WO2013017656A1

WO2013017656A1 - Antagonists of ribonucleases for treating obesity

Info

Publication number: WO2013017656A1
Application number: PCT/EP2012/065139
Authority: WO
Inventors: Martin BILBAN; Harald Esterbauer; Oswald WAGNER
Original assignee: Medizinische Universität Wien
Priority date: 2011-08-02
Filing date: 2012-08-02
Publication date: 2013-02-07

Abstract

The present invention relates to antagonists of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with an increase in adipocyte number or size, such as overweight, or obesity and the like. Also methods for assessing the activity of candidate antagonists of RNAse A family members, uses of cells, tissue or a non-human animal in such screening methods and a kit for carrying out the method are subject of the present invention. Moreover, the present invention relates to agonists of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with a decrease in adipocyte number. The disease may be lipodystrophy, like hereditary lipodystrophy, drug- induced lipodystrophy, lipodystrophy caused or induced by AIDS/HIV therapy, or traumatic lipodystrophy. The disease may also be is cachexia or a disease associated with disturbed energy storage.

Description

Antagonists of Ribonucleases for treating obesity

The present invention relates to antagonists of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with an increase in adipocyte number or size, such as overweight, or obesity and the like. Also methods for assessing the activity of candidate antagonists of RNAse A family members, uses of cells, tissue or a non-human animal in such screening methods and a kit for carrying out the method are subject of the present invention. Moreover, the present invention relates to agonists of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with a decrease in adipocyte number. The disease may be lipodystrophy, like hereditary lipodystrophy, drug- induced lipodystrophy, lipodystrophy caused or induced by AIDS/HIV therapy, or traumatic lipodystrophy. The disease may also be cachexia or a disease associated with disturbed energy storage.

Obesity is a condition where excess body fat accumulates to such an extent that one's health may be affected; see Arner (2010) Biochem and Biophys Res Comm 396, 101 - 104. Especially in developed countries obesity is increasing and constitutes a major health problem, as obesity also enhances the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes; see Spalding (2008) Nature 453, 783-787.

Established treatments of obesity include weight loss regimes, such as diets (i.e. calory restriction or change of food composition/content) exercise, bariatric surgery and/or use of drugs to prevent or delay intestinal uptake of nutrients. Yet, these treatments are cumbersome and associated with further health risks. For example, drugs or substances interfering with cellular metabolism (e.g. insulin) and substances interfering with food intake and satiety either via the central nervous system or via modulation of satiety signals (e.g. appetite suppressants (anorectics) - prevent absorption of fat in gut) are often associated with severe side effects, e.g. sleep disorders, hypertension, damage to the heart and haemorrhagic stroke. The treatment regimes disclosed in the art primarily aim at decreasing adipocyte size but fail to reduce adipocyte number; see Arner (2010), loc. cit. Adipocytes are the cells that primarily compose adipose tissue and are specialized in storing energy as fat. Yet, the fat mass in humans is the product of both adipocyte volume and adipocyte number. A particular form of overweight/obesity is hyperplastic overweight/obesity (increase in adipocyte number); see Arner (2010), loc. cit.

As many obese and almost all severely obese individuals have more than the average number of adipocytes (i.e. suffer from hyperplastic obesity), treatment of overweight/obesity should also target adipocyte number.

Thus, the technical problem underlying the present invention is the provision of means and methods for the medical intervention of diseases associated with an increase in adipocyte number, such as overweight/obesity and related disorders.

The technical problem is solved by provision of the embodiments characterized in the claims.

Accordingly, the present invention relates to an antagonist of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with an increase in adipocyte number. In other words, the present invention relates to an antagonist of a Ribonuclease being a member of the RNAse A family, wherein the antagonist is for use in treating a disease associated with an increase in adipocyte number. The antagonist may be a selective antagonist of a Ribonuclease being a member of the RNAse A family.

Pathological conditions associated with an increase in adipocyte number are well known in the art; one example is hyperplastic obesity. A corresponding increased number of adipocytes compared to a control (e.g. a healthy person or a subject/individual with normal weight (for example BMI below 25 kg/m²) can be easily determined. For example, a biopsy sample or a sample after liposuction may be taken and the number and size of adipocytes be counted e.g after Oil Red-0 (OrO) staining. An "increase in adipocyte number" may, for example, be a 20 % higher adipocyte number compared to the control. in the present invention ribonucleases of the RNAse A family have unexpectedly been identified as key regulators of preadipocyte differentiation. It is shown herein that antagonists of such RNAse A-like Ribonucleases prevent differentiation of preadipocytes and thus interfere with the formation of mature, differentiated adipocytes. Thereby, the number of adipocytes is dramatically decreased; see Figure 3.

Adipocytes are well known as cells capable of storing lipids. An increase in adipocyte number (i.e. hyperplasia) is found in overweight and obese individuals. It is clear that the inhibition of preadipocyte differentiation/adipocyte formation contributes to a better control of adipo genesis (i.e. the process of cell differentiation by which preadipocytes become adipocytes). Hence, the inhibition of adipocyte formation and reduction in the overall amount or number of adipocytes by the antagonists of the present invention provides a potent means in the treatment of overweight, obesity, or secondary disorders related thereto, such as diabetes. The herein provided antagonists are particularly useful in situations or pathological conditions where adipocyte hyperplasia is present, i.e. when the number of adipocytes is increased, the proliferation rate of (pre)adipocytes is increased and/or the differentiation rate of preadipocytes is increased. Hyperplasia is, in particular, correlated with obesity severity and is most marked in severely obese individuals (see Arner (2010), loc. cit.), The treatment of severely obese patients is, therefore, particularly envisaged in context of the present invention.

The appended examples show that murine EAR-1, -2 and -10 are strongly expressed in preadipocytes (3T3-L1 cell line and murine preadipocytes); also human R Ase 1 is strongly expressed in human adipose tissue. More importantly, it has been found herein for the first time that these R Ases promote adipogeneses as evidenced by the induction of the expression of adipo genie genes and the increased number of adipocytes (demonstrated by OrO-staining); see Figures 5 and 6. It is believed that the contribution of a Ribonuclease to adipogenesis correlates with its endogenous expression level or activity in (pre) adipocytes and/or (white) adipose tissue. As murine EAR-1, -2, -10 as well as human RNAse 1 have been found to be strongly expressed, these RNases are the primary targets of the herein provided antagonists. Due to the high homology level (at least within one RNAse A subfamily such as the murine EAR family or the human RNAses 1 to 13, it is, however, believed that further members of the RNAse A family promote adipogenesis. Accordingly, it is envisaged herein further members of the RNAse A (sub)family and preferably all members that are expressed in an overweight/adipose patient should be targeted. Preferably, only those Ribonucleases are targeted that are expressed in (pre) adipocytes/adipose tissue. Even more preferably, the herein provided antagonists only inhibit the Ribonucleases at the site of their expression in the patient, i.e. in adipose tissue, and/or (pre)adipocytes. Human RNAse 1 mRNA is predominantly expressed in adipose tissue, brain, heart, lung and testes, see Figure 16. Further, it is believed that an inhibitor directed against a specific Ribonuclease of a RNAse A (sub)family (e.g. RNAse 1) will likewise be effective against related Ribonucleases due to their high level of homology (e.g. RNAse 2 or RNAse 3). in the appended examples a loss of function preadipocyte 3T3-L1 cell line was used, wherein the expression of Eosinophil-associated Ribonucleases (or short„EAR") EARl, EAR2 and EAR10 is decreased. The 3T3-L1 cell line is a recognized model cell line of preadipocytes; see Poulos (2010) Exp Biol Med 235: 1 185-93. Differentiation of the 3T3-L1 cell line (i.e. the development/differentiation of preadipocytes) into mature adipocytes was induced by a standard hormonal mixture. Unexpectedly, preadipocytes of the EAR-Loss of function 3T3-L1 cell line only differentiated to a very limited extent into mature adipocytes upon induction. The above results were confirmed with an antagonist of human RNAse 1 . Figure 14 demonstrates successful RNASEl mRNA silencing in siRNASEl (cells treated with a siRNA oligo targeting RNASEl), but not siCtrl (cells treated with a control siRNA) treated SVF cells. An exemplary nucleotide sequence encoding siRNA targeting human RNASEl is shown in SEQ ID No.49 (AC C A A ATG ATG AGGCGC CGGA AT AT), whereas SEQ ID No.50 shows the RNA sequence of an siRNA targeting human RNASEl to be used in accordance with the present invention (ACCAAAUGAUGAGGCGCCGGAAUAU). The qPCR data show RNASEl mRNA expression on day 7 following induction with the differentiation cocktail. It was found that knock-down of RNASEl affects adipocyte differentiation, as measured by OrO staining after induction of differentiation of siCtrl or siRNASEl -treated SVF cells with a standard proadipogenic cocktail. At day 7 post-induction of differentiation, cells were fixed and stained with OrO to judge adipocyte content. Figure 15 shows quantitative staining with ORO, as measured with spectrophotometry. The intensity of ORO staining in RNASEl knockdown cells (siRNASEl) was significantly diminished by approximately 50% as compared with si Ctrl-treated cells, which shows that RNASEl is required for adipocyte differentiation and that inhibition of RNAse 1 inhibits adipocyte differentiation.

Thus, it was found herein that the inhibition of Ribonucleases of the RNAse A family prevents differentiation of preadipocytes, and, accordingly, decreases the number of adipocytes. As explained in detail above, differentiation/proliferation of preadipocytes and/or increase in the number of adipocytes is a decisive factor in the development or progress of diseases associated with a disturbance of adipocyte formation, such as overweight or obesity. Hence, preadipocyte differentiation/proliferation and/or the number/amount of adipocytes is directly linked to these diseases. Accordingly, it is clear that the inhibition of these mechanisms by the herein described antagonists inevitably affects the formation and number of adipocytes. Therefore, the herein described antagonists provide a potent means to interfere with the development and/or progress of the above diseases as well as of secondary diseases related thereto (such as diabetes).

The most common diseases to be treated in accordance with this invention are overweight (or "pre-obesity" which are used interchangeably herein) and/or obesity. Accordingly, the present invention relates in particular to means and methods for the medical intervention in an overweight or obese subject, in particular human patients. Overweight and obesity are defined as abnormal or excessive fat accumulation that may impair health. Body mass index (BMI) is a simple index of weight-for-height that is commonly used to classify overweight and obesity in adults. It is defined as a person's weight in kilograms divided by the square of his height in meters (kg/m²).

An "overweight" patient is often defined as having a body mass index (BMI) above 25 kg/m². In context of the present invention, "overweight" is preferably defined as a body mass index (BMI) between 25 to 30 kg/m² and "obesity" is preferably defined as a body mass index (BM) of higher than 30 kg/m . "Severe obesity" is usually defined as a body mass index (BM) of 40 kg//m² and higher than 40 kg/m². These definitions are in line with the present definition of the WHO: according to the WHO, a BMI greater than or equal to 25 is overweight and a BMI greater than or equal to 30 is obesity.

According to WHO, raised BMI is a major risk factor for noncommunicable diseases such as cardiovascular diseases (mainly heart disease and stroke), diabetes, musculoskeletal disorders (especially osteoarthritis - a highly disabling degenerative disease of the joints) and some cancers (endometrial, breast, and colon). The risk for these noncommunicable diseases increases with the increase in BMI. Accordingly, the present invention provides also means for treating or preventing the above secondary disorders and diseases, in that the BMI of overweight/obese subjects/patients can be reduced to normal levels (usually below 25 kg/m²). In one advantageous aspect, the antagonists of the present invention also allow the treatment of overweight or obese children. It is known in the art that childhood obesity is associated with a higher chance of obesity, premature death and disability in adulthood. In addition to increased future risks, obese children experience breathing difficulties, increased risk of fractures, hypertension, early markers of cardiovascular disease, insulin resistance and psychological effects. Accordingly, the treatment of childhood obesity is highly beneficial also in that the development of adult overweight/obesity can be prevented and/or in that the above- mentioned secondary disorders/diseases may be treated or prevented.

ΒΜΪ provides the most useful population-level measure of overweight and obesity as it is the same for both sexes and for all ages of adults. However, it should be considered a rough guide because it may not correspond to the same degree of fatness in different individuals. In certain medically indicated cases, it is therefore envisaged that also patients with a BMI below 25 kg/m² are to be treated with the herein provided antagonists in order to reduce their body weight. In the same vein, not every subject/patient with a high BMI (e.g. between 25 to 30 kg/m or higher than 30 kg/m ) is an "obese" or "overweight" patient - it is well known that individuals with greater than average muscle mass (e.g. certain athletes (like bodybuilders)) will have a higher BMI without having abnormal or excessive fat accumulation.

Therefore, the disease/disorder to be treated herein may in the alternative or in addition be characterized by the presence of 20 % or more body fat in the subject/patient. For example, a body fat percentage of 25 % or more may be characteristic for an overweight/obese man, and a body fat percentage of 32 % or more may be characteristic for an overweight obese woman. It is known in the art that a person's body fat percentage is the total weight of the person's fat divided by the person's weight.

The body's fat consists of essential body fat and storage body fat. Essential body fat is necessary to maintain life and reproductive functions. Essential fat is usually 3%-5% in men, and 8-12% in women. Storage body fat consists of fat accumulation in adipose tissue, part of which protects internal organs in the chest and abdomen.

The table below describes different percentages that are often used in the art to characterize the percentage of essential fat and the percentage of total fat in men and women: Description Women Men

Essential fat 10-13% 2-5%

Athletes 14-20% 6-13%

Fitness 21-24% 14-17%

Average 25-31% 18-24%

Obese 32%+ 25%+

The percentage of storage fat or extra fat as denoted herein may be calculated from the above given exemplary values. Yet, it is often difficult to exactly determine the percentage of essential fat and of storage fat. Therefore, the total fat percentage is routinely determined/estimated and used in the art in order to classify a subject/patient as overweight/obese. Appropriate measurement techniques are known in the art and include Near-infrared interactance or Dual energy X-ray absorptiometry (DXA). Also multicompartment models can be used; these models can include DXA measurement of bone, plus independent measures of body water and body volume. Various other components may be independently measured, such as total body potassium. Also in- vivo neutron activation can quantify all the elements of the body and use mathematical relations among the measured elements in the different components of the body (fat, water, protein, etc.) to develop simultaneous equations to estimate total body composition, including body fat. Also body average density measurement can be used to determine a subject/patients body fat percentage: this technique involves the measurement of a person's average density (total mass divided by total volume) and the application of a formula to convert that to body fat percentage. Bioelectrical impedance analysis is also a well known technique to estimate body fat percentage. Also anthropometric methods (measurements made of various parameters of the human body, such as circumferences of various body parts or thicknesses of skinfolds) may be used. Because most anthropometric formulas such as the Durnin-Womersley skinfold method, the Jackson-Poiiock skinfold method, and the US Navy circumference method, estimate body density, the body fat percentage is obtained by applying a second formula, such as the Siri or Brozek formula. Further, Skinfold methods may applied and the body fat percentage may even be calculated from the BML These and other methods are well known and can be deduced from reviews like Lee (2008) Curr Opin Clin Nutr Metab Care 11(5), 566-572 and Gallagher (2008) Int J Body Compos Res 6(4): 141-148 which are incorporated in their entirety herein. Preferably, the body fat percentage of a male patient/subject to be treated is at least 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 % and more preferably, at least 25 %. The body fat percentage of a female patient/subject to be treated is at least at least 25 ¾, 26 %, 27 %, 28 %, 29 %, more preferably 30 %, 31 % and even more preferably at least 32 %. The identification of obese patients according to the body fat percentage (for example determined according to the bioelectrical impedance criterion) may be especially advantageous in individuals having a BMI of below 30 kg/m²; according to the bioelectrical impedance criterion a man may be considered obese in case of a body fat percentage of at least 25 % and a woman may be considered obese in case of a body fat percentage of at least 30 %; see Frankenfield (2001) Nutrition 17:26-30 which is incorporated in its entirety herein. Upper limits of body fat percentage will have to be calculated on an individual basis; yet, typically body fat percentage does not exceed about 60 % even in severely obese subjects/patients.

Further, disorders which involve higher levels of triglycerides in the blood of a patient may be treated in accordance with the present invention. The recommended level of triglycerides (in a normal range) is in males 40-160 mg/dL and in females 35 to 135 rng/dL. However, in Germany also "higher levels" are tolerated on being normal; e.g. 250 mg dL. Accordingly, higher levels of triglycerides to be treated with the herein provided antagonists are preferably above 150 mg/dL, more preferably above 200 mg/dL and most preferably above 250 mg dL.

Accordingly, the present invention provides for a new medical use of Ribonuclease antagonists for treating obesity, and/or eating disorders leading to increased BMI body fat percentage/body weight/body mass as defined herein above. Also envisaged are disorders related to higher or pathologically high BMI/body fat percentage/body weight due to the use of drugs (like corticosteroids, antipsychotic drugs, antidepressants, particularly tricyclic antidepressants, oral contraceptives, etc.).

According to the International Statistical Classification of Diseases and Related Health Problems (10th Revision, Version for 2007) issued by the World Health Organization, the following diseases and disorders relate to obesity: E66 Obesity

Excludes: adiposogenital dystrophy ( E23.6 )

lipomatosis:

•NOS ( E88.2 )

• dolorosa [Dercum] ( E88.2 )

Prader-Willi syndrome ( Q87.1 )

E66.0 Obesity due to excess calories

E66.1 Drug-induced obesity

Use additional external cause code (Chapter XX), if desired, to identify drug. E66.2 Extreme obesity with alveolar hypoventilation

Pickwickian syndrome E66.8 Other obesity

Morbid obesity E66.9 Obesity, unspecified

Simple obesity NOS

The treatment of the above diseases and disorders is therefore also envisaged in context of the present invention. In accordance with the above, the invention relates to the use of Ribonuclease antagonists in the treatment or prevention of diseases/disorders related to, caused by or leading to higher or pathologically high body weight/BMI/body fat percentage.

The antagonists of the present invention may also be used to decrease adipocyte volume. Accordingly, the antagonists may be used in the treatment of a disease that is associated with an increase in adipocyte volume. As discussed above, overweight and obese individuals show an increase in adipocyte volume or, in other words, such individuals suffer from hypertrophic overweight/obesity. It is known in the art that hypertrophy is characteristic of all overweight and obese individuals; see Araer (2010), loc. cit. Accordingly, the herein provided antagonists may be used in the treatment of a disease associated with an increase in adipocyte volume, especially overweight and/or obesity. Preferably, the disease to be treated is hypertrophic overweight or hypertrophic obesity. As discussed above, known treatment regimens primarily aim at decreasing adipocyte size. Accordingly, such known treatment regimes are envisaged herein in context of co-therapy with the Ribonuciease inhibitors of the present invention. Such co-therapeutic approaches may be particularly advantageous as they target both the adipocyte volume and adipocyte number. Exemplary conventional therapies are described herein above and include weight loss regimes, such as diets (i.e. calory restriction or change of food composition/content) and exercise, bariatric surgery and/or use of drugs to prevent or delay intestinal uptake of nutrients.

In accordance with this invention it is also envisaged that Ribonuciease antagonists are employed in the medical intervention of secondary disorders related to a (pathological) increase of body weight/BMI body fat percentage. These "secondary disorders" may comprise, but are not limited to diabetes type 2, high blood pressure (hypertension), cardiovascular diseases, cancer, problems with sexual function and disorder of the muscular or bone system, and lipid disorders (such as hypertriglyceridemia and hypercholesterolemia). Problems with sexual function may comprise libido problems, penile dysfunction as well as FSAD (Female Sexual Arousal Disorder). Also dyslipidaemia may be a "secondary disorder". Also envisaged is the treatment of secondary disorders like are growth hormone deficiency, partial growth hormone deficiency or neuro-secretory dysfunction of growth hormone secretion.

Secondary disorders of the metabolism linked to higher body weight/body mass/BMI/body fat percentage and to be treated by the admimstration of Ribonuciease antagonists may also comprise, but are not limited to, glycogen storage diseases, lipid storage diseases (like Gaucher or Niemann Pick), endocrine disorders (like Cushings, hypothyroidism, insulinomas, lack of growth hormone, diabetes, adrenogenital syndrome, diseases of the adrenal cortex), tumors and metastases (such as craniopharyngeomas), Prader-Willi syndrome, Down syndrome and genetic diseases and syndromes (like, e.g., hyperlipoproteinemias, hypothalamic disorders, Frohlich syndrome or empty sella syndrome).

As mentioned, the antagonist to be used herein may be a selective antagonist of a Ribonuciease being a member of the RNAse family. In other words, the selective antagonist to be used in accordance with the present invention selectively inhibits/antagonizes (these terms can be used interchangeably herein) a Ribonuclease being a member of the RNAse family i.e. it primarily inhibits a Ribonuclease being a member of the RNAse family and does substantially not inhibit other proteins or compounds. The selective inhibitors shows, for example, a stronger Ribonuclease inhibition than inhibition of a protein which is not a Ribonuclease. Preferably, the selective inhibitors show at least a 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold,35 fold or 40 fold (or higher) stronger Ribonuclease inhibition than inhibition of a protein which is not a Ribonuclease, wherein the higher values are preferred. The selective inhibitors may show an up to 100 fold stronger Ribonuclease inhibition than inhibition of a protein which is not a Ribonuclease.

Selectivity expresses the biologic fact that at a given compound concentration enzymes (or proteins) are affected to different degrees. In the case of enzymes selective inhibition can be defined as preferred inhibition by a compound at a given concentration. Or in other words, an enzyme (or protein) is selectively inhibited over another enzyme (or protein) when there is a concentration which results in inhibition of the first enzyme (or first protein) whereas the second enzyme (or second protein) is not affected. To compare compound effects on different enzymes it is crucial to employ similar assay formats, such as an assay for measurement of a Ribonuclease activity as described in more detail below. Appropriate assays are also described in Rosenberg (1995), J Biol Chem. 270(14):7876-81 (reference 1); Rosenberg (1995) J. Biol Chem. 270(37):21539-44 (reference 2); Rosenberg (1997) Nucleic Acids Res. 25(17):3532-6 (reference 3); or Domachowske (1998) Nucleic Acids Res. 26(14):3358-63.

Assay for measurement of a Ribonuclease activity: The Ribonuclease assay was described previously in detail (see references 1, 2, 3, or 4). Briefly, the concentration of perchloric acid soluble ribonucleotides generated from acid-precipitable yeast tRNA (Sigma, St Louis, MO) in 40 mM sodium phosphate, pH 7.5 will be measured spectrophotometrically at 260 nm. Ribonuclease activity (pmol/min) at single enzyme/substrate concentrations will be determined for increasing concentrations of selective antagonists and depicted in bar graphs.

1 : Rosenberg HF. Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. J Biol Chem. 1995 Apr 7;270(14):7876-81 , 2: Rosenberg HF, Dyer D. Eosinophil cationic protein and eosinophil-derived neurotoxin. Evolution of novel function in a primate ribonuclease gene family. J. Biol Chem. 1995 Sep 15;270(37):21539-44.

3: Rosenberg HF, Dyer KD. Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity.

Nucleic Acids Res. 1997 Sep l;25(17):3532-6.

4: Domachowske JB, Dyer KD, Adams AG, Leto TL, Rosenberg HF. Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. Nucleic Acids Res. 1998 Jul 15;26(14):3358-63.

The ratio of IC50 values of selective inhibitors of a Ribonuclease, being a member of the RNAse A family, determined according to an appropriate assay (e.g. the assays described above for a compound which is a selective Ribonuclease inhibitor, for example, a selective Ribonuclease 1 inhibitor/ or other assays known in the art) and IC50 values of inhibitors which are not selective for a Ribonuclease (like Ribonuclease 1) determined according to an appropriate comparative assay (e.g. the assays described above for an compound which is not a selective Ribonuclease inhibitor, for example, not a selective Ribonuclease 1 inhibitor/ or other assays known in the art) is about 1 : 10 or lower. More preferred is a ratio of below 1 :15, 1 :20, 1:30, 1:35 or 1:40, 1 :50, 1:60, 1:70, 1:80, 1:90 or 1 :100 or even lower.

In accordance with the above, the selective antagonist to be used herein may be a selective antagonist of one particular Ribonuclease being a member of the RNAse family as disclosed herein, i.e. it primarily inhibits one particular Ribonuclease (like Ribonuclease 1) and does substantially not inhibit other Ribonucleases. The definitions and explanations given herein above in terms of "selective inhibitor'V'selective antagonist" as well as the definitions and explanations below on "Ribonucleases which are a member of the RNAse A family" apply, mutatis mutandis, in this context.

In one aspect of the present invention an antagonist of a Ribonuclease being a member of the RNAse A family is provided for use in treating a disease associated with an increase in adipocyte number, wherein the antagonist is a selective antagonist of a Ribonuclease being a member of the RNAse A family, and wherein said Ribonuclease being a member of the RNAse A family is a human RNAse 1, wherem said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24. The present invention provides an antagonist of a Ribonuclease being a member of the RNAse A family, wherein the antagonist is for use in treating a disease associated with an increase in adipocyte number, wherein the antagonist is a selective antagonist of a Ribonuclease being a member of the RNAse A family, and wherein said Ribonuclease being a member of the RNAse A family is a human RNAse 1, wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24.

The foilowing relates to "Ribonucleases which are a member of the RNAse A family" as used in context of the present invention. The term "Ribonuclease", "RNAse", "Ribonuclease A", "Ribonuclease A-like", "RNAse A-like" used herein denotes a member of the well known RNAse A family. In particular, the term refers to a polypeptide with an activity specific for Ribonucleases of the RNAse A family, and in particular, a proadipogenic activity, as described herein and shown in the appended example.

Exemplary "Ribonucleases which are a member of the RNAse A family" are murine Eosinophil-associated Ribonuclease ("EAR") or human Ribonucleases such as human RNase 1 as shown in SEQ ID NO. 24. These non-limiting exemplary ribonucleases are described herein below in more detail, Preferably, the Ribonucleases are expressed, more preferably overexpressed, in adipose tissue and/or (pre)adipocytes as determined on mRNA or protein level. Corresponding experiments showing a high expression of EAR-1, -2, -10 and/or RNAse 1 are provided in the appended examples. It is preferred that the antagonists target Ribonucleases which are expressed in adipose tissue and/or (pre)adipocytes and more preferably those Ribonucleases with a high expression level, like EAR-1, -2, -10 and/or human RNAse 1.

The term ^Eosinophil-associated Ribonuclease" or„EAR" denotes a vertebrate RNase family belonging to the larger ribonuclease A superfamily. To date 1 1 mouse EAR genes (EAR 1 to 7, EAR 10 to 12 and EAR 14 are known. In particular, the term„EAR" refers to a polypeptide with an activity specific for Eosinophil-associated Ribonuclease, and in particular, a proadipogenic activity, as described herein and shown in the appended example. Eosinophil- associated Ribonucleases (EAR) are well known in the art and, inter alia, described in Rosenberg (2001), loc. cit; Cho (2005), loc. cit. and Rosenberg (2008a), loc. cit.

Similarly, the term„human Ribonuclease" denotes the human RNase family belonging to the larger ribonuclease A superfamily. To date 13 human Ribonuclease genes (RNAse 1 to 13) are known. In particular, the term„ human Ribonuclease " refers to a polypeptide with an activity specific for human Ribonucleases, and in particular, a proadipogenic activity, as described herein and shown in the appended example. Ribonucleases of the RNAse A family are well known in the art as described in the following. Since the bovine pancreatic ribonuclease was first purified, the RNAse A superfamily, of which RNase A is the prototype, has been one of the most intensively studied objects in biochemistry, structural biology and enzymology. The molecular evolution of this family has, however, been a matter of debate. Cho (2005) Genomics 85, 208-220 investigate the evolutionary origin and relationship of members of the ribonuclease A superfamily identified in human, mouse, rat and chicken genomes. This group established that Eosinophil-associated Ribonuclease (EAR) represent a well-established group in this superfamily. Furthermore, Cho (2005), loccit. hypothesize that the superfamily started off as a host-defense mechanism during early vertebrate evolution.

