Use of an ankrd50 gene inhibitor in the preparation of a medicament for treating cancer

Abstract

The present application relates to the use of an ANKRD50 gene inhibitor for the preparation of a medicament for the treatment of cancer. The present application also relates to nucleic acid molecules, nucleic acid constructs, viral vectors, viral particles for use as ANKRD50 gene inhibitors, and to pharmaceutical compositions and combinations for the treatment of cancer.

Description

Technical Field

[0001] This invention relates to the field of gene therapy technology, and more specifically to the use of ANKRD50 gene inhibitors in the preparation of drugs for treating cancer. Background Technology

[0002] Pancreatic cancer is insidiously progressive, highly malignant, and has an extremely poor prognosis, making it one of the deadliest malignant tumors. The main pathological type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), which accounts for over 90% of pancreatic malignancies. It ranks fourth in mortality among cancers, with a five-year survival rate of less than 6%. The etiology of pancreatic cancer is not fully understood, and current knowledge of its malignant biological characteristics is limited, resulting in poor effectiveness of existing treatments. Therefore, early detection remains crucial for achieving the best prognosis in pancreatic cancer; however, early diagnosis remains extremely challenging, and patients with advanced pancreatic cancer often have a poor prognosis due to the loss of surgical options.

[0003] Therefore, developing new molecular mechanisms and effective therapeutic targets is an urgent priority for pancreatic cancer treatment, and it is of great significance for optimizing cancer treatment strategies and detecting tumor progression. Summary of the Invention

[0004] This application was made in view of the above-mentioned issues, and the correlation between ANKRD50 (Ankyrin repeat domain 50) gene expression and cancer, especially pancreatic cancer, was discovered, thereby completing the present invention.

[0005] In view of this, in a first aspect, this application provides the use of an ANKRD50 gene inhibitor in the preparation of a medicament for treating cancer in subjects in need.

[0006] Furthermore, ANKRD50 gene inhibitors refer to molecules or preparations that inhibit the ANKRD50 gene, prepared or screened using the ANKRD50 gene as a target.

[0007] Furthermore, the ANKRD50 gene is derived from humans, and its NCBI Gene number is 57182.

[0008] Furthermore, the aforementioned inhibitory effects include, but are not limited to, inhibiting ANKRD50 gene activity, or inhibiting RNA, proteins, and other small molecules transcribed or expressed by the ANKRD50 gene. In other words, ANKRD50 gene inhibitors also include molecules or preparations that target RNA, proteins, and other small molecules transcribed or expressed by the ANKRD50 gene and exhibit inhibitory effects on these RNA, proteins, and other small molecules.

[0009] In some implementations, the cancer is pancreatic cancer.

[0010] In some implementations, the cancer is pancreatic ductal carcinoma.

[0011] In some implementations, the ANKRD50 gene inhibitor is in the form of a nucleic acid molecule, a nucleic acid construct, a viral vector, a viral particle, an antibody, or a small molecule compound.

[0012] In some implementations, the ANKRD50 gene inhibitor is in the form of a nucleic acid molecule.

[0013] In some implementations, the nucleic acid molecule is double-stranded RNA or shRNA.

[0014] Furthermore, the target sequence of the ANKRD50 gene for the action of the above nucleic acid molecules is shown in SEQ ID No:1 or SEQ ID No:2.

[0015] Secondly, this application provides nucleic acid molecules, including double-stranded RNA or shRNA, for use as inhibitors of the ANKRD50 gene.

[0016] Furthermore, the double-stranded RNA or shRNA contains nucleotide sequences that can hybridize with the ANKRD50 gene under harsh conditions.

[0017] In some embodiments, the double-stranded RNA includes a first strand and a second strand, wherein the first and second strands are complementary to form an RNA dimer, and the sequence of the first strand is identical to the target sequence of the ANKRD50 gene.

[0018] Furthermore, the double-stranded RNA is a small interfering RNA (siRNA).

[0019] In some implementations, the shRNA includes a sense strand and an antisense strand, and a stem-loop structure connecting the sense strand and the antisense strand, the sequences of the sense strand and the antisense strand are complementary, and the sequence of the sense strand is identical to the target sequence of the ANKRD50 gene.

[0020] Furthermore, the nucleotide sequence of double-stranded RNA (such as siRNA) or shRNA is as shown in any of the sequences in SEQ ID No.:3-14.

[0021] Thirdly, this application provides a nucleic acid construct for use as an inhibitor of the ANKRD50 gene, including the nucleic acid molecule of the second aspect of this application.

[0022] Furthermore, the nucleic acid construct of this application contains a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule and is capable of expressing the shRNA. Furthermore, the nucleic acid construct is obtained by cloning the gene fragment encoding the shRNA in the nucleic acid molecule into a vector.

[0023] In some implementations, the nucleic acid construct may also include a vector and an optional promoter sequence.

[0024] Fourthly, this application provides a viral vector for use as an inhibitor of the ANKRD50 gene, which includes the nucleic acid construct of the third aspect of this application.

[0025] Fifthly, this application provides viral particles for use as an inhibitor of the ANKRD50 gene, which include the viral vector of the fourth aspect of this application.

[0026] Sixthly, this application provides a pharmaceutical composition for treating cancer, including one or more ANKRD50 gene inhibitors.

[0027] Furthermore, the pharmaceutical composition may include other drugs for treating cancer.

[0028] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0029] Seventhly, this application provides a combination of drugs, including an ANKRD50 gene inhibitor and one or more other drugs for treating cancer (preferably pancreatic cancer, more preferably pancreatic ductal carcinoma).

