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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0016—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • H—ELECTRICITY
  • H02—GENERATION; CONVERSION OR DISTRIBUTION OF ELECTRIC POWER
  • H02K—DYNAMO-ELECTRIC MACHINES
  • H02K7/00—Arrangements for handling mechanical energy structurally associated with dynamo-electric machines, e.g. structural association with mechanical driving motors or auxiliary dynamo-electric machines
  • H02K7/18—Structural association of electric generators with mechanical driving motors, e.g. with turbines
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present invention relates to lifespan, longevity and age-related diseases. More particularly, the invention relates to variants of sirtuin 6 (SIRT6), and therapeutic uses thereof, for the regulation of ageing and for treating and/or preventing age-related diseases.
• SIRT6 sirtuin 6
• age-related diseases include, e.g., cardiovascular diseases, strokes, hypertension, cancer, type 2 diabetes, Parkinson’s disease, Alzheimer’s disease and other dementia, chronic obstructive pulmonary disease (COPD), osteoarthritis, osteoporosis, cataract, age-related macular degeneration, hearing loss.
• cardiovascular diseases e.g., cardiovascular diseases, strokes, hypertension, cancer, type 2 diabetes, Parkinson’s disease, Alzheimer’s disease and other dementia, chronic obstructive pulmonary disease (COPD), osteoarthritis, osteoporosis, cataract, age-related macular degeneration, hearing loss.
• COPD chronic obstructive pulmonary disease
• SIRTs sirtuins
• SIRT6 encoded by the SIRT6 gene regulates the expression of telomere reverse transcriptase required for telomere elongation, as well as deacetylate histone 3 lysine 9 (H3K9) and deacetylate histone 3 lysine 56 (H3K56) resulting in maintaining the telomeric integrity.
• SIRT6 gene was shown to be recruited to the damaged sites and promote DNA repair through deacetylating the repair proteins such as poly (ADP-ribose) polymer-ase (PARP)-l, Ku70, NBS, and Wemer (WRN) helicase.
• SIRT6 acts as a transcriptional regulator to suppress gene expression by stabilizing the chromatin structure.
• SIRT6 modulates cellular senescence through the deacetylation of a variety of signaling molecules such as FOXO and p53. SIRT6 regulates the RelA subunit of NFKB by modifying the cellular senescence-related gene expression. Finally, SIRT6 also reduces cytotoxicity and therefore decreases the damage that causes premature senescence.
• SIRT6 may constitute a good target to prevent and/or treat age-related diseases.
• a first aspect of the invention relates to an isolated nucleic acid molecule encoding a variant of sirtuin 6 (SIRT6) having at least 75% identity with sequence SEQ ID NO: 1, the variant having at least one mutation selected in the group comprising or consisting of a substitution N308K and a substitution A313S with respect to sequence
• the nucleic acid molecule is of sequence selected in the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• Another aspect of the invention pertains to an isolated polypeptide encoded by a nucleic acid molecule as defined herein.
• polypeptide is of sequence selected in the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
• a further aspect of the invention relates to a vector comprising the isolated nucleic acid molecule as defined herein.
• the vector is a viral vector, in particular an adeno- associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an adenoviral vector, a retroviral vector, or a herpes virus vector.
• AAV adeno-associated viral vector
• exo-AAV exosome-associated AAV vector
• adenoviral vector adenoviral vector
• retroviral vector a retroviral vector
• herpes virus vector a viral vector, in particular an adeno-associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an adenoviral vector, a retroviral vector, or a herpes virus vector.
• the invention relates to a suspension comprising a vector as defined herein.
• the invention also pertains to a cell expressing the polypeptide as defined herein, the cell being preferably transfected with an isolated nucleic acid molecule, or a vector as defined herein.
• Another aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising (i) an isolated nucleic acid molecule, or an isolated polypeptide, or a vector as defined herein, and (ii) a pharmaceutically acceptable excipient.
• the invention relates to the use of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector as defined herein, in the regulation of ageing, and/or senescence, and/or lifespan in an individual, preferably a mammalian individual, more preferably a human individual.
• a further aspect of the invention pertains to the use of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according as defined herein, in the repair of double strand break in a cell, preferably a mammalian cell, more preferably a human cell.
• the invention also relates to an isolated nucleic acid molecule, or an isolated polypeptide, or a vector as defined herein, for use in the prevention and/or the treatment of an age-related disease.
• the individual is a mammalian individual, preferably a human individual.
• the age-related disease is selected in the group comprising or consisting of progeria, Wemer syndrome, neurodegenerative disease, Alzheimer's disease, cancer, cardiovascular disease, obesity, type 2 diabetes, hypercholesterolemia, hypertension, ocular disorders, cataracts, glaucoma, osteoporosis, blood clotting disorders, arthritis, hearing loss and stroke.
• the invention also relates to a kit comprising (i) an isolated nucleic acid molecule, or an isolated polypeptide, or a vector as defined herein, and (ii) means to administer the isolated nucleic acid molecule, the isolated polypeptide, or the vector.
• “About”, when preceding a figure, means plus or less 10% of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed. [0029] “Comprise” is intended to mean “contain”, “encompass” and “include”. In some embodiments, the term “comprise” also encompasses the term “consist of’.
• “Sirtuin 6” also referred to as “SIRT6”, is intended to refer to the polypeptide with the Entrez Gene number 51548, and also non-limitatively relates to the NAD- Dependent Protein Deacetylase Sirtuin-6, Regulatory Protein SIR2 Homolog 6, SIR2- Like Protein 6, SIR2L6, Sirtuin (Silent Mating Type Information Regulation 2, S. Cerevisiae, Homolog) 6, Sirtuin (Silent Mating Type Information Regulation 2 Homolog) 6, Sir2-Related Protein Type 6, Sirtuin Type 6 and EC 2.3.1.286.
• isolated refers to a nucleic acid molecule or polypeptide a that is removed from the initial biological context that has allowed to generate this nucleic acid molecule or polypeptide a.
• the biological context comprises at least a cell, or one or more enzyme(s).
• Nucleic acid also referred to as “polynucleotide”, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• “Nucleic acid” or “polynucleotide” include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double- stranded RNA, and RNA that is a mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
• Nucleic acid refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the term “nucleic acid” or “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• nucleic acid or “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
• Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
• Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acid residues other than the 20 gene-encoded amino acid residues.
• Age-related disease refers to the physiological or pathological conditions that correlate with an individual getting older or prematurely older, and which prevalence is above the average prevalence observed within the general population.
• age-related diseases include progeria, Wemer syndrome, neurodegenerative diseases, cardiovascular diseases, strokes, hypertension, cancer, type 2 diabetes, Parkinson’s disease, dementia, chronic obstructive pulmonary disease (COPD), osteoarthritis, osteoporosis, cataract, age-related macular degeneration, hearing loss, and the like.
• “Suspension” refers to a liquid mixture in which the active principle, such as the nucleic acid molecules, polypeptides, or vectors according to the invention, is/are floating in a liquid medium.
• “Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder, in particular an age-related disease. Those in need of treatment include those already with said disorder as well as those prone to develop the disorder or those in whom the disorder is to be prevented.
• An individual is successfully "treated” for an age-related disease if, after receiving a therapeutic amount of the active principle, in particular the nucleic acid molecules, polypeptides, or vectors according to the present invention, the individual shows observable and/or measurable reduction in or absence of one or more of the symptoms associated with the age-related disease; reduced morbidity and mortality, and improvement in quality of life issues.
• the active principle in particular the nucleic acid molecules, polypeptides, or vectors according to the present invention.
• Preventing refers to keeping from happening, and/or lowering the chance of the onset of, or at least one adverse effect or symptom of, an age-related disease, disorder or condition associated with a deficiency in or absence of an organ, tissue or cell function.
• “Therapeutically efficient amount” refers to the level or the amount of the active principle that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of an age-related disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of an age-related disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of an age-related disease, disorder, or condition; (4) reducing the severity or incidence of an age-related disease, disorder, or condition; or (5) curing an age-related disease, disorder, or condition.
• a therapeutically effective amount may be administered prior to the onset of an age-related disease, disorder, or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after the onset of an age-related disease, disorder, or condition, for a therapeutic action.
• a therapeutically effective amount of the active principle is an amount that is effective in reducing at least one symptom of an age-related disease, disorder or condition.
• “Individual” refers to an animal, preferably a mammal, more preferably a human.
• the individual is a male.
• the individual is a female.
• an individual may be a “patient”, i.e. a warm blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of cancer.
• the individual is an adult (for example a subject above the age of 18).
• the individual is a child (for example a subject below the age of 18).
• Figure 1 is a scheme showing the screening strategy for variants in genome maintenance (GM) genes in Ashkenazi Jewish (AJ) centenarians.
• FIG. 2 is a plot showing the purified SIRT6 proteins turn-over rate. SILAC analysis on HEK293 cells expressing SIRT6 variants.
• Figure 3 is a graph showing the thermostability of purified SIRT6 proteins (WT: wild type SIRT6 polypeptide; Cent: SIRT6 variant polypeptide with N308K and A313S mutations). Data represents two replicates with two technical replicates each using SIRT6 from the Roc and Ichor preps.
• Figures 4A-4B are a combination of graphs. Fig. 4A shows the fluorescence lifetime x measured from FLT signals by fitting the data to Equation 1 (see Methods).
• Each of the two different SIRT6 biosensors shows a decreased fluorescence lifetime compared to the GFP-only control, indicating highly significant FRET.
• the intra-GFP biosensor shows a greater lifetime change (more FRET, shorter distance R, more structural sensitivity).
