Use of nucleic acid in preparation of drug for preventing and treating cachexia and administration method therefor

Abstract

Use of a nucleic acid in the preparation of a drug for preventing and treating cachexia and an administration method therefor. The nucleic acid regulates in vivo cachexia-related factors by means of an oligonucleotide that the nucleic acid comprises or produces, initiates an autologous reprogramming state, regulates the activation / silencing of a key nucleic acid factor to enable a non-coding RNA regulatory signal programming network, and achieves the purpose of a single non-coding RNA regulating multiple signaling pathways, thereby enabling the establishment of new homeostasis from a dysregulated physiological state.

Description

Application and administration methods of nucleic acids in the preparation of drugs for the prevention and treatment of cachexia

[0001] This invention claims priority to Chinese Patent Application No. 202410817427.4, filed on June 24, 2024, entitled “Application and method of application of nucleic acid in preparation of drug for prevention and treatment of cachexia”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedical technology, and specifically relates to the application and administration method of nucleic acids in the preparation of drugs for the prevention and treatment of cachexia. Background Technology

[0003] Cachexia is a complex metabolic syndrome characterized by the consumption of the body's own tissues. It can occur in various diseases, such as cancer, AIDS, severe trauma, postoperative conditions, malabsorption, and sepsis. The most common type is cancer-associated cachexia, also known as tumor cachexia. Key clinical symptoms of cachexia include systemic inflammation progression, a hypoxic tumor microenvironment leading to a glycolytic metabolic pattern marked by the Warburg effect, insomnia, loss of appetite, weight loss due to muscle wasting, general weakness, and metabolic abnormalities.

[0004] Regarding cancer cachexia, the General Office of the National Health Commission of China issued the "Guidelines for Clinical Diagnosis and Treatment of Cancer Cachexia (2020 Edition)," which points out that there is a lack of treatment options and the treatment effects are poor. Randomized clinical trials have shown that appetite stimulants such as progestin analogs, 5-hydroxytryptamine (5-HT) antagonists, and anti-inflammatory drugs such as cyclooxygenase-2 (COX-2) inhibitors cannot completely reverse the abnormal state of cancer cachexia and are accompanied by risks such as thromboembolism. Furthermore, anti-tumor treatments such as radiotherapy and chemotherapy also exacerbate a key symptom of cachexia: muscle wasting.

[0005] Muscle loss and weight reduction are important indicators in the diagnosis and treatment of cachexia. Currently, reversing weight loss in patients with cancer cachexia, thereby aiding in the treatment of the condition and improving the quality of life for end-stage cancer patients, is a hot research topic in cancer treatment centered on patient interests. Summary of the Invention

[0006] To address at least some of the problems in the prior art, this invention demonstrates that the nucleic acid sequence of the invention can restore normal A2AR expression and treat cachexia. A seed sequence is creatively selected, and the nucleic acid sequence is designed to act as a "sponge," restoring the function of the A2AR gene in cachexia patients for prevention and treatment. This invention designs and prepares a nucleic acid sequence capable of preventing and treating cachexia, which contains or can be produced in vivo as oligonucleotides and exists in cells and the circulatory system, simultaneously restoring A2AR gene expression, further regulating the expression of related pathway proteins, and ultimately significantly altering cachexia. In vitro experiments demonstrate that the nucleotide sequence of this invention can restore the A2AR gene, and in vivo experimental models confirm the efficacy of this oligonucleotide in treating cachexia. Specifically, this invention includes the following:

[0007] In a first aspect, the present invention provides an oligonucleotide, said oligonucleotide being an oligonucleotide of the following (1) and / or (2):

[0008] (1) Oligonucleotides with sequences as shown in any of SEQ ID NO.1-42;

[0009] (2) The oligonucleotides in (1) above that have been modified with nucleotides and have the same function.

[0010] Specifically, the oligonucleotide is a sequence as shown in any one of SEQ ID NO. 1, 2, 4, 5, 9, 13, 14, 15, 19, 22, 23, 25, 26, 30, 34, 35, 36 or 40.

[0011] Specifically, the nucleotide modification includes modification of phosphate groups, ribose groups, and / or conjugated links; wherein, the modification of phosphate groups refers to modification of the oxygen in the phosphate group, including thiolation and boronization, thereby stabilizing the structure of nucleic acids; the modification of ribose groups refers to modification of the 2'-hydroxyl group in the ribose group, including the introduction of methoxy or fluorine at the 2'-hydroxyl position of the ribose group, thereby making it difficult for ribonuclease to cleave nucleic acids and increasing the stability of nucleic acids; the conjugated link modification includes small molecule ligands, antibodies, peptides, or carbon chains.

[0012] In another aspect, the present invention provides an expression vector comprising any of the oligonucleotides described above.

[0013] In some embodiments, the expression vector is an animal cell expression vector.

[0014] In some embodiments, the expression vector is a recombinant plasmid, an AAV viral vector, a lentiviral vector, or a retroviral vector.

[0015] In another aspect, the present invention provides the use of nucleic acids in the preparation of medicaments for the prevention and treatment of cachexia, wherein the nucleic acids comprise or are capable of producing oligonucleotides of (1) and / or (2) below:

[0016] (1) Oligonucleotides with sequences as shown in any one of SEQ ID NO.1-42;

[0017] (2) The oligonucleotides in (1) above that have been modified with nucleotides and have the same function.

[0018] In some embodiments, according to the application described in the present invention, the nucleotide modification includes modification of phosphate groups and / or ribose groups and / or conjugated links;

[0019] The modification of the phosphate group refers to any modification of the phosphate group, including thiomodification and boronization, to stabilize the structure of nucleic acids.

[0020] The modification of the ribosome group refers to any modification to the ribosome group, including the introduction of a methoxy group or fluorine at the 2'-hydroxyl position of the ribosome group, thereby making it difficult for ribonuclease to cleave nucleic acids and increasing the stability of nucleic acids.

[0021] The conjugated linking modification includes small molecule ligands, antibodies, peptides, carbon chains, and other molecules.

[0022] In some embodiments, according to the application described in the present invention, the nucleic acid capable of producing (1) and / or (2) oligonucleotides is an animal cell expression vector.

[0023] In some embodiments, according to the application described in the present invention, the vector used to generate the oligonucleotides (1) and / or (2) is a recombinant plasmid, an AAV viral vector, a lentiviral vector, or a retroviral vector.

[0024] In some embodiments, according to the application described in the invention, the oligonucleotide is contained in extracellular vesicles.

[0025] In some embodiments, according to the application described in the present invention, the dosage form of the drug includes injections, powders for injection, tablets, ointments, capsules, granules, aerosols, sprays, or powder inhalers.

[0026] In some embodiments, according to the application described in the invention, the drug is administered via systemic administration, including subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration, or sustained-release administration.

[0027] In some embodiments, according to the application described in the invention, the drug is designed to be administered at a dose of 4 μg-60 μg / person / dose, based on a body weight of 60 kg.

[0028] In some embodiments, according to the application described in the invention, the drug is designed to be administered at an amount of 0.005-1 μg / kg body weight of the oligonucleotide.

[0029] In some embodiments, according to the application described in this invention, the drug is designed to achieve an effective concentration of 1 × 10⁻⁶ oligonucleotides in the in vivo circulation system. 4 -1×10 9 Copy / μL, or the drug is designed to achieve an effective concentration of the oligonucleotide in the target organ of 1×10⁻⁶. 4 -1×10 9 Copy / mg.

[0030] In some embodiments, according to the application described in the present invention, the prevention and treatment include: preventing, alleviating, reversing, improving, or curing cachexia or its symptoms.

[0031] In some embodiments, according to the application described in the present invention, the cachexia includes one or more of the following stages: pre-cachexia, cachexia stage, and refractory cachexia stage caused by tumor development.

[0032] In some implementations, the tumor includes solid tumors or non-solid tumors;

[0033] The solid tumors include: gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, esophageal cancer, liver cancer, breast cancer, melanoma, osteosarcoma, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, bladder cancer, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, laryngeal cancer, kidney cancer, head and neck tumors, testicular cancer, thyroid cancer, or brain cancer.

