents are denaturants, this
denaturation is not irreversible and renaturation may occur upon removal
(by dialysis, for example) or dilution of the denaturant, allowing
re-formation of the immunologically and/or biologically active protein of
interest. After solubilization, the protein can be separated from other
bacterial proteins by standard separation techniques.
Alternatively, it is possible to purify proteins from bacteria periplasm.
Where the protein is exported into the periplasm of the bacteria, the
periplasmic fraction of the bacteria can be isolated by cold osmotic shock
in addition to other methods known to those of skill in the art (see,
Ausubel et al., supra). To isolate recombinant proteins from the periplasm,
the bacterial cells are centrifuged to form a pellet. The pellet is
resuspended in a buffer containing 20% sucrose. To lyse the cells, the
bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM
MgSO.sub.4 and kept in an ice bath for approximately 10 minutes. The cell
suspension is centrifuged and the supernatant decanted and saved. The
recombinant proteins present in the supernatant can be separated from the
host proteins by standard separation techniques well known to those of
skill in the art.
B. Purification of Proteins from Insect Cells
Proteins can also be purified from eukaryotic gene expression systems as
described in, e.g., Fernandez and Hoeffler, Gene Expression Systems
(1999). In some embodiments, baculovirus expression systems are used to
isolate Archipelin proteins or other proteins of the invention.
Recombinant cabulaoviruses are generally generated by replacing the
polyhedrin coding sequence of a baculovirus with a gene to be expressed
(e.g., an Archipelin polynucleotide). Viruses lacking the polyhedrin gene
have a unique plaque morphology making them easy to recognize. In some
embodiments, a recombinant baculovirus is generated by first cloning a
polynucleotide of interest into a transfer vector (e.g., a pUC based
vector) such that the polynucleotide is operably linked to a polyhedrin
promoter. The transfer vector is transfected with wildtype DNA into an
insect cell (e.g., Sf9, Sf21 or BT1-TN-5B1-4 cells), resulting in
homologous recombination and replacement of the polyhedrin gene in the
wildtype viral DNA with the polynucleotide of interest. Virus can then be
generated and plaque purified. Protein expression results upon viral
infection of insect cells. Expressed proteins can be harvested from cell
supernatant if secreted, or from cell lysates if intracellular. See, e.g.,
Ausubel et al. and Fernandez and Hoeffler, supra.
C. Standard Protein Separation Techniques for Purifying Proteins
1. Solubility Fractionation
Often as an initial step, and if the protein mixture is complex, an
initial salt fractionation can separate many of the unwanted host cell
proteins (or proteins derived from the cell culture media) from the
recombinant protein of interest. The salt used for fractionation can be,
e.g., ammonium sulfate. Ammonium sulfate precipitates proteins by
effectively reducing the amount of water in the protein mixture. Proteins
then precipitate on the basis of their solubility. The more hydrophobic a
protein is, the more likely it is to precipitate at lower ammonium sulfate
concentrations. A typical protocol is to add saturated ammonium sulfate to
a protein solution so that the resultant ammonium sulfate concentration is
between 20 30%. This will precipitate the most hydrophobic proteins. The
precipitate is discarded (unless the protein of interest is hydrophobic)
and ammonium sulfate is added to the supernatant to a concentration known
to precipitate the protein of interest. The precipitate is then
solubilized in buffer and the excess salt removed if necessary, through
either dialysis or diafiltration. Other methods that rely on solubility of
proteins, such as cold ethanol precipitation, are well known to those of
skill in the art and can be used to fractionate complex protein mixtures.
2. Size Differential Filtration
Based on a calculated molecular weight, a protein of greater and lesser
size can be isolated using ultrafiltration through membranes of different
pore sizes (for example, Amicon or Millipore membranes). As a first step,
the protein mixture is ultrafiltered through a membrane with a pore size
that has a lower molecular weight cut-off than the molecular weight of the
protein of interest. The retentate of the ultrafiltration is then
ultrafiltered against a membrane with a molecular cut off greater than the
molecular weight of the protein of interest. The recombinant protein will
pass through the membrane into the filtrate. The filtrate can then be
chromatographed as described below.
3. Column Chromatography
The proteins of interest can also be separated from other proteins on the
basis of their size, net surface charge, hydrophobicity and affinity for
ligands. In addition, antibodies raised against proteins can be conjugated
to column matrices and the proteins immunopurified. All of these methods
are well known in the art.
It will be apparent to one of skill that chromatographic techniques can be
performed at any scale and using equipment from many different
manufacturers (e.g., Pharmacia Biotech).
IV. Detection of Gene Expression
Those of skill in the art will recognize that detection of expression of
Archipelin polynucleotides has many uses. For example, as discussed
herein, detection of Archipelin levels in a patient is useful for
diagnosing diabetes or a predisposition for at least some of the
pathological effects of diabetes.
A variety of methods of specific DNA and RNA measurement using nucleic
acid hybridization techniques are known to those of skill in the art (see,
Sambrook, supra). Some methods involve an electrophoretic separation
(e.g., Southern blot for detecting DNA, and Northern blot for detecting
RNA), but measurement of DNA and RNA can also be carried out in the
absence of electrophoretic separation (e.g., by dot blot). Southern blot
of genomic DNA (e.g., from a human) can be used for screening for
restriction fragment length polymorphism (RFLP) to detect the presence of
a genetic disorder affecting an Archipelin polypeptide of the invention.
The selection of a nucleic acid hybridization format is not critical. A
variety of nucleic acid hybridization formats are known to those skilled
in the art. For example, common formats include sandwich assays and
competition or displacement assays. Hybridization techniques are generally
described in Hames and Higgins Nucleic Acid Hybridization, A Practical
Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci.
U.S.A., 63:378 383 (1969); and John et al. Nature, 223:582 587 (1969).
Detection of a hybridization complex may require the binding of a signal
generating complex to a duplex of target and probe polynucleotides or
nucleic acids. Typically, such binding occurs through ligand and anti-ligand
interactions as between a ligand-conjugated probe and an anti-ligand
conjugated with a signal. The binding of the signal generation complex is
also readily amenable to accelerations by exposure to ultrasonic energy.
The label may also allow indirect detection of the hybridization complex.
For example, where the label is a hapten or antigen, the sample can be
detected by using antibodies. In these systems, a signal is generated by
attaching fluorescent or enzyme molecules to the antibodies or in some
cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice
and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry
and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985),
pp. 9 20).
The probes are typically labeled either directly, as with isotopes,
chromophores, lumiphores, chromogens, or indirectly, such as with biotin,
to which a streptavidin complex may later bind. Thus, the detectable
labels used in the assays of the present invention can be primary labels
(where the label comprises an element that is detected directly or that
produces a directly detectable element) or secondary labels (where the
detected label binds to a primary label, e.g., as is common in
immunological labeling). Typically, labeled signal nucleic acids are used
to detect hybridization. Complementary nucleic acids or signal nucleic
acids may be labeled by any one of several methods typically used to
detect the presence of hybridized polynucleotides. The most common method
of detection is the use of autoradiography with .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P-labeled probes or the like.
Other labels include, e.g., ligands which bind to labeled antibodies,
fluorophores, chemiluminescent agents, enzymes, and antibodies which can
serve as specific binding pair members for a labeled ligand. An
introduction to labels, labeling procedures and detection of labels is
found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd
ed., Springer Verlag, N.Y. (1997); and in Haugland Handbook of Fluorescent
Probes and Research Chemicals, a combined handbook and catalogue Published
by Molecular Probes, Inc. (1996).
In general, a detector which monitors a particular probe or probe
combination is used to detect the detection reagent label. Typical
detectors include spectrophotometers, phototubes and photodiodes,
microscopes, scintillation counters, cameras, film and the like, as well
as combinations thereof. Examples of suitable detectors are widely
available from a variety of commercial sources known to persons of skill
in the art. Commonly, an optical image of a substrate comprising bound
labeling moieties is digitized for subsequent computer analysis.
Most typically, the amount of, for example, an Archipelin RNA is measured
by quantitating the amount of label fixed to the solid support by binding
of the detection reagent. Typically, the presence of a modulator during
incubation will increase or decrease the amount of label fixed to the
solid support relative to a control incubation which does not comprise the
modulator, or as compared to a baseline established for a particular
reaction type. Means of detecting and quantitating labels are well known
to those of skill in the art.
In some embodiments, the target nucleic acid or the probe is immobilized
on a solid support. Solid supports suitable for use in the assays of the
invention are known to those of skill in the art. As used herein, a solid
support is a matrix of material in a substantially fixed arrangement.
A variety of automated solid-phase assay techniques are also appropriate.
