Polynucleotide, pharmaceutical composition, and use thereof

Abstract

A polynucleotide, a pharmaceutical composition, and a use thereof. Multiple polynucleotide sequences targeting the 3'-untranslated region of mRNA of a lamin A mutant are newly designed. The expression level of a lamin A mutant (such as progerin) can be reduced at a lamin A mRNA stage, while the normal transcription of lamin C is not affected, therefore toxic side effects are reduced, thereby having broad prospects in the aspect of preventing or treating diseases (especially early aging) related to lamin A mutation.

Description

Polynucleotides, pharmaceutical compositions and uses thereof

[0001] This application claims priority to the Chinese invention patent application with application number CN202310688123.8, filing date June 9, 2023, and invention titled "Polynucleotides, pharmaceutical compositions and uses thereof." Technical Field

[0002] The present application relates to the field of biomedicine, and in particular to polynucleotides, pharmaceutical compositions and uses thereof that are effective in treating certain diseases. Background Art

[0003] Lamin A protein is part of the scaffolding structure that connects the cell nucleus and plays an important role in nuclear structure and function. It has been reported that Lamin A mutations can disrupt peripheral chromatin with specific epigenetic and molecular features, leading to the misexpression of genes normally expressed in other cell types and causing congenital diseases such as progeria, familial dilated cardiomyopathy, muscular dystrophy, and lipotrophy syndrome.

[0004] Hutchinson-Gilford Syndrome (HGPS), also known as premature aging syndrome (PGS), is a rare, fatal genetic disease. According to statistics, one in every four to eight million newborns worldwide suffers from progeria. Progeria is characterized by a body aging process that is five to ten times faster than normal. Patients have an elderly appearance, and their organs rapidly deteriorate, leading to a decline in physiological function. Symptoms include thin stature, hair loss, and delayed tooth eruption. Children with the disease typically live only between seven and 20 years of age, with the majority succumbing to age-related diseases such as cardiovascular disease.

[0005] Existing research has documented a possible cause of progeria: mutations in the LMNA gene, encoding lamin A, lead to a 150-nucleotide deletion in exon 11, containing the zinc metalloproteinase 24 (ZMPSTE24) cleavage site. This prevents the normal cleavage of the farnesylated C-terminus of lamin A, resulting in the formation of permanently farnesylated progerin, which is also the cause of the gradual degeneration of cellular structure and function in children with progeria. Mutations in the ZMPSTE24 gene also lead to the formation of permanently farnesylated lamin A, causing similar aging symptoms.

[0006] Currently, only one drug, Lonafarnib (trade name: Zokinvy), has been approved globally for the treatment of progeria. Lonafarnib is an oral farnesyltransferase inhibitor involved in the protein prenylation process. By inhibiting the prenylation 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 issues. Therefore, there is a need to develop safer and more effective drugs for the treatment of progeria.

[0007] Summary of the Invention

[0008] In order to solve and / or overcome the technical problems existing in the background technology,

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

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

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

[0012] Another object of the present application is a method for preventing and / or treating diseases associated with lamin A mutation.

[0013] In order to achieve the above technical objectives, the first aspect of the present application provides a polynucleotide, which comprises the following sequence or a polynucleotide fragment complementary to its fragment:

[0014] (i) the sequence shown in SEQ ID NO: 11; and / or

[0015] (ii) A polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO: 11.

[0016] In some preferred embodiments, the polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof:

[0017] (i) the sequence shown in SEQ ID NO: 1; and / or

[0018] (ii) A polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO: 1.

[0019] In some preferred embodiments, the polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof:

[0020] (i) the sequence shown in SEQ ID NO: 10; and / or

[0021] (ii) A polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO: 10.

[0022] In some preferred embodiments, the polynucleotide comprises at least one polynucleotide fragment selected from the group consisting of:

[0023] (i) a polynucleotide fragment as shown in SEQ ID NO: 2-9; and

[0024] (ii) a polynucleotide fragment having no less than 80% homology to SEQ ID NO: 2-9.

[0025] In some preferred embodiments, the polynucleotide is selected from at least one of the following groups:

[0026] (i) a polynucleotide as shown in SEQ ID NO: 2-9; and

[0027] (ii) a polynucleotide having no less than 80% homology to SEQ ID NO: 2-9.

[0028] In some preferred embodiments, the polynucleotide is a polynucleotide as shown in SEQ ID NO: 5 or SEQ ID NO: 7.

[0029] In some preferred embodiments, the length of the polynucleotide is not less than 10 bp, further preferably not less than 15 bp, more preferably 18-22 bp, for example 20 bp.

[0030] In some preferred embodiments, the length of the polynucleotide is no more than 100 bp.

[0031] In some preferred embodiments, the polynucleotide is obtained by isolation.

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

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

[0034] The fourth aspect of the present application provides a use of the polynucleotide described in the first aspect of the present application, or the small nucleic acid molecule described in the second aspect of the present application, or the pharmaceutical composition described in the third aspect of the present application, wherein the use comprises at least one of the following:

[0035] (i) preventing and / or treating diseases associated with lamin A mutation;

[0036] (ii) preparing drugs for preventing and / or treating diseases associated with lamin A mutation;

[0037] (iii) reducing the expression level of a lamin A mutant (preferably reducing the expression level of a progerin protein);

[0038] (iv) reducing the number of senescence-positive cells;

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

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

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

[0042] In some preferred embodiments, the lamin A mutation-related disease is a disease caused by a mutation in lamin A mRNA at one or more positions selected from the following: 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 A mRNA 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-associated disease is selected from at least one of dilated cardiomyopathy and conduction disorders (DCM-CD), muscular dystrophy, Charcot-Marie-Tooth disease type 2 (CMT2), familial partial lipodystrophy (FPLD), striated muscle laminopathy, progeroid syndrome, Emery-Dreyfus 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 application provides a method for preventing and / or treating a disease associated with lamin A mutation, the method comprising the steps of:

[0046] A therapeutically effective amount of the polynucleotide described in the first aspect of this application, or the small nucleic acid molecule described in the second aspect of this application, or the pharmaceutical composition described in the third aspect of this application is administered to the subject.

[0047] The sixth aspect of the present application provides a method for non-therapeutic in vitro (i) reducing the expression level of a lamin A mutant, or (ii) reducing the number of senescence-positive cells, or (iii) reducing the number of abnormal nuclear cells, the method comprising the steps of:

[0048] The biological tissue is contacted in vitro non-therapeutically with the polynucleotide described in the first aspect of the present application, or the small nucleic acid molecule described in the second aspect of the present application, or the pharmaceutical composition described in the third aspect of the present application.

