WO2015044379A1 - A dyrk1a polypeptide for use in preventing or treating metabolic disorders - Google Patents

Abstract

The invention relates to relates to a dual-specificity tyrosine phosphorylation-regulated kinase DYRKIA polypeptide or a functional equivalent thereof for use in preventing or treating a metabolic disorder in a patient in need thereof. The invention also relates to a DYRKIA polypeptide or a functional equivalent thereof for improving β-cell proliferation and/or function. The invention further relates to the use of a DYRKIA polypeptide or a functional equivalent thereof for reducing fat accumulation and/or adipogenesis.

Description

A DYRKIA POLYPEPTIDE FOR USE IN

PREVENTING OR TREATING METABOLIC DISORDERS

FIELD OF THE INVENTION:

The invention relates to the use of a dual- specificity tyrosine phosphorylation- regulated kinase DYRKIA polypeptide or a functional equivalent thereof for improving β-cell proliferation and/or function. The invention also relates to the use of a dual-specificity tyrosine phosphorylation-regulated kinase DYRKIA polypeptide or a functional equivalent thereof for reducing fat accumulation and/or adipogenesis. This opens the field of a new treatment for preventing or treating metabolic disorders such as diabetes and obesity.

BACKGROUND OF THE INVENTION:

Type 2 Diabetes Mellitus (T2DM) is characterized by high plasma glucose levels due to insulin resistance and impaired insulin secretion. It is a multigenic progressive disease influencing pancreatic beta cell adaptation to enhance insulin secretion in response to increased insulin resistance or body weight {Ferrannini, 2010} . In type 2 diabetic patients, both beta cell function and beta cell mass decrease {Butler, 2003; Ashcroft, 2012; Rahier, 2008} . Proliferation, differentiation, and apoptosis are fundamental cellular processes that regulate beta cell mass. These processes need to be tightly regulated and coordinated to produce the correct numbers of each of the different islet cell types of the mature islet. Alterations in these processes result in an altered beta cell mass and cause T2DM {Sachdeva, 2009} . Thus, understanding the underlying mechanisms that regulate beta cell mass is critical to define the susceptibility to the development of T2DM.

The dual-specificity tyrosine phosphorylation-regulated kinase DYRKIA (also named minibrain/MNB/YAKl) is a protein kinase from a family of proteins strongly conserved across evolution {Aranda, 2011 } . Mouse DyrklA and its drosophila homo log Minibrain (mnb) are implicated in growth during development {Becker, 1999; Tejedor, 2011; Fotaki, 2002} . Human DYRKIA maps within the Down's syndrome (DS) critical region on chromosome 21 (HSA21) {Delabar, 1993} . Interestingly, truncating mutations of human DYRKIA results in abnormal development with microcephaly and intra-uterine growth retardation (IUGR) leading to obesity {Oegema, 2010} . Despite the importance of DYRKIA in neural development, information is not available on a role of DYRKIA in beta cell development and function. - -

SUMMARY OF THE INVENTION:

In a first aspect, the invention relates to a dual-specificity tyrosine phosphorylation- regulated kinase DYRKIA polypeptide or a functional equivalent thereof for use in preventing or treating a metabolic disorder in a patient in need thereof.

In a second aspect, the invention also relates to a nucleic acid encoding a polypeptide for use for use in preventing or treating a metabolic disorder in a patient in need thereof.

In a third aspect, the invention further relates to a pharmaceutical composition comprising a DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof and a pharmaceutically acceptable carrier for use in preventing or treating a metabolic disorder in a patient in need thereof.

In a fourth aspect, the invention relates to a method for improving proliferation of a population of pancreatic β-cells in vitro or ex vivo, comprising a step of contacting said population with a culture medium comprising an effective amount of DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof.

In a fifth aspect, the invention relates to a method for improving proliferation of a pancreatic β-cell transplant, comprising a step of contacting said transplant with a culture medium comprising an effective amount of DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof.

In a sixth aspect, the invention relates to a DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use in a method for improving proliferation and/or function of pancreatic β-cells in a patient in need thereof.

In a seventh aspect, the invention relates to a DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use in a method for reducing fat accumulation and/or adipogenesis in a patient in need thereof.

In a last aspect, the invention relates to a DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use in a method for reducing excessive body weight in a patient in need thereof. DETAILED DESCRIPTION OF THE INVENTION:

The invention is based on the discovery that the dual-specificity tyrosine phosphorylation-regulated kinase DYRKIA controls the proliferation of pancreatic β-cells. Accordingly, the inventors have shown that DYRKIA is expressed in pancreatic islets and that change in Dyrkl A gene dosage in the mouse strongly modulates glycemia and circulating insulin levels. Specifically, their experiments in DyrklA haploinsufficient mice (DyrklA^+/ mice) showed severe glucose intolerance, reduced beta cell mass and decreased beta cell proliferation.

Conversely, DyrklA trisomic mice show glucose tolerance, improved beta cell mass and increased beta cell proliferation. Specifically, their experiments in mice overexpressing DyrklA under the control of its own regulatory sequences {mBACTgDyrkIA) showed that these mice exhibit decreased glucose levels and hyperinsulinemia in the fasting state. Improved glucose tolerance is observed in these mice as early as 4 weeks of age. Up- regulation of DyrklA in beta cells induces expansion of beta cell mass through increased proliferation and cell size. Up-regulation of DyrklA in beta cells induces expansion of beta cell mass through increased proliferation and cell size. Moreover, DyrklA upregulation shows resistance to High Fat diabetes induction. Importantly, mBACTgDyrkIA mice are protected against high fat diet-induced beta cell failure through increase in beta cell mass and insulin sensitivity. Additionally, the inventors have demonstrated that DyrklA modulation control directly fat accumulation.

Taken together, the present studies show the crucial role of the DYR 1A pathway in the regulation of beta cell mass and carbohydrate metabolism in vivo and these results show the crucial role of the DYRK1A pathway in the regulation of beta cell mass and carbohydrate metabolism in vivo and indicate that DYR 1A is a critical kinase for beta cell growth and modulation of fat accumulation. Accordingly, activating the DYR 1A pathway could thus represent an innovative way to increase functional beta cell mass.

Therapeutic methods and uses: The invention provides methods and compositions (such as pharmaceutical compositions) for use in preventing or treating a metabolic disorder in a patient in need thereof. The invention also provides methods and compositions for use in improving proliferation and/or function of pancreatic β-cells in vitro or ex vivo as well in a patient in need thereof. The invention also provides methods and compositions for use in reducing fat accumulation in a patient in need thereof.

In a first aspect, the invention relates to a dual-specificity tyrosine phosphorylation- regulated kinase DYRK1A polypeptide or a functional equivalent thereof for use in preventing or treating a metabolic disorder in a patient in need thereof. - -

The term "DYRKIA" has its general meaning in the art and refers to dual-specificity tyrosine phosphorylation-regulated kinase 1A of that is a serine/threonine kinase that autophosphorylates on tyrosine residues. The protein contains a nuclear localization signal and has been localized to the splicing-factor compartment (nuclear speckles), but it is also present in the cytoplasm. DYRKIA displays a broad substrate spectrum including transcription factors, splicing factors and synaptic proteins. The naturally occurring human DYRKIA gene has a nucleotide sequence as shown in Genbank Accession number NM 001396 and the naturally occurring human DYRKIA protein has an aminoacid sequence of 763 amino acids as shown in GenBank database under accession number NP 001387 and is shown as follows (SEQ ID NO: 1):

MHTGGETSACKPSSVRLAPSFSFHAAGLQMAGQMPHSHQYSDRRQPNISDQQVSALS YSDQIQQPLTNQVMPDIVMLQRRMPQTFRDPATAPLRKLSVDLIKTYKHINEVYYAK KKRRHQQGQGDDSSHKKERKVYNDGYDDDNYDYIVKNGEKWMDRYEIDSLIGKGS FGQVVKAYDRVEQEWVAIKIIKNKKAFLNQAQIEVRLLELMNKHDTEMKYYIVHLK RHFMFRNHLCLVFEMLSYNLYDLLRNTNFRGVSLNLTRKFAQQMCTALLFLATPELS IIHCDLKPENILLCNPKRSAIKIVDFGSSCQLGQRIYQYIQSRFYRSPEVLLGMPYDLAI DMWSLGCILVEMHTGEPLFSGANEVDQMNKIVEVLGIPPAHILDQAPKARKFFEKLP DGTWNLKKTKDGKREYKPPGTRKLHNILGVETGGPGGRRAGESGHTVADYLKFKDL ILRMLDYDPKTRIQPYYALQHSFFKKTADEGTNTSNSVSTSPAMEQSQSSGTTSSTSSS SGGSSGTSNSGRARSDPTHQHRHSGGHFTAAVQAMDCETHSPQVRQQFPAPLGWSG TEAPTQVTVETHPVQETTFHVAPQQNALHHHHGNSSHHHHHHHHHHHHHGQQALG NRTRPRVYNSPTNSSSTQDSMEVGHSHHSMTSLSSSTTSSSTSSSSTGNQGNQAYQNR PVAANTLDFGQNGAMDVNLTVYSNPRQETGIAGHPTYQFSANTGPAHYMTEGHLT MRQGADREESPMTGVCVQQSPVASS

The term "polypeptide" means herein a polymer of amino acids having no specific length. Thus, peptides, oligopeptides and proteins are included in the definition of "polypeptide" and these terms are used interchangeably throughout the specification, as well as in the claims. The term "polypeptide" does not exclude post-translational modifications that include but are not limited to phosphorylation, acetylation, glycosylation and the like.

A "native sequence" polypeptide refers to a polypeptide having the same amino acid sequence as a polypeptide derived from nature. Thus, a native sequence polypeptide can have the amino acid sequence of naturally-occurring polypeptide from any eukaryote organism such as yeast, chicken and mammals (including human). Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally-occurring allelic variants of the polypeptide.

A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the polypeptide. Ordinarily, a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the native sequence polypeptide.

By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.

In the frame of the present application, the percentage of identity is calculated using a global alignment (i.e., the two sequences are compared over their entire length). Methods for comparing the identity and homology of two or more sequences are well known in the art. The "needle" program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk world wide web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS: :needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

Polypeptides consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%), 97%), 98%) or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence. The polypeptide consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to an allelic variant of the reference sequence. It may for example only comprise substitutions compared to the reference sequence. The substitutions preferably correspond to conservative substitutions as indicated in the table below.

As used herein, the term "functional equivalent of the DYR 1 A polypeptide" includes the variants and the fragments of the polypeptide to which it refers (i.e. the DYR 1A polypeptide) and that retain the biological activity and the specificity of the parent polypeptide. Therefore, the "functional equivalent of the DYRK1A polypeptide" includes variants and fragments of the polypeptide represented by SEQ ID NO: 1.