For instance, very large scale immobilized polymer arrays (VLSIPS.TM.),
available from Affymetrix, Inc. in Santa Clara, Calif. can be used to
detect changes in expression levels of a plurality of genes involved in
the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor
et al. (1991) Science, 251: 767 777; Sheldon et al. (1993) Clinical
Chemistry 39(4): 718 719, and Kozal et al. (1996) Nature Medicine 2(7):
Detection can be accomplished, for example, by using a labeled detection
moiety that binds specifically to duplex nucleic acids (e.g., an antibody
that is specific for RNA-DNA duplexes). One example uses an antibody that
recognizes DNA-RNA heteroduplexes in which the antibody is linked to an
enzyme (typically by recombinant or covalent chemical bonding). The
antibody is detected when the enzyme reacts with its substrate, producing
a detectable product. Coutlee et al. (1989) Analytical Biochemistry
181:153 162; Bogulavski (1986) et al. J. Immunol. Methods 89:123 130;
Prooijen-Knegt (1982) Exp. Cell Res. 141:397 407; Rudkin (1976) Nature
265:472 473, Stollar (1970) PNAS 65:993 1000; Ballard (1982) Mol. Immunol.
19:793 799; Pisetsky and Caster (1982) Mol. Immunol. 19:645 650; Viscidi
et al. (1988) J. Clin. Microbial. 41:199 209; and Kiney et al. (1989) J.
Clin. Microbiol. 27:6 12 describe antibodies to RNA duplexes, including
homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA
hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville,
In addition to available antibodies, one of skill in the art can easily
make antibodies specific for nucleic acid duplexes using existing
techniques, or modify those antibodies which are commercially or publicly
available. In addition to the art referenced above, general methods for
producing polyclonal and monoclonal antibodies are known to those of skill
in the art (see, e.g., Paul (ed) Fundamental Immunology, Third Edition
Raven Press, Ltd., NY (1993); Coligan Current Protocols in Immunology
Wiley/Greene, N.Y. (1991); Harlow and Lane Antibodies. A Laboratory Manual
Cold Spring Harbor Press, NY (1989); Stites et al. (eds.) Basic and
Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,
Calif., and references cited therein; Goding Monoclonal Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, N.Y., (1986);
and Kohler and Milstein Nature 256: 495 497 (1975)). Other suitable
techniques for antibody preparation include selection of libraries of
recombinant antibodies in phage or similar vectors (see, Huse et al.
Science 246:1275 1281 (1989); and Ward et al. Nature 341:544 546 (1989)).
Specific monoclonal and polyclonal antibodies and antisera will usually
bind with a K.sub.D of at least about 0.1 .mu.M, preferably at least about
0.01 .mu.M or better, and most typically and preferably, 0.001 .mu.M or
The nucleic acids used in this invention can be either positive or
negative probes. Positive probes bind to their targets and the presence of
duplex formation is evidence of the presence of the target. Negative
probes fail to bind to the suspect target and the absence of duplex
formation is evidence of the presence of the target. For example, the use
of a wild type specific nucleic acid probe or PCR primers may serve as a
negative probe in an assay sample where only the nucleotide sequence of
interest is present.
The sensitivity of the hybridization assays maybe enhanced through use of
a nucleic acid amplification system that multiplies the target nucleic
acid being detected. Examples of such systems include the polymerase chain
reaction (PCR) system and the ligase chain reaction (LCR) system. Other
methods described in the art are the nucleic acid sequence based
amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase
systems. These systems can be used to directly identify mutants where the
PCR or LCR primers are designed to be extended or ligated only when a
selected sequence is present. Alternatively, the selected sequences can be
generally amplified using, for example, nonspecific PCR primers and the
amplified target region later probed for a specific sequence indicative of
An alternative means for determining the level of expression of the
nucleic acids of the present invention is in situ hybridization. In situ
hybridization assays are well known and are generally described in Angerer
et al, Methods Enzymol. 152:649 660 (1987). In an in situ hybridization
assay, cells, preferentially human pancreatic cells such as islet cells,
are fixed to a solid support, typically a glass slide. If DNA is to be
probed, the cells are denatured with heat or alkali. The cells are then
contacted with a hybridization solution at a moderate temperature to
permit annealing of specific probes that are labeled. The probes are
preferably labeled with radioisotopes or fluorescent reporters.
V. Immunological Detection of Archipelin
In addition to the detection of Archipelin genes and gene expression using
nucleic acid hybridization technology, one can also use immunoassays to
detect Archipelin polypeptides. Immunoassays can be used to qualitatively
or quantitatively analyze Archipelin. A general overview of the applicable
technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual
A. Antibodies to Target Proteins
Methods for producing polyclonal and monoclonal antibodies that react
specifically with a protein of interest are known to those of skill in the
art (see, e.g., Coligan, supra; and Harlow and Lane, supra; Stites et al.,
supra and references cited therein; Goding, supra; and Kohler and Milstein
Nature, 256:495 497 (1975)). Such techniques include antibody preparation
by selection of antibodies from libraries of recombinant antibodies in
phage or similar vectors (see, Huse et al., supra; and Ward et al.,
supra). For example, in order to produce antisera for use in an
immunoassay, the protein of interest or an antigenic fragment thereof, is
isolated as described herein. For example, a recombinant protein is
produced in a transformed cell line. An inbred strain of mice or rabbits
is immunized with the protein using a standard adjuvant, such as Freund's
adjuvant, and a standard immunization protocol. Alternatively, a synthetic
peptide derived from the sequences disclosed herein and conjugated to a
carrier protein can be used as an immunogen.
Polyclonal sera are collected and titered against the immunogen protein in
an immunoassay, for example, a solid phase immunoassay with the immunogen
immobilized on a solid support. Polyclonal antisera with a titer of
10.sup.4 or greater are selected and tested for their crossreactivity
against non-Archipelin proteins or even other homologous proteins from
other organisms, using a competitive binding immunoassay. Specific
monoclonal and polyclonal antibodies and antisera will usually bind with a
K.sub.D of at least about 0.1 mM, more usually at least about 1 .mu.M,
preferably at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M
For preparation of antibodies, e.g., recombinant, monoclonal, or
polyclonal antibodies, many technique known in the art can be used (see,
e.g., Kohler & Milstein, Nature 256:495 497 (1975); Kozbor et al.,
Immunology Today 4: 72 (1983); Cole et al., pp. 77 96 in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current
Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory
Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice
(2d ed. 1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes encoding a
monoclonal antibody can be cloned from a hybridoma and used to produce a
recombinant monoclonal antibody. Gene libraries encoding heavy and light
chains of monoclonal antibodies can also be made from hybridoma or plasma
cells. Random combinations of the heavy and light chain gene products
generate a large pool of antibodies with different antigenic specificity
(see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the
production of single chain antibodies or recombinant antibodies (U.S. Pat.
No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic mice, or
other organisms such as other mammals, may be used to express humanized or
human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology
10:779 783 (1992); Lonberg et al., Nature 368:856 859 (1994); Morrison,
Nature 368:812 13 (1994); Fishwild et al., Nature Biotechnology 14:845 51
(1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg &
Huszar, Intern. Rev. Immunol. 13:65 93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and heteromeric Fab
fragments that specifically bind to selected antigens (see, e.g.,
McCafferty et al., Nature 348:552 554 (1990); Marks et al, Biotechnology
10:779 783 (1992)). Antibodies can also be made bispecific, i.e., able to
recognize two different antigens (see, e.g., WO 93/08829, Traunecker et
al., EMBO J. 10:3655 3659 (1991); and Suresh et al., Methods in Enzymology
121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two
covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No.
4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
Methods for humanizing or primatizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as import residues,
which are typically taken from an import variable domain. Humanization can
be essentially performed following the method of Winter and co-workers
(see, e.g., Jones et al., Nature 321:522 525 (1986); Riechmann et al.,
Nature 332:323 327 (1988); Verhoeyen et al., Science 239:1534 1536 (1988)
and Presta, Curr. Op. Struct. Biol. 2:593 596 (1992)), by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a human
antibody. Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence from a
non-human species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from analogous sites in rodent antibodies.
A number of proteins of the invention comprising immunogens may be used to
produce antibodies specifically or selectively reactive with the proteins
of interest. Recombinant protein is an exemplary immunogen for the
production of monoclonal or polyclonal antibodies. Naturally occurring
protein may also be used either in pure or impure form. Synthetic peptides
made using the protein sequences described herein may also be used as an
immunogen for the production of antibodies to the protein. Recombinant
protein can be expressed in eukaryotic or prokaryotic cells and purified
as generally described supra. The product is then injected into an animal
capable of producing antibodies. Either monoclonal or polyclonal
antibodies may be generated for subsequent use in immunoassays to measure
Methods of production of polyclonal antibodies are known to those of skill
in the art. In brief, an immunogen, preferably a purified protein, is
mixed with an adjuvant and animals are immunized. The animal's immune
response to the immunogen preparation is monitored by taking test bleeds
and determining the titer of reactivity to the Archipelin of interest.
