Sirtuin 6 variants for the treatment of nonalcoholic fatty liver disease

Abstract

The present invention relates to variants of sirtuin 6 for the treatment of non-alcoholic fatty liver disease.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing and / or treating non-alcoholic fatty liver disease (NAFLD), particularly non-alcoholic steatohepatitis (NASH), which comprises a variant of SIRT6. [Background technology]

[0002] Fat accumulation in the liver (steatosis) is generally promoted by excessive alcohol consumption. Nonalcoholic fatty liver disease (NAFLD) is a general term for excessive accumulation of fat in the liver that is not related to excessive alcohol consumption.

[0003] Currently, non-alcoholic fatty liver disease affects approximately 20% of the general population. Steatosis is most often isolated (approximately 80% of cases). It is, moreover, a benign condition with a very low risk of complications. In the remaining 20% ​​of cases, steatosis causes hepatocyte damage (swelling of hepatocytes) and inflammation of the liver parenchyma: this is steatohepatitis or NASH (short for "non-alcoholic steatohepatitis").

[0004] Steatohepatitis represents an aggressive form of the disease because it promotes the accumulation of liver fibrosis in the liver. It is graded into 5 stages (0 to 4), with stage 4 corresponding to cirrhosis. Of all patients with steatosis, less than 5% have precirrhotic fibrosis, and 1% have cirrhosis. Although these percentages seem small, considering the frequency of steatosis, they ultimately represent a significant number of people. Summary of the Invention

[0005] The present invention therefore relates to an isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity to the sequence of SEQ ID NO: 1 for use in the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD), wherein the variant has at least one mutation comprising or selected from the group consisting of the substitution N308K and the substitution A313S relative to the sequence of SEQ ID NO: 1.

[0006] In one embodiment, the nucleic acid molecule is of a sequence selected from the group comprising or consisting of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0007] The present invention also relates to an isolated polypeptide encoded by the above-mentioned nucleic acid molecule for use in the prevention and / or treatment of NAFLD.

[0008] In one embodiment, the polypeptide is of a sequence selected from the group comprising or consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0009] The present invention further relates to a vector comprising the above-mentioned isolated nucleic acid molecule for use in the prevention and / or treatment of NAFLD.

[0010] In one embodiment, 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 herpesviral vector.

[0011] The present invention further relates to a suspension comprising the above-mentioned vector for use in the prevention and / or treatment of NAFLD.

[0012] The present invention further relates to a cell expressing the polypeptide for use as described above, wherein the cell is preferably transfected with the isolated nucleic acid molecule for use as described above, or the vector for use as described above, for use in the prevention and / or treatment of NAFLD.

[0013] Another object of the present invention is a pharmaceutical composition comprising (i) the above-mentioned isolated nucleic acid molecule, or the above-mentioned isolated polypeptide, or the above-mentioned vector, and (ii) a pharmaceutically acceptable excipient, for use in the prevention and / or treatment of NAFLD.

[0014] Another object of the present invention is an isolated acidic nucleic acid molecule for use as described above, an isolated polypeptide for use as described above, a vector for use as described above, a suspension for use as described above, a cell for use as described above, or a pharmaceutical composition for use as described above for the prevention and / or treatment of stage 1 or stage 2 NAFLD.

[0015] In one embodiment, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition is for the prevention and / or treatment of stage 1 NAFLD.

[0016] In one embodiment, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition is for the prevention and / or treatment of stage 2 NAFLD.

[0017] In one embodiment, the NASH is stage 1. In another embodiment, the NASH is stage 2. In one embodiment, the NASH is stage 3.

[0018] The present invention also relates to a method for preventing and / or treating non-alcoholic fatty liver disease (NAFLD), the method comprising administering to a patient in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity to SEQ ID NO: 1 (the variant having at least one mutation including or selected from the group consisting of the substitution N308K and the substitution A313S relative to SEQ ID NO: 1), or an isolated polypeptide encoded thereby, or a pharmaceutical composition comprising the same.

[0019] In one embodiment, the NAFLD is stage 1 or 2.

[0020] In one embodiment, the isolated nucleic acid molecule is comprised in a vector, preferably a viral vector, more preferably an adeno-associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an adenoviral vector, a retroviral vector, or a herpesviral vector.

[0021] In one embodiment, the methods of the invention further comprise administering to the patient another therapeutic agent.

[0022] A further object of the present invention relates to the use of an isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity with the sequence of SEQ ID NO: 1 for the manufacture of a pharmaceutical composition for the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD), wherein the variant has at least one mutation comprising or selected from the group consisting of the substitution N308K and the substitution A313S with respect to the sequence of SEQ ID NO: 1.

[0023] In one embodiment, the NAFLD is stage 1 or 2.

[0024] definition In the present invention, the following terms have the following meanings:

[0025] The term "about" preceding a number means the value of that number plus or minus 10%. It is to be understood that the value to which the term "about" refers is itself also specifically and preferably disclosed.

[0026] "Comprise" is intended to mean "contain," "encompass," and "include." In some embodiments, the term "comprise" also encompasses the term "consist of."

[0027] "Sirtuin 6," also referred to as "SIRT6," is intended to refer to the polypeptide having Entrez gene number 51548, and relates, without limitation, to NAD-dependent protein deacetylase sirtuin 6, regulatory protein SIR2 homolog 6, SIR2-like protein 6, SIR2L6, sirtuin (silent mating type signal regulation 2, S. cerevisiae, homolog) 6, sirtuin (silent mating type signal regulation 2 homolog) 6, Sir2-related protein type 6, sirtuin type 6, and EC 2.3.1.286.

[0028] "Isolated" refers to a nucleic acid molecule or polypeptide that has been removed from the original biological context that allowed it to be produced. In practice, the biological context includes at least one cell, or one or more enzymes.

[0029] "Nucleic acid" is also referred to as "polynucleotide" and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Nucleic acid" or "polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. Furthermore, "nucleic acid" or "polynucleotide" refers to triple-stranded regions containing RNA or DNA, or both RNA and DNA. The term "nucleic acid" or "polynucleotide" also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications have been made to DNA and RNA; thus, "nucleic acid" or "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short polynucleotides, often referred to as oligonucleotides.

[0030] "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 isomers. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acid residues other than the 20 gene-encoded amino acid residues.

[0031] A "suspension" refers to a liquid mixture in which an active ingredient, such as a nucleic acid molecule, polypeptide, or vector according to the present invention, is suspended in a liquid medium.

[0032] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and preventative or preventative measures, the purpose of which is to prevent or slow down (alleviate) the targeted pathological condition or disorder, particularly liver-related disease, particularly NAFLD, NASH, or cirrhosis. Those in need of treatment include those already with the disorder, as well as those prone to the disorder or those for which the disorder is to be prevented. An individual is successfully treated for liver-related disease, particularly NAFLD, NASH, or cirrhosis, if, after receiving a therapeutic dose of an active ingredient, particularly a nucleic acid molecule, polypeptide, or vector according to the present invention, the individual shows an observable and / or measurable reduction or lack of one or more symptoms associated with liver-related disease, particularly NAFLD, NASH, or cirrhosis, reduced morbidity and mortality, and improved quality of life. The above parameters for assessing successful treatment and disease improvement can be easily measured by routine procedures familiar to the physician or attending physician.

[0033] "Preventing" refers to preventing the occurrence and / or reducing the likelihood of occurrence of a liver-related disease, (particularly NAFLD, NASH or cirrhosis), a disorder or condition associated with a deficiency or lack of organ, tissue or cellular function, or reducing at least one adverse effect or symptom thereof.

[0034] "Individual" refers to an animal, preferably a mammal, more preferably a human. In one embodiment, the individual is male. In another embodiment, the individual is female. In one embodiment, the subject can be a "patient", i.e., a warm-blooded animal, more preferably a human, who is waiting to receive medical treatment, or is receiving medical treatment, or has been the subject of medical treatment, or is the subject, or will be the subject of medical treatment, or is being monitored for the development of liver-related disease, particularly NAFLD, NASH or cirrhosis. In one embodiment, the individual is an adult (e.g., a subject over 18 years old). In another embodiment, the individual is a child (e.g., a subject under 18 years old). [Brief explanation of the drawings]

[0035] [Figure 1A-1B] This is a set of photographs and graphs showing stably transfected IHH cells overexpressing SIRT6 allelic variants. (A) Representative immunofluorescence images of Katushka2S staining showing lentiviral transfection of empty vector and all three SIRT6 variants (WT, N308K, and N308K / A313S) in IHH cells. (B) Histograms show quantification of protein expression ratios of SIRT6 / GAPDH, H3K56Ac / histone 3, and H3K9Ac / histone 3 in IHH-transfected cells (N=5-7) compared to the control group. Data are presented as mean ± SEM. *p<0.05 compared to the empty group. [Figure 2] This graph shows that SIRT6 overexpression does not affect the pAKT / AKT ratio in IHH. The histogram shows the quantification of the protein expression ratio pAKT(ser473) / AKT compared with the control group with or without 30 minutes of 100 nM insulin treatment. These results reveal that protein levels are elevated in all groups after insulin stimulation, but there is no difference between the groups themselves (N=6). All data are expressed as mean ± SD. [Figure 3A-3B] Scatter plots showing metabolite profiling. (A) Score scatter plot of the PCA model of human hepatocyte samples. The first and second components are shown. Model diagnosis (A=3; R2X=0.677; Q2X=0.278). Ellipses represent the 95% confidence intervals from Hotelling's T2 test. (B) Score scatter plot of the PCA model of culture medium extracts. Model diagnosis (A=3; R2X=0.662; Q2X=0.115). Ellipses represent the 95% confidence intervals from Hotelling's T2 test. [Figure 4A-4B]Heatmap representation showing amino acid profiling. (A) Heatmap representation of amino acid and derivative changes for comparison between human hepatocyte groups. Color code represents log2 (fold change). Student's t-test p-values: *p<0.05, **p<0.01, ***p<0.001. (B) Heatmap representation of amino acid and derivative changes for comparison between culture medium groups. Color code represents log2 (fold change). Student's t-test p-values: *p<0.05, **p<0.01, ***p<0.001. [Figures 5A-5I] Graphs showing lipid profiling. (A) Heatmap representation of changes in saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) for comparison between IHH strains. Color codes represent log2 (fold change). (B) Boxplots of 18:1n-9 in IHH. (C) Boxplots of 20:3n-9 in hepatocytes. (D-I) Boxplots of PE(0:0 / 18:2) (D), PE(0:0 / 22:6) (E), PE(18:1 / 0:0) (F), PE(0-16:0 / 0:0) (G), PE(0:0 / 15:0) (H), and PE(0:0 / 16:1) (I) in IHH. Student's t-test p-values: not significant (ns), *p<0.05, **p<0.01, ***p<0.001. [Figures 6A-6C] Graphs showing that SIRT6 overexpression reduced basal collagen levels in IHH / LX2 spheroids. (A) Histograms show quantification of the percentage of collagen content in the spheroid structure. Collagen levels were significantly reduced in the N308K / A313S group compared to the empty, WT, and NK308K groups. (B) Quantification of soluble collagen content in the conditioned medium of different groups. All groups overexpressing one of the SIRT6 variants showed a significant decrease of approximately 30% in soluble collagen levels compared to the empty group. (C) mRNA levels of αSMA, COL1A1, TIMP1, vimentin, and MMP2 in the five spheroid groups. Data are presented as mean ± SEM. *p<0.05 vs. empty group. **p<0.01 vs. empty group. §p<0.05 vs. WT and N308K groups. #p<0.05 vs. all other groups. [Figure 7] Graphs showing amino acid profiling in hepatocytes. Heatmaps representing binary comparisons between hepatocyte populations for each metabolite. Heatmap color codes for log2 (fold change) and Student's t-test p-values ​​are shown at the bottom of the figure. [Figure 8] Graphs showing amino acid profiling in culture media. Heatmaps representing binary comparisons between culture media groups for each metabolite. Heatmap color codes for log2 (fold change) and Student's t-test p-values ​​are shown at the bottom of the figure. [Figures 9A-9F] Graphs showing amino acid profiling in hepatocytes. Boxplots of A) threonine, B) asparagine, C) aspartate, D) proline, E) arginine, and F) citrulline levels in hepatocytes. Student's t-test p-values: ns, p>0.05; *, p<0.05; **, p<0.01; ***p<0.001. [Figures 10A-10J] Graphs showing amino acid profiling in culture media. Boxplots of A) glutamate, B) asparagine, C) aspartate, D) arginine, E) cystine, F) serine, G) cystathionine, H) aminoadipic acid, I) citrulline, and J) S-sulfocysteine ​​in culture media groups. Student's t-test p-values: ns, p>0.05; *, p<0.05; **, p<0.01; ***p<0.001; ****p<0.0001. [Figures 11A-11C] Graphs showing lipid profiling. (A) Heat map representation of the effect of carbon and double bond number on diglyceride changes for comparison between WT and empty vector-transfected hepatocyte groups. (B) Heat map representation of the effect of carbon and double bond number on triglyceride changes for comparison between WT and empty vector-transfected hepatocyte groups. (A-B) Color code represents log2 (fold change). The x-axis represents the number of carbons, and the y-axis represents the number of double bonds. (C) Box plots of TG(58:2) and TG(60:3) in IHH. Student's t-test p-values: ns, p>0.05; *, p<0.05; **, p<0.01. [Figures 12A-12B] Graphs showing lipid profiling in IHH. Box plots of SM(32:1) (A) and SM(42:3) (B) in IHH. Student's t-test p-values: ns, p>0.05; *, p<0.05; **, p<0.01. [Figures 13A-13D] Graphs showing lipid profiling in hepatocytes. Box plots of Cer(d18:1 / 20:0) (A), Cer(d18:1 / 21:0) (B), Cer(d18:1 / 22:0) (C), and Cer(d18:1 / 18:0) (D) in hepatocytes. Student's t-test p-values: ns, p>0.05; *, p<0.05; **, p<0.01. [Figures 14A-14C] 14A-14C are histograms showing quantification of mRNA and protein expression during transient expression of SIRT6 and SIRTcent by AAV in LX-2 cells (astrocytes) using targeting constructs for clinical use. Figures 14A-14B show RNA expression. Figure 14C shows protein expression. [Figures 15A-15E] Figure 15 shows histograms and photographs showing quantification of mRNA and protein expression upon transient expression of SIRT6 and SIRTcent in organoids (stellate cells / hepatocytes) using targeting constructs for clinical use. Figure 15A shows RNA expression in spheroids. Figures 15B-15C show basal conditions. Figures 15D-15E show fibrotic conditions. [Figures 16A-16B] 1 is a histogram showing mRNA expression of several genes in primary hepatic stellate cells in healthy or NASH states, with or without expression of SIRT6wt or SIRT6cent. [Figure 17] 1 is a histogram showing RNA expression of genes related to lipid metabolism in IHH cells with or without expression of SIRT6wt or SIRT6cent. [Figures 18A-18D]Figure 1 shows the different patterns of gene expression in the tested groups (CTL (AAV-Luc), AAV-SIRT6-WT, and AAV-SIRT6-Cent). 56 genes are differentially expressed in AAV-SIRT6-WT and AAV-SIRT6-Cent treated cells. [Figure 19] 1 depicts an analysis of differentially expressed pathways between AAV-SIRT6-WT and AAV-SIRT6-Cent treated cells. [Figures 20A-20E] 20A and 20B are histograms showing histone post-translational modifications (PTMs) by SIRT6 WT and SIRT6cent in 3T3-L1 adipocytes. Figure 20A relates to histone H4 G4KGGKGLGKGGAKR17. Figure 20B relates to histone H3.1 / H3.3 K18QLATKAAR26. Figure 20C relates to histone H3.1 / H3.3 K9STGGKAPR17. Figure 20D relates to histone H3.1 K27SAPATGGVKKPHR40. Figure 20E relates to histone H3.3 K27SAPSTGGVKKPHR40. [Figures 21A-21I] 21A-21F show mouse body weight and weight gain, and FIG. 21G-21I show mouse organ weights. [Figures 22A-22D] 1 is a histogram showing hematological tests in the HF / DEN mouse model. [Figures 23A-23D] 1 is a histogram showing the protein expression levels of SIRT6 and B-catenin in the HF / DEN mouse model. [Figures 24A-24D] 24A and 24B show graphs and histograms illustrating the biodistribution in a HF / DEN mouse model, respectively, of the reporter (LUC), and of SIRT6c. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to an isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity to the sequence of SEQ ID NO: 1 for use in the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or cirrhosis, wherein the variant has at least one mutation comprising or selected from the group consisting of the substitution N308K and the substitution A313S relative to the sequence of SEQ ID NO: 1.