A polypeptide "fragment", as used herein, refers to a biologically active polypeptide that is shorter than a reference polypeptide (e.g. a fragment of the DYRK1A polypeptide). Thus, the polypeptide according to the invention encompasses polypeptides comprising or consisting of fragments of DYRK1 A, provided the fragments are biologically active. In the frame of the invention, the biologically active fragment may for example comprise at least 450, 500, 550, 600, 650, 700 or 750 consecutive amino acids of the DYRK1A polypeptide.

By "biological activity" of a functional equivalent of the DYRK1A polypeptide is meant (i) the capacity to improve β-cell proliferation; and/or (ii) the capacity to increase the production of insulin; and/or (iii) the capacity to increase the secretion of insulin; and/or (iv) the capacity to restore glucose tolerance in vivo (e.g. in a model of diabetic mice); and/or (v) the capacity to improve insulin resistance; and/or (vi) the capacity to reduce fat accumulation and/or adipogenesis (e.g. in a model of obese mice). - -

The skilled in the art can easily determine whether a functional equivalent of the DYRKIA polypeptide is biologically active. To check whether the newly generated polypeptides improve β-cell proliferation or increase insulin secretion in the same way than the initially characterized polypeptide DYRKIA (a polypeptide consisting of the sequence depicted in SEQ ID NO: 1) a proliferation assay, a real-time PCR quantification assay or an immunofluorescence staining (see in Example) may be performed with each polypeptide. Additionally, a time-course and a dose-response performed in vitro or in vivo (e.g. by using a model of diabetic mice) will determine the optimal conditions for each polypeptide.

In one embodiment, the polypeptides of the invention may comprise a tag. A tag is an epitope-containing sequence which can be useful for the purification of the polypeptides. It is attached to by a variety of techniques such as affinity chromatography, for the localization of said peptide or polypeptide within a cell or a tissue sample using immuno labeling techniques, the detection of said polypeptide by immunoblotting etc. Examples of tags commonly employed in the art are the GST (glutathion-S-transferase)-tag, the FLAG™-tag, the Strep- tag™, V5 tag, myc tag, His tag (which typically consists of six histidine residues), etc.

In another embodiment, the polypeptides of the invention may comprise chemical modifications improving their stability and/or their biodisponibility. Such chemical modifications aim at obtaining polypeptides with increased protection of the polypeptides against enzymatic degradation in vivo, and/or increased capacity to cross membrane barriers, thus increasing its half-life and maintaining or improving its biological activity. Any chemical modification known in the art can be employed according to the present invention. Such chemical modifications include but are not limited to:

- replacement(s) of an amino acid with a modified and/or unusual amino acid, e.g. a replacement of an amino acid with an unusual amino acid like Nle, Nva or Orn; and/or

- modifications to the N-terminal and/or C-terminal ends of the peptides such as e.g. N- terminal acylation (preferably acetylation) or desamination, or modification of the C- terminal carboxyl group into an amide or an alcohol group;

- modifications at the amide bond between two amino acids: acylation (preferably acetylation) or alkylation (preferably methylation) at the nitrogen atom or the alpha carbon of the amide bond linking two amino acids; - modifications at the alpha carbon of the amide bond linking two amino acids such as e.g. acylation (preferably acetylation) or alkylation (preferably methylation) at the alpha carbon of the amide bond linking two amino acids.

- chirality changes such as e.g. replacement of one or more naturally occurring amino acids (L enantiomer) with the corresponding D-enantiomers;

- retro-inversions in which one or more naturally-occurring amino acids (L-enantiomer) are replaced with the corresponding D-enantiomers, together with an inversion of the amino acid chain (from the C-terminal end to the N-terminal end);

- azapeptides, in which one or more alpha carbons are replaced with nitrogen atoms; and/or - betapeptides, in which the amino group of one or more amino acid is bonded to the β carbon rather than the a carbon.

Another strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained- release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel bio materials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri- functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e- amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half- life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold- limiting glomular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintaining circulatory half- life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery. In still another embodiment, the polypeptides of the invention may be fused to a heterologous polypeptide (i.e. polypeptide derived from an unrelated protein).

In one embodiment, the heterologous polypeptide is a peptide capable of being internalized into a cell.

Such peptides capable of being internalized into a cell used herein have in their respective primary amino acid sequences (that is, over their entire length) at least 25%, preferably at least 30% positively charged amino acid residues. The term "positively charged amino acids" (herein also referred to as "basic amino acids"), as used herein, denotes the - - entirety of lysine (K), histidine (H), and arginine (R) residue present in a particular peptide. In particular embodiments, the peptides used herein comprise in their respective primary amino acid sequences at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%) positively charged amino acid residues.

The term "capable of being internalized into a cell", as used herein, refers to the ability of the peptides to pass cellular membranes (including inter alia the outer "limiting" cell membrane (also commonly referred to as "plasma membrane"), endosomal membranes, and membranes of the endoplasmatic reticulum) and/or to direct the passage of a given agent or cargo through these cellular membranes. Such passage through cellular membranes is herein also referred to as "cell penetration". Accordingly, peptides having said ability to pass through cellular membranes are herein referred to as "cell-penetrating peptides" (CPP) also called "protein transduction domains" (PTD), "membrane translocation sequences" (MTS) or "translocating peptides". In the context of the invention, any possible mechanism of internalization is envisaged including both energy-dependent (i.e. active) transport mechanisms (e.g., endocytosis) and energy-independent (i.e. passive) transport mechanism (e.g., diffusion). As used herein, the term "internalization" is to be understood as involving the localization of at least a part of the peptides that passed through the plasma cellular membrane into the cytoplasma (in contrast to localization in different cellular compartments such as vesicles, endosomes or in the nucleus).

The peptides used in the invention are internalized into a cell with an efficacy being at least 80%, preferably at least 90% of the internalization efficacy of the TAT peptide having the amino acid sequence GRKKRRQRRRPQ (SEQ ID NO: 2). In other words, the functional activity of the peptides is characterized in comparison to a reference peptide (TAT represents the "gold standard" with regard to cell-penetrating peptides). In specific embodiments, the peptides used herein are internalized with an efficacy being 80%>, 85%, 90% or 95% of the internalization efficacy of the TAT peptide. In specific preferred embodiments, the peptides used herein are internalized with at least the same efficacy (i.e. 100%) as the TAT peptide.

In one particular embodiment, the cell-penetrating peptide is the HIV transactivator of transcription (TAT) peptide represented by SEQ ID NO: 2 (Vives et al, J. Biol. Chem., 272, 16010-16017, 1997) : GRKKRRQRRRPQ - -

The term "internalization efficacy", as used herein, is to be understood in a broad sense. The term does not only refer to the extent to which a peptide used in the invention passes through the plasma membrane of cells (i.e. the internalization behavior per se) but also to the efficiency by which the peptide directs the passage of a given agent or cargo (e.g. the DYR 1A polypeptide of the invention) through the cell plasma membrane (i.e. its transfection capability; herein also referred to as "trans fectivity"). Numerous methods of determining the internalization behavior and/or transfection capability of a given peptide are established in the art, for example, by attaching a detectable label (e.g. a fluorescent dye) to the peptide (and/or to the cargo to be transfected) or by fusing the peptide with a reporter molecule, thus enabling detection once cellular uptake of the peptide occurred, e.g., by means of FACS analysis or via specific antibodies (see, e.g., Ausubel, F.M. et al. (2001) Current Protocols in Molecular Biology, Wiley & Sons, Hoboken, NJ, USA). The skilled person is also well aware how to select the respective concentration ranges of the peptide and, if applicable, of the cargo to be employed in such methods, which may depend on the nature of the peptide, the size of the cargo, the cell type used, and the like.

Cell-penetrating peptides (such as hydrophilic CPPs and amphiphilic CPPs) are well known in the art and other CPPs useful in the invention include for example: - Penetratin or Antennapedia PTD represented by SEQ ID NO: 3

RQIKWFQNRRMKWK

- SynB 1 represented by SEQ ID NO : 4

RGGRLSYSRRRFSTSTGR

Transportan represented by SEQ ID NO: 5

GWTLNSAGYLLGKINLKALAALAKKIL

- Pep- 1 represented by SEQ ID NO : 6

KET WWET W WTE WS QPKKKR V

- MAP represented by SEQ ID NO: 7

KLALKLALKLALALKLA

- Polyarginines RxN (4<N< 17) and Polylysines KxN (4<N< 17)

In one embodiment, the peptide capable of being internalized into a cell is fused to the N-terminus or the C-terminus of the DYR 1 A polypeptide of the invention, directly or via a peptide spacer. This complex is produced by making a fusion in frame of a nucleotide . sequence encoding the DYRK1A polypeptide of the invention to a nucleotide sequence encoding the peptide/protein cargo, and expressing the resulting chimeric gene using standard recombinant DNA techniques. As used herein, the terms "fused" and "fusion" are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature. Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in- frame linker sequence.

As used herein, the term "fusion protein" means a protein comprising a first polypeptide linearly connected, via peptide bonds, to a second, polypeptide.

As used herein, the term "DYRKl A fusion protein" refers to a polypeptide comprising the DYRKIA polypeptide or a functional equivalent thereof fused to heterologous polypeptide. The DYRKIA fusion protein will generally share at least one biological property in common with the DYRKIA polypeptide (as described above).

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of a DYRKIA polypeptide or functional equivalents thereof, or a DYRKIA fusion protein such as a DYRK1A-CPP polypeptide or use in accordance with the invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others (e.g. HEK 293 cells). Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

Moreover, it should be noted that the majority of protein-based biopharmaceuticals bare some form of post-translational modification which can profoundly affect protein properties relevant to their therapeutic application. Protein glycosylation represents the most common modification (about 50% of human proteins are glycosylated). Glycosylation can introduce considerable heterogeneity into a protein composition through the generation of different glycan structures on the proteins within the composition. Such glycan structures are made by the action of diverse enzymes of the glycosylation machinery as the glycoprotein transits the Endoplasmatic Reticulum (ER) and the Golgi-Complex (glycosylation cascade). The nature of the glycan structure(s) of a protein has impact on the protein's folding, stability, life time, trafficking, pharmaco-dynamics, pharmacokinetics and immunogenicity. The glycan structure has great impact on the protein's primary functional activity. Glycosylation can affect local protein structure and may help to direct the folding of the polypeptide chain. One important kind of glycan structures are the so called N-glycans. They are generated by covalent linkage of an oligosaccharide to the amino (N)-group of asparagin residues in the consensus sequence NXS/T of the nascent polypeptide chain. N-glycans may further participate in the sorting or directing of a protein to its final target: the N-glycan of an antibody, for example, may interact with complement components. N-glycans also serve to stabilize a glycoprotein, for example, by enhancing its solubility, shielding hydrophobic patches on its surface, protecting from proteolysis, and directing intra-chain stabilizing interactions. Glycosylation may regulate protein half-life, for example, in humans the presence of terminal sialic acids in N-glycans may increase the half-life of proteins, circulating in the blood stream.

