WO2023174265A1 - 一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用 - Google Patents

一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用 Download PDF

Info

Publication number
WO2023174265A1
WO2023174265A1 PCT/CN2023/081330 CN2023081330W WO2023174265A1 WO 2023174265 A1 WO2023174265 A1 WO 2023174265A1 CN 2023081330 W CN2023081330 W CN 2023081330W WO 2023174265 A1 WO2023174265 A1 WO 2023174265A1
Authority
WO
WIPO (PCT)
Prior art keywords
dmp
mir533
raav
cells
mice
Prior art date
Application number
PCT/CN2023/081330
Other languages
English (en)
French (fr)
Inventor
王进科
罗涛
汤海林
Original Assignee
东南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东南大学 filed Critical 东南大学
Publication of WO2023174265A1 publication Critical patent/WO2023174265A1/zh

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0014Skin, i.e. galenical aspects of topical compositions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/06Antipsoriatics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/14Type of nucleic acid interfering N.A.
    • C12N2310/141MicroRNAs, miRNAs
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011Details
    • C12N2750/14011Parvoviridae
    • C12N2750/14111Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14121Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011Details
    • C12N2750/14011Parvoviridae
    • C12N2750/14111Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011Details
    • C12N2750/14011Parvoviridae
    • C12N2750/14111Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14132Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011Details
    • C12N2750/14011Parvoviridae
    • C12N2750/14111Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of inflammatory gene therapy biotechnology, and specifically relates to a recombinant adeno-associated virus and its application in the treatment of inflammatory diseases.
  • Inflammation is the body's protective response to infection and physical injury. A moderate inflammatory response helps maintain homeostasis in the body.
  • abnormal inflammatory responses can lead to inflammatory diseases.
  • some infectious agents can cause systemic inflammation that may lead to sepsis, cytokine release syndrome, acute respiratory distress syndrome, and even multiple organ failure.
  • Some persistent infections lead to chronic inflammation and a high risk of cancer, such as hepatitis and liver cancer caused by the hepatitis B virus.
  • Many chronic inflammations are caused by abnormal changes in adaptive immunity, leading to various autoimmune diseases, such as arthritis, inflammatory bowel disease, lupus, psoriasis, dermatitis, asthma, multiple sclerosis, and steatohepatitis. and even atherosclerosis, diabetes, neurodegenerative diseases, and inflammatory aging.
  • many abnormal changes in innate immunity can induce various autoinflammatory diseases. Therefore, abnormal inflammation poses a widespread and serious threat to human health.
  • cytokine monoclonal antibodies such as pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ , IL-1 ⁇ , IL-5, IL-6, IL-12, IL-17A , IL-17F, IL-23 and anti-inflammatory cytokines IL-4, IL-10, IL-11, IL-13, TGF ⁇
  • antibodies or antagonists of cytokine receptors such as IL-6R, IL-5R ⁇ , IL-4R ⁇
  • CD antibodies such as CD4, CD14, CD19, CD20, CD38, CD40.
  • JAK small molecule inhibitors (JAK1, JAK2, JAK3, TYK2) are rapidly developing promising new anti-inflammatory drugs. There is no doubt that current anti-inflammatory drugs have greatly benefited patients. However, anti-inflammatory drugs still face several key challenges, such as primary non-response, drug resistance or loss of response, relapse, and multiple side effects. Therefore, current treatment methods are far from meeting the needs of clinical treatment of inflammatory diseases.
  • NF- ⁇ B is a family of sequence-specific DNA-binding transcription factors, including RelA/p65, p50, p52, RelB, and c-Rel, which play a key regulatory role in inflammation.
  • activated NF- ⁇ B mainly RelA-p50 heterodimer
  • NF- ⁇ B can directly regulate the expression of a variety of inflammatory genes, including adhesion factors (such as ICAM-1, VCAM-1), cytokines (such as IL-1 ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-8, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-23, TNF ⁇ , IFN ⁇ , IFN ⁇ ) and chemokines (such as CCL5, CCL17, CCL19, CCL20, CCL22, CCL23, CCL28).
  • adhesion factors such as ICAM-1, VCAM-1
  • cytokines such as IL-1 ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-8, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-23, TNF ⁇ , IFN ⁇ , IFN ⁇
  • chemokines such as CCL5, CCL17, CCL19, CCL20, CCL22, CCL
  • the DMP-miR533 plasmid vector can sense and inhibit intracellular NF- ⁇ B activity on cells cultured in vitro.
  • the plasmid vector itself cannot yet be directly used for in vivo administration in animals, which means that this species Plasmid vectors cannot achieve transfection of cells in vivo and control of NF- ⁇ B activity.
  • inflammation itself is not the behavior of certain cells outside the body, but a biological process at the level of mammalian living bodies. physiological or pathological reactions. Therefore, it is still unknown whether DMP-miR533, which can sense and control NF- ⁇ B activity in cultured cells in vitro, can control NF- ⁇ B activity and achieve inflammation treatment in living mammals.
  • the present invention provides a new recombinant adeno-associated virus, which can treat inflammatory diseases in living mammals by inhibiting intracellular NF- ⁇ B activity, and is expected to be used to prepare new biological Drugs for the treatment of inflammatory diseases in humans.
  • the present invention also provides the construction method and application of the recombinant adeno-associated virus.
  • the present invention proposes a recombinant adeno-associated virus for the treatment of inflammatory diseases.
  • the recombinant adeno-associated virus rAAV-DMP-miR533 uses adeno-associated virus as a vector and contains one to multiple copies.
  • the functional DNA fragment DMP-miR; the functional DNA fragment DMP-miR is composed of two functional elements DMP and miR, wherein DMP is an NF- ⁇ B specific promoter, and the miR encodes a protein that can target NF- ⁇ B mRNA microRNA.
  • the DMP is an NF- ⁇ B specific promoter, consisting of an NF- ⁇ B decoy agent and a minimal promoter.
  • the DMP includes various sequences of NF- ⁇ B decoy agents and minimal promoters.
  • the sequence of the DMP (SEQ ID NO.1) is 5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CTA GAG GGT ATA TAA TGG AAG CTC GAC TTC CAG-3' .
  • the miRs include various artificially designed microRNAs encoding the NF- ⁇ B family members RELA, RELB or CREL.
  • the miR encodes an artificial microRNA targeting RELA, a member of the NF- ⁇ B family; preferably, the miR encodes miR533, where the sequence of miR533 (SEQ ID NO.2) is 5'-CAA AGA TGG GAT GAG AAA GGA-3 '.
  • the expressed miR after the expressed miR is processed and matured by the intracellular microRNA maturation system, it can bind to NF- ⁇ B mRNA in the cytoplasm, thereby inhibiting the expression of NF- ⁇ B protein; the microRNA maturation system processing and maturation means that the initially expressed miR must undergo These systems (some proteins and their complexes) must be cut and processed into mature miRs before they can exert their functions; the main steps in this process include the initial transcription product of miR, pri-miRNA, being processed by the Drosh-DGCR8 complex into pre-miRNA; pre-miRNA is assisted by Exportin 5 protein from the nucleus into the cytoplasm; pre-miRNA is further processed into miRNA in the cytoplasm by the Dicer-TRBP complex.
  • the adeno-associated virus includes adeno-associated viruses of various serotypes, such as any one from AAV1 to AAV9.
  • the adeno-associated virus is AAV2.
  • the construction method of the recombinant adeno-associated virus for the treatment of inflammatory diseases includes the following steps:
  • pAAV-DMP-miR533 and two helper plasmids (pAAV-Helper and pAAV-RC; Stratagene) was used to transfect 293T cells. After the cells were cultured, the cells and culture medium were collected and frozen and thawed. Pure chloroform was added to the cell lysis solution after freezing and thawing. After shaking, NaCl was added to the mixture and shaken until the NaCl was dissolved. Collect the supernatant after centrifugation, add PEG8000 and shake until it dissolves. Centrifuge and discard the supernatant, then dissolve the precipitate. Then add DNase and RNase to the dissolved precipitate. The reaction mixture is incubated at room temperature and then extracted. , collect the aqueous phase containing the purified virus. After quantifying the virus, aliquot it and store it at -80°C for later use. The obtained virus is named rAAV-DMP-miR533.
  • inflammatory diseases include various types of inflammation-related diseases, such as inflammation caused by infection, spontaneous inflammation, autoimmune diseases, neurodegenerative diseases, cancer, etc.
  • the inflammatory diseases include acute colitis, psoriasis and arthritis.
  • the rAAV-DMP-miR533 of the present invention can prepare new safe anti-inflammatory reagents that can be administered in a variety of administration methods.
  • the rAAV-DMP-miR533 can be mixed with reagents such as petroleum jelly to prepare external application reagents or drugs for the treatment of skin inflammation such as psoriasis.
  • reagents such as petroleum jelly
  • This method of administration greatly facilitates the treatment of local skin inflammation.
  • the present invention prepares a recombinant adeno-associated virus rAAV-DMP-miR533, in which DMP is an NF- ⁇ B specific promoter, which is formed by connecting the NF- ⁇ B decoy agent and the minimal promoter; wherein, miR533 can encode the target NF- ⁇ B.
  • Artificial microRNA of ⁇ B RELA Experimental studies have shown that rAAV-DMP-miR533 virus has good therapeutic effects on dextran sodium sulfate-induced acute colitis, imiquimod-induced psoriasis and collagen-induced arthritis in mice, and in these It has a good safety profile in the treatment of inflammation model mice.
  • the present invention is expected to provide a new technology and new reagent for the treatment of various inflammatory diseases.
  • DMP in the present invention is an NF- ⁇ B specific promoter, consisting of an NF- ⁇ B decoy and a minimal promoter.
  • MiR533 is an artificial microRNA targeting NF- ⁇ B RELA. It has been previously demonstrated that DMP-miR533 can sense and control NF- ⁇ B activity in cells cultured in vitro. Transfection of DMP-miR533 can cause NF- ⁇ B overactivated cells to undergo apoptosis, but has little effect on normal cells.
  • DMP-miR533 is packaged in adeno-associated virus (AAV) to solve its in vivo delivery problem, and the recombinant AAV (recombinant AAV, rAAV) packaged with DMP-miR533 is used to demonstrate DMP -Whether miR533 can control inflammation in the body and whether it is safe.
  • AAV adeno-associated virus
  • rAAV recombinant AAV
  • the inhibitory effect of rAAV-DMP-miR533 on inflammatory cells in vitro was first evaluated in vitro, and the successful packaging of rAAV-DMP-miR533, its ability to infect cells, and its ability to inhibit excessive activation of NF- ⁇ B in cells were demonstrated. of inhibition.
  • rAAV-DMP-miR533 was then used to treat mice with three typical inflammatory diseases, including dextran sulfate sodium (DSS)-induced acute colitis, imiquimod (IMQ)-induced psoriasis and Collagen-induced arthritis. This demonstrates the feasibility, reliability and safety of rAAV-DMP-miR533 in treating inflammation in living mammals.
  • DSS dextran sulfate sodium
  • IMQ imiquimod
  • rAAV-DMP-miR533 can cause inflammatory cells to undergo apoptosis, thereby eradicating inflammatory cells. Inflammatory cells can secrete proinflammatory cytokines, thereby exacerbating and worsening the inflammatory process. rAAV-DMP-miR533 can remove the source of inflammation by eradicating inflammatory cells.
  • This mechanism differs from current anti-inflammatory strategies that rely on antibodies or antagonists of cytokines and their receptors. Most of the direct target genes of NF- ⁇ B are cytokines, and inhibiting cytokines with antibodies can only temporarily neutralize the cytokines they produce, but cannot eradicate the inflammatory source that produces them.
  • cytokine antibodies to treat inflammatory diseases has a high recurrence rate.
  • the pleiotropic and redundant binding of many cytokines to their receptors can limit the efficacy of a single neutralizing agent. This is why inflammatory diseases are resistant to current treatment strategies.
  • Agents of cytokines may vary from person to person, resulting in low patient response rates for most current treatments.
  • rAAV-DMP-miR533 inhibited all inflammation-related cytokines by eradicating inflammatory cells.
  • rAAV-DMP-miR533 significantly reduced serum levels of two major pro-inflammatory factors, TNF- ⁇ and IL-6, at both the mRNA and protein levels. This reduction in the levels of major inflammatory factors in the blood is extremely important for treating local inflammatory symptoms and eliminating the systemic pathological effects of inflammatory factors. Therefore, rAAV-DMP-miR533 provides a new strategy with more broad-spectrum anti-inflammatory effects, and this tool may overcome some of the key challenges of current anti-inflammatory treatments, such as low response, drug resistance, relapse, and side effects.
  • the present invention has the following advantages:
  • the current inflammatory treatment reagents are mainly antibodies against various cytokines (such as TNF- ⁇ antibodies) and emerging JAK small molecule inhibitors. These therapeutic reagents have key clinical problems of low response rate and drug resistance.
  • cytokines such as TNF- ⁇ antibodies
  • JAK small molecule inhibitors have key clinical problems of low response rate and drug resistance.
  • AAV AAV genome delivery vector
  • the DNA fragment DMP-miR533 invented by the applicant earlier was packaged into a safe gene delivery vector AAV, and a new AAV particle—rAAV-DMP-miR53 was produced.
  • the anti-inflammatory effects of rAAV-DMP-miR533 at cellular and in vivo levels were systematically studied.
  • rAAV-DMP-miR533 had excellent anti-inflammatory effects both in vitro and in vivo.
  • this rAAV showed good inflammatory therapeutic effects in three typical inflammatory mouse models, including acute colitis models of dextran sulfate sodium (DSS)-induced acute colitis in mice, imiquine Mott (IMQ)-induced psoriasis model and collagen-induced mouse arthritis model.
  • IMQ imiquine Mott
  • rAAV-DMP-miR533 also demonstrated good biological safety during in vivo treatment. Therefore, the rAAV-DMP-miR53 developed in the present invention is expected to be used to prepare new biological drugs for the treatment of human inflammatory diseases.
  • rAAV-DMP-miR533 Compared with traditional NF- ⁇ B small molecule inhibitors, decoys and siRNA, the advantage of rAAV-DMP-miR533 is that it can avoid excessive inhibition of NF- ⁇ B activity in normal cells. This is the fundamental reason why traditional NF- ⁇ B small molecule inhibitors, decoys and siRNA cannot become drugs.
  • BAY 11-7082 a typical small molecule NF- ⁇ B inhibitor, causes significant apoptosis in both inflammatory cells and normal cells.