When appropriately high titers of antibody to the immunogen are obtained,
blood is collected from the animal and antisera are prepared. Further
fractionation of the antisera to enrich for antibodies reactive to the
protein can be done if desired (see, Harlow and Lane, supra).
Monoclonal antibodies may be obtained using various techniques familiar to
those of skill in the art. Typically, spleen cells from an animal
immunized with a desired antigen are immortalized, commonly by fusion with
a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511 519
(1976)). Alternative methods of immortalization include, e.g.,
transformation with Epstein Barr Virus, oncogenes, or retroviruses, or
other methods well known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by various
techniques, including injection into the peritoneal cavity of a vertebrate
host. Alternatively, one may isolate DNA sequences which encode a
monoclonal antibody or a binding fragment thereof by screening a DNA
library from human B cells according to the general protocol outlined by
Huse et al., supra.
Once target protein specific antibodies are available, the protein can be
measured by a variety of immunoassay methods with qualitative and
quantitative results available to the clinician. For a review of
immunological and immunoassay procedures in general see, Stites, supra.
Moreover, the immunoassays of the present invention can be performed in
any of several configurations, which are reviewed extensively in Maggio
Enzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra;
and Harlow and Lane, supra.
Immunoassays to measure target proteins in a human sample may use a
polyclonal antiserum which was raised to the protein (e.g., SEQ ID NO:2,
SEQ ID NO:7 and SEQ ID NOs:9 14) at least partially encoded by a sequence
described herein (e.g., SEQ ID NO:1 and SEQ ID NO:8) or a fragment
thereof. This antiserum is selected to have low cross-reactivity against
non-Archipelin proteins and any such cross-reactivity is removed by
immunoabsorption prior to use in the immunoassay.
Polyclonal antibodies that specifically bind to an Archipelin of interest
from a particular species can be made by subtracting out cross-reactive
antibodies using Archipelin homologs. In an analogous fashion, antibodies
specific to a particular Archipelin (e.g., the human Archipelin
polypeptide) can be obtained in an organism with multiple Archipelin genes
by subtracting out cross-reactive antibodies using other Archipelin.
B. Immunological Binding Assays
In some embodiments, a protein of interest is detected and/or quantified
using any of a number of well known immunological binding assays (see,
e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For
a review of the general immunoassays, see also Asai Methods in Cell
Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY
(1993); Stites, supra. Immunological binding assays (or immunoassays)
typically utilize a "capture agent" to specifically bind to and often
immobilize the analyte (in this case an Archipelin of the present
invention or antigenic subsequences thereof). The capture agent is a
moiety that specifically binds to the analyte. In some embodiments, the
capture agent is an antibody that specifically binds, for example, an
Archipelin polypeptide of the invention. The antibody (e.g., anti-Archipelin
antibody) may be produced by any of a number of means well known to those
of skill in the art and as described above.
Immunoassays also often utilize a labeling agent to specifically bind to
and label the binding complex formed by the capture agent and the analyte.
The labeling agent may itself be one of the moieties comprising the
antibody/analyte complex. Thus, the labeling agent may be a labeled
Archipelin polypeptide or a labeled anti-Archipelin receptor antibody.
Alternatively, the labeling agent may be a third moiety, such as another
antibody, that specifically binds to the antibody/protein complex.
In some embodiments, the labeling agent is a second antibody bearing a
label. Alternatively, the second antibody may lack a label, but it may, in
turn, be bound by a labeled third antibody specific to antibodies of the
species from which the second antibody is derived. The second antibody can
be modified with a detectable moiety, such as biotin, to which a third
labeled molecule can specifically bind, such as enzyme-labeled
Other proteins capable of specifically binding immunoglobulin constant
regions, such as protein A or protein G, can also be used as the label
agents. These proteins are normal constituents of the cell walls of
streptococcal bacteria. They exhibit a strong non-immunogenic reactivity
with immunoglobulin constant regions from a variety of species (see,
generally, Kronval, et al. J. Immunol., 111:1401 1406 (1973); and
Akerstrom, et al. J. Immunol., 135:2589 2542 (1985)).
Throughout the assays, incubation and/or washing steps may be required
after each combination of reagents. Incubation steps can vary from about 5
seconds to several hours, preferably from about 5 minutes to about 24
hours. The incubation time will depend upon the assay format, analyte,
volume of solution, concentrations, and the like. Usually, the assays will
be carried out at ambient temperature, although they can be conducted over
a range of temperatures, such as 10.degree. C. to 40.degree. C.
1. Non-Competitive Assay Formats
Immunoassays for detecting proteins of interest from tissue samples may be
either competitive or noncompetitive. Noncompetitive immunoassays are
assays in which the amount of captured analyte (in this case the protein)
is directly measured. In one "sandwich" assay, for example, the capture
agent (e.g., anti-Archipelin antibodies) can be bound directly to a solid
substrate where it is immobilized. These immobilized antibodies then
capture the Archipelin present in the test sample. The Archipelin thus
immobilized is then bound by a labeling agent, such as a second anti-Archipelin
receptor antibody bearing a label. Alternatively, the second antibody may
lack a label, but it may, in turn, be bound by a labeled third antibody
specific to antibodies of the species from which the second antibody is
derived. The second can be modified with a detectable moiety, such as
biotin, to which a third labeled molecule can specifically bind, such as
2. Competitive Assay Formats
In competitive assays, the amount of target protein (analyte) present in
the sample is measured indirectly by measuring the amount of an added
(exogenous) analyte (i.e., the Archipelin of interest) displaced (or
competed away) from a capture agent (i.e., anti antibody) by the analyte
present in the sample. In one competitive assay, a known amount of, in
this case, the protein of interest is added to the sample and the sample
is then contacted with a capture agent, in this case an antibody that
specifically binds to the Archipelin of interest. The amount of Archipelin
bound to the antibody is inversely proportional to the concentration of
Archipelin present in the sample. In some embodiments, the antibody is
immobilized on a solid substrate. The amount of the Archipelin bound to
the antibody may be determined either by measuring the amount of subject
protein present in a Archipelin protein/antibody complex or,
alternatively, by measuring the amount of remaining uncomplexed protein.
The amount of Archipelin protein may be detected by providing a labeled
Archipelin protein molecule.
A hapten inhibition assay is another exemplary competitive assay. In this
assay, a known analyte, in this case the target protein, is immobilized on
a solid substrate. A known amount of anti-Archipelin antibody is added to
the sample, and the sample is then contacted with the immobilized target.
In this case, the amount of anti-Archipelin antibody bound to the
immobilized Archipelin is inversely proportional to the amount of
Archipelin protein present in the sample. Again, the amount of immobilized
antibody may be detected by detecting either the immobilized fraction of
antibody or the fraction of the antibody that remains in solution.
Detection may be direct where the antibody is labeled or indirect by the
subsequent addition of a labeled moiety that specifically binds to the
antibody as described above.
Immunoassays in the competitive binding format can be used for
cross-reactivity determinations. For example, the protein encoded by the
sequences described herein can be immobilized on a solid support. Proteins
are added to the assay which compete with the binding of the antisera to
the immobilized antigen. The ability of the above proteins to compete with
the binding of the antisera to the immobilized protein is compared to that
of the protein encoded by any of the sequences described herein. The
percent cross-reactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10% cross-reactivity
with each of the proteins listed above are selected and pooled. The
cross-reacting antibodies are optionally removed from the pooled antisera
by immunoabsorption with the considered proteins, e.g., distantly related
The immunoabsorbed and pooled antisera are then used in a competitive
binding immunoassay as described above to compare a second protein,
thought to be perhaps a protein of the present invention, to the immunogen
protein. In order to make this comparison, the two proteins are each
assayed at a wide range of concentrations and the amount of each protein
required to inhibit 50% of the binding of the antisera to the immobilized
protein is determined. If the amount of the second protein required is
less than times the amount of the protein partially encoded by a sequence
herein that is required, then the second protein is said to specifically
bind to an antibody generated to an immunogen consisting of the target
3. Other Assay Formats
In some embodiments, western blot (immunoblot) analysis is used to detect
and quantify the presence of an Archipelin of the invention in the sample.
The technique generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support (such as, e.g., a
nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and
incubating the sample with the antibodies that specifically bind the
protein of interest. For example, the anti-Archipelin antibodies
specifically bind to the Archipelin on the solid support. These antibodies
may be directly labeled or alternatively may be subsequently detected
using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that
specifically bind to the antibodies against the protein of interest.
