Use of polynucleotide sequence

Abstract

The present invention relates to use of a polynucleotide. The in vitro administration of the polynucleotide sequence set forth in SEQ ID NO: 5 to somatic cells of naturally aged subjects can significantly reduce the expression of aging markers, DNA damage markers, and presenilin in the somatic cells, demonstrating potential efficacy in preventing or delaying aging.

Description

Uses of polynucleotide sequences

[0001] Cross-reference declaration

[0002] This application claims priority to Chinese patent application No. 202411822868X, filed on December 10, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedicine, and particularly to the use of polynucleotides. Background Technology

[0004] Lamin A protein is part of the scaffold structure of the cell nucleus and plays an important role in the structure and function of the nucleus. Previous literature has reported that mutations in Lamin A can disrupt peripheral chromatin with specific epigenetic and molecular characteristics, leading to the misexpression of genes normally expressed in other cell types, resulting in congenital diseases such as progeria, familial dilated cardiomyopathy, muscular dystrophy, and lipodystrophy syndrome.

[0005] Hutchinson-Gilford Syndrome (HGPS), also known as childhood progeria, is a rare and fatal genetic disorder. It is estimated that one in every 4 to 8 million newborns worldwide has HGPS. HGPS is characterized by a 5 to 10 times faster aging process than normal, resulting in an elderly appearance and rapid organ decline, leading to a decrease in physiological function. Symptoms include short stature, hair loss, and delayed tooth eruption. Children with HGPS typically live only to between 7 and 20 years of age, with the majority dying from age-related diseases such as cardiovascular disease.

[0006] Existing research has documented a possible cause of progeria, which is a 150-nucleotide deletion in exon 11, containing the zinc metalloproteinase 24 (ZMPSTE24) cleavage site, due to a mutation in the LMNA gene encoding lamin A. This prevents the normal cleavage of the farnesylated C-terminus of Lamin A, resulting in the production of permanently farnesylated progerin, a protein that causes the gradual degeneration of cellular structure and function in children with progeria. Similarly, mutations in the ZMPSTE24 gene can also lead to the formation of permanently farnesylated Lamin A protein, triggering similar aging symptoms.

[0007] Currently, only one drug, Lonafarnib (trade name: Zokinvy), has been approved globally for the treatment of progeria. Lonafarnib is an oral farnesyltransferase inhibitor that participates in the isoprenelation process of proteins. By inhibiting the isoprenelation of progerin, Zokinvy can reduce the accumulation of progerin in the cell nucleus. However, clinical studies have shown that this drug does not enable patients to achieve a normal lifespan and has serious safety concerns. Therefore, there is still a need in the field to develop safer and more effective drugs for the treatment of progeria.

[0008] Natural aging is a spontaneous and inevitable process that occurs naturally in organisms over time. It is a complex natural phenomenon characterized by structural degeneration, functional decline, and reduced adaptability and resistance. In the current global wave of aging, extending lifespan, delaying aging, and improving healthspan are urgent issues that need to be addressed. In recent years, due to advances in research on the mechanisms of aging, gene-editing-based drugs to combat natural aging have attracted attention. Lamin A is one of the important molecular mechanisms driving natural aging in the cell nucleus. During natural aging, the expression and function of Lamin A gradually become disordered, including its aberrant splicing to produce trace amounts of progerin. These changes disrupt nuclear membrane integrity, leading to genomic instability and heterochromatin loss, thereby inducing cellular senescence. However, there are currently no effective gene therapy drugs on the market to delay aging; therefore, there is an urgent need in this field to develop gene therapy-based drugs to delay aging. Summary of the Invention

[0009] The purpose of this invention is to provide a polynucleotide.

[0010] Another object of the present invention is a pharmaceutical composition.

[0011] Another object of the present invention is the use of the above-mentioned polynucleotide or pharmaceutical composition.

[0012] Another object of the present invention is to provide a method for preventing and / or treating diseases related to laminin A mutations.

[0013] Another object of the present invention is to provide the use of a polynucleotide in the preparation of a drug for delaying aging.

[0014] To address the aforementioned technical problems, the first aspect of the present invention provides an isolated polynucleotide, wherein the isolated polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof:

[0015] (i) the sequence shown in SEQ ID NO:11; and / or

[0016] (ii) A polynucleotide sequence formed by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO:11.

[0017] In some preferred embodiments, the isolated polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof:

[0018] (i) the sequence shown in SEQ ID NO:1; and / or

[0019] (ii) A polynucleotide sequence formed by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO:1.

[0020] In some preferred embodiments, the isolated polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof:

[0021] (i) the sequence shown in SEQ ID NO:10; and / or

[0022] (ii) A polynucleotide sequence formed by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO:10.

[0023] In some preferred embodiments, the isolated polynucleotide comprises at least one polynucleotide fragment selected from the group consisting of:

[0024] (i) polynucleotide fragments as shown in SEQ ID NO:2-9; and

[0025] (ii) A polynucleotide fragment with at least 80% homology to SEQ ID NO:2-9.

[0026] In some preferred embodiments, the isolated polynucleotide is selected from at least one of the following groups:

[0027] (i) polynucleotides as shown in SEQ ID NO:2-9; and

[0028] (ii) Polynucleotides with at least 80% homology to SEQ ID NO:2-9.

[0029] In some preferred embodiments, the isolated polynucleotide is a polynucleotide as shown in SEQ ID NO:5 or SEQ ID NO:7.

[0030] In some preferred embodiments, the length of the isolated polynucleotide is not less than 10 bp, more preferably not less than 15 bp, more preferably 18-22 bp, for example 20 bp.

[0031] In some preferred embodiments, the length of the isolated polynucleotide is no more than 100 bp.

[0032] A second aspect of the present invention provides a small nucleic acid molecule containing the polynucleotide described in the first aspect of the present invention, wherein the small nucleic acid molecule is siRNA, ASO, shRNA or miRNA.

[0033] A third aspect of the present invention provides a pharmaceutical composition comprising the polynucleotide described in the first aspect of the present invention or the small nucleic acid molecule described in the second aspect of the present invention, and a pharmaceutically acceptable carrier.

[0034] A fourth aspect of the present invention provides the use of the polynucleotide described in the first aspect of the present invention, or the small nucleic acid molecule described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention, for:

[0035] (i) Prevention and / or treatment of diseases associated with laminin A mutations;

[0036] (ii) To prepare drugs for the prevention and / or treatment of diseases related to laminin A mutations;

[0037] (iii) Reduce the expression level of lamin A mutant (preferably reduce the expression level of senescence protein);

[0038] (iv) Reduce the number of senescent positive cells; and / or

[0039] (v) Reduce the number of abnormal nuclei.

[0040] In some preferred embodiments, the lamin A mutation is a mutation of lamin AmRNA in exons 11 and / or 12.

[0041] In some preferred embodiments, the lamin A mutation-related disease is a disease caused by mutations in exons 11 and / or 12 of lamin AmRNA.

