WO2006015532A1 - Virus recombinant comprenant un gene bpi et solution pharmaceutique contenant le virus et qui utilise celui-ci - Google Patents

Virus recombinant comprenant un gene bpi et solution pharmaceutique contenant le virus et qui utilise celui-ci Download PDF

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WO2006015532A1
WO2006015532A1 PCT/CN2005/000986 CN2005000986W WO2006015532A1 WO 2006015532 A1 WO2006015532 A1 WO 2006015532A1 CN 2005000986 W CN2005000986 W CN 2005000986W WO 2006015532 A1 WO2006015532 A1 WO 2006015532A1
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gene
bpi
fcyl
gnb
recombinant virus
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PCT/CN2005/000986
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English (en)
Chinese (zh)
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Jindong Chen
Yunqing An
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Capital University Of Medical Sciences
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Priority to CN200580000538.1A priority Critical patent/CN100529065C/zh
Publication of WO2006015532A1 publication Critical patent/WO2006015532A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4742—Bactericidal/Permeability-increasing protein [BPI]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates to the field of gene therapy for diseases of mammalian Gram-negative bacteria (GNB) and/or GNB-like pathogens.
  • the present invention relates to a recombinant virus comprising a viral vector and a genetic construct selected from the group consisting of: 1) a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof; and 2) a human BPI gene or A chimeric gene of a functional fragment gene thereof, or a degenerate sequence thereof, wherein the human immunoglobulin heavy chain constant region Fc gene or an allelic gene thereof is further ligated at the 3' end of the human BPI gene or a functional fragment thereof gene.
  • the invention further relates to the use of a genetic construct comprising a human BPI gene or a functional fragment thereof for the preparation of a pharmaceutical composition for the treatment of a diseased gene therapy in a mammalian GNB and/or GNB-like pathogen.
  • the invention further relates to a method of genetically treating a disease with GNB and/or GNB-like pathogens using the recombinant virus or pharmaceutical composition. Background of the invention
  • GNB Gram-negative bacteria
  • Bactericidal/permeablity increasing protein is a cationic antibacterial protein with a molecular weight of about 55KD found in polymorphonuclear neutrophils (PMNs) for the first time in 1978.
  • the antibacterial protein consists of 456 amino acid residues, and its N-terminal fragment binds GNB's lipopolysaccharide (LPO) and lipid A with high affinity, neutralizing endotoxin and directly killing GNB [ Weiss, et al. 253: 2664 (1978)] [Weiss, et al. Blood, 69: 652 (1987)] [Ooi, et al" J. Exp. Med.
  • BPI and its functional fragments have the following major biological functions: broad-spectrum killing or inhibition of GNB, GNB-like pathogens (such as Chlamydia, Rickettsia and spirochetes) and fungi, and higher eukaryotic cells
  • GNB GNB-like pathogens
  • Non-toxic side effects neutralizes endotoxin (LPS), inhibits LPS-mediated proinflammatory cytokines (such as TNF-0U IL- ⁇ ) and other inflammatory mediators
  • LPS lipotoxin
  • cytokines such as TNF-0U IL- ⁇
  • other inflammatory mediators combined with antibiotics has significant synergistic sterilization Effect [Elsbach, et al. Curr Opin Immun. 10: 45 (1998)] [Beamer, ASM News. 68: 543 (2002)].
  • the Ig-like recombinant protein is a recombinant protein produced by recombinant DNA technology and chimeric by a functional protein and an immunoglobulin (Ig) heavy chain constant region (Fc) fragment.
  • the recombinant protein not only has the unique biological function of a certain functional protein, but also has the characteristics of good immunoglobulin stability and long half-life in vivo [Hodges, et al. Antimicrob Agents Chemother. 35: 2580 (1991)].
  • An Ig-like recombinant protein consisting of BPI or an N-terminal functional fragment thereof and an immunoglobulin Fc fragment or an allelic thereof (hereinafter referred to as a BPI-Fc recombinant protein) has both dual functions of BPI and Fc, and they not only have a medium It also has endotoxin and broad-spectrum killing effects on GNB. It also has biological properties such as longer serum half-life, activation of complement and mediating opsonophagocytosis.
  • Fc-activated complement and mediating opsonophagocytosis can rapidly produce non-specific bactericidal effects ( ⁇ 1 hour, independent of bacterial resistance) [An Yunqing, Foundation of Medical Immunology, Beijing Science and Technology Press , first edition, September 1998, ISDN 7-5304-2124-7]; Therefore, BPI-Fc recombinant protein can rapidly kill GNB (including GNB-like pathogens) through the dual action of BPI and Fc, and can overcome the clinical drug resistance problem. Therefore, BPI-Fc recombinant protein is expected to be a new and highly effective anti-infective substance better than BPI.
  • Gene therapy is a method of introducing a functional gene into an organ, tissue, or cell for expression of a disease.
  • Gene therapy is an effective means of maintaining or regulating the expression of a functional protein in an effective therapeutic concentration in vivo.
  • Gene delivery systems for gene therapy are classified into viral vectors and non-viral vectors.
  • virus-free viral vector gutless viral vector
  • AAV adeno-associated virus
  • mini-Ad Adenovirus microcarriers
  • HSV amplicon vectors are all virus-free gene viral vectors.
