Targeting of L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain (HADHSC) in disorders of glucose homeostasis

FIELD OF THE INVENTION

The invention provides methods, uses, agents, pharmaceutical formulations and kits for the treatment of disorders of glucose homeostasis and/or for modulating the endogenous production of insulin in subjects. The invention also relates to methods, uses, agents, compositions and kits for modulating the endogenous insulin production by isolated cells or tissues. More in particular, the invention teaches to target L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain (HADHSC). In addition, the invention also provides screening assays to identify agents that modulate the expression and/or the activity of HADHSC and can be useful in the above aspects of the invention.

BACKGROUND OF THE INVENTION

Precise regulation of circulating glucose levels, i.e., physiologically adequate glucose homeostasis, is essential for proper functioning and health of organisms, and it is well- documented that disturbances of glucose homeostasis can hallmark and/or contribute to the aetiology of several prevalent diseases.

For example, the diagnosis of fasting hyperglycaemia (i.e., impaired fasting glucose or IFG) or impaired glucose tolerance (IGT) in a subject suggests a greatly elevated risk of developing diabetes mellitus and in fact signifies a condition increasingly referred to as prediabetes.

Moreover, an excessively increased blood glucose level is also a major feature and a probable aetiological factor of clinical diabetes, which is a devastating condition that can be associated - with a range of complications affecting various organs throughout the body; for example, with various microvascular diseases including, e.g., retinopathy, nephropathy, neuropathy, etc., leading to blindness, kidney failure, etc.

Elevated circulating glucose levels also hallmark and plausibly contribute to the development of metabolic syndrome, i.e., a cluster of abnormalities that tend to co-occur in a subject and represent major risk factors for the development of coronary artery disease (CAD), such as premature atherosclerotic vascular disease. These abnormalities may involve, apart from impaired blood glucose, also truncal obesity, high serum low density lipoprotein (LDL)

cholesterol levels, low serum high density lipoprotein (HDL) cholesterol levels, high serum triglyceride levels, and high blood pressure (hypertension).

Importantly, the incidence of diabetes (particularly type 2 diabetes), metabolic syndrome and other hyperglycaemia-associated conditions continues to rise dramatically in developed societies, largely as a consequence of energy- and carbohydrate-rich diets, sedentary lifestyle, obesity and further risk factors.

On the other hand, hypoglycaemia, i.e., abnormally low circulating glucose levels, such as reactive or fasting hypoglycaemia, can cause neuroglycopenia characterised by symptoms such as, e.g., hunger, nervousness, perspiration, shakiness, dizziness, light-headedness, sleepiness, confusion, difficulty speaking, feeling anxious or weak, etc., and may even bring about a permanent damage to the brain.

Consequently, there exists an urgent need to devise novel and/or improved manners to combat disorders of glucose homeostasis, such as, e.g., the ones above.

Insulin plays a central role in glucose homeostasis and causes a reduction in the circulating glucose levels, generally by increasing the uptake, metabolism and/or storage of glucose in cells of peripheral tissues, most prominently the adipose and muscle tissues. Accordingly, useful therapeutic interventions in disorders of glucose homeostasis may impinge on the endogenous insulin production, thereby advantageously increasing or decreasing the circulating glucose levels. Insulin is a polypeptide hormone synthesised and secreted by the beta (β) cells of the Islets of Langerhans of the pancreas. The production of insulin in β cells responds to the presence and levels of circulating nutrients, in particular glucose. A central pathway that signals the presence and levels of circulating glucose in β cells is believed to involve the glucose transporter which mediates the uptake of glucose by β cells, the enzyme glucokinase (GK) which ensures intracellular phosphorylation of glucose in β cells over a broad range of glucose concentrations, the increased ATP vs. ADP concentration resulting from catabolism of nutrients, esp. glucose by β cells, and an effector system of ATP/ADP-gated K ⁺ channels which translates the high intracellular ATP vs. ADP ratio into channel closing and membrane depolarisation, in turn causing influx of Ca ²⁺ ions through voltage-gated Ca ²⁺ channels and insulin secretion by β cells.

Consequently, the players involved in the above pathway are considered potential targets for therapeutic modulation of insulin secretion, and therapeutic control of glucose homeostasis in disorders thereof, e.g., in diabetes. By means of example, sulfonylurea class of compounds, which induce closing of the ATP/ADP-gated K ⁺ channels, are prescribed in type 2 diabetes. Yet, given the increasing incidence of glucose homeostasis disorders, there exists a need to identify further cellular targets playing a role in the control of endogenous insulin production by β cells, and to provide agents and methods that desirably impinge on such novel targets and can be therapeutic in glucose homeostasis disorders.

L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain (HADHSC) (EC.1.1.1.35) is a part of the mitochondrial beta-oxidation pathway of fatty acids. It catalyses the reversible dehydrogenation of 3-hydroxyacyl-CoAs to their corresponding 3-ketoacyl-CoAs with concomitant reduction of NAD+ to NADH.

In humans, loss-of-function mutations in the HADHSC gene on chromosome 4q22-q26 have been associated with familial hyperinsulinemic hypoglycaemia of infancy which is characterised, among others, by excessive beta cell activation under fasting conditions (Clayton et al. 2001. J Clin Invest 108: 457-65; Molven et al. 2004. Diabetes 53: 221- 7). However, since these mutations in HADHSC occurred and presumably affected beta- oxidation in essentially all tissues, this prior art could not discriminate whether the HADHSC mutations had any intrinsic effect on pancreatic β cells or whether the hyperinsulinemia was secondary to general effects of the HADHSC mutations in other tissues. Moreover, the prior art also did not teach whether the lack of HADHSC would show any further effects in conditions where the subjects had increased blood glucose levels and where their insulin production by β cells was thus presumably potentiated. Also, due to the loss-of-function nature of the above HADHSC mutations, the prior art did not teach whether hyperinsulinemia would result in case that the lack of HADHSC was not complete, e.g., in conditions permitted beta-oxidation to take place.

SUMMARY OF THE INVENTION

In aspects, the present invention provides uses, methods, assays, agents, compositions and kits that address the above needs in the art.

More specifically, the invention surprisingly realises that agents that modulate the expression or the activity of L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain (HADHSC) can be useful in therapy of disorders of glucose homeostasis.

It should be noted that recently HADHSC has been renamed to "L-3-hydroxyacyl-CoA dehydrogenase" or "HADH", i.e., without the qualifier "short chain". Accordingly, reference anywhere in this specification to L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain or HADHSC should be read as encompassing a reference to the newly accepted designations L-3-hydroxyacyl-CoA dehydrogenase and HADH, respectively.

In addition, the invention also discloses that agents modulating the expression or the activity of HADHSC can control endogenous insulin production by isolated cells or tissues, preferably by isolated pancreatic β cells or tissues comprising such.

More in particular, as shown in the experimental section the present inventors surprisingly realised that HADHSC mRNA and protein is highly expressed in pancreatic β cells and cell lines as compared to a wide variety of other tissues, including even tissues with high rates of mitochondrial beta-oxidation of fatty acids (liver, muscle, kidney). Moreover, specifically in pancreatic β cells and cell lines, the expression of HADHSC is disproportionate to the expression of other enzymes of the beta-oxidation pathway.

Accordingly, the high expression of HADHSC specifically in pancreatic β can advantageously impose a degree of selectivity on treatments that target HADHSC, such that adverse effects of said treatments in other tissues, including tissues with high rate of mitochondrial beta- oxidation, would be minimised.

Hence, hypothesising that this striking expression pattern may signify an intrinsic role of HADHSC in pancreatic β cells, the inventors performed experiments in which they modulated HADHSC in pancreatic β cells or cell lines and followed the effect of such HADHSC modulation on the insulin production by the said cells. Again surprisingly, the modulation of HADHSC affected production of (i.e., the biosynthesis and/or secretion of) insulin by the said cells and even affected nutrient-responsiveness (esp. glucose-responsiveness) of insulin production by the β cells. This shows that HADHSC modulation can be useful even in non- fasting, basal conditions, i.e., conditions where β cells are already exposed to nutrients, esp. to glucose, as may be observed, e.g., in disorders characterised by hyperglycaemia.

Moreover, the effects were observed even at low levels of HADHSC modulation (e.g., reduction or inhibition) when there appeared to be no toxic side-effects.

The present invention integrates the above relevant realisations in its diverse aspects.

Hence, in an aspect the invention concerns an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC, for use as a medicament, particularly to treat a disorder of glucose homeostasis and/or to modulate the endogenous production of insulin.

In a further aspect the invention relates to the use of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC, for the preparation of a medicament for the treatment of a disorder of glucose homeostasis.

In a related aspect the invention relates to the use of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC, for the preparation of a medicament for modulating the endogenous production of insulin.

In an aspect the invention provides a method for treating a disorder of glucose homeostasis in a subject needing said therapy, comprising administering to the said subject a therapeutically effective amount of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC.

In a related aspect the invention provides a method for modulating the endogenous production of insulin in a subject needing said modulating, comprising administering to the said subject a therapeutically effective amount of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC.

In a further aspect, the invention provides a method comprising: (1 ) identifying or generating an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC; and (2) using the said agent for the preparation of a medicament for the treatment of a disorder of glucose homeostasis.

In a related aspect, the invention provides a method comprising: (1 ) identifying or generating an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC; and (2) using the said agent for the preparation of a medicament for modulating the endogenous production of insulin. In another aspect, the invention discloses a method for treating a disorder of glucose homeostasis in a subject needing said therapy, comprising: (1 ) identifying or generating an

agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC; and (2) administering to the said subject a therapeutically effective amount of the said agent.

In a related aspect, the invention discloses a method for modulating the endogenous production of insulin in a subject needing said modulating, comprising: (1 ) identifying or generating an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC; and (2) administering to the said subject a therapeutically effective amount of the said agent.

In a further aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC, or a pharmaceutically acceptable salt of said agent. Said pharmaceutical composition may commonly also comprise one or more of pharmaceutically acceptable buffers, carriers, excipients, stabilisers, etc.

In a further aspect, the invention provides kits comprising the above agent(s) or pharmaceutical composition(s) alongside other reagent(s), composition(s) or device(s) generally useful in the treatment of disorders of glucose homeostasis.

In still further aspect, the invention provides an assay to select, from a group of test agents, a candidate agent potentially useful as a therapeutic in the treatment of a disorder of glucose homeostasis and/or in modulating the endogenous production of insulin, said assay comprising determining whether a tested agent (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC.

The said assay may further comprise monitoring the effect, e.g., therapeutic effect, of the so- selected candidate agent when administered to an in vitro or in vivo model of the disorder of glucose homeostasis, e.g., a cellular, tissue or organism model, e.g., a non-human animal model, preferably a non-human mammal model. Otherwise, the said assay may comprise use of the so-selected candidate agent for the preparation of a composition for administration to and monitoring the effect, e.g., therapeutic effect, in a non-human animal model, preferably a non-human mammal model, of the disorder of glucose homeostasis.

The said assay may otherwise or in addition further comprise monitoring the effect, e.g., effect on the endogenous production of insulin, of the so-selected candidate agent when administered to an in vitro or in vivo model, e.g., a cellular, tissue or organism model, e.g., a

non-human animal model, preferably a non-human mammal model (e.g., as above). Else, the said assay may comprise use of the so-selected candidate agent for the preparation of a composition for administration to and monitoring the effect, e.g., effect on the endogenous production of insulin, in a non-human animal model, preferably a non-human mammal model (e.g., as above).

In a further related aspect, the invention concerns the use of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC to modulate endogenous production of insulin in an isolated cell or tissue, preferably isolated pancreatic β cells or tissues comprising such. As well as a corresponding method for modulating endogenous production of insulin in an isolated cell or tissue, preferably in isolated pancreatic β cells or tissues comprising such, comprising exposing the said isolated cell or tissue to an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC, such that modulation of endogenous production of insulin in the isolated cell or tissue is achieved. As well as the corresponding use of an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC for the manufacture of a medicament to modulate endogenous production of insulin in an isolated cell or tissue, preferably isolated pancreatic β cells or tissues comprising such.

For example, it is a known phenomenon that isolated pancreatic β cells in tissue culture gradually diminish insulin production (e.g., biosynthesis and/or secretion thereof), presumably due to their constant exposure to (high) glucose in such conditions. Accordingly, agents that modulate, and in particular reduce the expression or activity of HADHSC, can sustain insulin production in such cells. This can advantageously allow to improve the metabolic state of isolated β cells or tissues comprising such, e.g., before their transplantation to a recipient subject in clinical settings, or during their study in research settings, etc. The invention also concerns the ensuing particularly preferred, yet exemplary and non-limiting embodiments of the above aspects.

In an embodiment, the agent (a) can reduce the expression of HADHSC and/or (b) can reduce the activity of HADHSC.

In a related embodiment, the agent (a) can reduce the expression of HADHSC and/or (b) can reduce the activity of HADHSC, and is employed in disorders of glucose homeostasis which involve hyperglycaemia.

In a related embodiment, the agent (a) can reduce the expression of HADHSC and/or (b) can reduce the activity of HADHSC, and is employed in disorders of glucose homeostasis chosen from prediabetes; diabetes type 1 or 2, preferably diabetes type 2; or metabolic syndrome.

In a related embodiment, the agent (a) can reduce the expression of HADHSC and/or (b) can reduce the activity of HADHSC is employed for increasing endogenous insulin production.

In an embodiment, the agent that can reduce the expression of HADHSC is an antisense agent, preferably, an antisense oligonucleotide; or a ribozyme; or an agent capable of causing RNA interference.

In an embodiment, the agent that can reduce the activity of HADHSC is a polypeptide or protein; an antibody, preferably an intrabody; a peptide; a peptidomimetic; an aptamer; a chemical substance, preferably an organic molecule, more preferably a small organic molecule; a lipid; a carbohydrate; a nucleic acid, etc.

In an embodiment, the agent that can reduce the activity of HADHSC binds to HADHSC.

In an embodiment, the agent (a) can increase the expression of HADHSC and/or (b) can increase the activity of HADHSC.

In a related embodiment, the agent (a) can increase the expression of HADHSC and/or (b) can increase the activity of HADHSC, and is employed in disorders of glucose homeostasis which involve hypoglycaemia.

In a related embodiment, the agent (a) can increase the expression of HADHSC and/or (b) can increase the activity of HADHSC, and is employed in disorders of glucose homeostasis chosen from hyperinsulinemia or neuroglycopenia.

In a related embodiment, the agent (a) can increase the expression of HADHSC and/or (b) can increase the activity of HADHSC and is employed to reduce endogenous insulin production. In an embodiment, the agent that can increase the expression of HADHSC is a recombinant nucleic acid encoding HADHSC or a functional variant or fragment thereof.

In an embodiment, the agent that can increase the activity of HADHSC is a polypeptide or protein; an antibody, preferably an intrabody; a peptide; a peptidomimetic; an aptamer; a chemical substance, preferably an organic molecule, more preferably a small organic molecule; a lipid; a carbohydrate; a nucleic acid, etc.

In an embodiment, the agent that can increase the activity of HADHSC binds to HADHSC.