As used herein, the term "glycoprotein" refers to any protein having one or more N- glycans attached thereto. Thus, the term refers both to proteins that are generally recognized in the art as a glycoprotein and to proteins which have been genetically engineered to contain one or more N-linked glycosylation sites. As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N- acetylglucosamine linkage to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N- acetylglucosamine (GlcNAc) and sialic acid (e.g., N- acetyl-neuraminic acid (NANA)). The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues post-trans lationally in the Golgi apparatus for N~linked glycoproteins. A number of yeasts, for example, Pichia pastoris, Yarrowia lipolytica and

Saccharomyces cerevisiae are recently under development to use the advantages of such systems but to eliminate the disadvantages in respect to glycosylation. Several strains are under genetical development to produce defined, human-like glycan structures on a protein. Methods for genetically engineering yeast to produce human-like N- glycans are described in U.S. Patent Nos. 7,029,872 and 7,449,308 along with methods described in U.S. Published Application Nos. 20040230042, 20050208617, 20040171826, 20050208617, and 20060286637. These methods have been used to construct recombinant yeast that can produce therapeutic glycoproteins that have predominantly human-like complex or hybrid N- glycans thereon instead of yeast type N-glycans. As previously described, human-like glycosylation is primarily characterized by "complex" N-glycan structures containing N-acetylglusosamine, galactose, fucose and/or N-acetylneuraminic acid. Thus, several strains of yeasts have been genetically engineered to produce glycoproteins comprising one or more human-like complex or human-like hybrid N-glycans such as GlcNAcMan3GlcNAc2. As used herein, the term "metabolic disorder" refers to disorders, diseases, and conditions that are caused or characterized by abnormal energy use or consumption within the body. Examples of metabolic disorders include overweight, obesity, and diabetes.

As used herein, the term "diabetes" refers to the broad class of metabolic disorders characterized by impaired insulin production and glucose tolerance.

Diabetes includes type 1 and type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, impaired fasting glycaemia and impaired glucose tolerance. Type 1 diabetes is also known as Insulin Dependent Diabetes Mellitus (IDDM or T1DM). The terms are used interchangeably herein. Type 2 diabetes is also known as Non-Insulin- Dependent Diabetes Mellitus (NIDDM or T2DM) even it usually lead to insulin treatment.

In one embodiment, the metabolic disorder is Type 1 diabetes (T1DM).

In one embodiment, the metabolic disorder is Type 2 diabetes (T2DM).

In one particular embodiment, the patient in need thereof is an obese patient.

In another particular embodiment, the patient in need thereof is a lean patient.

Accordingly, a lean patient is an otherwise healthy subject with a BMI lesser than or equal to 25 kg/m² or even lesser or equal to 20 kg/m².

Thus, a lean patient does not show an excessive triglyceride accumulation.

As used herein, the term "obesity" refers to a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems. Body mass index (BMI), a measurement which compares weight and height, defines individuals as overweight or as suffering from excessive body weight (pre-obese) if their BMI is between 25 and 30 kg/m², and overtly obese when their BMI is greater than 30 kg/m². There is increased risk of comorbidities for individuals with a BMI between 25.0 to 29.9, and moderate to severe risk of co-morbidities for individuals with a BMI greater than 30.

It should be further noted that obesity is often accompanied by excess fat storage in tissues other than adipose tissue, including liver and skeletal muscle, which may lead to local insulin resistance and may stimulate inflammation, as in steatohepatitis. In addition, obesity changes the morphology and composition of adipose tissue, leading to changes in protein production and secretion (including several pro -inflammatory mediators).

In one particular embodiment, the obesity is abdominal obesity.

As used herein, the term "a patient in need thereof refers to a subject that has been diagnosed with a metabolic disorder (such as diabetes and/or obesity), or one that is at risk of developing any of these disorders. Patients in need of treatment for instance include those that have suffered an injury, disease, or surgical procedure affecting the pancreas, or individuals otherwise impaired in their ability to make insulin. Alternatively, patients in need of treatment may be for instance those that have been diagnosed as with overweight and obesity.

Such patients may be any mammal, e.g., human, dog, cat, horse, pig, sheep, bovine, mouse, rat or rabbit (preferably a human).

Alternatively, a nucleic acid encoding a polypeptide of the invention (such as the DYR 1A polypeptide as shown in SEQ ID NO: 1) or a vector comprising such nucleic acid or a host cell comprising such expression vector may be used in preventing or treating a metabolic disorder in a patient in need thereof.

A nucleic acid encoding a polypeptide of the invention (such as the DYR 1A polypeptide as shown in SEQ ID NO: 1) or a vector comprising such nucleic acid or a host cell comprising such expression vector may also be used in improving proliferation and/or function of pancreatic β-cells in a patient in need thereof.

A nucleic acid encoding a polypeptide of the invention (such as the DYR 1A polypeptide as shown in SEQ ID NO: 1) or a vector comprising such nucleic acid or a host cell comprising such expression vector may further be used in reducing fat accumulation in a patient in need thereof.

Accordingly, another aspect of the invention relates to a nucleic acid encoding an amino acids sequence comprising SEQ ID NO: 1 as described here above for use in preventing or treating a metabolic disorder in a patient in need thereof. In one embodiment, said nucleic acid encoding an amino acid sequence consisting on

SEQ ID NO: 1.

Nucleic acids of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination(s).

Another aspect of the invention is an expression vector comprising a nucleic acid sequence encoding an amino sequence comprising SEQ ID NO: 1 as described here above for use in preventing or treating a metabolic disorder in a patient in need thereof. - -

In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of a nucleic acid to the cells and preferably to cancerous cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences of interest. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

Another aspect of the invention is a host cell comprising an expression vector as described here above for use in preventing or treating a metabolic disorder. According to the invention, examples of host cells that may be used are human pancreatic beta cells (particularly those obtained from the subject to be treated).

The means by which the vector carrying the gene may be introduced into the cells include, but are not limited to, microinjection, electroporation, transduction, or transfection using DEAE-dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art.

In a particular embodiment, polypeptides, nucleic acids, expression vector or host cells of the invention are used advantageously for preventing or treating diabetes.

Another aspect of the invention relates to a method for preventing or treating a metabolic disorder in a patient in need thereof comprising a step of administering to said patient a therapeutically effective amount of a polypeptide or a functional equivalent thereof as described above, or a nucleic acid of the invention, or an expression vector of the invention or a host cell of the invention.

In one embodiment, the invention relates to a method for preventing or treating a metabolic disorder comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of SEQ ID NO: 1 as above described.

As used herein, the term "therapeutically effective amount" is intended for a minimal amount of active agent, which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount of the active agent" to a subject is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the subject.

As used herein, the term "treating" a disorder or a condition refers to reversing, alleviating or inhibiting the process of one or more symptoms of such disorder or condition.

As used herein, the term "preventing" a disorder or a condition refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a disorder or a condition or phenotype, including a symptom. Another aspect of the invention relates to the use a DYRK1A polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof in a method for improving proliferation and/or function of pancreatic β-cells in a patient in need thereof. The invention also relates to a method for improving proliferation and/or function of pancreatic β-cells in a patient in need thereof comprising a step of administering to said patient a therapeutically effective amount of a polypeptide or a functional equivalent thereof as described above, or a nucleic acid of the invention, or an expression vector of the invention or a host cell of the invention.

As used herein, "improving cell proliferation" refers to an increase in the number of cells, as compared to a control, e.g., the number of cells in the absence of treatment. Improved cell proliferation can be expressed as a comparative value, e.g., twice as many cells are present if cell proliferation is improved two-fold. In some embodiments, cell proliferation is improved by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, as compared to control levels. In some embodiments, cell survival is by two-, three-, four-, five-, or ten-fold of control levels.

Another aspect of the invention relates to a method for reducing fat accumulation and/or adipogenesis in a patient in need thereof comprising a step of administering to said patient a therapeutically effective amount of a polypeptide or functional equivalent thereof as described above, or a nucleic acid of the invention, or an expression vector of the invention or a host cell of the invention.

In one embodiment, the fat accumulation consists of unwanted localized fat deposits characterized by excess subcutaneous adipose tissue or abdominal visceral fat accumulation

Another aspect of the invention relates to a method for reducing excessive body weight in a patient in need thereof comprising a step of administering to said patient a therapeutically effective amount of a polypeptide or functional equivalent thereof as described above, or a nucleic acid of the invention, or an expression vector of the invention or a host cell of the invention.

In another aspect, the invention relates to an inhibitor of a miR A reducing the expression level of DYRK1A for use in preventing or treating a metabolic disorder in a - patient in need thereof. In one embodiment, the metabolic disorder is Type 1 diabetes (T1DM), Type 2 diabetes (T2DM) or obesity as previously described.

In one embodiment, an inhibitor of a miRNA reducing the expression level of DYRK1A is miR-199b or miR-1296 as respectively described in {da Costa Martins, 2010} and {Zhang, 2012} .

The term "miRNAs" (also called "miR") has its general meaning in the art and refers to microRNA molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported. miRNAs are each processed from a longer precursor RNA molecule ("precursor miRNA"). Precursor miRNAs are transcribed from non- protein-encoding genes. The precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved in animals by a ribonuclease Ill-like nuclease enzyme called Dicer. The processed miRNA is typically a portion of the stem. The processed miRNA (also referred to as "mature miRNA") become part of a large complex to down-regulate a particular target gene.

All the miRNAs pertaining to the invention are known per se and sequences of them are publicly available from the data base http://microrna.sanger.ac.uk/sequences/.

The human miRNAs of the invention are listed in Table A:

Table A: list of the human miRNAs according to the invention As used herein, the term "inhibitor of miRNA" refers to any molecule or compound that decreases or reduces the expression and/or activity of miRNA, or at least one precursor thereof. This inhibition should, as a consequence, prevent or treat a metabolic disorder, i.e. by improving proliferation and/or function of a pancreatic β-cell and/or by reducing fat accumulation and/or adipogenesis. - -

In one embodiment of the invention, the said inhibitor of a given miRNA reducing the expression level of DYRKl A is an oligonucleotide of 8-49 nucleotides in length having a sequence targeted to the said given miRNA, said miRNA being preferably selected from the group comprising miR- 199b or miR- 1296.

