U.S. patent application number 10/580601 was filed with the patent office on 2008-10-02 for method for preventing and treating diabetes using neurturin.
Invention is credited to Matthias Austen, Ulrike Burk.
Application Number | 20080241106 10/580601 |
Document ID | / |
Family ID | 34626383 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241106 |
Kind Code |
A1 |
Austen; Matthias ; et
al. |
October 2, 2008 |
Method for Preventing and Treating Diabetes Using Neurturin
Abstract
The present invention relates generally to methods for
preventing and/or treating pancreatic disorders, particularly those
related to diabetes, by administering a neurturin protein
product.
Inventors: |
Austen; Matthias;
(Gottingen, DE) ; Burk; Ulrike; (Gottingen,
DE) |
Correspondence
Address: |
Millen, White, Zelano & Branigan
Arlington Courthouse Plaza 1, 2200 Clarendon Boulevard, Suite 1400
Arlington
VA
22201
US
|
Family ID: |
34626383 |
Appl. No.: |
10/580601 |
Filed: |
November 29, 2004 |
PCT Filed: |
November 29, 2004 |
PCT NO: |
PCT/EP04/13534 |
371 Date: |
May 25, 2006 |
Current U.S.
Class: |
424/93.7 ;
435/325; 435/366 |
Current CPC
Class: |
C12N 2506/02 20130101;
C12N 5/0676 20130101; C12N 2501/13 20130101; A61P 3/10 20180101;
A61K 45/06 20130101; A61K 38/1709 20130101; A61P 3/08 20180101;
G01N 33/507 20130101; A61K 38/185 20130101 |
Class at
Publication: |
424/93.7 ;
435/366; 435/325 |
International
Class: |
C12N 5/08 20060101
C12N005/08; C12N 5/06 20060101 C12N005/06; A61K 35/39 20060101
A61K035/39 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2003 |
EP |
03027383.3 |
Claims
1. Use of a neurturin product and/or a modulator/effector thereof
for the manufacture of a medicament to stimulate and/or induce the
differentiation of insulin producing cells from progenitor
cells.
2. The use of claim 1, wherein the progenitor cells are stem
cells.
3. The use of claim 1, wherein the stem cells are embryonic or
somatic stem cells.
4. The use of claim 1, wherein the stem cells are of mammalian
origin, preferably of human origin, with the proviso that the use
of human embryos is excluded.
5. The use of claim 1, wherein the progenitor cells have been
transfected with a pancreatic gene, particularly the Pax4 gene.
6. The use of a neurturin product and/or a modulator/effector
thereof for the manufacture of a medicament to promote the
protection, survival and/or regeneration of insulin producing
cells.
7. The use of claim 6, wherein the insulin producing cells are
beta-cells.
8. The use of claim 6, wherein the insulin producing cells are of
mammalian origin, preferably of human origin.
9. The use of claim 6, wherein the insulin producing cells have
been transfected with a pancreatic gene, particularly the Pax4
gene.
10. The use of claim 1 for the prevention or treatment of a disease
going along with impaired beta-cell function.
11. The use of claim 10 for the treatment of beta-cell degeneration
in patients suffering from diabetes type I, LADA, or progressed
diabetes type II.
12. The use of claim 10 for the prevention of beta-cell
degeneration in patients at risk to develop beta-cell degeneration,
like for example but not limited to patients suffering from
diabetes type I or II, or LADA in early stages.
13. The use of claim 1, wherein a neurturin product or a
modulator/effector thereof that influences the expression level or
function of a neurturin product is administered to a patient (i) as
a pharmaceutical composition e.g. enterally, parenterally or
topically directly to the pancreas, (ii) via implantation of
neurturin protein product expressing cells, and/or (iii) via gene
therapy.
14. The use of claim 13, wherein the neurturin product or
modulator/effector thereof is administered in combination with
another pharmaceutical composition useful to treat beta-cell
degeneration, for example but not limited to hormones, growth
factors, or immune modulating agents.
15. The use of claim 1, wherein the neurturin product is a protein
including purified natural, synthetic or recombinant neurturin and
variants thereof.
16. The use of claim 15 wherein variants are selected from
insertion, substitution, deletion variants and/or chemically
modified derivatives, for example but not limited to hybrids of
neurturin and other TGF-beta proteins preferably from the
GDNF-family.
17. The use of claim 15, wherein the neurturin product is selected
from proteins or peptides substantially homologous to the human
neurturin precursor protein having the amino acid sequence
published as GenBank Accession Number NP.sub.--004549 and/or to the
mature neurturin protein product that results from the cleavage of
the neurturin protein precursor published as GenBank Accession
number NP.sub.--004549.
18. The use of claim 1, wherein the neurturin product is a nucleic
acid, e.g. RNA and/or DNA encoding a neurturin protein product.
19. The use of claim 1, wherein the neurturin product is selected
from neurturin homodimers or heterodimers of a neurturin protein
product and another protein, wherein the other protein preferably
belongs to the GDNF-family or a nucleic acid coding therefor.
20. The use of claim 1, wherein the neurturin product is of
mammalian origin, preferrably human origin.
21. The use of claim 1, wherein the differentiation of progenitor,
e.g. stem cells into insulin-producing cells in vitro comprises a)
optionally activating one or more pancreatic genes in progenitor
cells, b) optionally aggregating said cells to form embryoid
bodies, c) cultivating said cells or embryoid bodies in specific
differentiation media containing neurturin protein product and d)
identifying and optionally selecting insulin-producing cells.
22. The use of claim 21, wherein the neurturin-treated insulin
producing cells are (i) capable of a response to glucose and/or
(ii) capable of expressing glucagon.
23. The use of claim 21, wherein the neurturin-treated insulin
producing cells are capable of normalizing blood glucose levels
after transplantation into mice.
24. The use of claim 1, wherein an effective amount of in vitro
neurturin-treated cells are transplanted to a patient in need.
25. The use of claim 1, comprising a stimulation of neurturin
expression, wherein cells from a patient in need that have been
modified to produce and secrete a neurturin protein product in
vitro are re-implanted into the patient and/or wherein cells of a
patient in need are modified to produce and secrete a neurturin
protein product in vivo.
26. The use of claim 1 in combination with at least one further
other pharmaceutical agent.
27. The use of claim 26 in combination with at least one further
pharmaceutical agent suitable for the treatment or prevention of
pancreatic diseases and/or obesity and/or metabolic syndrome.
28. The use of claim 27 in combination with at least one further
pharmaceutical agent suitable for stimulating and/or inducing the
differentiation of insulin producing cells from progenitor
cells.
29. The use of claim 26 in combination with at least one further
pharmaceutical agent which has an immunosuppressive activity.
30. A method for differentiating or regenerating cells into
functional pancreatic cells, the method comprising: (a) cultivating
cells capable of being differentiated or regenerated into
pancreatic cells in the presence of an effective amount of
neurturin in vitro (b) allowing the cells to develop, to
differentiate and/or to regenerate at least one pancreatic
function; and (c) optionally preparing an effective amount of the
differentiated or regenerated pancreatic cells for transplantation
into a patient in need thereof.
31. The method of claim 30, wherein the patient in need is a human
individual.
32. The method of claim 30, wherein the patient in need has (a)
functionally impaired, (b) reduced numbers and/or (c) functionally
impaired and reduced numbers of pancreatic cells.
33. The method of claim 30, wherein said patient in need is a type
I diabetic patient or type II diabetic patient or LADA patient.
34. The method of claim 30, wherein the pancreatic cells are
insulin-producing cells.
35. The method of claim 30, wherein the pancreatic cells are
beta-cells of the pancreatic islets.
36. The method of claim 30, wherein the cells in step (a) are
selected from embryonic stem cells, adult stem cells, or somatic
stem cells.