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

[0050] Compared with the prior art, this application has at least the following advantages:

[0051] The present application designs a variety of polynucleotide sequences targeting the 3' untranslated region of lamin A mutant mRNA, which can reduce the expression level of lamin A mutants (such as progerin) at the Lamin A mRNA stage without affecting the normal transcription of Lamin C. Therefore, the toxic side effects are lower and the application has broad prospects for preventing or treating diseases related to lamin A mutations (especially progeria).

[0052] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] One or more embodiments and the corresponding figures in the accompanying drawings are used for exemplary description, and these exemplary descriptions do not constitute limitations on the embodiments.

[0054] FIG1 is a schematic diagram of the genotype identification results of HGPS children according to an embodiment of the present application;

[0055] FIG2 is a schematic diagram of qRT-PCR detection of progerin expression levels in fibroblasts of children according to an embodiment of the present application;

[0056] FIG3 is a diagram of the PBMC reprogramming model according to an embodiment of the present application;

[0057] FIG4 is a schematic diagram of IFA detection of iPSCs cell phenotypes according to an embodiment of the present application, ns, indicates P>0.05; *, indicates P<0.05; ***, indicates P<0.001;

[0058] FIG5 is a schematic diagram of the cardiomyocyte differentiation process according to an embodiment of the present application;

[0059] FIG6 is a graph showing the mRNA expression levels of genes related to myocardial differentiation at different stages detected by qRT-PCR according to an embodiment of the present application;

[0060] FIG7 is a graph showing the results of IFA detection of cTnT (observing myocardial fiber length) on day 11 of myocardial differentiation according to an embodiment of the present application;

[0061] FIG8 is a graph showing the results of IFA detection of Lamin A / C (karyotype observation) on day 11 of myocardial differentiation according to an embodiment of the present application;

[0062] FIG9 is a schematic diagram of the cardiomyocyte passage process according to an embodiment of the present application;

[0063] FIG10 is a diagram showing the morphology of cardiomyocytes of different generations observed under a bright field microscope according to an embodiment of the present application;

[0064] FIG11 is a diagram of SA-β-Gal senescence staining of cardiomyocytes of different passages according to an embodiment of the present application, wherein the percentage value represents the positive rate of senescence staining cells, and P (Passage) represents the number of passages;

[0065] FIG12 is a schematic diagram of IFA detection of cTnT (observation of myocardial fiber length) in different batches of myocardial passages according to an embodiment of the present application, where P (Passage) represents the number of passages;

[0066] FIG13 is a schematic diagram of IFA detection of Lamin A / C (observation of karyotype) in different batches of myocardial passages according to an embodiment of the present application, where P (Passage) represents the number of passages;

[0067] Figure 14 is a schematic diagram of the design of a progerin gamper targeting ASO according to an embodiment of the present application;

[0068] FIG15 is a diagram showing the effect of transfection of ASO_U5-U8 on the expression of progerin mRNA in c.1822G>A cardiomyocytes detected by qRT-PCR according to the examples of the present application, ***, indicating P<0.001;

[0069] FIG16 is a graph showing the effect of transfection of ASO_U5-U8 on the expression of progerin mRNA in c.1824C>T cardiomyocytes detected by qRT-PCR according to the examples of the present application, ***, indicating P<0.001;

[0070] FIG17 is a graph showing the effect of the dose of transfected ASO_U7 and U9 on the expression of progerin mRNA in c.1822G>A cardiomyocytes detected by qRT-PCR in the examples of the present application, ***, indicating P<0.001;

[0071] FIG18 is a graph showing the effect of the dose of transfected ASO_U7 and U9 on the expression of progerin mRNA in c.1824C>T cardiomyocytes detected by qRT-PCR in the examples of the present application, ***, indicating P<0.001;

[0072] FIG19 is a diagram showing the effect of the dose of transfected ASO_U7 and U9 on the expression of progerin protein in c.1822G>A cardiomyocytes detected by Western blot in the examples of the present application;

[0073] FIG20 is a graph showing the effect of the dose of transfected ASO_U7 and U9 on the expression of progerin protein in c.1824C>T cardiomyocytes detected by Western blot in the examples of the present application;

[0074] FIG21 is an IFA test of the effects of ASO_U7 and U9 on myocardial fibers and karyotypes of classic c.1824C>T and non-classic c.1822G>A according to the embodiment of the present application (MNA / C staining method);

[0075] FIG22 shows the effects of ASO_U7 and U9 on myocardial fibers and karyotypes of classic c.1824C>T and non-classic c.1822G>A detected by IFA (cTnT staining method) according to the examples of the present application;

[0076] FIG23 is a diagram showing SA-β-Gal cell senescence staining according to an embodiment of the present application;

[0077] FIG24 is a statistical diagram of the proportion of abnormal nuclei in cardiomyocytes according to an embodiment of the present application, ***, indicating P<0.001;

[0078] FIG. 25 is a statistical diagram of the proportion of SA-β-Gal-positive cells according to the examples of the present application; ***, indicates P<0.001.

[0079] FIG26 is a graph showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of cardiac progerin mRNA in LMNA G608G premature aging mice detected by qRT-PCR in the examples of the present application, ***, indicating P<0.001;

[0080] FIG27 is a graph showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of progerin mRNA in the liver of LMNA G608G premature aging mice detected by qRT-PCR in the examples of the present application, ***, indicating P<0.001;

[0081] FIG28 is a diagram showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of cardiac progerin protein in LMNA G608G premature aging mice detected by Western blot in the examples of the present application;

[0082] FIG29 is a diagram showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of progerin protein in the liver of LMNA G608G premature aging mice detected by Western blot in the examples of the present application;

[0083] Figure 30 is an ELISA method to detect the effect of subcutaneous injection of ASO_U7 and U9 on the expression of alanine aminotransferase, i.e., alanine aminotransferase (ALT), in the liver of LMNA G608G premature aging mice;

[0084] Figure 31 is an ELISA method to detect the effect of subcutaneous injection of ASO_U7 and U9 on the expression of aspartate aminotransferase, i.e., aspartate aminotransferase (AST), in the liver of LMNA G608G premature aging mice;

[0085] Figure 32 is an ELISA method to detect the effect of subcutaneous injection of ASO_U7 and U9 on the expression of uric acid (UA) in the kidneys of LMNA G608G premature aging mice;

[0086] Figure 33 is an ELISA method to detect the effect of subcutaneous injection of ASO_U7 and U9 on the expression of creatinine (Crea) in the kidneys of LMNA G608G premature aging mice;

[0087] Figure 34 is a graph showing the effect of subcutaneous injection of ASO_U7 and U9 on the expression of urea nitrogen (Urea) in the kidneys of LMNA G608G premature aging mice detected by ELISA.

[0088] Figure 35 is a schematic diagram of the process of subcutaneous injection of ASO_U7 and PBS control group into the progeria mouse model.

