U.S. patent application number 13/774097 was filed with the patent office on 2014-07-31 for use of pleitrophin for preventing and treating pancreatic diseases and/or obesity and/or metabolic syndrome.
The applicant listed for this patent is EVOTEC INTERNATIONAL GMBH. Invention is credited to Ulrike BURK, Daria ONICHTCHOUK.
Application Number | 20140213510 13/774097 |
Document ID | / |
Family ID | 34130024 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140213510 |
Kind Code |
A1 |
ONICHTCHOUK; Daria ; et
al. |
July 31, 2014 |
USE OF PLEITROPHIN FOR PREVENTING AND TREATING PANCREATIC DISEASES
AND/OR OBESITY AND/OR METABOLIC SYNDROME
Abstract
The present invention discloses the protein pleitrophin secreted
by the developing pancreas, and polynucleotides, which identify and
encode this protein. The invention also relates to the use of these
sequences in the diagnosis, study, prevention, and treatment of
pancreatic diseases (e.g. diabetes), obesity, and/or metabolic
syndrome.
Inventors: |
ONICHTCHOUK; Daria;
(Goettingen, DE) ; BURK; Ulrike; (Goettingen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EVOTEC INTERNATIONAL GMBH |
Hamburg |
|
DE |
|
|
Family ID: |
34130024 |
Appl. No.: |
13/774097 |
Filed: |
February 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10564769 |
Jun 12, 2006 |
8501473 |
|
|
PCT/EP04/07917 |
Jul 15, 2004 |
|
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13774097 |
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Current U.S.
Class: |
514/4.8 ;
514/6.7; 514/6.8; 514/6.9 |
Current CPC
Class: |
A61K 38/18 20130101;
C12N 15/8509 20130101; A61P 3/10 20180101; C07K 14/475 20130101;
C12N 2506/02 20130101; G01N 2500/00 20130101; G01N 2333/475
20130101; A01K 67/0271 20130101; A61P 3/00 20180101; A01K 2267/0362
20130101; A61P 3/04 20180101 |
Class at
Publication: |
514/4.8 ;
514/6.8; 514/6.9; 514/6.7 |
International
Class: |
C07K 14/475 20060101
C07K014/475 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2003 |
EP |
03016169.9 |
Claims
1.-39. (canceled)
40. A method for lowering blood glucose level in a subject in need
thereof, comprising administering to said subject a medicament
comprising an acceptable carrier and a human pleiotrophin
polypeptide or a functional fragment thereof.
41. The method according to claim 40, wherein said human
pleiotrophin polypeptide is (a) a polypeptide comprising the
sequence set forth in SEQ ID NO: 2, or an isoform, fragment or
variant of the polypeptide of SEQ ID NO: 2; (b) a polypeptide
encoded by the polynucleotide comprising the sequence set forth in
SEQ ID NO: 1; (c) a polypeptide which is encoded by a
polynucleotide which, as a result of the genetic code, is
degenerate with the polynucleotide sequence of (b); (d) a
polypeptide which is encoded by a polynucleotide which hybridizes
at 50.degree. C. in a solution containing 1.times.SSC and 0.1% SDS
to the polynucleotide sequence of (b) or (c); (e) a polypeptide
which is at least 85% identical to the polypeptide of (a); (f) a
polypeptide that differs from the polypeptide of (a) to (e) by a
mutation in the polynucleotide sequence encoding said polypeptide,
wherein said mutation causes an alteration, deletion, duplication
or premature stop in the encoded polypeptide.
42. The method according to claim 40, comprising administering a
human pleiotrophin polypeptide comprising the sequence set forth in
SEQ ID NO: 2.
43. The method according to claim 40, wherein said subject is
suffering from diabetes.
44. The method according to claim 43, wherein the diabetes is
insulin dependent diabetes mellitus or non-insulin dependent
diabetes mellitus.
45. The method according to claim 40, wherein said human
pleiotrophin polypeptide is (a) a polypeptide comprising the
sequence set forth in SEQ ID NO: 2 or a functional fragment
thereof; (b) a polypeptide encoded by the polynucleotide comprising
the sequence set forth in SEQ ID NO: 1; (c) a polypeptide which is
encoded by a polynucleotide which, as a result of the genetic code,
is degenerate with the polynucleotide sequence of (b); or (d) a
polypeptide which is at least 95% identical to the polypeptide of
(a).
46. The method according to claim 40, wherein said human
pleiotrophin polypeptide is a polypeptide comprising the sequence
set forth in SEQ ID NO: 2 or a functional fragment comprising 6 to
50 contiguous amino acids of said SEQ ID NO: 2.
47. A method for increasing insulin production by pancreatic .beta.
cells or generating functional pancreatic cells that express
glucagon in a subject, comprising administering to said subject a
medicament comprising an acceptable carrier and a human
pleiotrophin polypeptide or a functional fragment thereof.
48. The method according to claim 47, wherein said insulin
production or said glucagon expression is increased in said subject
in presence of glucose.
49. The method according to claim 47, wherein said insulin
production is increased by a factor of two in said subject in the
presence of glucose when compared to the insulin production in said
subject in the absence of glucose.
50. The method according to claim 47, wherein said insulin
production is increased by a factor of three in said subject in the
presence of glucose when compared to the insulin production in said
subject in the absence of glucose.
51. A method for regulation of body weight in a subject in need
thereof, comprising administering to said subject a medicament
comprising an acceptable carrier and a human pleiotrophin
polypeptide or a functional fragment thereof.
52. A method according to claim 52, comprising regulating body
weight in a subject suffering from obesity.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/564,769 filed on Jun. 12, 2006, which is the national phase
of PCT/EP04/07917 filed on Jul. 15, 2004, the disclosures in which
are incorporated by reference herein in their entirety.
[0002] This invention relates to the use of low molecular weight
DG001 proteins, to the use of polynucleotides encoding these, and
to the use of effectors/modulators thereof in the diagnosis, study,
prevention, and treatment of pancreatic diseases (e.g. diabetes
mellitus), obesity and/or metabolic syndrome and to the use in
regeneration of tissues such as pancreatic tissues and others.
[0003] Many human proteins serve as pharmaceutically active
compounds. Several classes of human proteins that serve as such
active compounds include hormones, cytokines, cell growth factors,
and cell differentiation factors. Most proteins that can be used as
a pharmaceutically active compound fall within the family of
secreted proteins. Secreted proteins are generally produced within
cells at rough endoplasmic reticulum, are then exported to the
golgi complex, and then move to secretory vesicles or granules,
where they are secreted to the exterior of the cell via exocytosis.
Examples for commercially used secreted proteins are human insulin,
thrombolytic agents, interferons, interleukins, colony stimulating
factors, human growth hormone, transforming growth factor beta,
tissue plasminogen activator, erythropoietin, and various other
proteins. Receptors of secreted proteins, which are membrane-bound
proteins, also have potential as therapeutic or diagnostic agents.
It is, therefore, important for developing new pharmaceutical
compounds to identify secreted proteins that can be tested for
activity in a variety of animal models. Thus, in light of the
pervasive role of secreted proteins in human physiology, a need
exists for identifying and characterizing novel functions for human
secreted proteins and the genes that encode them. This knowledge
will allow one to detect, to treat, and to prevent medical
diseases, disorders, and/or conditions by using secreted proteins
or the genes that encode them.
[0004] The pancreas is an essential organ possessing both an
exocrine function involved in the delivery of enzymes into the
digestive tract and an endocrine function by which various hormones
are secreted into the blood stream. The exocrine function is
assured by acinar and centroacinar cells that produce various
digestive enzymes and intercalated ducts that transport these
enzymes in alkaline solution to the duodenum. The functional unit
of the endocrine pancreas is the islet of Langerhans. Islets are
scattered throughout the exocrine portion of the pancreas and are
composed of four cell types: alpha-, beta-, delta- and PP-cells,
reviewed for example in Kim S. K. and Hebrok M., (2001) Genes Dev.
15: 111-127. Beta-cells produce insulin, represent the majority of
the endocrine cells and form the core of the islets, while
alpha-cells secrete glucagon and are located in the periphery.
Delta-cells and PP-cells are less numerous and secrete somatostatin
and pancreatic polypeptide, respectively.
[0005] Early pancreatic development has been well studied in
different species, including chicken, zebrafish, and mice (for an
detailed review, see Kim & Hebrock, 2001, supra). The pancreas
develops from distinct dorsal and ventral anlagen. Pancreas
development requires specification of the pancreas structure along
both anterior-posterior and dorsal-ventral axes. A number of
transcription factors, which are critical for proper pancreatic
development have been identified (see Kim & Hebrok, 2001,
supra; Wilson M. E. et al. Mech Dev. 120: 65-80).
[0006] In postnatal/adult humans, the acinar and ductal cells
retain a significant proliferative capacity that can ensure cell
renewal and growth, whereas the islet cells become mostly
mitotically inactive. This is in contrast to rodents where
beta-cell replication is an important mechanism in the generation
of new beta cells. It has been, suggested, that during embryonic
development, pancreatic islets of Langerhans originate from
differentiating duct cells or other cells with epithelial
morphology (Bonner-Weir S, and Sharma A., (2002) J. Pathol. 197:
519-526; Gu G. et al., (2003) Mech Dev. 120: 35-43). In adult
humans, new beta cells arise in the vicinity of ducts (Butler A. E.
et al., (2003) Diabetes 52: 102-110; Bouwens L. and Pipeleers D.
G., (1998) Diabetologia 41: 629-633). However, also an intra-islet
location or an origin in the bone marrow has been suggested for
precursor cells of adult beta cells (Zulewski H. et al., (2001)
Diabetes 50: 521-533; lanus A. et al., (2003) J Olin Invest. 111:
843-850). Pancreatic islet growth is dynamic and responds to
changes in insulin demand, such as during pregnancy or during the
increase in body mass occurring during childhood. In adults, there
is a good correlation between body mass and islet mass (Yoon K. H.
et al., (2003) J Clin Endocrinol Metab. 88: 2300-2308). Pancreatic
beta-cells secrete insulin, which is stimulated by high blood
glucose levels. Insulin amongst other hormones plays a key role in
the regulation of the fuel metabolism. Insulin leads to the storage
of glycogen and triglycerides and to the synthesis of proteins. The
entry of glucose into muscles and adipose cells is stimulated by
insulin. In patients who suffer from diabetes mellitus the amount
of insulin produced by the pancreatic islet cells is too low,
resulting in elevated blood glucose levels (hyperglycemia). In
diabetes type 1 beta cells are lost due to autoimmune destruction.
In type 2 diabetic patients, liver and muscle cells loose their
ability to respond to normal blood insulin levels (insulin
resistance). High blood glucose levels (and also high blood lipid
levels) lead to an impairment of beta-cell function and to an
increase in beta-cell apoptosis. It is interesting to note that the
rate of beta-cell neogenesis does not appear to change in type 2
diabetics (Butler et al., 2003, supra), thus causing a reduction in
total beta-cell mass over time. Eventually the application of
exogenous insulin becomes necessary in type 2 diabetics.
[0007] Improving metabolic parameters such as blood sugar and blood
lipid levels (e.g. through dietary changes, exercise, medication or
combinations thereof) before beta cell mass has fallen below a
critical threshold leads to a relatively rapid restoration of beta
cell function. However, after such a treatment the pancreatic
endocrine function would remain impaired due to the only slightly
increased regeneration rate.
[0008] In type 1 diabetics, the lifespan of pancreatic islets is
dramatically shortened due to autoimmune destruction. Treatments
have been devised which modulate the immune system and may be able
to stop or strongly reduce islet destruction (Raz I. et al., (2001)
Lancet 358: 1749-1753; Chatenoud L. et al., (2003) Nat Rev Immunol.
3: 123-132). However, due to the relatively slow regeneration of
human beta cells such treatments could only be fully successful at
improving the diabetic condition if they are combined with an agent
which can stimulate beta cell regeneration. Thus, both for type 1
and type 2 diabetes (early and late stages) there is a need to find
novel agents which stimulate beta cell regeneration.
[0009] Diabetes is a very disabling disease, because medications do
not control blood sugar levels well enough to prevent swinging
between high and low blood sugar levels. Patients with diabetes are
at risk for major complications, including diabetic ketoacidosis,
end-stage renal disease, diabetic retinopathy and amputation. There
are also a host of related conditions, such as metabolic syndrome,
obesity, hypertension, heart disease, peripheral vascular disease,
and infections, for which persons with diabetes are at
substantially increased risk. The treatment of these complications
contributes to a considerable degree to the enormous cost which is
imposed by diabetes on health care systems world wide.
[0010] Obesity is one of the most prevalent metabolic disorders in
the world. It is still a poorly understood human disease that
becomes as a major health problem more and more relevant for
western society. Obesity is defined as a body weight more than 20%
in excess of the ideal body weight, frequently resulting in a
significant impairment of health. Obesity may be measured by body
mass index, an indicator of adiposity or fatness. Further
parameters for defining obesity are waist circumferences, skinfold
thickness and bioimpedance. It is associated with an increased risk
for cardiovascular disease, hypertension, diabetes mellitus type
II, hyperlipidaemia and an increased mortality rate. Obesity is
influenced by genetic, metabolic, biochemical, psychological, and
behavioral factors and can be caused by different reasons such as
non-insulin dependent diabetes, increase in triglycerides, increase
in carbohydrate bound energy and low energy expenditure (Kopelman
P. G., (2000) Nature 404: 635-643).
[0011] The concept of `metabolic syndrome` (syndrome x,
insulin-resistance syndrome, deadly quartet) was first described
1966 by Camus and reintroduced 1988 by Reaven (Camus JP, 1966, Rev
Rhum Mal Osteoartic 33: 10-14; Reaven et al. 1988, Diabetes, 37:
1595-1607). Today, metabolic syndrome is commonly defined as
clustering of cardiovascular risk factors like hypertension,
abdominal obesity, high blood levels of triglycerides and fasting
glucose as well as low blood levels of HDL cholesterol. Insulin
resistance greatly increases the risk of developing the metabolic
syndrome (Reaven, 2002, Circulation 106: 286-288). The metabolic
syndrome often precedes the development of type II diabetes and
cardiovascular disease (Lakka H. M., 2002, JAMA 288: 2709-2716).
