WO2020083933A1

WO2020083933A1 - Fusion polypeptides and therapeutic applications thereof

Info

Publication number: WO2020083933A1
Application number: PCT/EP2019/078759
Authority: WO
Inventors: Melroy Xavier MIRANDA
Original assignee: Mexav Biotech Ag
Priority date: 2018-10-23
Filing date: 2019-10-22
Publication date: 2020-04-30

Abstract

Isolated fusion polypeptide comprising a first amino acid sequence characterized by at least (≥)85% sequence identity to SEQ ID NO 01 -SEQ NO 04, SEQ ID 23 or SEQ ID 08 said first amino acid sequence and/or said isolated fusion protein having an NAD+-dependent deacetylase function identical to SIRT1 isoform 1, isoform 2, isoform b or isoform c or the catalytic domain thereof, said first amino acid sequence being directly or indirectly chemically linked to at least one of second amino acid sequence comprising a fragment crystallizable (Fc) region or an albumin, or a post- translationally modified derivative thereof, for use in a method of treatment amelioration, mitigation, slowing, arresting, reversing or prevention of an age-related diseases or of a condition selected from the group consisting of: mitochondrial-related diseases/disorders, metabolic disorders, neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, but also dyslipidemia, hyperlipidemia, diabetes, metabolic syndrome, inflammation, apoptosis, neurodegeneration, cancer, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, and obesity-related conditions.

Description

TITLE

FUSION POLYPEPTIDES AND THERAPEUTIC APPLICATIONS THEREOF

TECHNICAL FIELD

The present invention relates to fusion polypeptides, in particular fusion polypeptide involving Sirtuin- 1 as well as further polypeptide fragments, including Fc regions or albumin, e.g. interacting with the neonatal Fc receptor. The proposed fusion polypeptide are shown to allow effective treatment or prevention of an age-related diseases or of a condition in particular selected from the group consisting of: mitochondrial-related diseases/disorders, metabolic disorders. neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, cancer but also dyslipidemia, hyperlipidemia, diabetes, autoimmunity, inflammation, apoptosis, oxidative stress and neurodegeneration.

PRIOR ART

WO-A-03061681 relates to the use of nucleic acid and amino acid sequences of Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase, glutamate dehydrogenase, kraken homolog, sirtuin 1. escargot homolog, human KIAA1585 protein, CG1 1940 homolog, dappled homolog, CG 1 1753 homolog, human KIAA0095 protein, formin-binding protein 21 , and/or homologous proteins in pharmaceutical compositions, and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders related to body- weight regulation and thermogenesis.

WO-A-2004055169 relates to modulation of cytochrome c acetylation, e.g., with a SIR polypeptide, which enables interventions that modulate lifespan regulation and cell proliferation, e.g., by modulating apoptosis and/or mitochondrial function such as respiration.

WO-A-2017207733 relates to the use of an isolated polypeptide comprising an amino acid sequence which has a NAD+-dependent deacetylase function identical to S1RT1 and is provided for use in a method of treatment or prevention of dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, diabetes, metabolic syndrome and obesity-related conditions. It further relates to a method ot determining acute coronary syndrome or determining the risk of a patient to develop coronary artery disease, comprising the quantification of SIRT1 expression levels.

WO-A-2009140562 provides SIRT1 polymorphic variants having a substitution at amino acid residue 107 or nucleotide 373. In certain embodiments, the Sirtl polypeptide variants have a L107P substitution and the nucleic acid variants have a T373C substitution. Genetic and/or biochemical testing is proposed to identify whether a patient carries one of the disclosed polymorphic variants. Based on the polymorphic variant the patient carries, a medical practitioner may administer an appropriate therapy, such as a sirtuin activator. WO-A-2016131892 relates to antibodies reactive to acetylated PCSK9, particularly to PCSK9 acetylated in specific positions, and related reagents. It also relates to a method of reducing LDL-cholesterol level in a patient in need thereof, said method comprising administering to the subject an effective amount of an antibody binding to acetylated PCSK9 as well as to a method of treating a cholesterol related disorder in a patient or hypercholesterolemia in need thereof, said method comprising administering to the subject an effective amount of an antibody binding to acetylated PCSK9.

SUMMARY OF THE INVENTION

Definitions: In the present specification, "SIRT1 " stands for Sirtuin- 1 (silent mating type information regulation 2 homologue 1). SIRT1 is a highly conserved protein deacetylase that requires NAD+ (nicotinamide adenine dinucleotide) as a co-substrate. The deacetylation of acetyl-lysines by Sirtuin 1 is coupled with NAD+ hydrolysis, producing nicotinamide and an acetyl-ADP ribose compound. Sirtuin 1 also exhibits NAD+- dependent histone deacetylase activity.

In the present specification, "recombinant SIRT1 " or "rSIRTI " refers to a SIRT1 polypeptide that has been produced using biotechnological methods. In certain embodiments, the term refers to human SIRT1 isoform 1 (Uniprot Q96EB6.1 , called isoform a in NCBI database with NCBI Reference Sequence NP_036370.2). In certain embodiments, the term refers to human SIRT1 isoform 2 (Uniprot Q96EB6-2) or isoform b (NCBI Reference Sequence NP_001 135970.1) or isoform c (NCBI Reference Sequence NP_001300978.1) or isoform d (GenBank Sequence with NCBI identifier AAH 12499.1) as detailed below.

In the present specification, the terms“sequence identity’" and“percentage of sequence identity" refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment ot sequences for comparison may be conducted by the local homology algorithm ot Smith and Waterman, Adv. Appl. Math. 2:482 (1 981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

An example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 1 1 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1 .- 2; Gap costs: Linear. Unless otherwise stated, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

In the present specification, the term low density lipoprotein (LDL) relates to a class of plasma lipoproteins with a high proportion of lipid, including cholesterol, cholesterol esters and triglycerides. LDL includes primarily apolipoprotein B-100 and apolipoprotein E. LDL incorporates and transports cholesterol in the plasma. The term LDL may be used in a context-dependent manner to designate cholesterol bound to LDL particles.

In the present specification, LDLR relates to low density lipoprotein receptor.

In the present specification, the terms acetylation and deacetylation are used in their meaning known in the art of biochemistry and cell biology; it refers to a modification of proteins, where acetyl groups are covalently attached to or removed from lysine residues within the protein. This modification is known to affect the properties and functions of the proteins. Acetylation of proteins is catalyzed by acetyltransferases, whereas deacetylation is catalyzed by deacetylases.

In the present specification, the term cardiovascular disease (CVD) is used to classify conditions that affect the heart, heart valves, blood, and vasculature of the body, particularly diseases affected by an aberrant plasma level of lipids, more particularly by an aberrant level of cholesterol, LDL, HDL and/or VLDL. Cardiovascular diseases that can be modified by the invention include endothelial dysfunction, coronary artery disease (CAD), angina pectoris, myocardial infarction, acute coronary syndrome (ACS), atherosclerosis, congestive heart failure, hypertension, cerebrovascular disease, stroke, transient ischemic attacks, deep vein thrombosis, peripheral artery disease, cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Particular diseases that are amenable to treatment according to the invention include atherosclerosis-associated endothelial dysfunction, atherosclerosis- associated coronary artery disease (CAD), atherosclerosis-associated angina pectoris, atherosclerosis-associated myocardial infarction, atherosclerosis-associated acute coronary syndrome (ACS), atherosclerosis-associated congestive heart failure, atherosclerosis- associated hypertension, atherosclerosis-associated cerebrovascular disease, stroke, atherosclerosis-associated transient ischemic attacks, atherosclerosis-associated deep vein thrombosis, atherosclerosis-associated peripheral artery disease, atherosclerosis-associated cardiomyopathy, atherosclerosis-associated arrhythmias, aortic stenosis, and atherosclerosis-associated aneurysm.

More particularly, diseases that are amenable to treatment according to the invention include dyslipidemia-associated endothelial dysfunction, dyslipidemia-associated coronary artery disease (CAD), dyslipidemia-associated angina pectoris, dyslipidemia-associated myocardial infarction, dyslipidemia-associated acute coronary syndrome (ACS), dyslipidemia-associated congestive heart failure, dyslipidemia-associated hypertension, dyslipidemia-associated cerebrovascular disease, stroke, dyslipidemia-associated transient ischemic attacks, dyslipidemia-associated deep vein thrombosis, dyslipidemia-associated peripheral artery disease, dyslipidemia-associated cardiomyopathy, dyslipidemia- associated arrhythmias, aortic stenosis, and dyslipidemia-associated aneurysm. Preferably, the following diseases are amenable to treatment according to the present invention: dyslipidemia, hyperlipidemia, diabetes, insulin resistance, hypertension, metabolic syndrome, inflammation, apoptosis, neurodegeneration. depression, cancer, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), autoimmune disease, steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, and obesity-related conditions.

Within the context of the present specification, the term coronary artery disease (CAD) is used synonymously with the term ischemic heart disease (IHD) and refers to conditions that are caused by decreased blood flow in the coronary arteries. CAD comprises a group of diseases including stable angina, unstable angina, myocardial infarction and sudden cardiac death. A particularly relevant CAD indication for treatment with the invention is ST-Elevation Myocardial Infarction (STEMI).

In the present specification, the term acute coronary syndrome (ACS) is used to describe an acute condition that usually occurs as a result of myocardial infarction or unstable angina.

According to a first aspect of the present invention, an isolated fusion polypeptide is proposed comprising

a first amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 , SEQ ID NO 02, SEQ ID NO 03, SEQ ID NO 04, SEQ ID 23 or SEQ ID 08,

said first amino acid sequence and/or said isolated fusion polypeptide having an NAD+- dependent deacetylase function identical to SIRT1 isoform 1 , isoform 2, isoform b, isoform c or isoform d

or to the catalytic domain of any of these S1RT1 isoforms (which is normally given by 244-489 of SIRT1 isoform 1 and 244-453 of SIRT1 isoform 2, 1- 194 of SIRT1 isoform b, 12- 186 of SIRT1 isoform c, and 62-297 of isoform d), or a minimum common catalytic sequence stretch of these isoforms,

said first amino acid sequence being directly or indirectly chemically linked to

at least one of a second amino acid sequence comprising a fragment crystallizable (Fc) region or an albumin, or a post-translational ly modified derivative of any of these two, for use in a method of treatment, amelioration, mitigation, slowing, arresting or reversing or prevention of

an age-related disease or of a condition selected from the group consisting of: mitochondrial-related diseases/disorders, metabolic disorders, neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, dyslipidemia, hyperlipidemia, diabetes, metabolic syndrome, inflammation, apoptosis, neurodegeneration, oxidative stress and cancer, hypercholesterolemia, autoimmunity, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, depression, and obesity-related conditions. Preferably diseases related to dyslipidemia, diabetes, insulin resistance, tatty liver disease, neurodegeneration, preferably Parkisons and Alzheimers, and cancer, preferably pancreatic cancer, are treated.

As for age related diseases these include conditions such as: frailty; bone density loss; bone mineral density loss; weight loss; muscular atrophy; muscular degeneration; decline in muscle mass; decline in muscle strength; decline in hand strength; decline in leg strength; decline in physical fitness; decline in movement; decline in freedom of movement; decline in quality of life assessment; decline in ejection fraction; decline in exercise capacity; decline in learning; decline in learning capacity; decline in memory; decline in intellectual quotient; cognitive deterioration; forgetfulness; decline in cognitive capacity; decline in cognitive function; decline in synaptic plasticity; decline in synaptic function; cellular senescence; chronic kidney disease (CKD); chronic kidney disease - mineral and bone disorder (CKD-MBD); polycystic kidney disease (PKD); autosomal dominant polycystic kidney disease (ADPKD); acute kidney injury (AKI); acute tubular necrosis (ATN); acute allergic interstitial nephritis (AAIN); glomerulonephritis; kidney disease; renal failure; Alport Syndrome; nonoliguric renal failure; alcoholism; hyperphosphatemia; muscular dystrophy (MS); type 1 diabetes; type 2 diabetes; cardiovascular disease (CVD); cardiovascular calcification; cerebrovascular insufficiency; vascular calcification; coronary artery disease; heart failure; left ventricular hypertrophy; uremic cardiomyopathy; abnormalities in blood pressure; salt-sensitive hypertension; tissue calcification; calcific atherosclerotic plaque burden; calcinosis; familial tumoral calcinosis; cancer; one or more tumors; myelin-related diseases; demyelinating diseases; neurodegenerative disease; neurovascular diseases; progressive supranuclear palsy (PSP); Pompe disease; Niemann- Pick disease; microgliosis; Farber disease (FD); bone mass diseases; osteoporosis; osteopenia; osteopenia (particularly loss of BMD of cortical bone); pulmonary emphysema; pulmonary fibrosis; cystic fibrosis, idiopathic (i.e., cause unknown) pulmonary fibrosis, radiation-induced lung injury, cirrhosis, biliary atresia, atrial fibrosis, endomyocardial fibrosis, (old) myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive capsulitis, skin atrophy; thymic atrophy; accumulation of renal interstitial matrix; glomerulosclerosis; anemia; albuminuria; proteinuria; infertility; Alzheimer's disease; Parkinson's Disease; dementia; vascular dementia; amyotrophic lateral sclerosis (ALS); motor neuron disease (MND); atrial fibrillation; chronic obstructive pulmonary disease (COPD); fibromyalgia; adult onset diabetes; arthritis; rheumatoid arthritis; osteoarthritis; glaucoma; cataracts; macular degeneration; multiple sclerosis (MS); lupus; ulcerative colitis; cachexia; obesity; vitamin D-related conditions; bone diseases; bone diseases through bone remodeling; stem cell depletion; sea sickness; space adaptation syndrome (SAS); nausea; vertigo; nonalcoholic steatohepatitis (NASH), cirrhosis of the liver and alcoholic steatohepatitis.

More generally speaking the proposed system is for use in a method of treatment, amelioration, mitigation, slowing, arresting or reversing or prevention of at least one of the following conditions:

A "mitochondrial-related diseases/disorders" characterized by malfunction of the mitochondria. A mitochondrial-related disease or disorder includes a muscle structure disorder, a neuronal activation disorder, a muscle fatigue disorder, a muscle mass disorder, a metabolic disease, a cancer, a vascular disease, an ocular vascular disease, a muscular eye disease, or a renal disease. In some embodiments, a "mitochondrial-related disease or disorder" is selected from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal ischemia/reperfusion injury (IRI), Duchenne & Becker muscular dystrophy, diabetes (type 1 or type II), obesity, and sarcopenia. In another embodiment, a "mitochondrial-related disease or disorder" is selected from Alpers's Disease, CPEO-Chronic progressive external ophthalmoplegia, Kearns-Sayra Syndrome (KSS), Leber Hereditary Optic Neuropathy (LHON), MELAS -Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke- like episodes, MERRF-Myoclonic epilepsy and ragged-red fiber disease, NARP-neurogenic muscle weakness, ataxia, and retinitis pigmentosa, Pearson Syndrome, platinum-based chemotherapy induced ototoxicity, Cockayne syndrome, xeroderma pigmentosum A, Wallerian degeneration, and HIV-induced lipodystrophy and peroxisomal diseases like X-linked adrenoleukodystrophy. In certain embodiments, the proposed SIRT1 fusion polypeptide may be useful for treatment mitochondrial myopathies. Mitochondrial myopathies range from mild, slowly progressive weakness of the extraocular muscles to severe, fatal infantile myopathies and multisystem encephalomyopathies. Some syndromes have been defined, with some overlap between them. Established syndromes affecting muscle include progressive external ophthalmoplegia, the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy, cardiac conduction defects, cerebellar ataxia, and sensorineural deafness), the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), the MERFF syndrome (myoclonic epilepsy and ragged red fibers), limb-girdle distribution weakness, and infantile myopathy (benign or severe and Fatal).

Sports performance refers to the ability of the athlete's muscles to perform when participating in sports activities. Enhanced sports performance, strength speed and endurance are measured by an increase in muscular contraction strength, increase in amplitude of muscle contraction, shortening of muscle reaction time between stimulation and contraction. Athlete refers to an individual who participates in sports at any level and who seeks to achieve an improved level of strength, speed and endurance in their performance, such as, for example, body builders, bicyclists, long distance runners, short distance runners, etc. Enhanced sports performance in manifested by the ability to overcome muscle fatigue, ability to maintain activity for longer periods of time, and have a more effective workout.

Fat-related metabolic disorders amenable to treatment with the SIRT1 fusion polypeptide include disorders in which (i) increased fat storage, reduced fat mobilization, and/or reduced fat burning is desired, and (ii) other disorders in which reduced fat storage, increased fat mobilization and/or increased fat burning is desired. Examples of the first category of disorders include, e.g., anorexia nervosa, wasting, AIDS-related weight loss, bulimia, cachexia. Examples of the latter category include, e.g., obesity, cardiovascular disease, osteoarthritis. The classification of other disorders (e.g., infertility, increased surgical risk, pregnancy complications) may depend on the weight of the subject, e.g., whether the subject is over- or underweight.

Obesity-related disease” and“Fat-related metabolic disorder” include, but are not limited to, anorexia nervosa, wasting, AIDS-related weight loss, bulimia, cachexia, lipid disorders including hyperlipidemia and hyperuricemia, insulin resistance, noninsulin dependent diabetes mellitus (NIDDM, or Type II diabetes), insulin dependent diabetes mellitus (1DDM or Type I diabetes), diabetes-related complications including microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions (including diabetic nephropathy), cardiovascular disease (including cardiac insufficiency, coronary insufficiency, and high blood pressure), atherosclerosis, atheromatous disease, stroke, hypertension, Syndrome X, gallbladder disease, osteoarthritis, sleep apnea, forms of cancer such as uterine, breast, colon, colorectal, pancreatic, kidney, and gallbladder, high cholesterol levels, complications of pregnancy, menstrual irregularities, hirsutism, muscular dystrophy, infertility, a weight-related disorder (characterized by a subject being over or under weight, e.g., being within the top or bottom 25th percentile of body mass index) and increased surgical risk. In preferred embodiments, a treated or diagnosed subject is a mammal, preferably a human.

