Sirna specifically inhibiting CD36 gene expression and use thereof

Abstract

Provided are an siRNA specifically inhibiting CD36 gene expression and the use thereof. Particularly, provided are siRNA, shRNA, a recombinant vector, a recombinant lentivirus, and a host cell that specifically inhibit CD36 gene expression, and the use thereof in the preparation of a drug for inhibiting the CD36 gene expression / treating CD36-related diseases.

Description

siRNAs that specifically inhibit CD36 gene expression and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an siRNA that specifically inhibits CD36 gene expression and its application. The siRNA targets CD36 and can be used to prevent or treat fibrotic diseases. Background Technology

[0002] CD36 is a scavenger receptor expressed in multiple cell types. It plays a crucial role in lipid metabolism, mediating lipid uptake, immune recognition, inflammation, molecular adhesion, and apoptosis. It is associated with angiogenesis, inflammatory responses, atherosclerotic thrombotic diseases, and metabolic disorders such as diabetes and obesity. Reports have shown that CD36 in hepatocytes can protect mice from diet-induced liver fibrosis in metabolic dysfunction-related fatty liver disease. The role of CD36 in the pathogenesis of pulmonary fibrosis has also been partially reported; CD36-mediated uptake of apoptotic AEC2 by lung macrophages is one of the possible drivers of pulmonary fibrosis.

[0003] Pulmonary fibrosis is the end-stage clinical manifestation of many interstitial lung diseases with different etiologies. It is characterized by persistent alveolar damage, fibroblast proliferation, and massive extracellular matrix (ECM) deposition, which leads to varying degrees of inflammation and fibrosis in the alveoli and interstitium, resulting in lung structural destruction and respiratory failure. Therefore, it is also known as interstitial lung disease.

[0004] Idiopathic pulmonary fibrosis (IPF) is the most common and severe chronic inflammatory interstitial lung disease of unknown etiology. Clinically, it presents as progressive dyspnea accompanied by a persistent dry cough. The disease is usually progressive, with a median survival of approximately 2.8 years and a 5-year survival rate of less than 50%. Most patients die from respiratory failure and secondary lung infections. IPF has been reported worldwide, and its incidence has been steadily increasing in recent years without significant geographical or racial differences. Patients are mostly middle-aged and elderly, typically developing the disease between 50 and 70 years of age, and it is rare in children. Given the large number of clinical cases, the prevalence and incidence of IPF are difficult to estimate; it is estimated to occur in 15-250 out of 100,000 people, with approximately 34,000 new cases annually depending on country, age, and sex. Currently, lung transplantation is the only treatment that can prolong the survival of patients with pulmonary fibrosis (IPF). The "Chinese Expert Consensus on the Diagnosis and Treatment of IPF" recommends pirfenidone, nintedanib, antacids, and N-acetylcysteine ​​as primary drug treatments for IPF. Among these, only pirfenidone and nintedanib are approved as effective treatments for IPF. While these drugs can slow the decline in lung function, they cannot reverse disease progression, and a significant proportion of patients have poor treatment responses. Furthermore, their specific pharmacological mechanisms are not fully understood. Therefore, elucidating the mechanisms of pulmonary fibrosis development, exploring new potential drug targets, and developing effective, relatively safe, and affordable drugs for pulmonary fibrosis are of significant social and medical importance. Summary of the Invention

[0005] The purpose of this invention is to provide a siRNA that specifically inhibits CD36 gene expression, its shRNA, and its applications.

[0006] In a first aspect, the present invention provides an siRNA for inhibiting CD36 gene expression, comprising an interference target sequence and a protective base sequence, wherein the interference target sequence comprises a nucleotide sequence selected from the following:

[0007] 5'-CCAGGGUAAGACACAGUGAUA-3' (SEQ ID NO: 1);

[0008] 5'-GCCAUAAUUGAGUUCCUUAUAAA-3' (SEQ ID NO: 2);

[0009] 5'-CGGAUCUGAAAUCGACCUUAA-3' (SEQ ID NO: 3);

[0010] 5'-GCAGGUCACAUAUUGGUCAA-3' (SEQ ID NO: 4);

[0011] 5'-GCCAAGCUAUUGCGACAUGAU-3' (SEQ ID NO: 5);

[0012] 5'-GAAGUUACAUAUUAGGCCAUA-3' (SEQ ID NO: 6);

[0013] 5'-CCGACGUUAAUCUGAAAGGAA-3' (SEQ ID NO:7);

[0014] 5'-GCCAUAAUCGACACAUAUAAA-3' (SEQ ID NO:8);

[0015] 5'-CCUGCUUAUCCAGAAGACAAU-3' (SEQ ID NO:9);

[0016] 5'-CCAUUGGUGAUGAGAAGGCAA-3' (SEQ ID NO: 10);

[0017] 5'-AGAACCUAUUGAUGGAUUAAA-3' (SEQ ID NO: 11);

[0018] 5'-ACGGCUGCAGGUCAACCUA-3' (SEQ ID NO: 12);

[0019] 5'-GGAUCUGAAAUCGACCUUA-3' (SEQ ID NO: 13);

[0020] 5'-GCUAUUGCGACAUGAUUAA-3' (SEQ ID NO: 14);

[0021] 5'-GCACCACUGUGUACAGACA-3' (SEQ ID NO: 15).

