Application of nucleic acid in preparation of medicine for preventing and treating hematite and application method of nucleic acid

Abstract

The invention discloses an application of nucleic acid in preparation of a medicine for preventing and treating hemagglutinin and an application method. According to the nucleic acid disclosed by the invention, nucleic acid factors related to in-vivo malignant fluid are regulated through oligonucleotides contained in or generated by the nucleic acid, an autologous reprogramming state is started, and by regulating activity / silence of key nucleic acid factors, a non-coding RNA regulation and control signal programming network is realized, and the purpose of regulating and controlling a plurality of signal channels by a single non-coding RNA is realized; and thus, a new steady state of the maladjustment physiological state is reconstructed. Compared with other nucleic acid drugs, the drug taking the nucleic acid as the active component has the advantages that the dosage is extremely small in the prevention and treatment process, the drug safety is ensured on the basis of achieving the drug effect, and the current situation of traditional plasmid / nucleic acid drug administration is subverted. In addition, the nucleic acid provided by the invention has no specific reaction, and has relatively high in-vivo safety.

Description

[0001] This application is a divisional application with an application date of June 24, 2024, an application number of CN202410817427.4, and an invention name of "Application and administration method of nucleic acids in the preparation of drugs for the prevention and treatment of cachexia", which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and particularly relates to the application and administration method of nucleic acid in the preparation of drugs for preventing and treating cachexia. Background Art

[0003] Cachexia is a complex metabolic clinical syndrome in which the body's own tissues are consumed. It can occur in a variety of diseases, such as tumors, AIDS, severe trauma, postoperatively, malabsorption and sepsis. The most common of these is tumor-associated cachexia, also known as tumor cachexia. The hallmark clinical symptoms of cachexia include progression of systemic inflammation, glycolytic metabolic pattern marked by the Warburg effect caused by hypoxia in the tumor microenvironment, insomnia, loss of appetite, weight loss caused by muscle wasting, systemic failure and metabolic abnormalities. In response to tumor cachexia, the General Office of the National Health Commission of China issued the "Guidelines for the Clinical Diagnosis and Treatment of Tumor Cachexia (2020 Edition)", which pointed out that there is a lack of treatment methods related to tumor cachexia and the treatment effect is poor. Clinical randomized trials have shown that appetite stimulants including progesterone analogs, 5-hydroxytryptamine (5-HT) antagonists, and anti-inflammatory drugs including cyclooxygenase-2 (COX-2) inhibitors cannot completely reverse the abnormal state of cancer cachexia, and are accompanied by risks such as thromboembolism. In addition, anti-tumor treatments such as radiotherapy and chemotherapy also aggravate the adverse reaction of muscle wasting, which is an important symptom of cachexia.

[0004] Muscle consumption and weight loss are important indicators for the diagnosis and treatment of cachexia. Currently, reversing the weight loss of patients with tumor cachexia, thereby helping to treat tumor cachexia and improving the quality of life of patients with terminal tumors, is a hot topic in tumor treatment research centered on the interests of patients. Summary of the invention

[0005] In order to solve at least some of the problems in the above-mentioned prior art, the present invention designs and prepares a nucleic acid that can be used to prevent and treat cachexia, which contains or can produce oligonucleotides in vivo, and stably exists in cells and the circulatory system while directly inhibiting cachexia-related nucleic acid factors, and further regulates the expression of related pathway proteins in a direct or indirect manner, and ultimately significantly changes cachexia. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, there is provided a use of a nucleic acid in the preparation of a drug for preventing and treating cachexia, wherein the nucleic acid comprises or is capable of producing the following oligonucleotides (1) and / or (2):

[0007] (1) an oligonucleotide whose sequence is shown in any one of SEQ ID No. 1-42;

[0008] (2) The oligonucleotides as described in (1) above which have been modified with nucleotides and have the same function.

[0009] In certain embodiments, according to the use of the present invention, the nucleotide modification comprises modifying the phosphate group and / or the ribose group;

[0010] The modification of the phosphate group refers to the modification of the oxygen in the phosphate group, including thiolation modification and borane modification, so as to stabilize the structure of the nucleic acid;

[0011] The modification of the ribose group refers to the modification of the 2'-hydroxyl group in the ribose group, including the introduction of a methoxy group or a fluorine group at the 2'-hydroxyl position of the ribose group, thereby making it difficult for ribonucleases to cleave nucleic acids and increasing the stability of nucleic acids.

[0012] In certain embodiments, according to the use of the present invention, the nucleic acid capable of producing the oligonucleotides (1) and / or (2) is an animal cell expression vector.

[0013] In certain embodiments, according to the application of the present invention, the vector used for the nucleic acid capable of producing (1) and / or (2) oligonucleotides is a recombinant plasmid, an AAV viral vector or a lentiviral vector.

[0014] In certain embodiments, according to the use of the present invention, the oligonucleotide is contained in an extracellular vesicle.

[0015] In certain embodiments, according to the use of the present invention, the dosage form of the drug includes injection, powder for injection, tablet, paste, capsule, granule, aerosol, spray or powder inhalation.

[0016] In certain embodiments, according to the use of the present invention, the drug is administered by systemic administration, including subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration or sustained release administration.

[0017] In certain embodiments, according to the use of the present invention, the medicament is designed to administer the oligonucleotide in an amount of 4 μg-60 μg / person / time based on 60 kg body weight.

[0018] In certain embodiments, according to the use of the present invention, the medicament is designed to be able to administer the oligonucleotide in an amount of 0.005-1 μg / kg body weight.

[0019] In certain embodiments, according to the use of the present invention, the drug is designed to enable the effective concentration of the oligonucleotide in the in vivo circulation system to be 1×10 4 -1×10 9 copies / μl, or the drug is designed to enable the effective concentration of the oligonucleotide in the target organ to be 1×10 4 -1×10 9 copies / mg.

[0020] In certain embodiments, according to the use of the present invention, the prevention and treatment comprises one of the following:

[0021] a. Prevent, alleviate, reverse, improve or cure cachexia or its symptoms;

[0022] b. Inhibit one or more of the proliferation, migration, invasion, and cell cycle changes of cells in the lesion;

[0023] c. Promote apoptosis, lysis or absorption of cells in the lesion.

[0024] In certain embodiments, according to the use of the present invention, the cachexia includes one or more of the pre-cachexia, cachexia and cachexia refractory stages caused by tumor development.

[0025] In certain embodiments, according to the use of the present invention, the symptoms of cachexia include muscle wasting, weight loss, mental depression, insomnia, systemic exhaustion, metabolic abnormalities, decreased appetite and / or decreased activity.