Examples of neurodegenerative and/or neuroinflammation diseases amenable to treatment with the proposed S1RT1 fusion polypeptide include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewy body disease, chorea-acanthocytosis, primary lateral sclerosis, ocular diseases (ocular neuritis), chemotherapy-induced neuropathies (e.g., from vincristine, paclitaxel, bortezomib), diabetes-induced neuropathies, Friedreich's ataxia, dementia (including Lewy Body disease, mild cognitive impairment (MCI), Primary Senile Degenerative Dementia, Alzheimer Type Senile Dementia and Alzheimer Type Dementia), Parkinsonian disorders (including Lewy Body disease and Parkinsonism-linked to chromosome 17 (FTDP-17)), progressive supranuclear palsy (also known as Steele-Richardson-Olszewski Syndrome or Disease, Progressive Supranuclear Ophthalmoplegia), Pick's disease and corticobasal degeneration. Multiple sclerosis (MS), including relapsing MS and monosymptomatic MS, and other demyelinating conditions, such as, for example, chronic inflammatory demyelinating polyneuropathy (CIDP), or symptoms associated therewith.

Diabetic neuropathies amenable to treatment with the proposed SIRT1 fusion polypeptide, which are neuropathic disorders that are associated with diabetes mellitus. Relatively common conditions which may be associated with peripheral neuropathy, diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy.

PNS diseases treatable with the proposed SIRT1 fusion polypeptide include: diabetes, leprosy, Charcot-Marie-Tooth disease, Guillain-Barre syndrome and Brachial Plexus Neuropathies (diseases of the cervical and first thoracic roots, nerve trunks, cords, and peripheral nerve components of the brachial plexus.

In another embodiment, the proposed SIRT1 fusion polypeptide is used to treat or prevent a polyglutamine disease. Exemplary polyglutamine diseases include Spinobulbar muscular atrophy (Kennedy disease), Huntington's Disease (FID), Dentatorubral-pallidoluysian atrophy (Haw River syndrome), Spinocerebellar ataxia type 1 , Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (Machado-Joseph disease), Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, and Spinocerebellar ataxia type 17.

Accordingly, the present invention provides anticoagulation and antithrombotic treatments with the proposed SIRT1 fusion polypeptide aiming at inhibiting tissue factor and the formation of blood clots in order to prevent or treat blood coagulation disorders such as myocardial infarction, stroke, loss of a limb by peripheral artery disease or pulmonary embolism.

In another embodiment, the proposed SIRTl fusion polypeptide is used to treat or prevent allergies and respiratory conditions, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome, and any chronic obstructive pulmonary disease (COPD). The compounds may be used to treat chronic hepatitis infection, including hepatitis B and hepatitis C.

Additionally, the proposed S1RT1 fusion polypeptide can be used to treat autoimmune diseases and/or inflammation associated with autoimmune diseases such as organ-tissue autoimmune diseases (e.g., Raynaud's syndrome), inflammatory bowel disease, scleroderma, myasthenia gravis, transplant rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosis, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), and Grave's disease.

in certain aspects of the invention, the vision impairment amenable to treatment with the proposed SIRTl fusion polypeptide is caused by damage to the optic nerve or central nervous system. In particular embodiments, optic nerve damage is caused by high intraocular pressure, such as that created by glaucoma. In other particular embodiments, optic nerve damage is caused by swelling of the nerve, which is often associated with an infection or an immune (e.g., autoimmune) response such as in optic neuritis.

In certain aspects of the invention, the vision impairment amenable to treatment with the proposed SIRTl fusion polypeptide is caused by retinal damage. In particular embodiments, retinal damage is caused by disturbances in blood flow to the eye (e.g., arteriosclerosis, vasculitis). In particular embodiments, retinal damage is caused by disruption of the macula (e.g., exudative or non-exudative macular degeneration).

Exemplary retinal diseases amenable to treatment with the proposed SIRTl fusion polypeptide include Exudative Age Related Macular Degeneration, Nonexudative Age Related Macular Degeneration, Retinal Electronic Prosthesis and RPE Transplantation Age Related Macular Degeneration, Acute Multifocal Placoid Pigment Epitheliopathy, Acute Retinal Necrosis, Best Disease, Branch Retinal Artery Occlusion, Branch Retinal Vein Occlusion, Cancer Associated and Related Autoimmune Retinopathies, Central Retinal Artery Occlusion, Central Retinal Vein Occlusion. Central Serous Chorioretinopathy, Eales Disease, Epimacular Membrane, Lattice Degeneration, Macroaneurysm, Diabetic Macular Edema, Irvine-Gass Macular Edema, Macular Hole, Subretinal Neovascular Membranes, Diffuse Unilateral Subacute Neuroretinitis, Nonpseudophakic Cystoid Macular Edema, Presumed Ocular Histoplasmosis Syndrome, Exudative Retinal Detachment, Postoperative Retinal Detachment, Proliferative Retinal Detachment, Rhegmatogenous Retinal Detachment, Tractional Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis, Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy, Background Diabetic Retinopathy, Proliferative Diabetic Retinopathy, Hemoglobinopathies Retinopathy, Purtscher Retinopathy, Valsalva Retinopathy, Juvenile Retinoschisis, Senile Retinoschisis, Terson Syndrome and White Dot Syndromes.

Other exemplary diseases amenable to treatment with the proposed S1RT1 fusion polypeptide include ocular bacterial infections (e.g. conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viral infections (e.g. Ocular Herpes Simplex Virus, Varicella Zoster Virus, Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as well as progressive outer retinal necrosis secondary to HIV or other HIV-associated and other immunodeficiency-associated ocular diseases. In addition, ocular diseases include fungal infections (e.g. Candida choroiditis, histoplasmosis), protozoal infections (e.g. toxoplasmosis) and others such as ocular toxocariasis and sarcoidosis.

Muscular dystrophy amenable to treatment with the proposed SIRT1 fusion polypeptide refers to a family of diseases involving deterioration of neuromuscular structure and function, often resulting in atrophy of skeletal muscle and myocardial dysfunction, such as Duchenne muscular dystrophy. In certain embodiments, sirtuin 1 protein may be used for reducing the rate of decline in muscular functional capacities and for improving muscular functional status in patients with muscular dystrophy.

The fusion polypeptide (protein) may consist of the two amino acid sequences, which are then directly linked to each other, however the fusion polypeptide may contain further elements such as, for example, a linker element between the two amino acid sequences (indirect connection), or further stretches attached to one or both ends of the fusion polypeptide. Preferably the fusion polypeptide consists of the two amino acid sequences, optionally connected by way of a linker sequence.

Furthermore it should be noted that the first and second amino acid sequence may also be given by sequences or in which not more than 5, preferably not more than 3, or 2 or 1 amino acid is removed from these sequences at one or both ends thereof.

The fusion polypeptide (protein) may further include an IgG or albumin tag that binds to FcRN its endogenous neonatal Fc receptor called, FcRn (half-life extension) or Clq (immune response). It has a multitude of biological and immunological functions. The most recognized of these functions is the FcRn-mediated recycling and transcytosis process that results in the extraordinarily long, ~ 21 day serum presence of IgG and albumin in humans. A peptide or compound bound to the Fc portion of an immunoglobulin (IgG) or to albumin will be recycled (and stored) intermittently by being internalized by the FcRn receptor. Thus, a protein or compound which has a short hall-life in the blood can be tagged with an FcRn ligand (example, IgG or albumin), which would increase its half- life. This can also be pH-dependent. Furthermore Fc-tags increase the molecular weight of the protein which prevents its excretion through the kidneys. A monomeric Fc-fragment adds 25kDa. Unless specifically designed Fc-fragments are expressed as dimers, thus adding 50kDa to the protein size.

Fc-fragments of use in the present invention can be from different immunoglobulins. Normally, IgG fragments are used due to their antibody properties. In IgG there are different sub-types - IgGl , IgG2, IgG3 and IgG4. For fusion polypeptides (for half-life extensions), one may use Fc-fragments from IgGl and IgG4 specifically. IgG3, IgGl or IgG2 (mainly IgG3) is used when an antibody-dependent cell mediated cytotoxicity is required (immune response). For example, antibodies targeting cancer or vaccination. By mutating the Fc-fragment at particular sites it is possible to increase the Fc-fragment binding to FcRn. Thus increasing the half-life of a drug beyond natural Fc-tagged proteins. Other factors that determine the effect of the Fc-fragment are the allotype of the immunoglobulin, its glycosylation at N297, fucosylation status, galactosylation and sialylation. Each modification influences the half-life and particularly the immunomodulatory activity of the Fc-fragment.

Of note, there are also other receptor sub-types of the Fcy-receptor family (FcyRI, FcyRIIa, FcyRIIb/IIc, FcyRIIIa, FcyRIIIb) which have different affinities for each of the immunoglobulin sub-types (IgGl, IgG2, IgG3 and IgG4). Alternate receptors also include FcRL, TRIM21 and DC-SIGN. The Fc-tail is in particular influencing the therapeutic window (increasing the halt-life). The tagging of SIRT1 with Fc or albumin causes its binding and recycling by FcRn receptors expressed mainly on epithelial and endothelial cells. Thus increasing its half-life. While recombinant SIRT1 maybe degraded in typically at least 30 min, or at least 60 min., the proposed SIRT1 fusion polypeptide is active for much longer.

Furthermore it is also assumed that Fc-tagging to the C -terminal protects it from proteases by blocking the binding of proteases to the C -terminal.

Preferably the Fc-tag is on the C -terminal because for the N-terminal preliminary studies show that may have different activity. That may mean that the N-terminal is required for internalization in cells or activity.

Linkers can improve orientation, expression, stability and activity of fusion proteins (between the Fc or albumin tag and the protein). In addition to flexible, rigid and cleavable linkers, there are also chemical linkers usually used for the pegylated-tagged variants of a protein.

According to a first preferred embodiment the fusion polypeptide is present as a homodimer, as a homo monomer with an additional second amino acid sequence of a fragment crystallizable (Fc) region and/or an albumin, or a post-translationally modified derivative thereof without attached first amino acid sequence being directly or indirectly chemically linked, or as a monomer.

In case of a homodimer, the system is present in the form of a dimer with two of such fusion polypeptides, typically both having the same structure, and which are typically linked by way of the second amino acid sequence, for example by hydrogen bonds, or by chemical bonds, typically mediated by post-translational modifications and/or by way of disulphide bonds.

In case of a Dimer, there is one fusion polypeptide as defined above, to which an additional second amino acid sequence in the form of a fragment crystallizable (Fc) region or an albumin, or a post-translationally modified derivative thereof, preferably having the same structure as the second amino acid sequence of the fusion polypeptide, is bound. This binding can again be by way of hydrogen bonds, or by chemical bonds, for example mediated by post-translational modifications and/or by way of disulphide bonds.

The fusion polypeptide may comprise a further amino acid sequence forming a third domain, wherein the third domain can by one selected from the group consisting of: a secretion signal domain, in particular in the form of an serum albumin preprotein, an Ig kappa chain V-III region MOPC, an IgK H Ig kappa chain V-III region VG precursor or a modified albumin signal SEQ ID 21 , an Fc region of immunoglobulin or a part thereof, albumin, an albumin-binding polypeptide, Pro/Ala/Ser(PAS), a C -terminal peptide (CTP) of the b-subunit of human chorionic gonadotropin, polyethyleneglycol(PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethylstarch (HES), an albumin-binding small molecule, and a combination or a post-translationally modified derivative thereof.

Such common secretion signals can be Serum albumin preproprotein (NPJ30468), lg kappa chain V-III region MOPC (Accession number XP_003514704), or IgK H Ig kappa chain V-III region VG precursor (Accession number P04433) or a modified albumin signal according to Seq ID 21.

Such further linked secretory signal peptides function as sorting signals. Signal peptides are located at the N-terminal of proteins and their length normally ranges between 14-30 amino acids. They have a tripartite structure, consisting of a hydrophobic core, flanked by an N-terminal and C-terminal. During translocation across the endoplasmic reticulum membrane, signal peptidases cleave the signal peptide at its C-terminal resulting in the protein entering the secretory pathway. Changes between 2-A amino acids of the signal peptide sequence can result in new cleavage sites and alter the expression-secretion efficiency, for example of antibody fragments. The fusion of different signal peptides in a target protein may result in the formation of different mRNA transcripts which vary in their stability and secondary structure, thus significantly influencing the amounts of the respective precursor proteins that are produced and secreted.

Said second amino acid sequence can be a fragment crystallizable (Fc) region which is based on immunoglobulin, in particular based on at least one of IgGl, IgG2, IgG3 or IgG4, preferably having a sequence according to any of SEQ ID NO 05 - SEQ ID NO 07 or an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity, wherein the fragment crystallizable (Fc) region can be post-translationally modified.

Said first amino acid sequence can be characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 with at least one of the following or a combination of the following mutations: S27E; S47E; T154E; S159E; S162E; T530E; S540E; S545E; S682E; T719D; K444S; E151 M; D298M; D305M; D348M; R179Y; R394Y; D434M; or D720M. According to yet another preferred embodiment, said first amino acid sequence is characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 and has at least one phosphorylation mutation and at least one of the following sites: S 14, S26, S27, S47, SI 59, SI 62, S172, S173, T344, S442, T530, S538-S540, S535, S538-S540, T544, S545, T719, or S747.

Further preferably, said first amino acid sequence is characterized by at least (>)95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 08.

Said second amino acid sequence can preferably be characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 05 with at least one of the following or a combination of the following mutations: Ll 17V; Ll 18A; P214S; T133Q; or M311L.

Said second amino acid sequence, preferably having a sequence according to any of SEQ ID NO 05 - SEQ ID NO 07, or an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%. >99%, >99.5%, >99.9% sequence identity. Such a system, which can be post-translationally modified, can be linked to the C-terminus and/or the N-terminus of the first amino acid sequence.

According to a further preferred embodiment the fusion polypeptide can be characterised in that between said first amino acid sequence and said second amino acid sequence there is no linker element or there is a linker element, which linker element is preferably a flexible, rigid or cleavable linker element, and which is more preferably selected from at least one of the following systems or a system based on these elements: (GGGGS)n, (G)n, (EAAAK)n, (XP)n, or disulphide, wherein n is in the range of 1-15, preferably in the range of 3-8, and wherein X can be any amino acid, preferably A. Other suitable linkers include: (Gly)5-Ser-(Gly)3-Ser-(Gly)4-Ser, (Gly)4-Ser-(Gly)4-Ser- (Gly)4-Ser, (Gly)3-LyS-(Gly)4, (Gly)3-Asn-Gly-Ser-(Gly)2, (Gly)3-Cys-(Gly)4 and Gly-Pro-Asn. Non-peptide linkers are also contemplated by the present invention. For example, alkyl linkers can be used. These alkyl linkers can further be substituted by any non-sterically hindering group, including, but not limited to, a lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g.. Cl, Br), CN, NH2, or phenyl. An exemplary non-peptide linker is a polyethylene glycol linker, wherein the linker has a molecular weight of 100 to 5000 kD, for example, 100 to 500 kD.

The post translational modification can be preferably selected from the group consisting of phosphorylation, glycosylation, fucosylation, galactosylation, lipidation, lipoylation, acetylation, acylation sulfonylation sulfinylation or sulphenylation, and combinations thereof.

According to a second aspect of the present invention it relates to a dosage form comprising the fusion polypeptide according to any oi the preceding claims tor use in a method of treatment or prevention of a condition selected from the group consisting of mitochondrial-related diseases/disorders, metabolic disorders, neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, dyslipidemia, hyperlipidemia, diabetes, metabolic syndrome, inflammation, apoptosis, neurodegeneration, cancer, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, and obesity- related conditions. The dosage form may be provided for parenteral, peroral, transdermal or transmucosal administration, in particular further including a pharmaceutically acceptable carrier.

According to a third aspect of the present invention it relates to a combination medicament, preferably in a unit dose form, comprising a fusion polypeptide as given above as well as a further therapeutically active or synergistic compound, preferably selected to be a SIRT1 activator or NAD+ supplement, more preferably selected from the group consisting of niacin (vitamin B3), nicotinamide mono-nucleotide, nicotinamide riboside (NR) and resveratrol. Further preferably such a combination medicament, preferably in a unit dose form, is comprising a fusion polypeptide as given above as well as at least one of the following types of medicaments: Inhibitors of a-amino-P-carboxymuconate-e- semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of a-amino-p-carboxymuconate- -semialdehyde in the de novo NAD+ synthesis pathway; Inhibitors of PARP-l ; Inhibitors of CD38; Activators of Nicotinamide riboside kinase 1, 2; Activators of Nicotinamide mononucleotide adenylyltransferase 1 or 2 or 3 (Na/NMNAT- 1,2,3); Activators of Nicotinic acid adenine dinucleotide synthase (NADS), AMPK activators like Metformin.

