WO2006015532A1

WO2006015532A1 - A recombinant virus comprising bpi gene and a pharmaceutical composition containing the virus and uses thereof

Info

Publication number: WO2006015532A1
Application number: PCT/CN2005/000986
Authority: WO
Inventors: Jindong Chen; Yunqing An
Original assignee: Capital University Of Medical Sciences
Priority date: 2004-08-13
Filing date: 2005-07-05
Publication date: 2006-02-16
Also published as: CN1839200A; CN100529065C; CN1733911A

Abstract

The present invention discloses a recombinant virus, which comprises a viral vector, and a gene construct selected from the following: 1) a human BPI gene or a functional fragment gene thereof, or a degenerate Sequence thereof, and 2) a chimeric gene comprising a human BPI gene or a functional fragment gene thereof, or a degenerate Sequence thereof, wherein the chimeric gene further comprises a Fc constant region gene or its allelic gene of a heavy chain of a human immunoglobulin in the 3' end of the human BPI gene or the functional fragment gene thereof. This invention also discloses the use of the gene construct comprising the human BPI gene or functional fragment gene thereof in the preparation of the pharmaceutical composition of gene therapy of the GNB and/or GNB like pathogen infection in Mammal. The present invention further provides a gene therapy method of the GNB and/or GNB like pathogen infection using the recombinant virus or the pharmaceutical composition.

Description

Recombinant virus containing BP I gene and pharmaceutical composition containing the same and use thereof

The present invention relates to the field of gene therapy for diseases of mammalian Gram-negative bacteria (GNB) and/or GNB-like pathogens. In particular, 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

Bacterial infection is a common clinical disease. There are many cases of severe infection and sepsis. The mortality rate is 30-50%. More than half of the infections with Gram-negative bacteria (GNB) develop into sepsis and cause internal disease. Endotoxin is associated with toxic shock death [Dahlberg, et al. J Surg Res. 63: 44 (1996)] [Lazaron, et al. Urol Clin North Am. 26: 687 (1999)] [Balk, et al. Crit Care Clin. 16: 179 (2000)]. Antibiotics have antibacterial action, but can not neutralize endotoxin. Sometimes GNB, which is highly sensitive to antibiotics, will promote endotoxin shock after a large number of deaths. It is currently treated with anti-lipopolysaccharide and anti-inflammatory cytokine monoclonal antibody. The result is not ideal [Mccloskey, et al. Ann Intern Med. 121: 1 (1994)] [Vincent, et al. Clin Infect Dis. 34: 1084 (2002)] In addition, bacterial resistance problems also pose great difficulties for clinical anti-infective treatment, and antibiotic treatment sometimes does not work. Therefore, the development of an anti-infective drug that can broadly kill GNB and neutralize endotoxin while overcoming drug resistance has become a hot research topic at home and abroad.

Bactericidal/permeablity increasing protein (BPI) 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. 174: 649 (1991)] [Weiss, et J. Clin Invest. 90: 1122 (1992). In 1989, Gray et al. successfully cloned BPI cDNA from human neutrophils, and subsequently recombinant BPI and its N-terminal functional fragment were successfully expressed in succession [Gray, et al. J Biol Chem. 264: 9505 (1989)] [Gazzano-Santoro, et al. Infect. Immunol. 60: 4754 (1992)] [Horwitz, et al. Protein Expr Purif. 8: 28 (1996)] ₀