[0026] Another aspect of the present invention provides a method for regulating cell proliferation in vitro, comprising the step of contacting in vitro cells with the oligonucleotide of the present invention. Preferably, the in vitro cells include tumor cells or immortalized cells.

[0027] Another aspect of the present invention provides a method for administering a nucleic acid-containing drug, wherein the nucleic acid comprises or is capable of producing the following oligonucleotides (1) and / or (2):

[0028] (1) an oligonucleotide whose sequence is shown in any one of SEQ ID No. 1-42;

[0029] (2) oligonucleotides as described in (1) above which have been modified with nucleotides and have the same function;

[0030] The method comprises systemically administering the drug.

[0031] In certain embodiments, the method for administering the nucleic acid-containing drug according to the present invention comprises systemic administration by subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration or sustained release administration.

[0032] In certain embodiments, according to the method for administering a nucleic acid-containing drug of the present invention, the administration of the drug can deliver the oligonucleotide into the body in an amount of 4 μg-60 μg / person / time based on 60 kg body weight.

[0033] In certain embodiments, according to the method for administering a nucleic acid-containing drug of the present invention, the administration of the drug can deliver the oligonucleotide into the body in an amount of 5 μg-50 μg / person / time based on 60 kg body weight.

[0034] In certain embodiments, according to the method for administering a nucleic acid-containing drug of the present invention, the administration of the drug can make the effective concentration of the oligonucleotide in the in vivo circulatory system be 1×10 4 -1×10 9 copies / μl, or the effective concentration of the oligonucleotide in the target organ is 1×10 4 -1×10 9 copies / mg.

[0035] The nucleic acid of the present invention regulates the nucleic acid factors related to cachexia in the body through the oligonucleotides it contains or produces, initiates the reprogramming state of the body, and realizes the non-coding RNA regulatory signal programming network by regulating the activity / silence of key nucleic acid factors, so as to achieve the purpose of regulating multiple signal pathways with a single non-coding RNA, thereby achieving a new steady state of rebuilding the disordered physiological state. The drug with the nucleic acid of the present invention as the active ingredient has a very small dosage compared to other nucleic acid drugs in the prevention and treatment process, and ensures the safety of medication on the basis of achieving drug efficacy, subverting the status quo of traditional plasmid / nucleic acid drug administration. In addition, the nucleic acid of the present invention has no specific reaction and has a high in vivo safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The expression results of exemplary oligonucleotides in SK-Hep-1 cell line are shown in FIG.

[0037] Figure 2 The effect of exemplary oligonucleotides on the expression of A2AR in MKN45 cells.

[0038] Figure 3 Shown are the effects of exemplary oligonucleotides on H292 cell proliferation.

[0039] Figure 4 The effect of exemplary oligonucleotides on nude mice with tumor cachexia. In the figure, (a) food intake; (b) water intake.

[0040] Figure 5 The effects of exemplary oligonucleotides on nude mice with tumor cachexia are shown in Figures 1 and 2. (a) weight growth rate; (b) tumor volume; (c) tumor number; and (d) tumor inhibition rate. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] Experimental methods for which specific conditions are not indicated in the specific examples are generally carried out according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0045] Studies have found that cells actively secrete "nucleic acid factors" to the extracellular space through exocrine mechanisms, which globally change the body's immunity and metabolism in vivo. This type of actively secreted "nucleic acid factors" participate in the in vivo reprogramming and system regulation process, just like physiological regulatory factors such as cytokines and chemical factors. In addition, nucleic acid factors may involve multiple signaling pathways in patients with tumors, such as PTEN, MEK3, TWIST1, and EZH2, and are related to multiple functions such as cell proliferation, cell cycle, angiogenesis, invasion, cell stemness, and chemotherapy resistance. The present invention solves the imbalance of the physiological state of cachexia patients by regulating the expression of cachexia-related nucleic acid factors, improves the symptoms of cachexia, especially tumor cachexia, and improves the quality of life of patients.

[0046] In the present invention, the term "cachexia" refers to a complex metabolic clinical syndrome in which the patient's own tissue is consumed. This disease type is characterized by loss of appetite, weight loss, systemic failure and metabolic abnormalities, and can occur in a variety of diseases, such as neovascular disease, tumors, AIDS, severe trauma, postoperative, malabsorption and sepsis. Cachexia recognized in the art is characterized by persistent skeletal muscle consumption, with or without loss of adipose tissue, and conventional nutritional therapy cannot completely alleviate it. The only drug currently officially listed for cachexia is Anamorelin produced by Ono Pharmaceutical Co., Ltd. in Japan, which achieves the purpose of improving cachexia by increasing appetite, but its application for listing in the European Union has not been approved, and it is still in Phase I clinical trial in my country.

[0047] In the present invention, the term "oligonucleotide" refers to a molecule consisting of a plurality of linked nucleotides, each nucleotide comprising a sugar (e.g., ribose or deoxyribose) and an organic base linked thereto. Organic bases include pyrimidines (e.g., cytosine (C), thymine (T) or uracil (U)) or purines (e.g., adenine (A) or guanine (G)). Depending on the sugar group, oligonucleotides are divided into DNA and RNA. In the present invention, oligonucleotides may not include any modification or may be modified. Modification types include base modifications, sugar modifications, and backbone modifications, etc. In certain embodiments, the oligonucleotides of the present invention include phosphorothioate backbone modifications, so that the oligonucleotides are resistant to the effects of nucleases and improve the stability of the oligonucleotides. The preparation method of the oligonucleotides of the present invention is not limited, and may be in vitro synthesis or may be obtained by biotechnology or bioengineering methods. These methods are also known in the art.

[0048] In certain embodiments, the oligonucleotides of the present invention are oligonucleotides capable of inhibiting cachexia-related nucleic acid factors. Exemplarily, the oligonucleotides of the present invention have the sequences shown in the following SEQ ID No. 1-42:

[0049]

[0050]

[0051] In the present invention, the term "nucleic acid factor" refers to nucleic acids related to cachexia or regulating its occurrence and development, especially small nucleic acid molecules. Generally, these nucleic acid factors can participate in tumor cell metabolism and regulate the development of tumor cachexia.

[0052] In the present 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 acid of the present invention is not limited, including long-chain nucleic acids or short-chain nucleic acids. The single-stranded long-chain nucleic acid is generally composed of more than 200 nucleotides, that is, the single-stranded length is more than 200nt. Short-chain nucleic acids are sometimes also referred to as oligonucleotides, which are generally single-stranded at this time, usually composed of 5-200 nucleotides in length, that is, 5-200nt in length.