In one aspect, provided herein are combination therapies for treating, preventing, or managing a metabolic disorder or a cardiovascular disorder comprising administering a therapeutically effective amount of a SIRT1 protein variant described herein (e.g., Fc fusion protein such as mutl SIRTl-Fc, (SEQ ID 14)) and one or more additional therapeutically active agents (e.g., therapeutic agents for metabolic disorders or cardiovascular disorders). Non-limiting examples of other therapeutically active agents lor use in combination with SIRT1 polypeptide variants provided herewith include obesity therapies (e.g., phentermine/topiramate, orlistat, lorcaserin, liraglutide, bupropion/naltrexone), high blood pressure therapies (e.g., diuretics, beta-blockers, alpha- blockers, ACE inhibitors, Angiotensin II Receptor Blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodialators, and combinations), diabetic therapies (e.g., insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonist, DPP-4 inhibitors, glucagon-like peptides, FGF21 variants, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics), NAFLD/NASH and cardiovascular therapies (e.g.. statins, fibrates, aspirin, anticoagulants).

In one aspect, provided herein are combination therapies for treating, preventing, or managing a metabolic disorder or a cardiovascular disorder comprising administering a therapeutically effective amount of an SIRT1 polypeptide variant described herein (e.g., Fc fusion protein such as mut2SIRTl-Fc, (SEQ-ID 28) and one or more therapeutically active agents selected from the following: amiloride (Midamor), bumetanide (Bumex), chlorthalidone (Hygroton), chlorothiazide (Diuril), furosemide (Lasix), hydrochlorothiazide or HCTZ (Esidrix, Hydrodiuril, Microzide), indapamide (Lozol), metolazone (Mykrox, Zaroxolyn), spironolactone (Aldactone), triamterene (Dyrenium), Acebutolol (Sectral), Atenolol (Tenormin), Betaxolol (Kerlone), Bisoprolol (Zebeta), Carteolol (Cartrol), Metoprolol (Lopressor, Toprol XL), Nadolol (Corgard), Nebivolol (Bystolic), Penbutolol (Levatol), Pindolol (Visken), Propranolol (Inderal), Sotalol (Betapace), Timolol (Blocadren), Doxazosin (Cardura), Prazosin (Minipress), Terazosin (Hytrin), Benazepril (Lotensin), Captopril (Capoten), Enalapril (Vasotec), Fosinopril (Monopril), Lisinopril (Prinivil, Zestril), Moexipril (Univasc), Perindopril (Aceon), Quinapril (Accupril), Ramipril (Altace), Trandolapril (Mavik), Norvasc (amlodipine), Plendil (felodipine), DynaCirc (isradipine), Cardene (nicardipine), Procardia XL, Adalat (nifedipine), Cardizem, Dilacor, Tiazac, Diltia XL (diltiazem), Sular (Nisoldipine), Isoptin, Calan, Verelan, Covera-HS (verapamil), Capoten (captopril), Vasotec (enalapril). Prinivil, Zestril (lisinopril), Lotensin (benazepril), Monopril (fosinopril), Altace (ramipril), Accupril (quinapril), Aceon (perindopril), Mavik (trandolapril), Univasc (moexipril), Atacand (candesartan), Avapro (irbesartan), Benicar (olmesartan), Cozaar (losartan), Diovan (valsartan), Micardis (telmisartan), Teveten (eprosartan), Chlorthalidone (Hygroton), Chlorothiazide (Diuril), Hydrochlorothiazide or HCTZ (Esidrix, Hydrodiuril, Microzide), Indapamide (Lozol), Metolazone (Mykrox, Zaroxolyn), Amiloride (Midamor), Bumetanide (Bumex), Furosemide (Lasix), Spironolactone (Aldactone), Triamterene (Dyrenium), Acebutolol (Sectral), Atenolol (Tenomiin), Betaxolol (Kerlone), Bisoprolol (Zebeta, Ziac), Carteolol (Cartrol), Carvedilol (Coreg). Labetalol (Normodyne, Trandate), Metoprolol (Lopressor, Toprol-XL), Nadolol (Corgard), Nebivolol (Bystolic), Penbutolol (Levatol), Pindolol (Visken), Propanolol (Inderal), Sotalol (Betapace), Timolol (Blocadren), fibric acid derivatives, niacin, and omega- 3 fatty acids, fenofibrate, gemfibrozil, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, pramlintide, acarbose (Precose), miglitol (Glyset), metformin, bromocriptine, alogliptin, linagliptin, saxagliptin, sitagliptin, albiglutide (Tanzeum), dulaglutide (Trulicity), exenatide (Byetta), exenatide extended-release (Bydureon), liraglutide (Victoza), nateglinide (Starlix), repaglinide (Prandin), repaglinide- metformin (Prandimet), dapagliflozin (Farxiga), dapagliflozin-metformin (Xigduo XR), canagliflozin (Invokana), canagliflozin-metformin (Invokamet), empagliflozin (Jardiance), empagliflozin-linagliptin (Glyxambi), empagliflozin-metformin (Synjardy), sotagliflozin, tofogliflozin, remogliflozin, luseogliflozin, ipragliflozin, atigliflozin, bexagliflozin, henagliflozin, licogliflozin, glimepiride (Amaryl), glimepiride-pioglitazone (Duetact), glimeperide-rosiglitazone (Avandaryl), gliclazide, glipizide-metformin (Metaglip), glyburide (DiaBeta, Glynase, Micronase), glyburide-metformin (Glucovance), chlorpropamide (Diabenese), tolazamide (Tolinase), tolbutamide (Orinase, Tol-Tab), rosiglitazone (Avandia), rosiglitazone- glimepiride (Avandaryl), rosiglitizone-metformin (Amaryl M),pioglitazone (Actos). pioglitazone-alogliptin (Oseni), pioglitazone-glimepiride (Duetact), and pioglitazone - metformin (Actoplus Met, Actoplus Met XR).

In some embodiments, the sodium glucose transporter (SGLT) inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, ertugliflozin, sotagliflozin, tofogliflozin, remogliflozin, luseogliflozin, ipragliflozin, atigliflozin, bexagliflozin. henagliflozin, licogliflozin, and a pharmaceutically acceptable salt of any of these. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is dapagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is empagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is canagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is ertugliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is licogliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is dapagliflozin or a pharmaceutically acceptable salt thereof. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is empagliflozin or a pharmaceutically acceptable salt thereof. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is canagliflozin or a pharmaceutically acceptable salt thereof. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is ertugliflozin or a pharmaceutically acceptable salt thereof. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is licogliflozin or a pharmaceutically acceptable salt thereof.

In specific aspects, methods provided herein comprising administering an SIRT1 polypeptide variants are for use as an adjunct to diet (e.g., healthy diet, calorie restricted diet), exercise, and/or other lifestyle modifications.

In order to deliver drug, e.g., SIRT1 polypeptides disclosed herein, at a predetermined rate such that the drug concentration can be maintained at a desired therapeutically effective level over an extended period, a variety of different approaches can be employed. In one example, a hydrogel comprising a polymer such as a gelatin (e.g., bovine gelatin, human gelatin, or gelatin from another source) or a naturally-occurring or a synthetically generated polymer can be employed. Any percentage of polymer (e.g., gelatin) can be employed in a hydrogel, such as 5, 10, 15 or 20%. The selection of an appropriate concentration can depend on a variety of factors, such as the therapeutic profile desired and the pharmacokinetic profile of the therapeutic molecule.

Examples of polymers that can be incorporated into a hydrogel include polyethylene glycol ("PEG"), polyethylene oxide, polyethylene oxide-co-polypropylene oxide, co-polyethylene oxide block or random copolymers, polyvinyl alcohol, poly(vinyl pyrrolidinone), poly(amino acids), dextran, heparin, polysaccharides, polyethers and the like.

Another factor that can be considered when generating a hydrogel formulation is the degree of crosslinking in the hydrogel and the crosslinking agent. In one embodiment, cross-linking can be achieved via a methacrylation reaction involving methacrylic anhydride. In some situations, a high degree of cross-linking may be desirable while in other situations a lower degree of crosslinking is preferred. In some cases a higher degree of crosslinking provides a longer sustained release. A higher degree of crosslinking may provide a firmer hydrogel and a longer period over which drug is delivered.

Any ratio of polymer to crosslinking agent (e.g., methacrylic anhydride) can be employed to generate a hydrogel with desired properties. For example, the ratio of polymer to crosslinker can be, e.g., 8:1, 16: 1, 24: 1, or 32: 1. For example, when the hydrogel polymer is gelatin and the crosslinker is methacrylate, ratios of 8: 1 , 16: 1, 24: 1 , or 32: 1 methyacrylic anhydride: gelatin can be employed.

Furthermore the present invention according to a further aspect relates to a method for treatment or prevention of a patient diagnosed or predicted of a condition selected from the group consisting of: mitochondrial-related diseases/disorders, metabolic disorders, neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, dyslipidemia, hyperlipidemia, diabetes, metabolic syndrome, inflammation, apoptosis, neurodegeneration, cancer, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), autoimmune diseases, pancreatitis, renal lipid deposition, and obesity-related conditions using a fusion polypeptide or a dosage form or a combination medicament according to any of the preceding claims in a therapeutically effective amount.

According to yet another aspect the present invention relates to a polynucleotide comprising a gene or cell line expressing a fusion polypeptide according to any of the preceding claims.

Furthermore, according to yet another aspect of the invention, it relates to a method of producing one or more fusion polypeptide as detailed above comprising:

(a) transforming a host cell with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide according to any of the preceding claims; and

(b) causing the host cell to express the fusion polypeptide;

(c) when the fusion polypeptide is expressed in a host cell, the nucleotide sequence encoding the fusion polypeptide may also code for a secretory signal sequence that will permit the polypeptide to be secreted.

(d) subjecting, if needed, a fluid comprising the said fusion polypeptide to Protein A or Protein G affinity chromatography or an ion exchange chromatography, wherein elution is preferably carried out at a pH ranging from 2.8 to 4.5, and the eluate obtained contains the purified fusion polypeptide.

Further embodiments of the invention are laid down in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings.

Fig. 1 shows overexpression of therapeutic SIRT1 increases AKG phosphorylation. HepG2 cells were transfected with empty vector (pcDNA3.1), wild-type S1RT1 (pCMV-wtSIRTl), secretory wild-type SIRT1 (pCMV-sec-wtSIRTl) and secretory therapeutic SIRT1 (pCMV-sec- mutSIRTl) for 24h. Representative western blot of AKT, p-AKT (ser473) and b-actin in HepG2 cell lysates showing an increase in relative p-AKT (ser473) expression in secretory mutant SIRTl transfected cells compared to empty vector, wild-type SIRTl and secretory wild-type SIRTl .

Fig. 2 shows overexpression of therapeutic SIRTl increases ABCA1 and LDL-R expression. HepG2 cells were transfected with empty vector (pcDNA3.1), wild-type SIRTl (pCMV-wtSIRTl), secretory wild-type SIRTl (pCMV- sec-wtSIRTl) and secretory therapeutic SIRTl (pCMV-sec-mut-SIRTl ) for 24h. Representative western blot of ABCA1, LDLR, PCSK9 and b-actin in HepG2 cell lysates showing an increase in LDLR and ABCA1 expression and a decrease in PCSK9 expression in secretory mutant SIRTl transfected cells compared to empty vector, wild-type SIRTl and secretory wild-type SIRTL

Fig. 3 shows overexpression of therapeutic SIRTl protects against LPS-induced cholesterol efflux. HepG2 cells were transfected with empty vector (pcDNA3. l), wild-type SIRTl (pCMV-wtSIRTl), secretory wild-type SIRTl (pCMV-sec-wtSIRTl ) and secretory therapeutic SIRTl (pCMV-sec- mutSIRTl) for 24h. Cells were treated with l OOng/mL LPS for 4h posttransfection. Representative western blot of ABCA1 , LDLR, PCSK9 and b- actin in HepG2 cell lysates showing a significant increase in ABCA1 and a decrease in LDLR expression in secretory mutant SIRTl transfected cells compared to empty vector, wild-type SIRTl and secretory wild-type SIRTL Fig. 4 shows overexpression of therapeutic SIRTl protects against LPS-induced inflammation. HepG2 cells were transfected with empty vector (pcDNA3.l), wild-type SIRTl (pCMV-wtSIRTl). secretory wild-type SIRTl (pCMV-sec-wtSIRTl) and secretory therapeutic SIRTl (pCMV-sec- mutSIRTl) for 24h. Cells were treated with lOOng/mL LPS for 4h posttransfection. Representative western blot oi 1CAM1 , TNFa, p-AKT (ser473), AKT and b-actin in HepG2 cell lysates showing a significant decrease in ICAM1, TNFa and p-AKT expression in secretory mutant S1RT1 transfected cells compared to empty vector, wild-type S1RT1 and secretory wild-type SIRT1.

Fig. 5 shows overexpression of therapeutic S1RT1 protects against LPS-induced apoptosis. HepG2 cells were transfected with empty vector (pcDNA3.1 ), wild-type SIRT1 (pCMV-wtSIRTl), secretory wild-type SIRT1 (pCMV- sec-wtS!RTl) and secretory therapeutic SIRT1 (pCMV-sec-mutSIRTl ) for 24h. Cells were treated with lOOng/mL LPS for 4h post-transfection. Representative western blot of ICAM1, TNFa, p-AKT (ser473), AKT and b-actin in HepG2 cell lysates showing a significant decrease in ICAM1 , TNFa and p-AKT expression in secretory mutant SIRT1 transfected cells compared to empty vector, wild-type SIRT1 and secretory wild-type S1RT1.

Fig. 6 shows recombinant SIRT1 aggregation. Recombinant protein was isolated from CHO-K1 cells transiently expressing SIRTl-his tag and SIRT1 fusion protein. Recombinant protein was treated with and without DTT at the indicated time-points. Samples were separated on an SDS-page gel as indicated. Western blot of samples were probed against anti-SIRTI antibody. Aggregates and monomers were marked based on calculated molecular weight.

Fig. 7 shows recombinant fusion mutant SIRT1 aggregation. Recombinant protein was isolated from CFlO-Kl cells transiently expressing SIRTl -his tag and SIRT1 fusion protein. Recombinant protein was treated with and without DTT at the indicated time-points. Samples were separated on an SDS-page gel as indicated. Western blot of samples were probed against anti-SIRTI antibody. Aggregates and monomers were marked based on calculated molecular weight.

Fig. 8 shows increased cytotoxic effect of secreted SIRT1 and its variants on pancreatic cancer cells. Panc-1 cells were transfected with empty vector (pcDNA3.1), wild-type SIRT1 (pCMV-wtSIRTl). secretory wild-type SIRT1 (pCMV-sec-wtSIRTl ) and secretory therapeutic SIRT1 (pCMV-sec- mutSIRTl) for 24h in a 96-well plate. Cells were treated with 50 pg/mL Gemcitabine and vehicle (PBS) for 24h post-transfection. Cells treated with Triton-X was used as positive control. Representative bar graph of LDH activity in cells following gemcitabine and vehicle treatment. Data presented as percent of positive control.

Fig. 9 shows recombinant SIRT1 and its variants protect neuroblastoma SH-SY5Y cells from MPP⁺ induced necrosis. SH-SY5Y neuroblastoma cells were treated with recombinant wild-type SIRT1 and secretory therapeutic SIRT1 variants for 2h in a 96-well plate. Cells were treated with MPP⁺ iodide and vehicle (PBS) for 2h. (A) Representative bar graph of LDH activity in cells following MPP⁺ iodide and vehicle treatment. Data presented as percent of vehicle treated control. (B) Representative western blots of SH-SY5Y lysates for ABCA1 , phospho-mTOR, LDLR, SIRT1 , p-AKT, p-AMPK and b-actin;

Fig. 10 shows treatment of recombinant SIRT1 and its variants on endothelial activation. Human aortic endothelial cells were pretreated with recombinant SIRT1 and variants (l pg each) for 10 min and co-stimulated with l pg/mL LPS for 3h. Representative western blot of adhesion molecules TNFa, ICAM-1 , VCAM-1 , p-mTOR and b-actin showing a decrease in VCAM-1 and ICAM-1 and a decrease in mTOR phosphorylation upon treatment with SIRT1 and its variants;

Fig. 1 1 shows recombinant SIRT1 and its variants protect human aortic endothelial cells (HAECs) from LPS-induced ROS production. HAECs were treated with recombinant wild-type SIRT1 and therapeutic SIRT1 variants (l pg each) in the presence of LPS for 2h in a 96-well plate. Representative bar graph of ROS activity in cells following LPS and vehicle treatment. Data presented as arbitrary units of luminescence;

Fig. 12 shows recombinant SIRT1 and its variants protect human aortic endothelial cells (HAECs) from TNFa-induced glucose sensitivity. HAECs were treated with recombinant wild-type SIRT1 and therapeutic SIRT1 variants (l pg) for 2h in a 96-well plate. Cells were treated with TNFa and vehicle (PBS) during the 2h. Representative bar graph of glucose uptake in cells following TNFa and vehicle treatment. Data presented as arbitrary units of luminescence;

Fig. 13 shows the effect of recombinant SIRT1 and its variants on primary human differentiated skeletal muscle cells on insulin-induced glucose sensitivity. Differentiated skeletal muscles were treated with recombinant wild-type SIRT1 and therapeutic SIRT1 variants (l pg each) for 2h in a 96-well plate. Cells were treated with vehicle (PBS) and insulin for 30min in the presence of SIRT1. Representative bar graph of glucose uptake in cells following vehicle and insulin treatment. Data presented as percent of positive control;

Fig. 14 shows FPLC chromatogram of recombinant fc fusion SIRT1 ;

Fig. 15 shows FPLC chromatogram of recombinant fc fusion mutant SIRT1 ;

Fig. 16 shows the effect of different proteases on recombinant SIRT1 and activity;