[0034] The non-solid tumors include leukemia or lymphoma.

[0035] In some implementations, the tumor includes stomach cancer, lung cancer, colorectal cancer, pancreatic cancer, and liver cancer.

[0036] In some embodiments, according to the application described in the present invention, the symptoms of cachexia include muscle wasting, weight loss, lethargy, insomnia, general weakness, metabolic abnormalities, decreased appetite and / or reduced activity.

[0037] In another aspect, the present invention provides a method for in vitro regulation of cell proliferation, comprising the step of contacting in vitro cells with the oligonucleotides described in the present invention. Preferably, the in vitro cells comprise tumor cells or immortalized cells.

[0038] In another aspect, the present invention provides a method for administering a nucleic acid-containing drug, wherein the nucleic acid comprises or is capable of producing oligonucleotides of (1) and / or (2) below:

[0039] (1) Oligonucleotides with sequences as shown in any one of SEQ ID NO.1-42;

[0040] (2) The oligonucleotides in (1) above that have undergone nucleotide modification and have the same function;

[0041] The method includes administering the drug via systemic administration.

[0042] In some embodiments, the method of administration of the nucleic acid-containing drug according to the present invention includes systemic administration by subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration, or sustained-release administration.

[0043] In some embodiments, according to the method of administration of the nucleic acid-containing drug according to the present invention, the drug is administered in a dose of 4 μg-60 μg / person / dose, based on a body weight of 60 kg, to deliver the oligonucleotide into the body.

[0044] In some embodiments, according to the method of administration of the nucleic acid-containing drug according to the present invention, the drug is capable of delivering the oligonucleotide into the body at a dose of 5 μg-50 μg / person / dose, based on a body weight of 60 kg.

[0045] In some embodiments, according to the method of administering a nucleic acid-containing drug according to the present invention, the drug enables the oligonucleotide to achieve an effective concentration of 1 × 10⁻⁶ in the in vivo circulation system. 4 -1×10 9 The number of copies / μL, or the concentration of the oligonucleotide effectively in the target organ, is 1 × 10⁻⁶. 4 -1×10 9 Copy / mg.

[0046] The nucleic acid of this invention regulates cachexia-related nucleic acid factors in vivo through its contained or generated oligonucleotides, initiating a self-reprogramming state. By regulating the activation / silencing of key nucleic acid factors, it achieves non-coding RNA regulation of signaling programming networks, enabling a single non-coding RNA to regulate multiple signaling pathways, thereby restoring a new homeostasis of disordered physiological states. Drugs using the nucleic acid of this invention as the active ingredient require extremely small dosages compared to other nucleic acid drugs during prevention and treatment, ensuring efficacy while maintaining safety, thus revolutionizing the traditional plasmid / nucleic acid drug administration. Furthermore, the nucleic acid of this invention exhibits no specific reactions and high in vivo safety. Attached Figure Description

[0047] Figure 1 illustrates the effect of exemplary oligonucleotide expression plasmids on the expression of target oligonucleotides in SK-Hep-1, HepG2, and H292 cell lines. In the figure, (a) SEQ ID NO.19 oligonucleotide expression plasmid in the SK-Hep-1 cell line; (b) SEQ ID NO.5 oligonucleotide expression plasmid in the HepG2 cell line; and (c) SEQ ID NO.5 oligonucleotide expression plasmid in the H292 cell line.

[0048] Figure 2 shows the effect of exemplary oligonucleotides on A2AR mRNA expression levels in different cell lines. In the figure, (a) changes in A2AR mRNA expression levels over time in the MKN45 cell line; (b) the fold upregulation of A2AR mRNA in the three different cell lines compared to the control group.

[0049] Figure 3 shows the effect of exemplary oligonucleotides on the expression level of A2AR protein in CFPAC-1, CW-2, and BxPC-3 cell lines. In the figure, (a) A2AR protein expression in CFPAC-1 and CW-2 cell lines was upregulated by fold compared to the control group; in the figure, (b) A2AR protein expression in CW-2 and BxPC-3 cell lines was upregulated by fold compared to the control group; and in the figure, (c) A2AR protein expression in BxPC-3 and SW620 cell lines was upregulated by fold compared to the control group.

[0050] Figure 4 shows the effect of modified exemplary oligonucleotides on the expression level of A2AR protein in SW620 and CFPAC-1 cell lines.

[0051] Figure 5 shows the effects of exemplary oligonucleotides on lung cancer cachexia in nude mice. In the figure, (a) A2AR protein expression level in tumor tissue; (b) weight gain rate; (c) food intake; (d) water intake; (e) tumor volume; (f) number of tumors; and (g) tumor inhibition rate.

[0052] Figure 6 illustrates the effects of exemplary oligonucleotides on lung cancer cachexia in nude mice. In the figure, (a) food intake; (b) water intake; (c) weight gain in the oligonucleotide group compared to the control group; (d) number of tumors; (e) tumor volume; and (f) tumor inhibition rate.

[0053] Figure 7 illustrates the effects of exemplary oligonucleotides on nude mice with pancreatic cancer cachexia. In the figure, (a) survival rate; (b) food intake; (c) clinical observation score; and (d) weight gain rate.

[0054] Figure 8 shows the effects of exemplary oligonucleotides on nude mice with colorectal cancer cachexia. In the figure, (a) is the rate of weight gain; and (b) is the tumor inhibition rate. Detailed Implementation

[0055] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0056] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0057] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0058] Experimental methods not specified in the specific embodiments are generally performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0059] Research has found that cells actively secrete "nucleic acid factors" extracellularly through exocytosis, globally altering the body's immunity and metabolism. These actively secreted "nucleic acid factors," like cytokines and chemical factors, participate in in vivo reprogramming and systemic regulation processes. This invention refers to the mechanism of endogenous "nucleic acid factors" in drug delivery, restoring the suppressed expression of genes in cachexia, resolving the physiological imbalance in cachexia patients, improving cachexia symptoms, especially tumor cachexia, and enhancing patients' quality of life.

[0060] In this invention, the term "cachexia" refers to a complex metabolic clinical syndrome in which the patient's own tissues are consumed. This type of disease is characterized by decreased appetite, weight loss, general weakness, and metabolic abnormalities, and can occur in various diseases, such as cardiovascular disease, cancer, AIDS, severe trauma, postoperative conditions, malabsorption, and sepsis. Cachexia is generally recognized in the art as being characterized by persistent skeletal muscle wasting, with or without adipose tissue loss, and cannot be completely relieved by conventional nutritional therapy. Currently, the only officially marketed drug for cachexia is Anamorelin, manufactured by Ono Pharmaceutical Co., Ltd. in Japan. It aims to improve cachexia by increasing appetite, but its marketing application in the European Union has not been approved, and it is currently in Phase I clinical trials in my country.

[0061] In this invention, the term "oligonucleotide" refers to a molecule composed of multiple linked nucleotides, each nucleotide containing a sugar (e.g., ribose or deoxyribose) and an organic base linked thereto. The organic base includes pyrimidines (e.g., cytosine (C), thymine (T), or uracil (U)) or purines (e.g., adenine (A) or guanine (G)). Based on the different sugar groups, oligonucleotides are classified as DNA and RNA. In this invention, oligonucleotides may be unmodified or modified. Modification types include base modifications, sugar modifications, and backbone modifications. In some embodiments, the oligonucleotides of this invention contain a phosphate thioester backbone modification, thereby making the oligonucleotide resistant to the effects of nucleases and improving its stability. The preparation method of the oligonucleotides of this invention is not limited; they can be synthesized in vitro or obtained through biotechnology or bioengineering methods. Specific methods are also known in the art.