[0053] In certain embodiments, the nucleic acid of the present invention may include the oligonucleotides of the present invention. 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, may include two, three, more than three or more oligonucleotides, and in the case of multiple oligonucleotides shown in the present invention, the connection mode between the oligonucleotides is not particularly limited, for example, a suitable spacer sequence may be used to connect to the oligonucleotide. Exemplarily, the nucleic acid comprises at least one oligonucleotide selected from SEQ ID No. 1-42.

[0054] In certain embodiments, the nucleic acid of the present invention is a nucleic acid molecule capable of producing the oligonucleotide of the present invention, such as a nucleic acid that produces the oligonucleotide of the present invention by transcription. In this case, the nucleic acid includes a nucleic acid expressing a sequence shown in any one of SEQ ID No.1-21. In certain embodiments, the nucleic acid can be part of an expression vector, and "expression vector" refers to a nucleic acid construct produced by recombination or synthesis, which carries a series of specific nucleic acid elements capable of transcribing a specific gene in a host cell, such as a promoter element, an enhancer element, and a selection element. Typically, gene expression is placed under the control of certain regulatory elements, such as constitutive or inducible promoters. In the present invention, the vector can be a plasmid, a phagemid, a 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 oligonucleotide of the present invention can be operably connected to one or more elements of the vector, such as a promoter element. "Operably connected" is used to describe the connection between a regulatory element and a gene or its coding region. That is, gene expression is usually placed under the control of certain regulatory elements, and a gene or coding region is "operably connected" to a regulatory element, meaning that the gene or coding region is controlled or affected by the regulatory element.

[0055] In the present invention, oligonucleotides can be used for the prevention and treatment of cachexia in the form of "extracellular nucleic acids". The oligonucleotides are isolated pure nucleic acid molecules and can stably exist in the extracellular environment, such as but not limited to blood, saliva, gastric juice, etc. This can not only have a therapeutic effect on in situ tumors, but also has important significance for metastatic tumors or distal tumors, because the oligonucleotides of the present invention can reach the target (target nucleic acid) through the circulatory system, so that this new systemic treatment scheme can replace chemotherapeutic drugs, thereby avoiding the toxic side effects of chemotherapeutic drugs to patients, so that patients can benefit from the treatment of cachexia.

[0056] The term "isolated" refers to a substance and / or entity that is separated from at least some of the components with which it was originally associated (whether in nature or / or in an experimental setting), and / or (2) is artificially produced, prepared and / or manufactured. An isolated substance and / or entity can 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 with which it was originally associated. In some embodiments, the purity of the isolated 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%. A substance is "pure" if it is substantially free of other components. Furthermore, an "isolated cell" refers to a cell that is not contained in a multicellular organism.

[0057] The present invention also provides a method for reversing, preventing, improving or treating cachexia or a related condition in a subject in need thereof, comprising the step of administering a therapeutically effective amount of a nucleic acid to a subject in need thereof, wherein the nucleic acid comprises or is capable of producing an oligonucleotide shown in any one of SEQ ID No. 1 to 42. It is understood that the method can be therapeutic or preventive.

[0058] In a preferred embodiment of the application and method according to the present invention, the vector used to produce the nucleic acid of the oligonucleotide sequence includes a recombinant plasmid, an AAV viral vector or a lentiviral vector. In a specific embodiment, the vector used to produce the nucleic acid of the oligonucleotide shown in any one of SEQ ID No. 1-21 is an animal cell expression vector.

[0059] It is understood that the nucleic acid or oligonucleotide of the present invention may be further contained in a suitable delivery carrier, such delivery carriers are known in the art, for example, including but not limited to: extracellular vesicles, especially exosomes, microvesicles, etc.; nanoparticles such as chitosan, polylactic-co-glycolic acid (PLGA), dendritic macromolecules; typical liposomes, etc. In a preferred embodiment, the oligonucleotide of the present invention is contained in an extracellular vesicle, such as an exosome.

[0060] In a preferred embodiment, the method of the present invention comprises the step of administering to a subject an animal cell expression vector, wherein the vector is capable of producing an oligonucleotide shown in any one of SEQ ID No. 1-21.

[0061] In a preferred embodiment, the step of administering cells transformed with a vector producing the oligonucleotide sequence to a subject is included. In one aspect, the plasmid of the present invention is introduced into cells by any suitable method for introducing a vector into cells (e.g., gene transfer), such as transfection and transduction, without particular limitation. In a specific embodiment, the recombinant plasmid constructed by the present invention has the sequence shown in SEQ ID NO.46.

[0062] In a preferred embodiment, the step of administering to a subject an extracellular vesicle, particularly an exosome (or exosome-like particle), containing the oligonucleotide sequence, wherein at least part of the membrane of the exosome or extracellular vesicle can be directly derived from a cell or prepared by artificial synthesis. The particle size of the exosome or extracellular vesicle is not particularly limited, and it can have a particle size of micrometers or even nanometers.

[0063] In a most preferred embodiment, the purpose of reversing, preventing, improving or treating cachexia or its related disorders is achieved by administering a therapeutically effective amount of a recombinant plasmid producing an oligonucleotide shown in any one of SEQ ID No. 1-21 to a subject in need thereof.

[0064] In the present invention, the drug comprising nucleic acid or oligonucleotide may further include a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable" refers to a carrier, excipient or stabilizer that is non-toxic to cells or mammals exposed to the dose and concentration used or has an acceptable toxicity level determined by 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 include 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 polyvinyl pyrrolidone; amino acids; carbohydrates, such as glucose, mannose or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium and nonionic surfactants, such as Tween and polyethylene glycol (PEG).

[0065] Without being bound by any theory, the oligonucleotide of the present invention may have a nucleotide sequence shown in any one of SEQ ID No. 1-42 or a sequence obtained by chemical modification. In addition, variant sequences of the above sequences are also within the scope of protection of the present invention. The term "variant sequence" refers to an oligonucleotide sequence that is not completely identical to the oligonucleotide sequence of the present invention, but retains one or more functional features of the oligonucleotide sequence of the present invention. In some aspects, the variant sequence of the oligonucleotide sequence or the chemically modified sequence retains all the functional features of the oligonucleotide sequence, such as but not limited to the binding activity to the target gene, the inhibitory activity to the target expression or the inhibitory activity to the target cell, the regulatory activity to the downstream signaling pathway of the target regulation and / or the efficacy of reversing, preventing, improving or treating cachexia. Preferably, the oligonucleotide obtained after modification does not cause the oligonucleotide to lose the function of inhibiting the nucleic acid factor and / or the function of inhibiting its expression. The regulatory activity of the downstream signaling pathway of the target regulation further includes regulating the expression of related proteins, especially regulating the expression of proteins such as A2AR, and finally significantly changing the glycolytic metabolic state unique to cachexia.