[0022] In another preferred embodiment, the protective base sequence in the siRNA may refer to the two nucleotide sequences at the 3' end of the siRNA: TT; the two TT bases are in a single-stranded suspended state, and their function is to enhance the stability of the siRNA double-stranded complex.

[0023] In another preferred embodiment, the interference target sequence of the siRNA is selected from: SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0024] In a second aspect, the present invention provides an shRNA that inhibits CD36 gene expression, said shRNA containing the structure shown in Formula II from its 5' to 3' ends: a1-b1-c-b2-a2 (Formula II)

[0025] In formula II,

[0026] b1 is the interference target sequence of the siRNA as described in any one of claims 1, b2 is a nucleotide sequence that is completely complementary to b1; c is a loop region sequence located between b1 and b2, and the loop region sequence is not complementary to b1 and b2;

[0027] a1 and a2 are either empty or optional nucleotide sequences consisting of 4-5 bases.

[0028] In another preferred embodiment, the ring sequence can be selected from: UCUGUC, UGCGGG, UCAAAC, UUUC, UGUC, UCGA, UGG, GCGCUU, GCGCUUGG, etc.

[0029] In another preferred embodiment, a1 is UGCUG, GGCC; and / or a2 is CAGG, CAGGA, or UUUUUG.

[0030] In another preferred embodiment, the DNA sequence of the shRNA is selected from the group consisting of:

[0031] 5'-CCGG-CCAGGGTAAGACACAGTGATA-CTCGAG-TATCACTGTGTCTTACCCTGG-TTTTTG-3' (SEQ ID NO: 16);

[0032] 5'-CCGG-GCCATAATTGAGTCCTATAAA-CTCGAG-TTTATAGGACTCAATTATGGCTTTTTG-3' (SEQ ID NO: 17);

[0033] 5'-CCGG-CGGATCTGAAATCGACCTTAA-CTCGAG-TTAAGGTCGATTTCAGATCCG-TTTTTG-3' (SEQ ID NO: 18);

[0034] 5'-CCGG-GCAGGTCAACATATTGGTCAA-CTCGAG-TTGACCAATATGTTGACCTGCTTTTTG-3' (SEQ ID NO: 19);

[0035] 5’-CCGG-GCCAAGCTATTGCGACATGAT-CTCGAG-ATCATGTCGCAATAGCTTGGC-TTTTTG-3’(SEQ ID NO:20);

[0036] 5’-CCGG-GAAGTTACATATTAGGCCATA-CTCGAG-TATGGCCTAATATGTAACTTC-TTTTTG-3’(SEQ ID NO:21);

[0037] 5’-CCGG-CCGACGTTAATCTGAAAGGAA-CTCGAG-TTCCTTTCAGATTAACGTCGG-TTTTTG-3’(SEQ ID NO:22);

[0038] 5’-CCGG-GCCATAATCGACACATATAAA-CTCGAG-GCCATAATCGACACATATAAA-TTTTTG-3’(SEQ ID NO:23);

[0039] 5’-CCGG-CCTGCTTATCCAGAAGACAAT-CTCGAG-ATTGTCTTCTGGATAAGCAGG-TTTTTG-3’(SEQ ID NO:24);

[0040] 5’-CCGG-CCATTGGTGATGAGAAGGCAA-CTCGAG-TTGCCTTCTCATCACCAATGG-TTTTTG-3’(SEQ ID NO:25);

[0041] 5’-CCGG-AGAACCTATTGATGGATTAAA-CTCGAG-TTTAATCCATCAATAGGTTCT-TTTTTG-3’(SEQ ID NO:26);

[0042] 5’-CCGG-ACGGCTGCAGGTCAACCTA-CTCGAG-TAGGTTGACCTGCAGCCGT-TTTTTG-3’(SEQ ID NO:27);

[0043] 5’-CCGG-GGATCTGAAATCGACCTTA-CTCGAG-TAAGGTCGATTTCAGATCC-TTTTTG-3’(SEQ ID NO:28);

[0044] 5'-CCGG-GCTATTGCGACATGATTAA-CTCGAG-TTAATCATGTCGCAATAGC-TTTTTG-3' (SEQ ID NO: 29);

[0045] 5'-CCGG-GCACCACTGTGTACAGACA-CTCGAG-TGTCTGTACACAGTGGTGC-TTTTTG-3' (SEQ ID NO: 30).

[0046] In a third aspect, the present invention provides a recombinant vector for inhibiting CD36 gene expression, comprising an adeno-associated virus (AAV) as a vector and a DNA sequence encoding the shRNA of the present invention.

[0047] In another preferred embodiment, a macrophage-specific promoter (such as SP146) is introduced into the recombinant vector to control the expression of shRNA, thereby enabling specific knockdown of CD36 in macrophages.

[0048] In another preferred embodiment, the AAV virus also contains a green fluorescent protein (GFP) sequence, which can be used to indicate the targeting of AAV.