[0037] As used herein, the phrase "at least 75% identity" includes 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.

[0038] The level of identity of two polypeptides can be determined by using any one of the known algorithms available in the state of the art. By way of example, the percentage amino acid identity can be determined using the CLUSTAL W software (version 1.83), with the parameters set as follows: -Slow / Accurate alignment: (1) Gap open penalty: 10.00; (2) Gap extension penalty: 0.1; (3) Protein weight matrix: BLOSUM; -Fast / approximate alignment: (4) Gap penalty: 3; (5) K-tuple (word) size: 1; (6) Number of top diagonals: 5; (7) Window size: 5; (8) Scoring method: Percent.

[0039] Within the scope of the present invention, SEQ ID NO: 1 refers to the 361 amino acid residue sequence of the wild-type SIRT6 polypeptide. In fact, the substitutions N308K and A313S refer to mutations in the codons encoding the natural 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.

[0040] Within the scope of the present invention, SEQ ID NO: 21 refers to the 1,068 nucleotide (bp) sequence of the wild-type SIRT6 polypeptide.

[0041] In some embodiments, the naturally occurring Asn (N) amino acid residue at position 308 in a SIRT6 polypeptide is encoded by the codon "aac" at positions 922-924 of SEQ ID NO: 21. In some embodiments, the naturally occurring Ser (S) amino acid residue at position 313 in a SIRT6 polypeptide is encoded by the codon "gcc" at positions 937-939 of SEQ ID NO: 21.

[0042] In a particular embodiment, the N308K substitution is represented by mutation of the codon "aac" at positions 922 to 924 of SEQ ID NO: 21 to codon "aag" or "aaa", preferably to codon "aag". In other words, the N308K substitution is represented by mutation of the nucleotide "c" at position 924 of SEQ ID NO: 21 to nucleotide "g" or nucleotide "a", preferably to nucleotide "g".

[0043] In certain embodiments, the A313S substitution is represented by a mutation of the codon "gcc" at positions 937 to 939 of SEQ ID NO: 21 to a codon selected from the group consisting of the codons "tcc", "tct", "tca" and "tcg", preferably to the codon "tcc". In other words, the A313S substitution is represented by one or two mutations selected from the group consisting of: a mutation of nucleotide "g" at position 937 of SEQ ID NO:21 to nucleotide "t"; a mutation of nucleotide "g" at position 937 of SEQ ID NO:21 to nucleotide "t"; and a mutation of nucleotide "c" at position 939 of SEQ ID NO:21 to nucleotide "t"; a mutation of nucleotide "g" at position 937 of SEQ ID NO:21 to nucleotide "t"; and a mutation of nucleotide "c" at position 939 of SEQ ID NO:21 to nucleotide "a"; a mutation of nucleotide "g" at position 937 of SEQ ID NO:21 to nucleotide "t"; and a mutation of nucleotide "c" at position 939 of SEQ ID NO:21 to nucleotide "g".

[0044] In certain embodiments, the isolated polypeptide that is a variant of SIRT6 has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. In some embodiments, the nucleic acid molecule is of a sequence selected from the group comprising or consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0045] As used herein, SEQ ID NO: 6 refers to the nucleic acid sequence of a variant of SIRT6 with an N308K substitution, in particular with a mutation of the codon "aac" at positions 922 to 924 of SEQ ID NO: 21 to the codon "aag".

[0046] As used herein, SEQ ID NO: 7 refers to the nucleic acid sequence of a variant of SIRT6 with an A313S substitution, in particular with a mutation of the codon "gcc" at positions 922-924 of SEQ ID NO: 21 to the codon "tcc".

[0047] As used herein, SEQ ID NO: 8 refers to the nucleic acid sequence of a variant of SIRT6 with N308K and A313S substitutions, in particular with a mutation of the codon "aac" at positions 922-924 of SEQ ID NO: 21 to the codon "aag", and a mutation of the codon "gcc" at positions 922-924 of SEQ ID NO: 21 to the codon "tcc".

[0048] In some embodiments, a variant of SIRT6 encoded by an isolated nucleic acid molecule defined herein may have additional mutations compared to a wild-type SIRT6 polypeptide.

[0049] In some embodiments, the variant of SIRT6 encoded by the isolated nucleic acid molecule defined herein has deacylase and / or mono ADP-ribosyltransferase (mADPr) activity.

[0050] In practice, deacylase activity and mono-ADP-ribosyltransferase (mADPr) activity can be assayed according to any suitable method in the art or the method adapted therefrom.For example, deacylase activity can be assayed by contacting SIRT6 variant with histone in vitro in the presence of NAD+, MgCl2, DTT, and using anti-H3K9ac antibody and anti-H3K18ac antibody to carry out Western blot analysis.For example, mono-ADP-ribosyltransferase (mADPr) activity can be assayed by contacting SIRT6 variant with PARP1 in vitro in the presence of ZnCl2, MgCl2, NAD+, DTT, salmon sperm DNA, and using anti-PADPR antibody to carry out Western blot analysis.

[0051] In one embodiment, the SIRT6 variant has a deacylase activity of up to about 90%, more preferably up to about 50%, and even more preferably up to about 25% of that of wild-type SIRT6 (SEQ ID NO: 1). Within the scope of the present invention, the expression "up to 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.

[0052] In some embodiments, the SIRT6 variant has a maximum of at least 100%, preferably at least about 200%, more preferably at least about 300% mono-ADP ribosyltransferase (mADPr) activity compared to wild-type SIRT6 (SEQ ID NO: 1). Within the scope of the present invention, the expression "at least about 100%" includes about 100%, 120%, 140%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750% or more.

[0053] In some embodiments, the nucleic acid molecule is a single-stranded or double-stranded nucleic acid molecule. In some embodiments, the nucleic acid molecule is DNA or RNA.

[0054] In one embodiment, the nucleic acid molecule is an RNA molecule.In some embodiments, the nucleic acid molecule is an RNA molecule selected from the list comprising messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), circular RNA (circRNA) and long non-coding RNA (lncRNA).In certain embodiments, the nucleic acid molecule is mRNA.

[0055] In another embodiment, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is a DNA molecule selected from the list comprising a genomic DNA (gDNA) or a complementary DNA (cDNA) molecule.

[0056] Nonalcoholic fatty liver disease (NAFLD) is characterized by the abnormal accumulation of triglycerides in hepatocytes, which is associated with metabolic syndrome, obesity, and high calorie intake, and usually does not involve steatosis secondary to other causes.

[0057] NAFLD comprises a wide range of liver pathologies, among which two main pathologies can be distinguished: steatosis alone or with minimal lobular inflammation (nonalcoholic fatty liver or NAFL), and nonalcoholic steatohepatitis (NASH).

[0058] In some embodiments, the isolated nucleic acid molecule according to the present invention is for preventing and / or treating NAFLD. There are four stages of NAFLD: Stage 1 is simple fatty liver or steatosis, Stage 2 is nonalcoholic steatohepatitis (NASH), Stage 3 is fibrosis, and Stage 4 is cirrhosis.

[0059] In some embodiments, the isolated nucleic acid molecule according to the present invention is for preventing and / or treating stage 1 or stage 2 NAFLD. In some embodiments, the isolated nucleic acid molecule according to the present invention is for preventing and / or treating non-fibrotic NAFLD. In some embodiments, the isolated nucleic acid molecule according to the present invention is for preventing and / or treating non-fibrotic stage NAFLD.

[0060] In some embodiments, the isolated nucleic acid molecule according to the present invention is for preventing and / or treating NASH (ie, stage 2 of NAFLD).

[0061] NASH is characterized by the accumulation of fat (steatosis), liver cell damage due to fat accumulation, apoptosis, inflammation, and fibrosis. There are three main stages of NASH. Stage 1 (mild) is when the liver contains more than 5% fat (steatosis), inflammation occurs, and the liver is larger than normal. Typically, in stage 1, the liver continues to function normally but may be impaired. This is also called compensated cirrhosis or NASH without fibrosis. Stage 2 (moderate) is when scarring (fibrosis) begins to appear in addition to the features of stage 1. Fibrosis can be classified as F1-F4. Stage 2 NASH is accompanied by fibrosis levels F1-F3. When patients reach this stage, the liver begins to deteriorate, leading to liver failure. This is also called NASH with fibrosis. Stage 3 (severe) is the most severe stage of NASH, and the disease progresses to cirrhosis or liver cancer. At this point, the only option is a liver transplant.

[0062] In some embodiments, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell, or pharmaceutical composition is for preventing and / or treating Stage 1, Stage 2, or Stage 3 NASH. In some embodiments, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell, or pharmaceutical composition is for preventing and / or treating Stage 1 or Stage 2 NASH. In some embodiments, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell, or pharmaceutical composition is for preventing and / or treating Stage 1 NASH. In some embodiments, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell, or pharmaceutical composition is for preventing and / or treating Stage 2 NASH. In some embodiments, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell, or pharmaceutical composition is for preventing and / or treating liver cirrhosis.

[0063] In some embodiments, the isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition is for preventing and / or treating stage 1 or stage 2 NAFLD and stage 1 or stage 2 NASH.

[0064] The present invention relates to isolated polypeptides encoded by nucleic acid molecules for use in the prevention and / or treatment of NAFLD.