  • Non-viral vectors are safe, but the efficiency of gene introduction is low. With the development of new high-efficiency non-viral vector technologies and materials (such as targeting vectors, nanocarriers, etc.), the application of such vectors in gene therapy will be more The more [Manfred, et al. DDT, 7(8): 479 (2002)] [Molas, et al. Curr Gene Ther. 3: 468 (2003)].
  • a recombinant virus comprising a viral vector and a genetic construct selected from the group consisting of:
  • a chimeric gene comprising a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof, wherein the human immunoglobulin heavy chain constant region Fc gene is further ligated at the 3' end of the human BPI gene or a functional fragment thereof gene or Its allele gene.
  • Another aspect of the invention relates to the use of a recombinant virus of the invention for the preparation of a pharmaceutical composition for gene therapy of a mammalian GNB and/or GNB-like pathogen (e.g., Chlamydia, Rickettsia and spirochete) infectious diseases, Wherein the mammal comprises a human.
  • a mammalian GNB and/or GNB-like pathogen e.g., Chlamydia, Rickettsia and spirochete
  • a further aspect of the invention relates to a pharmaceutical composition for GNB and/or GNB-like pathogen infection disease gene therapy comprising the recombinant virus of the invention, and a pharmaceutically acceptable carrier.
  • a further aspect of the invention relates to a pharmaceutical composition for GNB and/or GNB-like pathogen infection disease gene therapy comprising a pharmaceutically acceptable non-viral vector, and a genetic construct selected from the group consisting of: 1) a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof; and 2) a chimeric gene comprising a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof, wherein the human BPI gene or a functional fragment thereof thereof
  • the 3, terminus further links the human immunoglobulin heavy chain constant region Fc gene or its allele gene.
  • the invention further relates to a method for gene therapy of a GNB and/or GNB-like pathogen-infected disease comprising: administering a therapeutically effective amount of the recombinant virus of the invention or the pharmaceutical composition to a patient.
  • the method of gene therapy further comprises the step of administering a known antibiotic compound to a patient before, simultaneously or after administration of a therapeutically effective amount of the recombinant virus or the pharmaceutical composition of the present invention.
  • a recombinant virus comprising a viral vector and a genetic construct selected from the group consisting of:
  • a chimeric gene comprising a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof, wherein the human immunoglobulin heavy chain constant region Fc gene is further ligated at the 3' end of the human BPI gene or a functional fragment thereof gene or Its allele gene.
  • Viral vectors have advantages of high gene introduction efficiency as a gene therapy vector, and retrovirus vectors and adenoviral vectors have been widely used in gene therapy clinical research. However, they have limitations, such as the ability to induce immune response and safety in the body; specifically, the integration of the retroviral vector with the host genome may lead to cell carcinogenesis; the adenoviral vector does not integrate with the host genome, but because of its Viral proteins cause immune and inflammatory reactions that can exacerbate GNB and/or GNB-like pathogen infections, and their use is limited.
  • the virus-free gene viral vector not only has the advantages of high gene introduction efficiency, but also has the advantages of small cytotoxicity, immunotoxicity and good safety, and is the preferred carrier for gene therapy of GNB and/or GNB-like pathogen infection diseases.
  • the adeno-associated virus vector, the adenovirus microcarrier, and the HSV amplicon vector are all virus-free gene viral vectors. Specifically, the adeno-associated viral vector has the characteristics of being integrated with the host genome and having a long duration; and the third generation adenoviral vector-adenovirus microcarrier (mini-Ad) is deleted all or large.
  • adenoviral genes retain only the ITR and packaging signal sequences, have no integration with the host genome, mediate high expression levels of target genes, and have short durations; they are clinically useful in GNB and/or GNB-like pathogen infection disease gene therapy Applications will have their own advantages and respective applications, all within the scope of the claimed invention.
  • the adeno-associated viral vector (AAV vector) has the advantages of good safety, low immunogenicity, good stability and wide host range, and is recognized as the safest viral vector in gene therapy and vaccine research. It has received extensive attention.
  • the adeno-associated virus currently found has eight serotypes of AAV1 - AAV8, which are mainly distinguished by the difference in capsid proteins, and thus have different infection efficiencies for different tissues and cells.
  • the common AAV1 - AAV6 serotypes are isolated in humans and primates, with AAV2 being the most well studied.
  • AAV1 and AAV5 type vectors have a good application prospect [Davidson, et al. Proc. Natl. Acad. Sci. 97: 3428 (2000)].
  • the widely used AAV vectors are AAV2 type vectors based on AAV2 type, and have good transduction efficiency for various tissue cells, such as muscle, retina, liver, neuron cells and the like.
  • the AAV2 vector has a low transfection efficiency for some tissue cells, and 85% of the normal population has antibodies against AAV2, which causes a problem of transduction efficiency and a sharp decrease in therapeutic gene expression levels [Halbert, et al J Virol. 74: 1524 (2000)].
  • AAV3 AAV5 type vectors and their hybrid vectors appear.
  • the transduction efficiency of the AAV1 vector in tissues other than nerve tissue, such as muscle tissue and liver is generally higher.