[0089] FIG36 is a graph showing changes in body weight of premature aging mouse models (male) after subcutaneous injection of ASO_U7 (Ctrl=PBS).

[0090] FIG37 is a graph showing changes in body weight of premature aging mouse models (female) after subcutaneous injection of ASO_U7 (Ctrl=PBS).

[0091] FIG38 is a graph showing the effect of ASO_U7 injection on progerin mRNA expression in the liver of a progeria mouse model.

[0092] FIG39 is a graph showing the effect of ASO_U7 injection on the expression of progerin mRNA in the heart of a progeria mouse model.

[0093] FIG40 is a graph showing the effect of ASO_U7 injection on the expression of progerin mRNA in the arteries of a progeria mouse model.

[0094] Figure 41 is a graph showing the effect of ASO_U7 injection on the expression of progerin protein in the liver of a premature aging mouse model.

[0095] Figure 42 is a graph showing the effect of ASO_U7 injection on the expression of progerin protein in the heart of a progeria mouse model.

[0096] FIG43 is a graph showing the effect of ASO_U7 injection on the expression of progerin protein in the arteries of a progeria mouse model.

[0097] Figure 44 shows the HE staining results of the arteries of the ASO_U7-injected premature aging mouse model.

[0098] Figure 45 is a diagram showing the results of Masson staining of arterial fibrosis in the premature aging mouse model injected with ASO_U7.

[0099] Figure 46 is a diagram showing the results of Masson staining of cardiac fibrosis levels in the premature aging mouse model injected with ASO_U7.

[0100] Figure 47 is a diagram showing the Masson staining results of renal fibrosis levels in the premature aging mouse model injected with ASO_U7.

[0101] Figure 48 is a graph showing the results of a lifespan test on a premature aging mouse model (male) after subcutaneous injection of ASO_U7.

[0102] Figure 49 is a graph showing the results of a lifespan test on a premature aging mouse model (female) after subcutaneous injection of ASO_U7. DETAILED DESCRIPTION

[0103] In order to enable those skilled in the art to better understand and implement the technical solutions of the present application, before the embodiments, the applicant first provides the technical application background, technical application environment and technical terms that may be involved in the specific implementation of the present application, which the applicant considers necessary. It should be understood that these explanations should not be regarded as limitations on the present application.

[0104] Mutations in the LMNA gene can lead to abnormal splicing of pre-Lamin A mRNA, 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 lipoatrophy syndrome. In their research, the applicant found that by reducing Lamin A mRNA in mice while retaining Lamin C mRNA levels, all indicators of the mice were normal and no abnormal symptoms occurred. Therefore, Lamin C can partially compensate for the function of Lamin A in cells. In particular, in tissues where Lamin A expression is relatively low, such as brain tissue, the main form of Lamin A is Lamin C. Therefore, reducing the expression of Lamin A mutants will not affect the level of Lamin C, will not affect the normal function of brain tissue, and will improve the symptoms and occurrence and development of the disease.

[0105] Based on the above findings, the applicants have conducted extensive and in-depth research and designed a variety of antisense oligonucleotide sequences targeting the 3' untranslated region of lamin A mutant mRNA (e.g., progerin mRNA). These antisense oligonucleotide sequences are capable of reducing the expression of lamin A mutants at the lamin A mRNA stage. Therefore, they 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, Charcot-Marie-Tooth disease type 2, familial lipodystrophy, rhabdomyosarcoma, progeroid syndrome, Emery-Dreyfus muscular dystrophy, insulin resistance syndrome, progeria, limb-girdle muscular dystrophy, congenital muscular dystrophy, and Werner syndrome, while not affecting lamin C levels and improving safety. In a preferred example of the present application, the newly designed antisense oligonucleotide sequences can reduce the expression of progerin and significantly inhibit the occurrence of progeria without affecting lamin C levels or the normal function of brain tissue. Compared with previous treatments, such as Zokinvy and other methods of reducing Lamin A mutants, it has higher safety and shows great prospects as a drug for preventing or treating progeria.

[0106] polynucleotides

[0107] This application relates to polynucleotides. The polynucleotides of this application can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The polynucleotides of this application are designed to include polynucleotide fragments complementary to a target gene or a fragment thereof. As used herein, the term "a polynucleotide / gene comprising a gene X fragment" refers to a polynucleotide sequence / gene sequence that, in addition to containing the gene X sequence, also includes other polynucleotide fragments / gene fragments / bases before and / or after the gene X sequence. 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.

[0108] As used herein, the term "polynucleotide fragment" or "fragment" or "gene fragment" refers to a partial sequence of the polynucleotide / gene, that is, the sequence of the polynucleotide fragment 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 AAAGGG, AAAG, AGGGTTT, etc.

[0109] As used herein, the term "gene is within XX" refers to a gene sequence that is a gene sequence in the XXX region or a partial fragment thereof. In one embodiment of the present application, the target gene is within the 3' untranslated region (3'UTR) of lamin A mRNA or lamin mutant mRNA (e.g., presenilin mRNA), that is, the target gene is the 3' untranslated region (3'UTR) of lamin A mRNA or a partial fragment thereof, or the target gene is the 3' untranslated region (3'UTR) of lamin mutant mRNA or a partial fragment thereof. In one embodiment of the present application, the target gene is (i) a sequence as shown in SEQ ID NO: 11; and / or (ii) a polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides of the sequence shown in SEQ ID NO: 11. In one embodiment of the present application, the target gene is within the sequence as shown in SEQ ID NO: 11.

[0110] As used in this application, 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 in this application, the term "mutant" refers to an amino acid sequence obtained by mutation of the amino acid sequence of a certain protein at any position, or an amino acid sequence obtained by translation of the mRNA of a mutated protein. In this application, "lamin A mutant" refers to an amino acid sequence obtained by substitution, deletion and / or addition of one or more amino acids at any position in the amino acid sequence of lamin, or an amino acid sequence obtained by translation of the mutated lamin A mRNA. In a preferred embodiment of this application, "lamin A mutant" is an amino acid sequence obtained by translation of a sequence obtained by mutation of lamin A mRNA in exon 11 and / or 12. In a preferred embodiment of this application, "lamin A mutant" is a lamin A The amino acid sequence obtained by translating the sequence obtained by mutation of one or more positions selected from the following mRNAs: 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.19 30; 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 application, the "lamin A mutant" is an amino acid sequence obtained by translating the sequence obtained by mutation of one or more positions selected from the following positions of lamin A mRNA: c 1821; c.1822; c.1824; c.1968; c.1968+1; c.1968+1G>A; c1968+2; C1968+2; c.1968+5; and C.1968+5, and one or more mutations selected from these positions will lead to diseases associated with progerin.