• Fig 4B shows the FRET efficiency E and the distance R (in Angstrom; A) were determined from lifetime data (Equations. 2 and 3, Methods), revealing that the distance R, measure for the intra-GFP biosensor, was significantly greater for the centSIRT6 by 3.0 ⁇ 0.4 A.
• Figure 5A-5B are a combination of graphs.
• Fig.5A shows the Michaelis-Menten kinetic parameters using differential concentrations of myristoylated peptide.
• Fig. 5B shows the Michaelis-Menten kinetic parameters using differential concentrations of NAD+. Reactions were conducted in triplicate. Closed circles: WT; closed squares: Cent.
• Figure 6 is a graph showing the tryptophan fluorescence curves for SIRT6 variants with titrated concentrations of NAD+.
• Figure 7A-7B is a combination of plots showing the deacetylase activity on H3K9ac (Fig. 7A) and H3K18ac (Fig. 7B) residues, with reduced activity in centSIRT6 allele (the deacetylase activity is inversely proportional to the relative abundance of acetylated compounds).
• Figure 8A-8B is a combination of plots showing the deacetylation kinetics on H3K9ac (Fig. 8A) and H3K18ac (Fig. 8B) residues of SIRT6 variants.
• Figure 9A-9B is a combination of plots showing the in vitro deacetylation rates on H3K9ac (Fig. 9A) and H3K18ac (Fig. 8B) residues of purified SIRT6 proteins with histones purified from HeLa cells.
• Figure 10 is a plot showing the quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles, which did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. The average and standard deviation of three different preps is plotted.
• Figure 11 is a photograph showing the whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot.
• Cu cumate
• PQ paraquat.
• Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines.
• Figure 12 is a plot showing the self-ribosylation of SIRT6 using radiolabeled NAD+. Recombinant SIRT6 was incubated with P32-labeled NAD+ and then run on an SDS-PAGE. Signal was measured using a Phospholmager. All experiments were repeated at least three times; error bars show s.d. Significance was determined by Student’s t-test, two tailed. Asterisk indicate p ⁇ 0.05. Asterisks indicate significance over HPRT control.
• Figure 13 is a plot showing the self-ribosylation of SIRT6 with titration of NAD+.
• mADPr-specific antibody was used to detect ribosylated wild type SIRT6 (closed circles) and centSIRT6 (closed squares) proteins. Plot was fit the Michaelis-Menten equation with Kaleidagraph software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6 and maximal signal was ⁇ 2x greater for centSIRT6 compared to the wild type SIRT6.
• Figure 14 is a graph showing the activation of PARP1 by SIRT6 variants.
• SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly-ADPr antibody. Poly-ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly-ADPr signal in whole lanes for each sample. All experiments were repeated at least three times; error bars show s.d. Significance was determined by Student’s t-test, two tailed. Asterisk indicate p ⁇ 0.05. Lower asterisks indicate significance over HPRT control. Upper asterisks indicated significance over wild type SIRT6.
• Figure 15 is a graph showing the qRT-PCR analysis of LINE1 expression in cumate-inducible SIRT6 fibroblasts.
• Primers assessed both 5’ (ORF1; closed bars) and 3’ (ORF2; dashed bars) LINE1 sequences from the LlMdAl family of active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. All experiments were repeated at least three times. Error bars represent s.d. Significance was determined by Student’ s t-test, two tailed, unless otherwise stated. Asterisk indicate p ⁇ 0.05.
• Figure 16A-16B is a combination of graphs showing the stimulation of NHEJ (Fig. 16A) and HR (Fig. 16B) by SIRT6 variants. Reporter cell lines were co-transfected with SIRT6-expressing plasmid, I-Scel plasmid, and DsRed transfection control. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry.
• Figure 17 is a graph showing the basal gH2AC foci in cumate-inducible SIRT6 fibroblasts under uninduced (white bars) or induced (black bars) conditions. Foci were scored in at least 80 cells per condition.
• Asterisk indicates significant difference from the wild type SIRT6 (p ⁇ 0.05).
• Figure 19A-19B is a combination of plots showing the oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining 48 hours after exposure.
• FIG. 20A-20C is a combination of graphs.
• Fig. 20A is showing the number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours.
• HCA2 are normal human foreskin fibroblasts a: Control (CT); b: WT; c: N308K SIRT6 variant; d: A313S SIRT6 variant; e: Cent (N308K A313S variant). Asterisk indicates significant difference from the wild type SIRT6 (p ⁇ 0.05).
• Fig. 20B-20C are showing the apoptosis staining of cancer cell lines HT1080 (Fig.
• FIG. 21 is a photograph showing the immunoprecipitation (IP) experiments on lysates from cumate-induced fibroblasts expressing wild type (WT) or centSIRT6 (Cent) alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 hours prior to IP. The IP experiments were repeated three times. One representative set of IPs is shown.
• WT wild type
• Cent centSIRT6
• Figure 22 is a graph showing SIRT6 IP followed by Western blot with antibodies to LMNA, in which CentSIRT6 (Cent) shows stronger interaction with LMNA compared to the wild type SIRT6 (WT). Quantification of the IP experiment shown in Figure 21.
• Figure 23 is a graph showing LMNA IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6, in which LMNA shows enhanced interaction with centSIRT6 (Cent) compared to the wild type (WT). Quantification of the IP experiment shown in Figure 21.
• Figure 24 is a graph showing the quantification of the IP experiment shown in
• FIG. 21 SIRT6 IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues shows that centSIRT6 displays enhanced mADPr.
• Figure 25 is a graph showing the quantification of the IP experiment shown in Figure 21.
• IP with mADPr antibody using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA shows that LMNA displays enhanced mADPr signal in cells expressing centSIRT6 (Cent), as compared to wild type (WT).
• Figure 26 is a scheme showing protein-protein interactions profiles of SIRT6 and LMNA.
• SIRT6 and LMNA are colored dark grey and featured as central points in two opposing nodes of interactions.
• a third node (upper middle) shows interaction partners that are shared by SIRT6 and LMNA.
• Proteins whose interactions are enhanced by the centSIRT6 allele are colored light grey. Proteins that interacted equally with wild type and centSIRT6 alleles are uncolored.
• H15 is a special case because it showed increased interaction with the centSIRT6 and decreased interaction with LMNA in the presence of the centSIRT6 allele. Proteins known to be ribosylated in previous reports are shown as hexagons.
• Figure 27A-27B is a combination of histograms.
• Fig. 27A shows SIRT6 expression in cumate-inducible telomerase-immortalized HCA2 human fibroblast cell lines.
• SIRT6 alleles were integrated in the genome of SIRT6 knockout HCA2 cells using PiggyBac Transposon Vector System. Different dosages of cumate and resulting SIRT6 abundance were used to determine the dose needed to achieve equivalent SIRT6 expression for each cell line. Cells were normalized by count and total protein. Subsequent experiments utilizing cumate-inducible SIRT6 fibroblasts were controlled for SIRT6 abundance using these data.
• FIG.27B shows qRT-PCR expression analysis of SIRT6 using standardized dosages of cumate (dosage corresponds to boxes in Fig. 27A).
• Figure 28 is a histogram showing Self-ribosylation of SIRT6 using biotin- labeled NAD+. Recombinant SIRT6 was incubated with NAD+ conjugated with a biotin residue and then run on an SDS-PAGE. Each allele was assessed relative to its Ohr time point and normalized to SIRT6 total protein loading controls.
• Figure 29A-29F is a combination of photographs, graphs and histograms showing the analysis of CRISPR human MSC cell lines.
• Fig. 29A and 29B show DNA double strand repair efficiency in wild type and centSIRT6 (“Cent #1” and “Cent #2”) hMSCs.
• DSB repair reporter constructs were integrated into hMSCs. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as radio of GFP+/DsRed+ positive cells.
• Fig. 29C shows cell viability in MMS-treated wild type and cents IRT6 (“Cent”) hMSCs.
• Fig. 29D shows immunofluorescence staining of 53BP1 in wild type and centSIRT6 (“Cent”) hMSCs under MMS treatment. Numbers of 53BP1 foci in the nuclei of wild type and centSIRT6 hMSCs with or without MMS (0.25 mM) treatment were quantified. >600 nuclei from 10 images were scored.
• Fig. 29E shows qRT-PCR analysis of SIRT6 expression in wild type and centSIRT6 (“Cent #1” and “Cent #2”) hMSCs (P2).
• Figure 30A-30C is a combination of photograph and histograms illustrating overexpression of SIRT6 (wild type, SIRT6 N308K or centSIRT6 (“Cent”)) in human Werner Syndrome immortalized fibroblasts.
• Fig. 30A shows a proliferation assay of the same cells, using Hoechst fluorescence staining at 24h, 48h and 72h. Data are showed as mean + SEM.
• Fig. 30C shows that telomerase activity assay identified a significant decrease in cents IRT6 group (“Cent”) when compared to not transfected control cells.
• Figure 31A-31B is a combination of photograph and histogram showing that SIRT6 overexpression (wild type, SIRT6 N308K or centSIRT6 (“Cent”)) induces cell death in hepatocellular carcinoma cell lines, after 12 days LV transfection and 3 days after hygromycin selection.
• Fig. 31A shows that in human hepatocellular carcinoma (HCC) cells, SIRT6 WT/SIRT6 N308K/centSIRT6 (“Cent”) overexpression was lethal, with a larger effect observed for SIRT6 N308K/centSIRT6 (“Cent”).
• Fig. 31B represents the quantification of cell viability illustrated in FIG. 31A.