[0042] In some preferred embodiments, the lamin A mutation-related disease is a disease caused by mutations in lamin A RNA at one or more of the following locations: c.1711; c.1713; c.1714; c.1718; c.1733; c.1744; c.1745; c.1748; c.1751; c.1756; c.1762; c.1772; c.1774; c.178 6;c.1804;c.1821;c.1822;c.1824;c.1851;c.1868;c.1871;c.1892;c.1904;c.1916;c.1928 ; c.1930; c.1931; c.1940; c.1960; c.1961; c.1968; c.1968+1; c.1968+2; c.1968+5; and c.1975.

[0043] In some preferred embodiments, the lamin A mutation-related disease is a disease caused by one or more mutations in lamin Am RNA selected from the following: c.1711A>T; c.1713C>A; c.1714A>T; c.1714insCTGC; c.1718C>T; c.1733A>T; c.1744C>T; c.1745G>A; c.1748C>T; c.1751G>A; c.1756G>A; c.1762T>C; c.1772G>T; c.1774G>A; c.1786G>A; c.1804G>A ;c.1821G>A; c.1822G>A; c.1824C>T; c.1851C>T; c.1868C>G; c.1871G>A; c.1892G>A; c.1904G>A; c.1916A>G; c.1928C>A; c.1930C>T ; c.1931G>A; c.1940T>G; c.1960C>T; c.1961dup; c.1968G>A; c.1968+1G>A; c.1968+2T>C; c.1968+5G>A; c.1968+5G>C; and c.1975dup.

[0044] In some preferred embodiments, the lamin A mutation-related disease is selected from at least one of dilated cardiomyopathy and conduction disorders (DCM-CD), muscular dystrophy, peroneal muscular dystrophy type 2 (CMT2), familial partial lipopathy (FPLD), astriated muscle laminopathy, progeroid syndrome, edema-derived muscular dystrophy (EDMD), insulin resistance syndrome (IRS), progeria, limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (L-CMD), and Werner's syndrome, preferably progeria.

[0045] A fifth aspect of the present invention provides a method for preventing and / or treating diseases associated with lamin A mutations, the method comprising the steps of:

[0046] The subject is given a therapeutically effective amount of the polynucleotide described in the first aspect of the present invention, or the small nucleic acid molecule described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention.

[0047] A sixth aspect of the present invention provides a method for (i) reducing the expression level of laminin A mutant, or (ii) reducing the number of senescent positive cells, or (iii) reducing the number of abnormal nuclei in vitro without therapeutic intervention, the method comprising the steps of:

[0048] In vitro, non-therapeutic contact is made between biological tissues and the polynucleotides described in the first aspect of the invention.

[0049] In some preferred embodiments, the biological tissue is a population of cardiomyocytes from a progeria patient.

[0050] A seventh aspect of the present invention provides the use of a polynucleotide sequence, as shown in SEQ ID NO:5 (ASO_U7), for:

[0051] (a) Slows down natural aging;

[0052] (b) To prepare drugs that delay natural aging;

[0053] (c) In vitro, non-therapeutic reduction of the expression levels of somatic senescence markers;

[0054] (d) In vitro, non-therapeutic reduction of expression levels of somatic DNA damage markers; and

[0055] (e) Non-therapeutic reduction of somatic senescence protein expression levels in vitro.

[0056] In some preferred embodiments, the somatic cells are cardiomyocytes.

[0057] In some preferred embodiments, the aging biomarker is SA-β-gal.

[0058] In some preferred embodiments, the DNA damage marker is γH2AX.

[0059] In some preferred embodiments, the somatic cells are derived from naturally aging subjects.

[0060] In some preferred embodiments, the subject is a person who is at least 70 years old (preferably at least 80 years old, more preferably at least 85 years old, and even more preferably at least 90 years old).

[0061] In some preferred embodiments, the somatic cells are obtained by reprogramming PBMCs into iPSCs and then differentiating them.

[0062] The eighth aspect of the present invention provides a method for delaying natural aging, characterized in that the method includes the step of administering to a subject a therapeutically effective amount of a polynucleotide sequence as shown in SEQ ID NO:5 or a combination of drugs containing thereas.

[0063] In some preferred embodiments, the administration of the polynucleotide sequence as shown in SEQ ID NO:5, or a combination of drugs containing it, is by injection, preferably subcutaneous injection.

[0064] Compared with the prior art, the present invention has at least the following advantages:

[0065] This invention introduces a variety of novel polynucleotide sequences targeting the 3' untranslated region of lamin A mutant mRNA, which can reduce the expression level of lamin A mutants (such as senescence protein) at the Lamin AmRNA stage without affecting the normal transcription of Lamin C. Therefore, it has lower toxicity and shows great promise for the prevention or treatment of diseases related to lamin A mutations (such as natural aging).

[0066] In this invention, the polynucleotide sequence shown in SEQ ID NO:5, when applied in vitro to the somatic cells of naturally aging subjects, can significantly reduce the expression of aging markers, DNA damage markers, and progeria proteins in somatic cells.

[0067] The polynucleotide sequence shown in SEQ ID NO:5 in this invention has the potential therapeutic effect of preventing or delaying natural aging.

[0068] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0069] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0070] Figure 1 is a schematic diagram of the genotype identification results of HGPS patients according to an embodiment of the present invention;

[0071] Figure 2 is a schematic diagram of qRT-PCR detection of progerin expression level in fibroblasts of children according to an embodiment of the present invention;

[0072] Figure 3 is a diagram of the PBMC reprogramming mode according to an embodiment of the present invention;

[0073] Figure 4 is a schematic diagram of IFA detection of iPSC cell phenotype according to an embodiment of the present invention. ns indicates P>0.05; * indicates P<0.05; *** indicates P<0.001.

[0074] Figure 5 is a schematic diagram of the cardiomyocyte differentiation process according to an embodiment of the present invention;

[0075] Figure 6 is a graph showing the mRNA expression levels of genes related to different stages of myocardial differentiation detected by qRT-PCR according to an embodiment of the present invention.

[0076] Figure 7 is a graph showing the results of IFA detection of cTnT (observation of myocardial fiber length) on day 11 of myocardial differentiation according to an embodiment of the present invention;

[0077] Figure 8 is a graph showing the results of IFA detection of Lamin A / C (observed karyotype) on day 11 of myocardial differentiation according to an embodiment of the present invention;

[0078] Figure 9 is a schematic diagram of the cardiomyocyte passage process according to an embodiment of the present invention;

[0079] Figure 10 is a microscopic bright-field observation of the morphology of cardiomyocytes of different generations according to an embodiment of the present invention;

[0080] Figure 11 is a SA-β-Gal senescence staining map of cardiomyocytes of different passages according to an embodiment of the present invention. The percentage values ​​represent the positive rate of senescent staining cells, and P (Passage) represents the number of passages.

[0081] Figure 12 is a schematic diagram of IFA detection of different batches of myocardial passage cTnT (observation of myocardial fiber length) in an embodiment of the present invention, where P (Passage) represents the number of passages;

[0082] Figure 13 is a schematic diagram of IFA detection of different batches of Lamin A / C (observed karyotype) in myocardial passage according to an embodiment of the present invention, where P (Passage) represents the number of passages;

[0083] Figure 14 is a schematic diagram of a targeted progerin gamper form ASO design according to an embodiment of the present invention;

[0084] Figure 15 is a graph showing the effect of qRT-PCR detection of ASO_U5-U8 transfection on progerin mRNA expression in c.1822G>A cardiomyocytes according to an embodiment of the present invention. *** indicates P<0.001.

