GENETICALLY MODIFIED MESENCHYMAL STEM CELL EXPRESSING KLOTHO
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.
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 INVENTIONMesenchymal 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).
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.
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 β-glucuronidase (EC number 3.2.1.31) capable of hydrolyzing steroid β-glucuronides.
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).
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).
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.
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.
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).
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).
Background on Chronic Kidney Disease (CKD):
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).
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).
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.
Background on Cardiovascular Disease:
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 H2O2 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).
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 (Martín-Núñez, M. (2014) World J Cardiol. 6(12): 1262-1269).
Background on Morbus Alzheimer (AD):
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 6th 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 (Aβ 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-α. 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).
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).
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).
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).
Background on Multiple Sclerosis (MS):
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.
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).
Background on Amyotrophic Lateral Sclerosis (ALS):
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.
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).
Background on Parkinson Disease (PD):
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.
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).
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 INVENTIONIn 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.
This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.
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.
One preferred amino acid sequence of Klotho is available under accession number BAA23382 from the NCBI database. Said amino acid sequence corresponds to the α-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.
Moreover the invention also relates to further isoforms of the Klotho gene and encompasses β-Klotho and γ-Klotho.
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.
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.
SEQ ID NO 4 relates to a sequence of the human β-Klotho gene (cDNA) with the naturally occurring sequence.
SEQ ID NO 5 relates to a sequence of the human γ-Klotho gene (cDNA) with the naturally occurring sequence
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.
The invention therefore encompasses a modified MSC as described herein comprising a nucleic acid molecule selected from the group consisting of:
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;
b) a nucleic acid molecule which is complementary to a nucleotide sequence in accordance with a);
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%;
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
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).
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 β-glucuronidase activity, or via is functional effects described in the examples below. Appropriate assays for determining β-glucuronidase activity, or for assaying the desired biological functional effects described herein, and as shown in the examples, are known to a skilled person.
To measure the β-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 μg of purified secreted Klotho-protein. The reaction is carried out in a final volume of 100 μl. 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.
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:
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.
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.
In one embodiment the genetically modified cell as described herein are characterised in that said cell are CD34 negative.
In one embodiment the genetically modified cell as described herein are characterised in that said cell are human cell.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In a preferred embodiment the genetically modified mesenchymal stem cell as described herein is characterised in that the constitutive promoter is the EFS promoter.
In a preferred embodiment the genetically modified mesenchymal stem cell as described herein is characterised in that the constitutive promoter is the PGK promoter.
In a preferred embodiment the genetically modified mesenchymal stem cell as described herein is characterised in that the constitutive promoter is the EF1alpha promoter.
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.
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.
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.
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.
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.
In one embodiment the genetically modified mesenchymal stem cell as described herein is characterised in that the promoter is the Tie2 promoter.
In one embodiment the genetically modified mesenchymal stem cell as described herein is characterised in that the promoter is the RANTES promoter.
In one embodiment the genetically modified mesenchymal stem cell as described herein is characterised in that the promoter is the HSP70 promoter.
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.
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.
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.
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,
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.
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.
In a further aspect the invention relates to the genetically modified mesenchymal stem cell as described herein for use as a medicament.
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.
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
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).
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In one embodiment of the invention the neurodegenerative disease is Alzheimer's disease (AD).
In one embodiment of the invention the neurodegenerative disease is Multiple sclerosis (MS).
In one embodiment of the invention the neurodegenerative disease is Huntington's disease.
In one embodiment of the invention the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS).
In one embodiment of the invention the neurodegenerative disease is Parkinson's disease.
In one embodiment of the invention the neurodegenerative disease is Schizophrenia.
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.
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.
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.
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).
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.
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.
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.
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.
In one embodiment the present invention is directed to the treatment of sepsis via administration of the Klotho-modified MSCs as described herein.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 INVENTIONAll cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.
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.
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.
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.
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.
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.
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).
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.
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.
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.
As used herein, a “therapeutically effective number of cells” includes, without limitation, the following amounts and ranges of amounts: (i) from about 1×102 to about 1×108 cells/kg body weight; (ii) from about 1×103 to about 1×107 cells/kg body weight; (iii) from about 1×104 to about 1×106 cells/kg body weight; (iv) from about 1×104 to about 1×105 cells/kg body weight; (v) from about 1×105 to about 1×106 cells/kg body weight; (vi) from about 5×104 to about 0.5×105 cells/kg body weight; (vii) about 1×103 cells/kg body weight; (viii) about 1×104 cells/kg body weight; (ix) about 5×104 cells/kg body weight; (x) about 1×105 cells/kg body weight; (xi) about 5×105 cells/kg body weight; (xii) about 1×106 cells/kg body weight; and (xiii) about 1×107 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.
