U.S. patent application number 15/553954 was filed with the patent office on 2018-02-08 for genetically modified mesenchymal stem cell expressing klotho.
The applicant listed for this patent is apceth GmbH & Co. KG. Invention is credited to Christine Gunther, Felix Hermann, Manfred Stangl.
Application Number | 20180037868 15/553954 |
Document ID | / |
Family ID | 55453145 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180037868 |
Kind Code |
A1 |
Gunther; Christine ; et
al. |
February 8, 2018 |
GENETICALLY MODIFIED MESENCHYMAL STEM CELL EXPRESSING KLOTHO
Abstract
A genetically modified mesenchymal stem cell (MSC) includes an
exogenous nucleic acid that includes a Klotho encoding region
operably linked to a promoter or promoter/enhancer combination. The
MSCs can be used for the treatment of cancer, organ fibrosis, renal
failure, age-related changes of organs or organ systems,
arteriosclerosis, and neurodegenerative diseases, such as
Alzheimer's disease (AD), Multiple sclerosis (MS), Huntington's
disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease,
and Schizophrenia, as well as dementia, diabetes mellitus, sepsis
and autoimmune diseases and autoimmune-related diseases.
Inventors: |
Gunther; Christine;
(Munchen, DE) ; Stangl; Manfred; (Sauerlach,
DE) ; Hermann; Felix; (Munchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
apceth GmbH & Co. KG |
Munchen |
|
DE |
|
|
Family ID: |
55453145 |
Appl. No.: |
15/553954 |
Filed: |
February 26, 2016 |
PCT Filed: |
February 26, 2016 |
PCT NO: |
PCT/EP2016/054091 |
371 Date: |
August 25, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 35/28 20130101; A61K 38/47 20130101; C12N 2501/73 20130101;
C07K 14/435 20130101; A61K 2035/124 20130101; A61K 38/00 20130101;
C12N 2510/00 20130101; C12Y 302/01031 20130101; C12N 5/0663
20130101; C12N 9/2402 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; A61K 48/00 20060101 A61K048/00; A61K 38/47 20060101
A61K038/47; C12N 9/24 20060101 C12N009/24; A61K 35/28 20060101
A61K035/28 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2015 |
EP |
15156974.6 |
Sep 18, 2015 |
EP |
15185805.7 |
Oct 22, 2015 |
EP |
15191062.7 |
Claims
1. A genetically modified mesenchymal stem cell comprising an
exogenous nucleic acid comprising a Klotho-encoding region operably
linked to a promoter or promoter/enhancer combination, wherein the
genetically modified mesenchymal stem cell exhibits increased
Klotho expression compared to an unmodified mesenchymal stem
cell.
2. The genetically modified cell according to claim 1, wherein the
exogenous nucleic acid is comprised in a viral vector.
3. The genetically modified cell according to claim 1, wherein the
promoter is a constitutive promoter.
4. The genetically modified cell according to claim 1, wherein the
constitutive promoter is the EFS, PGK or EF1alpha promoter.
5.-9. (canceled)
10. The genetically modified cell according to claim 1, wherein the
Klotho encoding region encodes a protein according to one of SEQ ID
NO 6 to 10, or wherein the Klotho encoding region comprises or
consists of a sequence according to SEQ ID NO 1 to 5.
11. The genetically modified cell according to claim 1, wherein the
Klotho encoding region encodes for a secreted form of the Klotho
protein.
12. (canceled)
13. The genetically modified cell according to claim 1, wherein the
secreted form the Klotho protein possess an amino acid sequence
with an identity of at least 80% to SEQ ID NO 8, or an amino acid
sequence according to SEQ ID NO 8.
14. (canceled)
15. A method of treating a patient comprising introducing a
therapeutically effective number of genetically modified cells
according to claim 1 into the bloodstream of the patient.
16. The method according to claim 15, wherein said therapeutically
effective number of genetically modified cells is introduced
subcutaneously.
17. The method according to claim 15, wherein said therapeutically
effective number of genetically modified cells is administered
intrathecally.
18. (canceled)
19. The method according to claim 15, wherein the patient is
treated for a neurodegenerative disease.
20. The method according to claim 15, wherein the patient is
treated for cancer.
21. The method according to claim 15, wherein the patient is
treated for organ fibrosis.
22. The method according to claim 15, wherein the patient is
treated for renal disease.
23. The method according to claim 22, wherein the Klotho encoding
region encodes for a secreted form of the Klotho protein, and the
genetically modified mesenchymal stem cell exhibits increased
expression of said secreted from of Klotho protein compared to an
unmodified mesenchymal stem cell.
24. The method according to claim 15, wherein the patient is
treated for age-related changes of organs or organ systems.
25. The method according to claim 15, wherein the patient is
treated to slow, reverse and/or inhibit ageing.
26. The method according to claim 15, wherein the patient is
treated for arteriosclerosis.
27. The method according to claim 15, wherein the patient is
treated for dementia.
28. The method according to claim 15, wherein the patient is
treated for diabetes mellitus.
29. The method according to claim 15, wherein the patient is
treated for erectile dysfunction.
30. The method according to claim 15, wherein the patient is
treated for autoimmune diseases or autoimmune-related diseases.
31. The method according to claim 15, wherein the patient is
treated for an inflammatory disease of the lung.
32. The method according to claim 15, wherein the patient is
treated for sepsis.
33.-39. (canceled)
Description
[0001] The invention relates to a genetically modified mesenchymal
stem cell (MSC), wherein said stem cell comprises an exogenous
nucleic acid comprising a Klotho encoding region operably linked to
a promoter or promoter/enhancer combination. The invention relates
to the medical use of said MSCs for the treatment of cancer, organ
fibrosis, renal failure, age-related organ pathologies,
arteriosclerosis, neurodegenerative diseases, such as Alzheimer's
disease (AD), Multiple sclerosis (MS), Huntington's disease,
Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease, and/or
Schizophrenia, or dementia, diabetes mellitus, sepsis, erectile
dysfunction, cardio vascular diseases and autoimmune diseases.
BACKGROUND OF THE INVENTION
[0002] Mesenchymal stem cells (MSCs) are cells of non-hematopoietic
origin that reside in the bone marrow and other tissues. MSCs are
commonly considered to be multipotent adult progenitor cells that
have the ability to differentiate into a limited number of cell
lineages, such as osteoblasts, chondrocytes, and adipocytes.
Studies have been conducted on the use of MSCs as a therapeutic
entity based on this capacity to differentiate directly into these
end-stage phenotypes, including the use of MSCs to promote or
augment bone repair and for the repair of cartilage defects
(Vilquin and Rosset, Regenerative Medicine 2006: 1, 4, p 589, and
Veronesi et al, Stem Cells and Development 2013; 22, p 181). The
isolation and cultivation of MSCs for a number of therapeutic
indications has been described and represents a promising approach
towards treating inflammation-associated disorders (for example WO
2010/119039).
[0003] MSCs are known to exhibit immune evasive properties after
administration to a patient. MSCs have been shown to exhibit a
beneficial immune modulatory effect in cases of transplantation of
allogeneic donor material (Le Blanc et al, Lancet 2004: 363, p
1439), thereby reducing a potentially pathogenic alloreactivity and
rejection. Furthermore, MSCs are known to exhibit anti-tumorigenic
effects, for example against Kaposi's sarcoma (Khakoo et al, J Exp
Med 2006: 203, p 1235). MSCs treatment can also play a therapeutic
role in wound healing. The therapeutic delivery of MSCs can be
performed via systemic injection, followed by MSC homing to and
engraftment within sites of injury (Kidd et al, Stem Cells 2009:
27, p 2614). Although it is clear that MSCs have a regenerative
effect on injured tissue, their use as a delivery vehicle for
therapeutic proteins of interest has not yet been fully
explored.
[0004] The human Klotho gene encodes a type-1 transmembrane protein
of 1012 amino acids, which can also be expressed as a secreted form
by alternative splicing. Both forms have biological activity
including regulatory effects on general metabolism. Klotho is a
.beta.-glucuronidase (EC number 3.2.1.31) capable of hydrolyzing
steroid .beta.-glucuronides.
[0005] Loss of Klotho in mice results in the early appearance of
several pathological phenotypes that resemble human aging including
a short lifespan, infertility, arteriosclerosis/vascular
calcification, osteoporosis, skin atrophy, lung emphysema, acute
kidney injury, chronic kidney disease, renal fibrosis, diabetes and
cancer (Kuro-o, M. et al. Nature 1997, 390, p 45). In contrast,
overexpression of Klotho has been shown to increase the life-span
of mice (Kurosu, H. et al. Science 2005, 309, p 1829).
[0006] Klotho was detected in 1997 by Makoto Kuro-o, who found that
mice missing Klotho exhibited syndromes that resemble human ageing,
including a short lifespan. Klotho has been shown to be involved in
the suppression of several ageing phenotypes. A defect in klotho
gene expression in the mouse results in infertility,
arteriosclerosis, skin atrophy, osteoporosis and emphysema (Kuro-o,
M., et al. (1997), Nature 390, 45-51). The Klotho protein is most
highly expressed in the kidney, brain and pituitary gland, and is
present in lower levels within skeletal muscle, the urinary
bladder, the ovary and the testes (Avin, K. G., et al. (2014),
Frontiers in physiology 5, 189).
[0007] The Klotho protein exists in two forms: membrane Klotho and
secreted Klotho. Membrane Klotho functions as a receptor for a
hormone that regulates excretion of phosphate and synthesis of
active vitamin D in the kidney. Secreted Klotho functions as a
humoral factor with pleiotropic activities, including suppression
of growth factor signaling, suppression of oxidative stress, and
regulation of ion channels and transporters.
[0008] FGF23, a member of the fibroblast growth factor (FGF) family
was identified to be elevated in patients with autosomal dominant
hypophosphatemic rickets (ADHR). Thus FGF23 functions as a
phosphaturic hormone and a counter-regulatory hormone for vitamin D
(calcitriol) in a Klotho-dependent manner. Hyperphosphatemia leads
to stenosis of blood vessels, myocardial infarction, stroke and a
major shortening in life expectancy in patients with chronic kidney
disease (CKD). The absence of FGF-signaling in the kidney results
in increased serum levels of phosphate. It has been found that
secreted and the membrane bound form of Klotho forms a complex
together with the FGF-receptor and thereby increases
FGF23-dependent signaling (Kurosu et al. Journal of Biological
2006), 281(10) pp. 6120-61). Klotho's function as a cofactor for
FGF23 signaling is important for the regulation of the phosphate
serum levels.
[0009] Furthermore, Klotho functions through the modulation of
various signaling pathways including that of insulin growth factor
1 (IGF-1). One effect of Klotho is the increase in cellular
resistance to oxidative stress, which is involved in many different
pathological processes. It has been shown that through the
modulation of the cellular response to oxidative stress Klotho also
acts protectively in the context of neurodegenerative diseases such
as Alzheimer's Disease (Zeldich, E. et al. J Biol Chem 2014,
289(35):24700) and diabetes mellitus (Lin, Y. et al. Diabetes 2014,
DB140632). Klotho also has been suggested to be a repressor of
collagen synthesis and therefore might be beneficial in the context
of fibrosis (Ghosh, A. K. et al. Exp Biol Med 2013,
238(5):461).
[0010] It has been shown that Klotho expression is silenced in
several kinds of cancer cells, which is associated with enhanced
cell growth and the formation of cancer metastasis (Camilli et al.
Pgment Cell Melanoma Res 2011, 24(1), p 75; Wang et al, Am J Cancer
Res 2011, 1(1):111, Lee et al, Molecular Cancer 2010, 9:109). In
contrast, overexpression of Klotho in cancer cells can inhibit cell
growth and can promotes apoptosis of cancer cells (Chen, B. et al.
J of Exp and Clin Cancer Res 2010, 29:99). Furthermore, Klotho
up-regulation indirectly stimulated by administration of
renin-angiotensin system inhibitors or other compounds can lead to
suppression of renal fibrosis (Ming Chang Hu et al, Contrib Nephrol
2013, 180:47) and there appears to be a correlation between low
Klotho expression in diabetic rat models (Meng Fu Cheng et al,
Journal of Biomedicine and Biotechnology, 2010, 513853).
[0011] Background on Chronic Kidney Disease (CKD):
[0012] CKD is a growing international health problem, affecting
more than 26 million Americans. In patients with CKD renal Klotho
RNA is decreased. This clinical observation was confirmed in
numerous preclinical models, showing that unilateral nephrectomy
and contralateral ischemia reperfusion injury downregulates renal
klotho protein and mRNA expression. The same reduction in klotho
expression was shown in a chronic glomerulonephritis model. Klotho
overexpression improved renal function and ameliorated renal
histology in this model (Hu, M. C., et al. (2011), Journal of the
American Society of Nephrology: 22, 124-136, Haruna, Y., et al.
(2007) PNAS, 104, 2331-2336).
[0013] In CKD patients, there is a very high prevalence coronary
artery calcification, which increases cardiovascular morbidity and
mortality. The klotho FGF 23 axis plays an important role in
vascular mineralization (Stompor, T. (2014) World journal of
cardiology 6, 115-129). Increased cardiovascular (CV) morbidity and
mortality is well documented in chronic kidney disease (CKD). In a
survey among 1,120,295 adults in the San Francisco Bay Area a
strong correlation between renal function (estimated glomerular
filtration rate, GFR) and cardiovascular events was found (Go, A.
S., et al. (2004) The New England journal of medicine 351,
1296-1305).
[0014] In the Renal Research Institute (RRI)-CKD Study of adults
with moderate to severe CKD (Stages 3-5), enrolled between June
2000 and February 2006 (n=834) the authors found that heart rate
variability is predictive for clinical outcome and cardiovascular
disease (CVD) (Chandra, P., et al. (2012) official publication of
the European Dialysis and Transplant Association--European Renal
Association 27, 700-709). Chronic kidney disease (CKD) is therefore
a major risk factor for cardiovascular disease leading to increased
morbidity and shortening of lifespan. Klotho expression is markedly
reduced in kidneys from patients suffering from CKD. Restoring
Klotho expression by infusion of MSC-Klotho may improve kidney
function and thereby reduce the risk for cardiovascular death.
[0015] Background on Cardiovascular Disease:
[0016] Cardiovascular disease (CVD) is a prevalent condition in
general population and the first cause of death overall. Klotho has
been proposed as a key regulator of the development of CVD. In the
few clinical studies made, it has been observed a relationship
between low levels of soluble Klotho and the occurrence and
severity of CVD, as well as a reduction of cardiovascular risk when
they are high. Also, different polymorphisms of human Klotho gene
have been related to the incidence of cardiovascular events.
Moreover, several experimental studies indicate that this protein
acts in the maintenance of vascular homeostasis (Yamamoto M. et
al., J Biol Chem. 2005; 280: 38029-38034). Klotho improves
endothelial dysfunction through promotion of NO production and
mediates anti-inflammatory and anti-aging effects such as
suppression of adhesion molecules expression, attenuation of
nuclear factor-kappa B or inhibition of Wnt signaling. Klotho
regulates expression levels of the endothelial NO synthase (eNOS).
Six et al recently observed that attenuation mediated by Klotho of
FGF23 or phosphate-induced vasoconstriction is abolished by adding
nitro-L-arginine, a competitive inhibitor of NOS. Moreover, they
observed that exposure of HUVECs to Klotho increased NO production
and induced eNOS phosphorylation and iNOS expression.
Interestingly, Klotho was able to increase H.sub.2O.sub.2
production in cultured human VSMCs (HVSMCs), which suggests a more
complex effect of this protein on the regulation of vascular tone
through mediation of a ROS/NO balance (Six I et al. (2014), PLoS
One. 2014; 9:e93423).
[0017] Furthermore, this protein is related to the attenuation of
vascular calcification as well as prevention of cardiac
hypertrophy. The expression of this protein in the vascular wall
implies a new scenario for the treatment of vascular disorders.
Klotho protein is therefore related to CVD and plays a role in the
maintenance of functional vascular integrity (Martin-N nez, M.
(2014) World J Cardiol. 6(12): 1262-1269).
[0018] Background on Morbus Alzheimer (AD):
[0019] Neurodegenerative diseases, especially Morbus Alzheimer
(AD), are increasing in the western world. The Alzheimer's
association estimates that in the USA Alzheimer's is the 6.sup.th
leading cause of death, that every 67 seconds someone is diagnosed
with Alzheimer's and that the cost for medical treatment and
caregiving for these patients will exceed 1.1 trillion US $ by
2050. AD is characterized through the loss of neurons and synapses,
but also through the generation of neurotoxic amyloid beta peptides
(A.beta. plaques) and their deposition along with neurofibrillary
tangle formation. There is growing evidence, that the deposition of
amyloid is the central hallmark of the disease. Activated
astrocytes start an inflammatory reaction by producing
proinflammatory cytokines like II-6, II-1 and TNF-.alpha.. All this
starts with an improper reaction to oxidative stress, accumulation
of oxygen free radicals, hyperglycemia and insulin resistance
(Rosales-Corral, S., et al. (2015) Oxidative medicine and cellular
longevity, 985845).
[0020] Recently it was shown, that in the cerebrospinal fluid of AD
patients, the concentration of the anti-aging protein klotho is
significantly lower than in younger patients or in old patients
without AD (Semba, R. D., et al. (2014) Neuroscience letters 558,
37-40).
[0021] In the brain, Klotho protein is localized at the choroid
plexus, where the protein is dominantly localized at the apical
plasma membrane of ependymal cells. In kl-/- mouse brain, reduction
of synapses was evident in the hippocampus, suggesting a role of
Klotho as a humoral factor in the cerebrospinal fluid. Klotho
protein in the kidney is localized at the distal renal tubules (Li
S A, et al. (2004) Cell Structure and Function 29, 91-99).
[0022] Chen et al demonstrated, that loss of Klotho expression
leads to cognitive deficits. They found significant effects of
Klotho on oligodendrocyte functions, including induced maturation
of rat primary oligodendrocytic progenitor cells (OPCs) in vitro
and myelination. Klotho increased OPC maturation. In vivo studies
of Klotho knock-out mice and control littermates revealed that
knock-out mice have a significant reduction in major myelin protein
and gene expression. By immunohistochemistry, the number of total
and mature oligodendrocytes was significantly lower in Klotho
knock-out mice. At the ultrastructural level, Klotho knock-out mice
exhibited significantly impaired myelination of the optic nerve and
corpus callosum (Chen, C. D., et al. (2013) The Journal of
neuroscience 33, 1927-1939).
[0023] Background on Multiple Sclerosis (MS):
[0024] MS is a complex disease of the CNS that is characterized by
heterogeneous pathologies composed of both inflammatory and
neurodegenerative components. The most common histopathological
feature at early stages of the disease includes intermittent
episodes of acute inflammation within patches of white matter,
resulting in demyelination. Myelin is critical for maintaining
efficient axonal conduction and oligodendrocytes, the myelin
producer and maintainer of axonal health within the CNS, are
damaged or destroyed in MS patients. Endogenous oligodendrocyte
precursor cells (OPCs) are found to be universally dispersed within
the human CNS and can be found in high density within some subacute
lesions during early stages of MS.
[0025] Progressive MS is the latest stage of the disease,
characterized by a gradual worsening of symptoms without remission.
Severe neurological impairments dramatically reduce the quality of
life for the individual, and this is mainly attributed to expanding
cortical lesions impacting motor function. Pathologically, there is
widespread axonal degeneration and grey matter neuropathy. Diffuse
white and gray matter inflammation has been reported, correlating,
in part, to global microglial activation as well as the presence of
T cells, B cells and myelin-laden macrophages. Furthermore, there
is an overall failure of OPCs to efficiently remyelinate damaged
white and gray matter areas, dramatically reducing the possibility
for recovery (Chang, A., et al. (2002) The New England journal of
medicine 346, 165-173). Klotho enhances the maturation of OPCs into
mature oligodendrocytes (Chen, C. D., et al. (2013) The Journal of
neuroscience 33, 1927-1939).
[0026] Background on Amyotrophic Lateral Sclerosis (ALS):
[0027] ALS is a neurodegenerative disease of motor neurons with no
effective treatment. Wnt signaling plays important roles in nervous
system development and function, including axon guidance, synapse
formation and plasticity and has also been associated with
neurodegenerative diseases, including Alzheimer disease, Parkinson
disease and ALS. Axon degeneration is an important step in disease
progression.
[0028] The mechanisms underlying motor neuron cell death and axonal
degeneration in ALS remain elusive. Tury et al found, that two
non-canonical Wnt signaling components, aPKC and Ryk, which are
important regulators of axon growth and plasticity in both
developing embryos and in adult nervous system, were clearly up
regulated in the spinal cord of SOD1 (G93A) mice, providing
evidence that Wnt signaling is altered in ALS and might be involved
in disease etiology and pathogenesis (Tury, A., et al. (2014)
Developmental neurobiology 74, 839-850). A coimmunoprecipitation
study indicated that soluble klotho binds to various Wnt family
members, including Wnt1, Wnt3, Wnt4, and Wnt5a, suppresses Wnt
transcription, and inhibits Wnt biological activity in the skin. An
overexpression of klotho effectively antagonizes the activity of
endogenous and exogenous Wnt, which induces accelerated cell
senescence both in vitro and in vivo (Liu, H., et al. (2007)
Science 317, 803-806).
[0029] Background on Parkinson Disease (PD):
[0030] PD is a progressive neurodegenerative disorder clinically
characterized by the cardinal symptoms of resting tremor,
bradykinesia, cogwheel rigidity, and postural instability.
Responsiveness to L-3,4-dihydroxyphen-ylalanine (L-DOPA) and brain
imaging distinguish PD from other disorders. The pathological
hallmarks of PD are loss of dopaminergic cells in the substantia
nigra pars compacta and subsequent loss of dopamine innervation in
the striatum. Motor symptoms are the most obvious consequence of
this nigrostriatal neurodegeneration. However, not only the basal
ganglia but also other parts of the central nervous system as well
as the autonomic nervous system are affected. A wide range of
resulting non-motor symptoms can affect the patient's quality of
life. There is also a broad consensus that neurodegenerative
processes in PD start many years before the actual onset of
clinical symptoms. The phenotypical over-lapping between familial
and idiopathic PD is sufficient to dissect the commonly involved
pathways. These include mitochondrial dysfunction, oxidative
stress, protein misfolding, protein degradation, protein
aggregation, and inflammation.
[0031] Kosakai et al demonstrated that the number of
tyrosine-hydroxylase-positive dopaminergic neurons in the
substancia nigra pars compacta and the ventral tegmental area and
the striatal dopamine level in klotho-insufficient mice were
significantly decreased in age-dependent fashion. These phenotypic
features were completely rescued by vitamin D restriction,
indicating that abnormal increase in active vitamin D biosynthesis
by Klotho insufficiency induces degeneration of dopaminergic
neurons (Kosakai, A., et al. (2011) Brain research 1382,
109-117).
[0032] Due to its diverse functional roles in multiple organ
systems and its potential beneficial effect in various diseases,
novel treatments incorporating Klotho administration may represent
promising therapeutic approaches. Until the present time the
therapeutic administration of Klotho remains largely unexplored.
Effective therapeutic regimes, especially those enabling local
administration or local expression of Klotho in disease-affected
physiological niches, or methods of providing continual Klotho
protein expression in vivo, have not been described in the art.
SUMMARY OF THE INVENTION
[0033] In light of the prior art the technical problem underlying
the present invention is to provide alternative and/or improved
means for the treatment of diseases, particularly cancer, organ
fibrosis, renal failure, age-related organ pathologies,
arteriosclerosis, dementia, neurodegenerative diseases, such as
Alzheimer's disease (AD), Multiple sclerosis (MS), Huntington's
disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease,
and/or Schizophrenia, diabetes mellitus, sepsis, erectile
dysfunction or autoimmune diseases.
[0034] This problem is solved by the features of the independent
claims. Preferred embodiments of the present invention are provided
by the dependent claims.
[0035] The invention therefore relates to a genetically modified
mesenchymal stem cell, wherein said stem cell comprises an
exogenous nucleic acid comprising a Klotho encoding region operably
linked to a promoter or promoter/enhancer combination. Such MSCs
may be referred to as "Klotho-modified MSCs" or "MSC-Klotho". A
Klotho encoding region relates to any nucleic acid sequence that
encodes any given Klotho protein, encompassing but not limited to
those Klotho protein variants described herein.
[0036] One preferred amino acid sequence of Klotho is available
under accession number BAA23382 from the NCBI database. Said amino
acid sequence corresponds to the .alpha.-Klotho isoform.
Corresponding nucleic acid sequences that encode Klotho may be
provided by a person skilled in the art of molecular biology or
genetics. The use of sequence variants of Klotho that exhibit
functional analogy to the unmodified human form is also encompassed
by the present invention.
[0037] Moreover the invention also relates to further isoforms of
the Klotho gene and encompasses .beta.-Klotho and
.gamma.-Klotho.
[0038] Preferred sequences of the invention relate to those
provided below in Table 1. SEQ ID NO 1 relates to a sequence of the
complete human Klotho gene (cDNA) with the naturally occurring
sequence.
[0039] SEQ ID NO 2 relates to a codon optimized sequence (for
translation in human cells) of the complete human Klotho gene
(cDNA), as most preferred according to the present invention. The
encoded amino acid sequence for SEQ ID NO 1 and SEQ ID NO 2 are
identical. SEQ ID NO 3 relates to a sequence of the soluble form of
the Klotho gene (cDNA) with the naturally occurring sequence.
[0040] SEQ ID NO 4 relates to a sequence of the human .beta.-Klotho
gene (cDNA) with the naturally occurring sequence.
[0041] SEQ ID NO 5 relates to a sequence of the human
.gamma.-Klotho gene (cDNA) with the naturally occurring
sequence
[0042] Further sequence variants are hereby incorporated in the
invention that exhibit an alternative nucleic acid sequence to SEQ
ID NO 1-5 but encode the same or a corresponding or functionally
analogous amino acid sequence. Sequence variants obtained via using
degeneracy of the genetic code are included. Sequence optimized
nucleic acid sequences of those sequences provided herein are also
included within the scope of the invention.
TABLE-US-00001 TABLE 1 Preferred Klotho sequences. SEQ ID NO 1:
Human ATGCCCGCCAGCGCCCCGCCGCGCCGCCCGCGGCCGCCGC Klotho full length
CGCCGTCGCTGTCGCTGCTGCTGGTGCTGCTGGGCCTGGGC (membrane-bound) cDNA-
GGCCGCCGCCTGCGTGCGGAGCCGGGCGACGGCGCGCAGA Sequence (Pubmed:
CCTGGGCCCGTTTCTCGCGGCCTCCTGCCCCCGAGGCCGCG NM_004795.3)
GGCCTCTTCCAGGGCACCTTCCCCGACGGCTTCCTCTGGGCC Signal peptide (Pos.