The control of blood lipid levels and blood glucose levels is
essential for the treatment of the metabolic syndrome (see, for
example, Santomauro A. T. et al., (1999) Diabetes, 48:
1836-1841).
[0012] The molecular factors regulating food intake and body weight
balance are incompletely understood. Even if several candidate
genes have been described which are supposed to influence the
homeostatic system(s) that regulate body mass/weight, like leptin
or the peroxisome proliferator-activated receptor-gamma
co-activator, the distinct molecular mechanisms and/or molecules
influencing obesity or body weight/body mass regulations are not
known.
[0013] There is a need in the prior art for the identification of
candidate genes that are specifically expressed in early
development in certain pancreatic tissues. These genes and the
thereby encoded proteins can provide tools to the diagnosis and
treatment of severe pancreatic disorders and related diseases.
Therefore, this invention describes secreted proteins that are
specifically expressed in pancreatic tissues early in the
development. The invention relates to the use of these genes and
proteins in the diagnosis, prevention and/or treatment of
pancreatic dysfunctions, such as diabetes, and other related
diseases such as obesity and/or metabolic syndrome. These proteins
and genes are especially useful in regeneration processes, such as
regeneration of the pancreas cells.
[0014] In this invention, we disclose a secreted factor referred to
as DG001 which is involved in pancreas development, regeneration,
and in the regulation of energy homeostasis. DG001 corresponds to
human pleiotrophin, a member of the cytokine/growth factor family
of proteins, which is a secreted heparin-binding cytokine that
signals diverse functions involved with angiogenesis,
neuorogenesis, cell migration, and mesoderm-epithelial interactions
(for reviews, see, Deuel T. F. et al., (2002) Arch Biochem Biophys.
397: 162-171; Zhang N. and Deuel T. F, (1999) Curr Opin Hematol. 6:
44-50). Pleiotrophin is developmentally regulated and highly
conserved among species.
[0015] Pleiotrophin gene expression is found in cells in early
differentiation during different development periods and is
upregulated in cells with an early differentiation phenotype in
wound repair. Pleiotrophin expression also increases upon ischemic
injury.
[0016] In the non-cancerous state, pleiotrophin is very tightly
regulated, being expressed only in regions of the brain and
reproductive tract. Widespread deregulation of pleiotrophin is
found in many known human cancers or their derived cell lines. In
different human tumor cells, the pleiotrophin gene is strongly
expressed. Expression of the pleiotrophin gene in tumor cells in
vivo accelerates growth and stimulates tumor angiogenesis. In
addition to this regulation of tumor cells, pleiotrophin is causing
differential growth of osteoblasts and cranial nerve cells.
[0017] Pleiotrophin is the first ligand of any of the known
transmembrane tyrosine phosphatases. A chondroitin sulfate
proteoglycan (protein-tyrosine phosphatase zeta, PTPzeta) was
identified as a receptor for pleiotrophin which is inactivated by
pleiotrophin. Pleiotrophin thus regulates both normal cell
functions and different pathological conditions at many levels. It
signals these functions through a transmembrane tyrosine
phosphatase.
[0018] Accordingly, certain uses of DG001 have been described in
several patent applications. For example, DG001 was suggested to be
used to inhibit cellular growth and control angiogenesis, useful
for controlling post-surgical bleeding (EP535337-A2), cancer
(EP535337-A2), such as prostate cancer (WO 00/055174), or brain
tumors (WO 02/76510), for treatment and diagnosis of osteoporosis
(WO 92/00324), for nerve repair and for treating neurodegenerative
disorders (EP474979-A; WO 99/53943; dementia (WO 92/00324), cardiac
(coronary artery disease, ischaemic heart disease), kidney and
inflammatory disorders (WO 99/53943, WO 00/35473), diabetic
peripheral vasculopathies and peripheral atherosclerotic disease
(WO 99/53943), or to inhibit the infectivity of Herpesviridae virus
(EP569703-A2).
[0019] Accordingly, the present invention relates to a secreted
protein with novel functions in the human metabolism, regeneration,
and pancreatic developmental processes. The present invention
discloses specific genes and proteins encoded thereby and
effectors/modulators thereof involved in the regulation of
pancreatic function and metabolism, especially in pancreas diseases
such as diabetes mellitus, e.g. insulin dependent diabetes mellitus
and/or non-insulin dependent diabetes mellitus, and/or metabolic
syndrome, obesity, and/or related disorders such as coronary heart
disease, eating disorder, cachexia, hypertension,
hypercholesterolemia (dyslipidemia), liver fibrosis, and/or
gallstones. Further, the present invention relates to specific
genes and proteins encoded thereby and effectors/modulators thereof
involved in the modulation, e.g. stimulation, of pancreatic
development and/or the regeneration of pancreatic cells or tissues,
e.g. cells having exocrinous functions such as acinar cells,
centroacinar cells and/or ductal cells, and/or cells having
endocrinous functions, particularly cells in Langerhans islets such
as alpha-, beta-, delta- and/or PP-cells, more particularly
beta-cells.
[0020] In this invention, we used a screen for secreted factors
expressed in developing mammalian (mouse) pancreas, as described in
more detail in the Examples section (see Example 1). This screen
identified DG001 as secreted factor expressed in developing mouse
pancreas. The present invention describes mammalian DG001 proteins
and the polynucleotides encoding these, in particular human DG001,
as being involved in the conditions and processes mentioned
above.
[0021] The present invention relates to DG001 polynucleotides
encoding polypeptides with novel functions in the development and
regeneration of pancreatic tissues and thus in mammalian pancreatic
diseases (e.g. diabetes), and also in body-weight regulation,
energy homeostasis, and obesity, fragments of said polynucleotides,
polypeptides encoded by said polynucleotides or fragments thereof.
The invention also relates to vectors, host cells and recombinant
methods for producing the polypeptides and polynucleotides of the
invention. The invention also relates to effectors/modulators of
DG001 polynucleotides and/or polypeptides, e.g. antibodies,
biologically active nucleic acids, such as antisense molecules,
RNAi molecules or ribozymes, aptamers, peptides or low-molecular
weight organic compounds recognizing said polynucleotides or
polypeptides.
[0022] DG001 homologous proteins and nucleic acid molecules coding
therefore are obtainable from vertebrate species. Particularly
preferred are nucleic acids encoding the human DG001 protein and
variants thereof. The invention particularly relates to a nucleic
acid molecule encoding a polypeptide contributing to regulating the
energy homeostasis and the mammalian metabolism, wherein said
nucleic acid molecule comprises [0023] (a) the nucleotide sequence
of human DG001 (SEQ ID NO: 1) and/or a sequence complementary
thereto, [0024] (b) a nucleotide sequence which hybridizes at
50.degree. C. in a solution containing 1.times.SSC and 0.1% SDS to
a sequence of (a), [0025] (c) a sequence corresponding to the
sequences of (a) or (b) within the degeneration of the genetic
code, [0026] (d) a sequence which encodes a polypeptide which is at
least 85%, preferably at least 90%, more preferably at least 95%,
more preferably at least 98% and up to 99.6% identical to the amino
acid sequences of the human DG001 protein (SEQ ID NO: 2), [0027]
(e) a sequence which differs from the nucleic acid molecule of (a)
to (d) by mutation and wherein said mutation causes an alteration,
deletion, duplication and/or premature stop in the encoded
polypeptide or [0028] (f) a partial sequence of any of the
nucleotide sequences of (a) to (e) having a length of 15-25 bases,
preferably 25-35 bases, more preferably 35-50 bases and most
preferably at least 50 bases.
[0029] The function of the mammalian DG001 in mammalian metabolism
was validated by analyzing the expression of the transcripts in
different tissues and by analyzing the role in adipocyte
differentiation (see Example 3 and FIGS. 2A-2E for more detail). In
mouse models of insulin resistance and/or diabetes expression,
DG001 is strongly down-regulated in metabolic active tissue (WAT)
which is supporting an essential role of DG001 in the regulation of
the mammalian metabolism, particularly in processes related to,
obesity, diabetes, or metabolic syndrome (FIG. 2B). In addition,
expression of DG001 mRNA is significantly down-regulated in WAT in
mice showing symptoms of diabetes, lipid accumulation, and high
plasma lipid levels, if fed a high fat diet. Further, the
expression of DG001 is up-regulated in BAT and muscle tissues in
these mice (FIG. 2C). Thus, DG001 protein is playing a role in the
regulation of metabolism, particularly energy homeostasis and
thermogenesis.
[0030] Further, we show (see Examples) that the DG001 protein has
to be down-regulated at a very early stage of adipose
differentiation (until day 4) and upregulated during the late stage
in order for the preadipocyctes to differentiate into mature
adipocyctes. With regard to changes in expression intensity during
the differentiation of preadipocytes to adipocytes, a slight
reduction in relative signal intensity can be observed for DG001
during the in vitro differentiation program of 3T3-L1 (see FIG.
2D). The results are suggesting a role of DG001 as modulator of
adipogenesis.
[0031] Microarrays are analytical tools routinely used in
bioanalysis. A microarray has molecules distributed over, and
stably associated with, the surface of a solid support. The term
"microarray" refers to an arrangement of a plurality of
polynucleotides, polypeptides, antibodies, or other chemical
compounds on a substrate. Microarrays of polypeptides,
polynucleotides, and/or antibodies have been developed and find use
in a variety of applications, such as monitoring gene expression,
drug discovery, gene sequencing, gene mapping, bacterial
identification, and combinatorial chemistry. One area in particular
in which microarrays find use is in gene expression analysis (see
Example 4). Array technology can be used to explore the expression
of a single polymorphic gene or the expression profile of a large
number of related or unrelated genes. When the expression of a
single gene is examined, arrays are employed to detect the
expression of a specific gene or its variants. When an expression
profile is examined, arrays provide a platform for identifying
genes that are tissue specific, are affected by a substance being
tested in a toxicology assay, are part of a signaling cascade,
carry out housekeeping functions, or are specifically related to a
particular genetic predisposition, condition, disease, or
disorder.
[0032] Microarrays may be prepared, used, and analyzed using
methods known in the art (see for example, Brennan, T. M. et al.
(1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al., PCT
application WO 95/251116; Shalon, D. et al., PCT application WO
95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:
2150-2155; Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662).
Various types of microarrays are well known and thoroughly
described in Schena, M., ed. (1999; DNA Microarrays: A Practical
Approach, Oxford University Press, London).
[0033] Oligonucleotides or longer fragments derived from any of the
polynucleotides described herein may be used as elements on a
microarray. The microarray can be used in transcript imaging
techniques which monitor the relative expression levels of large
numbers of genes simultaneously as described below. The microarray
may also be used to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, to monitor progression/regression of disease
as a function of gene expression, and to develop and monitor the
activities of therapeutic agents in the treatment of disease. In
particular, this information may be used to develop a
pharmacogenomic profile of a patient in order to select the most
appropriate and effective treatment regimen for that patient. For
example, therapeutic agents which are highly effective and display
the fewest side effects may be selected for a patient based on
his/her pharmacogenomic profile.
[0034] As determined by microarray analysis, DG001 shows
differential expression in 3T3-L1 cells. A strong up-regulation is
observed concerning the expression of DG001 during adipocyte
differentiation (see FIG. 2E). The DG001 protein in preadipocyctes
has the potential to enhance adipose differentiation at a very
early stage. Therefore, the DG001 protein might play an essential
role in adipogenesis. The results are suggesting a role of DG001 in
the regulation in human metabolism, for example, as
effector/modulator (for example, enhancer) of adipogenesis. Thus,
DG001 is a strong candidate for the manufacture of a pharmaceutical
composition and a medicament for the treatment of conditions
related to human metabolism, such as diabetes, obesity, and/or
metabolic syndrome.
[0035] This invention further shows that DG001 induces the
differentiation of insulin-producing cells and is thus a target for
the treatment of diabetes. In connection with the present
invention, the term "progenitor cells" relates to undifferentiated
cells capable of being differentiated into insulin producing cells.
The term particularly includes stem cells, i.e. undifferentiated or
immature embryonic, adult, or somatic cells that can give rise to
various specialized cell types. The term "stem cells" can include
embryonic stem cells (ES) and primordial germ cells (EG) cells of
mammalian, e.g. human or animal origin. Isolation and culture of
such cells is well known to those skilled in the art (see, for
example, Thomson et al., (1998) Science 282: 1145-1147; Shamblott
et al., (1998) Proc. Natl. Acad. Sci. USA 95: 13726-13731; U.S.
Pat. No. 6,090,622; U.S. Pat. No. 5,914,268; WO 00/27995;
Notarianni et al., (1990) J. Reprod. Fert. 41: 51-56; Vassilieva et
al., (2000) Exp. Cell. Res. 258: 361-373). Adult or somatic stem
cells have been identified in numerous different tissues such as
intestine, muscle, bone marrow, liver, and brain. WO 03/023018
describes a novel method for isolating, culturing, and
differentiating intestinal stem cells for therapeutic use. In the
pancreas, several indications suggest that stem cells are also
present within the adult tissue (Gu and Sarvetnick, (1993)
Development 118: 33-46; Bouwens, (1998) Microsc Res Tech 43:
332-336; Bonner-Weir, (2000) J. Mol. Endocr. 24: 297-302).