In-depth studies have confirmed that recombinant 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 Non-toxic side effects; neutralizes endotoxin (LPS), inhibits LPS-mediated proinflammatory cytokines (such as TNF-0U IL-Ιβ) and 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)]. Further, according to the mechanism of action of BPI, which overcomes GNB clinical drug resistance [Mannion, et al J.Clin Investig 85 :... 853 (1990)] 0 Thus, recombinant BPI and functional fragments expected for clinical anti-infective therapy Provide a new way. In recent years, recombinant human BPI N-terminal functional fragment (rBPKJr) has achieved encouraging results in the study of animal models such as GNB pneumonia, GNB sepsis, chronic exudative otitis and post-limb ischemia-reperfusion injury. [Cazzola, et al. Curr Opin Pulm Med, 10: 204 (2004)] [Lin, et al. Antimicrob Agents Chemother. 40: 65 (1996)] [Nell, et al. Infect. Immun. 68: 2992 (2000 )] [Harkin, et al. J. Vase. Surg. 33: 840 (2001)], treatment of infectious diseases such as meningococcal bacteremia, traumatic blood loss and its complications, and complications after partial hepatectomy A certain effect has also been achieved [Levin, et al. Lancet. 356: 961 (2000)] [Demetriades, et al. J. Trauma. 46: 667 (1999)] [Wiezer, et al. Shock. 10: 161 (1998) )] [Beamer, ASM News. 68: 543 (2002)]. However, in clinical applications, recombinant BPI and its functional fragments have been greatly affected by the following major problems: (1) The short half-life in vivo (<60 minutes) is difficult to maintain effective therapeutic concentration; (2) It takes a long time to exert the bactericidal effect (>4 hours); (3) The treatment dose is large and the cost is high. In order to solve the above problems, we started the research of BPI-Fcyl recombinant antibacterial protein from 1996.

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. In addition, according to immunological principles, 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.

Viral vector gene introduction efficiency is high, and it is widely used in clinical research of gene therapy, but there are safety problems [Anderson, Nature, 392: 25 (1998)]. The virus-free viral vector (gutless viral vector) has good safety and is an important development direction of viral vectors [Sakhuja, et al. Hum Gene Ther. 14: 243 (2003)] ₀ adeno-associated virus (AAV) vector, Adenovirus microcarriers (mini-Ad) and 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)].

Since the first US clinical trial of human gene therapy in 1990, Dr. Anderson, a US scientist, according to the Recombinant DNA Advisory Committee, as of March 2004, there were 619 clinical trials for gene therapy. Involved in the prevention and treatment of malignant tumors, hereditary diseases, cardiovascular diseases, autoimmune diseases, neuroendocrine diseases and AIDS, but no reports of gene therapy for bacterial infections [http: 〃 www4.od, nih.gov/oba/rac /protocol.pdf].

The present inventors have for the first time clearly stated that bacterial infection diseases can be treated with gene therapy. Concept, and first applied AAV2-BPI-Fcyl recombinant adeno-associated virus containing BPI functional fragment gene in the treatment of mouse GNB infection. Summary of invention

In one aspect of the invention, 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;

2) 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 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. In a specific embodiment of the invention, 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. Detailed description of the invention

The scope and content of the present invention will be apparent to those skilled in the art from the following description of the present invention; therefore, the appended claims and any modifications in the future are within the scope of the present invention. .

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. Some 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. The order of infection efficiency of various serotype AAV-type vectors in mouse liver and muscle tissues is AAV1>AAV5>AAV3>AAV2>AAV4, and the order in rat retina is AAV5>AAV4>AAV1>AAV2>AAV3 Among them, 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. However, 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)]. In order to obtain higher efficiency of gene introduction, many researchers are working on a new AAV vector based on different serotypes of AAV. One of the main directions is to use AAV virus shells of other serotypes; AAV2 and AAV1 have good applications. , AAV3, AAV5 type vectors and their hybrid vectors appear. Compared with the AAV2 vector, the transduction efficiency of the AAV1 vector in tissues other than nerve tissue, such as muscle tissue and liver, is generally higher. [Bernd, et al. J Virol. 77: 2768 (2003)] ₀ 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. 6: 510 (2002)] „ Currently, various serotype AAV vectors are all “hybrid” vectors (except AAV5), namely: 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. For example, 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)].

Accordingly, one of ordinary skill in the art will recognize that various different serotype 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. The field of gene therapy for Gram-negative bacteria (GNB) and/or GNB-like pathogens. Specifically, 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. In the method of the present invention, an AAV2-type adeno-associated virus vector is preferably used.

Although the safety of viral vectors has yet to be further verified, it has been widely used in clinical 2/3 or more programs. Therefore, adeno-associated virus vectors and adenovirus vectors Both body, herpes simplex virus vectors and retroviral vectors are within the scope of the invention. In view of the fact that viral gene products can aggravate the immune and inflammatory responses of GNB and/or GNB-like pathogen-infected diseases; therefore, virus-free gene viral vectors are preferred in the present invention.

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.

In addition, with the progress of various novel viral vectors, 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.

In the present 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.