The role of eosinophils and Eosinophil-associated Ribonuclease (EAR) in host defense has been postulated prior to Cho (2005), loc. cit., in the art; see Rosenberg (2001) J. Leukocyte Biol. 70, 691-698. In this document the antiviral activity of EARs, in particular against the respiratory syncytial virus and the pneumonia vims of mice, is described. These viruses are single- stranded RNA virus pathogens and it is speculated in Rosenberg (2001) that EARs interact with a yet to be identified cellular target that is itself responding to a virus. Further, the authors of this document opine that the dysregulation of eosinophil mediated host defense may lead to asthma and reactive airways disease. Rosenberg (2008a) J. Leucocyte Biol. 83, 1079- 1087 summarizes the present understanding of the function of RNases in host defense. Inter alia, the role of eosinophil RNases (such as murine EARs and human RNase 2 (Eosinophil- derived neurotoxin, EDN) and human RNase 3 (Eosinophil cationic protein, ECP) is described in this paper. It is proposed therein that the anti-pathogenic acitivity of these proteins does not necessarily reside in their RNase activity; Rosenberg (2008a) propose that the toxicity to pathogens and the bactericidal activity may not be dependent on RNase activity. It is speculated that such RNases may rather play a role in antiviral host defense as chemoattractant agents and as endogenous ligands for TLR (Toll-like receptor).

EDN (which is the human ortholog of murine EAR1 and EAR2; see Rosenberg (2008), loccit.) is also a known neurotoxin; see Rosenberg (2008b), Curr Pharm Biotechnol 9(3), 135- 140. For example, EDN has been described as early as in the 1930s as causing a neurotoxic syndrome (the Gordon syndrome). The same antiviral activity as observed for EDN is confirmed in this document for murine EARs 1 and 2. Further, Rosenberg (2008b) describes that both EDN and EAR2 have a chemoattractant activity for immature dendritic cells and speculate that this capacity of activating antigen presenting cells (like dendritic cells) and the function as endogenous ligand for TLR2 might link innate and acquired immune responses. It is clear from the above that Ribonucleases of the RNAse A family may, at most, be involved in the immune response.

However, none of these prior art documents describes or proposes that such Ribonucleases might have a putative role in preadipocyte differentiation. For example, Rosen (2006), Mol Cell Biol 7, 885-896 provides an extensive review on the mechanisms involved in adipocyte differentiation, inter alia, the peroxisome proHferator-activated receptor γ (PPARy) and CCAT-enhancer-binding proteins (C/EBPs) are described as crucial transcription factors that promote adipogenesis. Yet, also Rosen (2006) is silent on a potential role of RNAses in adipocyte formation. in sum, none of the prior art document speculates on a role of RNAse A-like Ribonucleases in the pathogenesis of overweight/obesity. In the present invention, a link between Ribonucleases of the RNAse A family and adipogenesis/the number of adipocytes has, for the first time been established. Accordingly, the present invention provides for the first time means for the use of antagonists of RNAse A-like Ribonucleases in the treatment of diseases associated with an increase in adipocyte number, like overweight and/or obesity.

The diverse members of the RNAse A family are well known in the art. Exemplary members (as well as their nucleic acid and amino acid sequences) are described herein. However, the teaching of the present invention can readily be applied to other members of the RNAse A family by a skilled person. Accordingly, all members of the RNAse A family can be used in context of the present invention and the explanations given herein for exemplary members apply, mutatis mutandis, also to these other members.

Preferably, antagonists of murine EARl, murine EAR2 and/or murine EAR 10 (and/or their orfhologues, especially primate, and particularly human orthologues) are used in accordance with the present invention. The use of antagonists of human RNAse 1 is preferred in context of the present invention. Also antagonists of other human RNAses, like RNase 2 (also known as Eosinop I-Derived Neurotoxin, EDN) or human RNase 3 (Eosinophil Cationic Protein, ECP) may be employed in context of the present invention. As described in the above- mentioned documents, RNase 2 and RNase 3 are orthologues of murine EAR-1 and EAR-2. As documented in the appended examples, the knockdown of, inter alia, murine EAR-1 and EAR-2 and/or EAR-10 demonstrated that inhibitors of members of the EAR family, such as EAR-1, EAR-2 and/or EAR-10 can successfully be used in the treatment of diseases associated with a in increase in adipocyte number/disturbance of adipocyte formation, like overweight, obesity and the like.

Exemplary members of the RNAse A family are polypeptides comprising the amino acid sequence shown in SEQ ID NO: 2 (murine EAR-1), SEQ ID NO: 4 (murine EAR-2) and SEQ ID NO: 16 (murine EAR-10) as well as human RNAses such as the human RNAse 1 (amino acid sequence shown in SEQ ID NO. 24). Methods for determining the activity of such polypeptides are described herein further below.

Accordingly, the present invention relates to an antagonist of a Ribonuciease being a member of the RNAse A family for use in treating a disease associated with an increase in adipocyte number, wherein said Ribonuciease being a member of the RNAse A family is a human RNAse 1, wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24. In other words, the present invention provides an antagonist of a Ribonuciease being a member of the RNAse A family, wherein the antagonist is for use in treating a disease associated with an increase in adipocyte number, wherein said Ribonuciease being a member of the RNAse A family is a human RNAse 1 , wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24.

The EAR protein may be encoded by a nucleic acid sequence shown in SEQ ID NO: 1 (murine EAR-1), SEQ ID NO: 3 (murine EAR-2) and SEQ ID NO: 15 (murine EAR-10). A human RNAse may be encoded by a nucleic acid sequence as shown in SEQ ID NO: 23 (human RNase 1). The sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 (murine EAR-1), SEQ ID NO: 3 and SEQ ID NO: 4 (murine EAR-2), SEQ ID NO: 15 and SEQ ID NO: 16 (murine EAR-10), SEQ ID NO: 23 and SEQ ID NO: 24 (human RNase 1) refer to the gene encoding murine and human members of the RNAse A family and the murine or human RNAse A protein itself, respectively. As mentioned, the present invention is not limited to the use of murine or human members of the RNAse A family (such as the specific members cited above) or (a) functional fragment(s) thereof), but relates also to the medical use of antagonists (or agonists) of (a) orthologous or homologous member(s) of the RNAse A family (or (a) functional fragment(s) thereof).

The terms "orthologous'V'homologous" are described herein below. For example, further murine or human members of the RNAse A family may be used or rat or chicken members of the RNAse A family may be used in context of the present invention. Though specific sequences are provided herein, the respective sequences can also been deduced from public databases. For example, the amino acid sequence of human RNAse 1 can be deduced from the NCBI database (accession number NP_002924).

In context of the present invention, it is preferred that antagonists of or against (a) member(s) of the human RNAse A family (such as RNAse 1 or (a) functional fragment(s) thereof) are used in the treatment of humans suffering from (a) disease(s) associated with an increase in adipocyte number or disturbance of adipocyte formation. Correspondingly, antagonists of or against (a) member(s) of the murine EAR family (such as EAR- 1, EAR-2 and/or EAR- 10 or (a) functional fragment(s) thereof) are preferably used in the treatment of mice suffering from (a) disease(s) associated with a disturbance of adipocyte formation. Accordingly, it is preferred that antagonists of or against (a) member(s) of the RNAse A family (or against (a) functional fragment(s) thereof) to be used in the treatment of a specific organism (e.g. human, mouse or pig) specifically antagonize the member(s) of the RNAse A family of said specific organism (e.g. human, mouse or pig, respectively).

However, it is also within the scope of the present invention that the specific antagonists of or against (a) member(s) of the RNAse A family of a specific organism as described above may also be used in the treatment of closely related organisms; for example, antagonists of (a) member(s) of the human RNAse A family may be used in the treatment of a primate (like a chimpanzee), and vice versa. It is also envisaged that the antagonists of (a) member(s) of the RNAse A family of a specific organism may also be used in the treatment of distantly related organisms; for example, antagonists of (a) member(s) of the human RNAse A family may be used in the treatment of a mouse, and vice versa. Closely related organisms may, in particular, be organisms that form a subgroup of a species, e.g. different races of a species. Also organisms that belong to a different species but can be subgrouped under a common genus can be considered as closely related. Less closely related organisms belong to different genera subgrouped under one family. Distantly related organisms belong to different families. The taxonomic terms "race", "species", "genus", "family" and the like are well known in the art and can easily be derived from standard textbooks. Based on the teaching provided in the present invention are skilled person is therefore easily in the position to identify "closely related" or "distantly related" organisms.

A person skilled in the art is capable of identifying and/or isolating member(s) of the RNAse A family as defined herein and in particular as defined in sections (a) to (f) of the below- described specific aspect of the present invention or a nucleic acid molecule encoding said member of the RNAse A family of a specific organism (e.g. human, mouse, pig, guinea pig, rat, and the like) using standard techniques. Again, it is to be understood that antagonists of or against (a) member(s) of the RNAse A family (or of a functional fragment thereof), wherein said member is derived or can be isolated from human RNAse (e.g. RNAse 1), murine EAR or derived from members of the RNAse A family isolated from further organisms (e.g. pig, guinea pig, rat, and the like) is to be used in accordance with the present invention, in particular in the treatment of (a) disease associated with a disturbance of adipocyte formation such as an increase in adipocyte number.

As used herein the terms "RNAse A'VRNAse A of human origin" refer in particular to (a) protein(s) as found in the human body which can accordingly be isolated from a sample obtained from a human. The term "RNAse A (or a functional fragment thereof) derived from human RNase A" refers in particular to "human RNAse A'VRNAse A of human origin" which is modified as described herein below (e.g. by way of substitution, deletion and/or insertion of (an) amino acid(s)). Said modified polypeptide may also form part of a fusion protein. The explanations given herein above in respect of "human RNAse A'VRNAse A of human origin" apply, mutatis mutandis, to "murine EAR'VEAR of murine origin" and RNAse A isolated from other organisms, such as pigs, guinea pigs, rats, and the like.

The use of antagonists of (a) member(s) of the RNAse family (or antagonists of (a) (functional fragments) thereof) as described and defined herein in the treatment of economically, agronomically or scientifically important organisms is envisaged herein. Scientifically or experimentally important organisms include, but are not limited to, mice, rats, rabbits, guinea pigs and pigs. Yet, the treatment of (a) human(s) with antagonists of (a) member(s) of the human RNAse A family (in particular EDP and/or ECP, i.e. in general RNAse A of human origin or derived from human RNAse) or a functional fragment thereof is preferred in context of the present invention. Nucleic acid and amino acid sequences of specific and preferred members of the RNAse A family are described herein further below.

The term "antagonist of a Ribonuclease/RNAse being a member of the RNAse A family" or "inhibitor of a Ribonuciease/RNAse being a member of the RNAse A family" means in context of the present invention a compound capable of fully or partially preventing or reducing the physiologic activity and/or expression level of (a) such a Ribonuclease. The terms "antagonist" or "inhibitor" are used interchangeably herein.

In the context of the present invention said antagonist may, therefore, prevent, reduce, inhibit or inactivate the physiological activity of a Ribonuclease upon binding of said compound/substance (i.e. antagonist/inhibitor) to said Ribonuclease, Binding of an "antagonist/inhibitor" to a Ribonuclease may compete with or prevent the binding of a substrate of the Ribonuclease. Such an exemplary substrate, is, RNA. As used herein, the term "antagonist" also encompasses competitive antagonists, (reversible) non-competitive antagonists or irreversible antagonist, as described, inter alia, in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. Such an inhibition can be measured by determining substrate (e.g. RNA) turnover by the RNAses. For example, if RNA is not processed at all, the physiologic activity is completely inhibited.

An "antagonist" or "inhibitor" of a Ribonuclease may also be capable of preventing the function of a Ribonuclease by preventing/reducing the expression of the nucleic acid molecule encoding for said Ribonuclease. Thus, an antagonist/inhibitor of a Ribonuclease may lead to a decreased expression level of the Ribonuclease (e.g. decreased level of an Ribonuclease mRNA and/or of Ribonuclease protein); this may be reflected in a decreased Ribonuclease activity. The decreased activity and/or expression level can be measured/detected by the herein described methods. An "antagonist/inhibitor of a Ribonuclease being a member of the RNAse A family" may, for example, interfere with transcription of (an) Ribonuclease gene(s), processing (e.g. splicing, export from the nucleus and the like) of the gene product(s) (e.g. unspliced or partially spliced mRNA) and/or translation of the gene product (e.g. mature mRNA). The "antagonist/inhibitor of a Ribonuclease being a member of the RNAse A family" may also interfere with further modification (like glycosylation or phosphorylation) of the polypeptide/protein encoded by the Riboncuclease gene(s) and thus completely or partially inhibit the activity of the a Ribonuclease protein(s) as described herein above. Furthermore, the "antagonist/inhibitor a Ribonuclease being a member of the RNAse A family" may interfere with interactions of the Ribonuclease protein(s) with other proteins (thus, for example, interfering with the activity of complexes involving Ribonuclease protein(s)) or, in general, with its synthesis, e.g. by interfering with upstream steps of Ribonuclease expression or with signalling pathways in which the Ribonuclease is involved. Depending on the mode of action, such antagonists may, for example, be denoted "sequestering antagonists" or "signalling antagonists".

In sum, the herein described Ribonuclease antagonist/inhibitor will, accordingly, lead to a decrease or reduction of Riboncuclease expression level and/or activity, and thereby reduce preadipocyte proliferation/differentiation into adipocytes (adipocyte formation) and/or reduce adipocyte number. As shown in the appended examples (see Figure 3), the antagonists to be used herein are capable of strongly decreasing preadipocyte proliferation/differentiation into adipocytes (adipocyte formation) and/or reducing adipocyte number. Preferably, the preadipocyte proliferation/differentiation into adipocytes (adipocyte formation) and/or adipocyte number is reduced by at least 10 ¾, 20 %, 30 %, 40 %, 50 %, 60 %, more preferably, at least 70 %, 75 % and most preferably, at least 80 % compared to the previous state (i.e. prior to treatment with the antagonists).

Preferably, the antagonist(s) is(are) miRNA, dsRNA, siRNA, shRNA, sfRNA, anti- Ribonuclease antisense molecules, extracellular binding-partners, small (binding) molecules, aptamers, intramers, or antibody molecule such as a full antibody (immunoglobulin), a F(ab)- fragment, a F(ab)₂-fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody, a bispecific single chain antibody or a cross-cloned antibody. Preferred herein as antagonists are siRNAs, wherein said siRNA consists of a nucleic acid molecule comprising at least ten contiguous bases having a sequence as shown in the sequence of SEQ ID NO: 50 or encoded by the sequence shown in SEQ ID NO. 49. siRNA targeted against human RNAses of the RNAseA family (such as RNAse 1 and other members of said family) are commercially available and may be obtained, for example, from Invitrogen under catalog ID: HSS 109255). A negative control siRNA matched for GC-content is available under oligo ID: 12935-200 from Invitrogen.

An exemplary nucleotide sequence encoding siRNA targeting human RNASE 1 is shown in SEQ ID No.49 (ACCAAATGATGAGGCGCCGGAATAT), whereas SEQ ID No.50 shows the RNA sequence of an siRNA targeting human RNASE 1 to be used in accordance with the present invention (ACCAAAUGAUGAGGCGCCGGAAUAU).

Preferably, up to 10 % of the contiguous bases of the above-mentioned nucleic acid-molecule are non-complementary. The nucleic acid molecule may further comprise at least one base at the 5' end and/or at least one base at the 3' end. The nucleic acid molecule preferably consists of a molecule as shown in SEQ ID No.50 (ACCAAAUGAUGAGGCGCCGGAAUAU).

Also preferred herein as antagonists are miRNAs, wherein said miRNA consists of a nucleic acid molecule comprising at least ten contiguous bases having a sequence as shown in the sequence of miR-V2M 34242 (SEQ ID NO: 48). miRNA targeted against murine EAR 10 is commercially available and may be obtained from Open Biosystems under cat, no. RMM1766-96883532 in plasmid pSM2. Herein below detailed information is provided concerning miRNAs against murine EAR10.

7388, 0220,

6624, U72031, U72032

Mus musculus

SM2423-b -7

pS 2c

pSHAG-MAGIC2

Retroviral

Chloramphenicol (Concentration: 25 μ§ ιη1, Resistant Range: 25-25 μ^ι^ηΐ)

Kanamycin (Concentration: 25 ^ηιΐ, Resistant Range: 25-25 μ^ι^ηΐ)

U6 5' TGT GGA AAG GAC GAA ACA CC

MSCV-retroviral

The miRNA can be used to target/inhibit the Ribonucleases as shown or encoded by the following sequences which can be retrieved under the respective accession numbers: NM_ 001012766, NM_007894_? NM_007895, NM_017388, NM_0531 12, BC065391, BC094626, BC117060, BC140220, BC146516, BC 148661, BC152970, BC153204, BC156624, U72031, U72032. Corresponding exemplary sequences of Ribonucleases are also provided herein below.

Preferably, up to 10 % of the contiguous bases of the above-mentioned nucleic acid-molecule are non-complementary. The nucleic acid molecule may further comprise at least one base at the 5' end and/or at least one base at the 3' end. The nucleic acid molecule preferably consists of a molecule as shown in SEQ ID NO: 48 (miR-V2MM3 242). miRNAs, siRNAs and the like against the Ribonucleases are either commercially available or can be obtained by routine techniques.

However, not only the above described antagonists can successfully be used in the treatment of the herein defined associated with an increase in adipocyte number or disturbance of adipocyte formation, such as overweight/obesity and the like. Further exemplary antagonists to be used in accordance with the invention are chemical compounds and small compounds. A non-limiting example of such a compound is the well-known RNasin (RNAse inhibitor). RNasin® Ribonuclease Inhibitor is commercially available, for example from Promega. RNasin inhibits a broad spectrum of RNAses (amongst them RNAse A, RNAse B, and RNAse C). It is a 50 kDa protein that exerts its inhibitory effect by binding noncovalently to RNAses in a 1: 1 ratio; see Blackburn, P. and Moore, S. (1982) In: The Enzymes, Vol. XV, Part B; Blackburn, P. et al. (1977) J. Biol. Chem. 252, 5904-10. As shown in the appended examples, inhibition of EARs by RNAsin resulted in a pronounced decrease of key adipogenic marker genes (CEBP/a, PPARg, KLF15 and FABP4). This provides experimental proof that also this antagonist of Ribonucleases can be successfully used in the treatment of diseases associated with an increase in adipocyte number or a disturbance of adipocyte formation, such as overweight or obesity, and the like.

Also envisaged herein as antagonists of Ribonucleases is an antibody. Such an antibody antagonizing/inhibiting these Ribonucleases may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-stranded antibody or an antibody fragment, such as a Fab fragment or a fragment produced by a Fab expression library.

The antibody to be used in this context is preferably an antibody specifically recognizing a Ribonuclease protein or specifically recognizing a protein interacting with/binding to a Ribonuclease protein. These antibodies may be generated using the full-length Ribonuclease protein or fragments thereof as antigen in routine protocols. Preferably, the catalytic domain is used as antigen. The antibody may also specifically recognize a Ribonuclease agonist, i.e. a compound enhancing Ribonuclease activity and/or expression level. Preferably, the antibody is an "anti-Ribonuclease antibody", i.e. an antibody that specifically recognizes the Ribonucleases as defined herein. Through its ribonuclease inhibitory/antagonising capacity (preferably the antagonist inhibits generally RNAse activity of the herein described Ribonucleases) the antibody is useful in the treatment of the herein described diseases, such as diseases associated with an increase in adipocyte number or a disturbance of adipocyte formation. The anti-ribonuclease antibody to be used in accordance with, the invention can be obtained by known methods. Preferably, a monoclonal antibody is to be used. In particular antibodies of mammalian origin are useful in context of the invention. Such antibodies can be produced by a hybridoma and/or by a host transformed with an expression vector containing genetically engineered antibody genes. The antibody inhibits ribonuclease activity as described herein above in context of antagonists of Ribonucleases that are members of the RNAse A family.

The following relates to the production of antibodies, which are to be used in accordance with the present invention.

The hybridoma can be produced using a ribonuclease protein (or fragment thereof) as an antigen, thus eliciting an immune response preferably in a mammal, fusing the resulting immune cells with known parent cells by a known cell fusion method and screening monoclonal antibody-producing cells by a known screening method.

For example, the anti-Ribonuclease antibody may be produced as follows.

The Ribonuclease gene may be produced by biotechno logical means known in the art. The nucleic acid sequence encoding a Ribonuclease protein (or fragment thereof) may be inserted into a known expression vector, the vector may be introduced into an appropriate host cell and the thereby produced Ribonuclease protein is used as an antigen in immunization. Preferably, organisms and, in particular mammals used in the immunization process are compatible with the parent cell for use in cell fusion. Thus, such mammals usually include but are not limited to, rodents such as mice, rats, hamsters and the like.

The immunization procedure with the Ribonuclease protein (or fragment thereof) is carried out using a routine method. For example, immunization involves the intraperitoneal or subcutaneous administration of the Ribonuclease protein (or fragment thereof) to the mammal. A Ribo iclease protein (or fragment thereof) which has been diluted and suspended in an appropriate amount of phosphate buffered saline (PBS) or physiological saline and the like may be mixed with an appropriate amount of a common adjuvant, for example Freund's complete adjuvant. After being emulsified, the mixture is administered to a mammal preferably several times every 4 to 21 days.

After immunization has been carried out and after the increase of the respective antibody level in the serum has been confirmed, the immune cells are isolated from the mammal and are subjected to cell fusion. Preferably, the immune cells are isolated from the spleen.

Mammalian myeloma cells (i.e. parent cells which are subjected to cell fusion with the above- mentioned immune cells) include but are not limited to cell lines such as P3X63Ag8.653) (J. Immunol. (1979) 123: 1548-1550), P3X63Ag8U.l (Current Topics in Microbiology and Immunology (1978) 81 : 1-7), NS-1 (Kohler, G. and Milstein, C. , Eur. J. Immunol. (1976) 6: 511-519), MPC-11 (Margulies, D. H. et al. Cell (1976) 8: 405-415), SP2/0 (Shulman, M. et al. Nature (1978) 276: 269-270), FO (de St. Groth, S. F. et al, J. Immunol. Methods (1980) 35: 1-21), S194 (Trowbridge, I.S., J. Exp. Med. (1978) 148: 313- 323), R210 (Galfre, G. et al. Nature (1979) 277: 131-133) and the like.

A known method describing the above cell fusion is, for example, given in Milstein et al. (Kohler, G. and Milstein, C, Methods Enzymol. (1981) 73: 3-46). The cell fusion may be carried out in the presence of a cell fusion accelerator, such as polyethylene glycol (PEG), Sendai virus (HVJ) and the like. In addition, an adjuvant such as dimethyl sulfoxide, may be added to enhance efficiency of the fusion. The preferred ratio of myeloma cells and the immune cells in fusion is 1 to 10. Exemplary culture media to be used in the above cell fusion include RP Mil 640 medium and MEM culture medium and the like. Such media allow the growth of the above myeloma cell lines. Further, a serum supplement such as fetal calf serum (FCS) may be added.

In cell fusion, predetermined amounts of the above immune cells and the myeloma cells are mixed well in the above culture liquid, to which a PEG solution previously heated to about 37 °C, for example a PEG solution with a mean molecular weight of about 1000 to 6000, is added at a concentration of 30 to 60% (w/v) and mixed to obtain the desired fusion cells (hybridomas). In order to remove agents which interfere with the growth of hybridomas, the cell culture may be centrifuged, the supernatant removed and the cells resuspended in appropriate culture liquid. These steps may be repeated several times so as to remove the undesirable component as completely as possible.

The hybridoma can then be selected by cultivation in a standard selection medium, for example, in the HAT culture medium (a culture liquid containing hypoxanthine, aminopterin, and thymidine). Cultivation in said HAT culture medium is continued generally for a period of time which is sufficient to kill cells other than the desired hybridoma (for example, non-fused cells). This selection process lasts generally several days to several weeks. Hybridomas that produce the desired antibody are screened by a known dilution method and the antibody is then monoclonally cloned.

In the alternative to the above described method, a hybridoma can be obtained by in vitro methods. For example, human lymphocytes may be sensitized in vitro with Ribonuclease protein (or fragment thereof) or Ribonuclease protein (or fragment thereof) -presenting cells, and the resulting sensitized B lymphocytes are fused with a human myeloma cell, for example U266, in order to obtain the human antibody specifically binding to the Ribonuclease antigen. Furthermore, a transgenic animal having a repertoire of all human antibody genes is immunized with the antigen or the antigen-presenting cells to obtain the human antibody in the method described above (see International Patent Application WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

The thus generated monoclonal antibody-producing hybridomas can be subcultured under known culture conditions or can be stored for a prolonged period of time in liquid nitrogen. Monoclonal antibodies may be obtained as follows from the above hybridoma. For example, hybridomas are cultivated according to routine methods and antibodies are obtained from the supernatant. Alternatively, the hybridoma can be administered to and grown in a mammal compatible with said hybridoma and the antibodies are obtained as ascites. The first method is indicated in cases where high-purity antibodies are to be obtained, whereas the latter methods is useful for a large scale production of antibodies. Also a recombinant antibody can be used in accordance with the invention. Such a recombinant antibody may be produced by cloning the antibody of the hybridoma and integrating the antibody gene into a suitable vector. This vector can then be introduced into a host for producing the recombinant antibody; see, for example, Carl, A.K., Borrebaeck, and James, W. Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by Macmillan Publishers Ltd. 1990).

For example, mRNA encoding the variable (V) region of the antibody is isolated from the hybridoma. The isolation of mRNA is conducted by preparing total RNA using, for example, a known method such as the guanidine ultracentrifuge method (Chirgwin, J.M. et al., Biochemistry (1979) 18, 5294-5299), the AGPC method (Chmczynski, P. et al, (1987) 162, 156-159), and then mRNA is purified from the total RNA using the mRNA Purification kit (manufactured by Pharmacia) and the like. Alternatively, mRNA can be directly prepared using the Quick Prep mRNA Purification Kit (manufactured by Pharmacia). cDNA of the V region of antibody may be synthesized from the mRNA thus obtained using a reverse transcriptase. cDNA may be synthesized using the AMV Reverse Transcriptase First- strand cDNA Synthesis Kit and the like. Alternatively, for the synthesis and amplification of cDNA, the 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and the 5'-RACE method (Frohman, M.A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 8998-9002; Belyavsky, A. et al, Nucleic Acids Res. (1989) 17, 2919-2932) that employs polymerase chain reaction (PCR) may be used. The desired DNA fragment is purified from the PCR product obtained and may be Hgated to vector DNA. Moreover, a recombinant vector is constructed therefrom and is then introduced into E. coli etc., from which colonies are selected to prepare the desired recombinant vector. The base sequence of the desired DNA may be confirmed by a known method such as the dideoxy method.

Once the DNA encoding the V region of the antibody has been obtained, it may be ligated to DNA encoding the constant region (C region) of the antibody, which is then integrated into an expression vector. Alternatively, the DNA encoding the V region of the antibody may be integrated into an expression vector, which already contains DNA encoding the C region of the antibody. In order to produce the antibody for use in the present invention, the antibody gene is integrated as described below into an expression vector, whereby the antibody gene may then be expressed under the control of a regulatory region, for example an enhancer and/or a promoter. Subsequently, the expression vector may be transformed into a host cell and the antibody can then be expressed therein.

In accordance with the present invention, an artificially altered recombinant antibody such as a chimeric antibody and a humanized antibody can be used in order to lower the likelihood of a potential immune response in the human body to the antibody. Such antibodies can be produced according to known methods.