Hence, in further particularly preferable aspects and embodiments the invention also provides the subject matter as disclosed in any one of (I) to (VIII) below:

(I) the use of an agent that is able to reduce the expression of HADHSC, said agent chosen from an antisense agent, an antisense oligonucleotide, a ribozyme, or an agent capable of causing RNA interference, for the preparation of a medicament for the treatment of a disorder of glucose homeostasis chosen from hyperglycaemia, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), prediabetes, diabetes type 1 , diabetes type 2 and metabolic syndrome; (II) a method comprising: (1 ) identifying or generating an agent that can reduce the expression of HADHSC, said agent chosen from an antisense agent, an antisense oligonucleotide, a ribozyme, or an agent capable of causing RNA interference; and (2) using said agent for the preparation of a medicament for the treatment of a disorder of glucose homeostasis chosen from hyperglycaemia, IGT, IFG, prediabetes, diabetes type 1 , diabetes type 2 and metabolic syndrome;

(III) the use as set forth in (I) above or the method as set forth in (II) above, wherein said agent that can reduce the expression of HADHSC is an agent capable of causing RNA interference with HADHSC;

(IV) the use or method as set forth in (III) above, wherein the agent capable of causing RNA interference with HADHSC is chosen from short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA);

(V) the use or method as set forth in any of (III) or (IV) above, wherein the agent capable of causing RNA interference is produced by chemical synthesis, by enzymatic synthesis or recombinantly expressed from a vector in a cell;

(VI) the use or method as set forth in any of (IV) or (V) above, wherein the siRNA has at least 90% sequence identity with HADHSC mRNA;

(VII) the use or method as set forth in (Vl) above, wherein the siRNA targets a sequence in human HADHSC mRNA corresponding to any of the sequences represented by SEQ ID NO: 4 to SEQ ID NO: 26, as listed in Figure 15, in human HADHSC cDNA;

(VIII) the use or method as set forth in (VII) above, wherein the siRNA contains no more than 1 sequence variation per 10 basepairs compared to its respective HADHSC target sequence.

In additional particularly preferable aspects and embodiments the invention also provides the subject matter as disclosed in any one of (IX) to (XV) below: (IX) an assay to select, from a group of test agents, a candidate agent potentially useful as a therapeutic in the treatment of a disorder of glucose homeostasis chosen from hyperglycaemia, IGT, IFG, prediabetes, diabetes type 1 , diabetes type 2 and metabolic syndrome, said assay comprising determining whether a tested agent (a) reduces the expression of HADHSC and/or (b) reduces the activity of HADHSC; (X) the assay as set forth in (IX) above, comprising: (a) providing a cell expressing HADHSC or a functional variant or functional fragment thereof, (b) introducing to said cell a test agent, and (c) determining the expression of said HADHSC or functional variant or functional fragment thereof, thereby identifying whether the test agent reduces said expression;

(XI) the assay as set forth in (IX) above, comprising: (a) combining HADHSC or a functional variant or functional fragment thereof and a test agent, and (b) detecting whether said test agent reduces the activity of said HADHSC or functional variant or functional fragment thereof;

(XII) the assay as set forth in (Xl) above, wherein the HADHSC or functional variant or functional fragment thereof is in solution, affixed to a solid support, born on a cell surface, or located intracellular^;

(XIII) the assay as set forth in any of (IX), (Xl) or (XII) above, comprising: (a) assessing the ability of a test agent to bind to HADHSC or to a functional variant or functional fragment thereof, and (b) testing whether a test agent that binds to HADHSC or to functional variant or functional fragment thereof, reduces the activity of HADHSC; (XIV) the assay as set forth in any of (IX) to (XIII) above, further comprising monitoring the effect of the test agent on endogenous production of insulin;

(XV) the assay as set forth in any of (IX) to (XIV) above, further comprising use of the selected candidate agent for the preparation of a composition for administration to and monitoring the therapeutic effect thereof in a non-human animal model, preferably a non- human mammal model, of said disorder of glucose homeostasis.

These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims.

BRIEF DESCRIPTION OF FIGURES Figure 1 illustrates representative human HADHSC sequences.

Figure 2 illustrates a representative human HADHSC cDNA sequence.

Figure 3 illustrates a schematic overview of the organization of beta-oxidation pathway, as proposed by Liang et al. 2001 (Biochem Soc Trans 29: 279-282).

Figure 4 illustrates mRNA expression levels of HADHSC mRNA in various tissues vis-a-vis other components of the beta-oxidation pathway.

Figure 5 summarises data as in Figure 4 in table format.

Figure 6 illustrates HADHSC protein expression in various tissues.

Figure 7 illustrates siRNA mediated downregulation of HADHSC in INS1 β cells.

Figure 8 illustrates insulin production in cells with siRNA mediated downregulation of HADHSC in dependence on glucose or fatty acids in INS1 β cells.

Figure 9 illustrates shRNA mediated downregulation of HADHSC analysed on mRNA and protein level in INS1 β cells.

Figure 10 illustrates the effect of shRNA mediated downregulation of HADHSC on insulin biosynthesis and secretion in INS1 β cells. Figure 11 illustrates the effect of shRNA mediated downregulation of HADHSC on NAD and FAD metabolism in INS1 β cells.

Figure 12 illustrates the effect of siRNA and shRNA mediated downregulation of HADHSC on glucose oxidation in INS1 β cells.

Figure 13 illustrates HADHSC downregulation by siRNA in FACS isolated primary rat β cells. Figure 14 illustrates the effect of siRNA mediated HADHSC downregulation on insulin production in FACS isolated primary rat β cells.

Figure 15 summarises preferred siRNA target sequences as predicted in human HADHSC cDNA sequence annotated under GenBank accession number NM_005327 (also shown in Figure 2 as SEQ ID NO: 3) using various algorithms.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a cell" refers to one or more than one cells.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +1-5% or less, even more preferably +/-1 % or less, and still more preferably +/-0.1 % or less from the specified value, insofar such variations are appropriate to perform in the disclosed invention.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

Hence, aspects of the invention concern agents that can modulate the expression of HADHSC, as well agents that can modulate the activity of HADHSC, and uses of such agents, in particular in therapy, especially in the treatment of disorders of glucose homeostasis and in modulating endogenous insulin production, as set out in the Summary section.

As used herein, the term "agent" broadly refers to any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biological macromolecule), a combination or mixture thereof, a sample of undetermined composition, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Preferred though non-limiting "agents" include nucleic acids, oligonucleotides, ribozymes, polypeptides or proteins, a peptides, peptidomimetics, antibodies and fragments

and derivatives thereof, aptamers, chemical substances, preferably organic molecules, more preferably small organic molecules, lipids, carbohydrates, polysaccharides, etc., and any combinations thereof.

The terms "polypeptide" and "protein" are used interchangeably herein and generally refer to a polymer of amino acid residues linked by peptide bonds, and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, polypeptides, dimers (hetero- and homo- ), multimers (hetero- and homo-), and the like, are included within the definition. Both full- length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, etc. Furthermore, for purposes of the present invention, the terms also refer to such when including modifications, such as deletions, additions and substitutions (e.g., conservative in nature), to the sequence of a native protein or polypeptide.

The term "peptide" as used herein preferably refers to a polypeptide as used herein consisting essentially of <50 amino acids, e.g., <45 amino acids, preferably <40 amino acids, e.g., <35 amino acids, more preferably <30 consecutive amino acids, e.g., <25, <20, <15, <10 or <5 amino acids.

The term "nucleic acid" as used herein means a polymer of any length composed essentially of nucleotides, e.g., deoxyribonucleotides and/or ribonucleotides. Nucleic acids can comprise purine and/or pyrimidine bases, and/or other natural, chemically or biochemically modified (e.g., methylated), non-natural, or derivatised nucleotide bases. The backbone of nucleic acids can comprise sugars and phosphate groups, as can typically be found in RNA or DNA, and/or one or more modified or substituted (such as, 2'-O-alkylated, e.g., 2'-O-methylated or 2'-0-ethylated; or 2'-O,4'-C-alkynelated, e.g., 2'-O,4'-C-ethylated) sugars or one or more modified or substituted phosphate groups. For example, backbone analogues in nucleic acids may include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene (methylimino), 3'- N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs).

The term "nucleic acid" further specifically encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, gene, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids. The terms "ribonucleic acid" and "RNA" as used herein mean a polymer of any length composed of ribonucleotides. The terms "deoxyribonucleic acid" and

"DNA" as used herein mean a polymer of any length composed of deoxyribonucleotides. The term "DNA/RNA hybrid" as used herein mean a polymer of any length composed of one or more deoxyribonucleotides and one or more ribonucleotides.

A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised. A nucleic acid can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term "oligonucleotide" as used herein denotes single stranded nucleic acids (nucleotide multimers) of greater than 2 nucleotides in length and preferably up to 200 nucleotides in length, more preferably from about 10 to about 100 nucleotides in length, even more preferably from about 12 to about 50 nucleotides in length. Oligonucleotides can be synthesised by any method known in the art, e.g., by chemical or biochemical synthesis, e.g., solid phase phosphoramidite chemical synthesis, or by in vitro or in vivo expression from recombinant nucleic acid molecules, e.g., bacterial or retroviral vectors.

The term "can", as in, e.g., "can modulate the expression" or "can modulate activity", is synonymous to "is capable of" and signifies that an entity, e.g., an agent, has the ability to achieve the recited effect or action, e.g., when administered to a patient or to a relevant in vitro or in vivo model system, as opposed to achieving the recited effect or action at the exact time of the recitation (which may but need not be the case).

HADHSC

The terms "L-3-hydroxyacyl-CoA dehydrogenase, short-chain" or "HADHSC" and variants thereof refer herein to a mitochondrial enzyme (EC.1.1.1.35) which catalyses the reversible dehydrogenation of 3-hydroxyacyl-CoAs to their corresponding 3-ketoacyl-CoAs with concomitant reduction of NAD+ to NADH (for review see Yang et al. FEBS J. 2005 Oct;272(19):4874-83.). HADHSC has been reported to preferentially act on short- to medium- chain 3-hydroxyacyl-CoA molecules, e.g., preferably ranging from 4 to 10 carbons, and is therefore also known as "medium and short chain L-3-hydroxyacyl-CoA dehydrogenase" or "IWSCHAD". Other synonyms occasionally used for HADHSC include inter alia HAD, HADH, HADSC and HHF4.

Although the terms "short chain 3-hydroxyacyl-CoA dehydrogenase" or "SCHAD" have been at times used to denote HADHSC, these former terms under present nomenclature refer to a different mitochondrial enzyme known as type 10 17β-hydroxysteroid dehydrogenase (HSD10), type 2 hydroxyacyl-CoA dehydrogenase (HADH2) or EC 1.1.1.178 (see Yang et al. supra for details on nomenclature). Hence, the terms SCHAD, HSD10 and HADH2 as presently employed in the art are not synonymous to HADHSC et seq. as defined above.

Recently HADHSC has been renamed to "L-3-hydroxyacyl-CoA dehydrogenase" or "HADH", i.e., without the qualifier "short chain". Accordingly, reference anywhere in this specification to L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain or HADHSC should be read as encompassing a reference to the newly accepted designations L-3-hydroxyacyl-CoA dehydrogenase and HADH, respectively.

The above terms HADHSC and synonyms thereof encompass such proteins from any organism where found, and particularly from animals, preferably vertebrates, more preferably mammals, including humans and non-human mammals. The terms HADHSC and synonyms thereof as used herein refer to said enzymes when forming part of a living organism, organ, tissue, and/or cell, as well as when at least partly isolated therefrom, reconstituted, etc. The terms also encompass HADHSC when one, more or all of its parts have been expressed using recombinant DNA technology.

HADHSC and synonyms as used herein refer to polypeptides with a "native" sequence, i.e., polypeptides of which the primary sequence is the same as that of an HADHSC derived from nature. A skilled person understands that the native sequence of HADHSC may differ between different species due to genetic divergence between such species. Moreover, the native sequence of HADHSC may differ between or even within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, the native sequence of HADHSC may differ between or even within different individuals of the same species due to post-transcriptional modifications, e.g., differential splicing, RNA editing, etc. Accordingly, all HADHSC sequences found in nature, and preferably those defining biologically active molecules, are considered native.

Exemplary HADHSC include, without limitation, human HADHSC with primary amino acid sequence as annotated under Uniprot/Swissprot (http://www.expasy.org/) accession number

Q16836, including isoform 1 (Q16836-1 also shown in Figure 1 as SEQ ID NO: 1 ) and isoform 2 (Q16836-2 also shown in Figure 1 as SEQ ID NO: 2) generated due to alternative

splicing. A skilled person can appreciate that the above human HADHSC sequences may include signal peptides which mediate sorting of the proteins to mitochondria and may be (at least partly) absent from mature proteins. For example, the Uniprot/Swissprot entry for human HADHSC isoform 1 (Q16836-1 ) specifies a signal peptide composed of amino acids 1-12 as shown in SEQ ID NO: 1.

The human HADHSC protein is encoded by the respective HADH gene on chromosome 4q22-q26 annotated under Entrez GenelD 3033 (http://www.ncbi.nlm.nih.gov/entrez).

By "encoded" or "encoding" is meant that a nucleic acid sequence or its part corresponds, by virtue of the genetic code (of an organism in question, preferably mammalian, e.g., human), to a particular amino acid sequence, e.g., the amino acid sequence of a particular polypeptide or protein. By means of example, a nucleic acid sequence "encoding" a particular polypeptide or protein may include naturally-occurring genomic, hnRNA, pre-mRNA, mRNA (or therefrom obtained cDNA) for the said polypeptide or protein, or may include recombinant counterparts or variants of such naturally-occurring nucleic acid sequences. By nucleic acid sequence encoding the HADHSC protein, or any (preferably functional) variant or fragment thereof, is meant a nucleic acid sequence that corresponds, by virtue of the genetic code (of an organism in question, preferably mammalian, e.g., human), to the amino acid sequences of the said HADHSC, variants or fragments. By means of example and not limitation, a nucleic acid sequence encoding the HADHSC protein may include the respective, native genomic, hnRNA, pre-mRNA, mRNA (or therefrom obtained cDNA) sequences for the said protein, or may include recombinant counterparts or variants of such native nucleic acid sequences.

A skilled person understands that native nucleic acid sequences encoding HADHSC may differ between different species due to genetic divergence between such species. Moreover, the native nucleic acid sequences encoding HADHSC may differ between or even within different individuals of the same species due to normal genetic diversity (variation) within a given species, or due to post-transcriptional modifications (e.g., splicing). Accordingly, all nucleic acid sequences encoding HADHSC found in nature, and preferably those encoding biologically functional polypeptide molecules, are considered native. Exemplary cDNA sequence encoding HADHSC include, without limitation, human HADHSC cDNA sequence as annotated in the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) under

accession number NM_005327 (version NM_005327.2), also shown in Fig. 2 as SEQ ID NO: 3.

Addition human cDNA sequences encoding HADHSC or a portion thereof, are annotated in the UniGene transcriptome database (http://www.ncbi.nlm.nih.gov/UniGene) under accession number Hs.438289. These cDNA sequences may possibly contain sequence variations reflecting sequences differences between native nucleic acids (e.g., genomic, mRNA, etc.) encoding human HADHSC.

Further exemplary HADHSC include HADHSC homologues, preferably orthologues, from other organisms, such as, e.g., from other vertebrates, preferably other mammals, such as, e.g., mammals listed elsewhere in this specification. By means of example and not limitation, an HADHSC orthologue from dog is annotated in NCBI GenBank under accession number XP_535685 (protein) and XM_535685.2 (cDNA); from rat under accession number NP_476534.1 (protein) and NM_057186.1 (cDNA); from mouse under accession number NP_032238.1 (protein) and NM_008212.1 (cDNA); etc. In a preferred embodiment, the HADHSC of invention is human HADHSC

Agents modulating the activity of HADHSC

Hence, in an aspect of the invention agents can modulate the activity of HADHSC.

The term "modulate" has its common meaning, is synonymous with, e.g., "alter", "change" or "vary", and in particular encompasses both inhibition and activation of HADHSC activity. Agents that can inhibit or activate one or more aspects of HADHSC biological activity when HADHSC is exposed to said agents are referred to, respectively, as HADHSC "inhibitors" or "activators". These terms may also refer to that administration of an agent to an in vitro system, cell, tissue or an organism comprising HADHSC biological activity, preferably to a patient, will reduce or increase, respectively, one or more aspects of the said HADHSC biological activity than if the said agent had not been administered.