  • AAV5-type vectors are more efficient at transducing airway epithelial cells, muscles, neurons, glial cells, and retinal tissue than AAV2-type vectors. Retinal, brain and islets show better infection efficiency, one of the reasons for this difference is that AAV5 is different from the AAV2 virus cell receptor; and there are very few antibodies against AAV5 in the human population; therefore, the AAV5 type vector is more Suitable as a gene therapy vector for these target tissues [Jason, et al. Molecular Therapy.
  • a hybrid AAV vector having the infection characteristics of each serotype can be obtained by using the ITR of AAV2 and the rep protein of all or part of AAV2, respectively, by replacing the cap protein of other serotype AAV.
  • the “hybrid” vector has the following advantages: First, various carrier cell forests constructed for packaging the AAV2 vector can be continuously used, thereby greatly simplifying the process of "replacement of the AAV vector”; second, the use of the study is clear and clinically The safety of AAV2's ITR can be checked to avoid the risk of using other serotypes of ITR.
  • the AAV1/2 hybrid vector is an IAR and exogenous gene expression cassette in which AAV2 is packaged in the outer shell of the AAV1 virus, and has the characteristics of high infection efficiency of wild type AAV1 and good safety of AAV2 [Hildinger, et al. J Virol. 75: 6199 (2001)].
  • AAV vectors and hybrid vectors thereof can be selected for different target tissues, as a modification and improvement of a particular embodiment of the invention, for use in a mammal of the present invention.
  • various vectors derived from adeno-associated viruses of different serotypes including existing AAV2, AAV1, AAV3, AAV5 type adeno-associated virus vectors and hybrid vectors thereof, are within the scope of the present invention. in.
  • an AAV2-type adeno-associated virus vector is preferably used.
  • virus-free gene viral vectors are preferred in the present invention.
  • the viral vector may be selected from an adenovirus vector, a herpes simplex virus vector and a retroviral vector, or a virus-free viral vector, including an adeno-associated virus vector, an adenovirus microcarrier, and HSV amplification.
  • Subcarrier Preferably, it is a widely used AAV2 type adeno-associated virus vector.
  • the novel viral vector obtained can be used as a BPI gene or a chimeric gene thereof as long as the recombinant BPI gene or the BPI chimeric gene of the present invention can be successfully delivered.
  • the use of vectors for GNB and/or GNB-like pathogen infection disease gene therapy is within the scope of the claimed invention.
  • the functional fragment gene of the human BPI gene is 1) a polynucleotide sequence encoding a BPI ⁇ functional fragment of the human BPI protein of 1 to 199 amino acids as shown in SEQ ID NO: 1, wherein The amino acid sequence of the amino acid sequence of SEQ ID NO: 1 may optionally be substituted with Ala or Ser, or 2) the polynucleoside of the BPIW93 functional fragment encoding the C-terminally truncated 6 amino acid of the amino acid fragment of 1) Acid sequence.
  • the BPI chimeric gene refers to a 3, end of the human BPI gene further chimeric to various immunoglobulin heavy chain constant region Fc genes, also referred to as a BPI-Fc chimeric gene. Its corresponding expression of BPI-Fc recombinant protein has the dual functions of BPI and Fc, which has the functions of neutralizing endotoxin and broad-spectrum killing GNB, and has long half-life in vivo, good stability and activation of complement, mediating conditioning and phagocytosis.
  • the biological properties of anti-infective immunity can enhance bactericidal action by Fc-activated complement and mediating opsonophagocytosis. Theofan et al [U.S.
  • Patent 5,643,570 (1997) demonstrated that BPI-Fc recombinant protein has the effect of neutralizing endotoxin and killing GNB, and can protect mice against GNB. Infection with endotoxin; however, there is no mention in the patent that BPI-Fc enhances bactericidal action by Fc-activated complement and mediates opsonophagocytosis.
  • the Fc gene is known to include various immunoglobulin heavy chain constant region Fc genes or allelic genes thereof, wherein Cyl, Cy2 and Cy3 have dual functions of activating complement and mediating opsonophagocytosis, and Cod and Ca2 mediating opsonophagocytosis Features.
  • the human immunoglobulin heavy chain constant region Fc gene is selected from one of the following: Cyl, Cy2, Cy3, Cal and Ccx2 genes.
  • the present inventors used a BPI-Fcyl chimeric gene in which a human BPIw 99 functional fragment gene and a human Fcyl gene were chimeric as a therapeutic gene, and confirmed that the BPI-Fcyl recombinant protein has both dual functions of BPI and Fc, that is, not only has a neutralization
  • An expression control element refers to a promoter that drives gene expression, an enhancer, some regulatable sequences or elements, and a poly A sequence. They can drive or regulate the expression of a gene of interest, such as the recombinant BPI gene or the BPI-Fc chimeric gene of the present invention, in a host cell, thereby achieving the purpose of treating GNB and/or GNB-like pathogens.
  • Expression control elements that can be used in the present invention include, but are not limited to, viral promoters (such as CMV, SV40 promoter), housekeeping gene promoters (such as dihydrofolate reduction promoters), tissue/cell specific promoters (eg, A promoter of creatine kinase in muscle), an inducible promoter (such as a promoter of a metallothionein gene, a steroid-induced promoter), and the like.