1-52) GTGGGCAGCGCCGCCTACCAGACCGAGGGCGGCTGGCAGCA
GCACGGCAAGGGTGCGTCCATCTGGGATACGTTCACCCACCA
CCCCCTGGCACCCCCGGGAGACTCCCGGAACGCCAGTCTGC
CGTTGGGCGCCCCGTCGCCGCTGCAGCCCGCCACCGGGGAC
GTAGCCAGCGACAGCTACAACAACGTCTTCCGCGACACGGAG
GCGCTGCGCGAGCTCGGGGTCACTCACTACCGCTTCTCCATC
TCGTGGGCGCGAGTGCTCCCCAATGGCAGCGCGGGCGTCCC
CAACCGCGAGGGGCTGCGCTACTACCGGCGCCTGCTGGAGC
GGCTGCGGGAGCTGGGCGTGCAGCCCGTGGTCACCCTGTAC
CACTGGGACCTGCCCCAGCGCCTGCAGGACGCCTACGGCGG
CTGGGCCAACCGCGCCCTGGCCGACCACTTCAGGGATTACG
CGGAGCTCTGCTTCCGCCACTTCGGCGGTCAGGTCAAGTACT
GGATCACCATCGACAACCCCTACGTGGTGGCCTGGCACGGCT
ACGCCACCGGGCGCCTGGCCCCCGGCATCCGGGGCAGCCC
GCGGCTCGGGTACCTGGTGGCGCACAACCTCCTCCTGGCTC
ATGCCAAAGTCTGGCATCTCTACAATACTTCTTTCCGTCCCAC
TCAGGGAGGTCAGGTGTCCATTGCCCTAAGCTCTCACTGGAT
CAATCCTCGAAGAATGACCGACCACAGCATCAAAGAATGTCAA
AAATCTCTGGACTTTGTACTAGGTTGGTTTGCCAAACCCGTAT
TTATTGATGGTGACTATCCCGAGAGCATGAAGAATAACCTTTC
ATCTATTCTGCCTGATTTTACTGAATCTGAGAAAAAGTTCATCA
AAGGAACTGCTGACTTTTTTGCTCTTTGCTTTGGACCCACCTT
GAGTTTTCAACTTTTGGACCCTCACATGAAGTTCCGCCAATTG
GAATCTCCCAACCTGAGGCAACTGCTTTCCTGGATTGACCTTG
AATTTAACCATCCTCAAATATTTATTGTGGAAAATGGCTGGTTT
GTCTCAGGGACCACCAAGAGAGATGATGCCAAATATATGTATT
ACCTCAAAAAGTTCATCATGGAAACCTTAAAAGCCATCAAGCT
GGATGGGGTGGATGTCATCGGGTATACCGCATGGTCCCTCAT
GGATGGTTTCGAGTGGCACAGAGGTTACAGCATCAGGCGTGG
ACTCTTCTATGTTGACTTTCTAAGCCAGGACAAGATGTTGTTG
CCAAAGTCTTCAGCCTTGTTCTACCAAAAGCTGATAGAGAAAA
ATGGCTTCCCTCCTTTACCTGAAAATCAGCCCCTAGAAGGGAC
ATTTCCCTGTGACTTTGCTTGGGGAGTTGTTGACAACTACATT
CAAGTAGATACCACTCTGTCTCAGTTTACCGACCTGAATGTTT
ACCTGTGGGATGTCCACCACAGTAAAAGGCTTATTAAAGTGGA
TGGGGTTGTGACCAAGAAGAGGAAATCCTACTGTGTTGACTTT
GCTGCCATCCAGCCCCAGATCGCTTTACTCCAGGAAATGCAC
GTTACACATTTTCGCTTCTCCCTGGACTGGGCCCTGATTCTCC
CTCTGGGTAACCAGTCCCAGGTGAACCACACCATCCTGCAGT
ACTATCGCTGCATGGCCAGCGAGCTTGTCCGTGTCAACATCA
CCCCAGTGGTGGCCCTGTGGCAGCCTATGGCCCCGAACCAA
GGACTGCCGCGCCTCCTGGCCAGGCAGGGCGCCTGGGAGAA
CCCCTACACTGCCCTGGCCTTTGCAGAGTATGCCCGACTGTG
CTTTCAAGAGCTCGGCCATCACGTCAAGCTTTGGATAACGATG
AATGAGCCGTATACAAGGAATATGACATACAGTGCTGGCCACA
ACCTTCTGAAGGCCCATGCCCTGGCTTGGCATGTGTACAATG
AAAAGTTTAGGCATGCTCAGAATGGGAAAATATCCATAGCCTT
GCAGGCTGATTGGATAGAACCTGCCTGCCCTTTCTCCCAAAA
GGACAAAGAGGTGGCTGAGAGAGTTTTGGAATTTGACATTGG
CTGGCTGGCTGAGCCCATTTTCGGCTCTGGAGATTATCCATG
GGTGATGAGGGACTGGCTGAACCAAAGAAACAATTTTCTTCTT
CCTTATTTCACTGAAGATGAAAAAAAGCTAATCCAGGGTACCT
TTGACTTTTTGGCTTTAAGCCATTATACCACCATCCTTGTAGAC
TCAGAAAAAGAAGATCCAATAAAATACAATGATTACCTAGAAGT
GCAAGAAATGACCGACATCACGTGGCTCAACTCCCCCAGTCA
GGTGGCGGTAGTGCCCTGGGGGTTGCGCAAAGTGCTGAACT
GGCTGAAGTTCAAGTACGGAGACCTCCCCATGTACATAATATC
CAATGGAATCGATGACGGGCTGCATGCTGAGGACGACCAGCT
GAGGGTGTATTATATGCAGAATTACATAAACGAAGCTCTCAAA
GCCCACATACTGGATGGTATCAATCTTTGCGGATACTTTGCTT
ATTCGTTTAACGACCGCACAGCTCCGAGGTTTGGCCTCTATCG
TTATGCTGCAGATCAGTTTGAGCCCAAGGCATCCATGAAACAT
TACAGGAAAATTATTGACAGCAATGGTTTCCCGGGCCCAGAAA
CTCTGGAAAGATTTTGTCCAGAAGAATTCACCGTGTGTACTGA
GTGCAGTTTTTTTCACACCCGAAAGTCTTTACTGGCTTTCATAG
CTTTTCTATTTTTTGCTTCTATTATTTCTCTCTCCCTTATATTTTA
CTACTCGAAGAAAGGCAGAAGAAGTTACAAATAG SEQ ID NO 2: Human
ATGCCCGCCAGCGCCCCTCCAAGAAGGCCTAGACCTCCTCCA Klotho full length
CCTAGCCTGAGCCTGCTGCTGGTGCTGCTGGGACTGGGAGG (membrane-bound) cDNA-
AAGAAGGCTGAGAGCCGAACCTGGGGATGGCGCCCAGACAT Sequence as preferably
GGGCCAGATTCTCTAGACCACCCGCCCCTGAAGCCGCCGGA used in the constructs of
CTGTTTCAGGGAACCTTCCCCGATGGCTTCCTGTGGGCCGTG the present invention.
The GGATCTGCCGCCTATCAGACTGAAGGGGGCTGGCAGCAGCA sequence has been
codon- CGGCAAGGGCGCCTCTATCTGGGACACCTTCACCCACCATCC optimized and has
an TCTGGCCCCACCCGGCGACAGCAGAAATGCTTCTCTGCCTCT additional HA-Tag
(3037- GGGAGCCCCCAGCCCTCTGCAGCCTGCTACAGGGGATGTGG 3063; underlined
text in the CCAGCGACAGCTACAACAACGTGTTCCGGGACACAGAGGCCC sequence)
for detection at TGCGGGAACTGGGCGTGACCCACTACAGATTCAGCATCAGCT the
3'-end of the sequence, GGGCCAGAGTGCTGCCCAATGGCTCTGCCGGCGTGCCCAAT
STOP (3064-3072; AGAGAGGGCCTGCGGTACTACCGGCGGCTGCTGGAAAGACT provided
in bold text within GAGAGAACTGGGAGTGCAGCCCGTCGTGACCCTGTACCATTG the
sequence) GGACCTGCCCCAGAGACTGCAGGATGCCTATGGCGGCTGGG Signal peptide
(Pos. 1-52) CCAATAGAGCCCTGGCCGACCACTTCAGAGACTACGCCGAGC
TGTGCTTCCGGCACTTTGGCGGCCAAGTGAAGTACTGGATCA
CCATCGACAACCCCTACGTGGTGGCCTGGCACGGCTATGCCA
CAGGCAGACTGGCCCCTGGCATCAGAGGAAGCCCTAGACTG
GGCTACCTGGTGGCCCACAATCTGCTGCTGGCCCACGCTAAA
GTGTGGCACCTGTACAACACCAGCTTCCGGCCTACACAGGGC
GGCCAGGTGTCCATTGCCCTGAGCAGCCACTGGATCAACCCC
AGACGGATGACCGACCACAGCATCAAAGAGTGCCAGAAAAGC
CTGGACTTCGTGCTGGGATGGTTCGCCAAGCCCGTGTTCATC
GACGGCGACTACCCCGAGAGCATGAAGAACAACCTGTCCAGC
ATCCTGCCCGACTTCACCGAGAGCGAGAAGAAGTTCATCAAG
GGCACCGCCGATTTCTTCGCCCTGTGCTTCGGCCCTACCCTG
AGCTTCCAGCTGCTGGACCCCCACATGAAGTTCAGACAGCTG
GAAAGCCCCAACCTGCGGCAGCTGCTGAGCTGGATCGACCT
GGAATTCAACCACCCCCAGATTTTCATCGTGGAAAACGGCTG
GTTCGTGTCCGGCACCACCAAGAGGGACGACGCCAAGTACAT
GTATTACCTGAAAAAGTTTATCATGGAAACCCTGAAGGCCATC
AAGCTGGACGGCGTGGACGTGATCGGCTACACAGCCTGGTC
CCTGATGGACGGCTTCGAGTGGCACCGGGGCTACTCTATCAG
ACGGGGCCTGTTCTACGTGGACTTCCTGAGCCAGGACAAGAT
GCTGCTGCCTAAGAGCAGCGCCCTGTTTTACCAGAAGCTGAT
CGAGAAGAACGGCTTCCCACCCCTGCCCGAGAACCAGCCTCT
GGAAGGCACCTTCCCCTGCGATTTTGCCTGGGGCGTGGTGGA
CAACTACATCCAGGTGGACACCACCCTGTCCCAGTTCACCGA
CCTGAACGTGTACCTGTGGGACGTGCACCACAGCAAGCGGCT
GATTAAGGTGGACGGGGTCGTGACCAAGAAGCGGAAGTCCTA
CTGCGTGGACTTTGCCGCCATCCAGCCCCAGATTGCCCTGCT
GCAGGAAATGCACGTGACACACTTCCGGTTCTCCCTGGACTG
GGCCCTGATCCTGCCACTGGGCAATCAGAGCCAAGTGAACCA
CACCATTCTGCAGTACTACAGATGCATGGCCTCCGAGCTGGT
GCGCGTGAACATCACACCTGTGGTGGCCCTGTGGCAGCCCAT
GGCCCCTAATCAGGGACTGCCTAGACTGCTGGCTAGACAGGG
CGCCTGGGAGAACCCTTACACCGCCCTGGCCTTTGCCGAGTA
CGCCCGGCTGTGTTTCCAGGAACTGGGGCACCACGTGAAGCT
GTGGATCACAATGAACGAGCCCTACACCCGGAACATGACCTA
CAGCGCCGGACATAACCTGCTGAAGGCCCACGCCCTGGCTT
GGCATGTGTACAACGAGAAGTTCCGGCACGCCCAGAACGGCA
AGATCAGTATCGCCCTGCAGGCCGACTGGATCGAGCCCGCCT
GTCCCTTCAGCCAGAAAGACAAAGAGGTGGCCGAGCGGGTG
CTGGAATTCGACATTGGATGGCTGGCCGAGCCCATCTTCGGC
AGCGGCGATTACCCCTGGGTCATGCGGGACTGGCTGAACCA
GCGGAACAACTTCCTGCTGCCTTACTTTACCGAGGATGAGAA
GAAACTGATCCAGGGGACCTTCGACTTCCTGGCCCTGAGCCA
CTACACCACAATCCTGGTGGACAGCGAGAAAGAGGACCCCAT
CAAGTACAACGACTACCTGGAAGTGCAGGAAATGACCGACAT
CACCTGGCTGAATAGCCCCTCCCAGGTGGCCGTGGTGCCTTG
GGGACTGAGAAAGGTGCTGAATTGGCTGAAGTTTAAGTACGG
CGACCTGCCCATGTACATCATCAGCAACGGCATCGACGATGG
CCTGCACGCCGAGGACGATCAGCTGCGGGTGTACTACATGCA
GAACTACATCAACGAGGCCCTGAAAGCCCACATCCTGGACGG
CATCAACCTGTGCGGCTACTTCGCCTACAGCTTCAACGACCG
GACCGCCCCTAGATTCGGCCTGTACAGATACGCCGCCGACCA
GTTCGAGCCCAAGGCCAGCATGAAGCACTACCGGAAGATCAT
CGACAGCAATGGCTTCCCTGGCCCCGAGACACTGGAACGGTT
CTGCCCCGAGGAATTCACCGTGTGTACCGAGTGCAGCTTCTT
CCACACCAGAAAGTCCCTGCTGGCTTTTATCGCCTTCCTGTTC
TTCGCCAGCATCATCTCCCTGTCCCTGATCTTCTACTACAGCA
AGAAGGGCAGACGGTCCTACAAGTACCCCTACGACGTGCCCG ACTACGCCTGATGATGA SEQ ID
NO 3: Human ATGCCCGCCAGCGCCCCGCCGCGCCGCCCGCGGCCGCCGC Klotho
(secreted) cDNA- CGCAGTCGCTGTCGCTGCTGCTGGTGCTGCTGGGCCTGGGC Sequence
(Pubmed: GGCCGCCGCCTGCGTGCGGAGCCGGGCGACGGCGCGCAGA AB009667.2)
CCTGGGCCCGTTTCTCGCGGCCTCCTGCCCCCGAGGCCGCG Signal peptide (Pos.
1-52) GGCCTCTTCCAGGGCACCTTCCCCGACGGCTTCCTCTGGGCC
GTGGGCAGCGCCGCCTACCAGACCGAGGGCGGCTGGCAGCA
GCACGGCAAGGGTGCGTCCATCTGGGACACGTTCACCCACCA
CCCCCTGGCACCCCCGGGAGACTCCCGGAACGCCAGTCTGC
CGTTGGGCGCCCCGTCGCCGCTGCAGCCCGCCACCGGGGAC
GTAGCCAGCGACAGCTACAACAACGTCTTCCGCGACACGGAG
GCGCTGCGCGAGCTCGGGGTCACTCACTACCGCTTCTCCATC
TCGTGGGCGCGAGTGCTCCCCAATGGCAGCGCGGGCGTCCC
CAACCGCGAGGGGCTGCGCTACTACCGGCGCCTGCTGGAGC
GGCTGCGGGAGCTGGGCGTGCAGCCCGTGGTCACCCTGTAC
CACTGGGACCTGCCCCAGCGCCTGCAGGACGCCTACGGCGG
CTGGGCCAACCGCGCCCTGGCCGACCACTTCAGGGATTACG
CGGAGCTCTGCTTCCGCCACTTCGGCGGTCAGGTCAAGTACT
GGATCACCATCGACAACCCCTACGTGGTGGCCTGGCACGGCT
ACGCCACCGGGCGCCTGGCCCCCGGCATCCGGGGCAGCCC
GCGGCTCGGGTACCTGGTGGCGCACAACCTCCTCCTGGCTC
ATGCCAAAGTCTGGCATCTCTACAATACTTCTTTCCGTCCCAC
TCAGGGAGGTCAGGTGTCCATTGCCCTAAGCTCTCACTGGAT
CAATCCTCGAAGAATGACCGACCACAGCATCAAAGAATGTCAA
AAATCTCTGGACTTTGTACTAGGTTGGTTTGCCAAACCCGTAT
TTATTGATGGTGACTATCCCGAGAGCATGAAGAATAACCTTTC
ATCTATTCTGCCTGATTTTACTGAATCTGAGAAAAAGTTCATCA
AAGGAACTGCTGACTTTTTTGCTCTTTGCTTTGGACCCACCTT
GAGTTTTCAACTTTTGGACCCTCACATGAAGTTCCGCCAATTG
GAATCTCCCAACCTGAGGCAACTGCTTTCCTGGATTGACCTTG
AATTTAACCATCCTCAAATATTTATTGTGGAAAATGGCTGGTTT
GTCTCAGGGACCACCAAGAGAGATGATGCCAAATATATGTATT
ACCTCAAAAAGTTCATCATGGAAACCTTAAAAGCCATCAAGCT
GGATGGGGTGGATGTCATCGGGTATACCGCATGGTCCCTCAT
GGATGGTTTCGAGTGGCACAGAGGTTACAGCATCAGGCGTGG
ACTCTTCTATGTTGACTTTCTAAGCCAGGACAAGATGTTGTTG
CCAAAGTCTTCAGCCTTGTTCTACCAAAAGCTGATAGAGAAAA
ATGGCTTCCCTCCTTTACCTGAAAATCAGCCCCTAGAAGGGAC
ATTTCCCTGTGACTTTGCTTGGGGAGTTGTTGACAACTACATT
CAAGTAAGTCAGCTGACAAAACCAATCAGCAGTCTCACCAAGC CCTATCACTAG SEQ ID NO
4: Human .beta.- ATGAAGCCAGGCTGTGCGGCAGGATCTCCAGGGAATGAATGG Klotho
cDNA-Sequence ATTTTCTTCAGCACTGATGAAATAACCACACGCTATAGGAATA (Pubmed:
NM_175737.3; CAATGTCCAACGGGGGATTGCAAAGATCTGTCATCCTGTCAG bp 98-3232)
CACTTATTCTGCTACGAGCTGTTACTGGATTCTCTGGAGATGG
AAGAGCTATATGGTCTAAAAATCCTAATTTTACTCCGGTAAATG
AAAGTCAGCTGTTTCTCTATGACACTTTCCCTAAAAACTTTTTC
TGGGGTATTGGGACTGGAGCATTGCAAGTGGAAGGGAGTTGG
AAGAAGGATGGAAAAGGACCTTCTATATGGGATCATTTCATCC
ACACACACCTTAAAAATGTCAGCAGCACGAATGGTTCCAGTGA
CAGTTATATTTTTCTGGAAAAAGACTTATCAGCCCTGGATTTTA
TAGGAGTTTCTTTTTATCAATTTTCAATTTCCTGGCCAAGGCTT
TTCCCCGATGGAATAGTAACAGTTGCCAACGCAAAAGGTCTG
CAGTACTACAGTACTCTTCTGGACGCTCTAGTGCTTAGAAACA
TTGAACCTATAGTTACTTTATACCACTGGGATTTGCCTTTGGCA
CTACAAGAAAAATATGGGGGGTGGAAAAATGATACCATAATAG
ATATCTTCAATGACTATGCCACATACTGTTTCCAGATGTTTGGG
GACCGTGTCAAATATTGGATTACAATTCACAACCCATATCTAGT
GGCTTGGCATGGGTATGGGACAGGTATGCATGCCCCTGGAGA
GAAGGGAAATTTAGCAGCTGTCTACACTGTGGGACACAACTT
GATCAAGGCTCACTCGAAAGTTTGGCATAACTACAACACACAT
TTCCGCCCACATCAGAAGGGTTGGTTATCGATCACGTTGGGA
TCTCATTGGATCGAGCCAAACCGGTCGGAAAACACGATGGAT
ATATTCAAATGTCAACAATCCATGGTTTCTGTGCTTGGATGGTT
TGCCAACCCTATCCATGGGGATGGCGACTATCCAGAGGGGAT
GAGAAAGAAGTTGTTCTCCGTTCTACCCATTTTCTCTGAAGCA
GAGAAGCATGAGATGAGAGGCACAGCTGATTTCTTTGCCTTTT
CTTTTGGACCCAACAACTTCAAGCCCCTAAACACCATGGCTAA
AATGGGACAAAATGTTTCACTTAATTTAAGAGAAGCGCTGAAC
TGGATTAAACTGGAATACAACAACCCTCGAATCTTGATTGCTG
AGAATGGCTGGTTCACAGACAGTCGTGTGAAAACAGAAGACA
CCACGGCCATCTACATGATGAAGAATTTCCTCAGCCAGGTGCT
TCAAGCAATAAGGTTAGATGAAATACGAGTGTTTGGTTATACT
GCCTGGTCTCTCCTGGATGGCTTTGAATGGCAGGATGCTTAC
ACCATCCGCCGAGGATTATTTTATGTGGATTTTAACAGTAAAC
AGAAAGAGCGGAAACCTAAGTCTTCAGCACACTACTACAAACA
GATCATACGAGAAAATGGTTTTTCTTTAAAAGAGTCCACGCCA
GATGTGCAGGGCCAGTTTCCCTGTGACTTCTCCTGGGGTGTC
ACTGAATCTGTTCTTAAGCCCGAGTCTGTGGCTTCGTCCCCAC
AGTTCAGCGATCCTCATCTGTACGTGTGGAACGCCACTGGCA
ACAGACTGTTGCACCGAGTGGAAGGGGTGAGGCTGAAAACAC
GACCCGCTCAATGCACAGATTTTGTAAACATCAAAAAACAACT
TGAGATGTTGGCAAGAATGAAAGTCACCCACTACCGGTTTGCT
CTGGATTGGGCCTCGGTCCTTCCCACTGGCAACCTGTCCGCG
GTGAACCGACAGGCCCTGAGGTACTACAGGTGCGTGGTCAGT
GAGGGGCTGAAGCTTGGCATCTCCGCGATGGTCACCCTGTAT
TATCCGACCCACGCCCACCTAGGCCTCCCCGAGCCTCTGTTG
CATGCCGACGGGTGGCTGAACCCATCGACGGCCGAGGCCTT
CCAGGCCTACGCTGGGCTGTGCTTCCAGGAGCTGGGGGACC
TGGTGAAGCTCTGGATCACCATCAACGAGCCTAACCGGCTAA
GTGACATCTACAACCGCTCTGGCAACGACACCTACGGGGCGG
CGCACAACCTGCTGGTGGCCCACGCCCTGGCCTGGCGCCTC
TACGACCGGCAGTTCAGGCCCTCACAGCGCGGGGCCGTGTC
GCTGTCGCTGCACGCGGACTGGGCGGAACCCGCCAACCCCT
ATGCTGACTCGCACTGGAGGGCGGCCGAGCGCTTCCTGCAG
TTCGAGATCGCCTGGTTCGCCGAGCCGCTCTTCAAGACCGGG
GACTACCCCGCGGCCATGAGGGAATACATTGCCTCCAAGCAC
CGACGGGGGCTTTCCAGCTCGGCCCTGCCGCGCCTCACCGA
GGCCGAAAGGAGGCTGCTCAAGGGCACGGTCGACTTCTGCG
CGCTCAACCACTTCACCACTAGGTTCGTGATGCACGAGCAGC
TGGCCGGCAGCCGCTACGACTCGGACAGGGACATCCAGTTTC
TGCAGGACATCACCCGCCTGAGCTCCCCCACGCGCCTGGCT
GTGATTCCCTGGGGGGTGCGCAAGCTGCTGCGGTGGGTCCG
GAGGAACTACGGCGACATGGACATTTACATCACCGCCAGTGG
CATCGACGACCAGGCTCTGGAGGATGACCGGCTCCGGAAGT
ACTACCTAGGGAAGTACCTTCAGGAGGTGCTGAAAGCATACC
TGATTGATAAAGTCAGAATCAAAGGCTATTATGCATTCAAACTG
GCTGAAGAGAAATCTAAACCCAGATTTGGATTCTTCACATCTG
ATTTTAAAGCTAAATCCTCAATACAATTTTACAACAAAGTGATC
AGCAGCAGGGGCTTCCCTTTTGAGAACAGTAGTTCTAGATGC
AGTCAGACCCAAGAAAATACAGAGTGCACTGTCTGCTTATTCC
TTGTGCAGAAGAAACCACTGATATTCCTGGGTTGTTGCTTCTT
CTCCACCCTGGTTCTACTCTTATCAATTGCCATTTTTCAAAGGC
AGAAGAGAAGAAAGTTTTGGAAAGCAAAAAACTTACAACACAT
ACCATTAAAGAAAGGCAAGAGAGTTGTTAGCTAA SEQ ID NO 5: Human .gamma.-
ATGAAGCCAGTGTGGGTCGCCACCCTTCTGTGGATGCTACTG Klotho cDNA-Sequence
CTGGTGCCCAGGCTGGGGGCCGCCCGGAAGGGGTCCCCAG Homo sapiens lactase like
AAGAGGCCTCCTTCTACTATGGAACCTTCCCTCTTGGCTTCTC (LCTL), transcript
variant 1, CTGGGGCGTGGGCAGTTCTGCCTACCAGACGGAGGGCGCCT cDNA
GGGACCAGGACGGGAAAGGGCCTAGCATCTGGGACGTCTTC (Pubmed: NM_207338.3,
ACACACAGTGGGAAGGGGAAAGTGCTTGGGAATGAGACGGC bp: 133-1836)
AGATGTAGCCTGTGACGGCTACTACAAGGTCCAGGAGGACAT
CATTCTGCTGAGGGAACTGCACGTCAACCACTACCGATTCTCC
CTGTCTTGGCCCCGGCTCCTGCCCACAGGCATCCGAGCCGA
GCAGGTGAACAAGAAGGGAATCGAATTCTACAGTGATCTTATC
GATGCCCTTCTGAGCAGCAACATCACTCCCATCGTGACCTTG
CACCACTGGGATCTGCCACAGCTGCTCCAGGTCAAATACGGT
GGGTGGCAGAATGTGAGCATGGCCAACTACTTCAGAGACTAC
GCCAACCTGTGCTTTGAGGCCTTTGGGGACCGTGTGAAGCAC
TGGATCACGTTCAGTGATCCTCGGGCAATGGCAGAAAAAGGC
TATGAGACGGGCCACCATGCGCCGGGCCTGAAGCTCCGCGG
CACCGGCCTGTACAAGGCAGCACACCACATCATTAAGGCCCA
CGCCAAAGCCTGGCATTCTTATAACACCACGTGGCGCAGCAA
GCAGCAAGGTCTGGTGGGAATTTCATTGAACTGTGACTGGGG
GGAACCTGTGGACATTAGTAACCCCAAGGACCTAGAGGCTGC
CGAGAGATACCTACAGTTCTGTCTGGGCTGGTTTGCCAACCC
CATTTATGCCGGTGACTACCCCCAAGTCATGAAGGACTACATT
GGAAGAAAGAGTGCAGAGCAAGGCCTGGAGATGTCGAGGTTA
CCGGTGTTCTCACTCCAGGAGAAGAGCTACATTAAAGGCACA
TCCGATTTCTTGGGATTAGGTCATTTTACTACTCGGTACATCAC
GGAAAGGAACTACCCCTCCCGCCAGGGGCCCAGCTACCAGA
ACGATCGTGACTTGATAGAGCTGGTTGACCCAAACTGGCCAG
ATCTGGGGTCTAAATGGCTATATTCTGTGCCATGGGGATTTAG
GAGGCTCCTTAACTTTGCTCAGACTCAATACGGTGATCCTCCC
ATATATGTGATGGAAAATGGAGCATCTCAAAAATTCCACTGTA
CTCAATTATGTGATGAGTGGAGAATTCAATACCTTAAAGGATA
CATAAATGAAATGCTAAAAGCTATAAAAGATGGTGCTAATATAA
AGGGGTATACTTCCTGGTCTCTGTTGGATAAGTTTGAATGGGA
GAAAGGATACTCAGATAGATATGGATTCTACTATGTTGAATTTA
ACGACAGAAATAAGCCTCGCTATCCAAAGGCTTCAGTTCAATA
TTACAAGAAGATTATCATTGCCAATGGGTTTCCCAATCCAAGA
GAGGTGGAAAGTTGGTACCTCAAAGCTTTGGAAACTTGCTCTA
TCAACAATCAGATGCTTGCTGCAGAGCCCTTGCTAAGTCACAT
GCAAATGGTTACGGAGATCGTGGTACCCACTGTCTGCTCCCT
CTGTGTCCTCATCACTGCTGTTCTACTAATGCTCCTCCTGAGG AGGCAGAGCTGA
[0043] The invention therefore encompasses a modified MSC as
described herein comprising a nucleic acid molecule selected from
the group consisting of:
[0044] a) a nucleic acid molecule comprising a nucleotide sequence
that encodes a Klotho protein, preferably according to a protein
sequence of SEQ ID NO 6 to 10, whereby the nucleotide sequence is
preferably a sequence according to SEQ ID NO 1, 2, 3, 4 or 5;
[0045] b) a nucleic acid molecule which is complementary to a
nucleotide sequence in accordance with a);
[0046] c) a nucleic acid molecule comprising a nucleotide sequence
having sufficient sequence identity to be functionally
analogous/equivalent to a nucleotide sequence according to a) or
b), comprising preferably a sequence identity to a nucleotide
sequence according to a) or b) of at least 70%, 80%, preferably
90%, more preferably 95%;
[0047] d) a nucleic acid molecule which, as a consequence of the
genetic code, is degenerated into a nucleotide sequence according
to a) through c); and/or
[0048] e) a nucleic acid molecule according to a nucleotide
sequence of a) through d) which is modified by deletions,
additions, substitutions, translocations, inversions and/or
insertions and functionally analogous/equivalent to a nucleotide
sequence according to a) through d).