[0062] In some embodiments, the oligonucleotides of the present invention are genes whose expression was suppressed under cachexia. The oligonucleotide "seed" sequence is: 5'→3': UCUGUGCC or TCTGTGCC; its reverse complementary sequence is: 5'→3': GGCACAGA; correspondingly, there may be a random 0-8 nt before the designed "seed" sequence and a random 6-15 nt after it. Exemplarily, the oligonucleotides of the present invention have the sequences shown in SEQ ID NO. 1-42:

[0063] In this invention, the term "nucleic acid" refers to a linear fragment of single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be produced from any source. The length of the nucleic acids in this invention is not limited, and includes both long-chain and short-chain nucleic acids. Long-chain nucleic acids generally consist of more than 200 nucleotides per strand, i.e., a single-chain length of more than 200 nt. Short-chain nucleic acids are sometimes also called oligonucleotides, which are generally in single-chain form and typically consist of 5-200 nucleotides in length, i.e., a length of 5-200 nt.

[0064] In some embodiments, the nucleic acid of the present invention may include the oligonucleotides described herein. For example, the nucleic acid of the present invention may be in the form of a nucleic acid construct. The nucleic acid of the present invention may include at least one oligonucleotide of the sequence shown herein, for example, it may include two, three, more than three or more oligonucleotides. In the case of multiple oligonucleotides shown herein, the linkage between the oligonucleotides is not particularly limited, for example, suitable spacer sequences may be used to link the oligonucleotides. Exemplarily, the nucleic acid comprises at least one oligonucleotide selected from SEQ ID NO. 1-42.

[0065] In some embodiments, the nucleic acid of the present invention is a nucleic acid molecule capable of producing the oligonucleotides of the present invention, for example, the oligonucleotides of the present invention are produced by transcription of the nucleic acid. In this case, the nucleic acid includes a nucleic acid expressing the sequence shown in any one of SEQ ID NO. 1-21. In some embodiments, the nucleic acid may be part of an expression vector, which refers to a recombinant or synthetically produced nucleic acid construct carrying a specific set of nucleic acid elements capable of transcribing a specific gene in a host cell, such as promoter elements, enhancer elements, and selection elements. Typically, gene expression is under the control of certain regulatory elements, such as constitutive or inducible promoters. In the present invention, the vector may be a plasmid, phage particle, virus (including but not limited to adenovirus, lentivirus), and other types of vectors derived from viral or bacterial sources. The gene sequence used to express the oligonucleotides of the present invention may be operatively linked to one or more elements of the vector, such as promoter elements. "Operationally linked" is used to describe the connection between a regulatory element and a gene or its coding region. That is, gene expression is typically under the control of certain regulatory elements, and "operatively linked" a gene or coding region to a regulatory element means that the gene or coding region is controlled or influenced by the regulatory element.

[0066] In this invention, oligonucleotides can be used in the form of "extracellular nucleic acids" for the prevention and treatment of cachexia. The oligonucleotides are isolated pure nucleic acid molecules that can exist stably in the extracellular environment, such as, but not limited to, blood, saliva, and gastric juice. This not only has therapeutic effects on in situ tumors, but also has important significance for metastatic or distant tumors, because the oligonucleotides of this invention can reach the target (target nucleic acid) through the circulatory system. This allows this novel systemic treatment to replace radiotherapy and chemotherapy drugs, thereby avoiding the toxic side effects of radiotherapy and chemotherapy on patients and enabling patients to benefit from the treatment of cachexia.

[0067] The term "separated" means a substance and / or entity isolated from at least some of the components originally associated with it (whether in nature or / or in an experimental setting), and / or artificially produced, prepared, and / or manufactured. The separated substance and / or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components originally associated with it. In some embodiments, the purity of the separated reagent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100%. If a substance is substantially free of other components, the substance is "pure". In addition, "isolated cells" refers to cells that are not included in multicellular organisms.

[0068] The present invention also provides a method for reversing, preventing, improving, or treating cachexia or related conditions in a subject in need, comprising the step of administering a therapeutically effective amount of nucleic acid to the subject in need, wherein the nucleic acid comprises or is capable of producing oligonucleotides as shown in any one of SEQ ID NO. 1-42. It is understood that the method can be therapeutic or preventative.

[0069] In a preferred embodiment of the application and method according to the present invention, the vector used to generate the nucleic acid containing the oligonucleotide sequence includes a recombinant plasmid, an AAV viral vector, a lentiviral vector, or a retroviral vector. In a specific embodiment, the vector used to generate the nucleic acid containing the oligonucleotide shown in any one of SEQ ID NO. 1-21 is an animal cell expression vector.

[0070] It is understood that the nucleic acids or oligonucleotides of the present invention may be further contained in suitable delivery carriers known in the art, such delivery carriers including, but not limited to: extracellular vesicles, particularly exosomes, microvesicles, etc.; nanoparticles such as chitosan, polylactic-co-glycolic acid copolymer (PLGA), dendritic macromolecules, etc.; typical liposomes, etc. In a preferred embodiment, the oligonucleotides of the present invention are contained in extracellular vesicles, such as exosomes.

[0071] In a preferred embodiment, the method of the present invention includes the step of administering an animal cell expression vector to a subject, wherein the vector is capable of producing oligonucleotides as shown in any one of SEQ ID NO. 1-21.

[0072] In a preferred embodiment, the step includes administering to a subject cells transformed with the vector that produces the oligonucleotide sequence. In one aspect, the introduction of the plasmid of the present invention into cells can be performed by any suitable method for introducing a vector into cells (e.g., gene transfer), such as transfection and transduction, and is not particularly limited thereto.

[0073] In a preferred embodiment, the method includes administering to a subject extracellular vesicles containing the oligonucleotide sequence, particularly exosomes (or exosome-like particles), wherein at least a portion of the membrane of the exosome or extracellular vesicle may be derived directly from cells or prepared artificially. The particle size of the exosome or extracellular vesicle is not particularly limited and may have a micrometer-scale or even nanometer-scale particle size.

[0074] In a preferred embodiment, cachexia or related conditions are reversed, prevented, improved or treated by administering a therapeutically effective amount of a recombinant plasmid containing any of the oligonucleotides shown in SEQ ID NO. 1-21 to a subject in need.

[0075] In this invention, pharmaceuticals comprising nucleic acids or oligonucleotides may further include pharmaceutically acceptable excipients. The term "pharmaceutically acceptable" refers to a carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to the doses and concentrations used, or has a level of toxicity that is acceptable to those skilled in the art. A non-limiting example of a pharmaceutically acceptable excipient is an aqueous pH buffer solution. Pharmaceutically acceptable excipients may also comprise one or more of the following: antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming antiions, such as sodium; and nonionic surfactants, such as Tween and polyethylene glycol (PEG).

[0076] Free from any theoretical constraints, the oligonucleotides of this invention may have the nucleotide sequences shown in any one of SEQ ID NO. 1-42 or sequences obtained by chemical modification thereof. Furthermore, variant sequences of the above sequences are also within the scope of protection of this invention. The term "variant sequence" refers to an oligonucleotide sequence that is not completely identical to the oligonucleotide sequence of this invention, but retains one or more functional characteristics of the oligonucleotide sequence of this invention. In some aspects, the oligonucleotide sequence or its chemically modified variant sequence retains all the functional characteristics of the oligonucleotide sequence, such as, but not limited to, binding activity to target genes, inhibition of target expression or inhibition of target cells, regulatory activity of downstream signaling pathways regulated by the target, and / or the efficacy of reversing, preventing, improving, or treating cachexia. Preferably, the modified oligonucleotides do not result in the loss of the function of the oligonucleotide in inhibiting nucleic acid factors and / or the loss of its function in inhibiting their expression. Regulatory activity of downstream signaling pathways regulated by the target further includes regulating the expression of related proteins, particularly proteins such as A2AR, ultimately significantly altering the glycolytic metabolic state characteristic of cachexia.

[0077] In this invention, the inhibitory activity of oligonucleotides on target expression or on target cells can be determined by methods known in the art, and there are no particular limitations. For example, real-time quantitative PCR, Northern blotting, and other methods can be used to quantify nucleic acid factors. As another example, CCK8 assay can be used to detect the inhibitory rate of oligonucleotides on target cells; the oligonucleotides of this invention all exhibit high inhibitory rates on target cells. Furthermore, the oligonucleotides of this invention, as shown in SEQ ID NO. 1-42, have a significant inhibitory effect on the expression of target genes (nucleic acid factors) and / or the function of nucleic acid factors.