[0030] Furthermore, the aforementioned ANKRD50 gene inhibitor is administered simultaneously or sequentially with other drugs. In some embodiments, the ANKRD50 gene inhibitor is administered before or after the administration of other drugs / therapeutic agents, such as standard drugs / therapeutic agents.

[0031] In other respects, this application also provides a method for treating a subject suffering from a condition mediated by ANKRD50 gene expression (e.g., ANKRD50 gene-related diseases or conditions). The method includes administering a therapeutically effective amount of the ANKRD50 gene inhibitor of the present invention to the subject, thereby inhibiting the expression of the ANKRD50 gene in cells.

[0032] In some implementations, the subject is a human being.

[0033] In some implementations, the subject has cancer, particularly pancreatic cancer, and more particularly pancreatic ductal carcinoma.

[0034] In some embodiments, the expression of the ANKRD50 gene in cells is inhibited, resulting in a reduction of the protein level of ANKRD50 gene expression in the serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95% compared to before the administration of the ANKRD50 gene inhibitor.

[0035] In some implementations, inhibiting the expression of the ANKRD50 gene in cells manifests in the following ways: inhibiting the proliferation rate of cancer cells, inhibiting glucose uptake by cancer cells, promoting apoptosis of cancer cells, and inhibiting the formation of cancer cell clones. Attached Figure Description

[0036] Figure 1 Experimental results are shown to analyze the intrinsic driving force of liquid-liquid phase separation in ANKRD50.

[0037] Figure 2 Experimental results showing the expression of ANKRD50 in pancreatic ductal adenocarcinoma tissue are presented.

[0038] Figure 3 The in vitro experimental results show the effect of interfering with ANKRD50 expression on the proliferation of pancreatic cancer cells.

[0039] Figure 4 The in vivo experimental results show the effect of interfering with ANKRD50 expression on pancreatic cancer progression. Detailed Implementation

[0040] To provide a clear and consistent understanding of the terminology used in this specification, some definitions are provided below. Furthermore, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] When used in conjunction with the term "comprising" in the claims and / or description, the word "a" can mean "one," but it is also known to mean "one or more," "at least one," and "one or more." Similarly, the word "another" can mean at least a second or more.

[0042] The term “or” is used herein to mean the term “and / or” and may be used interchangeably with the term “and / or” unless the context clearly indicates otherwise. For example, “justice chain or antisense chain” is understood to mean “justice chain or antisense chain, or justice chain and antisense chain”.

[0043] As used in this specification and claims, the terms “comprising,” “having,” “including,” and “containing” are inclusive or open-ended and do not exclude additional, unstated elements or process steps.

[0044] ANKRD50

[0045] The terms “ANKRD50 (Ankyrin repeat domain 50)” and “ANKRD50 protein” are used interchangeably and refer to protein 50 of the ankyrin repeat domain. “ANKRD50” or “ANKRD50 gene” indicates the gene encoding ANKRD50. The sequence of the human ANKRD50 gene can be found at NCBI Gene:57182. More information about ANKRD50 can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=ANKRD50.

[0046] The terms “target sequence,” “target nucleic acid,” or “target mRNA” refer to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA products of RNA processing as primary transcription products. For example, the sequence of the human ANKRD50 mRNA transcript can be found, for example, in GenBank accessions NM_020337.3 or NM_001167882.2.

[0047] As of the date of this application, the entire contents of each of the aforementioned GenBank login numbers are incorporated herein by reference.

[0048] The term "ANKRD50 gene inhibitor" refers to a molecule or preparation that has an inhibitory effect on the ANKRD50 gene or its transcribed or expressed RNA and protein, prepared or screened using the ANKRD50 gene or its transcribed or expressed RNA and protein as a target.

[0049] The above-mentioned inhibitory effects include, but are not limited to: inhibiting the activity of the ANKRD50 gene, or inhibiting the activity of RNA, proteins and other small molecules transcribed or expressed by the ANKRD50 gene.

[0050] The term “inhibition” and similar expressions refer to reduction or effective cessation and can be used interchangeably with “reduce,” “silence,” “downgrade,” “curb,” and other similar terms, and include any level of inhibition.

[0051] This application provides the use of an ANKRD50 gene inhibitor in the preparation of a medicament for treating cancer in subjects of need. In some embodiments, the cancer is pancreatic cancer, particularly pancreatic ductal carcinoma.

[0052] In this context, "treatment" for any disease or condition refers to improving at least one disease or condition. In some embodiments, "treatment" refers to improving at least one bodily parameter, which may or may not be perceptible to the patient. In some embodiments, "treatment" refers to suppressing a disease or condition physically (e.g., stabilization of obvious symptoms), physiologically (e.g., stabilization of physical parameters), or both. In some embodiments, "treatment" refers to improving the quality of life of a subject in need or reducing the symptoms or side effects of a disease.

[0053] This application focuses particularly on the association between ANKRD50 expression and pancreatic cancer. Studies have shown that patients with diabetes have a 2.08 times higher risk of developing pancreatic cancer than those without diabetes, and that diabetic patients may also have poorer pathological features and longer-term outcomes. Furthermore, pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC), is known to exhibit increased glucose uptake. To identify key drivers of glucose uptake, the applicant first performed a Gene Set Enrichment Analysis (GSEA), obtaining 175 gene clusters potentially involved in the regulation of glucose transmembrane transport (https: / / www.gsea-msigdb.org / gsea / index.jsp). Subsequently, genotype expression (GTEx) sequence data of PDAC tumor and normal tissues from The Cancer Genome Atlas (TCGA) were analyzed, along with paired gene expression profiles of PDAC tumor tissues and adjacent non-tumor tissues from the GEO database (GSE15471 and GSE28735), and the expression profiles of these genes in normal and tumor tissues were compared. Ultimately, ANKRD50 was identified as a potential gene of interest that promotes PDAC progression by regulating glucose transport. The applicant also discovered that under high sugar conditions, ANKRD50 aggregates into granular aggregates to form ANKRD50 phase-separated bodies, and further analysis revealed the key sequence in ANKRD50 that regulates this process (i.e., SEQ ID No.:15).