  • Experimental studies of the present invention show that rAAV-DMP-miR533 can induce significant apoptosis of cancer cells (a typical inflammatory cell) with excessive activation of NF- ⁇ B, but has almost no effect on normal cells.
  • rAAV-DMP-miR533 can induce apoptosis of inflammatory normal cells. This shows that rAAV-DMP-miR533 can specifically act on inflammatory cells. This high selectivity is of great significance for improving in vivo application and reducing side effects. Therefore, rAAV-DMP-miR533 overcomes the side effect defects of traditional NF- ⁇ B inhibitors and has clinical translation potential.
  • AAV is a safe gene delivery tool with the advantages of low immunogenicity, non-pathogenicity, no genome insertion, and long-term stable expression. It has been approved for human clinical gene therapy.
  • the experimental research of the present invention shows that, under the premise of the dosage used, rAAV-DMP-miR533 does not show biological toxicity to mice with three inflammatory diseases.
  • three high-dose intravenous administrations of rAAV-DMP-miR533 to arthritic CIA mice had no effect on the mice's serum biochemical indicators and spleen.
  • the widely used anti-arthritis drug MTX showed significant toxic side effects on the liver and spleen of mice.
  • rAAV-DMP-miR533 can be mixed with petroleum jelly and applied externally to treat psoriasis. This method of administration greatly facilitates the treatment of local skin inflammation. This discovery has not been reported internationally and is an important innovation in the administration route of rAAV gene therapy. Therefore, rAAV-DMP-miR533 is a new safe anti-inflammatory agent that can be administered in multiple delivery modes. Although the potential clinical application of rAAV-DMP-miR533 will still be challenged by pre-existing or in vivo antibodies that neutralize AAV vectors, because these antibodies can block AAV administration and re-administration, this is also the case. Common barriers to current AAV-based therapies.
  • an AAV-based gene therapy was simultaneously developed for the treatment of inflammatory diseases.
  • AAV-based gene therapy is mainly used to treat human genetic diseases.
  • several AAV-based gene therapies have been launched into clinical trials, such as treating inflammatory diseases, especially autoimmune diseases such as rheumatoid arthritis, by expressing IFN- ⁇ or TNFR-IgG1Fc fusion proteins.
  • these treatments still only target one inflammation-related cytokine.
  • rAAV-DMP-miR533 directly targets the inflammatory center NF- ⁇ B itself.
  • the treatment of three typical inflammatory diseases shows that rAAV-DMP-miR533 based on AAV gene therapy provides a broader therapeutic strategy for inflammatory diseases.
  • Figure 1 shows plasmids and viral vectors. Plasmid maps of pAAV-MCS, pAAV-DMP-NT, pAAV-DMP-miR533, pAAV-CMV-EGFP, pAAV-DMP-miR533-CMV-EGFP, and their packaging into recombinant AAV (rAAV).
  • the prepared rAAVs were named rAAV-MCS, rAAV-DMP-NT, rAAV-DMP-miR533, rAAV-CMV-EGFP and rAAV-DMP-miR533-CMV-EGFP.
  • Figure 2 is a schematic diagram of inflammation treatment by rAAV-DMP-miR533.
  • A Schematic diagram of inflammation treatment by rAAV-DMP-miR533.
  • DMP decoy-minimal promoter
  • Pol II RNA polymerase II
  • RISC RNA-induced silencing complex.
  • FIG. 3 shows the treatment of inflammatory cells with pAAV-DMP-miR533.
  • HT-29 cells were transfected with various plasmids and then cultured for 24, 48 and 72 hours respectively.
  • A Representative fluorescence images of HT-29 cells after acridine orange (OA) staining. Scale bar: 100 ⁇ m.
  • Blank group, MCS and miR533 cells were transfected with Lipofectamine, pAAV-MCS and pAAV-DMP-miR533 respectively;
  • TNF- ⁇ cells induced by TNF- ⁇ (cells were induced with TNF- ⁇ at a final concentration of 10ng/mL for 1 hour );
  • TNF- ⁇ +MCS and TNF- ⁇ +miR533 cells induced by TNF- ⁇ transfected with pAAV-MCS and pAAV-DMP-miR533 respectively.
  • Figure 4 shows the treatment of inflammatory cells with rAAV-DMP-miR533-CMV-EGFP.
  • HL7702 cells were induced with TNF- ⁇ as needed and then infected with various rAAVs for 48 hours.
  • A Cell fluorescence image. Scale bar: 100 ⁇ m.
  • C Representative flow cytometry analysis of apoptosis.
  • TNF- ⁇ TNF- ⁇ induced cells (cells induced with TNF- ⁇ at a final concentration of 10ng/mL 1 hours);
  • FIG. 5 shows the flow cytometry analysis of EGFP fluorescence intensity.
  • HL7702 cells were induced with or without TNF- ⁇ at a final concentration of 10 ng/mL for 1 hour and then infected with various viruses for 48 hours.
  • MCS, miR533, EGFP and miR533-EGFP cells were infected with phosphate buffered saline (PBS), rAAV-MCS, rAAV-DMP-miR533, rAAV-CMV-EGFP and rAAV-DMP-miR533-CMV-EGFP respectively; TNF - ⁇ +MCS, TNF- ⁇ +miR533, TNF- ⁇ +EGFP, TNF- ⁇ +miR533-EGFP: rAAV-MCS, rAAV-DMP-miR533, rAAV-CMV-EGFP and rAAV-DMP-miR533-CMV were used respectively.
  • FIG. 6 shows colitis mice treated with rAAV-DMP-miR533.
  • a mouse model of colitis was established by dextran sulfate sodium (DSS) induction and treated by intravenous injection of rAAVs (i.v.).
  • DSS dextran sulfate sodium
  • i.v. intravenous injection of rAAVs
  • A Schematic diagram of the construction and treatment of DSS-induced acute colitis mouse model.
  • B Blood stains around the mouse anus.
  • C Body weight of mice.
  • D Mouse colon.
  • F H&E stained sections of representative colon samples. The black box indicates the magnified area. Scale bars: 200 ⁇ m (10 ⁇ ) and 100 ⁇ m (20 ⁇ ).
  • (I) qPCR detection of NF- ⁇ B RELA and its target gene expression in colon samples (n 6 mice).
  • mice In the blank group, mice drank normal water and were treated with PBS; in the DSS group, the mice drank water containing 3% DSS (DSS-induced mice) and were treated with PBS; in the MCS group, DSS-induced mice were treated with rAAV-MCS; for miR533, rAAV-DMP-miR533 treatment of DSS-induced mice. ns, no significant difference.
  • Figure 7 shows the establishment of a colitis mouse model by dextran sulfate sodium (DSS) induction and treatment by intravenous injection of rAAVs (i.v.) (biological replicate 2).
  • A Schematic diagram of the construction and treatment of DSS-induced acute colitis mouse model.
  • B Blood stains around the mouse anus.
  • C Body weight of mice.
  • D Mouse colon.
  • F H&E stained sections of representative colon samples. The black box indicates the magnified area. Scale bars: 200 ⁇ m (10 ⁇ ) and 100 ⁇ m (20 ⁇ ).
  • mice drank normal water and were treated with PBS; in the DSS group, the mice drank water containing 3% DSS (DSS-induced mice) and were treated with PBS; in the MCS group, DSS-induced mice were treated with rAAV-MCS; for miR533, rAAV-DMP-miR533 treatment of DSS-induced mice.
  • Figure 8 shows the intravenous injection of rAAV-DMP-miR533 to treat psoriasis mice.
  • the psoriasis mouse model was established by induction with imiquimod (IMQ) and treated by intravenous injection of rAAVs (iv).
  • IMQ imiquimod
  • iv intravenous injection of rAAVs
  • A Schematic diagram of the construction and treatment of psoriasis mouse model.
  • B Visual image of mouse back skin.
  • C H&E stained sections of representative skin samples. Scale bars: 200 ⁇ m (10 ⁇ ) and 100 ⁇ m (20 ⁇ ).
  • mice smeared with petroleum jelly were treated with PBS; MCS, mice induced by IMQ were treated with rAAV-MCS; miR533, mice induced by IMQ were treated with rAAV-DMP-miR533. ns, no significant difference.
  • Figure 9 shows the H&E pathological analysis and gene expression detection of relevant tissues of IMQ-induced psoriasis mice treated with rAAV-DMP-miR533 tail vein injection.
  • A H&E stained sections of all skin samples in different groups (blank group, MCS, miR533).
  • B Detection of NF- ⁇ B and its target gene expression in skin samples by qPCR.
  • Figure 10 shows the efficacy of rAAV-DMP-miR533 subcutaneous injection (i.h.) and skin administration (ad us.ext.) (external application) on psoriasis mouse models. These experiments were conducted with only one mouse.
  • A Photographs of mouse back skin on days 0, 6, and 12.
  • B Typical tissue section of H&E stained skin sample.
  • C ELISA detects TNF- ⁇ and IL-6 in serum.
  • D qPCR detects the expression of NF- ⁇ B and its target genes in skin samples.
  • F subcutaneous injection (i.h.); T, dermal administration (ad us.ext.).
  • Figure 11 shows the topical administration of rAAV-DMP-miR533 to the skin to treat psoriasis mice.
  • the psoriasis mouse model was established by induction with imiquimod (IMQ) and treated with rAAVs administered via skin application (topical application).
  • IMQ imiquimod
  • A Schematic diagram of the construction and treatment of psoriasis mouse model.
  • B Visual image of mouse back skin.
  • D H&E stained sections of representative skin samples. Scale bars: 200 ⁇ m (10 ⁇ ) and 100 ⁇ m (20 ⁇ ).
  • mice smeared with petroleum jelly were treated with PBS; MCS, mice induced by IMQ were treated with rAAV-MCS; for miR533, mice induced by IMQ were treated with rAAV-DMP-miR533. ns, not significant.
  • Figure 12 shows the treatment of imiquimod (IMQ)-induced psoriasis mouse model by administering rAAV-DMP-miR533 via skin application.
  • IMQ imiquimod
  • AAV-DMP-miR533 H&E stained sections of all skin samples in different groups (blank group, MCS, miR533).
  • B TNF- ⁇ and IL-6 mRNA expression levels in skin samples.
  • Figure 13 shows the effect of pAAV-DMP-miR533 on CT-26 cell apoptosis and viability.
  • CT-26 cells were transfected with various plasmids and cultured for 24, 48, and 72 h respectively.
  • A Representative images of flow cytometry analysis of apoptosis.
  • Lipo cells treated with Lipofectamine 2000.
  • NT cells transfected with pAAV-DMP-NT; miR533, cells transfected with pAAV-DMP-miR533.
  • Figure 14 shows the effect of pAAV-DMP-miR533 on the apoptosis and viability of NIH-3T3 cells.
  • NIH-3T3 was induced with or without TNF- ⁇ (final concentration 10 ng/mL) for 1 hour before transfection.
  • NIH-3T3 cells were then transfected with various plasmids and cultured for 24, 48 and 72 hours respectively.
  • A Representative images of flow cytometry analysis of apoptosis.
  • NT cells transfected with pAAV-DMP-NT; miR533, cells transfected with pAAV-DMP-miR533.
  • Figure 15 shows the detection of NF- ⁇ B and its target gene expression in CT26 and NIH-3T3 cells.
  • Lipo cells treated with lipo2000.
  • NT cells transfected with pAAV-NT. miR533, cells transfected with pAAV-DMP-miR533.
  • Figure 16 shows the treatment of arthritic mice with rAAV-DMP-miR533.
  • a mouse model of arthritis was established through collagen induction and treated by intravenous injection of rAAVs (iv).
  • A Schematic diagram of CIA mouse model construction and treatment. PBS, healthy group treated with phosphate buffered saline.
  • B Representative pictures of different groups of front and rear paws.
  • C Visual diagram of mouse spleen.
  • PBS group normal mice treated with PBS
  • CIA group CIA mice treated with PBS
  • MTX group CIA mice treated with methotrexate (MTX)
  • NT group CIA mice treated with rAAV-NT Mouse
  • miR533 group CIA mice treated with rAAV-DMP-miR533.
  • ns no significant difference.
  • NT non-target microRNA.
  • Figure 17 shows the treatment of arthritic mice with rAAV-DMP-miR533.
  • Figure 18 shows the treatment of arthritic mice with rAAV-DMP-miR533.
  • a collagen-induced arthritis (CIA) mouse model was established by collagen induction and treated by intravenous (i.v.) rAAVs.
  • A Representative H&E stained sections of all mouse ankle joints. Scale bar: 50 ⁇ m.
  • PBS normal mice treated with phosphate buffered saline (PBS); CIA, CIA mice treated with PBS; MTX, CIA mice treated with methotrexate (MTX); NT, treated with rAAV-NT CIA mice;miR533, CIA mice treated with rAAV-DMP-miR533. ns, not significant. NT, no target.
  • Figure 19 shows the treatment of arthritic mice with rAAV-DMP-miR533.
  • a collagen-induced arthritis (CIA) mouse model was established by collagen induction and treated by intravenous (i.v.) rAAVs.
  • A Representative H&E stained sections of major organs. Scale bar: 100 ⁇ m.
  • PBS normal mice treated with phosphate buffered saline (PBS); CIA, CIA mice treated with PBS; MTX, CIA mice treated with methotrexate (MTX); NT, treated with rAAV-NT CIA mice;miR533, CIA mice treated with rAAV-DMP-miR533.
  • NT no target.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • BUN blood urea nitrogen
  • Cr creatinine
  • UA uric acid.
  • Figure 20 shows the effects of pAAV-DMP-miR533 and pAAV-DMP-miR533-5 on the apoptosis and viability of CT-26 and NIH-3T3 cells.
  • Cells were transfected with various plasmids and then cultured for 24, 48, and 72 hours.
  • A Representative fluorescence images of OA-stained HL7702 cells.
  • pAAV-DMP-miR533 contains a single copy of DMP-miR533.
  • pAAV-DMP-miR533-5 contains five copies of DMP-miR533.
  • microRNA sequences targeting human or mouse RELA were designed on the BLOCK-iT TM RNAi Designer website ( https://rnaidesigner.thermofisher.com/rnaiexpress/ ) (Table 1 and Table 2).
  • the DMP-miR533 fragment was amplified from the pDMP-miR533 vector, and then ligated into the pAAV-MCS vector (VPK-410, Stratagene) using MluI (upstream) and XbaI (downstream) restriction enzyme sites to construct pAAV- DMP-miR533 ( Figure 1).
  • the CMV-EGFP fragment was amplified with a pair of primers, and its upstream MluI and downstream EocRI restriction sites were from pEGFP-C1 (Clontech), and then the CMV-EGFP fragment was cloned into pAAV-MCS to obtain the vector pAAV-CMV-EGFP ( figure 1).
  • the DNA fragments were PCR amplified using Hieff TM PCR Master Mix (With Dye) (Yeasen).
  • the DNA fragments amplified by PCR were purified and recovered using agarose gel electrophoresis and AxyPrep DNA gel extraction kit (Axygen).
  • the digestion ligation reaction contained appropriate restriction endonucleases (ThermoFisher Scientific) and T4 DNA ligase (ThermoFisher Scientific).
  • Plasmid pAAV-DMP-miR533-CMV-EGFP was obtained by constructing the CMV-EGFP fragment into the pAAV-DMP-miR533 vector (Fig. 1).
  • the miR-NT fragment was synthesized according to the sequence of plasmid pcDNA TM 6.2-GW/EmGFP-miR-Neg (Thermo Fisher Scientific) and inserted into pDMP-miR to obtain the pDMP-NT plasmid vector.
  • the DMP-NT fragment was copied from pDMP-NT and inserted into pAAV-MCS to obtain the pAAV-DMP-NT vector (Fig. 1). All plasmids including pAAV-MCS, pAAV-DMP-NT, pAAV-CMV-EGFP, pAAV-DMP-miR533, pAAV-DMP-miR533-CMV-EGFP, pAAV-Helper and pAAV-RC were transfected into E. coli DH5 ⁇ respectively. (Tiangen) and purified using EndoFree Plasmid kit (CWBio). All plasmids were verified by DNA sequencing. The oligonucleotides and primers used in this example were synthesized by Sangon Biotech (Shanghai, China) (Table 2 and Table 3).
  • HEK-293T human fetal kidney cells
  • HT-29 human colon cancer cells
  • CT-26 small Mouse colon cancer cells
  • HL7702 human normal liver cells
  • NIH-3T3 mouse embryonic fibroblasts
  • HT-29, CT-26, and HL7702 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco).
  • RPMI Roswell Park Memorial Institute
  • NIH-3T3 and HEK-293T were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco).
  • DMEM Dulbecco's Modified Eagle Medium
  • All media were supplemented with fetal bovine blood at a final content of 10% (HyClone), 100 units/mL penicillin (Thermo Fisher), and 100 ⁇ g/mL streptomycin (Thermo Fisher). All cells were cultured in a 37°C humidified incubator containing 5% CO2 .