[0049] In a fourth aspect, the present invention provides a recombinant lentivirus that inhibits CD36 gene expression, which is obtained by cloning a DNA sequence encoding the shRNA of the present invention into the lentiviral plasmid PLKO.1 to obtain a recombinant plasmid, and then co-transfecting the recombinant plasmid with the packaging plasmid psPAX2 and the envelope plasmid pMD2 into mammalian cells (such as HEK293T cells).

[0050] In a fifth aspect, the present invention provides a host cell comprising at least one selected from the present invention's siRNA, shRNA, their coding sequences, the present invention's recombinant vector, and the present invention's recombinant lentivirus.

[0051] The present invention does not particularly limit the type of host cell. For example, it can be the cell used to prepare the shRNA and other sequences of the present invention, such as the HEK293T cell described in the embodiments of this invention; or it can be a cell whose CD36 is knocked down by the above-mentioned sequences of the present invention, such as the lung macrophages described in the embodiments of this invention, and includes, but is not limited to, macrophage cell lines of various species and primary macrophages of various species.

[0052] In a sixth aspect, the present invention provides the use of siRNA as described in the first aspect, shRNA as described in the second aspect, recombinant vector as described in the third aspect, recombinant lentivirus as described in the fourth aspect, or host cell as described in the fifth aspect in the preparation of medicaments for the prevention or treatment of CD36-related diseases.

[0053] In another preferred embodiment, the disease is a fibrotic disease, such as pulmonary fibrosis, liver fibrosis, myocardial fibrosis, or kidney fibrosis.

[0054] In another preferred embodiment, the disease is pulmonary fibrosis.

[0055] In a seventh aspect, the present invention provides a method for specifically inhibiting CD36 gene expression, comprising the step of specifically inhibiting CD36 gene expression using the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA.

[0056] In another aspect, the present invention provides a method for treating pulmonary fibrosis, comprising administering a therapeutically effective amount of the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA to a subject in need.

[0057] In some implementations, the subject may be a mammal, including but not limited to humans, rats, dogs, pigs, cattle, sheep, horses, etc.

[0058] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0059] Figure 1 shows the knockdown efficiency of different siRNAs for CD36;

[0060] Figure 2 shows the effect of CD36 knockdown on macrophages and adeno-associated virus transfection on lung injury in mice;

[0061] Figure 3 shows the effects of CD36 knockdown on macrophages and adeno-associated virus transfection on lung fibrosis markers and lung function in mice.

[0062] Figure 4 shows the effect of CD36 knockdown adeno-associated virus transfection targeting macrophages on M2 polarization of mouse lung macrophages. Detailed Implementation

[0063] Through extensive and in-depth research, including numerous screenings and tests, the inventors have developed a specific siRNA and shRNA that inhibit CD36 gene expression, along with their applications. Using single-cell sequencing technology, the inventors discovered that CD36 expression is significantly upregulated in pulmonary fibrosis macrophages. Knocking down or reducing CD36 gene expression in mouse lung macrophages effectively protects mice from tissue damage and fibrosis. This invention further designs multiple siRNAs and shRNAs that specifically knock down CD36. By using adeno-associated virus (AAV) to deliver the shRNA specifically targeting mouse macrophages, this invention can be used to treat pulmonary fibrosis. This invention is based on these findings.

[0064] the term

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0067] As used herein, the term “room temperature” or “normal temperature” refers to a temperature of 4–40°C, preferably 25 ± 5°C.

[0068] siRNA

[0069] siRNA (small interfering RNA) is a class of small RNA molecules approximately 20-25 nucleotides long. siRNA inhibits the expression of specific genes within cells through the RNA interference (RNAi) pathway. siRNA consists of two complementary single-stranded RNA molecules that form a double-stranded structure with a 3' overhang.

[0070] Specifically, Ming provides a class of siRNAs that inhibit CD36 gene expression, with the following interference target sequences:

[0071] 5'-CCAGGGUAAGACACAGUGAUA-3' (SEQ ID NO: 1);

[0072] 5'-GCCAUAAUUGAGUUCCUUAUAAA-3' (SEQ ID NO: 2);

[0073] 5'-CGGAUCUGAAAUCGACCUUAA-3' (SEQ ID NO: 3);

[0074] 5'-GCAGGUCACAUAUUGGUCAA-3' (SEQ ID NO: 4);

[0075] 5'-GCCAAGCUAUUGCGACAUGAU-3' (SEQ ID NO: 5);

[0076] 5'-GAAGUUACAUAUUAGGCCAUA-3' (SEQ ID NO: 6);

[0077] 5'-CCGACGUUAAUCUGAAAGGAA-3' (SEQ ID NO:7);

[0078] 5'-GCCAUAAUCGACACAUAUAAA-3' (SEQ ID NO:8);

[0079] 5'-CCUGCUUAUCCAGAAGACAAU-3' (SEQ ID NO:9);

[0080] 5'-CCAUUGGUGAUGAGAAGGCAA-3' (SEQ ID NO: 10);

[0081] 5'-AGAACCUAUUGAUGGAUUAAA-3' (SEQ ID NO: 11);

[0082] 5'-ACGGCUGCAGGUCAACCUA-3' (SEQ ID NO: 12);

[0083] 5'-GGAUCUGAAAUCGACCUUA-3' (SEQ ID NO: 13);

[0084] 5'-GCUAUUGCGACAUGAUUAA-3' (SEQ ID NO: 14);

[0085] 5'-GCACCACUGUGUACAGACA-3' (SEQ ID NO: 15).