[0054] SEQ ID No.:15:

[0055]

[0056] The term "subject" can refer to any human or non-human animal. In some embodiments, the subject is a human.

[0057] The aforementioned "ANKRD50 gene inhibitor" can be in the form of nucleic acid molecules, nucleic acid constructs, viral vectors, viral particles, antibodies, or small molecule compounds.

[0058] Nucleic acid molecules

[0059] In some embodiments, the ANKRD50 gene inhibitor of this application is in the form of a nucleic acid molecule. That is, this application provides a nucleic acid molecule for use as an ANKRD50 gene inhibitor.

[0060] The term "nucleic acid molecule" refers to a polymer of RNA or DNA that is single-stranded or double-stranded and optionally contains synthetic, non-natural, or modified nucleotide bases.

[0061] In some embodiments, the nucleic acid molecule of this application is double-stranded RNA or shRNA.

[0062] The term "double-stranded RNA (dsRNA)" refers to a complex of ribonucleic acid molecules, which is a double-stranded structure having two antiparallel and substantially complementary nucleic acid strands, with "sense" (or "positive") and "antisense" orientations relative to the target RNA. In some embodiments of the present invention, dsRNA triggers the degradation of the target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference (or RNAi).

[0063] The term "short hairpin RNA (shRNA)" refers to an artificial RNA molecule with a tight hairpin loop, which can be used to silence target genes via RNA interference (RNAi). It consists of two short inverted repeat sequences. shRNA cloned into an shRNA expression vector comprises two short inverted repeat sequences separated by a loop sequence, forming a hairpin structure.

[0064] The term "small interfering RNA (siRNA)" in this paper refers to a bioactive agent containing RNA that mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. It guides the specific degradation of mRNA sequences through a process called RNA interference (RNAi). siRNA regulates (e.g., inhibits) gene expression in cells, such as those of a subject (e.g., a mammalian subject, such as a human).

[0065] In some implementations, the ANKRD50 gene target sequence for the nucleic acid molecules is as shown in SEQ ID No.:1 ( NM_ 020337.3 , Homo sapiens ankyrin repeat domain containing 50 (ANKRD50), transcript variant 1, mRNA) or SEQ ID No.: 2 ( NM_001167882.2, Homo sapiens ankyrin repeatdomain containing 50 (ANKRD50), transcript variant 2, mRNA).

[0066] The nucleic acid molecules of this application, acting on the ANKRD50 gene target sequence, can inhibit the expression of the ANKRD50 gene in pancreatic cancer cells. Inhibition of ANKRD50 gene expression refers to a reduction in the amount or level of RNA transcripts (e.g., ANKRD50 mRNA) or proteins encoded by the inhibited gene and / or a reduction in the amount or level of said gene activity in cells, cell populations, samples, or subjects compared to an appropriate reference (e.g., reference cells, cell populations, samples, or subjects). As used herein, “inhibition of ANKRD50 gene expression” means a reduction in the amount or level of ANKRD50 mRNA and / or ANKRD50 in cells, cell populations, samples, or subjects compared to an appropriate reference (e.g., reference cells, cell populations, samples, or subjects), such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0067] In some implementations, the ANKRD50 gene target sequence for the action of nucleic acid molecules is shown in SEQ ID No.:1.

[0068] Double-stranded RNA consists of two complementary RNA strands that hybridize to form a double-stranded structure under conditions where the dsRNA will be used (e.g., under physiological conditions). One strand of the dsRNA (the antisense strand) includes complementary regions that are substantially complementary to, and usually perfectly complementary to, the target sequence. The target sequence may be derived from the mRNA sequence formed during ANKRD50 gene expression.

[0069] In some implementations, the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the ANKRD50 gene under harsh conditions.

[0070] In some embodiments, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand being complementary to form an RNA dimer, and the sequence of the first strand being identical to the target sequence of the ANKRD50 gene.

[0071] In some implementations, the double-stranded RNA is a small interfering RNA (siRNA).

[0072] In some implementations, the shRNA contains a nucleotide sequence capable of hybridizing with the ANKRD50 gene under harsh conditions.

[0073] In some implementations, the shRNA comprises a sense strand and an antisense strand, and a stem-loop structure connecting the sense strand and the antisense strand, the sequences of the sense strand and the antisense strand are complementary, and the sequence of the sense strand is identical to the target sequence of the ANKRD50 gene.

[0074] In some implementations, the nucleotide sequence (5'-3') of siRNA or shRNA is shown in either of the following sequences:

[0075] SEQ ID No.:3 1291GCCCTATGTACTGAACTCT ORF

[0076] SEQ ID No.:4 1722GCTATTGCTTCTCTGTTCT ORF

[0077] SEQ ID No.:5 2080GCAAAGGTTCAGCCTATTT ORF

[0078] SEQ ID No.:6 2368GCCAAGAATTTAACACCAT ORF

[0079] SEQ ID No.:7 2468GGAATGGTACACCTGTCAG ORF

[0080] SEQ ID No.:8 2999GAGGACACGAGGATATTGT ORF

[0081] SEQ ID No.:9 3205GCAAGTAAAGGGCACGCAT ORF

[0082] SEQ ID No.:10 3697GCTTCACAAGAGGGTCATT ORF

[0083] SEQ ID No.:11 3939AAATGGTGCAAACGTAGAA ORF

[0084] SEQ ID No.:12 4420GTGCAGTCATTAACAATTA ORF

[0085] SEQ ID No.:13 5184ACGAACAATGCAAGATAGA ORF

[0086] SEQ ID No.:14 5251AGCCTTAAACAAGCTCTGA ORF

[0087] In one specific embodiment, the nucleotide sequence (5'-3') of the siRNA is shown in SEQ ID No.:6.