• Figure 32 is a graph showing that SIRT6 overexpression (wild type, SIRT6
• N308K or centSIRT6 (“Cent”) increase differentiation of 3T3-L1 pre-adipocytes (quantification of immunofluorescence).
• Figure 33A-33E is a combination of photograph and histograms showing the effect of SIRT6 overexpression (wild type, SIRT6 N308K and centSIRT6 (“Cent”)) in hepatic stellate cells (LX2).
• Fig. 33A shows the viability of LX2 cells after 24h of transfection with SIRT6 (WT or mutants).
• Fig. 33B-33E shows gene expression (mRNA) in LX2 cells overexpressing or not SIRT6 (WT or mutants) after 24h TGF-b 20ng treatment for Vimentin (Fig. 33B), TIMP1 (Fig. 33C), COL1A1 (Fig. 33D) and aSMA (Fig.
• Figure 34A-34C is a combination of histograms showing the effect of SIRT6 overexpression (wild type, SIRT6 N308K and centSIRT6 (“Cent”)) on spheroids made from Immortalized Human Hepatocytes (IHH) and human hepatic stellate cells (LX2). Spheroids are formed from IHH cells and with 5% the total cell mass of LX2.
• Fig. 34A shows the quantification of collagen I in IHH-LX2 spheroids overexpressing or not centSIRT6 (“Cent”) (quantification of immunofluorescence).
• 34B-34C shows IHH- LX2 spheroids gene expression relative to GAPDH for aSMA, COL1A1, TIMP1, VIMENTIN and MMP2, either in absence of Free Faty Acids (FFA) (Fig. 34B) or in presence of FFA (Fig. 34C) (* p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001 versus respective control).
• FFA Free Faty Acids
• the inventors used herein an alternative, candidate functional association approach to directly identify rare longevity gene variants enriched in the genome of a cohort of Ashkenazi Jewish (AJ) centenarians.
• AJ Ashkenazi Jewish
• the inventors performed targeted sequencing of 301 genes involved in Genome Maintenance (GM), a major longevity assurance system, in 496 AJ centenarians and 572 controls.
• GM Genome Maintenance
• SIRT6 a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme-6, involved in both DNA double-strand breaks (DSB) repair and longevity in model organisms.
• mADPr mono-ADP ribosyl transferase
• the inventors selected two genetically linked variants in the same allele (N308K and A313S), for further functional analysis. Characterization of this SIRT6 centenarian allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA DSB repair, and more robust cancer cell killing compared to the wild type allele.
• centSIRT6 displayed weaker deacetylase activity compared to the wild type. Conversely, its mADPr activity was strongly enhanced. FRET-based analysis demonstrated the centSIRT6 had a more open conformation. centSIRT6 displayed a stronger interaction with Earn in A/C (LMNA), which correlated with enhanced ribosylation of LMNA.
• LMNA Earn in A/C
• This invention relates to an isolated nucleic acid molecule encoding a variant of sirtuin 6 (SIRT6) having at least 75% identity with sequence SEQ ID NO: 1, the variant having at least one mutation selected in the group comprising or consisting of a N308K substitution and an A313S substitution with respect to sequence SEQ ID NO: 1.
• SIRT6 sirtuin 6
• the expression “at least 75% identity” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identity.
• the level of identity of 2 polypeptides may be performed by using any one of the known algorithms available from the state of the art.
• the amino acid identity percentage may be determined using the CLUSTAL W software (version 1.83), the parameters being set as follows: - for slow/accurate alignments: (1) Gap Open Penalty: 10.00; (2) Gap Extension
• the isolated nucleic acid molecule encodes a variant of SIRT6 having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, most preferably at least 96%, 97%, 98% or 99% identity with sequence SEQ ID NO: 1, the variant having at least one mutation selected in the group comprising or consisting of a N308K substitution and an A313S substitution with respect to sequence SEQ ID NO: 1.
• sequence SEQ ID NO: 1 refers to the 361 amino acid residues sequence of wild type SIRT6 polypeptide.
• substitutions N308K and A313S refer to the mutations of the codon encoding the naturally occurring Asn (N) amino acid residue at position 308 in the SIRT6 polypeptide, and the Ser (S) amino acid residue at position 313 in the SIRT6 polypeptide, respectively.
• sequence SEQ ID NO: 5 refers to the 1,068 nucleotides (bp) sequence of wild type SIRT6 polypeptide.
• the naturally occurring Asn (N) amino acid residue at position 308 in the SIRT6 polypeptide is encoded by codon “aac” at positions 922 to 924 of SEQ ID NO: 5.
• the naturally occurring Ser (S) amino acid residue at position 313 in the SIRT6 polypeptide is encoded by codon “gcc” at positions 937 to 939 of SEQ ID NO: 5.
• the N308K substitution is represented by a mutation of codon “aac” at positions 922 to 924 of SEQ ID NO: 5 into codon “aag” or codon “aaa”, preferably into codon “aag”.
• the N308K substitution is represented by a mutation of nucleotide “c” at positions 924 of SEQ ID NO: 5 into nucleotide “g” or nucleotide “a”, preferably into nucleotide “g”.
• the A313S substitution is represented by a mutation of codon “gcc” at positions 937 to 939 of SEQ ID NO: 5 into a codon selected in a group consisting of codons “tcc”, “tct”, “tea” and “teg”, preferably codon “tcc”.
• the A313S substitution is represented by one or two mutation(s) selected in a group consisting of a mutation of nucleotide “g” at positions 937 of SEQ ID NO: 5 into nucleotide “t”; a mutation of nucleotide “g” at positions 937 of SEQ ID NO: 5 into nucleotide “t” and of nucleotide “c” at positions 939 of SEQ ID NO: 5 into nucleotide “t”; a mutation of nucleotide “g” at positions 937 of SEQ ID NO: 5 into nucleotide “t” and of nucleotide “c” at positions 939 of SEQ ID NO: 5 into nucleotide “a”; and a mutation of nucleotide “g” at positions 937 of SEQ ID NO: 5 into nucleotide “t” and of nucleotide “c” at positions 939 of SEQ ID NO: 5 into nucleotide “g”. [0088] In
• sequence SEQ ID NO: 6 refers to the nucleic acid sequence of the variant of SIRT6 with N308K substitution, in particular, with mutation of codon “aac” at positions 922 to 924 of SEQ ID NO: 5 into codon “aag”.
• sequence SEQ ID NO: 7 refers to the nucleic acid sequence of the variant of SIRT6 with A313S substitution, in particular, with mutation of codon “gcc” at positions 922 to 924 of SEQ ID NO: 5 into codon “tcc”.
• sequence SEQ ID NO: 8 refers to the nucleic acid sequence of the variant of SIRT6 with N308K and A313S substitutions, in particular, with mutation of codon “aac” at positions 922 to 924 of SEQ ID NO: 5 into codon “aag” and with mutation of codon “gcc” at positions 922 to 924 of SEQ ID NO: 5 into codon “tcc”.
• the variant of SIRT6 encoded by the isolated nucleic acid molecule as defined herein has a deacylase and/or mono-ADP ribosyl transferase (mADPr) activity.
• deacylase activity and mono-ADP ribosyl transferase (mADPr) activity may be assayed accordingly to any suitable from the state in the art, or a method adapted therefrom.
• deacylase activity may be assayed by contacting in vitro the variant of SIRT6 with histones, in the presence of NAD + , MgCh, DTT and performing a Western blot analysis using anti-H3K9ac and anti-H3K18ac antibodies.
• mono-ADP ribosyl transferase (mADPr) activity may be assayed by contacting in vitro the variant of SIRT6 with PARP1, in the presence of ZnCh, MgCh, NAD + , DTT, salmon sperm DNA and performing a Western blot analysis using anti-PADPR antibodies.
• the variant of SIRT6 has at most about 90%, preferably at most about 50%, more preferably at most about 25% deacylase activity as compared to wild type SIRT6 (of sequence SEQ ID NO: 1).
• the expression “at most about 90%” encompasses about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less.
• the variant of SIRT6 has at most least 100%, preferably at least about 200%, more preferably at least about 300% mono-ADP ribosyl transferase (mADPr) activity as compared to wild type SIRT6 (of sequence SEQ ID NO: 1).
• the expression “at least about 100%” encompasses about 100%, 120%, 140%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750% or more.
• Another aspect of the invention relates to an isolated polypeptide encoded by a nucleic acid molecule according to the instant invention.
• This invention relates to an isolated polypeptide being a variant of SIRT6 having at least 75% identity with sequence SEQ ID NO: 1, the variant having at least one mutation selected in the group comprising or consisting of a substitution N308K and a substitution A313S with respect to sequence SEQ ID NO: 1.
• the expression “at least 75% identity” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity.
• the isolated polypeptide being a variant of SIRT6 has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, most preferably at least 96%, 97%, 98% or 99% identity with sequence SEQ ID NO: 1, the variant having at least one mutation selected in the group comprising or consisting of a substitution N308K and a substitution A313S with respect to sequence SEQ ID NO: 1.
• the polypeptide is of sequence selected in the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
• sequence SEQ ID NO: 2 refers to the amino acid sequence of the variant of SIRT6 with N308K substitution.
• the polypeptide of sequence SEQ ID NO: 2 is encoded by a nucleic acid molecule of sequence SEQ ID NO: 6.
• sequence SEQ ID NO: 3 refers to the amino acid sequence of the variant of SIRT6 with A313S substitution.
• the polypeptide of sequence SEQ ID NO: 3 is encoded by a nucleic acid molecule of sequence SEQ ID NO: 1
• sequence SEQ ID NO: 4 refers to the amino acid sequence of the variant of SIRT6 with N308K and A313S substitutions.