[0036] Embryonic stem cells can be isolated from the inner cell
mass of pre-implantation embryos (ES cells) or from the primordial
germ cells found in the genital ridges of post-implanted embryos
(EG cells). When grown in special culture conditions such as
spinner culture or hanging drops, both ES and EG cells aggregate to
form embryoid bodies (EB). EBs are composed of various cell types
similar to those present during embryogenesis. When cultured in
appropriate media, EB can be used to generate in vitro
differentiated phenotypes, such as extraembryonic endoderm,
hematopoietic cells, neurons, cardiomyocytes, skeletal muscle
cells, and vascular cells. We have previously described a method
that allows EB to efficiently differentiate into insulin-producing
cells (as described in patent application PCT/EP02/04362, published
as WO 02/086107 and by Blyszczuk et al., (2003) Proc. Natl. Acad
Sci USA 100: 998-1003, which are incorporated herein by
reference).
[0037] The results shown in FIG. 3 clearly demonstrate an induction
of the differentiation of insulin-producing cells by DG001. Thus,
DG001 can induce the differentiation of beta-cells and is therefore
a target for therapeutic uses in the treatment of diabetes, for
example, when regeneration of cells is required.
[0038] Before the present invention is described, it is understood
that all technical and scientific terms used herein have the same
meanings as commonly understood by one of ordinary skill in the art
to which this invention belongs.
[0039] In the present invention the term "beta-cell regeneration"
refers to an at least partial restoration of normal beta-cell
function by increasing the number of functional insulin secreting
beta-cells and/or by restoring normal function in functionally
impaired beta-cells.
[0040] The invention also encompasses polynucleotides that encode
the proteins of the invention and homologous proteins. Accordingly,
any nucleic acid sequence, which encodes the amino acid sequences
of the proteins of the invention and homologous proteins, can be
used to generate recombinant molecules that express the proteins of
the invention and homologous proteins. In a particular embodiment,
the invention encompasses a nucleic acid encoding DG001. It will be
appreciated by those skilled in the art that as a result of the
degeneracy of the genetic code, a multitude of nucleotide sequences
encoding the proteins, some bearing minimal homology to the
nucleotide sequences of any known and naturally occurring gene, may
be produced. The invention contemplates each and every possible
variation of nucleotide sequence that can be made by selecting
combinations based on possible codon choices.
[0041] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those of the polynucleotide encoding
the proteins of the invention, under various conditions of
stringency. Hybridization conditions are based on the melting
temperature (Tm) of the nucleic acid binding complex or probe, as
described in Wahl & Berger (1987: Methods Enzymol. 152:
399-407) and Kimmel (1987; Methods Enzymol. 152: 507-511), and may
be used at a defined stringency. Preferably, hybridization under
stringent conditions means that after washing for 1 h with
1.times.SSC and 0.1% SDS at 50.degree. C., preferably at 55.degree.
C., more preferably at 62.degree. C. and most preferably at
65.degree. C., particularly for 1 h in 0.2.times.SSC and 0.1% SDS
at 50.degree. C., preferably at 55.degree. C., more preferably at
62.degree. C. and most preferably at 65.degree. C., a positive
hybridization signal is observed. Altered nucleic acid sequences
encoding the proteins which are encompassed by the invention
include deletions, insertions or substitutions of different
nucleotides resulting in a polynucleotide that encodes the same or
a functionally equivalent protein.
[0042] The encoded proteins may also contain deletions, insertions
or substitutions of amino acid residues, which produce a silent
change and result in functionally equivalent proteins. Deliberate
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
biological activity of the protein is retained. Furthermore, the
invention relates to peptide fragments of the proteins or
derivatives thereof such as cyclic peptides, retro-inverso peptides
or peptide mimetics having a length of at least 4, preferably at
least 6 and up to 50 amino acids.
[0043] Also included within the scope of the present invention are
alleles of the genes encoding the proteins of the invention and
homologous proteins. As used herein, an `allele` or `allelic
sequence` is an alternative form of the gene, which may result from
at least one mutation in the nucleic acid sequence. Alleles may
result in altered mRNAs or polypeptides whose structures or
function may or may not be altered. Any given gene may have none,
one or many allelic forms. Common mutational changes, which give
rise to alleles, are generally ascribed to natural deletions,
additions or substitutions of nucleotides. Each of these types of
changes may occur alone or in combination with the others, one or
more times in a given sequence.
[0044] The nucleic acid sequences encoding DG001 and homologous
proteins may be extended utilizing a partial nucleotide sequence
and employing various methods known in the art to detect upstream
sequences such as promoters and regulatory elements.
[0045] In order to express a biologically active protein, the
nucleotide sequences encoding the proteins or functional
equivalents, may be inserted into appropriate expression vectors,
i.e., a vector which contains the necessary elements for the
transcription and translation of the inserted coding sequence.
Methods, which are well known to those skilled in the art, may be
used to construct expression vectors containing sequences encoding
the proteins and the appropriate transcriptional and translational
control elements. Regulatory elements include for example a
promoter, an initiation codon, a stop codon, a mRNA stability
regulatory element, and a polyadenylation signal. Expression of a
polynucleotide can be assured by (i) constitutive promoters such as
the Cytomegalovirus (CMV) promoter/enhancer region, (ii) tissue
specific promoters such as the insulin promoter (see, Soria et al.,
2000, Diabetes 49: 157), SOX2 gene promoter (see Li et al., (1998)
Curr. Biol. 8: 971-974), Msi-1 promoter (see Sakakibara et al.,
(1997) J. Neuroscience 17: 8300-8312), alpha-cardia myosin heavy
chain promoter or human atrial natriuretic factor promoter (Klug et
al., (1996) J. Clin. Invest 98: 216-224; Wu et al., (1989) J. Biol.
Chem. 264: 6472-6479) or (iii) inducible promoters such as the
tetracycline inducible system. Expression vectors can also contain
a selection agent or marker gene that confers antibiotic resistance
such as the neomycin, hygromycin or puromycin resistance genes.
These methods include in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination. Such
techniques are described in Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y. and Ausubel, F. M. et al. (1989) Current Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y.
[0046] In a further embodiment of the invention, natural, modified
or recombinant nucleic acid sequences encoding the proteins of the
invention and homologous proteins may be ligated to a heterologous
sequence to encode a fusion protein.
[0047] A variety of expression vector/host systems, as known in the
art, may be utilized to contain and express sequences encoding the
proteins or fusion proteins. These include, but are not limited to,
micro-organisms such as bacteria transformed with recombinant
bacteriophage, plasmid or cosmid DNA expression vectors; yeast
transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (e.g., baculovirus,
adenovirus, adeno-associated virus, lentiverus, retrovirus); plant
cell systems transformed with virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with
bacterial expression vectors (e.g., Ti or PBR322 plasmids); or
animal cell systems.
[0048] The presence of polynucleotide sequences of the invention in
a sample can be detected by DNA-DNA or DNA-RNA hybridization and/or
amplification using probes or portions or fragments of said
polynucleotides. Nucleic acid amplification based assays involve
the use of oligonucleotides or oligomers based on the sequences
specific for the gene to detect transformants containing DNA or RNA
encoding the corresponding protein. As used herein
`oligonucleotides` or `oligomers` refer to a nucleic acid sequence
of at least about 10 nucleotides and as many as about 60
nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides, which can be used as a probe or
amplimer.
[0049] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting polynucleotide sequences
include oligo-labeling, nick translation, end-labeling of RNA
probes, PCR amplification using a labeled nucleotide, or enzymatic
synthesis. These procedures may be conducted using a variety of
commercially available kits (Pharmacia & Upjohn, (Kalamazoo,
Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp.,
(Cleveland, Ohio).
[0050] The presence of DG001 in a sample can be determined by
immunological methods or activity measurement. A variety of
protocols for detecting and measuring the expression of proteins,
using either polyclonal or monoclonal antibodies specific for the
protein or reagents for determining protein activity are known in
the art. Examples include enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), and fluorescence activated cell
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
the protein is preferred, but a competitive binding assay may be
employed. These and other assays are described, among other places,
in Hampton, R. et al. (1990; Serological Methods, a Laboratory
Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983;
J. Exp. Med. 158: 1211-1226).
[0051] Suitable reporter molecules or labels, which may be used,
include radionuclides, enzymes, fluorescent, chemiluminescent or
chromogenic agents as well as substrates, co-factors, inhibitors,
magnetic particles, and the like.
[0052] Host cells transformed with nucleotide sequences encoding a
protein of the invention may be cultured under conditions suitable
for the expression and recovery of said protein from cell culture.
The protein produced by a recombinant cell may be secreted or
contained intracellularly depending on the sequence or/and the
vector used. As will be understood by those of skill in the art,
expression vectors containing polynucleotides, which encode the
protein may be designed to contain signal sequences, which direct
secretion of the protein through a prokaryotic or eukaryotic cell
membrane. Other recombinant constructions may be used to join
sequences encoding the protein to nucleotide sequence encoding a
polypeptide domain, which will facilitate purification of soluble
proteins. Such purification facilitating domains include, but are
not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAG
extension/affinity purification system (Immunex Corp., Seattle,
Wash.) The inclusion of cleavable linker sequences such as those
specific for Factor XA or Enterokinase (Invitrogen, San Diego,
Calif.) between the purification domain and the desired protein may
be used to facilitate purification.
[0053] The data disclosed in this invention show that the DG001
nucleic acids and proteins and effector/modulator molecules thereof
are useful in diagnostic and therapeutic applications implicated,
for example, but not limited to, pancreatic diseases (such as
diabetes mellitus, e.g. insulin dependent diabetes mellitus and/or
non-insulin dependent diabetes mellitus), obesity, metabolic
syndrome, eating disorder, cachexia, hypertension, coronary heart
disease, hypercholesterolemia (dyslipidemia), and/or gallstones.
Further, the data show that the DG001 nucleic acids and proteins
and effector/modulator molecules thereof are useful for the
modulation, e.g. stimulation, of pancreatic development and/or the
regeneration of pancreatic cells or tissues, e.g. cell having
exocrinous functions such as acinar cells, centroacinar cells
and/or ductal cells, and/or cells having endocrinous functions,
particularly cells in Langerhans islets such as alpha-, beta-,
delta- and/or PP-cells, more particularly beta-cells. Hence,
diagnostic and therapeutic uses for the proteins of the invention
nucleic acids and proteins of the invention are, for example but
not limited to, the following: (i) tissue regeneration in vitro and
in vivo (regeneration for all these tissues and cell types
composing these tissues and cell types derived from these tissues),
(ii) small molecule drug target, (iii) antibody target
(therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv)
diagnostic and/or prognostic marker, (v) protein therapy, (vi) gene
therapy (gene delivery/gene ablation), and/or (vii) research
tools.
[0054] According to this invention the DG001 product may be
administered [0055] i) as a pharmaceutical composition e.g.
enterally, parenterally or topically, preferably directly to the
pancreas, [0056] ii) via implantation of DG001 protein product
expressing cells, and/or [0057] iii) via gene therapy as described
in more detail below.
[0058] Further, the DG001 expression level in a patient may be
influenced by a DG001 modulator/effector administered [0059] i) as
a pharmaceutical composition e.g. enterally, parenterally or
topically, preferably directly to the pancreas, [0060] ii) via cell
based therapy, and/or [0061] iii) via gene therapy as described in
more detail below.
[0062] The DG001 product or the DG001 modulator/effector, i.e. a
pharmaceutically active substance influencing, particularly
increasing the DG001 expression level or function may be
administered in the above described manner alone or in combination
with another pharmaceutical composition useful to treat beta-cell
degeneration, for example hormones, growth factors or immune
modulating agents.
[0063] A DG001 product or a modulator/effector thereof may be
administered in patients suffering from a disease going along with
impaired beta-cell function, for example but not limited to
diabetes type 1, LADA, or progressed diabetes type 2. It is further
contemplated that a DG001 product or the modulator/effector thereof
may be administered preventively to patients at risk to develop
beta-cell degeneration, like for example but not limited to
patients suffering from diabetes type 2 or LADA in early stages. A
variety of pharmaceutical formulations and different delivery
techniques are described in further detail below.
[0064] The present invention also relates to methods for
differentiating progenitor cells into insulin-producing cells in
vitro comprising [0065] (a) activating one or more pancreatic genes
in a progenitor, e.g. stem cell (optional step, particularly if
embryonic stem cells are used) [0066] (b) aggregating said cells to
form embryoid bodies (optional step, particularly if embryonic stem
cells are used) [0067] (c) cultivating embryoid bodies or
cultivating adult stem cells (e.g., duct cells) in specific
differentiation media containing a DG001 protein product and/or a
modulator/effector thereof under conditions wherein beta-cell
differentiation is significantly enhanced, and [0068] (d)
identifying and selecting insulin-producing cells.
[0069] Activation of pancreatic genes may comprise transfection of
a cell with pancreatic gene operatively linked to an expression
control sequence, e.g. on a suitable transfection vector, as
described in WO 03/023018, which is herein incorporated by
reference. Examples of preferred pancreatic genes are Pdx1, Pax4,
Pax6, neurogenin 3 (ngn3), Nkx 6.1, Nkx 6.2, Nkx 2.2, HB 9,
BETA2/Neuro D, Isl 1, HNF1-alpha, HNF1-beta and HNF3 of human or
animal origin. Each gene can be used individually or in combination
with at least one other gene. Pax4 is especially preferred.
[0070] DG001 products, e.g. DG001 protein or nucleic acid products,
are preferably produced via recombinant techniques because such
methods are capable of achieving high amounts of protein at a great
purity, but are not limited to products expressed in bacterial,
plant, mammalian, or insect cell systems.