[0085] Figure 16 is a graph showing the effect of qRT-PCR detection of ASO_U5-U8 transfection on progerin mRNA expression in c.1824C>T cardiomyocytes according to an embodiment of the present invention. *** indicates P<0.001.

[0086] Figure 17 is a graph showing the effect of qRT-PCR detection of the dosage of ASO_U7 and U9 transfected on the expression of progerin mRNA in c.1822G>A cardiomyocytes according to an embodiment of the present invention. *** indicates P<0.001;

[0087] Figure 18 is a graph showing the effect of qRT-PCR detection of the dosage of ASO_U7 and U9 transfected on the expression of progerin mRNA in c.1824C>T cardiomyocytes according to an embodiment of the present invention. *** indicates P<0.001;

[0088] Figure 19 is a Western blot diagram showing the effect of the dosage of ASO_U7 and U9 transfection on the expression of progerin protein in c.1822G>A cardiomyocytes according to an embodiment of the present invention.

[0089] Figure 20 is a Western blot diagram showing the effect of ASO_U7 and U9 transfection doses on progerin protein expression in c.1824C>T cardiomyocytes according to an embodiment of the present invention.

[0090] Figure 21 illustrates the effects of IFA detection of ASO U7 and U9 on classic c.1824C>T and non-classical c.1822G>A myocardial fibers and karyotypes according to an embodiment of the present invention (MNA / C staining method);

[0091] Figure 22 illustrates the effects of IFA detection of ASO U7 and U9 on classic c.1824C>T and non-classical c.1822G>A myocardial fibers and karyotypes according to an embodiment of the present invention (cTnT staining method);

[0092] Figure 23 is a diagram of SA-β-Gal cell senescence staining according to an embodiment of the present invention;

[0093] Figure 24 is a schematic diagram of the proportion of abnormal nuclei in cardiomyocytes according to an embodiment of the present invention, ***, indicating P<0.001;

[0094] Figure 25 shows the statistical proportion of SA-β-Gal positive cells according to an embodiment of the present invention; *** indicates P<0.001.

[0095] Figure 26 is a graph showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of progerin mRNA in the heart of G608G premature aging mice by qRT-PCR according to an embodiment of the present invention. *** indicates P<0.001.

[0096] Figure 27 is a graph showing the effect of qRT-PCR on the expression of progerin mRNA in the liver of G608G premature aging mice by subcutaneous injection of ASO_U7 and U9 according to an embodiment of the present invention. *** indicates P<0.001.

[0097] Figure 28 is a Western blot diagram showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of progerin protein in the heart of G608G premature aging mice according to an embodiment of the present invention.

[0098] Figure 29 is a Western blot diagram showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of progerin protein in the liver of G608G premature aging mice according to an embodiment of the present invention.

[0099] Figure 30. Effects of subcutaneous injection of ASO_U7 and U9 on the expression of alanine aminotransferase (ALT) in the liver of G608G premature aging mice as detected by ELISA.

[0100] Figure 31. Effect of subcutaneous injection of ASO_U7 and U9 on the expression of aspartate aminotransferase (AST) in the liver of G608G premature aging mice as detected by ELISA.

[0101] Figure 32. Effects of subcutaneous injection of ASO_U7 and U9 on renal uric acid (UA) expression in G608G premature aging mice as detected by ELISA.

[0102] Figure 33. Effects of subcutaneous injection of ASO_U7 and U9 on renal creatinine (Crea) expression in G608G premature aging mice as detected by ELISA.

[0103] Figure 34. Effects of subcutaneous injection of ASO_U7 and U9 on renal urea nitrogen (Urea) expression in G608G premature aging mice as detected by ELISA.

[0104] Figure 35. Schematic diagram of the in vitro reprogramming of PBMCs into iPSCs;

[0105] Figure 36 Schematic diagram of iPSCs observed under a microscope, with iPSCs of a 30-year-old on the left and iPSCs of a 90-year-old on the right;

[0106] Figure 37 Schematic diagram of iPSCs differentiating into cardiomyocytes under a microscope. The left is cardiomyocytes differentiated from iPSCs in a 30-year-old, and the right is cardiomyocytes differentiated from iPSCs in a 90-year-old.

[0107] Figure 38 Schematic diagram of iPSCs differentiating into cardiomyocytes with SA-β-gal staining;

[0108] Figure 39 Schematic diagram of γH2AX immunofluorescence staining of iPSCs differentiating into cardiomyocytes;

[0109] Figure 40 Schematic diagram of progerin immunofluorescence staining of iPSCs differentiating into cardiomyocytes;

[0110] Figure 41 Schematic diagram of SA-β-gal staining after ASO_U7 treatment of cardiomyocytes differentiated from iPSCs in a 90-year-old patient;

[0111] Figure 42 Schematic diagram of γH2AX immunofluorescence staining after ASO_U7 treatment of iPSCs differentiated into cardiomyocytes in a 90-year-old patient;

[0112] Figure 43 Schematic diagram of progerin immunofluorescence staining after ASO_U7 treatment of iPSCs differentiated from cardiomyocytes in a 90-year-old patient. Detailed Implementation

[0113] Mutations in the LMNA gene can lead to abnormal splicing of pre-Lamin AmRNA, resulting in the accumulation of lamin A mutants (such as progerin or other Lamin A mutants), causing congenital diseases such as progeria, familial dilated cardiomyopathy, muscular dystrophy, and lipodystrophy syndrome. In our research, we discovered that reducing Lamin AmRNA levels while preserving Lamin C mRNA levels in mice resulted in normal indicators and no abnormal symptoms. Therefore, Lamin C can partially compensate for the function of Lamin A in cells. This is particularly true in tissues where normal Lamin A expression is relatively low, such as brain tissue, where Lamin C is the primary form. Therefore, reducing the expression of Lamin A mutants does not affect Lamin C levels or the normal function of brain tissue, while simultaneously improving disease symptoms and progression.

[0114] Based on the above findings, the inventors, through extensive and in-depth research, designed various antisense oligonucleotide sequences targeting the 3' untranslated region of lamin A mutant mRNA (e.g., presenilin mRNA). These sequences can reduce the expression of lamin A mutants at the Lamin AmRNA stage, and therefore can be used to prevent or treat various diseases directly or indirectly caused by lamin A mutations, such as dilated cardiomyopathy and conduction disorders, muscular dystrophy, peroneal muscular atrophy type 2, familial lipodystrophy, rhabdomyosarcoma, progeria-like syndrome, d'Arcy-Dereyfus muscular dystrophy, insulin resistance syndrome, progeria, limb-girdle muscular dystrophy, congenital muscular dystrophy, and Werner syndrome, while not affecting Lamin C levels, thus improving safety. In a preferred embodiment of the invention, the newly designed antisense oligonucleotide sequence can reduce the expression of presenilin, significantly inhibiting the occurrence of progeria, while not affecting Lamin C levels or normal brain function. Compared to previous treatments, such as Zokinvy and other methods for reducing Lamin A mutants, it has a higher safety profile and shows great promise as a drug for the prevention or treatment of progeria.