[0086] In addition, the siRNA also includes a positive strand that is completely complementary to the interference target sequence.

[0087] In another preferred embodiment, the siRNA also includes two unpaired bases (protective base sequences) such as TT at the 3' end of the antisense / sense strand; these two bases are in a single-stranded dangling state, which enhances the stability of the siRNA double-stranded complex and may contribute to its recognition and function.

[0088] Experiments have shown that the siRNA of the present invention can specifically inhibit CD36 gene expression, and the siRNAs containing SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 have particularly excellent CD36 inhibition effects.

[0089] shRNA

[0090] shRNA (short hairpin RNA) is a synthetic RNA molecule used to trigger the RNA interference (RNAi) pathway. shRNA is processed into siRNA precursors within the cell, ultimately achieving gene silencing; therefore, shRNA is a precursor molecule of siRNA.

[0091] shRNA is characterized by a hairpin structure, which includes a stem region (the double-stranded region formed by the base pairing of two complementary RNA strands) and a loop region (the portion connecting the two complementary RNA strands). Typically, the stem region of shRNA is about 19-29 nucleotides long, while the loop region consists of a few nucleotides (usually 4-8) and connects the two parts of the stem region.

[0092] Specifically, this paper provides a class of shRNAs based on the aforementioned siRNAs that inhibit CD36 gene expression. The shRNAs contain the structure shown in Formula II from the 5' to 3' ends: a1-b1-c-b2-a2 (Formula II)

[0093] In formula II,

[0094] b1 is a nucleotide sequence that can be expressed in cells as the CD36 siRNA described above; b2 is a nucleotide sequence that is substantially complementary or completely complementary to b1; c is a loop region sequence located between b1 and b2, and the loop region sequence is not complementary to b1 and b2.

[0095] a1 and a2 are either empty or optional nucleotide sequences consisting of 4-5 bases.

[0096] Typically, the ring sequence can be selected from: UCUGUC, UGCGGG, UCAAAC, UUUC, UGUC, UCGA, UGG, GCGCUU, GCGCUUGG, etc.

[0097] The a1 is UGCUG or GGCC; and / or the a2 is CAGG, CAGGA, or UUUUUG.

[0098] The structure shown in Formula II, after being transfected into cells, forms the secondary structure shown in Formula I:

[0099] Preferably, the present invention is also intended to include a DNA sequence capable of expressing the shRNA of the present invention.

[0100] In another preferred embodiment, the DNA sequence of the shRNA is selected from the group below, and after transcription / cleavage, a short fragment having the hairpin structure described above is formed to obtain the shRNA of the present invention:

[0101] 5'-CCGG-CCAGGGTAAGACACAGTGATA-CTCGAG-TATCACTGTGTCTTACCCTGG-TTTTTG-3' (SEQ ID NO: 16);

[0102] 5'-CCGG-GCCATAATTGAGTCCTATAAA-CTCGAG-TTTATAGGACTCAATTATGGC-TTTTTG-3' (SEQ ID NO: 17);

[0103] 5'-CCGG-CGGATCTGAAATCGACCTTAA-CTCGAG-TTAAGGTCGATTTCAGATCCG-TTTTTG-3' (SEQ ID NO: 18);

[0104] 5'-CCGG-GCAGGTCAACATATTGGTCAA-CTCGAG-TTGACCAATATGTTGACCTGC-TTTTTG-3' (SEQ ID NO: 19);

[0105] 5'-CCGG-GCCAAGCTATTGCGACATGAT-CTCGAG-ATCATGTCGCAATAGCTTGGC-TTTTTG-3' (SEQ ID NO: 20);

[0106] 5'-CCGG-GAAGTTACATATTAGGCCATA-CTCGAG-TATGGCCTAATATGTAACTTC-TTTTTG-3' (SEQ ID NO: 21);

[0107] 5'-CCGG-CCGACGTTAATCTGAAAGGAA-CTCGAG-TTCCTTTCAGATTAACGTCGG-TTTTTG-3' (SEQ ID NO: 22);

[0108] 5’-CCGG-GCCATAATCGACACATATAAA-CTCGAG-GCCATAATCGACACATATAAA-TTTTTG-3’(SEQ ID NO:23);

[0109] 5’-CCGG-CCTGCTTATCCAGAAGACAAT-CTCGAG-ATTGTCTTCTGGATAAGCAGG-TTTTTG-3’(SEQ ID NO:24);

[0110] 5’-CCGG-CCATTGGTGATGAGAAGGCAA-CTCGAG-TTGCCTTCTCATCACCAATGG-TTTTTG-3’(SEQ ID NO:25);

[0111] 5’-CCGG-AGAACCTATTGATGGATTAAA-CTCGAG-TTTAATCCATCAATAGGTTCT-TTTTTG-3’(SEQ ID NO:26);

[0112] 5’-CCGG-ACGGCTGCAGGTCAACCTA-CTCGAG-TAGGTTGACCTGCAGCCGT-TTTTTG-3’(SEQ ID NO:27);

[0113] 5’-CCGG-GGATCTGAAATCGACCTTA-CTCGAG-TAAGGTCGATTTCAGATCC-TTTTTG-3’(SEQ ID NO:28);

[0114] 5’-CCGG-GCTATTGCGACATGATTAA-CTCGAG-TTAATCATGTCGCAATAGC-TTTTTG-3’(SEQ ID NO:29);

[0115] 5’-CCGG-GCACCACTGTGTACAGACA-CTCGAG-TGTCTGTACACAGTGGTGC-TTTTTG-3’(SEQ ID NO:30).