[0111] As used in this application, the terms "presenilin," "progerin," and "progerin" are used interchangeably.

[0112] In a preferred embodiment of the present application, the "lamin A mutant" is an amino acid sequence obtained by translating the sequence obtained by lamin A mRNA undergoing one or more mutations 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>C; c.1824G>A; c.1826G>A; c.1830G>A; c.1831G>A; c.1832G>A; c.1833 .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.193 1G>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 application, the "lamin A mutant" is an amino acid sequence obtained by translating the sequence obtained by lamin A mRNA undergoing one or more mutations selected from the following: c1821G>A; c.1822G>A; c.1824C>T; c.1968G>A; c.1968+1G>C; c.1968+1G>A; c1968+2T>C; C1968+2T>A; c.1968+5G>A; and C.1968+5G>C.

[0113] In a preferred embodiment of the present application, the target gene is (i) the sequence shown in SEQ ID NO: 1; and / or (ii) a polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO: 1.

[0114] In a more preferred embodiment of the present application, the target gene is (i) the sequence shown in SEQ ID NO: 10; and / or (ii) a polynucleotide sequence formed by substitution, deletion, or addition of one or more nucleotides in 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 of lamin A mutant (preferably presenilin) ​​mRNA and protein.

[0115] 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 of the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAGT. In one embodiment of the present application, the polynucleotide sequence is reverse complementary to the sequence shown in SEQ ID NO: 1. In one embodiment of the present application, the polynucleotide sequence is reverse complementary to the sequence shown in a fragment of SEQ ID NO: 1. In one embodiment of the present application, the polynucleotide sequence is reverse complementary to a polynucleotide sequence formed by substituting, deleting or adding one or more nucleotides from the sequence shown in SEQ ID NO: 1. In one embodiment of the present application, the polynucleotide sequence is reverse complementary to a fragment of a polynucleotide sequence formed by substituting, deleting or adding one or more nucleotides from the sequence shown in SEQ ID NO: 1. In one embodiment of the present application, the polynucleotide sequence is reverse complementary to the sequence shown in SEQ ID NO: 10. In one embodiment of the present application, the polynucleotide sequence is reverse complementary to the sequence shown in a fragment of SEQ ID NO: 10. In one embodiment of the present application, 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: 10. In one embodiment of the present application, 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: 10.

[0116] In a preferred embodiment of the present application, the polynucleotide includes at least one polynucleotide fragment selected from the following groups: (i) a polynucleotide fragment as shown in SEQ ID NO: 2-9; and (ii) a polynucleotide fragment with a homology of not less than 80% (preferably not less than 90%, more preferably not less than 95%, more preferably not less than 98%, and more preferably not less than 99%) to SEQ ID NO: 2-9.

[0117] In another preferred embodiment of the present application, the polynucleotide is selected from at least one of the following groups: (i) a polynucleotide as shown in SEQ ID NO: 2-9; and (ii) a polynucleotide with a homology of not less than 80% (preferably not less than 90%, more preferably not less than 95%, more preferably not less than 98%, more preferably not less than 99%) to SEQ ID NO: 2-9.

[0118] In another preferred embodiment of the present application, the polynucleotide is at least one selected from the group consisting of: (i) a polynucleotide as shown in SEQ ID NOs: 3-7; and (ii) a polynucleotide having a homology of not less than 80% (preferably not less than 90%, more preferably not less than 95%, more preferably not less than 98%, and more preferably not less than 99%) to SEQ ID NOs: 3-7. Compared to other polynucleotide sequences, these polynucleotide sequences have a higher knockdown efficiency for lamin A mutant (preferably presenilin) ​​mRNA and lamin A mutant.

[0119] In another preferred embodiment of the present application, the polynucleotide is the polynucleotide set forth in SEQ ID NO: 5; or the polynucleotide set forth in SEQ ID NO: 7. The polynucleotides set forth in SEQ ID NOs: 5 and 7 have excellent specificity and low toxicity, reducing the expression of progerin mRNA and protein in cardiomyocytes by at least 50% (preferably 70% or more, and even more preferably 80% or more), without targeting other genomes, and are suitable for subsequent human trials.

[0120] In the present application, polynucleotides are preferably provided in an isolated form, more preferably purified to homogeneity. As used herein, the term "isolated" refers to a nucleic acid that is separated from at least one other component (e.g., nucleic acid) present in its natural source. In one embodiment, the nucleic acid is found only in the presence of, if any, solvents, buffers, ions, or other components normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids present in their natural source.

[0121] As used herein, the terms "homology" and "identity" are used interchangeably and refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be measured by the following method. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is arranged, and the number of positions containing identical nucleotides or amino acid residues in the arranged polynucleotide or polypeptide is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the arranged polynucleotide or polypeptide. A polynucleotide can be different in one position, for example, according to comprising different nucleotides (i.e., replacement or variation) or deletions of nucleotides (i.e., insertion or deletion of one or two nucleotides in a polynucleotide). A polypeptide can be different in one position, for example, by containing an amino acid (i.e., replacement or variation) or deletion of an amino acid (i.e., insertion of an amino acid or amino acid deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of amino acid residues in a polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0122] In the present application, the above-mentioned polynucleotides or fragments thereof can be prepared using conventional methods in the art. For example, they can be obtained using PCR amplification, recombinant methods or artificial synthesis methods. For PCR amplification, primers can be designed based on the disclosed relevant nucleotide sequences, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each step together in the correct order. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities using recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. Artificial synthesis of the relevant sequence is particularly suitable for when the fragment length is relatively short. Generally, fragments with very long sequences can be obtained by first synthesizing multiple small fragments and then connecting them.

[0123] The length of the polynucleotide in this application is preferably not less than 10 bp, more preferably not less than 15 bp, more preferably 18-22 bp, for example 20 bp, and the best effect is achieved when the length is 20 bp.

[0124] small nucleic acid molecules

[0125] The present application also relates to small nucleic acid molecules comprising the above polynucleotides, wherein the small nucleic acid molecules are siRNA, ASO, shRNA or miRNA; preferably ASO.

[0126] As used herein, the term "antisense oligonucleotide" or "ASO (antisense oligonucleotide)" refers to a polynucleotide or fragment thereof that is complementary to a specific target gene according to the principle of base complementarity to inhibit or block the expression of a specific target gene, and the target gene can be a DNA or RNA sequence. In order to enhance the stability, targeting, and improve metabolic capacity of antisense oligonucleotides, the antisense oligonucleotides herein may also include chemical modifications, such as modification of the oxygen atom of the phosphate bond by phosphorothioate, modification of the 2' position of the ribose by an alkyl group (e.g., a methyl group), LNA modification, etc. In order to promote the entry of ASOs into cells or improve the targeting of ASOs to organs or tissues in vivo, the antisense oligonucleotides herein may also include terminal Chol and GalNAc modifications. The front end and / or rear end of the polynucleotide or fragment thereof complementary to the target gene in the antisense oligonucleotides herein may optionally further include a short sequence of 1-5 (more preferably 3-5) arbitrary bases, and the bases in these short sequences are preferably chemically modified. Those skilled in the art can obtain the antisense oligonucleotides in this application by conventional methods in the art, such as chemical synthesis.