Other assay formats include liposome immunoassays (LIA), which use
liposomes designed to bind specific molecules (e.g., antibodies) and
release encapsulated reagents or markers. The released chemicals are then
detected according to standard techniques (see, Monroe et al. (1986) Amer.
Clin. Prod. Rev. 5:34 41).
The particular label or detectable group used in the assay is not a
critical aspect of the invention, as long as it does not significantly
interfere with the specific binding of the antibody used in the assay. The
detectable group can be any material having a detectable physical or
chemical property. Such detectable labels have been well-developed in the
field of immunoassays and, in general, most labels useful in such methods
can be applied to the present invention. Thus, a label is any composition
detectable by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., Dynabeads.TM.), fluorescent dyes
(e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P),
enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others
commonly used in an ELISA), and colorimetric labels such as colloidal gold
or colored glass or plastic (e.g., polystyrene, polypropylene, latex,
The label may be coupled directly or indirectly to the desired component
of the assay according to methods well known in the art. As indicated
above, a wide variety of labels may be used, with the choice of label
depending on the sensitivity required, the ease of conjugation with the
compound, stability requirements, available instrumentation, and disposal
Non-radioactive labels are often attached by indirect means. The molecules
can also be conjugated directly to signal generating compounds, e.g., by
conjugation with an enzyme or fluorescent compound. A variety of enzymes
and fluorescent compounds can be used with the methods of the present
invention and are well-known to those of skill in the art (for a review of
various labeling or signal producing systems which may be used, see, e.g.,
U.S. Pat. No. 4,391,904).
Means of detecting labels are well known to those of skill in the art.
Thus, for example, where the label is a radioactive label, means for
detection include a scintillation counter or photographic film as in
autoradiography. Where the label is a fluorescent label, it may be
detected by exciting the fluorochrome with the appropriate wavelength of
light and detecting the resulting fluorescence. The fluorescence may be
detected visually, by means of photographic film, by the use of electronic
detectors such as charge coupled devices (CCDs) or photomultipliers and
the like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting reaction
product. Finally simple colorimetric labels may be detected directly by
observing the color associated with the label. Thus, in various dipstick
assays, conjugated gold often appears pink, while various conjugated beads
appear the color of the bead.
Some assay formats do not require the use of labeled components. For
instance, agglutination assays can be used to detect the presence of the
target antibodies. In this case, antigen-coated particles are agglutinated
by samples comprising the target antibodies. In this format, none of the
components need to be labeled and the presence of the target antibody is
detected by simple visual inspection.
VI. Screening for Modulators of Archipelin
Modulators of Archipelin, i.e. agonists or antagonists or agents of
Archipelin activity or modulators of Archipelin polypeptide or
polynucleotide expression, are useful for treating a number of human
diseases, including diabetes. Administration of Archipelin agonists or
agents that increase expression of Archipelin can be used to treat
diabetic patients. For example, insufficient Archipelin due to functional
impairment of islets may contribute to some of the pathologies associated
with diabetes. Thus, restoration of Archipelin ameliorates some of these
Conversely, under conditions of islet hyperactivity, such as occurs in an
insulin resistant states, islet expansion may lead to overproduction of
Archipelin. Overproduction leads to a different set of deleterious
physiological effects that can be relieved by Archipelin antagonists.
Archipelin agonists or antagonists may have beneficial physiological
effects in diabetes whether or not the endogenous level of the peptide is
A. Methods for identifying Modulators of Archipelin
A number of different screening protocols can be utilized to identify
agents that modulate the level of expression or activity of Archipelin in
cells, particularly mammalian cells, and especially human cells. In
general terms, the screening methods involve screening a plurality of
agents to identify an agent that modulates the activity of Archipelin by
binding to Archipelin, preventing an inhibitor from binding to Archipelin
or activating expression of Archipelin, for example.
1. Archipelin Binding Assays
Preliminary screens can be conducted by screening for compounds capable of
binding to Archipelin, as at least some of the compounds so identified are
likely Archipelin activators. The binding assays usually involve
contacting an Archipelin protein with one or more test compounds and
allowing sufficient time for the protein and test compounds to form a
binding complex. Any binding complexes formed can be detected using any of
a number of established analytical techniques. Protein binding assays
include, but are not limited to, methods that measure co-precipitation,
co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration
on Western blots (see, e.g., Bennet, J. P. and Yamamura, H. I. (1985)
"Neurotransmitter, Hormone or Drug Receptor Binding Methods," in
Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61
89) as well as phage display and other binding assays known to those of
skill in the art. The Archipelin protein utilized in such assays can be
naturally expressed, cloned or synthesized Archipelin. In some
embodiments, two hybrid assays, or other expression-based in vivo binding
assays can be used. See, e.g., Fields, et al., Nature 340(6230):245 6
Binding assays are also useful, e.g., for identifying endogenous proteins
that interact with Archipelin. For example, receptors that bind Archipelin
can be identified in binding assays.
2. Expression Assays
Certain screening methods involve screening for a compound that
up-regulates the expression of Archipelin. Such methods generally involve
conducting cell-based assays in which test compounds are contacted with
one or more cells expressing Archipelin and then detecting an increase or
decrease in Archipelin expression (either transcript or translation
product). Some assays are performed with pancreatic islet cells, or other
cells, that express endogenous Archipelin.
Archipelin expression can be detected in a number of different ways. As
described herein, the expression level of Archipelin in a cell can be
determined by probing the mRNA expressed in a cell with a probe that
specifically hybridizes with a transcript (or complementary nucleic acid
derived therefrom) of Archipelin. Probing can be conducted by lysing the
cells and conducting Northern blots or without lysing the cells using in
situ-hybridization techniques (see above). Alternatively, Archipelin
protein can be detected using immunological methods in which a cell lysate
is probe with antibodies that specifically bind to Archipelin.
Other cell-based assays are reporter assays conducted with cells that do
not express Archipelin. Certain of these assays are conducted with a
heterologous nucleic acid construct that includes a Archipelin promoter
that is operably linked to a reporter gene that encodes a detectable
product. A number of different reporter genes can be utilized. Some
reporters are inherently detectable. An example of such a reporter is
green fluorescent protein that emits fluorescence that can be detected
with a fluorescence detector. Other reporters generate a detectable
product. Often such reporters are enzymes. Exemplary enzyme reporters
include, but are not limited to, .beta.-glucuronidase, CAT (chloramphenicol
acetyl transferase; Alton and Vapnek (1979) Nature 282:864 869),
luciferase, .beta.-galactosidase and alkaline phosphatase (Toh, et al.
(1980) Eur. J. Biochem. 182:231 238; and Hall et al. (1983) J. Mol. Appl.
In these assays, cells harboring the reporter construct are contacted with
a test compound. A test compound that either modulates the activity of the
promoter by binding to it or triggers a cascade that produces a molecule
that modulates the promoter causes expression of the detectable reporter.
Certain other reporter assays are conducted with cells that harbor a
heterologous construct that includes a transcriptional control element
that activates expression of Archipelin and a reporter operably linked
thereto. Here, too, an agent that binds to the transcriptional control
element to activate expression of the reporter or that triggers the
formation of an agent that binds to the transcriptional control element to
activate reporter expression, can be identified by the generation of
signal associated with reporter expression.
The level of expression or activity can be compared to a baseline value.
As indicated above, the baseline value can be a value for a control sample
or a statistical value that is representative of Archipelin expression
levels for a control population (e.g., healthy individuals not having or
at risk for type 1 or type 2 diabetes). Expression levels can also be
determined for cells that do not express Archipelin as a negative control.
Such cells generally are otherwise substantially genetically the same as
the test cells.
A variety of different types of cells can be utilized in the reporter
assays. As stated above, certain cells are nerve cells that express an
endogenous Archipelin. Cells not expressing Archipelin can be prokaryotic,
but preferably are eukaryotic. The eukaryotic cells can be any of the
cells typically utilized in generating cells that harbor recombinant
nucleic acid constructs. Exemplary eukaryotic cells include, but are not
limited to, yeast, and various higher eukaryotic cells such as the COS,
CHO and HeLa cell lines.
Various controls can be conducted to ensure that an observed activity is
authentic including running parallel reactions with cells that lack the
reporter construct or by not contacting a cell harboring the reporter
construct with test compound. Compounds can also be further validated as
Compounds that are initially identified by any of the foregoing screening
methods can be further tested to validate the apparent activity.
Preferably such studies are conducted with suitable animal models. The
basic format of such methods involves administering a lead compound
identified during an initial screen to an animal that serves as a model
for humans and then determining if Archipelin is in fact modulated. The
animal models utilized in validation studies generally are mammals of any
kind. Specific examples of suitable animals include, but are not limited
to, primates, mice and rats.