[0066] In the present invention, the inhibitory activity of oligonucleotides for target expression or target cells can be measured by methods known in the art, and this is not particularly limited. For example, methods such as real-time fluorescence quantitative PCR and Northern Blotting can be used to quantify nucleic acid factors. For another example, CCK8 can be used to detect the inhibition rate of oligonucleotides on target cells, and the oligonucleotides of the present invention have a high inhibition rate on target cells. In addition, the oligonucleotides of the present invention as shown in the SEQ ID No.1-42 sequence have a significant inhibitory effect on the expression of the target gene (nucleic acid factor) and / or the function of the nucleic acid factor. It can be understood by those skilled in the art that the GC content can be optimized according to the sequence shown in SEQ ID No.1-42 of the present invention to further improve the affinity of the oligonucleotides to the target sequence, thereby affecting its inhibition efficiency.

[0067] In the present invention, chemical modification of oligonucleotides to improve the stability, activity or half-life of oligonucleotides is known in the art. The oligonucleotides of the present invention use nucleotide groups (or nucleotide residues) as basic structural units, wherein the nucleotide groups contain phosphate groups, ribose groups and bases, and preferably, the oligonucleotides contain at least one modified nucleotide group. The modified nucleotide groups do not cause the oligonucleotides to inhibit the function of nucleic acid factors and / or the loss of their expression.

[0068] In the present invention, the modified nucleotide group is a nucleotide group in which the phosphate group and / or the ribose group are modified. For example, the modification of the phosphate group refers to the modification of the oxygen in the phosphate group, including but not limited to thio modification, borane modification, etc., such as replacing the oxygen in the phosphate group with sulfur and borane, respectively. The modification of the phosphate group can stabilize the structure of the nucleic acid and maintain the high specificity and high affinity of the base pairing. Preferably, the thio modified nucleotide group refers to a nucleotide group in which all the non-bridging oxygen atoms in the phosphodiester bonds are replaced with sulfur atoms.

[0069] The modification of the ribose group refers to the modification of the 2'-hydroxyl (2'-OH) in the ribose group. After introducing certain substituents such as methoxy or fluorine at the 2'-hydroxyl position of the ribose group, it is difficult for ribonucleases to cut nucleic acids, thereby increasing the stability of nucleic acids and making nucleic acids more resistant to nuclease hydrolysis. Modifications of the 2'-hydroxyl in nucleotide pentoses include 2'-fluoro modification (2'-fluro 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), 2'-deoxy modification (2'-Deoxy modification), etc.

[0070] In the present invention, the variant sequence of the oligonucleotide sequence may include an oligonucleotide having the same function after one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in any one of SEQ ID No. 1-42, and any designated sequence from the above sequences may be used as a basis to determine whether the variant sequence has the same function as the designated sequence under different circumstances. In a specific embodiment, the 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.

[0071] In certain embodiments, the insertion of nucleotides includes 5' and / or 3' terminal insertions, as well as intrasequence insertions of single or multiple residues (including at least 1, 2, 3 nucleotides).

[0072] In certain embodiments, the deletion of nucleotides refers to removing one or more residues from the oligonucleotide sequence. These deletion variants are usually prepared in the following manner: the nucleotides in the DNA encoding the oligonucleotides are subjected to site-specific mutagenesis, thereby producing the DNA encoding the variants, and thereafter expressing the DNA in recombinant cell culture. Alternatively, the deletion variants can be easily prepared by in vitro synthesis.

[0073] In certain embodiments, the displacement of nucleotides is wherein removed sequence for example at least 1 to at least 3 nucleotides and different nucleotides have been inserted in its position. Although the site for introducing sequence variation is predetermined, the mutation itself need not be predetermined. For example, in order to optimize the performance of the mutation at a given site, random mutagenesis can be carried out at the target region, and the expressed substitution variants can be screened with regard to the optimal combination of desired activity. The technology for carrying out substitution mutations at predetermined sites in DNA with known sequence is well-known.

[0074] In the present invention, nucleotide substitution is usually a single residue, nucleotide insertion is usually about 1-10 residues, and nucleotide deletion is usually about 1-30 residues.

[0075] In the present invention, the variant sequence of the oligonucleotide sequence may have 95%, such as 96%, 97%, 98%, or even 99% or more homology with the nucleotide sequence shown in any one of SEQ ID No. 1-42 and have the same function. Similarly, it can be determined based on any designated sequence from the above sequences whether the variant sequence with the above homology has the same function as the designated sequence.

[0076] To determine sequence homology (also sometimes referred to as "identity"), sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment over the full length of the sequences being compared.

[0077] The dosage form of the medicine or composition comprising the nucleic acid or oligonucleotide of the present invention is not particularly limited, and it can be selected from injection, powder for injection, tablet, paste, capsule, granule, aerosol, spray or powder inhalation. In a preferred embodiment, the dosage form of the medicine or composition comprising the nucleic acid or oligonucleotide of the present invention is injection.

[0078] In the present invention, the administration route of the drug or composition is not particularly limited, including but not limited to systemic administration in the form of subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration or sustained release administration.

[0079] In a preferred embodiment, the drug or composition is an injection suitable for parenteral administration, especially intravenous administration. In a most preferred embodiment, the purpose of reversing, improving or treating cachexia or its related conditions is achieved by administering a therapeutically effective amount of a recombinant plasmid vector comprising an oligonucleotide to a subject in need thereof by intravenous administration.

[0080] In the present invention, the term "prevention" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) the progression of undesirable physiological changes or disorders, such as cachexia. Compared with an untreated reference group under the same conditions, the degree of relief or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100%, measured by any standard technique. Beneficial or desired clinical results include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete). Those in need of treatment include those who already have cancer or cachexia-related diseases or those who need to prevent or improve cancer or cachexia-related diseases.

[0081] The term "subject" used in the present invention refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, and the like, that are about to receive a specific treatment. Typically, "subject" and "patient" are used interchangeably in the present invention and both refer to a human subject.