• the polypeptide of sequence SEQ ID NO: 4 is encoded by a nucleic acid molecule of sequence SEQ ID NO: 8.
• the polypeptide is a recombinant polypeptide.
• the term “recombinant polypeptide” refers to a polypeptide encoded by an engineered nucleic acid and synthesized upon transformation of said engineered nucleic acid into a microorganism or transfection in an eukaryotic cell for synthesis purposes.
• a further aspect of the invention relates to a vector comprising the isolated nucleic acid molecule according to the invention.
• the vector in a minicircle nucleic acid encompasses non-viral vectors that merely comprise a gene expression cassette and are free of viral and/or bacterial backbone DNA elements from standard plasmids.
• Plasmid is intended to refer to a small extra-genomic DNA molecule, most commonly found as circular double stranded DNA molecules that may be used as a cloning vector in molecular biology, to make and/or modify copies of DNA fragments up to about 15 kb ( i.e ., 15,000 base pairs). Plasmids may also be used as expression vectors to produce large amounts of proteins of interest encoded by a nucleic acid sequence found in the plasmid downstream of a promoter sequence.
• the term “cosmid” refers to a hybrid plasmid that contains cos sequences from Lambda phage, allowing packaging of the cosmid into a phage head and subsequent infection of bacterial cell wherein the cosmid is cyclized and can replicate as a plasmid.
• Cosmids are typically used as cloning vector for DNA fragments ranging in size from about 32 to 52 kb.
• bacterial artificial chromosome refers to extra-genomic nucleic acid molecule based on a functional fertility plasmid that allows the even partition of said extra-genomic DNA molecules after division of the bacterial cell.
• BACs are typically used as cloning vector for DNA fragment ranging in size from about 150 to 350 kb.
• the vector comprising the nucleic acid molecule encoding a variant of SIRT6 may be in the form of a plasmid, in particular resulting from the cloning of a nucleic acid of interest into a nucleic acid vector.
• non- limitative suitable nucleic acid vectors are pBluescript vectors, pET vectors, pETduet vectors, pGBM vectors, pBAD vectors, pUC vectors.
• the plasmid is a low copy plasmid. In one embodiment, the plasmid is a high copy plasmid.
• the vector is a viral vector.
• the viral vector is selected in a group comprising or consisting of an adenovirus; an adeno- associated virus (AAV); an exosome-associated AAV (exo-AAV); an alphavirus; a herpesvirus ; a retrovirus , such as , e.g. , a lentivirus or a non- integrative lend virus ; vaccinia virus; a baculovirus; or virus like particles such as, e.g., particles derived from Hepatitis B virus, Parvoviridae, Retroviridae, Flaviviridae, Paramyxoviridae or bacteriophages.
• the vector is a viral vector, in particular an adeno- associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an adenoviral vector, a retroviral vector, or a herpes virus vector.
• AAV adeno-associated viral vector
• exo-AAV exosome-associated AAV vector
• adenoviral vector adenoviral vector
• retroviral vector a retroviral vector
• herpes virus vector a viral vector, in particular an adeno-associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an adenoviral vector, a retroviral vector, or a herpes virus vector.
• the adeno-associated viral vector is AAV serotype 2 or AAV serotype 5.
• the vector in particular the viral vector, is an exo-AAV vector.
• exo-AAV refers to a vector wherein an adeno-associated virus (AAV) vector, or parts thereof, is associated with an extracellular vesicle (also referred to as exosome), wherein the AAV vector is partially fused, embedded or internalized in the extracellular vesicle.
• the extracellular vesicle may express specific proteins or markers, for example for targeting purposes.
• the vector, in particular the viral vector does not cross the blood-brain barrier. In some alternative embodiments, the vector, in particular viral vector crosses the blood-brain barrier.
• the choice of the viral vector may depend of the organ or tissue that is targeted.
• a vector that crosses the blood-brain barrier may be preferably selected.
• a vector that does not cross the blood-brain barrier may be preferably selected.
• assessing whether a vector, in particular a viral vector, crosses the blood/brain barrier may be performed by any suitable method acknowledged from the state of the art, or a method adapted therefrom.
• this assessment may be performed by the means of one of the two gold-standard experimental measures of blood/brain barrier permeability, namely, (1) logBB, which is intended to measure the concentration of a compound in the brain divided by concentration in the blood; and (2) logPS, which measures the permeability surface-area product.
• logBB which is intended to measure the concentration of a compound in the brain divided by concentration in the blood
• logPS which measures the permeability surface-area product.
• the vector in particular the viral vector, comprises a promoter sequence suitable for gene expression in mammalian individuals, preferably in human individuals.
• Non-limitative examples of promoter sequence suitable for gene expression in mammalian individuals, preferably in human individuals, include CMV (human cytomegalovirus) promoter, EFla (human elongation factor 1 alpha) promoter, SV40 (Simian vacuolating virus 40) promoter, PGK1 (phosphoglycerate kinase) promoter, Ubc (human ubiquitin C) promoter, and the like.
• the promoter sequence is preferably the CMV promoter.
• the vector in particular the viral vector, further comprises a nucleic acid sequence that facilitates the nuclear localization of the polypeptide encoded by the nucleic acid molecule according to the invention into a target recipient cell.
• these nuclear localization signal NLS have been abundantly discussed in the state of the art.
• the vector in particular the viral vector, may comprise a S/MAR (for scaffold/matrix attachment region) nucleic acid sequence.
• S/MAR nucleic acid sequence also referred to SAR (for scaffold-attachment region) or MAR (for matrix-associated region)
• SAR for scaffold-attachment region
• MAR for matrix-associated region
• the S/MAR nucleic acid sequence may serve as an origin of replication.
• vectors comprising a S/MAR nucleic acid sequence may behave as extra-chromosome in a transfected cell and be advantageously transmitted to its progeny.
• suitable S/MAR nucleic acid sequence may be determined as described in Narwade etal. (Nucleic Acids Research, 2019; 47(14):7247-7261).
• the invention relates to a suspension comprising a vector according to the invention.
• the suspension further comprises a fluid including one or more ingredients selected from the group consisting of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and a combination thereof.
• the suspension is formulated for intravenous infusion.
• the suspension formulated for intravenous infusion may comprise saline (e.g., 0.9% NaCl); lactated Ringers; 5% dextrose; a colloid, such as, e.g., albumin; the like; and any combination thereof.
• the invention pertains to a cell expressing the polypeptide, the cell being preferably transfected with an isolated nucleic acid molecule, or a vector according to the instant invention.
• the cell is a eukaryote cell, preferably an animal cells, more preferably a mammalian cell.
• mammalian cell includes non human mammalian cells and human cells. In some embodiments, the cell is a human cell.
• the cell is selected in the group comprising or consisting of nerve cells, bone cells, breast cells, red blood cells, white blood cells, cartilage cells, epithelial cells, endothelial cells, skin cells, muscle cells, bladder cells, kidney cells, liver cells, prostate cells, cervix cells, ovarian cells, pulmonary cells, retinal cells, conjunctival cells, corneal cells, fat cells, and the like.
• the cell according to the invention has been transfected with an isolated nucleic acid molecule according to the invention, or transduced with an isolated nucleic acid molecule according to the invention, or contacted with the vector or the suspension containing the nucleic acid molecule according to the invention. Therefore, the cell contains the nucleic acid molecules either integrated or not in its genome.
• the vector is an expression system
• the nucleic acid molecule encoding the variant of SIRT6 is present within the cell in a form allowing its expression and its final location, i.e., the cell nucleus and cytoplasm.
• Another aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising (i) an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention, and (ii) a pharmaceutically acceptable excipient.
• a suitable pharmaceutically acceptable carrier includes any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
• suitable pharmaceutically acceptable carriers may include, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and a mixture thereof.
• pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells.
• auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells. The preparation and use of pharmaceutically acceptable carriers are well known in the art.
• One aspect of the invention relates to the use of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention, in the regulation of ageing, and/or senescence, and/or lifespan in an individual, preferably a mammalian individual, more preferably a human individual.
• the term “regulating the senescence” is intended to refer to the process of decreasing, reducing or stopping the senescence mechanism, in particular premature senescence.
• the term “regulating the lifespan” is intended to refer to the process of increasing or improving the lifespan, in other words increasing or improving the duration of the life time course.
• the invention further relates to a method for the regulation of ageing, and/or senescence, and/or lifespan in an individual, preferably a mammalian individual, more preferably a human individual, in need thereof, comprising the administration of a therapeutically efficient amount of an isolated nucleic acid molecule, or an polypeptide isolated, or a vector according to the instant invention.
• a further aspect of the invention pertains to the use of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention, in the repair of double strand breaks in a cell, preferably a mammalian cell, more preferably a human cell.
• the invention relates also to a method for the repair of double strand breaks in a cell, preferably a mammalian cell, more preferably a human cell, comprising the administration of a therapeutically efficient amount of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention.
• repair of double strand breaks refers to the process allowing to recover DNA integrity by repair DNA damages consisting in the occurrence of a break in both strands of the double strand DNA nucleic acid.
• repair mechanisms such as non-homologous end joining (NHEJ) and homologous repair (HR).
• efficiency of double strand breaks repair may be assessed by any suitable method from the state of the art, or a method adapted therefrom.
• double strand break repairs efficiency may be assessed by the methods described in Seluanov etal. (J Vis Exp, 2010; doi: 10.3791/2002).