[0115] In addition, the inventors have discovered that the newly designed antisense oligonucleotide sequence can effectively delay natural aging, and multiple aging indicators show significant differences compared with the control group, such as reduced SA-β-gal and γH2AX.

[0116] Polynucleotides

[0117] This invention relates to polynucleotides. The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The polynucleotides of this invention are designed to include a polynucleotide fragment complementary to a target gene or a segment thereof. As used herein, the term "a polynucleotide / gene including a fragment of gene X" means that the overall sequence of the polynucleotide / gene contains, in addition to the sequence of gene X, other polynucleotide fragments / gene fragments / bases preceding and / or following gene X. For example, the polynucleotide sequence ATCG-X-CCTC includes gene X as a fragment, and the sequence also includes other fragments such as ATCG and CCTC.

[0118] As used herein, the terms “polynucleotide fragment” or “fragment” or “gene fragment” refer to a portion of the sequence of the polynucleotide / gene. That is, the fragment sequence of the polynucleotide should be the same as the original polynucleotide portion. For example, if the original polynucleotide sequence is AAAGGGTTT, the polynucleotide sequence can be any of the following: AAAGGG, AAAG, AGGGTTT, etc.

[0119] As used herein, the term "gene within XX" refers to a gene sequence that is a gene sequence of region XXX or a partial fragment thereof. In one embodiment of the invention, the target gene is located within the 3' untranslated region (3'UTR) of lamin AmRNA or a mutant lamin mRNA (e.g., early aging protein mRNA), i.e., the target gene is the 3' untranslated region (3'UTR) of lamin AmRNA or a partial fragment thereof, or the target gene is the 3' untranslated region (3'UTR) of a mutant lamin mRNA or a partial fragment thereof. In one embodiment of the invention, the target gene is (i) the sequence shown in SEQ ID NO:11; and / or (ii) a polynucleotide sequence formed by substituting, deleting, or adding one or more nucleotides to the sequence shown in SEQ ID NO:11. In one embodiment of the invention, the target gene is located within the sequence shown in SEQ ID NO:11.

[0120] As used herein, the term "mutation" refers to the substitution, deletion, and / or addition of one or more amino acids in an amino acid sequence, or the substitution, deletion, and / or addition of one or more polynucleotides in a polynucleotide sequence. As used herein, the term "mutant" refers to the amino acid sequence obtained by mutating the amino acid sequence of a protein at any position, or the amino acid sequence obtained by translating the mRNA of a mutated protein. In this invention, "lamin A mutant" refers to the amino acid sequence obtained by substituting, deleting, and / or adding one or more amino acids at any position in the amino acid sequence of lamin, or the amino acid sequence obtained by translating the mutated lamin AmRNA. In a preferred embodiment of this invention, the "lamin A mutant" is the amino acid sequence obtained by translating the sequence obtained by mutating the lamin AmRNA in exons 11 and / or 12. In a preferred embodiment of the present invention, the "lamin A mutant" is the amino acid sequence obtained by translating a sequence resulting from a mutation at one or more of the following positions in lamin A RNA: c.1711; c.1713; c.1714; c.1718; c.1733; c.1744; c.1745; c.1748; c.1751; c.1756; c.1762; c.1772; c.1774; c. .1786;c.1804;c.1821;c.1822;c.1824;c.1851;c.1868;c.1871;c.1892;c.1904;c.1916;c.19 28; c.1930; c.1931; c.1940; c.1960; c.1961; c.1968; c.1968+1; c.1968+2; c.1968+5; and c.1975. In a preferred embodiment of the present invention, the “lamin A mutant” is the amino acid sequence obtained by translating a sequence resulting from a mutation in lamin AmRNA at one or more of the following positions: c1821; c1822; c1824; c1968; c1968+1; c1968+1G>A; c1968+2; C1968+2; c1968+5; and C1968+5. Mutations at one or more of these positions will lead to diseases associated with progeria proteins.

[0121] As used in this invention, the terms "progerin", "premature aging protein" and "progerin" are used interchangeably.

[0122] In a preferred embodiment of the present invention, the "lamin A mutant" is the amino acid sequence obtained by translating a sequence resulting from one or more mutations in lamin AmRNA selected from the following: c.1711A>T; c.1713C>A; c.1714A>T; c.1714insCTGC; c.1718C>T; c.1733A>T; c.1744C>T; c.1745G>A; c.1748C>T; c.1751G>A; c.1756G>A; c.1762T>C; c.1772G>T; c.1774G>A; c.1786G>A; c.180 4G>A; c.1821G>A; c.1822G>A; c.1824C>T; c.1851C>T; c.1868C>G; c.1871G>A; c.1892G>A; c.1904G>A; c.1916A>G; c.1928C>A; c.1930C >T; c.1931G>A; c.1940T>G; c.1960C>T; c.1961dup; c.1968G>A; c.1968+1G>A; c.1968+2T>C; c.1968+5G>A; c.1968+5G>C; and c.1975dup. In a preferred embodiment of the present invention, the "lamin A mutant" is an amino acid sequence obtained by translating a sequence resulting from one or more mutations in the lamin AmRNA selected from the following: c1821G>A; c1822G>A; c1824C>T; c1968G>A; c1968+1G>C; c1968+1G>A; c1968+2T>C; C1968+2T>A; c1968+5G>A; and C1968+5G>C.

[0123] In a preferred embodiment of the present invention, the target gene is (i) the sequence shown in SEQ ID NO:1; and / or (ii) a polynucleotide sequence formed by substituting, deleting or adding one or more nucleotides to the sequence shown in SEQ ID NO:1.

[0124] In a more preferred embodiment of the present invention, the target gene is (i) the sequence shown in SEQ ID NO:10; and / or (ii) a polynucleotide sequence formed by substituting, deleting, or adding one or more nucleotides to the sequence shown in SEQ ID NO:10. The polynucleotide designed to target the sequence shown in SEQ ID NO:10 more significantly reduces the expression levels of lamin A mutant mRNA and protein (preferably senescent protein).

[0125] As used herein, the terms "sequence complement" and "reverse sequence complement" are used interchangeably and refer to a sequence that is in the opposite direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAGT. In one embodiment of the invention, the polynucleotide sequence is reverse complementary to the sequence shown in SEQ ID NO:1. In one embodiment of the invention, the polynucleotide sequence is reverse complementary to the sequence shown in the fragment of SEQ ID NO:1. In one embodiment of the invention, the polynucleotide sequence is reverse complementary to a polynucleotide sequence formed by substitution, deletion, or addition of one or more nucleotides of the sequence shown in SEQ ID NO:1. In one embodiment of the invention, the polynucleotide sequence is reverse complementary to a fragment of a polynucleotide sequence formed by substitution, deletion, or addition of one or more nucleotides of the sequence shown in SEQ ID NO:1. In one embodiment of the invention, the polynucleotide sequence is reverse complementary to the sequence shown in SEQ ID NO:10. In one embodiment of the invention, the polynucleotide sequence is reverse complementary to the sequence shown in the fragment of SEQ ID NO:10. In one embodiment of the present invention, the polynucleotide sequence is anticomplementary to a polynucleotide sequence formed by substitution, deletion, or addition of one or more nucleotides of the sequence shown in SEQ ID NO:10. In another embodiment of the present invention, the polynucleotide sequence is anticomplementary to a fragment of a polynucleotide sequence formed by substitution, deletion, or addition of one or more nucleotides of the sequence shown in SEQ ID NO:10.