[0088] In one specific embodiment, the nucleotide sequence (5'-3') of siRNA and / or shRNA is shown in SEQ ID No.:9.

[0089] Nucleic acid constructs

[0090] In some embodiments, the ANKRD50 gene inhibitor of this application is in the form of a nucleic acid construct. That is, this application provides a nucleic acid construct for use as an ANKRD50 gene inhibitor.

[0091] The term "nucleic acid construct" refers to non-naturally occurring nucleic acids produced using recombinant DNA technology. In particular, a nucleic acid construct is a nucleic acid molecule that has been modified to contain nucleic acid sequence segments combined or joined in a manner not found in nature.

[0092] The nucleic acid construct of this application is an ANKRD50 gene inhibitor, containing a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule, and capable of expressing the shRNA.

[0093] In some embodiments, the nucleic acid construct is obtained by cloning a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule into a vector. Therefore, in some embodiments, the nucleic acid construct also includes a vector, such as a lentiviral vector, vector type LV2 (U6 / Puro).

[0094] In some embodiments, the nucleic acid construct also contains a promoter sequence. The term "promoter" refers to a regulatory component that guides the transcription of the nucleic acid to which it is operably linked. A promoter can regulate both the transcription rate and efficiency of the operably linked nucleic acid. A promoter can also be operably linked to other regulatory components that enhance or repress promoter-dependent transcription of the nucleic acid. These regulatory components include, but are not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly function to regulate the amount of transcription from the promoter, including, for example, enhancers and silencers. The promoter is located near the transcription start site of the operably linked gene or coding sequence, on the same strand, and upstream of the DNA sequence (towards the 5' region of the positive strand).

[0095] In some implementations, the nucleic acid construct may include additional regulatory components, such as enhancer sequences, introns, microRNA targeting sequences, multi-connector sequences that facilitate DNA fragment insertion into the vector, and / or splicing signal sequences.

[0096] The aforementioned nucleic acid construct can be referred to as the "ANKRD50 gene interference nucleic acid construct". After being packaged into infectious viral particles by a virus, it infects tumor cells and then transcribes the shRNA described in this invention. Through enzyme digestion and other steps, siRNA is finally obtained, which is used to specifically silence the expression of the ANKRD50 gene.

[0097] Viral vector

[0098] In some embodiments, the ANKRD50 gene inhibitor of this application is in the form of a viral vector. That is, this application provides a viral vector for use as an ANKRD50 gene inhibitor.

[0099] The term "viral vector" refers to the nucleic acid portion of a virus particle, which can be packaged in a capsid.

[0100] Viral vectors typically contain at least (i) a nucleic acid construct; and (ii) all or part of a viral genome, such as inverted terminal repeat sequences of a viral genome.

[0101] The term "inverted terminal repeat" (ITR) refers to the nucleotide sequence located at the 5' end of the virus (5' ITR) and the nucleotide sequence located at the 3' end of the virus (3' ITR), which contains palindromic sequences and can fold over to form a T-shaped hairpin structure that acts as a primer during DNA replication initiation. It is also required for viral genome integration into the host genome, rescue from the host genome, and capsidation of viral nucleic acids into mature virions. A cis-ITR is required for vector genome replication and its packaging into viral particles.

[0102] In some implementations, the viral vector contains the 5' ITR and 3' ITR of the virus.

[0103] In some implementations, the virus is selected from parvoviruses (especially adeno-associated viruses), adenoviruses, alpha viruses, retroviruses (especially gamma retroviruses and lentiviruses), herpesviruses, especially lentiviruses.

[0104] In some implementations, the viral vector comprises 5'ITR and 3'ITR independently selected from lentiviruses.

[0105] Virus particles

[0106] In some embodiments, the ANKRD50 gene inhibitor of this application is in the form of viral particles. That is, this application provides viral particles for use as an ANKRD50 gene inhibitor.

[0107] The term "viral particle" refers to a viral particle that is infectious and usually has replication defects, comprising (i) a viral vector including packaging and (ii) a capsid.

[0108] In some implementations, the capsid is formed from capsid proteins of lentiviruses.

[0109] Viral particles carrying viral vectors and nucleic acid constructs as described herein can be produced using conventional methods and protocols selected by taking into account the structural characteristics of the viral particles to be produced.

[0110] Typically, viral particles can be produced in host cells, more specifically in specific virus-producing cells (packaging cells), which are transfected with nucleic acid constructs or viral vectors in the presence of helper vectors or viruses or other DNA constructs.

[0111] The term "packaging cell" refers to a cell or cell line that can be transfected with a nucleic acid construct or viral vector and provides all the missing functions required for the complete replication and packaging of the viral vector in a trans configuration. Packaging cells can express these missing viral functions constitutively or inducibly. Packaging cells can be adhesive or suspension cells.

[0112] In some implementations, packaging is performed with the assistance of packaging plasmids or cell lines.

[0113] Antibodies and small molecules

[0114] In some embodiments, the ANKRD50 gene inhibitor of this application is in the form of an antibody or a small molecule compound that specifically binds to a polypeptide transcribed and translated from ANKRD50.