[0065] The present invention relates to an isolated polypeptide which is a variant of SIRT6 having at least 75% identity with the sequence of SEQ ID NO: 1 for the prevention and / or treatment of NAFLD, wherein the variant has at least one mutation including or selected from the group consisting of substitution N308K and substitution A313S with respect to the sequence of SEQ ID NO: 1.

[0066] As used herein, the phrase "at least 75% identity" includes 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.

[0067] In certain embodiments, the isolated polypeptide that is a variant of SIRT6 has at least 75%, 80%, 85%, 90%, 95% identity to the sequence of SEQ ID NO: 1, and the variant has at least one mutation selected from the group consisting of an N308K substitution and an A313S substitution with respect to the sequence of SEQ ID NO: 1.

[0068] In certain embodiments, the isolated polypeptide that is a variant of SIRT6 has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. In some embodiments, the polypeptide is of a sequence selected from the group comprising or consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0069] As used herein, SEQ ID NO: 2 refers to the amino acid sequence of a variant of SIRT6 with an N308K substitution. In some embodiments, the polypeptide of SEQ ID NO: 2 is encoded by a nucleic acid molecule of SEQ ID NO: 6.

[0070] As used herein, SEQ ID NO: 3 refers to the amino acid sequence of a variant of SIRT6 with an A313S substitution. In some embodiments, the polypeptide of SEQ ID NO: 2 is encoded by a nucleic acid molecule of SEQ ID NO: 7.

[0071] As used herein, SEQ ID NO: 4 refers to the amino acid sequence of a variant of SIRT6 with an N308K substitution and an A313S substitution. In some embodiments, the polypeptide of SEQ ID NO: 4 is encoded by a nucleic acid molecule of SEQ ID NO: 8. In certain embodiments, the polypeptide is a recombinant polypeptide. As used herein, the term "recombinant polypeptide" refers to a polypeptide that is encoded by an engineered nucleic acid and synthesized upon transformation of the engineered nucleic acid into a microorganism for synthesis or transfection into a eukaryotic cell.

[0072] The present invention further relates to a vector comprising the aforementioned isolated nucleic acid molecule for use in the prevention and / or treatment of NAFLD.

[0073] In some embodiments, the vector comprising the isolated nucleic acid molecule is selected from the list including or consisting of a minicircle nucleic acid, a plasmid, a cosmid, a bacteriophage, a bacterial artificial chromosome, a viral vector, linear DNA, enzymatic DNA, and doggybone DNA.

[0074] In some embodiments, the vector comprising the isolated nucleic acid molecule is selected from the list comprising a minicircle nucleic acid, a plasmid, a cosmid, a bacteriophage, or a bacterial artificial chromosome or a viral vector.

[0075] As used herein, the term "minicircle nucleic acid" encompasses non-viral vectors that simply contain a gene expression cassette and do not contain viral and / or bacterial backbone DNA elements derived from standard plasmids.

[0076] As used herein, the term "plasmid" is intended to refer to small extragenomic DNA molecules, most commonly found as circular double-stranded DNA molecules that can be used in molecular biology as cloning vectors to create 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 nucleic acid sequences found in the plasmid downstream of a promoter sequence.

[0077] As used herein, the term "cosmid" refers to a hybrid plasmid containing a cos sequence from lambda phage, allowing packaging of the cosmid into the phage head and subsequent infection of bacterial cells, where the cosmid can be circularized and replicated as a plasmid. Cosmids are typically used as cloning vectors for DNA fragments ranging in size from about 32 to 52 kb.

[0078] As used herein, the term "bacterial artificial chromosome" or "BAC" refers to an extragenomic nucleic acid molecule based on a functional fertility plasmid that allows for uniform partitioning of the extragenomic DNA molecule after bacterial cell division. BACs are typically used as cloning vectors for DNA fragments ranging in size from approximately 150 to 350 kb.

[0079] As used herein, the term "enzymatic DNA" refers to a synthetic, linear, double-stranded, closed-end DNA molecule. As used herein, the term "doggybone DNA" refers to a minimal, linear, double-stranded, covalently closed DNA structure.

[0080] In practice, the vector comprising the nucleic acid molecule encoding a SIRT6 variant may be in the form of a plasmid, particularly obtained by cloning the nucleic acid of interest into a nucleic acid vector. In some embodiments, non-limiting suitable nucleic acid vectors are pBluescript vector, pET vector, pETduet vector, pGBM vector, pBAD vector, pUC vector. In one embodiment, the plasmid is a low-copy plasmid. In one embodiment, the plasmid is a high-copy plasmid.

[0081] In some embodiments, the vector is a viral vector.In some embodiments, the viral vector comprises or is selected from the group consisting of adenovirus, adeno-associated virus (AAV), exosome-associated AAV (exo-AAV), exosome, alphavirus, herpesvirus, retrovirus, for example, lentivirus or non-integrating lentivirus, vaccinia virus, baculovirus, virus-like particle, for example, hepatitis B virus, particle derived from Parvoviridae, Retroviridae, Flaviviridae, Paramyxoviridae or bacteriophage.In one embodiment, the exosome comprises at least one DNA molecule, RNA molecule and / or protein, preferably the variant of SIRT6 according to the present invention or the nucleic acid encoding it.

[0082] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group comprising or consisting of adenovirus, adeno-associated virus (AAV), exosome-associated AAV (exo-AAV), alphavirus, herpesvirus, retrovirus, such as lentivirus or non-integrating lentivirus, vaccinia virus, baculovirus, virus-like particle, such as hepatitis B virus, particle derived from Parvoviridae, Retroviridae, Flaviviridae, Paramyxoviridae, or bacteriophage.

[0083] In some embodiments, the viral vectors of the present invention are selected from the group comprising or consisting of adeno-associated viral vectors (AAV), exosome-associated AAV vectors (exo-AAV), exosomes, adenoviral vectors, retroviral vectors, lentiviruses, and herpes viral vectors.

[0084] In some embodiments, the viral vectors of the present invention are selected from the group comprising or consisting of adeno-associated viral vectors (AAV), exosome-associated AAV vectors (exo-AAV), adenoviral vectors, retroviral vectors, lentivirus, and herpesviral vectors.

[0085] In some embodiments, the viral vector of the invention is an adeno-associated viral vector (AAV), preferably AAV serotype 2 or AAV serotype 5.

[0086] In certain embodiments, vector, particularly viral vector, is exo-AAV vector.As used herein, exo-AAV refers to the vector that adeno-associated virus (AAV) vector or its part is associated with extracellular vesicle (also called exosome), and AAV vector is partially fused with, embedded in, or internalized in extracellular vesicle.Extracellular vesicle can express specific protein or marker, for example, for the purpose of targeting.

[0087] In a particular embodiment, the vector is an exosome, preferably an exosome comprising a payload or cargo, more preferably an exosome comprising a DNA molecule, an RNA molecule and / or a protein, even more preferably a variant of SIRT6 according to the present invention or a nucleic acid encoding same.

[0088] In another embodiment, the viral vector is a retrovirus or a lentivirus. In a preferred embodiment, the viral vector is a lentivirus.

[0089] In certain embodiments, the vector, particularly a viral vector, does not cross the blood-brain barrier, hi some alternative embodiments, the vector, particularly a viral vector, does cross the blood-brain barrier.

[0090] In some embodiments, the vector, particularly the viral vector, comprises a promoter sequence suitable for gene expression in a mammalian individual, preferably a human individual.

[0091] Non-limiting examples of promoter sequences suitable for gene expression in mammalian individuals, preferably human individuals, include the CMV (human cytomegalovirus) promoter, the EF1α (human elongation factor 1α) promoter, the SV40 (simian vacuolating virus 40) promoter, the PGK1 (phosphoglycerate kinase) promoter, the UbC (human ubiquitin C) promoter, the ColA2 promoter, the Col1A1 promoter, the Col3A1 promoter, and the like.

[0092] In certain embodiments, the promoter sequence is preferably the EF1α promoter.

[0093] In some embodiments, the vector, particularly 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 the target recipient cell. Indeed, these nuclear localization signals (NLS) are much discussed in the state of the art.

[0094] The present invention further relates to a suspension comprising the vector for use in the prevention and / or treatment of NAFLD.

[0095] In one aspect, the present invention relates to a suspension comprising a vector according to the present invention.

[0096] In some embodiments, the suspension further comprises a fluid comprising one or more components selected from the group consisting of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof.

[0097] In certain embodiments, the suspension is formulated for intravenous infusion. In practice, suspensions formulated for intravenous infusion may include saline (e.g., 0.9% NaCl), lactated Ringer's solution, 5% dextrose, colloids such as albumin, and the like, and any combination thereof.

[0098] The present invention further relates to cells expressing the polypeptide, which cells are preferably transfected with the isolated nucleic acid molecule or vector for use in the prevention and / or treatment of NAFLD.

[0099] In certain embodiments, the cell is a eukaryotic cell, preferably an animal cell, and more preferably a mammalian cell. As used herein, "mammalian cell" includes non-human mammalian cells and human cells. In some embodiments, the cell is a human cell.

[0100] In some embodiments, the cells are selected from the group comprising or consisting of nerve cells, bone cells, breast cells, red blood cells, white blood cells, chondrocytes, epithelial cells, endothelial cells, skin cells, muscle cells, bladder cells, kidney cells, liver cells, prostate cells, cervical cells, ovarian cells, lung cells, retinal cells, conjunctival cells, corneal cells, adipocytes, and the like.

[0101] It is understood that the cells according to the present invention are transfected with the isolated nucleic acid molecule according to the present invention, transduced with the isolated nucleic acid molecule according to the present invention, or contacted with a vector or suspension containing the nucleic acid molecule according to the present invention. Thus, the cells contain the nucleic acid molecule integrated or not integrated into their genome. In fact, since the vector is an expression system, the nucleic acid molecule encoding the SIRT6 variant is present in the cell in a form that allows its expression and its final location, i.e., in the cell nucleus and cytoplasm.

[0102] The present invention further relates to a pharmaceutical composition comprising (i) an isolated nucleic acid molecule, or an isolated polypeptide, or a vector, and (ii) a pharmaceutically acceptable excipient, for use in the prevention and / or treatment of NAFLD.

[0103] In some embodiments, suitable pharmaceutically acceptable carriers according to the present invention include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. In certain embodiments, suitable pharmaceutically acceptable carriers may include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and mixtures thereof. In some embodiments, pharmaceutically acceptable carriers may further contain small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which improve the shelf life or effectiveness of the cells. The preparation and use of pharmaceutically acceptable carriers are well known in the art.

[0104] In some embodiments, a nucleic acid molecule, polypeptide, vector, suspension, or pharmaceutical composition according to the invention is administered to an individual in need thereof by any suitable route, i.e., by transdermal administration, orally, topically, or parenterally, for example, by injection, including subcutaneous, intravenous, intraarterial, intramuscular, intraocular, and intraauricular administration.

[0105] In certain embodiments, nucleic acid molecules, polypeptides, vectors, suspensions, or pharmaceutical compositions according to the present invention are administered to an individual in need thereof by transdermal administration. In certain embodiments, nucleic acid molecules, polypeptides, vectors, suspensions, or pharmaceutical compositions according to the present invention are associated with compositions that enable and / or facilitate transdermal administration, for example, by increasing skin affinity or increasing skin barrier penetration. In some embodiments, transdermal administration allows for sustained release of nucleic acid molecules, polypeptides, vectors, suspensions, or pharmaceutical compositions according to the present invention.

[0106] In a particular embodiment, the nucleic acid molecule, polypeptide, vector, suspension or pharmaceutical composition according to the invention is administered to an individual in need thereof by intravenous administration, in particular by intravenous infusion or injection.

[0107] Within the scope of the present invention, the therapeutically effective amount of the nucleic acid molecule, polypeptide, vector, suspension or pharmaceutical composition according to the present invention to be administered may be determined by a physician or a person skilled in the art with appropriate authority and may be adjusted appropriately within the course of treatment.

[0108] In certain embodiments, the therapeutically effective amount administered may depend on various parameters, including the material selected for administration, whether the administration is a single dose or multiple doses, and individual parameters such as age, physical condition, size, weight, sex, and the severity of the age-related disease being treated.

[0109] In certain embodiments, a therapeutically effective amount of an isolated polypeptide according to the present invention, or a pharmaceutical composition comprising an isolated polypeptide, 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.

[0110] Within the scope of the present invention, the expression "about 0.001 mg to about 3,000 mg" means 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, 0.0510 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg , 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 20m g, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 55 0mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1,000mg, 1,100mg, 1,150mg, 1,20015mg, 1,250mg , 1,300mg, 1,350mg, 1,400mg, 1,450mg, 1,500mg, 1,550mg, 1,600mg, 1,650mg, 1,700mg, 1,750mg, 1,800mg, 1,85 Includes 0mg, 1,900mg, 1,950mg, 2,000mg, 2,100mg, 2,150mg, 2,200mg, 2,250mg, 2,300mg, 2,350mg, 2,400mg, 2,450mg, 2,500mg, 2,550mg, 2,600mg, 2,650mg, 2,700mg, 2,750mg, 2,800mg, 2,850mg, 2,900mg, 2,950mg, and 3,000mg.