Purified recombinant SIRT1 and fusion variants (5pg/ml each) were incubated with (a) vehicle, (b) cathepsin B, (c) caspase 3, (d) caspase 1 and (e) caspase 7. Representative bar graphs show difference in activity upon incubation with vehicle and different proteases;

Fig. 17 shows that therapeutic recombinant mutant SIRT1 fusion protein treatment reduces epididymal white adipose tissue in NASH mice. Eight-week-old NASH mice were fed a NASH diet for 3 months and treated with recombinant SIRT1 and its fusion protein variants for the last 2 weeks. Representative bar graph shows that mutant SIRT1 fusion protein reduces weight of epididymal white adipose tissue. Weight represented as percent body weight;

Fig. 18 shows that therapeutic recombinant mutant SIRT1 fusion protein treatment improve glucose metabolism in NASH mice. Eight-week-old NASH mice were fed a NASH diet for 3 months and treated with recombinant SIRT1 and its fusion protein variants for the last 2 weeks. Representative bar graph shows that wild-type and mutant SIRT1 fusion protein improve fasting glucose levels in mice. Blood glucose, mmol/L;

Fig. 19 shows that therapeutic recombinant mutant SIRT1 fusion protein treatment improves insulin resistance in NASH mice. Eight-week-old NASH mice were fed a NASH diet for 3 months and treated with recombinant SIRT1 and its fusion protein variants for the last 2 weeks. Representative bar graph shows that mutant SIRT1 fusion protein reduces serum insulin levels; Fig. 20 shows that therapeutic recombinant mutant S1RT1 fusion protein treatment improves insulin resistance in NASH mice. Eight-week-old NASH mice were fed a NASH diet for 3 months and treated with recombinant S1RT1 and its fusion protein variants for the last 2 weeks. Representative bar graph shows that mutant SIRT1 fusion protein reduces serum insulin levels and HOMA-IR, while wtSIRTl-fc increases HOMA-B levels. Fasting insulin and fasting glucose was used to calculate HOMA-IR;

Fig. 21 shows that therapeutic recombinant mutant SIRT1 fusion protein treatment reduces cholesterol in NASH mice. Eight-week-old NASFI mice were fed a NASH diet for 3 months and treated with recombinant SIRT1 and its fusion protein variants for the last 2 weeks. Representative bar graph shows that mutant SIRT1 fusion protein reduces total cholesterol and LDL-cholesterol. LDL, low-density lipoprotein, mmol/L;

Fig. 22 shows that therapeutic recombinant mutant SIRT1 fusion protein treatment reduces serum creatinine levels in NASH mice. Eight-week-old NASH mice were fed a NASH diet for 3 months and treated with recombinant S1RT1 and its fusion protein variants for the last 2 weeks. Representative bar graph shows that mutant SIRT1 fusion protein reduces serum creatinine levels

Fig. 23 shows that therapeutic recombinant SIRT1 fusion protein treatment protects against body weight loss in LPS-induced neuroinflammation model. Six week-old C57B16 mice were challenged with 1 mg/kg EPS on Day 0 and treated with wtSIRTl-Fc and mut2SIRTl-Fc for three days. Mice were weighed before LPS-treatment and on Day 4. Representative bar graph shows that wild-type and mutant SIRT1 fusion proteins prevent LPS- induced weight loss.

Fig. 24 shows that therapeutic recombinant S1RT1 fusion protein treatment protects against LPS-associated anhedonia. Six week-old C57B16 mice were challenged with lmg/kg LPS on Day 0 and treated with wtSIRTl-Fc and mut2SIRTl-Fc for three days. Sucrose preference was determined at 15h and 21 h post-LPS injections. Representative bar graph shows that mut2SIRTl-Fc treatment protects against LPS-associated anhedonia at 15h, while wtSIRTl-Fc protects at 21 h.

Fig. 25 shows the effect of SIRT1 treatment on LPS-induced NFkB activation in neuronal SH-SY5Y cells. Cells were treated with lOOng/mL LPS for 6h. Representative western blot of ac-p65, p65 and b-actin in SH-SY5Y cell lysates showing a significant increase in acetylated p65 in wild-type SIR. G 1 , wtSIRTl-Fc and mut2SIRTl-Fc compared to vehicle treated control and untreated controls.

Fig. 26 shows the effect of SIRT1 treatment on NAD+ levels in cell skeletal muscle cells. Primary skeletal muscle cells were treated with l pg/ml recombinant SIRT1 and its therapeutic variants for 4h. Representative bar graph shows that treatment of cells with recombinant SIRT1 or its therapeutic variants increases NAD+ levels in cells compared to vehicle treated controls

Fig. 27 shows the effect of cysteine mutations on SIRT1 activity. SIRT1 activity assay was performed using wild-type SIRT1 and different cysteine mutants. Representative bar graph shows that mutation of cysteine with alanine at C160, C268. C374 and C501 increases activity compared to recombinant SIRT1, while mutants at C502, C574, C623 and C671 have reduced activity compared to recombinant SIRT1.

DESCRIPTION OF PREFERRED EMBODIMENTS

List of mutations on SIRT1 :

Fc fragment mutations:

Explanation of SIRTl mutations: S27E

Serines 27 and 47, both residues were identified by mass spectrometry as putative S1RT1 phosphorylation sites. The high levels of SIRT1 protein in cancer reflect abnormal stabilization of the SIRT1 protein and that this was proposed to be mediated via JNK2- dependent phosphorylation of SIRT1 at S27. JNK2-dependent phosphorylation of SIR 1 1 at S27P stabilizes the SIRT1 protein.

S47E

Cyclin-dependent kinase 5 (CDK5) promotes SIRT1 S47 phosphorylation and endothelial senescence. CDK5 acts as an upstream kinase and promotes S47 phosphorylation. Hyperphosphorylation at this residue abolishes the anti-senescent and anti-inflammatory activity of SIRT1 in endothelial cells. Mutation of this single amino acid residue to the phospho-mimetic form S47D abolishes the antisenescence and anti-inflammatory activity of SIRT1 (However, based on results and experiments this may not be universally true), whereas replacing the serine residue with non-phosphorylable alanine enhances the antisenescence and growth-promoting effects of this protein. Hyperphosphorylated S1RT1 is accumulated mainly in the nucleus and shows a distinctive pattern of intranuclear localization. The non-phosphorylable mutant (S47A) SIRT1 is found to be around the nuclear rim and is widely dispersed throughout the cytoplasm. JNK1 also phosphorylates mouse SIRT1 protein in vitro and in vivo at Ser46 (Ser47 in human SIRT1 ). The phosphorylation induces SIRT1 enzyme activity and then induces protein degradation for SIRT1. Phosphorylation by mammalian target of rapamycin complex 1 (mTORCl) at Ser- 47 was found to inhibit SIRT1 deacetylation activity.

T154E

Phosphorylation of SIRT1 at T154 improves its binding to cytochrome C and modulates cell apoptosis.

S159E

Evolutionary similarity exists between SIRT1 and SIRT3 for this site. Studies show that phosphorylation of S1RT3 at this site activates SIRT3.

S162E

Analysis of phosphorylation sites shows that DNAPK has a binding site for S1RT1 at S162. DNA-PK is known to be activated in response to many types of DNA damage treatment, including IR, CPT and UV. It is required for non-homologous end joining (NHEJ) pathway of DNA repair. Mice deficient in the catalytic subunit of DNA-PK have a shorter lifespan and show an earlier onset of numerous aging related pathologies than corresponding wild-type littermates. The murine S154 refers to human S162. The protein kinase CK2 phosphorylation sites have been identified in murine SIRT1 , including SI 54, S649, S651, and S683. Given the fact that the CK2 activity was decreased in aging mice, the declined SIRT1 function in aging mice was due to compromised phosphorylation at S154. Old mouse endothelial cells (MECs)-mediated compromised SI 54 phosphorylation, reduced estrogen receptor- beta (ER expression, and the subsequent endothelial dysfunction in aging mice. Reduced ER expression is due to compromised phosphorylation of amino acid S154 in SIRT1, and single-mutant SIRT1-C152(D) restores this effect in aging mice. A single-mutant SIRT1-C152(D) restored the reduced ERP- expression in the endothelium with minimized reactive oxygen species generation and DNA damage and increased mitochondrial function and fatty acid metabolism. It also restored the association of SIRT1 with PPARy in old MECs. In high-fat diet aging mice, the endothelium-specific delivery of ERP or SIRT1-C152(D) on the vascular wall reduced the circulating lipids with ameliorated vascular damage, including the restored vessel tension and blood pressure.

S172A

In murine a phospho-defective S164A-SIRT1 mutant promotes fatty acid oxidation and improves glucose metabolism. A murine phospho-mimetic S164D-SIRT1 fails to provide beneficial effects. Furthermore, SIRT1 is hyperphosphorylated at S164 (humans SI 72) in obese fatty livers.

T344E

Phosphorylation of SIRT1 at T344 improves substrate selectivity. AMPK phosphorylates SIRT1 at T344. The phosphorylation modification prevents SIRT1 from deacetylating p53 and inhibiting it’s activation in cancer cells. Phosphorylation however, prevents the binding of DBC1 to SIRT1 and modulates lipid metabolism in cells.

S442E

Metformin binds to SIRT1 at S442 and phosphorylated SIRT1. Mutation at S442 significantly enhances deacetylase activity of SIRT1 and leads to an increase fatty acid utilization independently of changes in NAD+ concentration.

T530E

T530 of human SIRT1 (equivalent to T522 of mouse SIRT1) is a target of cyclin B/cdkl and JNK1 kinase. Human SIRT1 is phosphorylated by JNK1 on three sites: Ser27, Ser47, and Thr530 and this phosphorylation of SIRT1 increases its nuclear localization and enzymatic activity. Triple mutation of SIRT1 at S27, S47 and T530 to alanine (Mt-SIRTl) shows no significant difference in p53 acetylation in cells transfected with either WT- hSIRT or Mt-SIRTl when treated with H202 (to induce oxidative stress), indicating no dependence on SIRT1 phosphorylation state. However, expression of Mt-SIRTl causes only a modest decrease in acetylated H3 (Ac-H3) demonstrating substrate specificity for this response. In addition, treatment of cells with H202 resulted in a significant decrease in Ac-H3 and this effect was blocked in the presence of Mt-SIRTl , even in the presence of JNK activator. Furthermore, lack of phosphorylation of SIRT1 (shown by addition of JNK inhibitor) prevents its nuclear localisation only during H202 stimulation.

Two anti-apoptotic members of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family, DYRK1 A and DYRK3, directly bind and phosphorylate SIRT1 on threonine 522. DYRK family of protein kinases have been increasingly recognized as key regulators of cell proliferation and apoptosis. SIRT1 is selectively activated by the pro- survival DYRK family members through phosphorylation at Thr522 (mouse equivalent of human T530). Introduction of a negatively charged phosphate group at Thr522, a site that localizes within a conserved hinge region of SIRT1 linking the core and C-terminal domains, likely induces an open conformation. This conformational change does not affect the intrinsic catalytic ability of SIRT1. Instead, it repositions the inhibitory C-terminal domain, increasing the release of deacetylated products after the reaction is complete, thereby enhancing catalytic turnover and subsequent rounds of activity. Furthermore, an increase in SIRT1 activity is found, demonstrated by an increase in deacetylation of p53, and also inhibits cell apoptosis. In cancer cells expressing wild-type p53, S1RT1 promotes cellular senescence and limits cell proliferation. However, in cells containing mutant p53, overexpression of SIRT1 promotes cell survival through p53-independent mechanisms. T530-pSIRTl was found to bind origins and interact with a group of replication-associated proteins including MCM2, DDK, ORC2, RPA1 and PCNA.

Phosphorylation of SIRT1 at T522 inhibits adipogenesis in vitro. Dephosphorylation of SIRT1, thereby reduction in SIRT1 activity, is required for normal adipogenesis in vitro. Phosphorylation of SIRT1 at T522 enhances hepatic fatty acid oxidation, causes a mild reduction in total body fat and protects mice from high-fat diet induced dyslipidemia and hepatic steatosis.

S540 S1RT1 is phosphorylated by Cyclin B/Cdkl at T530 and S540. No eflects ot the T530A/S540A double mutant on deacetylase activity of S1RT1 using Fluor-de lys assay were observed. However, wild-type but not T530A/S540A double mutant, rescued the slower growth rate and the deficit in S-phase of the cell cycle, which were observed in SIRT1-/- embryonic stem (ES) cells, suggesting phosphorylation at T530 and S540 is required of ES cell proliferation.

S545E

SIRT1 is described to be phosphorylated at S545. S1RT1 has recognition motifs for Cdkl , Cdk5, ERK1, GSK3, IKK, PDK, PKA at S545.

S682E

SIRT1 is phosphorylated at Ser682 by Homeodomain-interacting protein kinase 2 (H1PK2). Ser682 phosphorylation of SIRT1 negatively regulates the interaction of SIRT1 with its endogenous activator, Active regulator of SIRT1 (AROS) and modulates the acetylation status of p53. In consequence, severe DNA damage, triggers SIRT1 Ser682 phosphorylation and results in the dissociation of the SIRT1-AROS complex. Loss of SIRT1-AROS interaction is expected to result in a drop in SIRT1 activity, thereby facilitating efficient p53 acetylation upon DNA damage.

T719D

RE Gy regulates hepatic cellular steatosis in a SirTl- and autophagy-dependent manner. Ubiquitin-independent REGy-proteasome and autophagy-lysosome systems are major proteolytic pathways. REGy inhibits SirTl -induced conversion of LC3-I to LC3-II, thus inhibits SIRT1 -induced autophagy. SirTl and autophagy are both required for the protective effect against hepatic steatosis in REGy-deficient mice. Glucose deprivation in HepG2 cells were shown to stimulate SirTl phosphorylation at T530 and T719, but not SI 4. AICAR (AMPK activator) treatment was shown to decrease REGy-SirTl binding and a concomitant increase in SirTl phosphorylation at both, T530 and T719. SirTl mutation in T530D but not in T719D has been shown to decrease SirTl-REGy interaction, while the phospho-mimetic SirTl-T530D mutant had an increased association with Atg5, thus activating autophagy. Glucose starvation stimulates SirTl phosphorylation mainly at T530 via AMPK, and this modification causes dissociation of SirTl -REGy coupled with increased association of SirTl- Atg5/7, contributing to liberation of SirTl to activate autophagy.

NAD+ mutation K444S

Mutation (K444) at a conserved residue on SIRT1, made the protein active in low NAD+ conditions. For example, in the presence of NAD+, K444E S1RT1 mutant is catalytically more active than WT (higher kcat) and has a significantly lower Km for both substrates. Mutant K444E is much more resistant to inhibition by SIRT1 inhibitor, suramin (IC50 ot 68.02 mM), compared to wild-type SIRT1 (IC50 of 2.99 mM). It also causes a minor increase in sensitivity to nicotinamide - inhibition of mutant SIRT1 (IC50 of 34.27 mM) by nicotinamide compared to wild-type SIRT1 (IC50 of 43.05 pM). Furthermore, mutant SIRT1 protein has increased thermostability i vitro and is also significantly more stable in cells than WT protein. Arginine 275 was proposed to form a bond with mutant K444E (charge-charge interaction) and increase the catalytic cleft stability. Thus the lysine group is replaced with serine since serine can also form a hydrogen bond with arginine at 275 and improve protein stability.

The analysis of the mutants predicts a decreased DDQ, suggesting increased rigidity of the catalytic core. Increased catalytic site rigidity can enhance stability and catalysis of some enzymes. Using SIRT1 structure (PDB 4i5i) and ELASPIC to determine DDΰ of SIRT1 I received the following results - for K444Q was 0.732, for K444E was 0.159 and for K444S is 0.248 suggesting that K444S improves rigidity of the catalytic core.

Cleavage sites

As per Expasy peptide cutter algorithm and using SIRT1 amino acid sequence - R179, R394, D434 and D720 were predicted to be cleavage recognition sites for some proteases. R179 and R394 were predicted to be cleavage sites for thrombin and were mutated to tyrosine (Y) to prevent thrombin cleavage.

Aspartic acid, D434 was predicted to be the cleavage site for caspase-3/caspase-7, while D720 was predicted to be the cleavage site for caspase-1. A study describes the cleavage site of SIRT1 in mice (D142A, D426A and D710A). Human S1RT1 is not cleaved by any enzyme at D 142. The mutant SIRT1 D142A was resistant to in vitro caspase-l cleavage, demonstrating that this is the cleavage site recognized by caspase-l .

Using the concept of graph-based structural signatures to study and predict the impact ot single-point mutations on protein stability, we used Cutoff Scanning Matrix (mCSM) algorithm which encodes the distance patterns between atoms to represent protein residue environments. We used the crystal structure PDB 5btr and PDB 4zzh to assess the effect ot point mutations on all the residues of SIRT1 within PDB 5btr and 4zzh crystal structure. Based on these analysis we identified point-mutations which do not affect the stability of the protein and which can be substituted to prevent SIRT1 cleavage. For example, the amphipathic Arginine has an aromatic side chain can interact directly with the side chain of the polar amino acid Tyrosine. Thus given that arginine mutation to tyrosine can improve protein stability as per mCSM and arginine can interact with tyrosine, we mutated R179 and R394 to Y.

Crystal structure of SIRT1 used for in silico analysis.