[0115] The term "small molecule compound" generally refers to organic or inorganic compounds with simple molecular formulas and small molecular weights, typically below 1 kDa. For example, antibodies usually have molecular weights above 150 kDa, while nucleic acid molecules (such as siRNA and shRNA) typically have molecular weights of approximately 13-25 kDa. Due to their relatively small molecular size, small molecule compounds can more easily penetrate biological membranes, bind to intracellular targets (such as enzymes and receptors), and readily exert their effects in vivo. Common small molecule compounds include drug molecules, such as aspirin, which has a molecular weight of 180 Da.

[0116] Pharmaceutical compositions and formulations

[0117] The present invention also provides a pharmaceutical composition comprising the ANKRD50 gene inhibitor described herein, and optionally a pharmaceutically acceptable excipient. In some embodiments, one or more pharmaceutically acceptable excipients (including mediators, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition comprising the ANKRD50 gene inhibitor to form a pharmaceutical formulation or pharmaceutical composition suitable for in vivo delivery to a subject (e.g., a human).

[0118] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, or dosage forms that are suitable for contact with tissues of human and animal subjects within the limits of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0119] The term “pharmaceuticalally acceptable excipient” refers to pharmaceutically acceptable materials, compositions or media, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, magnesium talc, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulation materials (involving the carrying or transport of an ANKRD50 gene inhibitor from one organ or part of the body to another organ or part of the body).

[0120] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, alcohol-soluble gluten, carbonate, and phosphate. In some embodiments, the buffered solution is a phosphate-buffered saline solution.

[0121] The pharmaceutical composition of this application is not particularly limited in form and may be in the form of solid, liquid, gel, semi-liquid or aerosol.

[0122] The dosage form of the above-mentioned pharmaceutical composition is any clinically or pharmaceutically acceptable dosage form, such as, but not limited to, powder, injection, capsule, oral liquid, tablet, drop, or spray.

[0123] In some embodiments, the pharmaceutical composition is in the form of a drug, which also includes instructions for use.

[0124] Furthermore, the instructions for use include information on the use of the drug, such as the effective dosage required to treat cancer, the frequency of administration, and the intervals between doses.

[0125] "Effective dose" refers to the amount of ANKRD50 gene inhibitor that, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, is sufficient to treat or prevent a disease. The "effective dose" will vary depending on the ANKRD50 gene inhibitor, the disease and its severity, and the age and weight of the subject with the disease to be treated or prevented. When administered alone to an individual, the therapeutically effective dose refers only to that component. When administered in combination, the effective dose refers to the combined amount of the active ingredients that cause the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. The effective dose of the therapeutic agent will result in an increase of at least 10% in diagnostic criteria or parameters, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0126] In another aspect, the present invention provides a pharmaceutical combination comprising an ANKRD50 gene inhibitor and at least one other drug (therapeutic agent) for treating cancer, preferably pancreatic cancer, more preferably pancreatic ductal carcinoma.

[0127] In some implementations, other drugs may be small molecule drugs, antibodies, antibody fragments, or other dsRNA, siRNA, etc.

[0128] Delivery and Use of ANKRD50 Gene Inhibitors

[0129] The ANKRD50 gene inhibitor of the present invention can be delivered to cells, such as cells in a subject (e.g., a subject with a metabolic disorder), in a variety of ways. For example, delivery can be performed by contacting cells with the ANKRD50 gene inhibitor of the present invention in vitro or in vivo. In vivo delivery can also be performed directly by administering to a subject a composition (e.g., a pharmaceutical composition) containing an ANKRD50 gene inhibitor (e.g., dsRNA, shRNA, or siRNA). Alternatively, in vivo delivery can be performed indirectly by administering at least one vector encoding and directing the expression of the ANKRD50 gene inhibitor.

[0130] In one implementation, the cells are pancreatic cells.

[0131] Another aspect of the invention relates to a method for reducing the expression and / or activity of the ANKRD50 gene in a subject, comprising administering an ANKRD50 gene inhibitor of the invention to the subject. In some embodiments, the method comprises administering a therapeutically effective amount of the ANKRD50 gene inhibitor of the invention to the subject, thereby inhibiting or reducing the expression of the ANKRD50 gene inhibitor gene in the subject (e.g., cells in the subject). In some embodiments, the method comprises contacting cells with a double-stranded RNA agent of the invention, such that the expression of the ANKRD50 gene is inhibited or reduced in the cells. In some such embodiments, the mRNA transcript of a target gene, such as the ANKRD50 gene, is degraded in the subject or cells, thereby inhibiting or reducing the expression of the ANKRD50 gene in the subject or cells.

[0132] In another aspect, the present invention relates to a method for treating a subject with pancreatic cancer, comprising administering to the subject a therapeutically effective amount of the ANKRD50 gene inhibitor disclosed herein, thereby treating the subject.

[0133] In some embodiments of the present invention, inhibiting the expression of the ANKRD50 gene in a subject or cell reduces the ANKRD50 protein level in the subject's serum by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0134] In some embodiments of the invention, the method further includes determining ANKRD50 levels in samples from a subject, such as in blood, serum, pancreatic tissue, or adipose tissue samples. ANKRD50 levels in samples from a subject can be measured before, during, and / or after administration of an ANKRD50 gene inhibitor (e.g., to monitor efficacy or treatment efficiency, to monitor ANKRD50 mRNA and / or protein levels before, during, or after treatment, etc.).

[0135] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific embodiments indicate preferred embodiments of the invention, these descriptions and embodiments are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in citations, are incorporated herein by reference in their entirety.

[0136] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0137] Example

[0138] The present invention will be more readily understood by referring to the following embodiments, which are used to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way.