[0111] In certain embodiments, an isolated polypeptide or a pharmaceutical composition comprising an isolated polypeptide according to the present invention may be at a dosage level sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, 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, about 0.01 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 10 mg / kg, more preferably from about 1 mg / kg to about 25 mg / kg of a subject's body weight per day. Within the scope of the present invention, the expression "about 0.001 mg / kg to about 100 mg / kg" means 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 , 0.1mg / kg, 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0.9mg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70mg / kg, 80mg / kg, 90mg / kg and 100mg / kg.

[0112] In some embodiments, a therapeutically effective amount of an isolated nucleic acid molecule, vector, or pharmaceutical composition according to the invention is about 10 per ml. 1 ~about 10 15 In practice, a therapeutically effective dose is in the range of about 10 1 , 5×10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5, 5×10 5 , 10 6 , 5×10 6 , 107, 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 , 5×10 12 , 10 13 , 5×10 13 , 10 14 , 5×10 14 and 10 15 In certain embodiments, a therapeutically effective amount is 1 cm 3 Approximately 10 per 1 ~about 10 15 This is a copy, 1cm 3 Approximately 10 per 1 , 5×10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 , 5×10 12 , 10 13 , 5×10 13 , 10 14 , 5×10 14 and 10 15 In some embodiments, the therapeutically effective amount is about 10 per dose. 1 ~about 10 15copies, which is about 10 per dose 1 , 5×10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 , 5×10 12 , 10 13 , 5×10 13 , 10 14 , 5×10 14 and 10 15 Includes copy.

[0113] The present invention also relates to the use of an isolated nucleic acid molecule, an isolated polypeptide, or a vector according to the present invention for the preparation or manufacture of a medicament for the prevention and / or treatment of NAFLD.

[0114] In some further aspects, the present invention relates to a method for the prevention and / or treatment of NAFLD in an individual in need thereof, comprising administering a therapeutically effective amount of an isolated nucleic acid molecule, isolated polypeptide, or vector according to the present invention.

[0115] In certain embodiments, the isolated nucleic acid molecule, isolated polypeptide, vector, suspension, or pharmaceutical composition according to the present invention is co-administered or sequentially administered with a drug suitable for preventing and / or treating NAFLD, in particular stage 1 NAFLD, and stage 1 and stage 2 NASH.

[0116] As used herein, the term "co-administered" refers to simultaneous administration of active ingredients. As used herein, the term "sequentially administered" refers to administration of a first active ingredient before or after administration of a second active ingredient.

[0117] Another aspect of the present invention relates to a kit comprising (i) an isolated nucleic acid, isolated polypeptide molecule, vector, or suspension according to the present invention, and (ii) a means for administering the isolated nucleic acid molecule, isolated polypeptide, vector, or suspension.

[0118] In some embodiments, the means for administering the isolated nucleic acid molecule, isolated polypeptide, vector, or suspension comprises a syringe or catheter.

[0119] In certain embodiments, the individual in need thereof is a mammalian individual, preferably a human individual.

[0120] In some embodiments, the individual has or is at risk of having NAFLD, particularly stage 1 or stage 2 NAFLD, more particularly stage 1 or stage 2 NASH.

[0121] In some embodiments, a variant of SIRT6 according to the present invention downregulates the expression of one or more of the following genes: αSMA, TIMP1, TP63, and COL1A1. In some embodiments, a variant of SIRT6 according to the present invention downregulates the β-catenin pathway. In some embodiments, a variant of SIRT6 according to the present invention downregulates the glucocorticoid pathway.

[0122] As used herein, "downregulate" means to decrease or reduce the mRNA and / or protein expression of a gene in a cell and / or tissue by at least 1%, 5%, 10%, 50%, or more compared to an untreated cell or tissue.

[0123] In some embodiments, a variant of SIRT6 according to the present invention upregulates expression of one or more of the following genes: MMP2, FN1, LOXL2, PDGFRb, FABP5, SGMS1.

[0124] As used herein, "upregulate" means to increase or enhance the mRNA and / or protein expression of a gene in a cell and / or tissue by at least 1%, 5%, 10%, 50%, or more compared to an untreated cell or tissue.

[0125] Example The present invention is further illustrated by the following examples. Example 1: Overexpression of SIRT6 variants in immortalized human hepatocytes (IHH) material and method cell culture A human hepatocyte cell line (IHH), isolated and immortalized by lentiviral transduction of SV40 T antigen and hTERT, was incubated in 1×10-6 M dexamethasone, 1×10 -12 Cells were maintained in phenol red-free Dulbecco's modified Eagle's medium (DMEM / F-12) containing human insulin (Humalog, Lilly), 10% FBS, and 1% penicillin / streptomycin. Cell culture medium was changed every 2 days, and cells were subcultured using TrypLE Express when they reached 90% confluence.

[0126] Immunoblotting analysis Briefly, cells were harvested using TrypLE Express, washed with 1x PBS, and centrifuged at 300 g. The supernatant was discarded, and the resulting pellet was resuspended in 1x RIPA lysis buffer (20-188, Millipore, USA) supplemented with Halt™ protease and phosphatase inhibitor cocktail (100X, ThermoFisher) and lysed on ice (4°C) for 30 min with vigorous vortexing every 10 min. The sample was then centrifuged at 10,000 g for 10 min at 4°C, the supernatant was transferred to a new microtube, and the protein concentration was measured using the Pierce™ BCA Protein Assay Kit (23225, ThermoFisher) according to the manufacturer's instructions. Equal amounts of protein sample (at least 20 μg) were mixed with 1× Laemmli sample buffer (1610747, 4×, Bio-Rad), heated to 95°C for 5 minutes, and cooled on ice. Equal amounts of protein (40 μl) were then loaded onto a 10% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (4568034, Bio-Rad) and separated by electrophoresis at 120 V for 45 minutes. Protein transfer was performed onto a PVDF membrane using a Trans-Blot Turbo RTA Mini 0.45 μm LF PVDF Transfer Kit (1704274, Bio-Rad) and a Bio-Rad Trans-Blot Turbo Transfer System at 1.3 A and 25 V for 10 minutes. The membranes were then blocked with 5% bovine serum albumin (BSA, P6154, BioWest) in TBST buffer (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20) for at least 30 min and incubated with specific primary antibodies (see below) diluted in TBST blocking solution at the appropriate dilution. After three washes in TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies diluted in TBST blocking buffer.After three additional washes with TBST, protein levels were detected using Clarity Western ECL Substrate (1705061, Bio-Rad), and signals were detected using a Bio-Rad ChemiDoc XRS+ imaging system. For quantitative measurements, scanned membranes were analyzed using Image Lab™ software (Bio-Rad).

[0127] The following antibodies were used: Cell Signaling Technology (MA, USA) - rabbit anti-Akt (1:1000), rabbit anti-phospho-Akt (Ser473) (1:1000), rabbit anti-histone H3 (D1H2, 1:1000); Abcam (UK) - rabbit anti-collagen I (1:1000), rabbit anti-SIRT6 antibody (1:1000, EPR18463); Thermo Fisher Scientific (CA, USA) - mouse IgG1 GAPDH monoclonal HRP-conjugated antibody (1:2000), secondary goat anti-rabbit IgG HRP-linked (1:2000), and secondary goat anti-mouse IgG HRP-linked (1:2000).

[0128] result Figure 1A shows the signal of the far-red fluorescent protein Katushka2S, contained in the LV cassette, either alone or together with one of the SIRT6 versions (WT, N308K, or N308K / A313S) in the empty group, indicating successful infection. Katushka2S signal was not detected in the IHH control cells. SIRT6 protein expression was measured by Western blotting (Figure 1B), confirming a significant increase in SIRT6 levels in the LV-SIRT6-transfected group compared with either empty cells or CTL cells. SIRT6 is actively recruited to target gene promoters and represses gene transcription by removing acetylated H3K9 and H3K56 sites. Accordingly, in the SIRT6 overexpression (OE) group, the level of acetylated histone H3K56 was significantly reduced, while H3K9Ac showed a tendency to be reduced (Figure 1B), confirming that increased SIRT6 expression was accompanied by a concomitant increase in its deacetylase activity.

[0129] Example 2: Overexpression of a longevity variant of SIRT6 significantly alters the metabolomic profile of IHH without changes in insulin sensitivity. material and method Immunoblotting analysis See Example 1.

[0130] Metabolomics Metabolic profiling was performed by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Cell pellets or cell culture medium were resuspended / diluted in cold extraction solvent spiked with metabolites not detected in the unspiked cell extract (internal standard) and incubated at -20°C for 1 hour. The samples were then vortexed and centrifuged at 18,000 x g for 5 minutes at 4°C. The supernatant was collected and incubated at 4°C, while the cell pellet was resuspended in cold extraction solvent again and incubated at -20°C for an additional 1 hour. The samples were again vortexed and centrifuged at 18,000 x g for 5 minutes at 4°C. The supernatant was collected and pooled with the previous supernatant sample. The supernatant was then dried under vacuum, reconstituted in water, resuspended with agitation for 15 minutes, centrifuged at 18,000 x g for 5 minutes at 4°C, and transferred to vials for UHPLC-MS analysis. Two different types of quality control (QC) samples were used to assess data quality: (i) QC calibration samples to correct for inter- and intra-batch response factor variations, and (ii) QC validation samples to assess how much the data pre-processing procedures improved data quality. Randomized sample injections were performed, with each QC calibration and validation extract evenly dispersed throughout the batch run. A specific UHPLC-MS method was used.

[0131] Data normalization and quality control: A normalization factor for each metabolite was calculated by dividing the metabolite's intensity in each sample by the recorded intensity of the appropriate internal standard in the same sample. The most appropriate internal standard for each variable was defined as the one resulting in the smallest corrected relative standard deviation calculated from the QC calibration samples across all analytical batches. Generally, the best internal standard trend followed the similarity of chemical structure between the spiked compound and the endogenous variable. Robust linear regression (internal standard-corrected response as a function of sample injection order) was used to estimate any intra-batch drift not corrected by internal standard correction in the QC calibration samples. For all variables, the internal standard-corrected response in each batch was divided by its corresponding intra-batch drift trend, thereby expressing the normalized abundance values ​​of the study samples relative to the batch-average QC calibration serum sample (arbitrarily set to 1). All remaining zero-value sample injection variable responses in the corrected dataset were replaced with missing values ​​before generating the final dataset used for statistical analysis of the study samples.

[0132] Univariate Data Analysis: Univariate statistical analysis was also performed on each metabolite measured in hepatocyte and culture medium samples, calculating group percentage changes and Student's t-test p-values ​​(or Werhiest's test if unequal variances were found) for comparisons between groups: WT vs. Empty; N308K vs. Empty; N308K / A313S vs. Empty; N308K vs. WT; N308K / A313S vs. WT; and N308K / A313S vs. N308K. To aid in visualization of the results, heat maps were generated for each sample type displaying the results of the above comparisons. These heat maps display the log2 (fold change) of metabolites included in the analysis, along with Student's t-tests for the comparisons performed. For each metabolite, the change between subgroups was calculated as the base 2 logarithm of the fold change. Darker blue and red indicate greater decreases and increases in metabolite levels, respectively. These values ​​are accompanied by significance levels based on p-values ​​from a Student's t-test. Three levels of increasing significance were considered: p<0.05, p<0.01, and p<0.001.

[0133] result It is well known that alterations in the insulin receptor substrate PI3K (phosphoinositide 3-kinase) and AKT signaling pathways are closely related to metabolic disorders, hepatic steatosis, and insulin resistance. Therefore, we aimed to assess the levels of insulin-sensitive / activated PI3K / AKT pathways. Overexpression of SIRT6 and its lifespan variants in IHH cells was assessed by immunoblotting. After overnight serum and glucose starvation of IHH cells overexpressing or not overexpressing SIRT6 and its allelic variants, they were stimulated with human insulin solution (100 nM) for 30 minutes, and then pAKT (Ser473) protein levels were measured. No significant differences in pAKT (Ser473) levels were observed between conditions, either with or without insulin administration (Figure 2).

[0134] Furthermore, detailed metabolic profiling of IHH cells and their supernatants was performed upon overexpression of SIRT6 and its allelic variants. Using an ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) platform optimized for broad metabolome coverage, we were able to optimally profile: (1) fatty acylates, bile acids, steroids, and lysoglycerophospholipids; (2) glycerolipids, glycerophospholipids, sterol lipids, and sphingolipids; and (3) amino acids and their derivatives. A total of 296 and 282 metabolic signatures were detected in the analyzed cell pellet and culture medium samples, respectively.