  • viral promoters such as CMV, SV40 promoter
  • housekeeping gene promoters such as dihydrofolate reduction promoters
  • tissue/cell specific promoters eg, A promoter of creatine kinase in muscle
  • an inducible promoter such as a promoter of a metallothionein gene, a steroid-induced promoter
  • the CMV promoter and S V40 poly A on the AAV2 type adeno-associated virus vector pSNAV are used as expression control elements.
  • the recombinant virus is an AAV2 type recombinant adeno-associated virus comprising a BPI-Fcyl chimeric gene, wherein The amino acid sequence encoded by the BPI-Fcyl chimeric gene is shown in SEQ ID Np: 2, which is chimeric by the human functional fragment gene and the human Fcyl gene, and the 5' and 3' ends thereof are ligated to the CMV promoter and SV40 pol 'A expression control element.
  • a BPI-containing recombinant virus for GNB and/or GNB-like pathogen-infected disease gene therapy is prepared by: constructing a BPI-Fcyl chimeric gene in an AAV2-type adeno-associated virus vector pSNAV The pSNAV-ssBPI-Fcyl adeno-associated virus expression vector was obtained, wherein the 5th end of the BPI-Fcyl chimeric gene contains the BPI signal peptide DNA sequence, and the 5th and 3' ends thereof are ligated to the CMV promoter and SV40 poly, respectively.
  • a expression control element then the obtained pSNAV-ssBPI-Fcyl was transfected into BHK-21 cells to obtain a vector cell line, and the vector strain of recombinant type 1 herpes simplex virus HSV1-rc/AUL2 was used to produce AAV2-BPI-Fcyl recombinant virus;
  • the AAV2-BPI-Fcyl recombinant virus for gene therapy was prepared by purification and identification.
  • AAV2-BPI-Fcyl recombinant virus can also be produced by other methods such as classical double plasmid co-transfection plus co-toxication and Ad-free methods.
  • the present inventors confirmed that gene therapy using the AAV2-BPI-Fcyl recombinant virus can effectively protect mice challenged by GNB infection. Since human F1 cannot activate the mouse complement system, human Fcyl may only partially cross-mediated opsonophagocytic effects due to species cross-mediated mouse phagocytic cells; thus, it can be seen that AAV2-BPI- is used in the examples.
  • the material basis for the anti-infective protection of Fcyl recombinant virus for gene therapy is mainly BPI. Similar results can be obtained by using the BPI gene alone as a therapeutic gene.
  • BPI-Fc has the dual functions of BPI and Fc, which not only has the effect of neutralizing endotoxin and killing GNB, but also can rapidly exert bactericidal effect through Fc-activated complement and mediate conditioning phagocytosis among the same species, and greatly enhance the bactericidal effect.
  • the BPI-Fc chimeric gene is expected to be a more effective therapeutic gene than the BPI gene alone in gene therapy applications for GNB and/or GNB-like pathogen infection.
  • recombinant viruses containing the BPI gene or the BPI-Fc chimeric gene are within the scope of the invention.
  • Another aspect of the invention relates to the use of a recombinant virus according to the invention for the preparation of a pharmaceutical composition for the treatment of a mammalian GNB and/or GNB-like pathogen (such as Chlamydia, Rickettsia and spirochete);
  • the mammal is preferably a human.
  • BPI and its functional fragments have broad-spectrum killing or inhibition of GNB and GNB-like pathogens containing lipopolysaccharide structures such as Chlamydia, Rickettsia and spirochete [Elsbach, et al. Curr Opin Immun. 10: 45 (1998) )] [Beamer, ASM News.
  • GNB and/or GNB-like pathogens such as Chlamydia, Rickettsia and spirochetes
  • Infectious diseases can be treated by the gene therapy method of the present invention.
  • BPI has a killing or inhibiting effect on fungi, and has a neutralizing effect on endotoxin; therefore, the use of the recombinant virus for preparing a pharmaceutical composition for treating fungal infection diseases and endotoxin-related diseases in mammals should also be within the scope of the invention.
  • a further aspect of the invention relates to a pharmaceutical composition for GNB and/or GNB-like pathogen infection disease gene therapy comprising the recombinant virus of the invention, and a pharmaceutically acceptable carrier.
  • non-viral vector for gene introduction has the characteristics of good safety, easy preparation, and high-dose use, but the gene introduction efficiency is low and the application in vivo is difficult. It is encouraging that such vectors have developed rapidly, and although non-viral vectors for gene therapy are currently predominantly liposomes, some targeting carriers and nanomaterial carriers have also presented attractive prospects. Therefore, a non-viral vector can also be used in the preparation of the gene therapy drug of the present invention.
  • a further aspect of the invention relates to a pharmaceutical composition for GNB and/or GNB-like pathogen infection disease gene therapy comprising a pharmaceutically acceptable non-viral vector, and a genetic construct selected from the group consisting of: 1) a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof; and 2) a chimeric gene comprising a human BPI gene or a functional fragment thereof, or a degenerate sequence thereof, wherein the human BPI gene or its function
  • the 3' end of the fragment gene is further linked to the human immunoglobulin heavy chain constant region Fc gene or its allele gene.
  • the pharmaceutically acceptable non-viral vector is a liposome.
  • the invention further relates to a method for GNB and/or GNB-like pathogen infection disease gene therapy comprising: administering a therapeutically effective amount of a recombinant virus or a pharmaceutical composition of the invention to a patient.