37. The method of claim 31, wherein the cells in step (a) are of
mammalian origin, preferably human origin, with the proviso that
the use of human embryos is excluded.
38. The method of claim 30, wherein neurturin is added at
concentrations between 1 ng/ml and 500 ng/ml, preferably between 10
and 100 ng/ml, more preferably at about 50 ng/ml.
39. The method of claim 30, wherein the at least one pancreatic
function is selected from insulin production in response to glucose
and expression of glucagon.
40. A method for differentiating or regenerating cells into
functional pancreatic cells, the method comprising: preparing an
effective amount of a neurturin product or of cells capable of
expressing a neurturin product for administration to a patient in
need thereof.
41. The method of claim 40, wherein the neurturin product is a
neurturin protein product.
42. The method of claim 40, wherein the neurturin product is a
nucleic acid encoding a neurturin protein product.
43. The method of claim 40, wherein cells have been modified to
produce and secrete a neurturin protein product and are prepared
for transplantation into a suitable location in the patient.
44. A cell preparation comprising neurturin-treated functional
pancreatic cells obtainable by the method of claim 30.
45. A cell preparation comprising a neurturin product expressing
cells obtainable by the method of claim 30.
46. The preparation of claim 44, which is a pharmaceutical
composition.
47. The preparation of claim 44 for the treatment or prevention of
pancreatic diseases, particularly diabetes.
48. The preparation of claim 44 for administration by
transplantation or for use in a medical device.
49. The preparation of claim 44, which contains pharmaceutically
acceptable carriers, diluents, and/or additives.
50. The preparation of claim 44, which is a diagnostic
composition.
51. The preparation of claim 44, which is a therapeutic
composition.
52. The preparation of claim 44 for the manufacture of an agent for
the regeneration of pancreatic tissues or cells, particularly
pancreatic beta cells.
53. The preparation of claim 44 for application in vivo.
54. The preparation of claim 44 for application in vitro.
55. A method for identifying and/or characterizing compounds
capable of modulating the differentiation or regeneration of cells
into functional pancreatic, particularly insulin-producing cells
comprising: contacting a compound to be tested with cells under
conditions wherein the cells are capable of being differentiated or
regenerated into functional pancreatic cells in the presence of
neurturin and determining the effect of the compound on the
differentiation process.
56. The method of claim 55 comprising transfecting the cells with a
DNA construct containing a reporter gene under regulatory control
of a gene involved in beta-cell differentiation, contacting said
transfected cells with a compound to be tested and determining the
activity of the reporter gene.
57. The method of claim 54 comprising contacting embryoid bodies
which are cultivated in a differentiation medium enhancing
beta-cell differentiation with a compound to be tested and
determining differentiation into insulin-producing cells.
58. A method for identifying and/or characterizing compounds
capable of modulating the differentiation or regeneration of cells
into functional pancreatic, particularly insulin-producing cells
comprising: contacting a compound to be tested with cells under
conditions wherein the cells are capable of being differentiated or
regenerated into functional pancreatic cells and determining the
effect of the compound on the expression of neurturin.
59. Use of a preparation of neurturin expressing cells for the
treatment and prevention of diabetes.
60. The use of claim 59 for inducing the regeneration of pancreatic
cells.
61. The use of claim 60, wherein pancreatic cells are beta-cells of
the islets.
62. Use of a preparation of neurturin-treated cells for the
treatment and/or prevention of diabetes.
63. The use of claim 62 wherein the cells are differentiated
progenitor cells capable of insulin production.
Description
[0001] The present invention relates generally to methods for
preventing and/or treating pancreatic disorders, particularly those
related to diabetes, by administering a neurturin product.
BACKGROUND OF THE INVENTION
[0002] The pancreas is an exocrine gland that secretes digestive
enzymes directly into the digestive tract as well as an endocrine
gland that secretes hormones into the blood stream. The exocrine
function is assured by acinar and centroacinar cells that secrete
various digestive enzymes via intercalated ducts into 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 main cell types: alpha-,
beta-, delta- and PP-cells (reviewed for example in Kim &
Hebrok, 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.
Recently, cells producing the neuropeptide Ghrelin have been found
in pancreatic islets (Wierup et al., 2002, Regul Pept.
107:63-9).
[0003] Early pancreatic development has been well studied in
different species, including chicken, zebrafish, and mice (for a
detailed review, see Kim & Hebrok, 2001, supra). The pancreas
develops from distinct dorsal and ventral anlagen. Pancreas
development requires specification of the pancreas anlage along
both anterior-posterior and dorsal-ventral axes. A number of
transcription factors, that are critical for proper pancreatic
development have been identified (see Kim & Hebrok, 2001,
supra; Wilson et al., 2003, Mech Dev. 120: 65-80).
[0004] In 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 & Sharma, 2002, J
Pathol. 197: 519-526; Gu et al., 2003, Mech Dev. 120: 35-43). In
adult humans, new beta-cells arise in the vicinity of ducts (Butler
et al., 2003, Diabetes 52: 102-110; Bouwens & Pipeleers 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 et al., 2001, Diabetes 50: 521-533;
lanus et al., 2003, J Clin Invest. 111: 843-850). Pancreatic islet
growth is dynamic and responds to changes in insulin demand, for
example during pregnancy or due to changing body weight during
childhood. In adults, there is a good correlation between body mass
and islet mass (Yoon et al., 2003, J Clin Endocrinol Metab. 88:
2300-2308).
[0005] Pancreatic beta-cells secrete insulin in response to 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 type I or
LADA (latent autoimmune diabetes in adults (Pozzilli & Di
Mario, 2001, Diabetes Care. 8:1460-67) beta-cells are being
destroyed due to autoimmune attack. The amount of insulin produced
by the remaining pancreatic islet cells is too low, resulting in
elevated blood glucose levels (hyperglycemia). In diabetes type II
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) in turn 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 II 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 II diabetics.
[0006] 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.
[0007] In type I diabetics, where beta-cells are being destroyed by
autoimmune attack, treatments have been devised which modulate the
immune system and may be able to stop or strongly reduce islet
destruction (Raz et al., 2001, Lancet 358: 1749-1753; Chatenoud et
al., 2003, Nat Rev Immunol. 3: 123-132; Homann et al., Immunity.
2002, 3:403-15). However, due to the relatively slow regeneration
of human beta-cells such treatments can only be successful if they
are combined with agents that can stimulate beta-cell
regeneration.
[0008] Diabetes is a very disabling disease, because today's common
anti-diabetic drugs do not control blood sugar levels well enough
to completely prevent the occurrence of high and low blood sugar
levels. Out of range blood sugar levels are toxic and cause
long-term complications like for example renopathy, retinopathy,
neuropathy and peripheral vascular disease. There is also a host of
related conditions, such as obesity, hypertension, heart disease
and hyperlipidemia, for which persons with diabetes are
substantially at risk.
[0009] Apart from the impaired quality of life for the patients,
the treatment of diabetes and its long term complications presents
an enormous financial burden to our healthcare systems with rising
tendency. Thus, for the treatment of, type I and type II diabetes
as well as for latent autoimmune diabetes in adults (LADA) there is
a strong need in the art to identify factors that induce
regeneration of pancreatic insulin producing beta-cells. These
factors could restore normal function of the endocrine pancreas
once its function is impaired or event could prevent the
development or progression of diabetes type I, diabetes type II, or
LADA.
[0010] In this invention, we disclose a novel and so far unknown
use for the neurotrophic factor neurturin to stimulate the
formation or regeneration of insulin producing beta-cells and thus,
a use in the treatment and prevention of diabetes.
[0011] Neurotrophic factors are growth factors that regulate the
survival and maintain the phenotypic differentiation of certain
nerve and/or glial cell populations (Varon et al., 1978, Ann. Rev.