An aspect of HADHSC biological activity is the enzymatic activity thereof, i.e., the capacity of HADHSC to catalyse the reversible dehydrogenation of 3-hydroxyacyl-CoAs to their corresponding 3-ketoacyl-CoAs with concomitant reduction of NAD+ to NADH; accordingly, an HADHSC "inhibitor" may inhibit the enzymatic activity of HADHSC, and an HADHSC "activator" may activate the enzymatic activity of HADHSC. For example, an exemplary way of measuring / testing the level, inhibition or activation of the enzymatic activity of HADHSC

by an agent of interest is shown in, e.g., Example 4 or as described by, e.g., Hanley et al. 2005 (J Physiol 562(Pt 2): 307-18).

The terms "inhibit" and "activate" encompass any extents of, respectively, inhibition or activation. For example, inhibition of one or more aspects of HADHSC biological activity, e.g., preferably its enzymatic activity, may be by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by at least about 96%, 97%, 98%, 99% or even by 100%, when

HADHSC polypeptide is exposed to an inhibitor (preferably, when molar ratio HADHSC : inhibitor is 1 :1 ) or, e.g., as measured in gross mass and/or at the level of individual cells.

Preferably, such inhibition may be by less than about 80%, e.g., less than about 70%, preferably by less than about 60%, e.g., by less than about 50%, more preferably by less than about 45%, e.g., less than about 40%, and even more preferably by less than about 35% or by less than about 30%, when HADHSC polypeptide is exposed to an inhibitor (preferably, when molar ratio HADHSC : inhibitor is 1 :1) or, e.g., as measured in gross mass and/or at the level of individual cells.

Also preferably, such inhibition may be by between about 10% and about 70%, preferably between about 20% and about 60%, more preferably between about 20% and 40%, and even more preferably between about 20% and 30%, when HADHSC polypeptide is exposed to an inhibitor (preferably, when molar ratio HADHSC : inhibitor is 1 :1 ) or, e.g., as measured in gross mass and/or at the level of individual cells.

The inventors have observed that such moderate inhibition of HADHSC activity is advantageous. For example, such moderate inhibition of HADHSC is not likely to adversely affect beta-oxidation in β cells. On the other hand, the inventors have realised that already at such moderate levels of HADHSC inhibition, insulin production was markedly increased and more responsive to nutrients. Moreover, based on their data, the inventors hypothesise that such moderate inhibition is advantageously increases both (pro)insulin biosynthesis and the rate of insulin secretion by β cells.

For example, activation of one or more aspects of HADHSC biological activity, e.g., preferably its enzymatic activity, may be by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least

about 150%, 200%, 250%, 300%, 400% or by at least about 500%, when HADHSC is exposed to an activator (preferably, when molar ratio HADHSC : activator is 1 :1 ) or, e.g., as measured in gross mass and/or at the level of individual cells.

Preferably, an agent that can modulate, e.g., inhibit or activate, HAHDSC activity does so specifically, i.e., selectively.

The terms "specifically modulate" or "selectively modulate" reflect a situation when an agent modulates HADHSC activity without substantially modulating the activity of random, unrelated targets, e.g., polypeptides or proteins, e.g., enzymes, also exposed to the said agent. "Without substantially modulating" reflects that such modulation (e.g., inhibition or activation) of an unrelated target, if any, would be less than about 20%, e.g., less than 15%, preferably less than about 10%, e.g., less than about 5%, preferably less than about 4%, 3%, 2% or 1% of the normal activity of such unrelated target, i.e., its activity when not exposed to the said agent.

For example, an agent specifically modulating HADHSC activity preferably shows substantially no modulation of the activity of other cellular components, e.g., of other cellular proteins and particularly cellular enzymes, e.g., of other mitochondrial proteins and enzymes, and in particular enzymes involved in mitochondrial beta-oxidation of fatty acids, e.g., of long- chain 3-hydroxyaxyl-CoA dehydrogenase (LCHAD) or to SCHAD (i.e., HSD10 or HADH2 as explained above). Advantageously, such specific modulation reduces the potential effects of agents on receptors other than their specific target, thereby improving the selectivity of the treatment and reducing the chance of unwanted side-effects

An agent that modulates, e.g., inhibits or activates, one or more aspects of HADHSC activity, e.g., preferably its enzymatic activity, may preferably bind to HADHSC. The term "binding" as used herein generally refers to a physical association, preferably herein a non-covalent physical association, between molecular entities, e.g., between a "ligand" (generally referring to any agent, e.g., a substance or molecule) and a "receptor" (generally referring to any molecule). Preferably, a "receptor" may be a polypeptide or protein, such as, e.g., HADHSC or variants or fragments thereof. Preferably, a "ligand" may be, e.g., a polypeptide or protein, an antibody, a peptide, a peptidomimetic, an aptamer, a chemical

substance (preferably an organic molecule, more preferably a small organic molecule), a lipid, a carbohydrate, a nucleic acid, etc.

In preferred embodiments, an agent is capable of binding to native conformation of HADHSC.

The term "native conformation" is used to refer to a conformation substantially retaining the secondary and tertiary structure of the native state of a protein. By means of example, HADHSC is said to have native conformation if it substantially retains the secondary and tertiary structure of the respective native protein, preferably enzymatically active HADHSC.

Advantageously, an agent binding the native conformation of a target polypeptide is awaited to be particularly effective in vivo as well as in isolated cells or tissues, where the respective target protein, e.g., HADHSC, is expected to be mainly found in its native, or substantially native, conformation.

In preferred embodiments, an agent is capable of binding to HADHSC under physiological conditions. "Physiological conditions" are those conditions characteristic of an organism's (e.g., a subject's to-be-treated, e.g., an animal or human subject's) healthy or normal functioning.

In preferred embodiments of the aspects of the invention, an agent of the invention binds to HADHSC with high affinity.

As used herein, binding can be considered "high affinity" when the affinity constant (K _A ) of such binding is K _A > 1x10 ⁴ M ^"1 , preferably K _A > 1x10 ⁵ M ^"1 , even more preferably K _A > 1x10 ⁶ M ^{" 1} such as, e.g., K _A > 1x10 ⁷ M ^"1 , yet more preferably K _A > 1x10 ⁸ M ^"1 , even more preferably K _A > 1x10 ⁹ M- ¹ , e.g., K _A > 1x10 ¹⁰ M ^"1 , and most preferably K _A > 1x10 ¹¹ M ^"1 , e.g., K _A > 1x10 ¹² M ^"1 , K _A > 1x10 ¹³ M ^"1 , K _A > 1x10 ¹⁴ M ^"1 , K _A > 1x10 ¹⁵ M ^"1 or even higher, wherein K _A = [Ligand_Receptor]/[Ligand][Receptor]. Determination of K _A can be carried out by methods known in the art, such as, e.g., using equilibrium dialysis and Scatchard plot analysis. Advantageously, high-affinity binding allows to reduce the quantity of an agent required to achieve a therapeutic effect in a patient, owing to the comparably high strength of interaction between the agent and its molecular target.

In further preferred embodiments of the aspects of the invention, binding of an agent of the invention to HADHSC can be specific.

The term "specifically bind" or "specific binding" reflects a situation when a ligand binds to a given receptor more readily than it would bind to a random, unrelated receptor. For example, a ligand (agent) specifically binding to a polypeptide or protein (1 ) (e.g., HADHSC) preferably displays little or no binding to other polypeptides, including homologues or orthologues of the polypeptide or protein (1 ), under conditions where it would specifically bind the said polypeptide or protein (1 ). Under little or no binding is meant K _A < 1x10 ⁴ M ^"1 , preferably K _A < 1x10 ³ M ^~ \ more preferably K _A < 1x10 ² M ^~ \ yet more preferably K _A < 1x10 ¹ M ^~ \ e.g., K _A < 1 M ^" \ most preferably K _A « 1 M ^"1 , e.g., K _A < 1x10 ^"1 M ^"1 , K _A < 1x10 ^"2 M ^"1 , K _A < 1x10 ^"3 M ^"1 , K _A < 1 x10 ^"4 M ^"1 , K _A < 1 x10 ⁵ M \ K _A < 1 x10 ⁶ M ^"1 , or smaller. By means of example and not limitation, an agent specifically binding to HADHSC preferably shows little or no binding to other cellular components, e.g., to other cellular proteins and particularly cellular enzymes, e.g., to other mitochondrial proteins and enzymes, and in particular to other enzymes involved in mitochondrial beta-oxidation of fatty acids, e.g., to long-chain 3-hydroxyaxyl-CoA dehydrogenase (LCHAD) or to SCHAD (i.e., HSD10 or HADH2 as explained above).

Advantageously, such specific binding reduces the potential effects of agents on receptors other than their specific target, thereby improving the selectivity of the treatment and reducing the chance of unwanted side-effects.

In preferred embodiments, an agent capable of modulation (e.g., inhibiting or activating) HADHSC, and preferably also capable of binding to HADHSC, as defined above, can be chosen from the group consisting of a chemical substance, preferably an organic molecule, more preferably a small organic molecule; a peptide; a peptidomimetic; a polypeptide or protein; an antibody, including fragments and derivatives thereof; an aptamer; a lipid; a carbohydrate; or a nucleic acid, including an oligonucleotide. Such agents may be isolated or substantially isolated as defined herein.

Many of the above recited types of agents, e.g., chemical substances, peptides, aptamers, carbohydrates, or nucleic acids, are available in synthetic, combinatorial and natural product libraries, and can be selected therefrom using screening assays of the invention determining modulation of HADHSC activity by test agents and/or binding of test agents to HADHSC. In a preferred embodiment, an agent capable of modulating the activity of and/or binding to HADHSC is a chemical substance, preferably an organic molecule, more preferably a small organic molecule.

The terms "chemical substance" or "chemical compound" as used herein refer to their connotation in the art; the terms encompass substances consisting of two or more different chemically bonded chemical elements, with a fixed ratio determining the composition. The term includes both inorganic and organic compounds. The terms "organic compound" or "organic molecule" as used herein refer to their broad connotation in the art. The terms encompass organic molecules which are natural products, as well as which are semi- or fully synthesised.

The term "small organic molecule", as used herein, refers to organic compounds with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da.

In an embodiment, an agent, which is an inhibitor of HADHSC, is an organic molecule selected from the group comprising or consisting of a compound of formula (I): CH ₃ (CH ₂ ) _n - CHOH-(CH ₂ ) ₃ COOH, wherein n is between 0 and 20.

In preferred embodiments, n is between 0 and 15, preferably between 0 and 10, more preferably between 0 and 8, even more preferably between 2 and 6, and yet more preferably between 2 and 4. In preferred embodiments, n is even such that the total number of C-atoms in compound (I) is even. In further preferred embodiments, n is 0 (i.e., 5-hydroxyhexanoic acid), 2 (i.e., 5-hydroxyoctanoic acid), 4 (i.e., 5-hydroxydecanoic acid), 6 (5- hydroxydodecanoic acid), or 8 (5-hydroxytetradecanoic acid). In a particularly preferred embodiment, n is 4 (i.e., 5-hydroxydecanoic acid). 5-hydroxydecanoic acid was demonstrated to be a HADHSC inhibitor by Hanley et al. 2005 (supra). In a preferred embodiment, the -OH group on C ⁵ (where C ¹ corresponds to the C-atom of the carboxyl group) is in D-configuration. As demonstrated in Hanley et al. 2005, D-configuration of this -OH group is predicted to give a higher degree of HADHSC inhibition. In other embodiments, however, the -OH group on C ⁵ may be in L-configuration or the compound (I) may be a racemic mixture in this respect. In embodiments, the organic molecule is selected from the group comprising or consisting of stereoisomeric forms, racemic mixtures, prodrugs, esters, pharmaceutically acceptable salts

(e.g., salts of the carboxyl group) and metabolites of the compound of formula (I) (e.g., as shown by Hanley et al., compound (I) may be metabolised to CoA derivative of the carboxyl group, and further to a -OH derivative on C ³ atom, which can particularly inhibit HADHSC).

In further embodiments, the organic molecule is selected from the group comprising or consisting of further derivatives of compound of formula (I).

In embodiments, one or more atoms, preferably C-atoms, of the said derivatives is substituted with a functional group other than -H.

By means of preferred examples but not limitation, one or more of the atoms, preferably C- atoms, may be independently substituted with alkyl, preferably C1-4 alkyl, more preferably methyl or ethyl; hydroxyalkyl, preferably C1-2 hydroxyalkyl; alkyloxy, preferably Ci _-2 alkyloxy; formyl; alkylcarbonyloxy, preferably Ci _-2 alkylcarbonyloxy; hydroxy (preferably on C ³ ); oxo; mercapto; halo, preferably chloro; or the like.

In embodiments, one or more bonds between C-atoms of the said derivatives may be a double bond. Preferably a double bond may be between C-atoms C ⁶ or higher. In an embodiment, organic molecule, which is an inhibitor of HADHSC, is selected from the group comprising or consisting of salicylic acid, derivatives thereof, or stereoisomeric forms, racemic mixtures, prodrugs, esters, pharmaceutically acceptable salts (e.g., salts of the carboxyl group) and metabolites thereof.

In an embodiment, the organic molecule, which is an inhibitor of HADHSC, is selected from the group comprising or consisting of salicylate, acetyl salicylate, choline salicylate, salicylsalicylate, sodium salicylate, magnesium salicylate, choline magnesium trisalicylate, gentisate, sodium gentisate, hydroxyhippurate, salacetamide, salamidacetic acid, sulfasalazine, salicylamide, thiosalicylate, and sodium thiosalicylate.

In a preferred embodiment, the organic molecule, which is an inhibitor of HADHSC, is salicylate, gentisate or hydroxyippurate. These compounds have been reported as HADHSC inhibitors by, e.g., Glasgow et al. 1999 (BBA 1454: 115-125).

In a further preferred embodiment, an agent capable of modulating the activity of and/or binding to HADHSC is a peptidomimetic, esp. a peptidomimetic of a peptide that binds to HADHSC. As used herein, the term "peptidomimetic" refers to a non-peptide agent that is a topological analogue of a corresponding peptide. Methods of rationally designing peptidomimetics of

peptides are known in the art. For example, the rational design of three peptidomimetics based on the sulphated 8-mer peptide CCK26-33, and of two peptidomimetics based on the 11-mer peptide Substance P, and related peptidomimetic design principles, are described in Horwell 1995 (Trends Biotechnol 13: 132-134). Peptidomimetics often show improved properties, e.g., improved stability, greater resistance to hydrolysis, or easier delivery, than their corresponding peptides.

In a further preferred embodiment, an agent capable of modulating the activity of and/or binding to HADHSC is an aptamer.

The term "aptamer" as used herein refers to single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo-DNA/RNA or any analogue thereof, that specifically bind to and alter the biological activity of a target molecule, preferably of a polypeptide or protein, such as, e.g., HADHSC. Aptamers are capable of binding their respective targets under physiological conditions. Selection of aptamers in vitro allows rapid isolation of extremely rare oligos that have high specificity and affinity for specific proteins. Exemplary RNA aptamers are described in US 5,270, 163, Ellington and Szostak 1990 (Nature 346: 818-822), Tuerk and Gold 1990 (Science 249: 505-510), incorporated by reference herein. RNA aptamers can frequently discriminate finely among discrete functional sites of a protein, see Gold et al. 1995 (Annu Rev Biochem 64: 763-797).

In a preferred embodiment, an agent capable of modulating the activity of and/or binding to HADHSC is an antibody, including fragments and derivatives thereof.