• the nucleic acid molecule, the polypeptide, the vector, the suspension or the pharmaceutical composition according to the invention are intended to increase, improve or ameliorate the efficiency of the DSB repair in the target cell, as compared to the efficiency of the DSB repair in the absence of any treatment, as a reference level.
• DSB repair encompasses at least 1.2-fold increase as compared to a reference level.
• the term “at least 1.2-fold” encompasses, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000-fold, or more.
• the invention relates to an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention, for use in the prevention and/or the treatment of an age-related disease.
• the invention also relates to the to the use of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention, for the preparation or the manufacture of a medicament for the prevention and/or the treatment of an age-related disease.
• the invention pertains to a method for the prevention and/or the treatment of an age-related disease in an individual in need thereof, comprising the administration of a therapeutically efficient amount of an isolated nucleic acid molecule, or an isolated polypeptide, or a vector according to the instant invention.
• the individual in need thereof is a mammalian individual, preferably a human individual.
• the age-related disease is selected in the group comprising or consisting of progeria, Wemer syndrome, neurodegenerative disease, Alzheimer's disease, cancer, cardiovascular disease, obesity, type 2 diabetes, hypercholesterolemia, hypertension, ocular disorders, cataracts, glaucoma, osteoporosis, blood clotting disorders, arthritis, hearing loss and stroke.
• the age-related disease is progeria or Werner syndrome.
• the age-related disease is cancer.
• the cancer is a blood cancer or a solid cancer.
• blood cancer also referred to as “hematologic cancer”, encompasses any cancer involving uncontrolled proliferation of blood cells, in particular white blood cells.
• Blood cancers includes leukemia, lymphoma (Hodgkin and non- Hodgkin lymphomas) and myeloma.
• the cancer is a blood cancer.
• said cancer is a blood cancer selected in the group consisting of Hodgkin’s disease, immunoblastic lymphadenopathy, lymphoma, chronic lymphocytic leukemia, acute leukemia, and the like.
• solid cancer encompasses any cancer (also referred to as malignancy) that forms a discrete tumor mass, as opposed to cancers (or malignancies) that diffusely infiltrate a tissue without forming a mass.
• the cancer is a solid cancer.
• the solid cancer is selected in the group consisting of fibrosarcoma, melanoma, breast carcinoma, colon carcinoma, renal carcinoma, adrenocortical carcinoma, testicular teratoma, skin sarcoma, fibrosarcoma, lung carcinoma, adenocarcinoma, liver carcinoma ( i.e ., hepatocarcinoma), glioblastoma, prostate carcinoma, ovarian cancer and pancreatic carcinoma.
• the age-related disease is selected in the group comprising or consisting of Hutchinson Gilford progeria, Werner syndrome, Alzheimer's disease and cancer.
• the age-related disease is selected in the group comprising or consisting of Hutchinson Gilford progeria, Werner syndrome, Alzheimer's disease and fibrosarcoma. [0155] In some embodiments, the age-related disease is selected in the group comprising or consisting of Hutchinson Gilford progeria, Werner syndrome, Alzheimer's disease and hepatocarcinoma.
• the age-related disease is hepatocarcinoma. In one embodiment, the age-related disease is fibrosarcoma.
• the progeria is the Hutchinson Gilford progeria.
• the age-related disease is Werner syndrome.
• the nucleic acid molecule, the polypeptide, the vector, the suspension, or the pharmaceutical composition according to the invention is to be administered to an individual in need thereof by any suitable route, i.e., by a dermal administration, by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra ocular administration and an intra-auricular administration ⁇
• the nucleic acid molecule, the polypeptide, the vector, the suspension, or the pharmaceutical composition according to the invention is to be administered to an individual in need thereof by a dermal administration ⁇
• the nucleic acid molecule, the polypeptide, the vector, the suspension, or the pharmaceutical composition according to the invention is associated with a composition enabling and/or facilitating dermal administration, e.g., by increasing dermal tropism or by increasing dermal barrier penetration.
• the dermal administration enables a prolonged liberation of the nucleic acid molecule, the polypeptide, the vector, the suspension, or the pharmaceutical composition according to the invention.
• the nucleic acid molecule, the polypeptide, the vector, the suspension, or the pharmaceutical composition according to the invention is to be administered to an individual in need thereof by an intravenous administration, in particular by intravenous infusion or intravenous injection.
• the therapeutically effective amount of the nucleic acid molecule, the polypeptide, the vector, the suspension, or the pharmaceutical composition according to the invention, to be administered may be determined by a physician or an authorized person skilled in the art and can be suitably adapted within the time course of the treatment.
• the therapeutically effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual’s parameters including age, physical conditions, size, weight, gender, and the severity of the age-related disease to be treated.
• a therapeutically effective amount of the isolated polypeptide, or the pharmaceutical composition comprising the isolated polypeptide according to the invention, agent may range from about 0.001 mg to about 3,000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.
• the expression “from about 0.001 mg to about 3,000 mg” includes, from about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg,
• the isolated polypeptide or the pharmaceutical composition comprising the isolated polypeptide according to the invention may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.
• the expression “from about 0.001 mg/kg to about 100 mg/kg” includes about 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 20 mg/kg, 30
• the therapeutically efficient amount of the isolated nucleic acid molecule, vector, or pharmaceutical composition according to the invention is ranging from about 10 1 to about 10 15 copies per ml.
• a therapeutically efficient amount includes about 10 1 , 5xl0 ⁇ 10 2 , 5xl0 2 , 10 3 , 5xl0 3 , 10 4 , 5xl0 4 , 10 5 , 5xl0 5 , 10 6 , 5xl0 6 , 10 7 , 5xl0 7 , 10 s , 5xl0 8 , 10 9 , 5xl0 9 , 10 10 , 5xl0 10 , 10 11 , 5xl0 n , 10 12 , 5xl0 12 , 10 13 , 5xl0 13 , 10 14 , 5xl0 14 and 10 15 copies per ml.
• the therapeutically efficient amount is from about 10 1 to about 10 15 copies per cm 3 , which includes about 10 1 , 5X10 1 , 10 2 , 5xl0 2 , 10 3 , 5xl0 3 , 10 4 , 5xl0 4 , 10 5 , 5xl0 5 , 10 6 , 5xl0 6 , 10 7 , 5xl0 7 , 10 s , 5xl0 8 , 10 9 , 5xl0 9 , 10 10 , 5xl0 10 , 10 11 , 5xl0 n , 10 12 , 5xl0 12 , 10 13 , 5xl0 13 , 10 14 , 5xl0 14 and 10 15 copies per cm 3 .
• the therapeutically efficient amount is from about 10 1 to about 10 15 copies per dose, which includes about 10 1 , 5xl0 ⁇ 10 2 , 5xl0 2 , 10 3 , 5xl0 3 , 10 4 , 5xl0 4 , 10 5 , 5xl0 5 , 10 6 , 5xl0 6 , 10 7 , 5xl0 7 , 10 8 , 5xl0 8 , 10 9 , 5xl0 9 , 10 10 , 5xl0 10 , 10 11 , 5xlO n , 10 12 , 5xl0 12 , 10 13 , 5xl0 13 , 10 14 , 5xl0 14 and 10 15 copies per dose.
• the isolated nucleic acid molecule, isolated polypeptide, vector, suspension, or pharmaceutical composition according to the instant invention is to be co-administered, or sequentially administered, with a drug suitable for preventing and/or treating an age-related disease, in particular a disease selected in the group consisting of progeria, Werner syndrome, neurodegenerative disease, Alzheimer's disease, cancer, cardiovascular disease, obesity, type 2 diabetes, hypercholesterolemia, hypertension, ocular disorders, cataracts, glaucoma, osteoporosis, blood clotting disorders, arthritis, hearing loss and stroke.
• a drug suitable for preventing and/or treating an age-related disease in particular a disease selected in the group consisting of progeria, Werner syndrome, neurodegenerative disease, Alzheimer's disease, cancer, cardiovascular disease, obesity, type 2 diabetes, hypercholesterolemia, hypertension, ocular disorders, cataracts, glaucoma, osteoporosis, blood clotting disorders, arthritis, hearing loss and stroke.
• the term “co-administered” refers to a simultaneous administration of the active principles.
• the term “sequentially administered” refers to an administration of a first active principle before or after the administration of a second active principle.
• Another aspect of the invention relates to a kit comprising (i) an isolated nucleic acid, an isolated polypeptide molecule, a vector, or a suspension according to the instant invention, and (ii) means to administer the isolated nucleic acid molecule, the isolated polypeptide, the vector, or the suspension.
• the means to administer the isolated nucleic acid molecule, the isolated polypeptide, the vector, or the suspension include a syringe or a catheter.
• Another aspect of the invention relates to a method for increasing adipogenic differentiation in a subject, comprising administering an effective amount of a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleic acid molecule encoding thereof.
• the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the nucleic acid sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• Another aspect of the invention relates to an in vitro method for increasing adipogenic differentiation of a cell, or population of cells, comprising exposing the cell, or population of cells, to an effective amount of a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleic acid molecule encoding thereof.
• the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the nucleic acid sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• the cell, or population of cells is a pre- adipocyte.
• Another aspect of the invention relates to a method for decreasing a-SMA expression in a subject, comprising administering an effective amount of a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleic acid molecule encoding thereof.
• the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the nucleic acid sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• Another aspect of the invention relates to an in vitro method for decreasing a- SMA expression in a cell, or population of cells, comprising exposing the cell, or population of cells, to an effective amount of a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleic acid molecule encoding thereof.
• the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the nucleic acid sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• the cell, or population of cells is a pre- adipocyte.
