WO2021115442A1 - Arnsi ciblé pour ptp1b et précurseur de celui-ci et application - Google Patents

Arnsi ciblé pour ptp1b et précurseur de celui-ci et application Download PDF

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WO2021115442A1
WO2021115442A1 PCT/CN2020/135815 CN2020135815W WO2021115442A1 WO 2021115442 A1 WO2021115442 A1 WO 2021115442A1 CN 2020135815 W CN2020135815 W CN 2020135815W WO 2021115442 A1 WO2021115442 A1 WO 2021115442A1
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sirna
ptp1b
sequence
expression vector
mice
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PCT/CN2020/135815
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Chinese (zh)
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陈熹
余梦超
张辰宇
华诗雨
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南京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a targeted type of siRNA for PTP1B and its precursors and applications.
  • PTP1B is an important drug target for obesity and type 2 diabetes.
  • PTP1B -/- mice can resist weight gain induced by high fat, and at the same time show higher insulin sensitivity than normal mice;
  • PTP1B in the nervous system plays a major role in metabolic regulation and can simultaneously regulate insulin signaling pathways and leptin The signal pathway has a good control effect on body weight and blood sugar.
  • the development of PTP1B inhibitory molecules is mainly divided into two categories, small molecule inhibitors and RNA interference drugs.
  • RNA interference drugs have good specificity, but are unstable and easy Degradation and high production cost, there is also the problem of not being able to pass the blood-brain barrier.
  • the present invention provides a siRNA capable of regulating the activity or expression of PTP1B.
  • the first aspect of the present invention provides a precursor sequence whose 5'to 3'ends have the structure shown in formula I:
  • B1 is the required first ribonucleic acid sequence, wherein the first ribonucleic acid sequence includes the PTP1B siRNA sense strand sequence;
  • B2 is a sequence that is substantially complementary or completely complementary to B1, and B2 and C are not complementary;
  • C is the sequence of stem-loop structure
  • nucleotide sequence of the sense strand of the PTP1B siRNA is selected from the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or a combination thereof.
  • the precursor sequence is shown in SEQ ID NO.:5.
  • the second aspect of the present invention provides a polynucleotide which can be transcribed by a host to form the precursor sequence of the first aspect of the present invention.
  • the third aspect of the present invention provides an expression vector containing the precursor sequence according to the first aspect of the present invention or the polynucleotide according to the second aspect of the present invention.
  • the expression vector also contains a polynucleotide encoding a short peptide of rabies virus surface glycoprotein (RVG peptide).
  • the expression vector includes a viral vector and a non-viral vector.
  • the expression vector is a plasmid.
  • the fourth aspect of the present invention provides a pharmaceutical preparation, which contains:
  • the expression vector contains the precursor sequence described in the first aspect of the present invention or the polynucleotide described in the second aspect of the present invention, or the precursor sequence described in the first aspect of the present invention.
  • the expression vector also contains a polynucleotide encoding a short peptide of rabies virus surface glycoprotein (RVG peptide).
  • the formulation is a liquid dosage form.
  • the preparation is an injection.
  • the expression vector includes a plasmid.
  • the expression vector or plasmid contains a promoter, an origin of replication and a marker gene.
  • the expression vector contains an expression cassette for expressing PTP1B siRNA.
  • the expression cassette (ie polynucleotide) is double-stranded and has the following structure:
  • Promoter-attB1-optional RVG-optional tag protein (such as Lamp2b)-5' siRNA flanking region sequence-sequence shown in formula I-3' siRNA flanking region sequence-attB2.
  • the formulation is a liposome or exosomal formulation.
  • the fifth aspect of the present invention provides an siRNA for inhibiting the expression of the PTP1B gene.
  • the sense strand nucleotide sequence of the siRNA is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, or a combination thereof.
  • the sixth aspect of the present invention provides a pharmaceutical composition containing the precursor sequence according to the first aspect of the present invention, or the expression vector according to the third aspect of the present invention, and a pharmaceutically acceptable Carrier.
  • the pharmaceutical composition includes PTP1B siRNA plasmid.
  • the pharmaceutical composition is the expression vector of the third aspect of the present invention, preferably a plasmid containing the precursor sequence of the first aspect of the present invention.