[0127] As used herein, the term "siRNA" (Small interfering RNA, siRNA) refers to a small RNA molecule (approximately 21-25 nucleotides) that can be processed by Dicer (an enzyme in the RNase III family that is specific for double-stranded RNA) from its precursor (such as dsRNA, shRNA, etc.). It can also be chemically synthesized or produced by processing other proteins. siRNA is a key member of the siRISC (siRISC) complex, stimulating the rapid cleavage and degradation of target RNAs with complementary sequences, leading to silencing of the target gene, making it a key functional molecule in RNAi. Given a specific gene target sequence, those skilled in the art can design and obtain siRNAs specific to that target sequence using conventional methods.

[0128] As used herein, 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. They are involved in regulating the expression of numerous genes in plants and animals. To date, over 4,000 miRNA molecules have been discovered in plants, animals, and viruses. Most miRNA genes exist in the genome as single copies, multiple copies, or clusters. Each miRNA can regulate multiple target genes, and several miRNAs can also jointly regulate the same gene, forming a complex regulatory network. It is speculated that miRNAs regulate the expression of more than half of all human genes. MiRNAs exist in various forms, the most primitive of which is pri-miRNA. Pri-miRNAs are processed by Drosha to become pre-miRNAs, or miRNA precursors, approximately 50-90 nucleotides in length. Pre-miRNAs are then cleaved by the enzyme Dicer to become mature miRNAs of approximately 20-24 nucleotides in length. MiRNAs inhibit target gene expression primarily by inhibiting translation and accelerating mRNA deadenylation, a mechanism distinct from siRNA-mediated mRNA degradation.

[0129] One way to produce small interfering RNA (siRNA) in vivo is to clone the siRNA sequence as part of a "short hairpin" into a plasmid vector. When delivered into an animal, the hairpin sequence is expressed to form a double-stranded RNA (shRNA) with a top loop structure, which is recognized and processed by the Dicer protein in the cell to produce functional siRNA.

[0130] As used herein, a precursor is a special shRNA constructed as a backbone. The shRNA comprises, from the 5′ end to the 3′ end, the following: (a) a 5′ flanking sequence region; (b) a 5′ pairing siRNA region; (c) a top loop region; (d) a 3′ pairing siRNA region, wherein the 5′ pairing siRNA region and the 3′ pairing siRNA region form a double-stranded region; (e) a 3′ flanking sequence region; the shRNA produces an siRNA, and the nucleotide sequence of the siRNA corresponds to the 3′ pairing siRNA region or the 5′ pairing siRNA region.

[0131] In a broad sense, shRNA stands for short hairpin RNA. shRNA consists of two short reverse-complementary sequences separated by a loop sequence, forming a hairpin structure. Transcription is typically controlled by an endogenous RNA polymerase III promoter. Five to six Ts are attached to the end of the shRNA sequence, acting as a transcriptional terminator for RNA polymerase III. shRNA can also be transcribed from promoters of other RNA polymerases.

[0132] Pharmaceutical composition

[0133] The present application also relates to a pharmaceutical composition containing the polynucleotide of the present application. The pharmaceutical composition in the present application includes the polynucleotide of the present application, and a pharmaceutically acceptable carrier. In order to better enable the polynucleotide of the present application to be accurately delivered to a specific part of the subject, or to improve the bioavailability of the polynucleotide of the present application relative to the subject, or to reduce the toxicity of the polynucleotide of the present application relative to the subject, the pharmaceutically acceptable carrier is optionally a liposome (liposome), lipid nanoparticle (LNP, lipid nanoparticle), protamine, a high molecular polymer (such as polyethyleneimine), an inorganic nanoparticle, an exosome, a polymer matrix, etc.

[0134] In other preferred embodiments of the present application, the pharmaceutical composition comprises: the polynucleotide of the present application and a lipid nanoparticle shell encapsulating the polynucleotide. Preferably, the lipid nanoparticle shell is composed of at least one selected from PEG lipids, cholesterol, ionizable cationic lipids, and helper lipids. To enhance targeting, the lipid nanoparticle shell may be optionally modified with a surface modifier.

[0135] In some other preferred embodiments of the present application, the polynucleotide sequence is conjugated with the asialoglycoprotein receptor ligand (GalNAc) to form GalNac-X, wherein X represents the polynucleotide of the present application.

[0136] Use or indications

[0137] The present application also relates to the use of polynucleotides or pharmaceutical compositions for (i) preventing and / or treating diseases associated with lamin A mutations; (ii) preparing drugs for preventing and / or treating diseases associated with lamin A mutations; (iii) reducing the expression level of lamin A mutants; (iv) reducing the number of senescence-positive cells; and / or (v) reducing the number of abnormal nuclear cells.

[0138] In the present application, any mRNA sequence that can specifically bind to the polynucleotide sequence of the present application can be cleaved by RNase H1, resulting in a decrease in the expression level of the protein translated from the mRNA sequence. Therefore, the polynucleotide sequence of the present application targets the 3'UTR region of the lamin A mutant mRNA, degrading the lamin A mutant mRNA and thereby reducing the expression level of the lamin A mutant, thereby being able to treat diseases associated with lamin A mutations.

[0139] As used in this application, the term "lamin A mutation-related disease" may include any disease that is directly or indirectly caused by lamin A or lamin A mRNA mutation. Preferably, the lamin A mutation is a mutation in lamin A mRNA at exon 11 and / or 12. The lamin A mutation-related disease is preferably a disease caused by a mutation in lamin A mRNA at exon 11 and / or 12. In a preferred embodiment of the present application, the "lamin A mutation-related disease" is a disease caused by lamin A mRNA mutation. Diseases caused by mutations in one or more mRNA positions selected from the group consisting of: 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 aforementioned mutation positions can be found on the website http: / / www.umd.be / LMNA / . In a preferred embodiment of the present application, the "lamin A mutation-related disease" is caused by lamin A Diseases caused by one or more mutations in mRNA selected from the group consisting of: 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.182 2G>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.Exemplarily, the disease associated with lamin A mutation is selected from at least one of the lamin A mutation-associated diseases selected from dilated cardiomyopathy and conduction disorders, muscular dystrophy, Charcot-Marie-Tooth disease type 2, familial lipodystrophy, rhabdomyosarcoma, progeroid syndrome, Emery-Dreyfus muscular dystrophy, insulin resistance syndrome, progeria, limb-girdle muscular dystrophy, congenital muscular dystrophy and Werner syndrome, preferably progeria.