The term "targeted" means having a nucleotide sequence that will allow hybridization to a target nucleic acid to induce a desired effect. In certain embodiments, a desired effect is reduction and/or inhibition of a target nucleic acid. The term "hybridize" means the annealing of complementary nucleic acids that occurs through "nucleotide complementarity", i.e. the ability of two nucleotides to pair non- covalently via hydrogen bonding.

On some embodiments, miRNA inhibitor oligonucleotides are 8 to 49 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides in length, or any range within. In some embodiments, oligonucleotides according to the invention, are 10 to 20 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length, or any range within.

In certain embodiments, the oligonucleotide has a sequence that is complementary to a miRNA or a precursor thereof. In one embodiment of the composition of the invention, the said oligonucleotide is an antisense oligonucleotide that is at least partially complementary to the sequence of the target miRNA reducing the expression level of DYRKl A, said target miRNA being preferentially selected from miR- 199b or miR- 1296. The term "antisense oligonucleotide" refers to an oligonucleotide having a nucleotide sequence complementary to a specific nucleotide sequence (referred to as a sense sequence) and capable of hybridizing with the sense sequence. The term "complementarity" means the nucleotide pairing ability between a first nucleic acid and a second nucleic acid. In certain embodiments, an antisense oligonucleotide has a nucleotide sequence that is complementary to a miRNA or a precursor thereof, meaning that the sequence of the antisense oligonucleotide is a least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a miRNA or precursor thereof, or that the two sequences hybridize under stringent hybridization conditions. Accordingly, in certain embodiments the nucleotide sequence of the antisense oligonucleotide may have one or more mismatched base pairs with respect to its target miRNA or precursor sequence, and is capable of hybridizing to its target sequence. In certain embodiments, the antisense oligonucleotide has a sequence that is fully complementary to a miRNA or precursor thereof, meaning that the nucleotide sequence of the antisense oligonucleotide is 100% identical of the complement of a miRNA or a precursor thereof.

In one embodiment, the antisense oligonucleotide sequence is "fully complementary" to the sequence of the target miRNA

In certain embodiment, the antisense oligonucleotide according to the invention has a sequence that is partially or fully complementary to the sequence of the target miRNA.

In one embodiment, the antisense oligonucleotide comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones.

Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H.

A modified backbone is typically preferred to increase nuclease resistance. A modified backbone can also be preferred because of its altered affinity for the target sequence compared to an unmodified backbone. An unmodified backbone can be RNA or DNA. Another suitable antisense oligonucleotide comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone. PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of 7V-(2- aminoethyl)- glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. A further suitable backbone comprises a morpholmo nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6- membered morpholmo ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non- ionic phosphorodiamidate linkage.

In yet a further embodiment, an antisense oligonucleotide of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base- pairing but adds significant resistance to nuclease degradation.

A further suitable antisense oligonucleotide of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a -OH; -F; substituted or unsubstituted, linear or branched lower (CI -CIO) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-or N-alkynyl; 0-, S-, or N-allyl; O-alkyl-O-alkyl, - methoxy, -aminopropoxy; -aminoxy; methoxyethoxy; dimethylaminooxyethoxy; and - dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid.

An LNA is a modified RNA nucleotide wherein the ribose moiety of LNA nucleotide is modified with an extra bridge connecting 2' and 4' carbons. This enhances the base stacking and pre-organization, and significantly increases the thermal stability. This bridge "locks" the ribose in 3'-endo structural conformation, which is often found in A- form of DNA or RNA. LNA nucleotides used in the present invention can be mixed with DNA or RNA bases in the oligonucleotide whenever desired. - -

According to the invention, the said antisense oligonucleotide is selected in the group consisting of a ribonucleotide, a deoxyribonucleotide, a small RNA, an antagomir, a LNA, a CDNA, a PNA, a morpholino oligonucleotide or a combination thereof.

In another embodiment, the antisense oligonucleotide consists of an antagomir. Antagomirs are chemically engineered oligonucleotides which are used to silence endogenous miRNA. An antagomir is a small synthetic RNA or DNA that is perfectly complementary to the specific miRNA target with either mispairing at the cleavage site or some sort of base modification to inhibit cleavage. Usually, antagomirs have some sort of modification to make it more resistant to degradation and facilitate cellular internalization. It is unclear how antagomirization (the process by which an antagomir inhibits miRNA activity) operates, but it is believed to inhibit by irreversibly binding the miRNA. Antagomirs are used to constitutively inhibit the activity of specific miRNAs.

In an embodiment of the invention, the said antagomir comprises a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous nucleotides complementary to a miRNA, or a precursor thereof, the said miRNA having a sequence selected from the group consisting of SEQ ID NO: 8 or 9.

In other embodiment of the invention, the said antagomir can include 2'-0-methyl modified nucleotide, cholesterol group or any similar or equivalent modification.

In another aspect, the invention relates to an inhibitor of a miRNA reducing the expression level of DYRK1A (such as an antagomir of miR-199b or of miR-1296) for use in a method for improving proliferation and/or function of pancreatic β-cells.

In still another aspect, the invention relates to an inhibitor of a miRNA reducing the expression level of DYRK1A (such as an antagomir of miR-199b or of miR-1296) for use in a method for reducing fat accumulation and/or adipogenesis.

Pharmaceutical compositions:

Another aspect of the invention relates to a pharmaceutical composition for use in preventing or treating a metabolic disorder in a patient in need thereof comprising: a) an DYRKIA polypeptide or a functional equivalent thereof according to the invention; or

b) an acid nucleic according to the invention; or

c) an expression vector according to the invention; or

d) an host cell according to the invention; or

e) an inhibitor of a miRNA reducing the expression level of DYRKIA (such as an antagomir of miR-199b or of miR-1296)

f) and a pharmaceutically acceptable carrier.

Still another aspect of the invention relates to a pharmaceutical composition for use in improving pancreatic β-cells proliferation and function in a patient in need thereof comprising:

a) an DYRKIA polypeptide or a functional equivalent thereof according to the invention; or

b) an acid nucleic according to the invention; or

c) an expression vector according to the invention; or

d) an host cell according to the invention; or

e) an inhibitor of a miRNA reducing the expression level of DYRKIA (such as an antagomir of miR-199b or of miR-1296)

f) and a pharmaceutically acceptable carrier.

In one embodiment, said pharmaceutical composition comprises a DYRKIA polypeptide having the sequence SEQ ID NO: 1.

Any therapeutic agent of the invention as above described may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

To prepare pharmaceutical compositions, an effective amount of a polypeptide or a nucleic acid according to the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The polypeptides thereof or the nucleic acid according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents (e.g. parabens, chlorobutanol, phenol, sorbic acid, thimerosal). In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently used.

Pharmaceutical compositions of the invention may comprise an additional therapeutic agent.

In one embodiment, said additional therapeutic active agent is an anti-diabetic drug.

As used herein, the term "anti-diabetic drug" refers to any compound, natural or synthetic, which can reduce glucose levels in the blood and therefore is useful for preventing or treating diabetes. Typically, anti-diabetic drugs encompass (1) insulin, (2) agents that increase the amount of insulin secreted by the pancreas (e.g. glucagon-like peptide-1 (GLP-1) receptor agonists and sulfonylureas) (3) agents that increase the sensitivity of target organs to insulin (e.g. biguanides and thiazolidinediones), and (4) agents that decrease the rate at which glucose is absorbed from the gastrointestinal tract (e.g. alpha-glucosidase inhibitors).

Non-limiting examples of anti-diabetic drug (those above-mentioned as well as other anti-diabetic drugs. g. inhibitors of of dipeptidylpeptidase-IV (DDP-4); GPR40 receptor agonists) which can be used in combination and that are contemplated by the invention include but are not limited to those described, for example, in US 2012/0004166.

In another aspect, the invention also relates to a kit-of-part composition comprising a polypeptide or a derivative thereof, or a nucleic acid, or a vector, or a host cell, or an inhibitor of a miRNA reducing the expression level of DYRK1A (such as an antagomir of miR-199b or of miR-1296) according to the invention and an additional therapeutic active agent.

In still another aspect, the invention further relates to a kit-of-part composition comprising a polypeptide or a derivative thereof, or a nucleic acid, or a vector, or a host cell, or an inhibitor of a miRNA reducing the expression level of DYR IA (such as an antagomir of miR-199b or of miR-1296) according to the invention and an additional therapeutic active agent for use in preventing or treating a metabolic disorder. In one embodiment, said additional therapeutic active agent is an anti-diabetic drug as described above.

The terms "kit" or "combined preparation", as used herein, define especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can be varied. The combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 6, 12, 18 or 24 h before the second partner.

A culture medium and transplantation of pancreatic β-cells - -

In another aspect, the invention relates to a method for improving proliferation and/or function of a population of pancreatic β-cells in vitro or ex vivo, comprising a step of contacting said population with a culture medium comprising an effective amount of DYRKl A polypeptide or a functional equivalent thereof.

In another aspect, the invention relates to a method for improving proliferation and/or function of a population of pancreatic β-cells in vitro or ex vivo, comprising a step of contacting said population with a culture medium comprising an effective amount of an inhibitor of a miRNA reducing the expression level of DYRKl A (such as an antagomir of miR-199b or of miR-1296 as previously described).

As used herein, the term "culture medium" refers to a liquid medium suitable for the in vitro or ex vivo culture of mammalian pancreatic β-cell, and preferably human pancreatic β- cell.

As used herein, the terms "pancreatic β-cell", "β islet cells", "insulin producing cells" and similar terms refer a population of pancreatic endocrine cells found in the islets of Langerhans. β islet cells produce and secrete insulin and amylin into the bloodstream.

The culture medium used by the invention may be a water-based medium that includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which are needed for cell survival.

For example, a culture medium according to the invention may be a synthetic tissue culture medium such as the RPMI (Roswell Park Memorial Institute medium) or the CMRL- 1066 (Connaught Medical Research Laboratory) for human use, supplemented with the necessary additives as is further described below (Section Examples).

In a preferred embodiment, the culture medium of the invention is free of animal- derived substances. In a preferred embodiment, the culture medium of the invention consists essentially of synthetic compounds, compounds of human origin and water. Advantageously, said culture medium can be used for culturing cells according to good manufacturing practices (under "GMP" conditions).

In another aspect, the invention also relates to a method for improving proliferation and/or function of a pancreatic β-cell transplant in vitro or ex vivo, comprising a step of contacting said transplant with a culture medium comprising an effective amount of DYRKl A polypeptide or a functional equivalent thereof.