[0135] PCA analysis First, principal component analysis (PCA) of hepatocyte extracts was performed for four IHH cell lines: empty, WT, N308K, or N308K / A313S. The PCA score scatter plots showed separation of WT, N308K, and N308K / A313S samples when the first t[1] and second t[2] components were plotted, with WT being more separated than the other groups (Figure 3A). The t[1] and t[2] components explained 34.4% and 19.0% of the inter-sample variability, respectively. Similarly, PCA analysis of IHH culture medium samples was performed. However, the inter-group separation (Figure 3B) was not as clear as in the hepatocyte extracts (Figure 3A).

[0136] Common metabolite changes (heat map) More changes in metabolite levels were observed in the analysis of human hepatocytes (Figure 7) than in the comparisons made between culture medium groups (Figure 8). Most of the changes were observed between WT samples and the other groups, and fewer metabolites were altered between both mutant groups and when compared to hepatocytes transfected with empty vector (Figure 7). There was a significant decrease in the levels of most glycerophospholipids in cells transfected with the WT SIRT6 sequence compared to the empty vector (Figure 7), but this decrease was not observed in the culture medium (Figure 8).

[0137] It was also clear that a nearly complete profile of amino acids (AA) was increased in the mutant group compared with hepatocytes transfected with the WT SIRT6 sequence (Figure 7). However, little change in amino acid levels was observed in the culture medium (Figure 8). Increases in several fatty acids (FAs) and glycerophospholipids (especially lysophosphatidylethanolamine, LPE) were also detected in the comparison between hepatocytes transfected with the SIRT6 mutant and WT sequences, but more species were altered in the N308K group than in the N308K / A313S group (Figure 7). A decrease in ceramide was detected only in the N308K group compared with the WT group, but not in the N308K / A313S group (Figure 7). Several glycerophospholipids were also increased in the culture medium of the mutant group compared with the WT group, particularly ether-linked glycerophosphatidylcholine (ether-PC) in the comparison between N308K and WT (Figure 8).

[0138] amino acid The levels of several amino acids were reduced in hepatocytes transfected with the WT SIRT6 sequence compared to the empty vector: threonine, aspartate, glutamate, asparagine, proline, sarcosine, and hypotaurine (Figure 4A). Surprisingly, however, most of these metabolites were increased in the mutant group compared to the empty vector group (Figure 4A). Examples of some of these changes are included in the boxplots in Figure 9. It is noteworthy that almost the entire profile of amino acids and derivatives was increased in the mutant group compared to the WT group (Figure 4A). The only exception was the decreased level of arginine in the N308K / A313S group compared to the other groups (Figure 9). In contrast, few changes were detected between the mutant-transfected hepatocyte groups. Among them, decreased levels of arginine and lysine and increased levels of citrulline were found in the N308K / A313S group compared to the N308K group (Figures 4 and 9).

[0139] Changes in amino acid levels were also observed between the culture medium groups (Figure 4B). The most significant changes (low p-values) in amino acid levels in the culture medium of WT-transfected hepatocytes compared to the empty vector group were decreases in aspartate, glutamate, asparagine, and arginine (Figure 4A and Figure 10). These decreases were also confirmed in the cell pellets, except for arginine (Figure 4A), for which the decrease in arginine did not reach a p-value <0.05 (Figure 9). The levels of asparagine, aspartate, and glutamate were increased in the culture medium of mutant cells compared to WT samples (Figure 10), whereas arginine, as detected in hepatocytes, was decreased in N308K / A313S samples compared to the WT group (Figures 10 and 11, respectively). The levels of several amino acids also changed between both mutant groups: cystine, serine, cystathionine, aminoadipic acid, citrulline, and sulfocysteine ​​(Figure 10).

[0140] saturated fatty acids No changes were detected in the levels of saturated fatty acids (SFA) between hepatocyte groups (Figure 5A). However, several monounsaturated and polyunsaturated fatty acids, such as oleic acid (18:1n-9) and mead acid (20:3n-9), were significantly increased in hepatocytes from the N308K group compared to CTL (empty) and SIRT6WT, but to a lesser extent in hepatocytes from the N308K / A313S group (Figure 5A-C).

[0141] Glycerolipids With regard to glycerolipids, few changes were detected in diglyceride or triglyceride levels, except for a decrease in some unsaturated species in WT-transfected hepatocytes compared to the empty vector group, particularly in species with longer acyl chains. This can be easily visualized in the carbon plots presented in Figures 11A and 11B, which represent the effect of carbon number and double bond content on the increase or decrease of diglycerides and triglycerides in the WT group compared to the empty vector control group. Some of these triglycerides with longer acyl chains were also decreased in the N308K / A313S group compared to control hepatocytes (Figure 11C).

[0142] A decrease in the levels of most glycerophospholipids was found in cells transfected with the WT SIRT6 sequence compared to the empty vector (Figure 7), but little change was detected in the culture medium (Figure 8). However, an increase in lysophosphatidylethanolamine species (LPE) was detected in the comparison between mutant and WT transfected hepatocytes, although more species were altered in the N308K group than in the N308K / A313S group (Figure 5D-I).

[0143] Sphingolipids Regarding sphingolipids (ceramides and sphingomyelins), several sphingomyelins were decreased in WT-transfected hepatocytes compared with the empty vector group, but their levels were not different when comparing the mutant and empty groups (Figure 12). Ceramides tended to be increased in WT-transfected hepatocytes compared with the empty vector group, but only Cer(d18:1 / 22:0) reached a p-value <0.05 (Figure 13). Furthermore, a decrease in ceramide was detected only in the N308K group, but not in the N308K / A313S group, when compared with the WT group (Figure 13).

[0144] conclusion Overall, our data revealed significant metabolomic changes in IHH cells upon overexpression of SIRT6 WT and longevity-associated mutants (N308K and N308K / A313S) compared with control cells. In summary: (1) Nearly the complete amino acid profile was increased in the mutant group compared with hepatocytes transfected with the WT sequence. The increase in citrulline levels in IHH cells from the N308K / A313S group was noteworthy. (2) Increases in several unsaturated fatty acids and glycerophospholipids were also detected in N308K and N308K / A313S, although more species were altered in N308K. (3) Ceramides tended to be increased in WT-transfected hepatocytes compared with the control empty vector group. Furthermore, a decrease in ceramides was detected in the N308K group compared with WT hepatocytes, but not in the N308K / A313S group. (4) Little change was observed in diglyceride or triglyceride levels.

[0145] Example 3: Overexpression of the longevity variant of SIRT6 (N308K / A313S) inhibits collagen deposition and fibrotic gene expression in 3D spheroids formed by co-culture of IHH and human hepatic stellate cells. material and method cell culture The LX2 cell line was obtained from CLS-GmbH (Eppelheim, Germany). The cell line was cultured in high-glucose DMEM (1X) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2. The cell culture medium was changed every 2 days, and the cells were subcultured using TrypLE Express when they reached 90% confluence.

[0146] 3D spheroids To generate cell spheroids, cells were seeded at 10,000 viable cells per well in 96-well round-bottom ultra-low attachment plates (BIOFLOAT, faCellilate). Each IHH LV-transfected cell line (empty, WT, N308K, and N308K / A313S) and normal IHH (control CTL) were co-cultured with LX2 cells at a 20:1 ratio to mimic physiological proportions in the liver parenchyma. In the liver parenchyma, hepatocytes are the predominant cell type, with stellate cells accounting for only approximately 5%. Spheroids were cultured in DMEM medium supplemented as described above. Plates were incubated at 37°C in a humidified atmosphere of 5% CO2 for 5 days.

[0147] Microscopy and fluorescence imaging Spheroids were fixed directly on the culture plate with 4% PFA for 10 minutes and then transferred to a minitube. After washing in PBS, the spheroids were kept in 15% sucrose for 1 hour, embedded in tissue freezing medium (OCT), cut at 7 μm using a cryotome (Leica Microsystems) at -20°C, and stored at -80°C for further use. To evaluate the effect of SIRT6 variants and their overexpression on liver tissue fibrosis, histological sections of the spheroids were immunolabeled to detect collagen 1A. Slides were washed once with 1x PBS to dissolve the OCT and blocked in 1x PBS supplemented with 0.2% Tween-20 and 5% BSA.

[0148] The primary antibody, rabbit anti-collagen I (1:500, ab34710, Abcam) was diluted in DAKO antibody diluent (S202230-2, Agilent Technologies) and incubated overnight at room temperature in a humid chamber. After three washes with 1x PBS, a mixture of secondary antibody (1:500) donkey anti-rabbit IgG conjugated with Alexa Fluor™ 647 was applied and incubated for at least 1 hour. After three washes with 1x PBS, slides were counterstained with DAPI (1 μg / ml) solution for 15 minutes and embedded in aqueous hardening medium (Mowiol). After hardening (overnight at 4°C), images were captured using an Axioscan Z.1 (ZEISS) equipped with a Hamamatsu ORCA-Flash 4.0 camera. All immunofluorescence images were evaluated using the ImageJ software (NIH, USA) analysis program. Fibrosis was determined as the abundance of collagen 1A in spheroid samples and assessed as the % of total spheroid area depicted by DAPI fluorescence at 100x magnification when at least five spheroids per condition / cell line were used in three consecutive and independent experiments.

[0149] Soluble collagen measurement The spheroid-conditioned medium (CM) was collected and centrifuged at 1,000 × g. The cell solution was homogenized on ice using a pre-chilled Dounce homogenizer. After overnight incubation, the acidic solution was centrifuged at 10,000 × g for 15 minutes at 4 °C to pellet all debris, and the clear supernatant was transferred to a new microtube. Collagen concentration was measured using the Soluble Collagen Assay Kit® (ab241015, Abcam, Cambridge, UK) according to the manufacturer's instructions. Fluorescence was measured using an Agilent BioTek FLx800 microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

[0150] Quantitative real-time PCR Briefly, column separation techniques were used for mRNA isolation using the RNeasy Mini Kit (74106, Qiagen, Germany) according to the manufacturer's instructions. At least four biological replicates were prepared for each treatment group. Total RNA was quantified using a Nano Drop 1000 spectrophotometer (Thermo Fisher Scientific), and a total of 1 μg of isolated RNA was used to prepare cDNA using a High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific). Real-time PCR was performed with at least two technical replicates using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) and SYBR™ Select Master Mix (4472908, Thermo Fisher Scientific). PCR reactions were maintained in a volume of 10 μl, and 250 ng of cDNA was added to each well. The primer sequences used in this study are listed in Table 1. [Table 1]

[0151] result The liver parenchyma is composed of various cell types, with hepatocytes accounting for approximately 80% of the total liver mass, and the second most abundant hepatocyte type, hepatic stellate cells (HSCs), accounting for 5-8%. Crosstalk between these two major hepatic cell types, and HSC-mediated collagen deposition, primarily regulate the progression of fibrosis and inflammation in NAFLD / NASH. Therefore, based on our metabolomics data, we employed a 3D spheroid culture model of IHH and hepatic stellate cells (LX2) overexpressing or not overexpressing SIRT6 and its longevity-associated variants to investigate the in vitro interactions between the two major hepatic cell types. Spheroid culture allows cell-to-cell connectivity and communication, recreating an environment closer to the in vivo state than monolayer cell culture. IHH and LX2 were cocultured for 5 days in ultra-low attachment 96-well plates, then harvested and processed for analysis. Figure 6A shows quantitative analysis of spheroid sections using DAPI (nuclei) and COL1A1, revealing a significant decrease in collagen content in spheroids containing IHH overexpressing the N308K / A313S version of SIRT6 compared to all other groups (Figure 6A). Collagen released into the conditioned medium by the spheroids was then measured. The results show that in all groups overexpressing any of the SIRT6 variants, collagen levels were approximately 30% lower compared to the empty vector group (Figure 6B). The mRNA expression of key fibrosis gene markers in the spheroids was also analyzed. COL1A1 levels were significantly higher in the WT and N308K groups compared to the empty or N308K / A313S groups (Figure 6C). Furthermore, MMP2, another important marker of fibrosis, showed a tendency to decrease in all SIRT6 overexpression groups, with significantly lower levels in the N308K / A313S group compared to the empty group. Thus, longevity-associated SIRT6 variants confer basal antifibrotic effects in in vitro multilineage 3D liver spheroids.

[0152] Example 4: In vitro assay - fibrosis material and method cell line The LX2 human hepatic stellate cell line was obtained from CLS-GmbH (Eppelheim, Germany) and cultured in high-glucose (4.5 g / l) DMEM (1×) supplemented with 10% fetal bovine serum (FBS), 15 mM Hepes buffer (Biowest, France), glutamine, 1% penicillin / streptomycin solution, and 100 μg / ml normocin at 37°C and 5% CO .

[0153] Immortalized human hepatocytes (IHH) An immortalized human hepatocyte cell line (IHH) isolated by lentiviral transduction with SV40 T antigen and hTERT as previously described [De Gottardi A, Vinciguerra M et al., Lab Invest 2007] was maintained in phenol red-free Dulbecco's modified Eagle's medium (DMEM / F-12) containing 1 x 10-6 M dexamethasone, 1 x 10-12 M human insulin (Humalog, Lilly), 10% FBS, and 1% penicillin / streptomycin.