For example, a chimeric antibody can be obtained by ligating the obtained DNA encoding the V region of antibody to DNA encoding the C region of human antibody, which is then integrated into an expression vector and introduced into a host for production of the antibody (see European Patent Application EP 125023, and International Patent Application WO 96/02576). By this method chimeric antibodies for use in the present invention can be obtained.

Humanized antibodies can be produced by grafting the complementarity determining regions (CDRs) of an antibody of a mammal other than the human, for example a murine antibody, into a human antibody (thereby replacing the original human CDRs). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

Specifically, a DNA sequence which can be generated to ligate the CDR of a murine antibody to the framework region (FR) of a human antibody is synthesized from several divided oligonucleotides having overlapping by PCR. The thus obtained DNA is ligated to the DNA encoding the C region of a human antibody. Then the DNA is integrated into an expression vector, which is introduced into a host for antibody production (see European Patent Application EP 239400 and International Patent Application WO 92- 19759).

In CDR grafting, the complementarity determining region and/or FR may be further adapted to maintain antigen binding specifitiy. For example, amino acids in the framework region of the antibody variable region may be substituted to allow the complementarity determining region to form a functional antigen biding site (Sato, , et al, Cancer Res. (1993) 53, 851-856).

In the generation of chimeric antibodies or humanized antibodies, the C region of human antibody may be used, such as Cy, and Cyl , Cy2, Gy3, and Cy4, and the like. The C region of human antibody may be further modified to improve the stability of antibody or the production thereof.

Chimeric antibodies consist of the variable region of antibody derived from a mammal other than the human, whereas the C region is derived from a human antibody. In contrast, humanized antibodies consist of the complementarity determining region of an antibody derived from a mammal other than the human, wheras the framework region (FR) and the C region of the antibody are derived from a human antibody. Accordingly, the risk that such antibodies elicit an immune response in the human body has been reduced which makes such antibodies particularly useful in context of the present invention.

Antibody genes may be expressed and obtained according to known methods. For example, a promoter, an antibody gene to be expressed, and a poly A signal at 3' downstream thereof can be operably linked and introduced into a vector. A non-limiting examples of the promoter/enhancer are human cytomegalovirus immediate early promo ter/enhancer, viral promoters/enhancers such as promoters of retrovirus, polyoma virus, adenovirus, and simian virus 40 (SV40), and promoters/enhancers derived from mammalian cells such as human elongation factor l a (HEFla). Expression may be performed according to the method of Mulligan et al, (Nature(1979) 277, 108) when SV40 promoter/enhancer is used, or according to the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322) when HEFla promoter/enhancer is used.

When the antibody is to be produced in a prokaryotic system (e.g. in B.coli), a construct may be used which contains an operably linked promoter, a signal sequence for antibody secretion, an antibody gene to be expressed. Non-limiting examples of a promoter to be used in such a context are lacz promoter and araB promoter. The method according to Ward et al. (Nature ( 1098) 341 , 544-546; FASEB J. (1992) 6, 2422-2427) may be used when lacz promoter is used and the method according to Better et al. (Science (1988) 240, 1041-1043) may be used when araB promoter is used. The pelB signal sequence (Lei, S. P. et al, J. Bacteriol. (1987) 169, 4379) can be used to allow antibody secretion into the periplasm of E. coli. The structure of the antibody is to be appropriately refolded prior to its use (see, for example, WO 96/30394) after the antibody has been isolated from the periplasm. The following origins of replication can be used: origins of replication derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like. Furthermore expression vectors can include selectable markers such as the aminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene, E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene and the like for the amplification of the gene copy number in the host cell system.

The antibody may be prepared in in vitro or in vivo systems, for example using eukaryotic cells or prokaryotic cells. Exemplary eukaryotic cells to be used are animal cells, plant cells, and fungal cells. Non-limiting examples of animal cells are (1) mammalian cells such as CHO cells, COS cells, myeloma cells, baby hamster kidney (BH ) cells, HeLa cells, and Vero cells, (2) amphibian cells such as Xenopus oocytes, or (3) insect cells such as sf9, sf21, and Tn5. Known plant cells include, for example, those derived from Nicotiana tabacum, which may be subjected to callus culture. Known fungal cells include yeasts such as the genus Saccharomyces, more specifically Saccharomyces cerevisiae, or filamentous fungi such as the genus Aspergillus , more specifically Aspergillus niger. Exemplary prokaryotic ceils are bacterial cells such as Escherichia coli (E.coli), Bacillus subtilis and the like. The antibody can be obtained by introducing antibody genes into these cells and culturing the transformed cells in vitro. Exemplary culture liquid to be used include DMEM, MEM, RPMI1640, and IMDM, optionally supplemented with serum supplements such as fetal calf serum (FCS).

The antibodies may also be produced in in vivo systems, e.g. in animals (for example mammals or insects) or plants. Non-limiting examples of animals to be used in this context are mammals, goats, pigs, sheep, mice, and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). An exemplary insect that may be used is silkworm. For example, antibodies may be produced in vivo by implanting cells into which the antibody gene has been introduced into the abdominal cavity of an animal and the like. A non-limiting example of a plant that may be used in the production of antibodies is tobacco.

Antibody genes are introduced into these animals or plants, and the antibodies are produced in such animals or plants, and recovered. For example, an antibody gene is inserted into the middle of the gene encoding protein, which is inherently produced in the milk such as goat β casein to prepare fusion genes. DNA fragments containing the fusion gene into which the antibody gene has been inserted are injected into a goat embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat bom to the goat who received the embryo or offsprings thereof. In order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be given to the transgenic goat as appropriate. (Ebert, .M. et al., Bio/Technology (1994) 12, 699-702).

When silkworms are used, baculoviras into which the desired antibody gene has been inserted is infected to the silkworm, and the desired antibody can be obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594). Moreover, when tabacco is used, the desired antibody gene is inserted into an expression vector for plants, for example pMON 530, and then the vector is introduced into a bacterium such as Agrobacterium tumefaciens. The bacterium is then infected to tobacco such as Nicotianatabacum to obtain the desired antibody from the leaves of the tobacco (Julian, K. -C. Ma et al, Eur. J. Immunol (1994) 24, 131-138).

When antibody is produced in in vitro or in vivo production systems, as described above, DNA encoding the heavy chain (H chain) or the light chain (L chain) of antibody may be separately integrated into an expression vector and the hosts are transformed simultaneously, or DNA encoding the H chain and the L chain may be integrated into a single expression vector and the host is transformed therewith (see International Patent Application WO 94- 1 1523).

As mentioned, also fragments of antibodies can be used in context of the present invention, such as Fab, F(ab')2, Fv or (bispecific) single-chain Fv (scFv), for example scFv in which Fv's of H chain and L chain were ligated via a suitable linker. Antibodies may be treated with an enzyme, for example, papain or pepsin, to produce antibody fragments. In the alternative, genes encoding such antibody fragments may be constructed, and then introduced into an expression vector, which is expressed in a suitable host cell (see, for example, Co, M. S. et al, J. Immunol (1994) 152, 2968- 2976; Better, M. and Horwitz, A.H., Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. and Skerra, A. , Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc. ; Lamoyi, E., Methods in Enzymology (1986) 121, 652-663; ousseaux, J. et al, Methods in Enzymology (1986) 121, 663-669; Bird, R. E. et al, TIBTECH (1991) 9, 132-137). scFv can be obtained by ligating the V region of H chain and the V region of L chain of antibody. In the scFv, the V region of H chain and the V region of L chain are preferably ligated via a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. U. S.A. (1988) 85, 5879-5883). The V region of H chain and the V region of L chain in the scFv may be derived from any of the above- mentioned antibodies. As the peptide linker for ligating the V regions, any single-chain peptide comprising, for example, 12 - 19 amino acid residues may be used.

DNA encoding scFv can be obtained using DNA encoding the H chain or the H chain V region of the above antibody and DNA encoding the L chain or the L chain V region of the above antibody as the template by amplifying the portion of the DNA encoding the desired amino acid sequence among the above sequences by the PCR technique with the primer pair specifying the both ends thereof, and by further amplifying the combination of DNA encoding the peptide linker portion and the primer pair which defines that both ends of said DNA be ligated to the H chain and the L chain, respectively.

Once DNA encoding scFv is constructed, an expression vector containing it and a host transformed with said expression vector can be obtained by conventional methods, and scFv can be obtained using the resultant host by conventional methods. These antibody fragments can be produced by obtaining the gene thereof in a similar manner to that mentioned above and by allowing it to be expressed in a host. Also modified antibodies, i.e. antibodies associated with various molecules such as polyethylene glycol (PEG) can be used. These modified antibodies can be obtained by chemically modifying the antibodies by known methods.

Antibodies produced and expressed as described above can be separated from the inside or outside of the host cell and may be purified to homogeneity. Separation and purification of the antibody for use in the present invention may be accomplished by affinity chromatograph, e.g. using Protein A column or Protein G column. Examples of carriers used in the Protein A column are Hyper D, POROS, Sepharose F. F. and the like. Alternatively, methods for separation and purification conventionally used for proteins can be used. Separation and purification of the antibody for use in the present invention may be accomplished by combining, as appropriate, chromatography other than the above-mentioned affinity chromatography, filtration, ultrafiltration, salting-out, dialysis and the like. Chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel- filtration and the like. These chromatographies can be applied to HPLC. Alternatively, reverse-phase chromatography can be used.

The concentration of antibody can be determined by measurement of absorbance or by enzyme- linked immunosorbent assay (ELISA) and the like. When absorbance measurement is employed, the antibody for use in the present invention or a sample containing the antibody may be appropriately diluted with PBS and the absorbance measured at 280 nm, followed by calculation using the absorption coefficient of 1.35 OD at 1 mg/ml. When ELISA is used, measurement may be conducted as follows. Goat anti- human IgG (manufactured by TAGO) is diluted to 1 pg/ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a 96-well plate (manufactured by Nunc), and is incubated overnight at 4 °C to immobilize the antibody.

After blocking, 100 μΐ of appropriately diluted antibody for use in the present invention or a sample containing the antibody, or 100 μΐ of human IgG (manufactured by CAPPEL) (the standard) is added, and incubated at room temperature for 1 hour. After washing, 100 ul of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody (manufactured by BIO SOURCE) is added, and incubated at room temperature for 1 hour. After washing, the substrate solution is added and incubated, followed by the measurement of absorbance at 405 nm using the MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate the concentration of the antibody.

The term "specifically recognizing" means in accordance with this invention that the antibody molecule is capable of specifically interacting with and/or binding to at least two amino acids of the member of the RNAse A family as defined herein. Antibodies can recognize, interact and/or bind to different epitopes on the same target molecule. The term "specifically recognizing" may, therefore, also relate to the specificity of the antibody molecule, i.e. to its ability to discriminate between the specific regions of the Ribonuclease as defined herein. Preferably, the antibody to be used in accordance with the invention does not cross-react with (poly)peptides of similar structure (e.g. further members of the RNAse A family). Cross- reactivity of a panel of constructs under investigation may be tested, for example, by assessing binding of said panel of antibodies under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999) to the member of the RNAse A family as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. An exemplary polypeptide that is more structurally and/or functionally related is a further member of the RNAse A family. An exemplary polypeptide that is less structurally and/or functionally related is an RNAse that is not a member of the RNAse A family, or a non-RNAse (poly)peptide, that are preferably expressed in adipose tissue and/or (pre)adipocytes. Those constructs (i.e. antibodies, (bispecific) scFvs and the like) that bind to the member of the Ribonuclease A family but do not or do not essentially bind to any of the other (poly)peptides mentioned above are considered to be specific for the member of the RNAse A family and may be selected for further studies in accordance with the invention.

The term "specifically recognizing" relates not only to a linear epitope but may also relate to a conformational epitope, a structural epitope or a discontinuous epitope consisting of two regions of the Ribonuclease or parts thereof. In context of this invention, a conformational epitope is defined by two or more discrete amino acid sequences separated in the primary sequence which come together on the surface of the molecule when the polypeptide folds to the native protein (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell 61, 553-6). For example, the catalytic domain of the herein described member of the Ribonuclease A family may be considered as a "conformational epitope".

The term "discontinuous epitope" means in context of the invention non-linear epitopes that are assembled from residues from distant portions of the polypeptide chain. These residues come together on the surface when the polypeptide chain folds into a three-dimensional structure to constitute a conformational/structural epitope.

The antibodies of the present invention are also envisaged to specifically bind to/interact with a conformational/structural epitope(s) composed of and/or comprising the two regions of a Ribonuclease described herein or parts thereof as disclosed herein below.

Accordingly, specificity can be determined experimentally by methods known in the art and methods as disclosed and described herein. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. The term "CDR" as employed herein relates to "complementary determining region", which is well known in the art. The CDRs are parts of immunoglobulins and T cell receptors that determine the specificity of said molecules and make contact with specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1, CDR2 and CDR3 in each V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. H means the variable heavy chain and L means the variable light chain. The CDR regions of an Ig-derived region may be determined as described in Kabat (1991). Sequences of Proteins of Immunological Interest, 5th edit,, NiH Publication no. 91-3242 U.S. Department of Health and Human Services, Chothia (1987). J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883. in accordance with this invention, a framework region relates to a region in the V domain (VH or VL domain) of immunoglobulins and T-cell receptors that provides a protein scaffold for the hypervariable complementarity determining regions (CDRs) that make contact with the antigen. In each V domain, there are four framework regions designated FR1, FR2, FR3 and FR4. Framework 1 encompasses the region from the N-terminus of the V domain until the beginning of CDR1, framework 2 relates to the region between CDR1 and CDR2, framework 3 encompasses the region between CDR2 and CDR3 and framework 4 means the region from the end of CDR3 until the C-terminus of the V domain; see, inter alia, Jane way, Immunobiology, Garland Publishing, 2001 , 5th ed. Thus, the framework regions encompass all the regions outside the CDR regions in VH or VL domains. Furthermore, the term "transition sequence between a framework and a CDR region" relates to a direct junction between the framework and CDR region. In particular, the term "transition sequence between a framework and a CDR region" means the sequence directly located N- and C-terminally of the CDR regions or amino acids surrounding CDR regions. Accordingly, frameworks may also comprise sequences between different CDR regions. The person skilled in the art is readily in a position to deduce from a given sequence the framework regions, the CDRs as well as the corresponding transition sequences; see Kabat (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services, Chothia (1987). J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883. The following relates to exemplary Ribonucleases to be used in accordance with the present invention.

In one embodiment of the present invention, an antagonist against a Ribonuclease being a member of the RNAse A family is provided wherein said Ribonucleas is a human RNAse, in particular selected from the group consisting of RNAse 1 , RNAse 2, RNAse 3, RNAse 4/5, RNAse 6, RNAse 7, RNAse 8, RNAse 9, RNAse 10, RNAse 11, RNAse 12, and RNAse 13; or

wherein said Ribonuclease is a (murine) Eosinophil-associated Ribonuclease (EAR), preferably selected from the group consisting of EAR- 1, EAR-2 and EAR- 10.

As mentioned above, preferred herein is the use of antagonists of or against murine EAR-1, EAR-2 and EAR-10 and their human orthologues. Antagonists of or against human RNAse 1 are particularly preferred. Exemplary human RNAses to be used herein may have the following amino acid sequences:

RNAse 1 having an amino acid sequence as shown in SEQ ID NO: 24;

RNAse 2 having an amino acid sequence as shown in SEQ ID NO: 26;

RNAse 3 having an amino acid sequence as shown in SEQ ID NO: 28;

RNAse 4/5 having an amino acid sequence as shown in SEQ ID NO: 30;

RNAse 6 having an amino acid sequence as shown in SEQ ID NO: 32;

RNAse 7 having an amino acid sequence as shown in SEQ ID NO: 34;

RNAse 8 having an amino acid sequence as shown in SEQ ID NO: 36;

RNAse 9 having an amino acid sequence as shown in SEQ ID NO: 38;

RNAse 10 having an amino acid sequence as shown in SEQ ID NO: 40;

RNAse 11 having an amino acid sequence as shown in SEQ ID NO: 42;

RNAse 12 having an amino acid sequence as shown in SEQ ID NO: 44; or

RNAse 13 having an amino acid sequence as shown in SEQ ID NO: 46.

In a very preferred embodiment, the present invention relates to an antagonist of human RNAse 1 for use in treating a disease with an increase in adipocyte number, wherein said RNAse 1 is selected from the group consisting of

(a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 23;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:24; (c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:24;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional RNAse 1 or a fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional RNAse 1 or a fragment thereof; and

(f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d).

The above embodiment (and all corresponding definitions and explanations) applies mutatis mutandis to any other ribonucleases of the RNase A family to be used in accordance with the present invention, in particular to the herein above mentioned human RNAses, such as human RNase 2 and/or RNAse 3.

In a further preferred embodiment, the present invention relates to an antagonist of EAR- 10 for use in treating a disease with an increase in adipocyte number, wherein said EAR- 10 is selected from the group consisting of

(a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 15;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 16;

(c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO: 16;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional EAR- 10 or a fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional EAR- 10 or a fragment thereof; and

(f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d). The above embodiment (and all corresponding definitions and explanations) applies mutatis mutandis to any other ribonuclease of the RNase A family to be used in accordance with the present invention, in particular to the herein above mentioned murine RNAses, such as murine EAR-1 or EAR-2.

Accordingly, an antagonist of EAR-1 is provided for use in treating a disease with an increase in adipocyte number, wherein said EAR-1 is selected from the group consisting of

(a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2;

(c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:2;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional EAR-1 or a fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional EAR-1 or a fragment thereof; and

Also an antagonist of EAR-2 is envisaged and preferred herein in this context, wherein said EAR-2 is selected from the group consisting of

(a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 3;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 4;

(c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO: 4;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional EAR-2, or a fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional EAR-2, or a fragment thereof; and (f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degen erate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d).

The term "functional Ribonuclease" used in context of the present invention refers to a polypeptide having at least 60 % homology to a polypeptide as defined in section (a) to (d) of the above-described specific aspect of the present invention which has essentially the same biological activity as a polypeptide having 100 % homology to a polypeptide as indicated in section (a) to (d), i.e. a polypeptide being essentially identical to a polypeptide having an amino acid sequence as depicted in SEQ ID NO; 24, 16, 2 or 4. Methods for determining the activity of (a) polypeptide(s) are well known in the art and may, for example, be deduced from standard text books, such as Bioanalytik (Lottspeich/Zorbas (eds.), 1998, Spektrum Akademischer Verlag). Methods for determining the activity of a Ribonuclease that is a member of the RNAse A family are also described herein below.

it is of note that a (functional) Ribonuclease or a functional fragment thereof as described and defined herein may further comprise a heterologous polypeptide, for example, (an) amino acid sequence(s) for identification and/or purification of the recombinant protein (e.g. amino acid sequence from C-MYC, GST protein, FLAG peptide, HIS peptide and the like), an amino acid sequence used as reporter (e.g. green fluorescent protein, yellow fluorescent protein, red fluorescent protein, luciferase, and the like), or antibodies/antibody fragments (like scFV). A person skilled in the art knows that for determination of homology as described herein only a part of a polypeptide is to be used, whereby said part is Ribonuclease (or a functional fragment thereof). Also further compounds (e.g. toxins or antibodies or fragments thereof) may be attached to Ribonuclease (or a functional fragment thereof) by standard techniques. These compounds may, in particular, be useful in a medical setting as described herein, wherein Ribonuclease (or a functional fragment thereof) is used as agonist. A skilled person is aware of compounds to be used/attached in this context.

Methods and assays for determining the activity of "Ribonucleases" are described herein below and in the appended examples. These methods also allow determining whether a polypeptide can be considered as a "functional Ribonuclease". The activity exhibited by the following exemplary polypeptides can be considered as "reference activity" of a functional Ribonuclease: a polypeptide having an amino acid sequence as depicted in SEQ ID NO:24 ("human RNAse 1"), SEQ ID NO:2 ("murine EAR-1"), SEQ ID NO:4 ("murine EAR-2"), or SEQ ID NO :16 ("murine EAR- 10"). The measurement of the activity of such RNAses is well known in the art; see Cormier (2002) American Journal of Respiratory Cell and Mol Biol 27, 678 to 687. RNAse activity may be assessed as follows using the commercially available RNase Alert GC System (Ambion):

A reaction mixture containing 400 ng total protein (e.g. cell/tissue extract containing Ribonuclease or purified Ribonuclease), RNaseAlert buffer and 200 nM fluorescent RNA substrate in a total reaction volume of 500 μΐ is equilibrated at 37 °C for 10 minutes. The samples are excited at 490 nm and the emission at 520 nm is recorded using a fluorescent spectrometer (e.g. F-4500; Hitachi). The RNAse activity of a sample can be determined from a standard curve of the RNase activity generated with RNAse A (e.g. available from Ambion under Cat. #2271). This assay is linear over a range of 0-250 pg for up to 2 hours.

A person skilled in the art is in the position to modify this protocol or refer to further protocols in the art known in the measurement of RNAse activity.

The meanings of the term "polypeptide" and "nucleic acid sequence(s)/molecule(s)" are well known in the art and are used accordingly in context of the present invention. For example, "nucleic acid sequence(s)/molecule(s)" as used herein refer(s) to all forms of naturally occurring or recombinantly generated types of nucleic acids and/or nucleic acid sequences/molecules as well as to chemically synthesized nucleic acid sequences/molecules. This term also encompasses nucleic acid analogues and nucleic acid derivatives such as e. g. locked DNA, PNA, oligonucleotide thiophosphates and substituted ribo-oligonucleotides. Furthermore, the term "nucleic acid sequence(s)/molecules(s)" also refers to any molecule that comprises nucleotides or nucleotide analogues.

Preferably, the term "nucleic acid sequence(s)/molecule(s)" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The "nucleic acid sequence(s)/molecule(s)" may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded. "Nucleic acid sequence(s)/molecule(s)" also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.

Furthermore, the term "nucleic acid sequence(s)/molecule(s)" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., US 552571 1, US 4711955, US 5792608 or EP 302175 for examples of modifications). The nucleic acid molecule(s) may be single- or double-stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the nucleic acid molecule(s) may be genomic DNA, cDNA, mRNA, anti sense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Colestrauss, Science (1996), 1386-1389. Said nucleic acid molecule(s) may be in the form of a plasmid or of viral DNA or RNA. "Nucleic acid sequence(s)/molecule(s)" may also refer to (an) oligonucleotide(s), wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included.

The nucleic acid sequence of members of the RNAse A family of other species than the herein provided human and murine sequences of members of the RNAse A family can be identified by the skilled person using methods known in the art, e.g. by using hybridization assays or by using alignments, either manually or by using computer programs such as those mentioned herein below in connection with the definition of the term "hybridization" and degrees of homology.

In one embodiment, the nucleic acid sequence encoding for orthologs of human RNAse I is at least 40% homologous to the nucleic acid sequence as shown in SEQ ID NO. 23. More preferably, the nucleic acid sequence encoding for orthologs of human RNAse 1 is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homologous to the nucleic acid sequence as shown in SEQ ID NOs. 23, wherein the higher values are preferred. Most preferably, the nucleic acid sequence encoding for orthologues of R Ase 1 is at least 99% homologous to the nucleic acid sequence as shown in SEQ ID NO. 23. The term "orthologous protein" or "orthologous gene" as used herein refers to proteins and genes, respectively, in different species that are similar to each other because they originated from a common ancestor.

The same definitions given herein in respect of orthologues/homologs of human RNAse 1 apply, mutatis mutandis, to orthologues/homologs of further human RNAses of the RNAseA family (such as EDP and/or ECP), in particular the nucleic acid sequence of herein described human. RNAses as, for example, shown in SEQ ID NO: 25 (human RNAse 2) or 27 (human RNAse 3). The definitions and explanations also apply, mutatis mutandis, to other members of the RNAse A family, in particular murine RNAses of the EAR family, or members isolated/derived from further sources, like the herein described animal sources such as pigs or guinea pigs and the like. Hybridization assays for the characterization of orthologs of known nucleic acid sequences are well known in the art; see e.g. Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989). The term "hybridization" or "hybridizes" as used herein may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, e.g., in Sambrook (2001) loc. cit; Ausubel (1989) loc. cit, or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1 x SSC, 0.1% SDS at 65°C or 2 x SSC, 60°C, 0.1 % SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

In accordance with the present invention, the terms "homology" or "percent homology" or "identical" or "percent identity" or "percentage identity" or "sequence identity" in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides that are the same (preferably at least 40% identity, more preferably at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity, most preferably at least 99% identity), when compared and aligned for maximum correspondence over a window of comparison (preferably over the full length), or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 75% to 90% or greater sequence identity may be considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 nucleotides in length, more preferably, over a region that is at least about 50 to 100 nucleotides in length and most preferably, over a region that is at least about 800 to 1200 nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. MoL Biol. 2 5:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, 1S 4, and a comparison of both strands. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

In order to determine whether an nucleotide residue in a nucleic acid sequence corresponds to a certain position in the nucleotide sequence of e.g. SEQ ID NOs: 23, 15, 1 or 3, respectively, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned herein. For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1 97), loc. cit.; Altschul (1993), loc. cit; Altschul (1990), loc. cit), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:

% sequence identity x % maximum BLAST score

100

and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those, which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the art.

The explanations and definitions given herein above in respect of "homology of nucleic acid sequences" apply, mutatis mutandis, to "amino acid sequences" of members of the Ribonuclease A family, in particular an amino acid sequence as depicted in SEQ ID NO: 24 (human RNAse 1), SEQ ID NO: 26 (human RNAse 2), SEQ ID NO: 28 (human RNAse 3), SEQ ID NO: 16 (murine EAR 10), SEQ ID NO: 2 (murine EAR 1) or SEQ ID NO: 4 (murine EAR 2).

In one embodiment, the polypeptide to be used in accordance with the present invention has at least 40 % homology to the Ribonuclease polypeptide being a member of the Ribonuclease A family having the amino acid sequence as, for example, depicted in SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 2 or SEQ ID NO: 4, respectively. More preferably, the polypeptide has at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homology to the Ribonuclease polypeptide being a member of the Ribonuclease A family having the amino acid sequence as, for example, depicted in SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 2 or SEQ ID NO: 4, respectively, wherein the higher values are preferred. Most preferably, the polypeptide has at least 99% homology to the Ribonuclease polypeptide being a member of the Ribonuclease A family having the amino acid sequence as, for example, depicted in SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 2 or SEQ ID NO: 4, respectively.

The terms "complement", "reverse complement" and "reverse sequence" referred to herein are described in the following example: For sequence 5'AGTGAAGT3', the complement is 3'TCACTTCA5', the reverse complement is 3'ACTTCACT5' and the reverse sequence is 5 'TGAAGTGA3 ' .

The following relates to pharmaceutical compositions, which may comprise the antagonist described and defined herein above.

The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of the pharmaceutical composition for purposes herein is thus determined by such considerations. The skilled person knows that the effective amount of pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

For example, if said compound is a an nucleic acid molecule (e.g. an miRNA, siRNA, a nucleic acid encoding a Ribonuclease, and the like) the total pharmaceutically effective amount of pharmaceutical composition administered parentera!ly per dose will be in the range of about 1 μg /kg/day to 100 mg /kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg kg/day, and most preferably for humans between about 0.01 and 1 mg kg/day. The presently recommended dose for nucleic acid molecules lies in a range of between 8 and 80 mg per/kg/day. However, this dose may be further decreased subject to therapeutic discretion, in particular if concomitantly certain lipids are applied or if the nucleic acid molecule is subject to certain chemical modifications. If given continuously, the pharmaceutical composition is typically administered at a dose rate of about ^g kg/hour to about 40 μg kg/hour_s either by 1- 4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests, which are well known to the person skilled in the art.