Stability in silico analysis of SIRT1 using PDB 5BTR - Crystal structure of SIRT1 in complex with resveratrol and an AMC-containing peptide Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol (2015) Genes Dev. 29: 1316-1325; PubMed: 26109052; DOI: 10.1 10 l/gad.265462.1 15

Stability in silico analysis of SIRT1 using PDB 4ZZH - Crystal structure of SIRT1 SIRT1 /Activator Complex Crystallographic structure of a small molecule SIRT1 activator- enzyme complex. Dai, H., et. A1 (2015) Nat Comrnun 6: 7645-7645 ; PubMed: 26134520 DOI: l0.l038/ncomms8645

Stabilizing in silico determined mutations:

R179Y;R394Y; D434M

Mutations to improve stability of SIRT1

In silico analysis of SIRT1 was performed using crystal structure PDB 5BTR and 4ZZH and mCSM algorithm. A predicted AAG >1.8 was used as a cut-off for selecting a highly stable mutation: E151M; D298M; D305M; D348M.

Results from experiments:

Sirtuin 1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD+)-dependent deacetylase enzyme. Caloric restriction causes an increase in SIRT1 activity. Furthermore, S1RT1 transgenic overexpressing mice have a caloric-restriction like phenotype. Recent studies have shown reduced plasma S1RT1 levels in patients with non-alcoholic tatty liver disease. Alternatively, caloric restriction has also been shown to increase plasma SIR! 1 levels in healthy women. To assess the therapeutic effect of circulating SIR I 1. we have overexpressed wild-type SIRT1 protein with a secretory signal, an active mutant SIRTl -fc fusion protein (therapeutic SIRT1) with a secretory signal and compared its effects to overexpression of intracellular SIRT1 and an empty vector control in HepG2 cells. SIRT1 is known to deacetylate and activate PPARy. PPARy-dependent AKT phosphorylation in HepG2 cells causes an increase in glucose tolerance and insulin sensitivity. We thus investigated if therapeutic SIRT1 caused an increase in AKT phosphorylation and its activation. Indeed, therapeutic SIRT1 caused an increase in AKT phosphorylation at Ser473 compared to intracellular SIRT1, secretory S1RT1 and empty vector control (figure 1), suggesting an increase in insulin sensitivity in liver cells.

ATP-binding cassette transporter (ABC) A1 plays a critical role in cholesterol efflux. ABCA1 mediates the efflux of cellular cholesterol from cells to nascent HDL particles. Intracellular SIRT1 is a positive regulator of LXR and is known to increase ABCA1 protein expression. We therefore investigated if secreted SIRT1 increases ABCA1 expression. Both secreted wild type SIRT1 and therapeutic SIRT1 caused an increase in ABCA1 expression compared to overexpression of intracellular S1RT1 and empty vector control (figure 2). However, overexpression of therapeutic SIRT1 caused substantially higher levels of ABCA1 expression indicating an improved activity compared to wild-type SIRT1. In line with this we also observed an increase in LDLR expression and a decrease in PCSK9 protein expression. Thus therapeutic SIRT1 demonstrates a potent anti- dyslipidemic effect in HepG2 cells.

In macrophages, ABCA1 is known to modulate the removal of lipopolysaccharide and accelerate cell recovery. Furthermore, ABCA1 is also known to reduce LPS-dependent inflammatory effects. Additionally SIRT1 is known to decrease inflammation through an increase in p65 deacetylation. To assess if therapeutic SIRT1 could reduce LPS-dependent inflammatory effects, HepG2 cells overexpressing different variants of SIRT1 were incubated with LPS for 4h. Cells overexpressing therapeutic SIRT1 showed a significant increase in ABCA1 compared to secretory wild-type SIRT1, intracellular SIRT1 and empty vector control (figure 3). Correspondingly, LDLR is responsible for the uptake of LPS. We found that cells expressing therapeutic SIRT1 showed a minor decrease in LDLR expression with no change in PCSK9 expression. Indeed, we found that therapeutic SIRT1 caused a significant decrease in TNFa and ICAM1 expression compared to empty vector control indicating an anti-inflammatory effect (figure 4). Similar to therapeutic SIRT1, we found intracellular and secretory SIRT1 also to cause a decrease in TNFa and ICAM1 indicating a similar anti-inflammatory effect of SIRT1 upon LPS stimulation. Since LPS is known to cause an increase in AKT phosphorylation causing an increase in inflammatory signals, we assessed if therapeutic SIRT1 prevents AKT activation. Indeed, western blot analysis showed that upon LPS stimulation therapeutic SIRT1 prevents the activation of AKT by reducing its phosphorylation at ser473. Thus suggesting a mechanism through which therapeutic SIRT1 could modulate LPS-induced inflammation.

LPS is known to induce apoptosis in different types of cells. We assessed the effect of therapeutic SIRT1 on LPS-induced apoptosis in HepG2 cells. Similar to empty vector control, intracellular S1RT1 overexpression caused an increase in expression of pro- apoptotic protein BAX, with no change in expression of anti-apoptotic BCL2 protein or anti-oxidant SOD2 protein, indicating an increase in apoptosis upon LPS-stimulation (figure 5). Intracellular SIRT1 caused a decrease in cytochrome c levels compared to empty vector control. Overexpression of secreted wild-type SIRT1 caused an increase in Cytochrome c with no change in BAX/BCL2 ratio or SOD2 expression compared to empty vector control indicating no change in apoptosis. However, unlike intracellular and secretor wild-type SIRT1, expression of therapeutic SIRT1 caused an increase in BCL2 and a decrease in BAX expression indicating an anti-apoptotic effect in FlepG2 cells upon LPS stimulation. Furthermore therapeutic SIRT1 caused a significant increase in SOD2 expression compared to intracellular SIRT1, secreted wild-type S1RT1 and empty vector control.

Secretory form of SIRT1 forms aggregates during production

For large-scale production of recombinant SIRT1 and Fc-SIRTl, a secreted form of the protein is highly desirable. To produce a secretory form of S1RT1 , a signal peptide was added in S1RT1 sequence at the N-terminal. However, attempts to express high levels of recombinant SIRT1 or Fc-SIRTl in CHO cells or HEK293E cells consistently resulted in low yields. To assess if the reduced yields were a result of protein misfolding or formation of disulfide bonded aggregates, we performed SDS-page analysis and western blotting in the presence and absence of reducing agent, 1 ,4-dithiothreitol (DTT). Isolated samples were incubated and heated at 95 degrees at different time-points in DTT. Western blot was performed using an antibody targeting the C -terminal of SIR^'l 1. Western blot analysis showed that recombinant SIRT1 incubated in laemmeli buffer without D 1 1 expressed as an aggregate in solution (figure 6). Incubation of SIRT1 in DTT without any heating resulted in disaggregation of SIRT1 (as observed in lane 3) and recombinant SIRT1 being expressed as a monomer. Of note, secreted SIRT1 appearance as a double band may be due to protease cleavage or insufficient gylcosylation. Prolonged heating of recombinant SIRTlat 5mins, lOmins and 20mins resulted in the expression of only the SIRT1 monomer and a saturation of the disaggregation effect of DTT. Thus suggesting that recombinant SIRT1 forms a multimer (or aggregate) due to the formation of disulfide bridges at cysteine residues which can be disaggregated by reducing the cysteine groups with DTT. Similarly, Fc-SIRTl was expressed as an aggregate which could be separated on an SDS- page gel and disaggregated upon incubation with DTT. Heating the samples at different times points caused an additional disaggregation and formation of Fc-SIRTl monomer and Fc-SIRTl homodimer (figure 7).

Currently, the crystal structure of mammalian SirTl is unsolved. The lack of a definitive crystal structure of the full-length of SIRT1 makes it difficult to identify which of its 19 total cysteine residues might be responsible for disulfide bridge formation, thus making it susceptible to protein aggregation. However, the solvent accessibility of SIRT1 regions have been partly described by deuterium exchange combined with mass spectroscopy. Using in silico analysis with NetSurfP 1.1 for solvent accessibility prediction and existing deuterium exchange mass spectrometry data, we identified 10 surface-exposed cysteine residues (Table 1 and Table 2). Cys374 is present in the Zinc binding domain and its mutation to serine to form a non-reducible mutant has been shown to reduce SIRT1 deacetylase activity. Alternatively, an increase in reduction of thiol groups at Cys374 by APEl/Ref-1 has been shown to increase SIRT1 deacetylase activity. To assess the effect of surface-exposed cysteine on SIRT1 protein aggregation and its deacetylase activity, we performed site-directed mutagenesis of each site.

Table 1. List of cysteine residues identified at surface of SIRT1 structure using NetSurfP

Table 2. List of cysteine residues identified at surface of SIRT1 structure identified by deuterium exchange mass spectrometry

Effect of therapeutic recombinant SIRT1 on pancreatic cancer in vitro

SIRT1 plays an important role in human malignant progression, inducing cancer cell proliferation and metastasis by regulating downstream gene expressions. SIRT1 expression is known to be significantly upregulated in pancreatic cancer tissues and cell lines and promotes proliferation of pancreatic cells. However, contrastingly, activation of SIRT1 by SIRT1 agonists were effective in inhibiting pancreatic tumor xenograft growth in vivo and inhibited cell survival of cancer cells in vitro. Thus we explored the effects of intracellular and extracellular SIRT1 on pancreatic cancer using Panc-l cells in vitro. Overexpression of intracellular wild-type SIRT1 showed a trend towards a decrease cytotoxicity as shown by an increase in LDH activity (fig. 8). However, overexpression of secretory wild-type SIRT, secretory Fc-SIRTl and secretory Fc-mutSIRTl showed a significant increase in cytotoxicity compared to empty vector control. Overexpression of secretory Fc-mutSIRTl showed substantially higher levels of cytotoxicity compared to secretory wild-type SIRT and secretory Fc-SIRTl as well. Interestingly, treatment of Panc-l cells with 50 pg/mL Gemcitabine caused an increase in cytotoxicity in empty vector control transfected cells compared to vehicle treated cells. However, overexpression of secretory wild-type SIRT1, secretory Fc-SIRTl and secretory Fc-mutSIRTl showed a similar increase in cytotoxicity in Gemcitabine treated cells as vehicle treated cells, suggesting an upstream effect of SIRT1 its cytotoxic effect. Thus providing evidence that treatment ot cells with wild-type SIRT1 and its therapeutic variants, specifically Fc-mutSIRTl can be used as a therapeutic candidate to treat pancreatic cancer.

Effect of therapeutic recombinant SIRT1 on neurodegeneration in vitro models

Accumulating evidence indicates that increased production of amyloid is the primary event that leads to the formation of amyloid plaques in Alzheimer's disease (AD). Therefore, we focused on the effect of amyloid peptide Ab1-42 -induced cytotoxicity using differentiated SH-SY5Y cells as an in vitro model system. Amyloid beta is shown to have no remarkable toxic effect on undifferentiated SH-SY5Y cells, however, the viability of the neuron-like differentiated SH-SY5Y cells is significantly decreased by the amyloid beta 1-42 peptide treatment. Thus the effect of unaggregrated and oligomeric amyloid beta 1 -42 peptide was assessed in the presence recombinant SIRT1 and its variants in differentiated SH-SY5Y cells.

RA-differentiated cells are less vulnerable than undifferentiated cells to toxin-mediated cell death induced by agents including l-methyl-4-phenyl-l ,2,3,6-tetrahydropyridine (MPTP), or its metabolite, 1 -methyl-4-phenylpyridinium ion (MPP⁺). Thus we assessed the effect of MPTP metabolite, MPP⁺, on undifferentiated SH-SY5Y cells in the presence of recombinant SIRT1 and its therapeutic variants. Treatment of undifferentiated SH-SY5Y cells with MPP⁺ iodide caused an increases in necrosis as assessed by ApoTox-Glo assay. Cells treated with recombinant wild-type SIRT1, wild-type Fc-SIRTl and therapeutic Fc- mutSIRTl protected against MPP⁺-mduced toxicity compared to vehicle treated control (fig. 9A). Western blot of cells treated with recombinant proteins in the presence and absence of MPP⁺ for 2h found no cleavage of caspase 3, suggesting MPP⁺ treatment caused a non-apoptotic-induced cell death (fig. 9B). Treatment of cells with recombinant wild-type SIRT1 and its therapeutic variants caused an increase in LDLR and ABCAl expression, predominantly in Fc-mutSIRTl treated cells suggesting that treatment with therapeutic Fc-mutSIRTl would protect against Alzheimer’s disease. Studies have shown that LDLR overexpression dramatically reduces Ab aggregation and enhances Ab clearance from the brain extracellular space. Furthermore, Ab deposition increases in the absence of ABCAl in mouse models of Alzheimer’s disease while overexpression of ABCA1 decreases amyloid deposition. Interestingly, treatment of cells with MPP⁺ caused an increase in LDLR and ABCA1 expression compared to vehicle-treated controls. Epidemiological studies show that patients with single-nucleotide polymorphism in ABCA1 have an increased risk to Parkinson’s disease. Treatment with recombinant wild- type SIRT1 and its therapeutic variants in the presence of MPP⁺ caused a decrease in LDLR and ABCA1 expression showing a cytoprotective effect specific to MPP treatment. Since SIRT1 is proposed to mediate the effects of caloric restriction, we assessed the expression of components of the intracellular energy sensing pathways, specifically mTOR and its principal up-stream regulators, AMPK and Akt. Western blot analysis revealed that cells treated with recombinant wtSIRTl and its variants caused an increase in p-AMPK expression compared to vehicle-treated controls (fig. 9B). The PI3K/Akt pathway has been implicated in SH-SY5Y cell survival and its dysregulation has been observed in Parkinson’s disease, in both in vivo and in vitro models. Akt is a major promoter of neuron survival and is negatively associated with dopaminergic neurodegeneration in Parkinson^'s disease. mTORC2 activity is known to decrease with age in flies and increase long-term memory and is primarily responsible for phosphorylation of AKT at ser473. Unlike vehicle-treated, wild-type SIRT1 or wild-type Fc-SIRTl, treatment of cells with therapeutic Fc-mutSIRTl caused a significant increase in AKT (ser473) phosphorylation suggesting an activation of mTORC2. Correspondingly, inhibition of mTORCl signaling in Parkinson’s disease prevents L-DOPA-induced dyskinesia. In line with this we found a decrease in phosphorylation of mTORCl at ser2448 in cells treated with Fc-mutSIRTl suggesting an inhibition of mTORCl activation. Thus therapeutic Fc-mutSIRTl represents a promising candidate for treatment of Parkinson’s disease through an increase in mTORC2 activity. This was further confirmed with the addition recombinant SIRTT and therapeutic variants on SH-SY5Y cells in the presence of MPP⁺ and vehicle control. In the presence of MPP⁺, SH-SY5Y cells have been shown to cause phosphorylation of AMPK. In line with this, western blot analysis showed that MPP⁺ caused an increase in phosphorylation of AMPK compared to vehicle treated control, however, treatment with recombinant wild-type SIRT1 and wild-type Fc-SIRTl caused an increase in phosphorylation of AMPK, AKT and activation of mTORCl . In contrast, treatment of cells with therapeutic Fc-mutSIRTl caused a reduction in phosphorylation of mTORCl, AKT and AMPK in the presence of MPP⁺. Materials and methods:

Cell culture

Human hepatoma, HepG2 cells, mouse macrophages, RAW 247.6 cells and pancreatic cancer, Panc-l cells were purchased from EACC (Sigma). Cells were maintained in complete DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma), glutamax and nonessential amino acids in a humidified incubator with 5% C02 and 95% air at 37oC. The medium was refreshed every 2 days.

SH-SY5Y human neuroblastoma cells were purchased from EACC (Sigma). Cells were maintained in complete culture medium consisting of Ham's F 12 and Eagle^'s minimum essential medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), glutamax and nonessential amino acids in a humidified incubator with 5% C02 and 95% air at 37°C. The medium was refreshed every 2 days.

Primary human aortic endothelial cells pre-screened for VEGF and angiogenesis signaling were purchased from Cell applications (Sigma) and cultured in endothelial growth medium (Sigma).

Differentiation of SH-SY5Y cells

For differentiating SH-SY5Y cells into a neuronal phenotype, cells (density 25,000 cells/cm2) were seeded in complete DMEM/F12 medium, glutamax and nonessential amino acids containing 10 mM RA for three days in subdued light . At the third day, cells were then exposed to 10 mM RA and 50 ng/mL BDNF in DMEM/F12 medium and kept for another three days. After the 6-day differentiation protocol, the cells were exposed to different stimulants.

Induction of Insulin Resistance in HepG2 Cells

HepG2 cells were seeded on 24- well plates at 1 ^c 105 cells/well and incubated for 24h to reach maximal confluence. The cells were then incubated for 24h in serum-free DMEM containing 25 mmol/L d-glucose and 1 ^c 10-9 mol/L insulin. Cells were treated with 5pg recombinant SIRT1 and its therapeutic variants for atleast lh.

Neurite morphology analysis

SH-SY5Y cells were plated in 24-well plates at a density of 1 * 105 cells/mL. After incubation for 24h, the medium was refreshed with RA (10 mM) for 3 days, followed by BDNF (50ng/mL) and recombinant SIRT1 and its variants. After day 2 the medium was changed daily for 3 more days. After incubation for 5 days, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 3 min, washed with PBS, stained with Coomassie Brilliant Blue, and washed with PBS. The morphological changes in the cells were observed under a phase-contrast microscope. Those cells whose cell body diameters longer than twice of the diameter ot cell body were considered as neurite- bearing cells. The percentage of the cells with neurites in a particular culture was determined by counting at least 200 cells in each sample.

Stimulants for cell culture

Human aortic endothelial cells and human hepatoma HepG2 cells were treated with TNFa at a final concentration of 10 ng/mL and lipopolysaccharide of 100 ng/mL for 24h. Pancreatic cancer, Panel cells, were treated with Gemcitabine at final concentration of 50 pg/ L for 24h. Neuroblastoma, SH-SY5Y cells, were treated with MPP iodide (Sigma). A final concentration of MPP+ (1 mM) was used to treat SH-SY5Y cells with or without differentiation. Mouse macrophages, RAW 264.7 cells, were treated to a final concentration of 10 pg/mL of ox-LDL for 24h. For all experiments, cells were treated with recombinant SIRT1 and/or its therapeutic variants drug at a final concentration of atleast 5 pg/mL.