As used herein, the term "antibody" is used in its broadest sense and generally refers to any immunologic binding agent. The term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments. The term "antibody" is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro or in vivo.

In an embodiment, the antibody may be any of IgA, IgD, IgE, IgG, and IgM classes, and preferably IgG class antibody.

In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains (see, e.g., Hamers-Casterman et al. 1993. Nature 363: 446-448). Hence, in these immunoglobulins the heavy chain variable region, referred to as VHH, forms the entire CDR. These molecules and functional fragments and/or derivatives thereof are also included by the term "antibody" as used herein. Accordingly, in an embodiment, the antibody may be a camelid antibody as described above. In a preferred embodiment, the antibody is a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies offer the advantages of, e.g., selectively and reproducibly targeting a particular antigen and even a particular epitope within the said antigen, as well as reproducible production and titre, amongst others evident to a skilled person.

By means of example and not limitation, monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. 1975 (Nature 256: 495), or may be made by recombinant DNA methods (e.g., as in US 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624-628) and Marks et al. 1991 (J MoI Biol 222: 581-597), for example. In further embodiments, the monoclonal antibodies specifically include chimeric antibodies, primatised antibodies, and humanised antibodies.

In further embodiments, the antibody agent may be antibody fragments. Some advantages of such fragments include, e.g., smaller size, easier delivery, absence of effector domains, etc.

"Antibody fragments" comprise a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv and scFv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies. The above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art-established meaning. The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and

mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., Camelus bactήanus and Camelus dromaderius), llama (e.g., Lama paccos, Lama glama or Lama vicugna) or horse. Further without limitation, the variable region may be condricthoid in origin (e.g., from sharks).

A skilled person will understand that an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen. An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g., glycosylation, etc.).

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, New York, 1988, incorporated herein by reference). As well as methods to produce recombinant antibodies or fragments thereof. For examples of methods of the preparation and uses of monoclonal antibodies, see, e.g., US 5,688,681 ; US 5,688,657; US 5,683,693; US 5,667,781 ; US 5,665,356; US 5,591 ,628; US 5,510,241 ; US 5,503,987; US 5,501 ,988; US 5,500,345 and US 5,496,705; Skerra et al. 1993 (Curr Opinion in Immunol 5: 256-262); Plϋckthun 1992 (Immunol Revs 130: 151-188); McCafferty et al. 1990 (Nature 348: 552-554); Clackson et al. 1991 (Nature 352: 624-628); Marks et al. 1991 (J MoI Biol 222: 581-597); Marks et al. 1992 (BioTechnology 10: 779-783), Waterhouse et al. 1993 (Nuc Acids Res 21 : 2265-2266); US 4,816,567; Morrison et al. 1984 (PNAS 81 : 6851 ); incorporated by reference in their entirety. Examples of the preparation and uses of polyclonal antibodies are disclosed in US 5,512,282; US 4,828,985; US 5,225,331 and US 5,124,147 which are incorporated by reference in their entirety. For examples of methods for preparation of antibody fragments, see, e.g., Morimoto et al. 1992 (J Biochem Biophys Methods 24: 107- 117); Brennan et al. 1985 (Science 229: 81 ); Carter et al. 1992 (BioTechnology 10: 163-167); WO 93/16185; US 5,571 ,894; US 5,587,458; US 5,641 ,870; incorporated by reference in their entirety. EP 0 656 946 describes the isolation and uses of camelid immunoglobulins and is incorporated herein by reference.

Typically, production of antibodies according to the invention may comprise immunisation of a host animal, preferably a vertebrate, more preferably a mammal, with a suitable antigen.

As used herein, the term "antigen" denotes any substance capable of eliciting an immune response in a host, and in particular capable of eliciting a humoral response involving the production of antibodies specific for the said antigen. An antigen comprises one or more than one antigenic determinants or epitopes which may be the same or different. The term "antigenic determinant" or "epitope" refers to a site of an antigen that is complementary to an antigen-binding site of a corresponding antibody and thus capable of specifically interacting with the latter.

For particular purposes of the invention, an "antigen" can comprise, consist essentially of, or consist of HADHSC, fragments thereof (e.g., including >4, >5, >6, >8, >10, preferably >15, more preferably >20, even more preferably >25, >30, >40, >50, >100 or >500 consecutive amino acids thereof; or, e.g., including >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80% or >90% of the polypeptide sequence), variants thereof (e.g., including one or more amino acid deletions, additions and/or substitutions, preferably conservative substitutions, wherein sequence identity with the native protein or fragment thereof - e.g., as determined by NCBI BLAST sequence alignment algorithm - can be >50%, >60%, preferably >70%, more preferably >80%, even more preferably >90%, >95%, >99%), derivatives thereof (e.g., including derivations of one or more amino acid residues thereof, e.g., by glycosylation, phosphorylation, disulphide bridge, etc., wherein the fraction of modified amino acids vis-a-vis the native protein or fragment thereof or variant thereof can be <50%, <40%, <30%, preferably <20%, more preferably <10%, even more preferably <5%, e.g., <4%, <3%, <2% or <1 %) or genetic or chemical fusions of any of the above with heterologous presenting carriers, e.g., GST, HBc, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor etc., insofar the above induce production of antibodies specific for (one or more epitopes of) the native HADHSC. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the antigens.

Antigenic regions of proteins, esp. of HADHSC, can be identified using, e.g., standard antigenicity and hydropathy plots as calculated, for instance, using Hopp/Woods method for antigenicity profiles (Hopp et al. 1981. PNAS 78: 3824-3828) and the Kyte-Doolittle technique for hydropathy plots (Kyte et al. 1982. J MoI Biol 157: 105-132). Such prediction programs are also included in standard sequence analysis software, e.g., in the GCG™ v. 11.1.2 package from Accelrys.

Antibodies generated against HADHSC as inducing antigen can be tested for binding to HADHSC using methods well-known in the art, e.g., immunoprecipitation, affinity chromatography, ELISA, RIA, denaturing or non-denaturing immunoblotting, immunocytochemitry, immunohistochemistry, etc., such as to select antibodies having properties as above and useful in the methods of the invention. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al. 1980 (Anal Biochem 107: 220). Similarly, methods for isolation and purification of antibodies, e.g., affinity purification, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, salt precipitation, etc., are well-known in the art. In a particularly preferred embodiment, the antibody may be an intrabody. The term "intrabody" or "intrabodies" refer to intracellular^ expressed antibody constructs, usually single-chain Fv antibodies, directed against a target inside a cell, e.g., as described in Nam et al. 2002 (Methods MoI Biol 193: 301 ), der Maurr et al. 2002 (J Biol Chem 277(47): 45075) or Cohen 2002 (Methods MoI Biol 178: 367). The scFv gene can be transferred into cells, where scFv protein expression can modulate the activity of its target, e.g. HADHSC. Indeed, the scFv intrabody can be expressed in the cytoplasm and directed to any cellular compartment where it can target intracellular proteins and elicit specific biological effects. Intrabodies thus provide effective means for blocking or modulating the activity of proteins.

Agents modulating the level of expression of HADHSC In a further aspect, the agents of the invention can modulate the expression of HADHSC.

The term "modulate" has its common meaning, is synonymous with, e.g., "alter", "change" or "vary", and in particular encompasses both increasing and reducing HADHSC expression.

These terms may generally refer to that administration of an agent to an in vitro system, cell, tissue or an organism comprising HADHSC expression, preferably to a patient, will increase or reduce said HADHSC expression than if the said agent had not been administered.

When an agent, e.g., a substance or molecule, is said to "reduce the expression" of HADHSC, this generally means that administration of the said substance to a cell, tissue or an organism, causes HADHSC to be expressed at a level relatively lower than if the said substance had not been administered. Such reduction of expression can be observed and quantified, e.g., at the level of heterogeneous nuclear RNA (hnRNA), precursor mRNA (pre- mRNA), mRNA, cDNA and/or the protein of HADHSC. Suitable methods to detect and

quantify expression are known in the art and include, without limitation, Northern blotting, quantitative RT-PCR, Western blotting, ELISA, RIA, immunoprecipitation, etc. The term encompasses any extent of reduction of expression, such as, by way of example, reduction of expression by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by at least about 96%, 97%, 98%, 99% or even by 100%, e.g., as measured in gross mass and/or at the level of individual cells.

Preferably, such reduction of expression may be by less than about 80%, e.g., less than about 70%, preferably by less than about 60%, e.g., by less than about 50%, more preferably by less than about 45%, e.g., less than about 40%, and even more preferably by less than about 35% or by less than about 30%, e.g., as measured in gross mass and/or at the level of individual cells.

Also preferably, such reduction of expression may be by between about 10% and about 70%, preferably between about 20% and about 60%, more preferably between about 20% and 40%, and even more preferably between about 20% and 30%, e.g., as measured in gross mass and/or at the level of individual cells.

The inventors have observed that such moderate reduction expression of HADHSC is advantageous. For example, such moderate reduction of HADHSC expression is not likely to adversely affect beta-oxidation in β cells. On the other hand, the inventors have realised that already at such moderate levels of HADHSC downregulation, insulin production was markedly increased and more responsive to nutrients. Moreover, based on their data, the inventors hypothesise that such moderate inhibition is advantageously increases both (pro)insulin biosynthesis and the rate of insulin secretion by β cells. Conversely, when an agent, e.g., a substance or molecule, is said to "increase the expression" of HADHSC, this generally means that administration of the said substance to a cell, tissue or an organism, causes HADHSC to be expressed at a level higher than if the said substance had not been administered. Such increase in expression can be observed and quantified, e.g., at the level of heterogeneous nuclear RNA (hnRNA), precursor mRNA (pre- mRNA), mRNA, cDNA and/or the protein of HADHSC. Suitable methods to detect and quantify expression are known in the art and include, without limitation, Northern blotting, quantitative RT-PCR, Western blotting, ELISA, RIA, immunoprecipitation, etc. The term

encompasses any extent of increase of expression, such as, by way of example, increase by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, e.g., as measured in gross mass and/or at the level of individual cells.

Preferably, an agent that can modulate, e.g., reduce or increase, HAHDSC expression does so specifically, i.e., selectively.

The terms "specifically modulate" or "selectively modulate" reflect a situation when an agent modulates HADHSC expression without substantially modulating the expression of random, unrelated targets, e.g., polypeptides or proteins, e.g., enzymes, also exposed to the said agent. "Without substantially modulating" reflects that such modulation (e.g., increase or reduction) of expression of an unrelated target, if any, would be less than about 20%, e.g., less than 15%, preferably less than about 10%, e.g., less than about 5%, preferably less than about 4%, 3%, 2% or 1 % of the normal expression of such unrelated target, i.e., its expression when not exposed to the said agent.

For example, an agent specifically modulating HADHSC expression preferably shows substantially no modulation of the expression of other cellular components, e.g., of other cellular proteins and particularly cellular enzymes, e.g., of other mitochondrial proteins and enzymes, and in particular enzymes involved in mitochondrial beta-oxidation of fatty acids, e.g., of long-chain 3-hydroxyaxyl-CoA dehydrogenase (LCHAD) or to SCHAD (i.e., HSD10 or HADH2 as explained above).

Advantageously, such specific modulation reduces the potential effects of agents on cellular molecules other than their specific target, thereby improving the selectivity of the treatment and reducing the chance of unwanted side-effects

In preferred embodiments, an agent or ligand capable of modulating expression of HADHSC can be chosen from the group consisting of nucleic acids, oligonucleotides, ribozymes, polypeptides or proteins, e.g., transcription factors, peptides, peptidomimetics, antibodies and fragments and derivatives thereof, aptamers, chemical substances, preferably organic molecules, more preferably small organic molecules, lipids, carbohydrates, polysaccharides, etc., and any combinations thereof.

In a preferred embodiment, an agent or ligand capable of increasing expression of HADHSC can be a transcription factor positively regulating HADHSC expression, such as, e.g., preferably the transcription factor Foxa2 (Lantz et al. 2004. J Clin Invest 114: 512-20) or a functional variant or fragment thereof; or a recombinant nucleic acid encoding and capable of effecting the expression of such.

In a preferred embodiment, an agent or ligand capable of increasing expression of HADHSC is a recombinant nucleic acid encoding and capable of effecting the expression of HADHSC or a functional variant or functional fragment thereof in a cell.

Typically, such recombinant nucleic acid may comprise a region coding for HADHSC or a functional variant or functional fragment thereof operably linked to regulatory sequences effecting the transcription of the said coding sequences in a cell. As known in the art, such regulatory sequences may typically include a suitable promoter and optionally and preferably an enhancer, as well as suitable transcription termination sequences. Optionally, an inclusion of an intron may increase expression. The promoter may be constitutive or inducible. The promoter may be tissue-specific and may be preferentially active in pancreatic tissue, preferably in pancreatic beta cells.

The term "variant" of HADHSC, as used herein, refers to polypeptides the amino acid sequence of which is substantially identical (i.e., largely but not wholly identical) to a native sequence of HADHSC. "Substantially identical" refers to at least 85% identical, e.g., preferably at least 90% identical, e.g., at least 91 % identical, 92% identical, more preferably at least 93% identical, e.g., 94% identical, even more preferably at least 95% identical, e.g., at least 96% identical, yet more preferably at least 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical.

Sequence identity between two polypeptides can be determined by optimally aligning (optimal alignment of two protein sequences is the alignment that maximises the sum of pair-scores less any penalty for introduced gaps; and may be preferably conducted by computerised implementations of algorithms, such as "Gap", using the algorithm of Needleman and

Wunsch 1970 (J MoI Biol 48: 443-453), or "Bestfit", using the algorithm of Smith and

Waterman 1981 (J MoI Biol 147: 195—197), as available in, e.g., the GCG™ v. 11.1.2 package from Accelrys) the amino acid sequences of the polypeptides and scoring, on one hand, the number of positions in the alignment at which the polypeptides contain the same amino acid residue and, on the other hand, the number of positions in the alignment at which

the two polypeptides differ in their sequence. The two polypeptides differ in their sequence at a given position in the alignment when the polypeptides contain different amino acid residues at that position (amino acid substitution), or when one of the polypeptides contains an amino acid residue at that position while the other one does not or vice versa (amino acid insertion or deletion). Sequence identity is calculated as the proportion (percentage) of positions in the alignment at which the polypeptides contain the same amino acid residue versus the total number of positions in the alignment. Further suitable algorithms for performing sequence alignments and determination of sequence identity include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J MoI Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).

At least some of the differences between the amino acid sequences of a variant and of the naturally occurring HADHSC with which the variant is substantially identical, can involve amino acid substitutions. Preferably, at least 85%, e.g., at least 90%, more preferably at least 95%, e.g., 100% of the said differences can be amino acid substitutions, preferably conservative amino acid substitutions. The term "conservative substitution" as used herein denotes that one amino acid residue has been replaced by another, biologically similar amino acid residue. Non-limiting examples of conservative substitutions include the substitution of one hydrophobic amino acid residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as between arginine and lysine, between glutamic and aspartic acids or between glutamine and asparagine, and the like.

The "variant" of HADHSC, as used herein, also specifically includes polypeptides having a certain degree of similarity to HADHSC. Preferably, such variants can be at least 90% similar, e.g., preferably at least 91 % similar, e.g., at least 92% similar, 93% similar, more preferably at least 94% similar, e.g., 95% similar, even more preferably at least 96% similar, e.g., at least 97% similar, yet more preferably at least 98% similar, e.g., at least 99% similar.

Sequence similarity between two polypeptides can be determined by optimally aligning (see above) the amino acid sequences of the polypeptides and scoring, on one hand, the number of positions in the alignment at which the polypeptides contain the same or similar (i.e., conservatively substituted) amino acid residue and, on the other hand, the number of positions in the alignment at which the two polypeptides otherwise differ in their sequence.