[0071] Further, the data show that the DG001 nucleic acids and
proteins and effector/modulator molecules thereof are useful for
the modulation, e.g. stimulation, of pancreatic development and/or
for the regeneration of pancreatic cells or tissues, e.g. cells
having exocrinous functions such as acinar cells, centroacinar
cells and/or ductal cells, and/or cells having endocrinous
functions, particularly cells in Langerhans islets such as alpha-,
beta-, delta- and/or PP-cells, more particularly beta-cells.
[0072] For example, but not limited to, cDNAs encoding the proteins
of the invention and particularly their human homologues may be
useful in stimulating, enhancing or regulating the regeneration of
tissues, and the proteins of the invention and particularly their
human homologues may be useful when administered to a subject in
need thereof. By way of non-limiting example, the compositions of
the present invention will have efficacy for treatment of patients
suffering from, for example, pancreatic diseases (e.g. diabetes),
obesity, and/or metabolic syndrome as described above.
[0073] Beside diabetes, the compositions of the present invention
will also have efficacy for treatment of patients with other
pancreatic diseases such as pancreatic cancer, dysplasia, or
pancreatitis.
[0074] The DG001 nucleic acids and proteins and
effectors/modulators thereof are useful in diagnostic and
therapeutic applications implicated in various embodiments as
described below. For example, but not limited to, cDNAs encoding
the proteins of the invention and particularly their human
homologues may be useful in gene therapy, and the proteins of the
invention and particularly their human homologues may be useful
when administered to a subject in need thereof. By way of
non-limiting example, the compositions of the present invention
will have efficacy for treatment of patients suffering from, for
example, pancreatic diseases (e.g. diabetes), obesity, and/or
metabolic syndrome as described above.
[0075] DG001 product cell therapy, i.e. pancreatic implantation of
cells producing DG001 protein product, is also contemplated. This
embodiment involves implanting cells capable of synthesizing and
secreting a biologically active form of DG001 protein product into
patients. Such DG001 protein product-producing cells may be cells
that are natural producers of DG001 protein product or may be cells
that are modified to express the protein. Such modified cells
include recombinant cells whose ability to produce a DG001 protein
product has been augmented by transformation with a gene encoding
the desired DG001 protein product in a vector suitable for
promoting its expression and secretion. In order to minimize a
potential immunological reaction in patients being administered
DG001 protein product of a foreign species, it is preferred that
the cells producing DG001 protein product be of human origin and
produce human DG001 protein product. Likewise, it is preferred that
the recombinant cells producing DG001 protein product be
transformed with an expression vector containing a gene encoding a
human DG001 protein product. Implanted cells may be encapsulated to
avoid infiltration of surrounding tissue. Human or nonhuman animal
cells may be implanted in patients in biocompatible, semipermeable
polymeric enclosures or membranes that allow release of DG001
protein product, but that prevent destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissue.
[0076] Alternatively, DG001 protein product secreting cells may be
introduced into a patient in need intraportally via a percutaneous
transhepatic approach using local anaesthesia. Between 3000 and 100
000 equivalent differentiated insulin-producing cells per kilogram
body weight are preferably administered. Such surgical techniques
are well known in the art and can be applied without any undue
experimentation, see Pyzdrowski et al, 1992, New England J.
Medicine 327:220-226; Hering et al., Transplantation Proc.
26:570-571, 1993; Shapiro et al., New England J. Medicine
343:230-238, 2000.
[0077] In a further preferred embodiment, DG001 protein product can
be delivered directly to progenitor, e.g. stem cells in order to
stimulate the differentiation of insulin producing cells. For
example, protein delivery can be achieved by polycationic liposomes
(Sells et al. (1995) Biotechniques 19:72-76), Tat-mediated protein
transduction (Fawell et al. (1993) Proc. Natl. Acad. Sci. USA
91:664-668) and by fusing a protein to the cell permeable motif
derived from the PreS2-domain of the hepatitis-B virus (Oess and
Hildt (2000) Gene Ther. 7:750-758). Preparation, production and
purification of such proteins from bacteria, yeast or eukaryotic
cells are well known by persons skilled in the art. In this
embodiment of the invention, DG001 may be added preferably at
concentrations between 1 ng/ml and 500 ng/ml, more preferably
between 10 and 100 ng/ml, e.g. at about 50 ng/ml.
[0078] Further, the invention relates to a cell preparation
comprising differentiated progenitor cells, e.g. stem cells
exhibiting insulin production, particularly an insulin-producing
cell line obtainable by the method described above. The
insulin-producing cells may exhibit a stable or a transient
expression of at least one pancreatic gene involved in beta-cell
differentiation. The cells are preferably human cells that are
derived from human stem cells. For therapeutic applications the
production of autologous human cells from adult stem cells of a
patient is especially preferred. However, the insulin producing
cells may also be derived from non-autologous cells. If necessary,
undesired immune reactions may be avoided by encapsulation,
immunosuppression and/or modulation or due to non-immunogenic
properties of the cells.
[0079] The insulin producing cells of the invention preferably are
beta-like cells, i.e. they exhibit characteristics that closely
resemble naturally occurring beta-cells. Further, the cells of the
invention preferably are capable of a quick response to glucose.
After addition of 27.7 mM glucose, the insulin production is
enhanced by a factor of at least 2, preferably by a factor of at
least 3. Further, the cells of the invention are capable of
normalizing blood glucose levels after transplantation into
mice.
[0080] The invention further encompasses functional pancreatic
cells obtainable or obtained by the method according to the
invention. The cells are preferably of mammalian, e.g. human
origin. Preferably, said cells are pancreatic beta-cells, e.g.
mature pancreatic beta-cells or stem cells differentiated into
pancreatic beta-cells. Such pancreatic beta cells preferably
secrete insulin in response to glucose. Moreover, the present
invention provides functional pancreatic cell that express glucagon
in response to glucose. A preparation comprising the cells of the
invention may additionally contain cells with properties of other
endocrine cell types such as alpha-cells, delta-cells and/or
PP-cells. These cells are preferably human cells.
[0081] The cell preparation of the invention is preferably a
pharmaceutical composition comprising the cells together with
pharmacologically acceptable carriers, diluents and/or adjuvants.
The pharmaceutical composition is preferably used for the treatment
or prevention of pancreatic diseases, e.g. diabetes.
[0082] According to the present invention, the functional insulin
producing cells treated with DG001 may be transplanted preferably
intrahepatic, directly into the pancreas of an individual in need,
or by other methods. Alternatively, such cells may be enclosed into
implantable capsules that can be introduced into the body of an
individual, at any location, more preferably in the vicinity of the
pancreas, or the bladder, or the liver, or under the skin. Methods
of introducing cells into individuals are well known to those of
skill in the art and include, but are not limited to, injection,
intravenous or parenteral administration. Single, multiple,
continuous or intermittent administration can be effected. The
cells can be introduced into any of several different sites,
including but not limited to the pancreas, the abdominal cavity,
the kidney, the liver, the celiac artery, the portal vein or the
spleen. The cells may also be deposited in the pancreas of the
individual.
[0083] The methodology for the membrane encapsulation of living
cells is familiar to those of ordinary skill in the art, and the
preparation of the encapsulated cells and their implantation in
patients may be accomplished without undue experimentation. See,
e.g., U.S. Pat. Nos. 4,892,538, 5,011,472, and 5,106,627, each of
which is specifically incorporated herein by reference. A system
for encapsulating living cells is described in PCT Application WO
91/10425 of Aebischer et al., specifically incorporated herein by
reference. See also, PCP Application WO 91/10470 of Aebischer et
al., Winn et al., Exper. Neurol., 1 13:322-329, 1991, Aebischer et
al., Exper. Neurol., 11 1:269-275, 1991; Tresco et al., ASAIO,
38:17-23, 1992, each of which is specifically incorporated herein
by reference. Techniques for formulating a variety of other
sustained- or controlled-delivery means, such as liposome carriers,
bio-erodible particles or beads and depot injections, are also
known to those skilled in the art.
[0084] In another embodiment gene therapy ex vivo is carried out,
i.e. the patient's own cells are transformed ex vivo to produce a
DG001 protein product or a protein stimulating DG001 expression and
are directly reimplanted. For example, cells retrieved from the
patient are cultured and transformed with an appropriate vector.
After an optional propagation/expansion phase, the cells are
transplanted back into the same patient's body, particularly the
pancreas, where they produce and release the desired DG001 protein
product. Delivery by transfection and by liposome injections may be
achieved using methods, which are well known in the art. Any of the
therapeutic methods described above may be applied to any suitable
subject including, for example, mammals such as dogs, cats, cows,
horses, rabbits, monkeys, and most preferably, humans.
[0085] DG001 product gene therapy in vivo may also be carried out
by introducing the gene coding for a DG001 protein product into
targeted pancreas cells via local injection of a nucleic acid
construct or other appropriate delivery methods (Hefti, J.
Neurobiol., 25:1418-1435, 1994). For example, a nucleic acid
sequence encoding a DG001 protein product may be cloned to a
suitable vector, e.g. an adeno-associated virus vector or
adenovirus vector for delivery to the pancreas cells. Alternative
viral vectors include, but are not limited to, retrovirus, herpes
simplex virus and papilloma virus vectors. Physical transfer,
either in vivo or ex vivo as appropriate, may also be achieved by
liposome-mediated transfer, direct injection (naked DNA),
receptor-mediated transfer (ligand-DNA complex), electroporation,
calcium phosphate precipitation or microparticle bombardment (gene
gun).
[0086] Immunosuppressive drugs, such as cyclosporin, can also be
administered to the patient in need to reduce the host reaction
versus graft. Allografts using the cells obtained by the methods of
the present invention are also useful because a single healthy
donor could supply enough cells to regenerate at least partial
pancreas function in multiple recipients.
[0087] Administration of a DG001 protein product and/or
modulators/effectors thereof in a pharmaceutical composition to a
subject in need thereof, particularly a human patient, leads to an
at least partial regeneration of pancreatic cells. Preferably,
these cells are insulin producing beta-cells that will contribute
to the improvement of a diabetic state. With the administration of
this composition e.g. on a short term or regular basis, an increase
in beta-cell mass can be achieved. This effect upon the body
reverses the condition of diabetes partially or completely. As the
subject's blood glucose homeostasis improves, the dosage
administered may be reduced in strength. In at least some cases
further administration can be discontinued entirely and the subject
continues to produce a normal amount of insulin without further
treatment. The subject is thereby not only treated but may be cured
entirely of a diabetic condition. Further, beta cells or precursors
thereof may be treated in vitro and implanted or reimplanted into a
subject in need thereof. Further, other cells of the pancreas can
be regenerated in vivo and/or in vitro to cure a certain condition.
However, even moderate improvements in beta-cell mass can lead to a
reduced requirement for exogenous insulin, improved glycemic
control and a subsequent reduction in diabetic complications. In
another example, the compositions of the present invention will
also have efficacy for treatment of patients with other pancreatic
diseases such as pancreatic cancer, dysplasia, or pancreatitis, if
beta-cells are to be regenerated.
[0088] The nucleic acids of the invention or fragments thereof, may
further be useful in diagnostic applications, wherein the presence
or amount of the nucleic acids or the proteins are to be assessed.
Further antibodies that bind immunospecifically to the novel
substances of the invention may be used in therapeutic or
diagnostic methods.
[0089] For example, in one aspect, antibodies, which are specific
for the proteins of the invention and homologous proteins, may be
used directly as an effector/modulator, e.g. an antagonist or an
agonist, or indirectly as a targeting or delivery mechanism for
bringing a pharmaceutical agent to cells or tissue which express
the protein. The antibodies may be generated using methods that are
well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric single chain, Fab
fragments, and fragments produced by a Fab expression library.
Neutralising antibodies, (i.e., those which inhibit dimer
formation) are especially preferred for therapeutic use.
[0090] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with the protein or any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
It is preferred that the peptides, fragments or oligopeptides used
to induce antibodies to the protein have an amino acid sequence
consisting of at least five amino acids, and more preferably at
least 10 amino acids.
[0091] Monoclonal antibodies to the proteins may be prepared using
any technique that provides for the production of antibody
molecules by continuous cell lines in culture. These include, but
are not limited to, the hybridoma technique, the human B-cell
hybridoma technique, and the EBV-hybridoma technique (Kohler, G.
and Milstein C., (1975) Nature 256: 495-497; Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al., (1983)
Proc. Natl. Acad. Sci. 80: 2026-2030; Cole, S. P. et al., (1984)
Mol. Cell. Biochem. 62: 109-120).
[0092] In addition, techniques developed for the production of
`chimeric antibodies`, the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et
al (1984) Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314:
452-454). Alternatively, techniques described for the production of
single chain antibodies may be adapted, using methods known in the
art, to produce single chain antibodies specific for the proteins
of the invention and homologous proteins. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries (Kang A. S. et al., (1991) Proc. Natl.
Acad. Sci. 88: 11120-11123). Antibodies may also be produced by
inducing in vivo production in the lymphocyte population or by
screening recombinant immunoglobulin libraries or panels of highly
specific binding reagents as disclosed in the literature (Orlandi,
R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G.
and Milstein C., (1991) Nature 349: 293-299).
[0093] Antibody fragments, which contain specific binding sites for
the proteins may also be generated. For example, such fragments
include, but are not limited to, the F(ab')2 fragments which can be
produced by Pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of F(ab')2 fragments. Alternatively, Fab expression libraries may
be constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 246:1275-1281).
[0094] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding and immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between the protein and its
specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering
protein epitopes are preferred, but a competitive binding assay may
also be employed (Maddox, supra).
[0095] In another embodiment of the invention, the polynucleotides
or fragments thereof or nucleic acid effector/modulator molecules
such as antisense molecules, aptamers, RNAi molecules or ribozymes
may be used for therapeutic purposes. In one aspect, aptamers, i.e.
nucleic acid molecules, which are capable of binding to a protein
of the invention and modulating its activity, may be generated by a
screening and selection procedure involving the use of
combinatorial nucleic acid libraries.