Site directed mutagenesis

The cloned SIRTl construct was digested with Hindlll and BamHI restriction enzymes (New England Biolabs) and inserted into a digested pDSG-IBAwtl vector using a T4 ligase kit (Promega) according to the manufacturer's instructions. For site-directed mutagenesis of the cysteine residues within the SIRTl constructs, we used the Q5 Site- Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. Primers were used as listed in Table 3.

Protein production

The SIRTl constructs were efficiently expressed in suspension-adapted CHO-K1 cells by Evitria AG, Zurich, Switzerland, using an Evitria expression vector system. The cell-seed was grown in eviGrow medium, a chemically defined, animal-component free serum-free medium. Cells were transfected with eviFect, Evitria’ s custom-made, proprietary transfection reagent, and cells grown after transfection in eviMake2, an animal-component free, serum-free medium. Recombinant and fusion protein were isolated from supernatant. Recombinant SIRTl his-tag protein were purified via poly-histidine tag using immobilized metal affinity chromatography (GE), while Protein-A affinity chromatography (GE) was adopted for the SIRTl -Fc purification.

In HEK293E cells - The SIRTl constructs were cloned into pDSG-IBAwtl and pDSG- IBAwt2 vectors. Cells were seeded at a density of 1 *10⁶ cells/ml. After 24h, transfection was performed using a cationic polymer - polyethylenimine (PEI). Transfection was carried out at a concentration of 1.5mg/l plasmid DNA with the addition ol PEI at a 1 :3 ratio. 48h post-transfection hydrolysate feeds were added at a concentration oi 0.5% with volume doubling. After 72h post-transfection glucose feed and sodium valproic acid was added with a temperature shift to 32°C. Cells were cultured until cell viability reached 75%. Recombinant and fusion protein were isolated from supernatant. Recombinant SIRT1 his-tag protein were purified via poly-histidine tag using immobilized metal affinity chromatography (GE), while Protein-A affinity chromatography (GE) was adopted for the SIRTl-Fc purification.

Western blot

Total protein from tissues and cells was prepared as described and analysed using RIPA lysis buffer. Whole cell protein lysates were extracted using Laemmli SDS lysis buffer supplemented with protease/phosphatase inhibitor mixture. Lysates were sonicated for lOs prior to quantification using Bradford protein assay (Pierce). For each sample, 30 pg of protein were separated on SDS-PAGE prior to western blotting using either ECL substrate kit (Bio-rad).

LDIT assay

LDH released from cells was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). Cells were plated in 96-well plates at a density of 2 x 104 cells per well and treated with recombinant SIRT1 and therapeutic variants at a concentration of 250 ng/mL for atleast 1 h, 2h or 24h. The supernatant was collected to measure LDH release according to the manufacturer’s instructions. Supernatant of cells treated with 10% Triton- X was used as positive control. Supernatant from wells with no cells was used as negative control.

Caspase 3/7 activity and necrosis assay

Cells were plated in 96-well plates at a density of 2 x 104 cells per well and treated with recombinant SIRT1 and therapeutic variants at a concentration of 250 ng/mL for atleast I h, 2h or 24h. Necrosis and caspase 3/7 activity assays was conducted using an ApoTox-Glo Triplex Assay kit (Promega, Madison, WI, USA). Necrosis, determined by live-cell protease activity, was assessed by measuring fluorescence. Caspase 3/7 activity was analyzed by measuring luminescence with a microplate reader (Perkin Elmer) according to the manufacturer’s protocol. Reactive oxygen species (ROS) assay

Extracellular H202 formed in the culture media as a result ol toxic stimulants and drug treatment were detected and quantified using the ROS-GIo H202 assa^y (Promega). Briefly, cells plated onto black- walled 96-well plates at a density ol 2 x 104 cells per well and treated with stimulants with and without recombinant drug, before following the manufacturer's protocol. Luminescence intensity was quantified using a microplate reader (Perkin Elmer) and normalized to untreated wells.

Greiss reagent assay

The concentration of N02- that accumulated in the endothelial cell culture medium over 24 h was determined in a microplate assay using Griess reagent. Fifty microliters of the culture supernatant was mixed with an equal amount of 1% sulfanilamide in 5% phosphoric acid and incubated at room temperature for 5 min. Then 50 mΐ of 0.1%N-1- naphthylethylenediamine dihydrochloride in water was added, and the mixture was incubated for an additional 5 min.

NO concentrations were evaluated using the Griess Reagent System (Promega, Madison, WI) according to the manufacturer’s protocol.

Wound healing scratch migration assay

Human aortic endothelial cells (Passage 4) were grown to confluence in 24-well plates and starved for 2h. The media were changed to basic endothelial growth medium without FBS, supplemented with 1 pg/mL of SIRT1 variants, and a scratch (wound) was made across the monolayer using a sterile P200 pipette tip. Images of the wells were captured at fixed points to record the area of clearing at time 0 and 8 h, and Image.! software was used to quantitate the cleared area.

Dil-LDL uptake assay

HepG2 cells were maintained in MEM supplemented with 10% FBS. The cells were seeded in 96 well black plates at a density of 1 ^ 104 cells per well and grown to 70-80% confluence. Afterwards, cells were changed to serum-free Opti-MEM for 24h and followed by incubation with 2pg recombinant SIRT1 and its variants for 30min. Then, 20 pg/mL Dil-LDL (Alfa Aesar) was added and incubated at 37 °C in the dark for additional 4h. Cells were washed three times with PBS. and LDL uptake was determined on a fluorescence plate reader (Victor X3, Perkin Elmer).

NAD+/NADH ratio assay

Cells were plated at 4 x 10³ cells in 96-well white plates. Cells were treated with recombinant SIRT1 and its therapeutic variants. Changes in the NAD+/NADH ratio were evaluated using an NAD+/NADH-Glo assay kit (Promega). NAD+ (oxidized NAD) and NADH (reduced NAD) levels were individually assessed in lysate of primary skeletal muscle cells cultured in the same well by measuring luminescence according to the manufacturer’s protocol. Luminescence intensity was quantified using a microplate reader (Perkin Elmer) and normalized to untreated wells.

Statistics

All experiments were performed in at least triplicate with mean S.E. reported for each comparison group. The means were analyzed using either a two-tailed Student’s t test or a one-way analysis of variance followed by a Bonferroni post hoc test (Prism 6.0, GraphPad Software, San Diego, CA).

Further Results from Experiments:

Effect on SIRT1 on human aortic endothelial cells:

LPS is known to cause endothelial dysfunction. TLR4-mediated LPS recognition mechanisms involves the nuclear localization and activation of NF-kB, resulting in an increased expression of TNFa, VCAM-1 , and ICAM-1. Deficiency of SIRT1 increases microvascular inflammation, morbidity, and mortality in early sepsis, whereas the SIRT1 activator reversed the aforementioned effect, showing that SIRT1 plays a protective role in the development of sepsis. Treatment of human aortic endothelial cells with recombinant SIRT1 and fusion SIRT1 variants prevented the expression TNFa and VCAM-1 in the presence of LPS, while mutSIRTl-fc reduced the expression of ICAM-1 as well in the presence of LPS (Fig. 10). Treatment with wtSIRTl-fc (SEQ-ID 10) and mutSIRTl-fc (SEQ-ID 14) also reduced mTOR phosphorylation and activation, both in the presence and absence of LPS suggesting other protective effects independent of LPS. Incubation of HAECs with various doses of TNFa is known to increase phosphorylation of eNOS at the negative regulatory site (Thr495) and increase oxidative stress. Hyperglycemia is associated with the activation of various ROS-producing pathways and increased oxidant production in endothelial cells. TNFa-stimulation causes Rac-1 activation, NF-KB activation and ROS production. Treatment of HAECs with recombinant mutant S1RT1 fusion protein in presence of TNFa inhibited ROS production compared to cells treated with TNFa alone or SIRT1 and WTSIRTl-fc in presence of TNFa (Fig. 1 1). Furthermore, glucose uptake is noninsulin dependent in endothelial cells and occurs via GLUT1 , while TNFa treatment reduces glucose uptake in aortic endothelial cells, increasing risk oi insulin resistance. Treatment of HAECs with recombinant SIRT1 (SEQ-ID 1) and SIR! 1 variants (SEQ-1D 10, SEQ-ID 14) inhibited the TN Fa-dependent reduction in glucose uptake (Fig. 12), protecting cells against cytokine-induced endothelial dysfunction. T hus, recombinant SIRT1 and SIRT1 variants treatment caused not only a decrease in TNFa production in endothelial cells but also protected against the inflammatory effect of circulating TNFa on cells suggesting alternate method to protect inflammatory disease caused by NFkB activation and TNFa effect.

Glucose uptake in skeletal muscles

Treatment of skeletal muscle cells with recombinant SIRT1 and its fusion variants increased glucose uptake in skeletal muscle cells. Treatment with mutSIRTl-Fc (SEQ-ID 14) increased basal glucose uptake more than recombinant SIRT (SEQ-ID 1) and WTSIRTl-Fc (SEQ-ID 10) (Fig. 13). Surprisingly, in the presence of insulin, recombinant SIRT1 and WTSIRTl-Fc reduced glucose uptake in skeletal muscle cells, however, mutSIRTl-Fc did not reduce the effect of insulin-stimulated glucose uptake. Thus, SIRT1 treatment may protect against hyperglycemia, insulin resistance and related diseases.

FPLC of SIRTl-Fc proteins

FPLC purification of WTSIRTl-Fc (SEQ ID 10) (Fig. 14) shows two peaks at 8.2min and 8.49min suggesting differential protein glycosylation leading to glycovariants, while peak at 5.7min and 7.3min suggests formation of dimers and aggregate formation. Furthermore, peak at 12.46min and 13.71min suggest protein instability and degradation. However, FPLC of mut2SIRTl-Fc (SEQ-ID 28) (Fig. 15) improved glycosylation of the protein, demonstrated by a single peak at 8.41 min. Additionally, a dimer was observed at 5.6min with no degradation products of the protein. Thus, the modifications of mutSIRTl improves its stability and glycosylation.

Effect of protease on SIRT1 activity

SIRT1 activity was assessed using the SIRT-Glo luminescence assay. Interestingly, tagging SIRT1 at the C-terminal with Fc improved activity compared to full-length protein. However, mutSIRTl -Fc (SEQ-ID 14) showed a much higher level of activity compared to rSIRTl (SEQ-ID 1) or WTSIRTl-Fc (SEQ-ID 10) (Fig. 16a), demonstrating the improvement in activity due to mutations. Cathepsin B is known to be a TNFa-responsive protease that cleaves SIRT1 and requires the C-terminal unstructured motif, which is essential for cathepsin B docking onto and cleavage of SIRT1. Thus, the structure of SIRT1 is critical to cathepsin B-dependent cleavage. Surprisingly, cathepsin B-dependent cleavage increased rSIRTl activity compared to untreated protein, while tagging ot Fc at the C -terminal increased SIRT1 activity in both WTSIRTl-Fc (SEQ-ID 10) and mutSIRTl-Fc (SEQ-ID 14) compared to recombinant SIRT1 (SEQ-ID 1 ) (Fig. 16b), suggesting that tagging at C-terminal can modulate SIRT1 activity. Incubation of caspase 1 and caspase 7 reduced full-length S1RT1 and WTSIRTl-Fc activity but tailed to reduce mutSIRTl -Fc activity (Fig. 16c, 16d). Thus, mutations of cleavage-sensitive site in mutSIRTl-Fc protects against proteases like caspase 1 and caspase 7. Furthermore, incubation of SIRT1 variants caspase 3 reduced WTSIRTl-Fc activity but failed to reduce mutSIRTl-Fc activity (Fig. 16e). Thus, the mutations of SIRT1 and tagging at the C- terminal not only increase SIRT1 activity but also protects against cleavage.

Effect of SIRT1 on NASH mice

Genetic deletion of SIRT1 is known to increase fatty liver in mice. The spectrum of nonalcoholic liver disease (NAFLD) ranges from simple steatosis (SS) to non-alcoholic steato hepatitis (NASH). NASH is the more aggressive form of fatty liver disease. To assess the effect of SIRT1 on NAFLD, 8-week old NASH mice were fed high-fat, high- cholesterol, high-fructose diet for 12 weeks to develop NASFI and treated with recombinant SIRT1 (SEQ-ID 1) or its variants (SEQ-ID10, SEQ ID 28) for 2 weeks. Another variant of SIRT1 polypeptide with different mutations was developed for pilot studies. Mut2-SIRT1 (SEQ-ID 28) is a variant which is resistant to protease cleavage, has phosphomimetic mutations and improved stability and glycosylation. mut2SlRTl-Fc treatment reduced epididymal white adipose tissue in mice compared recombinant SIRT1 or WTSIRTl-Fc treatment (Fig. 17). Two weeks treatment of mice with WTSIRTl-Fc and mut2SIRTl-Fc was sufficient to cause a decrease in fasting blood glucose levels compared to fasting glucose levels before treatment, compared to vehicle treated mice (Fig. 18). Interestingly, WTSIRTl-Fc increased circulating insulin levels, suggesting an increase in insulin secretion, while mut2SIRTl-Fc reduced serum insulin levels in mice suggesting an increase in insulin sensitivity (Fig. 19). Lower insulin secretion associated with risk of CHD or myocardial infarction. HOMA-IR and HOMA-B% are formulas used to calculate insulin resistance and b-cell function. wtSIRTl-Fc caused an increase in FIOMA-IR and an increase in HOMA-B% compared to vehicle treated mice, suggesting an increase in beta- cell function and insulinotropic behavior (Fig. 20). Mut2SIRTl -Fc caused a reduction in HOMA-IR and no HOMA-B% suggesting an improvement in insulin resistance. Thus, the activity of S1RT1 can be modulated to improve beta-cell function or insulin resistance based on site-directed mutations. Thus, both wtSIRTl-Fc and mut2SlRTl -Fc improve glucose homeostasis via different mechanisms. Recombinant mut2SIRTl-Fc treatment was also found to reduce total-cholesterol, in particular LDL-cholesterol (Fig. 21), suggesting an improvement in dyslipidemia and a decrease in serum creatinine suggesting an improvement in nephropathy (Fig. 22).

Effect of SIRT1 treatment on LPS-induced neuroinflammation

Neuroinflammation is a key pathological event triggering and perpetuating the neurodegenerative process associated with many neurological diseases. Peripheral injection of LPS enhances some aspects of Alzheimer’s, ALS, Huntington disease, such as microglial alterations and vascular dysfunction. Findings from clinical studies indicate that inflammatory processes might also be involved in the pathogenesis of depression. Studies indicate that depressive-like behavior can be observed in the absence of sickness 24h after systemic LPS administration. Thus we assessed the effect of SIRT1 treatment on LPS- induced neuroinflammation and anhedonia. Mice treated with wtSIRTl-Fc (SEQ-ID 10) and mut2SIRTl -Fc (SEQ-ID 28) were protected against LPS-induced body weight loss (Fig. 23). To assess depression and anhedonic response of mice treated with LPS, sucrose preference test was performed. Mice treated with LPS had a reduced preference to sucrose water. Treatment of mice with mut2SIRTl-Fc protected against LPS-induced anhedonia and depression at 15h post-LPS treatment (Fig. 24). The effect of wtSIRTl-Fc treatment was delayed and was observed at 21 h post-LPS treatment. Several markers of glial activation such as major histocompatibility complex (MF1C) class II, complement receptors, and scavenger receptors are increased in brain during normal aging and neurodegeneration. Histology of brain sections showed a decrease in GFAP and 1BA1 staining in protein treated group compared to vehicle treated, indicating protection against astrocytes and microglial activation and neuroinflammation in mice. To assess if S1RT1 treatment deacetylates and reduces NFkB activation, we assessed the expression of acetylated p65, in the presence of LPS and upon treatment with wild-type SIRT1 and different fusion variants. Treatment of SH-SY5Y neuronal cells with LPS increased acetylated p65, while treatment with recombinant SIRT1 (SEQ-ID 1), wtSIRTl -Fc (SEQ- ID 10) and mut2SIRTl-Fc (SEQ-ID 28) reduced acetylated p65 levels, suggesting a decrease in p65 activity (Fig. 25). Thus treatment with SIRT1 and its fusion variants protect against neuroinflammation and depression. Effect of SIRT1 treatment of NAD levels

Metabolism of fatty acids and glucose by cells requires NAD+ function as a hydrogen/electron transfer molecule. Therefore, NAD+ plays a vital role in energy production. Muscle diseases have a negative effect on health, lifespan, and/or quality of life. Genetic muscle diseases result in a variety of ultrastructural defects in muscle cells and progressive loss of muscle mass and function via multiple different mechanisms. Inflammatory or metabolic diseases (such as diabetes, obesity, autoimmune diseases, cancer, and infections) can result in loss of skeletal muscle as well. Given the integration and interdependence of the nervous and muscular systems, neural disorders or injuries can also impair muscle tissue structure and function. Additionally, skeletal muscle is lost as a natural part of the aging process, and this loss is exacerbated in a condition called sarcopenia. With increasing age, however, NAD+ levels and sirtuin activity steadily decrease, and the decline is further exacerbated by obesity, inflammation and sedentary lifestyles. Cellular levels of NAD+ modulate skeletal muscle cell differentiation. We hypothesized that S1RT1 may modulate NAD+ regulating enzymes, through a feedback loop and increases NAD levels. To assess if SIRT1 protein treatment increase NAD+ levels, skeletal muscle cells were treated with vehicle, wild-type SIRT1 protein (SEQ-ID 1) and fusion variants (SEQ-ID 10, SEQ ID 14). Treatment with recombinant SIRT1 and fusion variants caused a significant increase in cellular NAD+ levels (Fig.26). Thus, SIRT protein treatment can be used as a method to increase cellular NAD+ levels.