[0154] Human primary hepatic stellate cells from a healthy donor (n=1) and from donors with NASH (n=2) were obtained from Lonza and cultured in human stellate cell growth medium (catalog number: MCST250) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin solution at 37°C and 5% CO2.

[0155] AAV2 / 5 transduction After reaching confluence, cells were split and seeded into 24-well plates with a growth surface area of ​​2 cm, seeding 50 x 10 cells / well. Immediately after seeding, cells were transduced with AAV2 / 5 containing SIRT6 constructs: AAV-LUC (luciferase), AAV-SIRT6 (WT), and SIRT6 (N308K / A313S) (longevity) at 10 vg in basal DMEM medium for 24 hours, after which fresh medium was added and the cells were cultured for an additional 96 hours.

[0156] qPCR Column separation technology was used for mRNA isolation using the RNeasy mini-Kit (Qiagen, Germany). At least four biological replicates were prepared for each treatment group. Total RNA was quantified using a NanoDrop 1000 spectrophotometer, and 1 μg of the isolated total RNA was used to prepare cDNA using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Real-time PCR was performed with at least two technical replicates using a StepOnePlus™ Real-Time PCR System and SYBR™ SelectMaster Mix. PCR reactions were maintained in a volume of 10 μl, and 250 ng of cDNA was added to each well. GeNorm was used for accurate normalization of qPCR data by geometric averaging of two internal control genes (actin, GAPDH).

[0157] Immunoblotting Cells were harvested using TrypLEExpress, washed with 1x PBS, and centrifuged at 300 g. The supernatant was discarded, and the resulting pellet was resuspended in 1x RIPA lysis buffer supplemented with Halt™ protease and phosphatase inhibitor cocktail (100X, ThermoFisher) and lysed on ice (4°C) for 30 min with vigorous vortexing every 10 min. Samples were then centrifuged at 10,000 g for 10 min at 4°C, the supernatant transferred to a new microtube, and protein concentration was measured using the Pierce™ BCA Protein Assay Kit (23,225, ThermoFisher) according to the manufacturer's instructions. Equal amounts of protein sample (at least 20 μg) were mixed with 1× Laemmli sample buffer (1,610,747, 4×, Bio-Rad) and heated to 95°C for 5 minutes. After cooling on ice, equal amounts of protein (40 μl) were loaded onto a 10% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (4,568,034, Bio-Rad) and separated by electrophoresis at 120 V for 45 minutes. Protein transfer was performed onto PVDF membranes using a Trans-Blot Turbo RTA Mini 0.45 μm LF PVDF Transfer Kit (1,704,274, Bio-Rad) and the Bio-Rad Trans-Blot Turbo Transfer System at 1.3 A and 25 V for 10 minutes. The membranes were then blocked with 5% bovine serum albumin (BSA, P6154, BioWest) 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 specific primary antibodies (see below) diluted in TBST blocking solution at the appropriate dilution. After three washes with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies diluted in TBST blocking buffer. After three additional washes with TBST, protein levels were detected using Clarity Western ECL Substrate (1705061, Bio-Rad), and signals were detected using a Bio-Rad ChemiDoc XRS+ imaging system.For quantitative measurements, scanned membranes were analyzed using Image Lab™ software (Bio-Rad).

[0158] Spheroids To generate cell spheroids, cells were seeded at 10,000 viable cells per well in 96-well round-bottom ultra-low attachment plates (BIOFLOAT, faCellilate). Each IHH AAV-transfected cell line (LUC, SIRT6wt, SIRT6cent) was co-cultured with LX2 cells at a 20:1 ratio to mimic physiological proportions in the liver parenchyma. In the liver parenchyma, hepatocytes are the predominant cell type, with hepatic stellate cells accounting for only approximately 5%. Spheroids were cultured in DMEM medium supplemented as described above. Plates were incubated at 37°C in a humidified atmosphere with 5% CO2 for 5 days. In a subset of spheroids, TGFbeta (10 ng / ml) was added for the final 48 hours of incubation.

[0159] Immunofluorescence analysis of spheroids Spheroids were fixed directly on the culture plate with 4% PFA for 10 minutes and then transferred to a minitube. After washing with PBS, spheroids were stored in 15% sucrose for 1 hour, embedded in tissue freezing medium (OCT), and then sectioned at 7 μm using a cryotome (Leica Microsystems) at -20°C and stored at -80°C for further use. To evaluate the effect of SIRT6 variants and their overexpression on liver tissue fibrosis, spheroid tissue sections were immunolabeled to detect collagen 1A. Slides were washed once with 1x PBS to dissolve the OCT and blocked with 1x PBS supplemented with 0.2% Tween-20 and 5% BSA. The primary antibody, rabbit anti-collagen I (1:500, ab34710, Abcam) was diluted in DAKO antibody diluent (S202230-2, Agilent Technologies) and incubated overnight at room temperature in a humid chamber. After washing three times with 1x PBS, a mixture of donkey anti-rabbit IgG secondary antibody (1:500) conjugated with Alexa Fluor™ 647 was applied and incubated for at least 1 hour. After washing three times with 1x PBS, slides were counterstained with DAPI (1 μg / ml) solution for 5 minutes and embedded in aqueous hardening medium (Mowiol). After hardening (overnight at 4°C), images were captured using an Axioscan Z.1 (ZEISS) equipped with a Hamamatsu ORCA-Flash 4.0 camera. All immunofluorescence images were evaluated using the ImageJ software (NIH, USA) analysis program. Fibrosis was determined as the abundance of collagen 1A in the spheroid samples and assessed as the percentage of the total spheroid area delineated by DAPI fluorescence at 100x magnification, provided that at least five spheroids per condition / cell line were used in three consecutive, independent experiments.

[0160] result [Table 2]

[0161] LX-2 cells The liver parenchyma is composed of various cell types, with hepatocytes accounting for approximately 80% of the total liver mass, and the second most abundant hepatocyte type, hepatic stellate cells (HSCs), accounting for 5-8%. Crosstalk between these two major hepatic cell types and HSC-mediated collagen deposition primarily controls the progression of fibrosis and inflammation in NAFLD / NASH.

[0162] The expression of several genes associated with liver fibrosis (see Table 2) was measured in the HSCLX2 cell line by qPCR (vimentin, aSMA, TIMP1, MMP2, FN1, LOXL2, PDGFRb, collagens (COL1A1, COL3A1, COL4A1, COL5A1, COL6A1)) or by immunoblotting (collagen, TIMP1, MMP2, FN1, and LOXL2). The results are shown in Figures 14A-C.

[0163] Although some changes in gene expression were observed at the mRNA level, the most significant results at the protein level showed an inhibition of collagen production and an increase in MMP2 (matrix metalloproteinase) in HSCs overexpressing SIRT6-WT or SIRT6cent, as well as a specific inhibition of TIMP1 in a SIRT6cent-dependent manner. Notably, AAV-mediated overexpression of SIRT6 and SIRT6cent was confirmed at both the mRNA and protein levels in LX2.

[0164] The imbalance of MMP2 / TIMP1 and the regulation of FN1, LOXL2 and collagen expression suggest a SIRT6-dependent hepatic antifibrotic effect in vitro, with the more pronounced effect being SIRT6cent-dependent.

[0165] Spheroids The liver parenchyma is composed of various cell types, with hepatocytes accounting for approximately 80% of the total liver mass, and the second most abundant hepatocyte type, hepatic stellate cells (HSCs), accounting for 5-8%. Crosstalk between these two major hepatic cell types and HSC-mediated collagen deposition primarily controls the progression of fibrosis and inflammation in NAFLD / NASH.

[0166] The expression of several genes related to liver fibrosis (vimentin, aSMA, TIMP1, MMP2, FN1, LOXL2, and collagen (COL1A1)) was measured by qPCR in the HSC LX2 cell line. AAV-mediated overexpression of SIRT6 variants was confirmed at the mRNA and protein levels in LX2 and spheroids. The results are shown in Figures 15A-E.

[0167] Although some changes in gene expression were observed at the mRNA level, the most significant result from protein-level analysis in the spheroid experiments was the inhibition of collagen production in spheroids overexpressing IHH-SIRT6-WT or SIRT6cent, suggesting an anti-fibrotic effect that was more pronounced when SIRT6cent was overexpressed. The anti-fibrotic effect was observed both in the presence and absence of TGF-β, a major pro-fibrotic factor.

[0168] Primary hepatic stellate cells The results are shown in Figures 16A and 16B. Several changes in gene expression were observed at the mRNA level. As expected, there was a general activation of profibrotic markers (aSMA, COL1A1, vimentin, TP63, and TGFbeta) in HSCs from NASH patients. As observed at the protein level in LX2 cells, the reduction in collagen and the imbalance in MMP2 / TIMP1 suggest a SIRT6-dependent antifibrotic effect in both healthy and NASH HSCs, with a more pronounced effect dependent on SIRT6cent. Interestingly, overexpression of SIRT6wt, and more notably, overexpression of SIRT6cent, significantly inhibited the mRNA level of p63, a newly recognized profibrotic factor.

[0169] The data summarizing fibrosis levels are summarized in Table 3 below.

[0170] [Table 3] JPEG2025541838000004.jpg130159

[0171] Example 5: In vitro assays - lipid metabolism material and method cell line Immortalized human hepatocytes (IHH) A human hepatocyte cell line (IHH), isolated and immortalized by lentiviral transduction with SV40 T antigen and hTERT as previously described [De Gottardi A, Vinciguerra M et al., Lab Invest 2007], was maintained in phenol red-free Dulbecco's modified Eagle's medium (DMEM / F-12) containing 1x10-6 M dexamethasone, 1x10-12 M human insulin (Humalog, Lilly), 10% FBS, and 1% penicillin / streptomycin.

[0172] AAV2 / 5 transduction After reaching confluence, cells were split and seeded into 24-well plates with a growth surface area of ​​2 cm, with 50 x 10 cells / well. Immediately after seeding, cells were infected with AAV2 / 5 containing SIRT6 constructs: AAV-LUC (luciferase), AAV-SIRT6 (WT), and SIRT6 (N308K / A313S) (longevity) at 10 vg in basal DMEM medium for 24 hours, after which fresh medium was added and the cells were cultured for an additional 96 hours.

[0173] qPCR Column separation technology was used for mRNA isolation using the RNeasy mini-Kit (Qiagen, Germany). At least four biological replicates were prepared for each treatment group. Total RNA was quantified using a NanoDrop 1000 spectrophotometer, and 1 μg of the isolated total RNA was used to prepare cDNA using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Real-time PCR was performed with at least two technical replicates using a StepOnePlus™ Real-Time PCR System and SYBR™ SelectMaster Mix. PCR reactions were maintained in a volume of 10 μl, and 250 ng of cDNA was added to each well. GeNorm was used for accurate normalization of qPCR data by geometric averaging of two internal control genes (actin, GAPDH).

[0174] result [Table 4]

[0175] The results are shown in Figure 17. The expression of several genes involved in hepatic lipid metabolism (see Table 4) was measured by qPCR in the IHH cell line (CD36, FASN, FABP5, DAGT1, ACC1, SGMS1). AAV-induced overexpression of SIRT6 variants was confirmed at the mRNA level.

[0176] qPCR analysis revealed elevated levels of genes involved in the regulation of lipid metabolism and NASH progression (FABP5, SGMS1, ACC1) in IHH cells overexpressing SIRT6wt or SIRT6cent compared with the control group.

[0177] The data summarizing lipid metabolism are summarized in Table 5 below. [Table 5]

[0178] Example 6: In vitro transcriptomics material and method cell line Immortalized human hepatocytes (IHH) A human hepatocyte cell line (IHH), isolated and immortalized by lentiviral transduction with SV40 T antigen and hTERT as previously described [De Gottardi A, Vinciguerra M et al., Lab Invest 2007], was maintained in phenol red-free Dulbecco's modified Eagle's medium (DMEM / F-12) containing 1x10-6 M dexamethasone, 1x10-12 M human insulin (Humalog, Lilly), 10% FBS, and 1% penicillin / streptomycin.

[0179] AAV2 / 5 transduction After reaching confluence, cells were split and seeded into 24-well plates with a growth surface area of ​​2 cm, with 50 x 10 cells / well. Immediately after seeding, cells were infected with AAV2 / 5 containing SIRT6 constructs: AAV-LUC (luciferase), AAV-SIRT6 (WT), and SIRT6 (N308K / A313S) (longevity) at 10 vg in basal DMEM medium for 24 hours, after which fresh medium was added and the cells were cultured for an additional 96 hours.