[0139] Unless otherwise defined or explicitly stated in the context, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention. Unless otherwise stated, all materials and instruments used in this invention are commercially available.

[0140] Example 1: Analysis of the intrinsic driving forces behind the liquid-liquid phase separation tendency of ANKRD50

[0141] Experiment 1.1: Using the machine learning method DeePhase (which, after training, can distinguish the tendency of different protein and peptide sequences to undergo isomorphic phase separation and output a tendency score for each input sequence; the higher the score, the more likely phase separation will occur; for detailed information on DeePhase, please refer to "Learning the molecular grammar of protein condensates from sequence") https: / / doi.org / 10.1073 / pnas.2019053118 Assess the phase separation tendency score of the ANKRD50 sequence and obtain Figure 1 Figure A in the diagram. Further research will be conducted to determine how some key physical characteristics of the ANKRD50 sequence (such as fluidity, reversibility, and fusion) define its phase separation tendency.

[0142] Results: DeePhase assessment revealed a phase separation tendency score of 0.82 for ANKRD50, indicating a high probability of phase separation. Further analysis... Figure 1 Figure A shows that the IDR near the C-terminus and a relatively large proportion of polar and cationic residues throughout the sequence contribute the most to the phase separation tendency fraction, especially in the low complexity and disordered regions. Furthermore, the presence of an intrinsically disordered sequence (IDR) in domain 3&4 (D3&4, ANKRD50-C-terminus) suggests that the driving force behind the phase separation process may reside in this region.

[0143] Experiment 1.2: EGFP-ANKRD50-F and EGFP-ANKRD50-C were overexpressed in Capan cells under a strong CMV promoter. The expression was observed using a Leica TCS SP8 laser confocal microscope (emission wavelength 509 nm, excitation wavelength 488 nm). The resulting ANKRD50 bodies were then subjected to a photobleaching and fluorescence recovery after photoextraction (FRAP) experiment (to obtain...). Figure 1 Figure C in the image shows the results observed under a Leica laser confocal microscope at 1, 2, and 3 seconds before and after bleaching, using the same conditions (emission wavelength 509 nm, excitation wavelength 488 nm). Figure 1 Figure B in the diagram.

[0144] Result: As Figure 1 As shown in Figure B, ANKRD50 forms granular aggregates (referred to as ANKRD50 bodies, i.e., ANKRD50 phase-separated bodies). Figure 1 As shown in Figure C, the recovery time of the ANKRD50 body is fast (average t). 1 / 2 =1.908 seconds), with a high movement score (80.09%), thus proving that the ANKRD50 body exhibits dynamic liquid-like behavior.

[0145] Experiment 1.3: Expression and purification of domain 3&4 (D3&4, A50-C-terminus) peptide of human ANKRD50. ANKRD50-D3&4 peptide solutions at concentrations of 1 μM, 10 μM, and 20 μM were treated with physiological saline at room temperature. Additionally, a 1 μM ANKRD50-D3&4 peptide solution was treated with 0%, 2.5%, 10%, and 20% Polyethylene Glycol 8000 (PEG) solutions. The phase separation characteristics were observed under a Leica laser confocal microscope (Leica TCS SP8) to obtain... Figure 1 The D and E diagrams in the diagram.

[0146] Result: As Figure 1 As shown in Figure D, the phase separation formation ability increases with increasing concentration of the ANKRD50 C-terminal (D3&4) peptides; simultaneously, the phase separation formation ability mediated by the ANKRD50 C-terminal peptides also increases with increasing concentration of Polyethylene Glycol 8000 (PEG). Furthermore, as... Figure 1As shown in Figure E, the phase-separated particles mediated by the ANKRD50-C-terminal D3&4 peptide can fuse. These results indicate that the ANKRD50 D3&4 peptide containing the IDR exhibits fluidity, reversibility, and fusion characteristics, confirming the liquid-liquid phase separation properties of ANKRD50. Therefore, ANKRD50-D3&4 can be considered a key sequence driving the formation of LLPS and regulating glucose transport by ANKRD50 (its nucleotide sequence is shown in SEQ ID No.:15).

[0147] Example 2: Verification of the relationship between ANKRD50 expression and pancreatic cancer

[0148] Experiment 2.1: Gene set enrichment analysis (GSEA) was performed on pancreatic ductal adenocarcinoma tissue and adjacent normal tissue. Using the GSEA website, keywords (such as GLUCOSE TRANSPORT, GLUCOSE_IMPORT, GLUCOSE-IMPORT-ACROSS-PLASMA, etc.) were entered to obtain gene sets related to glucose transmembrane transport regulation. Sequencing data from multiple pancreatic cancer GEO databases (GSE62165, GSE15471, GSE16515, and GSE28735) were used to validate gene expression and identify gene sets closely related to glucose transmembrane transport regulation in pancreatic cancer. Figure 2 Figure A in the diagram.

[0149] Results: Gene set enrichment analysis (GSEA) identified 175 gene clusters potentially involved in the regulation of glucose transmembrane transport, with the ANKRD50 gene being the target. The results of gene expression validation are as follows: Figure 2 As shown in Figure A, the expression level of ANKRD50 mRNA in pancreatic ductal adenocarcinoma tissue was significantly upregulated compared with that in adjacent normal tissue (***p<0.001).

[0150] Experiment 2.2: Survival analysis of a cohort of pancreatic ductal adenocarcinoma patients based on ANKRD50 expression was performed using the Cancer Genome Atlas (TCGA) database. ANKRD50 expression was divided into a low-expression group (samples scored "-, 0" and "+, 1") and a high-expression group (samples scored "++, 2" and "+++, 3") using a four-part classification. Figure 2 Figure B in the diagram.