• Another aspect of the invention relates to a method for decreasing collagen expression in a subject, comprising administering an effective amount of a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleic acid molecule encoding thereof.
• the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the nucleic acid sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• collagen is selected from the group comprising or consisting of type I collagen, type II collagen, type III collagen, type V collagen and type IX collagen.
• collagen is type I collagen.
• the type I collagen is type I alpha 1 collagen or type I alpha 2 collagen.
• the type I collagen is type I alpha 1 collagen, and is encoded by COL1A1 gene.
• Another aspect of the invention relates to an in vitro method for decreasing collagen expression in a cell, or population of cells, comprising exposing the cell, or population of cells, to an effective amount of a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a nucleic acid molecule encoding thereof.
• the nucleic acid molecule has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5 or 100% sequence identity with the nucleic acid sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
• the cell, or population of cells is a pre- adipocyte.
• collagen is selected from the group comprising or consisting of type I collagen, type II collagen, type III collagen, type V collagen and type IX collagen.
• collagen is type I collagen.
• the type I collagen is type I alpha 1 collagen or type I alpha 2 collagen.
• the type I collagen is type I alpha 1 collagen, and is encoded by COL1A1 gene.
• This study group consisted of 1,068 Ashkenazi Jewish (AJ) samples: 496 AJ centenarians and 572 AJ controls that were previously collected as part of longevity study at the Albert Einstein College of Medicine.
• a centenarian is defined as a healthy individual living independently at 95 years of age or older and a control is defined as an individual without a family history of unusual longevity; parents of controls survived to the age of 85 years or less.
• Informed written consent was obtained in accordance with the policy of the Committee on Clinical Investigations of the Albert Einstein College of Medicine. Genomic DNAs were extracted from blood samples and amplified using Illustra GenomiPhi V2 DNA Amplification kits (GE healthcare Life Sciences®).
• stage 1 301 candidate genes involved in genome maintenance were selected and sequenced in 51 centenarians and 51 controls. The complete list of 301 candidate genes is provided in Table 1.
• Table 1 List of 301 genome maintenance genes sequenced in first round
• Table 2 List of 122 genes showing significant association with longevity in gene-based UMINP analysis
• stage 2 106 candidate genes involved in DNA double-strand breaks repair were selected and sequenced in 496 centenarians and 572 controls. The list of 106 DSB repair genes is provided in Table 3. [0188] Table 3: List of 106 DSB repair genes sequenced in second round
• a 25-sample pooling target capture sequencing was performed. Briefly, 25 samples from the same group were pooled together for equimolar concentration and totally 20 pools of centenarians and 23 pools of controls were used to generate indexed libraries according to the Illumina® TruSeq DNA sample preparation low-throughput (LT) protocol. Captured libraries were further generated using SeqCap EZ Choice Enrichment Kit (Roche®) which enriched genomic regions covering exons, exon-intron junctions, and 2 kb proximal promoter sequences of the candidate DSB genes and sequenced by Illumina® HiSeq2000.
• LT Illumina® TruSeq DNA sample preparation low-throughput
• Sequencing reads were aligned using BWA and variants were called using the software CRISP (Comprehensive Read analysis for Identification of Single Nucleotide Polymorphisms), which was specifically developed to call variants in pooled DNA sequence data. All variants in the 1,068 samples were characterized and functionally annotated using ANNOVAR. To test for longevity associations, we performed different subtypes of SKAT (Sequencing Kernel Association Test) analyses, such as SKAT for rare variants, SKAT-C for all variants (common and rare), and SKAT-0 for variants with directionality (enrichment only in centenarians or in controls) (Table 4).
• SKAT Sequencing Kernel Association Test
• Table 4 List of 10 longevity-associated DSB repair genes with their centenarian- enriched missense variants (part 1) 1.3. Cell lines
• HEK293 SIRT6 overexpression lines were generated by transfecting HEK293 cells with linearized CMV-SIRT6 plasmids via jetPRIME transfection reagent and selecting stably integrated clones.
• telomerase-immortalized HCA2 human fibroblast cell lines containing integrated reporter constructs I9A and H15C.
• HT1080 and HeLa cell lines were used to assess anti-tumor activity.
• H3 (Abeam® ab500)-l:5,000
• H3K9ac (Abeam® ab4441)-l: 1,000
• H3K18ac (Abeam® abll91)-l:l,000
• b-tubulin (Abeam® ab6046 1:10,000
• SIRT6 Cell Signaling® #12486)4:1,000
• AbD33204 (Rabbit, REF)- 1:500
• Earn in A/C-l 1,000
• Abeam® abl08595, Millipore® 05-714
• gH2AC (Millipore® 05-636)
• PARPl(Abcam® ab227244)-l 1,000
• mADPr 1:500 (AbD33204 and AbD33205).
• IP Immunoprecipitation
• Transfections were carried out by plating cells at a density of 500,000 cells/10 cm plate two days prior to transfection. Transfections were carried out using the Amaxa® Nucleofector with Normal Human Dermal Fibroblast transfection solution following manufacture’s protocol. For cancer cell transfections, jetPRIME transfection reagent was used to deliver plasmids into cells.
• gH2AC immunostaining was carried out as previously described in Mao et al. (Science, 2011; Vol. 332:1443-1446).
• Anti- gH2AC antibody was purchased from Millipore® (05-636).
• Apoptosis in fibroblasts was measured using the Annexin V Staining Kit (Roche®).
• lysate was incubated with Ni2+ -NTA agarose overnight.
• Solution with beads was placed in gravity column and washed with lysis solution, followed by 2 volumes of wash buffer (lysis buffer + 30 mM imidazole).
• Cell pellets were lysed in buffer containing 8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.5, with protease inhibitor cocktail (Roche®). Pellets were vortexed for 30 s and sonicated 5x 10 s with 1 min rest on ice in between sonication steps. Lysate was centrifuged at 15,000 xg for 10 min and then supernatant was collected.
• the substrate for this reaction is a synthetic TNF-derived peptide first developed by Schuster et al. (Sci Rep, 2016; Vol. 6:22643) was synthesized by Genscript®. Reactions were performed with 150 mM NaCl, 20 mM Tris, 5% glycerol, lmM b- mercaptoethanol, 2 mM SIRT6, and substrate concentration ranges of 7-1000 mM NAD+ and 2-88 pM peptide. Reactions were performed at 37°C and fluorescence was measured using 310/405 nm excitation/emission spectra on a Tecan Spark 20M plate reader. Readings were taken every 30 s and initial rates were calculated from the relative fluorescence increase over a minimum of 6 min.
• Histones were purified from cumate SIRT6 fibroblasts (SIRT6 KO primary human foreskin fibroblasts constitutively expressing the catalytic subunit of telomerase as well as various alleles of SIRT6 via a cumate-inducible promoter) using an established acid extraction method followed by propionylation of lysines prior to trypsin digestion to enhance coverage.
• a data independent analysis (DIA) mass spectrometry (MS) method was employed for quantitation of modified peptides after samples were separated by nano-LC using EpiProfile® or, alternatively, after direct infusion followed by a one- minute acquisition and analysis with EpiProfileLite®.
• Samples were washed with 3 additional ml of extraction buffer and eluted with 100 pi of boiling elution buffer (5% SDS with 50 mM Tris-HCl pH 7.5). After removal of SDS with S-columns (Protifi®; Huntington, NY) and trypsin digestion, peptides were resuspended in MS-grade water and labeled with tandem mass tags (TMT 10-plex; Thermo Fisher Scientific®). Samples were resolved by nano-electrospray ionization on an Orbitrap Fusion Lumos MS instrument (Thermo Fisher Scientific®) in positive ion mode using a 30 cm home-made column packed with 1.8 pm C-18 beads to resolve the peptides.
• Orbitrap Fusion Lumos MS instrument Thermo Fisher Scientific®
• Solvent A was 0.1% formic acid and solvent B was 100% acetonitrile (CAN) with 0.1% formic acid.
• the length of the run was 2 h with a 90 min gradient.
• CID (35% collision energy) was used for MS2 fragmentation.
• HCD (60% collision energy) was used for MS3 detection of TMT groups.
• Other details of the run parameters may be found in the embedded run report of the RAW data file uploaded to the ProteomeXchange database. Peptide assignments were made using Proteome Discoverer and Sequest and MS3 ions were used for quantitation. False discovery rates (FDR) were estimated using a Decoy Database Search with Target FDR (strict) set to 0.01 and Target FDR (relaxed) set to 0.05.
• FDR False discovery rates
• Validation was based on q- value. In the consensus step, ions with a co-isolation threshold above 30% were excluded. Normalization between replicates was achieved using the total protein approach where peptide counts for a single protein were divided by the sum of all proteins in the lane. Proteins appearing in specific antibodies (either anti-SIRT6 or anti- Lamin A) compared to normal IgG serum with Student t-test p ⁇ 0.05) were considered as interactions. Similarly, proteins showing higher levels in centSIRT6 versus wild type SIRT6 were based upon p ⁇ 0.05.
• HEK293-6E cells were transfected using 293fectin® reagent according to the manufacturer’s instructions (Thermo Fisher Scientific®).
• the SIRT6 biosensors were expressed using mammalian expression vector pTT5 (National Research Council, Canada). After two days in culture, approximately 30 million were produced, generating transiently transfected cell lines expressing the biosensor at high levels. The cell line was maintained using FI 7 medium (Sigma Aldrich®) + (200 nM/mL) GlutaMAX®.
• GM genome maintenance
• GM plays a role in human extreme longevity
• a panel of 301 genome maintenance genes (Table 1) in pathways including nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), non-homologous end-joining (NHEJ), telomere maintenance, and genes involved in the regulation of these pathways, was assembled.