[0180] RNA-Seq Index libraries were prepared from 2 mg / ea of ​​purified RNA from IHH-LUC, IHH-SIRT6wt, and IHH-SIRT6cent cells (n=4 per condition) using the TruSeq Total Stranded RNA Sample Preparation Kit (Illumina, Cambridge, UK) according to the manufacturer's instructions. Libraries were quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) and pooled so that each index-tagged sample was present in equimolar amounts; the final concentration of the pooled sample was 2 nmol / L. The pooled samples were then subjected to cluster generation and sequencing using an Illumina HiSeq2500 system (Illumina, Cambridge, UK) in a 2x100 paired-end format at a final concentration of 8 pmol / L. Short reads were aligned to the GRCm38 genome assembly using STAR (version 2.5.1a). Stacked reads were counted with htseq-count. Normalization of read counts and their comparison were performed using the R package. Genes were considered to be differentially expressed between groups if their expression values ​​differed significantly (q-value ≤ 0.05) by more than two-fold. Pathway enrichment analysis was performed by using Ingenuity Pathway Analysis (QIAGEN Inc). All calculations were performed in R ver. 3.4.1 (R Core Team 2017).

[0181] Whole transcriptome analysis The whole transcriptome of IHH cells transduced with AAV-SIRT6cent and AAV-SIRT6wt was analyzed using Ingenuity Pathway Analysis (IPA).

[0182] result The results are shown in Figures 18A-18D. Large changes in gene expression levels were observed comparing SIRT6wt or SIRT6cent to the control (LUC) (as evidenced by the heatmap and volcano plot), but only 56 genes were differentially expressed in the comparison of SIRT6 variant overexpression (SIRT6wt vs. SIRT6cent), suggesting finely tuned transcriptional regulation.

[0183] Analysis of differentially expressed pathways between cells treated with AAV-SIRT6-WT and AAV-SIRT6-Cent is shown in FIG.

[0184] Down-regulation of the β-catenin pathway in IHH cells treated with AAV-SIRT6-Cent versus AAV-SIRT6-WT was demonstrated. Of the 21 genes down-regulated in AAV-SIRT6wt-treated versus AAV-SIRT6cent-treated cells, 12 genes are involved in the β-catenin pathway (PKP1, S100AB, SPRR2D, KRT1, KRT6C, A2M, KRT6B, KRT5, HLA-DRA, KRT6A, IGHG1, MIR205HG). See Table 6.

[0185] [Table 6]

[0186] TP63 was identified by software as a central gene regulating the β-catenin pathway in AAV-treated IHH.

[0187] Downregulation of the glucocorticoid pathway in IHH cells treated with AAV-SIRT6- versus Cent AAV-SIRT6-WT was demonstrated. Of the 21 genes downregulated in AAV-SIRT6wt- versus AAV-SIRT6cent-treated cells, 11 genes are involved in the glucocorticoid pathway (SPINK5, DSG1, SPRR2D, KLK5, KRT1, SPRR1B, KRT6C, CALML5, KRT6B, SPRR2E, SPEE2A). See Table 7.

[0188] [Table 7]

[0189] FOXC1 was identified by software as a central gene regulating the glucocorticoid pathway in AAV-treated IHH.

[0190] Of note, this analysis used the entire transcriptome, not just the 56 differentially expressed genes, but these genes were again highlighted in this analysis as the most important genes.

[0191] In conclusion, SIRT6cent is involved in the regulation of the β-catenin and glucocorticoid pathways in immortalized human hepatocytes.

[0192] Example 7: Post-translational modifications (PTMs) of histones by SIRT6WT and SIRT6cent in 3T3-L1 adipocytes The most potent enzymatic activity described for SIRT6 is its function as a histone deacetylase. Strong evidence suggests that SIRT6 targets gene promoters and represses gene transcription by removing acetylation at H3K9, H3K18, and H3K56 heterochromatin sites. Among all histone post-translational modifications (PTMs), acetylation and methylation are the two most well-studied, and they functionally interact to fine-tune transcriptional output. To gain insight into SIRT6-dependent epigenetic regulation during adipogenesis, we performed a comprehensive analysis of histone acetylation / methylation PTMs by mass spectrometry (LC-MS / MS) in 3T3-L1 differentiated adipocytes (AdiE, AdiWT, and AdiCent).

[0193] material and method Histone extraction from 3T3-L1 cells overexpressing LUC (ctl or AdiE), SIRT6wt (or AdiWT), or SIRT6cent (or AdiCent) The histone extraction protocol was adapted from a previous study [Cincarova, L., et al., A combined approach for the study of histone deacetylase inhibitors. Mol Biosyst, 2012, 8(11):2937-45.]. Each sample was run six times. Cells on culture plates were gently washed with cold PBS and collected in lysis buffer (80 mM NaCl, 20 mM EDTA (Bio-Rad, California, USA), 1% Triton X-100 (Carl Roth, Germany), 45 mM sodium butyrate, and 0.1 mM PMSF (Thermo Fisher Scientific)) and incubated on ice for 20 min. After centrifugation (20,000 g, 8 min, 4°C), the upper lipid layer was removed, and PBS was added to the remaining sample containing chromatin. After three additional washes with PBS (20,000 g, 10 min, 4°C), the pellet was resuspended in 250 μL of ice-cold H2SO4 (Penta, Czech Republic) and incubated with shaking at 4°C for 2 h. The supernatant, clarified by centrifugation (20,000 g, 10 min, 4°C), was diluted with 250 μL of 50% ice-cold trichloroacetic acid and incubated with shaking at 0°C for 30 min. The resulting precipitate was collected by centrifugation (20,000 g, 30 min, 4°C), washed with acetone containing 50 mM HCl (Penta), washed twice more with acetone, and dried at room temperature. The prepared histone extract was dissolved in 20 μL of water.

[0194] Chemical derivatization of histone extracts The volume of the histone extract was reduced to 5 μL in a vacuum concentrator, 5 μL of acetonitrile (ACN; Honeywell, USA) was added, and the sample was subjected to microwave-assisted histone derivatization using trimethylacetic anhydride (sequencing grade modified, Promega Corporation, Madison, WI, USA) according to a previously published procedure

[48] . The pH was adjusted to 8 with NH4OH, and 3 μL of derivatization reagent consisting of trimethylacetic anhydride (Merck Millipore, Burlington, MA, USA) and ACN in a 1:3 (v / v) ratio was added. The sample was incubated at room temperature with shaking for 5 h, after which the derivatization step was repeated, including a 16-h incubation. The sample then proceeded to two microwave-assisted histone derivatizations as follows: The sample volume was reduced to 5 μL in a vacuum concentrator, and 50% (v / v) ACN was added to a final volume of 12 μL. Each round included three derivatization subcycles, which consisted of adjusting the sample pH to 8 with NH4OH, adding 3 μL of derivatization reagent, and incubating twice for 1 min in a microwave oven at 350 W (with brief rotation between incubations). The microtube containing the sample was covered with a glass beaker during incubation in the microwave oven. After two complete rounds (a total of six reagent additions), the sample volume was reduced to 5 μl, and 0.3 μg of SOLu-trypsin (Merck) in 40 μL of 100 mM ammonium bicarbonate (ABC) was added. The sample was incubated at 37 °C for 4 h, after which an additional 0.3 μg of SOLu-trypsin was added and incubated for 12 h. The digested sample underwent two rounds of microwave-assisted derivatization as described above to label the N-termini of the newly released peptides. After the first round, samples were diluted to a final volume of 24 μL and completely dried after the second round. Derivatized histones were diluted with 0.1% TFA and desalted with Pierce C18 Spin Tips #84850 (Thermo Fisher Scientific).Peptides were sequentially eluted with 0.1% TFA in 50% ACN and 0.1% TFA in 75% ACN. Samples were dried in a vacuum concentrator to remove TFA and reconstituted in 0.1% FA (Honeywell) before LC-MS / MS analysis.

[0195] LC-MS / MS and histone peptide database search Chemically derivatized peptides were measured using an LC-MS / MS system consisting of an Ultimate 3000RS LC-nano system coupled to an Orbitrap Lumos Tribrid spectrometer (Thermo Fischer Scientific) equipped with a Digital PicoView 550 ion source (New Objective) and an Active Background Ion Reduction Device (ESI Source Solutions). Prior to LC separation, tryptic digests were concentrated online on a μPrecolumn C18 PepMap100 trap column (5 μm particles, 300 μm ID, 5 mm; Waters). Chromatographic separation was performed on an Aurora C18 analytical column (1.6 μm particle size, 75 μm internal diameter, 25 mm; Ion Opticks). The mobile phase consisted of 0.1% formic acid (A) in water and 0.1% formic acid (B) in 80% acetonitrile (ACN), with the following B ratios: 5% to 25% (0–20 min), 25% to 29% (20–30 min), 29% to 32% (30–40 min), 32% to 38% (40–55 min), 38% to 50% (55–75 min), 50% to 85% (75–85 min), followed by an isocratic wash of 85% B (85–95 min). The trapping column and column were equilibrated at 99:1 (mobile phase A:B, flow rate 500 nL / min) before sample injection into the sample loop. The outlet of the analytical column was directly connected to the ion source. MS data were acquired using a data-dependent strategy to select up to the top 10 precursors based on their abundance in the survey scan (m / z 350–2000). The resolution of the survey scan was 60,000, with a target of 4 × 10, one microscan, and a maximum injection time of 54 ms. HCD MS / MS spectra were acquired at a target of 5 × 10, with a resolution of 15,000. The maximum injection time for MS / MS was 22 ms. Dynamic exclusion was enabled for 60 s after acquisition of one MS / MS spectrum, and early expiration was disabled. The isolation window for MS / MS fragmentation was set to 1.6 m / z.

[0196] Evaluation of mass spectrometry data Raw mass spectrometry data files were analyzed using Proteome Discoverer software (Thermo Fisher Scientific, version 2.2.0.388) with an in-house Mascot search engine (Matrix Science, version 2.6.2) to compare acquired spectra with entries in the UniProtKB human database (version 2021_12, 20,594 protein sequences), the cRAP contaminant database, and the in-house histone human database (version 2019_10, 52 protein sequences). Mass tolerances for peptides and MS / MS fragments were 10 ppm and 0.03 Da (0.5 Da for cRAP), respectively. Semi-Arg-C was used as the enzyme specificity, allowing a maximum of two missed cleavages. For searches against the cRAP database, the variable modification settings were oxidation (M), deamidation (N, Q), acetylation (K), and trimethylacetylation (K, N-terminal, S, T, Y). For searches against the UniProtKB human database, these were trimethylacetylated (K, N-terminal, S, T, Y). For histone database searches, these were acetylated (K), methylated (K, R), di-methylated (K), trimethylated (K), phosphorylated (S, T), and trimethylacetylated (K, N-terminal, S, T, Y). Identification of selected histone peptides was manually verified and quantified from peak areas obtained from EICs using Skyline (64-bit, version 23.1.1.268 software), which included identification alignment between raw data files based on retention time and m / z.

[0197] The relative abundance of histone peptides was assessed using R scripts on the KNIME analysis platform, following previously published methodologies. The relative abundance of specific modified peptide forms was calculated from the ratio of each precursor peak area to the total area of ​​the respective peptide sequence. Peak areas corresponding to post-translationally modified forms of individual histone peptides were treated as constructs, and the Aitchison methodology based on log ratios was applied for statistical evaluation. Missing values ​​were first imputed by iterative least-squares regression, and areas were converted to relative abundances (percentages). To assess global acetylation or methylation, the acetylated and unacetylated forms of each peptide, and analogously, the methylated and unmethylated forms, were fused. These fused abundances were then IR-transformed and compared using Hotelling's T2 test to globally assess differences in their distributions. For comparison of all individual peptide forms, the log2 ratio (two-part ai-transformed) of the relative abundance of one form versus the sum of the relative abundances of all other forms was calculated for each peptide, and a t-test was applied to assess differences in each individual form. Note that in compositional data, the relative abundances of individual parts cannot be directly compared due to constant sum constraints that result in spurious negative correlations. Data analysis was performed in R version 3.6.3, using the Hotelling R package for composition and ilr and ai-transformations, respectively, and Hotelling's T 2 The Hotelling T2 test and its variants were used. R package version 1.0-5.

[0198] result The results are shown in Figure 20A and Table 8 for histone H4 G4KGGKGLGKGGAKR17, Figure 20B and Table 9 for histone H3.1 / H3.3 K18QLATKAAR26, Figure 20C and Table 10 for histone H3.1 / H3.3 K9STGGKAPR17, Figure 20D and Table 11 for histone H3.1 K27SAPATGGVKKPHR40, and Figure 20E and Table 12 for histone H3.3 K27SAPSTGGVKKPHR40.

[0199] [Table 8]

[0200] [Table 9]

[0201] [Table 10]

[0202] [Table 11]

[0203] [Table 12]

[0204] Overexpression of SIR6wt or SIRT6cent in 3T3-L1 cells resulted in altered acetylation profiles of histones H3.1, H3.2, and H4. This was reflected in significant differences between AdiWT and AdiCent in the levels of lysines (K) K5, K8, K12, and K16 in H4, K9, K18, K14, and K23 in H3.1, and K27, K36, and K37 in H3.3. This confirmed H3K9, H3K18, H3K27, H3K23, H3K14, and H3K36 histones as SIRT6 targets, while identifying H3K37, H4K5, H4K8, and H4K12 histones as potentially new SIRT6 targets not yet described in the literature.