Neurosience 1: 327-361; Thoenen et al., Science, 229:238-242,
1985). Nerve growth factor (NGF) was the first neurotrophic factor
to be identified and characterized (Levi-Montalcini et al., 1951,
J. Exp. Zool. 116:321). The second member of this family to be
discovered was brain-derived neurotrophic factor (Leibrock et al.,
1989, Nature 341:149-152).
[0012] Glial-derived neurotrophic factor (GDNF)--a neurotrophic
factor structurally unrelated to NGF--was discovered during a
search for factors crucial to the survival of midbrain dopaminergic
neurons, which degenerate in Parkinson's disease (Lin et al., 1993,
Science 260:1130-2). Sequence analysis revealed it to be a distant
member of the superfamily of transforming growth factor 8
(TGF-beta) factors.
[0013] Another neurotrophic factor that is structurally closely
related to GDNF and unrelated to NGF is neurturin (Kotzbauer et
al., 1996, Nature 384: 467-470). Neurturin, GDNF and two other
related factors (artemin and persephin) define a family of
neurotrophic factors referred to as TGF-beta-related neurotrophins.
These neurotrophic factors promote the survival of various neurons
including peripheral autonomic and sensory neurons as well as
central motor and dopamine neurons, and have been proposed as
therapeutic agents for neurodegenerative diseases (see review by
Takahashi, 2001, Cytokine Growth Factor Rev 12(4):361-73; see also,
for example, U.S. Pat. No. 6,090,778 and EP1005358B1, the
disclosures of which are hereby incorporated by reference).
TGF-beta-related neurotrophins signal through a unique two-receptor
complex consisting of a glycosylphosphatidylinositol-linked cell
surface molecule, the GDNF family receptor alpha (GFRalpha) and
receptor protein tyrosine kinase Ret.
[0014] Apart from the described functions in neuronal tissue
GDNF/RET signalling is crucial for the differentiation of certain
non-neuronal tissues. For example, GFRalpha1 and the Ret are both
necessary receptor components for ureteric bud outgrowth and
subsequent branching in the developing kidney (Cacalano et al.,
1998, Neuron 21:53-62; Tang et al., 1998, J Cell Biol. 142
(5):1337-45). Ret, GFRalpha-1 (the GDNF receptor), and GFRalpha-2
(the Neurturin receptor) are expressed by testicular germ cells,
while GDNF and Neurturin are expressed by Sertoli cells. Both GDNF
and Neurturin stimulate DNA synthesis in spermatogonia.
Furthermore, GFRalpha, GFRalpha ligands and co-receptors are
expressed in germ cell tumors and thus may act as paracrine factors
in spermatogenesis (Viglietto et al., 2000, Int J Oncol.
16(4):689-94).
[0015] Only recently, it was shown that the biology of GDNF
signalling is much more complex than originally assumed. GDNF
family ligands also signal through the neural cell adhesion
molecule NCAM. In cells lacking Ret, GDNF binds with high affinity
to the NCAM and GFRalpha1 complex (see review by Sariola &
Saarma, 2003, J Cell Sci. 116(Pt 19):3855-62). Signalling via the
c-met receptor kinases has also been demonstrated (see Popsueva et
al., 2003, J Cell Biol. 161(1):119-29).
[0016] Although it has been discussed in the prior art that
GDNF/RET signalling is crucial for the differentiation of neuronal
and certain non-neuronal tissues, it has not been disclosed that a
member of family of TGF-beta-related neurotrophins is involved in
the regeneration of pancreatic tissue. We found surprisingly, that
neurturin stimulates the formation or regeneration of insulin
producing pancreatic beta-cells which play an essential role in
diabetes. Thus, in this invention, we disclose the use of neurturin
in the treatment and prevention of diabetes.
SUMMARY OF THE INVENTION
[0017] The present invention relates to new methods for stimulating
and/or inducing the differentiation of progenitor cells, e.g. stem
cells into insulin-producing cells or for promoting the protection,
survival and/or regeneration of insulin producing cells using a
neurturin product and/or a modulator/effector thereof that
influences, particularly increases the expression level or function
of a neurturin protein product.
[0018] Thus, the present invention provides methods for treating
patients suffering from a disease caused by, associated with,
and/or accompanied by functionally impaired and/or reduced numbers
of pancreatic islet cells, particularly insulin producing
beta-cells, by administering a therapeutically effective amount of
a neurturin product or a compound that influences the neurturin
expression level or function. Functional impairment or loss of
pancreatic islet cells may be due to e.g. autoimmune attack such as
in diabetes type I or LADA, and/or due to cell degeneration such as
in progressed diabetes type II. The methods of the present
invention may also be used to treat patients at risk to develop
degeneration of insulin producing beta-cells to prevent the start
or progress of such process.
[0019] The neurturin product or the effector/modulator thereof may
be administered e.g. as a pharmaceutical composition, via
implantation of neurturin product expressing cells, and/or via gene
therapy.
[0020] Further, the invention relates to cell preparations
comprising neurturin-treated insulin producing cells or neurturin
expressing cells.
[0021] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon consideration
of the following description of the Figures and detailed
description of the invention which describes presently preferred
embodiments thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows the neurturin dependent induction of the
differentiation of insulin producing cells.
[0023] Mouse embryonic stem (ES) cells were differentiated to
insulin producing cells as described previously (patent application
PCT/EP02/04362, published as WO 02/086107, which is incorporated
herein by reference). In differentiated cells, the abundance of
insulin mRNA (FIGS. 1A and 1B) and of beta-cell glucose transporter
Glut2 mRNA (FIGS. 1C and 1D) was determined using quantitative
RT-PCR in two independent examples. 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. `wt ES` refers to unmodified mouse R1
embryonic stem (ES) cells; `Pax4 ES` 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; `Glut2 expression rel. to delta Ct36` refers
to expression of beta-cell glucose transporter; `ES` refers to
mouse embryonic stem cells, as described in Example 1; `control`
refers to the differentiation protocol as described in Example 2,
without any addition of neurturin; `EB+NTN` refers to the
differentiation protocol as described in Example 3, with the
addition of neurturin to embryoid bodies).
[0024] FIG. 2 shows the structure of the mouse mDG770 transgenic
construct. Shown is the rIP promoter (0.8 kb rat insulin II
promoter) as a thin line, the mouse DG770 cDNA (mDG770) as white
box, the hybrid-intron structure (hybrid-intron) as grey box and
the polyadenylation signal (bgh-polyA) as black box.
[0025] FIG. 3 shows pancreatic islets of mDG770 transgenic mice
with ectopic mDG770 expression. Taqman expression analysis on islet
cDNA isolated from two wild type and two transgenic littermates
using a mDG770 specific primer/probe pair. The data are presented
as fold mDG770 induction relative to wild type mDG770 expression in
islets.
[0026] FIG. 4 shows random fed blood glucose levels of DG770
transgenic mice (rIP-mDG770) compared to wild type mice (wt) on
high fat (HF) diet. Shown are blood glucose levels from random fed
male wild type mice (.diamond-solid., N=5) and rIP-mDG770
transgenic mice (.box-solid., N=6). The data are expressed as mean
blood glucose+/-standard deviation. The blood glucose levels are
significantly lower in DG770 transgenic mice; meaning that higher
expression of DG770 in pancreatic islets of mammals will lower the
blood glucose level.
DETAILED DESCRIPTION OF THE INVENTION
[0027] 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.
[0028] 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.