[0078] Those skilled in the art will understand that the GC content can be optimized according to the sequences shown in SEQ ID NO.1-42 of the present invention to further improve the affinity of oligonucleotides for target sequences, thereby affecting their inhibition efficiency.

[0079] In this invention, chemical modification of oligonucleotides to improve their stability, activity, or half-life is known in the art. The oligonucleotides of this invention use nucleotide groups (or nucleotide residues) as basic structural units, wherein the nucleotide groups contain phosphate groups, ribose groups, and bases. Preferably, the oligonucleotides contain at least one modified nucleotide group. The modified nucleotide group does not cause the oligonucleotide to lose its ability to inhibit the function of nucleic acid factors and / or their expression.

[0080] In this invention, the modified nucleotide group is a nucleotide group in which the phosphate group and / or ribose group are modified. For example, modification of the phosphate group refers to any modification to the phosphate group, including but not limited to thiomodification, boronization, etc., such as replacing the oxygen in the phosphate group with sulfur and borane, respectively. Modification of the phosphate group can stabilize the structure of nucleic acids and maintain high specificity and high affinity of base pairing. Preferably, the thiomodified nucleotide group refers to a nucleotide group in which all non-bridging oxygen atoms in the phosphodiester bonds are replaced with sulfur atoms.

[0081] Ribosyl modification refers to any modification to the ribose group. For example, introducing substituents such as methoxy or fluorine at the 2'-hydroxyl position of the ribose group makes it more difficult for ribonucleases to cleave nucleic acids, thereby increasing the stability of the nucleic acid and giving it stronger resistance to nuclease hydrolysis. Modifications to the 2'-hydroxyl group in the pentose of nucleotides include 2'-fluorine modification, 2'-methoxy modification (2'-OME), 2'-methoxyethyl modification (2'-MOE), 2'-2,4-dinitrophenol modification (2'-DNP modification), locked nucleic acid modification (LNA modification), 2'-amino modification (2'-Amino modification), and 2'-deoxy modification (2'-Deoxy modification), etc.

[0082] In this invention, the variant sequences of the oligonucleotide sequence may include oligonucleotides with the same function, formed by substitution, deletion, and / or addition of one or more nucleotides of the nucleotide sequence shown in any one of SEQ ID NO. 1-42. Whether the variant sequences have the same function as the specified sequence under different conditions can be determined based on any specified sequence from the above sequences. In a specific embodiment, substitution, deletion, and / or addition refers to the substitution, deletion, and / or addition of one or more bases selected from A, G, C, and T.

[0083] In some embodiments, nucleotide insertion includes 5' and / or 3' end insertion, as well as intra-sequence insertion of one or more residues (including at least 1, 2, or 3 nucleotides).

[0084] In some implementations, nucleotide deletion refers to the removal of one or more residues from an oligonucleotide sequence. These deletion variants are typically prepared by site-specific mutagenesis of nucleotides in DNA encoding the oligonucleotide, resulting in DNA encoding the variant, and subsequently expressing the DNA in recombinant cell culture. Alternatively, deletion variants can be conveniently prepared by in vitro synthesis.

[0085] In some embodiments, nucleotide substitution involves the removal of, for example, at least one to at least three nucleotides from a sequence and the insertion of a different nucleotide at its position. While the sites used to introduce sequence variations are predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of mutations at a given site, random mutagenesis can be performed at the target region, and expressed substitution variants can be screened for the optimal combination of desired activities. Techniques for substitution mutations at predetermined sites in DNA having a known sequence are well known.

[0086] In this invention, nucleotide substitution is typically a single residue, nucleotide insertion is typically about 1-10 residues, and nucleotide deletion is typically about 1-30 residues.

[0087] In this invention, the variant sequences of the oligonucleotide sequence can be nucleotide sequences that have 95%, for example 96%, 97%, 98%, or even 99% or more homology with and have the same function as the nucleotide sequences shown in any one of SEQ ID NO. 1-42. Similarly, it can be determined whether the variant sequence having the above homology has the same function as the specified sequence based on any specified sequence from the above sequences.

[0088] To determine sequence homology (sometimes also called "identity"), sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0089] The dosage form of the drug or composition containing the nucleic acid or oligonucleotide of the present invention is not particularly limited, and may be selected from injections, powders for injection, tablets, ointments, capsules, granules, aerosols, sprays, or powder inhalers. In a preferred embodiment, the dosage form of the drug or composition containing the nucleic acid or oligonucleotide of the present invention is an injection.

[0090] In this invention, the route of administration of the drug or composition is not particularly limited, including but not limited to systemic administration by subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration or sustained-release administration.

[0091] In a preferred embodiment, the drug or composition is an injectable dosage form suitable for parenteral administration, particularly intravenous administration. In a most preferred embodiment, cachexia or related conditions are reversed, improved, or treated by administering a therapeutically effective amount of a recombinant plasmid vector containing oligonucleotides intravenously to a subject in need.

[0092] In this invention, the term "prevention" refers to therapeutic treatments and preventative or preventative measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as the progression of cachexia. This degree of relief or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). Those requiring treatment include individuals already suffering from cancer or cachexia-related diseases, or those requiring prevention or improvement of cancer or cachexia-related diseases.

[0093] The term "subject" as used in this invention refers to any animal (such as a mammal), including but not limited to humans, non-human primates, rodents, and the like, who are about to receive a specific treatment. Generally, "subject" and "patient" are used interchangeably in this invention, both referring to the subject of the study.

[0094] The term "therapeutic effective dose" as used in this invention refers to a pharmaceutically effective dosage, that is, an amount of active drug (i.e., nucleic acid or oligonucleotide) sufficient to significantly improve the condition without causing serious side effects. The dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the therapeutic dose of this invention can vary widely.

[0095] Although therapeutic doses can vary widely, the inventors have discovered that the applied nucleic acid or oligonucleotide can achieve effective treatment of cachexia at low doses. For an individual, such as a person weighing 60 kg, the daily dose of the nucleic acid or oligonucleotide is typically 4 μg / person / dose to 60 μg / person / dose, preferably 5-50 μg / person / dose. Exemplary effective dosages include, for example, 5 μg / person / dose, 6 μg / person / dose, 7 μg / person / dose, 8 μg / person / dose, 9 μg / person / dose, 10 μg / person / dose, 11 μg / person / dose, 12 μg / person / dose, 13 μg / person / dose, 14 μg / person / dose, 15 μg / person / dose, 16 μg / person / dose, 17 μg / person / dose, 18 μg / person / dose, 19 μg / person / dose, 20 μg / person / dose, 21 μg / person / dose, 22 μg / person / dose, 23 μg / person / dose, 24 μg / person / dose, 25 μg / person / dose, 26 μg / person / dose, 27 μg / person / dose. The dosages are as follows: 28 μg / person / dose, 29 μg / person / dose, 30 μg / person / dose, 31 μg / person / dose, 32 μg / person / dose, 33 μg / person / dose, 34 μg / person / dose, 35 μg / person / dose, 36 μg / person / dose, 37 μg / person / dose, 38 μg / person / dose, 39 μg / person / dose, 40 μg / person / dose, 41 μg / person / dose, 42 μg / person / dose, 43 μg / person / dose, 44 μg / person / dose, 45 μg / person / dose, 46 μg / person / dose, 47 μg / person / dose, 48 μg / person / dose, 49 μg / person / dose, and 50 μg / person / dose. It can be administered as a single dose once daily, divided into multiple doses daily, or at intervals.