In the present invention, 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. In the present invention, 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 The action of toxins and kills GNB, and the ability to rapidly activate bactericidal effects through Fc-activated complement and mediating opsonophagocytosis, greatly enhances bactericidal action (see the Examples below).

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. Those skilled in the art will recognize that any expression control element that can be used to modulate the recombinant BPI gene or BPI-Fc chimeric gene of the present invention can be used in the present invention. In one embodiment of the invention, the CMV promoter and S V40 poly A on the AAV2 type adeno-associated virus vector pSNAV are used as expression control elements.

In a preferred embodiment of the invention, 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.

In one embodiment of the present invention, 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.

According to BPI-Fc, it 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. It can be expected that 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. Comprehensive Above, in GNB and/or GNB-like pathogen-infected disease gene therapy applications, 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. 68: 543 (2002)]; Furthermore, BPI-Fc can also produce non-specific killing effects on GNB-like pathogens containing lipopolysaccharide structures through Fc-activated complement and mediating opsonophagocytosis [An Yun Qing, Foundation of Medical Immunology, Beijing Science and Technology Press, September 1998, 1 edition, ISDN 7-5304-2124-7]; Therefore, 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. In addition, 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.

In addition, the 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. Accordingly, 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. In a specific embodiment of the invention, 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.

In a specific embodiment of the invention, 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. In a specific embodiment of the invention, the combination of AAV2-BPI-Fcyl recombinant virus and cefuroxime sodium exhibits a significant synergistic antibacterial effect in vivo.

In summary, 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. DRAWINGS

Figure 1. Schematic diagram of the gene structure of AAV2-BPI-Fcyl recombinant virus.

Figure 2. 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. Among them, 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). As can be seen, 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.

AAV2-BPI-Fcyl recombinantly infected CHO-K1 cell culture supernatant expressed BPI-Fcyl recombinant protein, showing a band at about 48 KD (lane 2); no virus-infected CHO-K1 cell culture supernatant was not expressed ( Lane 1).

Figure 5. Time-dependent relationship of BPI-Fcyl recombinant protein to LPS neutralization. Among them, 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 showed that BPI-Fcyl recombinant protein could effectively neutralize LPS, and the neutralization effect was significant after 60 min.

Figure 6. Dose relationship of BPI-Fcyl recombinant protein to LPS neutralization. Among them, the PBS+LPS group was used as the unneutralized LPS control group, and the BPI-Fcyl recombinant protein was used as the present. Bottom control group. The results showed that BPI-Fcyl recombinant protein had a significant dose-dependent relationship with LPS.

Figure 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.

Figure 8. Enhancement of complementation of BPI-Fcyl recombinant protein to kill GNB. The results showed that BPI-Fcyl recombinant protein significantly enhanced the killing effect of bacteria with the participation of complement, confirming that BPI-Fcyl recombinant protein can enhance the bactericidal effect by activating complement.

Figure 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.

Figure 10. Gel-electrophoresis map of BPI-Fcyl mRNA transcription in muscle tissue of mice infected with AAV2-BPI-Fcyl recombinant virus by RT-PCR. Among them, 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.

Figure 11. Immunohistochemical staining for expression of BPI-Fcyl recombinant protein in mouse muscle tissue infected with AAV2-BPI-Fcyl recombinant virus. In the muscle tissue sections of the AAV2-BPI-Fcyl recombinant virus experimental group, 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).

Figure 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.

Figure 13. Effect of AAV2-BPI-Fcyl recombinant virus gene therapy on serum endotoxin levels in mice with lethal bacterial infection. The results showed that, within AAV2-BPI-Fcyl gene therapy recombinant viral serum endotoxin levels detected at each time point were significantly lower than the PBS group (using paired t- test, t = 9.29, p = 0.001 ) 0

Figure 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.

Figure 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 γ 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-Ιβ, 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.

Example 1 Preparation of AAV2-BPI-Fcyl Recombinant Virus

1. Construction of pSNAV-ssBPI-Fcyl adeno-associated virus expression vector

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 amino acid sequence of the BPI-Fcyl recombinant protein encoded by the BPI-Fcyl chimeric gene is SEQ. ID NO: 2 is shown.

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.