• NER nucleotide excision repair
• BER base excision repair
• MMR mismatch repair
• HR homologous recombination
• NHEJ non-homologous end-joining
• telomere maintenance genes involved in the regulation of these pathways
• SIRT6 knockout mice show premature ageing and genomic instability whereas mice overexpressing SIRT6 display an extended lifespan (see Mostoslavsky et al. Cell, 2006; Vol. 124:315-329). Higher SIRT6 activity is associated with more efficient DSB repair and longer maximum lifespan across mammalian species.
• SIRT6 is involved in DNA repair, telomere maintenance, silencing of the repetitive elements including LINE1 retrotransposons, regulation of glucose homeostasis, inflammation and pluripotency, and tumor suppression.
• centenarian SIRT6 allele found enriched in centenarians contained two missense mutations within the highly flexible C-terminus, converting a polar asparagine 308 to a charged lysine (N308K) and a hydrophobic alanine 313 to a polar serine (A313S). Genotyping in centenarian carriers confirmed that these two SIRT6 variants occurred as linked double variants, which we named centSIRT6. This allele was very rare (minor allele frequency of 0.001%) in the whole exome sequence data of 50,726 adult participants of predominantly European ancestry in the Geisinger Health System. It was next tested whether centenarian SIRT6 allele alters the biological activities of SIRT6 protein.
• centSIRT6 has similar affinity for NAD+ to the wild type allele (Table 6). Overall, these data indicate that centSIRT6 confers a slight reduction in deacylase catalytic efficiency (as defined by Vmax /Km) (Table 6).
• centSIRT6 displayed significantly lower deacetylase activity on recombinant nucleosomes with saturated H3K9ac and H3K18ac modifications ( Figure 7A-7B). Additionally, centSIRT6 allele displayed significantly slower histone deacetylase kinetics ( Figure 8A-8B). Similar to the synthetic histones, the centSIRT6 allele deacetylated both H3K9ac and H3K18ac residues on nucleosomes purified from HeLa cells, at a significantly reduced rate compared to the wild type SIRT6 ( Figure 9A-9B). Taken together, the combined data from deacylase and histone deacetylase experiments indicate that the centSIRT6 variant has reduced deacylase/deacetylase activity.
• telomerase-immortalized human HCA2 fibroblast cell lines were generated in a SIRT6 KO background containing a cumate switch promoter driving expression of SIRT6 WT, N308K, A313S, and the centSIRT6 alleles (cumate SIRT6 fibroblasts). Each cell line was induced with cumate to drive equal levels of SIRT6 expression ( Figures 27A-27B). Histone post-translational modifications from purified histones were quantified from these cells by mass spectrometry using peptide standards as described previously (Sidoli et al.
• centSIRT6, N308K, and A313S SIRT6 alleles showed similar global histone post translational modification (PTM) levels at all sites known to be SIRT6 substrates including H3K9ac, H3K18ac, and H3K56ac ( Figure 10).
• PTM global histone post translational modification
• SIRT6 In addition to its deacetylase activity, SIRT6 has mono-ADP ribosylation (mADPr) activity, able to mono-ADP ribosylate itself and other proteins. mADPr activity is critical to SIRT6 role in DNA damage response and LINE1 repression.
• mADPr mono-ADP ribosylation
• two known SIRT6 substrates were used: PARP1 and SIRT6, itself.
• the centSIRT6 demonstrated a significantly higher auto-ribosylation rate with radio labeled NAD+ than the wild type SIRT6 ( Figure 12).
• centSIRT6 displayed a similar increase in self-mADPr compared to the wild type, suggesting a general increase that is not cumulative between the two SNPs ( Figures 12 and 28).
• an antibody with specificity to mADPr residues was utilized to examine the self-ribosylation efficiency using a titration of NAD+ (Bonfiglio et al. ; Cell, 2020; Vol. 183:1086-1102).
• centSIRT6 displayed almost 2-fold higher maximal mADPr activity and displayed a higher Km NAD of 142+29, compared to 106+45 for wild type SIRT6 ( Figure 13).
• centSIRT6 displays enhanced mADPr activity.
• centSIRT6 is more efficient at silencing LINE1 elements, which are functionally implicated in longevity.
• centSIRT6 enhances genome maintenance
• a knock-in human embryonic stem cells carrying centSIRT6 allele was generated.
• the two centenarian mutations were introduced using CRISPR CAS9, and confirmed by sequencing.
• Two independent knock-in clones were generated and differentiated into MSCs.
• the MSCs were then transfected with linearized NHEJ and HR GFP reporters.
• Cells harboring centSIRT6 allele showed higher efficiency of repair ( Figures 29A-29B). These cells were also showed increased survival and lower number of 53BP1 foci upon treatment with methyl methanesulfonate (MMS), a potent DNA-damaging agent (Figures 29C-29D).
• MMS methyl methanesulfonate
• Figures 29C-29D potent DNA-damaging agent
• centSIRT6 alleles were transfected into two common tumor cell lines, HT1080 fibrosarcoma cells and HeLa cells.
• the centSIRT6 showed ⁇ 2-fold fewer adherent surviving cells in both cancer cell lines ( Figure 20A).
• the centSIRT6 allele triggered at least 2-fold higher levels of apoptosis than the wild type SIRT6 allele ( Figure 20B-20C).
• centSIRT6 mutations are located in the flexible C-terminus of SIRT6 and may influence protein-protein interactions
• the interactomes of the centSIRT6 and the wild type alleles were compared.
• Antibodies against SIRT6 were used to immuno- precipitate SIRT6-interacting proteins from cumate SIRT6 fibroblasts and to analyze them by mass spectrometry with tandem-mass tags (TMT) to permit accurate quantitation.
• TMT tandem-mass tags
• Several interacting proteins were enriched by the centSIRT6 allele (Table 7).
• Table 7 Proteins showing stronger interaction with cents IRT6 compared to the wild type SIRT6.
• Proteins were prepared from cumate-induced SIRT6 cell lines and immnno- precipitated with SIRT6 antibody; rabbit pre- immune serum was used as a control. Prior to analysis by mass spectrometry, samples were labeled with tandem-mass tags.
• LMNA is a nuclear scaffold protein playing a central role in nuclear organization and is also implicated in ageing. Aberrant processing of LMNA results in human premature ageing syndrome, Hutchison Gilford Progeria, while LMNA SNPs have been identified in human centenarians.
• centSIRT6 allele may influence the interaction of LMNA with other proteins.
• LMNA from the cumate fibroblasts expressing either the wild type SIRT6 or centSIRT6 alleles were immuno-precipitated and quantitative protein interaction analysis using TMT labeling followed by mass spectrometry were performed. While many proteins showed similar interaction with LMNA, regardless of the SIRT6 allele present, a large group of proteins showed enhanced interaction with LMNA in the presence of centSIRT6 ( Figure 26).
• LMNA-interaction partners enhanced by sentSIRT6 included ELAVL1, FUS, PCBP2, SAFB, SRSF1, SRSF3, TFIP11, THRAP3, BCLAF1, and U2AFBP all proteins that facilitate the coordination of the DNA damage response with RNA processing.
• Many of the proteins identified in SIRT6 and LMNA IPs are ADP-ribosylated (shown as hexagons in Figure 26), suggesting that ADP-ribosylation by SIRT6 or SIRT6-activated PARP1 may play a role in mediating these interactions.
• centSIRT6 has enhanced capability to promote DNA repair and suppress LINE1 elements, which is likely mediated by its enhanced mADPr activity and stronger interaction with LMNA.
• SIRT6 mutations associated with enhanced mADPr activity suggest that increased SIRT6 activity benefits human longevity. While increased SIRT6 activity correlates with longer lifespan in model organisms, it has not been clear which of SIRT6’s enzymatic activities are central to longevity. Interestingly, loss of SIRT6 deacetylation activity in humans or SIRT6 knockout in monkeys lead to severe developmental phenotypes rather than premature ageing, suggesting that, at least in primates, deacetylation activity of SIRT6 is required for development but not necessarily for adult longevity.
• the centSIRT6 variant also shows enhanced binding to LMNA which may further promote its function in DNA repair and chromatin organization via direct stimulation of SIRT6 enzymatic activity, and by coordinating interactions of LMNA with other components of the LMNA complex.
• 3T3-L1, LX2, HepG2, Hep3B (all purchased from American Type Culture Collection - ATCC, Manassas) and WSL (purchased from The Coriell Institute for Medical Research, NJ) cells were grown in basal media cosnsisting of high glucose (4.5g/l) DMEM (LM-D 1108/500, Biosera ) supplemented with 10% bovine fetal calf serum (L9665, Sigma- Aldrich), 15 mM hepes buffer (L0180, Biowest), GlutaMAXTM Supplement (lOOx, 35050-038, Gibco), 1001 I/ml penicillin and 100 mg/ml streptomycin solution (L0022, Biowest), lOOnM sodium pyruvate (11360, Gibco) and non-essential amino acids (NEAA 11140, lOOx, Gibco) in 5% C02/humidified atmosphere at 37°C. The medium was routinely changed every 2 days and the cells were sub
• IHH cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Williams E medium (12551032, Gibco) containing 2g/l glucose and supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 pg/ml streptomycin solution, 20 rnlJ/ml insulin and 50 nM dexamethasone in 5% C02/humidified atmosphere at 37 °C. The cell culture medium was changed every 2 days and the cells were sub-cultured using TrypLE Express when reaching 90% confluence.