[0096] In some embodiments, the daily dose of the nucleic acid or oligonucleotide is 0.005 μg / kg to 1 μg / kg, preferably 0.006 to 1 μg / kg. Exemplary effective dosages include 0.006 μg / kg, 0.007 μg / kg, 0.008 μg / kg, 0.009 μg / kg, 0.01 μg / kg, 0.02 μg / kg, 0.03 μg / kg, 0.04 μg / kg, 0.05 μg / kg, 0.06 μg / kg, 0 .07μg / kg, 0.08μg / kg, 0.09μg / kg, 0.1μg / kg, 0.2μg / kg, 0.3μg / kg, 0.4μg / kg, 0.5μg / kg, 0.6μg / kg, 0.7μg / kg, 0.8μg / kg, 0.9μg / kg, 1μg / kg.

[0097] In this invention, the administration of the drug enables the oligonucleotide to achieve an effective concentration of 1 × 10⁻⁶ in the in vivo circulatory system. 4 -1×10 9 copies / μL, preferably 2×104 -1×10 8 copies / μL, preferably 2×10 4 -1×10 7 copies / μL, for example 2×10 4 3×10 4 4×10 4 5×10 4 6×10 4 7×10 4 8×10 4 9×10 4 1×10 5 2×10 5 3×10 5 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 Copy / μL.

[0098] In this invention, the administration of the drug enables the oligonucleotide to achieve an effective concentration in the target organ of 1 × 10⁻⁶ based on the weight of the corresponding target organ. 4 -1×10 9 Copies / mg, preferably 2×10 4 -1×10 8 Copies / mg, preferably 2×10 4 -1×10 7 copies / mg, for example 2×10 4 3×10 4 4×10 4 5×10 4 6×10 4 7×10 4 8×10 4 9×10 4 1×10 5 2×10 5 3×10 5 4×10 5 5×105 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 1×10 7 Copy / mg.

[0099] In this invention, the nucleic acid or oligonucleotide can also be combined with other drugs to prepare a combined-administration pharmaceutical composition. Other drugs may or may not be any therapeutic agent for cachexia, and are not particularly limited thereto. It should be noted that the selected other drugs do not react with the nucleic acid or oligonucleotide of this invention in any undesirable way, resulting in mutual inhibitory activity. When combined with other drugs, such as chemotherapeutic agents, immune checkpoint inhibitors, antitumor drugs, and immunomodulatory agents, such as anti-PD-1 antibodies and anti-PD-L1 antibodies, the dosage of the other drugs depends on the specific drug type, the type of cachexia, and the stage of development. Each dose can be 0.5 mg / kg to 30 mg / kg, preferably 1 to 20 mg / kg; for example, for a person weighing 60 kg, each dose can typically be 1 mg to 1500 mg, such as 50 mg to 1200 mg, or 100 mg to 800 mg, 150 mg to 600 mg, or 200 mg to 500 mg. mg; exemplary dosages per administration include 50 mg, 100 mg, 120 mg, 160 mg, 180 mg, 200 mg, 240 mg, 260 mg, 300 mg, 320 mg, 360 mg, 400 mg, 500 mg, 600 mg, 800 mg, 1000 mg, 1200 mg, etc.; dosing intervals, such as once every 3-7 days or once every 1-5 weeks, for example, once every 3 days, once every 5 days, once every week, once every 10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, etc. The specific dosage and frequency should take into account factors such as the route of administration and the patient's health condition, which can be determined by a skilled physician using routine techniques.

[0100] In this invention, the tumors include, but are not limited to, solid tumors or non-solid tumors. Solid tumors include, but are not limited to, breast cancer, prostate cancer, melanoma, osteosarcoma, neuroblastoma, pancreatic cancer, lung cancer, rhabdomyosarcoma, Ewing sarcoma, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, laryngeal cancer, gastric cancer, kidney cancer, head and neck tumors, esophageal cancer, testicular cancer, thyroid cancer, or brain cancer. Non-solid tumors include, but are not limited to, hematologic malignancies such as leukemia and lymphoma, as well as tumors of the nervous system.

[0101] Example 1

[0102] The following examples, using lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, and liver cancer—tumor cells with a high incidence of cachexia—illustrate the culturing, transfection, and collection of cells.

[0103] Cell resuscitation: Quickly remove the desired cell line from the liquid nitrogen tank, place the cryovial in a 37-40℃ water bath and gently agitate until only a small amount of ice remains, then remove it; transfer the cell suspension from the cryovial to a 15mL centrifuge tube containing complete culture medium, centrifuge at 1200rpm for 10min, then transfer to a biosafety cabinet and aspirate the supernatant; resuspend the cells with an appropriate amount of complete culture medium, and then transfer them to a culture flask, observing uniform cell dispersion under a microscope; subsequently, transfer the culture flask to a CO2 incubator and culture at 37℃ with 5% CO2 until the cell confluence reaches 80-90%, then passage the cells.

[0104] Cell passage: Observe the cells in the culture flask under a microscope. When the confluence reaches 80-90%, transfer them to a biosafety cabinet. Aspirate the supernatant and wash 2-3 times with PBS. After aspirating the PBS, add trypsin to digest the cells. Observe the cells under a microscope when they shrink to a round shape, indicating that digestion is complete. Add an appropriate amount of culture medium and use a pipette to blow off the cells and collect the cell slurry into a centrifuge tube. Centrifuge the cell slurry at 1200 rpm for 10 min and aspirate the supernatant. Resuspend the cells in an appropriate amount of culture medium, aliquot the cells into culture flasks at a certain ratio, and transfer the culture flasks to a cell culture incubator at 37°C and 5% CO2 for culture. When the cell number and confluence reach the experimental requirements, proceed with subsequent experiments.

[0105] Cell plating: When the cell confluence in the culture flask reaches 80-90%, transfer the cells to a biosafety cabinet. Aspirate the culture medium and wash 2-3 times with PBS. After aspirating the PBS, add an appropriate amount of trypsin for digestion until the cells shrink to a round shape, then stop digestion. Add an appropriate amount of culture medium and gently pipette the cells to detach them. Collect the cell suspension in a centrifuge tube and centrifuge at 1200 rpm for 10 min. Aspirate the supernatant and resuspend the cells in an appropriate amount of culture medium. Transfer 20 μL of the cell suspension to a 1.5 mL EP tube, add trypan blue at a 1:1 ratio and mix well. Transfer 10 μL of the suspension to a hemocytometer and count the cells. Based on the counting results, transfer a certain amount of cell suspension to a 12-well plate and incubate the 12-well plate overnight at 37°C with 5% CO2.

[0106] Cell transfection: Prepare two centrifuge tubes, A and B. Add basal culture medium and Lipo3000 to tube A, invert to mix, and let stand for 5 min. Add P3000 and plasmid (buffered solvent for the control group, oligonucleotide expression plasmid for the experimental group, which is a plasmid solution that generates the target oligonucleotide through transcription) to tube B, invert to mix. After preparation (Lipo3000:plasmid:P3000 = 1 μL:0.5 μg:1 μL), mix reagents A and B and let stand for 15 min. Add the above mixture to a 12-well plate, take site-specific photographs, and then transfer to a cell culture incubator for culture at 37°C and 5% CO2.

[0107] Cell collection: Cell growth status of each treatment group was observed at 3h, 6h, 24h, 48h, and 72h post-transfection. At the sampling time points, the supernatant in the 12-well plates was aspirated and washed with PBS; trypsin was added to the 12-well plates for digestion, and after digestion, an appropriate amount of complete culture medium was added and the cells were pipetted evenly. Cell suspension from each group was collected; the cells were centrifuged at 1200 rpm for 10 min, the supernatant was aspirated, and an appropriate amount of PBS was added for resuspending and aliquoting. The cell suspension was stored at -80℃ for subsequent experiments.

[0108] Example 2

[0109] Oligonucleotide expression plasmids were designed using example sequences, and the expression plasmids were validated in vitro to transcribe example oligonucleotides in various human tumor cell lines ZHONG.

[0110] Taking the sequences SEQ ID NO.5 and SEQ ID NO.19 as examples, oligonucleotide expression plasmids were designed and tumor cell lines with a high incidence of cachexia were used. In this embodiment, SK-Hep-1, HepG2 and H292 cell lines were used to verify that the oligonucleotide expression plasmids could be correctly transcribed to produce SEQ ID NO.5 and SEQ ID NO.19.