[0049] Functionally analogous sequences refer to the ability to
encode a functional klotho gene product. Functionally analogous
sequences refer to the ability to encode a functional Klotho gene
product and to enable the same or similar functional effect as
human Klotho. Klotho function may be determined by its
.beta.-glucuronidase activity, or via is functional effects
described in the examples below. Appropriate assays for determining
.beta.-glucuronidase activity, or for assaying the desired
biological functional effects described herein, and as shown in the
examples, are known to a skilled person.
[0050] To measure the .beta.-glucuronidase of Klotho, recombinant
protein is subjected to an in vitro reaction. In this reaction the
fluorescence-labeled substrate glucuronide is hydrolyzed by the
Klotho protein. The reaction buffer contains 0.5 mM
4-methylumbelliferyl (4Mu)-D-glucuronide (Sigma), 0.1 M sodium
citrate buffer, pH 5.5, 0.05M NaCl, 0.01% Tween 20, and 20 .mu.g of
purified secreted Klotho-protein. The reaction is carried out in a
final volume of 100 .mu.l. The enzymatic function of Klotho
correlates with an increase in fluorescence intensity which is
measured at several time points with a multi-label counter ARVOsx
(PerkinElmer Life Sciences) at an excitation wavelength of 360 nm
and an emission wavelength of 470 nm. Hydrolyzed products are
quantified on the basis of 4-methylumbelliferone fluorescence.
[0051] The nucleotide sequence according to SEQ ID NO 1 to 5 encode
a human Klotho protein of the amino acid sequences according to SEQ
ID NO 6 to 10, which are preferred in the present invention:
TABLE-US-00002 TABLE 2 Preferred Klotho sequences. SEQ ID NO 6:
Human MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWA Klotho full
length RFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGK (membrane-bound)
amino GASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSY acid sequence as
encoded NNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYY by SEQ ID 1
RRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHF
RDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGS
PRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWIN
PRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSIL
PDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNL
RQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIM
ETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQ
DKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVV
DNYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCV
DFAAIQPQIALLQEMHVTHFRFSLDWALILPLGNQSQVNHTILQYY
RCMASELVRVNITPVVALWQPMAPNQGLPRLLARQGAWENPYT
ALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHNLLKA
HALAWHVYNEKFRHAQNGKISIALQADWIEPACPFSQKDKEVAE
RVLEFDIGWLAEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEK
KLIQGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVQEMTDITWLN
SPSQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDDGLHAEDD
QLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYR
YAADQFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECS
FFHTRKSLLAFIAFLFFASIISLSLIFYYSKKGRRSYK SEQ ID NO 7: Human
MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWA Klotho full length
RFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGK (membrane-bound) amino
GASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSY acid sequence with
NNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYY additional HA-Tag
RRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHF (underlined text in the
RDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGS sequence) as encoded
by PRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWIN SEQ ID NO 2.
PRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSIL
PDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNL
RQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIM
ETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQ
DKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVV
DNYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCV
DFAAIQPQIALLQEMHVTHFRFSLDWALILPLGNQSQVNHTILQYY
RCMASELVRVNITPVVALWQPMAPNQGLPRLLARQGAWENPYT
ALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHNLLKA
HALAWHVYNEKFRHAQNGKISIALQADWIEPACPFSQKDKEVAE
RVLEFDIGWLAEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEK
KLIQGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVQEMTDITWLN
SPSQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDDGLHAEDD
QLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYR
YAADQFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECS
FFHTRKSLLAFIAFLFFASIISLSLIFYYSKKGRRSYKYPYDVPDYA SEQ ID NO 8: Human
MPASAPPRRPRPPPQSLSLLLVLLGLGGRRLRAEPGDGAQTWA Klotho (secreted) amino
RFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGK acid sequence as encoded
GASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSY by SEQ ID NO 3
NNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYY
RRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHF
RDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGS
PRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWIN
PRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSIL
PDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNL
RQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIM
ETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQ
DKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVV DNYIQVSQLTKPISSLTKPYH
SEQ ID NO 9: Human .beta.-
MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALI Klotho amino acid
LLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGI sequence as encoded by
GTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIF SEQ ID NO 4,
LEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLL
DALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYC
FQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYT
VGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSEN
TMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFS
EAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREA
LNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVL
QAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKE
RKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESV
LKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCT
DFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALR
YYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPST
AEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGA
AHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYA
DSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGL
SSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYD
SDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYIT
ASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLA
EEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQT
QENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKF WKAKNLQHIPLKKGKRVVS
SEQ ID NO 10: Human .gamma.-
MKPVWVATLLWMLLLVPRLGAARKGSPEEASFYYGTFPLGFSW Klotho Homo sapiens
GVGSSAYQTEGAWDQDGKGPSIWDVFTHSGKGKVLGNETADV lactase like (LCTL),
ACDGYYKVQEDIILLRELHVNHYRFSLSWPRLLPTGIRAEQVNKK transcript variant 1,
amino GIEFYSDLIDALLSSNITPIVTLHHWDLPQLLQVKYGGWQNVSMA acid sequence
as encoded NYFRDYANLCFEAFGDRVKHWITFSDPRAMAEKGYETGHHAPG by SEQ ID NO
5. LKLRGTGLYKAAHHIIKAHAKAWHSYNTTWRSKQQGLVGISLNC
DWGEPVDISNPKDLEAAERYLQFCLGWFANPIYAGDYPQVMKD
YIGRKSAEQGLEMSRLPVFSLQEKSYIKGTSDFLGLGHFTTRYITE
RNYPSRQGPSYQNDRDLIELVDPNWPDLGSKWLYSVPWGFRRL
LNFAQTQYGDPPIYVMENGASQKFHCTQLCDEWRIQYLKGYINE
MLKAIKDGANIKGYTSWSLLDKFEWEKGYSDRYGFYYVEFNDRN
KPRYPKASVQYYKKIIIANGFPNPREVESWYLKALETCSINNQMLA
AEPLLSHMQMVTEIVVPTVCSLCVLITAVLLMLLLRRQS
[0052] Protein modifications to the klotho protein, which may occur
through substitutions in amino acid sequence, and nucleic acid
sequences encoding such molecules, are also included within the
scope of the invention. Substitutions as defined herein are
modifications made to the amino acid sequence of the protein,
whereby one or more amino acids are replaced with the same number
of (different) amino acids, producing a protein which contains a
different amino acid sequence than the primary protein. The
substitution may not significantly alter the function of the
protein. Like additions, substitutions may be natural or
artificial. It is well known in the art that amino acid
substitutions may be made without significantly altering the
protein's function. This is particularly true when the modification
relates to a "conservative" amino acid substitution, which is the
substitution of one amino acid for another of similar properties.
Such "conserved" amino acids can be natural or synthetic amino
acids which because of size, charge, polarity and conformation can
be substituted without significantly affecting the structure and
function of the protein. Frequently, many amino acids may be
substituted by conservative amino acids without deleteriously
affecting the protein's function. In general, the non-polar amino
acids Gly, Ala, Val, lie and Leu; the non-polar aromatic amino
acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr,
Cys, Gin, Asn and Met; the positively charged amino acids Lys, Arg
and His; the negatively charged amino acids Asp and Glu, represent
groups of conservative amino acids. This list is not exhaustive.
For example, it is well known that Ala, Gly, Ser and sometimes Cys
can substitute for each other even though they belong to different
groups.
[0053] In one embodiment the genetically modified cell as described
herein are characterised in that said cell are obtained from bone
marrow, umbilical cord, adipose tissue, or amniotic fluid.
[0054] In one embodiment the genetically modified cell as described
herein are characterised in that said cell are CD34 negative.
[0055] In one embodiment the genetically modified cell as described
herein are characterised in that said cell are human cell.
[0056] Due to their ability to migrate to areas of disease, in
particular areas of inflammation, MSCs surprisingly represent a
suitable tool for the delivery of Klotho as a therapeutic agent.
Without being bound by theory, the MSCs of the present invention
represent a drug delivery tool or vehicle for effective delivery of
a therapeutic agent to the site of disease. According to a
preferred embodiment of the present invention the therapeutic agent
is Klotho protein expressed from an exogenous nucleic acid in said
MSCs.
[0057] The cells of the present invention therefore enable
beneficial and surprising therapeutic effects. Surprisingly, the
administration of the MSCs described herein leads to effective
migration to the site of disease after systemic, preferably
intravenous, administration of the cells. The MSCs are capable of
migration and potentially engraftment in areas of diseased tissue
including tumors and other inflamed tissue. The MSCs themselves
provide an anti-inflammatory signal beneficial to the disease
conditions included in the invention, in addition to the enhanced
local effect of Klotho from expression of the transgene present in
the Klotho-modified MSCs.
[0058] The combination of MSCs exhibiting increased Klotho
expression (compared to unmodified MSCs) with the treatment of a
cancer, organ fibrosis, renal failure, age-related changes of
organs or organ systems, arteriosclerosis, dementia, diabetes
mellitus, neurodegenerative disease and autoimmune diseases and
autoimmune-related diseases shows unexpected synergy. MSC homing to
areas of diseased tissue, in addition to the anti-inflammatory
properties of the MSCs themselves and the therapeutic effect of the
Klotho transgene provides a synergistic therapeutic effect greater
than the sum of each individual effect when considered in an
isolated fashion.
[0059] The Klotho-modified MSCs thereby avoid and/or minimize
potential side effects due to systemic administration of Klotho
protein or Klotho-encoding nucleic acid vectors. The use of MSCs as
vehicles for Klotho administration provides local production of
Klotho in diseased regions of the body due to the homing
capabilities of MSCs towards inflamed tissue.
[0060] Moreover MSCs are known to exhibit beneficial
immunomodulatory effects on subjects and in particular on subjects
that are inflicted with an inflammatory diseases and/or an unwanted
immune response. It was particular surprising that the genetically
modified MSCs maintain the beneficial immunomodulatory properties
of MSCs. A person skilled in the art could not have suspected this,
instead it would have been expected that the expression of Klotho,
especially at therapeutically effective amounts, would interfere
with the immunomodulatory properties of MSCs. However Klotho-MSCs
maintain a beneficial modulatory function in particular on immune
cells of the subject. Klotho-MSCs are therefore surprisingly
effective for the treatment of diseases associated with unwanted
inflammation and/or immune response. The expression of the
therapeutically effective Klotho together with the immunomodulatory
properties of the Klotho-MSCs yield to a synergistic therapeutic
effect greater than the sum of the individual effects of both
therapeutic agents when considered separately.
[0061] Moreover the Klotho-modified MSCs yield surprising
therapeutic effects due to a continuous production of Klotho. After
administration the Klotho-modified MSCs may act as bio pump or drug
factory that continuously provides Klotho protein to the subject.
Thereby the amount of Klotho can be held at a therapeutic level
over long periods. As stated herein, the homing capabilities of
Klotho-MSCs advantageously lead to a localized expression of Klotho
in diseased regions. However the expressed Klotho can also be
transported by the vascular system throughout the body of the
subject. Administered Klotho-MSCs therefore also contributes in a
systemic manner largely irrespective of the location of the MSCs
within the body of the subject. The continuous production of Klotho
by the Klotho-MSCs is particularly advantageous as the Klotho
protein may undergo degradation. Therefore a direct systematic
administration of the Klotho protein to a subject would have to be
carried out repeatedly at short intervals to maintain sufficient
therapeutic levels. It is surprising that the genetically modified
MSCs can overcome this obstacle by a continuous expression of
Klotho over periods of more than 7, or more than 10 days and even
more than 30 days. By acting as a bio pump the MSC-Klotho arrive at
stable levels of Klotho within a subject for more than 1 week and
even more than 1 month.
[0062] Particular stable levels of therapeutically effective Klotho
result from expressing Klotho under the control of a constitutively
active promotor. To this end the EFS, PGK and/or CMV promotor have
proved particularly suited and allow for a prolonged expression at
elevated levels.
[0063] Moreover, MSCs expressing Klotho proved to be very efficient
in delivering the therapeutic protein into the vascular system of a
patient. By means of the vascular system the Klotho proteins is
transported throughout the body of the subject. Advantageously
thereby therapeutic levels of Klotho can be established at
different organs such as the liver, kidney and/or lung that may be
inflicted by a disease. The administration of Klotho-MSCs is also
effective for the treatment of neurodegenerative diseases that
affect the brain. This is even the case if the Klotho-MSCs have not
been introduced nor migrated towards the affected brain regions. It
is therefore suspected that Klotho may advantageously pass the
blood-brain barrier. Methods for the genetic modification of MSCs
are known to those skilled in the art. Examples of suitable methods
for genetic modification of MSCs are disclosed in WO 2010/119039
and WO 2008/150368.
[0064] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterized in that the exogenous
nucleic acid comprises a viral vector, for example in the form of a
viral expression construct, more preferably a retroviral
vector.
[0065] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterized in that the exogenous
nucleic acid is or comprises a non-viral expression construct.
[0066] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterized in that the promoter or
promoter/enhancer combination is a constitutive promoter. In
another embodiment the genetically modified mesenchymal stem cell
as described herein is characterised in that the promoter or
promoter/enhancer combination is the CMV or EF2 promoter.
[0067] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the constitutive promoter is the EFS promoter.
[0068] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the constitutive promoter is the PGK promoter.
[0069] In a preferred embodiment the genetically modified
mesenchymal stem cell as described herein is characterised in that
the constitutive promoter is the EF1alpha promoter.
[0070] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that said promoter or
promoter/enhancer combination is an inducible promoter.
[0071] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
inducible upon differentiation of said cell
post-administration.
[0072] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
an inflammation-specific promoter, preferably wherein said promoter
is induced by inflammatory mediators or cytokines and/or induced
when the genetically modified mesenchymal stem cell comes into
proximity with inflamed tissue.
[0073] The inducible forms of the promoter are designed to exhibit
inflammation specific and/or localized expression of the Klotho
protein. In combination with the homing and/or migratory properties
of the MSCs, a synergistic effect is achieved, so that very little
Klotho protein is expressed or produced in areas in the body of the
subject distinct from the diseased tissue or organ.
[0074] In other embodiments, the expression of Klotho occurs in
administered MSCs in a location distinct from the disease site and
the protein is transported throughout the vascular system to the
area of disease within the patient.
[0075] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the Tie2 promoter.
[0076] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the RANTES promoter.
[0077] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that the promoter is
the HSP70 promoter.
[0078] It was surprising, in light of the prior art, that the
expression of the inducible promoters mentioned herein led to
sufficient expression of the therapeutic protein Klotho upon
appropriate stimulus at the site of inflammation. The promoters
provided herein show suitable inducible properties for quick and
strong expression of Klotho upon entering into proximity with
inflamed tissue.
[0079] In one embodiment the genetically modified mesenchymal stem
cell as described herein is characterised in that said cell further
comprises (iii) a selection marker gene operably linked to (iv) a
constitutive promoter or promoter/enhancer combination.
[0080] In one embodiment the genetically modified cell as described
herein is characterized in that the Klotho encoding region encodes
a protein comprising or consisting of a sequence according to one
of SEQ ID NO 6 to 10, wherein the Klotho encoding region preferably
comprises or consists of a sequence according to SEQ ID NO 1 to
5.
[0081] In further embodiments the Klotho encoding region encodes a
protein comprising or consisting of a sequence according to one of
SEQ ID NO 6 to 10, or a sequence of at least 70% sequence identity,
or at least 75%, 80%, 85%, 90% or 95% sequence identity, to one of
SEQ ID NO 6 to 10,
[0082] In one embodiment the genetically modified cell as described
herein is characterized in that the Klotho encoding region encodes
for a secreted form of the Klotho protein. Advantageously the
secreted form of Klotho is therapeutically particularly effective
when expressed by mesenchymal stem cells.
[0083] In one embodiment the genetically modified cell as described
herein is characterized in that the secreted form the Klotho
protein comprises or consists of an amino acid sequence with an
identity of at least 70%, preferably of at least 80%, 85%, 90% or
at least 95% to SEQ ID NO 8. It is particularly preferred that the
secreted Klotho protein has a sequence as laid out by SEQ ID No 8
or is functionally analogous to a protein with an amino acid
sequence as laid out by SEQ ID NO 8.
[0084] In a further aspect the invention relates to the genetically
modified mesenchymal stem cell as described herein for use as a
medicament.
[0085] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that said cell is administered by introducing a therapeutically
effective number of cells into the bloodstream of a patient.
[0086] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
by introducing a therapeutically effective number of said cells
subcutaneously. It may further be preferably to this end the
MSCs-Klotho are encapsulated by a biocompatible matrix and
transplanted together with the matrix, preferably
subcutaneously
[0087] In one embodiment the invention relates to the genetically
modified mesenchymal stem cell as described herein for use in
cosmetic applications or plastic surgery, for example by
introducing an effective number of said cells subcutaneously or
intradermally to improve tautness or fairness of skin (reduction of
wrinkles).
[0088] In one embodiment of the invention the genetically modified
mesenchymal stem cell as described herein is intended for use in
cosmetic applications or plastic surgery, such as by introducing an
effective number of said cells subcutaneously or intradermally to
augment skin volume (reduction of wrinkles).
[0089] In one embodiment of the invention the genetically modified
mesenchymal stem cell as described herein is intended for use in
treating hair loss by introducing an effective number of MSC-Klotho
cells subcutaneously or intradermally.
[0090] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
by introducing a therapeutically effective number of said cells to
a subject within a biocompatible matrix.
[0091] Preferred materials for the biocompatible matrix are
agarose, carrageenan, alginate, chitosan, gellan gum, hyaluronic
acid, collagen, cellulose and its derivatives, gelatin, elastin,
epoxy resin, photo cross-linkable resins, polyacrylamide,
polyester, polystyrene and polyurethane or polyethylene glycol
(PEG). It is further preferred that the biocompatible matrix is a
semi-permeable hydrogel matrix and the Klotho-MSCs are entrapped by
said matrix. Advantageously the biocompatible matrix allows for an
efficient diffusion of nutrients, oxygens and other biomolecules to
ensure a long lasting viability of the Klotho-MSCs, while
immobilizing the cells. Thereby the Klotho-MSCs can be concentrated
at preferred locations within the subject. For instance the
Klotho-MSCs can be transplanted subcutaneously and/or in proximity
of diseased regions of the subject i.e. the kidney for the
treatment of renal diseases. It is surprising that by introducing
encapsulated Klotho-MSCs, the cells function particularly
efficiently as bio pumps and provide a high level of therapeutic
Klotho to the subject. Firstly, the biocompatible matrix allows for
the release of Klotho. Secondly, the elevated concentration of
Klotho-MSCs within the microencapsulation promotes a feedback
mechanism that results in the augmented production of Klotho in
comparison to individually migrating Klotho-MSCs. The
administration of microencapsulated Klotho-MSCs therefore
constitutes a particular beneficial treatment of diseases that
benefit from stable elevated levels of Klotho.
[0092] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the cell is administered intrathecally. To this end it is
preferred that a therapeutically effective number of Klotho-MSCs
are introduced into the spinal canal preferably into the
subarachnoid space of subject. Thereby the Klotho-MSCs are capable
of reaching the cerebrospinal fluid. Intrathecally administered
Klotho-MSCs exhibit a surprisingly high viability and allow for a
particular continuous provision of therapeutically effective Klotho
protein to the brain region of a subject. It therefore preferred
that the Klotho-MSCs are administered intrathecally for the
treatment of neurodegenerative diseases. Most preferably the
Klotho-MSCs are administered intrathecally for the treatment of
Alzheimer's disease (AD), Multiple sclerosis (MS), Huntington's
disease, Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease,
and/or Schizophrenia.
[0093] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that is administered at least once per month, preferably at
least once per week. The administration of Klotho-MSCs with the
preferred periodicity proved to be well suited to maintain
therapeutically effective levels of Klotho throughout the treatment
of the subject.
[0094] Either isolated or repeated administration may lead to
beneficial effects, whereby prolonged or continued administration,
for example a long term administration, is also preferred. An
example of such long term application is that the MSCs are
administrated to the patient in multiple events, for example once
per week or once per month, for at least two weeks, at least three
weeks, at least four weeks, at least one month, at least two
months, at least three months, at least four months, at least five
months, at least six months, at least seven months, at least eight
months, at least nine months, at least ten months, at least eleven
months, at least one year, at least two years, or at least three
years, or enduringly. Administration may, for example, be carried
out as often as once per day, once per week, once every 7 to 14, or
7 to 21 days, or once per month, or once per two months, over a
time period as mentioned above.
[0095] In a further aspect the invention relates to the genetically
modified mesenchymal stem cell as described herein for use as a
medicament in the treatment of disease.
[0096] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is cancer.
[0097] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is organ fibrosis.
[0098] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is renal failure.
[0099] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is associated with or caused by an age-related
change in an organ or organ system physiology or function.
[0100] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is or is associated with arteriosclerosis.
[0101] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the diseases is dementia.
[0102] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is diabetes mellitus.
[0103] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is an autoimmune disease.
[0104] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease is a lung disease.
[0105] In a further aspect the invention relates to the genetically
modified mesenchymal stem cell as described herein for use as a
medicament in the treatment of an inflammatory disorder.
[0106] In further embodiments the genetically modified mesenchymal
stem cell for use as a medicament as described herein is
characterised in that the inflammatory disease is vasculitis,
nephritis, inflammatory bowel disease, rheumatoid arthritis and/or
Graft versus Host disease.
[0107] In a further aspect the invention relates to the genetically
modified mesenchymal stem cell as described herein for use as a
medicament in the treatment of chronic fibrosis. In one embodiment
the genetically modified mesenchymal stem cell for use as a
medicament as described herein is characterised in that the
inflammatory and/or chronic fibrotic disease is of the kidney,
liver and/or colon of a subject. It was surprising that the
localized expression of Klotho in fibrotic regions could lead to
enhanced therapeutic effect. MSCs could show unexpected migratory
properties towards fibrotic tissue and via expression of Klotho
lead to a reduction in the formation of fibrotic tissue.
[0108] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that said cell is administered by introducing a therapeutically
effective number of said cells to the blood stream of a patient,
thereby achieving delivery of Klotho protein expressed from said
cells locally in regions of disease and/or inflammation. The
administration of the MSCs of the present invention may also take
place via routes of delivery including inhalation, endoscopic
tissue injection, catheter-mediated tissue injection, cerebrospinal
fluid injection, intraperitoneal injection, subcutaneous injection,
intramuscular injection, optionally in combination with
introduction of said cells into the bloodstream of a patient.
[0109] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the disease to be treated is a neurodegenerative
disease.
[0110] In one embodiment of the invention the neurodegenerative
disease is Alzheimer's disease (AD).
[0111] In one embodiment of the invention the neurodegenerative
disease is Multiple sclerosis (MS).
[0112] In one embodiment of the invention the neurodegenerative
disease is Huntington's disease.
[0113] In one embodiment of the invention the neurodegenerative
disease is Amyotrophic Lateral Sclerosis (ALS).
[0114] In one embodiment of the invention the neurodegenerative
disease is Parkinson's disease.
[0115] In one embodiment of the invention the neurodegenerative
disease is Schizophrenia.
[0116] In a preferred embodiment the present invention is directed
to the treatment of neurodegenerative disease via administration of
the genetically modified cells described herein that comprise a
Klotho transgene. Administration may be provided systemically, such
as intravenously, due to the ability of MSCs to pass the
blood-brain barrier. Alternative methods of administration, such as
epidural injections or other modes of administration that do not
require passage over the blood brain barrier are encompassed within
the invention.
[0117] In particular the therapeutic approach is intended in early
stage patients with neurodegenerative disease that has not
progressed into severe or late stages of the disease.
[0118] Previous studies have described a sufficient safety profile
with respect to the administration of MSCs to patients with MS or
ALS. Immunomodulatory effects of the MSCs have also been described
(Karussis et al., Arch Neurol. 2010 67(19), 1187). These beneficial
effects could be enhanced by the use of a Klotho transgene in the
MSCs described herein.