In another aspect, the invention also relates to a method for improving proliferation and/or function of a pancreatic β-cell transplant in vitro or ex vivo, comprising a step of contacting said transplant with a culture medium comprising an effective amount of an inhibitor of a miRNA reducing the expression level of DYRKl A (such as an antagomir of miR-199b or of miR-1296 as previously described).

A "transplant" as used herein, refers to the introduction of cells into an individual (recipient or host). A "pancreatic β-cell transplant" refers to a transplant that includes β-cells, but is not necessarily composed entirely of β-cells. The transplanted cells can be introduced as an entire organ (e.g., a pancreas), a largely intact tissue sample (e.g., a tissue graft, like islet transplantation), or as a disaggregated population of cells (e.g., enriched for β- islet cells) or a transplant of purified β-cells. The introduced cells can be from another individual (allotransplantation) or from the same individual (autotransplantation). In some cases, cells are removed from an individual, cultured under favorable conditions, and replaced. In some cases, undifferentiated or partially differentiated cells can be cultured under appropriate conditions to differentiate into β -cells, and transplanted into an individual.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: Pancreatic expression of DyrklA. (A) qPCR analysis of DyrklA, insulin and amylase mRNA expression in pancreases at different stages of fetal development, in adult pancreas and adult islets. Data are shown as the mean ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005. (B) Immunoblot for DYRKl A and beta actin in pancreatic extracts at different stages of fetal development, in adult pancreas and adult islets.

Figure 2: Dyrkla+/- mice exhibit impaired glucose tolerance and defective insulin secretion. (A) Body length at 16 weeks in wild type and Dyrkla^+/ mice. (B) Body weight evolution in wild type and Dyrkla^+/ mice. (C) Blood glucose concentrations in overnight fasted wild type and Dyrkla^+/ mice at the indicated age. (D) Serum insulin concentrations in 6-hours-fasted mice at the indicated age. (E, F) i.p. glucose tolerance tests on 12-week-old (E) and 36-week-old (F) Dyrkla^+/ and wild type mice. (G) Insulin tolerance test in fed 12-week-old mice (H) In vivo insulin secretion in 12-week-old Dyrkla^+/ and wild type mice. Data are shown as the mean ± SEM of at least three independent experiments. *P < 0.05: **P < 0.01; ***P < 0.005.

Figure 3: Insulin secretion in DyrklA ^~ islets. (A, B) Glucose-induced insulin secretion and insulin content in isolated islets from DyrklA^+/ and control mice. (C) Realtime PCR quantification of DyrklA, Insulin, MafA, NeuroD, Pdxl, ZnT8 and Glut2 mRNA was performed on isolated islets from wild type and Dyrkl A^+/ mice. Data are shown as mean ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005. Figure 4: Islet morphometry in wild type and DyrklA ^~ animals. (A) Decreased pancreatic weight in 3-month-old DyrklA^+/ mice relative to control littermates. (B) Immunodetection of insulin (brown) in pancreatic sections of 12-week-old mice from wild type and DyrklA^+/~ mice. Hemalun staining (blue) was used to counterstain the tissue. Scale bar: 5 mm. (C) Immunohistochemical quantification of the insulin- stained area showed that the beta cell mass was reduced in DyrklA^+/ compared to control mice. Data are shown as mean ± SEM of at least three independent experiments. **P < 0.01.

Figure 5: Assessment of beta cell proliferation and size in wild type and DyrklA^{+ ~} mice. (A) Proliferative index in sections stained for Ki67 and insulin was established by measurements of at least 2,000 cells. (B) Quantification of beta cell size in islets from wild type and DyrklA^+/ mice. Data include measurements of at least 500 cells. Data are shown as mean ± SEM from at least three pancreases per condition. *P < 0.05. Figure 6: Normal food intake and fat accumulation in Dyrkla+/- mice. (A) Lack of effect of DyrklA haploinsufficiency on food intake in male mice at 16 weeks. (B) Perigonadal fat mass in wild type and Dyrkla^+/ male mice of 16 weeks. Data are shown as the mean ± SEM of at least three independent experiments, ***P < 0.005.

EXAMPLE 1: Dyrkla haploinsufficiency induces diabetes as a result of decreased beta cell mass.

Material & Methods

DyrklA+/- mice: The generation of DyrklA+/- mice is described elsewhere {Fotaki, 2002} . Wild type and Dyrkla mutant mice {Fotaki, 2002} were used in accordance with the French Animal Care Committee's guidelines. The mice were bred on a genetic CD1 background and raised on a 12-hour light/ 12-hour dark cycle. They were fed with a standard laboratory chow diet. The first day post coitum was taken as embryonic day 0.5 (E0.5). Body length was measured, from the tip of the nose to the anal base of the tail.

Metabolic Studies: Blood samples were collected from the tail vein. Whole glucose levels were measured using OneTouch Vita blood glucose meter (LifeScan, Milpitas, CA, USA). Plasma insulin levels were determined by ELISA kit (Bertin Pharma, Montigny le Bretonneux, France). Glucose tolerance tests were performed in 16-h fasted animals by intraperitoneal glucose injection (2 g/kg) as described previously {Rachdi, 2008} . Insulin tolerance tests were done in 6-h fasted mice followed by glucose measurements at 30, 60, and 120 min after intraperitoneal insulin injection using 0.75 units/kg.

Pancreatic buds culture: Gastrointestinal tracts from El 1.5 mouse embryos were dissected. They were then laid on 0.45 μιη filters (Millipore, Billerica, MA) at the air-medium interface in Petri dishes that contained RPMI-1640 medium (Invitrogen, Carsbad, CA) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES (10 mmol/1), L- glutamine (2 mmol/1), nonessential amino acids (lx; Invitrogen), and 10% heat-inactivated fetal calf serum {Attali, 2007} . The gastrointestinal tracts were incubated for 7 days at 37°C in a humidified 95% air-5%> C02 gas mixture. The medium was changed every second day.

Islet studies: Islets were isolated from 12-week-old mice by collagenase digestion followed by purification through a Histopaque gradient (Sigma-Aldrich, Saint Quentin Fallavier, France). Insulin secretion was assessed by static incubation of isolated islets in Millicell insert (Millipore). Following overnight culture in RPMI containing 5.6 mmol 1 glucose, islets were pre-incubated for 30 minutes in Krebs-Ringer medium containing 2.8 mmol/1 glucose. Groups of 50 islets in triplicate were next incubated in Krebs-Ringer medium containing 2.8 mmol/1 glucose or 20 mmol/1 glucose for 30 minutes. Secreted insulin was measured using an ultrasensitive mouse insulin ELISA (Mercodia AB, Uppsala, Sweden) and normalized by total insulin content from islets extracted with acid-ethanol (1.5% [vol/vol] HC1 in 75% [vol/vol] ethanol).

Immunohistochemistry and quantification: Pancreases were immersed in 10%> formalin and embedded in paraffin. Sections (4μιη-ΐ1ιίΰ1ί) were processed for immunohistochemistry using a previously described protocol {Rachdi, 2003} . Antibodies were used at the following dilutions: mouse anti-insulin (Sigma, 1 :2,000); rabbit anti-insulin (Dako, Trappes, France, 1 :2,000); mouse anti-glucagon (Sigma, 1 :2,000); rabbit anti-amylase (Sigma, 1 :300); mouse anti-Ki67 (BD Biosciences, Le Pont de Claix, France, 1 :20), mouse anti-p27^Kipl (BD Biosciences, 1 :200); mouse anti-E-cadherin (BD Biosciences, 1 :200). The fluorescent secondary antibodies were purchased from Jackson ImmunoResearch (Soham, UK). The nuclei were stained using the Hoechst 33342 fluorescent stain (0.3 mg/ml; Invitrogen).

For adult pancreata, quantification of insulin staining was performed on five equally separated sections. In each section, the beta cell area and the pancreatic area were determined using NIH Image J software. The percent area of beta cells in each pancreatic section was determined by dividing the area of all insulin-positive cells by the total surface area of the section. The beta cell mass was calculated by multiplying the pancreas weight by the % area of the beta cells {Rachdi, 2008} .

For cultured fetal pancreata, all images of the 40-60 sections of each pancreas were digitized using cooled three-charge-coupled-device cameras (C5810 or C7780; Hamamatsu, Massy, France) attached to a fluorescence microscope (Leitz DMRB; Leica, Nanterre, France). For every image, the areas of each immunostaining were quantified using NIH Image J software (vl .31 which is freely available at http://rsb.info.nih.gov/ij/index.html), and then summed in order to obtain the total area per explant in mm², as previously described {Guillemain, 2007} . - -

RNA extraction and real-time PCR: Total RNA was extracted using an RNeasy Microkit (Qiagen, Courtaboeuf, France), and then reverse transcribed using Superscript reagents (Invitrogen). Real-time PCR was performed with the 7300 Fast real-time PCR system (Applied Biosystems, Courtaboeuf, France) using a previously described protocol {Guillemain, 2007} . The oligonucleotide sequences are available upon request. Cyclophilin A was used as the internal reference control.

Western blotting: Protein lysates were subjected to immunoblotting as described {Rachdi, 2012 #19} . The following antibodies were used: DyrklA (Abnova, Taoyuan County, Taiwan, 1 : 1,000) and Actin (Sigma, 1 :2,000). Immunoblotting experiments were performed three times.

Statistical analysis: Quantitative data are presented as the mean ± S.E.M. from at least three independent experiments, unless indicated. Interactions among the variables were investigated by two-way analysis of variance, and an unpaired Student's t-test was used to compare the independent means. Statistical significance was set at 5%.

Results

DyrklA is expressed in the developing pancreas and enriched in adult islets.

The expression pattern of Dyrkla in the mouse pancreas being unknown, we first analyzed its expression during mouse pancreas development. Dyrkla mRNA was detected as early as El l . Expression increased at E13 and then decreased (Fig. 1A). Dyrkla mRNA levels were low in whole adult (3 months old) pancreas, but were enriched in adult islets (Fig. 1A). Amylase and Insulin expression patterns are shown as control (Fig. 1 A). At the protein level, DYRK1A expression was detected in mouse pancreas at El l, E13 and E15, its expression being under the detection limit at El 7. DYRK1A was also undetectable in whole adult (3 months old) pancreas, but enriched in adult islets (Fig. IB). These results indicate that DYRK1 A is expressed and developmentally regulated during pancreatic development.

Dyrkla haploinsufficient mice are glucose intolerant and hypoinsulinemic.