[0082] The term "therapeutically effective amount" as used in the present invention refers to an effective dosage considered pharmaceutically, 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, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dosage of the present invention can vary widely.

[0083] Although the therapeutic dose can vary widely, the inventors have found that the nucleic acid or oligonucleotide administered can achieve effective treatment of cachexia at low doses. For an individual, such as an individual with a body weight of 60 kg, the daily dosage of the nucleic acid or oligonucleotide is generally 4 μg / person / time-60 μg / person / time, preferably 5-50 μg / person / time. Exemplary effective dosages include 5 μg / person / time, 6 μg / person / time, 7 μg / person / time, 8 μg / person / time, 9 μg / person / time, 10 μg / person / time, 11 μg / person / time, 12 μg / person / time, 13 μg / person / time, 14 μg / person / time, 15 μg / person / time, 16 μg / person / time, 17 μg / person / time, 18 μg / person / time, 19 μg / person / time, 20 μg / person / time, 21 μg / person / time, 22 μg / person / time, 23 μg / person / time, 24 μg / person / time, 25 μg / person / time, 26 μg / person / time, 27 μg / person / time The dosage of the drug may be as follows: person / time, 28μg / person / time, 29μg / person / time, 30μg / person / time, 31μg / person / time, 32μg / person / time, 33μg / person / time, 34μg / person / time, 35μg / person / time, 36μg / person / time, 37μg / person / time, 38μg / person / time, 39μg / person / time, 40μg / person / time, 41μg / person / time, 42μg / person / time, 43μg / person / time, 44μg / person / time, 45μg / person / time, 46μg / person / time, 47μg / person / time, 48μg / person / time, 49μg / person / time, 50μg / person / time. It may be administered as a single dose once a day, may be administered in multiple doses a day, or may be used at intervals.

[0084] In certain embodiments, the daily dosage of the nucleic acid or oligonucleotide is 0.005 μg / kg-1 μg / kg, preferably 0.006-1 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.

[0085] In the present invention, the administration of the drug can make the effective concentration of the oligonucleotide in the body's circulatory system be 1×10 4 -1×10 9 Copies / μl, preferably 2×104 -1×10 8 copies / μl, preferably 2×10 4 -1×10 7 Copies / μl, e.g. 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 copies / μl.

[0086] In the present invention, the administration of the drug can make the effective concentration of the oligonucleotide in the target organ be 1×10 4 -1×10 9 copies / mg, preferably 2×10 4 -1×10 8 copies / mg, preferably 2×10 4 -1×10 7 copies / mg, e.g. 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 copies / mg.

[0087] In the present invention, the nucleic acid or oligonucleotide can also be combined with other drugs to prepare a combined pharmaceutical composition. The other drug may or may not be any therapeutic agent for cachexia, and there is no particular limitation on this. It should be noted that the selected other drugs do not react with the nucleic acid or oligonucleotide of the present invention in any unexpected manner to produce mutual inhibitory activity. When administered in combination with other drugs, such as chemotherapeutic agents, immune checkpoint inhibitors, anti-tumor drugs and immunomodulators, such as anti-PD-1 antibodies and anti-PD-L1 antibodies, the dosage of other drugs depends on the specific type of drug, the type and stage of cachexia, and the dosage per administration may be 0.5 mg / kg-30 mg / kg, preferably 1-20 mg / kg; for example, for a person weighing 60 kg, the dosage per administration may generally be 1 mg-1500 mg, such as 50 mg-1200 mg, or 100 mg-800 mg, 150 mg-600 mg or 200 mg-500 mg; exemplary dosages per administration are, for example, 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.; interval administration, administration frequency, for example, administration once every 3-7 days or once every 1-5 weeks, for example, administration once every 3 days, administration once every 5 days, administration once every 1 week, administration once every 10 days, administration once every 2 weeks, administration once every 3 weeks, administration once every 4 weeks, administration once every 5 weeks, etc. The specific dosage and administration frequency should take into account factors such as the route of administration and the patient's health status, which can be determined by skilled physicians based on routine skills.

[0088] In the present invention, the tumor includes but is not limited to solid tumors or non-solid tumors, and the solid tumor includes but is not limited to breast cancer, prostate cancer, melanoma, osteosarcoma, neuroblastoma, pancreatic cancer, lung cancer, rhabdomyosarcoma, Ewing's sarcoma, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, nasopharyngeal cancer, 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 tumors of the blood system such as leukemia and lymphoma, and tumors of the nervous system.

[0089] Example 1

[0090] The following uses lung cancer cells, gastric cancer cells and liver cancer cells as examples to exemplify the culture, transfection and collection of cells.

[0091] Cell recovery: quickly take out the desired cell line from the liquid nitrogen tank, put the cryovial into a 37-40℃ water bath and gently shake it until a small amount of ice is left, then take it out; transfer the cell solution in the cryovial to a 15ml centrifuge tube containing complete culture medium, centrifuge at 1200rpm for 10min, then transfer it to a biosafety cabinet, remove the supernatant; resuspend the cells with an appropriate amount of complete culture medium, and then transfer it to a culture flask, and observe that the cells are evenly dispersed under a microscope; then transfer the culture flask to a CO 2 Incubator, 37°C, 5% CO 2 The cells were cultured until the confluence reached 80-90% and then passaged.

[0092] Cell passaging: Observe the cells in the culture flask under a microscope, transfer them to a biosafety cabinet when the confluence is 80-90%, remove the supernatant and then add PBS to wash 2-3 times, remove PBS and add trypsin to digest the cells, and digestion is completed when the cell morphology shrinks to a round shape under a microscope; add an appropriate amount of culture medium and use a pipette to blow off the cells and collect the cell fluid into a centrifuge tube; centrifuge the cell fluid at 1200rpm for 10min and remove the supernatant; add an appropriate amount of culture medium to resuspend the cells, divide the cells into culture flasks according to a certain ratio and transfer the culture flasks to a cell culture incubator at 37℃ 5% CO 2 The cells were cultured under the appropriate conditions and subsequent experiments were performed when the cell number and confluence reached the experimental requirements.

[0093] Cell plating: When the cell confluence in the culture flask is 80-90%, transfer it to a biosafety cabinet, remove the culture medium and then add PBS to wash 2-3 times, remove PBS and add an appropriate amount of trypsin for digestion, and stop digestion when the cell morphology shrinks to a round shape; add an appropriate amount of culture medium and use a pipette to slowly blow the cells to make them fall off, collect the cell fluid into a centrifuge tube, centrifuge at 1200rpm for 10min; remove the supernatant and add an appropriate amount of culture medium to resuspend the cells. Pipette 20μl of cell suspension into a 1.5ml EP tube, add trypan blue to the EP tube at a ratio of 1:1 and mix well, pipette 10μl of suspension into a hemocytometer and count the cells. Calculate a certain amount of cell fluid into a 12-well plate according to the counting results, and transfer the 12-well plate to a cell culture incubator at 37℃5% CO 2 Overnight culture under the same conditions.