  • the pharmaceutical composition also includes other (a) prevention and/or treatment of obesity-related diseases; (b) prevention and/or treatment of cardiovascular-related diseases; and/or (c) prevention and/or Or drugs to treat diseases related to metabolic abnormalities.
  • the other (a) prevention and/or treatment of obesity related diseases; (b) prevention and/or treatment of cardiovascular related diseases; and/or (c) prevention and/or treatment of metabolic abnormalities related diseases
  • the drug is selected from the following group: Orris, metformin, captopril, carvedilol tablets, gliclazide, metformin, or a combination thereof.
  • the pharmaceutical composition is the expression vector of claim 3, preferably a plasmid containing the precursor sequence of claim 1.
  • the dosage form of the pharmaceutical composition includes:
  • the dosage form of the pharmaceutical composition also includes spray, aerosol, powder mist, volatile liquid, topical solution, lotion, pouring lotion, liniment, cataplasm, Plasters, rubber ointments, ointments, plasters, pastes, eye drops, nose drops, ophthalmic ointments, gargles, sublingual tablets or suppositories.
  • the dosage form is injection, preferably intravenous injection or intraperitoneal injection.
  • the administration method of the pharmaceutical composition includes oral, respiratory, injection, transdermal, mucosal or cavity administration; preferably, the administration method includes direct injection of plasmid.
  • the seventh aspect of the present invention provides the use of the siRNA of the sixth aspect of the present invention, the precursor sequence of the first aspect of the present invention, or the expression vector of the third aspect of the present invention, for preparing a composition or preparation.
  • the composition or preparation is used for (a) prevention and/or treatment of obesity related diseases; (b) prevention and/or treatment of cardiovascular related diseases; and/or (c) prevention and/or treatment of metabolic disorders related diseases.
  • the obesity-related disease is selected from the group consisting of obesity, hyperlipidemia, or a combination thereof.
  • the cardiovascular-related disease is selected from the group consisting of hypertension, atherosclerosis, or a combination thereof.
  • the metabolic abnormality-related disease is selected from the group consisting of diabetes, fatty liver, or a combination thereof.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the mammal includes a human or non-human mammal.
  • the non-human mammals include rodents (such as rats and rabbits), primates (such as monkeys).
  • the eighth aspect of the present invention provides a method of (a) preventing and/or treating obesity-related diseases; (b) preventing and/or treating cardiovascular-related diseases; and/or (c) preventing and/or treating metabolic disorders-related diseases
  • the method is to administer a safe and effective amount of the expression vector according to the third aspect of the present invention, the pharmaceutical preparation according to the fourth aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention to a desired subject, thereby (a ) Prevention and/or treatment of obesity related diseases; (b) prevention and/or treatment of cardiovascular related diseases; and/or (c) prevention and/or treatment of metabolic disorders related diseases.
  • the administered dose is 1-20 mg/kg, preferably, 5-10 mg/kg.
  • the application frequency is 12 hours to 72 hours, preferably, 12 hours to 24 hours.
  • the administration includes: oral, respiratory, injection, transdermal, mucosal or cavity administration;
  • the administration includes injection of plasmids.
  • Figure 1 is a skeleton diagram of the plasmid of the present invention.
  • Figure 2 is the in vitro interference efficiency detection and cytotoxicity detection of plasmid molecules; according to the method shown in Figure 1, four plasmids with different interference sequences are constructed, and the plasmid molecules with the highest interference efficiency are screened by cell experiments and their cytotoxicity is detected.
  • Figure 3 shows the distribution of siRNA expressed by plasmid molecules in different tissues and their inhibitory effects on PTP1B;
  • A 12 hours after plasmid injection, the results of in situ hybridization detection of siRNA in mouse liver tissue, blue fluorescence is DAPI, green Fluorescence is PTP1B siRNA, control refers to the control plasmid;
  • B 12 hours after injection of plasmid, in situ hybridization test results of siRNA in mouse hypothalamus, blue fluorescence is DAPI, green fluorescence is PTP1B siRNA;
  • C injected every two days Once the plasmid was injected seven times, the level of PTP1B in mouse liver and hypothalamus was detected by western.
  • Figure 4 shows the effect of plasmid molecules on the body weight, food intake, body length and fat content of obese mice induced by high fat.
  • the high-fat-induced obese mice were divided into groups, and the control plasmid or PTP1B siRNA/RVG plasmid was injected every two days for a period of 3 weeks. After the end, the mice's body weight and other indicators were measured.