[0140] Treatment

[0141] The present application also relates to a method for preventing and / or treating a disease associated with lamin A mutation, comprising the steps of administering to a subject a therapeutically effective amount of a polynucleotide of the present application, or a pharmaceutical composition of the present application. As a mode of administration, the polynucleotide of the present application can be administered directly to the subject as an ASO, or can be administered to the subject in the form of a pharmaceutical composition.

[0142] As used herein, the term "subject" is defined herein to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In certain embodiments, the subject is a human.

[0143] As used herein, the term "therapeutically effective amount" refers to the amount of the active ingredient sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound refers to the amount of a therapeutic agent that, when used alone or in combination with other therapies, provides a therapeutic effect in the treatment or management of a disease or disorder. The term "therapeutically effective amount" can include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. In a preferred embodiment of the present application, the dosage of the polynucleotide or pharmaceutical composition of the present application is preferably 1 to 500 mg / kg (the subject is a human), 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

[0014] The present invention provides an example of an agonist or anti-tumor agent comprising the following: a) an anti-tumor agent, wherein the anti-tumor agent is administered in an amount of at least 1 mg / kg, 2) an anti-tumor agent, and b) an anti-tumor agent. The present invention provides an example of an agonist or anti-tumor agent, wherein the anti-tumor agent is administered in an amount of at least 1 mg / kg, 3) an anti-tumor agent, and b) an anti-tumor agent. The present invention provides an example of an agonist or anti-tumor agent, wherein the anti-tumor agent is administered in an amount of at least 1 mg / kg, 4) an anti-tumor agent, and b) an anti-tumor agent.

[0144] The dosage of the polynucleotide or pharmaceutical composition of the present application is preferably 1 to 500 mg / kg (the subject is a mouse), for example 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or 100 mg / kg.

[0145] As used herein, the term "treating" refers to eradicating or ameliorating a disease or condition, or one or more symptoms associated with the 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 specific embodiments, the term refers to minimizing the spread or worsening of a disease or condition by administering one or more preventive or therapeutic agents to a subject suffering from the disease or condition. In some embodiments, the term refers to administering the polynucleotides of the present application, in combination with or without other additional active agents, after the onset of symptoms of a particular disease.

[0146] As used herein, the term "prevention" refers to preventing a disease or condition, or the onset, recurrence or spread of one or more symptoms associated with the 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 specific embodiment, the term refers to administering the polynucleotides provided herein in conjunction with or without other additional active agents before the onset of symptoms, with respect to patients with a risk of suffering from a disease or disorder as described herein. The term encompasses the suppression and reduction of the symptoms of a particular disease. In a specific embodiment, patients with a family history of a disease are specifically selected as candidates. In addition, patients with a history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" can be used interchangeably with the term "preventative treatment."

[0147] The administration route of the polynucleotide or pharmaceutical composition of the present application is not limited, and is preferably intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (eg, intestinal), intranasal or intraperitoneal.

[0148] The administration frequency of the polynucleotide or pharmaceutical composition of the present application is preferably once a day, twice a day, three times a day, 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.

[0149] Other in vitro applications

[0150] The present application also relates to a method for non-therapeutic in vitro (i) reducing the expression level of a lamin A mutant, or (ii) reducing the number of senescence-positive cells, or (iii) reducing the number of cells with abnormal nuclei, comprising the step of non-therapeutic in vitro contacting a biological tissue with the polynucleotide of the present application. As examples of non-therapeutic applications, these methods can be applied to scientific research and laboratory drug screening.

[0151] In some preferred embodiments, the biological tissue is a progeria patient myocardial cell group. In one embodiment of the present application, when the polynucleotides of the present application are contacted with a progeria patient myocardial cell group in vitro, the expression level of lamin A mutant (such as progerin) significantly decreases. In one embodiment of the present application, when the polynucleotides of the present application are contacted with a progeria patient myocardial cell group in vitro, the number of senescent positive cells significantly decreases. In one embodiment of the present application, when the polynucleotides of the present application are contacted with a progeria patient myocardial cell group in vitro, the number of malformed nuclear cells significantly decreases. In one embodiment of the present application, when the polynucleotides of the present application are contacted with a progeria patient myocardial cell group in vitro, the expression level of progerin significantly decreases.

[0152] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0153] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0154] Example 1

[0155] Identification of multi-genotype HGPS genotypes and acquisition of reprogrammed iPSCs

[0156] PBMCs from children with HGPS were collected and isolated, and DNA was extracted. The LMNA and ZMPSTE24 genes were amplified, and the pathogenic genotypes of the patients were determined by comparison with the normal human genome. Most patients had the classic HGPS genotype (c.1824C>T), but some atypical LMNA mutations, such as c.1579C>T, c.1822G>A, and c.168C>G, as well as the ZMPSTE24 mutation c.469C>T, were also detected (Figure 1). qRT-PCR was used to assess progerin mRNA expression in the patients. Results showed that a form of aberrantly spliced ​​LMNA mRNA encoding progerin protein was highly expressed in cells with both the classic c.1824C>T and the atypical c.1822G>A mutations (Figure 2). Other mutations do not produce progerin protein, but instead splice it into other proteins and cause the associated disease. Finally, we selected children with classic c.1824C>T, nonclassic c.1822G>A, and ZMPSTE24 mutant c.469C>T for follow-up study.

[0157] To obtain iPSCs of the above genotypes, the reprogramming plasmids pCXLE-hSK, pCXLE-hUL, and pCXLE-hOCT3 / 4 were transferred into PBMCs separated from the patient's blood by electroporation, and iPSC single clones were picked on the 20th day (Figure 3). The obtained iPSCs were serially passaged, and the cell karyotype and stemness were detected by IFA (Figure 4). The results showed that PBMCs of various HGPS genotypes could be reprogrammed into iPSCs. After serial passage, the karyotype and stemness of the cells did not differ significantly from those of normal human iPSCs. The iPSCs of HGPS patients did not show obvious DNA damage (p-γHA2X staining), which is consistent with previous reports. The cells at the iPSC stage of HGPS did not have obvious aging phenotypes.