[0094] Cell transfection: prepare two centrifuge tubes A and B. Add basal culture medium and lipo3000 to tube A, invert and mix, and let stand for 5 minutes; add p3000 and plasmid (the control group is buffer solvent, and the experimental group is oligonucleotide plasmid, which can generate the target oligonucleotide through transcription) to tube B and invert and mix. After the preparation is completed (Lipo3000: plasmid: P3000 = 1μl: 0.5μg: 1μl), mix the A and B reagents and let stand for 15 minutes. Add the above mixture to a 12-well plate, take a fixed-point photo, and transfer it to a cell culture incubator at 37℃ 5% CO 2 Cultivated under conditions.

[0095] Cell collection: The cell growth status of each treatment group was observed at 3h, 6h, 24h, 48h and 72h after transfection. At the sampling time point, the supernatant in the 12-well plate was removed and PBS was added for washing; trypsin was added to the 12-well plate for digestion, and after digestion, an appropriate amount of complete medium was added and the cells were blown evenly to collect the cell fluid of each group; centrifuged at 1200rpm for 10min, the supernatant was removed, and an appropriate amount of PBS was added for resuspending and aliquoting, and the cell fluid was stored in a -80℃ refrigerator for subsequent experiments.

[0096] Example 2

[0097] This example shows the expression of the target oligonucleotide (SEQ ID No. 19) by the oligonucleotide plasmid in the SK-Hep-1 cell line.

[0098] The SK-Hep-1 cell line was cultured, transfected and collected according to the method described in Example 1, and the collected cells were used to detect the intracellular expression of oligonucleotides.

[0099] Total RNA extraction: add an appropriate amount of RNAiso Plus to the cell sample, mix the system and let it stand at room temperature for 3-10 minutes; transfer the system to an enzyme-free centrifuge tube, use a pipette to blow the system until there is no precipitation; add an appropriate amount of chloroform to the system, mix and let it stand at room temperature for 3-10 minutes (the system is milky white); centrifuge at 12000rpm for 10-20 minutes at 4℃; transfer the upper aqueous phase to a new enzyme-free centrifuge tube, add an appropriate amount of isopropanol, mix the system and let it stand at room temperature for 3-10 minutes; centrifuge the mixture at 12000rpm for 5-20 minutes at 4℃, discard the supernatant and then add an appropriate amount of 75% ethanol, mix and centrifuge at 4℃ for 5-15 minutes; discard the supernatant and dry the precipitate at room temperature; add an appropriate amount of DEPC water to the tube to dissolve the precipitate; take an appropriate amount of solution for total RNA detection, and store the rest at -80℃.

[0100] RNA reverse transcription: prepare new RNase-free centrifuge tubes and label them; prepare the reverse transcription system according to the following ratios: 0.5μl DEPC water; 2μl AMV RT5xBuffer; 1μl dNTP Mixture; 0.5μl AMV reverse transcriptase; 2μl RT-1 (1μM); and 4μl template; transfer the system to a PCR instrument and perform the reverse reaction at 16℃ for 30min, 37℃ for 60min, 85℃ for 10min, and 40℃ for ∞.

[0101] qPCR quantitative detection: prepare the qPCR reaction system according to the ratio of DEPC water 5.9μl; Taq PCR Mix (2×) 10μl; T1-1 probe 1μl; F1-1 primer 0.5μl; R1-2 primer 1.5μl; Rox reference dye 0.1μl; template (reverse transcription product) 1μl; perform qPCR quantitative detection according to the procedure shown in Table 1.

[0102] Table 1 qPCR reaction procedure

[0103]

[0104] The reaction results are as follows Figure 1 shown.

[0105] The experimental results showed that the expression level of the target oligonucleotide increased 6 hours after the oligonucleotide plasmid was transfected into the liver cancer SK-Hep-1 cell line, and the expression level of the target oligonucleotide peaked 24 hours after transfection (the expression level was 1.86×10 5 copies / ng), and then the expression level of the target oligonucleotide gradually decreased, but even at 72h after transfection, its expression level was still high (expression level was 1.13×10 5This indicates that the oligonucleotide plasmid can enter the cancer cells and express the target oligonucleotide smoothly and continuously.

[0106] Example 3

[0107] This example shows the effect of the oligonucleotide plasmid expressing SEQ ID NO. 19 on the expression of the A2AR gene in the MKN45 cell line. Based on the molecular characteristics of the oligonucleotide and combined with the tumor inhibition effect of the oligonucleotide in vivo and in vitro, A2AR was selected as one of the candidate targets of the oligonucleotide in tumor cells, and the effect of the oligonucleotide on the expression of A2AR in MKN45 cells was explored (MKN45 cell culture, transfection and sample collection were performed as described in Example 1).

[0108] Candidate target: A2AR (SEQ ID NO. 43);

[0109] Method development: qPCR upstream and downstream primers were designed for the CDS region of the candidate target, and the sequences containing the designed upstream and downstream primer binding sites were introduced into the plasmid to form a recombinant plasmid as a standard.

[0110] Target Primers:

[0111] F: 5'-TGCTCGCTATCCCATTCG-3' (SEQ ID NO. 44);

[0112] R: 5'-GCGTGAGGACCAGGACAAA-3' (SEQ ID NO. 45).

[0113] The total RNA extraction and RNA sample reverse transcription process were the same as in Example 2.

[0114] Gene expression detection: The extracted total RNA samples were reverse transcribed (reverse transcription program: 37°C, 15 min; 85°C, 5 min) and the primers and standards were pretreated; the qPCR program was set up and run according to Table 2.

[0115] Table 2 qPCR reaction procedure (A2AR)

[0116]

[0117] The results of A2AR gene expression in the samples are as follows Figure 2 shown.

[0118] Depend on Figure 2It can be seen that 6 hours after oligonucleotide plasmid transfection, the expression of A2AR target in MKN45 cells increased significantly; 24 hours after transfection, the gene expression of A2AR target was adjusted back to a low level, but it was still detectable. The increased expression of A2AR in tumor cells can inhibit the accumulation of adenosine, thereby inhibiting tumor immune escape. This shows that the intervention of oligonucleotide plasmids can inhibit the immune escape of MKN45 cells to a certain extent, which indirectly proves the inhibitory effect of oligonucleotide plasmids on tumor cells.