If the effective compound is a protein (e.g. an antibody or a Ribonuclease protein as described herein), such proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 20 mg/kg body weight per dose, e.g. between 0.1 mg to 10 mg/kg body weight, e.g. between 0.5 mg to 5 mg/kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

Pharmaceutical compositions of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. Preferably the pharmaceutical compositions of the invention are administered parenterally. Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration, which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al, Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl ethacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030- 4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution, Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; countenons such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The components of the pharmaceutical composition ordinarily will be stored in unit or multi- dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.

The nucleic acid molecules may be delivered as follows: for example, the nucleic acid molecules can be injected directly into a cell, such as by microinjection. Alternatively, the molecules can be contacted with a cell, preferably aided by a delivery system. Useful delivery systems include, for example, liposomes and charged lipids. Liposomes typically encapsulate oligonucleotide molecules within their aqueous center. Charged lipids generally form lipid- oligonucleotide molecule complexes as a result of opposing charges.

These liposomes-oligonucleotide molecule complexes or lipid-oligonucleotide molecule complexes are usually internalized in cells by endocytosis. The liposomes or charged lipids generally comprise helper lipids, which disrupt the endosomal membrane and release the oligonucleotide molecules.

Other methods for introducing nucleic acid molecules into a cell include use of delivery vehicles, such as dendrimers, biodegradable polymers, polymers of amino acids, polymers of sugars, and oligonucleotide-binding nanoparticles. In addition, pluoronic gel as a depot reservoir can be used to deliver the anti-microRNA oligonucleotide molecules over a prolonged period. The above methods are described in, for example, Hughes et al., Drug Discovery Today 6, 303-315 (2001); Liang et al. Eur. J. Biochem. 269 5753-5758 (2002); and Becker et aL, In Antisense Technology in the Central Nervous System (Leslie, R. A., Hunter, A. J. & Robertson, H. A., eds), pp.147-157, Oxford University Press.

Targeting of nucleic acid molecules to a particular cell can be performed by any method known to those skilled in the art. For example, nucleic acid molecules can be conjugated to an antibody or ligand specifically recognized by receptors on the cell. For example, the ligand can be DDR2 (discoid domain receptor 2) expressed on fibrotic cells. Alternatively, an antibody to DDR2 (discoid domain receptor 2) can be employed.

In one embodiment, the present invention provides a method for treating the herein described diseases, such as diseases associated with a disturbance of adipocyte formation, e.g. an increase in adipocyte number as described herein, in a mammal in need thereof. Preferably, the mammal is a human. The method comprises administering into the mammal an effective amount of an antagonist of a member of the Ribonuclease A family as defined herein. The effective amount is determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. Also disclosed and envisaged herein is the use of an antagonist of a member of the Ribonuclease A family as defined herein for the preparation of a pharmaceutical composition for treating the herein described diseases, such as diseases associated with a disturbance of adipocyte formation, e.g. an increase in adipocyte number as described herein.

Also envisaged herein are methods for reducing the number, amount and/or proliferation of adipocytes or preadipocytes in a subject/patient (preferably a human patient). Methods for reducing overweight or obesity, high body weight or high BMI to a normal level in a subject/patient (preferably a human patient) are also subject- of the present invention.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the herein described diseases or symptom thereof and/or may be therapeutic in terms of partially or completely curing the herein described diseases and/or adverse effect attributed to such diseases. The term "treatment" as used herein covers any treatment of a disease in a subject and includes: (a) preventing such diseases from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease. The antagonist can be introduced into the mammal by any method, known to those in the art. For example, the above described methods for introducing the antagonist (if it is a nucleic acid molecule) into a cell can also be used for introducing the molecules into a mammal.

As mentioned above, it is envisaged herein that the above described and defined antagonists /nucleic acid molecules can also be applied in combination with conventional therapies of obesity such as all forms of diets (i.e. calory restriction or change of food composition/content), all forms of bariatric surgery, use of drugs to prevent or delay intestinal uptake of nutrients or drags/substances interfering with cellular metabolism (e.g. insulin) and substances interfering with food intake and satiety, either via the central nervous system or via modulation of satiety signals The additional therapy can also be selected to treat or ameliorate a side effect of one or more pharmaceutical compositions of the present invention. Such side effects include, without limitation, disturbances of immune system or vascularisation.

Moreover, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents can be administered at the same time. The one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents can also be prepared together in a single formulation.

A "patient" or "subject" for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In a preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.

As explained above, ribonucleases of the RNAse A family have unexpectedly been identified herein as key regulators of preadipocyte differentiation. In addition to the surprising finding that antagonists of Ribonucleases are useful in the treatment of diseases associated with a disturbance of adipocyte formation (e.g. increase in adipocyte number), it has been found that also agonists of Ribonuclases are useful in a medical context.

Accordingly, a further aspect of the present invention relates to an agonist of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with a decrease in adipocyte number. It is to be understood that the present invention relates to an agonist of a Ribonuclease being a member of the RNAse A family, wherein the agonist is for use in treating a disease associated with a decrease in adipocyte number. The agonist may be a selective agonist.

As demonstrated in the appended examples, overexpression of EAR- 10 in the model cell line 3T3-L1 led to a dramatically increased expression of adipocyte marker genes (such as aP2 and adiponectin). Increased adipogenesis was also shown by a pronounced increase in the number of adipocytes as evidenced by Oil red O (OrO) staining. As shown in the appended examples and explained herein above, overexpression of EAR- 10 accelerates differentiation of preadipocytes. The above results were confirmed with human RNAse 1. Figure 14 demonstrates successful RNASE3 mRNA silencing in siRNASEl (cells treated with a siRNA oligo targeting RNASEi), but not siCtrl (cells treated with a control siRNA) treated SVF cells. The qPCR data show RNASEI mRNA expression on day 7 following induction with the differentiation cocktail. It was found that knock-down of RNASEI affects adipocyte differentiation, as measured by OrO staining after induction of differentiation of siCtrl or siRNASEl -treated SVF cells with a standard proadipogenic cocktail. At day 7 post-induction of differentiation, cells were fixed and stained with OrO to judge adipocyte content. Figure 15 shows quantitative staining with ORO, as measured with spectrophotometry. The intensity of ORO staining in RNASEI knock-down cells (siRNASEl) was significantly diminished by approximately 50% as compared with siCtrl-treated cells, which shows that RNASEI is required for adipocyte differentiation. This demonstrates that stimulation increase of RNAse 1 expression or activity increases adipocyte differentiation and, similarly, increase in adipocyte volume.

It is clear that the stimulation increase of preadipocyte differentiation/adipocyte formation contributes to a better control of adipogenesis (i.e. the process of cell differentiation by which preadipocytes become adipocytes). The number of adipocytes (adipocytes are well known as cells capable of storing lipids) is directly linked with disorders associated with the de-novo formation of adipocytes and the differentiation of existing preadipocytes. Such diseases include the whole spectrum of lipodystrophies (genetic, drug induced, traumatic, etc.).

Hence, the promotion/enhancement of adipocyte formation and increase in the overall amount or number of adipocytes by the agonists of the present invention provides a potent means in the treatment of the above diseases, like lipodystrophies. The herein provided agonists are particularly useful in situations or pathological conditions where the number of adipocytes is decreased, the proliferation rate of (pre) adipocytes is decreased and/or the differentiation rate of preadipocytes is decreased.

The appended examples show that murine EAR-1, -2 and -10 are strongly expressed in preadipocytes (3T3-L1 cell line and murine preadipocytes); also human RNAse 1 is strongly expressed in human adipose tissue. More importantly, it has been found herein for the first time that these RNAses promote adipogenesis as evidenced by the induction of the expression of adipogenic genes and the increased number of adipocytes (demonstrated by OrO-staining); see Figures 5 and 6. It is believed that the contribution of a Ribonuclease to adipogenesis correlates with its endogenous expression level or activity in (pre)adipocytes and/or (white) adipose tissue. As murine EAR-1, -2, -10 as well as human RNAse 1 have been found to be strongly expressed, these RNases are the primary targets of the herein provided agonists. Due to the high homology level (at least within one RNAse A subfamily such as the murine EAR family or the human RNAses 1 to 13) it is, however, believed that further members of the RNAse A family promote adipogenesis. Accordingly, it is envisaged herein that further members of the RNAse A (sub)family and preferably all members that are expressed in an overweight/adipose patient should be targeted. Preferably, only those Ribonucleases are targeted that are expressed in (pre)adipocytes/adipose tissue. Even more preferably, the herein provided agonists only promote/enhance the Ribonucleases at the site of their expression in the patient, i.e. in adipose tissue, and/or (pre)adipocytes. Human RNASE 1 mRNA is predominantly expressed in adipose tissue, brain, heart, lung and testes (see Figure 16). Further, it is believed that an agonist of a specific Ribonuclease of a RNAse A (sub)family (e.g. RNAse 1) will likewise be effective for related Ribonucleases due to their high level of homology (e.g. RNase 2 or RNAse 3).

Due to their very high homology all members of the RNAse A family may be used in this context. Without being bound, it is believed that RNAse A family members may enhance differentiation of preadipocytes by their RNAse A activity. Thus, all RNA degrading proteins and substances are useful in the enhancement of adipocyte differentiation. As shown in the appended examples (see Figure 6), the agonists to be used herein are capable of strongly increasing preadipocyte proliferation/differentiation into adipocytes (adipocyte formation) and/or inducing adipocyte number. Preferably, the preadipocyte proliferation/differentiation into adipocytes (adipocyte formation) and/or adipocyte number is induced by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, more preferably, at least 70 %, 75 % and most preferably, at least 80 %, 90 % or 100 % compared to the previous state (i.e. prior to treatment with the agonists).

The agonists of the present invention may also be used to increase adipocyte volume. Accordingly, the antagonists may be used in the treatment of a disease that is associated with a decrease in adipocyte volume. This therapy is particularly indicated in patients suffering from underweight (e.g. BMl below 18.5). The term underweight generally refers to a human who is considered to be under a healthy weight. "Underweight" means weighing less than what is expected to be a healthy person (underweight = insufficiency of weight). The definition is usually made with reference to the body mass index (BMl). A BMl of less than 18.5 is usually referred to as underweight. This medical definition of underweight may differ from other uses of the term, such as those based on attractiveness overweight.

The treatment may particularly be useful for patients with eating disorders, HIV patients or cancer patients. These patients may suffer from underweight due to medicaments or other therapies. Often the therapy cannot be continued due to the weakness of the underweight patients. Therefore, it is necessary that these patient gain weigth in order to be able to be treated with the cancer, HIV etc. medicaments/therapy. In other words, the agonists provided herein may be particularly useful in cotherapy with the above mentioned therapies of HIV, cancer and so on.

Accordingly, the present invention relates to an agonist of a Ribonuclease being a member of the R Ase A family as defined herein for use in treating a disease associated with a decrease in adipocyte number, wherein the diseases may be lipodystrophy, in particular hereditary lipodystrophy, drug-induced lipodystrophy, preferably lipodystrophy caused or induced by AIDS/HIV therapy, or traumatic lipodystrophy, or wherein the disease may be cachexia or a disease associated with disturbed energy storage. Also Ribonuclease being a member of the RNAse A family can. be considered as an agonist (to be used in accordance with the present invention).

In context of agonists of a Ribonuclease being a member of the RNAse A family, the disease to be treated is preferably a disease where proliferation and/or differentiation of preadipocytes is prevented or completely blocked. Thus, the number and/or amount of adipocytes is decreased in such a pathological condition.

For example, in lipodystrophy, adipocytes are absent or at least drastically reduced. In subjects/patients suffering from lipodystrophy, lipids accumulate in muscle, liver and other locations which is believed to cause significant metabolic derangement, including insulin resistance and hepatosteatosis that leads to cirrhosis; see Rosen (2006), Nature Rev. 7, 885- 896.

In accordance with the above, the use of Ribonuclease agonists in the treatment of lipodystrophy represents a preferred embodiment of the present invention. Also the treatment of secondary disorders of a disease associated with a disturbance of adipocyte formation is envisaged herein, such as non-alcoholic fatty liver disease (NAFLD). The herein provided Ribonuclease agonists are also useful in situations where insulin resistance and/or hepatosteatosis (NAFLD) are induced. The above-mentioned lipodystrophies may be hereditary lipodystrophies. In the alternative or in addition, a patient may suffer from a lipodystrophy that is caused/induced by external factors, such as medicaments or drugs used in teatments. One well known example of a lipodystrophy induced by drugs or medicaments is lipodystrophy caused or induced by AIDS/HIV therapy. Generally, the diseases to be treated include the whole spectrum of lipodystrophies (genetic, drug induced, traumatic, and the like). Furthermore, various forms of cachexia and diseases associated with disturbed energy storage can be treated with agonists of a member of the RNAse A family.

The definitions and explanations given herein above for "a member of the RNAse A family" apply, mutatis mutandis, in this context.

Accordingly, the present invention relates to the following aspects:

Herein provided is an agonist of a Ribonuclease being a member of the RNAse A family, wherein the agonist is for use in treating a disease associated with a decrease in adipocyte number. The agonist may be a selective agonist.

The Ribonuclease may be a member of the RNAse A family. It may be a human RNAse, in particular selected from the group consisting of

1, RNAse 2, RNAse 3, RNAse 4/5, RNAse 6, RNAse 7, RNAse 8, RNAse 9, RNAse 10, RNAse 1 1, RNAse 12, and RNAse 13 or the Ribonuclease may be a member of the RNAse A family, like a Eosinophil-associated Ribonuclease (EAR), preferably selected from the group consisting of EAR-1, EAR-2 and EAR- 10.

The RNAse may one of the following RNAses:

RNAse 1 having an amino acid sequence as shown in SEQ ID NO: 24;

RNAse 2 having an amino acid sequence as shown in SEQ ID NO: 26;

RNAse 3 having an amino acid sequence as shown in SEQ ID NO: 28;

RNAse 4/5 having an amino acid sequence as shown in SEQ ID NO: 30;

RNAse 6 having an amino acid sequence as shown in SEQ ID NO: 32; RNAse 7 having an amino acid sequence as shown in SEQ ID NO: 34;

RNAse 8 having an amino acid sequence as shown in SEQ ID NO: 36;

RNAse 9 having an amino acid sequence as shown in SEQ ID NO: 38;

RNAse 10 having an amino acid sequence as shown in SEQ ID NO: 40;

RNAse 11 having an amino acid sequence as shown in SEQ ID NO: 42;

RNAse 12 having an amino acid sequence as shown in SEQ ID NO: 44; and

RNAse 13 having an amino acid sequence as shown in SEQ ID NO: 46.

The RNAse 1 may be selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:24;

(c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO:24;

The EAR-1 may be selected from the group consisting of

(a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO: 1 ;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2;

(i) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d). The EA -2 may be selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 4;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional EAR-2, or a fragment thereof; and

The EAR- 10 may be selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 16;

The disease associated with a decrease in adipocyte number may be lipodystrophy. The lipodystrophy may be hereditary lipodystrophy, drug-induced lipodystrophy, lipodystrophy caused or induced by AIDS HIV therapy, or traumatic lipodystrophy.

The disease associated with a decrease in adipocyte number may be cachexia or a disease associated with disturbed energy storage. Furthermore, the present invention provides a method for assessing the activity of a candidate molecule suspected of being an agonist of a Ribonuclease being a member of the RNAse A family, in particular of Ribonucleases as defined herein, comprising the steps of:

(a) contacting a cell, tissue or a non-human animal comprising a Ribonuclease of the RNAse A family with said candidate molecule;

(b) detecting an increase in activity of said Ribonuclease; and

(c) selecting a candidate molecule that increases activity of said Ribonuclease;

wherein an increase of the Ribonuclease activity is indicative for the capacity of the selected molecule to increase adipocyte number and/or increase adipocyte volume.

The term "agonist of a member of the RNAse A family" or "activator of a member of the RNAse A family" means in context of the present invention a compound capable of fully or partially stimulating or increasing the physiologic activity and/or expression level of (a) a member of the RNAse A family. The terms "agonist" or "activator" are used interchangeably herein.

As used herein, accordingly, the term "agonist" also encompasses partial agonists or co- agonists co-activators. In addition thereto, however, an "agonist" or "activator" of a member of the RNAse A family in the context of the present invention may also be capable of stimulating the function of the Ribonuclease by inducing/enhancing the expression of the nucleic acid molecule encoding for said receptor. Thus, an agonist/activator of a member of the Ribonuclease A family may lead to an increased expression level of the Ribonuclease (e.g. increased level of Ribonuclease mRNA, Ribonuclease protein); this may be reflected in an increased Ribonuclease activity. This increased activity can be measured/detected by the herein described methods.

An agonist/activator of the Ribonuclease in the context of the present invention may, accordingly, also encompass transcriptional activators of Ribonuclease expression that are capable of enhancing Ribonuclease function. The term "agonist" comprises partial agonists. As partial agonists the art defines candidate molecules that behave like agonists, but that, even at high concentrations, cannot activate a Ribonuclease to the same extent as a full agonist. Furthermore, the "agonist/activator of a Ribonuclease being a member of the RNAse A family" may have an effect on interactions of the Ribonuclease protein(s) with other proteins (thus, for example, having an effect on the activity of complexes involving Ribonuclease protein(s)) or, in general, with its synthesis, e.g. by having an effect on upstream steps of Ribonuclease expression or with signalling pathways in which the Ribonuclease is involved. Depending on the mode of action, such agonists may, for example, be denoted "sequestering antagonists" or "signalling antagonists".

Hence, the use of potent agonists/activators of a Ribonuclease will lead to a decrease or reduction of a Ribonuclease expression level and/or activity, and thereby increase adipocyte formation. In accordance with the above definition of "agonist" or "activator" also a Ribonuclease itself, the Ribonuclease being a member of the RNAse A family, can be considered as its own agonist activator. For example, overexpression of a Ribonuclease may lead to enhanced Ribonuclease activity, thus agonizing Ribonuclease function. Accordingly, it is preferred that a Ribonuclease as defined herein can be used for the treatment of a disease associated with a disturbance of adipocyte formation, for example, associated with a decrease in adipocyte number, such as lipodystrophy.

In accordance with the above, the agonist to be used herein may be a selective agonist of a Ribonuclease being a member of the RNAse family. In other words, the selective agonist to be used in accordance with the present invention selectively enliances/activates etc. (these terms can be used interchangeably herein) a Ribonuclease being a member of the RNAse family i.e. it primarily activates a Ribonuclease being a member of the RNAse family and does substantially not activate other proteins. The selective agonist shows, for example, a stronger Ribonuclease activation than activation of a protein which is not a Ribonuclease. Preferably, the selective agonist show at least a 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold or 40 fold (or higher) stronger Ribonuclease activation than activation of a protein which is not a Ribonuclease, wherein the higher values are preferred. The selective agonist may show an up to 100 fold stronger Ribonuclease activation than activation of a protein which is not a Ribonuclease.

Selectivity expresses the biologic fact that at a given compound concentration enzymes (or proteins) are affected to different degrees. In the case of enzymes (or proteins) selective activation can be defined as preferred activation by a compound at a given concentration. Or in other words, an enzyme (or protein) is selectively activated over another enzyme (or protein) when there is a concentration which results in activation of the first enzyme (or first protein) whereas the second enzyme (or second protein) is not affected. To compare compound effects on different enzymes it is crucial to employ similar assay formats, such as an assay for measurement of a Ribonuclease activity as described in more detail below. Appropriate assays are also described in Rosenberg (1995), J Biol Chem. 270(14):7876-81 (reference 1); Rosenberg (1995) J. Biol Chem. 270(37):21539-44 (reference 2); Rosenberg (1997) Nucleic Acids Res. 25(17):3532-6 (reference 3); or Domachowske (1998) Nucleic Acids Res. 26(14):3358-63.

Assay for measurement of a Ribonuclease activity. The Ribonuclease assay was described previously in detail (see references 1, 2, 3, 4). Briefly, the concentration of perchloric acid soluble ribonucleotides generated from acid-precipitable yeast tRNA (Sigma, St Louis, MO) in 40 mM sodium phosphate, pH 7.5 will be measured spectrophotometrically at 260 nm. Ribonuclease activity (pmol/min) at single enzyme/substrate concentrations will be determined for increasing concentrations of selective agonists and depicted in bar graphs.

1 : Rosenberg HF. Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. J Biol Chem. 1995 Apr 7;270(14):7876-81.

2: Rosenberg HF, Dyer KD. Eosinophil cationic protein and eosinophil-derived neurotoxin.

Evolution of novel function in a primate ribonuclease gene family. J. Biol Chem. 1995 Sep

15;270(37):21539-44.

Nucleic Acids Res. 1997 Sep l ;25(17):3532-6.

it is preferred herein that the ratio of EC₅₀ values of a selective agonist of a Ribonuclease, being a member of the RNAse A family, determined according to an appropriate assay (e.g. the assays described above for a compound which is a selective Ribonuclease agonist, for example, a selective Ribonuclease 1 agonist/ or other assays known in the art)) and EC50 values of agonists which are not selective for a Ribonuclease (like Ribonuclease 1) determined according to an appropriate comparative assay (e.g. the assays described above for a compound which is not a selective Ribonuclease agonist, for example, not a selective Ribonuclease 1 agonist/ or other assays known in the art)) is about 1 : 10 or higher. More preferred is a ratio of higher than 1 : 15, 1 :20, 1 :30, 1 :35 or 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90 or 1 : 100 or even higher. In accordance with the above, the selective agonist to be used herein may be a selective agonist of one particular Ribonuclease being a member of the RNAse family as disclosed herein, i.e. it primarily activates one particular Ribonuclease (like Ribonuclease 1) while it does substantially not activate other Ribonucleases. The definitions and explanations given herein above in terms of "selective agonist'V'selective activator" as well as the definitions and explanations on "Ribonucleases which are a member of the RNAse A family" apply, mutatis mutandis, in this context.

In this context, one preferred embodiment of the present invention refers to the use of human RNAse 1 (or its orthologoues).

Accordingly, the present invention relates to an agonist of a Ribonuclease being a member of the RNAse A family, for use in treating a disease associated with a decrease in adipocyte number, wherein the agonist is a selective agonist of a Ribonuclease being a member of the RNAse A family, and wherein said Ribonuclease being a member of the RNAse A family is human RNAse, RNAse 1 , wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24. In other words, the present invention relates to an agonist of a Ribonuclease being a member of the RNAse A family, wherein the agonist is for use in treating a disease associated with a decrease in adipocyte number, wherein the agonist is a selective agonist of a Ribonuclease being a member of the RNAse A family, and wherein said Ribonuclease being a member of the RNAse A family is human RNAse, RNAse 1, wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24.

The human RNAse 1 to be used in accordance with the invention (in particular in context of agonists of Ribonuclease for use in the treatment of a disease associated with a disturbance of adipocyte formation, for example, associated with a decrease in adipocyte number, such as lipodystrophy) may be

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 24;

(c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO: 24;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional human RNAse 1, or a fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional human RNAse 1 , or a fragment thereof; and

Accordingly, the present invention relates to an agonist of a Ribonuclease being a member of the RNAse A family, for use in treating a disease associated with a decrease in adipocyte number, wherein said Ribonuclease being a member of the RNAse A family is human RNAse 1, wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24. In other words, the present invention relates to an agonist of a Ribonuclease being a member of the RNAse A family, wherein the agonist is for use in treating a disease associated with a decrease in adipocyte number, wherein said Ribonuclease being a member of the RNAse A family is human RNAse 1 , wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24.

One further preferred embodiment of the present invention refers to the use of EAR- 10 (or its orthoiogoues). The EAR- 10 to be used in accordance with the invention (in particular in context of agonists of EAR for use in the treatment of a disease associated with a disturbance of adipocyte formation, such as lipodystrophy) may be

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 16;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional EAR- 10, or a fragment thereof; (e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional EAR- 10, or a fragment thereof; and

Details for ectopic expression of murine EAR10 are given below:

For ectopic murine EAR 10 we used the Full ORF Expression Clone

OCACo5052C 121 -pEF-DEST51

http://www.imagenes-bio.de/productinfo?CloneID=OCACo5052C 121 -pEF-DEST51

This plasmid contains the full murine EAR 10 sequence

http://www.ncbi.nlm.nih.gov/nuccore/147898142?report=genbank

cloned into the plasmid pEF-DEST51 :

htt : //www. imagenes-bio.de/info/vectors/pEF-DEST51 _pic . shtml

From this plasmid, the full EarlO-sequence containing the His and V5 flag was subcloned into the retroviral vector MSCV-hygro (Clontech) as described in the appended examples.

All the explanations given herein above in context of "antagonists of Ribonucleases being a member of the RNAse A family" apply, mutatis mutandis, also to the Ribonucleases to be used in context of "agonists of Ribonucleases being a member of the RNAse A family" (for example in context of Ribonucleases to be used as agonists or in context of methods for the identification of agonists). In particular, the explanations and definitions given herein above on the use of Ribonuclease homologues and orthologues, the pharmaceutical compositions comprising antagonists, and the administration modus are applicable in this context. Exemplary agonists useful in this context are extracellular binding-partners, small binding molecules, aptamers, intramers, and an antibody molecule such as a full antibody (immunoglobulin), a F(ab) -fragment, a F(ab)₂-fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody, a bispecific single chain antibody or a cross-cloned antibody. The explanations given herein above on the generation and use of antagonist antibodies apply, mutatis mutandis, to agonist antibodies.

In accordance with this, the present invention provides a method for treating the herein described diseases, such as diseases associated with a disturbance of adipocyte formation in a mammal in need thereof. Preferably, the mammal is a human. The method comprises administering into the mammal an effective amount of an agonist of Ribonuclease as defined herein. The effective amount is determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. Also disclosed and envisaged herein is the use of an agonist of Ribonuclase as defined herein above for the preparation of a pharmaceutical composition for treating the herein described diseases, such as diseases associated with a disturbance of adipocyte formation (e.g. decrease in adipocyte number).

Also envisaged herein are methods for increasing the number, amount and/or proliferation of adipocytes or preadipocytes in a subject/patient (preferably a human patient). Methods for increasing (low) body weight, low BMI or low body fat percentage to a normal level (e.g. BMI higher than 18.5 and lower or equal to 25) in a subject/patient (preferably a human patient) are also subject- of the present invention.

As explained above, the means and methods of the present invention are not limited to the use of the herein specifically described compounds for use in the treatment of a disease associated with a disturbance of adipocyte formation. A skilled person is readily in the position to determine whether a candidate compound is a potent antagonist (or agonist) of a Ribonuclease, and accordingly, an artisan will easily be capable of assessing the activity of compounds and/or capable of identifying further compounds to be used in the present invention.

The explanations given herein below refer to potential antagonists of a Ribonuclease. However, the respective explanations and definitions apply, mutatis mutandis, also to agonists of a Ribonuclease; i.e. the terms "antagonist", "antagonize", "inhibit" and the like is then to be replaced by "agonist", "agonize", "activate" and the like and the term "decrease" (and the like) is to be replaced by "increase" (and the like). In other words, such terms used in the context of "antagonists" are to be replaced by their respective opposite terms in context of "agonists". In particular, all explanations given herein above in respect of a Ribonuclease, members of the a Ribonuclease family, orthologues or homologues of (a) Ribonuclease(s) apply here, mutatis mutandis.

Therefore, the present invention relates to a method for assessing the activity of a candidate molecule suspected of being an antagonist a Ribonuclease comprising the steps of:

(a) contacting a cell, tissue or a non-human animal comprising a Ribonuclease with said candidate molecule; (b) detecting a decrease in Ribonuclease activity; and

(c) selecting a candidate molecule that decreases Ribonuclease activity;

wherein a decrease of the Ribonuclease activity is indicative for the capacity of the selected molecule to antagonise adipocyte formation.

Similarly, the present invention also relates to a method for assessing the activity of a candidate molecule suspected of being an agonist of a Ribonuclease comprising the steps of:

(a) contacting a cell, tissue or a non-human animal comprising a Ribonuclease with said candidate molecule;

(b) detecting an increase in Ribonuclease activity; and

(c) selecting a candidate molecule that increases Ribonuclease activity;

wherein an increase of the Ribonuclease activity is indicative for the capacity of the selected molecule to agonise adipocyte formation.