  • the method of gene therapy further comprises administering a known antibiotic compound before, simultaneously or after administration of a therapeutically effective amount of the recombinant virus or the pharmaceutical composition of the invention. In the patient's steps.
  • the combination of BPI or its functional fragments with antibiotics has a significant synergistic bactericidal effect [Elsbach, et al. Curr Opin Immun. 10: 45 (1998)] [Beamer, ASM News. 68: 543 (2002)], while the present invention
  • the recombinant virus or the pharmaceutical composition functions in vivo by expressing a protein of interest comprising human BPI or a functional fragment thereof.
  • the recombinant virus or the pharmaceutical composition of the present invention is also administered in combination with an antibiotic to have a synergistic bactericidal effect.
  • the combination of AAV2-BPI-Fcyl recombinant virus and cefuroxime sodium exhibits a significant synergistic antibacterial effect in vivo.
  • the recombinant virus, the pharmaceutical composition and the gene therapy method for gene therapy of the present invention have serious immune function to patients with severe infection, especially for tumor radiotherapy and chemotherapy, organ transplantation, AIDS disease and surgery, and are prone to serious diseases. Infected population has broad clinical application prospects.
  • Figure 1 Schematic diagram of the gene structure of AAV2-BPI-Fcyl recombinant virus.
  • FIG. 1 Gel electrophoresis pattern of BPI-Fcyl mRNA transcription in CHO-K1 cells infected with AAV2-BPI-Fcyl recombinant virus by RT-PCR. Among them, CHO-K1 cells infected with AAV2-BPI-Fcyl recombinant virus showed an expected DNA band of about 300 bp (lane 2), and no virus-infected CHO-K1 cells showed no corresponding amplification bands (lane 3). Lane 1 was pBR322 DNA. /Msp I molecular weight marker.
  • Figure 3 Detection of BPI-Fcyl recombinant protein expression in CHO-K1 cell culture supernatant by AAV2-BPI-Fcyl recombinant virus infection by spot assay.
  • CHO-K1 cell culture supernatants infected with AAV2-BPI-Fcyl recombinant virus with MOI values of 5 X 10 4 , 1 X 10 5 , 5 X 10 5 (vg/cell ) were (spot 1), (spot 2) and (spot 3), the positive control was 0.1 human IgG (spot 4).
  • the BPI-Fcyl recombinant protein was expressed in the supernatant of CHO-K1 cells infected with AAV2-BPI-Fcyl recombinant virus, and the expression level was positively correlated with the infection dose of AAV2-BPI-Fcyl recombinant virus; CHO- without viral infection K1 cell culture supernatant was not expressed (species.
  • Figure 4 Western Blot detection of AAV2-BPI-Fcyl recombinant virus-infected BHO-Fcyl recombinant protein expression in CHO-K1 cell culture supernatant.
  • FIG. 5 Time-dependent relationship of BPI-Fcyl recombinant protein to LPS neutralization.
  • the PBS+LPS group was used as the unneutralized LPS control group
  • the BPI-Fcyl recombinant protein was used as the background control group. The results showed that BPI-Fcyl recombinant protein could effectively neutralize LPS, and the neutralization effect was significant after 60 min.
  • FIG. 7 Bacterial growth turbidimetry assay for in vitro bactericidal action of BPI-Fcyl recombinant protein. Among them, PBS was the control group. The results showed that the BPI-Fcyl recombinant protein had a significant killing effect on Escherichia coli 0111:B4, and began to inhibit growth significantly after 4 hours, showing a dose-dependent relationship.
  • FIG. 9 Enhancement of leukocyte killing of GNB by BPI-Fcyl recombinant protein.
  • the results showed that the killing effect of BPI-Fcyl recombinant protein on bacteria was significantly enhanced by the participation of leukocytes (phagocytic cells), which confirmed that BPI-Fcyl recombinant protein can enhance the phagocytic killing effect of phagocytic cells by modulating the BPI-Fcyl recombinant protein.
  • FIG. 10 Gel-electrophoresis map of BPI-Fcyl mRNA transcription in muscle tissue of mice infected with AAV2-BPI-Fcyl recombinant virus by RT-PCR.
  • AAV2-BPI-Fcyl recombinant virus-infected mouse 1 (lane 2), mouse 2 (lane 3) and mouse 3 (lane 4) all showed an expected DNA cleavage band of about 300 bp; normal mouse control group
  • the corresponding amplification bands did not appear in mouse 1 (lane 5), mouse 2 (lane 6) and mouse 3 (lane 7); lane 1 was the pBR322 DNA/Msp I molecular weight marker.
  • FIG. 11 Immunohistochemical staining for expression of BPI-Fcyl recombinant protein in mouse muscle tissue infected with AAV2-BPI-Fcyl recombinant virus.
  • the muscle fibers were brown-yellow (Fig. lib), and no muscle fiber staining was observed in the muscle tissue sections of the normal mouse control group (Fig. 11a).
  • AAV2-EGFP recombination Green fluorescent protein expression was observed in the muscle tissue sections of the virus control group at the injection site (Fig. 11c).
  • FIG. 12 Effect of AAV2-BPI-Fcyl recombinant virus gene therapy on blood and organ bacterial content in mice with lethal bacterial infection.