[0111] Experimental methods:

[0112] The SK-Hep-1, HepG2, and H292 cell lines were cultured, transfected, and collected according to the method described in Example 1. The collected cells were used for RNA extraction, and the intracellular expression of oligonucleotides was detected.

[0113] qPCR quantitative detection: Prepare the qPCR reaction system according to the following ratio: DEPC water 5.9 μL; TaqPCR Mix (2×) 10 μL; T probe 1 μL; F primer 0.5 μL; R primer 1.5 μL; Rox reference dye 0.1 μL; template (reverse transcription product) 1 μL; and perform qPCR quantitative detection according to the procedure shown in Table 1.

[0114] Table 1 qPCR reaction procedure

[0115] The results showed that oligonucleotide expression plasmids designed using the example sequences could be transcribed into example oligonucleotides in various human tumor cell lines. The concentrations of the oligonucleotide sequences of the transcription products at 24 h are shown in Table 2 below.

[0116] After the expression plasmid enters the cell, it begins to express and generate oligonucleotide sequences. The concentration gradually accumulates and can be maintained for a relatively long time, remaining at a high concentration even after 48 hours. Different example sequences exhibit similar transcriptional accumulation and metabolic characteristics in different cell lines. As shown in Figure 1(a)(b)(c), the expression plasmid designed based on SEQ ID NO.19 can be efficiently transcribed in the SK-Hep-1 cell line, and the expression plasmid designed based on SEQ ID NO.5 can be efficiently transcribed in the HepG2 and H292 cell lines, with the concentration gradually accumulating over time. The transcription results of different sequences and cell lines are highly consistent.

[0117] Table 2. Oligonucleotide expression levels at 24h

[0118] Example 3

[0119] In advanced tumor states, A2AR gene expression is suppressed. Nucleic acid or oligonucleotide sequences with specific characteristics can restore A2AR gene expression, resulting in a higher level of A2AR gene mRNA abundance in human tumor cells.

[0120] Taking the oligonucleotide expression plasmid designed with the sequences of SEQ ID NO.5 and SEQ ID NO.19 as an example, this embodiment uses MKN45, H292 and HepG2 cell lines, which are tumor cell lines with a high incidence of cachexia, to verify that oligonucleotides containing seed sequences can restore A2AR gene expression.

[0121] Taking the oligonucleotide expression plasmid designed with the SEQ ID NO.19 sequence as an example, the experimental results are shown in Figure 2(a). The A2AR gene mRNA can be upregulated and restored within 48 hours, with the highest abundance at 6 hours.

[0122] Taking the oligonucleotide expression plasmid designed with the SEQ ID NO.5 sequence as another example, the experimental results are shown in Figure 2(b). In various cell lines, the expression of A2AR gene mRNA can be restored and upregulated within 48 hours.

[0123] Experimental methods: MKN45, H292, and HepG2 cell lines were cultured, transfected, and samples were collected according to the method described in Example 1. The collected cells were used for mRNA extraction, and the A2AR mRNA content was detected by qPCR using the following primers.

[0124] Primers:

[0125] F: 5'-TGCTCGCTATCCCATTCG-3' (SEQ ID NO. 43);

[0126] R: 5'-GCGTGAGGACCAGGACAAA-3' (SEQ ID NO. 44).

[0127] Example 4

[0128] After the A2AR gene expression was restored by nucleic acid or oligonucleotide sequences with the characteristics of the example, the abundance of A2AR protein in the cell was restored.

[0129] Taking the oligonucleotide expression plasmid designed with the sequences of SEQ ID NO.5, SEQ ID NO.15 and SEQ ID NO.19 as an example, using tumor cell lines with a high incidence of cachexia, this example uses CW-2, CFPAC-1, BxPC-3 and SW620 cell lines to verify that after the oligonucleotide containing the seed sequence restores the expression of A2AR gene mRNA, the abundance of A2AR protein also increases.

[0130] Using the oligonucleotide expression plasmid designed with the SEQ ID NO. 5 sequence as an example, the experimental results are shown in Figure 3(a). The expression level of A2AR protein in CFPAC-1 and CW-2 cells was significantly increased compared with the control group. Using the oligonucleotide expression plasmid designed with the SEQ ID NO. 19 sequence as an example, the experimental results are shown in Figure 3(b). The expression level of A2AR protein in CW-2 and BxPC-3 cells was significantly increased compared with the control group. Using the oligonucleotide expression plasmid designed with the SEQ ID NO. 15 sequence as an example, the experimental results are shown in Figure 3(c). The expression level of A2AR protein in BxPC-3 and SW620 cells was significantly increased compared with the control group.

[0131] Therefore, it can be seen that after the exemplary oligonucleotide restores A2AR mRNA expression, the expression level of A2AR protein also recovers and is upregulated.

[0132] Experimental methods: CW-2, CFPAC-1, and BxPC-3 cell lines were cultured, transfected, and collected according to the method described in Example 1. The collected cells were used for protein extraction, and the abundance of A2AR protein was detected by ELISA.

[0133] Example 5

[0134] This embodiment demonstrates that after chemical modification of the characteristic nucleic acid or oligonucleotide sequences studied in this example, the modified oligonucleotides can also restore the function of A2AR abundance.

[0135] Taking the sequence SEQ ID NO.5 as an example, it was modified by thiolation, fluorination, and methylation. Using tumor cell lines with a high incidence of cachexia, SW620 and CFPAC-1 cell lines were used in this example to verify that the chemically modified oligonucleotides with seed sequences could restore A2AR protein abundance. The A2AR protein expression results are shown in Figure 4. As shown in Figure 4, the expression level of A2AR protein in SW620 and CFPAC-1 cells was significantly higher than that in the control group. Therefore, it can be concluded that modifying the oligonucleotide sequence does not change its function of restoring A2AR abundance.

[0136] Modification

[0137] mA*fC*mUmGmCfCmUfGfUmCmUmGmUfGmCfCmUmGmUmG*mU*mU (m: methylation; *: thioation; f: fluorination).

[0138] Experimental Methods: SW620 and CFPAC-1 cell lines were cultured according to the method described in Example 1 and then transfected. Two centrifuge tubes, A and B, were prepared. Tube A contained basal culture medium and Lipo3000; tube B contained basal culture medium and modified oligonucleotides. The reagents in tubes A and B were mixed by inverting and allowed to stand before the mixture was added to the cells. Specific transfection conditions were as per the instructions of the commercial transfection kit. After transfection, the cells were cultured again and collected according to the method described in Example 1. The collected cells were used for protein extraction, and the abundance of A2AR protein was detected by ELISA.

[0139] Example 6

[0140] Drugs used to treat tumors, such as chemotherapy and radiotherapy, may induce or worsen cachexia. However, the oligonucleotides of this invention can treat cachexia while maintaining tumor stability and inhibiting tumor progression. This embodiment uses tumor cell inhibition rate to further confirm the potential of the proposed sequence series for treating tumor cachexia.

[0141] Examples 1-5 demonstrate that the nucleic acids or oligonucleotides with the characteristics of this invention can accurately restore A2AR gene expression. Using SEQ ID NO. 1, 2, 4, 5, 9, 13, 14, 15, and 19 as examples, we studied tumor cell inhibition rates using pancreatic cancer, colon cancer, lung cancer, and liver cancer cell lines with high rates of cachexia. The results show that the example sequences all exhibited certain inhibition rates, demonstrating their potential for treating tumor cachexia. Detailed inhibition rates are shown in Table 3 below.

[0142] Experimental Methods: H292, CFPAC-1, CW-2, SW620, BxPC-3, A549, H460, and HepG2 cell lines were cultured and transfected according to the method described in Example 1. After transfection, 10 μL of CCK8 reagent was added to the selected culture wells at appropriate time points, and then the cells were transferred to a cell culture incubator for culture at 37°C and 5% CO2. After 1-2 hours of culture, the OD values ​​in the corresponding wells were detected using a microplate reader. 450 value.

[0143] Table 3 shows the tumor cell inhibition rate of example sequences.