The two polypeptides otherwise differ in their sequence at a given position in the alignment when the polypeptides contain non-conservative amino acid residues at that position, or when one of the polypeptides contains an amino acid residue at that position while the other one does not or vice versa (amino acid insertion or deletion). Sequence similarity is calculated as the proportion (percentage) of positions in the alignment at which the polypeptides contain the same or similar amino acid residue versus the total number of positions in the alignment.

The term "functional variant" of HADHSC as used herein refers to a variant as defined above which at least partly retains its functionality, preferably enzymatic activity. For example, variant HADHSC would retain at least 20%, e.g., at least 30% or at least 40%, preferably at least 50%, e.g., at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%, e.g., at least 95% of its enzymatic activity, as measured by standard assays in the art.

The term "fragment" of HADHSC, as used herein, refers to a polypeptide that has an N- terminal and/or C-terminal deletion of one or more amino acid residues as compared to the HADHSC, or a variant (preferably a functional variant) thereof, but where the remaining primary sequence of the fragment is identical to the corresponding positions in the amino acid sequence of HADHSC, or a variant (preferably a functional variant) thereof.

For example, a fragment of HADHSC, or of a (preferably functional) variant thereof, may include a sequence of >5 consecutive amino acids, preferably >10 consecutive amino acids, more preferably >20 consecutive amino acids, even more preferably >30 consecutive amino acids, e.g., >40 consecutive amino acids, and most preferably >50 consecutive amino acids, e.g., >60, >70, >80, >90, >100, >200 or >500 consecutive amino acids of HADHSC, or of a (preferably functional) variant thereof.

A fragment of HADHSC, or of a (preferably functional) variant thereof, can also represent at least 80%, e.g., at least 85%, preferably at least 90%, more preferably at least 95% or even 99% of the amino acid sequence of HADHSC, or of a (preferably functional) variant thereof.

The term "functional fragment" of HADHSC, as used herein, refers to a fragment as defined above which at least partly retains its functionality. For example, functional fragment of HADHSC would retain at least 20%, e.g., at least 30% or at least 40%, preferably at least 50%, e.g., at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%, e.g., at least 95% of its enzymatic activity, as measured by standard assays in the art.

In preferred embodiments, an agent capable of reducing the expression of HADHSC can be chosen from the group consisting of a chemical substance, preferably an organic molecule, more preferably a small organic molecule; an antisense agent, e.g., an antisense oligonucleotide, a ribozyme, or an agent capable of causing RNA interference. In a preferred embodiment, an agent capable of reducing the expression of HADHSC is an antisense reagent, esp. an antisense oligonucleotide.

The term "antisense" as used herein refers to a molecule designed to interfere with gene expression and capable of specifically binding to a desired target polynucleotide sequence. Antisense molecules typically (but not necessarily) comprise an oligonucleotide or oligonucleotide analogue capable of specifically hybridising to the target sequence. Hence, the term "antisense" oligonucleotide refers to an oligonucleotide or oligonucleotide analogue comprising, consisting essentially of or consisting of a nucleic acid sequence that is complementary or substantially complementary (i.e., largely but not wholly complementary) to a sequence within genomic DNA, hnRNA, mRNA or cDNA, preferably mRNA or cDNA, encoding a protein of interest; such as, e.g., within the genomic DNA, hnRNA, mRNA or cDNA, preferably mRNA or cDNA, of HADHSC. "Substantially complementary" refers to at least 85% complementary, e.g., preferably at least 90% complementary, e.g., at least 91% complementary, 92% complementary, more preferably at least 93% complementary, e.g., 94% complementary, even more preferably at least 95% complementary, e.g., at least 96% complementary, yet more preferably at least 97% complementary, e.g., at least 98% complementary, and most preferably at least 99% complementary. It is contemplated that antisense oligonucleotide may be complementary or substantially complementary to any of the 5' untranslated region, the coding region and/or the 3' untranslated region of an mRNA or cDNA. Without being limited to any theory or mechanism, it is generally believed that the activity of antisense oligonucleotides depends on the binding of the oligonucleotide to the target nucleic acid, thus disrupting the function of the target, either by hybridization arrest (e.g., preventing the action of polymerases RNA processing) or by destruction of target RNA by RNase H (the ability to activate RNAse H when hybridised to RNA) resulting in inhibition of expression. In this and below references, the terms "hybridisation" or "hybridise" as used herein, refers to any process by which a strand of nucleic acid binds with a strand comprising complementary sequence(s) through base pairing, preferably involving hydrogen bonding, more preferably by

Watson-Crick base pairing interactions. Hybridisation can take place between distinct strands or within the same strand.

Hybridisation and the strength of hybridisation (i.e., the strength of the association between the nucleic acid strands) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the melting temperature of the formed hybrid, and the G:C ratio within the nucleic acids. In addition to sequence information, it is possible to determine if a nucleic acid has >85, >90, >95 or even >100% identity/complementarity by hybridisation at high stringency. "High stringency" conditions include conditions equivalent to the following exemplary conditions for binding or hybridisation at 65 ⁰ C in a solution consisting of 5xSSPE (43.8 g/l NaCI, 6.9 g/l NaH ₂ PO ₄ -H ₂ O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1 % SDS, δxDenhardt's reagent (50xDenhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma) and 100 μg/ml denatured salmon sperm DNA), followed by washing in a solution comprising 5xSSPE, 0.1 % SDS at 65 ⁰ C when a probe of about 500 nucleotides in length is employed. Other exemplary conditions for hybridisation at "high stringency" for nucleic acid sequences over approximately 50-100 nucleotides in length include conditions equivalent to hybridisation in 6xSSC at 45°C, followed by one or more washes in 0.2xSSC, 0.1 % SDS at 65°C. Numerous equivalent conditions may be employed to vary stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilised, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulphate, polyethylene glycol) are considered and the hybridisation solution may be varied to generate conditions of low or high stringency hybridisation different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridisation under conditions of high stringency (e.g., increasing the temperature of the hybridisation and/or wash steps, the use of formamide in the hybridisation solution, etc.). Guidance for performing hybridisation reactions can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6, 1989, and more recent updated editions, all of which are incorporated by reference. Typically, antisense agents suitable for the present invention may be capable of hybridising to their respective target at high stringency conditions. Such agents may hybridise specifically to the target under physiological conditions.

The terms "complementary" or "complementarity" as used herein with reference to nucleic acids, refer to the normal binding of polynucleotides under permissive salt (ionic strength) and temperature conditions by base pairing, preferably Watson-Crick base pairing. By means of example, complementary Watson-Crick base pairing occurs between the bases A and T, A and U or G and C. For example, the sequence A-G-T (i.e., 5'-A-G-T -3') is thus complementary to sequence A-C-T (i.e., 5'-A-C-T-3').

Complementarity between two single-stranded nucleic acid molecules may be "partial", such that only some nucleotides of the nucleic acids would bind when the strands hybridise, or it may be "complete", such that total complementarity exists between the single stranded molecules. By means of example, a relatively shorter nucleic acid strand would show total complementarity to a relatively longer nucleic acid strand, if the latter strand comprised a sequence fully complementary to the sequence of the former strand.

The "degree of complementarity" of a nucleic acid molecule (1 ) to a nucleic molecule (2) can be expressed as the proportion (percentage) of nucleotides of the nucleic acid (1 ) molecule that would be expected to match, i.e., form Watson-Crick base-pairing, with nucleotides of the nucleic acid molecule (2), when the said nucleic acid molecules (1) and (2) were hybridised, preferably in high stringency conditions.

In a further preferred embodiment, an agent capable of reducing the expression HADHSC is a ribozyme. The term "ribozyme" as used herein refers to a nucleic acid molecule, preferably an oligonucleotide or oligonucleotide analogue, capable of catalytically cleaving a polynucleotide. Preferably, a "ribozyme" may be capable of cleaving mRNA of a given polypeptide or protein, thereby reducing translation thereof; such as, preferably mRNA of HADHSC. Exemplary ribozymes contemplated herein include, without limitation, hammer head type ribozymes, ribozymes of the hairpin type, delta type ribozymes, etc. For teaching on ribozymes and design thereof, see, e.g., US 5,354,855, US 5,591 ,610, Pierce et al. 1998 (Nucleic Acids Res 26: 5093-5101), Lieber et al. 1995 (MoI Cell Biol 15: 540-551), and Benseler et al. 1993 (J Am Chem Soc 115: 8483-8484), incorporated herein by reference in their entirety.

In a yet further preferred embodiment, an agent capable of reducing the expression of HADHSC is capable of causing RNA interference with the respective transcripts, preferably mRNAs.

"RNA interference" or "RNAi" is a term initially applied to a phenomenon observed in plants and worms where double-stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner. Consequently, RNAi refers generally to the process of sequence- specific post-transcriptional gene silencing in animals mediated by short interfering nucleic acids (siNA), preferably by short interfering RNAs (siRNAs). RNAi provides a useful method of inhibiting gene expression in vitro or in vivo.

RNA interference agents may include any of short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against the expression of HADHSC.

In the present context, the expression "dsRNA" relates to double stranded RNA capable of causing RNA interference. In accordance with the present invention, any suitable double- stranded RNA fragment capable of directing RNAi or RNA-mediated gene silencing of a target gene can be used. As used herein, a "double-stranded ribonucleic acid molecule (dsRNA)" refers to any RNA molecule, fragment or segment containing two strands forming an RNA duplex, notwithstanding the presence of single stranded overhangs of unpaired nucleotides. The double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence (i.e. to at least a portion of the mRNA transcript) of the target gene to be down-regulated. The other strand of the double-stranded RNA is complementary to this target nucleotide sequence.

The double-stranded RNA need only be sufficiently similar to the mRNA sequence of the target gene to be down-regulated that it has the ability to mediate RNAi. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and a nucleotide sequence of the dsRNA sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs.

According to the invention, the "dsRNA" or "double stranded RNA", whenever said expression relates to RNA that is capable of causing interference, may be formed form two separate (sense and antisense) RNA strands that are annealed together. Alternatively, the dsRNA may have a foldback stem-loop or hairpin structure wherein the two annealed strands of the dsRNA are covalently linked. In this embodiment, the sense and antisense strands of the

dsRNA are formed from different regions of a single RNA sequence that is partially self- complementary.

As used herein, the term "RNAi molecule" is a generic term referring to double stranded RNA molecules including small interfering RNAs (siRNAs), hairpin RNAs (shRNAs), and other RNA molecules which can be cleaved in vivo to form siRNAs. RNAi molecules can comprise either long stretches of dsRNA identical or substantially identical to the target nucleic acid sequence or short stretches of dsRNA identical or substantially identical to only a region of the target nucleic acid sequence.

The subject RNAi molecules can be "small interfering RNAs" or "siRNAs." siRNA molecules are usually synthesized as double stranded molecules in which each strand is around 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length. The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, the siRNA molecules comprise a 3' hydroxyl group. In certain embodiments, the siRNA molecules can be generated by processing of longer double-stranded RNAs, for example, in the presence of the enzyme dicer.

Alternatively, the RNAi molecule is in the form of a hairpin structure, named as hairpin RNA or shRNA. The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Preferably, such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.

The present RNAi molecules may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties.

In some cases, at least one strand of the RNAi molecules has a 3' overhang from about 1 to about 6 nucleotides in length, and for instance from 2 to 4 nucleotides in length. More preferably, the 3' overhangs are 1-3 nucleotides in length. In certain embodiments, one strand has a 3' overhang and the other strand is blunt-ended or also has an overhang. The length of the overhangs may be the same or different for each strand. In order to further enhance the stability of the RNAi molecules, the 3' overhangs can be stabilized against degradation. In

one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi. For further details on design of siRNA agents, see, e.g., Elbashir et al. 2001 (Nature 411 : 494-501 ), or references in Example 3.

In a preferred embodiment, the invention relates to the use of an RNA sequence to prepare an RNAi molecule as defined herein, and preferably a siRNA molecule. Said RNAi molecule, preferably siRNA molecule, is characterized by one or more, and preferably by all of the following criteria:

- having at least 50% sequence identity, preferably at least 70% sequence identity, more preferred at least 80% sequence identity, even more preferred at least 90 % sequence identity with the target mRNA, e.g., mRNA for HADHSC, having a sequence which targets the exon area of the target gene; - showing a preference for targeting the 3' end of the target gene rather than for targeting the 5' end of the target gene.

In a further preferred embodiment, the siRNA molecule may be further characterized by one or more of the following criteria: having a nucleic acid length of between 15 to 25 nucleotides and preferably of between 18 to 22 nucleotides, and preferably of 19 nucleotides; having a GC content comprised between 30 and 50 %

- showing a TT(T) sequence at its 3' end; showing no secondary structure when adopting the duplex form; having a Tm (melting temperature) of lower than 20 ⁰ C - having the nucleotides indicated in Table A in the sequence of the nucleotides, wherein h is a, c, t/u but not g, and wherein d is a, g, t/u but not c, and wherein w is a or t/u, but not g or e:

Table A

Production of any above nucleic acid reagents, including antisense reagents, ribozymes and

RNAi molecules, can be carried out by chemical synthetic methods or by recombinant nucleic

5 acid techniques, e.g., expressed from a vector in a cell, e.g., a viral vector, a eukaryotic expression vector, a gene therapy expression vector (i.e., in vivo), etc., or enzymatically synthesized, e.g., by in vitro transcription from a DNA template using a T7 or SP6 RNA polymerase. The nucleic acid molecules may be produced enzymatically or by partial/total organic synthesis. Any modified ribonucleotide can be introduced by in vitro enzymatic or

10 organic synthesis.

Any above nucleic acid reagents, including antisense reagents, ribozymes and RNAi molecules, can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify nucleic acid reagents. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to 15 purify the molecules. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify the molecules.

The main obstacle to achieve in vivo gene silencing by nucleic acids, e.g., antisense, ribozyme or RNAi technologies, is delivery. To improve thermal stability, resistance to nuclease 0 digestion and to enhance cellular uptake of such tools, various approaches are applicable and are known to a skilled person. They include, e.g.: chemical modifications like locked nucleic acid (LNA), phosphonate substitution, phosphorothioate substitution, phosphorodithioate substitution, morpholino oligomers, 2'- fluoro substitution, 2'-O-methyl substitution, stabilized stealth™ RNAi (Invitrogen), etc. 5 - encapsulation in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticules or dendrimers, poly (lactic-Co- Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc.; co-injection with protective agent like the nuclease inhibitor aurintricarboxylic acid.

Delivery of agents

Depending on the precise nature of the agents, these may be delivered to cells in vivo or in vitro according to protocols commonly employed in the art. For example, organic compounds, peptides, or peptidomimetics may be delivered per se or commonly in conjunction with suitable excipients as detailed elsewhere in this specification.

By means of example, there are several well-known methods of introducing (ribo)nucleic acids (e.g., antisense, ribozymes or RNAi, recombinant nucleic acids encoding an agent, e.g., an shRNA, an HADHSC protein or an anti-HADHSC single-chain antibody or intrabody) into animal cells, any of which may be used in the present invention and which depend on the host. At the simplest, the nucleic acid can be directly injected into the target cell / target tissue. Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment ("gene gun" method), infection with viral vectors, electroporation, and the like. Other techniques or methods which are suitable for delivering (ribo)nucleic acid molecules to target cells include the continuous delivery of an such molecule as defined herein from poly (lactic-Co-Glycolic Acid) polymeric microspheres. Convection-enhanced delivery, as detailed by Kawakami et al. 2004 (J Neurosurg 101 : 1004-1011 ) of stabilized RNAi molecules as defined herein can also be used. Another possibility is the use of implantable drug-releasing biodegradable micropsheres, as those recently reviewed by Menei and Benoit 2003 (Acta Neurochir 88: 51 -55). It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used. In preferred embodiment, the delivery of the agents may be preferentially to pancreatic tissue, and more preferebly preferentially to beta-cells thereof. Suitable ways of targeting include, e.g., linkage of an agent to a ligand recognising a beta-cell specific surface molecule. E.g., select viral vectors may have or may be engineered to show preference towards beta-cells, as known in the art.