Further, the present invention relates to a method for identifying a candidate molecule suspected of being an antagonist of a Ribonuclease comprising the steps of:

(b) detecting a decrease in Ribonuclease activity; and

(c) selecting a candidate molecule that decreases Ribonuclease activity;

Again, the present invention relates also to a method for identifying a candidate molecule suspected of being an agonist of a Ribonuclease comprising the steps of:

(b) detecting an increase in Ribonuclease activity; and

(c) selecting a candidate molecule that increases Ribonuclease activity;

It is to be understood that the detected activity of a Ribonuclease is compared to a standard or reference value of a Ribonuclease activity. The standard/reference value may be detected in a cell, tissue, or non-human animal as defined herein, which has not been contacted with a potential Ribonuclease inhibitor or prior to the above contacting step. The decrease in the activity of the Ribonuclease upon contacting with (a) candidate molecule(s) may also be compared to the decrease in Ribonuclease activity induced by (a) routinely used reference compound(s). A skilled person is easily in the position to determine/assess whether the activity and/or expression of a Ribonuclease is (preferably statistically significant) increased.

In accordance with this invention, in particular the screening or identifying methods described herein, a cell, tissue or non-human animal to be contacted with a candidate molecule comprises the Ribonuclease as defined herein. For example said cell, tissue or non-human animal may express a Ribonuclease gene, in particular also (an) additional (copy) copies of a Ribonuclease gene, (a) Ribonuclease mutated gene(s), a recombinant Ribonuclease gene construct and the like. As explained herein below, the capability of a candidate molecule to inhibit/antagonize Ribonuclease may, accordingly, be detected by measuring the expression level of such gene products of Ribonuclease or of corresponding gene constructs (e.g. mRNA or protein), wherein a low expression level (compared to a standard or reference value) is indicative for the capability of the candidate molecule to act as inhibitor/antagonist.

The term "comprising Ribonuclease" may, for example, relate to to the Ribonuclease gene(s) or proteins known in the art and described herein, but also to a reporter construct which comprises the Ribonuclease (or a functional fragment thereof) and a "reporter". Exemplary reporters (reporter gene products), which can be used in the screening methods of the invention are luciferase, (green/red) fluorescent protein and variants thereof, EGFP (enhanced green fluorescent protein), RFP (red fluorescent protein, like DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue green fluorescent protein), YFP (yellow fluorescent protein), β-galactosidase or chloramphenicol acetyltransferase. The skilled person is readily in the position to generate and use also other reporters/reporter constructs, which can be employed in accordance with the present invention. The use of fusion proteins containing a Ribonuclease protein (or a functional fragment tiiereof) and a reporter gene product is also envisaged in the methods of the present invention.

Antagonists of a Ribonuclease may interfere with the transcription of the Ribonuclease or with the transcription of a reporter construct, in particular Ribonuclease fusion proteins. For example, the antagonists may bind to the promoter region of the Ribonuclease or of the fusion protein, thus preventing initiation of transcription or stopping the already initiated transcription process. The antagonists may also bind to/interfere with components of the transcription machinery, thereby effectively inhibiting initiation of transcription or continuation of transcription. Such an interference with the transcription of the Ribonuclease, Ribonuclease constructs or Ribonuclease fusion proteins by a candidate molecule will be reflected in a decreased transcription activity and hence, a reduced transcript level (e.g. unspliced/partially spliced/spliced mRNA). It is also envisaged herein that a reporter construct to be used herein comprises the promoter of a Ribonuclease linked to a reporter as described herein. Thus, activity of the Ribonuclease may be reflected in an activation of its promoter and, hence, in turn reflected in the change/decrease of the reporter signal associated with the reporter.

Due to the reduced transcript level also the level of the translated gene product (i.e. the protein level) will be decreased. The level of the above described fusion proteins preferably correlates with the signal strength of a detectable signal associated with the reporter gene product. Exemplary Ribonuclease fusion proteins are proteins comprising Ribonuclease (or a functional fragment thereof) and a reporter as described above (e.g. luciferase, (green/red) fluorescent protein and variants thereof, EGFP (enhanced green fluorescent protein), and the like).

Accordingly, a decrease in Ribonuclease (promoter) activity (which may, for example, be reflected in a decrease in the (Ribonuclease promoter) reporter signal) upon contacting the cell/tissue/non-human animal with a candidate molecule will indicate that the candidate molecule is indeed a Ribonuclease inhibitor/antagonist and, thus, capable of decreasing preadipocyte formation/proliferation. The candidate molecules which decrease Ribonuclease (promoter) activity as defined herein above are selected out of the candidate molecules tested, wherein those molecules are preferably selected which strongly decrease Ribonuclease (promoter) activity (reflected, for example, in a pronounced decrease in the reporter signal). It is assumed that the Ribonuclease antagonizing/inhibiting activity of a candidate molecule is the stronger the more the reporter signal is decreased.

It is envisaged in the context of the present invention (in particular the screening/identifying methods disclosed herein) that also cellular extracts can be contacted (e.g. cellular extracts comprising Ribonuclease as described and defined herein). For example, these cellular extracts may be obtained from the (transgenic/genetically engineered) cell(s), tissue(s) and/or non- human animal(s) to be used herein, in particular to be contacted with the candidate molecule. The use of such cellular extracts is particular advantageous since it allows the assessment of the activity of a candidate molecule in vitro. The assessing/screening methods taking advantage of such (cellular) extracts can, for example, be used in prescreening candidate molecules, wherein the molecules selected in such a prescreen are then subject to subsequent screens, for example in the cell-based methods disclosed herein, in particular in methods wherein a (transgenic) cell(s), tissue(s) and/or non-human animal(s) are contacted with a candidate molecule. In this context, it is accordingly preferred that the candidate molecule has been selected in the in vitro pre-screening method, described herein above and below.

It is to be understood that in a high throughput screening routinely, many (often thousands of candidate molecules) are screened simultaneously. Accordingly, in a (first) screen candidate molecules are selected, which decrease Ribonuclease activity.

Step (a) of the screening methods of the present invention, i.e. the "contacting step" may also be accomplished by adding a (biological) sample or composition containing said candidate molecule or a plurality of candidate molecules (i.e. various different candidate molecules) to the sample to be analyzed (e.g. (a) cell(s)/tissue(s)/non-human animal comprising Ribonuclease or a functional fragment thereof).

Generally, the candidate molecule(s) or a composition comprising containing the candidate molecule(s) may for example be added to a (transfected) cell, tissue or non-human animal comprising Ribonuclease. As defined and disclosed herein, the term "comprising Ribonuclease" refers not only to the Ribonuclease gene(s) or proteins known in the art and described herein, but also to reporter constructs comprising a reporter and Ribonuclease. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. Also reporter constructs comprising a promoter and/or enhancer region of Ribonuclease and a reporter as defined herein can be used in the screening/identifying methods. Accordingly, the cell(s), tissue(s) and/or non-human animals used in the context of the present invention, in particular in context of the screening/identifying methods can be stably or transiently transfected with the reporter constructs disclosed herein.

In particular the identification/assessment of candidate molecules which are capable of inhibiting/antagonizing Ribonuclease may be, inter alia, performed by transfecting an appropriate host with a nucleic acid molecule encoding Ribonuclease (or a functional fragment thereof) and contacting said host with (a) candidate molecule(s). The host (cell, tissue, non- human animal) can also be transfected with the above described reporter constructs, e.g. luciferase reporter constructs, such as, but not limited to, reporter constructs comprising a luciferase reporter. The host may comprise CHO-cell, HEK 293, HeLa, Cos 7, PC 12 or NIH3T3 cell, frog oocytes or primary cells like primary cardiomyocytes, fibroblasts, muscle, endothelial or embryonic stem cells. Alternatively, it is also possible to use cell lines stably transfected with a nucleic acid molecule encoding Ribonuclease or a functional fragment thereof. The explanations given herein above in respect of "cells" also apply to tissues/non- human animals comprising or derived from these cells. A sample to be analyzed may also be a biological, medical or pathological sample, for example fluids that comprise cells, tissues or cell cultures. Such a fluid may be a body fluid or also excrements and may also be a culture sample. The body fluids may comprise but are not limited to blood, serum, plasma, urine, saliva, synovial fluid, spinal fluid, cerebrospinal fluid, tears, stool and the like.

The (biological) sample or composition, comprising a plurality of candidate molecules are usually subject to a first screen. The samples/compositions tested positive in the first screen are often subject to subsequent screens in order to verify the previous findings and to select the most potent inhibitors/antagonists of the Ribonuclease. Upon multiple screening and selection rounds those candidate molecules will be selected which show a pronounced capacity to inhibit/antagonize Ribonuclease as defined and disclosed herein. For example, batches (i.e. compositions/samples) containing many candidate molecules will be rescreened and batches with no or insufficient inhibitory activity of candidate molecules be discarded without re- testing.

For example, if a (biological) sample or composition with many different candidate molecules is tested and one (biological) sample or composition is tested positive, then it is either possible in a second screening to screen, preferably after purification, the individual molecule(s) of the (biological) sample or composition. It may also be possible to screen subgroups of the (biological) sample or composition of the first screen in (a) subsequent screen(s). The screening of compositions with subgroups of those candidate molecules tested in previous screening rounds will thus narrow in on (an) potential potent Ribonuclease inhibitor(s). This may facilitate and accelerate the screening process in particular when a large number of molecules is screened. Accordingly, the cycle number of screening rounds is reduced compared to testing each and every individual candidate molecule in (a) first (and subsequent) screen(s) (which is, of course, also possible). Thus, depending on the complexity or the number of the candidate molecules, the steps of the screening method described herein can be performed several times until the (biological) sample or composition to be screened comprises a limited number, preferably only one substance which is indicative for the capacity of screened molecule to decrease the proliferation/differentiation rate of preadipocytes. The term "decrease in Ribonuclease activity" in step (b) of the screening method means that the "activity of the Ribonuclease" is reduced upon contacting the cell, tissue, or non-human animal comprising a Ribonuclease with the candidate molecule, preferably in comparison to a (control) standard or reference value, as defined herein.

Particularly preferred are optical measurement techniques that allow a resolution of fluorescence on the level of single cells or single cells of a tissue, preferably at the subcellular level. They may involve fluorescence, for example confocal microscopy, digital image recording, preferably a CCD camera and suitable picture analysis software. Preferably, step (b) is carried out after the measurement of a standard response by performing a control experiment. For example, the activity of a Ribonuclease is measured in a cell, tissue or a non- human animal comprising a Ribonuclease without contacting a candidate molecule in a first screen. In a second screen, after contacting the candidate molecule, the activity of the Ribonuclease is measured. A difference in the activities will indicate whether the tested candidate molecule is indeed an antagonist of a Ribonuclease and capable of decreasing preadipocyte differentiation/proliferation.

The activity of a Ribonuclease can be quantified by measuring, for example, the level of gene products (e.g. mRNA and/or protein of the Ribonuclease and said component, respectively) by any of the herein described methods, activities, or other cellular functions, like inter alia, the involvement in signalling pathways or changes in intracellular localization.

As mentioned, a "decreased Ribonuclease activity" and, accordingly, a decreased concentration/amount of Ribonuclease proteins in a sample may be reflected in a decreased expression of the corresponding gene(s) encoding the Ribonuclease protein(s). Therefore, a quantitative assessment of the gene product (e.g. protein or spliced, unspliced or partially spliced mRNA) can be performed in order to evaluate decreased expression of the corresponding gene(s) encoding the Ribonuclease protein(s). Also here, a person skilled in the art is aware of standard methods to be used in this context or may deduce these methods from standard textbooks (e.g. Sambrook, 2001, loc. cit.). For example, quantitative data on the respective concentration/amounts of mRNA from the Ribonuclease can be obtained by Northern Blot, Real Time PCR and the like. Preferably, the concentration/amount of the gene product (e.g. the herein above described Ribonuclease mRNA or Ribonuclease protein) may be decreased by at least about 10 %, 20 %, 30 %, 40 %, preferably by at least 50 %, 60 %, 70 %, 80 %, 90 %, 91%, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or even 100 % compared to a control sample. A decrease by 100 % means that no gene product is detectable by herein described routine methods. It is preferred herein that Ribonuclease proteins are (biologically) active or functional. Methods for determining the activity of a Ribonuclease are described herein above and shown in the appended example. Since the Ribonuclease proteins are preferably (biologically) active/functional (wherein it is preferred that at least 70 %, 75 %, preferably at least 80%, 85 %, 90 %, 95 %, 96, %, 97%, 98 % and most preferably, at least 99 % of Ribonuclease proteins of a sample a (biologically) active/functional), an decreased concentration/amount of Ribonuclease proteins in a sample reflects a decreased (biological) activity of the Ribonuclease protein.

As mentioned, a person skilled in the art is aware of standard methods to be used for determining or quantitating expression of a nucleic acid molecule encoding, for example, the Ribonuclease (or fragments thereof) as defined herein. For example, the expression can be determined on the protein level by taking advantage of immunoagglutination, immunoprecipitation (e.g. immunodiffusion, immunelectrophoresis, immune fixation), western blotting techniques (e.g. (in situ) immuno histochemistry, (in situ) immuno cytochemistry, affinity chromatography, enzyme immunoassays), and the like. Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on specific binding, e.g. of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). For example, concentration/amount of Ribonuclease proteins in a cell, tissue or a non-human animal can be determined by enzyme linked-immunosorbent assay (ELISA). Alternatively, Western Blot analysis or inimunohistochemical staining can be performed. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi -dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction.

Expression can also be determined on the nucleic acid level (e.g. if the gene product/product of the coding nucleic acid sequence is an unspliced/partially spliced/spliced mRNA) by taking advantage of Northern blotting techniques or PCR techniques, like in-situ PCR or Real time PCR. Quantitative determination of mRNA can be performed by taking advantage of northern blotting techniques, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques referred to above, like, for example, quantitative PCR techniques, such as Real time PCR. These and other suitable methods for detection and/or determination of the concentration/amount of (specific) mRNA or protein(s)/polypeptide(s) are well known in the ait and are, for example, described in Sambrook (2001), Ioc. cit.).

A skilled person is capable of determining the amount of mRNA or polypeptides/proteins, in particular the gene products described herein above, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of, for example, the mRNA or polypeptides/proteins to be determined. Accordingly, the activity of a Ribonuclease (or a functional fragment thereof) may be quantified based on the mRNA or protein level of the Ribonuclease or a functional fragment thereof and vice versa.

Genetic readout systems are also envisaged. Analogously, the activity of a Ribonuclease or of a functional fragment thereof may be quantified by any molecular biological method as described herein. A skilled person is also aware of standard methods to be used in determining the amount/concentration of Ribonuclease expression products (in particular the protein and the nucleic acid level of a Ribonuclease) in a sample or may deduce corresponding methods from standard textbooks (e.g. Sambrook, 2001).

In samples obtained from (a) cell(s), tissue or a cell culture(s) transfected with appropriate constructs or obtained from transgenic animals or cell cultures derived from a non-human animal(s), the concentration/amount of Ribonuclease protein can be determined by bioassays, if, for example, a Ribonuclease-inducible promoter is fused to a reporter gene. Apparently, decreased expression of the reporter gene/activity of the reporter gene product will reflect a decreased Ribonuclease activity, in particular a decreased concentration/amount of Ribonuclease protein. Alternatively, the effect of the Ribonuclease protein on the expression of (a) reporter gene(s) may be evaluated by determining the amount/concentration of the gene product of the reporter gene(s) (e.g. protein or spliced, unspliced or partially spliced mRNA). Further methods to be used in the assessment of mRNA expression of a reporter gene are within the scope of a skilled person and also described herein below.

Also in this context, reporter constructs comprising a promoter and/or enhancer region of Ribonuclease and a reporter as defined herein can be used in the screening/identifying methods. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. The use of such constructs in screening methods is also demonstrated in the appended examples. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. These and other reporters/reporter constructs/reporter signals are also described herein above and below.

The difference, as disclosed herein is statistically significant and a candidate molecule(s) is (are) selected, if the Ribonuclease activity (or of a corresponding reporter signal) is strongly decreased, preferably is very low or non-dectable. For example, the Ribonuclease activity (or of a corresponding reporter signal) may be decreased by at least 50%, 60%, 70%, 80%, more preferably by at least 90% compared to the (control) standard value. In a cell based method the ceils can be transfected with one or more constructs encoding a Ribonuclease or a functional fragment thereof as described above and optionally a reporter under the transcriptional control of the Ribonuclease or a functional fragment thereof as described above.

Preferably, the selected compound has a high Ribonuclease inhibiting/antagonizing activity. This can be reflected in the capacity of the Ribonuclease antagonist/inhibitor to potently decrease the activity of Ribonuclease.

The above detected difference between the activity of a Ribonuclease or the activity of a functional fragment of the Ribonuclease in a cell, tissue or a non-human animal contacted with said candidate molecule and the activity in the (control) standard value (measured e.g. in the absence of said candidate molecule) may be reflected by the presence, the absence, the increase or tire decrease of a specific signal in the readout system, as in the herein described fluorescence based system.

Preferably, candidate agents to be tested encompass numerous chemical classes, though typically they are organic compounds, preferably small (organic) molecules as defined herein above.

Candidate agents may also comprise functional groups necessary for structui'al interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

Exemplary classes of candidate agents may include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc.

As mentioned above, candidate agents are also found among other biomolecules including amino acids, fatty acids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

The reporter constructs for detecting Ribonuclease inhibition as described herein above may be comprised in a cell, tissue or a non-human animal. Methods for transfecting cells or tissues are known in the art. Accordingly, calcium phosphate treatment or electroporation may be used for transfecting cells or tissues to express said reporter constructs (see Sambrook (1989), loc. cit). Furthermore, nucleic acid molecules expressing said reporter constructs can be reconstituted into liposomes for delivery to target cells. As a further alternative, cells may be transduced to express specific reporter construct using genetically engineered viral vectors. In another preferred embodiment, the non-human animal comprising said reporter construct for detecting Ribonuclease inhibition is a transgenic non-human animal. The non-human organism to be used in the described screening assays is preferably selected from the group consisting of C. elegans, yeast, drosophiia, zebrafish, guinea pig, rat and mouse. The generation of such a transgenic animal is within the skill of a skilled artisan. Corresponding techniques are, inter alia, described in "Current Protocols in Neuroscience" (2001), John Wiley&Sons, Chapter 3.16. Accordingly, the invention also relates to a method for the generation of a non-human transgenic animal comprising the step of introducing a reporter construct for detecting Ribonuclease inhibition as disclosed herein into an ES-cell or a germ cell. The non-human transgenic animal provided and described herein is particular useful in screening methods and pharmacological tests described herein above. In particular the non- human transgenic animal described herein may be employed in drug screening assays as well as in scientific and medical studies wherein antagonists/inhibitors of Ribonuclease for the treatment of a disease associated with a disturbance of adipocyte formation are tracked, selected and/or isolated.

The transgenic/genetically engineered cell(s), tissue(s), and/or non-human animals to be used in context of the present invention, in particular, the screening/identifying methods, preferably comprise the herein described and defined reporter constructs. Also in this context, reporter constructs may comprise a promoter and/or enhancer region of a Ribonuclease and a reporter as defined herein. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. These and other reporters/reporter constructs/reporter signals are also described herein above and below.

In a further embodiment, the present invention relates to the use of a cell, tissue or a non- human animal for screening and/or validation of a compound suspected of being an antagonist of a Ribonuclease being a member of the RNAse A family. Correspondingly, the present invention relates to the use of a cell, tissue or a non-human animal for screening and/or validation of a compound suspected of being an agonist of a Ribonuclease being a member of the RNAse A family. Accordingly, herein envisaged is the use of a cell, tissue or a non-human animal for screening and/or validation of a compound suspected of being an antagonist of a Ribonuclease being a member of the RNAse A family.

The term "cell" as used in this context may also comprise a plurality of cell as well as cells comprised in a tissue. A cell to be used may, for example any cells of the adipose tissue. For example, a high and stable expression of a Ribonuclease may facilitate the detection of a decrease in Ribonuclease activity. Since wild-type cells have sometimes a low or unstable Ribonuclease expression, the use of (a) transgenic cell(s), tissue(s), non-human animal is particularly envisaged, if these cells have a high Ribonuclease expression (reflected in a high protein or mRNA level). (Transgenic) cells(s), tissue(s) and non-human animals to be used in accordance with the present invention are also described herein above.

The used non-human animal or cell may be transgenic or non transgenic. In this context the term "transgenic" particularly means that at least one of the Ribonuclease genes as described herein is overexpressed; thus the Ribonuclease activity in the non-human transgenic animal or a transgenic animal cell is enhanced. Generally, it is preferred herein that Ribonuclease is highly expressed in (a) cell(s), tissue(s), non-human animal to be used in the screening methods as described above.

The term "transgenic non-human-animal", "transgenic cell" or "transgenic tissue" as used herein refers to an non-human animal, tissue or cell, not being a human that comprises different genetic material of a corresponding wild-type animal, tissue or cell. The term "genetic material" in this context may be any kind of a nucleic acid molecule, or analogues thereof, for example a nucleic acid molecule, or analogues thereof as defined herein. The term "different" means that additional or fewer genetic material in comparison to the genome of the wild type animal or animal cell. An overview of different expression systems to be used for generating transgenic cell/animal refers for example to Methods in Enzymology 153 {1987), 385-516, in Bitter et al (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al, (Methods in Molecular Biology 75 (1997), 427-440).

In a preferred embodiment, the (transgenic) non-human animal or (transgenic) cell is or is derived from a mammal. Non-limiting examples of the (transgenic) non-human animal or derived (transgenic) cell are selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig and Drosophila.

Generally, the (transgenic) cell may be a prokaryotic or eukaryotic cell. For example, the (transgenic) cell in accordance with the present invention may be but is not limited to bacterial, yeast, fungus, plant or animal cell. In general, the transformation or genetically engineering of a cell with a nucleic acid construct or a vector can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.

The invention also relates to a kit useful for carrying out the methods as described herein comprising polynucleotides and/or antibodies capable of detecting the activity of a Ribonuclease as characterized above. The embodiments disclosed in this connection with the method of the present invention apply, mutatis mutandis, to the kit of the present invention. Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solution(s), wash solution(s) and/or remaining reagent(s) or material required in the pharmacological and drug screening assays or the like as describes herein. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.

In a preferred embodiment of the present invention the kit may be advantageously used for carrying out the method for detecting the Ribonuclease activity as described herein. Additionally, the kit of the present invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures, which are known to the person skilled in the art.

Similarly, kits are provided which comprise the candidate molecule as described herein, the nucleic acid molecule, the vector, the cell, tissue and/or a non-human transgenic animal of the invention. These kits provided herein are particularly useful in the methods of the present invention and in particular in the determination of the Ribonuclease activity. These kits as well as the methods provided herein are also useful in pharmaceutical screenings, also comprising "high-throughput" screening. The technical advantage of the herein described methods as well as the kits is the use of a Ribonuclease or a fragment thereof as a functional biosensor.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1. EAR-1 and EAR-2 are induced in adipogenesis. Upper panel: EAR-1 and EAR-2 mRNA expression during differentiation of 3T3-L1 cells into adipocytes. EAR-1 and EAR-2 mRNA expression was measured in postconfluent 3T3-L1 cells (DayO) and upon induction of differentiation by the addition of a mix of adipogenic inducers dexamethasone, IBMX, insulin and troglitazone (DMIT). Total RNA was isolated at the indicated time points and subjected to qPCR analysis. Lower panel: EAR-1 and EAR-2 mRNA expression during differentiation of primary preadipocytes into adipocytes as described for 3T3-L1 cells. EARl and EAR2 mRNA expression were normalized to the murine ARP housekeeper gene. Data are shown as mean ± SEM of three biological replicates. Figure 2. Stable EAR-1, -2 and -10 gene knockdown in 3T3-L1 cells. 3T3-L1 preadipocytes expressing a shRNAmir targeting EAR-1 , EAR-2 and EAR- 10 (miEAR) or Ctrl LMP vector (LMP) were transiently transfected with an expression plasmid for EAR10 fused to the V5 epitop. 36 hrs post transfection, protein lysates were prepared for western blotting with an anti-V5 antibody, beta-actin demonstrates equal protein loading.

Figure 3. Knockdown of EAR-1, -2 and -10 suppresses adipocyte differentiation.

Postconfluent 3T3-L1 preadipocytes expressing a shRNAmir targeting EAR-1 , EAR-2 and EAR- 10 (miEAR.) or ctri LMP vector (LMP) were induced to differentiate into adipocytes with DMIT. Oil red O staining of the cells was performed on d 7. Cells were fixed and stained with Oil Red O, and either the wells (upper panel) or a microscopic view of the wells (insert; lower panel) was photographed.

Figure 4. Knockdown of EAR-genes suppresses proadipogenic gene expression.

Postconfluent 3T3-L1 preadipocytes expressing a shRNAmir targeting EAR-1, EAR-2 and EAR- 10 (miEAR) or Ctrl LMP vector (LMP) were induced to differentiate into adipocytes with DMIT The cells were harvested at the indicated time points, and the relative mRNA levels of the indicated genes were measured by quantitative RT-PCR and normalized to the amount of the murine ARP gene. The data represent means ± SEM (n - 3).

Figure 5. Protein lysates were prepared from 3T3-L1 cells engineered to stably overexpress murine EAR- 10 fused to a V5-tag (MSCV_EAR10-V5) or empty vector expressing cells (MSCV) and immunoblotted with an anti-V5 antibody, beta-actin demonstrates equal protein loading.

Figure 6. Overexpression of EAR-10 enhances proadipogenic gene expression.

Postconfluent 3T3-LI preadipocytes engineered to stably express EAR-10 fused to a V5-tag (MSCV_EAR10-V5; filled bars) or empty vector (MSCVJiygro; open bars) were induced to differentiate into adipocytes with DMIT. (A) Oil red O staining of the cells was performed on d 7. Cells were fixed and stained with Oil Red O, and either the wells (upper panel) or a microscopic view of the wells was photographed. (B) The cells were harvested at the indicated time points, and the relative mRNA levels of the indicated genes were measured by quantitative RT-PCR and normalized to the amount of the murine ARP gene. The data represent means ± SEM (n = 3).

Figure 7. Blockade of the RNase activity of EAR-1, -2 and -10 diminishes adipocyte differentiation. Postconfluent 3T3-L1 cells were induced to differentiate into adipocytes with DMIT. RNAsin (filled bars) or vehicle (open bars) was added at day 0 to the cell culture medium and every 24hrs thereafter. RNA was harvested at dayO and day5 post differentiation and the relative mRNA levels of the indicated genes were measured by quantitative RT-PCR and normalized to the amount of the murine ARP gene. The data represent means ± SEM (n = 3).

Figure 8. Isolation procedure of the adipose depot stromal vascular fraction (SVF). The scheme illustrates the experimental steps of the isolation procedure of SVF from adipose tissue. The isolated SVF contains preadipocytes which can be cultivated and induced to differentiate into mature adipocytes upon induction with a proadipogenic cocktail (detailed ingredients are described in M&M).

Figure 9. RNASE1 mRNA expression throughout adipocyte differentiation. Human preadipocytes from subcutaneous (left panel) or visceral (right panel) adipose tissue were differentiated with a standard adipogenic cocktail. Total RNA was harvested at the indicated time points and RNASE1 mRNA levels was measured by quantitative RT-PCR and normalized to the amount of the human RPLPO/ARP gene. The data represent means ± SEM (n = 3).