Dyrkla ^~/_ mice could not be used in this study as they die between El 0.5 and El 3.5 {Fotaki, 2002} . As a result, we focused on DjrWa-haploinsufficient (Dyrkla^+/~) male mice. As previously described {Fotaki, 2002}, Dyrkla^+/ mice were smaller than controls (Fig. 2A). At week 4, they showed a significant reduction in body weight (Fig. 2B) {Fotaki, 2002} . Interestingly, at week 12, body weight was higher in Dyrkla^+/ mice than in controls and past 12 weeks, the body weight of Dyrkla^+/ mice continued to increase faster than that of control mice (Fig. 2A). This gain of weight that was not paralleled by an increase in food consumption (Fig. 6A) and was mainly due to an increased abdominal fat (Fig. 6B) as described previously {Waki, 2007}

Since DyrklA haploinsufficiency resulted in impaired glucose tolerance, and decreased insulin levels while insulin sensitivity was unchanged, we next concentrated on the function of Dyrkla in pancreatic beta cells.

Insulin secretion and gene expression in islets from Dyrkla^{+ ~} mice: Immunofluorescence staining for insulin and glucagon showed that islet architecture was conserved in DyrklA^+/ mice with insulin-positive cells in the core of the islet and non beta cells in the periphery. We next compared insulin secretion in isolated islets from DyrklA^+/ and wild type mice. At basal glucose levels (2.8 mM), insulin secretion did not differ between mutant and control islets (Fig. 3A). Glucose (20 mM) induced insulin secretion in islets, from wild type and Dyrkla^+/ mice (Fig. 3 A). However, total insulin content of islets from DyrklA^+/ mice was lower than control (Fig. 3B)

We next performed comparative gene expression analysis between islets from wild type and from DyrklA ^7" mice. We first confirmed the haploinsufficiency of DyrklA by qPCR (Fig. 3C). In islets from Dyrkla^+/~ mice, Insulin- 1 expression was decreased, as was the case for MafA and NeuroDl that encode two insulin gene transactivators {Olbrot, 2002} {Naya, 1997} (Fig. 3D). On the other hand, the expression of Pdxl, another insulin transactivator {Ahlgren, 1998} was not modulated by DyrklA defect (Fig. 3B). Finally, the expression of glucose transporter 2 (Glut-2) and Zinc transporter 8 (Znt8) that are involved in insulin secretion was not altered (Fig. 3C).

Beta cell mass is decreased in DyrklA haploinsufficient mice: We next determined whether the reduction in circulating insulin observed in DyrklA haploinsufficient mice was caused by a decreased beta cell mass. We first observed that the pancreatic weight at 12 weeks was lower in DyrklA haploinsufficient mice when compared to wild type mice (Fig. 4A). We next stained pancreatic sections for insulin and measured beta cell mass. Beta cell mass in 12-week-old DyrklA^+/" mice was 55.7% lower than in control mice (Fig. 4B and C). We next asked whether this pancreatic phenotype was pancreas autonomous. For this purpose, we cultured El 1.5 gastrointestinal tracts for seven days under conditions permissive for endocrine and acinar cell development {Attali, 2007} . Pancreatic acinar and endocrine cells developed and differentiate from wild type and Dyrkl a^+/~ gastrointestinal tracts. Acinar cell development, measured following amylase immunostaining, that represents the majority of the differentiated pancreatic tissue, showed a 47% decrease in Dyrkl a^+/~ pancreases. This paralleled the global decrease in pancreatic weight observed in vivo (Fig. 4A). Beta cell development, measured following insulin immunostaining, showed a major decrease in Dyrkl a^+/~ pancreases (91 > decrease when compared to wild type digestive tracts). Such in vitro experiments strongly suggest that DyrklA haploinsufficiency induced a decreased in beta cell number in a pancreas-autonomous fashion. Beta cell proliferation, apoptosis and size in DyrklA ^' and wild type mice: Beta cell proliferation, measured following Ki67 immunostaining showed a 2-fold decrease in 12- weeks old Dyrkl A^+/~ mice when compared to controls (Fig. 5 A), while the frequency of beta cell apoptosis as measured by TUNEL staining was similar between Dyrkl A^+/~ and wild type mice (data not shown). Beta cell size measurement demonstrated a 20% decrease in DyrklA^+/~ mice (Fig. 5B). Collectively, such results indicate that DyrklA haploinsufficiency reduces beta cell mass by decreasing cell proliferation and cell size.

Alterations in cell cycle progression in DyrklA ^' mice are associated with increased nuclear P27 accumulation: We and others previously demonstrated that the cyclin-dependent kinase inhibitor p27^&pl contributes to the regulation of beta cell mass {Rachdi, 2006;Uchida, 2005} and that its expression in beta cells is controlled, at least in part, by the transcription factor FOXOl {Uchida, 2005} . As FOXOl is one of the main targets of DyrklA {Woods, 2001 }, we assessed the expression of Cip/Kip inhibitors P27 by immunostaining. As expected, in wild type mice, very rare beta cells stained positive for P27 (1.05 ± 1%). Interestingly, this number sharply increased in Dyrkl a haploinsufficient mice, with 43.45 ±0.8 % beta cells positive for P27. Collectively, our data suggest that Dyrkl A decreases nuclear P27 levels and represses beta cell proliferation. This process results in decreased beta cell mass and consequently, in a defect in beta cell ability to regulate blood glucose. DISCUSSION:

In this study, we are providing evidence that Dyrkl A expression level is essential for the proper regulation of the pancreatic beta cell mass. We demonstrate that in mice, DyrklA haploinsufficiency leads to a decrease in beta cell number and size. This reduction of beta cell mass in DyrklA^+/~ mice gives rise to impaired glucose tolerance associated with hypoinsulinemia.

Our first set of data indicated that in mice, DyrklA haploinsufficiency caused a decrease in newborn body weight, which persisted until week 4 of postnatal life. Later on, DyrklA^+/" mice gained weight and became obese when compared to control mice. This defect was associated with glucose intolerance, hyperglycemia, hypoinsulinemia without insulin resistance. In human, DyrklA microdeletion causes a distinctive clinical syndrome with mental retardation and intrauterine growth retardation leading later on to obesity {Oegema, 2010} . Information on the metabolic status of such patients has not been described in detail. It will now be interesting to determine precisely the glycemic and insulinemic status of such patients, to determine whether the metabolic status observed in mice, is reproduced in Human.

DyrklA+/- mice are glucose intolerant and different arguments suggest that this phenotype is intrinsic to the expression and role of DyrklA in the pancreas. First, DyrklA is expressed during pancreatic development and enriched in pancreatic islets. Second, in vivo, beta cells from DyrklA+/- mice secreted insulin upon glucose challenge, but quantitatively less than wild type mice. Third, peripheral glucose assimilation or clearance, assessed by the insulin tolerance test was unchanged in DyrklA+/- mice when compared with controls. Fourth, insulin expression and content in isolated islets from Dyrkl A+/- mice was decreased when compared to controls. Finally, we compared beta cell development from wild type and DyrklA+/- mice in a previously described in vitro model that properly recapitulates pancreatic cell development {Attali, 2007} . This type of approach had previously been used to study in vitro beta cell development from transgenic mice {Bhushan, 2001 } {Wilson, 2005} {Fontaniere, 2008} . With this model, we demonstrated that the decreased beta cell mass observed in vivo in Dyrkl A+/- mice was reproduced in vitro. It will now be interesting to determine the pancreatic phenotype of mice with beta cell specific deletion of DyrklA when such mice will be available. - -

DyrklA ^" mice are glucose intolerant and hypoinsulinemic. This phenotype does not seem to be due to a qualitative defect in insulin secretion as in vitro, beta cells properly secrete insulin upon glucose challenge. In fact, this phenotype seems to be due to a decrease in beta cell mass as is the case in a number of other mouse models. For example, ablation of phosphoinositide-dependent protein kinase 1 in pancreatic beta cells results in decreased beta cell mass giving rise to hypoinsulinemia {Hashimoto, 2006} . Mice that over-express the cell cycle inhibitor p27KIPl in pancreatic beta cells represent another example. Such mice have a decreased beta cell mass and are glucose intolerant, hyperglycemic and hypoinsulinemic {Uchida, 2005; Rachdi, 2006} . Interestingly, beta cell proliferation is decreased in such mice that over-express p27KIPl and we found here that beta cell proliferation is decreased in DyrklA ^7" mice, while the expression of p27KIPl is induced. A link was recently reported between DYRKIA and P27. Specifically, in chick neuronal precursors, DYRKIA regulated cell growth and p27KIPl expression {Hammerle, 2011 } . However, while our data indicate that in the pancreas, Dyrkla negatively regulates p27KIPl expression, in chick neuronal precursors, DYRKIA induced p27KIPl expression by suppressing NOTCH signaling {Hammerle, 2011 } . Whether DyrklA controls the NOTCH pathway in the pancreas should be further studied. In yeast, Yakl, the Dyrk homolog has been studied in great details for its effects on cellular growth {Martin, 2004} . There, it was shown that the conserved target of rapamycin (TOR) signaling pathway negatively regulates YAK1 {Martin, 2004} . To the best of our knowledge, in mammals, no link has been described between DyrklA and mTOR. However, the pancreatic phenotypes observed in the present study resemble the ones observed in mice deficient in the mTOR pathway. Indeed mice exposed to rapamycin, an inhibitor of the mTOR pathway, have a decreased beta cell mass with decreased beta cell size and proliferation and are hypoinsulinemic {Yang, 2012} . Moreover, disruption of Tsc2 in pancreatic beta cells that activates the mTOR pathway, gives rise to mice with increased beta cell mass, increased beta cell size and proliferation, giving rise to hypoglycemia and hyperinsulinemia {Rachdi, 2008} . Such similarities are intriguing, and it could be interesting to further search for a link between DyrklA and mTOR in mammals.

In conclusion, we discovered in this study that DyrklA is a new regulator of beta cell proliferation through modulation of P27 nuclear accumulation. Such a pathway is important - - for proper regulation of glycemia as Dyrkla haploinsufficiency induces diabetes. Activating the Dyrkl A pathway might offer a novel approach to increase beta cell mass.

EXAMPLE 2: DyrklA induces pancreatic beta cell mass expansion and improves glucose tolerance.

Material & Methods

Animals: The generation of mBACTgDyrklA mice has previously been described {Guedj, 2012} . Wild-type and Dyrkla mutant mice were used in accordance with French Animal Care Committee guidelines. The mice were bred on a genetic C57B16J background and raised on a 12 h light-dark cycle. They were fed with a standard laboratory chow diet. The high- fat diet consisted of a synthetic high- fat diet with 60% kcal% fat (HFD) (D 12492; Research Diets, New Brunswick, NJ, USA). mBACTgDyrklA mice under HFD were fed for 12 weeks from the 4th week. The first day post coitum was taken as embryonic day 0.5 (E0.5).