[0096] In a further aspect, antisense molecules may be used in
situations in which it would be desirable to block the
transcription of the mRNA. In particular, cells may be transformed
with sequences complementary to polynucleotides encoding DG001 and
homologous proteins. Thus, antisense molecules may be used to
modulate/effect protein activity or to achieve regulation of gene
function. Such technology is now well known in the art, and sense
or antisense oligomers or larger fragments, can be designed from
various locations along the coding or control regions of sequences
encoding the proteins. Expression vectors derived from
retroviruses, adenovirus, herpes or vaccinia viruses or from
various bacterial plasmids may be used for delivery of nucleotide
sequences to the targeted organ, tissue or cell population.
Methods, which are well known to those skilled in the art, can be
used to construct recombinant vectors, which will express antisense
molecules complementary to the polynucleotides of the genes
encoding the proteins of the invention and homologous proteins.
These techniques are described both in Sambrook et al. (supra) and
in Ausubel et al. (supra). Genes encoding the proteins of the
invention and homologous proteins can be turned off by transforming
a cell or tissue with expression vectors, which express high levels
of polynucleotides that encode the proteins of the invention and
homologous proteins or fragments thereof. Such constructs may be
used to introduce untranslatable sense or antisense sequences into
a cell. Even in the absence of integration into the DNA, such
vectors may continue to transcribe RNA molecules until they are
disabled by endogenous nucleases. Transient expression may last for
a month or, more with a non-replicating vector and even longer if
appropriate replication elements are part of the vector system.
[0097] As mentioned above, modifications of gene expression can be
obtained by designing antisense molecules, e.g. DNA, RNA or nucleic
acid analogues such as PNA, to the control regions of the genes
encoding DG001 and homologous proteins, i.e., the promoters,
enhancers, and introns. Oligonucleotides derived from the
transcription initiation site, e.g., between positions -10 and +10
from the start site, are preferred. Similarly, inhibition can be
achieved using "triple helix" base-pairing methodology. Triple
helix pairing is useful because it cause inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature (Gee, J. E. et al., (1994) Gene 149: 109-114; Huber, B.
E. and Carr B. I., Molecular and Immunologic Approaches, Futura
Publishing Co., Mt. Kisco, N.Y.). The antisense molecules may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0098] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples, which may be used, include engineered
hammerhead motif ribozyme molecules that can be specifically and
efficiently catalyze endonucleolytic cleavage of sequences encoding
the proteins of the invention and homologous proteins. Specific
ribozyme cleavage sites within any potential RNA target are
initially identified by scanning the target molecule for ribozyme
cleavage sites which include the following sequences: GUA, GUU, and
GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides corresponding to the region of the target gene
containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0099] Nucleic acid effector/modulator molecules, e.g. antisense
molecules and ribozymes may be prepared by any method known in the
art for the synthesis of nucleic acid molecules. These include
techniques for chemically synthesizing oligonucleotides such as
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences. Such DNA sequences may be incorporated into a
variety of vectors with suitable RNA polymerase promoters such as
T7 or SP6. Alternatively, these cDNA constructs that synthesize
antisense RNA constitutively or inducibly can be introduced into
cell lines, cells or tissues. RNA molecules may be modified to
increase intracellular stability and half-life. Possible
modifications include, but are not limited to, the addition of
flanking sequences at the 5' and/or 3' ends of the molecule or
modifications in the nucleobase, sugar and/or phosphate moieties,
e.g. the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the backbone of the molecule.
This concept is inherent in the production of PNAs and can be
extended in all of these molecules by the inclusion of
non-traditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0100] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection
and by liposome injections may be achieved using methods, which are
well known in the art. Any of the therapeutic methods described
above may be applied to any suitable subject including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0101] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of DG001 nucleic acids and the proteins and homologous
nucleic acids or proteins, antibodies to the proteins of the
invention and homologous proteins, mimetics, agonists, antagonists
or inhibitors of the proteins of the invention and homologous
proteins or nucleic acids. The compositions may be administered
alone or in combination with at least one other agent, such as
stabilizing compound, which may be administered in any sterile,
biocompatible pharmaceutical carrier, including, but not limited
to, saline, buffered saline, dextrose, and water. The compositions
may be administered to a patient alone or in combination with other
agents, drugs or hormones. The pharmaceutical compositions utilized
in this invention may be administered by any number of routes
including, but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral,
topical, sublingual or rectal means.
[0102] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries, which facilitate
processing of the active compounds into preparations, which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0103] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art. For any compounds, the
therapeutically effective dose can be estimated initially either in
cell culture, assays, e.g., of preadipocyte cell lines or in animal
models, usually mice, rabbits, dogs or pigs. The animal model may
also be used to determine the appropriate concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans. A
therapeutically effective dose refers to that amount of active
ingredient, for example the DG001 nucleic acids or proteins or
fragments thereof or antibodies, which is sufficient for treating a
specific condition. Therapeutic efficacy and toxicity may be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED50 (the dose therapeutically
effective in 50% of the population) and LD50 (the dose lethal to
50% of the population). The dose ratio between therapeutic and
toxic effects is the therapeutic index, and it can be expressed as
the ratio, LD50/ED50. Pharmaceutical compositions, which exhibit
large therapeutic indices, are preferred. The data obtained from
cell culture assays and animal studies is used in formulating a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage from
employed, sensitivity of the patient, and the route of
administration. The exact dosage will be determined by the
practitioner, in light of factors related to the subject that
requires treatment. Dosage and administration are adjusted to
provide sufficient levels of the active moiety or to maintain the
desired effect. Factors, which may be taken into account, include
the severity of the disease state, general health of the subject,
age, weight, and gender of the subject, diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long-acting pharmaceutical
compositions may be administered every 3 to 4 days, every week or
once every two weeks depending on half-life and clearance rate of
the particular formulation. Normal dosage amounts may vary from 0.1
to 100,000 .mu.g, up to a total dose of about 1 g, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
employ different formulations for nucleotides than for proteins or
their inhibitors. Similarly, delivery of polynucleotides or
polypeptides will be specific to particular cells, conditions,
locations, etc.
[0104] In another embodiment, antibodies which specifically bind to
the proteins may be used for the diagnosis of conditions or
diseases characterized by or associated with over- or
under-expression of the proteins of the invention and homologous
proteins or in assays to monitor patients being treated with the
proteins of the invention and homologous proteins, or
effectors/modulators thereof, e.g. agonists, antagonists, or
inhibitors. Diagnostic assays include methods which utilize the
antibody and a label to detect the protein in human body fluids or
extracts of cells or tissues. The antibodies may be used with or
without modification, and may be labeled by joining them, either
covalently or non-covalently, with a reporter molecule. A wide
variety of reporter molecules, which are known in the art may be
used several of which are described above.
[0105] A variety of protocols including ELISA, RIA, and FACS for
measuring proteins are known in the art and provide a basis for
diagnosing altered or abnormal levels of gene expression. Normal or
standard values for gene expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibodies to the protein under conditions
suitable for complex formation. The amount of standard complex
formation may be quantified by various methods, but preferably by
photometric means. Quantities of protein expressed in control and
disease, samples e.g. from biopsied tissues are compared with the
standard values. Deviation between standard and subject values
establishes the parameters for diagnosing disease.
[0106] In another embodiment of the invention, the polynucleotides
specific for the DG001 proteins and homologous proteins may be used
for diagnostic purposes. The polynucleotides, which may be used,
include oligonucleotide sequences, antisense RNA and DNA molecules,
and PNAs. The polynucleotides may be used to detect and quantitate
gene expression in biopsied tissues in which gene expression may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess gene expression,
and to monitor regulation of protein levels during therapeutic
intervention.
[0107] In one aspect, hybridization with probes which are capable
of detecting polynucleotide sequences, including genomic sequences,
encoding the proteins of the invention and homologous proteins or
closely related molecules, may be used to identify nucleic acid
sequences which encode the respective protein. The hybridization
probes of the subject invention may be DNA or RNA and are
preferably derived from the nucleotide sequence of the
polynucleotide encoding the proteins of the invention or from a
genomic sequence including promoter, enhancer elements, and introns
of the naturally occurring gene. Hybridization probes may be
labeled by a variety of reporter groups; for example, radionuclides
such as 32P or 35S or enzymatic labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
[0108] Polynucleotide sequences specific for DG001 proteins and
homologous nucleic acids may be used for the diagnosis of
conditions or diseases, which are associated with the expression of
the proteins. Examples of such diseases include the pancreatic
diseases (e.g. diabetes), obesity, metabolic syndrome, and/or
others. Polynucleotide sequences specific for the DG001 proteins
and homologous proteins may also be used to monitor the progress of
patients receiving treatment for pancreatic diseases (e.g.
diabetes), obesity, and/or metabolic syndrome. The polynucleotide
sequences may be used qualitative or quantitative assays, e.g. in
Southern or Northern analysis, dot blot or other membrane-based
technologies; in PCR technologies; or in dip stick, pin, ELISA or
chip assays utilizing fluids or tissues from patient biopsies to
detect altered gene expression.
[0109] In a particular aspect, the DG001 nucleotide sequences may
be useful in assays that detect activation or induction of various
metabolic diseases or dysfunctions. The nucleotide sequences may be
labeled by standard methods, and added to a fluid or tissue sample
from a patient under conditions suitable for the formation of
hybridization complexes. After a suitable incubation period, the
sample is washed and the signal is quantitated and compared with a
standard value. The presence of altered levels of nucleotide
sequences encoding the proteins of the invention and homologous
proteins in the sample indicates the presence of the associated
disease. Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies, in
clinical trials or in monitoring the treatment of an individual
patient.
[0110] In order to provide a basis for the diagnosis of a disease
associated with expression of the DG001 proteins and homologous
proteins, a normal or standard profile for expression is
established. This may be accomplished by combining body fluids or
cell extracts taken from normal subjects, either animal or human,
with a sequence or a fragment thereof, which is specific for the
nucleic acids encoding the proteins of the invention and homologous
nucleic acids, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease. Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that, which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0111] With respect to pancreatic diseases (e.g. diabetes),
obesity, and/or metabolic syndrome, the presence of an unusual
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease or may
provide a means for detecting the disease prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this
type may allow health professionals to employ preventative measures
or aggressive treatment earlier thereby preventing the development
or further progression of the metabolic diseases and disorders.
[0112] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding the proteins of the invention and
homologous proteins may involve the use of PCR. Such oligomers may
be chemically synthesized, generated enzymatically or produced from
a recombinant source. Oligomers will preferably consist of two
nucleotide sequences, one with sense orientation
(5prime.fwdarw.3prime) and another with antisense
(3prime.rarw.5prime), employed under optimized conditions for
identification of a specific gene or condition. The same two
oligomers, nested sets of oligomers or even a degenerate pool of
oligomers may be employed under less stringent conditions for
detection and/or quantification of closely related DNA or RNA
sequences.
[0113] In another embodiment of the invention, the nucleic acid
sequences may also be used to generate hybridization probes, which
are useful for mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome or to a
specific region of the chromosome using well known techniques. Such
techniques include FISH, FACS or artificial chromosome
constructions, such as yeast artificial chromosomes, bacterial
artificial chromosomes, bacterial P1 constructions or single
chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood
Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.
FISH (as described in Verma et al. (1988) Human Chromosomes: A
Manual of Basic Techniques, Pergamon Press, New York, N.Y.). The
results may be correlated with other physical chromosome mapping
techniques and genetic map data. Examples of genetic map data can
be found in the 1994 Genome Issue of Science (265:1981f).
Correlation between the location of the gene encoding the proteins
of the invention on a physical chromosomal map and a specific
disease or predisposition to a specific disease, may help to
delimit the region of DNA associated with that genetic disease.
[0114] The nucleotide sequences of the subject invention may be
used to detect differences in gene sequences between normal,
carrier or affected individuals. An analysis of polymorphisms, e.g.
single nucleotide polymorphisms may be carried out. Further, in
situ hybridization of chromosomal preparations and physical mapping
techniques such as linkage analysis using established chromosomal
markers may be used for extending genetic maps. Often the placement
of a gene on the chromosome of another mammalian species, such as
mouse, may reveal associated markers even if the number or arm of a
particular human chromosome is not known. New sequences can be
assigned to chromosomal arms or parts thereof, by physical mapping.
This provides valuable information to investigators searching for
disease genes using positional cloning or other gene discovery
techniques. Once the disease or syndrome has been crudely localized
by genetic linkage to a particular genomic region, for example, AT
to 11q22-23 (Gatti, R. A. et al. (1988) Nature 336: 577-580), any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. The nucleotide
sequences of the subject invention may also be used to detect
differences in the chromosomal location due to translocation,
inversion, etc. among normal, carrier or affected individuals.
[0115] In another embodiment of the invention, the proteins of the
invention, their catalytic or immunogenic fragments or
oligopeptides thereof, an in vitro model, a genetically altered
cell or animal, can be used for screening libraries of compounds in
any of a variety of drug screening techniques. One can identify
effectors, e.g. receptors, enzymes, proteins, ligands, or
substrates that bind to, modulate or mimic the action of one or
more of the DG001 proteins of the invention. The protein or
fragment thereof employed in such screening may be free in
solution, affixed to a solid support, borne on a cell surface, or
located intracellulary. The formation of binding complexes, between
the DG001 proteins of the invention and the agent tested, may be
measured. Agents could also, either directly or indirectly,
influence the activity of the proteins of the invention.
[0116] In addition activity of the proteins of the invention
against their physiological substrate(s) or derivatives thereof
could be measured in cell-based or cell-free assays. Agents may
also interfere with posttranslational modifications of the protein,
such as phosphorylation and dephosphorylation, farnesylation,
palmitoylation, acetylation, alkylation, ubiquitination,
proteolytic processing, subcellular localization and degradation.