Figure 10 and 11 mRNA expression levels of human RNASE genes and well-known adipocyte-specific genes. Human visceral preadipocytes were induced to differentiate into adipocytes with a standard proadipogenic cocktail. RNA was harvested at the indicated time points and subjected to DNA Microarray analysis. The list shows normalized signal intensities (Figure 10) of the human RNASE gene family in addition to typical adipocyte genes. Fold change gene regulation in relation to day 0 is shown in Figure 1 1.

Figure 12. DNA Microarray expression profiling for typical adipocyte-specific genes as shown by a heatmap. Shades of red indicate distinct degrees of gene activation in relation to gene expression on day 0 before induction of differentiation.

Figure 13. Images of human primary preadipocytes isolated from subcutaneous white adipose tissue before and at the end of differentiation. Phase contrast images of subconfluent and confluent human preadipocytes (left and middle images, respectively). Mature adipocytes are indicated by Oil red O staining of accumulated intracellular lipid droplets (right image). The scheme below summarizes the DNA Microarray experimental setup.

Figure 14: Efficient knock-down of RNASEl in isolated human preadipocytes. Human preadipocytes were electroporated with a control siRNA (siCtrl) or an siRNA targeting human RNASEl (siRNASEl) and differentiated with a standard adipogenic cocktail for 7 days. RNA was harvested on day 7 post differentiation and the relative mRNA levels of the RNASEl gene was measured by quantitative RT-PCR and normalized to the amount of the human RPLPO/ARP gene. The data represent means ± SEM (n = 3; *P<0,05).

Fig. 15: Knockdown of RNASEl impairs human adipocyte differentiation. Human preadipocytes were electroporated with a control siRNA (siCtrl) or an siRNA targeting human

RNASEl (siRNASEl) and differentiated with a standard adipogenic cocktail. 7 Days later, the cells were fixed and stained with Oil Red 0. The dye was eluted with isopropanol. Bars represent optical density evaluated at 510nm. The data represent means ± SEM (n = 3; ***P<0.005).

Figure 16: RNASEl mRNA expression in human tissues. 1 μ total RNA per tissue from a human tissue RNA atlas (Ambion, Austin, TX, USA) was converted into cD A by reverse transcription using the RETROscript First Strand Synthesis Kit (Applied Biosystems, Foster City, CA, USA) for RT-PCR. Primers for human RNASEl were designed via the Primer3 software (http://frodo.wi.mit.edu/cgi~bin/primer3/primer3_www.cgi). Using the ABI Prism 7700 sequence detection system (PE Applied Biosystems, Warrington, UK), PC cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles at 94°C for 30 seconds, 60° C for 15 seconds and 72°C for 30 seconds and a 10 minutes terminal incubation at 72°C. Sequence Detector Software (SDS version 1.6.3, PE Applied Biosystems) was used to extract the PCR data, which were then exported to Excel (Microsoft, Redmond, WA) for further analyses. Data were analyzed according to the 2-AACT method. The RNA- amount of RPLP0/ARBP was used as an internal control.

The present invention is additionally described by way of the following illustrative non- limiting example that provide a better understanding of the present invention and of its many advantages.

Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001).

The Examples illustrate the invention.

Example 1.

Materials and Methods

Cell Culture

HEK-293FT cells (Invitrogen, Carlsbad, CA, USA) were maintained in DMEM supplemented with 10% FBS. 3T3-L1 preadipocytes were maintained and differentiated as described (Xu et al,, 2008). Briefly, 3T3-L1 preadipocytes were grown to confluence in DMEM supplemented with 10% calf serum, Two days after confluence, cells were supplied with differentiation medium (DMEM containing 10% FBS plus 1.7 μΜ insulin, 10 mM dexamethasone, and 0.5 mM 3-isobutyl-l-methylxanthine (DMIT)). Fort -eight hours after induction, cells were fed maintenance medium (DMEM containing 10% FBS plus 1.7 μΜ insulin), and the medium was replaced every 2 days.

Retrovirus Preparation and Infection

Retrovirus preparation and infection were performed as described (Bilban et al, 2008). Briefly, pMSCV, LMP empty vectors, or their derivatives containing specific cDNA or shRNAmir, along with vectors containing and reverse transcriptase (gag-pol) and VSV-G- expressing plasmids, was transfected into 293FT packaging cells with Lipofectamine 2000 (Invitrogen). Viral supernatant was collected 48 h after transfection, filtered through 0.45-m filters, and added to target cells for 12 h along with 8 g/ml Polybrene. Cells were selected with 4 g/ml puromycin or 400g/ml hygromycin tomakestable lines and were maintained in media containing appropriate antibiotics.

Construction of transgenic cell lines

Constitutive stable EAR10 knock-down in 3T3-L1 cells was generated by transduction with the microRNA (miRNA) adapted retroviral vector LMP (OpenBiosystems, Huntsville, AL, USA). shRNAmir (microRNA- adapted short hairpin RNA) against murine EAR10 generated in pSM2 vector (Open Biosystems, cat.no. RMM1766-96883532, directed against Gene Accession: NM_0531 12) was subcloned into the LMP vector with Xhol and EcoRI (Invitrogen, Carlsbad, CA) restriction enzymes. Confirmation was verified by restriction site analysis and sequencing. To produce murine stem cell virus (MSCV) particles, HEK293FT cells were transiently co -transfected with a vector containing the viral packaging proteins gag and pol, a vector containing env, and either LMP, or LMP-miHO (LMP containg miRNA against human HO-1). Lipofectamine 2000 (Invitrogen) reagent was used for transfection. Forty-eight hours after transfection, viral supernatants were collected, centrifuged at 1500 rpm for 3 min, filtered through a 0.4 μηα filter, supplemented with 8 μg/ml Polybrene (Sigma), and used to infect 3T3-L1 cells. Stable integrants were selected with puromycin (5 μg/ml) over a period of 2 weeks. EAR10 knock-down was verified by western blotting (Fig 2A). Real-time PCR

Total RNA (1 μg) was reverse transcribed into cDNA by MMLV enzyme (Promega, Mannheim, Germany) with random hexamers (1 g μg total RNA). The reaction mixture was incubated at 37°C for 45 minutes followed by 15 min at 45°C and 20 min at 70°C. All PCRs were performed using the SYBR Geen kit (BioRad, Hercules, CA, USA). Primers for selected genes were designed via the Primer 3 software (http://frodo.wi.mit.edu/cgi- bin primer3/primer3_www.cgi). Primers for EAR1 and EAR2 were used as described in Phipps et al, Blood, 2007 1 0: 1578-1586). Using the ABI Prism 7700 sequence detection system (PE Applied Biosystems, Warrington, UK), PCR cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles at 94°C for 30 seconds, 60°C for 1 seconds and 72°C for 30 seconds and a 10 minutes terminal incubation at 72°C. Sequence Detector Software (SDS version 1.6.3, PE Applied Biosystems) was used to extract the PCR data, which were then exported to Excel (Microsoft, Redmond, A) for further analyses. Data were analyzed according to the 2-D DCT (i.e. 2^"ΑΔα) method. The RNA-amount of RPLPO/ARBP was used as an internal control.

Western blot analyses

Western blot analyses were performed using standard protocols as recently done (10, 30). Equal amounts of protein lysates (35 μ ) were separated on 10% SDS/polyacrylamide (PAA) gels and transferred onto Polyvinylidene fluoride (PVDF) -membranes (GE Healthcare, Amersham, Buckinghamshire, UK). After blocking filters were incubated overnight (4°C) with monoclonal mouse antibodies against V5 epitope (1 :1000; SIGMA, USA), β-actin (1:5000; Abeam, Cambridge, MA, USA). After 1 h of treatment (room temperature) with secondary antibodies (anti-mouse Ig horseradish peroxidase linked, Amersham; 1 :50.000) signals were developed by using ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech).

Oil Red O Staining and Quantification of Lipid Accumulation in the Kinetics of adipocyte differentiation The accumulation of lipids signifying the formation of adipocytes was observed by staining the differentiated cells or EBs with Oil Red 0. Oil Red O stock solution (0.5%) was prepared in 60% triethyi phosphate and filtered in cellulose nitrate filters as described previously (24). The stock solution was diluted 6:4 in water and double filtered before use. Cells were washed before staining 2 h with the Oil Red O working solution and then washed with tap water. To measure the lipid accumulation of 3T3-L1 preadipocytes during differentiation, the Oil Red O dye was extracted with isopropyl alcohol by gentle shaking, and its absorbance was measured immediately at 510 nm (25, 26). Microscopy and Digital Image Generation. A D1X digital camera (Nikon) attached to a Microphot FX A upright microscope (Nikon) with a 63 dippable Zeiss objective and a 10 eyepiece was used to take pictures of Oil Red O-stained cells. NikonView5 software was used to transfer images. For pictures of Oil Red O-stained 3T3-L1 adipocytes, a lower magnification objective (40x) was used.

Results

It is generally accepted that the regulation of adipogenesis prevents obesity. However, the mechanisms controlling adipogenesis have not been completely defined in the art.

Expression of Eosinophil-associated ribonuclease A family member (EAR)-genes in adipocyte cells

A strong and early expression of EAR-genes EAR- 1,-2, and- 10 in the course of adipocyte differentiation in 3T3-L1 preadipocytes as well as primary preadipocytes isolated from perigonadal fat pads of C57/BL6 mice (denoted as 'SVF'= stromal vascular fraction) was noted. Preadipocyte differentiation was initiated with a standard cocktail consisting of insulin, dexamethasone, troglitazone and IBMX ('DMIT') and RNA isolated at indicated time points. Ear-mRNA levels were determined by real-time PCR (Figure 1). Sequence analysis of EAR 1,- 2, and -10 showed that primers for EAR1 (according to Phipps et al, 2007) also detect EAR10 and, to a lesser extent, EAR2, whereas primers for EAR2 have only weak homology to EAR1 and EARlO. EAR-Loss of function 3T3-L1 cell model

To investigate a potential adipogenic role of EAR-genes, a stable 3T3-L1 knockdown cell line (termed miEARlO) was established by retroviral transfection of a microRNA adapted vector. In addition to EAR10, this microRNA sequence also delets EAR-1 and -2. Figure 2 demonstrates successful EAR10 knockdown in miEARlO, but not control-infected ("LMP"; cells infected with empty vector) cells. The western blot was generated 36 hours post transfection of LMP and miEARlO cells with a V5-tagged EAR10 plasmid. For detection of EARiO an anti-V5 epitope antibody, which recognizes V5-tagged EAR10 was used to investigate if knock-down of EARIO affects adipocyte differentiation, differentiation of LMP and miEARlO 3T3-L1 cells was induced with a standard cocktail consisting of insulin, dexamethasone, troglitazone and IBMX. At day 7 post-induction with DMIT, cells were fixed and stained with OrO to judge adipocyte content. Figure 3 shows representative photographs of OrO stains of LMP and miEAR cell culture wells as well as microscopic magnifications, in addition to OrO staining, realtime PCR on selected proadipogenic genes was performed on day 0, 2, 3 and 7 of adipocyte differentiation. As shown in Figure 3 (right panel), upon induction with the differentiation cocktail, the expression of PPARy, adiponectin as well as FABP4 (= aP2) was strongly upregulated in LMP control cells, but diminished in cells lacking EAR10 (miEARlO). In addition, real-time PCR of EAR.1,2 and 10 at indicated time points confirmed successful knockdown. The The proadipogenic activity of Ear genes was confirmed through gain-of-function studies, which are described in the following.

EAR10 gain-of function 3T3-L1 cell model

To investigate whether overexpression of EAR10 promotes adipogenesis, V5-tagged Ear 10 was stably overexpressed using retroviral delivery into 3T3-L1 preadipocytes. Thus, V5- tagged EarlO was expressed under the control of a CMV promoter (3T3L1-MSCV-EAR10- V5). As a control, cells were transiently transfected with empty plasmid (3T3L1-MSCV- pcDNA). Total RNA was harvested at 0 hrs and day 3 post differentiation. Realtime-PCR of selected adipogenic genes demonstrated higher mRNA expression levels of PPARg, adiponectin as well as FABP4 in 3T3-L1 cells ectopically expressing EAR10 (Figure 5).

As shown in Fig. 5, EarlO- V5 protein levels were strongly increased in Earl0-V5 transgenic cells relative to cells transduced with control virus (pMSCV). Strikingly, overexpression of Earl0-V5 in these cells markedly increased adipogenesis (Fig. 6), as shown by OrO staining of neutral lipids. Adipocyte markers, such as FABP4/aP2 and adiponectin (Fig 6), were significantly increased in Earl0-V5 overexpressing cells, further confirming a proadipogenic effect.

Proadipogenic function of secreted, extracellular EAR10

It was further investigated if inhibition of endogenous expression of EAR- 1,-2,- 10 with RNAse inhibitor (RNAsin) affects expression of key adipogenic genes in the course of adipocyte differentiation. RNAsin was added at day 0 to the cell culture medium and every 24 hrs thereafter, RNA was harvested at day 0 and day 5 post differentiation and subjected to qPCR (Figure 7). Note that expression of PPARg, CEBP/a, LF15 and FABP4 was significantly lower in cells treated with the RNAse inhibitor.

Example 2.

The above experiments can, mutatis mutandis, be performed using human RNAses and corresponding means and methods. An exemplary protocol for RNAse 1 and inhibitors thereof is provided below.

Materials and Methods

Isolation of human preadipocytes

Subcutaneous adipose tissue is obtained from healthy individuals undergoing iipoaspiration. This study is approved by the ethics committee of the Medical University of Vienna and the General Hospital Vienna (EK no. 1 115/2010). Visceral preadipocytes were purchased from ZenBio (Research Triangle Park, NC, USA). See Figure 8 for an illustration of the isolation procedure. All subjects gave written informed consent before taking part in the study. Minced adipose tissue is washed in phosphate-buffered saline (PBS) and digested with 2 mg/ml collagenase type IV in Hanks' buffered salt solution (both obtained from Sigma Chemical Co., St. Louis, Mo.) supplemented with 50 ^^ηιΐ gentamicm (mvitrogen, Carlsbad, Calif.) for 1 hour at 37°C with constant shaking. Cells are filtered through a 250-μηι nylon filter and centrifuged for 10 minutes at 380 g. Red blood cells are lysed in hypotonic buffer, cells are centrifuged for 10 minutes at 380 g, and cell pellets are resuspended in Dulbecco's Modified Eagle Medium/Ham's F12 (Sigma), supplemented with 2 mM L-glutamine, 50 μ§/πι1 gentamicin (all from Invitrogen), and 10% fetal calf serum (HyClone; Thermo Scientific, Logan, Utah). After filtration through a 70-μηι nylon mesh, cells are incubated at 37°C in a humidified atmosphere with 5% carbon dioxide for 24 to 48 hours. Subsequently, nonadherent cells are removed by washing with phosphate-buffered saline, adherent cells trypsinized and, after washing/centrifugation, resuspended in preadipocyte cultutre medium

Human Adipocyte Differentiation and Oil Red O staining.

To induce adipocyte differentiation, cells are incubated in Dulbecco's Modified Eagle Medium/Ham's F12, 33 μΜ biotin, 17 μΜ pantothenate, 1 nM triiodothyronine, 100 nM dexamethasone (all from Sigma), 500 nM human insulin (Roche, Basel, Switzerland), 1 μΜ troglitazone(Sigma), and, for the first 3 days, 250 μΜ IBMX (Sigma). Differentiation media is replaced after 3 days with fresh differentiation medium (excluding dexamethasone and IBMX) and cells are differentiated for a further 7-10 days.

Oil Red. O Staining and Quantification of LipidAcciimulation was performed as described above for 3T3-L1 cells.

Human siRNA transfection:

Small interfering (si) RNA targeting human RNASE1 (catalog ID: HSS 109255) or negative control siRNA matched for GC-content (oligo ID: 12935-200), both from Invitrogen) are delivered into human primary preadipocytes by nucleofection (cat. No. VPE-1001, Amaxa, Lonza Bioscience) according to manufacturer's recommendations for nucleofection of human mesenchymal stem cells. Briefly, 6 ^χ 10⁵ preadipocytes will be nucleofected with siRNA (100 nmol/L) in the Human MSC Nucleofector Solution and program C-17.

DNA Microarray gene expression profiling

Total RNA was extracted from subconfluent culture using an RNeasy kit (Qiagen). Total RNA (200 ng) was then used for GeneChip analysis. Preparation of terminal-labeled cDNA, hybridization to genome-wide human Gene Level 1.0 ST GeneChips (Affymetrix, Santa Clara, C A, USA) and scanning of the arrays were carried out according to manufacturer's protocols https://www.affymetrix.com. MA Signal extraction, normalization and filtering was performed as described (http://www.bioconductor.org/)

Results

RNASE1 is the predominant RNASE expressed throughout human adipocyte differentiation. Human preadipocytes can readily be isolated for cell cultur from white adipose tissue by a combination of dissection and digestion with collagenase and DNase followed by progressive size filtration and centrifugation steps (summarized in Figure 8). RNASE 1 mRNA levels increase throughout differentiation of preadipocytes isolated form either visceral or subcutaneous human white adipose tissue (Figure 9). To determine which RNASE gene family member is expressed and regulated in human adipocyte differentiation, a DNA microarray screen was performed using RNA extracted from preadipocytes induced to differentiate into mature adipocytes at several time points. Inspection of the normalized signal intensities (reflecting mRNA levels) shows that RNASE1 is expressed in undifferentiated preadipocytes (i.e. day 0), increasing throughout the course of differentiation (Figure 10). Dynamic changes in mRNA expression levels of typical adipocyte genes throughout the time course validates the adipogenic differentiation of our preadipocytes (Figure 0). Fold changes in gene expression (relative to day 0) is depicted in Figure 11. Evaluation of the expression of well-known adipocyte specific genes validates our DNA Microarray screen (Figure 12). Undifferentiated preadipocytes have fibroblast-like morphologies and when induced to undergo terminal differentiation in adipogenic media, preadipocytes change into round cells filled with lipid droplets that can be readily stained with oil-red (Figure 13).

Pro-adipoigenic function of human RNASE1.

To investigate whether RNASE1 affects human adipocyte differentiation, an siRNA targeting human RNASE1 was delivered into human preadipocytes 36 hours before start of differentiation with a standard proadipogenic cocktail (see M&M). Figure 14 demonstrates successful RNASEl mRNA silencing in siRNASEl (ceils treated with a siRNA oligo targeting RNASEl), but not siCtrl (cells treated with a control siRNA) treated SVF cells. The qPCR data show RNASEl mRNA expression on day 7 days following induction with the differentiation cocktail. To investigate if knock-down of RNASE1 affects adipocyte differentiation, differentiation of siCtrl or siRNASEl -treated SVF cells was induced with a standard proadipogenic cocktail. At day 7 post-induction of differentiation, cells were fixed and stained with OrO to judge adipocyte content. Figure 15 shows quantitative staining with ORO, as measured with spectrophotometry. The intensity of ORO staining in RNASE1 knockdown cells (siRNASEl) was significantly diminished by approximately 50% as compared with siCtrl-treated cells, which shows that RNASEl is required for adipocyte differentiation.

The present invention refers to the following nucleotide and amino acid sequences:

The sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants.

Underlined and bold letters within sequences denote miRNA binding site (sense sequence).

SEQ ID No. 1 :

Nucleotide sequence encoding murine EAR1 (member 1 of mus musculus eosinophil- associated, ribonuclease A family); accession number NM_007894. The coding region ranges from nucleotide 69 to nucleotide 536. acattatccctgatttccaggacaaccagccctcagttccacgggagccacaaagcagac

agggaaacatgggtccgaagctgcttgagtcccgactttgtctcctgctgctgctaggac

ttgtcctaatgcttgcctcatgcctgggtcaaaccccttcccagaagtttgcj^Ltcca^

atatcaataataataccaacctccaatgtaatgttgaaatgatgcgtattaacagggcta gaagaacatgtaagggcttaaatacttttcttcatacaagttttgctaatgctgttggtg

tgtgtggaaaiccaagtggcttgtgcagtgacaagagaagtcaaaactgtcataatagtt

catctcgggtacatataactgtctgtaacatcaccagtcgggcaacaaattatacccaat

gcagataccaatcaagaagatcattggagtactacacagttgcctgtgaccccagaactc

cacaggacagtcccatgtatccagtggttccagttcacttggatgggacattttagctcc

agtgccagcactttgcaccttcgctcatctgctactgccgagatcatagtagtgaagaga

caattcctctgtctgc gctccatataacacatgtcctc gtgaaacctgtcacttactccc

tttaattttgctgaatagatcttttctaattaataaatacctttgcatacaaaaaaaaaa

aaaaaaaaaaaaaaaaaaa

SEQ ID No. 2:

Amino acid sequence of murine EARl (mus musculus eosinophil-associated, ribonuclease A family, member 1); accession number NP_031920.

MGPKLLESRLCLLLLLGLVLMLASCLGQTPSQKFAIQFIINNNTNLQCNVEMMRINRA

RRTCKGLNTFLHTSFANAVGVCGNPSGLCSDKRSQNCHNSSSRVHITVCNITSRATNY

TQCRYQSRRSLEYYTVACDPRTPQDSPMYPVVPVHLDGTF

SEQ ID No. 3:

Nucieotide sequence encoding murine EAR2 (member 2 of mus musculus eosinophil- associated, ribonuclease A family); accession number NM_007895, The coding region ranges from nucleotide 72 to nucleotide 542. acacatttatccctgatttccaggacaaccagccctaagttccacgggagccacaaagca

gacagggaaacatgggtccgaagctgcttgagtctcgactttgtctcctgctgctgctag

gacttgtcctaatgcttgcctcatgcctgggacaaaccccttcccagtggtttgcc tcc

a^cataicaataataatgccaacctccaatgtaatgttgaaatgcagcgtattaacaggt

ttagaagaacatgtaagggcttaaatacltttcttcatacaagttttgctaatgctgttg

gtgtgtgtggaaatccaagtggcttgtgcagtgacaatataagtagaaactgtcataata

gttcatctcgggtacgtataactgtctgtaacatcaccagtcggaggagaacaccttata

cccaatgcagataccaaccaagaagatcattggagtactacacagtigcctgtaacccca

gaactccacaggacagtcccatgtatccagtggttccagttcacttggatgggacatttt

agctccagtgccagcactttgcacctttgctcatctgctactgccgagatcatagtagtg aagagacaattcctctgtctgcgctccatatagcacatgtcctcgtgaaacctgtcactt

actccctttaattttgctgaatagatcttttctaattaataaaiacctttgcatac

SEQ ID No. 4;

Amino acid sequence of murine EAR2 (member 2 of mus musculus eosinophil-associated, ribonuclease A family); accession number NPJ331921.

MGPKLLESRLCLLLLLGLVLMLASCLGQTPSQWFAIQHINNNANLQCNVEMQRINRF

RRTCKGLNTFLHTSFANAVGVCGNPSGLCSDNISRNCHNSSSRVRITVCNITSRRRTPY

TQCRYQPRRSLEYYTVACNPRTPQDSPMYPVVPVHLDGTF

SEQ ID No. 5:

Nucleotide sequence encoding murine EAR3; accession number NM_017388. The coding region ranges from nucleotide 1 to nucleotide 471.

ATGGGTCCGAAGCTGCTTGAGTCCCGACTTTGTCTCCTGCTGCTGCTAAGACTTGT

CCTAATGCTTGCCTCATGCCTGGGACAAACCCCTTCCCGGTGGTTTGCCATCCAG

CATATCAATAATAATACCAACCTCCGATGTAATGTTGAAATGCTGCGTATTAACA

GGTTTAGAAGAACATGTAAGGGCTTAAATACTTTTCTTCATACAAGTTTTGCTAAT

GCTGTTGGTGTGTGTGGAAATCCAAGTGGCTTGTGCAGTGACAATATAAGTAGAA

ACTGTCATAATAGTTCATCTCGGGTACATATAACTGTCTGTAACATCACCAGTCGG

AGGAGAATACCTTATACCCAATGCAGATACCAACCAAGAAGATCAGTGGAGTACT

ACACAGTTGCCTGTAATCCCAGAACTTCACTGGACAGTCCCATGTATCCAGTGGTT

CCAGTTCACTTGGATGGGACATTTTAG

SEQ ID No. 6:

Amino acid sequence of murine EAR3; accession number NP_059084.

MGPKLLESRLCLLLLLRLVLMLASCLGQTPSRWFAIQHINNNTNLRCNVEMLRINRFR

RTCKGLNTFLHTSFANAVGVCGNPSGLCSDNISRNCHNSSSRVHITVCNITSRRRIPYT

QCRYQPRRSVEYYTVACNPRTSLDSPMYPVVPVHLDGTF

SEQ ID No. 7:

Nucleotide sequence of murine EAR4; accession number NM_001017422. The coding region ranges from nucleotide 1 to nucleotide 468. ATGGGTCCGAAGCTGCTTGAGTCCCGACTTTGTCTCCTGCTGCTGTTGGGACTTGT

CCTAATGCTTGCCTCATGCCAGGCACAAATCCTTTCCCAGAAGTTTTACACCCAGC

ATATCTATAATAGCACCTACCCCCGATGTGATGCTGTAATGAGGGTTGTTAACAG

GTATAGACCAAGATGTAAGGACATAAATACTTTTCTTCACACAAGTTTTGCTGATG

TTGTTGCTGTGTGTGGCCATCCAAATATCACCTGCAACAACTTGACAAGAAAAAA

TTGTCATGCTAGTTCATTTCAGGTATTTATAACTTTTTGTAACCTCACTATGCCGAC

AAGAATATGCACACAATGCAGATACCAAACGACAGGTTCAGTGAAGTACTACAG

AGTAGCCTGTGAGAACAGAACTCCACAAGACACTCCCATGTATCCAGTGGTTCCA

GTTCACTTGGATGGGACATTTTAG

SEQ ID No. 8:

Amino acid sequence of murine EAR4; accession number NP_001017422.

MGPKLLESRLCLLLLLGLVLMLASCQAQILSQKFYTQHIY STYPRCDAVMRVVNRY RPRC DINTFLHTSFADVVAVCGHPNITCNNLTR NCHASSFQVFITFCNLTMPTRICT QCRYQTTGSVKYYRVACENRTPQDTPMYPVVPVHLDGTF

SEQ ID No. 9:

Nucleotide sequence encoding murine EARS; accession number NM_019398. The coding region ranges from nucleotide 205 to nucleotide 672.