[0029] As used herein, the term "neurturin product" includes
neurturin protein products such as purified natural or synthetic
neurturin and variants thereof. Variants include insertion,
substitution and deletion variants and chemically modified
derivatives. Variants also include recombinant proteins, for
example but not limited to hybrids of neurturin and other TGF-beta
proteins (preferably from the GDNF-family). Also included are
proteins or peptides substantially homologous to the human
neurturin precursor protein having the amino acid sequence
published as GenBank Accession Number NP.sub.--004549. The term
"neurturin product" also includes polynucleotides (e.g. mRNA/DNA)
encoding the above described neurturin protein product. The term
"neurturin product" also includes neurturin homodimers or
heterodimers of a neurturin protein product and another protein,
wherein the other protein preferably belongs to the
GDNF-family.
[0030] The term "biologically active" as used herein means that the
neurturin product induces and/or stimulates the differentiation of
insulin producing cells from progenitor, e.g. stem cells and/or
promotes the protection, survival, or regeneration of insulin
producing cells, e.g. beta-cells. The biological activity of
neurturin products may be determined as described in the Examples
of the present application.
[0031] The term "substantially homologous" as used herein means
having a degree of homology to the biologically active human
neurturin product resulting from the cleavage of the neurturin
precursor having the amino acid sequence published as GenBank
Accession Number NP.sub.--004549 or to the human neurturin
precursor itself, that is preferably in excess of 70%, most
preferably in excess of 80%, and even more preferably in excess of
90% or 95%. The degree of homology between the mouse and the human
protein is about 91%, and it is contemplated that preferred
mammalian neurturin proteins will have a similarly high degree of
homology. Also included are proteins which are hybrids between
neurturin and another TGFbeta-protein, preferably another member of
the GDNF-family which retain the stimulatory effect on islet cell
formation found in Neurturin. The percentage of homology or
identity between a neurturin product and the human neurturin
protein or a precursor or a nucleic acid coding therefor may be
determined according to standard procedures, e.g. by using the
BLAST algorithm. Preferably, the percentage of homology or identity
is calculated as the percentage of nucleotide or amino acid
residues found in the smaller of the two sequences that align with
identical nucleotides or amino acid residues in the sequence being
compared, when four gaps in a length of 100 nucleotides or amino
acids may be introduced to assist in that alignment. Also included
as substantially homologous is any neurturin protein product which
may be isolated by virtue of cross-reactivity with antibodies to
the neurturin protein product or whose genes may be isolated
through hybridization with the gene or with segments of the gene
encoding the neurturin protein product.
[0032] 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 0027995; 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 &
Sarvetnick, 1993, Development 118:33-46; Bouwens, 1998, Microsc Res
Tech 43:332-336; Bonner-Weir, 2000, J. Mol. Endocr.
24:297-302).
[0033] 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(3):998-1003, which are incorporated herein by
reference).
[0034] The term `cultivation medium` means a suitable medium
capable of supporting growth and differentiation of stem cells. The
term `differentiation medium` means a suitable medium for inducing
the differentiation of stem cells into insulin-producing cells. The
term `terminal differentiation medium` means a suitable medium for
terminal differentiation of insulin-producing cells. Examples of
preferred media are described in WO/023018, which are herein
incorporated by reference.
[0035] In this invention, we disclose a novel and so far unknown
use for the neurotrophic factor neurturin to stimulate and/or
induce the formation or regeneration of insulin producing cells and
thus, a use in the treatment and prevention of diseases going along
with (e.g. caused by, associated with or accompanied by) impaired
beta-cell function, for example but not limited to diabetes
mellitus. More particularly, the diseases are diabetes type I,
diabetes type II or LADA.
[0036] The present invention is based on the surprising finding
that neurturin stimulates the differentiation of insulin producing
cells from stem cells in vitro. Thus, a therapeutically effective
amount of neurturin product may be administered to promote the
regeneration of pancreatic beta-cells or to promote the formation
of insulin-producing cells from stem cells or progenitor cells in
vitro or in vivo. The present invention further relates to
applications in the medical field that directly arise from the
method of the invention. Additionally, the present invention
relates to applications for the identification and characterization
of compounds with therapeutic medical effects or toxicological
effects that directly arise from the method of the invention.
[0037] According to this invention the neurturin product may be
administered [0038] i) as a pharmaceutical composition e.g.
enterally, parenterally or topically, preferably directly to the
pancreas, [0039] ii) via implantation of neurturin protein product
expressing cells, and/or [0040] iii) via gene therapy as described
in more detail below.
[0041] Further, the neurturin expression level in a patient might
be influenced by a neurturin modulator/effector administered [0042]
i) as a pharmaceutical composition e.g. enterally, parenterally or
topically, preferably directly to the pancreas, [0043] ii) via cell
based therapy, and/or [0044] iii) via gene therapy as described in
more detail below.
[0045] The neurturin product or the neurturin modulator/effector,
i.e. a pharmaceutically active substance influencing, particularly
increasing the neurturin 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.
[0046] A neurturin 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 I, LADA, or progressed diabetes type II. It is
further contemplated that a neurturin 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 II
or LADA in early stages. A variety of pharmaceutical formulations
and different delivery techniques are described in further detail
below.
[0047] The present invention also relates to methods for
differentiating progenitor cells into insulin-producing cells in
vitro comprising [0048] (a) activating one or more pancreatic genes
in a progenitor, e.g. stem cell (optional step, particularly if
embryonic stem cells are used) [0049] (b) aggregating said cells to
form embryoid bodies (optional step, particularly if embryonic stem
cells are used) [0050] (c) cultivating embryoid bodies or
cultivating adult stem cells (e.g., duct cells) in specific
differentiation media containing a neurturin protein product and/or
a modulator/effector thereof under conditions wherein beta-cell
differentiation is significantly enhanced, and [0051] (d)
identifying and selecting insulin-producing cells.
[0052] 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, IsI 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.
[0053] Neurturin products, e.g. neurturin 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.
Neurturin Protein Product
[0054] Recombinant neurturin protein product forms include
glycosylated and non-glycosylated forms of the protein. In general,
recombinant techniques involve isolating the genes encoding for
neurturin protein product, cloning the gene in suitable vectors
and/or cell types, modifying the gene if necessary to encode a
desired variant, and expressing the gene in order to produce the
neurturin protein product.
[0055] Alternatively, a nucleotide sequence encoding the desired
neurturin product may be chemically synthesized. It is contemplated
that a neurturin product may be expressed using nucleotide
sequences that vary in codon usage due to the degeneration of the
genetic code or allelic variations or alterations made to
facilitate production of the protein product by the selected
cell.
[0056] Kotzbauer et al., Nature 384:467-470, describe the
identification of a mouse cDNA and amino acid sequence and a human
cDNA and amino acid sequence for neurturin protein. The neurturin
products according to this invention may be isolated or generated
by a variety of means. Exemplary methods for producing neurturin
products useful are described in patent application WO 97/08196,
the disclosures of which are hereby incorporated by reference. Also
described are a variety of vectors, host cells, and culture growth
conditions for the expression of neurturin protein, as well as
methods to synthesize variants of neurturin protein product.
Additional vectors suitable for the expression of neurturin protein
product in E. coli are disclosed in Patent No. EP 0 423 980, the
disclosure of which is hereby incorporated by reference.
[0057] The molecular weight of purified neurturin indicates that in
its biologically active form the protein is a disulfide-bonded
dimer. The material isolated after expression in a bacterial system
is essentially biologically inactive, and exists as a monomer.
Refolding is necessary to produce the biologically active
disulfide-bonded dimer. Processes suitable for the refolding and
maturation of the neurturin expressed in bacterial systems are
substantially similar to those described in WO93/06116. Standard in
vitro assays for the determination of neurturin activity are also
substantially similar to those determining GDNF activity as
described in WO93/06116 and in U.S. application Ser. No.
08/535,681, and are hereby incorporated by reference.