  • the results show that AAV2-BPI-Fcyl is heavy
  • the bacterial content in the serum and organs of the group of virus-treated group was significantly lower than that of the PBS control group.
  • FIG. 14 Pathological examination of lethal bacterial infected mice with AAV2-BPI-Fcyl recombinant virus gene therapy. The results showed that compared with normal mice (column 14a), the bacteria attacked the dead mice (Fig. 14b), and the organs were congested with obvious vasodilation, which was consistent with the pathological changes caused by endotoxin shock; AAV2-BPI-Fc Y l Surviving mice in the recombinant virus gene therapy group (column in Figure 14c) showed only a small amount of congestion in each organ.
  • FIG. 15 Effect of AAV2-BPI-Fcyl recombinant virus gene therapy on the dynamic changes of endotoxin and proinflammatory cytokines in serum of lethal bacterial infection mice.
  • the results showed that after bacterial infection, the serum levels of endotoxin (A), IL-ip (B) and TNF-a (C) in the AAV2-BPI-FC ⁇ 1 recombinant virus experimental group were significantly lower than those in the PBS control group. Rats, and they all reached their respective peaks 12 hours after infection (experimental mice) and 18 hours (control mice), and their endotoxin and pro-inflammatory cytokines (IL- ⁇ , TNF-a) dynamics There is a correlation between changes.
  • the technical solution claimed in the present invention will be further described below in conjunction with the embodiments and the accompanying drawings.
  • Extract HL-60 cells (ATCC CCL-240) mRNA (refer to QIAGEN company Oligotex Direct mRNA Kit kit instructions); use Pl (5,-CCT GAA TTC GGT ACC ATG AG A GAG AAC ATG GCC A -3,, SEQ ID NO: 3) and P2 (5,-AGC TGG AAT TCA CGG AT-3', SEQ ID NO: 4) is a primer for routine RT-PCR amplification (refer to the Promega Access RT-PCR system kit instructions).
  • the main parameters of the experiment are as follows: After 45 minutes of reverse transcription at 48 ° C, 40 PCR reactions Cycle, where the anneal temperature is 51.5 °C. Approximately 300 bp amplified fragment signalBPI 300 containing the BPI signal peptide DNA sequence (SEQ ID NO: 5) was amplified by RT-PCR.
  • the signBPI 3 was double digested with EcoR I/Hinc II. .
  • the 107 bp EcoR I/Hinc II double-digested fragment signBPI was recovered; the self-constructed plasmid pBV-sBPI-FV/l was digested with Hinc II, and the 1284 bp fragment sBPI 1284 was recovered; the signBPI was routinely ligated with T4 DNA ligase. 1 () 7 and sBPI 1284 , using the reaction solution as a template, using P1 and P2 as primers, performing conventional PCR amplification.
  • the main parameters of the experiment are as follows: PCR reaction 30 cycles, wherein the renaturation temperature is 51.5 °C. Approximately 300 bp amplified fragment ssBPI 300 was obtained by PCR amplification.
  • the ssBPI 3 was digested with Kpn I/EcoR I. . 272 bp of Kpn I/EcoR I double-cleavage fragment ssBPI 272 was recovered; pBV-sBPI-Fcyl was digested with EcoR I/Sal I, and 1107 bp EcoR I/Sal I double-cut fragment BPI-Fcyl 1107 was recovered; ssBPI was 272 and BPI-FCY1 11()7 are ligated to pSNAV [Wu Zhijian et al., Acta Violologica Sinica, 16: 1 (2000)], Kpn I/Sal I double-cleavage site, transformation, screening, zymography and DNA By sequencing, the BPI-Fcyl chimeric gene containing the BPI signal peptide DNA sequence and its adeno-associated virus expression vector pSNAV-ssBPI-Fcyl were constructed accurately.
  • the pSNAV-ssBPI-Fcyl plasmid was deposited on August 9, 2004 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC, No. 13 North Section, Zhongguancun, Haidian District, Beijing, China) with a deposit number of 1205.
  • CGMCC China Microbial Culture Collection Management Committee
  • preparation step bare tube is as follows: pSNAV-ssBPI- Fcyl was transfected into BHK-21 cells, and the vector cell line carrying the BPI-Fcyl chimeric gene was screened by G418, and the vector strain of recombinant type 1 herpes simplex virus HSV1-rc/A UL2 was used to produce AAV2-BPI-Fcyl recombinant virus.
  • a high titer AAV2-BPI-Fcyl recombinant virus (with a titer of 5-10 ⁇ 10 12 vg/ml) was prepared for gene therapy research by purification and identification.
  • the gene structure of AAV2-BPI-Fcyl recombinant virus is shown in Figure 1.
  • Example 2 AAV2-BPI-Fc Y l recombinant virus mediated expression of BPI-Fcyl gene in CHO-K1 cells
  • AAV2-BPI-Fcyl recombinant virus infected CHO-K1 (ATCC CCL-61) cells Well-grown CHO-K1 cells were plated into 6-well plates at 1 ⁇ 10 5 cells/well in DMEM/10% fetal bovine serum. 37 in F12 medium.
  • RT-PCR was used to detect the transcription of BPI-Fcyl mRNA in CHO-K1 cells.
  • the mRNA of CHO-K1 cells was extracted and the expression of BPI-Fcyl was detected by routine RT-PCR.