[0116] Expression vector

[0117] Typically, the DNA sequence encoding the shRNA is located on an expression vector. Therefore, this invention also includes a vector containing a DNA sequence expressing the siRNA or shRNA described in this invention. The expression vector typically also contains a promoter, a replication origin, and / or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vectors required by this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as resistance to kanamycin, gentamicin, hygromycin, and ampicillin.

[0118] In this invention, 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, pCAMBIA3101+pUC-35S, pCMVp-NEO-BAN, pBI121, pBin438, pCAMBIA1301, pSV2, CMV4 expression vector, and pmiR-RB-Report. TM , pshOK-basic, mmu-mir 300-399miRNASelect TM pshRNA-copGFP Lentivector, GV317, GV309, GV253, GV250, GV249, GV234, GV233, GV232, GV201, GV159 or other GV series expression vectors.

[0119] In another preferred embodiment, in the expression vector, the promoter operably linked to the DNA expressing the shRNA includes constitutive promoters or tissue-specific promoters, and representative 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, GPIIb (IntegrinαIIb) promoter, ICAM-2 (CD102) promoter, MB (Myoglobin) promoter, NphsI (Nephrin) promoter, SPB promoter, SV40 / hAlb promoter, SYN1 promoter, WASP promoter or combinations thereof, LysM promoter, MCP-1 promoter, CD11b promoter, CD68 promoter, and SP146 promoter. Preferred promoters are those that are specifically activated in macrophages (such as LysM promoter, MCP-1 promoter, CD11b promoter, CD68 promoter, SP146 promoter).

[0120] Preferably, the present invention provides a recombinant vector for inhibiting CD36 gene expression, comprising an adeno-associated virus (AAV) as a vector and a nucleotide sequence encoding the shRNA of the present invention.

[0121] Specifically, a macrophage-specific promoter (such as SP146) is introduced into the recombinant vector to control the expression of shRNA, thereby enabling specific knockdown of CD36 in macrophages.

[0122] In another preferred embodiment, the AAV virus also contains a green fluorescent protein (GFP) sequence, which can be used to indicate the targeting of AAV.

[0123] The present invention also provides a recombinant lentivirus that inhibits CD36 gene expression, which is obtained by cloning the nucleotide sequence encoding the shRNA of the present invention into the lentiviral plasmid PLKO.1 to obtain a recombinant plasmid, and then co-transfecting the recombinant plasmid with the packaging plasmid psPAX2 and the envelope plasmid pMD2 into mammalian cells (such as HEK293T cells).

[0124] The present invention also provides a host cell comprising at least one selected from the present invention's siRNA, shRNA, DNA sequence encoding the same, the present invention's recombinant vector, and the present invention's recombinant lentivirus.

[0125] The present invention does not particularly limit the type of host cell. For example, it can be the cell used to prepare the shRNA and other sequences of the present invention, such as the HEK293T cell described in the embodiments of this invention; or it can be a cell whose CD36 is knocked down by the above-mentioned sequences of the present invention, such as the lung macrophages described in the embodiments of this invention, and includes, but is not limited to, macrophage cell lines of various species and primary macrophages of various species.

[0126] The expression vector of the present invention can be constructed using methods commonly used in the art.

[0127] Pharmaceutical Composition and Administration

[0128] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0129] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0130] The pharmaceutical compositions of the present invention contain 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 solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0131] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0132] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, microparticles, microvesicles, exosomes, shedding vesicles, nanocapsules / nanoparticles, β-cyclodextrin inclusion compounds, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0133] In this invention, the expression vector can be applied directly to the target, or the expression vector can be combined with a pharmaceutically acceptable carrier to form a drug for administration. Administration includes intravenous injection.

[0134] Treatment

[0135] This invention also provides a method for treating CD36 expression-related diseases, namely, applying a safe and effective amount of the expression vector or pharmaceutical composition of this invention to the desired subject, thereby treating CD36 activity-related diseases. Generally, "CD36 expression-related diseases" refer to patients suffering from said diseases where the expression level (or activity) of CD36 protein or mRNA is significantly different from the amount (or activity) E0 of CD36 in normal human tissues or body fluids, i.e., high expression. Preferably, "high expression" means E1 ≥ 1.5E0, more preferably E1 ≥ 2E0. Whether CD36 is highly expressed can be detected by conventional methods. Typically, diseases associated with high CD36 expression include (but are not limited to): cardiovascular diseases such as atherosclerosis, coronary artery disease (CAD), and hypertension; metabolic diseases such as type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic fatty liver disease (NAFLD); hematologic disorders such as thrombosis; Alzheimer's disease (AD); or cancer. In particular, this invention has unexpectedly revealed high CD36 expression in fibrotic diseases such as pulmonary fibrosis.