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.
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.
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.
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.
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.
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.
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.
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.
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 β-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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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-β1, mainly synthesized by T-cells during the healing process. TGF-β1 is secreted in a latent form associated with LAP (latency associated peptide). LAP is cleaved to allow the activation of TGF-β1 which is able to bind its receptors TGF-βR1 (transforming growth factor receptor-β1) and TGF-βR2. Therefore, there is a large pool of inactive TGF-β1 in the extracellular environment. Various agents can induce TGF-β1 activation: MMPs, reactive oxygen and nitrogen species (ROS and RNS), cytokines, or other stimuli such as ionizing radiation. The binding of TGF-β1 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).
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.
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.
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.
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.
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.
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.
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.
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.
Arteriosclerosis according to the present invention refers to, but is not limited to, one or more of, atherosclerosis, arteriosclerosis obliterans, and Monckeberg's arteriosclerosis.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
ExamplesThe 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.
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.
Preparation of Human Mesenchymal Stem Cells:
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).
Generation of Vectors for the Expression of Klotho:
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.
The promoters assessed during evaluation relate to the inducible promoters Tie2, RANTES or the HSP70 promoter, or the constitutive CMV or PGK promoters.
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.
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.
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).
Genetic Modification of Mesenchymal Stem Cells:
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:
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.
Viral vector supernatant in a final volume of 2 ml is added to each PLL-coated well. The number of particles should between 2×10e3 and 1×10e6 per well, which will result in multiplicity of infection of 0.25 and 10. The loaded plate is centrifuged for 2000×g, 30 min, 4° C. Afterwards the supernatant is discarded and 1×10e5 mesenchymal stem cells are seeded per well. The plates are incubated at 37° with 5% CO2 for further use.
Analysis of Transgene Expression in MSC:
Flow Cytometry:
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 μl BD Golgi Plug (Cat. No. 555029) per 1 ml Medium to enrich the expressed Klotho in the Golgi apparatus of the transduced cells.
Cells are incubated for 16 h at 37° 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.
A hemagglutinin-tag specific antibody labeled with Phycoerythrin (PE) (Milteny, 120-002-687) is used for detection of the expressed Klotho. 2×10e5 MSC are stained with 100 μl of antibody (1:75 diluted with Perm/wash solution, Becton Dickinson, 554723).
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).
ELISA to Determine Klotho Levels in the Supernatant:
Transduced MSC are seeded in 6 well plates (1×10e5 MSC per well). Transduced MSC, which carry the pac puromycin resistance gene, are enriched by puromycin selection. Puromycin (3 μg/ml medium) is added to the medium and cells are cultivated over a period of 5 days at 37° 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 1×10e5 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).
Klotho-Expressed in MSC Protects the Cells from H2O2-Induced Apoptosis:
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.
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.
Klotho is able to protect cells from the effects of reactive oxygen species:
Transduced and selected MSC (as described above) and non-transduced control MSC are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated 16 h at 37° C., 5° 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 H2O2 in the culture is between 10-100 μM. In addition, selected samples of MSC are not treated with H2O2 and serve as control. All samples are trypsinated to detach the MSC from the plates.
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 H2O2 show increased survival and reduced apoptosis in comparison to native MSC treated with H2O2.
Klotho Secreted from Transduced MSC Protects HUVEC Cells from H2O2-Induced Apoptosis:
Klotho is able to protect cells from the effects of reactive oxygen species:
Transduced and selected MSC (as described previously) and non-transduced control MSC are seeded into 6-well plates (50000-200000 cells per cm2). In addition, selected samples of MSC are not treated with H2O2 and serve as control. The cells are incubated 16-48 h at 37° C., 5° C. The supernatant is collected, filtered (0.25 μm) from the cells and stored.
Human umbilical vein endothelial cells (HUVEC) are seeded are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated for 16 h at 37° C. to let the cells attach to the plates. The cells are treated with Hydrogen peroxide (H2O2, Roth, cat. No. 8070.2). The final concentration of H2O2 in the culture is between 10-100 μM. 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.
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 H2O2.
Klotho-Expressed in MSC Increases the Production of Nitric Oxide (NO) in the MSC:
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.