  • B Effect of plasmid administration on food intake of obese mice
  • C Effect of plasmid administration on body length of obese mice
  • D Plasmid administration on mice The influence of gonadal fat weight; among them, * means p ⁇ 0.05, ** means p ⁇ 0.01, and *** means p ⁇ 0.005.
  • Figure 5 shows the effect of plasmid molecules on insulin sensitivity, glucose tolerance and leptin sensitivity in obese mice induced by high fat.
  • the experimental mice were tested for ITT, GTT, and leptin sensitivity after the administration, and the mice's serum leptin content and serum insulin content were also tested.
  • A Effect of plasmid administration on mouse insulin sensitivity
  • B Effect of plasmid administration on mouse glucose tolerance
  • C Effect of plasmid administration on mouse leptin sensitivity
  • C mouse body weight Change graph
  • D is the mouse food intake change graph
  • E the effect of plasmid administration on mouse serum leptin content
  • F the effect of plasmid administration on mouse serum insulin content
  • Figure 6 is a metabolic cage detecting the effect of plasmid molecules on oxygen consumption, respiratory exchange ratio, activity volume, and caloric production in obese mice induced by high fat.
  • AB the effect of plasmid administration on oxygen consumption in mice
  • A oxygen consumption line chart
  • B oxygen consumption statistics chart
  • CD the effect of plasmid administration on mouse respiratory exchange ratio
  • C respiratory exchange ratio line chart
  • D Respiratory exchange ratio statistical graph
  • EF Effect of plasmid administration on the activity of mice
  • E broken line graph of activity of mice
  • F statistical graph of activity of mice.
  • GH The effect of plasmid administration on heat production in mice
  • G Line chart of heat production in mice
  • H Statistics chart of heat production in mice; where * means p ⁇ 0.05, ** means p ⁇ 0.01, *** means p ⁇ 0.005.
  • Figure 7 shows the effects of plasmid molecules on the four blood lipids of obese mice induced by high fat.
  • AD the effect of plasmid administration on the blood lipid content of mice
  • A total cholesterol content
  • B triglyceride content
  • C high-density lipoprotein content
  • D low-density lipoprotein content
  • * means p ⁇ 0.05
  • ** means p ⁇ 0.01
  • *** means p ⁇ 0.005.
  • Fig. 8 shows the improvement of the fatty liver of high-fat-induced obese mice by plasmid molecules.
  • Figure 9 is the in vivo safety test of plasmid molecules.
  • A the effect of plasmid administration on the serum level of alanine aminotransferase in high-fat-induced obese mice
  • B the effect of plasmid administration on the serum level of aspartate aminotransferase in high-fat-induced obese mice.
  • the present invention prepares a precursor siRNA capable of efficiently expressing PTP1B siRNA for the first time.
  • the precursor siRNA of the present invention can efficiently express the siRNA after being processed by the host cell, thereby effectively avoiding the interference effect of the reverse complementary sequence of the target sequence on the function of the target sequence.
  • the precursor siRNA of the present invention can effectively express the PTP1B siRNA sequence in vivo, and has a more effective therapeutic effect on obesity-related diseases; cardiovascular-related diseases; and/or metabolic abnormalities-related diseases.
  • the inventor completed the present invention.
  • the term “about” may refer to a value or composition within an acceptable error range of a particular value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • the terms "host”, “subject”, and “desired subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, such as cows, horses, dogs, cats, pigs, sheep, goats, camels, rats, mice, hares, and rabbits.
  • Rabies virus glycoprotein is a neurophilic protein that can bind to acetylcholine receptors expressed by nerve cells.
  • Rabies virus is a single-stranded negative-stranded RNA virus of the Rhabdoviridae family and has an envelope. The virus mainly encodes glycoprotein G.
  • the G protein is anchored on the surface of the virus envelope in the form of a trimer, and can bind to receptors on the cell surface to mediate membrane fusion and allow the virus to invade cells.
  • G protein is the main antigen protein of rabies virus, which stimulates the body to produce neutralizing antibodies.
  • RVG peptide specifically binds to choline bodies expressed by neuronal cells, and RVG targets are expressed outside the cell membrane, guiding exosomes to pass through the blood-brain barrier and transport to nerve cells.