CAGCGCTCAGTTCCACGGGAGCCACAAAGCAGACTGGGTAAGACACTGACTGTCT

GGGCCTGGGACAGGAGGGGCAGTGATGGGACAGTGTCCAGGAGAAGTCCCACCG

TGGACCCACAGTGACAGCAGAGGGCCTTCAGGAAGAGATATGGAAAACTGAGGT

AGGGTGGAAGTAGTCACTTCTCTTCTGTCCTCACAGGAAACATGGGTCTGAAGCT

GCTTGAATCCAGACTTTGTCTCCTGCTGCTGCTGGGACTTGTCCTAACGCTTGTCTC

ATGCCAGCGACCAACCCCTTCCCAGAAGTTTGACATCCAGCATATCTATAAGAAA

TCCTCTCCCAAATGTGATGATGCAATGCGGGTCGTTAACAAGTATACAGGAAAAT

GTAAGGACTTGAATACTTTTCTTCATACAACTTTTGCTGATGTTGTCCGTGTGTGTC

AC A ATC C ACC C A AG ACTTGC A A AG AC GGGAC A AG TCC A A ATTGTC ATG AT AGTTC

ATCTAAGGTATCTGTAACTATCTGTAAACTCACAAAACGGGCAAGGAATTATACC

CACTGCAGATACAAAACAACAGGAGCAAAGAAGTCCTACACAGTTGCCTGTAACC

CCAGAACTCCAAAGGACCGTCCCACCTATCCAGTAGTTCCGGTTCACTTGGATCG

GCTATTTTAGCTCCAGTGCCAGCACTTTGTACCTTCCCTCATCTGCTACTGCCGAG

ATCATAGTAGTGAAGAACGAATTCCTCTGTCTGCGCTGCAT

SEQ ID No. 10:

Amino acid sequence of murine EARS; accession number NP_062271. MGLKLLESRLCLLLLLGLVLTLVSCQRPTPSQ FDIQHIYK SSPKCDDAMRVVNKYT

G CKDLNTFLHTTFADVVRVCHNPPKTC DGTSPNCHDSSSKVSVTICKLT RARNY

THC YKTTGA SYTVACNPRTPKDRPTYPVVPVHLDRLF

SEQ ID No. 1 1 :

Nucleotide sequence encoding murine EAR6; accession number NM_053111. The coding region ranges from nucleotide 68 to nucleotide 535,

CATTATCCCTGATTTCCAGGACAACCAGTGCTCAGTTCCACGGGAGCCACAACAC

AGACTGGGAAACATGGGTCCGAAGCTGCTTGAGTCCCAACTTTGTCTCCTGCTGAT

GCTGGGACTTGTCCTAATGCTTGCCTCATGCCAGAAACCAACCGCATCCCAGTGGT

TTGCTACCCAGCATATCACTTATAAAGCCAACCTCCAATGTAATGTTGAAATGCAG

GCTATTAACATGCATAGACCAAGATGTAAGGGCTTAAATACTTTTCTTCATACAAG

TTTTATTAATGTTGTTGGTGTGTGTAGCAATCCAAGTGGCTTGTGCAGCGACAAAA

TAAGTCAAAACTGTCATAATAGTTCGTCTCGGGTACCTATAACTGTCTGTAACCTC

ACAACTCCGAGAAGAAATTATACCCAATGCAGATACCAAACAAAGGGATCAGTG

GAGTACTACACAGTTGCCTGTGAGCCCAGAGTTGCATGGGATTGTCCCATCTATCC

AGTGGTTCCAGTTCACTTGGATGGGACATTTTAGCTCCAGTGCCAGCACTTTGTAC

CTTCGATCATCTGCTACTGCCAAGATCATAGTAGTGAAGAACCAATTCCTCTGTCT

GCGCTGCATATCAGCACCTGTCCTCATGAAACCTGTCCCTGACTCCCTFTAACTCA

GCTGAATAGATCTTTTCTAATTAATAAACACATTTGCATACAAAAAAAAAAAAAA

AAAAAAAAAAAAAA

SEQ ID No. 12:

Amino acid sequence of murine EAR6; accession number NP_444341.1

MGPKLLESQLCLLLMLGLVLMLASCQKPTASQWFATQHITYKANLQCNVEMQAINM HRPRC GLNTFLHTSFINVVGVCSNPSGLCSDKISQNCHNSSSRVPITVCNLTTPRRNY TQ CR YQT G S VE Y YT V ACEPRV A WDCPI YP V VP VHLDGTF

SEQ ID No. 13: Nucleotide sequence encoding murine EAR7; accession number N _017385 (Ear7 is not annotated in NCBI i.e. the EAR7 protein is identical to the EAR6 protein). The coding region ranges from nucleotide 1 to nucleotide 468.

ATGGGTCCGAAGCTACTTGAGTCCCAACTTTGTCTCCTGCTGATGCTGGGACTTGT

CCTAATGCTTGCCTCATGCCAGCGACCAACCGCATCCCAGTGGTTTGCCACCCAGC

ATATCACTTATAAAGCCAACCTCCAATGTAATGTTGAAATGCAGGCTATTAACAT

GCATAGACCAAGATGTAAGGGCTTAAATACTTTTCTTCATACAAGTTTTATTAATG

TTGTTGGTGTGTGTGGCAATCCAAGTGGCTTGTGCAGCGACAAAATAAGTCAAAA

CTGTCATAATAGTTCATCTCGGGTACCTATAACTGTCTGTAACCTCACAACTCCGG

GAAGAAATTATACCCAATGCAGATACCAAACAAAGGGATCAGTGGAGTACTACA

CAGTTGCCTGTGAGCCCAGAGTTGCATGGGATTGTCCCATCTATCCAGTGGTTCCA

GTTCACTTGGATGGGACATTTTAG

SEQ ID No. 14:

Amino acid sequence of murine EAR?; accession number NP_059081.

GP LLESQLCLLLMLGLVL LASCQRPTASQWFATQHITYKANLQCNVEMQAINM HRPRCKGLNTFLHTSFINVVGVCGNPSGLCSD ISQNCHNSSSRVPITVCNLTTPGRNY TQCRYQT GSVEYYTVACEPRVAWDCPIYPVVPVHLDGTF

SEQ ID No. 15:

Nucleotide sequence encoding murine EAR 10 (member of mus museums eosinophil- associated, ribonuclease A family); accession number NM_0531 12. The coding region ranges from nucleotide 1 to nucleotide 471. atgggtccaaagctgcttgagtcccgaatttgcctcctgctgctgctaggacttgtccta

atgcttgcctcatgcctggaacaaaccacttcccagtggtttaccatccagcatatcaat

aataatgccaacctccaatgtaatgttgaaatgcagcgtattaacaggtttagaagaaca

tgtaagggcttaaatacttttcttcatacaagttttgctaatgctgttggtgtgtgtgga

aatccaagtggcttgtgcagtgacaatataagtcaaaactgtcataatagttcatatcgg

gtacatataactgtctgtaacatcaccagitggaggagaacaccttatacccaatgcaga

taccaagctaaaagatcattggagtactacacagttgcctgtgaccccagaactccacag

gacagtcccatgtatccagtggttccagttcacttggatgggacattttag SEQ ID No. 16:

Amino acid sequence of murine EAR10 (member of mus muscuius eosinophil-associated, ribonuclease A family); accession number NP_444342, GP LI.ESRiCLLIJ GLVLMLASCLGQTTSQWFAIQHINNNANLQCNVEMQRINRFR

RTC GLNTFLHTSFANAVGVCGNPSGLCSDNISQNCHNSSYRVHITVCNITSWRRTPY

TQCRYQAKRSLEYYTVACDPRTPQDSPMYPVVPVHLDGTF

SEQ ID No. 17:

Nucleotide sequence encoding murine EARl l ; accession number NMJ3531 13. The coding region ranges from nucleotide 46 to nucleotide 513.

AGCCAGCGCTCAGTTCCACGGGAGCCACAAAGCAGACTGGGAAACATGGGTCTG

GAGCAACTTGAGTCTCGACTTTGTCTCCTGCTGCTGCTGGGACATGTCCTAATGCT

TGCCTCATGCCAGCCATTGACCCCCTCCCGGTGGTTTGACATCCAGCATATCTATA

ACAGAGCCTATCCCCGATGTGATGATGCAATGCGGGCCGTTAACAGTTACACAGG

AGTGTGTAAAGACATAAATACTTTrCTTCATACAACTTTTGCTAATGTTGTCCGTG

TGTGTCATAATCCACGTAAGATCTGCAAAAATGGGATAAGTAGAAATTGTCATGA

TAGTTCAAATCGGGTACAAGTAACTATCTGTATACTCACAACTCCGGCCAGTCATT

ATTCCAACTGCAGATACCGAACAACAAGATCAATGAAGTACTACACAGTTGCCTG

TGAC C CC AG A ACTCCTC AGG AC AGTC C C ATGT ATCC AGTGGTTC C AGTTC ACTTGG

ATGGGATATTTTAGCTCCAGTGCCAGCACTTTGTACCTTCATTCATCGGCTACTGC

CAAGATCATAGTTGGGAAGAACCGAGTCCTCTGTTTGCACTGCATGTCAGCATCT

GTCCTCATGAAACCTGTTGCTGACTCCCTTTAATTTAGCTGAACAAAAAGTTTCTA

ATCAATAAACACTTTTGCACACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

A. A.

SEQ ID No. 18:

Amino acid sequence of murine EAR1 1 ; accession number NP_444343.

MGLEQLESRLCLLLLLGHVL LASCQPLTPSRWFDIQHIYNRAYPRCDDAMRAVNSY TGVCKDINTFLHTTFA VVRVCHNPRKICKNGISRNCHDSSNRVQVTICILTTPASHYS NCRYRTTRSM YYTVACDPRTPQDSPMYPVVPVHLDGIF

SEQ ID No. 19: Nucleotide sequence encoding murine EAR12 (member of raus musculus eosinophil- associated, ribonuclease A family); accession number NM_001012766. The coding region ranges from nucleotide 1 to nucleotide 471.

atgggtccgaagctgcttgagtcccgactttgtctcctgttgttgctaggacttgtccta

atgcttgcctcatgcctgggacaaaccccttcccagtggtttgccatccagcatatcaat

aataatgccaacctccaatgtaatgttgaaatgctgcgtattaacaggtttagaagaaca

tgtaagggcttaaatacttttcttcatacaagttttgctaatgctgttggtgtgtgtgga

aatccaagtggcttgtgcagtgacaatataagtcaaaactgtcataatagttcatctcgg

gtacatataactgtctgtagcatcaccagtcggagaagaacaccttatacccaatgcaga

taccaaccaagaagatcagtggagtactacacagttgcctgtaatcccagaacttcactg

gacagtcccatgtatccagtggttccagttcacttggatgggacattttag

SEQ ID No. 20:

Amino acid sequence of murine EAR12 (member of mus musculus eosinophil- associated, ribonuclease A family); accession number NP_001012784

MGP LLESRLCLLLLLGLVLMLASCLGQTPSQWFAIQHIN NANLQCNVEMLRINRFR

RTCKGLNTFLHTSFANAVGVCGNPSGLCSDNISQNCHNSSSRVHITVCSITSRRRTPYT

QCRYQPRRSVEYYTVACNPRTSLDSPMYPVVPVHLDGTF

SEQ ID No. 21 :

Nucleotide sequence encoding murine EAR14; accession number NM_017389. The coding region ranges from nucleotide 1 to nucleotide 462

ATGAAGCTGCCTGAGTCCAGTCTTTGTCTCCTGCTGCTGTTGGGACTTGTCCTAAT

GCTTGCCTCATGCCAGGCACAAATCCTTTCCCAGAAGTTTTACACCGAGTATATCT

ATAATAGCACCTACCCCCGATGTGATGCTGTAATGAGGGTTGTTAACAGGTATAG

ACCAAGATGTAAGGACATAAATACTTTTCTTCACACAAGTTTTGCTGATGTTGTTG

CTGTGTGTGGCCATCCAAATATCACCTGCAACAACTTGACAAGAAAAAATTGTCA

TGCTAGTTCAT1TCAGGTATTTATAACTTTTTGTAACCTCACTACGCCGACAAGAA

TATGCACACAATGCAGATACCAAACGACAGGTTCAGTGAAGTACTACAGAGTAGC CTGTGAGAACAGAACTCCACAAGACACTCCCATCTATCCAGTGGTTCCAGTTCACT TGGATGGGACATTTTAG

SEQ ID No. 22:

Amino acid sequence of murine EAR14; accession number NP_059085

MKLPESSLCLLLLLGLVLMLASCQAQILSQKFYTEYIYNSTYPRCDAVMRVVNRYRPR

CKDINTFLHTSFADVVAVCGHPNITCNNLTR NCHASSFQVFITFCNLTTPTRICTQCR

YQTTGSVKYYRVACENRTPQDTPIYPVVPVHLDGTF

SEQ ID No. 23:

Nucleotide sequence encoding human RNASEl (homo sapiens ribonuclease, member 1 of RNase A family, (pancreatic)); accession number NM__198232. The coding region ranges from nucleotide 107 to nucleotide 577.

GTATAAGGTCCACACCCCGGGAGCTGAGTGATTGCAGAAACTGGCCTTCCATCTC

TCTCAGACACCAAGCTGCAGATCCAGGCTTTTCTGGGAAAGTGAGGCCACCATGG

CTCTGGAGAAGTCTCTTGTCCGGCTCCTTCTGCTTGTCCTGATACTGCTGGTGCTG

GGCTGGGTCCAGCCTTCCCTGGGCAAGGAATCCCGGGCCAAGAAATTCCAGCGGC

AGCATATGGACTCAGACAGTTCCCCCAGCAGCAGCTCCACCTACTGTAACCAAAT

GATGAGGCGCCGGAATATGACACAGGGGCGGTGCAAACCAGTGAACACCTTTGT

GCACGAGCCCCTGGTAGATGTCCAGAATGTCTGTTTCCAGGAAAAGGTCACCTGC

AAGAACGGGCAGGGCAACTGCTACAAGAGCAACTCCAGCATGCACATCACAGAC

TGCCGCCTGACAAACGGCTCCAGGTACCCCAACTGTGCATACCGGACCAGCCCGA

AGGAGAGACACATCATTGTGGCCTGTGAAGGGAGCCCATATGTGCCAGTCCACTT

TGATGCTTCTGTGGAGGACTCTACCTAAGGTCAGAGCAGCGAGATACCCCACCTC

CCTCAACCTCATCCTCTCCACAGCTGCCTCTTCCCTCTTCCTTCCCTGCTGTGAAAG

AAGTAACTACAGTTAGGGCTCCTATTCAACACACACATGCTTCCCTTTCCTGAGTC

CCATCCCTGCGTGATTTTGGGGGTGAAGAGTGGGTTGTGAGGTGGGCCCCATGTT

AACCCCTCCACTCTTTCTTTCAATAAAACGCAGTTGCAAACACCTGAA

SEQ ID No. 24:

Amino acid sequence of human RNASEl (homo sapiens ribonuclease, member 1 of RNase A family, (pancreatic)); accession number NP_002924. MALEKSLVRLLIXVLILLVLGWVQPSLGKESRA KFQRQHMDSDSSPSSSSTYCNQM MRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTC NGQGNCYKSNSSMHITDC RLTOGSRYPNCAYRTSP ERHIIVACEGSPYVPVHFDASVEDST

SEQ ID No. 25:

Nucleotide sequence encoding human RNASE2 (homo sapiens ribonuclease, member 2 of RNase A family, (liver, eosinophil-derived neurotoxin)); accession number NMJ302934. The coding region ranges from nucleotide 72 to nucleotide 557.

GCTGCCCCTGAACCCCAGAACAACCAGCTGGATCAGTTCTCACAGGAGCTACAGC

GCGGAGACTGGGAAACATGGTTCCAAAACTGTTCACTTCCCAAATTTGTCTGCTTC

TTCTGTTGGGGCTTCTGGCTGTGGAGGGCTCACTCCATGTCAAACCTCCACAGTTT

ACCTGGGCTCAATGGTTTGAAACCCAGCACATCAATATGACCTCCCAGCAATGCA

CCAATGCAATGCAGGTCATTAACAATTATCAACGGCGATGCAAAAACCAAAATAC

TTTCCTTCTTACAACTTTTGCTAACGTAGTTAATGTTTGTGGTAACCCAAATATGAC

CTGTCCTAGTAACAAAACTCGCAAAAATTGTCACCACAGTGGAAGCCAGGTGCCT

TTAATCCACTGTAACCTCACAACTCCAAGTCCACAGAATATTTCAAACTGCAGGTA

TGCGCAGACACCAGCAAACATGTTCTATATAGTTGCATGTGACAACAGAGATCAA

CGACGAGACCCTCCACAGTATCCGGTGGTTCCAGTTCACCTGGATAGAATCATCT

AAGCTCCTGTATCAGCACTCCTCATCATCACTCATCTGCCAAGCTCCTCAATCATA

GCCAAGATCCCATCTCTCCATATACTTTGGGTATCAGCATCTGTCCTCATCAGTCT

CCATACCCCTTCAGCTTTCCTGAGCTGAAGTGCCTTGTGAACCCTGCAATAAACTG

CTTTGCAAATTC

SEQ ID No. 26:

Amino acid sequence of human RNASE2 (homo sapiens ribonuclease, member 2 of RNase A family, (liver, eosinophil-derived neurotoxin)); accession number NP_002925.

MVPKLFTSQICLLLLLGLLAVEGSLHV PPQFTWAQWFETQHINMTSQQCTNAMQVI

NNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKTR NCHHSGSQVPLIHCNLTT

PSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRII

SEQ ID No. 27: Nucleotide sequence encoding human RNASE3 (homo sapiens ribonuclease, member 3 of RNase A family (eosinophil cationic protein)); accession number NM_002935. The coding region ranges from nucleotide 55 to nucleotide 537.

GAACAACCAGCTGGATCAGTTCTCACAGGAGCCACAGCTCAGAGACTGGGAAAC

ATGGTTCCAAAACTGTTCACTirCCAAATTTGTCTGCTTCTTCTGTTGGGGCTTATG

GGTGTGGAGGGCTCACTCCATGCCAGACCCCCACAGTTTACGAGGGCTCAGTGGT

TTGCCATCCAGCACATCAGTCTGAACCCCCCTCGATGCACCATTGCAATGCGGGC

A ATTA AC A ATT ATCG ATGGC GTTGC A A A A ACC A AA AT AC TTTTC TTC GT AC A ACTT

TTGCTAATGTAGTTAATGTTTGTGGTAACCAAAGTATACGCTGCCCTCATAACAGA

ACTCTCAACAATTGTCATCGGAGTAGATTCCGGGTGCCTTTACTCCACTGTGACCT

CATAAATCCAGGTGCACAGAATATTTCAAACTGCACGTATGCAGACAGACCAGGA

AGGAGGTTCTATGTAGTTGCATGTGACAACAGAGATCCACGGGATTCTCCACGGT

ATCCTGTGGTTCCAGTTCACCTGGATACCACCATCTAAGCTCCTGTATCAGCAGTC

CTCATCATCACTCATCTGCCAAGCTCCTCAATCATAGCCAAGATCCCATCCCTCCA

TGTACTCTGGGTATCAGCAACTGTCCTCATCAGTCTCCATACCCCTTCAGCTTTCCT

GAGCTGAAGTCCCTTGTGAACCCTGCAATAAACTGCTTTGCAAATTCA

SEQ ID No. 28:

Amino acid sequence of human RNASE3 (homo sapiens ribonuclease, member 3 of RNase A family (eosinophil cationic protein)); accession number NP_002926.

MVPKLFTSQICLLLLLGLMGVEGSLHARPPQFTRAQWFAIQHISLNPPRCTIAMRAINN YR RC NQNTFLRTTFANVVNVCGNQSIRCPHNRTLNNCHRSRFRVPLLHCDLINPG AQN1SNCTYADRPGRRFYVVACDNRDPRDSPRYPVVPVHLDTTI

SEQ ID No. 29:

Nucleotide sequence encoding human RNASE4/5 (homo sapiens angiogenin, member 5 of ribonuclease, RNase A family); accession number NM_00U45. The coding region ranges from nucleotide 121 to nucleotide 564.

TCCAGGTTCACACAACTGGAACCCATCTCCAGGAACAAACAGCTGGAACCCATCT

CCCGTTGAAGGGAAACTGCCAGATTTTTGTAAGATTCTTCCTCCTGGGAGCCTGTG

TTGGAAGAGATGGTGATGGGCCTGGGCGTTTTGTTGTTGGTCTTCGTGCTGGGTCT

GGGTCTGACCCCACCGACCCTGGCTCAGGATAACTCCAGGTACACACACTTCCTG

ACCCAGCACTATGATGCCAAACCACAGGGCCGGGATGACAGATACTGTGAAAGC

ATCATGAGGAGACGGGGCCTGACCTCACCCTGCAAAGACATCAACACATTTATTC

ATGGC AACAAGCGCAGCATCAAGGCCATCTGTGAAAACAAGAATGGAAACCCTC

ACAGAGAAAACCTAAGAATAAGCAAGTCTTCTTTCCAGGTCACCACTTGCAAGCT ACATGGAGGTTCCCCCTGGCCTCCATGCCAGTACCGAGCCACAGCGGGGTTCAGA

AACGTTGTTGTTGCTTGTGAAAATGGCTTACCTGTCCACTTGGATCAGTCAATTTT

CCGTCGTCCGTAACCAGCGGGCCCCTGGTCAAGTGCTGGCTCTGCTGTCCTTGCCT

TCCATTTCCCCTCTGCACCCAGAACAGTGGTGGCAACATTCATTGCCAAGGGCCCA

AAGAAAGAGCTACCTGGACCTTTTGTTTTCTGTTTGACAACATGTTTAATAAATAA

AAATGTCTTGATATCAGTAAGAA

SEQ ID No. 30:

Amino acid sequence of human RNASE4/5 (homo sapiens angiogenin, member 5 of ribonuclease, RNase A family); accession number NP_0G 1091046.

MVMGLGVLLLVFVLGLGLTPPTLAQDNSRYTHFLTQHYDA PQGRDDRYCESIMRR

RGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRIS SSFQVTTCKLHGGSPWPPC

QYRATAGFRNVVVACENGLPVHLDQSIFRRP

SEQ ID No. 31 :

Nucleotide sequence encoding human RNASE6 (homo sapiens ribonuclease, member k6 of RNase A family); accession number NM_005615. The coding region ranges from nucleotide 294 to nucleotide 746.

GTTTTCAGTCATGTTGTTTCTTTAAGTTGGTTTAGCCTTCCTGTATACATGTGATAA

TGATACTGTTTTAAAGAACAGGTGTGAAAGTCTAAGAGTTTAATCCAGACTCTGC

ACAAGAAGTTAATCTGCCAAACCCTAGGATTCTCCTTCTGAAAGCTCAGAATATTT

CTTC C C CTTC CCT AT AC AC AC AGGGCTCG AAGGGTT AGAG A A G A A AGGC AGAC A A

CAGCTTCTGAGCTTTGGACTAATCACAGCCTCTGTTCTCAGCAGGAGCCCCAACAC

TGAGACCAGAAAAGATGGTGCTATGCTTTCCTCTTCTTTTACTGCTGCTGGTTCTA

TGGGGACCAGTGTGTCCACTTCATGCTTGGCCTAAGCGTCTCACCAAGGCTCACTG

GTTTGAAATTCAGCATATACAGCCAAGTCCTCTCCAATGCAACAGGGCAATGAGT

GGCATCAACAATTATACCCAGCACTGTAAGCATCAAAATACCTTTCTGCATGACTC

TTTCCAGAATGTGGCTGCTGTCTGTGATTTGCTCAGCATTGTCTGCAAAAATCGTC

GGCACAACTGCCACCAGAGCTCAAAGCCTGTCAACATGACTGACTGCAGACTCAC

TTC AGG A A AGTATC C CC AGTGCC GCT AT AGTGCTGCTGCCC AGTAC A A ATTCTTC A

TTGTTGCCTGTGACCCCCCTCAGAAGAGCGATCCCCCCTACAAGTTGGTTCCTGTA

CACTTAGATAGTATTCTCTAAGGCATCCACTGCATTTCCTTTCATTTGACATAGCTT

CTGTTACAATTGCATCCATGTTTTCTTTTCTTTTGTTGATTTTCTTGTTCCCGTAGAA

GAAAGAAGAAAGGTGTTTGGAGAATTCGAGTGCCTAGGATGCCAGACCAGAGTT

GAGACAAAAAGAAATAAGATTATTTTCTGCTTTGTAGTTCTGTACTTTTCGAGAGA

AGGGAATAGGGAAGACAGCAAAGAAAGATTCAGATTTCTAACCCTGCAACTTTTG

CCAAGCTTTATTGCCCTGTGTTCACAGCAATAAAACCACTTCCTGCTGCATTCTAA

AAAA SEQ ID No. 32:

Amino acid sequence of human RNASE6 (homo sapiens ribonuclease, member k6 of RNase A family); accession number NP_005606.

MVLCFPLLLLLLVLWGPVCPLHAWP RLT AHWFEIQHIQPSPLQCNRAMSGINNYT QHCKHQNTFLHDSFQNVAAVCDLLSIVCKNRREINCHQSSKPVNMTDCRLTSGKYPQ CRYS A A AQ YKFFI V ACDPP QKS DPP YKL VP VH LD S ί L

SEQ ID No. 33:

Nucleotide sequence encoding human RNASE7 (homo sapiens ribonuclease, member 7 of RNase A family); accession number NM_032572. The coding region ranges from nucleotide 258 to nucleotide 728.

GTATTTGGGACATGTAAGTGGAGAGGCATGAACCTGATTCATTTCCTGATCCAGT

GATGCTCCCAGCCCACCCCCAAACAGACACAGCGTAGCCCGGGCCAGCTCTTAAG

GAGTTCAGGAGTGAGAAGAGGCCCTCAGAGATCTGACAGCCTAGGAGTGCGTGG

AC ACC AC CTC AGCCC ACTG AGC AGG AGTC AC AGC AC G A AGAC C A AGCGC A A AGC

GACCCCTGCCCTCCATCCTGACTGCTCCTCCTAAGAGAGATGGCACCGGCCAGAG

CAGGATTCTGCCCCCTTCTGCTGCTTCTGCTGCTGGGGCTGTGGGTGGCAGAGATC

CCAGTCAGTGCCAAGCCCAAGGGCATGACCTCATCACAGTGGTTTAAAATTCAGC

AC ATGC AGC C C AGC C CTC A AGC ATGC A ACTC AGC CATG A A A A AC ATT A AC A AGC A

CACAAAACGGTGCAAAGACCTCAACACCTTCCTGCACGAGCCTTTCTCCAGTGTG

GCCGCCACCTGCCAGACCCCCAAAATAGCCTGCAAGAATGGCGATAAAAACTGCC

ACCAGAGCCACGGGGCCGTGTCCCTGACCATGTGTAAGCTCACCTCAGGGAAGTA

TCCGAACTGCAGGTACAAAGAGAAGCGACAGAACAAGTCTTACGTAGTGGCCTGT

AAGCCTCCCCAGAAAAAGGACTCTCAGCAATTCCACCTGGTTCCTGTACACTTGG

ACAGAGTCCTTTAGGTTTCCAGACTGGCTTGCTCTTTGGCTGACCTTCAATTCCCTC

TCCAGGACTCCGCACCACTCCCCTACACCCAGAGCATTCTCTTCCCCTCATCTCTT

GGGGCTGTTCCTGGTTCAGCCTCTGCTGGGAGGCTGAAGCTGACACTCTGGTGAG

CTGAGC TCTA G AGGG ATGGCTTTTC ATCTTTTTGTTGCTGTTTTC CC AG ATGCTTAT

CCCCAAGAAACAGCAAGCTCAGGTCTGTGGGTTCCCTGGTCTATGCCATTGCACA

TGTCTCCCCTGCCCCCTGGCATTAGGGCAGCATGACAAGGAGAGGAAATAAATGG

AAAGGGGGCATATGGGATTTGTGGACACAGCTGTTTCTGTTCCTGAACTAGAAGT

CTTCCCCAGCTCTGACGTGGCAGTGAGGTGACCTGAAGGAAAGAAAAATATAAAT

AAATACCACTTCATATTTGTATAGAATCCTCTAATCCCTTGTGACATAGACTTGAC

AGGGATTGTATGCCTTCTTTATGGATGAGGAAATTAAGGTTTTAGAAAGCTTAATG

AATTAAAGAGCTTGTCTAATTAGTTAGTAGCAGAACCTGGACTTGAACCTAGGTC

TCCTTGCTCTAAATACAGTGTACCTTCTACTCTACCAGTTGCGCAAGAAAGAAGTC ACTGTTACAGAGGCAAGCGGTGAACTAGGTAAGAGTTCACTCATGAAGAAACGA

GTGCTCTGAAGAGCCAGTTACCCTGTGTTGGCTGCAATAAAGGTCATTACCTCTCT

AGCCAAAAAAAAAAAAAAA

SEQ ID No. 34:

Amino acid sequence of human RNASE7 (homo sapiens ribonuciease, member 7 of RNase A family); accession number NP__115961 APARAGFCPLLLLLLLGLWVAEIPVSAKPKGMTSSQWF IQHMQPSPQACNSAMK NINKHTKRCKDLNTFLHEPFSSVAATCQTP IACKNGDKNCHQSHGAVSLT CKLTS GKYPNCRYKE RQNKSYVVAC PPQKKDSQQFHLVPVHLDRVL

SEQ ID No. 35:

Nucleotide sequence encoding human RNASE8 (homo sapiens ribonuciease, member 8 of RNase A family); accession number NM_138331. The coding region ranges from nucleotide Ito nucleotide 465.