B. Modulators of Archipelin
The compounds tested as modulators of Archipelin can be any small chemical
compound, or a biological entity, such as a protein, sugar, nucleic acid
or lipid. Alternatively, modulators can be genetically altered versions of
an Archipelin gene or gene product. Typically, test compounds will be
small chemical molecules and peptides. Essentially any chemical compound
can be used as a potential modulator or ligand in the assays of the
invention, although most often compounds that can be dissolved in aqueous
or organic (especially DMSO-based) solutions are used. The assays are
designed to screen large chemical libraries by automating the assay steps
and providing compounds from any convenient source to assays, which are
typically run in parallel (e.g., in microtiter formats on microtiter
plates in robotic assays). It will be appreciated that there are many
suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich
(St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica
Analytika (Buchs, Switzerland) and the like.
In some embodiments, high throughput screening methods involve providing a
combinatorial chemical or peptide library containing a large number of
potential therapeutic compounds (potential modulator or ligand compounds).
Such "combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify those
library members (particular chemical species or subclasses) that display a
desired characteristic activity. The compounds thus identified can serve
as conventional "lead compounds" or can themselves be used as potential or
A combinatorial chemical library is a collection of diverse chemical
compounds generated by either chemical synthesis or biological synthesis,
by combining a number of chemical "building blocks" such as reagents. For
example, a linear combinatorial chemical library such as a polypeptide
library is formed by combining a set of chemical building blocks (amino
acids) in every possible way for a given compound length (i.e., the number
of amino acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical building
Preparation and screening of combinatorial chemical libraries is well
known to those of skill in the art. Such combinatorial chemical libraries
include, but are not limited to, peptide libraries (see, e.g., U.S. Pat.
No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487 493 (1991) and
Houghton et al., Nature 354:84 88 (1991)). Other chemistries for
generating chemical diversity libraries can also be used. Such chemistries
include, but are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random
bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines
(e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins,
benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA
90:6909 6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer.
Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217 9218 (1992)),
analogous organic syntheses of small compound libraries (Chen et al., J.
Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science
261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and
Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat.
No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature
Biotechnology, 14(3):309 314 (1996) and PCT/US96/10287), carbohydrate
libraries (see, e.g., Liang et al., Science, 274:1520 1522 (1996) and U.S.
Pat. No. 5,593,853), small organic molecule libraries (see, e.g.,
benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S.
Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat.
No. 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially
available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville
Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster
City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous
combinatorial libraries are themselves commercially available (see, e.g.,
ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3D
Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
C. Solid Phase and Soluble High Throughput Assays
In the high throughput assays of the invention, it is possible to screen
up to several thousand different modulators or ligands in a single day. In
particular, each well of a microtiter plate can be used to run a separate
assay against a selected potential modulator, or, if concentration or
incubation time effects are to be observed, every 5 10 wells can test a
single modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a single
plate can easily assay from about 100 to about 1500 different compounds.
It is possible to assay several different plates per day; assay screens
for up to about 6,000 20,000 different compounds are possible using the
integrated systems of the invention. More recently, microfluidic
approaches to reagent manipulation have been developed.
The molecule of interest can be bound to the solid state component,
directly or indirectly, via covalent or non covalent linkage, e.g., via a
tag. The tag can be any of a variety of components. In general, a molecule
that binds the tag (a tag binder) is fixed to a solid support, and the
tagged molecule of interest (e.g., Archipelin) is attached to the solid
support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular
interactions well described in the literature. For example, where a tag
has a natural binder, for example, biotin, protein A, or protein G, it can
be used in conjunction with appropriate tag binders (avidin, streptavidin,
neutravidin, the Fc region of an immunoglobulin, etc.) Antibodies to
molecules with natural binders such as biotin are also widely available
and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination
with an appropriate antibody to form a tag/tag binder pair. Thousands of
specific antibodies are commercially available and many additional
antibodies are described in the literature. For example, in one common
configuration, the tag is a first antibody and the tag binder is a second
antibody which recognizes the first antibody. In addition to
antibody-antigen interactions, receptor-ligand interactions are also
appropriate as tag and tag-binder pairs, such as agonists and antagonists
of cell membrane receptors (e.g., cell receptor-ligand interactions such
as transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin receptors and
antibodies, the cadherin family, the integrin family, the selectin family,
and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book
I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g.,
opiates, steroids, etc.), intracellular receptors (e.g., which mediate the
effects of various small ligands, including steroids, thyroid hormone,
retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids
(both linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies can all interact with various cell
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,
polyureas, polyamides, polyethyleneimines, polyarylene sulfides,
polysiloxanes, polyimides, and polyacetates can also form an appropriate
tag or tag binder. Many other tag/tag binder pairs are also useful in
assay systems described herein, as would be apparent to one of skill upon
review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve
as tags, and include polypeptide sequences, such as poly Gly sequences of
between about 5 and 200 amino acids (SEQ ID NO:53). Such flexible linkers
are known to those of skill in the art. For example, poly(ethylene glycol)
linkers are available from Shearwater Polymers, Inc., Huntsville, Ala.
These linkers optionally have amide linkages, sulfhydryl linkages, or
Tag binders are fixed to solid substrates using any of a variety of
methods currently available. Solid substrates are commonly derivatized or
functionalized by exposing all or a portion of the substrate to a chemical
reagent which fixes a chemical group to the surface which is reactive with
a portion of the tag binder. For example, groups which are suitable for
attachment to a longer chain portion would include amines, hydroxyl, thiol,
and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used
to functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well described in
the literature (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149 2154
(1963) (describing solid phase synthesis of, e.g., peptides); Geysen et
al., J. Immun. Meth. 102:259 274 (1987) (describing synthesis of solid
phase components on pins); Frank and Doring, Tetrahedron 44:60316040
(1988) (describing synthesis of various peptide sequences on cellulose
disks); Fodor et al., Science, 251:767 777 (1991); Sheldon et al.,
Clinical Chemistry 39(4):718 719 (1993); and Kozal et al., Nature Medicine
2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to substrates
include other common methods, such as heat, cross-linking by UV radiation,
and the like.
The invention provides in vitro assays for identifying, in a high
throughput format, compounds that can modulate the expression or activity
of Archipelin. Control reactions that measure Archipelin activity of the
cell in a reaction that does not include a potential modulator are
optional, as the assays are highly uniform. Such optional control
reactions are appropriate and increase the reliability of the assay.
Accordingly, in some embodiments, the methods of the invention include
such a control reaction. For each of the assay formats described, "no
modulator" control reactions which do not include a modulator provide a
background level of binding activity.
In some assays it will be desirable to have positive controls to ensure
that the components of the assays are working properly. At least two types
of positive controls are appropriate. First, a known activator of
Archipelin of the invention can be incubated with one sample of the assay,
and the resulting increase in signal resulting from an increased
expression level or activity of Archipelin determined according to the
methods herein. Second, a known inhibitor of Archipelin can be added, and
the resulting decrease in signal for the expression or activity of
Archipelin can be similarly detected. It will be appreciated that
modulators can also be combined with activators or inhibitors to find
modulators which inhibit the increase or decrease that is otherwise caused
by the presence of the known modulator of Archipelin.
D. Computer-Based Assays
Yet another assay for compounds that modulate the activity of Archipelin
involves computer assisted drug design, in which a computer system is used
to generate a three-dimensional structure of Archipelin based on the
structural information encoded by its amino acid sequence. The input amino
acid sequence interacts directly and actively with a pre-established
algorithm in a computer program to yield secondary, tertiary, and
quaternary structural models of the protein. Similar analyses can be
performed on potential receptors of Archipelin. The models of the protein
structure are then examined to identify regions of the structure that have
the ability to bind, e.g., Archipelin. These regions are then used to
identify polypeptides that bind to Archipelin.
The three-dimensional structural model of the protein is generated by
entering protein amino acid sequences of at least 10 amino acid residues
or corresponding nucleic acid sequences encoding a potential Archipelin
receptor into the computer system. The amino acid sequences encoded by the
nucleic acid sequences provided herein represent the primary sequences or
subsequences of the proteins, which encode the structural information of
the proteins. At least 10 residues of an amino acid sequence (or a
nucleotide sequence encoding 10 amino acids) are entered into the computer
system from computer keyboards, computer readable substrates that include,
but are not limited to, electronic storage media (e.g., magnetic
diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM),
information distributed by internet sites, and by RAM. The
three-dimensional structural model of the protein is then generated by the
interaction of the amino acid sequence and the computer system, using
software known to those of skill in the art.