  • siRNA refers to a class of RNA molecules that are processed from transcripts that can form siRNA precursors. Mature siRNA usually has 18-26 nucleotides (nt) (more specifically about 19-22 nt), and siRNA molecules with other numbers of nucleotides are not excluded. siRNA can usually be detected by Northern blotting.
  • Human-derived siRNA can be isolated from human cells.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not separated and purified, but the same polynucleotides or polypeptides are separated and purified from other substances that exist in the natural state. .
  • siRNA is generally produced by simulating miRNA production mechanism, and such siRNA can be processed from precursor RNA (Precursor RNA, Pre-RNA).
  • precursor RNA Precursor RNA, Pre-RNA
  • the precursor RNA can be folded into a stable stem-loop (hairpin) structure, and the length of the stem-loop structure is generally between 50-100 bp.
  • the precursor RNA can be folded into a stable stem-loop structure, and both sides of the stem of the stem-loop structure contain two substantially complementary sequences.
  • the precursor RNA can be natural or artificially synthesized.
  • the precursor siRNA is an artificially synthesized precursor siRNA, and the precursor siRNA has a structure shown in formula I:
  • B1 is the PTP1B siRNA sense strand sequence
  • B2 is a sequence complementary to B1 (including substantially complementary and complete complementary);
  • C is the stem-loop structure
  • the shown precursor siRNA can be processed in the host to form PTP1B siRNA.
  • the precursor miRNA forming the PTP1B siRNA can be spliced to generate the siRNA that regulates the PTP1B gene, that is, PTP1B siRNA (for example, SEQ ID NO.: 1, 2, 3, 4).
  • SEQ ID NO.: 2 GAUUAGUGUCAACUUCAAACC
  • the precursor RNA can be sheared to generate siRNA, and the siRNA can be substantially complementary to at least a part of the sequence of the mRNA encoding the gene.
  • B2 and B1 are basically complementary.
  • substantially complementary means that the sequence of nucleotides is sufficiently complementary to interact in a predictable manner, such as forming a secondary structure (such as a stem-loop structure).
  • two "substantially complementary" nucleotide sequences have at least 70% of the nucleotides complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; more preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • two sufficiently complementary molecules can have up to 40 unmatched nucleotides; preferably, up to 30 unmatched nucleotides; more preferably, up to 20 unmatched nucleosides Acid; More preferably, there are at most 10 unmatched nucleotides, such as 1, 2, 3, 4, 5, 8, 11 unmatched nucleotides.
  • the precursor sequence of the present invention is shown in SEQ ID NO.: 5:
  • SEQ ID NO.5 GCTAACTTCAGTGTCTGGACTCGTTTTGGCCACTGACTGACGAGTCCAGACTGAAGTTAGC.
  • the "stem-loop” structure is also referred to as the "hairpin” structure, which refers to a nucleotide molecule that can form a secondary structure including a double-stranded region (stem).
  • the double-stranded region is formed by two regions (located on the same molecule) of the nucleotide molecule, the two regions are arranged on both sides of the double-stranded part; it also includes at least one "loop” structure, including non-complementary nucleotides Molecules, that is, single-stranded regions.
  • the double-stranded portion of the nucleotide can maintain the double-stranded state.
  • insertions, deletions, substitutions, etc. can lead to non-complementarity in a small region or the small region itself forms a stem-loop structure or other forms of secondary structure.
  • the two regions can still be substantially complementary, and in the foreseeable Interaction occurs in the way to form the double-stranded region of the stem-loop structure.
  • the stem-loop structure is well-known to those skilled in the art. Usually, after obtaining a nucleic acid with a nucleotide sequence with a primary structure, those skilled in the art can determine whether the nucleic acid can form a stem-loop structure.
  • the "stem-loop structure” may exist at the end of the precursor siRNA shown in formula I.
  • C will form a fixed terminal stem-loop structure;
  • the “stem-loop structure” "It can also exist inside the precursor siRNA of formula I.
  • B1 and B2 are not completely complementary, the bases of B1 or B2 that are not complementary bound will form an internal loop.
  • the siRNA of the present invention refers to the microRNA of the siRNA family that inhibits PTP1B, and the microRNA of the siRNA family that inhibits PTP1B includes: siRNA that inhibits PTP1B or a modified siRNA derivative that inhibits PTP1B.