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.
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.
Klotho increases the production NO, an important intercellular messenger, in MSC:
Transduced and selected MSC (as described previously) and non-transduced control MSC are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated for 48 h at 37° C./5% CO2.
To evaluate NO production, 100 μL 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.
Klotho Secreted from Transduced MSC Induces Increased Nitric Oxide-Production in HUVEC Cells:
Transduced and selected MSC (as described previously) and non-transduced control MSC are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated for 48 h at 37° C./5% CO2. The supernatant is collected, filtered (0.25 μm) from the cells and stored.
Human umbilical vein endothelial cells (HUVEC) are seeded are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated for 16 h at 37° 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 μL 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.
Klotho Secreted from Transduced MSC Suppress TGF-Beta Signaling in Target Cells
Transduced and selected MSC (as described previously) and non-transduced control MSC are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated for 48 h at 37° C./5% CO2. The supernatant is collected, filtered (0.25 μm) from the cells and stored.
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.
Klotho Secreted from Transduced MSC Increases FGF-23 Signaling in Target Cells
Transduced and selected MSC (as described previously) and non-transduced control MSC are seeded into 6-well plates (5000-50000 cells per cm2). The cells are incubated for 48 h at 37° C./5% CO2. The supernatant is collected, filtered (0.25 μm) from the cells and stored.
293 cells, which express the FGF-receptor, are seeded in 6 well plates (5000 cells per cm2) 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×)). 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.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs:
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.
Experimental Approach:
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.
Two different Experiments are conducted in 6 groups.
Experiment A:
Group 1: Mice age 12-15 months are placed in metabolic cages once a week. No further treatment will be performed until they die.
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 1×106 MSCs once a month until death. All animals are placed in metabolic cages once a week.
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 1×106 Klotho-modified MSCs once a month until death. All animals are placed in metabolic cages once a week.
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.
Experiment B:
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.
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 1×106 MSCs once a month until death. Once a week ECG and heart rate variability are measured.
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 1×106 Klotho-modified MSCs once a month. Once a week ECG and heart rate variability are measured.
Results: Animals in group 3 show significant improved heart rate variability in comparison to animals of group 1 and 2.
MSC Expressing Klotho Improve Kidney Fibrosis in an Murine Model
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 (5×10̂5-2×10̂6 cells per mouse) or with PBS. The treatment is repeated every 3 days.
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.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Alzheimer's Disease (AD):
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 Aβ pathology in APPswe/PS1(M146V) double transgenic mice.
Experimental Setup
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.
Three different Experiments will be conducted in 18 groups.
Experiment A:
Mesenchymal stem cells from young donor animals, age 6 weeks, are genetically modified to express klotho. 1×106 MSC-Klotho are injected intravenously once a month.
Group 1 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 2 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 3 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax mice
Mice are injected with saline intravenously once a month.
Group 4 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 5 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 6 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax mice
1×106 of non-transduced mesenchymal stem cells from young donor animals (age 6 weeks) are injected intravenously into the mice once a month.
Group 7 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 8 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 9 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax mice
Cognitive tests are performed once a week over a 3 month period.
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.
Experiment B:
Mesenchymal stem cells from young donor animals, age 6 weeks, are genetically modified to express klotho. 1×106 MSC-Klotho are injected intrathecally once a month.
Group 1 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 2 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 3 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1 Lfa/Mmjax mice
Mice are injected with saline intrathecally once a month
Group 4 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 5 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 6 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1 Lfa/Mmjax mice
1×106 of non-transduced mesenchymal stem cells from young donor animals (age 6 weeks) are injected intravenously into the mice once a month.
Group 7 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 8 C3B6-Tg(APP695)3Dbo/Mmjax mice
Group 9 B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1 Lfa/Mmjax mice
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.
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 Aβ 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.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Multiple Sclerosis (MS):
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.
Experimental Setup:
A mouse model of autoimmune encephalomyelitis (EAE) is used. C57BL/6 (H-2b) mice are immunized with myelin oligodendrocyte protein (MOG35-55).
Two different Experiments are conducted in 6 groups
Experiment A:
Mesenchymal stem cells from young donor animals, age 6 weeks, are genetically modified to express klotho. 1×106 MSC-Klotho are injected intravenously once a week.
Group 1 C57BL/6 (H-2b) immunized with (MOG35-55)
Mice are injected with saline intravenously once a week
Group 2 C57BL/6 (H-2b) immunized with (MOG35-55)
1×106 of non-transduced mesenchymal stem cells from young donor animals (age 6 weeks) are injected intravenously into the mice once a month.