In another preferred embodiment, agents, esp. proteinaceous agents, e.g., intrabodies, may be provided with a mitochondrial signal sequence as known in the art, to facilitate their accumulation in mitochondria.

Disorders of glucose homeostasis The present invention concerns methods and agents useful for the treatment of disorders of glucose homeostasis.

"Disorders of glucose homeostasis" encompass all disorders in which disturbance of physiologically normal homeostasis of circulating glucose belongs to, i.e., is comprised amongst, symptoms of the said disorders, and/or belongs to aetiological factors of the said disorders. The diagnosis of a disorder of glucose homeostasis in a subject, i.e., the differentiation between physiologically normal vs. abnormal glucose homeostasis in the said subject, may be done by a number of methods well-known in clinical practice, including but not limited to measurement of fasting glucose level; and oral or intravenous glucose tolerance test. A typical fasting blood glucose test measures the concentration of glucose in blood, serum or plasma of a subject after a fasting period of usually at least 10-12 hours. The normal range of whole blood glucose concentrations (normoglycaemia), indicative of physiologically normal glucose homeostasis, in this test is between 60 mg/dL (3.0 mmol/L) and 110 mg/dL (5.6 mmol/L). A typical oral glucose tolerance test (OGTT) is carried out as follows: after an overnight fasting period (e.g., 10 to 12 hours), a subject drinks a solution containing a known amount of glucose; blood is drawn before the subject drinks the glucose solution, and blood is drawn again every 30 to 60 minutes after the glucose solution is consumed for up to 3 hours. The normal ranges of whole blood glucose concentrations in a 75-gram oral glucose tolerance test (normoglycaemia), indicative of physiologically normal glucose homeostasis, are: between 60 mg/dL (3.0 mmol/L) and 110 mg/dL (5.6 mmol/L) after fasting; less than 200 mg/dL (10.1 mmol/L) at 1 hour after consumption of the glucose solution; less than 140 mg/dL (7.1 mmol/L) at 2 hours after consumption of the glucose solution.

Accordingly, a disorder of glucose homeostasis is diagnosed when glucose concentrations in at least one and preferably both above tests, or analogous tests commonly employed in the

art, fall outside of the normoglycaemic ranges, e.g., as indicated above, at least on one occasion and preferably on two or more occasions.

Typically, a diagnosis of hypoglycaemia as a disorder of glucose homeostasis or as a symptom of said disorder is made if the measured glucose levels are below their normal ranges.

In embodiments disorders of glucose homeostasis encompass hypoglycaemia, diabetic hypoglycaemia, neuroglycopenia, hyperinsulinemic hypoglycaemia, hyperinsulinemic hypoglycaemia of infancy, partial pancreatectomy, and the like.

As explained above, agents that increase the expression level or activity of HADHSC can be particularly useful in the therapy of disorders of glucose homeostasis which involve hypoglycaemia, such as the disorders in the previous paragraph.

Agents that increase the expression level or activity of HADHSC can be particularly useful for reducing the endogenous production of insulin.

Typically, a diagnosis of hyperglycaemia as a disorder of glucose homeostasis or as a symptom of said disorder is made if the measured glucose levels are above their normal ranges.

By means of example and not limitation, in the above fasting blood glucose test, a diagnosis of impaired fasting glucose (IFG) is diagnosed when the whole blood glucose concentration of a subject is above 110 mg/dL (5.6 mmol/L) but less than 126 mg/dL (6.4 mmol/L). Diabetes mellitus may be diagnosed when the whole blood glucose concentration of a subject is above 126 mg/dL (6.4 mmol/L).

By means of example and not limitation, in the above OGTT, impaired glucose tolerance (IGT) is diagnosed when the whole blood glucose concentration of a subject at 2 hours after consumption of the glucose solution is higher than 140 mg/dL (7.1 mmol/L) but less than 200 mg/dL (10.1 mmol/L). Diabetes mellitus may be diagnosed when the whole blood glucose concentration of a subject at 2 hours after consumption of the glucose solution is higher than 200 mg/dL (10.1 mmol/L).

When at least one of IFG and IGT, and preferably both IFG and IGT, are diagnosed, the condition may be referred in the art as "prediabetes".

In embodiments disorders of glucose homeostasis thus encompass hyperglycaemia; IGT; IFG; prediabetes; diabetes, including diabetes types 1 and 2, preferably diabetes type 2; metabolic syndrome and the like.

As explained above, agents which reduce the expression level or activity of HADHSC can be particularly useful in the therapy of disorders of glucose homeostasis which involve hyperglycaemia, such as the disorders in the previous paragraph.

Agents that reduce the expression level or activity of HADHSC can be particularly useful for increasing the endogenous production of insulin.

The terms "metabolic syndrome", "insulin resistance syndrome" (IRS), or "syndrome X" may be used interchangeably herein and refer to a cluster of abnormalities which tend to co-occur in a subject and which represent major risk factors for the development of coronary artery disease (CAD), such as premature atherosclerotic vascular disease. These abnormalities in particular involve insulin resistance (impaired blood glucose), truncal obesity, high serum low density lipoprotein (LDL) cholesterol levels, low serum high density lipoprotein (HDL) cholesterol levels, high serum triglyceride levels, and high blood pressure (hypertension).

According to the National Cholesterol Education Program of NIH, diagnosis of metabolic syndrome is made in the presence of any three of the following abnormalities: truncal obesity, defined as waist circumference of more than 102 cm for men and more than 89 cm for women; high serum levels of triglycerides, i.e., 150 mg/dL or higher; low serum levels of HDL cholesterol, i.e., below 40 mg/dL for men and below 50 mg/dL for women; high blood pressure, i.e., 130/85 mm Hg or higher; impaired fasting glucose, i.e., 110 mg/dL or higher.

Primary diabetes mellitus (DM) is classified as type 1 diabetes (also called juvenile onset DM or insulin dependent diabetes mellitus, IDDM) and type 2 diabetes mellitus (also called non- insulin dependent diabetes mellitus, NIDDM). Type 1 diabetes is a hormone deficient state, in which the pancreatic beta cells appear to have been destroyed by the body's own immune defense mechanisms. The destruction of beta cells in type 1 diabetes leads to the inability to produce insulin, and thereby chronic insulin deficiency. Patients with type 1 diabetes have little or no endogenous insulin secretory capacity and develop extreme hyperglycemia. Type 1 diabetes was fatal until the introduction of insulin replacement therapy - first using insulins from animal sources, and more recently, using human insulin made by recombinant DNA technology.

Type 2 diabetes mellitus (referred to herein as "type 2 diabetes") is typically a chronic, lifelong (i.e., progressing over several decades) disease characterized by insulin resistance. In clinical terms, insulin resistance is present when normal or elevated blood glucose levels persist in the face of normal or elevated levels of insulin. Symptoms may include excessive thirst, frequent urination, hunger, and fatigue. Typically, type 2 diabetes may be diagnosed when the fasting blood glucose concentration of a subject is above 126 mg/dL (6.4 mmol/L) on two occasions. Type 2 diabetes may also be diagnosed using OGTT when the blood glucose concentration of a subject at 2 hours after consumption of the glucose solution is higher than 200 mg/dL (10.1 mmol/L). Hyperglycaemia associated with type 2 diabetes can sometimes be reversed or ameliorated by diet changes or weight loss which may at least partially restore the sensitivity of the peripheral tissues to insulin. Treatment of type 2 diabetes frequently does not require the use of insulin. Therapy in type 2 diabetes usually involves dietary therapy and lifestyle modifications, typically for 6-12 weeks in the first instance. Features of a diabetic diet include an adequate but not excessive total calorie intake, with regular meals, restriction of the content of saturated fat, a concomitant increase in the polyunsaturated fatty acid content, and an increased intake of dietary fiber. Lifestyle modifications include the maintenance of regular exercise, as an aid both to weight control and also to reduce the degree of insulin resistance. If after an adequate trial of diet and lifestyle modifications, fasting hyperglycemia persists, then a diagnosis of "primary diet failure" may be made, and either a trial of oral hypoglycaemic therapy or direct institution of insulin therapy may be required to produce blood glucose control and, thereby, to minimize the complications of the disease. Progression of type 2 diabetes is associated with increasing hyperglycemia coupled with a relative decrease in the rate of glucose-induced insulin secretion. Therefore, for example, in late-stage type 2 diabetes there may be an insulin deficiency.

The present inventors believe that the effect of the methods and agents of the invention are mediated by modulating the endogenous production of insulin thereby.

Accordingly, the methods and agents of the invention can also be employed to modulate the endogenous production of insulin. Treatment

The present invention also regards treating disorders of glucose homeostasis and/or modulating endogenous insulin production in a subject needing such therapy, comprising

administering a therapeutically effective amount of one or more above agent(s) of the invention.

Except when noted, "subject" or "patient" are used interchangeably and refer to animals, preferably vertebrates, more preferably mammals, and specifically includes human patients and non-human mammals. "Mammalian" subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet and experimental animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orang-utans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Accordingly, "subject" or "patient" as used herein means any mammalian patient or subject to which the compositions of the invention can be administered.

Preferred patients are human subjects. As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of a disorder of glucose homeostasis, e.g., diabetes type 2. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

As used herein, a phrase such as "a subject in need of treatment" includes subjects, such as mammalian subjects, that would benefit from treatment of a given condition, preferably a disorder of glucose homeostasis, e.g., as defined above. Such subjects will typically include, without limitation, those that have been diagnosed with the condition, those prone to have or develop the said condition and/or those in whom the condition is to be prevented.

The term "therapeutically effective amount" refers to an amount of a therapeutic substance or composition effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect and performance. Without limitation, a therapeutic endpoint may be adequate glycaemic control (e.g., HbA1c<7%), increased (or decreased, depending on the treatment) insulin secretion, etc.

The agent(s) of the invention may be used alone or in combination with any of the known therapies for disorders of glucose homeostasis, e.g., selected from the group comprising therapeutics for combating disorders involving hypoglycaemia as defined above, such as without limitation, diazoxide or octreotide; or therapeutics for combating disorders involving hyperglycaemia as defined above, such as without limitation, biguanides (e.g., metformin), thiazolidinediones (TZD), sulfonylurea and derivatives thereof, or incretin (GLP1 agonists).

The agent(s) of the invention can thus be administered alone or in combination with one or more active compounds. The latter can be administered before, after or simultaneously with the administration of the said agent(s). For example, doses can be adjusted to avoid toxicity resulting from excessive inhibition of beta-oxidation in other tissues with high HADHSC expression, such as liver and muscle. Monitoring of side effects is possible, by measuring urinary excretion of organic acids, and by LC-MS profiling of whole blood acyl-carnitine and 3-hydroxy-acyl distribution.

Optimal predicted combinations are HADHSC inhibitors plus TZD, or HADHSC inhibitors plus GLP1/incretin agonists as latter compounds are presumed to stimulate differentiated beta cell phenotype.

As will be appreciated by a skilled person, patients susceptible for therapy with the agents of the invention primarily include ones comprising at least residual beta cells.

Pharmaceutical preparations A further object of the invention are pharmaceutical preparations which comprise a therapeutically effective amount an agent or agent(s) of the invention as defined herein, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, i.e., one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilisers, etc. The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

The term "pharmaceutically acceptable salts" as used herein means an inorganic acid addition salt such as hydrochloride, sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline

earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of pharmaceutically acceptable ammonium salts are ammonium salt and tetramethylammonium salt. Examples of pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine, and phenylalanine.

The pharmaceutical composition according to the invention may further comprise at least one active compound, as defined above.

The pharmaceutical composition according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories. Administration can also be carried out parenterally, for example subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion. Other suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or the inhalative administration in the form of nasal sprays or aerosol mixtures, or, for example, microcapsules, implants or rods.

The preparation of the pharmaceutical compositions can be carried out in a manner known per se. To this end, the nucleic acid and/or the active compound, together with one or more solid or liquid pharmaceutical carrier substances and/or additives (or auxiliary substances) and, if desired, in combination with other pharmaceutically active compounds having therapeutic or prophylactic action, are brought into a suitable administration form or dosage form which can then be used as a pharmaceutical in human medicine. For the production of pills, tablets, sugar-coated tablets and hard gelatin capsules it is possible to use, for example, lactose, starch, for example maize starch, or starch derivatives, talc, stearic acid or its salts, etc. Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc. Suitable carriers for the preparation of solutions, for example of solutions for injection, or of emulsions or syrups are, for example, water, physiological sodium chloride solution, alcohols such as ethanol, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc. It is also possible to lyophilize the nucleic acid and/or the active compound and to use the resulting lyophilisates, for example, for preparing preparations for injection or infusion. Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.

The pharmaceutical preparations can also contain additives, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.

Preferably, the present composition is administered in a GLP/GMP solvent, containing or not cyclobetadextrine and/or similar compounds.

The dosage or amount of agents of the invention used, optionally in combination with one or more active compounds to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, it depends on the nature and the severity of the disorder to be treated, and also on the sex, age, weight and individual responsiveness of the human or animal to be treated, on the efficacy and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent(s) of the invention.

Without limitation, depending on the type and severity of the disease, a typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. A preferred dosage of the agent may be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks.

Where applicable, e.g., where the agent is a polypeptide, peptide, antibody, antisense agent, ribozyme or siRNA agent, the invention also contemplates administration thereof by gene therapy, according to effective techniques known in the art.

By way of example, the agents of the invention may be delivered at the site of pancreas.

In another embodiment, the invention provides a kit comprising a pharmaceutical composition according to the invention, and an active compound as defined herein, for simultaneous, separate or sequential administration to a subject in need thereof.

Screening assays

In an aspect, the invention provides assays to select, from a group of test agents, a candidate agent potentially useful as a therapeutic in the treatment of a disorder of glucose homeostasis, said assay comprising determining whether the tested agent (a) can modulate the activity of HADHSC or (b) can modulate the expression of HADHSC.

Preferred types of test agents in the screening assays are agents as described in the previous sections, including antisense agents, e.g., antisense oligonucleotides, ribozymes, agents potentially capable of causing RNA interference; polypeptides or proteins; antibodies; peptides, peptidomimetics, aptamers, chemical substances (preferably an organic molecules, more preferably a small organic molecules), lipids, carbohydrates, nucleic acids, etc. Some of said test agent types, e.g., chemical compounds, peptides, carbohydrates, etc., can be obtained from synthetic, combinatorial or natural product libraries. Other test agents may be designed with the knowledge of their target.

In an embodiment, the assays (drug screening assays or bioassays) include a step of assessing the test agent for its ability to modulate, e.g., increase or reduce the activity of HADHSC or of a functional variant or functional fragment thereof.

In a preferred embodiment, the assays (drug screening assays or bioassays) may include a step of assessing the test agent for its ability to bind to, preferably with high affinity, and even more preferably specifically bind to, HADHSC or to a functional variant or functional fragment thereof.

In an embodiment, a test agent which binds to HADHSC or to a functional variant or functional fragment thereof may be subsequently tested for its effect on the activity of HADHSC or of a functional variant or functional fragment thereof.