Moreover, agents could influence the dimerization or
oligomerization of the proteins of the invention or, in a
heterologous manner, of the proteins of the invention with other
proteins, for example, but not exclusively, docking proteins,
enzymes, receptors, or translation factors. Agents could also act
on the physical interaction of the proteins of this invention with
other proteins, which are required for protein function, for
example, but not exclusively, their downstream signaling.
[0117] Methods for determining protein-protein interaction are well
known in the art. For example binding of a fluorescently labeled
peptide derived from the interacting protein to the DG001 protein
of the invention, or vice versa, could be detected by a change in
polarisation. In case that both binding partners, which can be
either the full length proteins as well as one binding partner as
the full length protein and the other just represented as a peptide
are fluorescently labeled, binding could be detected by
fluorescence energy transfer (FRET) from one fluorophore to the
other. In addition, a variety of commercially available assay
principles suitable for detection of protein-protein Interaction
are well known In the art, for example but not exclusively,
AlphaScreen (PerkinElmer) or Scintillation Proximity Assays (SPA)
by Amersham. Alternatively, the interaction of the DG001 proteins
of the invention with cellular proteins could be the basis for a
cell-based screening assay, in which both proteins are
fluorescently labeled and interaction of both proteins is detected
by analysing cotranslocation of both proteins with a cellular
imaging reader, as has been developed for example, but not
exclusively, by Cellomics or EvotecOAl. In all cases the two or
more binding partners can be different proteins with one being the
protein of the invention, or in case of dimerization and/or
oligomerization the protein of the invention itself.
[0118] Of particular interest are screening assays for agents that
have a low toxicity for mammalian cells. The term "agent" as used
herein describes any molecule, e.g. protein or pharmaceutical, with
the capability of altering or mimicking the physiological function
of one or more of the proteins of the invention. Candidate agents
encompass numerous chemical classes, though typically they are
organic molecules, preferably small organic compounds having a
molecular weight of more than 50 and less than about 2,500 Daltons.
Candidate agents comprise functional groups necessary for
structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The candidate agents often comprise
carbocyclic or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups.
[0119] Candidate agents are also found among biomolecules including
peptides, saccharides, fatty acids, steroids, purines, pyrimidines,
nucleic acids and derivatives, structural analogs or combinations
thereof. Candidate agents are obtained from a wide variety of
sources including libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts are available
or readily produced. Additionally, natural or synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. Known pharmacological
agents may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs. Where the
screening assay is a binding assay, one or more of the molecules
may be joined to a label, where the label can directly or
indirectly provide a detectable signal.
[0120] Another technique for drug screening, which may be used,
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO 84/03564. In this method, as applied
to the proteins of the invention large numbers of different small
test compounds, e.g. aptamers, peptides, low-molecular weight
compounds etc., are provided or synthesized on a solid substrate,
such as plastic pins or some other surface. The test compounds are
reacted with the proteins or fragments thereof, and washed. Bound
proteins are then detected by methods well known in the art.
Purified proteins can also be coated directly onto plates for use
in the aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support. In another embodiment, one may
use competitive drug screening assays in which neutralizing
antibodies capable of binding the protein specifically compete with
a test compound for binding the protein. In this manner, the
antibodies can be used to detect the presence of any peptide, which
shares one or more antigenic determinants with the protein.
[0121] Compounds that bind DG001 proteins, e.g. antibodies, are
useful for the identification or enrichment of cells, which are
positive for the expression of the proteins of the invention, from
complex cell mixtures. Such cell populations are useful in
transplantation, for experimental evaluation, and as source of
lineage and cell specific products, including mRNA species useful
in identifying genes specifically expressed in these cells, and as
target for the identification of factors of molecules that can
affect them. Cells expressing the protein of the invention or which
have been treated with the protein of the invention are useful in
transplantation to provide a recipient with pancreatic islet cells,
including insulin producing beta cells; for drug screening;
experimental models of islet differentiation and interaction with
other cell types; in vitro screening assays to define growth and
differentiation factors, and to additionally characterize genes
involved in islet development and regulation; and the like. The
native cells may be used for these purposes, or they may be
genetically modified to provide altered capabilities. Cells from a
regenerating pancreas, from embryonic foregut, stomach and
duodenum, or other sources of pancreatic progenitor cells may be
used as a starting population. The progenitor cells may be obtained
from any mammalian species, e.g. equine, bovine, porcine, canine,
feline, rodent, e.g. mice, rats, hamster, primate, etc.
particularly human.
[0122] In another embodiment, in a high-throughput screening
method, the cells are transfected with a DNA construct, e.g. a
viral or non-viral vector containing a reporter gene, e.g. the lacZ
gene or the GFP gene, under regulatory control of a promoter of a
gene involved in for example beta-cell differentiation, e.g. a
promoter of a gene stimulation beta-cell differentiation,
preferably a Pax4 promoter. The transfected cells are divided into
aliquots and each aliquot is contacted with a test substance, e.g.,
DG001. The activity of the reporter gene corresponds to the
capability of the test compound to induce beta-cell
differentiation.
[0123] In a further embodiment, which may be combined with the
high-throughput screening as described above, a medium throughput
validation is carried out. Therein, the test compound is added to
stem cells being cultivated and the insulin production, is
determined. Following an initial high throughput assay, such as the
cell based assay outlined above where for example a Pax4 promoter
is used as marker for beta-cell regeneration, the activity of
candidate molecules to induce beta-cell differentiation is tested
in a validation assay comprising adding said compounds to the
culture media of the embryoid bodies. Differentiation into
insulin-producing cells is then evaluated, e.g. by comparison to
wild type and/or Pax4 expressing ES cells to assess the
effectiveness of a compound.
[0124] The nucleic acids encoding the DG001 proteins of the
invention can be used to generate transgenic cell lines and
animals. These transgenic non-human animals are useful in the study
of the function and regulation of the proteins of the invention in
vivo. Transgenic animals, particularly mammalian transgenic
animals, can serve as a model system for the investigation of many
developmental and cellular processes common to humans. A variety of
non-human models of metabolic disorders can be used to test
modulators of the protein of the invention. Misexpression (for
example, over-expression or lack of expression) of the protein of
the invention, particular feeding conditions, and/or administration
of biologically active compounds can create models of metabolic
disorders.
[0125] In one embodiment of the invention, such assays use mouse
models of insulin resistance and/or diabetes, such as mice carrying
gene knockouts in the leptin pathway (for example, ob (leptin) or
db (leptin receptor) mice), as described above. In addition to
testing the expression of the proteins of the invention in such
mouse strains (see Examples), these mice could be used to test
whether administration of a candidate modulator alters for example
lipid accumulation in the liver, in plasma, or adipose tissues
using standard assays well known in the art, such as FPLC,
colorimetric assays, blood glucose level tests, insulin tolerance
tests and others.
[0126] Transgenic animals may be made through homologous
recombination in non-human embryonic stem cells, where the normal
locus of the gene encoding the protein of the invention is altered.
Alternatively, a nucleic acid construct encoding the protein of the
invention is injected into oocytes and is randomly integrated into
the genome. Vectors for stable integration include plasmids,
retroviruses and other animal viruses, yeast artificial chromosomes
(YACs), and the like. The modified cells or animals are useful in
the study of the function and regulation of the protein of the
invention. For example, a series of small deletions and/or
substitutions may be made in the gene that encodes the protein of
the invention to determine the role of particular domains of the
protein, functions in pancreatic differentiation, etc.
[0127] Furthermore, variants of the genes of the invention like
specific constructs of interest include anti-sense molecules, which
will block the expression of the protein of the invention, or
expression of dominant negative mutations, which will block or
alter the expression of the proteins of the invention may be
randomly integrated into the genome. A detectable marker, such as
lac Z or luciferase may be introduced into the locus of the genes
of the invention, where upregulation of expression of the genes of
the invention will result in an easily detected change in
phenotype.
[0128] One may also provide for expression of the genes of the
invention or variants thereof in cells or tissues where it is not
normally expressed or at abnormal times of development. In
addition, by providing expression of the protein of the invention
in cells in which they are not normally produced, one can induce
changes in cell behavior.
[0129] DNA constructs for homologous recombination will comprise at
least portions of the genes of the invention with the desired
genetic modification, and will include regions of homology to the
target locus. DNA constructs for random integration do not need to
contain regions of homology to mediate recombination. Conveniently,
markers for positive and negative selection are included. DNA
constructs for random integration will consist of the nucleic acids
encoding the proteins of the invention, a regulatory element
(promoter), an intron and a poly-adenylation signal. Methods for
generating cells having targeted gene modifications through
homologous recombination are known in the art. For non-human
embryonic stem (ES) cells, an ES cell line may be employed, or
embryonic cells may be obtained freshly from a host, e.g. mouse,
rat, guinea pig, etc. Such cells are grown on an appropriate
fibroblast-feeder layer and are grown in the presence of leukemia
inhibiting factor (LIF).
[0130] When non-human ES or embryonic cells or somatic pluripotent
stem cells have been transfected, they may be used to produce
transgenic animals. After transfection, the cells are plated onto a
feeder layer in an appropriate medium. Cells containing the
construct may be selected by employing a selection medium. After
sufficient time for colonies to grow, they are picked and analyzed
for the occurrence of homologous recombination or integration of
the construct. Colonies that are positive may then be used for
embryo manipulation and morula aggregation. Briefly, morulae are
obtained from 4 to 6 week old superovulated females, the Zona
Pellucida is removed and the morulae are put into small depressions
of a tissue culture dish. The ES cells are trypsinized, and the
modified cells are placed into the depression closely to the
morulae. On the following day the aggregates are transferred into
the uterine horns of pseudopregnant females. Females are then
allowed to go to term. Chimeric offsprings can be readily detected
by a change in coat color and are subsequently screened for the
transmission of the mutation into the next generation
(F1-generation). Offspring of the F1-generation are screened for
the presence of the modified gene and males and females having the
modification are mated to produce homozygous progeny. If the gene
alterations cause lethality at some point in development; tissues
or organs can be maintained as allogenic or congenic grafts or
transplants, or in vitro culture. The transgenic animals may be any
non-human mammal, such as laboratory animal, domestic animals,
etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and
others. The transgenic animals may be used in functional studies,
drug screening, and other applications and are useful in the study
of the function and regulation of the proteins of the invention in
vivo.
[0131] Finally, the invention also relates to a kit comprising at
least one of [0132] (a) a nucleic acid molecule coding for a
protein of the invention or a functional fragment thereof; [0133]
(b) a protein of the invention or a fragment or an isoform thereof;
[0134] (c) a vector comprising the nucleic acid of (a); [0135] (d)
a host cell comprising the nucleic acid of (a) or the vector of
(c); [0136] (e) a polypeptide encoded by the nucleic acid of (a) or
the vector of (c); [0137] (f) a fusion polypeptide encoded by the
nucleic acid of (a) or the vector of (c); [0138] (g) an antibody,
an aptamer or another effector/modulator against the nucleic acid
of (a) or the polypeptide of (b), (e) or (f) and [0139] (h) an
anti-sense oligonucleotide of the nucleic acid of (a).
[0140] The kit may be used for diagnostic or therapeutic purposes
or for screening applications as described above. The kit may
further contain user instructions.
[0141] The Figures show:
[0142] FIGS. 1A and 1B together shows human DG001 nucleic acid and
protein.
[0143] FIG. 1A shows the nucleic acid sequence encoding the human
DG001 protein (SEQ ID NO: 1).
[0144] FIG. 1B shows the amino acid sequence (one-letter code) of
human DG001 protein (SEQ ID NO: 2).
[0145] FIGS. 2A-2E together shows the analysis of DG001 protein
expression in mammalian tissues. The relative RNA-expression is
shown on the Y-axis, in FIGS. 2A to 2C the tissues tested are given
on the X-axis. WAT refers to white adipose tissue, BAT refers to
brown adipose tissue. In FIG. 2D, the X-axis represents the time
axis. `d0` refers to day 0 (start of the experiment), `d2`-`d12`
refers to day 2 day 12 of adipocyte differentiation).
[0146] FIG. 2A shows the quantitative (real-time PCR) analysis of
DG001 expression in wild-type mouse tissues.
[0147] FIG. 2B shows the quantitative (real-time PCR) analysis of
DG001 expression in genetically obese mice (ob/ob-mice) and fasted
mice (fasted-mice) compared to wild-type mice (wt-mice).
[0148] FIG. 2C shows the quantitative (real-time PCR) analysis of
DG001 expression in mice fed with a high fat diet compared to mice
fed with a control diet.
[0149] FIG. 2D shows the quantitative (real-time PCR) analysis of
DG001 expression in mammalian fibroblast (3T3-L1) cells, during the
differentiation from preadipocytes to mature adipocytes.
[0150] FIG. 2E shows the microarray analysis of DG001 expression in
mammalian fibroblast (3T3-L1) cells, during the differentiation
from preadipocytes to mature adipocytes.
[0151] FIG. 3 shows the DG001 dependent induction of the
differentiation of insulin producing cells.
[0152] Mouse embryonic stem (ES) cells were differentiated as
described previously (patent application PCT/EP02/04362, published
as WO 02/086107, which is incorporated herein by reference). At the
end of the differentiation procedure, cells were harvested and
total RNA was isolated. The abundance of insulin mRNA (FIG. 3) was
determined using quantitative RT-PCR in an Applied Biosystems 7000
sequence detection device. Levels were normalized using 18S RNA as
control and a cycle number of 36 as reference. The numbers on the
vertical line refer to the abundance of the indicated transcripts
relative to an abundance for which 36 cycles are necessary for
detection. `R1` refers to unmodified mouse R1 embryonic stem (ES)
cells; `Pax4` refers to R1 mouse embryonic stem (ES) cells stably
transfected with a CMV-Pax4 expression construct; insulin
expression rel. to `.DELTA.Ct36` refers to expression of insulin in
FIG. 3; `control, supernatant 293 cells` refers to the
differentiation protocol as described in Example 6, with the
addition of supernatant of 293 cells without DG001; `DG001-enriched
supernatant` refers to the differentiation protocol as described in
Example 7, with the addition of DG001 enriched supernatant of 293
cells to differentiated cells.