[0119] Additional indications exist, that Klotho may show
therapeutic potential in treating neurodegenerative disease. Small
molecule enhancers of Klotho function have been suggested to show
therapeutic function in treating MS or other neurodegenerative
diseases (Abraham et al., Future Med Chem, 2012 Sep. 4:13, 1671),
klotho has been linked previously to myelination of the central
nervous system (Chen et al., J Neurosci. 2013 January 33(5):1927)
and reduced levels of klotho are found in Alzheimer's patients
(Semba et al., Neurosci. Lett. 2014 Jan. 13; 558: 37). Despite
these suggestions the use of a Klotho transgene in MSCs for
treatment of these medical conditions represents a surprising
result considering the difficulties and low expectation of success
in administering therapeutic transgenic products via cellular
therapy. Surprisingly, locally delivered Klotho via transgene
expression from a cellular MSC vehicle leads to a potential
therapeutic effect against neurodegenerative disease, when said
MSCs are administered either systemically (such as via i. v.
injections), or locally (such as via epidural or cranial
injection).
[0120] Various tests are available to the skilled person in order
to assess neurodegenerative disease and are therefore also suitable
to assess potential therapeutic effect of the klotho-MSCs described
herein. For example, attention tests may be conducted, comprising
pre-attentive tests, interrogating prepulse or inhibition,
attention tests such as interrogating orientation, multiple choice
responses, serial reaction tasks, go/no-go tests, or cognitive
testing, including interrogating object discrimination, social
transmission of food preference, transverse pattern tests, or
learning and memory tests, such as associative tests, for example
interrogating passive avoidance, one-way or two-way active
avoidance, or spatial/contextual tests, comprising the radial arm
maze, or morris water maze, or conditional emotional responses,
such as testing for conditioned taste aversion, potentiated
startle, or fear conditioning.
[0121] In a preferred embodiment the present invention is directed
to the treatment of heart diseases and kidney disease via
administration of the genetically modified cells described herein
that comprise a Klotho transgene. These two groups of numerous
medical indications are linked by the special feature of salt
deposits, or calcification, in the arteries and veins of particular
patients. Klotho is involved in phosphate metabolism, and thereby
in the processing of salts in the body of mammalian subjects. In
certain subjects, phosphate or calcium salts are not processed
correctly and this leads to deposition of salts in the arteries and
associated arterial, occlusive or cardiovascular disease. Such
diseases may be characterized by "hardening" of the arteries.
[0122] One object of the invention is therefore the treatment of
cardiovascular disease, circulatory disorders, arterial diseases
and/or ischemic obstructive or occlusive conditions or strokes,
preferably coronary heart diseases or peripheral arterial
obstructive diseases, atherosclerosis and/or
transplantation-induced sclerosis; a cerebral occlusive disease, or
renal occlusive disease.
[0123] Surprisingly, subjects with kidney malfunction, in
particular those in which the detection of creatine is not able to
indicate the presence of said kidney malfunction, are at increased
risk of suffering the heart or ischemic diseases described
herein.
[0124] In one embodiment the present invention is directed to the
treatment of sepsis via administration of the Klotho-modified MSCs
as described herein.
[0125] In one embodiment the present invention is directed to the
treatment of erectile dysfunction via administration of the
Klotho-modified MSCs as described herein. Klotho is known to play a
role in nitrogen monoxide (NO) regulation, thereby potentially
influencing erectile function.
[0126] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament are characterised in that the disease
is an age-related change of organs or organ systems. In one
embodiment the medical use of the MSCs described herein is directed
to the treatment of ageing as such.
[0127] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament are characterised in that the
mesenchymal stem cells are used as a medicament to treat and/or
prevent ageing or senescence. It is surprising that by
administering Klotho-MSCs biological ageing processes can be
effectively slowed, reversed and/or inhibited.
[0128] Klotho has been suspected to have positive anti-ageing
properties. However the genetically modified MSCs that express
Klotho exhibit a particularly effective anti-ageing function since
the Klotho-MSCs target cells, tissues and/or organs of a subject
inflicted by biological ageing. Thereby the Klotho-MSCs provide
locally therapeutically effective dose of Klotho to ageing affected
regions. Moreover Klotho-MSCs lead to the restoration and/or
rejuvenation of the cell population of the subject and in
particular of the stem cell population of the subject. The
therapeutic effect of Klotho towards senescence augments to a
surprising extent, when expressed by genetically modified MSCs.
[0129] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that the subject is human.
[0130] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that said genetically modified cells are allogeneic with respect
to the subject.
[0131] In one embodiment the genetically modified mesenchymal stem
cell for use as a medicament as described herein is characterised
in that said genetically modified cells are autologous with respect
to the subject.
[0132] In a further aspect of the invention the MSCs as described
herein may comprise an exogenous nucleic acid encoding a chemokine
ligand in combination with nucleic acid sequences suitable for
expression of said ligand. Chemokine-encoding sequences may be
either present in the same exogenous nucleic acid molecule that
encodes Klotho or in a separate exogenous nucleic acid. Multiple
integrated nucleic acid constructs or cassettes may be present in
the MSCs of the present invention, each carrying one or more genes
of interest, for example therapeutic genes, such as Klotho or other
genes involved in mobilization of the cells, such as a chemokine.
The invention therefore relates to the mesenchymal cells described
herein, wherein the exogenous nucleic acid preferably encodes an
inflammatory chemokine. Such chemokines are known to a skilled
person. Examples of inflammatory chemokines relate to CXCL-8, CCL2,
CCL3, CCL4, CCL5, CCL11 and CXCL10, CXCL1, CXCL2.
[0133] The invention further relates to a nucleic acid vector
comprising a region encoding a Klotho protein, said region operably
linked to a promoter or promoter/enhancer combination. In preferred
embodiments the promoter or promoter/enhancer combination is one of
those mentioned above.
[0134] The invention also relates to a method of delivering a
Klotho protein to a cell, or to a subject in need thereof, for
example in the context of a medical use of a Klotho protein,
comprising administering the nucleic acid vector as described
herein to a cell or subject, and/or administering a genetically
modified MSC comprising the nucleic acid vector to a subject with
one or more of the medical conditions disclosed herein The
invention also relates to a method for the genetic transformation
of an MSC, comprising the treatment of an MSC with a nucleic acid
vector as disclosed herein encoding a Klotho protein.
DETAILED DESCRIPTION OF THE INVENTION
[0135] All cited documents of the patent and non-patent literature
are hereby incorporated by reference in their entirety.
[0136] The "mesenchymal cells" disclosed herein (also referred to
in some embodiments as "mesenchymal stem cells" or "MSCs") can give
rise to connective tissue, bone, cartilage, and cells in the
circulatory and lymphatic systems. Mesenchymal stem cells are found
in the mesenchyme, the part of the embryonic mesoderm that consists
of loosely packed, fusiform or stellate unspecialized cells. As
used herein, mesenchymal stem cells include, without limitation,
CD34-negative stem cells.
[0137] In one embodiment of the invention, the mesenchymal cells
are fibroblast-like plastic adherent cells, defined in some
embodiments as multipotent mesenchymal stromal cells and also
include CD34-negative cells.
[0138] For the avoidance of any doubt, the term mesenchymal cell
encompasses multipotent mesenchymal stromal cells that also
includes a subpopulation of mesenchymal cells, MSCs and their
precursors, which subpopulation is made up of multipotent or
pluripotent self-renewing cells capable of differentiation into
multiple cell types in vivo.
[0139] As used herein, CD34-negative cell shall mean a cell lacking
CD34, or expressing only negligible levels of CD34, on its surface.
CD34-negative cells, and methods for isolating such cells, are
described, for example, in Lange C. et al., "Accelerated and safe
expansion of human mesenchymal stromal cells in animal serum-free
medium for transplantation and regenerative medicine". J. Cell
Physiol. 2007, Apr. 25.
[0140] Mesenchymal cells can be differentiated from hematopoietic
stem cells (HSCs) by a number of indicators. For example, HSCs are
known to float in culture and to not adhere to plastic surfaces. In
contrast, mesenchymal cells adhere to plastic surfaces. The
CD34-negative mesenchymal cells of the present invention are
adherent in culture.
[0141] The genetically modified cell(s) described herein may
comprise different types of carriers depending on whether it is to
be administered in solid, liquid or aerosol form, and whether it
need to be sterile for such routes of administration as injection.
The present invention can be administered intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intrarectally, topically, intratumorally,
intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically, intraocularally, orally, topically, locally,
inhalation (e.g., aerosol inhalation), injection, infusion,
continuous infusion, localized perfusion bathing target cells
directly, via a catheter, via a lavage, in cremes, in lipid
compositions (e.g., liposomes), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein
by reference).
[0142] The present invention encompasses treatment of a patient by
introducing a therapeutically effective number of cells into a
subject's bloodstream. As used herein, "introducing" cells "into
the subject's bloodstream" shall include, without limitation,
introducing such cells into one of the subject's veins or arteries
via injection. Such administering can also be performed, for
example, once, a plurality of times, and/or over one or more
extended periods. A single injection is preferred, but repeated
injections over time (e.g., quarterly, half-yearly or yearly) may
be necessary in some instances. Such administering is also
preferably performed using an admixture of CD34-negative cells and
a pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known to those skilled in the art and include,
but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate
buffer or 0.8% saline, as well as commonly used proprietary
cryopreservation media.
[0143] Administration may also occur locally, for example by
injection into an area of the subject's body in proximity to a
tumor disease. MSCs have been shown to migrate towards cancerous
tissue. Regardless, the local administration of the cells as
described herein may lead to high levels of the cells at their site
of action.
[0144] Additionally, such pharmaceutically acceptable carriers can
be aqueous or non-aqueous solutions, suspensions, and emulsions,
most preferably aqueous solutions. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions and suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's and fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers such as
Ringer's dextrose, those based on Ringer's dextrose, and the like.
Fluids used commonly for i.v. administration are found, for
example, in Remington: The Science and Practice of Pharmacy, 20th
Ed., p. 808, Lippincott Williams S-Wilkins (2000). Preservatives
and other additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases, and
the like.
[0145] As used herein, a "therapeutically effective number of
cells" includes, without limitation, the following amounts and
ranges of amounts: (i) from about 1.times.10.sup.2 to about
1.times.10.sup.8 cells/kg body weight; (ii) from about
1.times.10.sup.3 to about 1.times.10.sup.7 cells/kg body weight;
(iii) from about 1.times.10.sup.4 to about 1.times.10.sup.6
cells/kg body weight; (iv) from about 1.times.10.sup.4 to about
1.times.10.sup.5 cells/kg body weight; (v) from about
1.times.10.sup.5 to about 1.times.10.sup.6 cells/kg body weight;
(vi) from about 5.times.10.sup.4 to about 0.5.times.10.sup.5
cells/kg body weight; (vii) about 1.times.10.sup.3 cells/kg body
weight; (viii) about 1.times.10.sup.4 cells/kg body weight; (ix)
about 5.times.10.sup.4 cells/kg body weight; (x) about
1.times.10.sup.5 cells/kg body weight; (xi) about 5.times.10.sup.5
cells/kg body weight; (xii) about 1.times.10.sup.6 cells/kg body
weight; and (xiii) about 1.times.10.sup.7 cells/kg body weight.
Human body weights envisioned include, without limitation, about 5
kg, 10 kg, 15 kg, 30 kg, 50 kg, about 60 kg; about 70 kg; about 80
kg, about 90 kg; about 100 kg, about 120 kg and about 150 kg. These
numbers are based on pre-clinical animal experiments and human
trials and standard protocols from the transplantation of CD34+
hematopoietic stem cells. Mononuclear cells (including CD34+ cells)
usually contain between 1:23000 to 1:300000 CD34-negative
cells.
[0146] As used herein, "treating" a subject afflicted with a
disorder shall mean slowing, stopping or reversing the disorders
progression. In the preferred embodiment, treating a subject
afflicted with a disorder means reversing the disorders
progression, ideally to the point of eliminating the disorder
itself. As used herein, ameliorating a disorder and treating a
disorder are equivalent. The treatment of the present invention may
also, or alternatively, relate to a prophylactic administration of
said cells. Such a prophylactic administration may relate to the
prevention of any given medical disorder, or the prevention of
development of said disorder, whereby prevention or prophylaxis is
not to be construed narrowly under all conditions as absolute
prevention. Prevention or prophylaxis may also relate to a
reduction of the risk of a subject developing any given medical
condition, preferably in a subject at risk of said condition.
[0147] Typically, the term "inflammation" as used in its
art-recognized sense relates to a localized or systemic protective
response elicited by injury, infection or destruction of tissues
which serves to protect the subject from an injurious agent and the
injured tissue. Inflammation is preferably characterized by
fenestration of the microvasculature, leakage of the elements of
blood into the interstitial spaces, and migration of leukocytes
into the inflamed tissue, which may lead to an uncontrolled
sequence of pain, heat, redness, swelling, and loss of
function.
[0148] Inflammation can be classified as either acute or chronic.
Acute inflammation is the initial response of the body to harmful
stimuli and is achieved by the increased movement of plasma and
leukocytes (especially granulocytes) from the blood into the
injured tissues. A cascade of biochemical events propagates and
matures the inflammatory response, involving the local vascular
system, the immune system, and various cells within the injured
tissue. Prolonged inflammation, known as chronic inflammation,
leads to a progressive shift in the type of cells present at the
site of inflammation and is characterized by simultaneous
destruction and healing of the tissue from the inflammatory
process.
[0149] In some embodiments of the invention the MSCs as described
herein migrate towards physiological niches affected by a disease
condition, such as areas of inflammation, in order to impart their
therapeutic effect, for example in a local manner.
[0150] As used herein "cell migration" is intended to mean movement
of a cell towards a particular chemical or physical signal. Cells
often migrate in response to specific external signals, including
chemical signals and mechanical signals. Chemotaxis is one example
of cell migration regarding response to a chemical stimulus. In
vitro chemotaxis assays such as Boyden chamber assays may be used
to determine whether cell migration occurs in any given cell. For
example, the cells of interest may be purified and analyzed.
Chemotaxis assays (for example according to Falk et al., 1980 J.
Immuno. Methods 33:239-247) can be performed using plates where a
particular chemical signal is positioned with respect to the cells
of interest and the transmigrated cells then collected and
analyzed. For example, Boyden chamber assays entail the use of
chambers isolated by filters, used as tools for accurate
determination of chemotactic behavior. The pioneer type of these
chambers was constructed by Boyden (Boyden (1962) "The chemotactic
effect of mixtures of antibody and antigen on polymorphonuclear
leucocytes". J Exp Med 115 (3): 453). The motile cells are placed
into the upper chamber, while fluid containing the test substance
is filled into the lower one. The size of the motile cells to be
investigated determines the pore size of the filter; it is
essential to choose a diameter which allows active transmigration.
For modelling in vivo conditions, several protocols prefer coverage
of filter with molecules of extracellular matrix (collagen, elastin
etc.) Efficiency of the measurements can be increased by
development of multiwell chambers (e.g. NeuroProbe), where 24, 96,
384 samples are evaluated in parallel. Advantage of this variant is
that several parallels are assayed in identical conditions.
[0151] Alternatively, tissue samples may be obtained from subjects
(for example rodent models) after cell transplantation and assayed
for the presence of the cells of interest in particular tissue
types. Such assays may be of molecular nature, identifying cells
based on nucleic acid sequence, or of histological nature,
assessing cells on the basis of fluorescent markings after antibody
labeling. Such assays are also particularly useful for assessing
engraftment of transplanted cells. Assays for engraftment may also
provide information on cell migration, as to some extent the
engraftment is dependent on cell localization prior to
engraftment.
[0152] In some embodiments of the invention the MSCs as described
herein engraft in physiological niches affected by a disease
condition, such as areas of inflammation, in order to impart their
therapeutic effect, for example in a local manner.
[0153] As used herein "engraftment" relates to the process of
incorporation of grafted or transplanted tissue or cells into the
body of the host. Engraftment may also relate to the integration of
transplanted cells into host tissue and their survival and under
some conditions differentiation into non-stem cell states.
[0154] Techniques for assessing engraftment, and thereby to some
extent both migration and the biodistribution of MSCs, can
encompass either in vivo or ex vivo methods. Examples of in vivo
methods include bioluminescence, whereby cells are transduced to
express luciferase and can then be imaged through their metabolism
of luciferin resulting in light emission; fluorescence, whereby
cells are either loaded with a fluorescent dye or transduced to
express a fluorescent reporter which can then be imaged;
radionuclide labeling, where cells are loaded with radionuclides
and localized with scintigraphy, positron emission tomography (PET)
or single photon emission computed tomography (SPECT); and magnetic
resonance imaging (MRI), wherein cells loaded with paramagnetic
compounds (e.g., iron oxide nanoparticles) are traced with an MRI
scanner. Ex vivo methods to assess biodistribution include
quantitative PCR, flow cytometry, and histological methods.
Histological methods include tracking fluorescently labeled cells;
in situ hybridization, for example, for Y-chromosomes and for
human-specific ALU sequences; and histochemical staining for
species-specific or genetically introduced proteins such as
bacterial .beta.-galactosidase. These immunohistochemical methods
are useful for discerning engraftment location but necessitate the
excision of tissue. For further review of these methods and their
application see Kean et al., MSCs: Delivery Routes and Engraftment,
Cell-Targeting Strategies, and Immune Modulation, Stem Cells
International, Volume 2013 (2013).
[0155] Progenitor or multipotent cells, such as the mesenchymal
cells of the present invention, may therefore be described as
protein delivery vehicles, essentially enabling the localization
and expression of therapeutic gene products in particular tissues
or regions of the subject's body. Such therapeutic cells offer the
potential to provide cellular therapies for diseases that are
refractory to other treatments. For each type of therapeutic cell
the ultimate goal is the same: the cell should express a specific
repertoire of genes, preferably exogenous nucleic acids that code
for therapeutic gene products, thereby modifying cell identity to
express said gene product and provide a therapeutic effect, such as
an anti-inflammatory effect. The cells of the invention, when
expanded in vitro, represent heterogeneous populations that include
multiple generations of mesenchymal (stromal) cell progeny, which
lack the expression of most differentiation markers like CD34.
These populations may have retained a limited proliferation
potential and responsiveness for terminal differentiation and
maturation along mesenchymal and non-mesenchymal lineages.
[0156] As used herein the term "bio pump" or "drug factory"
preferably describe the function of Klotho-MSCs as a continuously
producing source of Klotho. By administering Klotho-MSCs to a
subject particularly stable levels of Klotho can be provided. In
the sense the bio pump, that is the Klotho-MSCs, allow for a
continuous supply that maintains Klotho levels at a particular
state, for example it may compensate for losses of Klotho for
instance due to a degeneration of the protein.
[0157] As used herein "inducible expression" or "conditional
expression" relates to a state, multiple states or system of gene
expression, wherein the gene of interest, such as the therapeutic
transgene, is preferably not expressed, or in some embodiments
expressed at negligible or relatively low levels, unless there is
the presence of one or more molecules (an inducer) or other set of
conditions in the cell that allows for gene expression. Inducible
promoters may relate to either naturally occurring promoters that
are expressed at a relatively higher level under particular
biological conditions, or to other synthetic promoters comprising
any given inducible element. Inducible promoters may refer to those
induced by particular tissue- or micro-environments or combinations
of biological signals present in particular tissue- or
micro-environments, or to promoters induced by external factors,
for example by administration of a small drug molecule or other
externally applied signal.
[0158] As used herein, in "proximity with" a tissue includes, for
example, within 5 mm, within 1 mm of the tissue, within 0.5 mm of
the tissue and within 0.25 mm of the tissue.
[0159] Given that stem cells can show a selective migration to
different tissue microenvironments in normal as well as diseased
settings, the use of tissue-specific promoters linked to the
differentiation pathway initiated in the recruited stem cell is
encompassed in the present invention and could in theory be used to
drive the selective expression of therapeutic genes only within a
defined biologic context. Stem cells that are recruited to other
tissue niches, but do not undergo the same program of
differentiation, should not express the therapeutic gene. This
approach allows a significant degree of potential control for the
selective expression of the therapeutic gene within a defined
microenvironment and has been successfully applied to regulate
therapeutic gene expression during neovascularization. Potential
approaches to such gene modifications are disclosed in WO
2008/150368 and WO 2010/119039, which are hereby incorporated in
their entirety.
[0160] As used herein, a "secreted" protein preferably refers to
those proteins capable of being directed to the endoplasmic
reticulum, the secretory vesicles, or the extracellular space as a
result of a signal sequence, as well as those proteins released
into the extracellular space without necessarily containing a
signal sequence. If the secreted protein is released into the
extracellular space, the secreted protein can undergo extracellular
processing. The release into the extracellular space can preferably
occur by many mechanisms, including exocytosis and proteolytic
cleavage.
[0161] As used herein, "nucleic acid" shall mean any nucleic acid
molecule, including, without limitation, DNA, RNA and hybrids or
modified variants thereof. An "exogenous nucleic acid" or
"exogenous genetic element" relates to any nucleic acid introduced
into the cell, which is not a component of the cells "original" or
"natural" genome. Exogenous nucleic acids may be integrated or
non-integrated, or relate to stably transfected nucleic acids.
[0162] Any given gene delivery method is encompassed by the
invention and preferably relates to viral or non-viral vectors, as
well as biological or chemical methods of transfection. The methods
can yield either stable or transient gene expression in the system
used.
[0163] Genetically modified viruses have been widely applied for
the delivery of genes into stem cells. Preferred viral vectors for
genetic modification of the MSCs described herein relate to
retroviral vectors, in particular to gamma retroviral vectors. The
gamma retrovirus (sometimes referred to as mammalian type C
retroviruses) is a sister genus to the lentivirus clade, and is a
member of the Orthoretrovirinae subfamily of the retrovirus family.
The Murine leukemia virus (MLV or MuLV), the Feline leukemia virus
(FeLV), the Xenotropic murine leukemia virus-related virus (XMRV)
and the Gibbon ape leukemia virus (GALV) are members of the gamma
retrovirus genus. A skilled person is aware of the techniques
required for utilization of gamma retroviruses in genetic
modification of MSCs. For example, the vectors described Maetzig et
al (Gammaretroviral vectors: biology, technology and application,
2001, Viruses June; 3(6):677-713) or similar vectors may be
employed. For example, the Murine Leukemia Virus (MLV), a simple
gammaretrovirus, can be converted into an efficient vehicle of
genetic therapeutics in the context of creating gamma
retrovirus-modified MSCs and expression of a therapeutic transgene
from said MSCs after delivery to a subject.
[0164] Genetically modified viruses have been widely applied for
the delivery of genes into stem cells. Adenoviruses may be applied,
or RNA viruses such as Lentiviruses, or other retroviruses.
Adenoviruses have been used to generate a series of vectors for
gene transfer cellular engineering. The initial generation of
adenovirus vectors were produced by deleting the El gene (required
for viral replication) generating a vector with a 4 kb cloning
capacity. An additional deletion of E3 (responsible for host immune
response) allowed an 8 kb cloning capacity. Further generations
have been produced encompassing E2 and/or E4 deletions.
Lentiviruses are members of Retroviridae family of viruses (M.
Scherr et al., Gene transfer into hematopoietic stem cells using
lentiviral vectors. Curr Gene Ther. 2002 February; 2(1):45-55).
Lentivirus vectors are generated by deletion of the entire viral
sequence with the exception of the LTRs and cis acting packaging
signals. The resultant vectors have a cloning capacity of about 8
kb. One distinguishing feature of these vectors from retroviral
vectors is their ability to transduce dividing and non-dividing
cells as well as terminally differentiated cells.
[0165] Non-viral methods may also be employed, such as alternative
strategies that include conventional plasmid transfer and the
application of targeted gene integration through the use of
integrase or transposase technologies. These represent approaches
for vector transformation that have the advantage of being both
efficient, and often site-specific in their integration. Physical
methods to introduce vectors into cells are known to a skilled
person. One example relates to electroporation, which relies on the
use of brief, high voltage electric pulses which create transient
pores in the membrane by overcoming its capacitance. One advantage
of this method is that it can be utilized for both stable and
transient gene expression in most cell types. Alternative methods
relate to the use of liposomes or protein transduction domains.
Appropriate methods are known to a skilled person and are not
intended as limiting embodiments of the present invention.
[0166] Cancer comprises a group of diseases that can affect any
part of the body and is caused by abnormal cell growth and
proliferation. These proliferating cells have the potential to
invade the surrounding tissue and/or to spread to other parts of
the body where they form metastasis. Worldwide, there were 14
million new cases of cancer and 8.2 million cancer related deaths
in 2012 (World Cancer Report 2014). The majority of cancers is
caused by environmental signals involving tobacco use, obesity and
infections among others, while around 5-10% are genetic cases.
Cancers can be classified into subcategories based on the cell of
origin. The most common subcategories are carcinomas from
epithelial cells, sarcomas from connective tissue and lymphomas and
leukemias from hematopoietic cells. Cancer is associated with a
high variety of local and systemic symptoms and cannot be cured in
many cases. In light of the high number of new cancer patients and
cancer related deaths novel treatment strategies are required.
[0167] Cancer according to the present invention refers to all
types of cancer or neoplasm or malignant tumors found in mammals,
including leukemias, sarcomas, melanomas and carcinomas. Examples
of cancers are cancer of the breast, pancreas, colon, lung,
non-small cell lung, ovary, and prostate.
[0168] Leukemias include, but are not limited to acute
nonlymphocytic leukemia, chronic lymphocytic leukemia, acute
granulocytic leukemia, chronic granulocytic leukemia, acute
promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,
a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,
bovine leukemia, chronic myelocytic leukemia, leukemia cutis,
embryonal leukemia, eosinophilic leukemia, Gross' leukemia,
hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic
leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic
leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic
leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid
leukemia, lymphosarcoma cell leukemia, mast cell leukemia,
megakaryocytic leukemia, micromyeloblastic leukemia, monocytic
leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid
granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia,
plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia,
Rieder cell leukemia, Schilling's leukemia, stem cell leukemia,
subleukemic leukemia, and undifferentiated cell leukemia.
[0169] Sarcomas include, but are not limited to a chondrosarcoma,
fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma,
osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma,
alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma,
chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor
sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma,
fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,
granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple
pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells,
lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,
Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,
malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic
sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and
telangiectaltic sarcoma.
[0170] Melanomas include, but are not limited to include, for
example, acral-lentiginous melanoma, amelanotic melanoma, benign
juvenile melanoma, Cloudman's melanoma, S91 melanoma,
Harding-Passey melanoma, juvenile melanoma, lentigo maligna
melanoma, malignant melanoma, nodular melanoma, subungal melanoma,
and superficial spreading melanoma.