[0119] Example 4

[0120] This example shows the effect of the oligonucleotide plasmid expressing SEQ ID NO. 19 on the proliferation of H292 cell line.

[0121] H292 cells were cultured according to the cell culture method in Example 1. During the cell plating stage, H292 cells were plated at 5.78×10 4 / well was inoculated into a 96-well plate for culture. During the cell transfection phase, the transfection reagent was prepared according to the ratio of Lipo3000: oligonucleotide plasmid: P3000 = 1 μl: 0.5 μg: 1 μl. After transfection, the 96-well plate was transferred to a cell culture incubator at 37°C and 5% CO 2 Cultivated under conditions.

[0122] CCK8 assay was performed on each treatment group at 3h, 6h, 24h, 48h and 72h. CCK8 reagent was added to the selected culture wells, and then transferred to a cell culture incubator at 37℃ and 5% CO 2 After 1 hour of culture, the OD in the corresponding wells was detected using an enzyme marker 450 The cell inhibition rate was calculated according to the test value:

[0123] Inhibition rate = [(Ac-As) / (Ac-Ab)] x 100%,

[0124] AS: absorbance of the experimental well (culture medium containing cells, CCK-8, and drug to be tested);

[0125] Ac: absorbance of control wells (culture medium containing cells, CCK-8, and no drug to be tested);

[0126] Ab: absorbance of blank wells (culture medium without cells, test drugs, or CCK-8).

[0127] The experimental results are as follows Figure 3As shown, the proliferation of H292 cells was inhibited 3 hours after oligonucleotide plasmid transfection. As the experiment progressed, the inhibition rate of oligonucleotide plasmid on H292 cells continued to increase, reaching 57.13% at 72 hours, indicating that oligonucleotide plasmid had an inhibitory effect on the proliferation of H292 cells.

[0128] Example 5

[0129] This example shows the inhibitory effect of the oligonucleotide plasmid expressing SEQ ID NO. 14 on the proliferation of H460 and HEPG2 cells.

[0130] H460 cells and HEPG2 were cultured according to the cell culture method in Example 1. During the cell plating stage, H460 cells and HEPG2 were plated at a rate of 1×10 4 The cells were inoculated into a 96-well plate at 100 μg / well for culture and transfection (the ratio of each reagent was the same as in Example 1). The 96-well plate was then transferred to a cell culture incubator at 37°C and 5% CO. 2 Cultivated under conditions.

[0131] CCK8 assay was performed for each treatment group at 72 h. 10 μl of CCK8 reagent was added to the selected culture wells, which were then transferred to a cell culture incubator at 37°C with 5% CO. 2 After culturing for 2 h, the OD in the corresponding wells was detected using an enzyme marker 450 value.

[0132] The results of the inhibition of the proliferation of H460 and HEPG2 cells by the oligonucleotide represented by SEQ ID NO.14 are shown in Table 3 below.

[0133] Table 3

[0134]

[0135] Example 6

[0136] This example shows the effect of the oligonucleotide plasmid expressing SEQ ID NO. 5 on the proliferation of BXPC3 cells and A549 cell lines.

[0137] BXPC3 cells and A549 cells were cultured according to the cell culture method in Example 1. During the cell plating stage, BXPC3 cells and A549 cells were plated at a rate of 1×10 4 The cells were inoculated into a 96-well plate at 100 μg / well for culture and transfection (the ratio of each reagent was the same as in Example 1). The 96-well plate was then transferred to a cell culture incubator at 37°C and 5% CO. 2 Cultivated under conditions.

[0138] CCK8 assay was performed for each treatment group at 72 h. 10 μl of CCK8 reagent was added to the selected culture wells, which were then transferred to a cell culture incubator at 37°C with 5% CO. 2 After culturing for 2 h, the OD in the corresponding wells was detected using an enzyme marker 450 value.

[0139] The results are shown in Table 4 below.

[0140] Table 4

[0141]

[0142] Example 7

[0143] This example shows the effect of oligonucleotide plasmids expressing SEQ ID NOs. 1, 2, 4, 9, 13 and 15 on the proliferation of H292 cells.

[0144] H292 cells were cultured according to the cell culture method in Example 1. During the cell plating stage, H292 cells were plated at a rate of 1×10 4 The cells were inoculated into a 96-well plate at 100 μg / well for culture and transfection (the ratio of each reagent was the same as in Example 1). The 96-well plate was then transferred to a cell culture incubator at 37°C and 5% CO. 2 Cultivated under conditions.

[0145] CCK8 assay was performed for each treatment group at 72 h. 10 μl of CCK8 reagent was added to the selected culture wells, which were then transferred to a cell culture incubator at 37°C with 5% CO. 2 After culturing for 2 h, the OD in the corresponding wells was detected using an enzyme marker 450 value.

[0146] The results are shown in Table 5 below.

[0147] Table 5

[0148]

[0149] Example 8

[0150] This example shows the effect of oligonucleotide plasmids on tumor-bearing nude mice.

[0151] Model establishment: 36 6-week-old BALB / C nude mice were purchased, half male and half female, and used to establish the "nude mouse cachexia model" after passing the quarantine and adaptive feeding. The cells cultured in Example 1 (H460 cell line) were cultured at 1×10 7 Cells · 200 μl -1 ·Only -1The nude mice were subcutaneously inoculated with an inoculum of 1.50 μg / mL (3 mice were used for subcutaneous tumor inoculation, and the rest were used for orthotopic tumor inoculation). After the inoculation, the nude mice were cultured in cages according to gender. When the subcutaneous tumor grew to an appropriate size, the mice were euthanized, and the tumor was removed under sterile conditions and used for orthotopic tumor inoculation.

[0152] After the tumor tissue is removed and trimmed, the nude mice used to establish the in situ tumor model are anesthetized and the tumor tissue is inoculated at the lower edge of the left rib arch (lung tissue). After the inoculation, they are transferred to the breeding cage and samples are taken regularly to observe the model establishment status until the model is successfully established (i.e., weight loss is greater than 10% or tumor metastasis occurs).