The amino acid sequence represents a primary structure that encodes the
information necessary to form the secondary, tertiary and quaternary
structure of the protein of interest. The software looks at certain
parameters encoded by the primary sequence to generate the structural
model. These parameters are referred to as "energy terms," and primarily
include electrostatic potentials, hydrophobic potentials, solvent
accessible surfaces, and hydrogen bonding. Secondary energy terms include
van der Waals potentials. Biological molecules form the structures that
minimize the energy terms in a cumulative fashion. The computer program is
therefore using these terms encoded by the primary structure or amino acid
sequence to create the secondary structural model.
The tertiary structure of the protein encoded by the secondary structure
is then formed on the basis of the energy terms of the secondary
structure. The user at this point can enter additional variables such as
whether the protein is membrane bound or soluble, its location in the
body, and its cellular location, e.g., cytoplasmic, surface, or nuclear.
These variables along with the energy terms of the secondary structure are
used to form the model of the tertiary structure. In modeling the tertiary
structure, the computer program matches hydrophobic faces of secondary
structure with like, and hydrophilic faces of secondary structure with
Once the structure has been generated, potential ligand binding regions
are identified by the computer system. Three-dimensional structures for
potential ligands are generated by entering amino acid or nucleotide
sequences or chemical formulas of compounds, as described above. The
three-dimensional structure of the potential ligand is then compared to
that of Archipelin to identify binding sites of Archipelin. Binding
affinity between the protein and ligands is determined using energy terms
to determine which ligands have an enhanced probability of binding to the
Computer systems are also used to screen for mutations, polymorphic
variants, alleles and interspecies homologs of genes encoding an
Archipelin polypeptide of the invention. Such mutations can be associated
with disease states or genetic traits. As described above, GeneChip.TM.
and related technology can also be used to screen for mutations,
polymorphic variants, alleles and interspecies homologs. Once the variants
are identified, diagnostic assays can be used to identify patients having
such mutated genes. Identification of the mutated Archipelin genes
involves receiving input of a first amino acid sequence of a Archipelin
(or of a first nucleic acid sequence encoding a Archipelin of the
invention), e.g., any amino acid sequence having at least 60%, optionally
at least 85%, identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, and SEQ ID NO:8, or conservatively modified versions
thereof. The sequence is entered into the computer system as described
above. The first nucleic acid or amino acid sequence is then compared to a
second nucleic acid or amino acid sequence that has substantial identity
to the first sequence. The second sequence is entered into the computer
system in the manner described above. Once the first and second sequences
are compared, nucleotide or amino acid differences between the sequences
are identified. Such sequences can represent allelic differences in
various Archipelin genes, and mutations associated with disease states and
VII. Compositions, Kits and Integrated Systems
The invention provides compositions, kits and integrated systems for
practicing the assays described herein using nucleic acids encoding the
Archipelin polypeptides of the invention, or Archipelin proteins, anti-Archipelin
The invention provides assay compositions for use in solid phase assays;
such compositions can include, for example, one or more nucleic acids
encoding an Archipelin immobilized on a solid support, and a labeling
reagent. In each case, the assay compositions can also include additional
reagents that are desirable for hybridization. Modulators of expression or
activity of an Archipelin of the invention can also be included in the
assay compositions. Solid supports include, e.g., petri plates, microtiter
dishes and microarrays.
The invention also provides kits for carrying out the assays of the
invention. The kits typically include a probe which comprises an antibody
that specifically binds to Archipelin or a polynucleotide sequence
encoding an Archipelin polypeptide, and a label for detecting the presence
of the probe. The kits may include several polynucleotide sequences
encoding Archipelin polypeptides of the invention. Kits can include any of
the compositions noted above, and optionally further include additional
components such as instructions to practice a high-throughput method of
assaying for an effect on expression of the genes encoding the Archipelin
polypeptides of the invention, or on activity of the Archipelin
polypeptides of the invention, one or more containers or compartments
(e.g., to hold the probe, labels, or the like), a control modulator of the
expression or activity of Archipelin polypeptides, a robotic armature for
mixing kit components or the like.
The invention also provides integrated systems for high-throughput
screening of potential modulators for an effect on the expression or
activity of the Archipelin polypeptides of the invention. The systems
typically include a robotic armature which transfers fluid from a source
to a destination, a controller which controls the robotic armature, a
label detector, a data storage unit which records label detection, and an
assay component such as a microtiter dish comprising a well having a
reaction mixture or a substrate comprising a fixed nucleic acid or
A number of robotic fluid transfer systems are available, or can easily be
made from existing components. For example, a Zymate XP (Zymark
Corporation; Hopkinton, Mass.) automated robot using a Microlab 2200
(Hamilton; Reno, Nev.) pipetting station can be used to transfer parallel
samples to 96 well microtiter plates to set up several parallel
simultaneous binding assays.
Optical images viewed (and, optionally, recorded) by a camera or other
recording device (e.g., a photodiode and data storage device) are
optionally further processed in any of the embodiments herein, e.g., by
digitizing the image and storing and analyzing the image on a computer. A
variety of commercially available peripheral equipment and software is
available for digitizing, storing and analyzing a digitized video or
digitized optical image, e.g., using PC (Intel x86 or Pentium
chip-compatible DOS.RTM., OS2.RTM. WINDOWS.RTM., WINDOWS NT.RTM. or
WINDOWS95.RTM. based computers), MACINTOSH.RTM., or UNIX.RTM. based (e.g.,
SUN.RTM. work station) computers.
One conventional system carries light from the specimen field to a cooled
charge-coupled device (CCD) camera, in common use in the art. A CCD camera
includes an array of picture elements (pixels). The light from the
specimen is imaged on the CCD. Particular pixels corresponding to regions
of the specimen (e.g., individual hybridization sites on an array of
biological polymers) are sampled to obtain light intensity readings for
each position. Multiple pixels are processed in parallel to increase
speed. The apparatus and methods of the invention are easily used for
viewing any sample, e.g., by fluorescent or dark field microscopic
VIII. Gene Therapy Applications
A variety of human diseases can be treated by therapeutic approaches that
involve stably introducing a gene into a human cell such that the gene is
transcribed and the gene product is produced in the cell. Diseases
amenable to treatment by this approach include inherited diseases,
including those in which the defect is in a single gene. Gene therapy is
also useful for treatment of acquired diseases and other conditions. For
discussions on the application of gene therapy towards the treatment of
genetic as well as acquired diseases, see, Miller Nature 357:455 460
(1992); and Mulligan Science 260:926 932 (1993).
In the context of the present invention, gene therapy can be used for
treating a variety of disorders and/or diseases in which Archipelin has
been implicated. For example, introduction by gene therapy of
polynucleotides encoding an Archipelin polypeptide of the invention can be
used to treat, e.g., diabetes.
A. Vectors for Gene Delivery
For delivery to a cell or organism, the nucleic acids of the invention can
be incorporated into a vector. Examples of vectors used for such purposes
include expression plasmids capable of directing the expression of the
nucleic acids in the target cell. In other instances, the vector is a
viral vector system wherein the nucleic acids are incorporated into a
viral genome that is capable of transfecting the target cell. In
embodiments, the nucleic acids can be operably linked to expression and
control sequences that can direct expression of the gene in the desired
target host cells. Thus, one can achieve expression of the nucleic acid
under appropriate conditions in the target cell.
B. Gene Delivery Systems
Viral vector systems useful in the expression of the nucleic acids
include, for example, naturally occurring or recombinant viral vector
systems. Depending upon the particular application, suitable viral vectors
include replication competent, replication deficient, and conditionally
replicating viral vectors. For example, viral vectors can be derived from
the genome of human or bovine adenoviruses, vaccinia virus, herpes virus,
adeno-associated virus, minute virus of mice (MVM), HIV, sindbis virus,
and retroviruses (including but not limited to Rous sarcoma virus), and
MoMLV. Typically, the genes of interest are inserted into such vectors to
allow packaging of the gene construct, typically with accompanying viral
DNA, followed by infection of a sensitive host cell and expression of the
gene of interest.
As used herein, "gene delivery system" refers to any means for the
delivery of a nucleic acid of the invention to a target cell. In some
embodiments of the invention, nucleic acids are conjugated to a cell
receptor ligand for facilitated uptake (e.g., invagination of coated pits
and internalization of the endosome) through an appropriate linking
moiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem. 263:14621
14624 (1988); WO 92/06180). For example, nucleic acids can be linked
through a polylysine moiety to asialo-oromucocid, which is a ligand for
the asialoglycoprotein receptor of hepatocytes.
Similarly, viral envelopes used for packaging gene constructs that include
the nucleic acids of the invention can be modified by the addition of
receptor ligands or antibodies specific for a receptor to permit
receptor-mediated endocytosis into specific cells (see, e.g., WO 93/20221,
WO 93/14188, and WO 94/06923). In some embodiments of the invention, the
DNA constructs of the invention are linked to viral proteins, such as
adenovirus particles, to facilitate endocytosis (Curiel et al., Proc.