[0171] Carcinomas include, but are not limited to acinar carcinoma,
acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,
carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar
carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma
basocellulare, basaloid carcinoma, basosquamous cell carcinoma,
bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic
carcinoma, cerebriform carcinoma, cholangiocellular carcinoma,
chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus
carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma
cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct
carcinoma, carcinoma durum, embryonal carcinoma, encephaloid
carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides,
exophytic carcinoma, carcinoma exulcere, carcinoma fibrosum,
gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,
carcinoma gigantocellulare, glandular carcinoma, granulosa cell
carcinoma, hair-matrix carcinoma, hematoid carcinoma,
hepatocellular carcinoma, Hurthle cell carcinoma, hyaline
carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma,
carcinoma in situ, intraepidermal carcinoma, intraepithelial
carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma,
large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare,
lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma
medullare, medullary carcinoma, melanotic carcinoma, carcinoma
molle, mucinous carcinoma, carcinoma muciparum, carcinoma
mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous
carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat
cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary
carcinoma, periportal carcinoma, preinvasive carcinoma, prickle
cell carcinoma, pultaceous carcinoma, renal cell carcinoma of
kidney, reserve cell carcinoma, carcinoma sarcomatodes,
schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti,
signet-ring cell carcinoma, carcinoma simplex, small-cell
carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle
cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticurn,
carcinoma telangiectodes, transitional cell carcinoma, carcinoma
tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma
villosum.
[0172] Additional cancers include, but are not limited to Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer,
lymphomas, thyroid cancer, esophageal cancer, genitourinary tract
cancer, malignant hypercalcemia, cervical cancer, endometrial
cancer, adrenal cortical cancer, and prostate cancer.
[0173] Fibrosis is the endpoint of many chronic inflammatory
diseases and is defined by an abnormal accumulation of
extracellular matrix components. The term fibrosis designates the
increase of fibrous connective tissue and material including
collagen and other extracellular matrix proteins in the parenchyma
of organs. This can occurs in multiple organs in response to
external stimulation such as injury, infection, inflammation.
Fibrosis can change the architecture and function of the affected
tissue, which can interfere with organ function and therefore lead
to pathology and even organ failure. Examples of Fibrosis include
pulmonary fibrosis, liver cirrhosis, myocardial and renal fibrosis
among others. New treatment options for this group of diseases are
needed to improve the condition of affected patients.
[0174] Organ fibrosis according to the present invention refers to,
but is not limited to, one or more of idiopathic pulmonary
fibrosis, endomyocardial fibrosis, old myocardial infarction,
atrial fibrosis, mediastinal fibrosis, myelofibrosis of the bone
marrow, retroperitoneal fibrosis, progressive massive fibrosis of
the lung, nephrogenic systemic fibrosis of the skin, crohn's
disease, keloid, scleroderma/systemic sclerosis, arthrofibrosis,
Peyronie's disease, Dupuytren's contracture, or adhesive
capsulitis.
[0175] Despite its slow progression, it leads to organ malfunction.
Fibrosis can affect almost any tissue. One of the main molecular
agents inducing fibrosis is TGF-.beta.1, mainly synthesized by
T-cells during the healing process. TGF-.beta.1 is secreted in a
latent form associated with LAP (latency associated peptide). LAP
is cleaved to allow the activation of TGF-.beta.1 which is able to
bind its receptors TGF-.beta.R1 (transforming growth factor
receptor-.beta.1) and TGF-.beta.R2. Therefore, there is a large
pool of inactive TGF-.beta.1 in the extracellular environment.
Various agents can induce TGF-.beta.1 activation: MMPs, reactive
oxygen and nitrogen species (ROS and RNS), cytokines, or other
stimuli such as ionizing radiation. The binding of TGF-.beta.1 to
its receptors activates the Smad (small mothers against
decapentaplegic homolog) signaling pathway which induces the
transcription of various genes, including genes encoding members of
the extracellular matrix (collagens mostly). It also activates the
differentiation of fibrocytes toward functional fibroblasts (Benoit
et al., Breakthrough Stem Cells International 2014, Article ID
340257, 26 pages).
[0176] The term renal failure describes a medical condition in
which the kidney is not functioning adequately to fulfil its
physiological functions. The two main forms are chronic kidney
disease and acute kidney injury. Acute kidney injury is defined by
a rapid loss of renal function within less than 3 months. Chronic
kidney disease is a progressive disease associated with gradual
loss of renal function over a period of several months to years
leading toward organ failure. It is estimated that in the US, 16.8%
of adults aged 20 years and older were affected during 1999 to
2004. The 3 major causes of chronic kidney disease are diabetes,
hypertension and glomerulonephritis. No specific treatment has been
shown to slow down chronic kidney disease and late stage patients
are treated with cost intensive renal replacement therapy involving
dialysis and transplantation. Similarly, acute kidney injury often
requires renal replacement therapy. Therefore, alternative
treatment strategies to cure or slow down progression of acute
kidney injury and chronic kidney disease are needed.
[0177] Renal failure according to the present invention refers to,
but is not limited to, one or more of, acute kidney injury, chronic
kidney disease, or acute-on-chronic renal failure.
[0178] In aging individuals, the function of almost every organ of
the body is declining due to age-related changes also in the
absence of pathology. These changes can be due to a variety of
reasons including loss of organ specific cell function or organ
specific cells. On the other hand, declining function of one organ
can affect the function of other organs of the body. Age-related
organ changes make older individuals less able to handle stress and
external challenges such as physical activity, drug treatment,
infections, and temperature changes, among many others. If the
aging process of the various organ systems of the body could be
slowed down this would increase the quality of life of aging people
and would benefit society as a whole.
[0179] Age-related changes of organs and organ systems according to
the present invention refers to, but is not limited to, one or more
of age-related changes of bones, joints, ears, muscles, body fat,
eyes, mouth, nose, skin, brain, nervous system, spinal cord, heart,
blood vessels, lung, intestine, stomach, colon, esophagus, kidney,
urinary tract, reproductive organs, breasts, endocrine system, bone
marrow, and immune system.
[0180] The MSCs of the present invention may therefore be used as
an anti-ageing agent, for example in the treatment of ageing, for
example senescence-related ageing. "Senescence-related ageing"
refers to senescence, meaning generally "to grow old", or "ageing".
Biological aging is the process of accumulative changes to
molecular and cellular structure that disrupts metabolism with the
passage of time, resulting in deterioration and death. Senescence
occurs both on the level of the whole organism (organismal
senescence) as well as on the level of its individual cells
(cellular senescence). The treatment of senescence (anti-ageing) is
one aspect of the present invention. The treatment of ageing, or
the treatment of senescence, relates in some embodiments to
slowing, reversing and/or inhibiting the ageing process from
occurring.
[0181] During aging the incidence of acute and chronic conditions
such as neurological disorders, diabetes, degenerative arthritis,
and even cancer rises within individuals, so that aging has been
termed the substrate on which age-associated diseases grow. The
invention therefore relates to prophylactic methods for preventing
diseases associated with ageing.
[0182] The molecular pathways underlying aging are not well
understood, as large individual heterogeneity of the biological
aging process is observed. These inter-individual differences are
proposed to derive from accumulation of stochastic damage that is
counteracted by genetically encoded and environmentally regulated
repair systems. At the level of molecules repair works by enzymatic
systems while on the cellular level it works by replication and
differentiation to maintain tissue homeostasis. However, the
replicative potential of somatic and adult stem cells is limited by
cellular senescence and recent evidence shows that counteracting
senescence or removing senescent cells delays the onset of
age-associated pathologies. The present invention therefore
provides means for the treatment and/or prevention and/or reduction
in risk of ageing as such, in addition to age-related medical
conditions.
[0183] The term arteriosclerosis describes the pathological
thickening, hardening and loss of elasticity of artery walls that
can lead to stenosis and subsequent insufficient blood supply of
downstream tissues resulting in ischemia. This process is often
associated with calcification of the arterial wall. There are
different types of arteriosclerosis that affect different
anatomical locations and have different etiologies. Atherosclerosis
is a specific type of arteriosclerosis, which is defined by the
accumulation of white blood cells in the artery wall and formation
of atheromatous plaques. Atherosclerosis is a chronic disease that
can remain asymptomatic for extended periods until lumen stenosis
of the affected artery occurs. Additionally, ruptures of
atherosclerotic lesions can lead to thrombus formation and
subsequent thromboembolism, which can lead to tissue
necrosis/infarction in all parts of the body. Dramatic examples of
such events are myocardial infarction and stroke, these
after-effects of atherosclerosis represent the most common cause of
death in industrialized countries and therefore improved treatment
strategies are urgently needed.
[0184] Arteriosclerosis according to the present invention refers
to, but is not limited to, one or more of, atherosclerosis,
arteriosclerosis obliterans, and Monckeberg's arteriosclerosis.
[0185] The terms circulatory disorders, cardiovascular disease,
artery or blood vessel conditions and/or ischemic obstructive or
occlusive diseases or conditions refer to states of vascular tissue
where blood flow is, or can become, impaired or altered from normal
levels. Many pathological conditions can lead to vascular diseases
that are associated with alterations in the normal vascular
condition of the affected tissues and/or systems. Examples of
vascular conditions or vascular diseases to which the methods of
the invention apply are those in which the vasculature of the
affected tissue or system is senescent or otherwise altered in some
way such that blood flow to the tissue or system is reduced or in
danger of being reduced or increased above normal levels. It refers
to any disorder in any of the various parts of the cardiovascular
system, which consists of the heart and all of the blood vessels
found throughout the body. Diseases of the heart may include
coronary artery disease, CHD, cardiomyopathy, valvular heart
disease, pericardial disease, congenital heart disease (e.g.,
coarctation, atrial or ventricular septal defects), and heart
failure. Diseases of the blood vessels may include
arteriosclerosis, atherosclerosis, hypertension, stroke, vascular
dementia, aneurysm, peripheral arterial disease, intermittent
claudication, vasculitis, venous incompetence, venous thrombosis,
varicose veins, and lymphedema.
[0186] It was a surprising aspect of the present invention that the
MSCs described herein localized in vivo to areas of atherosclerosis
after systemic administration. The MSCs produced in a localized
manner sufficient transgene to provide a therapeutic effect without
unwanted systemic and uncontrolled expression of said
transgene.
[0187] Neurodegenerative disease or neurodegeneration is a term for
medical conditions in which the progressive loss of structure or
function of neurons, including death of neurons, occurs. Many
neurodegenerative diseases, including ALS, Parkinson's,
Alzheimer's, and Huntington's, occur as a result of
neurodegenerative processes. Such diseases are commonly considered
to be incurable, resulting in progressive degeneration and/or death
of neuron cells. A number of similarities are present in the
features of these diseases, linking these diseases on a
sub-cellular level. Some of the parallels between different
neurodegenerative disorders include atypical protein assembly as
well as induced cell death.
[0188] Dementia is a group of brain diseases causing a gradual
decline of cognitive functions. Most of these diseases are chronic
neurodegenerative diseases and are associated with neurobehavioral
and/or neuropsychiatric symptoms that disable patients to
independently perform activities of daily live. Alzheimer's disease
is the most common form of dementia with 25 million affected
individuals worldwide in the year 2000. This number is expected to
increase to 114 million cases in 2050, unless preventive or
neuroprotective therapy approaches emerge.
[0189] Dementia according to the present invention refers to, but
is not limited to, one or more of, Alzheimer's disease, vascular
dementia, post-stroke dementia, Lewy body dementia, frontotemporal
dementia, Huntington's disease, and Creutzfeldt-Jakob disease.
[0190] Erectile dysfunction is a multifactorial disorder associated
with aging and a range of organic and psychogenic conditions,
including hypertension, hypercholesterolemia, diabetes mellitus,
cardiovascular disease, and depression. Nitric oxide (NO) is
believed to be an important vasoactive neurotransmitter and
chemical mediator of penile erection. Impaired NO bioactivity is a
pathogenic mechanism of erectile dysfunction. The efficacy of the
PDE-5 inhibitors in the treatment of erectile dysfunction serves to
illustrate the importance of the NO regulation in erectile
function, since these agents counteract the degradation of
NO-generated cGMP. However, not all patients respond to PDE-5
inhibitors, such that additional therapies are required (Burnett A
L, J Clin Hypertens 2006 December; 8(12Suppl4):53-62).
[0191] Diabetes mellitus is a group of chronic metabolic diseases
that are associated with high blood sugar levels over prolonged
periods, which can lead to severe complications including
cardiovascular diseases, stroke, kidney failure, foot ulcers and
damaged eyes. The two main subtypes are type 1 and type 2 diabetes
mellitus. Type 1 diabetes mellitus is characterized by the loss of
insulin-producing cells in the pancreas. It accounts for about 10%
of the diabetes cases in the US and Europe, mostly affects children
and is often associated with autoimmune pathologies. Type 2
diabetes mellitus is characterized by insulin resistance. Diabetes
mellitus represents a massive health issue with more than 350
million affected people in 2013 worldwide.
[0192] Diabetes mellitus according to the present invention refers
to, but is not limited to, one or more of, type 1 diabetes
mellitus, type 2 diabetes mellitus, gestational diabetes, and
latent autoimmune diabetes of adults.
[0193] Autoimmune diseases are a group a diseases that are caused
by an abnormal immune response of the body against specific
molecules or cells that are normally present in the body and should
therefore be tolerated by the immune system under physiological
conditions. The pathological reaction of the body's immune system
against its own components can lead to severe physical conditions.
A large number of diseases have been identified as being caused by
autoimmune reactions and many pathologies of unclear etiology are
suspected to have autoimmune components and are therefore termed
autoimmune-related diseases. Therefore, the development of
effective and specific treatment strategies for this group of
diseases is urgently needed.
[0194] Autoimmune diseases and autoimmune-related diseases
according to the present invention refers to, but is not limited
to, one or more of, acute disseminated encephalomyelitis, acute
necrotizing hemorrhagic leukoencephalitis, addison's disease,
agammaglobulinemia, alopecia areata, amyloidosis, ankylosing
spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome
(APS), autoimmune angioedema, autoimmune aplastic anemia,
autoimmune dysautonomia, autoimmune hepatitis, autoimmune
hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear
disease, autoimmune myocarditis, autoimmune oophoritis, autoimmune
pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic
purpura, autoimmune thyroid disease, autoimmune urticaria, axonal
& neuronal neuropathies, Balo disease, Behcet's disease,
bullous pemphigoid, cardiomyopathy, Castleman disease, Celiac
disease, chagas disease, chronic inflammatory demyelinating
polyneuropathy, chronic recurrent multifocal ostomyelitis,
Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal
pemphigoid, Crohn's disease, Cogans syndrome, Cold agglutinin
disease, Congenital heart block, Coxsackie myocarditis, CREST
disease, essential mixed cryoglobulinemia, demyelinating
neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's
disease (neuromyelitis optica), discoid lupus, Dressler's syndrome,
endometriosis, eosinophilic esophagitis, eosinophilic fasciitis,
erythema nodosum, experimental allergic encephalomyelitis, Evans
syndromej, fibrosing alveolitis, giant cell arteritis, giant cell
myocarditis, glomerulonephritis, Goodpasture's syndrome,
granulomatosis with polyangiitis, Graves' disease, Guillain-Barre
syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis,
hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis,
hypogammaglobulinemia, idiopathic thrombocytopenic purpura, IgA
nephropathy, IgG4-related sclerosing disease, inclusion body
myositis, interstitial cystitis, juvenile arthritis, juvenile
diabetes (Type 1 diabetes), juvenile myositis, Kawasaki syndrome,
Lambert-Eaton syndrome, leukocytoclastic vasculitis, Lichen planus,
Lichen sclerosus, ligneous conjunctivitis, linear IgA disease
(LAD), lupus (SLE), Lyme disease (chronic), Meniere's disease,
microscopic polyangiitis, mixed connective tissue disease, mooren's
ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia
gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia,
ocular cicatricial pemphigoid, pptic neuritis, palindromic
rheumatism, pediatric autoimmune neuropsychiatric disorders
associated with streptococcus, paraneoplastic cerebellar
degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg
syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral
uveitis), pemphigus, peripheral neuropathy, perivenous
encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis
nodosa, type I, II, & III autoimmune polyglandular syndromes,
polymyalgia rheumatica, polymyositis, postmyocardial infarction
syndrome, postpericardiotomy syndrome, progesterone dermatitis,
primary biliary cirrhosis, primary sclerosing cholangitis,
psoriasis, psoriatic arthritis. idiopathic pulmonary fibrosis,
pyoderma gangrenosum, pure red cell aplasia, Raynauds phenomenon,
reactive arthritis, reflex sympathetic dystrophy, Reiter's
syndrome, relapsing polychondritis, restless legs syndrome,
retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis,
sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's
syndrome, sperm & testicular autoimmunity, stiff person
syndrome, subacute bacterial endocarditis (SBE), susac's syndrome,
sympathetic ophthalmia, Takayasu's arteritis, temporal
arteritis/giant cell arteritis, thrombocytopenic purpura,
Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes,
ulcerative colitis, undifferentiated connective tissue disease,
uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, or
Wegener's granulomatosis.
[0195] In preferred embodiments the lung disease is selected from
an inflammatory or restrictive lung disease, a respiratory tract
infection, a malignant or benign tumor of the lung and/or a
pulmonary vascular disease or condition.
Examples
[0196] The invention is further described by the following
examples. These are not intended to limit the scope of the
invention. The experimental examples relate to the development of
technology that enables Klotho expression from genetically modified
MSCs. The examples further relate to trials in models suitable for
testing the treatment of various medical conditions.
[0197] In preferred embodiments the examples relate to the
preclinical development of a novel gene therapy product that
combines the multiple beneficial effects of Klotho in the context
of pathology with the immunomodulatory properties of primary human
mesenchymal stem cells (MSCs) for the treatment of the above
mentioned diseases.
[0198] Preparation of Human Mesenchymal Stem Cells:
[0199] Human cells are isolated from bone marrow by plastic
adherence and are cultured in growth medium e.g. FBS containing
DMEM as described by Pittinger, M. F. (2008) Mesenchymal stem cells
from adult bone marrow, In D. J. Prockop, D. G. Phinney, B. A.
Bunnell, Methods in Molecular Biology 449, Mesenchymal stem cells,
Totowa: Humana Press). Mouse cells are isolated according to
established methods. Methods for the isolation of MSCs from mice
are known in the art, for example as described in Soleimani (Nat
Protoc. 2009; 4(1):102-6) or Zhu (Nat Protoc. 2010; 5,
550-560).
[0200] Generation of Vectors for the Expression of Klotho:
[0201] The transgene expression cassettes comprising a promoter and
a coding region (e.g. cDNA) for human Klotho gene expression (full
length or soluble form) are constructed using standard cloning
techniques as described in Julia Lodge, Peter Lund, Steve Minchin
(2007) Gene Cloning, Ney York: Tylor and Francis Group.
[0202] The promoters assessed during evaluation relate to the
inducible promoters Tie2, RANTES or the HSP70 promoter, or the
constitutive CMV or PGK promoters.
[0203] In some embodiments the gene is fused with tag-sequences
(e.g. marker proteins/peptides like the hemagglutinin-tag or the
HIS-tag) to allow easy detection of expression of the Klotho
transgene (Hinrik Garoff, 1985, Annual Review of Cell Biology, Vol.
1: 403-445). In particular, examples are described below having
implemented the hemagglutinin-tagged version of Klotho for
detection of Klotho expression.
[0204] In some examples the signal peptide of the Klotho gene is
replaced by other Signal sequences. Furthermore, in some examples,
gene sequences may be employed that are codon optimized to allow
enhanced translation. The examples incorporating human Klotho
employed sequences according to SEQ ID NO 2 or 3.
[0205] The transgene is then inserted into a suitable vector system
(e.g. lentiviral or gamma-retroviral vector) by standard cloning
techniques. A suitable vector is for example described by Baum
(patent application EP 1757703 A2). The vector preferably comprises
a second transgene cassette consisting of a promoter, an IRES
sequence and a selectable marker gene (cell surface marker or
resistance gene, for example the pac gene to confer puromycin
resistance) to allow enrichment of genetically modified cells later
in the process (David P. Clark, Nanette J. Pazdernik, 2009,
Biotechnology: Applying the Genetic Revolution, London:
Elsevier).
[0206] Genetic Modification of Mesenchymal Stem Cells:
[0207] The transduction is performed with modifications as
described by Murray et al., 1999 Human Gene Therapy. 10(11):
1743-1752 and Davis et al., 2004 Biophysical Journal Volume 86
1234-1242. In detail:
[0208] 6-well cell culture plates (e.g. Corning) are coated with
Poly-L-Lysine (PLL) (e.g. Sigma-Aldrich, P4707-50ML): The PLL
solution (0.01%) is diluted to final concentration between 0.0001%
and 0.001% with PBS. 2 ml of the diluted PLL are used for each
well. The plate is incubated at least for 2 h at room temperature.
After incubation, the plates are washed carefully with PBS.
[0209] Viral vector supernatant in a final volume of 2 ml is added
to each PLL-coated well. The number of particles should between
2.times.10e3 and 1.times.10e6 per well, which will result in
multiplicity of infection of 0.25 and 10. The loaded plate is
centrifuged for 2000.times.g, 30 min, 4.degree. C. Afterwards the
supernatant is discarded and 1.times.10e5 mesenchymal stem cells
are seeded per well. The plates are incubated at 37.degree. with 5%
CO2 for further use.
[0210] Analysis of Transgene Expression in MSC:
[0211] Flow Cytometry:
[0212] To demonstrate that the Klotho is expressed MSCs
intracellular flow cytometry assays are performed. 3 days after
transduction, MSC medium is exchanged with medium containing 1
.mu.l BD Golgi Plug (Cat. No. 555029) per 1 ml Medium to enrich the
expressed Klotho in the Golgi apparatus of the transduced
cells.
[0213] Cells are incubated for 16 h at 37.degree. C. and are then
immunostained for the expression of the Klotho transgene. MSCs are
harvested. The cells are permeabilized using the BD
Cytofix/Cytoperm Cell Permeabilization/Fixation Solution (Becton
Dickinson, 554722) according to the manufacturer's instructions to
allow intracellular staining of the target Klotho protein.
[0214] A hemagglutinin-tag specific antibody labeled with
Phycoerythrin (PE) (Milteny, 120-002-687) is used for detection of
the expressed Klotho. 2.times.10e5 MSC are stained with 100 .mu.l
of antibody (1:75 diluted with Perm/wash solution, Becton
Dickinson, 554723).
[0215] Alternatively, antibodies directly directed against Klotho
are used according to the instruction of the manufacturer (e.g.
ProSci 45-810). The stained cells are washed and resuspended in
PBS. The cells are then analyzed on an FC500 flow cytometer
(Beckman Coulter).
[0216] ELISA to Determine Klotho Levels in the Supernatant:
[0217] Transduced MSC are seeded in 6 well plates (1.times.10e5 MSC
per well). Transduced MSC, which carry the pac puromycin resistance
gene, are enriched by puromycin selection. Puromycin (3 .mu.g/ml
medium) is added to the medium and cells are cultivated over a
period of 5 days at 37.degree. C. and 5% CO2 with medium exchanges
every 2 days to deplete non-transduced cells from the culture.
Afterwards, puromycin-free medium is used for the culture. MSC are
reseeded at a defined cell number of 1.times.10e5 cells per well in
a 6 well-plate and are incubated for 72 h. Medium is collected and
used for Klotho specific ELISA for quantification according to the
manufacturer's instructions (e.g. R&D Systems DY5334-05).
[0218] Klotho-Expressed in MSC Protects the Cells from
H.sub.2O.sub.2-Induced Apoptosis:
[0219] Imbalanced defense mechanisms against antioxidants, or
overproduction or incorporation of free radicals, leads to
neurodegeneration, by which neural cells suffer functional or
sensory loss in neurodegenerative diseases. Oxidative stress (OS)
leads to free radical attack on neural cells and contributes to
neuro-degeneration; imbalanced metabolism and excess reactive
oxygen species (ROS) generate a range of disorders such as
Alzheimer's disease, Parkinson's disease, aging and many other
neurodegenerative disorders (Uttara et al., Curr Neuropharmacol.
2009 March; 7(1): 65-7). The ability of Klotho to protect cells
from damage caused by reactive oxygen species represents a useful
therapeutic model for the treatment of neurodegenerative
disorders.
[0220] Oxidative stress markers are available in chronic kidney
disease (CKD) patients and have confirmed the long held belief that
CKD is a pro-oxidant state. Recent studies suggest that the link
between oxidative stress and inflammation in CKD is emerging as a
key process contributing to the pathogenesis of oxidative stress in
these patients (Massy et al., Semin Dial. 2009 July-August;
22(4):405-8). The ability of Klotho to protect cells from damage
caused by reactive oxygen species represents a useful therapeutic
model for the treatment of chronic kidney disease.
[0221] Klotho is able to protect cells from the effects of reactive
oxygen species:
[0222] Transduced and selected MSC (as described above) and
non-transduced control MSC are seeded into 6-well plates
(5000-50000 cells per cm.sup.2). The cells are incubated 16 h at
37.degree. C., 5.degree. C. Transduced and non-transduced cells are
subsequently treated with Hydrogen peroxide (H2O2, Roth, cat. No.
8070.2) for a period of 2-8 h. The final concentration of
H.sub.2O.sub.2 in the culture is between 10-100 .mu.M. In addition,
selected samples of MSC are not treated with H.sub.2O.sub.2 and
serve as control. All samples are trypsinated to detach the MSC
from the plates.
[0223] The survival of MSC is determined by flow cytometry. The
samples are subjected to the Dead Cell Apoptosis Kit (ThermoFisher
Scientific, V13241) according to the manufacturer's instructions.
The kit allows the detection of dead and apoptotic cells in the
samples by staining with Annexin V Alexa Fluor 488 and Propidium
Iodide (PI) (Vermes et al. (1995) Journal of Immunological Methods,
Volume 184, Issue 1, Pages 39-51). The samples are analyzed by Flow
cytometry. Klotho expressing MSC treated with H.sub.2O.sub.2 show
increased survival and reduced apoptosis in comparison to native
MSC treated with H.sub.2O.sub.2.
[0224] Klotho Secreted from Transduced MSC Protects HUVEC Cells
from H.sub.2O.sub.2-Induced Apoptosis:
[0225] Klotho is able to protect cells from the effects of reactive
oxygen species:
[0226] Transduced and selected MSC (as described previously) and
non-transduced control MSC are seeded into 6-well plates
(50000-200000 cells per cm.sup.2). In addition, selected samples of
MSC are not treated with H.sub.2O.sub.2 and serve as control. The
cells are incubated 16-48 h at 37.degree. C., 5.degree. C. The
supernatant is collected, filtered (0.25 .mu.m) from the cells and
stored.