[0058] Neurturin product variants are prepared by introducing
appropriate nucleotide changes into the DNA encoding the
polypeptide or by in vitro chemical synthesis of the desired
polypeptide. It will be appreciated by those skilled in the art
that many combinations of deletions, insertions, and substitutions
can be made resulting in a protein product variant presenting
neurturin biological activity.
[0059] Mutagenesis techniques for the replacement, insertion or
deletion of one or more selected amino acid residues are well known
to one skilled in the art (e.g., U.S. Pat. No. 4,518,584, the
disclosure of which is hereby incorporated by reference.)
[0060] Neurturin substitution variants have at least one amino acid
residue of the human or mouse neurturin amino acid sequence removed
and a different is residue inserted in its place. Such substitution
variants include allelic variants, which are characterized by
naturally occurring nucleotide sequence changes in the species
population that may or may not result in an amino acid change.
[0061] Chemically modified derivatives of neurturin protein
products also may be prepared by one of skill in the art given the
disclosures herein. The chemical moieties most suitable for
derivatization include water soluble polymers. A water soluble
polymer is desirable because the protein to which it is attached
does not precipitate in an aqueous environment, such as a
physiological environment. Preferably, the polymer will be
pharmaceutically acceptable for the preparation of a therapeutic
product or composition. One skilled in the art will be able to
select the desired polymer based on such considerations as whether
the polymer/protein conjugate will be used therapeutically, and if
so, the desired dosage, circulation time, resistance to
proteolysis, and other considerations. A particularly preferred
water-soluble polymer for use herein is polyethylene glycol.
Attachment at residues important for receptor binding should be
avoided if receptor binding is desired. One may specifically desire
an N-terminal chemically modified protein.
[0062] The present invention contemplates use of derivatives which
are prokaryote-expressed neurturin, or variants thereof, linked to
at least one polyethylene glycol molecule, as well as use of
neurturin, or variants thereof, attached to one or more
polyethylene glycol molecules via an acyl or alkyl linkage.
Pegylation may be carried out by any of the pegylation reactions
known in the art. See, for example: Focus on Growth Factors, 3
(2):4-10, 1992; EP 0 154 316, the disclosure of which is hereby
incorporated by reference; EP 0 401 384; and the other publications
cited herein that relate to pegylation.
[0063] The present invention also discloses use of derivatives
which are prokaryote-expressed neurturin, or variants thereof,
linked to at least one hydrophobic residue, for example fatty acid
molecule, as well as use of neurturin, or variants thereof,
attached to one or more hydrophobic residues. For example, patent
application published as WO 03/010185, which is hereby incorporated
by reference, describes a method for producing acylated
polypeptides in transformed host cells by expressing a precursor
molecule of the desired polypeptide which are then to be acylated
in a subsequent in vitro step.
Polynucleotides Encoding Neurturin Protein Product
[0064] The present invention further provides polynucleotides that
encode neurturin protein products, whether recombinantly produced
or naturally occurring.
[0065] A nucleic acid sequence encoding a neurturin protein
product, can readily be obtained in a variety of ways, including,
without limitation, chemical synthesis, cDNA or genomic library
screening, expression library screening, and/or PCR amplification
of cDNA. These methods and others useful for isolating such nucleic
acid sequences are set forth, for example, by Sambrook et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989), by Ausubel et
al., eds (Current Protocols in Molecular Biology, Current Protocols
Press, 1994), and by Berger and Kimmel (Methods in Enzymology:
Guide to Molecular Cloning Techniques, vol. 152, Academic Press,
Inc., San Diego, Calif., 1987). Chemical synthesis of a nucleic
acid sequence which encodes a neurturin protein product can also be
accomplished using methods well known in the art, such as those set
forth by Engels et al. (Angew. Chem. Intl. Ed., 28:716-734, 3 0
1989).
[0066] Included within the scope of this invention are neurturin
product polynucleotides with the native signal sequence and other
pre-pro sequences as well as polynucleotides wherein the native
signal sequence is deleted and replaced with a heterologous signal
sequence. The heterologous signal sequence selected should be one
that is recognized and processed, i.e., cleaved by a signal
peptidase, by the host cell. For prokaryotic host cells that do not
recognize and process the native neurturin signal sequence, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group of the alkaline phosphatase,
penicillinase, or heat-stable enterotoxin 11 leaders. For yeast
secretion, the native neurturin signal sequence may be substituted
by the yeast invertase, alpha factor, or acid phosphatase leaders.
In mammalian cell expression the native signal sequence is
satisfactory, although other mammalian signal sequences may be
suitable.
[0067] Expression and cloning vectors generally include a nucleic
acid sequence that enables the vector to replicate in one or more
selected host cells.
Neurturin Product Pharmaceutical Compositions
[0068] Neurturin product pharmaceutical compositions typically
include a therapeutically effective amount of a neurturin product
in admixture with one or more pharmaceutically and physiologically
acceptable formulation. In addition to the active ingredients,
neurturin product 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.), the disclosure of which is
hereby incorporated by reference.
[0069] Once the therapeutic composition has been formulated, it may
be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or dehydrated or lyophilized powder. Such
formulations may be stored either in a ready to use form or in a
form, e.g., lyophilized, requiring reconstitution prior to
administration. The optimal pharmaceutical formulations will be
determined by one skilled in the art depending upon considerations
such as the route of administration and desired dosage. Such
formulations may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of the present
neurturin proteins, variants and derivatives. Other effective
administration forms, such as slow-release formulations, inhalant
mists, or orally active formulations are also envisioned.
[0070] For example, in a sustained release formulation, the
neurturin product may be bound to or incorporated into particulate
preparations of polymeric compounds (such as polylactic acid,
polyglycolic acid, etc.) or liposomes.
Administration/Delivery of Neurturin Product
[0071] The neurturin product may be administered by any suitable
means, preferably enterally or parenterally or topically directly
to the pancreas, as known to those skilled in the art. The specific
dose may be calculated according to considerations of body weight,
body surface area or organ size. Further refinement of the
calculations necessary to determine the appropriate dosage for
treatment involving each of the above mentioned formulations is
routinely made by those of ordinary skill in the art and is within
the ambit of tasks routinely performed. Appropriate dosages may be
ascertained through use of the established assays for determining
dosages utilized in conjunction with appropriate dose-response
data. The final dosage regimen involved in a method for treating
the above described conditions will be determined by the attending
physician, considering various factors which modify the action of
drugs, e.g., the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors. As studies are conducted, further
information will emerge regarding the appropriate dosage levels for
the treatment of various diseases and conditions.
[0072] It is envisioned that the continuous administration or
sustained delivery of a neurturin product may be advantageous for a
given treatment. While continuous administration may be
accomplished via a mechanical means, such as with an infusion pump,
it is contemplated that other modes of continuous or near
continuous administration may be practiced. For example, chemical
derivatization or encapsulation may result in sustained release
forms of the protein having the effect of continuous presence, in
predictable amounts, based on a determined dosage regimen. Thus,
neurturin protein products include proteins derivatized or
otherwise formulated to effectuate such continuous
administration.
[0073] Neurturin product cell therapy, i.e. pancreatic implantation
of cells producing neurturin protein product, is also contemplated.
This embodiment would involve implanting cells capable of
synthesizing and secreting a biologically active form of neurturin
protein product into patients. Such neurturin protein
product-producing cells may be cells that are natural producers of
neurturin protein product or may be cells that are modified to
express the protein. Such modified cells include recombinant cells
whose ability to produce a neurturin protein product has been
augmented by transformation with a gene encoding the desired
neurturin protein product in a vector suitable for promoting its
expression and secretion. In order to minimize a potential
immunological reaction in patients being administered neurturin
protein product of a foreign species, it is preferred that the
cells producing neurturin protein product be of human origin and
produce human neurturin protein product. Likewise, it is preferred
that the recombinant cells producing neurturin protein product be
transformed with an expression vector containing a gene encoding a
human neurturin 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 neurturin protein product, but that prevent
destruction of the cells by the patient's immune system or by other
detrimental factors from the surrounding tissue.