Typically, an embodiment includes (a) combining (1) HADHSC, or a functional variant or functional fragment thereof and (2) a test agent, e.g., under conditions which allow for binding of the polypeptide (1) and test agent (2) to form a complex, and detecting the formation of a complex, in which the ability of the test agent (2) to interact with polypeptide (1) is indicated by the presence of the test agent in the complex. Formation of said complexes can be quantified, for example, using standard immunoassays. The embodiment may further comprise isolation and/or identification of the said test agent.

Typically, an embodiment includes (a) combining (1 ) HADHSC, or a functional variant or functional fragment thereof and (2) a test agent (optionally where the test agent binds to HADHSC or a functional variant or functional fragment thereof as determined above), and detecting whether the test agent (2) modulates, e.g., increases or reduces the activity of the polypeptide (1 ). Said activity can be quantified as known in the art and described herein.

The HADHSC or functional variants or functional fragments thereof used in such assays may be free in solution, affixed to a solid support, born on a cell surface, or located intracellular^. The method may use eukaryotic or prokaryotic host cells which natively express HADHSC, or which are transiently or stably transformed with recombinant nucleic acids expressing HADHSC variants, fragments or derivative thereof.

An assay of the effect of a test agent on HADHSC biological activity, e.g., enzymatic activity, may as well comprise contacting the test agent with a cell, tissue, organ or non-human model organism expressing HADHSC or functional variants or functional fragments thereof and having HADHSC activity, and assessing alteration in biological activity of the HADHSC. Suitable assessment method is described, e.g., in example 4.

In an embodiment, the assays (drug screening assays or bioassays) include a step of assessing the test agent for its ability to modulate, e.g., reduce or increase, the expression of HADHSC. In an embodiment, said assay comprises: (a) providing a cell expressing HADHSC, or optionally a functional variant or functional fragment thereof, (b) introducing to said cell a test agent, and (c) determining the expression of the HADHSC, or optionally a variant, derivative or fragment thereof, thereby identifying whether the test agent modulates the said expression. Expression can be quantified at various levels as described above. The embodiment may further comprise isolation and/or identification of the said test agent. The expression of HADHSC in the cell may be intrinsic to the cell or may be facilitated recombinantly, e.g., by transforming the said cell transiently or stably with a nucleic acid, e.g., cDNA, encoding HADHSC or a suitable variant, fragment or derivative thereof.

In embodiments, the assays are to select, from a group of test agents, a candidate agent potentially useful as a therapeutic in the treatment of a disorder of glucose homeostasis, as defined above. In embodiments, the assays are to select, from a group of test agents, a candidate agent potentially useful for modulating endogenous insulin production, as defined above.

In addition, the invention also relates to the agents identifiable by any of the herein described screening methods. Also, the present invention contemplates a method for the production of a composition comprising the steps of admixing an agent identifiable by the assays as described herein with a pharmaceutically acceptable carrier. It will be clear that the present invention contemplates a composition comprising an agent identifiable by any of the herein described methods. Moreover, the present invention contemplates the use of an agent identifiable by any of the herein described methods as medicament. Such agents are particularly suited for the treatment of disorders of glucose homeostasis.

Use in isolated cells or tissues As mentioned, in an aspect, the invention concerns using an agent that (a) can modulate the expression of HADHSC and/or (b) can modulate the activity of HADHSC for modulation of the endogenous production of insulin in an isolated cell or tissue, preferably isolated pancreatic β cells or tissues comprising such.

Preferably, the agent can reduce the expression of HADHSC or inhibit the activity of HADHSC, such as to obtain an increase in the endogenous insulin production.

Essentially, the above described preferred features regarding the type of agent, extent of modulation, etc. can be applied for this purpose.

The term "isolated" refers to cells or tissues that are not associated with one or more cells or tissues one or more cellular or tissue components with which the cell or tissue is associated in vivo. For example, an isolated cell or tissue may have been removed from its native environment, or may result from propagation, e.g., ex vivo propagation, of a cell that has been removed from its native environment.

Hence, the present use generally implies manipulation of the cells or tissues in vitro, i.e., outside, or external to, animal or human body or "ex vivo". By means of example and not limitation, addition of agents aiming at a reduction in HADHSC expression or activity of about 10% to about 60%, preferably from about 20 to about 50% (or further preferred extents as defined above) can increase insulin synthesis, thereby increasing cellular insulin stores. Preferably, such a treatment should be given either intermittently, or in a low dose in order to avoid excessive beta cell activation and loss of beta cell insulin content. By means of example, the use of about 50-250 μM salicylate can achieve such effect, and other agents discussed above may be employed to obtain an effect similar hereto.

The invention is further illustrated with examples that are not to be considered limiting.

EXAMPLES

Example 1 : Experimental procedures

Example 1a: Materials Rat INS1 832-13 β cells were from Dr. C. Newgard, and were cultured in 10% FCS (Hyclone), RPMI 1640 - Glutamax (Gibco, Invitrogen Corp., Carlsbad, California), supplemented with 10 mM HEPES, 1 mM sodium pyruvate, pencillin/streptomycine and 50 μM β-mercaptoethanol ("INS1 culture medium").

Rat β cells were cultured in Ham's F10 nutrient mixture (Gibco) supplemented with 0.5% BSA (Cohn Analog, Sigma), 2 mM glutamine, 10 mM glucose, penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2% FCS (Hyclone). D-[U- ¹⁴ C]-glucose (306-311 mCi/mmol; 1 mCi/5ml) was from Amersham Biosciences (New Jersey, USA). All other chemicals were obtained from Sigma- Aldrich (Schnelldorf, Germany).

Example 1 b: Real-time PCR and western blot analysis of rat beta cells and tissues Beta cells (90 ± 3% insulin-positive cells), and islet non-beta cells (typically 68 ± 9% glucagon-positive cells and < 10% β cells) were purified from bled, male Wistar rats (150- 25Og) as previously described (Pipeleers et al. 1985. Endocrinology 117: 806-816).

Total RNA was isolated from pancreatic endocrine cells within 4 hours of the start of the isolation, using RNAeasy (Qiagen) minicolums. The other rat tissues were isolated within 10 minutes of decapitation and bleeding of the rats, and snap frozen in liquid nitrogen, in RNAse- free micro tubes. These tissues included liver, total brain, pituitary, kidney, testis, lung, spleen, visceral white fat, quadriceps skeletal muscle. RNA from rat tissues, and from INS1 832-13, was extracted using the TRIzol protocol. Prior to quantitative PCR, RNA quality was verified (Agilent Bioanalyzer, minimal cut-off RIN > 8). Genomic DNA contamination was removed by DNAse using TURBO DNA-free (Ambion, Austin, Texas, U.S.A.) and reverse- transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA).

Targets were amplified from cDNA template on ABI Prism 7700 Sequence Detector using TaqMan Universal PCR Master Mix, sequence-specific primers and TaqMan MGB probe

(Applied Biosystems). All mRNA amplifications in rat tissues were done using following "Assays on Demand": acyl-CoA dehydrogenase, short chain (ACADS, Rn00574634_m1 ), acyl-CoA dehydrogenase, medium chain (ACADM, Rn00566390_m1 ), acyl-CoA dehydrogenase, long chain (ACADL, Rn00563121_m1 ) acetyl-CoA acyltransferase 2 (mitochondrial thiolase, Acaa2, Rn00590503_m1 ), acetyl-CoA acyltransferase 1 (peroxisomal thiolase, Acaai , Rn00560616_m1 ), peroxisome proliferator activated receptor α (PPAR α, Rn00566193_m1 ), L-3-hydroxyacyl-coenzymeA dehydrogenase, short chain (HADHSC, Rn00589352_m1). Expression of target genes were always normalized towards beta actin (δCt), and expressed versus a chosen calibrator (comparative δδCt method).

Example 1c: HADHSC protein expression analyzed by western blotting and immune staining

For western blotting, cells or tissues were homogenized in RIPA buffer containing protease inhibitors, sonicated and their protein content was measured spectrophotometrically by the Wallac/Victor (TM) Detector.

Typically, 15 μg protein was mixed with SDS containing sample buffer and separated by 12% SDS-PAGE. After electrophoresis, protein was transferred to a nitrocellulose membrane

(Schleicher and Schuell, Dassel, Germany) and incubated with chicken anti-human HADHSC

(A22261 ,Genway Biotech, Inc., San Diego, CA, USA) at 1 :4000 dilution. Horseradish peroxidase-linked Rabbit Anti-Chicken /Turkey IgG (H+L) (1/2000, 61-3120, Zymed

Laboratories, San Francisco, CA, USA) was used as second antibody and peroxidase activity was detected by enhanced chemiluminescence (Pierce ECL Western Blotting Substrate,

Perbio Science, UK ). The intensities of the bands of interest were quantified by Scion image software, expressed in arbitrary units of optical density (OD) and normalized by the intensity of β-actin from the same blot. For β-actin, we used actin (l-19)sc-1616 goat polyclonal IgG

(1/1000) and donkey anti-goat IgG-HRP sc-2020 HRP conjugate (both from Santa Cruz Biotechnology, CA, USA) as primary and secondary antibody respectively.

The Genway chicken anti-human HADHSC antibody was also used for immune staining of human and rat pancreas sections and rat sorted beta cells. Mitochondria were visualized using the Invitrogen/Molecular Probes antibody A6403 raised against human electron transport chain complex IV/COX (cytochrome c oxidase, subunit 1 ). Small tissues fragments were fixed for 30 minutes in 4 % formaldehyde followed by an overnight incubation in 30% sucrose, after snap freezing in liquid nitrogen the tissue was stored at -80 ⁰ C until cryosection into 5 μM sections. Dissociated rat islet cells were cultured overnight, fixed in 4% formol (15

min at 20 ⁰ C), permeabilised with 0.2% Triton X-100 (Sigma). Non-specific staining was blocked wit 10% donkey serum in PBS, followed by incubation overnight at 4°C with primary antibodies, 60 min with secondary antibodies (anti-mouse Cy3, anti-chicken Cy2, Jackson laboratories) and addition of Hoechst342 (sigma). Images were acquired on Axioplan (Carl Zeiss) microscope using Smartcapture VP software; digital processing was limited to a 5x5 soft enhance filter. Figures 3-4 were created using Photoshop software.

Example 1d: Lipid transfection and siRNA/shRNA-mediated expression silencing in INS1 832- 13 3 cells and primary rat beta cells siRNA transfections of INS1 cells. 15 x 10 ⁴ INS1 832-13 β-cells were plated in 1 ml_ INS1 culture medium in 24-well plates and cultured overnight prior to transfection with siRNA.

Lipid-transfection was done in opti-MEM reduced serum growth medium with Glutamax

(Gibco, Invitrogen) for 4 hours, using siLentFect Lipid Reagent (Biorad) and Dharmacon siRNA. Transfection was done in a total volume of 250 μl, at 20 nM siRNA (0.07 μg siRNA/well) and a Npid/siRNA ratio of 1 μl siLentFECT/0.07μg siRNA. SiRNA against rat HADHSC was Dharmacon's siGENOME SMARTpool reagent (M-091802-00), a pool of 4 different siRNAs. The silencing effect of the combined pool of 4 different siRNAs was always more powerful than the individual helices, which achieved mRNA silencing ranging from 45-

55 percent, individually (data not shown).

As control siRNA reagent, we used siGLO RISC-free siRNA (D-001600-01-20), a fluorescent siRNA with no known target genes and designed not to interact with the RISC complex.

Silencing efficiency was verified 24-48 hours after transfection, at the mRNA level (Taqman qPCR) and western blotting. Over 95% of siGLO transfected cells showed cytoplasmic, punctuate siRNA-associated fluorescence. In around 60% of cells, in addition a diffuse cytoplasmic red fluorescence was seen. Co-transfections with eGFP-coding plasmids resulted in microscopically detectable GFP expression in 65-70% of cells. Transfection did not result in overt cytotoxicity (as assessed by propidium-iodide/Hoechst staining), but slowed down cellular proliferation. Assuming that all INS1 cells in the cultures were cell cycling to the same extent, the number of cell cycles/24 (F) hours decreased from 0.53±0.21 for untransfected cells to 0.43±0.20 for siRNA-transfected cells (p<0.001 , n=8). No differences in cellular proliferation were observed between control siGLO lamin A/C, RISC-free siRNA or HADHSC SMART pool siRNA-treated cells.

siRNA transfection of primary rat beta cells. Transfection was initiated in FACS- purified rat beta cells shortly after isolation; beta cells were aggregated for 2 hours and cultured overnight in the presence of 50 nM siRNA-lipid complexes. The same siRNA reagents were used as in INS1 cells, but the transfection lipid for primary cells was Jetsi- ENDO (Polyplus, France). Again, over 85% of beta cells in the aggregates accumulated fluorescent siRNA: the siGLO-signal showed an intense punctuate distribution associated with a weaker diffuse cytoplasmic fluorescence.

Introduction of shRNAi-plasmids in INS1 cells. Short hairpin RNAi plasmids targeting rat HADHSC matched the criteria of both the Dharmacon (Reynolds et al. 2004. Nat Biotechnol 22: 326-330) and Whitehead (Yuan et al. 2004. Nucleic Acids Res 32, W130-134) siRNA design protocols. Sequences were: A3 plasmid: δ'-ATACAGTAGTGTTGGTGGA- 3'(SEQ ID NO: 27) (sense) and C6 plasmid: δ'-AGCGAGGCGATGCATCTAA-S' (SEQ ID NO: 28) (sense) targeting respectively nucleotides 151-170 and 704-722 of the rat HADHSC gene (NM_057186.1 ). These positions were at least 40 bp removed from a highly conserved L-3- hydroxyacyl-coA dehydrogenase motif around nucleotides 660-673. Sequence of scrambled control plasmid was δ'-GAGCATGCGAGCCATGCAC-S' (SEQ ID NO: 29) (sense). ShRNAi- plasmids were introduced into INS1 cells using the same protocol as for siRNA, using now 2 μl siLENTfect/μg DNA. Co-transfection with eGFP plasmids (weight ratio 1 :10 GFP/shRNA plasmids) indicated 65 ± 5% GFP expressing cells, as measured by FACS. ShRNAi-mediated HADHSC knockdown did not inhibit cell proliferation (F= 0.40 ± 0.07 and 0.43 ± 0.07 for scrambled and A3, respectively).

Example 1 e: Functional analysis of HADHSC silencing in INS1 832-13 3 cells and rat beta cells

Unless otherwise stated, INS1 cell function was studied in KRBH-based buffer, further designated as "Cell Medium" (116 mM NaCI, 1.8 mM CaCI2 2(H2O), 0.8 mM MgSO4 7(H2O),

5.4 mM KCI, 1 mM NaH2PO4 2(H2O), 26 mM NaHCO3 and 0.5% BSA). For static insulin secretion transfected and control INS1 cells were first washed with and pre-cultured for 1 hour in Cell Medium supplemented to 2 mM glucose prior to the start of the release. We monitored insulin secretion from 70-80% confluent INS1 cultures over 1 hour, in 1 ml. Cell Medium at the indicated nutrient concentrations. Released and cellular insulin was measured by radio-immunoassay.

Fatty acids (butyrate, caproic acid and palmitate) were added from a 10Ox stock in 90% ethanol. Palmitate was studied here at 500μM, in the presence of 0.5% Boehringer BSA.

After release, glucose (3-25 mM)-exposed cells were quickly dissociated with trypsin-EDTA, pooled and Bϋrker counted for further functional analysis. Insulin secretion by siRNA-treated primary rat beta cells was measured in a dynamic perifusion system, as described (Van Schravendijk et al. 1992. J Biol Chem 267: 21344- 21348).