  • HEK-293T was inoculated into a 75cm 2 culture flask at a density of 5 ⁇ 10 6 cells per flask and cultured for 24 hours.
  • Two helper plasmids pAAV-Helper and pAAV-RC; Stratagene
  • one pAAV plasmid pAAV-MCS, pAAV-DMP-NT, pAAV-DMP-miR533, pAAV- DMP-miR533-CMV-EGFP, pAAV-CMV-EGFP
  • the cells and culture medium were then placed in a 37°C water bath and incubated for 2 hours. The entire freezing and thawing process was repeated three times. Pure chloroform was added to the frozen and thawed cell lysate at a volume ratio of 1:10, and the mixture was shaken vigorously at 37°C for 1 hour. After shaking, add NaCl to the mixture to a final concentration of 1 mol and shake until NaCl is dissolved. The dissolution solution was centrifuged at 15,000 revolutions per minute (rpm) for 15 minutes at 4°C, then the supernatant was collected, PEG8000 with a final concentration of 10% (w/v) was added and shaken until it was dissolved.
  • rpm revolutions per minute
  • the reaction was centrifuged again at 15,000 rpm for 15 minutes at 4°C, the supernatant was discarded, and the pellet was dissolved in PBS. Then add DNase and RNase to the dissolved pellet, and the final concentration of nuclease is 1 ⁇ g/mL.
  • the reaction was incubated at room temperature for 30 minutes. Finally, the incubated reagent was extracted once with chloroform (1:1 volume), and the aqueous phase containing the purified virus was transferred to a new test tube.
  • AAV titers were determined by using primers AAV-F/R and qPCR detection (Table 3). After the virus is quantified, aliquot it and store it at -80°C for later use.
  • viruses obtained were named rAAV-MCS, rAAV-DMP-NT, rAAV-DMP-miR533, rAAV-DMP-miR533-CMV-EGFP and rAAV-CMV-EGFP.
  • NF- ⁇ B is widely overactivated in inflammatory cells.
  • this example designed a rAAV named rAAV-DMP-miR533 ( Figure 2A), where DMP is a promoter and consists of an NF- The ⁇ B decoy is composed of a minimal promoter, while miR533 encodes an artificial microRNA for targeting NF- ⁇ B RELA.
  • DMP-miR533 is transfected into human colon cancer cells (HT-29), mouse colon cancer cells (CT-26), TNF ⁇ -induced human normal liver cells (HL7702) and mouse embryonic fibroblasts (NIH-3T3), etc.
  • miR533 targeting human and mouse NF- ⁇ B RELA transcripts was designed and prepared respectively (Table 1). Human cells were treated with DMP-miR533 targeting human NF- ⁇ B RELA, and mouse cells and mice were treated with DMP-miR533 targeting mouse NF- ⁇ B RELA.
  • serotype AAV2 is used as the recombinant adeno-associated virus vector.
  • pAAV-MCS vector (VPK-410, Stratagene) was used to insert the functional DNA fragment DMP-miR, and two helper plasmids, pAAV-Helper and pAAV-RC, were used to co-transform 293T cells to prepare recombinant adeno-associated virus; the above three plasmids
  • the recombinant adeno-associated viruses packaged and prepared by the system are serotype AAV2 recombinant adeno-associated viruses because the helper plasmid pAAV-RC contains the Rep and Cap genes of AAV2.
  • AAV2 has a relatively wide range of tissue infectivity, so the constructed rAAV-DMP-miR533 can universally treat inflammation in different tissues and organs, such as inflammatory colitis in intestinal tissue and inflammatory colitis in skin tissue treated by the present invention. Inflammatory psoriasis, inflammatory arthritis located in joint tissue. If a serotype of AAV with a significant bias for tissue invasion is used, such as AAV9 that prefers neural tissue, when the constructed rAAV is used to treat inflammation in different tissues and organs, it is necessary to construct rAAV-DMP-miR533 of different serotypes. The process Cumbersome and costly. In addition, AAV2 is also one of the natural adeno-associated viruses.
  • Cells (HT-29, CT-26, HL7702 and NIH-3T3) (1 ⁇ 10 5 ) were seeded into 24-well plates and cultured overnight at 37°C and 5% CO2 . Then, the pAAV plasmid (500 ng/well) prepared in various Examples 1 was transfected into cells using Lipofectamine2000 (Thermo Fisher) according to the instructions. After transfection, cells were cultured for 24 hours, 48 hours, and 72 hours respectively. If necessary, normal cells were induced with TNF- ⁇ (Sigma-Aldrich) at a final concentration of 10 ng/mL for 1 hour before transfection.
  • TNF- ⁇ Sigma-Aldrich
  • the cells were then stained with acridine orange (Solarbio) according to the instructions, and the viable cells showed a uniform green color.
  • Cells were imaged with a fluorescence microscope (IX51, Olympus) and counted using Image-Pro Plus software.
  • Cell viability detection Cell Counting Kit-8 (CCK-8, Yeasen) was also used to measure and analyze cell viability.
  • Cells (HT-29, CT-26, HL7702 and NIH-3T3) (5 ⁇ 10 3 ) were seeded into a 96-well plate and cultured overnight at 37°C and 5% CO2 .
  • Various pAAV plasmids prepared in Example 1 (200 ng/well) were then transfected into cells using Lipofectamine 2000 (Thermo Fisher). After transfection, cells were cultured for 24 hours, 48 hours, and 72 hours respectively. If necessary, normal cells were induced with TNF- ⁇ (Sigma-Aldrich) at a final concentration of 10 ng/mL for 1 hour before transfection.
  • CCK-8 reagent (10 ⁇ L/well) was added to the cells and incubated for 1 hour, and the absorbance of the solution was measured at 450 nm using a microplate reader (BioTek).
  • Apoptosis detection Cells (HT-29, CT-26, HL7702 and NIH-3T3) (5 ⁇ 10 5 ) were seeded into a 6-well plate and cultured overnight at 37°C and 5% CO2 .
  • Various pAAV plasmids prepared in Example 1 (4 ⁇ g/well) were then transfected into cells using Lipofectamine 2000 (Thermo Fisher). After transfection, cells were cultured for 24 hours, 48 hours, and 72 hours respectively. If necessary, normal cells were induced with TNF- ⁇ (Sigma-Aldrich) at a final concentration of 10 ng/mL for 1 hour before transfection.
  • Apoptosis was then quantified by detection and analysis using AnnexinV-FITC/PI Apoptosis Detection Kit (Vazyme) and flow cytometry (Calibur, BD, USA) according to the manufacturer's instructions.
  • Cell EGFP fluorescence detection Cells (HT-29, CT-26, HL7702 and NIH-3T3) (5 ⁇ 10 3 ) were seeded into a 96-well plate and cultured at 37°C 5% CO 2 overnight. The cells were then infected with various rAAVs prepared in Example 1 (5 ⁇ 10 7 vg/well). After infection, cells were cultured for 48 hours. If necessary, normal cells were induced with TNF- ⁇ (Sigma-Aldrich) at a final concentration of 10 ng/mL for 1 hour before infection. EGFP fluorescence was imaged with a fluorescence microscope (IX51, Olympus) and quantitatively analyzed by flow cytometry (Calibur, BD, USA). At the same time, AnnexinV-FITC/PI apoptosis detection kit and flow cytometry were used to detect cell apoptosis.
  • RQ relative quantitation
  • HL7702 human normal liver cells
  • TNF- ⁇ a known NF- ⁇ B inducer
  • HL7702 cells that were not induced by TNF- ⁇ were used as a control.
  • AO staining of cells showed that pAAV-DMP-miR533 had no obvious effect on normal HL7702 cells, but after TNF- ⁇ induction, the number of HL7702 cells was significantly reduced ( Figure 3E).
  • rAAV-MCS adeno-associated viruses
  • rAAV-CMV-EGFP rAAV-DMP-miR533
  • rAAV-DMP-miR533-CMV-EGFP figure 1
  • the CMV-EGFP fragment was inserted into rAAV-DMP-miR533 to monitor the infection of cells by rAAV-DMP-miR533.
  • the empty virus rAAV-MCS which contains the CMV promoter and does not insert any target gene, was used as a negative control.
  • the packaged virus was used to infect TNF- ⁇ -treated and untreated HL7702 cells respectively.
  • DMP-miR533 can inhibit the expression of NF- ⁇ B RELA and further cause inflammatory cell apoptosis and reduced activity, indicating that rAAV-DMP-miR533 has a good anti-inflammatory effect in vitro.
  • DSS dextran sodium sulfate
  • MP dextran sodium sulfate
  • mice in the DSS+MCS and DSS+miR533 groups were intravenously injected with 100 ⁇ L of 1 ⁇ 10 10 vg/mL rAAV-MCS and rAAV-DMP-miR533, respectively. Measure your weight every day.
  • mice On the 8th day, the mice were sacrificed, and the colon from the anus to the ileocecum was separated and blood was collected. At the same time, the length of the colon of each mouse was measured. Colon tissue was used for preparation of paraffin sections and gene expression detection. Sections were stained with hematoxylin-eosin (H&E), imaged, and scored. The pathological score of the colon tissue was blindly scored by other technical personnel not related to the project according to four levels: 0 points, no obvious pathological changes; 1 point, focal inflammatory cell infiltration; 2 points, extensive inflammatory cell infiltration ; 3 points, diffuse inflammatory cell infiltration; 4 points, inflammatory cell infiltration, tissue degeneration and necrosis, and fibrous connective tissue hyperplasia.
  • H&E hematoxylin-eosin
  • TNF- ⁇ and IL-6 levels in serum According to the ELISA instructions, TNF- ⁇ (ab208348, abcam) and IL-6 ELISA kits (ab222503, abcam) were used to measure the levels of TNF- ⁇ and IL-6 in serum. Determination.
  • H&E hematoxylin and eosin
  • mice in the blank group were normal, with hard feces and no blood.
  • the mice induced by DSS gradually became dull and inactive.
  • the feces changed from normal to soft and soft.
  • the feces was obviously bloody, and the anus was still bleeding on the 7th day.
  • mice were then treated with various reagents.
  • the symptoms of DSS-induced mice treated with rAAV-MCS were similar to those of DSS-induced mice treated with PBS.
  • the feces of mice in both groups became soft and bloody.
  • DSS-induced mice treated with rAAV-miR533 showed a significant improvement in fecal status and a significant reduction in anal bleeding.
  • Dynamic measurement of body weight showed that mice with acute colitis treated with rAAV-MCS and PBS lost weight, while mice with acute colitis treated with rAAV-DMP-miR533 gained weight (Fig. 6C).
  • the mouse colon length measurement results showed that DSS induction resulted in shorter mouse colon length ( Figures 6D and 6E).
  • mice Two batches of animal experiments were conducted on male BALB/c (8 weeks; Cavens, China) psoriasis modeling mice.
  • Mice in the blank group were treated with Vaseline cream.
  • the mice in the MCS and miR533 groups were smeared with 5% w/w imiquimod (IMQ) (Sichuan Mingxin Pharmaceutical, China) every day on the shaved back area, at a dose of 62.5mg/each.
  • IMQ 5% w/w imiquimod
  • RT-qPCR and ELISA kits were then used to detect the expression levels of TNF- ⁇ and IL-6 in skin and serum samples, respectively.
  • Psoriasis model mice were treated with 100 ⁇ L of 1 ⁇ 10 10 vg/mL rAAV-DMP-miR533 by subcutaneous injection or application every day. After 6 days, the mice were sacrificed, and sample collection and detection were the same as in the previous intravenous injection experiment.
  • mice in the MCS and miR533 groups continued to be smeared with 5% w/w IMQ every day.
  • the blank group continued to apply Vaseline cream until euthanasia. All mice were sacrificed on day 12 and photographed, while dorsal skin and blood samples were collected.
  • mice's body weight and Psoriasis Area and Severity Index were monitored and recorded daily.
  • the erythema, scale and thickness on the skin of each mouse were independently scored from 0 to 4: 0, none; 1, slight; 2, moderate; 3, significant; 4, extremely significant.
  • the sum of the three indicators represents the severity of inflammation (score, 0-12).
  • mice were constructed using imiquimod (IMQ) induction and treated with a variety of reagents (Figure 8A). IMQ was continuously applied to the shaved back skin of mice. Six days later, symptoms such as redness, inflammation, itching, skin thickening, and silver scales appeared on the mouse skin ( Figure 8B). Subsequently, IMQ-induced psoriasis mice were intravenously injected with PBS, rAAV-MCS and rAAV-DMP-miR533.
  • IMQ imiquimod
  • an IMQ-induced psoriasis mouse was also treated with subcutaneous injection and skin application.
  • the results showed that both administration methods achieved similar therapeutic effects as the above intravenous injection (Figure 10), including skin recovery (Figure 10A and 10B), serum pro-inflammatory factors (Figure 10C), and NF- ⁇ B RELA and its targets
  • the expression of the gene was downregulated (Fig. 10D).
  • Collagen-induced arthritis (CIA) mouse model was established through double immunization.
  • mice were injected intradermally in the tail with equal volumes of chicken type II collagen solution (2 mg/mL) and complete Freund's adjuvant (2 mg/mL) (Chondrex, Redmond, WA, USA).
  • the chicken type II collagen solution was emulsified with incomplete Freund's adjuvant (Chondrex, Redmond, WA, USA), and a booster injection was given to the mouse tail at a position different from the first immunization.
  • All paws of each mouse were scored to characterize disease: 0, normal; 1, mild swelling and erythema limited to the midfoot and ankle; 2, mild swelling and erythema extending to the midfoot and ankle; 3, mild swelling and erythema extending to the midfoot and ankle. There is moderate swelling and erythema from the metatarsal joint to the ankle; 4. Severe swelling and erythema of the feet, ankles and fingers.
  • the clinical score for each mouse was the sum of the scores from all four paws.
  • mice's body weight and clinical scores were monitored and assessed every other day. Use vernier calipers to measure mouse ankle width, paw thickness, and tail thickness. All mice were euthanized and photographed 4 weeks after administration of MTX, NT or miR533, and serum samples from each group were collected for biochemical index testing. ELISA kits were used to detect the expression levels of TNF- ⁇ and IL-6 in hind paw and serum samples. Tissues including heart, liver, spleen, lung, and kidney were collected for H&E staining analysis. The spleens of all mice were photographed and weighed.
  • Ankle tissue was used for subsequent H&E staining analysis and gene expression detection, and its histopathology score was performed by other unrelated technicians based on the following four Blind grade scoring: 0, normal synovium; 1. Presence of synovial hypertrophy and cell invasion; 2. Presence of pannus and cartilage erosion; 3. Presence of cartilage and subchondral bone erosion; 4. Dysfunction and stiffness of the entire joint.
  • Micro-CT All DBA/1J mice were sacrificed and their paws (including ankle joints) were collected for micro-CT imaging (Micro-CT) using an in vivo micro-CT scanner (vivaCT 80, SCANCO Medical AG, Switzerland). -CT). Reconstruction and analysis of high-resolution tomographic images are performed in the SCANCO GPU Accelerated Reconstruction system.
  • non-cytotoxic pAAV-DMP-NT was packaged into AAV to prepare rAAV-DMP-NT, and rAAV-DMP-NT was used as the corresponding negative control to further evaluate the in vivo anti-inflammatory effect of rAAV-DMP-miR533.
  • RA Rheumatoid arthritis
  • CIA collagen-induced arthritis
  • a rAAV packaging plasmid pAAV-DMP-miR533-5 containing 5 copies of DMP-miR533 was also constructed.
  • CT-26 and NIH-3T3 were transfected in parallel with this plasmid and the rAAV packaging plasmid pAAV-DMP-miR533 containing a single copy of DMP-miR533.
  • the transfection method is the same as in Example 2. The effects of transfection of two plasmids on the apoptosis and viability of two types of cells were observed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Dermatology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