[0074] Alternatively, neurturin 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.
[0075] In a further preferred embodiment, neurturin 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, neurturin 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.
[0076] 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.
[0077] The insulin producing cells of the invention preferably
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.
[0078] 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.
[0079] 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.
[0080] According to the present invention, the functional insulin
producing cells treated with neurturin 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.
[0081] 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.
[0082] In another embodiment gene therapy ex vivo is envisioned,
i.e. the patient's own cells may be transformed ex vivo to produce
a neurturin protein product or a protein stimulating neurturin
expression and would be directly reimplanted. For example, cells
retrieved from the patent may be cultured and transformed with an
appropriate vector. After an optional propagation/expansion phase,
the cells can be transplanted back into the same patient's body,
particularly the pancreas, where they would produce and release the
desired neurturin 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.
[0083] Neurturin product gene therapy in vivo is also envisioned,
by introducing the gene coding for a neurturin 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 neurturin protein product may be contained in
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).
[0084] 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.
[0085] The neurturin nucleic acid and protein and
effectors/modulators thereof may be administered either as a
monotherapy or as a combination therapy with other pharmaceutical
agents. For example, they may be administered together with other
pharmaceutical agents suitable for the treatment or prevention of
pancreatic diseases and/or obesity and/or metabolic syndrome,
particularly with other pharmaceutical agents suitable for
stimulating and/or inducing the differentiation of insulin
producing cells from progenitor cells. Further, they may be
administered together with pharmaceutical agents which have an
immunosuppressive activity, e.g. antibodies, polypeptides and/or
peptidic or non-peptidic low molecular weight substances. Preferred
examples of immunosuppressive agents are listed in the following
Table 1.
TABLE-US-00001 TABLE 1 Exemplary agents for immune suppression
Names Mechanism 2-amino-1,3-propanediol derivatives Used for
preventing or treating chronic rejection in a patient receiving an
organ or tissue allo- or xenotransplant
2-amino-2[2-(4-octylphenyl)ethyl] Immunosuppression, from
accelerated lymphocyte homing propane-1,3-diol hydrochloride
40-O-(2-hydroxyethyl)-rapamycin, Sirolimus (rapamycin) derivative,
used for acute kidney rejection; SDZ-RAD, Everolimus reduces
rejection and graft vasculopathy following heart transplantation by
inhibiting cell proliferation 6-(3-dimethyl-aminopropionyl)
Immunosuppressing action useful also for treating autoimmune
forskolin disease 6-mercaptopurine (6-MP) Used to treat Crohn's
disease, inflammatory bowel disease and for organ transplant
therapy A-420983 Lck-inhibitor ABX-CBL (CBL-1) Mouse monoclonal AB
targeted against human T-cell, B cells, NK cells and monocytes, for
treatment of steroid-resistant graft vs host diseases, potential
use in treatment of inflammatory and autoimmune disorders Alefacept
(human LFA-3 IgG1 fusion Knocks out causative memory T-lymphocytes;
Used to treat psoriasis, protein) a T-cell mediated inflammatory
disorder Antisense ICAM-1 inhibitor (ISIS Mouse monoclonal AB
blocks white blood cell adhesion to T-cell 2302), Enlimomab, BIRR1,
surface molecule (ICAM-1r); treatment of kidney transplant
rejection Alicaforsen Antithymocyte immunoglobulin Anti-human
thymocyte, immunoglobulin; used in reversal of acute (ATGAM) kidney
transplant rejection and will likely be used off-label for
transplant induction therapy Azathioprine Treatment of rheumatoid
arthritis and prevention of kidney transplant rejection, and other
autoimmune or inflammatory disorders such as inflammatory bowel
disease Baohuoside-1 Flavonoid; inhibits lymphocyte activation; Ma
et al., Transplantation 78: 831-838, (2004) basiliximab Monoclonal
AB that binds to receptor sites on T-cells, preventing activation
by transplanted tissue (renal transplant) BMS-279700 Lck-inhibitor
BTI-322 Mouse derived monoclonal AB targeted to CD2 receptor; used
for prevention of first-time kidney rejection, and treatment of
resistant rejection Cladribine Antimetabolite and immunosuppressive
agent that is relatively selective for lymphocytes; used to treat
lymphoid malignancies, e.g., hairycell leukemia CP-690550 JAK-3
inhibitor Cyclophosphamide (CTX) Immunosuppressant for treatment of
arthritis and other auto-immune disorders and cancers Cyclosporine
(cyclosporin A, 11 amino acid cyclic peptide; blocks helper T-cell,
cyclosporin) immunosuppressant used in organ transplant therapy and
other immune diseases Daclizumab, HAT (Humanized Anti- Monoclonal
AB inhibits binding of IL-2 to IL-2 receptor by binding Tac), SMART
anti-Tac, anti-CD25, to IL-2 receptor, suppresses T-cell activity
against allografts (renal and humanized anti-IL2-receptor
transplant) Dexamethasone (Decadron, Dexone, An adrenocorticoid,
effective immunosuppressant in various Dexasone) disorders
DIAPEP-277 Immunomodulatory properties Dipeptide Boronic Acid
(DPBA) Proteasome inhibitor; Wu et al., Transplantation 78:
360-366, (2004) Docosahexaenoic acid (DHA) Immunosuppressant that
lowers the proportion of T cells expressing CD4 or CD8, blocks
antigen recognition process; Taku et al., Journal of Agricultural
and Food Chemistry 48: 1047, (2000) efalizumab T-cell modulator
that target T-cells through interactions with adhesion molecules on
endothelial cell surface, target migration of T- cells into the
skin and target activation of T-cells; Used to treat Psoriasis
Efomycine M Leukocyte adhesion inhibitor, Anti-inflammatory FTY720
(oral myriocin derivative) Alters lymphocyte infiltration into
grafted tissues; used for prevention of organ rejection in kidney
transplants Glatiramer acetate (co-polymer-1) Synthetic peptide
copolymer; decoy that mimics structure of myelin so immune cells
bind Copaxone instead of myelin; for multiple sclerosis Glial
fibrillary acidic protein (GFAP) Possesses immunosuppressive
activities in diabetic animal models; Winer et al., Nature Medicine
9: 198, (2003) Gusperimus (15-deoxyspergualin) Intravenous
immunosuppressant; suppresses production of cytotoxic T-cells,
neutrophils and macrophages HLA-B2702 peptide Human peptide, blocks
action of NK cells and T-cell mediated toxicities, used for
prevention of first kidney allograft rejection hu1124 (anti-CD11a)
Humanized monoclonal antibody; targets CD11a receptor on surface of
T cells to selectively inhibit immune system rejection of
transplanted organs hOKT31.gamma.(Ala-Ala) non Fc-binding humanized
anti CD3 antibody Infliximab Monoclonal AB, binds and inactivates
human TNFalpha; used to treat Crohn's disease and rheumatoid
arthritis Interferon Immunomodulatory properties ISAtx247 Used to
treat autoimmune diseases such as rheumatoid arthritis and
psoriasis isotretinoin Immunosuppressant, reduces ability of T
cells to proliferate in response to immune challenge. Vergelli et
al., Immunopharmacology, 31: 191, (1997) L-683,742: also described
as 31- Treatment of autoimmune diseases, infectious diseases and/or
desmethoxy-31-hydroxy-L-683,590 prevention of organ transplant
rejections Leflunomide (ARAVA) Antiinflammatory agent Medi-500
(T10B9) Intravenous monoclonal AB that targets human T-cells;
treats acute kidney rejection and graft-vs-host disease Medi-507
Intravenous humanized AB directed against CD2 T-cell; used to treat
corticosteroidresistant graft vs host disease and prevention of
kidney rejection Methotrexate Antimetabolite used to treat Crohn's
disease, severe psoriasis, and adult rheumatoid arthritis (and as
an anti-cancer drug) Mitoxantrone Antiproliferative effect on
cellular immune system including T-cells, B-cells and macrophages;
used to treat hormone-refractory prostate cancer, acute myelogenous
leukemia and multiple sclerosis mycophenolate mofetil Proliferation
of T and B lymphocytes by blocking the synthesis of purine
nucleotides; used in organ transplant therapy and inflammatory
bowel disease OKT4A Mouse monoclonal AB targeted against human CD4
T cell; used for prevention of kidney transplant rejection when
used in combination with other immunosuppressant drugs
Muromonab-CD3 Monoclonal AB that binds to receptor sites on
T-cells, preventing activation by transplanted tissue Prednisolone
Corticosteroid, suppresses inflammation associated with transplant
rejection Psora-4 Kv1.3-blocker Rifampicin Antibiotic; has
immunomodulatory properties Rituximab CD20 antibody S100.beta.