  • the main parameters of the experiment are as follows: Primers are P1 and P2. After 45 minutes of reverse transcription at 48 ° C, the PCR reaction was carried out for 40 cycles with a renaturation temperature of 51.5 ° C; RT-PCR amplification products were identified by 2% agarose gel electrophoresis. The results are shown in Figure 2: Pre-existing CHO-K1 cells infected with AAV2-BPI-Fcyl recombinant virus Approximately 300 bp DNA amplification band.
  • the BPI-Fcyl recombinant protein was expressed in the CHO-K1 cell culture supernatant infected with AAV2-BPI-Fcyl recombinant virus, and the expression level was positively correlated with the AAV2-BPI-Fcyl recombinant virus infection dose.
  • the cell culture supernatant was taken for routine Western Blot detection.
  • the method is as follows: The cell culture supernatant was subjected to 10% SDS-PAGE electrophoresis (+DTT) and transferred to a nitrocellulose membrane; TBST containing 5% BSA was used. After the blocking solution was blocked, the binding was sufficiently incubated in a 1:500-diluted solution of HRP-labeled goat anti-human IgG1 Fc antibody, and the membrane was washed 3-4 times with TBST and developed by a chemiluminescence kit.
  • AAV2-BPI-Fcyl recombinant virus mediates the BPI-Fcyl gene to express the target protein intact.
  • Example 3 Biological characteristics and functions of BPI-Fcyl recombinant protein
  • the PBS+LPS group was used as the unneutralized LPS control group, and the BPI-Fcyl recombinant protein was used as the background control group.
  • the results are shown in Figure 5: BPI-Fcyl recombinant protein can effectively neutralize LPS, and the neutralization effect is significant after 60 min.
  • Complement control group The sputum sputum broth was mixed with ⁇ fresh guinea pig serum, and the water bath was incubated at 37 ° C for 1 hour; (3) Recombinant protein control group: Take ⁇ bacteria solution and mix with BPI-Fcyl recombinant protein of different dilutions of ⁇ , and water bath at 37 °C for 3 hours; (4) Recombinant protein plus complement experimental group: Mix ⁇ bacteria solution with ⁇ different dilution BPI-Fcyl recombinant protein, and mix with ⁇ fresh guinea pig serum at 37 °C for 3 hours, 37 °C Water bath for 1 hour; the same as the bacterial control group. The results are shown in Figure 8. With the participation of complement, the BPI-Fcyl recombinant protein has a significantly enhanced killing effect on bacteria, confirming that BPI-Fcyl recombinant protein can enhance the bactericidal effect by activating complement.
  • Enhancement of killing of GNB by BPI-Fcyl recombinant protein by leukocytes The bacteria used in this experiment is E. coli 0111:B4, and the concentration of the bacteria is about 10 4 CFU/ml.
  • the experiment was set as follows. (1) Bacterial control group: Take ⁇ bacteria solution and ⁇ physiological saline mixed hook, 37 ° C water bath for 1 hour; evenly spread the bacteria solution on 4 plates, culture at 37 ° C for 16 hours, colony counts take the average.
  • White blood cell control group Take ⁇ bacteria liquid and ⁇ human blood white blood cells (lxlO 5 white blood cells/ml) and mix, 37 ° C water bath for 1 hour;
  • Recombinant protein control group The ⁇ broth was mixed with BPI-Fcyl recombinant protein of different dilutions of ⁇ , and water bath at 37 °C for 3 hours;
  • Recombinant protein plus leukocyte experimental group Take ⁇ bacteria solution and mix with BPI-Fcyl recombinant protein of different dilutions of ⁇ , 37.
  • AAV2-BPI-Fcyl recombinant virus mediates the expression of BPI-Fcyl gene in mice and its anti-infective effect in vivo
  • the AAV2-BPI-Fcyl recombinant virus (5 ⁇ 10 10 ⁇ ./100 ⁇ 1/each) was injected into the quadriceps of the right hind limb of Balb/c mice at 5-6 weeks old, and the expression of BPI-Fcyl gene was detected at 1-2 weeks.
  • the experiment consisted of a normal mouse control group injected with PBS and a control group of AAV2-EGFP recombinant virus.
  • RT-PCR detection of BPI-Fcyl mRNA transcription in mouse muscle tissue extraction of muscle tissue mRNA at the injection site of injected virus mice;
  • the method is briefly described as follows.
  • the muscle tissue of the injection site of the injected virus mouse is taken, and the paraffin-embedded section (4-5 ⁇ ) is laid flat on the slide, dewaxed with a cleaning agent, and then washed with ethanol and distilled water in sequence, and then used. After incubated with 3% hydrogen peroxide for 8 min, the endogenous peroxidase was removed. After washing with PBS, the tissue sections were blocked with blocking solution, and then labeled with 1:100 HRP labeled mouse anti-human IgG Fc antibody for 1 hour at 37 ° C, washed with PBS. Piece 3 The color was routinely developed with DAB chromogenic reagent, and tissue staining was observed under the microscope after sealing.
  • the muscle tissue of AAV2-EGFP control mice was frozen (8 ⁇ ), and the expression of green fluorescent protein in mouse muscle tissue was observed under fluorescent microscope. The results are shown in Figure 11.