[0136] The fibrotic diseases of this invention include (but are not limited to): pulmonary fibrosis, liver fibrosis, cardiac fibrosis, renal fibrosis, pancreatic fibrosis, skin fibrosis, intestinal fibrosis, ocular fibrosis, esophageal fibrosis, or combinations thereof, preferably pulmonary fibrosis, particularly idiopathic pulmonary fibrosis. In this invention, prevention and / or treatment of pulmonary fibrosis includes one or more features selected from the group consisting of: improving alveolar structure; reducing mesenchymal cell proliferation in lung tissue; reducing total cell count and LDH content in bronchoalveolar lavage fluid; improving inflammation (reducing inflammatory factors such as IL-1β, IL-6, and CCL2); reducing extracellular matrix proliferation and deposition; reducing hydroxyproline content; improving the molecular levels of fibrosis-related indicators (such as reducing hydroxyproline content, α-SMA, COL-1, and / or Fibronectin levels); and / or reducing lung injury and improving lung function, etc.

[0137] The main advantages of this invention include:

[0138] This invention provides a new class of siRNAs that specifically inhibit CD36, as well as their shRNAs, the DNA sequences encoding them, recombinant vectors, recombinant lentiviruses, or host cells.

[0139] The CD36 inhibitors of the present invention have excellent therapeutic effects in vivo, and can therefore be used to develop new drugs for the treatment of CD36-related diseases.

[0140] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0141] Example 1: Screening for CD36 siRNA knockdown targeting lung macrophages

[0142] Lipofectamine 3000 and 20 μM siRNA (1 μL:1 μL ratio) were added to 50 μL of Opti-MEM and mixed for 5 min, gently pipetting 3-5 times to mix. The siRNA and lipo3000 were thoroughly mixed and incubated at room temperature for 10 min, gently pipetting 3-5 times to mix. The culture medium was replaced with 50% Opti-MEM. The liposome complex was then added to lung macrophages MH-S and incubated for 12 hours before changing the medium.

[0143] After 48 hours, the knockdown efficiency was verified, and subsequent processing and testing were performed. Culture medium was aspirated from cell culture plates, washed twice with PBS, and 200 μL of Trizol was added to each well. The mixture was slowly shaken on a shaker to cover the cell surface and incubated on ice for 5 min. Cells and Trizol were scraped off with a pipette tip, and the mixture was continuously pipetted and collected into 1.5 mL RNase-free EP tubes. 40 μL of chloroform was added to each tube, and the mixture was vortexed. Subsequent steps were the same as for tissue RNA extraction. For RNA concentration determination, the NanoDrop wells were washed twice with DEPC water and wiped clean with lens paper. DEPC water was used as a blank control for zeroing. 1 μL of the sample to be tested was added to the NanoDrop well, and the data was measured and saved. Next step: reverse transcription. The RNA concentration was measured, and the volume required for 1 μg of RNA was calculated. 5X reaction solution was added, and the remainder was brought to 20 μL with water. The reaction was carried out at 37°C for 15 min, followed by inactivation at 85°C for 5 s, and reverse transcription was performed to form cDNA. Real-time quantitative PCR was then performed: the reaction system consisted of 5.5 μL of 2×SYBR, 5 μL of cDNA, and 0.5 μL of primers, for a total volume of 11 μL. Using GAPDH as the internal reference gene, ΔCt (target gene Ct - internal reference gene Ct) was calculated for each sample. The control group's ΔCt was used as the standard, and ΔΔCt (each sample's ΔCt - control group's ΔCt) was calculated. The relative expression level of the target gene mRNA in each sample was 2 - ΔΔCt.

[0144] As shown in Figure 1, all 15 siRNAs of the present invention have the effect of knocking down CD36, among which the siRNAs with sequences 2, 7, 8 and 13-15 can significantly knock down the expression level of CD36.

[0145] Example 2: Targeting lung macrophages to knock down CD36 slows down bleomycin-induced pulmonary fibrosis in mice

[0146] Animal model preparation: Male C57BL / 6J mice, aged 8-10 weeks, were anesthetized by intraperitoneal injection of 10% sodium pentobarbital. CD36-knockdown adeno-associated virus AAV-shCD36 (AAV-SP146-shCD36), targeting macrophages, was injected intratracheally. The shCD36 sequence is SEQ ID NO:28, with a titer of 1.0E+11 (VG / mL). Each mouse received 50 μL of virus via tracheal injection. Simultaneously, the control group received the same titer and volume of empty adeno-associated virus AAV-Scr. Two weeks after virus injection, bleomycin was injected intratracheally to establish the model. Mice injected with the same volume of saline via tracheal injection served as controls. Twenty-one days after administration of bleomycin or saline, mice were sacrificed, and the degree of pulmonary fibrosis was analyzed.