  • the nucleotide sequence of the siRNA that inhibits PTP1B is shown in SEQ ID NO.: 1-4, corresponding to si-1, si-2, si-3, and si-4, respectively. Particularly preferred is SEQ ID NO.: 4.
  • siRNA variants and derivatives in a broad sense can also include siRNA variants.
  • Those of ordinary skill in the art can use general methods to modify siRNAs that inhibit tyrosine kinases. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy -Glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification), etc.
  • a polynucleotide construct that can be processed into siRNA that can affect the expression of the corresponding mRNA after being introduced can be designed, that is, the polynucleotide construct can up-regulate the corresponding The amount of siRNA. Therefore, the present invention provides an isolated polynucleotide (construction), which can be transcribed into precursor RNA by human cells, and the precursor RNA can be sheared by human cells And expressed as the siRNA.
  • polynucleotide construct contains one or more structural units represented by formula II:
  • Seq forward is a nucleotide sequence that can be expressed in cells as the siRNA that inhibits PTP1B
  • Seq reverse is a nucleotide sequence that is substantially complementary to Seq forward
  • Seq reverse is a nucleotide sequence that can be expressed in cells Expressed as the nucleotide sequence of the siRNA
  • the Seq forward is a nucleotide sequence that is substantially complementary to the Seq forward
  • X is the spacer sequence between the Seq forward and the Seq reverse, and the spacer sequence It is not complementary to Seq forward and Seq reverse;
  • each structural unit can express the same or different siRNA
  • Seq forward , Seq reverse and X are defined as above;
  • the polynucleotide construct is located on an expression vector. Therefore, the present invention also includes a vector containing the siRNA or the polynucleotide construct.
  • the expression vector usually also contains a promoter, an origin of replication, and/or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vector required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as calamycin, gentamicin, hygromycin, and ampicillin resistance.
  • the expression vector is not particularly limited, and includes commercially available or conventionally prepared expression vectors.
  • Representative examples include (but are not limited to): pcDNATM6.2-GW / miR, pcDNA3, pMIR-REPORT miRNA, pAdTrack-CMV, pCMVp-NEO-BAN, pSV2, CMV4 expression vectors, pmiR-RB-Report TM, pshOK-basic, mmu-mir 300-399 miRNASelect TM , pshRNA-copGFP Lentivector, GV317, GV309, GV253, GV250, GV249, GV234, GV233, GV232, GV201, GV159 or other GV series eukaryotic expression vectors.
  • the promoter operatively connected to the expression of the precursor siRNA polynucleotide includes a constitutive promoter or a tissue-specific promoter, preferably a Pcmv promoter. In other words, these promoters are used to drive the expression of precursor siRNA.
  • promoters include (but are not limited to): Pcmv promoter, U6, H1, CD43 promoter, CD45 (LCA) promoter, CD68 promoter, Endoglin (CD105) promoter, Fibronectin promoter, Flt-1 (VEGFR-1) promoter, GFAP promoter, GPIlb (Integrin ⁇ IIb) promoter, ICAM-2 (CD102) promoter, MB (Myoglobin) promoter, NphsI (Nephrin) promoter, SPB promoter, SV40/hAlb promoter Promoter, SYN1 promoter, WASP promoter, or a combination thereof.
  • an effective amount or “effective dose” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • pharmaceutically acceptable ingredients are suitable for humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the administration mode.
  • the dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, and sustained-release agent.
  • it can be prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials).
  • the factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration The way and so on.
  • the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg/kg animal body weight (preferably 0.0001 mg-10 mg/kg animal body weight), satisfactory effects can be obtained.
  • several divided doses can be given every day, or the dose can be reduced proportionally.
  • the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, microparticles, microvesicles, exosomes, and exosomes. Shedding vesicles, nanocapsules (Nanocapsules/Nanoparticles), ⁇ -cyclodextriniclusion compound proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof.
  • the choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.
  • the expression vector can be directly administered to a subject, or the expression vector and a pharmaceutically acceptable carrier can be prepared into a drug combination before administration. Said administration includes intravenous injection.