[0158] Example 2

[0159] Construction and testing of HGPS myocardial differentiation model

[0160] Manipulating the Wnt pathway can rapidly and effectively induce the differentiation of iPSCs into cardiomyocytes. However, few methods have been reported for cardiomyocyte differentiation across multiple HGPS genotypes. We established a universal cardiomyocyte differentiation method for multiple HGPS genotypes by optimizing the starting cell density, the concentration of the GSK-3 inhibitor CHIR99021, and the Wnt pathway inhibitor Wnt-C59 (Figure 5). Some cells began beating on day 7 of differentiation (cardiac progenitor stage), and by day 9, 95% of cells were beating rhythmically. qRT-PCR analysis revealed that c.1824C>T and c.1822G>A cells began expressing progerin on day 7, with expression levels increasing with increasing differentiation time (Figure 6). Concomitantly, expression of the OCT4 gene, which represents cell stemness, gradually decreased, while expression of cardiac troponin T (cTnT) began to increase rapidly on day 5. Statistical analysis of cardiomyocyte beating frequency showed that the beating frequency of the WT group was significantly higher than that of the HGPS group. Lamin A / C and cTnT IFA staining results showed that HGPS did not show obvious nuclear deformities in the early stages of cardiomyocyte differentiation, and there was no significant difference in myocardial fiber length ( Figures 7 and 8 ).

[0161] Example 3

[0162] Phenotypic identification and aging index detection of HGPS passaged cardiomyocytes

[0163] To further investigate phenotypic changes in HGPS cardiomyocytes of different genotypes during passage, we serially passaged differentiated cardiomyocytes and analyzed them using SA-β-Gal staining, qRT-PCR, IFA, and counting beating frequency and cell proliferation rate. The results showed that HGPS cardiomyocytes of different genotypes began to exhibit varying degrees of senescence phenotypes with increasing passage number compared to the WT group, with the classic c.1824C>T phenotype being particularly pronounced (Figures 9 to 13).

[0164] Example 4

[0165] Design of progerin-targeted ASOs

[0166] As the primary form of nucleic acid drug development in clinical practice, antisense oligonucleotides (ASOs) have made significant progress in the treatment of rare diseases, driven by advances in gene sequencing, chemical modification, and delivery systems. Considering the serious safety issues with the FDA-approved small molecule drug Zokinvy for HGPS, ASOs are expected to become a new option for clinical treatment. In this example, some ASO sequences were designed that target the 3'UTR, as shown in SEQ ID NO:11. These sequences target progerin mRNA, reducing progerin protein expression and alleviating progerin-induced cell damage, thereby slowing the progression of premature aging. (A schematic diagram of the design of progerin-targeted ASOs is shown in Figure 14 ).

[0167] First, target gene sequences suitable for ASO design are determined. These target genes target partial fragments of the 3'UTR, as shown in SEQ ID NO: 1.

[0168] SEQ ID NO: 1:

[0169] Furthermore, the sequence suitable for designing ASO is the sequence shown in SEQ ID NO: 10.

[0170] SEQ ID NO: 10:

[0171] SEQ ID NO: 11:

[0172] The applicant designed multiple ASO sequences targeting 3'UTR fragments as shown in Table 1 below, and commissioned a company to synthesize the corresponding fragments, which were then dissolved in sterile water and set aside. Cardiomyocytes differentiated from iPSCs of patients with progeria of different genotypes were cultured at a rate of 5x10 5Cells were evenly plated in 6-well plates at a concentration of 100 μg / well. After 48 hours, beating cardiomyocytes were observed and ready for ASO transfection. Using Lip3000 transfection reagent, different ASOs were transfected into the corresponding cardiomyocytes at concentrations of 50 nM or 100 nM. Mock cells and a negative transfection control group were also set up. Cardiomyocytes were cultured for 48 hours after transfection before cell samples were collected. RNA samples were collected by aspirating the supernatant from the 6-well plates, gently washing twice with PBS, then adding 350 μl of TRK cell lysis buffer to each well. Total RNA was then extracted using the OMEGA RNA Extraction Kit (R6824-02) for analysis. Protein samples were collected by aspirating the supernatant from the 6-well plates, gently washing twice with PBS, then adding 200 μl of Beyotime Protein Lysis Buffer (RIPA) (P0013B) to each well. Lyse on ice for 15 minutes, centrifuge at 12,000 rpm at 4°C, collect the supernatant, and determine the protein concentration before analysis. Real-time fluorescence quantitative PCR was used to detect the expression of mRNAs such as progerin. Detection primers were designed based on the sequences of genes such as progerin. Total RNA was then reverse-transcribed into cDNA using the Novozymes Reverse Transcription Kit (R223-01). The expression of mRNAs such as progerin was then quantified using the Novozymes Dye-Based Fluorescence Quantification Kit (Q711-03), and statistical data was plotted. Western blot was used to detect protein expression: ACE gradient protein separation gel (B221212) was used for protein electrophoresis. 30 μg of sample protein was added to each well at 160 V for 90 minutes. Semi-dry transfer was then performed, followed by incubation with the corresponding antibodies. Protein expression was then recorded using a chemiluminescence analyzer.

[0173] Table 1

[0174] Example 5

[0175] Verification of the effect of ASO on reducing progerin mRNA expression

[0176] To investigate the effects of different ASOs in premature aging cardiomyocyte models, the inventors transfected cardiomyocytes with different ASOs, expressing either classic c.1824C>T or atypical c.1822G>A mutations. qRT-PCR results showed that ASOs U5-U11 (100 nM) significantly reduced progerin mRNA expression in cardiomyocytes, with ASOs U5-U9 reducing progerin mRNA expression by over 70% (Figures 15 and 16). Dose-dependent studies using ASOs U7 and U9, for example, demonstrated that 50 nM of ASOs U7 and U9 effectively reduced progerin mRNA and protein expression (Figures 17-20). IFA test 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 senescence-positive cells (Figures 21-25). The above results prove that ASO_U7 and U9 targeting progerin can significantly reduce the expression of progerin mRNA and slow down the process of cell senescence.

[0177] To further explore the effect of ASO on the progeria mouse model, we screened the ASO sequences designed above. We subcutaneously injected ASO_U7 and U9 (50 mg / kg) once a week into the LMNA G608G (c.1824C>T mutation) progeria mouse model, and set up a PBS group as a control group. After one month of continuous injection, the expression of progerin mRNA and protein in the heart and liver tissues of the mice was detected. It was found that both showed a certain inhibitory effect on the expression of progerin mRNA and protein (as shown in Figures 26-29, qRT-PCR and Western blot results showed that ASO_U7 and ASO_U9 can significantly reduce the expression of progerin mRNA and protein in the heart and liver of progeria mice).

[0178] Example 6

[0179] ASO specificity and toxicity

[0180] The study found that the specificity of U7 and U9 designed in the above embodiments was better than that of U5, U6 and U8, especially U7 had the best specificity.

[0181] The inventors collected serum from mice injected with PBS, ASO_U7, and U9 and analyzed liver and kidney biochemical markers. The experiments revealed that ASO_U7 had the highest specificity and lowest toxicity, making it more suitable for subsequent human trials. As shown in Figures 30-34 , the ASO_U7 group did not cause significant liver and kidney damage compared to the PBS group.