[0144] Based on the studies in Examples 1-6 above, it is confirmed that the nucleic acid or oligonucleotide sequences with the characteristics of this invention, as well as the nucleotide-modified oligonucleotides with the same function, can accurately restore the function of the A2AR gene in various tumor cells, further regulate the expression of related pathway proteins, and ultimately achieve the goal of treating cachexia. Furthermore, while treating cachexia, it also significantly inhibits tumor cell growth. This confirms the therapeutic potential of the sequences of this invention for treating cachexia.

[0145] Typical symptoms of cachexia include weight loss and anorexia, and the currently accepted causes are metabolic disorders and negative energy balance. To further demonstrate the efficacy of the nucleic acid or oligonucleotide therapy of this invention in treating tumor cachexia, we simultaneously confirmed in vivo that A2AR inhibition could be relieved. We then evaluated the efficacy of anorexia by eating and the efficacy of weight loss by body weight, using an in situ tumor model to conduct an in vivo study on the efficacy of cachexia treatment.

[0146] In vivo experimental results confirmed that the use of oligonucleotides with the specific characteristics of the example can relieve A2AR inhibition and restore expression in the in situ model, demonstrating a significant therapeutic effect on tumor cachexia.

[0147] In vivo studies, using the sequence SEQ ID NO.5 as an example, employed tumor cell lines with a high incidence of cachexia. Examples 7-10 of this invention established orthotopic cachexia models using lung cancer H460, pancreatic cancer BxPC-3, and colorectal cancer SW620 cell lines, respectively. Treatment with SEQ ID NO.5 yielded good efficacy in all cases. Detailed results are described in the respective examples.

[0148] Example 7

[0149] This embodiment confirms that nucleic acids or oligonucleotides with the characteristics of this invention can also restore A2AR expression in an in vivo model, treat lung cancer cachexia, improve food and water intake, reduce weight loss rate, maintain energy metabolism balance, and inhibit the progression of tumor cachexia.

[0150] Model establishment: Thirty-six 6-week-old BALB / c nude mice were purchased, quarantined, and acclimatized before being used to establish a "nude mouse cachexia model." The cells cultured in Example 1 (H460 cell line) were used at a concentration of 1×10⁻⁶. 7 Cells · 200 μL -1 ·Only -1 Four nude mice were subcutaneously inoculated with the appropriate amount of the inoculation solution. After inoculation, the nude mice were separated into different cages according to sex and cultured. When the subcutaneous tumors grew to an appropriate size, the mice were euthanized, and the tumors were removed under sterile conditions and used for in situ tumor inoculation.

[0151] After the tumor tissue is removed and trimmed, nude mice used for establishing the orthotopic tumor model will be anesthetized, and the tumor tissue will be inoculated at the lower edge of the left costal arch (lung tissue). After inoculation, the mice will be transferred to a rearing cage, and the model establishment status will be observed regularly until the model is successfully established (i.e., weight loss of more than 10% or tumor metastasis).

[0152] In vivo efficacy study: After successful model establishment, nude mice were injected via tail vein into each group with the control reagent and an oligonucleotide expression plasmid expressing the sequence SEQ ID NO.5. The dosage was 0.6 mg / kg (calculated as 2.7 μg / kg based on the oligonucleotide sequence), administered once daily for 7 days. During the administration process, the body weight, clinical status, and dietary indicators of the nude mice in each treatment group were observed and recorded. At the experimental endpoint, the nude mice were dissected, and the number and volume of tumors were observed and recorded. The abundance of A2AR protein in the tumor tissue was detected using ELISA.

[0153] The experimental results are shown in Figure 5. Figure 5(a) shows that the abundance of A2AR in the tumor tissue of the drug-treated group was significantly higher than that of the control group at the experimental endpoint. This confirms that the administration of oligonucleotide sequences in the in vivo model can also relieve the inhibition of A2AR and restore its expression. Figure 5(b) shows that the weight gain rate of the nude mice in the oligonucleotide group remained stable during the drug administration period, significantly alleviating the core symptoms of cachexia, and the weight at the experimental endpoint was significantly higher than that of the control group. Figures 5(c) and (d) show that from the second drug administration onwards, the food intake (except for the fourth drug administration) and water intake of the nude mice in the oligonucleotide group were higher than those in the control group. Figures 5(e), (f), and (g) show that at the end of the experiment, the tumor volume and tumor number of the nude mice in the experimental group were reduced by 81.39% and 62.11% respectively compared with the control group, and tumor progression was inhibited. In addition, observation at the cage edge during the experiment showed that the mice did not experience diarrhea, poor condition, lethargy, arched back, cold body, or death throughout the drug administration period.

[0154] This indicates that the example sequence in this invention can restore the expression of A2AR protein, significantly improve the weight, feeding and drinking behavior of cachectic nude mice, effectively alleviate metabolic disorder symptoms, inhibit tumor development, and comprehensively improve the survival status of cachectic mice.

[0155] Example 8

[0156] This embodiment demonstrates that nucleic acids or oligonucleotides with the characteristics of this invention can treat lung cancer cachexia in vivo. It improves food and water intake, reduces weight loss, maintains energy metabolic balance, and inhibits the progression of tumor cachexia.

[0157] Model establishment: Same as Example 7.

[0158] In vivo efficacy study: After successful model establishment, mice in each group were injected via tail vein with the control reagent and an oligonucleotide expression plasmid expressing the sequence SEQ ID NO.5. The dosage was 0.2 mg / kg (0.9 μg / kg calculated based on the oligonucleotide sequence), administered once daily for 7 days. During the administration process, the food and water intake of the nude mice were observed and recorded. Additionally, the body weight and the number and volume of tumors at the experimental endpoint were observed and recorded in 16 mice.

[0159] The experimental results are shown in Figure 6. Figure 6(a) and (b) show that from the second administration onwards, the food intake (except for the fourth administration) and water intake of the nude mice in the oligonucleotide group were higher than those in the control group; Figure 6(c) shows that the weight gain rate of the nude mice in the oligonucleotide group remained stable during the administration period, significantly alleviating the core symptoms of cachexia, and the weight at the end of the experiment was significantly higher than that of the control group; Figure 6(d), (e), and (f) show that at the end of the experiment, the tumor volume and number of the nude mice in the experimental group were reduced by 49.67% and 39.13% respectively compared with the control group, and tumor progression was inhibited.

[0160] This indicates that the example sequence in this embodiment can significantly improve the weight, feeding and drinking behavior of cachectic nude mice, effectively alleviate metabolic disorder symptoms, inhibit tumor development, and comprehensively improve the survival status of cachectic mice.

[0161] Example 9

[0162] This embodiment demonstrates that nucleic acids or oligonucleotides with the characteristics of this invention can treat pancreatic cancer cachexia, improve food intake, reduce weight loss rate, maintain energy metabolism balance, improve the survival rate during the cachexia period, and improve the survival status of cachexia patients.

[0163] Model establishment: 24 6-week-old BALB / C nude mice were purchased. After culturing the BxPC-3 cell line using the method shown in Example 1, the cells were directly inoculated into the pancreas tail of the nude mice.

[0164] In vivo efficacy study: After successful model establishment, nude mice were injected via tail vein into each group with the control reagent and the oligonucleotide expression plasmid expressing SEQ ID NO. 5. The dosage was 0.4 mg / kg, administered once daily for 14 days. During the administration process, the clinical status and cachexia indicators such as food intake of the nude mice in each treatment group were observed and recorded.

[0165] The experimental results are shown in Figure 7. Figure 7(a) shows that the survival rate of nude mice in the oligonucleotide group was better than that in the control group throughout the treatment, demonstrating that oligonucleotide treatment can reduce the risk of death from cachexia. Figure 7(b) shows that during the treatment period, the food intake of the oligonucleotide group continued to increase, and the difference widened significantly after the 8th administration, indicating a gradual recovery of metabolic homeostasis. Figure 7(c) shows that the clinical observation scores of nude mice in the oligonucleotide group were significantly better than those in the control group during the treatment period. Figure 7(d) shows that the weight gain rate of nude mice in the oligonucleotide group remained at a stable level, and the weight loss rate was significantly better than that in the control group.