[0126] In a preferred embodiment of the invention, the polynucleotide includes a polynucleotide fragment selected from at least one of the following groups: (i) a polynucleotide fragment as shown in SEQ ID NO:2-9; and (ii) a polynucleotide fragment having at least 80% homology to SEQ ID NO:2-9 (preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%).

[0127] In another preferred embodiment of the invention, the polynucleotide is selected from at least one of the following groups: (i) polynucleotides as shown in SEQ ID NO:2-9; and (ii) polynucleotides with at least 80% homology to SEQ ID NO:2-9 (preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%).

[0128] In another preferred embodiment of the invention, the polynucleotide is selected from at least one of the following: (i) polynucleotides as shown in SEQ ID NO:3-7; and (ii) polynucleotides with at least 80% homology to SEQ ID NO:3-7 (preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%). These polynucleotide sequences are more efficient at knocking down lamin A mutant (preferably early aging protein) mRNA and lamin A mutants compared to other polynucleotide sequences.

[0129] In another preferred embodiment of the invention, the polynucleotide is as shown in SEQ ID NO:5; or as shown in SEQ ID NO:7. The polynucleotides shown in SEQ ID NO:5 and 7 have good specificity and low toxicity, reducing the expression levels of aging protein mRNA and protein in cardiomyocytes by at least 50% (more preferably 70%, more preferably 80%), and do not target other genomes, making them suitable for subsequent human trials.

[0130] In this invention, polynucleotides are preferably provided in isolated form, and more preferably purified to homogenization. As used herein, the term "isolated" refers to nucleic acids isolated from at least one other component (e.g., nucleic acids) present in their natural sources. In one embodiment, nucleic acids are found only in (if any) solvents, buffers, ions, or other components normally present in their solution. The terms "isolated" and "purified" do not include nucleic acids present in their natural sources.

[0131] As used herein, the terms “homology” and “identity” are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residues, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0132] In this invention, the aforementioned polynucleotides or fragments thereof can be prepared using methods conventional in the art, such as PCR amplification, recombinant synthesis, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, followed by splicing the amplified fragments together in the correct order. Once the relevant sequences are obtained, recombinant synthesis can be used to obtain them in large quantities. This typically involves cloning them into a vector, transforming them into cells, and then isolating the relevant sequences from the proliferated host cells using conventional methods. Artificial synthesis is particularly suitable for synthesizing short fragments. Generally, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0133] The length of the polynucleotide in this invention is preferably not less than 10 bp, more preferably not less than 15 bp, more preferably 18-22 bp, for example 20 bp, with the best effect achieved when the length is 20 bp.

[0134] Small nucleic acid molecules

[0135] The present invention also relates to small nucleic acid molecules containing the above-mentioned polynucleotides, wherein the small nucleic acid molecules are siRNA, ASO, shRNA or miRNA; preferably ASO.

[0136] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a polynucleotide or fragment thereof that is complementary to a specific target gene according to the principle of base complementarity, thereby inhibiting or blocking the expression of the specific target gene, which can be a DNA or RNA sequence. To enhance the stability, targeting ability, and metabolic capacity of antisense oligonucleotides, the antisense oligonucleotides described herein may also include chemical modifications, such as modification of the oxygen atom of the phosphate ester bond with a thiophosphate ester, modification of the 2′ position of the ribose with an alkyl group (e.g., methyl), or LNA modification. To promote ASO entry into cells or improve the organ or tissue targeting of ASO in vivo, the antisense oligonucleotides described herein may also include terminal Chol or GalNAc modifications. Optionally, the front and / or back ends of the polynucleotide or fragment thereof complementary to the target gene in the antisense oligonucleotides described herein may also include a short sequence of 1-5 (more preferably 3-5) bases of any type, and the bases in these short sequences are preferably chemically modified. Those skilled in the art can obtain the antisense oligonucleotides of this invention using conventional methods, such as chemical synthesis.

[0137] As used herein, the term "siRNA" (Small interfering RNA) refers to a small RNA molecule (approximately 21-25 nucleotides) that can be processed from its precursors (such as dsRNA, shRNA, etc.) by Dicer (an enzyme in the RNase III family that is specific for double-stranded RNA), or synthesized chemically or produced by processing other proteins. siRNA is a major member of siRISC, stimulating the rapid cleavage and degradation of target RNA with its complementary sequence, leading to the silencing of the target gene, thus becoming a key functional molecule in RNAi. Given a specific gene target sequence, those skilled in the art can design and obtain siRNA targeting that target sequence using conventional methods.

[0138] As used in this article, the term "miRNA" (microRNA) refers to a class of non-coding single-stranded RNA molecules, approximately 20-24 nucleotides in length, encoded by endogenous genes, which participate in the regulation of expression of a large number of genes in plants and animals. To date, more than four thousand miRNA molecules have been discovered in plants, animals, and viruses. Most miRNA genes exist in the genome as single copies, multiple copies, or gene clusters. Each miRNA can regulate multiple target genes, and several miRNAs can also work together to regulate the same gene, forming a complex regulatory network. It is estimated that miRNAs regulate the expression of more than half of the genes in humans. miRNAs exist in various forms, the most primitive being pri-miRNA; after processing by Drosha, pri-miRNA becomes pre-miRNA, or miRNA precursor, approximately 50-90 nucleotides in length; pre-miRNA is then cleaved by the Dicer enzyme to become mature miRNA, approximately 20-24 nucleotides in length. miRNAs mainly inhibit target gene expression by suppressing translation and accelerating mRNA deadenylation, a mechanism different from siRNA-mediated mRNA degradation.

[0139] One way to generate small interfering RNA (siRNA) in vivo is to clone the siRNA sequence as part of a short hairpin into a plasmid vector. When introduced into an animal, the hairpin sequence is expressed, forming a double-stranded RNA (shRNA) with a terminal loop structure. This shRNA is then recognized and processed by the Dicer protein in the cell, producing a functional siRNA.

[0140] As used herein, a precursor is used to construct a specific shRNA with a backbone. The shRNA, from 5′ to 3′, comprises: (a) a 5′ flanking sequence region; (b) a 5′ paired siRNA region; (c) a apical loop region; (d) a 3′ paired siRNA region, wherein the 5′ paired siRNA region and the 3′ paired siRNA region form a double-stranded region; and (e) a 3′ flanking sequence region. The shRNA produces siRNA, and the nucleotide sequence of the siRNA corresponds to either the 3′ paired siRNA region or the 5′ paired siRNA region.

[0141] In a broad sense, shRNA is an abbreviation for short hairpin RNA. shRNA consists of two short, inversely complementary sequences separated by a terminal loop sequence, forming a hairpin structure. Transcription is typically controlled by the promoter of endogenous RNA polymerase III, with 5-6 T-termini attached to the end of the shRNA sequence as a transcription terminator for RNA polymerase III. shRNA can also be transcribed from the promoters of other RNA polymerases.