  • the muscle fibers of the AAV2-BPI-Fcyl recombinant virus experimental group showed brown-yellow expression in the muscle tissue sections, and no muscle fiber staining was observed in the muscle tissue sections of the normal mouse control group.
  • AAV2-EGFP recombinant virus Green fluorescent protein expression was observed in the muscle tissue sections of the control group at the injection site.
  • mice After 2 weeks of virus injection, the mice were subjected to ablation of the eyeballs, and 1/3 of them were prepared for anticoagulation and 2/3 to prepare serum. After the anticoagulation and serum were mixed in each group of mice (3/group), the target gene product in serum and its main biological effects were detected.
  • the modified gene-linked immunosorbent assay is used to detect the target gene product BPI-Fc ⁇ ⁇ chimeric protein in mouse serum.
  • the method is as follows: nitrocellulose with a diameter of 5 ⁇ m The filter discs were placed in ⁇ mouse serum and 10% albumin solution (blank test control) for 15 minutes; after natural drying (20 minutes), the filter discs were placed in uncoated ELISA.
  • AAV2-BPI-Fcyl recombinant virus gene therapy has a significant protective effect on the minimum lethal E. coli 0111:B4 infected mice.
  • Table 2 Protective effect of AAV2-BPI-Fcyl recombinant virus gene therapy on mice with minimal lethal E. coli 0111:B4 infection
  • Liver, spleen, kidney and small intestine sections of surviving mice were taken from normal Balb/c mice, bacterial challenged death mice and AAV2-BPI-Fcyl recombinant virus gene treatment group.
  • HE staining was performed to observe the pathology of each organ. change. The results are shown in Figure 14.
  • the bacteria attacked the dead mice, and the vasodilation was obvious, which was consistent with the pathological changes caused by endotoxin shock.
  • the AAV2-BPI-Fcyl recombinant virus gene treatment group survived. Only a small amount of congestion was observed in the organs of the mouse.
  • mice were removed from the eyeballs at different times after the lethal bacterial infection challenge.
  • the serum of each group (3/group) was mixed, and the endotoxin and reference ELISA in the mixed serum were detected according to the instructions of the sputum kit.
  • the kit purchased from R&D Systems Inc.) instructions for the detection of pro-inflammatory cytokine levels (repeated three times in the same experiment).
  • AAV2-BPI-Fc y l recombinant virus gene therapy can significantly reduce the levels of endotoxin and pro-inflammatory cytokines in serum and resist endotoxin toxic shock caused by lethal bacterial infection.
  • mice aged 5-6 weeks were randomly divided into four groups of 10 animals each.
  • One hour after infection of each group of mice with minimal lethal dose E. coli 0111:B4 intramuscular injections were given to mice in groups of 125 pg/mL, 500 g/mL, and 1000 pg/mL cefuroxime sodium (( ⁇ only).
  • mice were divided into the following four groups: PBS group, cefuroxime sodium group (25 ⁇ 8 /100 ⁇ 7), AAV2-BPI-Fcyl recombinant virus group, AAV2-BPI-Fcyl recombinant virus and cefuroxime sodium (25 ⁇ 100 ⁇ only).
  • PBS group cefuroxime sodium group
  • AAV2-BPI-Fcyl recombinant virus group AAV2-BPI-Fcyl recombinant virus
  • cefuroxime sodium 25 ⁇ 100 ⁇ only
  • the combined application group 10 mice per group. After infection with the minimum lethal dose of E. coli 0111:B4, the survival rate of each group of mice was observed within 72 hours (the same experiment was repeated twice).

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Abstract

Cette invention présente un virus recombinant, qui inclut un vecteur viral et un gène hybride sélectionné parmi la liste suivante : 1) Un gène BPI humain ou fragment de gène fonctionnel de celui-ci, ou une séquence dégénérée de celui-ci, et 2) un gène chimère comprenant un gène humain BPI ou un fragment de gène fonctionnel de celui-ci, ou une séquence dégénérée de celui-ci où le gène chimère est composé d’un gène de la région constante Fc ou de son gène allélique de la chaîne lourde de l’hémoglobine humaine de la 3e extrémité du gène BPI humain ou un fragment de gène fonctionnel de celui-ci. Cette invention met aussi en lumière l’emploi d’un gène hybride composé d’un gène BPI humain ou d’un fragment de gène fonctionnel de celui-ci dans la préparation d'une solution pharmaceutique de la thérapie génique de la bactériémie gram-négatif BGN et infection pathogène BGN similaire des mammifères. La présente invention fournit une méthode de thérapie génique supplémentaire du BGN et/ou de l’infection pathogène BGN similaire par l’emploi du virus recombinant ou d’une composition pharmaceutique.
PCT/CN2005/000986 2004-08-13 2005-07-05 Virus recombinant comprenant un gene bpi et solution pharmaceutique contenant le virus et qui utilise celui-ci WO2006015532A1 (fr)

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CN109762841B (zh) * 2018-11-19 2021-03-02 厦门联合安金生物工程有限公司 BPI-Fc融合蛋白及其用途

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WO2019215305A1 (fr) * 2018-05-09 2019-11-14 Universität Regensburg Protéine bactéricide/augmentant la perméabilité destinée à être utilisée dans une méthode d'immunisation, de préférence comme adjuvant dans un procédé de vaccination

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