• Williams E medium 12551032, Gibco
• the cells are kept in the maintenance media consisting of basal high-glucose DMEM media supplemented only with 25 nM dexamethasone and 0. lpg/ml insulin for the period of at least 4 days.
• Adipogenic differentiation and/or accumulation of intracellular lipids was evaluated using fluorescence labeling by BODIPY dye (1 pg/ml) for 30 minutes (ex/em 493/503) and DAPI counterstain 1 ug/mL for 30 min. Fluorescence was then captured by Agilent BioTek FLx800 Microplate Fluorescence Reader equipped with appropriate excitation/emission filters.
• Genomic RNA was isolated by column separation techniques using an RNeasy mini Kit (74106, Qiagen, Germany), according to manufacturer's instructions. At least 4 biological replicates were prepared for each treatment group. After quantification of isolated total RNA on NanoDrop 1000 spectrophotometer (ThermoFisher Scientific), lpg of total isolated RNA was used to prepare cDNA using a High-Capacity cDNA Reverse Transcription Kit (4368814, ThermoFisher Scientific). Real Time-PCR was performed in at least two technical replicates using a StepOnePlus TM Real-Time PCR System (Applied Biosystems) and SYBRTM Select Master Mix (4472908, ThermoFisher Scientific). The PCR reaction was held in lOul volume and 250 ng cDNA per well was used as the input quantity. The primer sequences used in this study are listed in Table 8.
• TIMP1 F ACCACCTTATACCAGCGTTATGA 17 R GGTGTAGACGAACCGGATGTC 18 VIMENTIN F AGTCCACTGAGTACCGGAGAC 19 R CATTTCACGCATCTGGCGTTC 20 MMP2 F TACAGGATCATTGGCTACACACC 21
• Quantitative RT-PCR was used to determine changes in average telomere length of FV transfected WSF cells and untreated WSF based on ScienCell's Relative Human Telomere Fength Quantification qPCR Assay Kit (#8909).
• the kit directly compares the average telomere length of the samples.
• the telomere primer set recognizes and amplifies telomere sequences.
• the single copy reference (SCR) primer set recognizes and amplifies a 100 bp-long region on human chromosome 17, and serves as reference for data normalization.
• the carefully designed primers ensure: (i) high efficiency for trustworthy quantification; and (ii) no non-specific amplification.
• Genomic DNA was isolated from using the Qiagen DNA Isolation Kit (Qiagen, Hilden, Germany). Each PCR reaction contained genomic DNA sample (0.01 pg/pF), telomere primer, 2x qPCR master mix, and nuclease-free water. Primer-probe real-time PCR was performed using StepOnePlusTM Real-Time PCR System (Applied Biosystems). All reactions were performed in duplicate, and template controls were included in each run.
• telomere length was calculated by following the manufacturer's instructions.
• telomere activity of FV WSF transfected cell lines and normal WSF cells was assessed by qPCR in StepOnePlusTM Real-Time PCR System (Applied Biosystems) using SYBR® Green assay kits (ScienCell’s Telomerase Activity Quantification qPCR Assay Kit [TAQ], Carlsbad, CA, USA) following the recommendations of the manufacturers. Three million cells were harvested for each sample. For each experiment, two controls were used: a negative telomerase and positive cell lysates (Cat #8928e).
• PCR was performed in a final volume of 20 pL using 1 pL of post-telomerase reaction sample, 2 pL of TPS, 10 pL of 2x qPCR FastStart Essential Green Master Mix (Roche Diagnostics International) and 7 pL of nuclease-free water.
• the PCR conditions were as follows: 95°C for 10 min followed by 36 cycles of: 95 °C for 20 sec, 52°C for 20 sec and 72°C for 45 sec. All reactions were run in three replicates. Data analysis was conducted according to manufacturer’s instructions.
• Equal amount of protein sample (at least 20pg) was mixed with lx Laemmli Sample buffer (1610747, 4x, Bio-Rad) and after boiling at 95°C for 5 min and cooling on ice, equal volume of proteins (40pl) was loaded on 10% Mini-PROTEAN® TGX Stain- Free TM Protein Gels (4568034, Bio-Rad) and separated by electrophoresis running at 120 volts for 45 minutes. Protein transfer was performed on PVDF membranes using Trans- Blot Turbo RTA Mini 0.45 pm LF PVDF Transfer Kit (1704274, Bio-Rad) and Bio-Rad Trans-Blot Turbo Transfer System at 1.3 A and 25V for 10 min.
• Membranes were then blocked with 5 % bovine serum albumin (BSA, P6154, BioWest) dissolved in TBST buffer (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1 % Tween 20) for at least 30 minutes and incubated with the specific primary antibodies (see below) diluted in TBST blocking solution, at appropriate dilutions. Following three washes in TBST buffer, membranes were incubated with a secondary antibody conjugated with horseradish peroxidase diluted in TBST blocking buffer.
• BSA bovine serum albumin
• Antibodies used in this study Cell Signaling Technology (MA, USA) - rabbit anti-Akt (1:1000), rabbit anti-Phospho-Akt (Ser473) (1:1000), rabbit anti Vimentin (1:1000), rabbit anti Histone H3 (D1H2, 1:1000), Abeam (UK) - mouse anti- -actin HRP conjugated antibody (AC-15; 1:2000), mouse anti-GAPDH monoclonal HRP conjugated antibody (1:2000), rabbit anti Collagen I (1:1000), rabbit anti aSMA (1:1000), rabbit anti SIRT6 antibody (1:1000, EPR18463), ThermoFischer Scientific (CA, USA) - mouse IgGl GAPDH monoclonal HRP conjugated antibody (1:2000), secondary goat Anti rabbit IgG HR P-1 inked (1:2000);
• BIOFLOATTM 96- well round bottom ultra-low attachment plates for cell culture at 10000 viable cells per well.
• Each IHH LV transfect cell line (CTL/EMPTY, WT, N308K and centSIRT6) was co-cultured with either normal LX2 or overexpressing centSirt6 LX2 cells with 20: 1 ratio, to reproduce the physiological proportion in the liver parenchyma, where hepatocytes are major cell type with only ⁇ 5% hepatic stellate cells.
• the spheroids were grown in basal high glucose DMEM media supplemented as described above.
• the plates were incubated for 3 days at 37°C in a humidified atmosphere at 5% C02, after which spheroids were treated or not with free fatty acid solution (FFA, L9655, Sigma- Aldrich) and kept in cultivation for 48 hours. Spheroids were then collected, washed twice with PBS, fixed in 4% paraformaldehyde + Eosin solution for 20 minutes, incubated in 10% sucrose for 20 minutes and incorporated into tissue freezing media (14020108926, Leica Biosystems, USA) for cutting and subsequent immuno-histological analysis.
• FFA free fatty acid solution
• Fibrosis i.e., collagene 1A abundance in spheroid samples, was evaluated as the % of the total spheroid area delineated by DAPI fluorescence at lOOx magnification, when at least 5 spheroids per each condition/cell line were used in three consecutive and independent experiments.
• SIRT6 wild type or SIRT6 carrying one or two mutations associated with human exceptional longevity were overexpressed in a variety of cell lines, and assessed how this affected their specific cellular functions.
• SIRT6 WT/SIRT6 N308K/centSIRT6 overexpression was lethal, with a larger effect observed for SIRT6 N308K/centSIRT6 ( Figure 31A-31B). Therefore, mutated SIRT6 may be useful for treating cancer.
• SIRT6 deficiency results in hypoglycemia and impairment of energy homeostasis, suggesting that SIRT6 could play an important role in regulating adipocyte differentiation.
• Aged adipocytes become hypertrophic, in particular under a high fat diet intake, becoming insulin resistant and producing a wide pattern of pro-inflammatory cytokines, contributing to the increase of circulating triglycerides and free fatty acids, which together affects other organs and results in the comorbidities typically associated with obesity.
• the increase of adipogenesis allows the formation of new adipocytes that are more sensitive to insulin, showing increased capacity of fatty storage and enhanced levels of adipokines, that contribute to metabolic homeostasis and lowering of inflammation levels.
• SIRT6 plays a role in the increase of adipogenic differentiation ( Figure 32), consistent with the role of this protein in energy homeostasis.
• the SIRT6 mutants (SIRT6 N308K and centSIRT6) retain this property, which is of interest for the treatment of ageing and ageing -related diseases.
• a-SMA expression is considered a reliable marker of hepatic stellate cell (HSC) activation and a key biomarker for liver fibrosis.
• HSCs hepatic stellate cell
• TGF-b Transforming Growth Factor beta
• SIRT6 WT/SIRT6 N308K/centSIRT6 overexpression did not affect cell viability or proliferation ( Figure 33A).
• TGFbeta Upon LX2 activation with TGFbeta, there was a blockage of the activation of some fibrogenic markers (vimentin, TIMP1) by lentiviral infection, irrespective of the genotype ( Figure 33B-33C).
• Collagen mRNA expression was increased by TGFbeta, with no effect of SIRT6 WT/SIRT6 N308K/centSIRT6 overexpression ( Figure 33D).
• centSIRT6 significantly decreased the mRNA expression of aSMA, a major fibrogenic marker (Figure 33E).
• centSIRT6 LX2 The lower gene expression levels of a-SMA in centSIRT6 LX2 compared to control suggests a potential anti-fibrotic effect of centSIRT6 in preventing liver fibrosis and/or promoting its resolution.
• Hepatic spheroids model [0266] Together with a-SMA, Collagen (COL1A1) represent one of the key fibrosis markers. Its production in liver is increased mainly by activated hepatic stellate cells.

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