[0227] Human umbilical vein endothelial cells (HUVEC) are seeded
are seeded into 6-well plates (5000-50000 cells per cm.sup.2). The
cells are incubated for 16 h at 37.degree. C. to let the cells
attach to the plates. The cells are treated with Hydrogen peroxide
(H.sub.2O.sub.2, Roth, cat. No. 8070.2). The final concentration of
H.sub.2O.sub.2 in the culture is between 10-100 .mu.M. Different
dilutions of the Klotho containing supernatant and control
supernatant is added to the HUVEC cells (2-4 ml volume). Samples
are incubated for 2-8 h. All samples are trypsinated to detach the
HUVEC cells from the plates.
[0228] The survival of HUVEC cells is determined by flow cytometry.
The samples are subjected to the Dead Cell Apoptosis Kit
(ThermoFisher Scientific, V13241) according to the manufacturer's
instructions. The kit allows the detection of dead and apoptotic
cells in the samples by staining with Annexin V Alexa Fluor 488 and
Propidium Iodide (PI) (Vermes et al. (1995) Journal of
Immunological Methods, Volume 184, Issue 1, Pages 39-51). The
samples are analyzed by Flow cytometry. HUVEC cells treated with
supernatant from Klotho-expressing MSCs show increased survival and
reduced apoptosis in comparison to untreated HUVECs, when cultured
in the presence of H.sub.2O.sub.2.
[0229] Klotho-Expressed in MSC Increases the Production of Nitric
Oxide (NO) in the MSC:
[0230] The overall production of nitric oxide (NO) is decreased in
chronic kidney disease (CKD) which contributes to cardiovascular
events and further progression of kidney damage. Interventions that
can restore NO production are likely to reduce the cardiovascular
complications of CKD as well as slowing the rate of progression
(Baylis, Am J Physiol Renal Physiol. 2008 January; 294(1):F1-9).
The ability of Klotho to restore NO levels represents a useful
therapeutic model for the treatment of kidney disease.
[0231] As discussed herein, nitric oxide (NO) is believed to be an
important vasoactive neurotransmitter and chemical mediator of
penile erection, whereby impaired NO bioactivity is a pathogenic
mechanism of erectile dysfunction. The ability of Klotho to restore
NO levels represents a useful therapeutic model for the treatment
of erectile dysfunction.
[0232] Nitric oxide (NO) is an intercellular messenger that
performs a number of functions, including neurotransmission,
vasodilatation, inhibition of platelet aggregation, and modulation
of leukocyte adhesion. NO has recently been shown to act as a
potent cytotoxic effector molecule as well as to play an important
role in the pathogenesis of organ-specific autoimmunity. NO may
also modulate the immune response by interfering with Th1/Th2
balance in autoimmune diseases (Singh et al., Immunol Res. 2000;
22(1):1-19). The ability of Klotho to restore NO levels represents
a useful therapeutic model for the treatment of autoimmune
disease.
[0233] Klotho increases the production NO, an important
intercellular messenger, in MSC:
[0234] Transduced and selected MSC (as described previously) and
non-transduced control MSC are seeded into 6-well plates
(5000-50000 cells per cm.sup.2). The cells are incubated for 48 h
at 37.degree. C./5% CO.sub.2.
[0235] To evaluate NO production, 100 .mu.L of supernatant from
each well of the culture plate is transferred to a new 96-well
plate. The same amount of Griess reagent (1% sulfanilamide, 0.1%
naphthylenediamine dihydrochloride, and 2.5% phosphoric acid) is
added to the supernatant. Nitrite concentrations in the
supernatants are obtained by linear regression analysis of the
standard curve by using serial double dilutions of sodium nitrite
from 200 mmol/L to the 11th dilution. Absorbance is determined at
540 nm by using a microplate reader (Spectramax 190--Molecular
Device, Sunnyvale, Calif.). The concentration of NO is higher in
samples collected from transduced MSC.
[0236] Klotho Secreted from Transduced MSC Induces Increased Nitric
Oxide-Production in HUVEC Cells:
[0237] Transduced and selected MSC (as described previously) and
non-transduced control MSC are seeded into 6-well plates
(5000-50000 cells per cm.sup.2). The cells are incubated for 48 h
at 37.degree. C./5% CO.sub.2. The supernatant is collected,
filtered (0.25 .mu.m) from the cells and stored.
[0238] Human umbilical vein endothelial cells (HUVEC) are seeded
are seeded into 6-well plates (5000-50000 cells per cm.sup.2). The
cells are incubated for 16 h at 37.degree. C. to let the cells
attach to the plates. Different dilutions of the Klotho containing
supernatant and control supernatant is added to the HUVEC cells
(2-4 ml volume). Samples are incubated for 6-48 h. To evaluate NO
production, 100 .mu.L of supernatant from each HUVEC well of the
culture plate is transferred to a new 96-well plate. The same
amount of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine
dihydrochloride, and 2.5% phosphoric acid) is added to the
supernatant. Nitrite concentrations in the supernatants are
obtained by linear regression analysis of the standard curve by
using serial double dilutions of sodium nitrite from 200 mmol/L to
the 11th dilution. Absorbance is determined at 540 nm by using a
microplate reader (Spectramax 190--Molecular Device, Sunnyvale,
Calif.). The concentration of NO is higher in HUVEC cells treated
with supernatant from Klotho-expressing MSCs.
[0239] Klotho Secreted from Transduced MSC Suppress TGF-Beta
Signaling in Target Cells
[0240] Transduced and selected MSC (as described previously) and
non-transduced control MSC are seeded into 6-well plates
(5000-50000 cells per cm.sup.2). The cells are incubated for 48 h
at 37.degree. C./5% CO.sub.2. The supernatant is collected,
filtered (0.25 .mu.m) from the cells and stored.
[0241] NRK52E renal epithelial cells are treated with the collected
supernatants for 30 min and then stimulated with TGF-beta (10
ng/ml) for 30 min. The cells are lysed and used for immunoblot
analysis. To detect activation of the TGF-beta signaling cascade in
the renal cells, an antibody against phosphorylated Smad2 (pSmad2)
or antibody that recognized Smad2 regardless of its phosphorylation
state (Smad2) is used. An increase in the fraction of
phosphorylated Smad2 in comparison to total Smad2 indicates
activation of TGF-beta signaling. Klotho containing supernatant
suppresses activation of TGF-beta signaling compared to cells that
are treated with supernatants devoid of Klotho or untreated
controls.
[0242] Klotho Secreted from Transduced MSC Increases FGF-23
Signaling in Target Cells
[0243] Transduced and selected MSC (as described previously) and
non-transduced control MSC are seeded into 6-well plates
(5000-50000 cells per cm.sup.2). The cells are incubated for 48 h
at 37.degree. C./5% CO.sub.2. The supernatant is collected,
filtered (0.25 .mu.m) from the cells and stored.
[0244] 293 cells, which express the FGF-receptor, are seeded in 6
well plates (5000 cells per cm.sup.2) and incubated overnight.
Medium is exchanged for Klotho-containing or Klotho-free
supernatant and the cells are incubated for 30 min. Afterward mouse
FGF23 (R179Q) (10 ng/ml) is added and the cells are incubated for
an additional 15 min. Cells are harvested and lysed using lysis
buffer (M PER Mammalian Protein Extraction Reagent) containing
inhibitors for phosphatase and proteinase (Halt Protease Inhibitor
Cocktail, EDTA Free (100.times.)). FGF signaling is determined by
immunoblot analysis using anti-phospho-FRS2a antibody (p-FRS2a),
anti-phospho-ERK1/2 antibody (p-ERK1/2), or anti-ERK1/2 antibody
(ERK1). Klotho containing supernatant activates FGF23 signaling
compared to cells that are treated with supernatants devoid of
Klotho or untreated controls.
[0245] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs:
[0246] The experiments described herein are based on the occurrence
that old mice lack klotho in the kidney, and that Klotho deficiency
in old mice can be restored by i.v. application of Klotho-modified
MSCs. In murine models mouse MSCs were employed, obtained by
methods as described above, and the mouse Klotho sequence was used.
Klotho-modified MSCs improve renal function, improve heart rate
variability and prolong lifespan in old mice.
[0247] Experimental Approach:
[0248] Mice at the age of 12-15 months are obtained from Jackson
Laboratories. All animals are placed in metabolic cages once a week
to measure renal function. After sacrifice, klotho expression in
the kidney will determined by immunohistology.
[0249] Two different Experiments are conducted in 6 groups.
[0250] Experiment A:
[0251] Group 1: Mice age 12-15 months are placed in metabolic cages
once a week. No further treatment will be performed until they
die.
[0252] Group 2: Mice age 12-15 months are treated with
non-transduced MSCs (Mesenchymal stem cells having been obtained
from young donor animals, age 6 weeks). They receive
1.times.10.sup.6 MSCs once a month until death. All animals are
placed in metabolic cages once a week.
[0253] Group 3: Mice age 12-15 months are treated with
Klotho-modified MSCs (Mesenchymal stem cells having been obtained
from young donor animals, age 6 weeks, are genetically modified
according to the protocols described herein to express transgenic
klotho). They receive 1.times.10.sup.6 Klotho-modified MSCs once a
month until death. All animals are placed in metabolic cages once a
week.
[0254] Results: Animals in group 3 show significant improved renal
function and live significantly longer in comparison to animals in
group 1 and 2. Klotho expression in the kidney is significantly
higher in animals in group 3 than in the other two groups.
[0255] Experiment B:
[0256] Group 1: Mice age 12-15 months are implanted with an ETA-F10
transmitter (DSI, St. Paul, Minn., USA). Once a week ECG and heart
rate variability are measured.
[0257] Group 2: Mice age 12-15 months are implanted with an ETA-F10
transmitter (DSI, St. Paul, Minn., USA). All animals are treated
with non-transduced MSCs (Mesenchymal stem cells having been
obtained from young donor animals, age 6 weeks). Treated mice
receive 1.times.10.sup.6 MSCs once a month until death. Once a week
ECG and heart rate variability are measured.
[0258] Group 3: Mice age 12-15 months are implanted with an ETA-F10
transmitter (DSI, St. Paul, Minn., USA). All animals are treated
with Klotho-modified MSCs (Mesenchymal stem cells having been
obtained from young donor animals, age 6 weeks, are genetically
modified to express klotho as described herein). Treated mice
receive 1.times.10.sup.6 Klotho-modified MSCs once a month. Once a
week ECG and heart rate variability are measured.
[0259] Results: Animals in group 3 show significant improved heart
rate variability in comparison to animals of group 1 and 2.
[0260] MSC Expressing Klotho Improve Kidney Fibrosis in an Murine
Model
[0261] To induce kidney fibrosis 129S1/SvImJ mice (7-10 weeks of
age) are used. The right ureter is surgically exposed and ligated
(unilateral ureteral obstruction, UUO). After surgery, mice are
injected intravenously or intraperitoneally with Klotho-expressing
MSC (5.times.10 5-2.times.10 6 cells per mouse) or with PBS. The
treatment is repeated every 3 days.
[0262] After 14 days post-surgery mice are sacrificed and the
kidney is prepared for histology. Markers of renal fibrosis, such
as the number of interstitial fibroblasts, interstitial volume,
expression for collagen I are all increased in UUO animals treated
with PBS only. In contrast, UUO animals that receive
Klotho-expressing MSC show markedly decreased levels of renal
fibrosis.
[0263] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Alzheimer's Disease
(AD):
[0264] The following examples describe approaches that demonstrate
that intravenous or intrathecally administered MSC-Klotho induce
maturation of oligodendrocytic progenitor cells (OPCs), intravenous
or intrathecally administered MSC-Klotho increases the number of
total oligodendrocytes, intravenous or intrathecally administered
MSC-Klotho improve myelination of oligodendrocytes, intravenous or
intrathecally administered MSC-Klotho reduces plaques in a mouse
model of APPswe/PS1(M146V) double transgenic mice, intravenous or
intrathecally administered MSC-Klotho restores cognition in a mouse
model of APPswe/PS1(M146V) double transgenic mice, intravenous or
intrathecally administered MSC-Klotho induce behavioral recovery by
elevating brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3) and vascular endothelial growth factor (VEGF) levels in the
brains of APPswe/PS1(M146V) double transgenic mice, and that
intravenous or intrathecally administered MSC-Klotho promote
activation of microglia that secrete neurotrophic agents and
results in cognitive improvements and a reduction in A.beta.
pathology in APPswe/PS1(M146V) double transgenic mice.
[0265] Experimental Setup
[0266] Three different mouse strains (C3B6-Tg(APP695)3Dbo/Mmjax,
C3B6-Tg(APP695)3Dbo/Mmjax, B6;129-Psen1tm1Mpm
Tg(APPSwe,tauP301L)1Lfa/Mmjax) resembling Alzheimer's disease are
employed.
[0267] Three different Experiments will be conducted in 18
groups.
[0268] Experiment A:
[0269] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express klotho. 1.times.10.sup.6
MSC-Klotho are injected intravenously once a month.
[0270] Group 1 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0271] Group 2 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0272] Group 3 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax
mice
[0273] Mice are injected with saline intravenously once a
month.
[0274] Group 4 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0275] Group 5 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0276] Group 6 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax
mice
[0277] 1.times.10.sup.6 of non-transduced mesenchymal stem cells
from young donor animals (age 6 weeks) are injected intravenously
into the mice once a month.
[0278] Group 7 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0279] Group 8 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0280] Group 9 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax
mice
[0281] Cognitive tests are performed once a week over a 3 month
period.
[0282] Results: Animals in groups 1-3 show significant improvement
of attention, learning and memory compared with animals in groups
4-6 and groups 7-9.
[0283] Experiment B:
[0284] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express klotho. 1.times.10.sup.6
MSC-Klotho are injected intrathecally once a month.
[0285] Group 1 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0286] Group 2 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0287] Group 3 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1 Lfa/Mmjax
mice
[0288] Mice are injected with saline intrathecally once a month
[0289] Group 4 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0290] Group 5 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0291] Group 6 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1 Lfa/Mmjax
mice
[0292] 1.times.10.sup.6 of non-transduced mesenchymal stem cells
from young donor animals (age 6 weeks) are injected intravenously
into the mice once a month.
[0293] Group 7 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0294] Group 8 C3B6-Tg(APP695)3Dbo/Mmjax mice
[0295] Group 9 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1 Lfa/Mmjax
mice
[0296] Three animals of each group will be sacrificed every week
for 3 months. Immunohistochemistry, electron microscopy, HPLC,
qRT-PCR and western blotting of brain tissue will be performed.
[0297] Results: Animals in groups 1-3 show significant improvement
in maturation of oligodendrocytic progenitor cells (OPCs),
increased number of total oligodendrocytes, improved myelination of
oligodendrocytes, reduced A.beta. plaques, elevation of
brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and
vascular endothelial growth factor (VEGF) levels, compared with
animals in groups 4-6 and groups 7-9.
[0298] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Multiple Sclerosis (MS):
[0299] The following examples describe approaches that demonstrate
that intravenous or intrathecally administered MSC-Klotho induce
maturation of oligodendrocytic progenitor cells (OPCs), intravenous
or intrathecally administered MSC-Klotho increases the number of
total oligodendrocytes, intravenous or intrathecally administered
MSC-Klotho improve myelination of oligodendrocytes, intravenous or
intrathecally administered MSC-Klotho reduce inflammation, and that
intravenous or intrathecally administered MSC-Klotho reduce number
of activated T- and B-cells in the brain of MS patients.
[0300] Experimental Setup:
[0301] A mouse model of autoimmune encephalomyelitis (EAE) is used.
C57BL/6 (H-2b) mice are immunized with myelin oligodendrocyte
protein (MOG.sub.35-55).
[0302] Two different Experiments are conducted in 6 groups
[0303] Experiment A:
[0304] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express klotho. 1.times.10.sup.6
MSC-Klotho are injected intravenously once a week.
[0305] Group 1 C57BL/6 (H-2b) immunized with (MOG.sub.35-55)
[0306] Mice are injected with saline intravenously once a week
[0307] Group 2 C57BL/6 (H-2b) immunized with (MOG.sub.35-55)
[0308] 1.times.10.sup.6 of non-transduced mesenchymal stem cells
from young donor animals (age 6 weeks) are injected intravenously
into the mice once a month.
[0309] Group 3 C57BL/6 (H-2b) immunized with (MOG.sub.35-55)
[0310] Experiment B:
[0311] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express klotho. 1.times.10.sup.6
MSC-Klotho are injected intrathecally once a week.
[0312] Group 4 C57BL/6 (H-2b) immunized with (MOG.sub.35-55)
[0313] Mice are injected with saline intrathecally once a week.
[0314] Group 5 C57BL/6 (H-2b) immunized with (MOG.sub.35-55)
[0315] 1.times.10.sup.6 of non-transduced mesenchymal stem cells
from young donor animals (age 6 weeks) are injected intrathecally
into the mice once a month.
[0316] Group 6 C57BL/6 (H-2b) immunized with (MOG.sub.35-55)
[0317] All animals in groups 1-6 are followed daily for paralysis
beginning in the tail and hind limbs and progressing to the
fore-limbs concurrent with weight loss. Once a week 3 animals from
each group are sacrificed and immunohistochemistry, electron
microscopy, HPLC, qRT-PCR and western blotting of brain tissue is
performed.
[0318] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Amyotrophic Lateral
Sclerosis (ALS):
[0319] The following examples describe approaches that demonstrate
that intravenous or intrathecally administered MSC-Klotho induce
maturation of oligodendrocytic progenitor cells (OPCs), intravenous
or intrathecally administered MSC-Klotho increases the number of
total oligodendrocytes, intravenous or intrathecally administered
MSC-Klotho improve myelination of oligodendrocytes, intravenous or
intrathecally administered MSC-Klotho reduce axon degeneration,
intravenous or intrathecally administered MSC-Klotho will inhibit
Wnt signaling.
[0320] Experimental Setup:
[0321] SOD1 (G93A) mice are utilized. Two different Experiments
will be conducted in 6 groups
[0322] Experiment A:
[0323] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express klotho. 1.times.10.sup.6
MSC-Klotho are injected intravenously once a week.
[0324] Group 1 SOD1 (G93A)
[0325] Mice are injected with saline intravenously once a week
[0326] Group 2 SOD1 (G93A)
[0327] 1.times.10.sup.6 of non-transduced mesenchymal stem cells
from young donor animals (age 6 weeks) are injected intravenously
into the mice once a month.
[0328] Group 3 SOD1 (G93A)
[0329] Experiment B:
[0330] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express klotho. 1.times.10.sup.6
MSC-Klotho are injected intrathecally once a week.
[0331] Group 4 SOD1 (G93A)
[0332] Mice are injected with saline intrathecally once a week
[0333] Group 5 SOD1 (G93A)
[0334] 1.times.10.sup.6 of non-transduced mesenchymal stem cells
from young donor animals (age 6 weeks) are injected intrathecally
into the mice once a month.
[0335] Group 6 SOD1 (G93A)
[0336] Once a week 3 animals from each group are sacrificed and
brain and spinal cord will be harvested. Immunohistochemistry,
electron microscopy, HPLC, qRT-PCR and western blotting of brain
and spinal cord are performed.
[0337] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Diabetes Type 1
[0338] The following examples describe approaches that demonstrate
that intravenous administered MSC-Klotho delay the onset of
diabetes type 1 in mice.
[0339] Experimental Setup:
[0340] To assess the effect of Klotho-MSCs on the development of
diabetes in vivo, a mouse model of cyclophosphamide-accelerated
type 1 diabetes is performed (adapted from Brode et al., The
Journal of Immunology 2006). NOD mice are obtained, where the
incidence of diabetes in female mice is 75% by 40 weeks of age. To
accelerate and synchronize diabetes, female 8-week-old NOD mice are
treated with a single i.p. injection of cyclophosphamide (CY) (200
mg/kg body weight in 0.9% normal saline). Mice are then randomly
divided into treatment and control groups.
[0341] One to five days after cyclophosphamide treatment each
animal receives 200 .mu.L of PBS by tail vain or intraperitoneal
injection. Mice are monitored weekly for hyperglycemia until they
become diabetic, as defined by two consecutive (>24 hr apart)
non-fasting blood glucose levels >240 mg/dl.
[0342] Group 1 NOD after cyclophosphamide treatment
[0343] One to five days after cyclophosphamide treatment animal
receives 200 .mu.L of 1.times.10.sup.6 MSCs (from young donor
animals, age 6 weeks) in 200 .mu.L PBS by tail vain or
intraperitoneal injection. Mice are monitored weekly for
hyperglycemia until they become diabetic, as defined by two
consecutive (>24 hr apart) non-fasting blood glucose levels
>240 mg/dl.
[0344] Group 2 NOD after cyclophosphamide treatment
[0345] Mesenchymal stem cells from young donor animals, age 6
weeks, are genetically modified to express Klotho. One to five days
after cyclophosphamide treatment animal receives 1.times.10.sup.6
Klotho-MSCs in 200 .mu.L PBS by tail vain or intraperitoneal
injection. Mice are monitored weekly for hyperglycemia until they
become diabetic, as defined by two consecutive (>24 hr apart)
non-fasting blood glucose levels >240 mg/dl.
[0346] Group 3 NOD after cyclophosphamide treatment
[0347] Mice of group 1 develop diabetes within 30 days, whereas the
onset of diabetes in mice treated with either MSCs (group 2) or
Klotho-MSCs (group 3) is delayed. Interestingly, for mice of group
3, the delay in development of diabetes is increased by 2 weeks
compared to group 2.
[0348] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Diabetes Type 2
[0349] The following examples describe approaches that demonstrate
that intravenous administered MSC-Klotho will improve glucose
metabolism diabetes type 2 in mice. A similar model is described in
Chen et al. (J Diabetes Research, 2015, Art ID 796912).
[0350] Experimental Setup:
[0351] C57BL/6 mice are fed standard (SD) or high fat (HFD) diet.
After 4 weeks mice are divided into 6 groups
[0352] Group 1: Mice fed with SD receive saline intravenously once
a week for 8 weeks
[0353] Group 2: Mice fed with HFD receive saline intravenously once
a week for 8 weeks
[0354] Group 3: Mice fed with SD receive 1.times.10.sup.6 MSC once
a week for 8 weeks
[0355] Group 4: Mice fed with HFD receive 1.times.10.sup.6 MSC once
a week for 8 weeks
[0356] Group 5: Mice fed with SD receive 1.times.10.sup.6
MSC-Klotho once a week for 8 weeks
[0357] Group 6: Mice fed with HFD receive 1.times.10.sup.6
MSC-Klotho once a week for 8 weeks
[0358] Mice in groups 4 and 6 show improved glucose metabolism
(hyperglycemia, hyperinsulinemia body weight and/or beta cell mass)
compared with mice in group 2.
[0359] Klotho-MSCs (group 6) show further improved glucose
metabolism compared with mice treated with non-transduced MSCs
(group 4).
[0360] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Chronic Renal Failure
[0361] C57/BL6 mice age 10 weeks are uninephrectomized and receive
a subcutaneous implantation of a 50 mg doxycorticosterone acetate
(DOCA) pellet. 7 days later mice are implanted subcutaneous with an
osmotic mini pump delivering 1.5 ng angiotensin II per minute and
per gram body weight for 2 weeks. A similar model is described in
Kirchhoff et al (Kidney International, 2008, 73, 643).
[0362] During the 4 following weeks mice receive either saline
intravenously or MSC intravenously or MSC-Klotho intravenously once
a week for 4 weeks. Blood pressure, albuminuria and serum
creatinine are monitored once a week
[0363] Group 1:
[0364] C57/BL6 with DOCA and angiotensin II receive intravenous
saline for 4 weeks.
[0365] Group 2:
[0366] C57/BL6 with DOCA and angiotensin II receive intravenous
1.times.10.sup.6 MSC once a week for 4 weeks.
[0367] Group 3:
[0368] C57/BL6 with DOCA and angiotensin II will receive
intravenous 1.times.10.sup.6 MSC-Klotho once a week for 4
weeks.
[0369] Mice in groups 2 and 3 show improved renal and cardial
function measured by serum creatinine, albuminuria, blood pressure
and heart rate variability compared to mice in group 1.
[0370] Group 3 mice show further improved renal and cardial
function measured by serum creatinine, albuminuria, blood pressure
and heart rate variability compared with mice in group 2.
[0371] Histological and electron microscopic changes of renal
architecture are improved in mice in groups 2 and 3 compared to
mice in group 1.
[0372] Studies in Animal Models for Interrogating Klotho Function
when Expressed from Transgenic MSCs in Parkinson Disease (PD):
[0373] Although pre-clinical models are yet to be established, the
inventors assert that intravenous or intrathecally administered
MSC-Klotho will reduce oxidative stress, intravenous or
intrathecally administered MSC-Klotho will reduce protein
misfolding, protein degradation, protein aggregation, intravenous
or intrathecally administered MSC-Klotho will reduce inflammation,
and that intravenous or intrathecally administered MSC-Klotho will
increase dopaminergic cells in the substantia nigra pars
compacta.
[0374] Additional experimentation on the basis of the approaches
described above is ongoing.