possesses immunosuppressive activities in diabetic animal models
Sirolimus, Rapamycin Immunosuppressant and potent inhibitor of
cytokine (e.g.IL-2)- dependent T-cell proliferation (kidney
transplant)
[0086] The combination therapy may comprise coadministration of the
medicaments during the treatment period and/or separate
administration of single medicaments during different time
intervals in the treatment period.
[0087] Administration of a neurturin 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 could be
cured entirely of a diabetic 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] In a further embodiment, the present invention allows the
production of cells for the identification and/or characterisation
of compounds which stimulate beta-cell differentiation, insulin
secretion and/or glucose response, more particularly of compounds
which increase the neurturin expression level or function. This
method is particularly suitable for in vivo testing for diagnostic
applications and drug development or screening. The compound of
interest is added to suitable cells and the neurturin expression or
function is determined. Alternatively, a compound of interest is
added to a neurturin-treated cell and the effect on cell
differentiation and/or insulin production is determined.
Preferably, differentiated insulin-producing cells used. Insulin
levels in treated cells can be determined, e.g. quantified by
Enzyme Linked Immunoabsorbent Assay (ELISA) or Radio Immuno Assay
(RIA). Using this method, a large number of compounds can be
screened and compounds that induce neurturin expression or support
the activity of neurturin leading to a beta-cell differentiation
and/or an increase in insulin secretion can be identified
readily.
[0089] 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
beta-cell differentiation, e.g. preferably a Pax4 promoter. The
transfected cells are divided into aliquots and each aliquot is
contacted with a test substance, e.g. candidate 1, candidate 2, and
candidate 3. The activity of the reporter gene corresponds to the
capability of the test compound to induce beta-cell
differentiation.
[0090] 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
cells being cultivated and the neurturin expression and/or the
insulin production is determined. Following an initial high
throughput assay, such as the cell based assay outlined above where
e.g. 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 cells to
assess the effectiveness of a compound.
EXAMPLES
[0091] A better understanding of the present invention and of its
many advantages will be had from the following examples, given by
way of illustration.
Example 1
Generation of ES Cells Expressing the Pax4 Gene
[0092] Mouse R1 ES 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).
[0093] ES cells were cultured in Dulbecco's modified Eagle's medium
containing 4.5 g/l glucose, 10.sup.-4 M beta-mercaptoethanol, 2 nM
glutamine, 1% non-essential amino acids, 1 nM Na-pyruvate, 15% FCS
and 500 U/ml leukaemia inhibitory factor (LIF). Briefly,
approximately 10.sup.7 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% CO.sub.2. These untreated and
undifferentiated ES cells were used as control the experiment shown
in FIG. 1 (referred to as `ES` in FIG. 1).
Example 2
Differentiation of ES Cells into Insulin-Producing Cells (Referred
to as `Control` in FIG. 1)
[0094] The ES cell line R1 (wild type, `wt ES` in FIG. 1) and ES
cells constitutively expressing Pax4 (`Pax4 ES` in FIG. 1) were
cultivated as embryoid bodies (EB) by the hanging drop method, 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, with
media as described below and in Table 2. 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 medium: Nutrient Mixture F-12 (DMEM/F12).
Example 3
Expression of Pancreas Specific Genes after Differentiation of ES
Cells into Insulin-Producing Cells
[0095] Expression levels of pancreas specific genes was measured by
semi-quantitative RT-PCR analysis. Differentiated wild type ES and
Pax4 ES cells were collected after embryoid body formation and
suspended in lysis buffer (4 M guanidinium thiocyanate, 25 mM
sodium citrate, pH 7; 0.5% sarcosyl, 0.1 M beta-mercaptoethanol).
Total RNA was isolated by the single step extraction method
described by Chomczynski & Sacchi, 1987, Anal. Biochem. 162:
156-159). mRNA was reverse transcribed using PolyT tail primer
Oligo d(T).sub.16 (PerkinElmer) and the resulting cDNA was
amplified using oligonucleotide primers complementary and identical
to transcripts of beta-cell glucose transporter Glut2 and insulin.
The house keeping gene beta-tubulin was used as internal standard.
Reverse transcription (RT) was performed with MuLV reverse
transcriptase (Perkin Elmer). Multiplex PCRs were carried out using
AmpliTaq DNA polymerase (Perkin Elmer) as described in Wobus at
al., 1997, supra. mRNA levels of genes encoding Glut2 and insulin
were analysed using the Dynalbeads mRNA DIRECT micro kit (Dynal)
according to the manufacturer's instructions.
[0096] One third of each PCR reaction was separated by
electrophorese. Ethidium bromide fluorescence signals of gels were
analyzed by a special software (TINA2.08e) The intensity of the
ethidium bromide fluorescence signals was determined from the area
under the curve for each peak and the data of target genes were
plotted as percentage changes in relation to the expression of the
housekeeping gene beta-tubulin.
[0097] Results show that markers for beta-cell differentiation
function were expressed at higher levels in Pax4.sup.+
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. 1). Expression of Glut2 in differentiated stem cells
indicates that hormone-producing cells are capable of responding to
glucose. Expression of substantial amounts of insulin in
differentiated stem cells indicates that differentiated cells show
a phenotype similar to beta-cells.
Example 4
Induction of Differentiation of Insulin-Producing Cells by
Neurturin (Referred to as EB+NTN in FIG. 1)
[0098] In order to study the effect of neurturin to induce
beta-cell differentiation in vitro, we have generated stable mouse
embryonic stem (ES) cells expressing Pax4 under the control of the
cytomegalovirus (CMV) early promoter/enhancer region as described
in Example 1. 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 formed in the presence of 50
ng/ml of neurturin solution in PBS-01% BSA. Embryoid bodies were
subsequently plated, again neurturin was added every second day
until day 12 and afterwards enzymatically dissociated, and
replated. After dissociation, cells were cultured in a
differentiation medium containing various growth factors (see Table
2 for more detail). Neurturin was obtained from RDI Research
Diagnostics INC, USA, Order Number RDI-4511. Under such conditions,
the expression of insulin was significantly induced by neurturin in
two independent examples (FIGS. 1A and 1B). In addition, the
addition of neurturin to the differentiation medium did
significantly enhance the expression of the glucose transporter
Glut-2 in two independent examples (FIGS. 1C and 1D). By
comparison, wild type ES cells did not contain any
insulin-producing cell at the same stage and only small numbers of
Glut-2 expressing cells. These data demonstrate that neurturin can
significantly promote and enhance ES cells differentiation into
insulin-producing cells compared to wild type ES cells.