提供了一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用,所述重组腺相关病毒以腺相关病毒为载体,包含一个到多个拷贝的功能性DNA片段DMP-miR;所述功能性DNA片段DMP-miR由两个功能元件DMP和miR构成,其中DMP为NF-κB特异性启动子,所述miR为编码可靶向NF-κB mRNA的microRNA。本发明的rAAV-DMP-miR533在小鼠上对葡聚糖硫酸钠诱导的急性结肠炎、咪喹莫特诱导的银屑病和胶原诱导的关节炎具有良好的治疗效果并具有良好的安全性,有望为多种炎症性疾病的治疗提供一种新技术及新试剂。

Description

一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用 技术领域
本发明涉及炎症基因治疗生物技术领域,具体涉及一种重组腺相关病毒及其在炎症性疾病治疗中的应用。
背景技术
炎症是机体对感染和身体损伤的保护性反应。适度的炎症反应有助于维持体内的稳态。然而,异常的炎症反应会导致炎症性疾病。例如,一些传染性病原体可导致全身炎症,可能导致败血症、细胞因子释放综合征、急性呼吸窘迫综合征,甚至多器官功能衰竭。一些持续性感染会导致慢性炎症以及癌症高风险诱发,例如乙型肝炎病毒引起的肝炎和肝癌。许多慢性炎症是由适应性免疫异常变化引起的,从而导致了各种自身免疫性疾病,如关节炎、炎性肠病、狼疮、银屑病、皮炎、哮喘、多发性硬化、脂肪性肝炎,甚至动脉粥样硬化、糖尿病、神经退行性疾病和炎性衰老。此外,先天免疫的许多异常变化还会诱发各种自身炎症性疾病。因此,异常炎症对人类健康构成了广泛而严重的威胁。
为了治疗炎症性疾病,这些疾病相关的分子机制被广泛研究。目前已经明确了它们的基础信号通路(例如NF-κB和JAK-STAT)以及主要的参与分子,其中许多潜在的靶点被尝试用于抗炎药物的开发。迄今为止,许多药物已被应用来治疗各种炎症性疾病,例如广泛使用的皮质类固醇(如糖皮质激素)。同时许多抗炎生物制剂也陆续被研发,例如细胞因子单克隆抗体(如促炎细胞因子TNF-α、IL-1α、IL-1β、IL-5、IL-6、IL-12、IL-17A、IL-17F、IL-23和抗炎细胞因子IL-4、IL-10、IL-11、IL-13、TGFβ)、细胞因子受体的抗体或拮抗剂(例如IL-6R、IL-5Rα、IL-4Rα)和CD抗体(例如CD4、CD14、CD19、CD20、CD38、CD40)。JAK小分子抑制剂(JAK1、JAK2、JAK3、TYK2)则是迅速发展起来的有前景的新型抗炎药。毫无疑问,目前的消炎药已经让患者受益匪浅。然而,抗炎药物仍面临几个关键性的挑战,比如原发性无应答、耐药或者响应丢失、复发和多种副作用。因此,当前的治疗方法还远远未能满足炎症性疾病临床治疗的需求。
NF-κB是一个序列特异性DNA结合转录因子家族,包括RelA/p65、p50、p52、RelB和c-Rel,在炎症中起关键调节作用。被多种诱导剂诱导之后,活化的NF-κB(主要是RelA-p50异二聚体)可以通过直接结合启动子和增强子来诱导靶基因的表达,从而参与细胞增殖、凋亡、先天免疫反应等过程。其中,NF-κB能直接调节多种炎症基因的表达,包括粘附因子(如ICAM-1、VCAM-1)、细胞因子(如IL-1α、IL-1β、IL-2、IL-6、IL-8、IL-10、IL-11、IL-12、IL-13、IL-15、IL-17、IL-23、TNFα、IFNβ、IFNγ)和趋化因子(例如CCL5、CCL17、CCL19、CCL20、CCL22、CCL23、CCL28)。诱骗寡核苷酸和小干扰核酸(siRNA)也曾被作为NF-κB的候选抑制剂。但是由于其活性不可控、递送困难、易降解,这两种NF-κB抑制剂在临床试验中也未能成功。为了克服它们的局限性,申请人前期开发了一种新的NF-κB抑制剂分子:一种结合了NF-κB诱骗子和microRNA序列的质粒载体DMP-miR533(Int.J.Biochem.Cell.Biol.2018,95:43-52;专利号ZL201710812983.2)。
申请人前期技术中已经在体外培养的细胞上初步论证DMP-miR533质粒载体可感知并抑制细胞内的NF-κB活性,但质粒载体本身目前还不能直接用于动物体内给药,也是说该种质粒载体无法实现体内细胞的转染和NF-κB活性的控制。此外,炎症本身不是体外某种细胞的行为,而是哺乳动物活体水平的一种生 理或病理反应。因此,在体外培养细胞中可感知和控制NF-κB活性的DMP-miR533是否能够在哺乳动物活体上控制NF-κB活性并实现炎症的治疗,仍是未知。此外,体外培养的细胞是一种非常单纯的人工可控的环境,与具有复杂生命系统的哺乳动物活体的体内生理及病理环境无法相提并论。这就是为什么很多富有希望的候选药物分子在临床实验上都无法成为药物的与原因。因此,为了论证和开发DMP-miR533在哺乳动物活体上控制NF-κB活性并实现炎症的治疗的可能性,为人类的炎症治疗探索新的技术。
发明内容
发明目的:针对现有炎症治疗存在的问题,本发明提供一种新型重组腺相关病毒,该病毒可通过抑制细胞内NF-κB活性实现哺乳动物活体上炎症疾病的治疗,有望用于制备新型生物药物,用于人类炎症性疾病的治疗。
本发明还提供所述重组腺相关病毒的构建方法和应用。
技术方案:为了实现上述目的,本发明提出一种用于炎症性疾病治疗的重组腺相关病毒,所述重组腺相关病毒rAAV-DMP-miR533以腺相关病毒为载体,其包含一个到多个拷贝的功能性DNA片段DMP-miR;所述功能性DNA片段DMP-miR由两个功能元件DMP和miR构成,其中DMP为NF-κB特异性启动子,所述miR为编码可靶向NF-κB mRNA的microRNA。
其中,所述DMP为NF-κB特异性启动子,由一个NF-κB诱骗子和一个最小启动子组成,该DMP包括各种序列的NF-κB诱骗子和最小启动子。
作为优选,所述DMP的序列(SEQ ID NO.1)为5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CTA GAG GGT ATA TAA TGG AAG CTC GAC TTC CAG-3'。
其中,所述miR包括编码靶向NF-κB家族成员RELA、RELB或CREL的各种人工设计microRNA。
作为优选,所述miR编码靶向NF-κB家族成员RELA的人工microRNA;作为优选,miR编码miR533,其中miR533的序(SEQ ID NO.2)为5′-CAA AGA TGG GAT GAG AAA GGA-3′。
其中,所述功能性DNA片段DMP-miR被重组腺相关病毒导入细胞后,其功能元件DMP可结合细胞核内的转录因子蛋白NF-κB,从而激活miR的表达。
进一步地,表达的miR经细胞内microRNA成熟系统加工成熟后,可在细胞质中结合NF-κB mRNA,从而抑制NF-κB蛋白的表达;所述microRNA成熟系统加工成熟是指最初表达的miR必须经过这些系统(一些蛋白及其复合物)剪切加工后变成成熟的miR才会发挥其功能;该过程的主要环节包括miR的最初转录产物pri-miRNA在细胞核内被Drosh-DGCR8复合物加工为pre-miRNA;pre-miRNA经Exportin 5蛋白辅助有细胞核进入细胞质;pre-miRNA在细胞质内经Dicer-TRBP复合物进一步加工为miRNA。
其中,所述腺相关病毒包括各种血清型的腺相关病毒,如AAV1到AAV9中的任意一种。
作为优选,所述腺相关病毒为AAV2。
本发明所述的用于炎症性疾病治疗的重组腺相关病毒的构建方法,包括如下步骤:
(1)从pDMP-miR533载体上扩增DMP-miR533,将其连接到pAAV-MCS载体上构建得到pAAV-DMP-miR533;
(2)使用pAAV-DMP-miR533和两种辅助质粒(pAAV-Helper和pAAV-RC; Stratagene)对293T细胞进行转染,细胞培养后,将细胞和培养基收集并冻融,将纯氯仿加入细胞冻融完的裂解液中,震荡后,向混合物中加入NaCl并摇动至NaCl溶解,离心后收集上清液,加入PEG8000并摇动至其溶解,离心弃去上清液,而后使沉淀溶解,然后将DNase和RNase加入溶解完的沉淀中,反应物在室温下温育,再进行萃取,收集含有纯化病毒的水相,病毒定量后,将其分装至-80℃保存备用,获得的病毒命名为rAAV-DMP-miR533。
本发明所述的用于炎症性疾病治疗的重组腺相关病毒在制备新型炎症性疾病治疗试剂中的应用。
进一步的,炎症性疾病包括各种类型炎症相关疾病,如感染引起的炎症、自发炎症、自身免疫病、神经退行性疾病、癌症等。
其中,所述炎症性疾病包括急性结肠炎、银屑病和关节炎。
本发明的rAAV-DMP-miR533能够制备以多种给药方式进行给药的新型安全抗炎试剂。
作为优选,所述rAAV-DMP-miR533可以通过与凡士林等试剂混合制备用于治疗银屑病等皮肤炎症的外用涂抹试剂或药物,这种给药方式极大便利了局部皮肤炎症的治疗。
本发明制备了一种重组腺相关病毒rAAV-DMP-miR533,其中DMP是一种NF-κB特异性启动子,由NF-κB诱骗子和最小启动子连接形成;其中miR533可编码靶向NF-κB RELA的人工microRNA。实验研究表明,rAAV-DMP-miR533病毒在小鼠上对葡聚糖硫酸钠诱导的急性结肠炎、咪喹莫特诱导的银屑病和胶原诱导的关节炎具有良好的治疗效果,并在这些炎症模型小鼠治疗中具有良好的安全性。本发明有望为多种炎症性疾病的治疗提供一种新技术及新试剂。
本发明中的DMP是一种NF-κB特异性启动子,由一个NF-κB诱骗子和一个最小启动子组成。MiR533是一种靶向NF-κB RELA的人工microRNA。前期已经证明DMP-miR533可以感知和控制体外培养细胞内的NF-κB活性。通过转染DMP-miR533可使NF-κB过度活化细胞发生凋亡,但对正常细胞几乎没影响。,在本发明中,将DMP-miR533包装在腺相关病毒(adeno-associated virus,AAV)中以解决其体内递送问题,并以包装了DMP-miR533的重组AAV(recombinant AAV,rAAV)在论证DMP-miR533是否具有控制体内炎症的作用以及是否具有安全性。本发明中,首先在体外评估了rAAV-DMP-miR533对体外炎性细胞的抑制作用,论证了rAAV-DMP-miR533的包装成功、对细胞的侵染能力以及对细胞内过度活化NF-κB活性的抑制。然后,将rAAV-DMP-miR533用于治疗具有三种典型炎症性疾病的小鼠,包括葡聚糖硫酸钠(DSS)诱发的急性结肠炎、咪喹莫特(IMQ)诱发的银屑病和胶原诱发的关节炎。以此论证rAAV-DMP-miR533在哺乳动物活体上治疗炎症的可行性、可靠性及安全性。
在机制方面,本发明发现rAAV-DMP-miR533可以使炎性细胞凋亡,从而根除炎性细胞。炎性细胞可以分泌促炎细胞因子,从而加剧和恶化炎症过程。rAAV-DMP-miR533可以通过根除炎性细胞从而去除炎症源。这种机制不同于目前依赖于细胞因子及其受体的抗体或拮抗剂的抗炎策略。NF-κB的直接靶基因大多数是细胞因子,而用抗体抑制细胞因子只能瞬时中和其产生的细胞因子,不能根除产生它们的炎性源。这就导致炎性细胞仍然可以持续产生细胞因子。因此,用细胞因子的抗体去治疗炎症性疾病复发率高。此外,许多细胞因子与其受体的多效性和冗余性结合会限制单一中和试剂的功效。这也是炎症性疾病对当下治疗策略具有抗性的原因。重要的是,作为特定炎症性疾病的关键参与者或关键调节 剂的细胞因子可能因人而异,导致了目前大多数治疗方法对患者应答率低。相比之下,rAAV-DMP-miR533通过根除炎性细胞而抑制了与炎症相关的所有细胞因子。例如,在三种炎症小鼠模型中,rAAV-DMP-miR533在mRNA和蛋白质水平上均显著降低了血清中两种主要的促炎因子TNF-α和IL-6的水平。这种血液中主要炎性因子水平的降低,对于治疗局部炎症症状和消除炎性因子的全身性病理作用具有极其重要的意义。因此,rAAV-DMP-miR533提供了一种更具广谱性抗炎的新策略,该工具可能克服当前抗炎治疗的一些关键挑战,如低响应、耐药、复发和副作用等。
有益效果:与现有技术相比,本发明具有如下优点:
目前的炎症治疗试剂主要是各种细胞因子的抗体(如TNF-α抗体)和新兴的JAK小分子抑制剂,这些治疗试剂存在应答率低和耐药的关键临床问题。为了突破现有的炎症治疗技术,发展基于AAV的新型炎症基因治疗新技术。本发明中将申请人前期发明的DNA片段DMP-miR533包装到安全的基因递送载体AAV中,制造了新型AAV颗粒——rAAV-DMP-miR53。系统研究了rAAV-DMP-miR533在细胞和活体水平上的抗炎作用。结果表明,rAAV-DMP-miR533在体外和体内均具有优异的抗炎作用。特别是,这种rAAV在三种典型的炎症小鼠模型中均表现出了良好的炎症治疗效果,包括急性结肠炎模型的葡聚糖硫酸钠(DSS)诱导的小鼠急性结肠炎、咪喹莫特(IMQ)诱导的银屑病模型和胶原诱导的小鼠关节炎模型。此外,在体内治疗过程中,rAAV-DMP-miR533还展现出了良好的生物安全性。因此,本发明研制的rAAV-DMP-miR53有望用于制备新型生物药物,用于人类炎症性疾病的治疗。
相对于传统的NF-κB小分子抑制剂、诱骗子和siRNA,rAAV-DMP-miR533的优势在于它可以避免对正常细胞中NF-κB活性的过度抑制。这是传统NF-κB小分子抑制剂、诱骗子和siRNA为什么不能成为药物的根本原因。例如,作为NF-κB抑制剂的典型小分子NF-κB抑制剂BAY 11-7082,会使炎性细胞和正常细胞均发生显著凋亡。本发明实验研究表明,rAAV-DMP-miR533能够诱导具有NF-κB过度活化的癌细胞(一种典型的炎性细胞)的显著凋亡,但对正常细胞几乎没有影响。但是当正常细胞被NF-κB诱导剂TNF-α诱导后成为炎性细胞时,rAAV-DMP-miR533则能诱导炎性化的正常细胞凋亡。说明rAAV-DMP-miR533能够特异性低作用于炎性细胞,这种高度选择性对于提高体内应用型和降低副作用具有重要意义。因此,rAAV-DMP-miR533克服了传统NF-κB抑制剂的副作用缺陷,具有临床转化潜力。