Metabolic studies: Metabolic studies were performed on male mice. At least eight mice were analyzed per group. Daily food intake was measured in 16-week old mice. Blood samples were collected from the tail vein. Blood glucose levels were measured using the OneTouch Vita blood glucose meter (LifeScan, Milpitas, CA, USA). Plasma insulin levels were determined by ELISA kit (ALPCO, Salem, NH, USA). Glucose tolerance tests were performed on mice fasted for 16h by glucose injection (2 g/kg, i.p.) as previously described {Rachdi, 2008} . Insulin tolerance tests were carried out on mice that had been fasted for 6h and glucose measurements were taken at 30, 60 and 120min after i.p. injection of insulin (0.75U/kg). Pyruvate tolerance tests were done on mice that had been fasted for 16h and then glucose measurements were taken at 30, 60 and 120min after i.p. injection of sodium pyruvate (1.5 g/kg; Sigma- Aldrich, St Quentin Fallavier, France).

Islet studies: Islets were isolated by collagenase digestion followed by purification through a Histopaque gradient (Sigma-Aldrich). Insulin secretion was assessed by static incubation of isolated islets in Millicell inserts (Millipore). Briefly, following overnight culture in RPMI medium containing 5.6mmol/l glucose, the islets were pre-incubated for lh in Krebs-Ringer medium containing 2.8mmol/l glucose. Groups of 50 islets in triplicate were then incubated in Krebs-Ringer medium containing 2.8 or 20mmol/l glucose for lh. Secreted insulin and the insulin contents were measured using an ultrasensitive mouse insulin ELISA (ALPCO). DNA content was measured using Quant-iT™ PicoGreen ® dsDNA Kit from Invitrogen (Saint Aubin, France). For cell proliferation analyses, islet were cultured 7 days and pulsed with 10 mM of bromodeoxyuridine (BrdU) for 1 h before fixation.

Western blotting; Protein lysates from pancreases at different stage of life were subjected to immunoblotting. The following antibodies were used: DyrklA (Abnova, Taipei City, Taiwan, 1 : 1,000) and Actin (Sigma, 1 :2,000). Immunoblotting experiments were performed three times. Quantification of the Western blots by densitometry was done using NIH Image J software and normalized against that of actin.

Immunohistochemistry and quantification: Pancreases were immersed in 10% formalin and embedded in paraffin. Sections

were processed for immunohistochemistry {Rachdi, 2003} . Antibodies were used at the following dilutions: mouse anti-insulin (1 :2,000; Sigma); rabbit anti-insulin (1 :2,000; Dako, Trappes, France); mouse anti-glucagon (1 :2,000; Sigma); mouse anti-Ki67 (1 :20; BD Biosciences, Le Pont de Claix, France) and mouse anti-BrdU (1 :4; Amersham, Courtaboeuf). The fluorescent secondary antibodies were from Jackson ImmunoResearch (Soham, UK). The nuclei were stained using the Hoechst 33342 fluorescent stain (0.3 mg/ml; Invitrogen).

Sections were digitized using cooled three-CCD cameras (C5810 or C7780;

Hamamatsu, Massy, France) attached to a fluorescence microscope (Leitz DMRB; Leica, Nanterre, France). Quantification of insulin staining was performed on five equally separated sections for adult pancreases and on every second slide for fetal pancreases. In each section, the beta cell area and the pancreatic area were determined using NIH Image J software (vl .31; freely available at htt ://rsb nfo .nih. ov ij index.html) . For adult pancreases, the percentage of the beta cell area in each pancreatic section was determined by dividing the total area of insulin-positive cells by the surface area of the section and the beta cell mass was calculated by multiplying the pancreas weight by the % area of beta cells. For fetal pancreases, immunostained areas were quantified using NIH Image J software on every image, and then summed to obtain the total area per explant in mm², as previously described {Rachdi, 2012} .

Beta cell size was calculated by dividing the beta cell area by the number of beta cell nuclei using NIH Image J software, as previously described {Rachdi, 2008} . RNA extraction and real-time PCR: Total RNA was extracted using an RNeasy Microkit (Qiagen, Courtaboeuf, France), and then reverse transcribed using Superscript reagents (Invitrogen). Real-time PCR was performed with the 7300 Fast real-time PCR system (Applied Biosystems, Courtaboeuf, France). The oligonucleotide sequences are available upon request. Cyclophilin A was used as the internal reference control.

Statistical analysis: Quantitative data are presented as the mean ± SEM from at least three independent experiments, unless indicated. Interaction between the variables was investigated by two-way analysis of variance, and an unpaired Student's t test was used to compare the independent means. Statistical significance was set at p< 0.05. Results: mBACTgDyrkIA mice are glucose tolerant and hyperinsulinemic:

We first examined whether upregulation of DYR IA resulted in metabolic alterations. There were no significant differences in body weight during the 24 weeks of observation. Interestingly, from week 28, body weight continued to increase in wild-type mice while it reached a plateau in mBACTgDyrkIA mice. This difference in weight growth rate was not linked to any variation in food consumption between the two genotypes but associated with a decreased white fat mass observed in aged mBACTgDyrkIA mice. Glycemia measurements indicate lower fasting blood glucose levels in mBACTgDyrkIA mice compared to wild type mice. Lower glycaemia was observed in 4-week old mBACTgDyrkIA mice and remained lower at all time points tested up to 48 weeks. This was associated with increased glucose tolerance, observed in intraperitoneal glucose tolerance tests. In parallel, mBACTgDyrkIA mice had higher plasma insulin levels than wild-type mice at all time points tested between weeks 4 and 48. Insulin secretory response following i.p. injection of glucose was then assessed. Glucose-stimulated insulin secretion was greater in mBACTgDyrkIA mice than wild-type mice. In contrast, peripheral glucose assimilation, or clearance, assessed by the insulin tolerance test, were unchanged compared to controls. Finally, after pyruvate administration the blood glucose levels were similar in both groups of mice indicating no defect in the liver in term of gluconeogenesis. Since Dyrkla upregulation resulted in improved glucose tolerance and increased insulin levels, while insulin sensitivity was unchanged, we next focused on the effect of DYRKIA on pancreatic beta cells. - -

Insulin secretion and gene expression in islets from mBACTgDyrklA mice:

In our study, when compared to wild type mice, mBACTgDyrklA mice show increased DyrklA expression levels in pancreatic samples at all tested experimental time points. This increased level of expression was observed also in other tissues expressing DyrklA. At the protein level, DYRK1A expression was increased in pancreas of mBACTgDyrklA mice compared to wild-type mice.

Immunofluorescence staining for insulin and glucagon indicates that the islet architecture is conserved in mBACTgDyrklA mice, with insulin-positive cells in the core of the islet and non-beta cells in the periphery. The glucagon/insulin ratio is also conserved in mBACTgDyrklA mice. We next compared insulin secretion in isolated islets from mBACTgDyrklA and wild-type mice. When expressed as percentage of insulin content, insulin secretion levels at un- stimulating (2.8mM) and stimulating (20mM) glucose levels are similar for mutant and control islets, demonstrating the functionality of islets from mBACTgDyrklA mice. Significantly, the total insulin content of islets from mBACTgDyrklA mice is higher than that of islets from wild-type mice. Thus, quantitatively, when reported per μg of islet protein or per μg of islet DNA, islets from mBACTgDyrklA mice secrete more insulin than islets from wild-type mice. Therefore, islets from mBACTgDyrklA mice are functional and they contain and secrete more insulin than islets from wild-type mice. Comparative gene expression analysis between islets from wild-type and mBACTgDyrklA mice confirm the upregulation of Dyrkla in mutant islets. Insulin, Glucagon and Somatostatin expression is increased in islets from mBACTgDyrklA mice. In parallel, Mafa and Neurodl expression, which encode two insulin gene transactivators, is higher, while the expression of Pdxl, another insulin transactivator, is not modulated by Dyrkla upregulation. Finally, the expression of Glut2 (also known as Slc2a2) and Znt8 (also known as Slc30a8), which are both involved in insulin secretion, is unchanged.

Increased beta cell mass, proliferation and size in mBACTgDyrklA mice:

In 16 week-old adult mice, pancreatic weight and beta cell area, measured following insulin immunostaining, are both higher in mBACTgDyrklA mice, showing a 2.16 fold increase in absolute beta cell mass when compared wild-type pancreases. Beta cell size measurement demonstrated a 34% increase in mBACTgDyrklA mice. The beta cell proliferation rate, measured as the percentage of insulin-positive cells that stained positive for Ki67, is also more than 2-fold higher in mBACTgDyrklA mice. Comparative gene expression analysis of genes involved in proliferation indicates that the expression of Ccndl, Ccnd2 and Ki67, three cell-cycle-related genes, is also higher in islets from mBACTgDyrklA mice.

Interestingly, quantification of insulin staining performed at El 7, shows that, beta cell mass is already higher in mBACTgDyrklA than wild-type mice during prenatal development. At this prenatal stage, beta cell proliferation, measured following Ki67 immunostaining, is also higher in mBACTgDyrklA mice compared with wild-type mice.

We next asked whether this phenotype was pancreas autonomous. For this purpose, we isolated and cultured islets for seven days before a pulse of BrdU. Brdu staining shows that in-vitro, beta cell proliferation is higher in mBACTgDyrklA mice compared with wild-type mice as is the case in-vivo. mBACTgDyrklA mice are resistant to high-fat diet-induced diabetes:

In mice, an HFD results in insulin resistance, hyperinsulinemia and increased beta cell mass. However, beta cell hyperplasia is not sufficient to prevent glucose intolerance. As mBACTgDyrklA mice have an increased beta cell mass associated with increased insulin secretion, we tested whether mBACTgDyrklA would combat high fat induced diabetes. Four- week old mBACTgDyrklA and wild type mice were fed either 60% fat or a normal diet for 12 weeks. On the HFD, wild type mice gain weight, while mBACTgDyrklA show a clear resistance to weight gain. After 12 weeks of HFD, wild type mice develop glucose intolerance, which is not the case in mBACTgDyrklA mice, which show a curve similar to control mice under a normal diet. Of note, while in wild type mice an HFD induces insulin resistance, this is not the case in mBACTgDyrklA mice. Consistent with their overall improved metabolic phenotype under HFD, mBACTgDyrklA mice have increased beta cell mass relative to wild type mice. Taken together these results indicate that mBACTgDyrklA mice resist against HFD-induced diabetes.