[0205] Example 8: In vivo assay in the HF / DEN model material and method Animal models 7-8 week old C57BL / 6N-Tyr <cbrd>Thirty-six male and 36 female albino mice (Charles River) were included in the study. Animals were fed a high-fat diet (HFD) (EF D12492, 60 kJ% fat (lard), ssniff Spezialdiaten GmbH-Germany) and 25 mg / kg of DEN toxin, or a control (CTL) diet (EF D12450B, 10 kJ% fat (lard / SBO), ssniff Spezialdiaten GmbH). All mice were weighed weekly throughout the study. Seven weeks after HFD / DEN induction, male and female mice were randomly divided into six experimental groups according to their sex: AAV-LUC (LUC), AAV-SIRT6wt (WT), and AAV-SIRT6cent (CEN), both of which were fed the CTL and HFD / DEN diets. Relative weight gain was calculated by comparing individual body weights on the day of AAV injection. The area under the curve was quantified.

[0206] After 9 weeks of AAV treatment, the mice were sacrificed, their organs were removed and weighed, and relative organ weights, normalized to their respective body weights, were calculated.

[0207] Hematological evaluation Hematological evaluation was performed on non-coagulated blood (containing EDTA) and measured immediately after blood collection / sampling from the axillary vessels during mouse sacrifice by anesthetic overdose (xylazine 20 mg / kg + ketamine 300 mg / kg) using a BC-2800Vet (Mindray, Shenzhen, PRC).

[0208] Protein expression levels of SIRT6 and β-catenin Snap-frozen liver tissue (up to 10 mg) was digested in ice-cold 1x RIPA buffer (supplemented with Halt™ protease and phosphatase inhibitor cocktail (100X), Thermofischer Scientific) and sonicated for 30 minutes with vortexing at 10-minute intervals. The tissue extract was then centrifuged at 20,000 g for 15 minutes at 4°C. The supernatant was transferred to a new tube, and protein concentration was measured using the Pierce BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific). For WB, 13 μg of total protein per sample was loaded onto a 10% unstained polyacrylamide SDS-PAGE gel (TGX™ FastCast™ Acrylamide Kit, Biorad) and run at 200 V for 35 minutes in a chilled water bath. Proteins were then electrotransferred to PVDF membranes using a Bio-Rad Trans-Blot Turbo RTA Mini 0.45 μm LF PVDF Transfer Kit (Bio-Rad) and a Bio-Rad Trans-Blot Turbo Transfer System at 1.3 A and 25 V for 7 minutes. The membranes were then blocked with 5% bovine serum albumin (BSA, P6154, BioWest) in TBST buffer (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20) for at least 30 minutes. The membranes were then incubated overnight at 4°C with primary antibody solutions: anti-vinculin (ab129002, rabbit mAb), anti-SIRT6 (ab191385, rabbit mAb EPR18463), and anti-β-catenin (8480S, rabbit mAb) (all at a dilution of 1:2000). The appropriate blots were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody diluted 1:2500 in TBST blocking buffer for at least 1 hour at room temperature. After three successive washes with TBST, protein levels were detected with Clarity™ Western ECL Substrate (1705060, Bio-Rad), and signals were detected with a Bio-Rad ChemiDoc XRS+ imaging system.For quantitative measurements, scanned membranes were analyzed using Image Lab™ software (Bio-Rad). SIRT6 and B-catenin expression / abundance were normalized to vinculin.

[0209] Biodistribution In vivo bioluminescence imaging (BLI) allows repeated assessment of reporter gene expression in tissues of live mice injected with an appropriate viral vector throughout the course of an experiment, without the need to sacrifice the animals. Highly sensitive reporter genes (e.g., Luc2) allow for the identification of the putative location of vector-infected tissues and the quantification of their expression levels. Therefore, we used the AAV8.CMV-Luc2 vector with an identical administration scheme to the AAV.CMV-SIRT6cent vector. Different administration schemes were used in groups E (2.5x10^12 vg / kg (medium dose), two injections), F (2.5x10^12 vg / kg (medium dose), two injections), and I (2.5x10^12 vg / kg (medium dose), two injections), with five mice per group. 12 vg / kg (medium dose, 1 injection), group G (1.0x10 13 vg / kg (high dose, 2 injections), group H (1.0x10 13 The virus was injected at a dose of 1000 mg / kg (high dose, single injection). LUC2 expression was monitored twice weekly in 20 mice using the IVIS Lumina II in vivo imaging system from day 7 to day 28 after viral vector injection.

[0210] Expression of the AAV8.CMV-SIRT6c vector in mouse tissues was analyzed by reverse transcription quantitative PCR (qPCR) 28 days after injection. Total RNA was isolated from the tissues and subjected to reverse transcription using oligo-dT(20) primers. The WPRE sequence was selected as the qPCR target sequence because it is present in the mature AAV vector-derived mRNA (both the SIRT6c and Luc2 vectors) and has no homologous sequence in the mouse genome or transcriptome. For absolute quantification, different dilutions of the SIRT6c-WPRE plasmid with known copy numbers were used as references, against which the Ct values ​​of the samples were converted to copy numbers per nanogram of total RNA.

[0211] Relative SIRT6 protein expression compared to β-tubulin was assessed by Western blot in tissue organs of mice 28 days after treatment with medium and high doses of AAV-SIRT6c or AAV-Luc.

[0212] result Mouse weight Although no differences were observed in female mice, body weight and relative weight gain were reduced in male mice fed the HFD / DEN diet and treated with AAV-SIRT6wt or AAV-SIRT6cent compared to mice receiving the same diet but treated with AAV-LUC instead (Figures 21A-F). This was also seen in quantification of the area under the curve for relative weight gain.

[0213] Organ weight No differences were observed in mice receiving the control diet and treated with AAV-LUC, AAV-SIRT6wt, or AAV-SIRT6cent (Figures 21G-21I). Increased lung and pancreas weights were observed in mice induced on the HFD / DEN diet and treated with AAV-SIRT6cent compared with mice receiving the same HFD / DEN diet but treated with AAV-SIRT6wt and AAV-LUC instead. More importantly, mice induced on the HFD / DEN diet and treated with AAV-SIRT6cent exhibited reduced liver weight compared with other treatment groups.

[0214] Hematological examination of males and females from the HFD / DEN model Within each blood parameter, males and females are shown for control versus HFD / DEN-induced mice (Figures 22A-22D). Treatment with AAV-SIRT6wt and AAV-SIRT6cent resulted in higher hemoglobin and red blood cell levels in HFD / DEN-induced male mice, while white blood cells (WBCs) and leukocytes were reduced in the same treatment groups.

[0215] Protein expression levels of SIRT6 and β-catenin The results are shown in Figures 23A to 23D.

[0216] Western blot analysis showed that SIRT6 levels were obviously increased for AAV-SIRT6wt and AAV-SIRT6cent in male and female mice at 9 weeks after AAV injection in the HFD-DEN treatment group.

[0217] Treatment of these mice with AAV-SIRT6wt and AAV-SIRT6cent reduced β-catenin levels in both males and females, and these observations were more pronounced in AAV-SIRT6cent-treated animals receiving the HFD / DEN diet.

[0218] Reporter (LUC) biodistribution BLI analysis showed that tail vein injection of the AAV Luc2 vector was 100% successful, with all 20 mice producing a BLI signal (Figures 24A-B). Furthermore, the highest levels of signal were recorded between 10 and 16 days postinjection, both in the anatomical region of the liver and throughout the body, consistent with the vector dose. After its peak, the overall BLI signal gradually decreased until the end of the 28-day period, although levels remained high in mice receiving higher vector doses.

[0219] Biodistribution of SIRT6c Twenty-eight days after two injections of high-dose AAV-SIRT6c, SIRT6c mRNA expression was observed primarily in the liver, with lower levels in the spleen and heart (Fig. 24C). However, at the medium dose, lower SIRT6c expression was observed in the liver, while similar expression was detected in the spleen (upper panel). Little expression was observed in the heart.

[0220] Twenty-eight days after a single injection of high-dose AAV-SIRT6c, SIRT6c mRNA expression was observed primarily in the liver and, at lower levels, in the heart of treated mice (Figure 24C). After a single injection of medium-dose AAV-SIRT6c, SIRT6c expression was observed primarily in the liver and, at lower levels compared to the high dose (lower panel).

[0221] Overexpression of SIRT6c protein was prominent in the livers of high- and medium-dose AAV-SIRT6c-treated mice at 28 days post-treatment compared with control mice treated with AAV-LUC, as shown by Western blot, with only minor levels in the spleens (Figure 24D).

[0222] array SEQ ID NO: 1 - Amino acid sequence of wild-type SIRT6 MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRAC RGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRINGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS SEQ ID NO:2 - Amino acid sequence of SIRT6N308K variant MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRAC RGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRIKGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS SEQ ID NO:3 - Amino acid sequence of SIRT6A313S variant MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVV FHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGELRD TILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRINGSIPSGPKQ25 EPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS SEQ ID NO: 4 - Amino acid sequence of SIRT6N308K / A313S variant MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRAC RGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRIKGSIPSGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS SEQ ID NO:5 - SIRT6 nucleic acid sequence SEQ ID NO:6 - Nucleic acid sequence of SIRT6 N308K variant SEQ ID NO:7 - Nucleic acid sequence of SIRT6A313S variant SEQ ID NO:8 - Nucleic acid sequence of SIRT6N308K / A313S variant SEQ ID NO: 21 - Nucleic acid sequence of wild-type SIRT6 < / cbrd>

Claims

1. An isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity to the sequence of SEQ ID NO: 1 for use in the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD), wherein the variant comprises or has at least one mutation selected from the group consisting of the substitution N308K and the substitution A313S relative to the sequence of SEQ ID NO:

1.

2. 2. The nucleic acid molecule of claim 1, which has a sequence selected from the group consisting of or including SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8.

3. 10. An isolated polypeptide encoded by the nucleic acid molecule of claim 1 for use in the prevention and / or treatment of NAFLD.

4. 4. The isolated polypeptide of claim 3, which is of a sequence selected from the group comprising or consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:

4.

5. A vector comprising the isolated nucleic acid molecule of claim 1 for use in the prevention and / or treatment of NAFLD.

6. The vector according to claim 5, which is a viral vector, in particular an adeno-associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an exosome, an adenoviral vector, a retroviral vector, or a herpesviral vector.

7. A suspension comprising the vector according to claim 5 for use in the prevention and / or treatment of NAFLD.

8. A cell expressing a polypeptide for use according to claim 3, preferably transfected with an isolated nucleic acid molecule for use according to claim 1 or a vector for use according to claim 5, for use in the prevention and / or treatment of NAFLD.

9. A pharmaceutical composition comprising (i) an isolated nucleic acid molecule described in claim 1, or an isolated polypeptide described in claim 3, or a vector described in claim 5, and (ii) a pharmaceutically acceptable excipient, for use in the prevention and / or treatment of NAFLD.

10. An isolated acidic nucleic acid molecule for use according to claim 1, an isolated polypeptide for use according to claim 3, a vector for use according to claim 5, a suspension for use according to claim 7, a cell for use according to claim 8 or a pharmaceutical composition for use according to claim 9 for the prevention and / or treatment of stage 1 or stage 2 NAFLD.

11. 11. The isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition of claim 10 for the prevention and / or treatment of stage 1 NAFLD.

12. 11. The isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition of claim 10 for the prevention and / or treatment of stage 2 NAFLD.

13. 13. The isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition of claim 12, wherein NASH is at stage 1.

14. 13. The isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition of claim 12, wherein NASH is at stage 2.

15. 13. The isolated acidic nucleic acid molecule, isolated polypeptide, vector, suspension, cell or pharmaceutical composition of claim 12, wherein NASH is at stage 3.

16. A method for preventing and / or treating non-alcoholic fatty liver disease (NAFLD), comprising administering to a patient in need thereof a therapeutically effective amount of an isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity to SEQ ID NO: 1, wherein the variant comprises or has at least one mutation selected from the group consisting of substitutions N308K and A313S relative to SEQ ID NO: 1, or an isolated polypeptide encoded thereby, or a pharmaceutical composition comprising the same.

17. 17. The method of claim 16, wherein the disease is stage 1 or 2 NAFLD.

18. 17. The method of claim 16, wherein the isolated nucleic acid molecule is comprised in a vector, preferably a viral vector, more preferably an adeno-associated viral vector (AAV), an exosome-associated AAV vector (exo-AAV), an exosome, an adenoviral vector, a retroviral vector, or a herpes viral vector.

19. 17. The method of claim 16, further comprising administering to the patient another therapeutic agent.

20. Use of an isolated nucleic acid molecule encoding a variant of Sirtuin 6 (SIRT6) having at least 75% identity to the sequence of SEQ ID NO: 1 for the manufacture of a pharmaceutical composition for the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD), wherein the variant has at least one mutation including or selected from the group consisting of substitutions N308K and A313S with respect to the sequence of SEQ ID NO:

1.

21. The use according to claim 20, wherein NAFLD is in stage 1 or 2.

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