[0142] Pharmaceutical Composition

[0143] This invention also relates to pharmaceutical compositions containing the polynucleotides of this invention. The pharmaceutical compositions of this invention include the polynucleotides of this invention and pharmaceutically acceptable carriers. To better enable precise delivery of the polynucleotides of this invention to specific sites in a subject, or to improve the bioavailability of the polynucleotides of this invention relative to a subject, or to reduce the toxicity of the polynucleotides of this invention relative to a subject, pharmaceutically acceptable carriers may optionally be liposomes, lipid nanoparticles (LNPs), protamine, polymers (e.g., polyethyleneimine), inorganic nanoparticles, exosomes, polymer matrices, etc.

[0144] In some other preferred embodiments of the present invention, the pharmaceutical composition comprises: the polynucleotide of the present invention and a lipid nanoparticle shell encapsulating thereon. Preferably, the lipid nanoparticle shell is composed of at least one selected from PEG lipids, cholesterol, ionizable cationic lipids, and auxiliary lipids. To increase targeting, the lipid nanoparticle shell may be modified with a surface modifier.

[0145] In some other preferred embodiments of the present invention, the polynucleotide sequence is conjugated with a desialyl glycoprotein receptor ligand (GalNAc) to form GalNac-X, where X represents the polynucleotide of the present invention.

[0146] Uses or indications

[0147] The present invention also relates to the use of polynucleotides or pharmaceutical compositions for (i) prevention and / or treatment of diseases associated with laminin A mutations; (ii) preparation of medicaments for prevention and / or treatment of diseases associated with laminin A mutations; (iii) reduction of the expression level of laminin A mutants; (iv) reduction of the number of senescent positive cells; and / or (v) reduction of the number of abnormal nuclei.

[0148] In this invention, any mRNA sequence that can specifically bind to the polynucleotide sequence of this invention can be cleaved by ribonuclease H1, resulting in a decrease in the expression level of the protein translated from that mRNA sequence. Therefore, the polynucleotide sequence of this invention targets the 3'UTR region of lamin A mutant mRNA, causing the lamin A mutant mRNA to degrade and thereby reducing the expression level of lamin A mutant, thus enabling the treatment of diseases related to lamin A mutations.

[0149] As used herein, the term "lamin A mutation-related disease" can include any disease directly or indirectly caused by mutations in lamin A or lamin AmRNA. Preferably, a lamin A mutation is a mutation in lamin A mRNA at exons 11 and / or 12. Lamin A mutation-related disease is preferably caused by mutations in lamin AmRNA at exons 11 and / or 12. In a preferred embodiment of the invention, "lamin A mutation-related disease" refers to a disease caused by mutations in lamin A... Diseases caused by mutations in mRNA at one or more of the following locations: c.1711; c.1713; c.1714; c.1718; c.1733; c.1744; c.1745; c.1748; c.1751; c.1756; c.1762; c.1772; c.1774; c.1786; c.1804; c.1821; c.1822; c.1824; c.1851; c.1868; c.1871; c.1892; c.1904; c.1916; c.1928; c.1930; c.1931; c.1940; c.1960; c.1961; c.1968; c.1968+1; c.1968+2; c.1968+5; and c.1975. The mutation locations mentioned above can be found at http: / / www.umd.be / LMNA / . In a preferred embodiment of the present invention, "lamin A mutation-related disease" refers to a disease caused by one or more mutations in lamin A RNA selected from the following: c.1711A>T; c.1713C>A; c.1714A>T; c.1714insCTGC; c.1718C>T; c.1733A>T; c.1744C>T; c.1745G>A; c.1748C>T; c.1751G>A; c.1756G>A; c.1762T>C; c.1772G>T; c.1774G>A; c.1786G>A; c.1804G> A; c.1821G>A; c.1822G>A; c.1824C>T; c.1851C>T; c.1868C>G; c.1871G>A; c.1892G>A; c.1904G>A; c.1916A>G; c.1928C>A; c.1930C> T; c.1931G>A; c.1940T>G; c.1960C>T; c.1961dup; c.1968G>A; c.1968+1G>A; c.1968+2T>C; c.1968+5G>A; c.1968+5G>C; and c.1975dup.For example, the disease associated with lamin A mutation is selected from at least one of dilated cardiomyopathy and conduction disorders, muscular dystrophy, peroneal muscular dystrophy type 2, familial lipodystrophy, rhabdomyosarcoma, progeria-like syndrome, daradryfoss muscular dystrophy, insulin resistance syndrome, progeria, limb-girdle muscular dystrophy, congenital muscular dystrophy, and Werner syndrome, preferably progeria.

[0150] Treatment

[0151] This invention also relates to methods for preventing and / or treating diseases related to lamin A mutations, comprising the steps of administering to a subject a therapeutically effective amount of the polynucleotide of the invention, or a pharmaceutical composition of the invention. As a method of administration, the polynucleotide of the invention may be administered directly to the subject as an ASO, or may be administered to the subject in the form of a pharmaceutical composition.

[0152] As used herein, the term "subject" is defined as including animals, such as mammals, including but not limited to primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a particular embodiment, the subject is a human.

[0153] As used herein, the term "therapeuticly effective amount" refers to the quantity of an effective ingredient sufficient to provide a therapeutic effect in the treatment or control of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with that disease or disorder. A therapeutically effective amount of a compound refers to the quantity of a therapeutic agent that, when used alone or in combination with other therapies, can provide a therapeutic effect in the treatment or control of a disease or disorder. The term "therapeuticly effective amount" may include quantities that improve overall therapy, reduce or avoid symptoms or causes of a disease or disorder, or enhance the therapeutic efficacy of another therapeutic agent. In a preferred embodiment of the invention, the dosage of the polynucleotide or pharmaceutical composition of the invention is preferably from 1 to 500 mg / kg (human subject), for example 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, etc. The dosages are 4.5 mg / kg, 5 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 0.6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or 100 mg / kg. When the subject is another non-human mammal, the corresponding dosage can be obtained according to dosage conversion methods well known in the art.

[0154] The preferred dosage of the polynucleotide or pharmaceutical composition of the present invention is 1 to 500 mg / kg (for mice), for example 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or 100 mg / kg.

[0155] As used herein, the term "treatment" refers to the eradication or improvement of a disease or condition, or one or more symptoms associated with such a disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be treated. In certain embodiments, the term refers to minimizing the spread or aggravation of a disease or condition by administering one or more preventative or therapeutic agents to a subject suffering from such a disease or condition. In some embodiments, the term refers to the administration of the polynucleotide of the present invention, with or without other additional active agents, after the onset of symptoms of a particular disease.

[0156] As used herein, the term "prevention" refers to the prevention of the onset, recurrence, or spread of a disease or condition, or one or more symptoms associated with such disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be prevented. In a particular embodiment, the term refers to the administration of the polynucleotide provided herein, with or without other additional active agents, to a patient at risk of developing the disease or disorder described herein, prior to the onset of symptoms. The term encompasses the suppression and reduction of symptoms of a particular disease. In a particular embodiment, patients with a family history of a disease are specifically considered candidates. Furthermore, patients with a history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be used interchangeably with the term "preventive treatment."

[0157] The route of administration of the polynucleotide or pharmaceutical composition of the present invention is not limited, but is preferably via intramuscular, subcutaneous, oral, intravenous, skin, mucosal (e.g., intestinal), nasal or peritoneal routes.