[0166] This demonstrates that nucleic acids or oligonucleotides with the characteristics of the example sequence in this embodiment can treat pancreatic cancer cachexia, continuously increasing food intake, stabilizing weight gain, improving clinical condition, and ultimately significantly improving the survival rate of the cachexia model. It confirms that the example sequence's treatment of cachexia focuses on metabolic regulation, achieving a dual improvement in quality of life and survival.

[0167] Example 10

[0168] This embodiment demonstrates that nucleic acids or oligonucleotides with the characteristics of this invention can treat colorectal cancer cachexia, reduce weight loss rate, maintain energy metabolism balance, and inhibit the development of colorectal cancer tumors.

[0169] Model establishment: Using the SW620 cell line, following the method in Example 7, after subcutaneous tumor implantation of cells in nude mice, tissue blocks were in situ inoculated on the serosal surface of the mesenteric side at 1 cm from the end of the cecum of nude mice.

[0170] In vivo efficacy study: After successful model establishment, nude mice were injected via tail vein into each group with the control reagent and the oligonucleotide expression plasmid expressing SEQ ID NO. 5. The dosage was 0.1 mg / kg, administered once daily for 14 days. During the administration process, the body weight of the nude mice in each treatment group was recorded, and on the day after the last administration, they were dissected to record the size of the in situ tumor.

[0171] The experimental results are shown in Figure 8. Figure 8(a) shows that the body weight of the nude mice in the oligonucleotide group consistently increased, and the growth rate was consistently higher than that of the control group. Figure 8(b) shows that at the end of the experiment, the tumor volume of the nude mice in the drug-treated group was reduced by 50.69% compared with the control group, and tumor progression was inhibited.

[0172] This demonstrates that the example sequence in this embodiment achieves a dual breakthrough in relieving cachexia symptoms and suppressing tumors in a highly metastatic colorectal cancer model.

[0173] Based on the studies in Examples 1-10 above, it is confirmed that the present invention creatively selects a seed sequence that can restore A2AR gene expression. A vector for stable transcription of this nucleic acid or oligonucleotide was designed, and in vivo and in vitro experiments verified that it can accurately restore the A2AR gene, regulate the expression of downstream pathway proteins, improve appetite, maintain or even increase weight, and ultimately reverse the pathological process of cachexia. In vivo models showed significant improvement in the core symptoms of cachexia (increased food intake, inhibited weight loss, and prolonged survival), providing a novel strategy for the treatment of cachexia.

[0174] This specification provides specific sequences of nucleic acids or oligonucleotides. In accordance with relevant regulations, this application also provides a computer-readable sequence listing. It should be noted that the sequences in the computer-readable sequence listing are for reference only. In the event of any discrepancy between the sequences in this specification and the sequences in the computer-readable sequence listing, the sequences in this specification shall prevail.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An oligonucleotide, characterized in that, The oligonucleotide is an oligonucleotide of the following (1) and / or (2): (1) Oligonucleotides with sequences as shown in any of SEQ ID NO.1-42; (2) The oligonucleotides in (1) above that have been modified with nucleotides and have the same function.

2. The oligonucleotide according to claim 1, characterized in that, The oligonucleotide is a sequence as shown in any one of SEQ ID NO. 1, 2, 4, 5, 9, 13, 14, 15, 19, 22, 23, 25, 26, 30, 34, 35, 36 or 40.

3. The oligonucleotide according to any one of claims 1-2, characterized in that, The nucleotide modification includes modification of phosphate groups, ribosome groups, and / or conjugated links; wherein, the modification of phosphate groups refers to modification of the oxygen in the phosphate group, including thiolation and boronization, thereby stabilizing the structure of nucleic acids; the modification of ribosome groups refers to modification of the 2'-hydroxyl group in the ribosome group, including the introduction of methoxy or fluorine at the 2'-hydroxyl position of the ribosome group, thereby making it difficult for ribonuclease to cleave nucleic acids and increasing the stability of nucleic acids; the conjugated link modification includes small molecule ligands, antibodies, peptides, or carbon chains.

4. An expression carrier, characterized in that, The expression vector contains the oligonucleotide as described in any one of claims 1-3, or can produce the oligonucleotide as described in claim 1 within the host.

5. The expression vector according to claim 4, characterized in that, The vectors include recombinant plasmids, AAV viral vectors, lentiviral vectors, or retroviral vectors.

6. The use of the oligonucleotide according to any one of claims 1-3 or the expression vector according to any one of claims 4-5 in the preparation of a drug for preventing cachexia.

7. The application according to claim 6, characterized in that, The prevention and treatment include: preventing, alleviating, reversing, improving, and curing cachexia or its symptoms.

8. The application according to claim 7, characterized in that, The cachexia includes one or more of the following stages caused by tumor development: pre-cachexia, cachexia, and refractory cachexia.

9. The application according to claim 8, characterized in that, The tumor may be a solid tumor or a non-solid tumor; The solid tumors include: gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, esophageal cancer, liver cancer, breast cancer, melanoma, osteosarcoma, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, bladder cancer, ovarian cancer, cervical cancer, nasopharyngeal carcinoma, laryngeal cancer, kidney cancer, head and neck tumors, testicular cancer, thyroid cancer, or brain cancer. The non-solid tumors include leukemia or lymphoma.

10. The application according to claim 9, characterized in that, The tumors include stomach cancer, lung cancer, intestinal cancer, pancreatic cancer, or liver cancer.

11. The application according to claim 6, characterized in that, Symptoms of cachexia include muscle wasting, weight loss, lethargy, insomnia, general weakness, metabolic abnormalities, decreased appetite, and / or reduced activity.

12. A pharmaceutical composition, characterized in that, It includes the oligonucleotide as described in any one of claims 1-3 or the expression vector as described in any one of claims 4-5.

13. The pharmaceutical composition according to claim 12, characterized in that, It also includes pharmaceutically acceptable carriers.

14. The pharmaceutical composition according to claim 13, characterized in that, The dosage forms of the drug include injections, powders for injection, tablets, ointments, capsules, granules, aerosols, sprays, or powder inhalers.

15. A method of administering a nucleic acid-containing drug, characterized in that, The nucleic acid includes or is capable of producing oligonucleotides of (1) and / or (2) below: (1) Oligonucleotides with sequences as shown in any one of SEQ ID NO.1-42; (2) The oligonucleotides in (1) above that have undergone nucleotide modification and have the same function; The method includes administering the drug via systemic administration.

16. The method of administering a nucleic acid-containing drug according to claim 15, characterized in that, Systemic administration includes subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration, or sustained-release administration.

17. The application method according to claim 15, characterized in that, The drug can deliver the oligonucleotide into the body at a dose of 4-60 μg / person / dose, based on a body weight of 60 kg.

18. The application method according to claim 17, characterized in that, The drug can deliver the oligonucleotide into the body at a dose of 5-50 μg / person / dose, based on a body weight of 60 kg.

19. The application method according to claim 15, characterized in that, The administration of the drug enables the oligonucleotide to achieve an effective concentration of 1 × 10⁻⁶ in the in vivo circulatory system. 4 -1×10 9 Copy / μL, or administration of the drug enables the oligonucleotide to achieve an effective concentration of 1×10⁻⁶ in the target organ. 4 -1×10 9 Copy / mg.

20. The application method according to claim 19, characterized in that, The administration of the drug enables the oligonucleotide to achieve an effective concentration of 2 × 10⁻⁶ in the in vivo circulatory system. 4 -1×10 8 Copy / μL, or administration of the drug enables the oligonucleotide to achieve an effective concentration of 2 × 10⁻⁶ in the target organ. 4 -1×10 8 Copy / mg.

21. A method for treating and / or preventing cachexia, characterized in that, The oligonucleotide of any one of claims 1-3, the expression vector of any one of claims 4-5, or the pharmaceutical composition of any one of claims 12-14, or the administration method of any one of claims 15-20, is administered to the patient.

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