BRIEF DESCRIPTION OF DRAWINGS
[0153] FIGS. 1A and 1B together shows human DG001 nucleic acid and
protein.
[0154] FIG. 1A shows the nucleic acid sequence encoding the human
DG001 protein (SEQ ID NO: 1).
[0155] FIG. 1B shows the amino acid sequence (one-letter code) of
human DG001 protein (SEQ ID NO: 2).
[0156] FIGS. 2A-2E together shows the analysis of DG001 protein
expression in mammalian tissues. The relative RNA-expression is
shown on the Y-axis. In FIGS. 2A to 2C the tissues tested are given
on the X-axis. WAT refers to white adipose tissue, BAT refers to
brown adipose tissue. In FIG. 2D, the X-axis represents the time
axis. `d0` refers to day 0 (start of the experiment), `d2`-`d12`
refers to day 2-day 12 of adipocyte differentiation.
[0157] FIG. 2A shows the quantitative (real-time PCR) analysis of
DG001 expression in wild-type mouse tissues.
[0158] FIG. 2B shows the quantitative (real-time analysis of DG001
expression in genetically obese mice (ob/ob-mice) and fasted mice
(fasted-mice) compared to wild-type mice (wt-mice).
[0159] FIG. 2C shows the quantitative (real-time PCR) analysis of
DG001 expression in mice fed with a high fat diet compared to mice
fed with a control diet.
[0160] FIG. 2D shows the quantitative (real-time PCR) analysis of
DG001 expression in mammalian fibroblast (3T3-L1) cells, during the
differentiation from preadipocytes to mature adipocytes.
[0161] FIG. 2E shows the microarray analysis of DG001 expression in
mammalian fibroblast (3T3-L1) cells, during the differentiation
from preadipocytes to mature adipocytes.
[0162] FIG. 3 shows that the markers for beta-cell differentiation
function were expressed at higher levels in Pax4+ differentiated ES
cells than in differentiated wild type ES cells demonstrating that
activation of a pancreatic developmental control gene renders
differentiation more efficient than for wild type ES cells.
[0163] The examples illustrate the invention:
EXAMPLE 1
Identification of Secreted Factors Expressed in Pancreas
[0164] A screen for secreted factors expressed in developing mouse
pancreas was carried out according to methods known by those
skilled, in the art (see, for example Pera E. M. and De Robertis E.
M., (2000) Mech Dev 96: 183-195) with several modifications.
Expression cDNA Library:
[0165] During organogenesis, the pancreatic bud is surrounded and
influenced by the associated mesenchyme. (see for example, Madsen
O. D. et al., (1996) Eur. J. Biochem. 242: 435-445 and Slack, J.
M., (1995) Development 121: 1569-1580). Recently, it was suggested,
that white adipocytes origin directly from mesenchymal cells
(Atanossova P. K., (2003) Folia Med. 45: 41-45). During
embryogenesis, the innervation and vascularization of the pancreas
can be observed. Therefore, the tissue used in the screen might
have contained besides pancreatic cells some adipocyte precursors,
blood vessels, as well as neuronal cells.
[0166] A mouse embryonic stage 9.5-15 pancreatic bud library was
prepared in pCMVSPORT-6 vector using SUPERSCRIPT Plasmid System
from Invitrogen according to the manufacturer's instructions. The
non-amplified library was electroporated into MaxEff DH10B cells
(Invitrogen).
Secretion Cloning
[0167] Bacterial clones were picked with sterile toothpicks from
agar plates and cultured in 96-deep-well microtiter plates in
LB-ampicillin (see Sambrook et al., supra). Aliquots of 8 cultures
were pooled, and plasmid DNA was isolated using the BioRobot-9600
apparatus according to the manufacturer's instructions (Qiagen;
QIAprep(r) Turbo BioRobot Kit. Human 293 cell culture cells were
cultured in 75 ml tissue culture flasks in DMEM and 10% fetal calf
serum. At 90-99% confluence, the cells were splitted at 1:3 ratio
and plated onto poly-D-lysine (Sigma) coated 96-well plates. Cells
were transfected with 100-500 ng plasmid using lipofectamine 2000
(Invitrogen). After 6 hours, the medium was exchanged for fresh
complete growth medium. 24 hours after transfection, the cells were
washed twice with DMEM without cysteine and methionine
(Invitrogen), supplemented with 1% dialysed Bovine serum (Sigma)
with 50 microgram per ml Heparin (Sigma) and glutamine. The cells
were labeled radioactively (`S35 Met-label`, from Hartmann Analytic
GmbH). After 12 hours, aliquots of the supernatants were harvested
in 96-well PCR plates and subjected to SDS gel electrophoresis in
precast 4.A-inverted.20% gradient polyacrylamide Criterion gels
(Biorad) under reducing conditions, using Criterion Dodeca Cell gel
running chamber (Biorad). The gels were fixed in 10% acetic acid,
25% isopropanol for 30 min, soaked 15-30 min in AMPLIFY reagent
(Amersham), dried and exposed to X-OMAT (AR) film (Kodak). Positive
clones were identified and regrown in 96-well-plates. DNA of
individual clones was prepared and used for transfection as
described above. If one of the clones yielded proteins of the same
size as that of the original pool, a positive clone was identified.
Positive clones were partially sequenced from the 5prime end
(SEQLAB, Goettingen).
EXAMPLE 2
Identification of the Human DG001 Homologous Nucleic Acid And
Protein
[0168] The term "polynucleotide comprising the nucleotide sequence
as shown in GenBank Accession number" relates to the expressible
gene of the nucleotide sequences deposited under the corresponding
GenBank Accession number. The term "GenBank Accession number"
relates to NCBI GenBank database entries (Ref.: Benson et al.,
Nucleic Acids Res. 28 (2000) 15-18).
[0169] DG001 homologous proteins and nucleic acid molecules coding
therefore are obtainable from insect or vertebrate species, e.g.
mammals or birds. Particularly preferred are nucleic acids
comprising human DG001 homologs. The following mouse sequence was
identified in the secreted factor screen: GenBank Accession Number
NM-008973 and GenBank Accession Number NP-032999.
[0170] Sequences homologous to mouse DG001 were identified using
the publicly available program BLASTP 2.2.3 of the non-redundant
protein data base of the National Center for Biotechnology
Information (NCBI) (see, Altschul et al., 1997, Nucleic Acids Res.
25: 3389-3402). The best human homolog of mouse DG001 is GenBank
Accession Number NM-002825 (SEQ ID NO: 1; see FIG. 1A) and GenBank
Accession Number NP-002816 (SEQ ID NO: 2; see FIG. 1B).
EXAMPLE 3
Expression of the Polypeptides in Mammalian Tissues
[0171] To analyse the expression of the polypeptides disclosed in
this invention in mammalian tissues, several mouse strains
(preferably mice strains C57B1/6J, C57B1/6 ob/ob and C57B1/KS db/db
which are standard model systems in obesity and diabetes research)
were purchased from Harlan Winkelmann (33178 Borchen, Germany) and
maintained under constant temperature (preferably 22YC), 40 percent
humidity and a light/dark cycle of preferably 14/10 hours. The mice
were fed a standard chow (for example, from ssniff Spezialitaten
GmbH, order number ssniff M-Z V1126-000). For the fasting
experiment ("fasted wild-type mice"), wild-type mice were starved
for 48 h without food, but only water supplied ad libitum (see, for
example, Schnetzler et al., (1993) J Clin Invest 92: 272-280,
Mizuno et al. (1996) Proc Natl Acad Sci 93: 3434-3438). In a
further experiment wild-type (wt) mice were fed a control diet
(preferably Altromin C1057 mod control, 4.5% crude fat) or high fat
diet (preferably Altromin C1057 mod. high fat, 23.5% crude fat).
Animals, were sacrificed at an age of 6 to 8 weeks. The animal
tissues, were isolated according to standard procedures known to
those skilled in the art, snap frozen in liquid nitrogen and stored
at -80.degree. C. until needed.
[0172] For analyzing the role of the proteins disclosed in this
invention in the in vitro differentiation of different mammalian
cell culture cells for the conversion of pre-adipocytes to
adipocytes, mammalian fibroblast (3T3-L1) cells (e.g., Green &
Kehinde, (1974) Cell 1: 113-116) were obtained from the American
Tissue Culture Collection (ATCC, Hanassas, Va., USA; ATCC-CL 173).
3T3-L1 cells were maintained as fibroblasts and differentiated into
adipocytes as described in the prior art (e.g., Qiu. et al., (2001)
J. Biol. Chem. 276: 11988-11995; Slieker et al., (1998) BBRC 251:
225-229). In brief, cells were plated in DMEM/10% FCS (Invitrogen,
Karlsruhe, Germany) at 50,000 cells/well in duplicates in 6-well
plastic dishes and cultured in a humidified atmosphere of 5% CO2 at
37.degree. C. At confluence (defined as day 0: d0) cells were
transferred to serum-free (SF) medium, containing DMEM/HamF12 (3:1;
Invitrogen), fetuin (300 .mu.g/ml; Sigma, Munich, Germany),
transferrin (2 .mu.g/ml; Sigma), pantothenate (17 .mu.M; Sigma),
biotin (1 .mu.M; Sigma), and EGF (0.8 nM; Hoffmann-La Roche, Basel,
Switzerland). Differentiation was induced by adding dexamethasone
(DEX; 1 .mu.M; Sigma), 3-methyl-isobutyl-1-methylxanthine (MIX; 0.5
mM; Sigma), and bovine insulin (5 .mu.g/ml; Invitrogen). Four days
after confluence (d4), cells were kept in SF medium, containing
bovine insulin (5 .mu.g/ml) until differentiation was completed. At
various time points of the differentiation procedure, beginning
with day 0 (day of confluence) and day 2 (hormone addition; for
example, dexamethasone and 3-isobutyl-1-methylxanthine), up to 10
days of differentiation, suitable aliquots of cells were taken
every two days.
[0173] RNA was isolated from mouse tissues or cell culture cells
using Trizol Reagent (for example, from Invitrogen, Karlsruhe,
Germany) and further purified with the RNeasy Kit (for example,
from Qiagen, Germany) in combination with an DNase-treatment
according to the instructions of the manufacturers and as known to
those skilled in the art. Total RNA was reverse transcribed
(preferably using Superscript II RNaseH--Reverse Transcriptase,
from Invitrogen, Karlsruhe, Germany) and subjected to Taqman
analysis preferably using the Taqman 2.times.PCR Master Mix (from
Applied Biosystems, Weiterstadt, Germany; the Mix contains
according to the Manufacturer for example AmpliTaq Gold DNA
Polymerase, AmpErase UNG, dNTPs with dUTP, passive reference Rox
and optimized buffer components) on a GeneAmp 5700 Sequence
Detection System (from Applied Biosystems, Weiterstadt,
Germany).
[0174] The following primer/probe combinations were used for the
TaqMan analysis (GenBank Accession Number NM-008973 for the mouse
DG001 sequence): Mouse DG001 forward primer (SEQ ID NO: 3): 5'-CAA
GTA CCA GTT CCA GGC TTG G-3'; mouse DG001 reverse primer (SEQ ID
NO: 4): 5'-GCT CGC TTC AGG CTG CC-3'; mouse DG001 Taqman probe (SEQ
ID NO: 5): (5/6-FAM) TGA CCT CAA TAC CGC CTT GAA GAC CAG AAC
(5/6-TAMRA).
[0175] The function of the mammalian DG001 in metabolism was
further validated by analyzing the expression of the transcripts in
different tissues and by analyzing the role in adipocyte
differentiation.
[0176] Expression profiling studies confirm the particular
relevance of DG001 as regulator of energy metabolism in mammals.
Quantitative PCR (Taqman) analysis revealed that DG001 is expressed
in several mammalian tissues, with highest expression levels in
hypothalamus and brain in wild type mice. In 5, addition, DG001 is
expressed in metabolic active tissue such as white adipose tissue
(WAT) and at lower levels in brown adipose tissue (BAT) in wild
type mice as depicted in FIG. 2A.
[0177] We used mouse models of insulin resistance and/or diabetes,
such as mice carrying gene knockouts in the leptin pathway (for
example, ob/ob (leptin) or db (leptin receptor/ligand) mice) to
study the expression of DG001. Such mice develop typical symptoms
of diabetes, show hepatic lipid accumulation and frequently have
increased plasma lipid levels (see Bruning et al, (1998) Mol. Cell.
2: 559-569). We found, for example, that the expression of DG001 is
down-regulated in metabolic active tissue (WAT) in genetically
induced obese mice (ob/ob) compared to wild type mice and strongly
up-regulated in liver in genetically induced obese mice (ob/ob) and
fasted mice compared to wild type mice (see FIG. 2B). Expression of
DG001 mRNA was also examined in susceptible wild type mice (for
example, C57B116) that show symptoms of diabetes, lipid
accumulation, and high plasma lipid levels, if fed a high fat diet.
In those mice, the expression of DG001 is down-regulated in WAT and
up-regulated in spleen and muscle supporting that DG001 is involved
in the regulation of mammalian metabolism (see FIG. 2C).
[0178] We show in this invention that the DG001 mRNA is decreased
at a very early stage of adipose differentiation (until day 4) and
increased during the late stage of differentiation into mature
adipocyctes (FIG. 2D). Therefore, the DG001 protein might play an
essential role in adipogenesis.