AAV是一种安全的基因传递工具,它具有免疫原性低、无致病性、无基因组插入、长期稳定表达等优点,已被获批用于人类临床基因治疗。本发明实验研究表明,在已使用剂量的前提下,rAAV-DMP-miR533对三种炎症性疾病的小鼠均没有显现出生物毒性。特别是,对关节炎CIA小鼠进行了三次高剂量的静脉给药,rAAV-DMP-miR533对小鼠的血清生化指标和脾脏均没有产生影响。相比之下,广泛使用的抗关节炎药物MTX,对小鼠肝脏和脾脏展示出了显著的毒副作用。此外,在本发明中,首次发现rAAV-DMP-miR533可以通过与凡士林混合外用涂抹治疗银屑病。这一给药方式极大便利了局部皮肤炎症的治疗。这一发现在国际上上没有报道,是对rAAV基因治疗给药途径的重要创新。因此,rAAV-DMP-miR533是一种能够以多种给药方式进行给药的新型安全抗炎试剂。虽然rAAV-DMP-miR533潜在的临床应用仍然会受到预先存在或体内能中和AAV载体的抗体的挑战,因为这些抗体能够阻断AAV的给药和再给药,这也是 目前基于AAV疗法的普遍障碍。然而,这一障碍可以通过一些新的方法来克服,例如利用内肽酶(例如Imlifidase,IdeS)或者IgG降解酶(IdeZ)去消除预先存在的抗AAV抗体,用CRISPR瞬时抑制内源性Myd88,以及使用具有TLR9抑制序列的AAV改造载体等。此外,其他非病毒载体,如脂质纳米粒(LNPs)也能被用于DMP-miR533的递送。此外,认为rAAV包装中残留的空壳rAAV也可能时一种有效rAAV抗体中和剂。
在本发明中,同时开发了一种基于AAV的基因疗法用于炎症性疾病的治疗。目前,基于AAV的基因疗法主要用于治疗人类遗传疾病。其中,几种基于AAV的基因疗法已开展了临床试验,例如通过表达IFN-β或TNFR-IgG1Fc的融合蛋白来治疗炎症性疾病,尤其是自身免疫性疾病,如类风湿性关节炎。然而,这些治疗仍然只针对一种炎症相关的细胞因子。相比之下,rAAV-DMP-miR533直接靶向炎症中枢NF-κB本身。三种典型炎症疾病的治疗表明,基于AAV基因治疗的rAAV-DMP-miR533为炎症性疾病提供了更广谱的治疗策略。
附图说明
图1为质粒和病毒载体。pAAV-MCS、pAAV-DMP-NT、pAAV-DMP-miR533、pAAV-CMV-EGFP、pAAV-DMP-miR533-CMV-EGFP的质粒图谱,及其包装成重组AAV(rAAV)。制备的rAAV分别命名为rAAV-MCS、rAAV-DMP-NT、rAAV-DMP-miR533、rAAV-CMV-EGFP和rAAV-DMP-miR533-CMV-EGFP。
图2为rAAV-DMP-miR533的炎症治疗示意图。(A)rAAV-DMP-miR533的炎症治疗示意图。DMP,诱骗子-最小启动子;Pol II,RNA聚合酶II;RISC,RNA诱导的沉默复合物。(B)炎性细胞和正常细胞中NF-κB的表达水平。具有NF-κB活性的癌细胞HT-29和CT-26被视为天然炎性细胞。正常细胞HL7702和NIH-3T3没有NF-κB活性。然而,当用促炎细胞因子TNF-α诱导时,这两种细胞成为了具有NF-κB活性的诱导性炎性细胞。通过qPCR检测NF-κB RELA的表达(n=3孔)。
图3为用pAAV-DMP-miR533处理炎性细胞。用各种质粒转染HT-29细胞,然后分别培养24、48和72小时。(A)吖啶橙(OA)染色后HT-29细胞的代表性荧光图。比例尺:100μm。(B)不同时间点的活细胞计数(n=3个图像)。使用Image-Pro Plus软件对吖啶橙染色后的荧光图像进行细胞计数。(C)CCK-8检测HT-29细胞的生长曲线(n=3孔)。(D)转染各种质粒48小时后,qPCR检测HT-29细胞中NF-κB及其靶基因的相对表达量(n=3孔)。RQ=2–ΔΔCt。RQ,相对定量。(E)吖啶橙染色后,HL7702细胞的代表性荧光图。比例尺:100μm。根据需要,对HL7702细胞使用TNF-α诱导,然后进行质粒转染。转染的细胞分别培养24、48和72小时。空白组、MCS和miR533:分别用Lipofectamine、pAAV-MCS和pAAV-DMP-miR533转染细胞;TNF-α:TNF-α诱导的细胞(用终浓度为10ng/mL的TNF-α诱导细胞1小时);TNF-α+MCS和TNF-α+miR533:分别用pAAV-MCS和pAAV-DMP-miR533转染的TNF-α诱导后的细胞。
图4为用rAAV-DMP-miR533-CMV-EGFP处理炎性细胞。根据需要,对HL7702细胞使用TNF-α诱导,然后用各种rAAV侵染48小时。(A)细胞荧光图。比例尺:100μm。(B)流式细胞术分析的细胞荧光强度(n=3孔)。(C)代表性细胞凋亡的流式细胞术分析图。(D)流式细胞术分析的细胞凋亡(n=3孔)。(E)CCK-8检测的细胞活力(n=3孔)。(F)通过qPCR检测病毒侵染48小时后细胞的NF-κB及其靶基因的表达(n=3孔)。空白组、MCS、miR533、EGFP和miR533-EGFP:分别用磷酸盐缓冲盐水(PBS)、rAAV-MCS、 rAAV-DMP-miR533、rAAV-CMV-EGFP和rAAV-DMP-miR533-CMV-EGFP侵染细胞;TNF-α:TNF-α诱导的细胞(用终浓度为10ng/mL的TNF-α诱导细胞1小时);TNF-α+MCS、TNF-α+miR533、TNF-α+EGFP、TNF-α+miR533-EGFP:分别用rAAV-MCS、rAAV-DMP-miR533、rAAV-CMV-EGFP和rAAV-DMP-miR533-CMV-EGFP侵染TNF-α诱导的细胞。
图5为EGFP荧光强度的流式细胞仪分析。将HL7702细胞用或不用10ng/mL终浓度的TNF-α诱导1小时,然后用各种病毒感染48小时。MCS、miR533、EGFP和miR533-EGFP:分别用磷酸盐缓冲液(PBS)、rAAV-MCS、rAAV-DMP-miR533、rAAV-CMV-EGFP和rAAV-DMP-miR533-CMV-EGFP侵染细胞;TNF-α+MCS、TNF-α+miR533、TNF-α+EGFP、TNF-α+miR533-EGFP:分别用rAAV-MCS、rAAV-DMP-miR533、rAAV-CMV-EGFP和rAAV-DMP-miR533-CMV-EGFP侵染TNF-α诱导的细胞。
图6为用rAAV-DMP-miR533治疗结肠炎小鼠。通过葡聚糖硫酸钠(DSS)诱导建立结肠炎小鼠模型,并通过静脉注射rAAVs(i.v.)进行治疗。(A)DSS诱导的急性结肠炎小鼠模型构建及治疗示意图。(B)小鼠肛门周围的血迹。(C)小鼠的体重。(D)小鼠的结肠。(E)小鼠的结肠长度(n=6只小鼠)。(F)代表性结肠样本的H&E染色切片。黑框表示放大区域。比例尺:200μm(10×)和100μm(20×)。(G)结肠样本的组织病理学评分(n=6只小鼠)。(H)ELISA检测血清中的TNF-α和IL-6水平(n=6只小鼠)。(I)qPCR检测结肠样本中NF-κB RELA及其靶基因的表达(n=6只小鼠)。空白组,小鼠饮用正常水并用PBS治疗;DSS组,小鼠饮用含3%DSS的水(DSS诱导小鼠)并用PBS治疗;MCS组,用rAAV-MCS治疗DSS诱导小鼠;miR533,用rAAV-DMP-miR533治疗DSS诱导的小鼠。ns,没有显著性差异。
图7为通过葡聚糖硫酸钠(DSS)诱导建立结肠炎小鼠模型,并通过静脉注射rAAVs(i.v.)进行治疗(生物学重复2)。(A)DSS诱导的急性结肠炎小鼠模型构建及治疗示意图。(B)小鼠肛门周围的血迹。(C)小鼠的体重。(D)小鼠的结肠。(E)小鼠的结肠长度(n=6只小鼠)。(F)代表性结肠样本的H&E染色切片。黑框表示放大区域。比例尺:200μm(10×)和100μm(20×)。(G)结肠样本的组织病理学评分(n=6只小鼠)。空白组,小鼠饮用正常水并用PBS治疗;DSS组,小鼠饮用含3%DSS的水(DSS诱导小鼠)并用PBS治疗;MCS组,用rAAV-MCS治疗DSS诱导小鼠;miR533,用rAAV-DMP-miR533治疗DSS诱导的小鼠。(H)ELISA检测血清中的TNF-α和IL-6水平(n=6只小鼠)。
图8为rAAV-DMP-miR533静脉注射治疗银屑病小鼠。银屑病小鼠模型通过咪喹莫特(IMQ)诱导建立,并通过静脉注射rAAVs(i.v.)进行治疗。(A)银屑病小鼠模型的构建及治疗示意图。(B)小鼠背部皮肤的直观图。(C)代表性皮肤样本的H&E染色切片。比例尺:200μm(10×)和100μm(20×)。(D)IMQ诱导6天后皮肤样本的组织病理学评分(n=3只小鼠)。(E)IMQ诱导12天后皮肤样本的组织病理学评分(空白组和MCS组,n=3只小鼠;miR533组,n=6只小鼠)。(F)ELISA检测血清中的TNF-α和IL-6水平(空白组和MCS组,n=3只小鼠;miR533组,n=6只小鼠)。(G)qPCR检测皮肤样本中TNF-α和IL-6的mRNA水平(空白组和MCS组,n=3只小鼠;miR533组,n=6只小鼠)。空白组,用PBS治疗经凡士林涂抹的小鼠;MCS,用rAAV-MCS治疗IMQ诱导的小鼠;miR533,用rAAV-DMP-miR533治疗IMQ诱导的小鼠。ns,没有显著性差异。
图9为IMQ诱导的银屑病小鼠经rAAV-DMP-miR533尾静脉注射治疗后相关组织的H&E病理分析和基因表达检测。(A)不同组(空白组、MCS、miR533)中所有皮肤样本的H&E染色切片。(B)通过qPCR检测皮肤样本中表达NF-κB及其靶基因的表达。
图10为rAAV-DMP-miR533皮下注射(i.h.)和皮肤给药(ad us.ext.)(外用)对银屑病小鼠模型的疗效。这些实验只用一只老鼠进行试验。(A)第0、6、12天小鼠背部皮肤的照片。(B)典型的H&E染色皮肤样本组织切片。(C)ELISA检测血清中的TNF-α和IL-6。(D)qPCR检测皮肤样本中NF-κB及其靶基因的表达。F,皮下注射(i.h.);T,皮肤给药(ad us.ext.)。
图11为rAAV-DMP-miR533皮肤外用给药治疗银屑病小鼠。银屑病小鼠模型通过咪喹莫特(IMQ)诱导建立,并通过皮肤涂抹给药(外用)rAAVs进行治疗。(A)银屑病小鼠模型的构建及治疗示意图。(B)小鼠背部皮肤的直观图。(C)银屑病面积和严重程度指数(PASI)评分(n=6只小鼠)。(D)代表性皮肤样本的H&E染色切片。比例尺:200μm(10×)和100μm(20×)。(E)皮肤样本的组织病理学评分(n=6只小鼠)。(F)小鼠脾直观图。(G)脾重(n=6只小鼠)。(H)每组小鼠平均体重(n=6只小鼠)。(I)ELISA检测血清中TNF-α和IL-6水平(n=6只小鼠)。空白组,用PBS治疗经凡士林涂抹的小鼠;MCS,用rAAV-MCS治疗IMQ诱导的小鼠;miR533,用rAAV-DMP-miR533治疗IMQ诱导的小鼠。ns,不显著。
图12为通过皮肤涂抹给药rAAV-DMP-miR533治疗咪喹莫特(IMQ)诱导的银屑病小鼠模型。(A)不同组(空白组、MCS、miR533)中所有皮肤样本的H&E染色切片。(B)皮肤样品中TNF-α和IL-6的mRNA表达水平。(C)通过qPCR检测皮肤样本中NF-κB及其靶基因的表达(n=6只小鼠)。
图13为pAAV-DMP-miR533对CT-26细胞凋亡和活力的影响。CT-26细胞经多种质粒转染后分别培养24、48、72h。(A)细胞凋亡流式细胞仪分析的代表性图像。(B)细胞凋亡(n=3孔)。(C)细胞活力(n=3孔)。Lipo,用Lipofectamine 2000处理的细胞。NT,用pAAV-DMP-NT转染的细胞;miR533,用pAAV-DMP-miR533转染的细胞。
图14为pAAV-DMP-miR533对NIH-3T3细胞凋亡和活力的影响。NIH-3T3在转染前用或不用TNF-α(终浓度为10ng/mL)诱导1小时。然后用各种质粒转染NIH-3T3细胞,然后分别培养24、48和72小时。(A)细胞凋亡流式细胞仪分析的代表性图像。(B)细胞凋亡(n=3孔)。(C)细胞活力(n=3孔)。NT,用pAAV-DMP-NT转染的细胞;miR533,用pAAV-DMP-miR533转染的细胞。
图15为CT26和NIH-3T3细胞中NF-κB及其靶基因表达检测。用pAAV-DMP-miR533转染48小时。根据需要,在转染前对NIH-3T3细胞使用终浓度为10ng/mL的TNF-α诱导1小时。用去PCR检测CT26(A)和NIH-3T3(B)细胞中NF-κB及其靶基因的表达。Lipo,用lipo2000处理的细胞。NT,用pAAV-NT转染的细胞。miR533,用pAAV-DMP-miR533转染的细胞。
图16为用rAAV-DMP-miR533治疗关节炎小鼠。通过胶原(collagen)诱导建立关节炎(CIA)小鼠模型,并通过静脉注射rAAVs进行治疗(i.v.)。(A)CIA小鼠模型构建及治疗示意图。PBS,用磷酸盐缓冲盐水处理的健康组。(B)不同组前后爪的代表性图片。(C)小鼠脾脏直观图。(D)脾重(n=6只小鼠)。(E)每组小鼠的平均体重(n=6只小鼠)。(F)关节炎严重程度的临床评分(n=6只 小鼠)。(G)爪子厚度(n=6只小鼠)。(H)脚踝厚度(n=6只小鼠)。(I)尾部宽度(n=6只小鼠)。(J)ELISA检测血清中TNF-α和IL-6水平(n=6只小鼠)。(K)qPCR检测后爪样本中TNF-α和IL-6的mRNA水平(n=6只小鼠)。(L)后爪与踝关节的micro-CT成像。轮廓区域展示了高分辨的micro-CT成像图。(M)代表性踝关节的H&E染色切片。比例尺:50μm。(N)踝关节的组织病理学评分(n=6只小鼠)。PBS组,用PBS治疗的正常小鼠;CIA组,用PBS治疗的CIA小鼠;MTX组,用甲氨蝶呤(MTX)治疗的CIA小鼠;NT组,用rAAV-NT治疗的CIA小鼠;miR533组,用rAAV-DMP-miR533治疗的CIA小鼠。ns,没有显著性差异。NT,无靶点的microRNA。