[0182] Example 7

[0183] Effects of ASO on Progeria Mouse Model

[0184] To further investigate the effects of ASOs on progeria mouse models, we tested the aforementioned ASO_U7. We administered ASO_U7 (50 mg / kg) subcutaneously once weekly to LMNA G608G progeria mice (3 males and 3 females), as shown in Figure 35. A PBS control group was also assigned for five consecutive months. The mice were weighed and weighed weekly before injection (Figures 36 and 37). Five months after injection, the mice were euthanized, and tissues and organs, including liver, heart, and arteries, were collected at low temperatures. Progerin mRNA and protein expression was measured in each tissue and organ. Concurrently, tissues and organs, including heart, arteries, and kidneys, were collected at room temperature, fixed with 4% paraformaldehyde, and subjected to histopathological examination (HE and Masson staining). Results showed that the ASO_U7-treated mice had significantly higher body weights than the PBS control group (Figures 36 and 37), significantly improving and reversing the weight loss seen in progeria mice.

[0185] mRNA detection results showed that ASO_U7 could significantly inhibit the expression of progerin mRNA in the mouse heart, liver, and arteries (Figures 38-40). Protein detection results showed that ASO_U7 could effectively inhibit the expression of progerin protein in the mouse liver, heart, and arteries (Figures 41-43).

[0186] Masson staining results showed that injection of ASO_U7 could effectively reduce the fibrosis levels in the arteries ( FIG. 45 ), hearts ( FIG. 46 ), and kidneys ( FIG. 47 ) of male and female progeria mice.

[0187] Example 8

[0188] Effect of ASO on lifespan in a progeria mouse model

[0189] To further explore the effect of ASO on the lifespan of progeria mouse models, we tested ASO_U7. We subcutaneously injected ASO_U7 17 mg / kg and 50 mg / kg once a week into the LMNA G608G progeria mouse model (6 males and 6 females) (the injection process is shown in Figure 35). At the same time, a PBS group was set as the control group. The death of mice was continuously observed and the lifespan of mice was recorded. The test results showed that injection of ASO_U7 can extend the lifespan of male (Figure 48) and female (Figure 49) progeria mice by at least 3 months, or even longer.

[0190] [Corrected 28.11.2024 according to Rule 91] Those skilled in the art will understand that the above-mentioned embodiments are specific examples for implementing the present application, and in actual applications, various changes can be made in form and details without departing from the spirit and scope of the present application.

Claims

1. A polynucleotide, characterized in that The length of the polynucleotide is not less than 10 bp, and the polynucleotide includes a polynucleotide fragment complementary to the following sequence or a fragment thereof: (i) the sequence shown in SEQ ID NO: 11; and / or (ii) A polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO:

11.

2. The polynucleotide according to claim 1, characterized in that The polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof: (i) the sequence shown in SEQ ID NO: 1; and / or (ii) A polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO:

1.

3. The polynucleotide according to claim 1, characterized in that The polynucleotide comprises a polynucleotide fragment complementary to the following sequence or a fragment thereof: (i) the sequence shown in SEQ ID NO: 10; and / or (ii) A polynucleotide sequence formed by substitution, deletion or addition of one or more nucleotides in the sequence shown in SEQ ID NO:

10.

4. The polynucleotide according to claim 1, characterized in that The polynucleotide comprises at least one polynucleotide fragment selected from the group consisting of: (i) a polynucleotide fragment as shown in SEQ ID NO: 2-9; and (ii) a polynucleotide fragment having no less than 80% homology to SEQ ID NO: 2-9.

5. The polynucleotide according to claim 1, characterized in that The polynucleotide is selected from at least one of the following groups: (i) a polynucleotide as shown in SEQ ID NO: 2-9; and (ii) a polynucleotide having no less than 80% homology to SEQ ID NO: 2-9.

6. The polynucleotide according to claim 5, characterized in that The polynucleotide is the polynucleotide shown in SEQ ID NO:5 or the polynucleotide shown in SEQ ID NO:

7.

7. The polynucleotide according to any one of claims 1 to 6, characterized in that The polynucleotide is obtained by isolation.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polynucleotide according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

9. Use of the polynucleotide according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 8, characterized in that: The use includes at least one of the following: (i) preventing and / or treating diseases associated with lamin A mutation; (ii) preparing drugs for preventing and / or treating diseases associated with lamin A mutation; (iii) reducing the expression level of lamin A mutants; (iv) reducing the number of senescence-positive cells; (v) Reduce the number of abnormal nuclear cells.

10. The use according to claim 9, characterized in that The lamin A mutation is that the lamin A mRNA is mutated in exon 11 and / or exon 12.

11. The use according to claim 9, characterized in that The lamin A mutation-related disease is a disease caused by mutation of lamin A mRNA in exon 11 and / or exon 12.

12. The use according to claim 11, characterized in that The lamin A mutation-related disease is a disease caused by a mutation in lamin A mRNA at one or more positions selected from the following: 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.

13. The use according to claim 12, characterized in that The lamin A mutation-related disease is a disease caused by one or more mutations in lamin A mRNA 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.182 2G>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.

14. The use according to claim 9, characterized in that The lamin A mutation-related disease is selected from at least one of dilated cardiomyopathy and conduction disorders, muscular dystrophy, Charcot-Marie-Tooth disease type 2, familial lipodystrophy, rhabdomyosarcoma, progeroid syndrome, Emery-Dreyfus muscular dystrophy, insulin resistance syndrome, progeria, limb-girdle muscular dystrophy, congenital muscular dystrophy and Werner syndrome.

15. A small nucleic acid molecule, characterized in that The small nucleic acid molecule contains the polynucleotide according to any one of claims 1 to 6 or claim 7.

16. The small nucleic acid molecule according to claim 15, characterized in that The small nucleic acid molecule is at least one of siRNA, ASO, shRNA or miRNA.

17. The small nucleic acid molecule according to claim 15, characterized in that ， 18. A method for preventing and / or treating diseases associated with lamin A mutation, characterized in that: The method comprises the steps of: A therapeutically effective amount of the polynucleotide of any one of claims 1 to 6, or the pharmaceutical composition of claim 8, or the small nucleic acid molecule of any one of claims 15 to 17 is administered to the subject.

19. A method for non-therapeutic in vitro reduction of the expression level of lamin A mutants, or reduction of the number of senescence-positive cells, or reduction of the number of abnormal nuclear cells, the method comprising the steps of: The biological tissue is contacted in vitro non-therapeutically with the polynucleotide according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 8, or the small nucleic acid molecule according to any one of claims 15 to 17.

20. The method according to claim 19, characterized in that The biological tissue is a myocardial cell population from patients with progeria.

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