[0147] As shown in Figure 2 (A and B), H&E staining of pathological sections revealed that, compared with the Scr virus-transduced saline control group mice, the bleomycin-induced model group mice exhibited disrupted alveolar structure and thickened pulmonary interstitium. The shCD36 adeno-associated virus-transduced model mice showed significantly improved alveolar structure, reduced mesenchymal cell proliferation in lung tissue, and alleviated lung injury. Compared with the Scr virus-transduced saline control group mice, the bleomycin-induced model group mice showed a significantly increased H&E index, while the shCD36 adeno-associated virus-transduced model mice showed a decreased H&E index, thus alleviating lung injury.

[0148] As shown in Figure 2 (C and D), the total protein and LDH content results indicate that, compared with the control group mice transduced with Scr virus, the total protein and LDH content of the bleomycin-induced model group mice were increased, while the total protein and LDH content of the shCD36 adeno-associated virus-transduced model mice were significantly reduced, alleviating lung injury.

[0149] In summary, the results of H&E staining, total protein in alveoli, and LDH content in Figure 2 show that, in bleomycin-induced pulmonary fibrosis model mice, mice transduced with shCD36 adeno-associated virus exhibited less lung damage compared to mice transduced with Scr virus.

[0150] As shown in Figures 3A and 3B, Masson staining, fibrosis scores, and collagen area of ​​the pathological sections revealed that, compared with the control group mice transduced with Scr virus, bleomycin-induced model mice showed significantly improved collagen deposition, and shCD36 adeno-associated virus-transduced model mice also showed significantly improved collagen deposition and alleviated lung injury. Compared with the control group mice transduced with Scr virus, the fibrosis score and collagen area of ​​the bleomycin-induced model mice were significantly increased, while those of the shCD36 adeno-associated virus-transduced model mice were significantly decreased, thus alleviating pulmonary fibrosis.

[0151] As shown in Figure 3C, the transcription results of fibrosis-related genes showed that, compared with the control group mice transduced by Scr virus, the levels of α-SMA, COL-1, and Fibronectin in the bleomycin-induced model group mice were significantly increased, while the levels of α-SMA, COL-1, and Fibronectin in the shCD36 adeno-associated virus transduced model group mice were significantly decreased, thus alleviating pulmonary fibrosis.

[0152] As shown in Figure 3D, the forced vital capacity (FVC) results indicate that, compared with the control group mice transduced with Scr virus, the FVC volume of the bleomycin-induced model group mice was significantly decreased, while the FVC volume of the shCD36 adeno-associated virus transduced model mouse group was significantly increased.

[0153] In summary, the Masson staining and transcription results of fibrosis-related genes in Figure 3 show that, in bleomycin-induced pulmonary fibrosis model mice, mice transduced with shCD36 adeno-associated virus exhibited significantly milder pulmonary fibrosis compared to mice transduced with Scr virus.

[0154] As shown in Figure 4A, the transcription results of fibrosis-related genes show that, compared with the control group mice transduced by Scr virus, the expression of CD206, Arg1, and Tgf-β in the bleomycin-induced model group mice was significantly increased, while the expression of CD206, Arg1, and Tgf-β in the shCD36 adeno-associated virus transduced model group mice was significantly decreased by more than 50%.

[0155] As shown in Figure 4B, the results of Tgf-β content in bronchoalveolar lavage fluid indicate that, compared with the control group mice transduced with Scr virus, the BALF Tgf-β content in the bleomycin-induced model group mice was significantly increased, while the shCD36 adeno-associated virus transduced model mouse group was able to significantly downregulate the BALF Tgf-β content to 100 pg / ml.

[0156] In summary, the transcription results of fibrosis-related genes and the BALF Tgf-β content results in Figure 4 show that, in bleomycin-induced pulmonary fibrosis model mice, mice transduced with shCD36 adeno-associated virus were able to inhibit macrophage M2 polarization compared with mice transduced with Scr virus.

[0157] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A siRNA that inhibits CD36 gene expression, comprising an interference target sequence and a protective base sequence, wherein, The interference target sequence comprises nucleotide sequences selected from the following: 5'-CCAGGGUAAGACACAGUGAUA-3' (SEQ ID NO: 1); 5'-GCCAUAAUUGAGUUCCUUAUAAA-3' (SEQ ID NO: 2); 5'-CGGAUCUGAAAUCGACCUUAA-3' (SEQ ID NO: 3); 5'-GCAGGUCACAUAUUGGUCAA-3' (SEQ ID NO: 4); 5'-GCCAAGCUAUUGCGACAUGAU-3' (SEQ ID NO: 5); 5'-GAAGUUACAUAUUAGGCCAUA-3' (SEQ ID NO: 6); 5'-CCGACGUUAAUCUGAAAGGAA-3' (SEQ ID NO:7); 5'-GCCAUAAUCGACACAUAUAAA-3' (SEQ ID NO:8); 5'-CCUGCUUAUCCAGAAGACAAU-3' (SEQ ID NO:9); 5'-CCAUUGGUGAUGAGAAGGCAA-3' (SEQ ID NO: 10); 5'-AGAACCUAUUGAUGGAUUAAA-3' (SEQ ID NO: 11); 5'-ACGGCUGCAGGUCAACCUA-3' (SEQ ID NO: 12); 5'-GGAUCUGAAAUCGACCUUA-3' (SEQ ID NO: 13); 5'-GCUAUUGCGACAUGAUUAA-3' (SEQ ID NO: 14); 5'-GCACCACUGUGUACAGACA-3' (SEQ ID NO: 15).