图17为用rAAV-DMP-miR533治疗关节炎小鼠。通过胶原诱导建立胶原诱导的关节炎(CIA)小鼠模型,并通过静脉注射(i.v.)rAAVs进行治疗。不同组(PBS、CIA、NT、MTX、miR533)中所有小鼠的前爪和后爪照片(n=6只小鼠;PBS,用磷酸盐缓冲盐水(PBS)治疗的正常小鼠;CIA,用PBS治疗的CIA小鼠;MTX,用甲氨蝶呤(MTX)治疗的CIA小鼠;NT,用rAAV-NT治疗的CIA小鼠;miR533,用rAAV-DMP-miR533治疗的CIA小鼠。ns,不显著。NT,无靶标。
图18为用rAAV-DMP-miR533治疗关节炎小鼠。通过胶原诱导建立胶原诱导的关节炎(CIA)小鼠模型,并通过静脉注射(i.v.)rAAVs进行治疗。(A)所有小鼠踝关节的代表性H&E染色切片。比例尺:50μm。(B)qPCR检测到后爪中NF-κB及其靶基因的表达(n=6只小鼠)。PBS,用磷酸盐缓冲盐水(PBS)治疗的正常小鼠;CIA,用PBS治疗的CIA小鼠;MTX,用甲氨蝶呤(MTX)治疗的CIA小鼠;NT,用rAAV-NT治疗的CIA小鼠;miR533,用rAAV-DMP-miR533治疗的CIA小鼠。ns,不显著。NT,无靶标。
图19为用rAAV-DMP-miR533治疗关节炎小鼠。通过胶原诱导建立胶原诱导的关节炎(CIA)小鼠模型,并通过静脉注射(i.v.)rAAVs进行治疗。(A)主要器官的代表性H&E染色部分。比例尺:100μm。(B)肝脏的血清生化指标(n=6只小鼠)。(C)肾脏血清生化指标(n=6只小鼠)。PBS,用磷酸盐缓冲盐水(PBS)治疗的正常小鼠;CIA,用PBS治疗的CIA小鼠;MTX,用甲氨蝶呤(MTX)治疗的CIA小鼠;NT,用rAAV-NT治疗的CIA小鼠;miR533,用rAAV-DMP-miR533治疗的CIA小鼠。ns,不显著。NT,无靶标。ALT,丙氨酸氨基转移酶;AST,天冬氨酸氨基转移酶;ALP,碱性磷酸酶;BUN,血尿素氮;Cr,肌酐;UA,尿酸。
图20为pAAV-DMP-miR533和pAAV-DMP-miR533-5对CT-26和NIH-3T3细胞凋亡和活力的影响。用各种质粒转染细胞,然后分别培养24、48和72小时。(A)OA染色的HL7702细胞的代表性荧光图像。(B)细胞活力(n=3孔)。pAAV-DMP-miR533含有单拷贝DMP-miR533。pAAV-DMP-miR533-5含有五拷贝DMP-miR533。(c)pAAV-DMP-miR533和pAAV-DMP-miR533-5对CT-26和NIH-3T3细胞凋亡和活力影响示意图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
实施例1
载体构建与病毒制备
载体构建:靶向人或鼠RELA的microRNA序列在BLOCK-iTTM RNAi Designer网站上(https://rnaidesigner.thermofisher.com/rnaiexpress/)设计(表1和 表2)。DMP-miR533片段从pDMP-miR533载体上扩增,然后使用MluI(上游)和XbaI(下游)限制性酶切位点将其连接到pAAV-MCS载体(VPK-410,Stratagene)上构建得到pAAV-DMP-miR533(图1)。CMV-EGFP片段用一对引物扩增,其上游MluI和下游EocRI酶切位点来自pEGFP-C1(Clontech),然后将CMV-EGFP片段克隆到pAAV-MCS中以获得载体pAAV-CMV-EGFP(图1)。使用HieffTM PCR Master Mix(With Dye)(Yeasen)对DNA片段进行PCR扩增。用琼脂糖凝胶电泳和AxyPrep DNA凝胶提取试剂盒(Axygen)对PCR扩增得到的DNA片段进行纯化回收。消化连接反应包含适当的限制性内切核酸酶(ThermoFisher Scientific)和T4DNA连接酶(ThermoFisher Scientific)。通过将CMV-EGFP片段构建到pAAV-DMP-miR533载体中得到质粒pAAV-DMP-miR533-CMV-EGFP(图1)。作为阴性对照载体,根据质粒pcDNATM 6.2-GW/EmGFP-miR-Neg(Thermo Fisher Scientific)的序列合成miR-NT片段,将其插入至pDMP-miR中,获得pDMP-NT质粒载体。从pDMP-NT拷贝DMP-NT片段并插入至pAAV-MCS中以获得pAAV-DMP-NT载体(图1)。所有质粒包括pAAV-MCS、pAAV-DMP-NT、pAAV-CMV-EGFP、pAAV-DMP-miR533、pAAV-DMP-miR533-CMV-EGFP、pAAV-Helper和pAAV-RC都分别转染至大肠杆菌DH5α(Tiangen)中并用EndoFree Plasmid试剂盒(CWBio)纯化。所有质粒均通过DNA测序验证。本实施例中使用的寡核苷酸和引物由Sangon Biotech(中国上海)合成(表2和表3)。
表1.miRNA的靶序列
表2.用于构建miRNA表达载体的寡核苷酸序列
细胞培养:实施例中使用的所有细胞系均来自中国科学院上海生命科学研究所细胞资源中心,包括HEK-293T(人胎肾细胞)、HT-29(人结肠癌细胞)、CT-26(小鼠结肠癌细胞)、HL7702(人正常肝细胞)、NIH-3T3(小鼠胚胎成纤维细胞)。HT-29、CT-26、HL7702细胞在Roswell Park Memorial Institute(RPMI)1640培养基(Gibco)中培养。NIH-3T3、HEK-293T在Dulbecco’s Modified Eagle Medium(DMEM)培养基(Gibco)中培养。所有培养基均添加终含量为10%的胎牛血 清(HyClone)、100单位/mL的青霉素(Thermo Fisher)和100μg/mL的链霉素(Thermo Fisher)。所有细胞均在含有5%CO2的37℃加湿培养箱中培养。
病毒制备:HEK-293T以每瓶5×106个细胞的密度接种到75cm2的培养瓶中,培养24小时。然后根据说明书使用Lipofectamine 2000(Thermo Fisher)对两种辅助质粒(pAAV-Helper和pAAV-RC;Stratagene)和一种pAAV质粒(pAAV-MCS、pAAV-DMP-NT、pAAV-DMP-miR533、pAAV-DMP-miR533-CMV-EGFP、pAAV-CMV-EGFP)进行共转染。细胞继续培养72小时后,将细胞和培养基收集并置于-80℃下保存过夜。随后将细胞和培养基置于37℃水浴锅中孵育2小时。整个冻融过程重复3次。按1:10体积比例将纯氯仿加入细胞冻融完的裂解液中,并使该混合物在37℃下剧烈振荡1小时。震荡完后,向混合物中加入NaCl至终浓度其为1摩尔并摇动至NaCl溶解。将溶解液置于4℃下以15,000转/分钟(rpm)离心15分钟,随后收集上清液,加入终浓度为10%(w/v)的PEG8000并摇动至其溶解。将反应物再在4℃下以15,000rpm离心15分钟,弃去上清液,而后使沉淀溶解在PBS中。然后将DNase和RNase加入溶解完的沉淀中,核酸酶终浓度为1μg/mL。反应物在室温下温育30分钟。最后将温育完的试剂用氯仿(1:1体积)萃取一次,把含有纯化病毒的水相转移到新的试管中。通过使用引物AAV-F/R和qPCR检测确定AAV的滴度(表3)。病毒定量后,将其分装至-80℃保存备用。获得的病毒分别命名为rAAV-MCS、rAAV-DMP-NT、rAAV-DMP-miR533、rAAV-DMP-miR533-CMV-EGFP和rAAV-CMV-EGFP。
NF-κB广泛过度活化于炎性细胞中,为了抑制NF-κB活性,本实施例设计了一个名为rAAV-DMP-miR533的rAAV(图2A),其中DMP是一个启动子,由一个NF-κB诱骗子和一个最小启动子组成,而miR533则编码一段人工microRNA用于靶向NF-κB RELA。当DMP-miR533转染人结肠癌细胞(HT-29)、小鼠结肠癌细胞(CT-26)、TNFα诱导的人正常肝细胞(HL7702)和小鼠胚胎成纤维细胞(NIH-3T3)等炎性细胞时(图2B),DMP将会与过度活化的NF-κB结合,从而激活miR533的转录。而当NF-κB被miR533干扰抑制时,炎性细胞因子(NF-κB靶基因),如TNF-α和IL-6表达量将会下调。因此,NF-κB的抑制可进一步导致炎性细胞凋亡。相比之下,在正常细胞如人正常肝细胞(HL7702)和小鼠胚胎成纤维细胞(NIH-3T3)(图2B)中,DMP-miR533由于缺乏NF-κB活性而无法发挥作用(图2A)。为了充分评估DMP-miR533在细胞和动物中的抗炎作用,设计并制备了分别靶向人和小鼠NF-κB RELA转录物的miR533(表1)。人细胞用靶向人NF-κB RELA的DMP-miR533处理,小鼠细胞和小鼠用靶向小鼠NF-κB RELA的DMP-miR533处理。
本实施例中采用血清型AAV2为重组腺相关病毒载体。本实例中用pAAV-MCS载体(VPK-410,Stratagene)插入功能性DNA片段DMP-miR,并使用两种辅助质粒pAAV-Helper和pAAV-RC共转化293T细胞制备重组腺相关病毒;上述三质粒系统所包装制备的重组腺相关病毒则为血清型AAV2重组腺相关病毒,因为在辅助质粒pAAV-RC上含有AAV2的Rep和Cap基因。AAV2具有比较广泛的组织侵染能力,这样所构建的rAAV-DMP-miR533就可以通用性地治疗发生不同组织器官的炎症,如本发明治疗的位于肠道组织的炎症结肠炎、位于皮肤组织的炎症银屑病、位于关节组织的炎症关节炎。如果使用某种组织侵染具有显著偏向性的血清型AAV,如偏向神经组织的AAV9,所构建的rAAV在治疗不同组织器官的炎症时,就必须构建不同血清型的rAAV-DMP-miR533,过程 繁琐且成本高。此外,AAV2也是天然腺相关病毒之一,经过长期于人类共存进化,人体对这种AAV具有极低的免疫原性,使其使用更安全。此外,AAV2的专利已经过期,在制备治疗试剂时不存在专利限制,有利于推广应用。
实施例2
炎性细胞处理
将细胞(HT-29、CT-26、HL7702和NIH-3T3)(1×105)接种到24孔板中,37℃ 5%CO2培养过夜。然后根据说明书,使用Lipofectamine2000(Thermo Fisher)将用各种实施例1制备的pAAV质粒(500ng/孔)转染至细胞内。转染后,细胞分别培养24小时、48小时、72小时。如果需要,在转染前,先用终浓度为10ng/mL的TNF-α(Sigma-Aldrich)诱导正常细胞1小时。随后根据说明书,使用吖啶橙(Solarbio)对细胞进行染色,其中活细胞呈现均匀的绿色。细胞用荧光显微镜(IX51,Olympus)成像,并用Image-Pro Plus软件计数。
细胞活力检测:同时使用Cell Counting Kit-8(CCK-8,Yeasen)测定并分析细胞活力。将细胞(HT-29、CT-26、HL7702和NIH-3T3)(5×103)接种到96孔板中,37℃ 5%CO2培养过夜。然后使用Lipofectamine 2000(Thermo Fisher)将各种实施例1制备的pAAV质粒(200ng/孔)转染至细胞内。转染后,细胞分别培养24小时、48小时、72小时。如果需要,在转染前,先用终浓度为10ng/mL的TNF-α(Sigma-Aldrich)诱导正常细胞1小时。最后往细胞中加入CCK-8试剂(10μL/孔)并孵育1小时,使用酶标仪(BioTek)在450nm处测量溶液吸光度。
细胞凋亡检测:将细胞(HT-29、CT-26、HL7702和NIH-3T3)(5×105)接种到6孔板中,37℃ 5%CO2培养过夜。然后使用Lipofectamine 2000(Thermo Fisher)将各种实施例1制备的pAAV质粒(4μg/孔)转染至细胞内。转染后,细胞分别培养24小时、48小时、72小时。如果需要,在转染前,先用终浓度为10ng/mL的TNF-α(Sigma-Aldrich)诱导正常细胞1小时。然后根据制造商的说明,通过使用AnnexinV-FITC/PI细胞凋亡检测试剂盒(Vazyme)和流式细胞术(Calibur,BD,USA)检测分析定量细胞凋亡。
细胞EGFP荧光检测:将细胞(HT-29、CT-26、HL7702和NIH-3T3)(5×103)接种到96孔板中,37℃ 5%CO2培养过夜。然后用各种实施例1制备的rAAV(5×107vg/孔)侵染细胞。侵染后,细胞培养48小时。如果需要,在侵染前,先用终浓度为10ng/mL的TNF-α(Sigma-Aldrich)诱导正常细胞1小时。EGFP荧光用荧光显微镜(IX51,Olympus)成像,并通过流式细胞术(Calibur,BD,USA)进行定量分析。同时使用AnnexinV-FITC/PI细胞凋亡检测试剂盒和流式细胞术检测细胞凋亡。
基因表达的定量PCR(qPCR)检测:按照说明书的实验方案,使用TRIzolTM(Invitrogen)从培养的细胞和小鼠组织中分离总RNA。随后用带有gDNA Eraser的PrimeScriptTM RT试剂盒(Takara)生成互补的DNA(cDNA)。往样品中混入Fast SYBR Green Master Mix(Roche),然后在ABI StepOne Plus仪器(Applied Biosystems)上,通过定量PCR(qPCR)检测cDNA上靶基因的表达量。每个样品均进行三次技术重复。相对mRNA转录水平计算为2–ΔCt or 2–ΔΔCt,其中,ΔCt=Cttarget-CtGADPH;ΔΔCt=ΔCttreatment–ΔCtcontrol。2–ΔΔCt也被定义为相对定量(RQ)。使用熔解曲线分析验证了所有qPCR引物(表3)的特异性。
表3.用于qPCR的引物序列