CO2 formation from glucose was measured in duplicate samples of 105 INS1 β cells, incubated for 2 h at 37 ⁰ C in 200 μl of Ham's F10 medium containing 0.5% BSA, 2 mM L- glutamine, 10 mM Hepes, and 1OmM D-glucose (5 μCi D-[U- ¹⁴ C]-glucose). After 2 h, the metabolism was stopped by injecting 20 μl HCI 1 N and 250 μl hydroxyhiamine (Packard Bioscience, Groningen, The Netherlands) was used to capture the produced ¹⁴ CO ₂ during 1 h at room temperature. D-[U- ¹⁴ C]-glucose oxidation rates were determined by liquid scintillation counting of the generated ¹⁴ CO ₂ . FACS-measurement of nutrient-associated changes in endogenous fluorescence for flavin (FAD and FMN, Argon laser excitation 488/ emission 530 nm) and NAD(P)H (UV-Argon laser excitation 351-363 nm/ emission 400-470 nm) was done following a 1 hour incubation at the indicated glucose concentrations, as described (Martens et al. 2005. J Biol Chem 280: 20389-96).

Example 1f: Statistical analysis Data are presented as means ± se of n independent experiments. Statistical analysis was performed using SPSS software for regression analysis and comparison of means. Statistical analysis was performed using analysis of variance (ANOVA) or paired Student's t-testing where appropriate. Differences were considered significant when p < 0.05.

Example 2: Results

Example 2a: Rat beta cells express high levels of HADHSC mRNA and protein: HADHSC expression in beta cells is disproportionate to their expression level of other beta-oxidation enzymes

We quantified mRNA expression levels of HADHSC and other beta-oxidation enzymes using Taqman real-time PCR. A schematic overview of the organization of this pathway, as proposed by Liang et al. 2001 (Biochem Soc Trans 29: 279-282) is shown in Fig. 3.

We simultaneously measured the relative mRNA levels of the mitochondrial enzymes ACADS, ACADM, ACADL, HADHSC and ACAA2 (see Liang et al. 2001 supra). Additionally, as marker for the peroxisomal beta-oxidation compartment we also quantified the mRNA level of ACAA1 , the peroxisomal thiolase, counterpart of mitochondrial ACAA2. We also measured the mRNA levels of PPAR alpha, the main transcriptional regulator of mitochondrial and peroxisomal fatty acid oxidation (Lalloyer et al. 2006. Diabetes 55: 1605-1613). The abundance of PPAR alpha mRNA is an order of magnitude lower than mRNA levels of metabolic enzymes (Fig. 4, separate Y axis on the right).

PPAR alpha expression is higher in tissues known to have high fatty acid oxidation rates, mainly liver, skeletal muscle and kidney than in the other tested tissues, and is associated with higher gene expression levels of the tested beta-oxidation enzymes (Fig. 4, mean ± se, n= 4-8).

Pancreatic endocrine cells (beta and islet non-beta) have a lower gene expression level of PPAR alpha, as well as of the beta-oxidation enzymes ACADS, ACADM, ACADL and ACAA2. On the other hand, their HADHSC mRNA-level is higher than that in all tested tissues (Fig 4).

Table 1 in Figure 5 (n=4-8) shows the relative mRNA expression level of HADHSC as compared to mitochondrial ACADM and ACAA2 and peroxisomal ACAA1 in various tissues (relative levels of HADHSC versus ACADM, ACAA2 and ACAA1 mRNA is calculated as 2 ^"δCt where δCt = (Ct value of HAHSC) - (Ct of other enzyme), both measured in the same run). These data disclose a marked unbalance between the expression level of HADHSC and other components of the beta-oxidation pathway, specifically in beta cells. Of note, this particular HADHSC expression pattern is also maintained in immortalized INS1 832-13 β cells, indicating that these rat beta cell lines are a suitable model to study the possible role of HADHSC in beta cell nutrient sensing.

HADHSC protein levels were measured using an affinity-purified chicken anti-human HADHSC antibody. This antibody, in rat tissues, detected a single protein band, at the expected molecular weight of 34 kDa (data not shown). HADHSC protein levels in purified beta cells were in the same range as the levels detected in the liver and heart muscle (Fig. 6). The expression in beta cells is also markedly higher than in the islet endocrine non-beta cell fraction (p<0.01 , n=4).

lmmunocytochemical double-staining using a monoclonal antibody raised against mitochondrial respiratory complex I subunit and against HADHSC, confirmed the expected mitochondrial localisation of HADHSC in β cells (not shown). lmmunohistochemical staining for HADHSC, using the affinity-purified polyclonal chicken anti- human antibody (Genway Inc., USA) confirmed high expression of HADHSC protein in rodent and human Langerhans islets, with low HADHSC protein expression in the surrounding exocrine. Within the islet, HADHSC protein expression was mainly confined to the insulin- expressing cells, with much lower levels in glucagon-expressing alpha cells (not shown).

Example 2b: RNAi-mediated suppression of HADHSC protein increases fractional insulin secretion induced by glucose in INS1 832-13 3 cells

We next examined the effect of selective suppression of HADSHC protein on insulin secretion by the glucose-responsive beta cell line INS1 832-13 (Hohmeier et al. 2000. Diabetes 49: 424-430). Two RNA interference strategies were tested for their effect on HADHSC protein expression, and on glucose-stimulated insulin secretion at 48-72 hours after initiation of knockdown.

Duplex siRNA-mediated HADHSC silencing. INS1 cells were transfected, with a pool of 4 different siRNAs against rat HADHSC (designated further as HADHSC siRNA), or with fluorescence-labelled (siGLO) control siRNA. Two control siRNAs were tested: (1 ) siGLO lamin A/C designed to target human/mouse lamin A/C - which was only partially active in rat cells (45 ± 8 % knockdown) - and (2) siGLO RISC free, a siRNA designed not to interact with the RISC complex. Both control siRNAs gave similar results, and could be used interchangeably.

At 48 hours after transfection, HADHSC siRNA resulted in 70% decrease in HADHSC mRNA vs. siGLO-transfected cells (Fig.7a, p<0.001 , n=8). The mRNA expression of ACADM, another enzyme of the mitochondrial beta-oxidation pathway was unaffected, indicating that HADSHC silencing was sequence-specific. This reduction resulted in a moderate (20%) but significant reduction of the HADHSC/beta-actin protein ratio (Fig.7b, p<0.05, n=8).

When subsequently tested for their glucose-induced insulin secretion (GSIS), the HADHSC suppressed cells exhibited moderate left- and upward shift of the GSIS concentration- response curve as compared to control-transfected cells, which was significant in the higher glucose range (12 and 25 mM, p<0.01 , n=8). Raising glucose from 3 to 12 mM caused a

similar fold change of insulin secretion in untransfected and siGLO-transfected cells, (2.67 ± 1.21 and 2.54 ± 1.21 fold respectively, p=0.41 , n=8); in HADHSC knockdown cells the fold induction was 3.11 ± 0.87 (n=8, p<0.05 versus siGLO and untransfected cells) (Figure 8a).

HADHSC-suppressed cells tended to be more responsive to short chain fatty acids: addition of butyrate (C4-fatty acid, 2 mM) in presence of 3 mM glucose caused a 2.12 ± 0.66 (p=0.06, n=8) higher secretion in HADHSC-suppressed cells as compared to a 1.64 ± 0.49 (p=0.30, n=8) in siGLO-cells (Fig. 8b).

Insulin secretion under depolarizing conditions (KCI 30 mM at 12 mM glucose) was comparable in HADHSC siRNA- and control-transfected cells (119 ± 41 and 112 ± 37 pg insulin/103 cells x h respectively, n=8, p=0.39).

Cellular insulin content at 48 hours did not differ between untransfected cells (921 ± 162 pg ins/103 cells, mean ± SD, n=5) and cells transfected with siGLO control (936 ± 174 pg ins/103 cells) or HADHSC siRNA (931 ± 195 pg ins/103 cells).

Plasmid-shRNAi mediated HADHSC knockdown. In order to achieve a better suppression of HADHSC protein expression, we designed pSUPER-basic vectors expressing shRNAi targeting rat HADHSC, and examined their effects at 48 and 72 hours after initiation of knockdown using two different plasmids (A3 and C6) designed against HADHSC; pSUPER-basic scrambled shRNAi served as control. A3- and C6-transfection suppressed HADHSC mRNA by 80 ± 1.5 and 60 ± 5 percent respectively when measured by qPCR at 72 hours (p<0.001 versus scrambled shRNAi, n=8). Again, silencing was sequence-specific as the mRNA levels of ACAA2, were unaffected (Fig. 9a). The more potent A3 plasmid suppressed HADHSC protein level by 50% and 65-79% at 48 and 72 hours, respectively (Fig. 9c, n=8, p<0.001 ) while C6 caused a less marked HADHSC downregulation which reached only significance after 72h. Both at 48 and 72h, A3 and C6- suppressed cells were investigated for their glucose-induced insulin secretory responses. At 48 hours, the 50% HADHSC knockdown by A3 increased moderately, but significantly (p<0.01 , n=8) insulin secretion in the lower glucose range (3-6 mM glucose). At 72 hours, the stimulatory effect waned - remaining only significant at 6 mM glucose. Surprisingly, the less potent C6 plasmid caused a robust stimulation of insulin secretion over the whole glucose range at 48 hours (p<0.05, n=4). Again, the stimulation was strongly diminished at 72 hours (Figure 10 a, b).

We reasoned that, if HADHSC suppression enhances insulin secretion, without parallel increase in insulin biosynthesis, loss of beta cell insulin content could ensue, affecting normal secretory function. Indeed, HADHSC knockdown by A3 depleted insulin content (40% reduction as compared to scrambled, p<0.01 , n=8, Fig. 10c) at 72 hours, when its correlated stimulatory effect on secretion was no longer clear, while it had no detectable effect at 48 hours.

C6, surprisingly, increased cellular insulin stores at 48 hours by 20% (p<0.05 versus scrambled, n=4), in parallel with its strong effects on insulin release at this time point. Over the next 24 hours however, C6-treated cells also lost insulin content (22% reduction in C6 insulin content at 72 versus 48 hours, p<0.05, n=4), indicating ongoing degranulation.

When insulin secretion was expressed as percent of cellular insulin content (fractional secretion) within each individual experiment (Fig. 10 d-e), HADHSC knockdown consistently increased the fractional secretion rate, both at 48 and 72 hours. In addition, the stimulatory effect was dependent on the degree of HADHSC knockdown, being more potent in the A3- than in the C6-transfected cells.

Example 2c: HADHSC suppression does not stimulate glucose-stimulated insulin secretion by acceleration of glucose metabolism in INS1 cells

Sustained suppression of fatty acid metabolism could result in increased glucose catabolic flux through pyruvate dehydrogenase, via a Randle effect (Randle 1998. Diabetes Metab Rev 14: 263-283). We thus examined if the secretory activation in HADHSC knockdown cells was associated with acceleration of glucose metabolism.

As a first attempt to correlate the biochemical changes induced by HADHSC knockdown and functional beta cell activation, we wished to exclude that HADHSC suppression increased fractional GSIS merely by accelerating glucose metabolism. Therefore, we measured glucose-induced redox changes of total cellular NAD(P)H, and mitochondrial riboflavins (FAD/FMN) as indicator for glucose metabolic rate and nutrient responsiveness in beta cells.

INS1 832-13 β cells showed a sigmoid glucose-NAD(P)H responsiveness that was quantitatively similar to that of freshly isolated primary cells (Martens et al. 2005 supra), and remained intact after lipid-based transfection. A3 plasmid-transfected cells showed slightly higher NAD(P)H levels from 6 mM glucose on as compared to scrambled-transfected cells

(Fig. 11a), but these changes did not reach significance except at 12 mM glucose (p <0.05, n=5).

Effects on the redox state of mitochondrial flavins, on the other hand, were more marked. As shown in Fig. 11b, severe, A3-mediated HADHSC knockdown, but not moderate C6- mediated knockdown, caused a marked increase in cellular levels of (oxidized) FAD/FMN. This reflects either (i) an increased baseline flavin oxidation (shifts from non-fluorescent reduced FADH2/FMNH2 to green fluorescent FAD/FMN), or alternatively (ii) increased total cellular FAD content. It was not explained by a differential glucose effect on flavin reduction, which was similar in scrambled-, C6- or A3-transfected cells. The increased flavin oxidation in A3- treated cells remained present after addition of rotenone, showing that the oxidation does not take not place at the FMN group of complex l-NADH-ubiquinol oxidoreductase (not shown).

In parallel, we directly measured CO ₂ formation from glucose in these cells: in line with its minor effects on glucose-induced NAD(P)H formation, HADHSC suppression by siRNA or shRNAi-plasmids exerted no effects on mitochondrial glucose oxidation, which is the major cellular source of NAD(P)H (Table 2 in Figure 12).

Example 2d: HADHSC siRNA-treatment also increases glucose-induced insulin secretion in FACS-purified primary rat beta cells

We extended our observations in INS1 β-cells to FACS-purified beta cells isolated from rat. Freshly isolated cells were re-aggregated and cultured in the presence 50 nM siRNA/cationic lipid complexes for 24 hours, followed by replacement of the siRNA-containing medium by normal culture medium.

72 hours after the initiation of this reverse transfection protocols, HADHSC siRNA-treated beta cells showed a moderate, 20 ± 4 percent reduction in HADHSC proteins, as illustrated by the dot plot and western in Fig. 13 a-b.

In parallel, we examined insulin secretion dynamically in a perifusion setting, with a step-wise increase in glucose concentration from 2.5 up to 20 mM. As shown in Fig. 14, HADHSC siRNA-treated beta cells showed higher secretory responsiveness to glucose from 5 mM glucose on up to 20 mM; these effects reached significance at 20 mM glucose, both prior and after addition of 10 nM glucagon (p<0.01 , HADHSC si versus siGLO control, n=4). Cellular insulin content did not differ significantly between the various conditions.

Example 3: Suitable siRNA target sequences in human HADHSC cDNA/mRNA

Figure 15 (Table 3) summarises preferred siRNA target sequences as predicted in human HADHSC cDNA sequence annotated under GenBank accession number NM_005327 (also shown in Figure 2 as SEQ ID NO: 3). These target sequences were predicted using siRNA prediction algorithms as taught by Dharmacon ("D") (Reynolds et al. 2004. Nat Biotechnol 22: 326-30), Invitrogen BLOCK-iT (TM) RNAi Designer ("B") (http://rnaidesigner.invitrogen.com/rnaiexpress), GC siRNA finder ("G") (Wang and Mu. 2004. Bioinformatics 20: 1818-20), and the Whitehead institute ("W") (Yuan et al. 2004. Nucleic Acids Res 32(Web Server issue): W130-4).

Example 4: Exemplary cell-free and beta cell-based screening assays for HADHSC activity modulators

Exemplary cell-based assay of HADHSC activity

Cell pellets of INS1 beta cells or primary human/rodent beta cells are resuspended in 25 mM phosphate, 0.2mM EDTA, 0.2% (vol/vol) Triton X-100 (pH 8.0), incubated on ice for 30 min, then centrifuged for 12,000 g, 10 min. HADHSC activity is then measured in the supernatant in the reverse direction by following the disappearance of NADH at 340 nm, in a reaction mixture containing 0.1 M potassium phosphate (pH 7.0)/0.1 mg/ml NADH, 0.3 mg/ml fatty acid-free BSA and 30 μM ketoacyl-coA (short chain, acetoacetylco-A) or 30 μM 3- ketopalmitoyl-coA . This assay can be performed either after pre-treatment of live cells with an agent to be tested, or after addition of the said agent to an extract of the said cells.

Cell-free assay for HADHSC activity

An enzymatic reaction as above is followed wherein the target enzyme is human/pig heart 3- hydroxyacyl-coA dehydrogenase obtained from commercial sources.