[0158] The preferred dosing frequency of the polynucleotide or pharmaceutical composition of the present invention is once daily, twice daily, three times daily, once every two days, once every three days, once every five days, once every week, twice every week, once every month, twice every month, three times every month, once every two months, once every three months, once every four months, once every five months, once every six months, or once every year.

[0159] Other in vitro application methods

[0160] This invention also relates to methods for (i) reducing the expression level of lamin A mutant, or (ii) reducing the number of senescent positive cells, or (iii) reducing the number of abnormal nucleated cells in vitro without therapeutic intervention, comprising the step of contacting biological tissue with the polynucleotides of this invention in vitro without therapeutic intervention. As an example of non-therapeutic application, it can be applied to scientific research and laboratory drug screening, etc.

[0161] In some preferred embodiments, the biological tissue is a progeria patient's cardiomyocyte population. In one embodiment of the invention, when the polynucleotides of the present invention are contacted in vitro with a progeria patient's cardiomyocyte population, the expression level of lamin A mutant (e.g., progerin) is significantly reduced. In one embodiment of the invention, when the polynucleotides of the present invention are contacted in vitro with a progeria patient's cardiomyocyte population, the number of senescent positive cells is significantly reduced. In one embodiment of the invention, when the polynucleotides of the present invention are contacted in vitro with a progeria patient's cardiomyocyte population, the number of abnormal nuclei is significantly reduced. In one embodiment of the invention, when the polynucleotides of the present invention are contacted in vitro with a progeria patient's cardiomyocyte population, the expression level of progerin is significantly reduced.

[0162] In some preferred embodiments, the biological tissue is a population of cardiomyocytes from naturally aging individuals.

[0163] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0164] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0165] Example 1

[0166] Multigenotypic HGPS genotyping and acquisition of reprogrammed iPSCs

[0167] DNA was extracted from PBMCs of HGPS patients after collection and isolation. LMNA and ZMPSTE24 genes were amplified, and the pathogenic genotype of the patients was determined by comparison with the genomes of normal individuals. Most patients had the classic HGPS genotype (c.1824C>T), but some non-classical LMNA mutant HGPS genotypes were also detected, such as c.1579C>T, c.1822G>A, and c.168C>G, as well as the ZMPSTE24 mutant c.469C>T (Figure 1). qRT-PCR was used to detect progerin mRNA expression in the patients, and the results showed high progerin mRNA expression only in classic c.1824C>T and non-classical c.1822G>A cells (Figure 2). Finally, we selected patients with classic c.1824C>T, non-classical c.1822G>A, and the ZMPSTE24 mutant c.469C>T for further research.

[0168] To obtain iPSCs with the aforementioned genotypes, reprogramming plasmids pCXLE-hSK, pCXLE-hUL, and pCXLE-hOCT3 / 4 were transduced into PMBCs isolated from the blood of infected children via electroporation, and iPSC monoclonal samples were picked on day 20 (Figure 3). The obtained iPSCs were continuously passaged, and karyotype and stemness were detected by IFA (Figure 4). The results showed that PBMCs with various HGPS genotypes could be reprogrammed into iPSCs. After continuous passage, the karyotype and stemness of the cells were not significantly different from those of normal human iPSCs. The iPSCs from HGPS patients did not show significant DNA damage (γHA2X staining), consistent with previous reports. The iPSC stage of HGPS patients did not exhibit a significant senescence phenotype.

[0169] Example 2

[0170] Construction and Detection of HGPS Myocardial Differentiation Model

[0171] Controlling the Wnt pathway can rapidly and effectively induce iPSCs to differentiate into cardiomyocytes; however, no method for differentiating HGPS multigenotype cardiomyocytes has been reported. We established a universal method for differentiating HGPS multigenotype cardiomyocytes by optimizing cell initiation density, the concentration of the GSK-3 inhibitor CHIR99021, and the concentration of the Wnt pathway inhibitor Wnt-C59 during the experimental process (Figure 5). On day 7 post-differentiation (cardiac progenitor stage), some cells exhibited beating, and by day 9, 95% of cells began rhythmic beating throughout the cell line. qRT-PCR analysis revealed that c.1824C>T and c.1822G>A cells began expressing progerin on day 7, and the expression level continued to increase with the extension of differentiation time (Figure 6). Simultaneously, the expression of the OCT4 gene, representing cell stemness, gradually decreased, while the expression level of cardiac troponin T (cTnT) began to increase rapidly on day 5. Statistical results of cardiomyocyte beating frequency showed that the beating frequency in the WT group was significantly higher than that in the HGPS group. Lamin A / C and cTnT IFA staining results showed that HGPS did not show obvious nuclear abnormalities in the early stage of cardiomyocyte differentiation, and there was no significant difference in myocardial fiber length (Figures 7 and 8).

[0172] Example 3

[0173] HGPS passaged cardiomyocyte phenotyping and aging marker detection

[0174] To further investigate the phenotypic changes of different HGPS cardiomyocyte genotypes during passage, we continuously passaged differentiated cardiomyocytes and detected changes using SA-β-gal staining, qRT-PCR, IFA, statistical analysis of beating frequency, and cell proliferation rate. The results showed that, compared to the WT group, different HGPS cardiomyocyte genotypes began to exhibit varying degrees of senescence phenotypes with increasing passage number, especially the classic c.1824C>T phenotype (Figures 9-13).

[0175] Example 4

[0176] Targeted progerin ASO design

[0177] As a primary form of nucleic acid drug development in clinical practice, antisense oligonucleotides (ASOs) have made significant progress in the treatment of rare diseases thanks to innovations in gene sequencing, chemical modification, and delivery systems. Considering the serious safety issues of the FDA-approved HGPS small molecule drug Zokinvy, ASOs hold promise as a new clinical treatment option. This embodiment designs some ASO sequences targeting the 3'UTR, as shown in SEQ ID NO:11. These sequences reduce the expression level of progerin protein by targeting progerin mRNA, thereby mitigating the damage progerin causes to cells and slowing down the premature aging process (a schematic diagram of the progerin-targeting ASO design is shown in Figure 14).

[0178] First, target gene sequences suitable for designing ASOs are identified. These target genes target partial segments of the 3'UTR, as shown in SEQ ID NO:1.

[0179] SEQ ID NO:1:

[0180] Furthermore, suitable sequences for designing ASOs are those targeting sequences such as those shown in SEQ ID NO:10.