Sequence CWU 1
1
1013039DNAHomo sapiens 1atgcccgcca gcgccccgcc gcgccgcccg cggccgccgc
cgccgtcgct gtcgctgctg 60ctggtgctgc tgggcctggg cggccgccgc ctgcgtgcgg
agccgggcga cggcgcgcag 120acctgggccc gtttctcgcg gcctcctgcc
cccgaggccg cgggcctctt ccagggcacc 180ttccccgacg gcttcctctg
ggccgtgggc agcgccgcct accagaccga gggcggctgg 240cagcagcacg
gcaagggtgc gtccatctgg gatacgttca cccaccaccc cctggcaccc
300ccgggagact cccggaacgc cagtctgccg ttgggcgccc cgtcgccgct
gcagcccgcc 360accggggacg tagccagcga cagctacaac aacgtcttcc
gcgacacgga ggcgctgcgc 420gagctcgggg tcactcacta ccgcttctcc
atctcgtggg cgcgagtgct ccccaatggc 480agcgcgggcg tccccaaccg
cgaggggctg cgctactacc ggcgcctgct ggagcggctg 540cgggagctgg
gcgtgcagcc cgtggtcacc ctgtaccact gggacctgcc ccagcgcctg
600caggacgcct acggcggctg ggccaaccgc gccctggccg accacttcag
ggattacgcg 660gagctctgct tccgccactt cggcggtcag gtcaagtact
ggatcaccat cgacaacccc 720tacgtggtgg cctggcacgg ctacgccacc
gggcgcctgg cccccggcat ccggggcagc 780ccgcggctcg ggtacctggt
ggcgcacaac ctcctcctgg ctcatgccaa agtctggcat 840ctctacaata
cttctttccg tcccactcag ggaggtcagg tgtccattgc cctaagctct
900cactggatca atcctcgaag aatgaccgac cacagcatca aagaatgtca
aaaatctctg 960gactttgtac taggttggtt tgccaaaccc gtatttattg
atggtgacta tcccgagagc 1020atgaagaata acctttcatc tattctgcct
gattttactg aatctgagaa aaagttcatc 1080aaaggaactg ctgacttttt
tgctctttgc tttggaccca ccttgagttt tcaacttttg 1140gaccctcaca
tgaagttccg ccaattggaa tctcccaacc tgaggcaact gctttcctgg
1200attgaccttg aatttaacca tcctcaaata tttattgtgg aaaatggctg
gtttgtctca 1260gggaccacca agagagatga tgccaaatat atgtattacc
tcaaaaagtt catcatggaa 1320accttaaaag ccatcaagct ggatggggtg
gatgtcatcg ggtataccgc atggtccctc 1380atggatggtt tcgagtggca
cagaggttac agcatcaggc gtggactctt ctatgttgac 1440tttctaagcc
aggacaagat gttgttgcca aagtcttcag ccttgttcta ccaaaagctg
1500atagagaaaa atggcttccc tcctttacct gaaaatcagc ccctagaagg
gacatttccc 1560tgtgactttg cttggggagt tgttgacaac tacattcaag
tagataccac tctgtctcag 1620tttaccgacc tgaatgttta cctgtgggat
gtccaccaca gtaaaaggct tattaaagtg 1680gatggggttg tgaccaagaa
gaggaaatcc tactgtgttg actttgctgc catccagccc 1740cagatcgctt
tactccagga aatgcacgtt acacattttc gcttctccct ggactgggcc
1800ctgattctcc ctctgggtaa ccagtcccag gtgaaccaca ccatcctgca
gtactatcgc 1860tgcatggcca gcgagcttgt ccgtgtcaac atcaccccag
tggtggccct gtggcagcct 1920atggccccga accaaggact gccgcgcctc
ctggccaggc agggcgcctg ggagaacccc 1980tacactgccc tggcctttgc
agagtatgcc cgactgtgct ttcaagagct cggccatcac 2040gtcaagcttt
ggataacgat gaatgagccg tatacaagga atatgacata cagtgctggc
2100cacaaccttc tgaaggccca tgccctggct tggcatgtgt acaatgaaaa
gtttaggcat 2160gctcagaatg ggaaaatatc catagccttg caggctgatt
ggatagaacc tgcctgccct 2220ttctcccaaa aggacaaaga ggtggctgag
agagttttgg aatttgacat tggctggctg 2280gctgagccca ttttcggctc
tggagattat ccatgggtga tgagggactg gctgaaccaa 2340agaaacaatt
ttcttcttcc ttatttcact gaagatgaaa aaaagctaat ccagggtacc
2400tttgactttt tggctttaag ccattatacc accatccttg tagactcaga
aaaagaagat 2460ccaataaaat acaatgatta cctagaagtg caagaaatga
ccgacatcac gtggctcaac 2520tcccccagtc aggtggcggt agtgccctgg
gggttgcgca aagtgctgaa ctggctgaag 2580ttcaagtacg gagacctccc
catgtacata atatccaatg gaatcgatga cgggctgcat 2640gctgaggacg
accagctgag ggtgtattat atgcagaatt acataaacga agctctcaaa
2700gcccacatac tggatggtat caatctttgc ggatactttg cttattcgtt
taacgaccgc 2760acagctccga ggtttggcct ctatcgttat gctgcagatc
agtttgagcc caaggcatcc 2820atgaaacatt acaggaaaat tattgacagc
aatggtttcc cgggcccaga aactctggaa 2880agattttgtc cagaagaatt
caccgtgtgt actgagtgca gtttttttca cacccgaaag 2940tctttactgg
ctttcatagc ttttctattt tttgcttcta ttatttctct ctcccttata
3000ttttactact cgaagaaagg cagaagaagt tacaaatag 303923072DNAHomo
sapiens 2atgcccgcca gcgcccctcc aagaaggcct agacctcctc cacctagcct
gagcctgctg 60ctggtgctgc tgggactggg aggaagaagg ctgagagccg aacctgggga
tggcgcccag 120acatgggcca gattctctag accacccgcc cctgaagccg
ccggactgtt tcagggaacc 180ttccccgatg gcttcctgtg ggccgtggga
tctgccgcct atcagactga agggggctgg 240cagcagcacg gcaagggcgc
ctctatctgg gacaccttca cccaccatcc tctggcccca 300cccggcgaca
gcagaaatgc ttctctgcct ctgggagccc ccagccctct gcagcctgct
360acaggggatg tggccagcga cagctacaac aacgtgttcc gggacacaga
ggccctgcgg 420gaactgggcg tgacccacta cagattcagc atcagctggg
ccagagtgct gcccaatggc 480tctgccggcg tgcccaatag agagggcctg
cggtactacc ggcggctgct ggaaagactg 540agagaactgg gagtgcagcc
cgtcgtgacc ctgtaccatt gggacctgcc ccagagactg 600caggatgcct
atggcggctg ggccaataga gccctggccg accacttcag agactacgcc
660gagctgtgct tccggcactt tggcggccaa gtgaagtact ggatcaccat
cgacaacccc 720tacgtggtgg cctggcacgg ctatgccaca ggcagactgg
cccctggcat cagaggaagc 780cctagactgg gctacctggt ggcccacaat
ctgctgctgg cccacgctaa agtgtggcac 840ctgtacaaca ccagcttccg
gcctacacag ggcggccagg tgtccattgc cctgagcagc 900cactggatca
accccagacg gatgaccgac cacagcatca aagagtgcca gaaaagcctg
960gacttcgtgc tgggatggtt cgccaagccc gtgttcatcg acggcgacta
ccccgagagc 1020atgaagaaca acctgtccag catcctgccc gacttcaccg
agagcgagaa gaagttcatc 1080aagggcaccg ccgatttctt cgccctgtgc
ttcggcccta ccctgagctt ccagctgctg 1140gacccccaca tgaagttcag
acagctggaa agccccaacc tgcggcagct gctgagctgg 1200atcgacctgg
aattcaacca cccccagatt ttcatcgtgg aaaacggctg gttcgtgtcc
1260ggcaccacca agagggacga cgccaagtac atgtattacc tgaaaaagtt
tatcatggaa 1320accctgaagg ccatcaagct ggacggcgtg gacgtgatcg
gctacacagc ctggtccctg 1380atggacggct tcgagtggca ccggggctac
tctatcagac ggggcctgtt ctacgtggac 1440ttcctgagcc aggacaagat
gctgctgcct aagagcagcg ccctgtttta ccagaagctg 1500atcgagaaga
acggcttccc acccctgccc gagaaccagc ctctggaagg caccttcccc
1560tgcgattttg cctggggcgt ggtggacaac tacatccagg tggacaccac
cctgtcccag 1620ttcaccgacc tgaacgtgta cctgtgggac gtgcaccaca
gcaagcggct gattaaggtg 1680gacggggtcg tgaccaagaa gcggaagtcc
tactgcgtgg actttgccgc catccagccc 1740cagattgccc tgctgcagga
aatgcacgtg acacacttcc ggttctccct ggactgggcc 1800ctgatcctgc
cactgggcaa tcagagccaa gtgaaccaca ccattctgca gtactacaga
1860tgcatggcct ccgagctggt gcgcgtgaac atcacacctg tggtggccct
gtggcagccc 1920atggccccta atcagggact gcctagactg ctggctagac
agggcgcctg ggagaaccct 1980tacaccgccc tggcctttgc cgagtacgcc
cggctgtgtt tccaggaact ggggcaccac 2040gtgaagctgt ggatcacaat
gaacgagccc tacacccgga acatgaccta cagcgccgga 2100cataacctgc
tgaaggccca cgccctggct tggcatgtgt acaacgagaa gttccggcac
2160gcccagaacg gcaagatcag tatcgccctg caggccgact ggatcgagcc
cgcctgtccc 2220ttcagccaga aagacaaaga ggtggccgag cgggtgctgg
aattcgacat tggatggctg 2280gccgagccca tcttcggcag cggcgattac
ccctgggtca tgcgggactg gctgaaccag 2340cggaacaact tcctgctgcc
ttactttacc gaggatgaga agaaactgat ccaggggacc 2400ttcgacttcc
tggccctgag ccactacacc acaatcctgg tggacagcga gaaagaggac
2460cccatcaagt acaacgacta cctggaagtg caggaaatga ccgacatcac
ctggctgaat 2520agcccctccc aggtggccgt ggtgccttgg ggactgagaa
aggtgctgaa ttggctgaag 2580tttaagtacg gcgacctgcc catgtacatc
atcagcaacg gcatcgacga tggcctgcac 2640gccgaggacg atcagctgcg
ggtgtactac atgcagaact acatcaacga ggccctgaaa 2700gcccacatcc
tggacggcat caacctgtgc ggctacttcg cctacagctt caacgaccgg
2760accgccccta gattcggcct gtacagatac gccgccgacc agttcgagcc
caaggccagc 2820atgaagcact accggaagat catcgacagc aatggcttcc
ctggccccga gacactggaa 2880cggttctgcc ccgaggaatt caccgtgtgt
accgagtgca gcttcttcca caccagaaag 2940tccctgctgg cttttatcgc
cttcctgttc ttcgccagca tcatctccct gtccctgatc 3000ttctactaca
gcaagaaggg cagacggtcc tacaagtacc cctacgacgt gcccgactac
3060gcctgatgat ga 307231650DNAHomo sapiens 3atgcccgcca gcgccccgcc
gcgccgcccg cggccgccgc cgcagtcgct gtcgctgctg 60ctggtgctgc tgggcctggg
cggccgccgc ctgcgtgcgg agccgggcga cggcgcgcag 120acctgggccc
gtttctcgcg gcctcctgcc cccgaggccg cgggcctctt ccagggcacc
180ttccccgacg gcttcctctg ggccgtgggc agcgccgcct accagaccga
gggcggctgg 240cagcagcacg gcaagggtgc gtccatctgg gacacgttca
cccaccaccc cctggcaccc 300ccgggagact cccggaacgc cagtctgccg
ttgggcgccc cgtcgccgct gcagcccgcc 360accggggacg tagccagcga
cagctacaac aacgtcttcc gcgacacgga ggcgctgcgc 420gagctcgggg
tcactcacta ccgcttctcc atctcgtggg cgcgagtgct ccccaatggc
480agcgcgggcg tccccaaccg cgaggggctg cgctactacc ggcgcctgct
ggagcggctg 540cgggagctgg gcgtgcagcc cgtggtcacc ctgtaccact
gggacctgcc ccagcgcctg 600caggacgcct acggcggctg ggccaaccgc
gccctggccg accacttcag ggattacgcg 660gagctctgct tccgccactt
cggcggtcag gtcaagtact ggatcaccat cgacaacccc 720tacgtggtgg
cctggcacgg ctacgccacc gggcgcctgg cccccggcat ccggggcagc
780ccgcggctcg ggtacctggt ggcgcacaac ctcctcctgg ctcatgccaa
agtctggcat 840ctctacaata cttctttccg tcccactcag ggaggtcagg
tgtccattgc cctaagctct 900cactggatca atcctcgaag aatgaccgac
cacagcatca aagaatgtca aaaatctctg 960gactttgtac taggttggtt
tgccaaaccc gtatttattg atggtgacta tcccgagagc 1020atgaagaata
acctttcatc tattctgcct gattttactg aatctgagaa aaagttcatc
1080aaaggaactg ctgacttttt tgctctttgc tttggaccca ccttgagttt
tcaacttttg 1140gaccctcaca tgaagttccg ccaattggaa tctcccaacc
tgaggcaact gctttcctgg 1200attgaccttg aatttaacca tcctcaaata
tttattgtgg aaaatggctg gtttgtctca 1260gggaccacca agagagatga
tgccaaatat atgtattacc tcaaaaagtt catcatggaa 1320accttaaaag
ccatcaagct ggatggggtg gatgtcatcg ggtataccgc atggtccctc
1380atggatggtt tcgagtggca cagaggttac agcatcaggc gtggactctt
ctatgttgac 1440tttctaagcc aggacaagat gttgttgcca aagtcttcag
ccttgttcta ccaaaagctg 1500atagagaaaa atggcttccc tcctttacct
gaaaatcagc ccctagaagg gacatttccc 1560tgtgactttg cttggggagt
tgttgacaac tacattcaag taagtcagct gacaaaacca 1620atcagcagtc
tcaccaagcc ctatcactag 165043135DNAHomo sapiens 4atgaagccag
gctgtgcggc aggatctcca gggaatgaat ggattttctt cagcactgat 60gaaataacca
cacgctatag gaatacaatg tccaacgggg gattgcaaag atctgtcatc
120ctgtcagcac ttattctgct acgagctgtt actggattct ctggagatgg
aagagctata 180tggtctaaaa atcctaattt tactccggta aatgaaagtc
agctgtttct ctatgacact 240ttccctaaaa actttttctg gggtattggg
actggagcat tgcaagtgga agggagttgg 300aagaaggatg gaaaaggacc
ttctatatgg gatcatttca tccacacaca ccttaaaaat 360gtcagcagca
cgaatggttc cagtgacagt tatatttttc tggaaaaaga cttatcagcc
420ctggatttta taggagtttc tttttatcaa ttttcaattt cctggccaag
gcttttcccc 480gatggaatag taacagttgc caacgcaaaa ggtctgcagt
actacagtac tcttctggac 540gctctagtgc ttagaaacat tgaacctata
gttactttat accactggga tttgcctttg 600gcactacaag aaaaatatgg
ggggtggaaa aatgatacca taatagatat cttcaatgac 660tatgccacat
actgtttcca gatgtttggg gaccgtgtca aatattggat tacaattcac
720aacccatatc tagtggcttg gcatgggtat gggacaggta tgcatgcccc
tggagagaag 780ggaaatttag cagctgtcta cactgtggga cacaacttga
tcaaggctca ctcgaaagtt 840tggcataact acaacacaca tttccgccca
catcagaagg gttggttatc gatcacgttg 900ggatctcatt ggatcgagcc
aaaccggtcg gaaaacacga tggatatatt caaatgtcaa 960caatccatgg
tttctgtgct tggatggttt gccaacccta tccatgggga tggcgactat
1020ccagagggga tgagaaagaa gttgttctcc gttctaccca ttttctctga
agcagagaag 1080catgagatga gaggcacagc tgatttcttt gccttttctt
ttggacccaa caacttcaag 1140cccctaaaca ccatggctaa aatgggacaa
aatgtttcac ttaatttaag agaagcgctg 1200aactggatta aactggaata
caacaaccct cgaatcttga ttgctgagaa tggctggttc 1260acagacagtc
gtgtgaaaac agaagacacc acggccatct acatgatgaa gaatttcctc
1320agccaggtgc ttcaagcaat aaggttagat gaaatacgag tgtttggtta
tactgcctgg 1380tctctcctgg atggctttga atggcaggat gcttacacca
tccgccgagg attattttat 1440gtggatttta acagtaaaca gaaagagcgg
aaacctaagt cttcagcaca ctactacaaa 1500cagatcatac gagaaaatgg
tttttcttta aaagagtcca cgccagatgt gcagggccag 1560tttccctgtg
acttctcctg gggtgtcact gaatctgttc ttaagcccga gtctgtggct
1620tcgtccccac agttcagcga tcctcatctg tacgtgtgga acgccactgg
caacagactg 1680ttgcaccgag tggaaggggt gaggctgaaa acacgacccg
ctcaatgcac agattttgta 1740aacatcaaaa aacaacttga gatgttggca
agaatgaaag tcacccacta ccggtttgct 1800ctggattggg cctcggtcct
tcccactggc aacctgtccg cggtgaaccg acaggccctg 1860aggtactaca
ggtgcgtggt cagtgagggg ctgaagcttg gcatctccgc gatggtcacc
1920ctgtattatc cgacccacgc ccacctaggc ctccccgagc ctctgttgca
tgccgacggg 1980tggctgaacc catcgacggc cgaggccttc caggcctacg
ctgggctgtg cttccaggag 2040ctgggggacc tggtgaagct ctggatcacc
atcaacgagc ctaaccggct aagtgacatc 2100tacaaccgct ctggcaacga
cacctacggg gcggcgcaca acctgctggt ggcccacgcc 2160ctggcctggc
gcctctacga ccggcagttc aggccctcac agcgcggggc cgtgtcgctg
2220tcgctgcacg cggactgggc ggaacccgcc aacccctatg ctgactcgca
ctggagggcg 2280gccgagcgct tcctgcagtt cgagatcgcc tggttcgccg
agccgctctt caagaccggg 2340gactaccccg cggccatgag ggaatacatt
gcctccaagc accgacgggg gctttccagc 2400tcggccctgc cgcgcctcac
cgaggccgaa aggaggctgc tcaagggcac ggtcgacttc 2460tgcgcgctca
accacttcac cactaggttc gtgatgcacg agcagctggc cggcagccgc
2520tacgactcgg acagggacat ccagtttctg caggacatca cccgcctgag
ctcccccacg 2580cgcctggctg tgattccctg gggggtgcgc aagctgctgc
ggtgggtccg gaggaactac 2640ggcgacatgg acatttacat caccgccagt
ggcatcgacg accaggctct ggaggatgac 2700cggctccgga agtactacct
agggaagtac cttcaggagg tgctgaaagc atacctgatt 2760gataaagtca
gaatcaaagg ctattatgca ttcaaactgg ctgaagagaa atctaaaccc
2820agatttggat tcttcacatc tgattttaaa gctaaatcct caatacaatt
ttacaacaaa 2880gtgatcagca gcaggggctt cccttttgag aacagtagtt
ctagatgcag tcagacccaa 2940gaaaatacag agtgcactgt ctgcttattc
cttgtgcaga agaaaccact gatattcctg 3000ggttgttgct tcttctccac
cctggttcta ctcttatcaa ttgccatttt tcaaaggcag 3060aagagaagaa
agttttggaa agcaaaaaac ttacaacaca taccattaaa gaaaggcaag
3120agagttgtta gctaa 313551704DNAHomo sapiens 5atgaagccag
tgtgggtcgc cacccttctg tggatgctac tgctggtgcc caggctgggg 60gccgcccgga
aggggtcccc agaagaggcc tccttctact atggaacctt ccctcttggc
120ttctcctggg gcgtgggcag ttctgcctac cagacggagg gcgcctggga
ccaggacggg 180aaagggccta gcatctggga cgtcttcaca cacagtggga
aggggaaagt gcttgggaat 240gagacggcag atgtagcctg tgacggctac
tacaaggtcc aggaggacat cattctgctg 300agggaactgc acgtcaacca
ctaccgattc tccctgtctt ggccccggct cctgcccaca 360ggcatccgag
ccgagcaggt gaacaagaag ggaatcgaat tctacagtga tcttatcgat
420gcccttctga gcagcaacat cactcccatc gtgaccttgc accactggga
tctgccacag 480ctgctccagg tcaaatacgg tgggtggcag aatgtgagca
tggccaacta cttcagagac 540tacgccaacc tgtgctttga ggcctttggg
gaccgtgtga agcactggat cacgttcagt 600gatcctcggg caatggcaga
aaaaggctat gagacgggcc accatgcgcc gggcctgaag 660ctccgcggca
ccggcctgta caaggcagca caccacatca ttaaggccca cgccaaagcc
720tggcattctt ataacaccac gtggcgcagc aagcagcaag gtctggtggg
aatttcattg 780aactgtgact ggggggaacc tgtggacatt agtaacccca
aggacctaga ggctgccgag 840agatacctac agttctgtct gggctggttt
gccaacccca tttatgccgg tgactacccc 900caagtcatga aggactacat
tggaagaaag agtgcagagc aaggcctgga gatgtcgagg 960ttaccggtgt
tctcactcca ggagaagagc tacattaaag gcacatccga tttcttggga
1020ttaggtcatt ttactactcg gtacatcacg gaaaggaact acccctcccg
ccaggggccc 1080agctaccaga acgatcgtga cttgatagag ctggttgacc
caaactggcc agatctgggg 1140tctaaatggc tatattctgt gccatgggga
tttaggaggc tccttaactt tgctcagact 1200caatacggtg atcctcccat
atatgtgatg gaaaatggag catctcaaaa attccactgt 1260actcaattat
gtgatgagtg gagaattcaa taccttaaag gatacataaa tgaaatgcta
1320aaagctataa aagatggtgc taatataaag gggtatactt cctggtctct
gttggataag 1380tttgaatggg agaaaggata ctcagataga tatggattct
actatgttga atttaacgac 1440agaaataagc ctcgctatcc aaaggcttca
gttcaatatt acaagaagat tatcattgcc 1500aatgggtttc ccaatccaag
agaggtggaa agttggtacc tcaaagcttt ggaaacttgc 1560tctatcaaca
atcagatgct tgctgcagag cccttgctaa gtcacatgca aatggttacg
1620gagatcgtgg tacccactgt ctgctccctc tgtgtcctca tcactgctgt
tctactaatg 1680ctcctcctga ggaggcagag ctga 170461012PRTHomo sapiens
6Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro Pro Ser 1
5 10 15 Leu Ser Leu Leu Leu Val Leu Leu Gly Leu Gly Gly Arg Arg Leu
Arg 20 25 30 Ala Glu Pro Gly Asp Gly Ala Gln Thr Trp Ala Arg Phe
Ser Arg Pro 35 40 45 Pro Ala Pro Glu Ala Ala Gly Leu Phe Gln Gly
Thr Phe Pro Asp Gly 50 55 60 Phe Leu Trp Ala Val Gly Ser Ala Ala
Tyr Gln Thr Glu Gly Gly Trp 65 70 75 80 Gln Gln His Gly Lys Gly Ala
Ser Ile Trp Asp Thr Phe Thr His His 85 90 95 Pro Leu Ala Pro Pro
Gly Asp Ser Arg Asn Ala Ser Leu Pro Leu Gly 100 105 110 Ala Pro Ser
Pro Leu Gln Pro Ala Thr Gly Asp Val Ala Ser Asp Ser 115 120 125 Tyr
Asn Asn Val Phe Arg Asp Thr Glu Ala Leu Arg Glu Leu Gly Val 130 135
140 Thr His Tyr Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro Asn Gly
145 150 155 160 Ser Ala Gly Val Pro Asn Arg Glu Gly Leu Arg Tyr Tyr
Arg Arg Leu 165 170 175 Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Pro
Val Val Thr Leu Tyr 180 185 190 His Trp Asp Leu Pro Gln Arg Leu Gln
Asp Ala Tyr Gly Gly Trp Ala 195 200 205 Asn Arg Ala Leu Ala Asp His
Phe Arg Asp Tyr Ala Glu Leu Cys Phe 210 215 220 Arg His Phe Gly Gly
Gln Val Lys Tyr Trp Ile Thr Ile Asp Asn Pro 225 230 235 240 Tyr Val
Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu Ala Pro Gly 245 250 255
Ile Arg Gly Ser Pro Arg Leu Gly Tyr Leu Val Ala His Asn Leu Leu 260
265 270 Leu Ala His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe Arg
Pro 275 280 285 Thr Gln Gly Gly Gln Val Ser Ile Ala Leu Ser Ser His
Trp Ile Asn 290 295 300 Pro Arg Arg Met Thr Asp His Ser Ile Lys Glu
Cys Gln Lys Ser Leu 305 310 315 320 Asp Phe Val Leu Gly Trp Phe Ala
Lys Pro Val Phe Ile Asp Gly Asp 325
330 335 Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Ile Leu Pro Asp
Phe 340 345 350 Thr Glu Ser Glu Lys Lys Phe Ile Lys Gly Thr Ala Asp
Phe Phe Ala 355 360 365 Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu
Leu Asp Pro His Met 370 375 380 Lys Phe Arg Gln Leu Glu Ser Pro Asn
Leu Arg Gln Leu Leu Ser Trp 385 390 395 400 Ile Asp Leu Glu Phe Asn
His Pro Gln Ile Phe Ile Val Glu Asn Gly 405 410 415 Trp Phe Val Ser
Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr Met Tyr 420 425 430 Tyr Leu
Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Lys Leu Asp 435 440 445
Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp Gly Phe 450
455 460 Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr Val
Asp 465 470 475 480 Phe Leu Ser Gln Asp Lys Met Leu Leu Pro Lys Ser
Ser Ala Leu Phe 485 490 495 Tyr Gln Lys Leu Ile Glu Lys Asn Gly Phe
Pro Pro Leu Pro Glu Asn 500 505 510 Gln Pro Leu Glu Gly Thr Phe Pro
Cys Asp Phe Ala Trp Gly Val Val 515 520 525 Asp Asn Tyr Ile Gln Val
Asp Thr Thr Leu Ser Gln Phe Thr Asp Leu 530 535 540 Asn Val Tyr Leu
Trp Asp Val His His Ser Lys Arg Leu Ile Lys Val 545 550 555 560 Asp
Gly Val Val Thr Lys Lys Arg Lys Ser Tyr Cys Val Asp Phe Ala 565 570