Effect of cysteine and disulfide bridges on SIRT1 activity

Cysteines in proteins frequently form disulfide bonds. We identified different cysteine groups (table 1, table 2) which were solvent accessible. To verify if replacement of these cysteines can improve protein stability or activity, we performed site-directed mutagenesis of SIRT1 (SEQ ID 1) at each cysteine group, replacing each cysteine group to alanine. Cysteine may also be replaced by serine or other amino acids to assess disulfide bond formation or thermostability. Replacing of cysteine to alanine would help deduce if the replaced SH-group was involved in disulphide bonding. Protein was purified using his-tag columns. Thus we assessed the S1RT1 activity of purified SIRT1 mutated at C67A, Cl 60 A, C268A, C374A, C501A, C502A, C574A, C623A and C671A. Deacetylation assay was performed using a peptide comprising 317-320 (QPKK) on p53 conjugated to aminoluciferin. The Zn2+-tetrathiolate of SIRT1 (C371 , C374, C395, and C398) consists of four surface-exposed cysteine residues that are conserved among all seven human sirtuins. Importantly, the vicinal nature of cysteines 371 and 374 makes these residues susceptible to intra-molecular disulfide bond formation by endogenous oxidants or oxidative stress. We found that mutation of C374 to alanine increases S1RT1 activity (Fig.27). Furthermore, we found that mutation of Cysteine at Cl 60, C268 and C501 also increased SIRT1 activity. While mutation of C574, C623 and C671 reduced S1RT1 activity, suggesting that cysteines at these sites are required for protein activity and maybe be subject to post-translation modifications or disulfide bond formation. Thus, SIR! 1 activity can be modulated by mutating the above cysteine groups to different amino acids. To be noted, the current assay assesses the activity of SIRT1 deacetylation of p53 substrate and that the activity may be different for different substrates.

These cysteine replacements by serine or other amino acids are useful not only for SIRT1 alone but in particular so for any of the fc linked variants as claimed and as given in the general specification.

MATERIAL AND METHODS

FPLC protein purification

Protein was purified by FPLC. Purity was determined by analytical size exclusion chromatography with an Agilent AdvanceBio SEC column (300A 2.7 um 7.8 x 300 mm) and DPBS as running buffer at 0.8 ml/min. The concentration was determined by measuring absorption at a wavelength of 280 nm. The extinction coefficient was calculated using a proprietary algorithm.

Pharmacokinetics

Animals

C57B1/6 mice fed a normal diet (Purina, 5C089 from age 8 weeks of age. Animals were randomly allocated into 6 groups. The pharmacokinetics (PK) of recombinant SIRT1 and fusion SIRT1 variants were evaluated in mice after an IV dose of 1 mg/kg and 0.3mg/kg. Blood was collected retro-orbitally into AXYGEN microtubes at 30mins, 2h, 24h, 48h, 96h and 120h post-dosing for SIRT1 and its variants (3 mice/time point/treatment). The resultant 20m1 serum was snap-frozen and stored at -80°C until analysis for SIRT1 serum ELISA. The pharmacokinetic parameters were estimated by noncompartmental modeling. SIRT1 ELISA

SIRT1 was quantified in serum harvested from wild-type mice using an Abeam SimpleStep ELISA kit (abl 71573). Serum was diluted 1 :2 and added to each well and processed as per manufacturer’s instructions. The plate was read at 450 nm.

Neuroinflammation model

Animals and treatment

C57BL/6J male mice (7-8 weeks old) were housed in a temperature, humidity and light controlled room. Each cage was treated with LPS (lmg/kg) and divided into lour treatment groups consisting of saline group (IP, control) and fusion SIRT1 groups (SEQ-ID 10, SEQ- ID 28). Each group consisted of eight male mice. LPS on day one while the mice received drug treatment over the duration of 3 days.

Sucrose preference test

The test was performed within 24 h after LPS administration to evaluate anhedonia. The procedure consisted of an adaptation period 24h before the test in which mice were trained to adapt to sucrose solution with two bottles of 1% (w/v) sucrose solution placed in each cage. Twenty-four hours later, sucrose solution in one bottle was replaced with tap water during 24 h. After this adaptation period, mice were deprived of water and food for further 2h before the dark cycle. For the sucrose preference test mice were housed in individual cages with free access to two bottles containing 200 ml of sucrose solution (2% w/v) and 200 ml of water. After 15h and 2lh, the volumes of consumed sucrose solution and water were recorded and the sucrose preference was calculated as follows: % sucrose consumption = sucrose consumption^ water + sucrose consumption) ^c 100.

Histology

Each mouse was perfused and brain was fixed with 3.7% formaldehyde. All specimens were embedded in paraffin and sliced into 4-pm thick sections. Sections were deparaffinized and then rehydrated, antigens were retrieved, and endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS. After the sections had been blocked with an 1HC blocking reagent for 1 h, they were incubated with anti-lbal antibody or anti-GFAP antibody in blocking reagent at 4°C overnight. Slides were then washed in PBS, incubated with species-specific biotinylated secondary antibody (1 :200) for 30 min, washed with PBS again, amplified consecutively with avidin-horseradish peroxidase (FIRP) and visualized by incubating them with 3,3'-diaminobenzidine tetrahydrochloride. All slides were counterstained with hematoxylin, dehydrated, and mounted. For negative controls, the procedure omitted the primary antibody.

NASH model

Animals All animal experiments were conducted according to internationally accepted principles for the care and use of laboratory animals. The animal protocol was designed to minimize pain or discomfort to the animals. Male mice were obtained from Jackson (USA) and housed in a controlled environment. 8-weeek old mice had ad libitum access to water and were led a diet high in fat (40%kcal fat), 20%kcal fructose and 2% cholesterol (D09100310, SYSE) for 12 weeks. Mice were treated during the last 2 weeks of the study. Body weight and food intake were measured weekly during the treatment period.

Drug treatment

Animals were stratified (n = 6 per group) based on fasting glucose levels to a mean of 12 mmol/1, and treated for 2 weeks with vehicle (saline, IP, TIW), mut2S!RTl-Fc (0.3 mg/kg, IP, TIW), WTSIRTl-Fc (0.3 mg/kg, IP, TIW). A terminal blood sample was collected from the tail vein in fasted mice and used for plasma biochemistry. Animals were sacrificed by cardiac puncture under isoflurane anesthesia. Blood samples were processed as described below.

Biochemical and histological analyses

Biochemical and histological analyses were performed following standard protocols. Mice were fasted (6h) prior to sacrifice and serum samples were obtained for clinical chemistry analysis (AU480 clinical analyzer, Beckman-Coulter; Brea, CA). Plasma analytes triglycerides (TG) and total cholesterol (TC), glucose (GLU), insulin (1NS), low density lipoprotein (LDL) and serum creatinine.

Statistical analyses

Data were analyzed using GraphPad Prism v6.02 software (GraphPad, La Jolla, CA, United States). All results are shown as mean ± SEM. A two-way ANOVA with Bonferroni’s post-hoc test was performed for body weight analysis. A one-way ANOVA with Dunnett’s post-hoc test was used for all other parameters. A p-value < 0.05 was considered statistically significant.

SIRTl-glo activity assay

SIRT1 activity was measured using a SIRT-Glo Assay kit (Promega), following the manufacturer’s instructions. For each reaction, protein was incubated at a concentration of 1 Lig/ml with PBS or different proteases - Caspase 1 , Caspase 3, Caspase 7, Cathepsin B (50 units) at room temperature for 30min. SIRTl-glo reagent was added at a 1 : 1 ratio and incubated for 45min and the product was measured via luminescence. SEQUENCES

Seq ID 1 - SIRT1 isoform 1 (Uniprot identifier: Q96EB6-1 , catalytic domain from position 254-489 in bold face)

MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLERSPGEPGGAA

PEREVP AAARGCPGAA AAALWREAEAEA AAAGGEQEAQ AT AAAGEGD GPGLQ

GPSREPPLADNLYDEDDDDEGEEEEEAAAAA1GYRDNLLFGDEIITNGFHSCESDEE

DRASHASSSDWTPRPRIGPYTFVQQHLM1GTDPRTILKDLLPET1PPPELDDMTLWQ

JVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGI

YARLAVDFPDLPDPQAMFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALS

DKEGKLLRNYTQNIDTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGD

IFNQVVPRCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVDLLIVIG

SSLKVRPVALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGE

YAKLCCNPVKLSEITEKPPRTQKELAYLSELPPTPLHVSEDSSSPERTSPPDSSVIVT

LLDQAAKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGE

KNERTSVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDD

VLSSSSCGSNSDSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGTDG

DDQEAINEA1SVKQEVTDMNYPSNKS

The minimum common catalytic sequence stretch which is common to all SIRT1 isoforms is given by the stretch defined by positions 316 - 453 in the isoform 1 sequence.

Seq ID 2 - SIRT1 isoform 2 (Uniprot identifier: Q96EB6-2, catalytic domain from position 254-453 in bold face)

MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLERSPGEPGGAA

PEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQ

GPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDEIITNGFHSCESDEE

DRASHASSSDWTPRPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLWQ

IVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGI

YARLAVDFPDLPDPQAMFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALS

DKEGKLLRNYTQNIDTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGD

IFNQVVPRCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVDLLIVIG

SSLKVRPVALIPSNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQSP

SLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEAISVKQEVTD MNYPSNKS

Seq ID 3 - S1RT1 isoform b (NAD-dependent protein deacetylase sirtuin-l isoform b [Homo sapiens], NCBI identifier NP_00l 135970.1, catalytic domain from position 1-194 in bold face)

MFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDT

LEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGDIFNQVVPRCPRCPADEP

LAIMKPEIVFFGENLPEQFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHE

VPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSEITEKP

PRTQKELAYLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQAAKSNDDLDVSES

KGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWP

NRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQ

SPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGTDGDDQEA1NEA1SVKQEVT

DMNYPSNKS

Seq ID 4 - SIRT1 isoform c (NAD-dependent protein deacetylase sirtuin-l isoform c [Homo sapiens], NCBI identifier NP_001300978.1, catalytic domain from position 12-186 in bold face)

MCLCSGRKTILEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDTLEQVAGIQ RIIQCHGSF AT ASCLICKYKVDCE AVRGDIFN Q VVPRCPRCP ADEPL AI M KPEI VFFGENLPEQFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHEVPQILINR

EPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSE1TEKPPRTQKEL

AYLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEEK

PQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQI

SRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPME

DESEIEEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEAISVKQEVTDMNYPSNK

S

Seq ID 5 -Fc fragment

DKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKT1S KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Seq ID 6 -vFc fragment

PCPAPEVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSD1

AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Seq ID 7 v2Fc fragment

DKTHTCP PCPAPEVAGG PSVFLFPPKP KDQLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC

LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW

QQGNVFSCSV LHEALHSHYT QKSLSLSPGK

Seq ID 8 Mut SIRT1

MADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD

MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ HLMIGTDPRT

ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NTIEDAVKLL

QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL

CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS

VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL

KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN

GLEDEPDVPE RAGGAGFGEM GDDQEAINEA ISVKQEVTDM NYPSNKS Seq ID 9 - SIRT1 isoform 1 Fc-N-Wt SIRT1

MDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK ADEAALALQ PGGSPSAAGA DREAASSPAG EPLRKRPRRD GPGLERSPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD EIITNGFHSC ESDEEDRASF1 ASSSDWTPRP RIGPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVIN1LSE PPKRKKRKDI NT1EDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFDIE YFRKDPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIDTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPRCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVDLLIVIG SSLKVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVHNELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPT PLHVSEDSSS PERTSPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES 1AEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QSPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGTD GDDQEAINEA ISVKQEVTDM NYPSNKS

Seq ID 10 - SIRT1 isoform 1 Fc-C-Wt SIRT1

MADEAALALQ PGGSPSAAGA DREAASSPAG EPLRKRPRRD GPGLERSPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD EIITNGFHSC ESDEEDRASH ASSSDWTPRP R1GPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFDIE YFRKDPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIDTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPRCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVDLLIVIG SSLKVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVHNELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPT PLHVSEDSSS PERTSPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QSPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGTD GDDQEAINEA ISVKQEVTDM NYPSNKS DKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MFIEALHNHYT QKSLSLSPGK

Seq ID 1 1 - SIRT1 isoform 1 vFc-N-Wt SIRT1

MDKTHTCP PCPAPEVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK ADEAALALQ PGGSPSAAGA DREAASSPAG EPLRKRPRRD GPGLERSPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD EIITNGFHSC ESDEEDRASH ASSSDWTPRP RIGPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIYINILSE PPKRKKRKDI NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFDIE YFRKDPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIDTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPRCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVDLLIVIG SSLKVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPT PLHVSEDSSS PERTSPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QSPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGTD GDDQEAINEA ISVKQEVTDM NYPSNKS Seq ID 12 - SIRT1 isoform 1 vFc-C-Wt S1RT1

M ADEAALALQ PGGSPSAAGA DREAASSPAG EPLRKRPRRD GPGLERSPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD EIITNGFHSC ESDEEDRASH ASSSDWTPRP RIGPYTFVQQ FILMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NT1EDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFDIE YFRKDPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIDTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPRCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVDLLIVIG SSLKVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPT PLHVSEDSSS PERTSPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QSPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGTD GDDQEAINEA ISVKQEVTDM NYPSNKS DKTFITCP PCPAPEVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Seq ID 13 - SIRT1 isoform 1 Fc-N-Mut S1RT1

MDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK ADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD MIIENGFHEC EEDEEDRASFI ASSSDWTPYP RIGPYTFVQQ FILMIGTDPRT ILKDLLPETJ PPPELDDMTL WQIVINILSE PPKRKKRKD1 NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGEM GDDQEAINEA ISVKQEVTDM NYPSNKS

Seq ID 14 - SIRT1 isoform 1 Fc-C-Mut SIRT1

MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ FILMIGTDPRT

ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NTIEDAVKLL

QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS

VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES 1AEQMENPDL

KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESE1EEFYN

GLEDEPDVPE RAGGAGFGEM GDDQEA1NEA ISVKQEVTDM NYPSNKS DKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK Seq ID 15 - SIRT1 isoform 1 vFc-N-Mut SIRT1

MDKTHTCP PCPAPEVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK ADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAA1 GYRDNLLFGD MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC FIRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGEM GDDQEAINEA ISVKQEVTDM NYPSNKS

Seq ID 16 - SIRT1 isoform 1 vFc-C-Mut SIRT1

MADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC FIRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGEM GDDQEAINEA ISVKQEVTDM NYPSNKS DKTHTCP PCPAPEVAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Seq ID 17 - SIRT1 isoform 1 v2Fc-N-Mut SIRT1 with linker

MDKTHTCP PCPAPEVAGG PSVFLFPPKP KDQLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV LHEALHSHYT QKSLSLSPGK GGGGSGGGGSGGGGS

ADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NT1EDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVHNELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGEM GDDQEA1NEA ISVKQEVTDM NYPSNKS Seq ID 18 - SIRT1 isoform 1 v2Fc-C-Mut SIRT1 with linker

MADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ HLMIGTDPRT

ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKD1 NTIEDAVKLL

VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES 1AEQMENPDL

GLEDEPDVPE RAGGAGFGEM GDDQEA1NEA ISVKQEVTDM NYPSNKS GGGGSGGGGSGGGGS DKTHTCP PCPAPEVAGG PSVFLFPPKP KDQLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV LHEALHSHYT QKSLSLSPGK

Seq ID 19 - SIRT1 isoform 1 v2Fc-N-Mut SIRT1 with linker and albumin secretion signal (signal fragment underlined):

MKWVTFISLLFLFSSAYS DKTHTCP PCPAPEVAGG PSVFLFPPKP KDQLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV LHEALHSHYT QKSLSLSPGK

GGGGSGGGGSGGGGS ADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ HLMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKDI NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGEM GDDQEAINEA ISVKQEVTDM NYPSNKS

Seq ID 20 - SIRT1 isoform 1 v2Fc-C-Mut SIRT1 with linker and albumin secretion signal (signal fragment underlined):

MKWVTFISLLFLPSSAYS ADEAALALQ PGGSPSAAGA DREAASEPAG EPLRKRPRRD GPGLEREPGE PGGAAPEREV PAAARGCPGA AAAALWREAE AEAAAAGGEQ EAQATAAAGE GDNGPGLQGP SREPPLADNL YDEDDDDEGE EEEEAAAAAI GYRDNLLFGD MIIENGFHEC EEDEEDRASH ASSSDWTPYP RIGPYTFVQQ FILMIGTDPRT ILKDLLPETI PPPELDDMTL WQIVINILSE PPKRKKRKD1 NTIEDAVKLL QECKKIIVLT GAGVSVSCGI PDFRSRDGIY ARLAVDFPDL PDPQAMFMIE YFRKMPRPFF KFAKEIYPGQ FQPSLCHKFI ALSDKEGKLL RNYTQNIMTL EQVAGIQRII QCHGSFATAS CLICKYKVDC EAVRGDIFNQ VVPYCPRCPA DEPLAIMKPE IVFFGENLPE QFHRAMKYDK DEVMLLIVIG SSLSVRPVAL IPSSIPHEVP QILINREPLP HLHFDVELLG DCDVIINELC HRLGGEYAKL CCNPVKLSEI TEKPPRTQKE LAYLSELPPE PLHVSEDSSE PERTEPPDSS VIVTLLDQAA KSNDDLDVSE SKGCMEEKPQ