[0151] Result: As Figure 2As shown in Figure B, patients with high ANKRD50 gene expression had worse overall survival (OS) and disease-free survival (DFS), p<0.05, indicating that high ANKRD50 gene expression is associated with poor prognosis in patients with pancreatic ductal carcinoma.

[0152] Experiment 2.3: Pancreatic ductal adenocarcinoma (PDAC) tissue and paired adjacent normal tissue were obtained from the Department of Hepatobiliary and Pancreatic Surgery, Renji Hospital, Shanghai Jiao Tong University. Immunohistochemical staining was performed on the PDAC tissue and paired adjacent normal tissue. The expression level of ANKRD50 was determined based on the staining intensity. Multiple observers were used to assess the consistency of the assessment. Figure 2 Figure C in the diagram.

[0153] Result: As Figure 2 As shown in Figure C, ANKRD50 protein expression was found to be significantly higher in human pancreatic cancer tissues than in paired adjacent normal tissues.

[0154] Experiment 2.4: Overall survival prognostic analysis was performed on patients in the low expression group and high expression group from Experiment 2.2, and the results were obtained. Figure 2 The D diagram in the image.

[0155] Result: As Figure 2 As shown in Figure D, pancreatic cancer patients with high ANKRD50 expression had shorter overall survival (p<0.05), further confirming that high ANKRD50 gene expression is associated with poor prognosis in patients with pancreatic ductal carcinoma.

[0156] Experiment 2.5: Immunohistochemical staining was performed on pancreatic tissue (NP) from normal mice, low-grade and high-grade pancreatic ductal intraepithelial neoplasia tissues (PanIN-1 / 2, PanIN-2 / 3), and pancreatic ductal adenocarcinoma tissue (PDAC) using a spontaneous pancreatic cancer KPC model. ANKRD50 expression levels were determined based on staining intensity, and the consistency of the assessments was evaluated by multiple observers. Figure 2 Figure E in the diagram.

[0157] Result: As Figure 2 As shown in Figure E, the expression levels of ANKRD50 protein in low-grade, high-grade pancreatic ductal intraepithelial neoplasia tissue and pancreatic ductal adenocarcinoma tissue of mice were significantly upregulated compared with normal mouse pancreatic tissue, and showed an increasing trend.

[0158] Conclusion: The above experimental results indicate that the expression of ANKRD50 is closely related to the development and prognosis of pancreatic cancer.

[0159] Example 3: In vitro experimental test of the effect of inhibiting ANKRD50 expression on the proliferation of pancreatic cancer cells.

[0160] Experiment 3.1: Pancreatic cancer cell lines PANC-1, AsPC-1, BXPC-3, SW1990, Capan-1, Patu8988, CFPAC-1, and the control cell line HPDE were selected. Western blotting (WB) was used to detect the expression level of ANKRD50 in each cell line. Figure 3 Figure A in the diagram.

[0161] Result: As Figure 3 As shown in Figure A, the expression level of ANKRD50 in pancreatic cancer cell lines was significantly higher than that in control cell lines. Furthermore, the AsPC-1 and BXPC-3 cell lines exhibited the highest relative expression of ANKRD50; therefore, these two cell lines were selected for subsequent functional experiments.

[0162] Experiment 3.2: ANKRD50 expression was silenced in AsPC-1 and BXPC-3 cells by interfering with siRNAs (denoted as siANKRD50-1 and siANKRD50-2, respectively) as shown in SEQ ID No.:6 and 9. ANKRD50 expression in cells of the ANKRD50 interference group and the control group (without siRNA interference, siNC) was further analyzed by qPCR and Western blotting to verify the interference efficiency of the siRNAs. The qPCR data output was the Ct value (Cyclethreshold) of the target gene and the internal reference gene, i.e., the number of cycles required for the fluorescence signal to reach the detection threshold. Each sample was subjected to at least three biological and technical replicates. The relative expression level was calculated using the 2^(-ΔΔCt) method. Substituting the ΔΔCt value into the following formula, the relative expression level of ANKRD50 was obtained: Relative expression level = 2 - ΔΔCt. Figure 3 Figures B and C in the diagram.

[0163] Result: As Figure 3 As shown in Figures B and C, in AsPC-1 and BXPC-3 cells, the expression level of ANKRD50 was significantly decreased in cells treated with siRNA interference compared with the control group (***p<0.001).

[0164] Experiment 3.3: CCK-8 cell viability assays were performed on cells from the interference group and the control group. Cell viability was reflected by measuring absorbance (OD value, wavelength 450 nm, after subtracting background signal from blank control wells). Higher OD values ​​indicated more viable cells. Each sample was subjected to at least three biological and technical replicates to obtain... Figure 3 The D diagram in the image.

[0165] Result: As Figure 3As shown in Figure D, the viability of AsPC-1 and BXPC-3 cells in the interference group was significantly lower than that in the control group (*p<0.05, **p<0.01), indicating that interfering with ANKRD50 expression can inhibit cell activity.

[0166] Experiment 3.4: Colony formation experiments were performed on cells from the interference group and the control group. The number of colonies was counted to reflect changes in cell proliferation capacity or viability; a cell colony is typically defined as a cluster containing >50 cells. At least three biological replicates were set up for each group to ensure the reliability of the experimental results. Images of the culture dishes were taken, and the number of colonies was counted manually or using software (ImageJ). Figure 3 Figure E in the diagram.

[0167] Result: As Figure 3 As shown in Figure E, the number of cell clones in the interference group was significantly lower than that in the control group (p<0.001), indicating that interfering with the expression of ANKRD50 can inhibit the clonal formation of pancreatic cancer cells.