[0099] The results shown in FIG. 1 clearly demonstrate a
significant induction of the differentiation of insulin-producing
(FIGS. 1A and 1B) and glucose responsive (FIGS. 1C and 1D) cells,
if neurturin is added to embryoid bodies. Thus, neurturin has a
strong inductive effect on the differentiation of insulin-producing
beta cells.
TABLE-US-00002 TABLE 2 Protocol for the induction of
differentiation of insulin-producing cells by Neurturin. Media B1,
B2 and B27 supplement, (NA-Niacinamide) are described in
Rolletschek et al., 2001, Mech. Dev. 105: 93-104. Stage of Coating
and Day Cultivation Medium Analysis 0 hanging drops Iscove + 15%
RNA (200 cells/drop) FCS addition of (ES cells) Neurturin (NTN) 50
ng/ml 1 2 EBs in Iscove + 15% suspension FCS + NTN, 50 ng/ml 3 4
plating of EBs Iscove + 10% gelatin FCS + NTN, coating 50 ng/ml RNA
(EBs) +1 medium change B1 medium + ornithine/ NTN, 50 ng/ml laminin
+2 coating +3 medium change +4 +5 medium change +6 +7 medium change
+8 dissociation B2 + B27 + RNA (1 .times. 6 NA + 10% FCS cm dish)
+9 medium change see above, withdraw FCS +10 +11 medium change +12
+13 medium change +14 +15 medium change +16 +17 medium change +18
+19 medium change +20 +21 medium change +22 +23 medium change +24
+25 medium change Immunofluorescence (IF); RNA +26 +27 medium
change +28 IF; RNA +29 medium change +30 +31 medium change +32 IF;
RNA
Example 5
Functional Characterization of the Differentiated Insulin-Producing
Cells
[0100] 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 was performed on the
differentiated cells. On the day of the assay, the differentiation
medium of 12 or 6 well plate was removed and the cells were washed
3 times with Krebs Ringer Bicarbonate Hepes Buffer (KRBH; 125 mM
NaCl, 4.7 mM KCl, 1 mM CaCl.sub.2, 1.2 mM KH.sub.2PO.sub.4, 1.2 mM
MgSO.sub.4, 5 mM NaHCO.sub.3, 25 mM Hepes, 0.1% BSA) supplemented
with 2.8 mM glucose. For pre-incubation cells were incubated in
KRBH+2.8 mM glucose for 2 hours at 37.degree. C. Afterwards cells
were incubated in 750 .mu.l up to 1 ml KRBH+2.8 mM glucose for 1
hour at 37.degree. C. The supernatant was kept for measurement of
basal insulin secretion. For the stimulated insulin release 750
.mu.l up to 1 ml KRBH containing 16.7 mM glucose was added to the
cells. After 1 hour incubation at 37.degree. C., the KRBH was
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
were determined by an Enzyme-Linked Immunosorbent Assay (ELISA) for
mouse insulin (Mercodia) and performed according to the manufacture
r's recommendations. An alternative medium for proper insulin
release was medium based on DMEM with glucose concentration of 1
g/l (Gibco) supplemented with non-essential amino acids (Gibco,
stock solution 1:100) and additional factors mentioned above. Such
medium can be applied 1 to 6 days before use of the cells.
[0101] A basal insulin secretion is expected when both wild type
and neurturin induced insulin-producing cells are cultured in low
glucose concentrations (2.8 mM). However, the neurturin induced
insulin-producing cells highly respond to glucose simulation. In
the presence of high glucose concentrations (16.7 mM), an increase
in insulin secretion is expected in neurturin ES derived
insulin-producing cells.
Example 6
Transplantation of Pax4 ES Derived Insulin-Producing Cells in STZ
Diabetic Mice
[0102] The therapeutic potential of neurturin 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.
[0103] Non-fasted Male BalbC mice were treated with STZ to develop
hyperglycaemia after STZ treatment. Mice were considered diabetic
if they had a blood glucose level above 10 mmol/l for more than 3
consecutive days. Cells were transplanted under the kidney capsule
and into the spleen of animals. The presence of the
insulin-producing cells was confirmed by immunohistological
analysis of the transplanted tissue. Results are expected to
demonstrate that the transplanted cells can normalise blood glucose
in diabetic animals.
[0104] 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.
Example 7
Generation of a mDG770 Transgenic Construct
[0105] A complete mDG770 Open Reading Frame (ORF) was cloned under
the control of the rat insulin promoter II (Lomedico et al., (1979)
Cell 18: 545-558) using the Gateway system (Invitrogen). For the
structure of the transgenic construct, see also FIG. 2.
Example 8
Generation of rIP-mDG770 Transgenic Mice
[0106] Transgenic construct DNA (see Example 7) was injected into
C57/BL6 x CBA embryos (Harlan Winkelmann, Borchen, Germany) using
standard techniques (see, for example, Brinster et al. (1985),
Proc. Natl. Acad. Sci. USA 82: 4438-4442). The mDG770 transgene
(see Example 7) was expressed under the control of the rat insulin
promoter II (Lomedico et al., supra) using techniques known to
those skilled in the art (for example, see, Gunnig et al. (1987),
Proc. Natl. Acad. Sci. USA 84, 4831-4835). Using this technique,
several independent founderlines were generated.
Example 9
Genotype Analysis of rIP-mDG770 Transgenic Mice
[0107] Genotyping was performed by PCR using genomic DNA isolated
from the tail tip. To detect the mDG770 transgene a transgene
specific forward primer (5' tgc tat ctg tct gga tgt gcc 3' and a
mDG770 transgene specific reverse primer (5' aag gac acc tcg tcc
tca tag 3') was used.
Example 10
mDG770 Expression Analysis via TaqMan Analysis
[0108] The expression of the mDG770 transgene in islets was
monitored by TaqMan analysis. For this analysis, 25 ng cDNA derived
from pancreatic islet RNA isolated from transgenic mice and their
littermates and a mDG770 specific primer/probe pair were used to
detect endogenous as well as transgenic mDG770 expression (mDG770-1
forward primer: 5' GCC TAT GAG GAC GAG GTG TCC 3', mDG770 reverse
primer: 5' AGC TCT TGC AGC GTG TGG T 3', mDG770 probe: 5' TCC TGG
ACG TGC ACA GCC GC 3'). TaqMan analysis was performed using
standard techniques known to those skilled in the art. Ectopic
transgene expression was detected in 3 of 4 rIP-mDG770 transgenic
founderlines analysed. The two founderlines showing highest
transgene expression levels were used for further analysis. For the
level of mDG70 expression in islets of a transgenic animal compared
to a wild-type animal, see also FIG. 3.
Example 11
Analytical Procedures Performed in mDG770 Transgenic Mice
[0109] 3 to 6 mice were housed per cage and were provided with food
ad libitum. Metabolic blood parameters were determined using venous
blood isolated from tail vein or via retroorbital bleeding. Blood
glucose values were determined using One Touch blood glucose meters
(LifeScan, Germany). mDG770 transgenic mice exhibit reduced random
fed blood glucose levels (rIP-mDG770), compared to wild type (wt)
mice, see also FIG. 4.
Sequence CWU 1
1
5121DNAArtificialPrimer 1tgctatctgt ctggatgtgc c
21221DNAArtificialPrimer 2aaggacacct cgtcctcata g
21342DNAArtificialPrimer 3gcctatgagg acgaggtgtc cgcctatgag
gacgaggtgt cc 42419DNAArtificialPrimer 4agctcttgca gcgtgtggt
19520DNAArtificialprimer 5tcctggacgt gcacagccgc 20
* * * * *