  • the present invention also provides a method of (a) preventing and/or treating obesity-related diseases; (b) preventing and/or treating cardiovascular-related diseases; and/or (c) preventing and/or treating diseases related to metabolic abnormalities, That is, a safe and effective amount of the expression vector or pharmaceutical composition of the present invention is administered to a desired subject, thereby (a) preventing and/or treating obesity-related diseases; (b) preventing and/or treating cardiovascular-related diseases; and/or (c) Preventing and/or treating diseases related to metabolic abnormalities.
  • the present invention first developed a siRNA sequence specifically designed for PTP1B, which can effectively inhibit the expression of PTP1B in the liver and hypothalamus, and can also (a) prevent and/or treat obesity-related diseases; (b) prevent and/or Treat cardiovascular-related diseases; and/or (c) prevent and/or treat metabolic disorders-related diseases.
  • the precursor siRNA of the present invention can effectively avoid overexpression of the target sequence while also overexpressing the reverse complementary sequence of the target sequence, thereby effectively avoiding the interference effect of the reverse complementary sequence of the target sequence on the function of the target sequence.
  • the present invention combines the precursor PTP1B siRNA with the RVG polypeptide to more effectively (a) prevent and/or treat obesity-related diseases; (b) prevent and/or treat cardiovascular-related diseases; and/or (c ) Prevent and/or treat diseases related to metabolic abnormalities.
  • the cell proliferation experiment uses CCK-8 reagent to detect. Plant the cells into a six-well plate according to the passage ratio, and perform nucleic acid transfection when the cell density is appropriate. When the cells are changed, the cells are digested and counted. Resuspend the cells in 2% DMEM at a ratio of 5000-10000 cells according to 100 ⁇ L of suspension per well, set 6 replicates for each sample, and plant 5 96-well plates according to time points. At the 12th, 24th, 36th, 48th, and 60th hours after laying the slabs, one board was taken out.
  • mice Before starting the experiment, the mice must be fasted for 6 hours; Note: All the litter, trough, and drinking water of the mice must be replaced during fasting to prevent the experiment results from being affected. 2. When fasting for about 5.5 hours, weigh all the mice to be tested and calculate the insulin dosage; Note: The insulin dosage of normal mice, high-fat mice, and ob/ob mice are: 0.75U/kg, 1U respectively /kg, 1U/kg 3. Separate the mice to be tested, do not confuse them, and then cut the tail tip to measure the blood glucose value in sequence after 6 hours, and record the data. At this time, the data is 0min. 4.
  • Insulin was administered intraperitoneally according to body weight, and all mice were injected at a uniform rate within 15 minutes; 5. Time was calculated from the end of the insulin injection of the first mouse, and the mice were measured at 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes. Blood glucose was recorded and recorded; 6. After the experiment, the mice were returned to the cage and feed was added. 7. Each mouse uses its own blood glucose at 0 min as 100%, and calculates the percentage of blood glucose at each other time point. The percentages of each group of mice at the same time point are averaged to draw the ITT blood glucose curve.
  • GTT Glucose Tolerance
  • Leptin sensitivity experiment 1. Place the mice to be tested separately, 1 in each cage, add quantitative feed, and adapt for 2 days; 2. Start two days before the formal experiment, measure the weight of each mouse at 8:30 every morning And appetite. 3. The body weight and food intake were measured at 8:30 on the day of the formal experiment (day 0) and on the first day. Each mouse was intraperitoneally injected with diluted leptin at a dose of 0.5 mg/kg at 9:00 am and 7:00 pm, respectively. 4. No more leptin was given from day 2 to day 5, and weight and food intake were only measured at 8:30 in the morning. 5. Take the average body weight and food intake of the first two days of the experiment as 100%, and then take the percentages for each day's data, and draw the curve for the average percentage of mice in the group.
  • Serum insulin enzyme-linked immunosorbent assay enzyme linked immunosorbent assay, elisa
  • enzyme linked immunosorbent assay enzyme linked immunosorbent assay
  • Serum leptin enzyme-linked immunosorbent assay Reagent preparation: before the test, take out all reagents and return to room temperature; take an appropriate amount of 20 ⁇ concentrated lotion, dilute with distilled water to 1 ⁇ lotion, mix well and take an appropriate amount of 10 ⁇ concentrated for detection Buffer, dilute with distilled water to 1 ⁇ detection buffer, mix according to the total number of samples (samples and standards), dilute and concentrate the antibody with 1 ⁇ detection buffer at a ratio of 1:100, and use within 30 min. According to the total number of samples, use 1 ⁇ The detection buffer should be diluted according to the horseradish peroxidase-labeled streptavidin provided in the 1:100 dilution kit, and samples from different sources must be used within 30 minutes.