Natl. Acad. Sci. U.S.A. 88:8850 8854 (1991)). In other embodiments,
molecular conjugates of the instant invention can include microtubule
inhibitors (WO/9406922), synthetic peptides mimicking influenza virus
hemagglutinin (Plank et al., J. Biol. Chem. 269:12918 12924 (1994)), and
nuclear localization signals such as SV40 T antigen (WO93/19768).
Retroviral vectors are also useful for introducing the nucleic acids of
the invention into target cells or organisms. Retroviral vectors are
produced by genetically manipulating retroviruses. The viral genome of
retroviruses is RNA. Upon infection, this genomic RNA is reverse
transcribed into a DNA copy which is integrated into the chromosomal DNA
of transduced cells with a high degree of stability and efficiency. The
integrated DNA copy is referred to as a provirus and is inherited by
daughter cells as is any other gene. The wild type retroviral genome and
the proviral DNA have three genes: the gag, the pol and the env genes,
which are flanked by two long terminal repeat (LTR) sequences. The gag
gene encodes the internal structural (nucleocapsid) proteins; the pol gene
encodes the RNA directed DNA polymerase (reverse transcriptase); and the
env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs serve to
promote transcription and polyadenylation of virion RNAs. Adjacent to the
5' LTR are sequences necessary for reverse transcription of the genome
(the tRNA primer binding site) and for efficient encapsulation of viral
RNA into particles (the Psi site) (see, Mulligan, In: Experimental
Manipulation of Gene Expression, Inouye (ed), 155 173 (1983); Mann et al.,
Cell 33:153 159 (1983); Cone and Mulligan, Proceedings of the National
Academy of Sciences, U.S.A., 81:6349 6353 (1984)).
The design of retroviral vectors is well known to those of ordinary skill
in the art. In brief, if the sequences necessary for encapsidation (or
packaging of retroviral RNA into infectious virions) are missing from the
viral genome, the result is a cis acting defect which prevents
encapsidation of genomic RNA. However, the resulting mutant is still
capable of directing the synthesis of all virion proteins. Retroviral
genomes from which these sequences have been deleted, as well as cell
lines containing the mutant genome stably integrated into the chromosome
are well known in the art and are used to construct retroviral vectors.
Preparation of retroviral vectors and their uses are described in many
publications including, e.g., European Patent Application EPA 0 178 220;
U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504 512 (1986); Mann et
al., Cell 33:153 159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA
81:6349 6353 (1984); Eglitis et al. Biotechniques 6:608 614 (1988); Miller
et al. Biotechniques 7:981 990 (1989); Miller (1992) supra; Mulligan
(1993), supra; and WO 92/07943.
The retroviral vector particles are prepared by recombinantly inserting
the desired nucleotide sequence into a retrovirus vector and packaging the
vector with retroviral capsid proteins by use of a packaging cell line.
The resultant retroviral vector particle is incapable of replication in
the host cell but is capable of integrating into the host cell genome as a
proviral sequence containing the desired nucleotide sequence. As a result,
the patient is capable of producing, for example, an Archipelin
polypeptide of interest and thus restore the cells to a normal phenotype.
Packaging cell lines that are used to prepare the retroviral vector
particles are typically recombinant mammalian tissue culture cell lines
that produce the necessary viral structural proteins required for
packaging, but which are incapable of producing infectious virions. The
defective retroviral vectors that are used, on the other hand, lack these
structural genes but encode the remaining proteins necessary for
packaging. To prepare a packaging cell line, one can construct an
infectious clone of a desired retrovirus in which the packaging site has
been deleted. Cells comprising this construct will express all structural
viral proteins, but the introduced DNA will be incapable of being
packaged. Alternatively, packaging cell lines can be produced by
transforming a cell line with one or more expression plasmids encoding the
appropriate core and envelope proteins. In these cells, the gag, pol, and
env genes can be derived from the same or different retroviruses.
A number of packaging cell lines suitable for the present invention are
also available in the prior art. Examples of these cell lines include Crip,
GPE86, PA317 and PG13 (see Miller et al., J. Virol. 65:2220 2224 (1991)).
Examples of other packaging cell lines are described in Cone and Mulligan
Proceedings of the National Academy of Sciences, USA, 81:6349 6353 (1984);
Danos and Mulligan Proceedings of the National Academy of Sciences, USA,
85:6460 6464 (1988); Eglitis et al. (1988), supra; and Miller (1990),
Packaging cell lines capable of producing retroviral vector particles with
chimeric envelope proteins may be used. Alternatively, amphotropic or
xenotropic envelope proteins, such as those produced by PA317 and GPX
packaging cell lines may be used to package the retroviral vectors.
In some embodiments of the invention, an antisense nucleic acid is
administered which hybridizes to a gene encoding an Archipelin of the
invention or to a transcript thereof. The antisense nucleic acid can be
provided as an antisense oligonucleotide (see, e.g., Murayama et al.,
Antisense Nucleic Acid Drug Dev. 7:109 114 (1997)). Genes encoding an
antisense nucleic acid can also be provided; such genes can be introduced
into cells by methods known to those of skill in the art. For example, one
can introduce a gene that encodes an antisense nucleic acid in a viral
vector, such as, for example, in hepatitis B virus (see, e.g., Ji et al.,
J. Viral Hepat. 4:167 173 (1997)), in adeno-associated virus (see, e.g.,
Xiao et al., Brain Res. 756:76 83 (1997)), or in other systems including,
but not limited, to an HVJ (Sendai virus)-liposome gene delivery system
(see, e.g., Kaneda et al., Ann. NY Acad. Sci. 811:299 308 (1997)), a
"peptide vector" (see, e.g., Vidal et al., CR Acad. Sci III 32:279 287
(1997)), as a gene in an episomal or plasmid vector (see, e.g., Cooper et
al., Proc. Natl. Acad. Sci. U.S.A. 94:6450 6455 (1997), Yew et al. Hum
Gene Ther. 8:575 584 (1997)), as a gene in a peptide-DNA aggregate (see,
e.g., Niidome et al., J. Biol. Chem. 272:15307 15312 (1997)), as "naked
DNA" (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466), in lipidic
vector systems (see, e.g., Lee et al., Crit Rev Ther Drug Carrier Syst.
14:173 206 (1997)), polymer coated liposomes (U.S. Pat. Nos. 5,213,804 and
5,013,556), cationic liposomes (Epand et al., U.S. Pat. Nos. 5,283,185;
5,578,475; 5,279,833; and 5,334,761), gas filled microspheres (U.S. Pat.
No. 5,542,935), ligand-targeted encapsulated macromolecules (U.S. Pat.
Nos. 5,108,921; 5,521,291; 5,554,386; and 5,166,320).
C. Pharmaceutical Formulations
When used for pharmaceutical purposes, the vectors used for gene therapy
are formulated in a suitable buffer, which can be any pharmaceutically
acceptable buffer, such as phosphate buffered saline or sodium
phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and
other buffers known to the ordinarily skilled artisan such as those
described by Good et al. Biochemistry 5:467 (1966).
The compositions can additionally include a stabilizer, enhancer or other
pharmaceutically acceptable carriers or vehicles. A pharmaceutically
acceptable carrier can contain a physiologically acceptable compound that
acts, for example, to stabilize the nucleic acids of the invention and any
associated vector. A physiologically acceptable compound can include, for
example, carbohydrates, such as glucose, sucrose or dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins or other stabilizers or excipients. Other
physiologically acceptable compounds include wetting agents, emulsifying
agents, dispersing agents or preservatives, which are particularly useful
for preventing the growth or action of microorganisms. Various
preservatives are well known and include, for example, phenol and ascorbic
acid. Examples of carriers, stabilizers or adjuvants can be found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Philadelphia, Pa., 17th ed. (1985).
D. Administration of Formulations
The formulations of the invention can be delivered to any tissue or organ
using any delivery method known to the ordinarily skilled artisan. In some
embodiments of the invention, the nucleic acids of the invention are
formulated in mucosal, topical, and/or buccal formulations, particularly
mucoadhesive gel and topical gel formulations. Exemplary permeation
enhancing compositions, polymer matrices, and mucoadhesive gel
preparations for transdermal delivery are disclosed in U.S. Pat. No.
E. Methods of Treatment
The gene therapy formulations of the invention are typically administered
to a cell. The cell can be provided as part of a tissue, such as an
epithelial membrane, or as an isolated cell, such as in tissue culture.
The cell can be provided in vivo, ex vivo, or in vitro.