[0153] In vivo efficacy study: After the model was successfully established, the nude mice were randomly divided into 2 groups, half male and half female. The control reagent and oligonucleotide plasmid injection were injected into each group of mice by tail vein injection. The dosage was 0.2 mg / kg (the dosage was 0.9 μg / kg calculated according to the oligonucleotide sequence), the frequency of administration was 1 time / 2 days, and the administration was 7 times. During the administration process, the clinical status, food intake, drinking water and other cachexia indicators of the nude mice in each treatment group were observed and recorded.

[0154] The experimental results are as follows Figure 4 As shown. From the second administration, the food intake of the experimental group (oligonucleotide) ( Figure 4 (a)), water intake ( Figure 4 (b)) were higher than those of the control group (CK). Except for the fourth administration, the food intake of the control group was higher than that of the experimental group. At other time points, the food intake and water intake of the experimental group were higher than those of the control group. It can be seen that the intervention of oligonucleotide plasmids can increase the food intake and water intake of nude mice with tumor cachexia and help alleviate cachexia symptoms.

[0155] Example 9

[0156] This example shows the effect of oligonucleotide plasmids on nude mice with tumor cachexia.

[0157] The tumor cachexia nude mouse model was established in the same manner as in Example 8.

[0158] In vivo efficacy study: After the model was successfully established, the nude mice were randomly divided into 2 groups, half male and half female. The control reagent and oligonucleotide plasmid injection were injected into each group of mice by tail vein injection. The dosage was 0.6 mg / kg, the frequency of administration was 1 time / 2 days, and the administration was 7 times. During the administration process, the clinical status, dietary indicators, tumor indicators, etc. of the nude mice in each treatment group were observed.

[0159] The experimental results are as follows Figure 5 As shown in the experimental results, after nude mice with tumor cachexia were injected with oligonucleotide plasmids, Figure 5From (a), we can see that the weight growth rate of nude mice in the experimental group (oligonucleotide) has always remained at a relatively stable level. The key indicator of cachexia, namely weight loss, has been alleviated to a great extent, and the weight has maintained a steady growth. By the end of the experiment, the weight of nude mice in the experimental group was significantly higher than that of nude mice in the control group (CK). Figure 5 As shown in (b), (c) and (d), at the end of the experiment, the tumor volume and number of nude mice in the experimental group were reduced by 81.39% and 62.11% respectively compared with those in the control group, and tumor progression was inhibited. In addition, observations at the side of the cage during the experiment showed that the mice did not have diarrhea, poor condition, poor spirits, hunched backs, cold bodies or death during the entire medication process, indicating that the oligonucleotide plasmids have improved the survival status of nude mice with tumor cachexia.

[0160] The specific sequences of nucleic acids or oligonucleotides are provided in this specification, and according to relevant regulations, this application also provides a sequence table in computer-readable form. It should be noted that the sequences in the sequence table in computer-readable form are for reference only. In the event that the sequences in this specification are inconsistent with the sequences in the sequence table in computer-readable form, the sequence content in this specification shall prevail.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of nucleic acid in the preparation of a drug for preventing and treating cachexia, characterized in that: The nucleic acid comprises or is capable of producing the following oligonucleotides (1) and / or (2): (1) an oligonucleotide whose sequence is shown in any one of SEQ ID No. 1-42; (2) The oligonucleotides as described in (1) above which have been modified with nucleotides and have the same function.

2. The use according to claim 1, characterized in that: The nucleotide modification includes modification of the phosphate group and / or the ribose group; The modification of the phosphate group refers to the modification of the oxygen in the phosphate group, including thiolation modification and borane modification, so as to stabilize the structure of the nucleic acid; The modification of the ribose group refers to the modification of the 2'-hydroxyl group in the ribose group, including the introduction of a methoxy group or a fluorine group at the 2'-hydroxyl position of the ribose group, thereby making it difficult for ribonucleases to cleave nucleic acids and increasing the stability of nucleic acids.

3. The use according to claim 1, characterized in that: The nucleic acid capable of producing the oligonucleotides (1) and / or (2) is an animal cell expression vector.

4. The use according to claim 1, characterized in that: The vector used to produce the nucleic acid of oligonucleotide (1) and / or (2) is a recombinant plasmid, AAV virus vector or lentiviral vector.

5. The use according to claim 1, characterized in that: The oligonucleotides are contained in extracellular vesicles.

6. The use according to claim 1, characterized in that: The dosage form of the drug includes injection, powder for injection, tablet, paste, capsule, granule, aerosol, spray or powder inhalation.

7. The use according to claim 1, characterized in that: The control comprises one of the following: a. Prevent, alleviate, reverse, improve or cure cachexia or its symptoms; b. Inhibit one or more of the proliferation, migration, invasion, and cell cycle changes of cells in the lesion; c. Promote apoptosis, lysis or absorption of cells in the lesion.

8. The use according to claim 1, characterized in that: The cachexia includes one or more of the pre-cachexia, cachexia stage and cachexia refractory stage caused by tumor development.

9. The use according to claim 1, characterized in that: Symptoms of cachexia include muscle wasting, weight loss, mental depression, insomnia, systemic exhaustion, metabolic abnormalities, decreased appetite and / or decreased activity.

10. A method for administering a nucleic acid-containing drug, characterized in that: The nucleic acid comprises or is capable of producing the following oligonucleotides (1) and / or (2): (1) an oligonucleotide whose sequence is shown in any one of SEQ ID No. 1-42; (2) oligonucleotides as described in (1) above which have been modified with nucleotides and have the same function; The method comprises systemically administering the drug.

11. The method for administering a nucleic acid-containing drug according to claim 10, characterized in that: This includes systemic administration by subcutaneous injection, intramuscular injection, intravenous administration, oral administration, inhalation administration or sustained release administration.

12. The application method according to claim 10, characterized in that The administration of the drug can deliver the oligonucleotide into the body in an amount of 4 μg-60 μg / person / time based on 60 kg body weight.

13. The application method according to claim 10, characterized in that The administration of the drug can deliver the oligonucleotide into the body in an amount of 5 μg-50 μg / person / time based on 60 kg body weight.

14. The application method according to claim 10, characterized in that The administration of the drug can make the effective concentration of the oligonucleotide in the body's circulatory system be 1×10 4 -1×10 9 copies / μl, or the administration of the drug can make the effective concentration of the oligonucleotide in the target organ be 1×10 4 -1×10 9 copies / mg.

15. The application method according to claim 10, characterized in that The administration of the drug can make the effective concentration of the oligonucleotide in the body's circulatory system be 2×10 4 -1×10 8 copies / μl, or the administration of the drug can make the effective concentration of the oligonucleotide in the target organ be 2×10 4 -1×10 8 copies / mg.

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