Group 3 C57BL/6 (H-2b) immunized with (MOG35-55)
Experiment B:
Mesenchymal stem cells from young donor animals, age 6 weeks, are genetically modified to express klotho. 1×106 MSC-Klotho are injected intrathecally once a week.
Group 4 C57BL/6 (H-2b) immunized with (MOG35-55)
Mice are injected with saline intrathecally once a week.
Group 5 C57BL/6 (H-2b) immunized with (MOG35-55)
1×106 of non-transduced mesenchymal stem cells from young donor animals (age 6 weeks) are injected intrathecally into the mice once a month.
Group 6 C57BL/6 (H-2b) immunized with (MOG35-55)
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.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Amyotrophic Lateral Sclerosis (ALS):
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.
Experimental Setup:
SOD1 (G93A) mice are utilized. Two different Experiments will be conducted in 6 groups
Experiment A:
Mesenchymal stem cells from young donor animals, age 6 weeks, are genetically modified to express klotho. 1×106 MSC-Klotho are injected intravenously once a week.
Group 1 SOD1 (G93A)
Mice are injected with saline intravenously once a week
Group 2 SOD1 (G93A)
1×106 of non-transduced mesenchymal stem cells from young donor animals (age 6 weeks) are injected intravenously into the mice once a month.
Group 3 SOD1 (G93A)
Experiment B:
Mesenchymal stem cells from young donor animals, age 6 weeks, are genetically modified to express klotho. 1×106 MSC-Klotho are injected intrathecally once a week.
Group 4 SOD1 (G93A)
Mice are injected with saline intrathecally once a week
Group 5 SOD1 (G93A)
1×106 of non-transduced mesenchymal stem cells from young donor animals (age 6 weeks) are injected intrathecally into the mice once a month.
Group 6 SOD1 (G93A)
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.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Diabetes Type 1
The following examples describe approaches that demonstrate that intravenous administered MSC-Klotho delay the onset of diabetes type 1 in mice.
Experimental Setup:
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.
One to five days after cyclophosphamide treatment each animal receives 200 μL 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.
Group 1 NOD after cyclophosphamide treatment
One to five days after cyclophosphamide treatment animal receives 200 μL of 1×106 MSCs (from young donor animals, age 6 weeks) in 200 μL 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.
Group 2 NOD after cyclophosphamide treatment
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 1×106 Klotho-MSCs in 200 μL 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.
Group 3 NOD after cyclophosphamide treatment
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.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Diabetes Type 2
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).
Experimental Setup:
C57BL/6 mice are fed standard (SD) or high fat (HFD) diet. After 4 weeks mice are divided into 6 groups
Group 1: Mice fed with SD receive saline intravenously once a week for 8 weeks
Group 2: Mice fed with HFD receive saline intravenously once a week for 8 weeks
Group 3: Mice fed with SD receive 1×106 MSC once a week for 8 weeks
Group 4: Mice fed with HFD receive 1×106 MSC once a week for 8 weeks
Group 5: Mice fed with SD receive 1×106 MSC-Klotho once a week for 8 weeks
Group 6: Mice fed with HFD receive 1×106 MSC-Klotho once a week for 8 weeks
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.
Klotho-MSCs (group 6) show further improved glucose metabolism compared with mice treated with non-transduced MSCs (group 4).
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Chronic Renal Failure
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).
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
Group 1:
C57/BL6 with DOCA and angiotensin II receive intravenous saline for 4 weeks.
Group 2:
C57/BL6 with DOCA and angiotensin II receive intravenous 1×106 MSC once a week for 4 weeks.
Group 3:
C57/BL6 with DOCA and angiotensin II will receive intravenous 1×106 MSC-Klotho once a week for 4 weeks.
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.
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.
Histological and electron microscopic changes of renal architecture are improved in mice in groups 2 and 3 compared to mice in group 1.
Studies in Animal Models for Interrogating Klotho Function when Expressed from Transgenic MSCs in Parkinson Disease (PD):
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.
Additional experimentation on the basis of the approaches described above is ongoing.
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)
Type: Application
Filed: Feb 26, 2016
Publication Date: Feb 8, 2018
Inventors: Christine Günther (Munchen), Manfred Stangl (Sauerlach), Felix Hermann (Munchen)
Application Number: 15/553,954