数据统计分析:所有数据均表示为平均值±标准偏差(SD),并通过GraphPad Prism 8.0软件进行统计分析和图表绘制。使用双尾Student’s T检验确定两组之间的统计差异。根据数据,三组或更多组则通过使用单向或双向方差分析(ANOVA)与Tukey’s或Sidak’s多重比较配对进行统计分析检验。p<0.05的差异被认为具有统计学意义。
结果:为了评估DMP-miR533系统杀死炎性细胞的可行性,首先选用NF-κB过度活化的人结肠癌细胞(HT-29)作为实验对象,用pAAV-DMP-miR533转染24小时至72小时。细胞的吖啶橙(AO)染色显示pAAV-DMP-miR533对HT-29细胞具有显著的细胞毒性(图3A和3B)。细胞生长曲线还显示HT-29细胞的生长被pAAV-DMP-miR533显著抑制(图3C)。同时,pAAV-MCS(空病毒载体)对细胞的活力和生长均不产生影响(图3B和3C)。为进一步验证pAAV-DMP-miR533的体外干扰效率,通过qPCR检测了NF-κB RELA及其靶基因的表达。结果表明,这些基因的表达被pAAV-DMP-miR533显著抑制,但在Lipofectamine(空白组)和pAAV-MCS组无变化(图3D)。这些数据表明pAAV-DMP-miR533可以通过敲低NF-κB及其靶基因的表达来显著抑制NF-κB过度活化的细胞生长。
为了进一步探索pAAV-DMP-miR533是否对炎症有影响,用已知的NF-κB诱导物TNF-α诱导人正常肝细胞(HL7702),构建细胞炎性模型。同时以未用TNF-α诱导的HL7702细胞作为对照。细胞的AO染色显示pAAV-DMP-miR533对正常HL7702细胞没有明显影响,但经TNF-α诱导后,HL7702的细胞数量大幅减少(图3E)。这些结果表明pAAV-DMP-miR533可以引起炎性细胞死亡,但对非炎性的细胞作用不明显。
为了在体内应用,将不同的DNA片段包装到腺相关病毒(AAV)中,以此来构建rAAV-MCS、rAAV-CMV-EGFP、rAAV-DMP-miR533和rAAV-DMP-miR533-CMV-EGFP(图1)。同时将CMV-EGFP片段插入rAAV-DMP-miR533以监测rAAV-DMP-miR533对细胞的侵染情况。以包含CMV启动子,不插入任何目的基因的空病毒rAAV-MCS作为阴性对照。首先将包装好的病毒分别侵染TNF-α处理和未处理的HL7702细胞。通过流式细胞术分析EGFP的表达和细胞凋亡情况。结果表明,EGFP在rAAV-DMP-miR533-CMV-EGFP侵染的HL7702细胞与rAAV-CMV-EGFP侵染的细胞中具有相似的表达量(图4A和4B;图5),这说明制备的病毒可以有效地侵染细胞。此外,rAAV-DMP-miR533和rAAV-DMP-miR533-CMV-EGFP均能诱导被TNF-α诱导的HL7702细胞显著凋亡;然而,相同的侵染对正常的HL7702细胞影响甚微(图4C和4D)。细胞活力测定进一步显示,只有rAAV-DMP-miR533和rAAV-DMP-miR533-CMV-EGFP的侵染能导致TNF-α诱导的HL7702细胞活力大幅下降(图4E)。
为了进一步验证rAAV-DMP-miR533感染炎性细胞凋亡的原理,用qPCR技术检测了HL7702细胞中NF-κB RELA及其靶基因的表达。结果表明,TNF-α显 著诱导了NF-κB RELA及其靶基因的表达(图4F)。但是rAAV-DMP-miR533和rAAV-DMP-miR533-CMV-EGFP的侵染能够逆转这一现象(图4F)。总之,DMP-miR533可以抑制NF-κB RELA的表达并进一步导致炎性细胞凋亡以及活力降低,表明rAAV-DMP-miR533具有良好的体外抗炎作用。
实施例3
结肠炎模型的构建与治疗
将购自Cavens(中国)的BALB/c小鼠随机分为4组(n=6),包括空白组、葡聚糖硫酸钠(DSS)、DSS+MCS和DSS+miR533组。空白组小鼠饮水,其他3组小鼠的饮用水中含有3%葡聚糖硫酸钠(DSS)(M.W=36000-50000)(MP)。在饮用含3%DSS的水后的第3天和第5天,DSS+MCS和DSS+miR533组小鼠分别静脉注射100μL 1×1010vg/mL rAAV-MCS和rAAV-DMP-miR533。每天测量体重。第8天处死小鼠,分离肛门至回盲部的结肠并采血,同时测量每只小鼠的结肠长度。结肠组织用于石蜡切片的制备和基因表达检测。切片用苏木精-伊红(H&E)染色、成像和评分。结肠组织的病理学评分由其他与项目无关的技术人员根据四个等级进行盲评打分:0分,无明显病理变化;1分,局灶性炎症细胞浸润;2分,广泛的炎性细胞浸润;3分,弥漫性炎症细胞浸润;4分,炎症细胞浸润,组织变性坏死,纤维结缔组织增生。
血清中TNF-α和IL-6水平的测定:根据ELISA说明书,使用TNF-α(ab208348,abcam)和IL-6ELISA试剂盒(ab222503,abcam)对血清中TNF-α和IL-6的水平进行测定。
组织切片的制备及苏木精和伊红(H&E)染色:对包括心脏、肝脏、脾脏、肺、肾脏在内的小鼠组织依次进行解剖,石蜡包埋,切片,苏木精和伊红(H&E)染色处理。简而言之,首先将组织切块,然后将其在室温下置于4%多聚甲醛溶液(Sangon Biotech,China)中固定过夜。随后,取出固定好的标本,依次完成脱钙,石蜡包埋,组织切片,苏木精染色溶液(C0107,Beyotime)和曙红染色溶液(C0109,Beyotime)染色实验步骤。最后用显微镜(IX51,Olympus)对制备的载玻片成像拍照。组织病理学评分由其他无关的研究人员盲评打分。
基因表达的qPCR检测:同实施例2。
数据统计分析:同实施例2。
结果:为了评估DMP-miR533的体内抗炎作用,葡聚糖硫酸钠(DSS)被用于小鼠结肠炎模型的诱导构建(图6A)。如图6B所示,每天对4组小鼠的精神状态和大便状态进行观察,发现空白组小鼠一切正常,粪便硬实,无血。而DSS诱导的小鼠逐渐变得迟钝和不活跃,饮用3%DSS后的第3天,粪便性状由正常变为湿软,第5天粪便明显带血,第7天肛门仍然出血。这些症状表明DSS诱导的急性结肠炎小鼠建模成功。随后用各种试剂处理小鼠。用rAAV-MCS处理的DSS诱导小鼠与用PBS处理的DSS诱导小鼠的症状相似,两组小鼠粪便均变软,出现血便。而用rAAV-miR533治疗后的DSS诱导小鼠粪便状态明显改善和肛门出血显著减少。体重的动态测量显示,用rAAV-MCS和PBS处理的急性结肠炎小鼠体重减轻,而用rAAV-DMP-miR533治疗后的急性结肠炎小鼠体重增加(图6C)。小鼠结肠长度测量结果表明DSS诱导导致小鼠结肠长度变短(图6D和6E)。只有经rAAV-DMP-miR533的治疗才能使小鼠结肠长度恢复(图6D和6E)。结肠组织H&E染色切片进一步显示,DSS诱导后小鼠结肠出现了明显的炎症病理损伤,如黏膜结肠隐窝消失、杯状细胞丢失、细胞变性、明显致密淋巴细胞浸润、中性粒细胞和浆细胞明显浸润等。然而,rAAV-DMP-miR533的治疗 逆转了这些损伤,例如小鼠结肠具有相对完整的粘膜层结构、明显的隐窝结构和极为少量的中性粒细胞和浆细胞浸润(图6F和6G)。血清中典型促炎因子的定量结果表明,DSS能诱导血清中TNF-α和IL-6的大幅升高(图6H)。rAAV-DMP-miR533则显著降低了这两个因子的水平(图6H)。此外,DSS还使NF-κB RELA及其靶基因过度激活表达(图6I);同样地,rAAV-DMP-miR533的治疗大幅下调了这些基因的表达(图6I)。此外,上述动物实验,还开展了另一个独立生物学重复并获得了相似的结果(图7)。总之,这些数据充分表明rAAV-DMP-miR533在DSS诱导的急性结肠炎小鼠中具有显著的体内抗炎作用。
实施例4
银屑病模型的构建与治疗
对雄性BALB/c(8周;Cavens,中国)银屑病造模小鼠开展了两批动物实验。首先将小鼠背部进行剃毛处理(剃毛面积约2.5cm×2.5cm),之后随机分为3组(n=6),包括空白组、MCS和miR533组。空白组小鼠用凡士林乳膏涂抹处理。MCS和miR533组小鼠的背部剃毛处每天涂抹5%w/w咪喹莫特(IMQ)(中国四川明欣药业),剂量为62.5mg/每只。
第一批动物实验中,IMQ给药6天后,空白组和MCS组各处死3只小鼠(n=3),采集皮肤和血液样本。MCS组(n=3)和miR533组(n=6)的剩余小鼠分别静脉注射100μL 1×1010vg/mL rAAV-MCS和rAAV-DMP-miR533。MCS和miR533组的小鼠每天仍保持5%w/w的IMQ涂抹。空白组的小鼠继续采用凡士林乳膏处理。持续6天后,处死所有小鼠并收集皮肤和血液样品,随后分别采用RT-qPCR和ELISA试剂盒检测皮肤和血清样品中TNF-α和IL-6的表达水平。同时,在另一只银屑病模型小鼠(n=1)上尝试了rAAV-DMP-miR533的皮下给药(i.h.)和外用药(外用)(ad us.ext.)治疗。银屑病模型小鼠每天进行皮下注射或涂抹100μL 1×1010vg/mL rAAV-DMP-miR533治疗。持续6天,处死小鼠,样品采集和检测与之前的静脉注射实验相同。
在5%w/w IMQ造模后的第二批动物实验中,分别对MCS组和miR533组小鼠(n=6)的背部剃毛区域涂抹100μL的1×1010vg/mL rAAV-MCS和rAAV-DMP-miR533,持续6天。类似地,MCS和miR533组的小鼠每天继续涂抹5%w/w IMQ。空白组一直涂抹凡士林乳膏直至安乐死。在第12天处死所有小鼠并拍照,同时收集背部皮肤和血液样品。每天监测和记录小鼠的体重和银屑病面积和严重程度指数(PASI)。每只小鼠皮肤上的红斑、鳞屑和厚度从0到4分别进行独立评分:0,无;1、轻微;2、适中;3、显著;4、极为显著。三个指标的总和表示炎症的严重程度(评分,0-12)。
血清中TNF-α和IL-6水平的测定:同实施例3。
组织切片的制备及H&E染色:同实施例3。
基因表达的qPCR检测:同实施例2。
数据统计分析:同实施例2。
结果:为了进一步证实rAAV-DMP-miR533的体内抗炎作用,使用咪喹莫特(IMQ)诱导构建小鼠银屑病模型,并用多种试剂尝试治疗(图8A)。在小鼠剃毛的背部皮肤上连续涂抹IMQ,六天后小鼠皮肤出现了发红、发炎、发痒、皮肤增厚以及银色鳞屑等症状(图8B)。随后分别对IMQ诱导的银屑病小鼠静脉注射PBS、rAAV-MCS和rAAV-DMP-miR533。结果表明,rAAV-MCS的治疗没有病理损伤恢复的效果;而rAAV-DMP-miR533的治疗使小鼠受损的皮肤明显接近了PBS处理的健康小鼠(健康组)(图8B)。皮肤组织切片的H&E染色也 显示出IMQ诱导的小鼠皮肤有明显的脓肿、角化过度和炎症细胞浸润等病理特征;而rAAV-DMP-miR533的处理能显著使皮肤恢复(图8C、8D和8E;图9A)。血清促炎因子的检测表明IMQ诱导后,TNF-α和IL-6水平升高(图8F)。而rAAV-DMP-miR533的治疗大幅降低了血清中这两个因子的水平(图8F)。皮肤中基因表达的检测表明,IMQ显著激活了TNF-α、IL-6、NF-κB RELA及其靶基因的表达(图8G和图9B)。但经rAAV-DMP-miR533治疗后,这些基因则被显著抑制(图8G和图9B)。这些数据说明rAAV-DMP-miR533的静脉注射对小鼠银屑病具有不错的疗效。
为了寻找其他给药方式,还对一只IMQ诱导的银屑病小鼠分别进行了皮下注射和皮肤涂抹给药治疗。结果表明,两种给药方式均获得了与上述静脉注射相似的治疗效果(图10),包括皮肤恢复(图10A和10B)、血清促炎因子(图10C)以及NF-κB RELA及其靶基因的表达下调(图10D)。考虑到更好的治疗效果和给药便利性,选取皮肤涂抹给药方式进行了放大治疗,包含更多的小鼠个体(n=6)(图11A)。银屑病小鼠分别用凡士林和混合在凡士林中的rAAV-DMP-miR533进行连续6天皮肤给药治疗(图11A)。结果显示,rAAV-DMP-miR533皮肤涂抹给药方式获得了良好的治疗效果,包括皮肤外观恢复(图11B)、低银屑病面积和严重程度指数(PASI)(图11C)、皮肤组织结构愈合(图11D和11E;图12A),脾肿大改善(图11F和11G),体重增加(图11H),血清中TNF-α和IL-6水平降低(图11I),以及皮肤组织中TNF-α,IL-6(图12B)和NF-κB RELA及其靶基因显著下调(图12C)。总之,这些结果充分证明rAAV-DMP-miR533在IMQ诱导的银屑病小鼠中具有良好的体内抗炎疗效。
实施例5
关节炎模型的构建与治疗
30只雄性的DBA/1J小鼠(8周;Cavens,中国)被随机分为5组,其中一组仅注射PBS(n=6)作为健康组对照。通过双重免疫建立胶原诱导关节炎(CIA)小鼠模型。对于第一次免疫,小鼠在尾部皮内注射等体积的鸡II型胶原溶液(2mg/mL)和完全弗氏佐剂(2mg/mL)(Chondrex,Redmond,WA,USA)。第一次免疫21天后,用不完全弗氏佐剂(Chondrex,Redmond,WA,USA)乳化鸡II型胶原溶液,在与第一次免疫不同的位置对小鼠尾部进行免疫加强注射。对每只小鼠的所有爪子进行评分来定性疾病:0,正常;1、轻度肿胀和红斑局限于中足和踝关节;2、轻度肿胀和红斑延伸至足中部和踝关节;3、从跖骨关节到脚踝有中度肿胀和红斑;4、脚、脚踝和手指严重肿胀和红斑。每只小鼠的临床评分是四只爪子的评分总和。
CIA小鼠分为四个实验组:(1)CIA组:CIA小鼠注射PBS作为对照组(iv;n=6);(2)MTX治疗组:CIA小鼠注射MTX(1mg/kg),隔日给药,共6次(iv;n=6);(3)NT治疗组:CIA小鼠每隔一天静脉注射rAAV-NT(1×109vg/小鼠;n=6),共3次;(4)miR533治疗组:CIA小鼠每隔一天静脉注射rAAV-DMP-miR533(1×109vg/小鼠;n=6),共3次。小鼠的体重和临床评分每隔一天进行监测和评估。用游标卡尺测量小鼠脚踝宽度、爪子厚度和尾部厚度。所有小鼠在MTX或NT或miR533给药4周后被安乐死并拍照,同时采集各组血清标本进行生化指标检测。采用ELISA试剂盒检测后爪和血清样品中TNF-α和IL-6的表达水平。收集包括心脏、肝脏、脾脏、肺和肾脏在内的组织用于H&E染色分析。对所有小鼠的脾脏进行拍照和称重。踝关节组织用于后续的H&E染色分析和基因表达检测,其组织病理学评分由其他无关的技术人员根据以下四个 等级盲评打分:0,正常滑膜;1、存在滑膜肥大和细胞侵袭;2、存在血管翳和软骨糜烂;3、存在软骨和软骨下骨侵蚀;4、整个关节的功能障碍和僵硬。
血清中TNF-α和IL-6水平的测定:同实施例3。
组织切片的制备及H&E染色:同实施例3。本实验还额外对小鼠爪子(包括踝关节)组织进行解剖处理。
组织切片的制备及H&E染色:同实施例2。
基因表达的qPCR检测:同实施例2。
数据统计分析:同实施例2。
微型CT(Micro-CT):处死所有DBA/1J小鼠并收集它们的爪子(包括踝关节),使用体内微型CT扫描仪(vivaCT 80,SCANCO Medical AG,Switzerland)对其进行微型CT成像(Micro-CT)。高分辨率断层图像的重建和分析在SCANCO GPU Accelerated Reconstruction系统中进行。
结果:在上述细胞和小鼠实验中,pAAV-MCS和rAAV-MCS分别用作pAAV-DMP-miR533和rAAV-DMP-miR533的阴性对照。为了给DMP-miR533提供更合适的阴性对照,构建了一种新的载体DMP-NT,该载体编码了一个对人类和小鼠基因组均无靶标(NT)的microRNA。为了评估该载体,小鼠结肠癌细胞(CT26)分别用pAAV-DMP-miR533和pAAV-DMP-NT转染。结果显示pAAV-DMP-miR533对CT26的细胞生长产生了显著抑制并诱导了细胞凋亡(图13A-13C);而pAAV-DMP-NT对细胞的生长和凋亡没有显著影响(图13A-13C)。为了进一步评估这两种载体,用这两种载体分别转染正常小鼠胚胎成纤维细胞(NIH-3T3)。结果表明,两种载体均未诱导细胞发生明显的细胞凋亡和生长抑制(图14A-14C)。然而,当细胞被TNF-α诱导后,pAAV-DMP-miR533会诱导细胞产生显著凋亡和生长抑制(图14A-14C)。pAAV-DMP-NT仍然对受到TNF-α诱导的细胞没有影响(图14A-14C)。qPCR检测还显示pAAV-DMP-miR533显著抑制了CT26和TNF-α诱导的NIH-3T3的NF-κB RELA及其靶基因的表达(图15A和15B);而pAAV-DMP-NT对两种细胞中的基因表达均没有影响(图15A和15B)。总之,这些结果表明DMP-miR533通过抑制NF-κB活性产生了体外抗炎作用。此外,将无细胞毒性的pAAV-DMP-NT包装到AAV中来制备rAAV-DMP-NT,并以rAAV-DMP-NT作为对应的阴性对照来进一步评估rAAV-DMP-miR533的体内抗炎作用。
类风湿性关节炎(RA)是一种以关节病变为主的慢性自身免疫性疾病。通过双重免疫建立了胶原诱导的关节炎(CIA)小鼠模型,用于RA治疗研究(图16A)。在初次免疫后的第28天,CIA小鼠被随机分为4组(n=6),分别静脉注射PBS、甲氨蝶呤溶液(MTX),rAAV-DMP-NT,和rAAV-DMP-miR533。健康组小鼠仅静脉注射PBS作为健康对照(n=6)。在第46天,对所有小鼠实施安乐死。爪子(包括踝关节)的成像显示,rAAV-DMP-miR533获得了比MTX更好的治疗效果(图16B和图17)。同时,rAAV-DMP-miR533的治疗使CIA小鼠恢复了正常的脾脏体积和重量,而MTX治疗后小鼠脾肿大的情况比CIA模型鼠更加严重(图16C和16D)。此外,rAAV-DMP-miR533治疗后小鼠体重维持稳定,MTX治疗则使小鼠体重减轻(图16E)。小鼠病理变化的动态测量显示,rAAV-DMP-miR533和MTX的治疗均显著改善了临床评分(图16F)、爪子厚度(图16G)和脚踝宽度(图16H)。值得注意的是,只有rAAV-DMP-miR533的治疗使小鼠尾部宽度接近健康小鼠(图16I)。CIA模型组中,血清促炎因子TNF-α和IL-6水平显著升高;rAAV-DMP-miR533和MTX均能显着降低血清中这两种 因子的水平(图16J)。但rAAV-DMP-miR533具有比MTX更好的疗效(图16J)。这些治疗效果也通过后爪组织中这两种因子mRNA的表达水平得到证实(图16K)。更令人信服的是,小鼠后爪的Micro-CT成像显示,CIA小鼠的踝关节和指关节具有严重的骨侵蚀现象(图16L)。而经rAAV-DMP-miR533治疗后,小鼠骨侵蚀现象得到大大改善,且疗效明显优于MTX(图16L)。H&E染色和关节组织病理学评估的结果也支持了这一结论(图16M和16N;图18A)。CIA小鼠具有大量的血管翳、严重的骨破坏、广泛的软骨损伤、炎症细胞浸润等病理特征,rAAV-DMP-miR533和MTX治疗均显著改善了这些病理变化。相比之下,rAAV-DMP-miR533的治疗更为突出。此外,rAAV-DMP-miR533的治疗还显著抑制了前爪中NF-κB RELA及其靶基因的表达(图18B)。MTX的治疗则不调控这些基因的表达(图18B),表明炎性细胞仍然存在。值得关注的是,在上述所有实验中,rAAV-DMP-NT均没有显示出治疗效果(图16和图18)。主要器官(心脏、肝脏、脾脏、肺和肾脏)的H&E切片染色表明rAAV-DMP-miR533明显改善了CIA建模所致的组织损伤,尤其是肺损伤(图19A)。而MTX则不显著,甚至对肝脏和脾脏造成了一定损伤和坏死(图19A)。最后,对在第46天收集的血清进行生化指标检测,数据进一步表明rAAV-DMP-miR533对这些生化指标没有产生影响,具有良好的生物安全性(图19B)。而经MTX治疗后,小鼠血清中ALT、AST和ALP普遍升高,表明其具有肝毒性(图19C)。总之,这些结果证明了rAAV-DMP-miR533在胶原诱导的关节炎小鼠中具有良好的体内抗炎作用。
实施例6
多拷贝DMP-miR533的抗炎效果
为了进一步探讨增加DMP-miR533拷贝数对rAAV-DMP-miR533抗炎效果的影响,还构建了一个含有5拷贝DMP-miR533的rAAV包装质粒pAAV-DMP-miR533-5。用此质粒和含有单拷贝DMP-miR533的rAAV包装质粒pAAV-DMP-miR533平行转染CT-26和NIH-3T3。转染方法同实施例2。观察两种质粒转染对两种细胞凋亡和活力的影响。结果表明,相较于pAAV-DMP-miR533,pAAV-DMP-miR533-5进一步显著提高了对炎性细胞CT-26的促凋亡效果和抑制其生长活力(图20),而仍然对正常细胞NIH-3T3的凋亡和活力不产生显著影响(图20)。由此可以推断用pAAV-DMP-miR533-5包装的含有5拷贝DMP-miR533的rAAV-DMP-miR533病毒,在体内应有更好的抗炎效果。