The formulations can be introduced into the tissue of interest in vivo or
ex vivo by a variety of methods. In some embodiments of the invention, the
nucleic acids of the invention are introduced into cells by such methods
as microinjection, calcium phosphate precipitation, liposome fusion, or
biolistics. In further embodiments, the nucleic acids are taken up
directly by the tissue of interest.
In some embodiments of the invention, the nucleic acids of the invention
are administered ex vivo to cells or tissues explanted from a patient,
then returned to the patient. Examples of ex vivo administration of
therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci.
USA 93(6):2414 9 (1996); Koc et al., Seminars in Oncology 23 (1):46 65
(1996); Raper et al., Annals of Surgery 223(2):116 26 (1996); Dalesandro
et al., J. Thorac. Cardi. Surg., 11(2):416 22 (1996); and Makarov et al.,
Proc. Natl. Acad. Sci. USA 93(1):402 6 (1996).
IX. Administration and Pharmaceutical Compositions
Modulators of Archipelin (e.g., agonists, including Archipelin
polypeptides, and antagonists) can be administered directly to the
mammalian subject for modulation of Archipelin signaling in vivo.
Administration is by any of the routes normally used for introducing a
modulator compound into ultimate contact with the tissue to be treated and
well known to those of skill in the art. Although more than one route can
be used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than another
The compounds of the present invention can also be used effectively in
combination with one or more additional active agents depending on the
desired target therapy (see, e.g., Turner, N. et al. Prog. Drug Res.
(1998) 51: 33 94; Haffner, S. Diabetes Care (1998) 21: 160 178; and
DeFronzo, R. et al. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4). A
number of studies have investigated the benefits of combination therapies
with oral agents (see, e.g., Mahler, R., J. Clin. Endocrinol. Metab.
(1999) 84: 1165 71; United Kingdom Prospective Diabetes Study Group: UKPDS
28, Diabetes Care (1998) 21: 87 92; Bardin, C. W., (ed.), Current Therapy
In Endocrinology And Metabolism, 6th Edition (Mosby--Year Book, Inc., St.
Louis, Mo. 1997); Chiasson, J. et al., Ann. Intern. Med. (1994) 121: 928
935; Coniff, R. et al., Clin. Ther. (1997) 19: 16 26; Coniff, R. et al.,
Am. J. Med. (1995) 98: 443 451; and Iwamoto, Y. et al., Diabet. Med.
(1996) 13 365 370; Kwiterovich, P. Am. J. Cardiol (1998) 82(12A): 3U 17U).
These studies indicate that modulation of diabetes and hyperlipidemia,
among other diseases, can be further improved by the addition of a second
agent to the therapeutic regimen. Combination therapy includes
administration of a single pharmaceutical dosage formulation which
contains an Archipelin modulator of the invention and one or more
additional active agents, as well as administration of an Archipelin
modulator and each active agent in its own separate pharmaceutical dosage
formulation. For example, an Archipelin modulator and a thiazolidinedione
can be administered to the human subject together in a single oral dosage
composition, such as a tablet or capsule, or each agent can be
administered in separate oral dosage formulations. Where separate dosage
formulations are used, an Archipelin modulator and one or more additional
active agents can be administered at essentially the same time (i.e.,
concurrently), or at separately staggered times (i.e., sequentially).
Combination therapy is understood to include all these regimens.
Still another example of combination therapy can be seen in modulating
diabetes (or treating diabetes and its related symptoms, complications,
and disorders), wherein the AKRIC modulators can be effectively used in
combination with, for example, sulfonylureas (such as chlorpropamide,
tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase,
glimepiride, and glipizide), biguanides (such as metformin), a PPAR beta
delta agonist, a ligand or agonist of PPAR alpha such as
thiazolidinediones (such as ciglitazone, pioglitazone (see, e.g., U.S.
Pat. No. 6,218,409), troglitazone, and rosiglitazone (see, e.g., U.S. Pat.
No. 5,859,037)); dehydroepiandrosterone (also referred to as DHEA or its
conjugated sulphate ester, DHEA-SO4); antiglucocorticoids; TNF.alpha.
inhibitors; .alpha.-glucosidase inhibitors (such as acarbose, miglitol,
and voglibose), amylin and amylin derivatives (such as pramlintide, (see,
also, U.S. Pat. Nos. 5,902,726; 5,124,314; 5,175,145 and 6,143,718,
6,136,784)), insulin secretogogues (such as repaglinide, gliquidone, and
nateglinide (see, also, U.S. Pat. Nos. 6,251,856; 6,251,865; 6,221,633;
6,174,856)), insulin, as well as the active agents discussed above for
The pharmaceutical compositions of the invention may comprise a
pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers
are determined in part by the particular composition being administered,
as well as by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions of the present invention (see, e.g.,
Remington's Pharmaceutical Sciences, 17.sup.th ed. 1985)).
The modulators (e.g., agonists or antagonists) of the expression or
activity of the Archipelin, alone or in combination with other suitable
components, can be prepared for injection or for use in a pump device.
Pump devices (also known as "insulin pumps") are commonly used to
administer insulin to patients and therefore can be easily adapted to
include compositions of the present invention. Manufacturers of insulin
pumps include Animas, Disetronic and .MiniMed.
The modulators (e.g., agonists or antagonists) of the expression or
activity of the Archipelin, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can be "nebulized")
to be administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable propellants, such as dichlorodifluoromethane,
propane, nitrogen, and the like.
Formulations suitable for administration include aqueous and non-aqueous
solutions, isotonic sterile solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation isotonic,
and aqueous and non-aqueous sterile suspensions that can include
suspending agents, solubilizers, thickening agents, stabilizers, and
preservatives. In the practice of this invention, compositions can be
administered, for example, orally, nasally, topically, intravenously,
intraperitoneally, or intrathecally. The formulations of compounds can be
presented in unit-dose or multi-dose sealed containers, such as ampoules
and vials. Solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described. The modulators can
also be administered as part a of prepared food or drug.
The dose administered to a patient, in the context of the present
invention should be sufficient to effect a beneficial response in the
subject over time. The optimal dose level for any patient will depend on a
variety of factors including the efficacy of the specific modulator
employed, the age, body weight, physical activity, and diet of the
patient, on a possible combination with other drugs, and on the severity
of the case of diabetes. It is recommended that the daily dosage of the
modulator be determined for each individual patient by those skilled in
the art in a similar way as for known insulin compositions. The size of
the dose also will be determined by the existence, nature, and extent of
any adverse side-effects that accompany the administration of a particular
compound or vector in a particular subject.
In determining the effective amount of the modulator to be administered a
physician may evaluate circulating plasma levels of the modulator,
modulator toxicity, and the production of anti-modulator antibodies. In
general, the dose equivalent of a modulator is from about 1 ng/kg to 10
mg/kg for a typical subject.
For administration, Archipelin modulators of the present invention can be
administered at a rate determined by the LD-50 of the modulator, and the
side-effects of the inhibitor at various concentrations, as applied to the
mass and overall health of the subject. Administration can be accomplished
via single or divided doses.
X. Diagnosis of Diabetes
The present invention also provides methods of diagnosing diabetes or a
predisposition of at least some of the pathologies of diabetes. Diagnosis
involves determining the level of Archipelin in a patient and then
comparing the level to a baseline or range. Typically, the baseline value
is representative of Archipelin in a healthy (i.e., non-diabetic) person.
As discussed above, variation of levels (either high or low) of Archipelin
from the baseline range suggests that the patient is either diabetic or at
risk of developing at least some of the pathologies of diabetes. Variation
van be, e.g., at least 5%, 10%, 20%, 50%, 200%, 400%, 500%, or 1000% or
more of a baseline value or range. In some embodiments, the level of
Archipelin are measured by taking a blood sample from a patient and
measuring the amount of Archipelin in the sample using any number of
detection methods, such as those discussed herein. For instance, fasting
and fed blood or urine levels can be tested.
Glucose tolerance tests can also be used to detect the effect of glucose
levels on Archipelin levels. In glucose tolerance tests, the patient's
ability to tolerate a standard oral glucose load is evaluated by assessing
serum and urine specimens for glucose levels. Blood samples are taken
before the glucose is ingested, glucose is given by mouth, and blood or
urine glucose levels are tested at set intervals after glucose ingestion.
Claim 1 of 10 Claims
1. A method of determining
islet cell-specific activity in an individual, the method comprising,
detecting the level of a polypeptide comprising SEQ ID NO:9 in a sample
wherein the said sample comprises human blood from the individual, and
determining islet cell-specific activity in the individual by comparing the
level of the polypeptide in the sample to a baseline value or range
associated with a known islet cell activity wherein said islet cell activity
is associated with the ability of the islet cell to produce said
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