EVQTSRNVES IAEQMENPDL KNVGSSTGEK NERTSVAGTV RKCWPNRVAK

EQISRRLDGN QYLFLPPNRY IFHGAEVYSD SEDDVLSSSS CGSNSDSGTC QEPSLEEPME DESEIEEFYN GLEDEPDVPE RAGGAGFGEM GDDQEAINEA

1SVKQEVTDM NYPSNKS GGGGSGGGGSGGGGS DKTHTCP PCPAPEVAGG

PSVFLFPPKP KDQLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPASIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV LHEALHSHYT QKSLSLSPGK

Seq ID 21 - Modified albumin signal:

MKWVTFISLLFLFSSSSRA

Seq ID 22 - Albumin secretory signal:

MKWVTFISLLFLFSSAYS

Seq ID 23 - SIRT1 isoform d (NAD-dependent protein deacetylase sirtuin-1 isoform c [Homo sapiens], GenBank Sequence with NCBI identifier AAH12499.1, catalytic domain from position 62-297 in bold face):

MIGTDPRTILKDLLPETIPPPELDDMTLWQIVIN1LSEPPKRKKRKDINTIEDAVKLL

QECKKIIVLTGAGVSVSCGIPDFRSRDGIYARLAVDFPDLPDPQAMFDIEYFRK

DPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDTLEQVAGIQR

IIQCHGSFATASCLICKYKVDCEAVRGALFSQVVPRCPRCPADEPLAIMKPEIV

FFGENLPEQFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHEVPQILINRE

PLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSEITEKPPRTQKELA

YLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEEKP

QEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQIS

RRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPME

DESEIEEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEA1SVKQEVTDMNYPSNK

S

Seq ID 24 - SIRT1 isoform 1 Mut2-SIRT1

MADEAALALQPGGSPSAAGADREAASEPAGEPLRKRPRRDGPGLEREPGEPGGAA

PEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQ

GPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDMIITNGFHSCEEDE

EDRASHAASSDWTPYPRIGPYTFVQQHLMIGTDPRTILKDLLPET1PPPELDDMTW

QIVINILSEPPKRKKRKDlNTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIY

ARLAVDFPDLPDPQAMFM1EYFRKMPRPFFKFAKE1YPGQFQPSLCHKFIALSDKE

GKLLRNYTQNIMTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGDIFNQVV PYCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVMLLIVIGSSLKVRPV

ALIPSS1PHEVPQIL1NREPLPHLHFDVELLGDCDVHNELCHRLGGEYAKLCCNPVK

LSEITE PPRTQKELAYLSELPPEPLYVSEDSSSPERTSPPDSSV1VTLLDQAAKSND

DLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGT

VRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNS

DSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGDMGDDQEAI EAI

SYKQEVTDMNYPSNKS

Seq ID 25 - SIRT1 isoform 1 Fc-N-Mut2-SIRT1

MDKTHTCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGKADEAALALQPGGSPSAAGADREAASEPAGEPLRKRPRRDGPGLEREP

GEPGGAAPEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEG

DNGPGLQGPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDMIITNGF

HSCEEDEEDRASHAASSDWTPYPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPEL

DDMTLWQIVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDF

RSRDGIYARLAVDFPDLPDPQAMFMIEYFRKMPRPFFKFAKE1YPGQFQPSLCHKF1

ALSDKEGKLLRNYTQNIMTLEQVAG1QRIIQCHGSFATASCLICKYKVDCEAVRGD

IFNQVVPYCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVMLLIV1GSSL

KVRPVALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLC

CNPVKLSEITEKPPRTQKELAYLSELPPEPLYVSEDSSSPERTSPPDSSVIVTLLDQA

AKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERT

SVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSS

CGSNSDSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGDMGDDQE

AINEAISVKQEVTDMNYPSNKS

Seq ID 26 - SIRT1 isoform 1 Fc-C-Mut2-SIRTl

MADEAALALQPGGSPSAAGADREAASEPAGEPLRKRPRRDGPGLEREPGEPGGAA

PEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQ

GPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDMIITNGFHSCEEDE

EDRASHAASSDWTPYPRIGPYTFVQQHLMIGTDPRT1LKDLLPET1PPPELDDMTLW Q1V1NILSEPPKRK RKDINT1EDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIY

ARLAVDFPDLPDPQAMFMIEYFRKMPRPFFKFAKEIYPGQFQPSLCHKFIALSDKE

GKLLRNYTQNIMTLEQVAGIQRI1QCHGSFATASCLICKYKVDCEAVRGDIFNQVV

PYCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVMLLIVIGSSLKVRPV

ALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVIJNELCHRLGGEYAKLCCNPVK

LSEITEKPPRTQKELAYLSELPPEPLYVSEDSSSPERTSPPDSSVIVTLLDQAAKSND

DLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGT

VRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNS

DSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGDMGDDQEAINEA1

SVKQEVTDMNYPSNKSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW

LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSYMHEALHNFIYTQKSLSLSPGK

Seq ID 27 - SIRT1 isoform 1 Fc-N-Mut2-SIRTl WITFI LINKER

MDKTHTCPPCPAPEVAGGPSVFLFPPKPKDTLMlSRTPEVTCVVVDVSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK.CKVSNKA

LPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGKGGGGSGGGGSGGGGSADEAALALQPGGSPSAAGADREAASEPAGEP

LRKRPRRDGPGLEREPGEPGGAAPEREVPAAARGCPGAAAAALWREAEAEAAAA

GGEQEAQATAAAGEGDNGPGLQGPSREPPLADNLYDEDDDDEGEEEEEAAAAAI

GYRDNLLFGDMIITNGFHSCEEDEEDRASHAASSDWTPYPRIGPYTFVQQHLMIGT

DPRTILKDLLPETIPPPELDDMTLWQIVINILSEPPKRKKRKDINTIEDAVKLLQECK

KIIVLTG AGV SV SCGIPDFRSRDGIY ARLAVDFPDLPDPQAMFMIE YFRKMPRPFFK

FAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIMTLEQVAG1QR1IQCHGSFAT

ASCLICKYKVDCEAVRGDIFNQVVPYCPRCPADEPLAIMKPEIVFFGENLPEQFHRA

MKYDKDEVMLLIVIGSSLKVRPVALIPSSIPHEVPQILINREPLPHLHFDVELLGDCD

VIINELCHRLGGEYAKLCCNPVKLSEITEKPPRTQKELAYLSELPPEPLYVSEDSSSP

ERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMEN

PDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRY1F

HGAEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPMEDESE1EEFYNGLEDEPDVP ERAGGAGFGDMGDDQEAINEAISVKQEVTDMNYPSNKS

Seq ID 28 - SIRT1 isoform 1 Fc-C-Mut2-SIRTl WITH LINKER

MADEAALALQPGGSPSAAGADREAASEPAGEPLRKRPRRDGPGLEREPGEPGGAA

PEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQ

GPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDMIITNGFHSCEEDE

EDRASHAASSDWTPYPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLW

QIVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCG1PDFRSRDGIY

ARLAVDFPDLPDPQAMFMIEYFRKMPRPFFKFAKEIYPGQFQPSLCHKFIALSDKE

GKLLRNYTQNIMTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGDIFNQVV

PYCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVMLLIVIGSSLKVRPV

ALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVI1NELCHRLGGEYAKLCCNPVK

LSEITEKPPRTQKELAYLSELPPEPLYVSEDSSSPERTSPPDSSV1VTLLDQAAKSND

DLDV SESKGCMEEKPQEV QTSRN VESIAEQMENPDLKN V GSST GEKNERTSV AGT

VRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNS

DSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGDMGDDQEAINEAI

SVKQEVTDMNYPSNKSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Seq ID 29 - SIRT1 isoform 1 v2Fc-C-Mut2-SIRTl with linker

MADEAALALQPGGSPSAAGADREAASEPAGEPLRKRPRRDGPGLEREPGEPGGAA

PEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQ

GPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDMIITNGFHSCEEDE

EDRASHAASSDWTPYPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLW

QIVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIY

ARLAVDFPDLPDPQAMFMIEYFRKMPRPFFKFAKEIYPGQFQPSLCHKFIALSDKE

GKLLRNYTQNIMTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGD1FNQVV

PYCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVMLLIVIGSSLKVRPV

ALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVK

LSEITEKPPRTQKELAYLSELPPEPLYVSEDSSSPERTSPPDSSVIVTLLDQAAKSND DLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGT

VRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNS

DSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGDMGDDQEAINEAI

SVKQEVTDMNYPSNKSGGGGSGGGGSGGGGSDKTHTCPPCPAPEVAGGPSVFLFP

PKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPP

SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

Seq ID 30 - SIRT1 isoform 1 v2Fc-N-Mut2 SIRT1 with linker

MDKTHTCPPCPAPEVAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKF

N W YVDGVEVHN AKTKPREEQ YN STYRV V S VLT VLHQD WLNG KE YKCKV SNKA

LPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLFIEALHSHYTQK

SLSLSPGKGGGGSGGGGSGGGGSADEAALALQPGGSPSAAGADREAASEPAGEPL

RKRPRRDGPGLEREPGEPGGAAPEREVPAAARGCPGAAAAALWREAEAEAAAAG

GEQEAQATAAAGEGDNGPGLQGPSREPPLADNLYDEDDDDEGEEEEEAAAAAIG

YRDNLLFGDMIITNGFHSCEEDEEDRASHAASSDWTPYPRIGPYTFVQQHLMIGTD

PRTILKDLLPETIPPPELDDMTLWQIVINILSEPPKRKKRKDINT1EDAVKLLQECKKI

IVLTGAGVSVSCGIPDFRSRDGIYARLAVDFPDLPDPQAMFMIEYFRKMPRPFFKFA

KEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIMTLEQVAGIQRIIQCHGSFATAS

CLICKYKVDCEAVRGDIFNQVVPYCPRCPADEPLAIMKPEIVFFGENLPEQFHRAM

KYDKDEVMLLIVIGSSLKVRPVALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVI

INELCHRLGGEYAKLCCNPVKLSEITEKPPRTQKELAYLSELPPEPLYVSEDSSSPER

TSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPD

LKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHG

AEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPER

AGGAGFGDMGDDQEAINEAISVKQEVTDMNYPSNKS

Claims

1. An isolated fusion polypeptide comprising

a first amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, or >99.9% sequence identity to SEQ ID NO 01, SEQ ID NO 02, SEQ ID NO 03, SEQ ID NO 04, SEQ ID 23 or SEQ ID 08 said first amino acid sequence and/or said isolated fusion polypeptide having an NAD⁺-dependent deacetylase function identical to SIRT1 isoform 1 , isoform 2, isoform b, isoform b or isoform d or the catalytic domain thereof,

said first amino acid sequence being directly or indirectly chemically linked to at least one of: a second amino acid sequence comprising a fragment crystallizable Fc region, or an albumin, or a post-translationally modified derivative of any of these two,

for use in a method of treatment, amelioration, mitigation, slowing, arresting or reversing or prevention of a condition selected from the group consisting of: mitochondrial diseases/disorders, metabolic disorders, neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, dyslipidemia, hyperlipidemia, diabetes, metabolic syndrome, inflammation, apoptosis, autoimmunity, oxidative stress, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, and fatty liver disease, depression, neurodegeneration, preferably Parkinson^'s and Alzheimer's, and cancer, preferably pancreatic cancer.

2. A fusion polypeptide according to claim 1, wherein it is present as a homodimer, or as a dimer of said fusion polypeptide with an additional second amino acid sequence a fragment crystallizable Fc region and/or an albumin, or a post- translationally modified derivative thereof without attached first amino acid sequence being directly or indirectly chemically linked, or as a monomer.

3. A fusion polypeptide according to any of the preceding claims, wherein the fusion polypeptide comprises a further amino acid sequence forming a third domain, wherein the third domain is one selected from the group consisting ot: a secretion signal domain, in particular in the form ol an serum albumin preprotein, an lg kappa chain V-III region MOPC, an IgK H Ig kappa chain V-III region VG precursor or a modified albumin signal SEQ ID 21, an Fc region ot immunoglobulin or a part thereof, albumin, an albumin-binding polypeptide, Pro/Ala/Ser(PAS), a C-terminal peptide (CTP) of the b-subunit of human chorionic gonadotropin, polyethyleneglycol(PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethylstarch (HES), an albuminbinding small molecule, and a combination or a post-translational ly modified derivative thereof.

4. A fusion polypeptide according to any of the preceding claims, wherein said second amino acid sequence is a fragment crystallisable Fc region is based on immunoglobulin, in particular based on at least one of IgG 1 , Ig2, IgG3 or IgG4, preferably having a sequence according to any of SEQ ID NO 05 - SEQ ID NO 07 or an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity, wherein the fragment crystallisable Fc region can be post-translationally modified.

5. A fusion polypeptide according to any of the preceding claims, wherein said first amino acid sequence is characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 with at least one of the following or a combination of the following mutations, wherein X stands for a mutated amino acid different from the original one: S27X; S47X; T154X; S159X; S162X; T530X; S540X; S545X; S682X; T719X; K444X; El 5 IX; D298X; D305X; D348X; R179X; R394X; D434X; D720X, in particular R179X; R394X; D434X; or D720X, wherein preferably there is at least one of the following or a combination of the following mutations: S27E; S47E; T154E; S159E; S162E; T530E; S540E; S545E; S682E; T719D; K444S; E151M; D298M; D305M; D348M; R179Y; R394Y; D434M; or D720M, in particular R179Y; R394Y; D434M; H533Y, D720M;

and/or wherein said first amino acid sequence is characterized by at least (>)85%, >87_.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 01 and has at least one phosphorylation mutation and at least one of the following sites: S14, S26, S27, S47, SI 59. S162, S172, S173. T344, S442, T530, S538-S540, S535, S538-S540, T544, S545, T719, or S747;

and/or wherein said first amino acid sequence is characterized by at least (>)95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 08.

6. A fusion polypeptide according to any of the preceding claims, wherein said second amino acid sequence is characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity to SEQ ID NO 05 with at least one of the following or a combination of the following mutations: Ll 17V; Ll 18A; P214S; T133Q; or M31 1 L.

7. A fusion polypeptide according to any of the preceding claims, wherein said second amino acid sequence, preferably having a sequence according to any of SEQ ID NO 05 - SEQ ID NO 07, or an amino acid sequence characterized by at least (>)85%, >87.5%, >90%, >92%, >94%, >95%, >96%, >97%, >98%, >99%, >99.5%, >99.9% sequence identity, which can be post-translationally modified, is linked to the C-terminus, to the N-Terminus, or both, of the first amino acid sequence.

8. A fusion polypeptide according to any of the preceding claims, wherein between said first amino acid sequence and said second amino acid sequence there is no linker element or there is a linker element, which is preferably a flexible, rigid or cleavable linker element, and which is more preferably selected from at least one of the following systems or a system based on these elements: (GGGGS)n, (G)n, (EAAAK)n, (XP)n, or disulphide, wherein n is in the range of 1-15, preferably in the range of 3-8, and wherein X can be any amino acid, preferably A.

9. A fusion polypeptide according to any of the preceding claims, wherein the post translational modification is selected from the group consisting of phosphorylation, glycosylation, fucosylation, galactosylation. lipidation, lipoylation, acetylation, acylation suifonylation sulfmylation or sulphenylation, and combinations thereof.

10. A dosage form comprising the fusion polypeptide according to any of the preceding claims for use in a method of treatment, amelioration, mitigation, slowing, arresting, reversing or prevention of a condition selected from the group consisting of: mitochondrial diseases/disorders, metabolic disorders, neurodegenerative diseases, polyglutamine diseases, anticoagulation and antithrombotic conditions, allergies and respiratory conditions, autoimmune diseases, vision impairment, dyslipidemia, hyperlipidemia, diabetes, metabolic syndrome, inflammation, apoptosis, autoimmunity, neurodegeneration, including Alzheimers, Parkinson’s, Huntington^'s, oxidative stress, hypercholesterolemia, atherosclerosis, cardiovascular disease (CVD), steatohepatitis (fatty liver disease), pancreatitis, renal lipid deposition, depression, and/or for enhancing NAD levels.

1 1. A dosage form according to claim 10, for parenteral, peroral, transdermal or transmucosal administration, in particular further including a pharmaceutically acceptable carrier.

12. A combination medicament, preferably in a unit dose form, comprising a fusion polypeptide according to any of the preceding claims as well as a further therapeutically active or synergistic compound, preferably selected to be a SIRT1 activator or NAD⁺ supplement, more preferably selected from the group consisting of niacin (vitamin B3), nicotinamide mono nucleotide, nicotinamide ribosome (NR) and resveratrol.

13. A polynucleotide comprising a gene or cell line expressing a fusion polypeptide according to any of the preceding claims.

14. A method of producing one or more fusion polypeptide according to any of the preceding claims comprising:

(b) causing the host cell to express the fusion polypeptide

(c) when the fusion polypeptide is expressed in a host cell, the nucleotide sequence encoding the fusion polypeptide preferably coding for a secretory signal sequence that permits the polypeptide to be secreted.

(d) preferably subjecting a fluid comprising the said fusion polypeptide to Protein A or Protein G affinity chromatography or an ion exchange chromatography, wherein elution is preferably carried out at a pH ranging from 2.8 to 4.5, and the eluate obtained contains the purified fusion polypeptide.