EXAMPLE 4
Microarray Analysis of the Differential Expression of Transcripts
of the Proteins of the Invention in Mammalian Tissues
[0179] RNA preparation from murine 3T3-L1 cells was done as
described in Example 3. The target preparation, hybridization, and
scanning was performed as described in the manufactures manual (see
Affymetrix Technical Manual, 2002, obtained from Affinetrix, Santa
Clara, USA).
[0180] The expression analysis (using Affymetrix GeneChips) of the
DG001 gene using 3T3-L1 differentiation clearly shows an
up-regulation of human. DG001 in adipocytes, confirming the 3T3-L1
differentiation data obtained with the TaqMan method described in
Example 3.
EXAMPLE 5
Generation of ES Cells Expressing the Pax4 Gene
[0181] Mouse R1ES cells (Nagy et al., (1993) Proc. Natl. Acad. Sci.
USA 90: 8424-8428) were electroporated with the Pax4 gene under the
control of the CMV promoter and the neomycin resistance gene under
the control of the phosphoglycerate kinase I promoter (pGK-1).
[0182] ES cells were cultured in Dulbecco's modified Eagle's medium
containing 4.5 g/l glucose, 104 M beta-mercaptoethanol, 2 nM
glutamine, 1% non-essential amino acids, 1 nM Na-pyruvate, 20%
fetal calf serum (FCS) and 500 U/ml leukaemia inhibitory factor
(LIF). Briefly, approximately 107 ES cells resuspended in 0.8 ml
phosphate buffered saline (PBS) were subjected to electroporation
with 25 .mu.g/ml of linearized expression vector (Joyner, Gene
Targeting: A Practical Approach, Oxford University Press, New York,
1993). Five minutes after electroporation, ES cells were plated on
petri dishes containing fibroblastic feeder cells previously
inactivated by treatment with 100 .mu.g/ml mitomycin C. One day
after electroporation, culture medium was changed to medium
containing 450 .mu.g/ml G418. Resistant clones were separately
isolated and cultured 14 days after applying the selection medium.
Cells were always cultured at 37.degree. C., 5% CO2. These
untreated and undifferentiated ES cells were used as control the
experiment.
EXAMPLE 6
Differentiation of ES Cells into Insulin-Producing Cells (Referred
to as `Control, Supernatant 293 Cells` in FIG. 3)
[0183] The ES cell line R1 (wild type, `R1` in FIG. 3) and ES cells
constitutively expressing Pax4 (`Pax4` in FIG. 3) were cultivated
as embryoid bodies (EB) by the hanging drop method, as described in
patent application PCT/EP02/04362, published as WO 02/086107, which
is incorporated herein by reference, with media as described below
and in Table 1. The embryoid bodies were allowed to form in hanging
drop cultures for 2 days and then transferred for three days to
suspension cultures in petri dishes. At day 5, EBs were plated
separately onto gelatin-coated 6 cm cell culture dishes containing
a differentiation medium prepared with a base of Iscove modified
Dulbecco's medium. After dissociation and replating at day 14 cells
were cultured up to 40 days in the differentiation medium prepared
with a base of Dulbecco's modified Eagle's medium: Nutrient Mixture
F-12 (DMEM/F12) with the addition of supernatant of 293 cells
without DG001.
EXAMPLE 7
Expression of Pancreas Specific Genes after Differentiation Of ES
Cells into Insulin-Producing Cells
[0184] Expression levels of pancreas specific genes was measured by
Taqman analysis as described in Example 3. Total RNA was isolated
from undifferentiated R1 and Pax4+ ES cells (control ES cells) at
day 0 and differentiated R1ES and Pax4+ ES cells at day 40. RNA
preparation was done as described in Example 3 without using Trizol
reagent.
[0185] Results show that markers for beta-cell differentiation
function were expressed at higher levels in Pax4+ differentiated ES
cells than in differentiated wild type ES cells demonstrating that
activation of a pancreatic developmental control gene renders
differentiation more efficient than for wild type ES cells (FIG.
3). Expression of substantial amounts of insulin in differentiated
stem cells indicates that differentiated cells show a phenotype
similar to beta-cells.
EXAMPLE 8
Induction of Differentiation of Insulin-Producing Cells by DG001
(Referred to as `DG001-Enriched Supernatant` in FIG. 3)
[0186] In order to study the effect of DG001 to induce beta-cell
differentiation in vitro, stable mouse embryonic stem (ES) cells
expressing the Pax4 under the control of the cytomegalovirus (CMV)
early promoter/enhancer region were generated as described in
Example 5. Pax4 and wild type ES cells were then cultured in
hanging drops or spinner cultures to allow the formation of
embryoid bodies. Embryoid bodies were subsequently plated,
enzymatically dissociated, and replated. After dissociation, cells
were cultured in a differentiation medium containing various growth
factors (see Table 1 for more detail). Additionally DG001 enriched
supernatant of 293 cells was added every second day until day 40.
Under such conditions, the expression of insulin was induced by
DG001 (FIG. 3). By comparison, wild type ES cells did contain only
very small numbers of insulin-producing cells at the same stage.
These data demonstrate that DG001 can significantly promote and
enhance ES cells differentiation into insulin-producing cells
compared to wild type ES cells.
[0187] The results shown in FIG. 3 clearly demonstrate a
significant induction of the differentiation of insulin-producing
cells, if DG001 is added on later stages of differentiation. Thus,
DG001 has a strong inductive effect on the differentiation of
insulin-producing beta cells.
[0188] Media B2 and B27 are described in Rolletschek et al., (2001)
Mech. Dev. 105: 93-104.
TABLE-US-00001 Stage of Coating and Day Cultivation Medium Analysis
0 hanging drops Iscove + 20% FCS RNA (ES cells) (600 cells/drop) 1
2 EBs in Iscove + 20% FCS suspension 3 4 5 plating of EBs Iscove +
20% FCS gelatin coating RNA (EBs) +1 ornithine/ +2 laminin coating
+3 +4 +5 +6 +7 +8 +9 dissociation N2 + B27 + NA + 10% FCS RNA (1
.times. 6 cm dish) +10 medium change N2 + B27 + NA + DG001 enriched
supernatant +11 +12 medium change +DG001 enriched supernatant +13
+14 medium change +DG001 enriched supernatant +15 +16 medium change
+DG001 enriched supernatant +17 +18 medium change +DG001 enriched
supernatant +19 +20 medium change +DG001 enriched supernatant +21
+22 medium change +DG001 enriched supernatant +23 +24 medium change
+DG001 enriched supernatant +25 +26 medium change +DG001 enriched
supernatant +27 +28 medium change +DG001 enriched supernatant +29
+30 methum change +DG001 enriched supernatant +31 +32 medium change
+DG001 enriched RNA supernatant
EXAMPLE 9
Functional Characterisation of the Differentiated Insulin-Producing
Cells
[0189] One important property of beta-cells is glucose responsive
insulin secretion. To test whether the Pax4 derived
insulin-producing cells possessed this glucose responsive property,
an in vitro glucose responsive assay can be performed on the
differentiated cells. On the day of the assay, the differentiation
medium of 12 or 6 well plate is removed and the cells are washed 3
times with Krebs Ringer Bicarbonate Hepes Buffer (KRBH; 125 mM
NaCl, 4.74 mM KCl, 1 mM CaCl.sub.2, 1.2 mM KH2PO4, 1.2 mM MgSO4, mM
NaHCO3, 25 mM Hepes, pH 7.4 and 0.1% BSA) supplemented with 2.8 mM
Glucose. For preincubation cells are incubated in KRBH+2.8 mM
Glucose for 2 hours at 37.degree. C. Afterwards cells are incubated
in 500 ml KRBH+2.8 mM Glucose for 1 hour and the supernatant is
then kept for measurement of basal insulin secretion. For the
stimulated insulin release 500 ml KRBH containing 27.7 mM glucose
are added to the cells. After 1 hour incubation at 37.degree. C.,
the supernatant is recovered for measurement of glucose-induced
insulin secretion and the cells were extracted with acid-ethanol
(see also Irminger, J.-C. et al., 2003, Endocrinology 144:
1368-1379). Insulin levels are determined by an Enzyme-Linked
Immunosorbent Assay (ELISA) for mouse insulin (Mercodia) and
performed according to the manufacturer's recommendations.
EXAMPLE 10
Transplantation of Pax4 ES Derived Insulin-Producing Cells In STZ
Diabetic Mice
[0190] The therapeutic potential of DG001 induced insulin-producing
cells to improve and cure diabetes can be investigated by
transplanting the cells into streptozotocin induced diabetic mice.
Streptozotocin is an antibiotic which is cytotoxic to beta-cells
when administered at certain dosage (see Rodrigues et al.:
Streptozotocin-induced diabetes, in McNeill. (ed) Experimental
Models of Diabetes, CRC Press LLC, 1999). Its effect is rapid,
rendering an animal severely diabetic within 48 hours.
[0191] Non-fasted Male BalbC mice can be treated with STZ to
develop hyperglycaemia after STZ treatment. Mice are considered
diabetic if they have a blood glucose level above 10 mmol/l for
more than 3 consecutive days. Cells are transplanted under the
kidney capsule and into the spleen of animals. The presence of the
insulin-producing cells can be confirmed by immunohistological
analysis of the transplanted tissue. Results are expected to
demonstrate that the transplanted cells can normalise blood glucose
in diabetic animals.
[0192] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
511300DNAhumangene(1)..(1300)nucleic acid sequence encoding the
human DG001 protein 1ctctccctcc ctcgcccagc cttcgtcctc ctggcccgct
cctctcatcc ctcccattct 60ccatttccct tccgttccct ccctgtcagg gcgtaattga
gtcaaaggca ggatcaggtt 120ccccgccttc cagtccaaaa atcccgccaa
gagagcccca gagcagagga aaatccaaag 180tggagagagg ggaagaaaga
gaccagtgag tcatccgtcc agaaggcggg gagagcagca 240gcggcccaag
caggagctgc agcgagccgg gtacctggac tcagcggtag caacctcgcc
300ccttgcaaca aaggcagact gagcgccaga gaggacgttt ccaactcaaa
aatgcaggct 360caacagtacc agcagcagcg tcgaaaattt gcagctgcct
tcttggcatt cattttcata 420ctggcagctg tggatactgc tgaagcaggg
aagaaagaga aaccagaaaa aaaagtgaag 480aagtctgact gtggagaatg
gcagtggagt gtgtgtgtgc ccaccagtgg agactgtggg 540ctgggcacac
gggagggcac tcggactgga gctgagtgca agcaaaccat gaagacccag
600agatgtaaga tcccctgcaa ctggaagaag caatttggcg cggagtgcaa
ataccagttc 660caggcctggg gagaatgtga cctgaacaca gccctgaaga
ccagaactgg aagtctgaag 720cgagccctgc acaatgccga atgccagaag
actgtcacca tctccaagcc ctgtggcaaa 780ctgaccaagc ccaaacctca
agcagaatct aagaagaaga aaaaggaagg caagaaacag 840gagaagatgc
tggattaaaa gatgtcacct gtggaacata aaaaggacat cagcaaacag
900gatcagttaa ctattgcatt tatatgtacc gtaggctttg tattcaaaaa
ttatctatag 960ctaagtacac aataagcaaa aacaaaaaga aaagaaaatt
tttgtagtag cgttttttaa 1020atgtatacta tagtaccagt aggggcttat
aataaaggac tgtaatctta tttaggaagt 1080tgacttatag tacatgataa
atgatagaca attgaggtaa gttttttgaa attatgtgac 1140attttacatt
aaattttttt tacatttttt gggcagcaat ttaaatgtta tgactatgta
1200aactacttct cttgttaggt aatttttttc acctagattt ttttcccaat
tgagaaaaat 1260atatactaaa caaaaaaaaa aaaaaaaaaa aaaaaaaaaa
13002168PRThumanamino acid sequence of human DG001 protein 2Met Gln
Ala Gln Gln Tyr Gln Gln Gln Arg Arg Lys Phe Ala Ala Ala1 5 10 15Phe
Leu Ala Phe Ile Phe Ile Leu Ala Ala Val Asp Thr Ala Glu Ala 20 25
30Gly Lys Lys Glu Lys Pro Glu Lys Lys Val Lys Lys Ser Asp Cys Gly
35 40 45Glu Trp Gln Trp Ser Val Cys Val Pro Thr Ser Gly Asp Cys Gly
Leu 50 55 60Gly Thr Arg Glu Gly Thr Arg Thr Gly Ala Glu Cys Lys Gln
Thr Met65 70 75 80Lys Thr Gln Arg Cys Lys Ile Pro Cys Asn Trp Lys
Lys Gln Phe Gly 85 90 95Ala Glu Cys Lys Tyr Gln Phe Gln Ala Trp Gly
Glu Cys Asp Leu Asn 100 105 110Thr Ala Leu Lys Thr Arg Thr Gly Ser
Leu Lys Arg Ala Leu His Asn 115 120 125Ala Glu Cys Gln Lys Thr Val
Thr Ile Ser Lys Pro Cys Gly Lys Leu 130 135 140Thr Lys Pro Lys Pro
Gln Ala Glu Ser Lys Lys Lys Lys Lys Glu Gly145 150 155 160Lys Lys
Gln Glu Lys Met Leu Asp 165322DNAArtificial SequenceDescription of
Artificial Sequence primer 5`-3 3caagtaccag ttccaggctt gg
22417DNAArtificial SequenceDescription of Artificial Sequence
primer 5`-3 4gctcgcttca ggctgcc 17530DNAArtificial
SequenceDescription of Artificial Sequence probe 5tgacctcaat
accgccttga agaccagaac 30
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