[0181] SEQ ID NO:10:

[0182] SEQ ID NO:11:

[0183] The inventors designed multiple ASO sequences targeting the 3'UTR fragment as shown in Table 1 below, and commissioned a company to synthesize the corresponding fragments, which were then dissolved in sterile water for later use. Cardiomyocytes differentiated from iPSCs of patients with different genotypes of premature aging were divided into groups of 5 x 10-1. 5The concentration of ASO was evenly distributed into each well of a 6-well plate. After 48 hours, cardiomyocytes were observed to begin beating, and transfection with ASO was prepared. Using Lip3000 transfection reagent, different concentrations of ASO (50 nM or 100 nM) were transfected into the corresponding cardiomyocytes. A mock cell group and a negative control group for the transfection reagent were also set up. After transfection, cardiomyocytes were cultured for another 48 hours before cell samples were collected. RNA sample collection: The supernatant in the 6-well plate was aspirated, and the cells were gently washed twice with PBS. Then, 350 μL of TRK cell lysis buffer was added to each well. Total RNA was extracted using the OMEGA RNA Extraction Kit (R6824-02) for analysis. Protein sample collection: The supernatant in the 6-well plate was aspirated, and the cells were gently washed twice with PBS. Then, 200 μL of Beyotime protein-specific cell lysis buffer RIPA (P0013B) was added to each well. The cells were lysed on ice for 15 minutes, then centrifuged at 12,000 rpm at 4°C. The supernatant was collected, and the protein concentration was determined for analysis. Real-time quantitative PCR was used to detect the expression of progerin and other mRNAs. Primers were designed based on the sequences of progerin and other genes. Total RNA was reverse transcribed into cDNA using the Novizan reverse transcription kit (R223-01). The expression levels of progerin and other mRNAs were then quantified using the Novizan dye-based quantitative PCR kit (Q711-03), and the data were plotted. Western blot was used to detect protein expression: Protein electrophoresis was performed using an ACE gradient protein separating gel (B221212). 30 μg of protein was added to each well, and the electrophoresis was performed at 160 V for 90 minutes. Semi-dry transfer was then performed, followed by incubation with the corresponding antibody. The protein was then exposed using a chemiluminescence analyzer, and the protein expression levels were recorded.

[0184] Table 1

[0185] Example 5

[0186] Verification of the effect of ASO in reducing progerin mRNA expression

[0187] To investigate the effects of different ASOs in a premature aging cardiomyocyte model, the inventors transfected classical c.1824C>T and non-classical c.1822G>A cardiomyocytes with different types of ASOs. qRT-PCR results showed that ASO_U5-U11 (100 nM) significantly reduced the expression of protein mRNA in cardiomyocytes, with ASO_U5-U9 reducing protein mRNA expression by more than 70% (Figures 15 and 16). Taking ASO_U7 and ASO_U9 as examples, dose-dependent experiments demonstrated that 50 nM ASO_U7 and ASO_U9 effectively reduced protein mRNA and protein expression levels (Figures 17-20). IFA results showed that the number of abnormal nuclei was significantly reduced after transfection with ASO_U7 and U9. SA-β-gal staining results also proved that transfection with U7 and U9 could reduce the number of senescent positive cells (Figures 21-25). The above results demonstrate that ASO U7 and U9, which target progerin, can significantly reduce progerin mRNA expression and slow down the cellular senescence process.

[0188] To further investigate the effects of ASO in the premature aging mouse model, we screened the designed ASO sequences. In the G608G premature aging mouse model, we subcutaneously injected ASO_U5-U9 (50 mg / kg) once a week, with the PBS group serving as the control group. After one month of continuous injection, we detected the expression of progerin mRNA and protein in the heart and liver tissues of the mice. We found that both ASO_U5 and ASO_U9 showed a certain inhibitory effect on the expression of progerin mRNA and protein (exemplarily shown in Figures 26-29; qRT-PCR and Western blot results showed that both ASO_U7 and ASO_U9 could significantly reduce the expression of progerin mRNA and protein in the heart and liver of premature aging mice).

[0189] Example 6

[0190] ASO specificity and toxicity

[0191] The study found that U5, U9, and U11 designed in the above embodiments had the best specificity, while U8 had relatively poor specificity and high toxicity.

[0192] The inventors collected serum from mice in the PBS and ASO_U5-U9 injection groups and performed biochemical tests on liver and kidney-related indicators. Experiments showed that ASO_U7 and ASO_U9 exhibited the best specificity and lowest toxicity, making them more suitable for subsequent human trials. As exemplarily shown in Figures 30-34, compared to the PBS injection group, the ASO_U7 and ASO_U9 groups did not cause significant liver and kidney damage.

[0193] Example 7

[0194] Establishment of in vitro aging cell model

[0195] We collected blood from individuals aged 30 and 90, isolated PBMCs, and then reprogrammed them into iPSCs (in vitro cellular cells) via in vitro reprogramming (Figure 35) (Figure 36) for subsequent aging-related detection. To further examine the aging phenotypes of iPSCs from different age groups, we differentiated iPSCs from 30 and 90-year-old individuals into cardiomyocytes using a laboratory-established method (Figure 37) and performed aging phenotype detection. The results showed that cardiomyocytes differentiated from iPSCs of different ages exhibited different aging phenotypes. For example, cardiomyocytes differentiated from iPSCs of 90-year-old individuals had a higher proportion of positive staining for the aging markers SA-β-gal (Figure 38), γH2AX (Figure 39), and progerin (Figure 40), indicating that the cardiomyocytes differentiated from iPSCs of 90-year-old individuals were more aged and exhibited typical aging phenotypes. These results demonstrate that cardiomyocytes differentiated from iPSCs of different age groups can be used for research related to aging therapy.

[0196] Example 8

[0197] ASO_U7's effect on delaying normal aging was tested.

[0198] To investigate the effects of ASO_U7 on aging, we transfected 100 nm of ASO_U7 into cardiomyocytes differentiated from iPSCs of 90-year-old elderly individuals, with the untransfected group serving as the control. Aging markers were measured 72 hours post-transfection. The results showed that ASO_U7 treatment significantly reduced the proportion of cells positive for SA-β-gal (Figure 41), γH2AX (Figure 42), and progerin (Figure 43), indicating that ASO_U7 has a good effect in slowing down aging.

[0199] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. The use of isolated polynucleotide sequences, characterized in that, The polynucleotide, as shown in SEQ ID NO:5 (ASO_U7), is used for: (a) Slows down natural aging; (b) To prepare drugs that delay natural aging; (c) In vitro, non-therapeutic reduction of the expression levels of somatic senescence markers; (d) Non-therapeutic reduction of the expression levels of somatic DNA damage markers in vitro; and / or (e) Non-therapeutic reduction of somatic senescence protein expression levels in vitro.

2. The use of the polynucleotide sequence according to claim 1, characterized in that, The somatic cells are cardiomyocytes.

3. The use of the polynucleotide sequence according to claim 1, characterized in that, The aging marker is SA-β-gal.

4. The use of the polynucleotide sequence according to claim 1, characterized in that, The DNA damage marker is γH2AX.

5. The use of the polynucleotide sequence according to claim 1 or 2, characterized in that, The somatic cells were derived from naturally aging subjects.

6. The use of the polynucleotide sequence according to claim 5, characterized in that, The subjects were individuals aged 70 years or older (preferably 80 years or older, more preferably 85 years or older, and even more preferably 90 years or older).

7. The use of the polynucleotide sequence according to claim 6, characterized in that, The somatic cells were obtained by reprogramming PBMCs into iPSCs and then differentiating them.

8. A method for preventing and / or treating natural aging, characterized in that, The method includes the steps of administering a therapeutically effective amount of a polynucleotide sequence, such as that shown in SEQ ID NO:5, or a combination of drugs containing such a sequence to a subject.

9. The method as described in claim 8, characterized in that, The administration of the polynucleotide sequence as shown in SEQ ID NO:5, or a combination of drugs containing it, is by injection, preferably subcutaneous injection.

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