575 Ala Ile Gln Pro Gln Ile Ala Leu Leu Gln Glu Met His Val Thr His
580 585 590 Phe Arg Phe Ser Leu Asp Trp Ala Leu Ile Leu Pro Leu Gly
Asn Gln 595 600 605 Ser Gln Val Asn His Thr Ile Leu Gln Tyr Tyr Arg
Cys Met Ala Ser 610 615 620 Glu Leu Val Arg Val Asn Ile Thr Pro Val
Val Ala Leu Trp Gln Pro 625 630 635 640 Met Ala Pro Asn Gln Gly Leu
Pro Arg Leu Leu Ala Arg Gln Gly Ala 645 650 655 Trp Glu Asn Pro Tyr
Thr Ala Leu Ala Phe Ala Glu Tyr Ala Arg Leu 660 665 670 Cys Phe Gln
Glu Leu Gly His His Val Lys Leu Trp Ile Thr Met Asn 675 680 685 Glu
Pro Tyr Thr Arg Asn Met Thr Tyr Ser Ala Gly His Asn Leu Leu 690 695
700 Lys Ala His Ala Leu Ala Trp His Val Tyr Asn Glu Lys Phe Arg His
705 710 715 720 Ala Gln Asn Gly Lys Ile Ser Ile Ala Leu Gln Ala Asp
Trp Ile Glu 725 730 735 Pro Ala Cys Pro Phe Ser Gln Lys Asp Lys Glu
Val Ala Glu Arg Val 740 745 750 Leu Glu Phe Asp Ile Gly Trp Leu Ala
Glu Pro Ile Phe Gly Ser Gly 755 760 765 Asp Tyr Pro Trp Val Met Arg
Asp Trp Leu Asn Gln Arg Asn Asn Phe 770 775 780 Leu Leu Pro Tyr Phe
Thr Glu Asp Glu Lys Lys Leu Ile Gln Gly Thr 785 790 795 800 Phe Asp
Phe Leu Ala Leu Ser His Tyr Thr Thr Ile Leu Val Asp Ser 805 810 815
Glu Lys Glu Asp Pro Ile Lys Tyr Asn Asp Tyr Leu Glu Val Gln Glu 820
825 830 Met Thr Asp Ile Thr Trp Leu Asn Ser Pro Ser Gln Val Ala Val
Val 835 840 845 Pro Trp Gly Leu Arg Lys Val Leu Asn Trp Leu Lys Phe
Lys Tyr Gly 850 855 860 Asp Leu Pro Met Tyr Ile Ile Ser Asn Gly Ile
Asp Asp Gly Leu His 865 870 875 880 Ala Glu Asp Asp Gln Leu Arg Val
Tyr Tyr Met Gln Asn Tyr Ile Asn 885 890 895 Glu Ala Leu Lys Ala His
Ile Leu Asp Gly Ile Asn Leu Cys Gly Tyr 900 905 910 Phe Ala Tyr Ser
Phe Asn Asp Arg Thr Ala Pro Arg Phe Gly Leu Tyr 915 920 925 Arg Tyr
Ala Ala Asp Gln Phe Glu Pro Lys Ala Ser Met Lys His Tyr 930 935 940
Arg Lys Ile Ile Asp Ser Asn Gly Phe Pro Gly Pro Glu Thr Leu Glu 945
950 955 960 Arg Phe Cys Pro Glu Glu Phe Thr Val Cys Thr Glu Cys Ser
Phe Phe 965 970 975 His Thr Arg Lys Ser Leu Leu Ala Phe Ile Ala Phe
Leu Phe Phe Ala 980 985 990 Ser Ile Ile Ser Leu Ser Leu Ile Phe Tyr
Tyr Ser Lys Lys Gly Arg 995 1000 1005 Arg Ser Tyr Lys 1010
71021PRTHomo sapiens 7Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg
Pro Pro Pro Pro Ser 1 5 10 15 Leu Ser Leu Leu Leu Val Leu Leu Gly
Leu Gly Gly Arg Arg Leu Arg 20 25 30 Ala Glu Pro Gly Asp Gly Ala
Gln Thr Trp Ala Arg Phe Ser Arg Pro 35 40 45 Pro Ala Pro Glu Ala
Ala Gly Leu Phe Gln Gly Thr Phe Pro Asp Gly 50 55 60 Phe Leu Trp
Ala Val Gly Ser Ala Ala Tyr Gln Thr Glu Gly Gly Trp 65 70 75 80 Gln
Gln His Gly Lys Gly Ala Ser Ile Trp Asp Thr Phe Thr His His 85 90
95 Pro Leu Ala Pro Pro Gly Asp Ser Arg Asn Ala Ser Leu Pro Leu Gly
100 105 110 Ala Pro Ser Pro Leu Gln Pro Ala Thr Gly Asp Val Ala Ser
Asp Ser 115 120 125 Tyr Asn Asn Val Phe Arg Asp Thr Glu Ala Leu Arg
Glu Leu Gly Val 130 135 140 Thr His Tyr Arg Phe Ser Ile Ser Trp Ala
Arg Val Leu Pro Asn Gly 145 150 155 160 Ser Ala Gly Val Pro Asn Arg
Glu Gly Leu Arg Tyr Tyr Arg Arg Leu 165 170 175 Leu Glu Arg Leu Arg
Glu Leu Gly Val Gln Pro Val Val Thr Leu Tyr 180 185 190 His Trp Asp
Leu Pro Gln Arg Leu Gln Asp Ala Tyr Gly Gly Trp Ala 195 200 205 Asn
Arg Ala Leu Ala Asp His Phe Arg Asp Tyr Ala Glu Leu Cys Phe 210 215
220 Arg His Phe Gly Gly Gln Val Lys Tyr Trp Ile Thr Ile Asp Asn Pro
225 230 235 240 Tyr Val Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu
Ala Pro Gly 245 250 255 Ile Arg Gly Ser Pro Arg Leu Gly Tyr Leu Val
Ala His Asn Leu Leu 260 265 270 Leu Ala His Ala Lys Val Trp His Leu
Tyr Asn Thr Ser Phe Arg Pro 275 280 285 Thr Gln Gly Gly Gln Val Ser
Ile Ala Leu Ser Ser His Trp Ile Asn 290 295 300 Pro Arg Arg Met Thr
Asp His Ser Ile Lys Glu Cys Gln Lys Ser Leu 305 310 315 320 Asp Phe
Val Leu Gly Trp Phe Ala Lys Pro Val Phe Ile Asp Gly Asp 325 330 335
Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Ile Leu Pro Asp Phe 340
345 350 Thr Glu Ser Glu Lys Lys Phe Ile Lys Gly Thr Ala Asp Phe Phe
Ala 355 360 365 Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu Leu Asp
Pro His Met 370 375 380 Lys Phe Arg Gln Leu Glu Ser Pro Asn Leu Arg
Gln Leu Leu Ser Trp 385 390 395 400 Ile Asp Leu Glu Phe Asn His Pro
Gln Ile Phe Ile Val Glu Asn Gly 405 410 415 Trp Phe Val Ser Gly Thr
Thr Lys Arg Asp Asp Ala Lys Tyr Met Tyr 420 425 430 Tyr Leu Lys Lys
Phe Ile Met Glu Thr Leu Lys Ala Ile Lys Leu Asp 435 440 445 Gly Val
Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp Gly Phe 450 455 460
Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr Val Asp 465
470 475 480 Phe Leu Ser Gln Asp Lys Met Leu Leu Pro Lys Ser Ser Ala
Leu Phe 485 490 495 Tyr Gln Lys Leu Ile Glu Lys Asn Gly Phe Pro Pro
Leu Pro Glu Asn 500 505 510 Gln Pro Leu Glu Gly Thr Phe Pro Cys Asp
Phe Ala Trp Gly Val Val 515 520 525 Asp Asn Tyr Ile Gln Val Asp Thr
Thr Leu Ser Gln Phe Thr Asp Leu 530 535 540 Asn Val Tyr Leu Trp Asp
Val His His Ser Lys Arg Leu Ile Lys Val 545 550 555 560 Asp Gly Val
Val Thr Lys Lys Arg Lys Ser Tyr Cys Val Asp Phe Ala 565 570 575 Ala
Ile Gln Pro Gln Ile Ala Leu Leu Gln Glu Met His Val Thr His 580 585
590 Phe Arg Phe Ser Leu Asp Trp Ala Leu Ile Leu Pro Leu Gly Asn Gln
595 600 605 Ser Gln Val Asn His Thr Ile Leu Gln Tyr Tyr Arg Cys Met
Ala Ser 610 615 620 Glu Leu Val Arg Val Asn Ile Thr Pro Val Val Ala
Leu Trp Gln Pro 625 630 635 640 Met Ala Pro Asn Gln Gly Leu Pro Arg
Leu Leu Ala Arg Gln Gly Ala 645 650 655 Trp Glu Asn Pro Tyr Thr Ala
Leu Ala Phe Ala Glu Tyr Ala Arg Leu 660 665 670 Cys Phe Gln Glu Leu
Gly His His Val Lys Leu Trp Ile Thr Met Asn 675 680 685 Glu Pro Tyr
Thr Arg Asn Met Thr Tyr Ser Ala Gly His Asn Leu Leu 690 695 700 Lys
Ala His Ala Leu Ala Trp His Val Tyr Asn Glu Lys Phe Arg His 705 710
715 720 Ala Gln Asn Gly Lys Ile Ser Ile Ala Leu Gln Ala Asp Trp Ile
Glu 725 730 735 Pro Ala Cys Pro Phe Ser Gln Lys Asp Lys Glu Val Ala
Glu Arg Val 740 745 750 Leu Glu Phe Asp Ile Gly Trp Leu Ala Glu Pro
Ile Phe Gly Ser Gly 755 760 765 Asp Tyr Pro Trp Val Met Arg Asp Trp
Leu Asn Gln Arg Asn Asn Phe 770 775 780 Leu Leu Pro Tyr Phe Thr Glu
Asp Glu Lys Lys Leu Ile Gln Gly Thr 785 790 795 800 Phe Asp Phe Leu
Ala Leu Ser His Tyr Thr Thr Ile Leu Val Asp Ser 805 810 815 Glu Lys
Glu Asp Pro Ile Lys Tyr Asn Asp Tyr Leu Glu Val Gln Glu 820 825 830
Met Thr Asp Ile Thr Trp Leu Asn Ser Pro Ser Gln Val Ala Val Val 835
840 845 Pro Trp Gly Leu Arg Lys Val Leu Asn Trp Leu Lys Phe Lys Tyr
Gly 850 855 860 Asp Leu Pro Met Tyr Ile Ile Ser Asn Gly Ile Asp Asp
Gly Leu His 865 870 875 880 Ala Glu Asp Asp Gln Leu Arg Val Tyr Tyr
Met Gln Asn Tyr Ile Asn 885 890 895 Glu Ala Leu Lys Ala His Ile Leu
Asp Gly Ile Asn Leu Cys Gly Tyr 900 905 910 Phe Ala Tyr Ser Phe Asn
Asp Arg Thr Ala Pro Arg Phe Gly Leu Tyr 915 920 925 Arg Tyr Ala Ala
Asp Gln Phe Glu Pro Lys Ala Ser Met Lys His Tyr 930 935 940 Arg Lys
Ile Ile Asp Ser Asn Gly Phe Pro Gly Pro Glu Thr Leu Glu 945 950 955
960 Arg Phe Cys Pro Glu Glu Phe Thr Val Cys Thr Glu Cys Ser Phe Phe
965 970 975 His Thr Arg Lys Ser Leu Leu Ala Phe Ile Ala Phe Leu Phe
Phe Ala 980 985 990 Ser Ile Ile Ser Leu Ser Leu Ile Phe Tyr Tyr Ser
Lys Lys Gly Arg 995 1000 1005 Arg Ser Tyr Lys Tyr Pro Tyr Asp Val
Pro Asp Tyr Ala 1010 1015 1020 8549PRTHomo sapiens 8Met Pro Ala Ser
Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro Gln Ser 1 5 10 15 Leu Ser
Leu Leu Leu Val Leu Leu Gly Leu Gly Gly Arg Arg Leu Arg 20 25 30
Ala Glu Pro Gly Asp Gly Ala Gln Thr Trp Ala Arg Phe Ser Arg Pro 35
40 45 Pro Ala Pro Glu Ala Ala Gly Leu Phe Gln Gly Thr Phe Pro Asp
Gly 50 55 60 Phe Leu Trp Ala Val Gly Ser Ala Ala Tyr Gln Thr Glu
Gly Gly Trp 65 70 75 80 Gln Gln His Gly Lys Gly Ala Ser Ile Trp Asp
Thr Phe Thr His His 85 90 95 Pro Leu Ala Pro Pro Gly Asp Ser Arg
Asn Ala Ser Leu Pro Leu Gly 100 105 110 Ala Pro Ser Pro Leu Gln Pro
Ala Thr Gly Asp Val Ala Ser Asp Ser 115 120 125 Tyr Asn Asn Val Phe
Arg Asp Thr Glu Ala Leu Arg Glu Leu Gly Val 130 135 140 Thr His Tyr
Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro Asn Gly 145 150 155 160
Ser Ala Gly Val Pro Asn Arg Glu Gly Leu Arg Tyr Tyr Arg Arg Leu 165
170 175 Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Pro Val Val Thr Leu
Tyr 180 185 190 His Trp Asp Leu Pro Gln Arg Leu Gln Asp Ala Tyr Gly
Gly Trp Ala 195 200 205 Asn Arg Ala Leu Ala Asp His Phe Arg Asp Tyr
Ala Glu Leu Cys Phe 210 215 220 Arg His Phe Gly Gly Gln Val Lys Tyr
Trp Ile Thr Ile Asp Asn Pro 225 230 235 240 Tyr Val Val Ala Trp His
Gly Tyr Ala Thr Gly Arg Leu Ala Pro Gly 245 250 255 Ile Arg Gly Ser
Pro Arg Leu Gly Tyr Leu Val Ala His Asn Leu Leu 260 265 270 Leu Ala
His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe Arg Pro 275 280 285
Thr Gln Gly Gly Gln Val Ser Ile Ala Leu Ser Ser His Trp Ile Asn 290
295 300 Pro Arg Arg Met Thr Asp His Ser Ile Lys Glu Cys Gln Lys Ser
Leu 305 310 315 320 Asp Phe Val Leu Gly Trp Phe Ala Lys Pro Val Phe
Ile Asp Gly Asp 325 330 335 Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser
Ser Ile Leu Pro Asp Phe 340 345 350 Thr Glu Ser Glu Lys Lys Phe Ile
Lys Gly Thr Ala Asp Phe Phe Ala 355 360 365 Leu Cys Phe Gly Pro Thr
Leu Ser Phe Gln Leu Leu Asp Pro His Met 370 375 380 Lys Phe Arg Gln
Leu Glu Ser Pro Asn Leu Arg Gln Leu Leu Ser Trp 385 390 395 400 Ile
Asp Leu Glu Phe Asn His Pro Gln Ile Phe Ile Val Glu Asn Gly 405 410
415 Trp Phe Val Ser Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr Met Tyr
420 425 430 Tyr Leu Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Lys
Leu Asp 435 440 445 Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu
Met Asp Gly Phe 450 455 460 Glu Trp His Arg Gly Tyr Ser Ile Arg Arg
Gly Leu Phe Tyr Val Asp 465 470 475 480 Phe Leu Ser Gln Asp Lys Met
Leu Leu Pro Lys Ser Ser Ala Leu Phe 485 490 495 Tyr Gln Lys Leu Ile
Glu Lys Asn Gly Phe Pro Pro Leu Pro Glu Asn 500 505 510 Gln Pro Leu
Glu Gly Thr Phe Pro Cys Asp Phe Ala Trp Gly Val Val 515 520 525 Asp
Asn Tyr Ile Gln Val Ser Gln Leu Thr Lys Pro Ile Ser Ser Leu 530 535
540 Thr Lys Pro Tyr His 545 91044PRTHomo sapiens 9Met Lys Pro Gly
Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe 1 5 10 15 Phe Ser
Thr Asp Glu Ile Thr Thr Arg Tyr Arg Asn Thr Met Ser Asn 20 25 30
Gly Gly Leu Gln Arg Ser Val Ile Leu Ser Ala Leu Ile Leu Leu Arg
35
40 45 Ala Val Thr Gly Phe Ser Gly Asp Gly Arg Ala Ile Trp Ser Lys
Asn 50 55 60 Pro Asn Phe Thr Pro Val Asn Glu Ser Gln Leu Phe Leu
Tyr Asp Thr 65 70 75 80 Phe Pro Lys Asn Phe Phe Trp Gly Ile Gly Thr
Gly Ala Leu Gln Val 85 90 95 Glu Gly Ser Trp Lys Lys Asp Gly Lys
Gly Pro Ser Ile Trp Asp His 100 105 110 Phe Ile His Thr His Leu Lys
Asn Val Ser Ser Thr Asn Gly Ser Ser 115 120 125 Asp Ser Tyr Ile Phe
Leu Glu Lys Asp Leu Ser Ala Leu Asp Phe Ile 130 135 140 Gly Val Ser
Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro 145 150 155 160
Asp Gly Ile Val Thr Val Ala Asn Ala Lys Gly Leu Gln Tyr Tyr Ser 165
170 175 Thr Leu Leu Asp Ala Leu Val Leu Arg Asn Ile Glu Pro Ile Val
Thr 180 185 190 Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Gln Glu Lys
Tyr Gly Gly 195 200 205 Trp Lys Asn Asp Thr Ile Ile Asp Ile Phe Asn
Asp Tyr Ala Thr Tyr 210 215 220 Cys Phe Gln Met Phe Gly Asp Arg Val
Lys Tyr Trp Ile Thr Ile His 225 230 235 240 Asn Pro Tyr Leu Val Ala
Trp His Gly Tyr Gly Thr Gly Met His Ala 245 250 255 Pro Gly Glu Lys
Gly Asn Leu Ala Ala Val Tyr Thr Val Gly His Asn 260 265 270 Leu Ile
Lys Ala His Ser Lys Val Trp His Asn Tyr Asn Thr His Phe 275 280 285
Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp 290
295 300 Ile Glu Pro Asn Arg Ser Glu Asn Thr Met Asp Ile Phe Lys Cys
Gln 305 310 315 320 Gln Ser Met Val Ser Val Leu Gly Trp Phe Ala Asn
Pro Ile His Gly 325 330 335 Asp Gly Asp Tyr Pro Glu Gly Met Arg Lys
Lys Leu Phe Ser Val Leu 340 345 350 Pro Ile Phe Ser Glu Ala Glu Lys
His Glu Met Arg Gly Thr Ala Asp 355 360 365 Phe Phe Ala Phe Ser Phe
Gly Pro Asn Asn Phe Lys Pro Leu Asn Thr 370 375 380 Met Ala Lys Met
Gly Gln Asn Val Ser Leu Asn Leu Arg Glu Ala Leu 385 390 395 400 Asn
Trp Ile Lys Leu Glu Tyr Asn Asn Pro Arg Ile Leu Ile Ala Glu 405 410
415 Asn Gly Trp Phe Thr Asp Ser Arg Val Lys Thr Glu Asp Thr Thr Ala
420 425 430 Ile Tyr Met Met Lys Asn Phe Leu Ser Gln Val Leu Gln Ala
Ile Arg 435 440 445 Leu Asp Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp
Ser Leu Leu Asp 450 455 460 Gly Phe Glu Trp Gln Asp Ala Tyr Thr Ile
Arg Arg Gly Leu Phe Tyr 465 470 475 480 Val Asp Phe Asn Ser Lys Gln
Lys Glu Arg Lys Pro Lys Ser Ser Ala 485 490 495 His Tyr Tyr Lys Gln
Ile Ile Arg Glu Asn Gly Phe Ser Leu Lys Glu 500 505 510 Ser Thr Pro
Asp Val Gln Gly Gln Phe Pro Cys Asp Phe Ser Trp Gly 515 520 525 Val
Thr Glu Ser Val Leu Lys Pro Glu Ser Val Ala Ser Ser Pro Gln 530 535
540 Phe Ser Asp Pro His Leu Tyr Val Trp Asn Ala Thr Gly Asn Arg Leu
545 550 555 560 Leu His Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro
Ala Gln Cys 565 570 575 Thr Asp Phe Val Asn Ile Lys Lys Gln Leu Glu
Met Leu Ala Arg Met 580 585 590 Lys Val Thr His Tyr Arg Phe Ala Leu
Asp Trp Ala Ser Val Leu Pro 595 600 605 Thr Gly Asn Leu Ser Ala Val
Asn Arg Gln Ala Leu Arg Tyr Tyr Arg 610 615 620 Cys Val Val Ser Glu
Gly Leu Lys Leu Gly Ile Ser Ala Met Val Thr 625 630 635 640 Leu Tyr
Tyr Pro Thr His Ala His Leu Gly Leu Pro Glu Pro Leu Leu 645 650 655
His Ala Asp Gly Trp Leu Asn Pro Ser Thr Ala Glu Ala Phe Gln Ala 660
665 670 Tyr Ala Gly Leu Cys Phe Gln Glu Leu Gly Asp Leu Val Lys Leu
Trp 675 680 685 Ile Thr Ile Asn Glu Pro Asn Arg Leu Ser Asp Ile Tyr
Asn Arg Ser 690 695 700 Gly Asn Asp Thr Tyr Gly Ala Ala His Asn Leu
Leu Val Ala His Ala 705 710 715 720 Leu Ala Trp Arg Leu Tyr Asp Arg
Gln Phe Arg Pro Ser Gln Arg Gly 725 730 735 Ala Val Ser Leu Ser Leu
His Ala Asp Trp Ala Glu Pro Ala Asn Pro 740 745 750 Tyr Ala Asp Ser
His Trp Arg Ala Ala Glu Arg Phe Leu Gln Phe Glu 755 760 765 Ile Ala
Trp Phe Ala Glu Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ala 770 775 780
Ala Met Arg Glu Tyr Ile Ala Ser Lys His Arg Arg Gly Leu Ser Ser 785
790 795 800 Ser Ala Leu Pro Arg Leu Thr Glu Ala Glu Arg Arg Leu Leu
Lys Gly 805 810 815 Thr Val Asp Phe Cys Ala Leu Asn His Phe Thr Thr
Arg Phe Val Met 820 825 830 His Glu Gln Leu Ala Gly Ser Arg Tyr Asp
Ser Asp Arg Asp Ile Gln 835 840 845 Phe Leu Gln Asp Ile Thr Arg Leu
Ser Ser Pro Thr Arg Leu Ala Val 850 855 860 Ile Pro Trp Gly Val Arg
Lys Leu Leu Arg Trp Val Arg Arg Asn Tyr 865 870 875 880 Gly Asp Met
Asp Ile Tyr Ile Thr Ala Ser Gly Ile Asp Asp Gln Ala 885 890 895 Leu
Glu Asp Asp Arg Leu Arg Lys Tyr Tyr Leu Gly Lys Tyr Leu Gln 900 905
910 Glu Val Leu Lys Ala Tyr Leu Ile Asp Lys Val Arg Ile Lys Gly Tyr
915 920 925 Tyr Ala Phe Lys Leu Ala Glu Glu Lys Ser Lys Pro Arg Phe
Gly Phe 930 935 940 Phe Thr Ser Asp Phe Lys Ala Lys Ser Ser Ile Gln
Phe Tyr Asn Lys 945 950 955 960 Val Ile Ser Ser Arg Gly Phe Pro Phe
Glu Asn Ser Ser Ser Arg Cys 965 970 975 Ser Gln Thr Gln Glu Asn Thr
Glu Cys Thr Val Cys Leu Phe Leu Val 980 985 990 Gln Lys Lys Pro Leu
Ile Phe Leu Gly Cys Cys Phe Phe Ser Thr Leu 995 1000 1005 Val Leu
Leu Leu Ser Ile Ala Ile Phe Gln Arg Gln Lys Arg Arg 1010 1015 1020
Lys Phe Trp Lys Ala Lys Asn Leu Gln His Ile Pro Leu Lys Lys 1025
1030 1035 Gly Lys Arg Val Val Ser 1040 10567PRTHomo sapiens 10Met
Lys Pro Val Trp Val Ala Thr Leu Leu Trp Met Leu Leu Leu Val 1 5 10
15 Pro Arg Leu Gly Ala Ala Arg Lys Gly Ser Pro Glu Glu Ala Ser Phe
20 25 30 Tyr Tyr Gly Thr Phe Pro Leu Gly Phe Ser Trp Gly Val Gly
Ser Ser 35 40 45 Ala Tyr Gln Thr Glu Gly Ala Trp Asp Gln Asp Gly
Lys Gly Pro Ser 50 55 60 Ile Trp Asp Val Phe Thr His Ser Gly Lys
Gly Lys Val Leu Gly Asn 65 70 75 80 Glu Thr Ala Asp Val Ala Cys Asp
Gly Tyr Tyr Lys Val Gln Glu Asp 85 90 95 Ile Ile Leu Leu Arg Glu
Leu His Val Asn His Tyr Arg Phe Ser Leu 100 105 110 Ser Trp Pro Arg
Leu Leu Pro Thr Gly Ile Arg Ala Glu Gln Val Asn 115 120 125 Lys Lys
Gly Ile Glu Phe Tyr Ser Asp Leu Ile Asp Ala Leu Leu Ser 130 135 140
Ser Asn Ile Thr Pro Ile Val Thr Leu His His Trp Asp Leu Pro Gln 145
150 155 160 Leu Leu Gln Val Lys Tyr Gly Gly Trp Gln Asn Val Ser Met
Ala Asn 165 170 175 Tyr Phe Arg Asp Tyr Ala Asn Leu Cys Phe Glu Ala
Phe Gly Asp Arg 180 185 190 Val Lys His Trp Ile Thr Phe Ser Asp Pro
Arg Ala Met Ala Glu Lys 195 200 205 Gly Tyr Glu Thr Gly His His Ala
Pro Gly Leu Lys Leu Arg Gly Thr 210 215 220 Gly Leu Tyr Lys Ala Ala
His His Ile Ile Lys Ala His Ala Lys Ala 225 230 235 240 Trp His Ser
Tyr Asn Thr Thr Trp Arg Ser Lys Gln Gln Gly Leu Val 245 250 255 Gly
Ile Ser Leu Asn Cys Asp Trp Gly Glu Pro Val Asp Ile Ser Asn 260 265
270 Pro Lys Asp Leu Glu Ala Ala Glu Arg Tyr Leu Gln Phe Cys Leu Gly
275 280 285 Trp Phe Ala Asn Pro Ile Tyr Ala Gly Asp Tyr Pro Gln Val
Met Lys 290 295 300 Asp Tyr Ile Gly Arg Lys Ser Ala Glu Gln Gly Leu
Glu Met Ser Arg 305 310 315 320 Leu Pro Val Phe Ser Leu Gln Glu Lys
Ser Tyr Ile Lys Gly Thr Ser 325 330 335 Asp Phe Leu Gly Leu Gly His
Phe Thr Thr Arg Tyr Ile Thr Glu Arg 340 345 350 Asn Tyr Pro Ser Arg
Gln Gly Pro Ser Tyr Gln Asn Asp Arg Asp Leu 355 360 365 Ile Glu Leu
Val Asp Pro Asn Trp Pro Asp Leu Gly Ser Lys Trp Leu 370 375 380 Tyr
Ser Val Pro Trp Gly Phe Arg Arg Leu Leu Asn Phe Ala Gln Thr 385 390
395 400 Gln Tyr Gly Asp Pro Pro Ile Tyr Val Met Glu Asn Gly Ala Ser
Gln 405 410 415 Lys Phe His Cys Thr Gln Leu Cys Asp Glu Trp Arg Ile
Gln Tyr Leu 420 425 430 Lys Gly Tyr Ile Asn Glu Met Leu Lys Ala Ile
Lys Asp Gly Ala Asn 435 440 445 Ile Lys Gly Tyr Thr Ser Trp Ser Leu
Leu Asp Lys Phe Glu Trp Glu 450 455 460 Lys Gly Tyr Ser Asp Arg Tyr
Gly Phe Tyr Tyr Val Glu Phe Asn Asp 465 470 475 480 Arg Asn Lys Pro
Arg Tyr Pro Lys Ala Ser Val Gln Tyr Tyr Lys Lys 485 490 495 Ile Ile
Ile Ala Asn Gly Phe Pro Asn Pro Arg Glu Val Glu Ser Trp 500 505 510
Tyr Leu Lys Ala Leu Glu Thr Cys Ser Ile Asn Asn Gln Met Leu Ala 515
520 525 Ala Glu Pro Leu Leu Ser His Met Gln Met Val Thr Glu Ile Val
Val 530 535 540 Pro Thr Val Cys Ser Leu Cys Val Leu Ile Thr Ala Val
Leu Leu Met 545 550 555 560 Leu Leu Leu Arg Arg Gln Ser 565
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