2. The siRNA as described in claim 1, characterized in that, The interference target sequences of the siRNA are selected from: SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:

15.

3. A shRNA that inhibits CD36 gene expression, said shRNA containing the structure shown in Formula II from its 5' to 3' ends: a1-b1-c-b2-a2 Formula II In formula II, b1 is the interference target sequence of the siRNA as described in any one of claims 1, b2 is a nucleotide sequence that is completely complementary to b1; c is a loop region sequence located between b1 and b2, and the loop region sequence is not complementary to b1 and b2; a1 and a2 are either empty or optional nucleotide sequences consisting of 4-5 bases.

4. The shRNA as described in claim 3, characterized in that, The DNA sequence of the shRNA is selected from the following group: 5’-CCGG-CCAGGGTAAGACACAGTGATA-CTCGAG-TATCACTGTGTC TTACCCTGG-TTTTTG-3’(SEQ ID NO:16); 5’-CCGG-GCCATAATTGAGTCCTATAAA-CTCGAG-TTTATAGGACTCAATTATGGCTTTTTG-3’(SEQ ID NO:17); 5’-CCGG-CGGATCTGAAATCGACCTTAA-CTCGAG-TTAAGGTCGATTTCAGATCCG-TTTTTG-3’(SEQ ID NO:18); 5’-CCGG-GCAGGTCAACATATTGGTCAA-CTCGAG-TTGACCAATATGTTGACCTGCTTTTTG-3’(SEQ ID NO:19); 5’-CCGG-GCCAAGCTATTGCGACATGAT-CTCGAG-ATCATGTCGCAAT AGCTTGGC-TTTTTG-3’(SEQ ID NO:20); 5’-CCGG-GAAGTTACATATTAGGCCATA-CTCGAG-TATGGCCTAATATGTAACTTC-TTTTTG-3’(SEQ ID NO:21); 5’-CCGG-CCGACGTTAATCTGAAAGGAA-CTCGAG-TTCCTTTCAGATTAACGTCGG-TTTTTG-3’(SEQ ID NO:22); 5’-CCGG-GCCATAATCGACACATATAAA-CTCGAG-GCCATAATCGACACATATAAA-TTTTTG-3’(SEQ ID NO:23); 5’-CCGG-CCTGCTTATCCAGAAGACAAT-CTCGAG-ATTGTCTTCTGGATAAGCAGG-TTTTTG-3’(SEQ ID NO:24); 5’-CCGG-CCATTGGTGATGAGAAGGCAA-CTCGAG-TTGCCTTCTCATCACCAATGG-TTTTTG-3’(SEQ ID NO:25); 5’-CCGG-AGAACCTATTGATGGATTAAA-CTCGAG-TTTAATCCATCAATAGGTTCT-TTTTTG-3’(SEQ ID NO:26); 5’-CCGG-ACGGCTGCAGGTCAACCTA-CTCGAG-TAGGTTGACCTGCAGCCGT-TTTTTG-3’(SEQ ID NO:27); 5'-CCGG-GGATCTGAAATCGACCTTA-CTCGAG-TAAGGTCGATTTCAGATCC-TTTTTG-3' (SEQ ID NO: 28); 5'-CCGG-GCTATTGCGACATGATTAA-CTCGAG-TTAATCATGTCGCAATAGC-TTTTTG-3' (SEQ ID NO: 29); 5'-CCGG-GCACCACTGTGTACAGACA-CTCGAG-TGTCTGTACACAGTGGTGC-TTTTTG-3' (SEQ ID NO: 30).

5. A recombinant vector for inhibiting CD36 gene expression, comprising an adeno-associated virus (AAV) as a vector and a DNA sequence encoding the shRNA of the present invention.

6. The recombinant vector as described in claim 5, characterized in that a macrophage-specific promoter (such as SP146) is introduced into the recombinant vector to control the expression of shRNA, thereby achieving specific knockdown of CD36 in macrophages.

7. A recombinant lentivirus that inhibits CD36 gene expression, which is obtained by cloning a DNA sequence encoding the shRNA of the present invention into the lentiviral plasmid PLKO.1 to obtain a recombinant plasmid, and then co-transfecting the recombinant plasmid with the packaging plasmid psPAX2 and the envelope plasmid pMD2 into mammalian cells (such as HEK293T cells).

8. A host cell comprising at least one selected from the siRNA of claim 1 or 2, the shRNA of claim 3 or 4, their coding sequences, the recombinant vector of claim 5 or 6, and the recombinant lentivirus of claim 7.

9. The use of the siRNA of claim 1 or 2, the shRNA of claim 3 or 4, their coding sequences, the recombinant vector of claim 5 or 6, the recombinant lentivirus of claim 7, or the host cell of claim 8 in the preparation of a medicament for the prevention or treatment of CD36-related diseases.

10. The use as described in claim 9, characterized in that, The disease in question is fibrosis.

11. The use as described in claim 10, characterized in that, The fibrotic diseases are selected from the following group: pulmonary fibrosis, liver fibrosis, myocardial fibrosis, or renal fibrosis.

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