ATGGCACCGGCCAGAGCAGGATGCTGCCCCCTGCTGCTGCTGCTTCTGGGGCTGT

GGGTGGCAGAGGTCCTAGTCAGAGCCAAGCCCAAGGACATGACATCATCTCAGTG

GTTTAAAACTCAGCATGTGCAGCCCAGCCCTCAAGCATGCAACTCAGCCATGAGC

ATCATCAATAAGTACACAGAACGGTGCAAAGACCTCAACACCTTCCTGCACGAGC

CCTTCTCCAGTGTGGCCATCACCTGCCAGACCCCCAACATAGCCTGCAAGAATAG

CTGTAAAAACTGCCACCAGAGCCACGGGCCCATGTCCCTGACCATGGGTGAGCTC

ACCTCAGGGAAGTACCCAAACTGCAGGTACAAAGAGAAGCACCTGAACACACCT

T AC AT AGTGGC CTGTG ACCCTC C AC A AC AGGGTGAC C C AGGGT ACCC ACTTGTTC

CTGTGCACTTGGATAAAGTTGTCTAA

SEQ ID No. 36:

Amino acid sequence of human RNASE8 (homo sapiens ribonuciease, member 8 of RNase A family); accession number NP_612204.

MAPARAGCCPLLLLLLGLWVAEVLVRAKP DMTSSQWFKTQHVQPSPQACNSAMSII NKYTERCKDLNTFLHEPFSSVAITCQTPNIAC NSCKNCHQSHGPMSLTMGELTSGKY PNCRYKEKHLNTPYIVACDPPQQGDPGYPLVPVHLDKVV SEQ ID No. 37:

Nucleotide sequence encoding human RNASE9 (homo sapiens ribonuclease, member 9 of RNase A family (non-active, i.e. presumably no RNAse activity) accession number NM_001 1 10359. The coding region ranges from nucleotide 108 to nucleotide 725.

GCTCTACCTCTTGGACTTCCAACTTCTGTGAGTCAACATAGTGCCTCTTGCATTTA

AGAAACATCTCATCACACTCTTATCAGCATTGAGTTTTGTCTGCAGGAAAAATGAT

GAGAACTCTCATCACCACACACCCACTGCCCCTGCTTCTATTGCCGCAGCAGCTGC

TGCAGCTGGTGCAGTTTCAAGAGGTGGATACAGATTT GATTTCCCAGAAGAAGA

TAAAAAAGAAGAATTTGAAGAGTGTTTGGAAAAATTTTTTAGTACAGGGCCCGCC

AGACCACCTACCAAAGAAAAAGTCAAAAGACGTGTCCTTATTGAACCTGGAATGC

CACTAAATCATATAGAGTACTGTAACCATGAAATCATGGGAAAAAATGTTTACTA

CAAACACCGTTGGGTGGCAGAACATTACTTCCTTCTTATGCAATATGACGAGCTCC

AAAAAATCTGTTACAACAGAT TGTGCCATGTAAGAATGGAATTAGGAAATGTAA

CAGGAGCAAAGGTCTTGTAGAAGGAGTGTATTGTAATT AACAGAAGCATTTGAA

ATACCAGCGTGTAAATACGAATCACTTTATAGGAAGGGCTACGTCCTTATCACTTG

TTCATGGCAAAATGAAATGCAAAAACGTATTCCTCATACTATAAATGATCTCGTG

GAGCCACCTGAACACAGAAGTTTCCTCAGTGAGGATGGTGTCTTTGTCATATCGCC

CTAGCAGAGCGTTCTCTGAGAGCTTAGGGTGGGAGGAATATTTCTCTCCATACTG

ATCCTTATAGATCAAAGTTGAGAATGGTAATTTTGCTTCCATTCACTTTAATCACC

CACCTTCCTTTCTTCCTTTCCCAACACCTCACAAAAGCACCTTAGGCTAGGTTCAC

AGACGGTGCTCTGAGATGTGGTCTCATGCAGGTCCCCTAAGTTAGCATGCCTTGA

ATGTGT AC GG AT AC AG ATGCCTGC AGTGGA AGG ATG AC C AGC ACC A AG AGG AA

AAGGAAATGGTTGCCTGAAGTGTGCTCCAAAGACACATCCATGTGATTCTCTAGT

TCAGCAGAAAGAATAAAAATTGCTTCAATCTAAAAAAAAAAAAAAAAAAAA

SEQ ID No. 38:

Amino acid sequence of human R ASE9 (homo sapiens ribonuclease, member 9 of RNase A family (non-active; i.e. presumably no RNAse activity); accession number NP_001 103827

M RTLITTHPLPLLLLPQQLLQLVQFQEVDTDFDFPEED KEEFEECLE FFSTGPARP PTKEKVKRRVLIEPGMPLNHIEYCNHEIMG NVYYKHRWVAEHYFLLMQYDELQKI CYNRFVPC NGIR C RS GLVEGVYCNLTEAFEIPACKYESLYRKGYVLITCS QNE MQKPJPHTINDLVEPPEHRSFLSEDGVFVISP

SEQ ID No. 39: Nucleotide sequence encoding human RNASEIO (homo sapiens ribonuclease, member 10 of RNase A family (non-active; i.e. presumably no RNAse activity); accession number NM_001012975. The coding region ranges from nucleotide 1 to nucleotide 651.

ATGAAGCTGAATCTGGTGCAGATCTTTTTCATGTTGCTGATGCTGCTGCTGGGCCT

GGGGATGGGCCTGGGGTTGGGACTTCATATGGCTACAGCAGTCTTGGAGGAGAGT

GATCAACCGCTCAATGAATTTTGGTCCAGTGACTCACAGGACAAAGCTGAGGCCA

CTGAGGAGGGAGACGGCACCCAAACCACAGAAACGCTGGTGCTTAGCAACAAAG

AAGTGGTGCAACCTGGCTGGCCAGAAGATCCCATCCTCGGTGAAGATGAGGTTGG

GGGTAACAAGATGCTCAGAGCCTCAGCTCTCTTTCAGAGCAACAAAGACTATCTT

AGGCTTGACCAGACAGATAGAGAATGCAATGATATGATGGCACACAAGATGAAG

GAGCCCAGTCAGAGTTGCATAGCCCAGTATGCATTCATCCATGAGGATCTAAACA

CAGTCAAAGCTGTCTGTAACAGTCCTGTCATTGCCTGTGAGCTCAAGGGGGGAAA

ATGTCACAAAAGCTCCCGACCTTTTGATTTGACATTGTGCGAGCTGTCCCAACCAG

ACCAGGTCACTCCTAACTGCAATTACCTAACTTCTGTTATAAAAAAGCACATTATT

ATAACCTGTAATGACATGAAGCGCCAGTTACCAACTGGACAATGA

SEQ ID No. 40:

Amino acid sequence of human RNASEIO (homo sapiens ribonuclease, RNase A family, 10 (non-active, i.e. presumably no RNAse activity); accession number NP_001012993.

MKLNLVQIFFMLLMLLLGLGMGLGLGLHMATAVLEESDQPLNEF SSDSQDKAEAT EEGDGTQTTETLVLSN EVVQPGWPEDPILGEDEVGGNKMLRASALFQSNKDYLRLD QTDRECNDMMAHKMKEPSQSCIAQYAFIHEDLNTV AVCNSPVIACELKGG CH SS RPFDLTLCELSQPDQVTPNCNYLTSVIKKHIIITCNDM RQLPTGQ

SEQ ID No. 41 :

Nucleotide sequence encoding human RNASEl l (homo sapiens ribonuclease, RNase A family, 1 1 (non-active, i.e. presumably no RNAse activity); accession number NM_145250. The coding region ranges from nucleotide 185 to nucleotide 784.

TGAGATCTCTTCTCTCAATGGCATTGGAGCTGGCTGTGCCTGAGGCAGACCTGGAC

CGTGGACATGGGGCAATGCCTTGAGCGGAAGGGGAAGCCACTGAATTTTGGGTGT

CACCAGGTAAACAGAGCCCTCAGCATCTGAATAGAAACTGAACAGGAACAGAAG

AGATTACACTACATCTGAGATGGAGACCTTTCCTCTGCTGCTGCTCAGCCTGGGCC

TGGTTCTTGCAGAAGCATCAGAAAGCACAATGAAGATAATTAAAGAAGAATTTAC

AGACGAAGAGATGCAATATGACATGGCAAAAAGTGGCCAAGAAAAACAGACCAT

TGAGATATTAATGAACCCGATCCTGTTAGTTAAAAATACCAGCCTCAGCATGTCC A AGGATG AT ATGTCTTC C AC ATT ACTG AC ATTC AGA AGTTT AC ATT AT A ATG AC C C

CAAGGGAAACAGTTCGGGTAATGACAAAGAGTGTTGCAATGACATGACAGTCTG

GAGAAAAGTTTCAGAAGCAAACGGATCGTGCAAGTGGAGCAATAACTTCATCCGC

AGCTCCACAGAAGTGATGCGCAGGGTCCACAGGGCCCCCAGCTGCAAGTTTGTAC

AGAATCCTGGCATAAGCTGCTGTGAGAGCCTAGAACTGGAAAATACAGTGTGCCA

GTTCACTACAGGCAAACAATTCCCCAGGTGCCAATACCATAGTGTTACCTCATTAG

AGAAGATATTGACAGTGCTGACAGGTCATTCTCTGATGAGCTGGTTAGTTTGTGGC

TCTAAGTTGTAAATCCCACAGAGCTTTAGGACTAGGGTCTTACTAAAGAAGGACC

TCTTCTTGTTCATTCTTGTTTAAACCTTTCCTTAATATCTACTCTTTAGCACTATAGT

GAACTCCTGATTATTTATTCTAACTGGAGGAGTGAAAAATCCAAAATTGTGGATA

ATTCAATTAAAAGTTATGACTGATACTGGCGTGCAGATTCTTCTCTCTCTATCTCTC

TCTCTGTGTGGGGGGAGGGTGGGGAGGGTGTAAAACAAACTGAATTTCATCCTAG

CTCAGCTATAAAGCCAAGAAACAGAATCCACCTAACTAGGCAGAATTAAGGAAA

CAAGGACTAAAGCCAATATGGACAATGAAAAGCTAATTGTACTGCCAATGATACT

ATTCAGAAAAATAGTGAATGAGTGTGACATATATTATTATCACTACTCTCTTCAAA

GGGTATGTGCTCACACTTGTGTGAATTGATGTATGCACATAACAAAGAGTCTGCCT

GTCCTTCAGGAAATGCCAGAGGAAAAAGATTCTTTGGCTGAGCTGATGCTCTCAG

TGTCAATAGCTCCAGCCCTATGCAATCTCCATTCTTCCACCCCCATCTCTATTTCAC

AGGTTGCTGTAAAGAGAAGAAAATTCTTATTGTAATCTTTATTCAAGCCAAGTCTT

GATACCCTGGGGCTGTGGGAGAGGAATGGCAAGCCATGGAAGAATAGAGGAGCC

AGAAAGATGGGAATAGAAAGTCTGTGCTAAGTACCAGGCAGGACTGGTCTTCAAC

CTTGATGTCTTATGATACTGTAGTTAATTITGTTCACTACCAGCTATTTATTTGCAA

ACGTTCGTGGTTCAACATTCTGCTTAAAGTATGGACAGTTATCTTCTTCATCGATG

TGTGGCCATATGTTTCTATTCCTCTCCTTAAATCCTCCCTCCATCCTGAGACCACTC

TTTCTTAATGTTCTAATTTTAACTAAATGCATGTAAATGTATGTAAAAA

SEQ ID No. 42:

Amino acid sequence of human RNASE11 (homo sapiens ribonuclease, RNase A family, 11 (non-active, i.e. presumably no RNAse activity); accession numberNP_660293.

METFPLLLLSLGLVLAEASESTM IIKEEFTDEEMQYDMA SGQEKQTIEILMNPILLV KNTSLSMSKDDMSSTLLTFRSLHYNDPKGNSSGNDKECCNDMTVWRKVSEANGSC WSNNFiRSSTEVMRRVHRAPSCKFVQNPGISCCESLELENTVCQFTTGKQFPRCQYHS VTSLEKILTVLTGHSLMSWLVCGSKL SEQ ID No. 43:

Nucleotide sequence encoding human RNASE12 (homo sapiens ribonuclease, member 12 of RNase A family (non-active); accession number NM_001024822. The coding region ranges from nucleotide 101 to nucleotide 544

GGGGTTGGAGCAGGAGGAATCTGACTGTCCTTTGCTACTTTCTATCTTCCCTTACT

CAACAGCAGTCAAAGCCAAAGATGCGAAGTCACTCTTACCTCTGATGATAATAAT

GGTGATAATTTTCTTGGTGCTTCTGTTCTGGGAAAATGAGGTGAATGATGAAGCA

GTGATGTCAACTTTAGAACACTTGCATGTGGACTACCCTCAGAATGACGTTCCCGT

TCCTGCAAGGTACTGCAACCACATGATCATACAAAGAGTTATCAGGGAACCTGAC

CACACTTGTAAAAAGGAGCATGTCTTCATCCATGAGAGGCCTCGAAAAATCAATG

GTATTTGCATTTCTCCCAAGAAGGTTGCTTGCCAAAACCTTTCGGCCATTTTCTGCT

TTCAGAGTGAGACAAAGTTCAAAATGACAGTCTGTCAGCTCATTGAAGGCACAAG

ATACCCTGCCTGCAGGTACCACTATTCCCCCACAGAGGGGTTTGTTCTTGTCACTT

GTGATGACTTGAGGCCAGATAGTTTCCTTGGCTATGTTAAATAACTCAAGATCAGC

TC C CG AGTCTG AGATCTCTTCTCTC A ATGGC ATTGG AGCTGGCTGTGC CTG AGGC A

GACCTGGACCGTGGACATGGGGCAATGCCTTGAGCGGAAGGGGAAGCCACTGGT

AATTAATTTATCCTTCCTGTATTGCTGGGTTGGGATTGTTTTATTCTGCTTCAATAA

AATAATCTTTACTGAATTAAAAAAA

SEQ ID No. 44:

Amino acid sequence of human RNASE12 (homo sapiens ribonuclease, member 12 of RNase A family (non-active, i.e. presumably no RNAse activity); accession number NP_001019993 IIMVIIFLVLLFWENEVNDEAVMSTLEHLHVDYPQNDVPVPARYCNHMIIQRVIREP

DHTCKKEHVFIHERPRKINGICISPKKVACQNLSAIFCFQSET FKMTVCQLIEGTRYPA

CRYHYSPTEGFVLVTCDDLRPDSFLGYV

SEQ ID No. 45:

Nucleotide sequence encoding human RNASE13 (homo sapiens ribonuclease, member 13 of RNase A family (non-active, i.e. presumably no RNAse activity); accession number NMJ301012264. The coding region ranges from nucleotide 139 to nucleotide 609.

ATTTTTACCTAGAAACCTTTCTAGCCTCTCCTCATCTCTTGACTGTCTTGGCCTTGG CAAGAAGAATACACGTCCCTACCCAGCTATCTTCAGCTCCTGTCCTTCCTCCCAGC TGCCAGAGAATTGTCAGCAGGAGGAATGGCACCAGCTGTGACCCGGCTCCTTTTC CTCCAGCTTGTTCTGGGGCCAACTCTGGTCATGGACATCAAGATGCAGATTGGCA GCAGGAACTTCTATACCTTAAGCATTGACTATCCCAGGGTTAACTACCCAAAGGG

TTTCCGGGGCTATTGTAATGGTCTGATGTCCTATATGCGAGGCAAGATGCAAAATT

CAGATTGCCCAAAGATCCATTATGTGATACATGCCCCTTGGAAGGCCATCCAGAA

GTTCTGCAAGTATAGTGACAGCTTCTGTGAGAATTACAATGAATACTGCACACTC

ACCCAGGATTCCCTCCCCATCACGGTCTGCTCCCTGAGCCACCAACAGCCACCCAC

TAGCTGCTACTACAATAGCACCCTAACCAACCAGAAGCTCTACCTACTCTGCTCCC

GCAAGTATGAAGCTGATCCAATAGGTATCGCTGGTCTCTATTCGGGAATTTAATTC

CTGAGCCACTCCCCAACCTCTACCACCACAGGCCAGCTGTCTCCATTTCCAACACT

CTGATCCCTGTAACAAGACTTCCTTCCTGACTTACGAGGCAGGCAGGGTCCCCTCC

CAAGAGACCAGGTGGGTGCGAAAATGTCCAGGGCTTTGATCACCATGCAAAGTGG

TGCTATCTTCTAGGTTCTTTCCAGCTTGTTTACCCCCTCTTCGCCCCCACCTCCTCA

TCATCCTTCCTCTCCCAGAGACAGTAAAACCCACCCAAGAAGTTGACTGTGTCCA

GTTGGCAGTGACTGTCCACCCACTGGGCCAGATCCTAAGTCCTGGATGGAGGCCC

AGGGCCTCAGCTCACACCATGGCAACAGGCACCAGCTCCGAGTCTCACCTCCCCA

TCCCCACCAGCCTTCTCATGTCAGCCTCCCCCAGTTCCCTTATAATTCCCTCCACTC

ACCCACCTACTCAGGGGATATGGCTAGCTATAGGAACCTCCCTGAGGCCCTCAGA

CTCATGTTAGCCTCACTTCCCCACAACCCCCAGTTTCCAGCCTGCTTCTCTCTGTGA

AATCATGGTTTCTCCCTTCATATAACTGTCCTCTCCTGCTTCCTCTTACCATGGAGC

CT AG GACTGTCTCCC CC AC ACTCCC A AC A AC C GTAGTC AC CCTGC CTTCCTTTTTC

CAACCTCTTTTTCTTCCAGTCTTCTTACCTTGGCTCACTGGCATTTCAAGGTATCTT

CTCTCACCCATTCCCCAGAGGTTCCTGCTTCCCTTCTCTTCCAGAAGCTCCTCCCAA

ATCCCCCAGACTCCAGAGTCTGCCCCTGACCAGGCAGCGAATGAGGCAGGCCCGG

CTCTAATCGAACATAAATTTCTAAAACCACATCTGAGTATGTAAGATGTAACCATT

GGGGGAAACTGGGTGAAGGGCACACGAGCCCCCTCTGTACCATTTTTTGTAACTT

CCTGTCAACCCATAATCAATTCAAAATAAAAAGTTAATAACACA

SEQ ID No. 46:

Amino acid sequence of human RNASE 13 (homo sapiens ribonuclease, member 13 of RNase A family (non-active, i.e. presumably no RNAse activity); accession number NP_001012264.

MAPAVTRLLFLQLVLGPTLVMDIKMQIGSRNFYTLSIDYPRVNYPKGFRGYCNGLMS

YMRGKMQNSDCPKIHYVIHAPWKAIQ FCKYSDSFCENYNEYCTLTQDSLPITVCSLS

HQQPPTSCYYNSTLTNQKLYLLCSRKYEADPIGIAGLYSGI

SEQ ID No. 47:

Nucleotide sequence encoding murine mature miR-V2MM_34242,

TTATTGATATGCTGGATGG SEQ ID No.48:

RNA sequence of murine mature miR-V2MMJ34242.

UU AUUGAU AUGCU GGAUGG

SEQ ID No.49:

Nucleotide sequence encoding/of siRNA targeting human RNASEl : ACCAAATGATGAGGCGCCGGAATAT

SEQ ID No.50:

RNA sequence of siRNA targeting human RNASEl ;

ACCAAAUGAUGAGGCGCCGGAAUAU

Claims

An antagonist of a Ribonuclease being a member of the R Asc A family for use in treating a disease associated with an increase in adipocyte number.

The antagonist of claim 1 , wherein the antagonist is a selective antagonist of a Ribonuclease being a member of the RNAse A family.

The antagonist of claim 1 or 2, wherein said antagonist is selected from the group consisting of miRNA, dsRNA, siRNA, shRNA, stRNA, anti-RNAse A antisense molecules, extracellular binding-partners, small binding molecules such as RNAsin, aptamers, intramers, and an antibody molecule such as a full antibody (immunoglobulin), a F(ab)-fragmcnt, a F(ab)₂- fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody- construct, a synthetic antibody, a bispecific single chain antibody or a cross-cloned antibody.

The antagonist of claim 3, wherein said siRNA consists of a nucleic acid molecule comprising at least ten contiguous bases having a sequence as shown in the sequence of SEQ ID NO: 50, wherein, in particular, up to 10 % of the contiguous bases are non- complementary and which may, optionally, further comprise at least one base at the 5' end and/or at least one base at the 3' end; or

wherein said miRNA consists of a nucleic acid molecule comprising at least ten contiguous bases having a sequence as shown in the sequence of miR-V2MM 34242 (SEQ ID NO: 48), wherein, in particular, up to 10 % of the contiguous bases are non- complementary and which may, optionally, further comprise at least one base at the 5' end and/or at least one base at the 3' end.

The antagonist of claim 4, wherein said nucleic acid molecule consists of a molecule as shown in SEQ ID NO: 50; or wherein said nucleic acid molecule consists of a molecule as shown in SEQ ID NO: 48 (miR- V2MM 34242).

6. The antagonist of any one of claims 1 to 5, wherein said disease associated with an increase in adipocyte number and/or an increase in adipocyte volume is overweight (pre-obesity), in particular overweight defined as a body mass index (BMI) between 25 to 30 kg/m² of the subject to be treated,

7. The antagonist of any one of claims 1 to 5, wherein said disease associated with an increase in adipocyte number and/or an increase in adipocyte volume is obesity, in particular obesity defined as a body mass index (BMI) of higher than 30 kg/m² of the subject to be treated.

8. The antagonist of any one of claims 1 to 7, wherein said disease associated with an increase in adipocyte number is hyperplastic obesity,

9. The antagonist of any one of claims 1 to 8, wherein said disease is characterized as 20% or more body fat in the subject to be treated.

10. The antagonist of any one of claims 1 to 9,

wherein said Ribonuclease being a member of the RNAse A family is a human RNAse, in particular selected from the group consisting of RNAse 1, RNAse 2, RNAse 3, RNAse 4/5, RNAse 6, RNAse 7, RNAse 8, RNAse 9, RNAse 10, RNAse 11, RNAse 12, and RNAse 13

or

wherein said Ribonuclease being a member of the RNAse A family is a Eosinophil - associated Ribonuclease (EAR), preferably selected from the group consisting of EAR- 1 , EAR- 2 and EAR- 10.

11. The antagonist of claim 10,

wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24;

wherein said RNAse 2 has an amino acid sequence as shown in SEQ ID NO: 26;

wherein said RNAse 3 has an amino acid sequence as shown in SEQ ID NO: 28;

wherein said RNAse 4/5 has an amino acid sequence as shown in SEQ ID NO: 30; wherein said RNAse 6 has an amino acid sequence as shown in SEQ ID NO: 32;

wherein said RNAse 7 has an amino acid sequence as shown in SEQ ID NO: 34;

wherein said RNAse 8 has an amino acid sequence as shown in SEQ ID NO: 36;

wherein said RNAse 9 has an amino acid sequence as shown in SEQ ID NO: 38; wherein said RNAse 10 has an amino acid sequence as shown in SEQ ID NO; 40; wherein said RNAse 1 1 has an amino acid sequence as shown in SEQ ID NO: 42; wherein said RNAse 12 has an amino acid sequence as shown in SEQ ID NO: 44; and wherein said RNAse 13 has an amino acid sequence as shown in SEQ ID NO: 46.

12. The antagonist of claim 10, wherein said RNAse 1 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:24;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of

(a) to (d), whereby said polypeptide is a functional RNAse 1 or a fragment thereof; and

(f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence o a nucleic acid molecule as defined in (a), (c) and (d).

13. The antagonist of claim 10, wherein said EAR-1 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2;

(c) a polypeptide encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO: 2;

14. The antagonist of claim 10, wherein said EAR-2 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 4;

(f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d),

15, The antagonist of claim 10, wherein said EAR- 10 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 16;

16. Method for assessing the activity of a candidate molecule suspected of being an antagonist of a Ribonuclease being a member of the RNAse A family, in particular of Ribonucleases as defined in any of claims 10 to 15, comprising the steps of;

(b) detecting a decrease in activity of said Ribonuclease; and

(c) selecting a candidate molecule that decreases activity of said Ribonuclease; wherein a decrease of the Ribonuclease activity is indicative for the capacity of the selected molecule to antagonise an increase in adipocyte number and/or an increase in adipocyte volume.

17. An agonist of a Ribonuclease being a member of the RNAse A family for use in treating a disease associated with a decrease in adipocyte number,

18. An agonist of claim 17, wherein the agonist may be a selective agonist of a Ribonuclease being a member of the RNAse A family.

19. The agonist of claim 17 or 18,

or

wherein said Ribonuclease being a member of the RNAse A family is a Eosinophil- associated Ribonuclease (EAR), preferably selected from the group consisting of EAR- l, EAR-2 and EAR- 10.

20. The agonist of claim 19,

wherein said RNAse 1 has an amino acid sequence as shown in SEQ ID NO: 24;

wherein said RNAse 2 has an amino acid sequence as shown in SEQ ID NO: 26;

wherein said RNAse 3 has an amino acid sequence as shown in SEQ ID NO: 28;

wherein said RNAse 4/5 has an amino acid sequence as shown in SEQ ID NO: 30; wherein said RNAse 6 has an amino acid sequence as shown in SEQ ID NO; 32;

wherein said RNAse 7 has an amino acid sequence as shown in SEQ ID NO: 34;

wherein said RNAse 8 has an amino acid sequence as shown in SEQ ID NO: 36; wherein said RNAse 9 has an amino acid sequence as shown in SEQ ID NO: 38;

wherein said RNAse 10 lias an amino acid sequence as shown in SEQ ID NO: 40; wherein said RNAse 1 1 has an amino acid sequence as shown in SEQ ID NO: 42; wherein said RNAse 12 has an amino acid sequence as shown in SEQ ID NO; 44; and wherein said RNAse 13 lias an amino acid sequence as shown in SEQ ID NO: 46,

21. The agonist of claim 19, wherein said RNAse 1 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 24;

22. The agonist of claim 19, wherein said EAR-1 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO:2;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is a functional EAR-1 or a fragment thereof; and (f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) and (d).

23. The agonist of claim 19, wherein said EAR-2 is selected from the group consisting of

(a) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO; 3;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 4;

24. The agonist of claim 1 , wherein said EAR- 10 is selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 16;

25. The agonist of any one of claims 17 to 24, wherein said disease associated with a decrease in adipocyte number is lipodystrophy.

26. The agonist of claim 25, wherein said lipodystrophy is hereditary lipodystrophy, drug- induced lipodystrophy, lipodystrophy caused or induced by AIDS/HIV therapy, or traumatic lipodystrophy.

27. The agonist of any one of claims 17 to 24, wherein said disease associated with a decrease in adipocyte number is cachexia or a disease associated with disturbed energy storage.

28. Method for assessing the activity of a candidate molecule suspected of being an agonist of a Ribonuclease being a member of the RNAse A family, in particular of Ribonucleases as defined in any of claims 17 to 24, comprising the steps of:

(a) contacting a cell, tissue or a non-human animal comprising a Ribonuclease of the RN Ase A family with said candidate molecule;

(b) detecting an increase in activity of said Ribonuclease; and

(c) selecting a candidate molecule that increases activity of said Ribonuclease; wherein an increase of the Ribonuclease activity is indicative fo the capacity of the selected molecule to increase adipocyte number and/or increase adipocyte volume.