[0168] Conclusion: The results of the above in vitro cell experiments indicate that ANKRD50 is closely related to the proliferation and colony formation of pancreatic ductal adenocarcinoma cells, and interfering with the expression of ANKRD50 can inhibit the proliferation and colony formation of pancreatic cancer cells.

[0169] Example 4: In vivo experimental test of the effect of inhibiting ANKRD50 expression on pancreatic cancer in mice.

[0170] Based on the results of previous in vitro cell function experiments, the effect of ANKRD50 on the progression of pancreatic ductal adenocarcinoma was further investigated in mice.

[0171] Experiment 4.1: A silencing ANKRD50 cell lines were constructed by infecting AsPC-1 and BXPC-3 cells with a lentivirus stably expressing interfering ANKRD50 shRNA (as shown in SEQ ID No. 9). A subcutaneous pancreatic cancer tumor model was established by subcutaneously seeding control cells (shNC) of AsPC-1 and BXPC-3 and ANKRD50 interfering cells (shANKRD50) into the subcutaneous tissue around the groin of nude mice. After 7 weeks, the subcutaneous tumors were harvested for observation and their volume and weight were measured.

[0172] Result: As Figure 4 As shown in Figures A and B, the tumor volume and weight in the interference group were significantly smaller than those in the control group (**p<0.01, ***p<0.001). Tumor volume change curves revealed that tumor growth in the interference group was significantly slower than in the control group (**p<0.01, ***p<0.001).

[0173] Experiment 4.2: An orthotopic pancreatic cancer model was constructed. Using the orthotopic pancreatic seeding method, AsPC-1 and BXPC-3 interference group and control group cells were inoculated into the orthotopic pancreas of mice, respectively. The progression of the orthotopic tumor was periodically monitored using small animal imaging, and subcutaneous tumors were harvested for observation and testing of their volume, weight, and survival rate.

[0174] Results: Imaging results are as follows Figure 4 As shown in Figures C and F, tumor progression in the interference group was significantly slower than in the control group. The prognosis of the mice was statistically analyzed, and tumor samples were extracted for testing; the results are as follows. Figure 4 As shown in Figures D, E, G, and H, the tumor volume and weight in the interference group were significantly smaller than those in the control group, and the interference group had a better survival rate than the control group. This indicates that interfering with ANKRD50 expression can significantly inhibit the progression of pancreatic carcinoma in situ and relatively improve the prognosis of pancreatic carcinoma in situ (*p<0.05, **p<0.01, ***p<0.001).

[0175] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. Use of an ANKRD50 gene inhibitor in the preparation of a medicament for the treatment of cancer, preferably pancreatic cancer, more preferably pancreatic ductal carcinoma, in subjects in need, preferably in humans, wherein the ANKRD50 gene inhibitor refers to a molecule or preparation that has an inhibitory effect on the ANKRD50 gene, prepared or screened with the ANKRD50 gene as a target.

2. The use according to claim 1, wherein, The ANKRD50 gene inhibitor is in the form of nucleic acid molecules, nucleic acid constructs, viral vectors, viral particles, antibodies, or small molecule compounds, preferably in the form of nucleic acid molecules.

3. The use as described in claim 1 or 2, wherein, When the ANKRD50 gene inhibitor is in the form of a nucleic acid molecule, the nucleic acid molecule is a double-stranded RNA or shRNA.

4. The use according to claim 3, wherein, The target sequence of the ANKRD50 gene for the action of the nucleic acid molecule is shown in any one of SEQ ID No.:1-2, preferably as shown in SEQ ID No.:

1.

5. A nucleic acid molecule used as an inhibitor of the ANKRD50 gene, wherein, The nucleic acid molecules include: A double-stranded RNA containing a nucleotide sequence capable of hybridizing with the ANKRD50 gene under stringent conditions, wherein preferably, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand being complementary to form an RNA dimer, and the sequence of the first strand being identical to the target sequence of the ANKRD50 gene; or The shRNA contains a nucleotide sequence capable of hybridizing with the ANKRD50 gene under harsh conditions. Preferably, the shRNA comprises a sense strand and an antisense strand, and a stem-loop structure connecting the sense strand and the antisense strand, the sequences of the sense strand and the antisense strand being complementary, and the sequence of the sense strand being identical to the target sequence of the ANKRD50 gene.

6. The nucleic acid molecule according to claim 5, wherein, The double-stranded RNA is a small interfering RNA (siRNA); and / or The nucleotide sequence of the double-stranded RNA or the shRNA is shown in any of SEQ ID No.:3-14.

7. A nucleic acid construct for use as an inhibitor of the ANKRD50 gene, comprising the nucleic acid molecule of claim 5 or 6.

8. The nucleic acid construct according to claim 7, wherein the nucleic acid construct further includes a vector and an optional promoter sequence.

9. A viral vector for use as an inhibitor of the ANKRD50 gene, comprising the nucleic acid construct of claim 7 or 8.

10. A viral particle used as an inhibitor of the ANKRD50 gene, comprising the viral vector according to claim 9.

11. A pharmaceutical composition for treating cancer, preferably pancreatic cancer, more preferably pancreatic ductal carcinoma, wherein, The pharmaceutical composition includes an ANKRD50 gene inhibitor, optionally at least one other drug for treating cancer, preferably pancreatic cancer, more preferably pancreatic ductal carcinoma, and optionally a pharmaceutically acceptable excipient.

12. A drug combination comprising an ANKRD50 gene inhibitor and at least one other drug for treating cancer, preferably pancreatic cancer, more preferably pancreatic ductal carcinoma, wherein the ANKRD50 gene inhibitor is preferably administered simultaneously or sequentially with the other drug.

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