EXAMPLE 3: Adipogenesis is regulated by the kinase DYRK1A. Material & Methods

Animals: The generation of mBACTgDyrklA and DyrklA^+/" mice has previously been described. Wild-type and Dyrkla mutant mice were used in accordance with French Animal Care Committee guidelines. The mice were raised on a 12 h light-dark cycle. They were fed with a standard laboratory chow diet supplemented or not with DYRKIA inhibitor derived from EGCG (epigallocatechin-gallate).

Immunohistochemistry and quantification: Fat pads were immersed in 10% formalin and embedded in paraffin. Sections (4μιη-ΐ1ιίΰ1ί) were processed for trichrome staining.

Sections were digitized using cooled three-CCD cameras (C5810 or C7780; Hamamatsu, Massy, France) attached to a fluorescence microscope (Leitz DMRB; Leica, Nanterre, France). Adipocyte cell size was calculated by dividing the adipocyte area by the number of adipocyte nuclei using NIH Image J software.

Statistical analysis: Quantitative data are presented as the mean ± SEM from at least three independent experiments, unless indicated. Interaction between the variables was investigated by two-way analysis of variance, and an unpaired Student's t test was used to compare the independent means. Statistical significance was set at p< 0.05. Results

DYRKIA is a key factor regulating adipogenesis.

We next examined whether the expression level of DyrklA affect fat cell size within the adipose tissue. Fat cell sizes from adipose tissue sections were determined in 16 week old DyrklA^+/~ and wild-type animal by automated image analysis. Histological examination clearly showed an increase in adipocyte cell size in DyrklA haploinsufficient mice.

Inhibitor of DYRKIA induces obesity.

In mice, one month diet supplemented with EGCG derived inhibitor of DyrklA results in an increased white fat mass. As mBACTgDyrkIA mice have a lowest white fat mass we tested the effect of the DYRKIA inhibitor. We clearly observed a loss of the protective effect against fat accumulation observed in the mBACTgDyrkIA mice under DYRKIA inhibitor treatment. These results suggest that DYRKIA regulates the adipogenesis.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Ferrannini E (2010) The stunned beta cell: a brief history. Cell Metab 11 : 349-352

Butler AE, Janson J, Bonner- Weir S, Ritzel R, Rizza RA, Butler PC (2003) Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52: 102-110 Ashcroft FM, Rorsman P (2012) Diabetes mellitus and the beta cell: the last ten years.

Cell 148: 1160-1171

Rahier J, Guiot Y, Goebbels RM, Sempoux C, Henquin JC (2008) Pancreatic beta-cell mass in European subjects with type 2 diabetes. Diabetes Obes Metab 10 Suppl 4: 32-42

Sachdeva MM, Staffers DA (2009) Minireview: Meeting the demand for insulin: molecular mechanisms of adaptive postnatal beta-cell mass expansion. Mol Endocrinol 23 : 747-758

Aranda S, Laguna A, de la Luna S (2011) DYR family of protein kinases: evolutionary relationships, biochemical properties, and functional roles. FASEB J 25:449-462 Becker W, Joost HG (1999) Structural and functional characteristics of Dyrk,a novel subfamily of protein kinases with dual specificity. Prog Nucleic Acid Res Mol Biol 62: 1-17 Tejedor FJ, Hammerle B (2011) MNB/DYRK1A as a multiple regulator of neuronal development. FEBS J 278: 223-235

Fotaki V, Dierssen M, Alcantara S, et al. (2002) DyrklA haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice. Mol Cell Biol 22: 6636-6647

Delabar JM, Theophile D, Rahmani Z, et al. (1993) Molecular mapping of twenty- four features of Down syndrome on chromosome 21. Eur J Hum Genet 1 : 114- 124

Oegema R, de Klein A, Verkerk AJ, et al. (2010) Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRKIA. Mol Syndromol 1 : 113-120

Atouf F, Czernichow P, Scharfmann R (1997) Expression of neuronal traits in pancreatic beta cells. Implication of neuron-restrictive silencing factor/repressor element silencing transcription factor, a neuron-restrictive silencer. J Biol Chem 272: 1929-1934

Rorsman P (1997) The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia 40: 487-495 van Arensbergen J, Garcia-Hurtado J, Moran I, et al. (2010) Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program. Genome Res 20: 722-732

Rachdi L, Balcazar N, Osorio-Duque F, et al. (2008) Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1- dependent manner. Proc Natl Acad Sci U S A 105: 9250-9255

Attali M, Stetsyuk V, Basmaciogullari A, et al. (2007) Control of beta-cell differentiation by the pancreatic mesenchyme. Diabetes 56: 1248-1258

Rachdi L, Marie JC, Scharfmann R (2003) Role for VPAC2 Receptor-Mediated Signals in Pancreas Development. Diabetes 52: 85-92.

Rachdi L, Balcazar N, Elghazi L, et al. (2006) Differential effects of p27 in regulation of beta-cell mass during development, neonatal period, and adult life. Diabetes 55: 3520-3528

Guillemain G, Filhoulaud G, Da Silva-Xavier G, Rutter GA, Scharfmann R (2007) Glucose is necessary for embryonic pancreatic endocrine cell differentiation. J Biol Chem 282: 15228-15237

Rachdi L, Aiello V, Duvillie B, Scharfmann R (2012) L-leucine alters pancreatic beta- cell differentiation and function via the mTor signaling pathway. Diabetes 61 : 409-417

Waki H, Park KW, Mitro N, et al. (2007) The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARgamma expression. Cell Metab 5: 357-370 Olbrot M, Rud J, Moss LG, Sharma A (2002) Identification of beta-cellspecific insulin gene transcription factor RIPE3bl as mammalian MafA. Proc Natl Acad Sci U S A 99: 6737- 6742

Naya FJ, Huang HP, Qiu Y, et al. (1997) Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD deficient mice. Genes Dev 11 : 2323-2334

Ohlsson H, Karlsson K, Edlund T (1993) IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 12: 4251-4259

Uchida T, Nakamura T, Hashimoto N, et al. (2005) Deletion of Cdknlb ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia in diabetic mice. Nat Med 11 : 175-182

Georgia S, Bhushan A (2006) p27 Regulates the transition of beta-cells from quiescence to proliferation. Diabetes 55: 2950-2956 Woods YL, Rena G, Morrice N, et al. (2001) The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site. Biochem J 355: 597-607

Bhushan A, Itoh N, Kato S, et al. (2001) FgflO is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128: 5109-5117

Wilson ME, Yang KY, Kalousova A, et al. (2005) The HMG box transcription factor Sox4 contributes to the development of the endocrine pancreas. Diabetes 54: 3402-3409

Fontaniere S, Duvillie B, Scharfmann R, Carreira C, Wang ZQ, Zhang CX (2008) Tumour suppressor menin is essential for development of the pancreatic endocrine cells. J Endocrinol 199: 287-298

Hashimoto N, Kido Y, Uchida T, et al. (2006) Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of loss of beta cell mass. Nat Genet 38: 589-593

Hammerle B, Ulin E, Guimera J, Becker W, Guillemot F, Tejedor FJ (2011) Transient expression of Mnb/Dyrkla couples cell cycle exit and differentiation of neuronal precursors by inducing p27KIPl expression and suppressing NOTCH signaling. Development 138: 2543-2554

Martin DE, Soulard A, Hall MN (2004) TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell 119: 969-979

Yang SB, Lee HY, Young DM, et al. (2012) Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity. J Mol Med (Berl) 90: 575-585

Guedj F, Pereira PL, Najas S, Barallobre M-J, Chabert C, Souchet B, Sebrie C, Verney C, Herault Y, Arbones M, et al. (2012) DYR IA: a master regulatory protein controlling brain growth. Neurobiol Dis; 46: 190-203.

Zhang Y, Liao JM, Zeng SX, Lu H (2011) p53 downregulates Down syndrome- associated DYRKIA through miR-1246. EMBO Rep. Jun 3;12(8):811-7.

da Costa Martins PA, Salic K, Gladka MM, Armand AS, Leptidis S, el Azzouzi H, Hansen A, Coenen-de Roo CJ, Bierhuizen MF, van der Nagel R, van Kuik J, de Weger R, de Bruin A, Condorelli G, Arbones ML, Eschenhagen T, De Windt LJ.(2010) MicroRNA-199b targets the nuclear kinase Dyrkla in an auto-amplification loop promoting calcineurin/NFAT signalling. Nat Cell Biol. Dec; 12(12): 1220-7.

Claims

CLAIMS:

1. A dual- specificity tyrosine phosphorylation-regulated kinase DYRKIA polypeptide or a functional equivalent thereof for use in preventing or treating a metabolic disorder in a patient in need thereof.

2. The DYRKIA polypeptide for use according to claim 1, wherein the DYRKIA polypeptide has the sequence represented by SEQ ID NO: 1.

3. The DYRKIA polypeptide for use according to claims 1 or 2, wherein said DYRKIA polypeptide is fused to a cell penetrating peptide (CPP).

4. The DYRKIA polypeptide for use according to any one claims 1 to 3, wherein said CPP is the TAT peptide represented by SEQ ID NO: 2.

5. The DYRKIA polypeptide for use according to any one claims 1 to 4, wherein said metabolic disorder is diabetes or obesity.

6. The DYRKIA polypeptide for use according to any one claims 1 to 5, wherein diabetes is type 2 diabetes mellitus (T2DM).

7. The DYRKIA polypeptide for use according to any one claims 1 to 6, wherein said patient is a lean patient.

8. The DYRKIA polypeptide for use according to any one claims 1 to 5, wherein diabetes is type 1 diabetes mellitus (T1DM).

9. A nucleic acid encoding a polypeptide for use according to any one claims 1 to 8.

10. A pharmaceutical composition comprising a DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof and a pharmaceutically acceptable carrier for use according to any one claims 1 to 8.

11. A method for improving proliferation and/or function of a population of pancreatic β- cells in vitro or ex vivo, comprising a step of contacting said population with a culture medium comprising an effective amount of DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof.

12. A method for improving proliferation and/or function of a pancreatic β-cell transplant in vitro or ex vivo, comprising a step of contacting said transplant with a culture medium comprising an effective amount of DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof.

13. A DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use in a method for improving proliferation and/or function of pancreatic β-cells in a patient in need thereof.

14. A DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use in a method for reducing fat accumulation and/or adipogenesis in a patient in need thereof.

15. The DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use according to claim 14, wherein the fat accumulation is abdominal fat accumulation.

16. A DYRKIA polypeptide or a functional equivalent thereof, or a polynucleotide encoding thereof for use in a method for reducing excessive body weight in a patient in need thereof.

17. An inhibitor of a miRNA reducing the expression level of DYRKIA for use in preventing or treating a metabolic disorder in a patient in need thereof.

18. The inhibitor for use according to claim 17, wherein said inhibitor is an antag

miR-199b or of miR-1296.