Claims (13)

  1. 一种用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述重组腺相关病毒rAAV-DMP-miR533以腺相关病毒为载体,其包含一个到多个拷贝的功能性DNA片段DMP-miR;所述功能性DNA片段DMP-miR由两个功能元件DMP和miR构成,其中DMP为NF-κB特异性启动子,所述miR为编码可靶向NF-κB mRNA的microRNA。
  2. 根据权利要求1所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述DMP为NF-κB特异性启动子,由一个NF-κB诱骗子和一个最小启动子组成,该DMP包括各种序列的NF-κB诱骗子和最小启动子。
  3. 根据权利要求2所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述DMP的序列为5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CTA GAG GGT ATA TAA TGG AAG CTC GAC TTC CAG-3'。
  4. 根据权利要求1所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述miR包括编码靶向NF-κB家族成员RELA、RELB或CREL的microRNA。
  5. 根据权利要求4所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述miR编码靶向NF-κB家族成员RELA的人工microRNA;所述miR编码miR533,其中miR533的序列为5′-CAA AGA TGG GAT GAG AAA GGA-3′。
  6. 根据权利要求1所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述功能性DNA片段DMP-miR被重组腺相关病毒导入细胞后,其功能元件DMP可结合细胞核内的转录因子蛋白NF-κB,从而激活miR的表达。
  7. 根据权利要求3所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,表达的miR经细胞内microRNA成熟系统加工成熟后,可在细胞质中结合NF-κB mRNA,从而抑制NF-κB蛋白的表达。
  8. 根据权利要求1所述的用于炎症性疾病治疗的重组腺相关病毒,其特征在于,所述腺相关病毒包括各种血清型的腺相关病毒AAV1到AAV9中的任意一种。
  9. 一种权利要求1所述的用于炎症性疾病治疗的重组腺相关病毒的构建方法,其特征在于,包括如下步骤:
    (1)从pDMP-miR533载体上扩增DMP-miR533,将其连接到pAAV-MCS载体上构建得到pAAV-DMP-miR533;
    (2)使用pAAV-DMP-miR533和两种辅助质粒pAAV-Helper和pAAV-RC对293T细胞进行转染,细胞培养后,将细胞和培养基收集并冻融,将纯氯仿加 入细胞冻融完的裂解液中,震荡后,向混合物中加入NaCl并摇动至NaCl溶解,离心后收集上清液,加入PEG8000并摇动至其溶解,离心弃去上清液,而后使沉淀溶解,然后将DNase和RNase加入溶解完的沉淀中,反应物在室温下温育,再进行萃取,收集含有纯化病毒的水相,病毒定量后,将其分装至-80℃保存备用,获得的病毒命名为rAAV-DMP-miR533。
  10. 一种权利要求1所述的用于炎症性疾病治疗的重组腺相关病毒在制备新型炎症性疾病治疗试剂中的应用。
  11. 根据权利要求10所述的应用,其特征在于,所述炎症性疾病包括感染引起的炎症、自发炎症、自身免疫病、神经退行性疾病、癌症。
  12. 根据权利要求10所述的应用,其特征在于,所述炎症性疾病优选包括急性结肠炎、银屑病和关节炎。
  13. 根据权利要求12所述的应用,其特征在于,所述重组腺相关病毒rAAV-DMP-miR533通过与凡士林混合在制备治疗皮肤炎性疾病外用试剂或者药物中的应用。
PCT/CN2023/081330 2022-03-14 2023-03-14 一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用 WO2023174265A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210249034.9 2022-03-14
CN202210249034.9A CN115161289B (zh) 2022-03-14 2022-03-14 一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用

Publications (1)

Publication Number Publication Date
WO2023174265A1 true WO2023174265A1 (zh) 2023-09-21

Family

ID=83483983

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/081330 WO2023174265A1 (zh) 2022-03-14 2023-03-14 一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用

Country Status (2)

Country Link
CN (1) CN115161289B (zh)
WO (1) WO2023174265A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117599071B (zh) * 2024-01-19 2024-04-09 中国中医科学院中药研究所 土贝母苷甲在制备用于治疗银屑病的药物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1609224A (zh) * 2004-09-17 2005-04-27 中国人民解放军第二军医大学 携带α-黑素细胞刺激素基因的重组腺相关病毒表达载体与用途
US20140296154A1 (en) * 2011-05-06 2014-10-02 Yale University Method of preventing development of psoriatic lesions
CN108066763A (zh) * 2017-12-21 2018-05-25 陈敏 Pcsk9抑制剂在制备治疗t细胞介导的炎症免疫性疾病药中的应用
CN108220336A (zh) * 2017-12-14 2018-06-29 东南大学 基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达及应用
US20220023301A1 (en) * 2020-07-27 2022-01-27 ImmuneTarget, Inc. METHODS OF TREATING AUTOIMMUNE DISEASES WITH SMALL MOLECULE NF-kB INHIBITORS

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013070A1 (en) * 1996-09-25 1998-04-02 Cell Genesys, Inc. Non-invasive administration of adeno-associated viral vectors
CN1142272C (zh) * 2000-02-02 2004-03-17 中国人民解放军军事医学科学院百环生物医学研究中心 携带人肝细胞生长因子基因的重组腺病毒的用途
JP2005506334A (ja) * 2001-10-04 2005-03-03 スミスクライン・ビーチャム・コーポレイション NF−κB阻害剤
WO2006073727A2 (en) * 2004-12-21 2006-07-13 Monsanto Technology, Llc Recombinant dna constructs and methods for controlling gene expression
GB0909198D0 (en) * 2009-05-28 2009-07-15 Queen Mary & Westfield College Methods and composition for treating NF-kappa B mediated disorders
CN102719556A (zh) * 2011-03-29 2012-10-10 北京五加和分子医学研究所有限公司 基于AAV载体的高通量miRNA活性检测方法及其应用
WO2016115671A1 (zh) * 2015-01-20 2016-07-28 安徽医科大学 Imo4基因表达的抑制剂在制备银屑病外用型治疗药物中的用途
KR101889140B1 (ko) * 2015-10-12 2018-08-17 연세대학교 산학협력단 p65의 전사 조절 도메인과 단백질 운반 도메인을 포함하는 신규 융합 단백질 및 이의 용도
CN105435244A (zh) * 2015-12-02 2016-03-30 重庆高圣生物医药有限责任公司 Lin28a基因过表达腺相关病毒在促进创伤修复中的应用
CN107299099A (zh) * 2017-07-07 2017-10-27 中国人民解放军第四军医大学 可抑制角蛋白17表达的SiRNA序列及应用
CN107365785B (zh) * 2017-09-11 2020-02-18 东南大学 一种调控细胞内NF-κB活性的基因表达载体及其调控方法和应用
CN108410893B (zh) * 2018-02-27 2020-07-31 东南大学 一种由NF-κB启动的肿瘤细胞特异效应基因表达载体及其表达产物和应用
JP2020189828A (ja) * 2019-05-17 2020-11-26 国立大学法人 岡山大学 外用剤組成物
CA3171959A1 (en) * 2020-02-18 2021-08-26 Research Institute At Nationwide Children's Hospital Aav-mediated targeting of mirna in the treatment of x-linked disorders
US20230321278A1 (en) * 2020-07-10 2023-10-12 President And Fellows Of Harvard College Aav vector delivery systems

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1609224A (zh) * 2004-09-17 2005-04-27 中国人民解放军第二军医大学 携带α-黑素细胞刺激素基因的重组腺相关病毒表达载体与用途
US20140296154A1 (en) * 2011-05-06 2014-10-02 Yale University Method of preventing development of psoriatic lesions
CN108220336A (zh) * 2017-12-14 2018-06-29 东南大学 基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达及应用
CN108066763A (zh) * 2017-12-21 2018-05-25 陈敏 Pcsk9抑制剂在制备治疗t细胞介导的炎症免疫性疾病药中的应用
US20220023301A1 (en) * 2020-07-27 2022-01-27 ImmuneTarget, Inc. METHODS OF TREATING AUTOIMMUNE DISEASES WITH SMALL MOLECULE NF-kB INHIBITORS

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Master's Thesis", 30 May 2020, SOUTHEAST UNIVERSITY, CN, article TANG, HAILIN: "Research on New Technology of Gene Therapy for Inflammation Based on Transcription Factor NF-κB", pages: 1 - 81, XP009548946, DOI: 10.27014/d.cnki.gdnau.2020.001237 *

Also Published As

Publication number Publication date
CN115161289A (zh) 2022-10-11
CN115161289B (zh) 2023-12-05

Similar Documents

Publication Publication Date Title
CN107075515B (zh) C/EBPα组合物和使用方法
JP4709545B2 (ja) 修飾された低分子干渉rna分子および使用方法
US8138161B2 (en) Modified small interfering RNA molecules and methods of use
ES2743600T3 (es) Métodos de tratamiento de los trastornos inflamatorios vasculares
US7928083B2 (en) H19 silencing nucleic acid agents for treating rheumatoid arthritis
JP2015518710A (ja) ヘモグロビン遺伝子ファミリー発現を調節するための組成物及び方法
US20210260168A1 (en) Compositions and methods of fas inhibition
WO2023174265A1 (zh) 一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用
CN110420331B (zh) Alkbh5抑制物在治疗病毒感染性疾病中的应用
JP2008525029A (ja) 遺伝子サイレンシングに有用なhbvおよびhcv保存配列
JP2018517704A (ja) 筋萎縮性側索硬化症(ALS)を処置するためのmiR−155阻害剤
CN113249380B (zh) 靶向covid-19新冠病毒的反义寡核苷酸、natac嵌合分子及其应用
Fang et al. MicroRNA-31-3p is involved in substance P (SP)-associated inflammation in human colonic epithelial cells and experimental colitis
EP2596806A1 (en) Method for prevention of colectomy
US9084808B2 (en) Modified small interfering RNA molecules and methods of use
CN116555257A (zh) 一种抑制奥秘克戎Nsp13基因的siRNA及其应用
Mao et al. Long-term and efficient inhibition of hepatitis B virus replication by AAV8-delivered artificial microRNAs
CN111433360B (zh) 靶向ckip-1的双链rna分子及其用途
Luo et al. An AAV-Based NF-κB-Targeting Gene Therapy (rAAV-DMP-miR533) to Inflammatory Diseases
JP2005505283A (ja) Stat−1依存的遺伝子の発現の調節
JP7525578B2 (ja) C/EBPα小分子活性化RNA組成物
WO2020248771A9 (zh) 基于hsa-miR-320a的糖尿病早期预警和/或诊断试剂盒的制备方法、防治糖尿病的药物及其筛选方法和制备方法
WO2024059904A1 (en) Anti-fibrotic microrna composition
CN116694637A (zh) 一种抑制新冠病毒刺突糖蛋白基因的siRNA及其应用
WO2021125932A1 (es) Composición farmacéutica para el tratamiento de enfermedades que cursan con fibrosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23769767

Country of ref document: EP

Kind code of ref document: A1