  • the measurement needs to be diluted with 1 ⁇ detection buffer.
  • the normal mice and high-fat induced mice used in the experiment were diluted 10-20 times when detecting the serum leptin content.
  • the mouse leptin standard is diluted with a certain amount of distilled water to form a 8000pg/mL standard.
  • After standing for 20 minutes, it is diluted with the standard diluent at a 2-fold ratio to form 0, 62.5, 125, 250, 500, 1000, Standard product gradients of 2000, 4000, pg/mL; 2. Remove the unused slats, put them back in the aluminum foil bag and re-seal the seal; 3.
  • the four plasmid molecules were respectively transfected into the mouse liver cancer cell line Hepa1-6 with a confluence of 60-70%. After 30 hours, the expression level of PTP1B in the cells was detected by a western experiment. The results show that PTP1B si-4 has the highest interference efficiency with PTP1B protein ( Figure 2A-B).
  • the plasmid with the highest interference efficiency and the control plasmid were transfected into Hepa1-6 with a confluency of 60-70%, and the cells were digested 6-8 hours later and counted. Resuspend the cells in 2% DMEM at a ratio of 5,000 cells in 100 ⁇ L of suspension per well, set 6 replicates for each sample, and plant 4 96-well plates according to time points. At 0, 12, 24, and 36 hours, one plate was taken out. Aspirate the culture solution, add 100 ⁇ L of diluted CCK-8 reagent (the ratio of cck-8 reagent to 2% DMEM culture solution is 1:9), and then put it back into the incubator for incubation.
  • the control plasmid and PTP1B siRNA-4/RVG plasmid were injected into the tail vein of normal mice at a dose of 10 mg/kg; 12 hours later, the mice were sacrificed, and the liver tissue and hypothalamus tissue of the mice were taken for in situ hybridization experiments. .
  • the sequence that is completely complementary to the PTP1B siRNA-4 sequence is added with green fluorescent modification as a detection probe to indicate the distribution of siRNA-4 in tissue sections; the results show that in the liver tissue of mice There is a large number of siRNA-4 distribution (Figure 3A), and siRNA-4 can also be detected in hypothalamic tissues, proving that PTP1B siRNA-4/RVG plasmid does have a brain-targeting effect (Figure 3B).
  • the high-fat-induced obese mice were equally divided into two groups according to their body weight, and the control plasmid and PTP1B siRNA-4/RVG plasmid were injected at a dose of 10 mg/kg respectively.
  • the injection is once every two days for a total of three weeks.
  • the body weight and food intake were measured every two days, and other metabolic related indicators were tested after the end of the administration.
  • Hyperlipidemia is also an important sign of obesity.
  • the four items of blood lipids in mice were tested, and it was found that the total cholesterol, triglycerides, and low-density lipoprotein of mice injected with PTP1B siRNA-4/RVG plasmid decreased significantly (Figure 7A-B, D), while the "scavenger" high density The lipoprotein content increased to a certain extent ( Figure 7C). This result indicates that the metabolism of glucose and lipids in mice is improved, and the accumulation of blood lipids is reduced.
  • injection of PTP1B siRNA-1/RVG plasmid, PTP1B siRNA-2/RVG plasmid, PTP1B siRNA-3/RVG plasmid can also obtain the similar effect of PTP1B siRNA-4/RVG plasmid.

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Abstract

L'invention concerne un ARNsi ciblé pour PTP1B et un précurseur de celui-ci et une application, et plus particulièrement, l'invention concerne une séquence précurseur d'ARNsi et un ARNsi produit à partir de celle-ci. L'ARNsi et un précurseur de celui-ci peuvent efficacement : (A) prévenir et/ou traiter des maladies liées à l'obésité ; (b) prévenir et/ou traiter des maladies cardiovasculaires ; et/ou (c) prévenir et/ou traiter des maladies liées au trouble métabolique.
PCT/CN2020/135815 2019-12-13 2020-12-11 Arnsi ciblé pour ptp1b et précurseur de celui-ci et application WO2021115442A1 (fr)

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