2. Production and preparation of AAV2-BPI-Fcyl recombinant virus

Prepared by Benyuan Zhengyang Gene Technology Co., Ltd., preparation method Literature [Wu Zhijian et al., Chinese Science (C), 31: 423 (2001)] [Wu Xiaobing, et al. Chinese Science Bulletin. 46: 485 (2001)], 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. C, 5% C0 ₂ in culture after 12 hours; with serum-free DMEM is gently rinsed twice / F12 medium, respectively MOI of ^{^{^{5xl0 4, lxl0 5, 5xl0 5}}} (vg / cell) of AAV2-BPI- Fcyl recombinant virus was infected with CHO-K1 cells in serum-free DMEM/F12 culture medium; after 1 hour of culture, DMEM/F12 medium containing 10% fetal bovine serum was further cultured at 37 ° C, 5 % C0 ₂ for 48- After 72 hours, the expression of BPI-Fcyl gene in CHO-K1 cells was examined; the test was carried out with no virus-infected CHO-K1 cell control group.

1. 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.

2. Dot-Blot assay for expression of BPI-Fcyl recombinant protein in CHO-K1 cell culture supernatant

3 μl of the cell culture supernatant was spotted on a nitrocellulose membrane (experimental 0.1 μ _δ standard human IgG spot was used as a positive control); after blocking with 5% BSA in TBST blocking solution, HRP containing 1:500 dilution The goat anti-human IgG1 Fc antibody (purchased from Shenzhen Jingmei Bioengineering Co., Ltd.) was fully incubated and combined, washed 3-4 times with TBST, and developed with a chemiluminescence kit (refer to Pierce's kit instructions) . The result is shown in Figure 3. 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.

3. Western Blot detection of BPI-Fcyl recombinant protein expression in CHO-K1 cell culture supernatant

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. The results are shown in Figure 4: The BPI-Fcyl recombinant protein was expressed in the culture supernatant of AAV2-BPI-Fcyl recombinantly infected CHO-K1 cells, and a band was displayed at about 48 KD.

It can be seen that the 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

1. Neutralization of LPS by BPI-Fcyl recombinant protein: (1), take 200μ1 BPI-Fcyl recombinant protein (25pg/ml) and mix with 200μ1 LPS solution (0.5 Eu/ml), incubate in 37°C water bath, sample ΙΟΟμΙ every 30min; put in test tube without pyrogen Inside, then refer to the 鲎 kit (purchased from Shanghai Yihua Clinical Medical Technology Co., Ltd.) operating instructions, add 50μ1 鲎 reagent to mix, 37 ° C water bath for 25 minutes; add 50μ1 chromogenic matrix 鲎 tripeptide to mix, 37 ° C Water bath for 3 minutes; add azo reagent to mix and develop color, and measure OD _545nm value. 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.

(2), respectively, 50μ1 different dilutions of BPI-Fcyl recombinant protein and 50μ1 LPS (0.5 Eu / ml) were mixed, incubated in 37 ° C water bath for 60 min, refer to the 鲎 kit operation instructions, add 50μ1 鲎 reagent mixed hook , 37. C water bath for 25 minutes; add 50μ1 coloring matrix 鲎 tripeptide mixed hook, 37 ° C water bath for 3 minutes; add azo reagent to mix and develop color, measure OD _545nm value. 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 6: BPI-Fcyl recombinant protein neutralized LPS in a dose-dependent manner.

2. In vitro bactericidal effect of BPI-Fcyl recombinant protein:

Determined by bacterial growth turbidimetry, as follows: Take ΙΟΟμΙ E. coli 0111:B4 (CMCC(B) No. 44101-9) bacterial solution (lxl0 ⁶ CFU/ml), respectively, with different concentrations of ΙΟΟμΙ (6pg/ml) After mixing with BPI-Fcyl recombinant protein of 0.6 pg/ml), it was added to 4 ml of LB medium and shake cultured at 37 ° C, and 600 μl was measured every 2 hours to measure OD _{60 () nm} value; The results are shown in Figure 7: BPI-Fcyl recombinant protein showed significant killing effect on E. coli 0111:B4 (significant inhibition of growth after 4 hours) and showed a dose-dependent relationship.

3. Complement enhances the killing effect of BPI-Fcyl recombinant protein on GNB: In this experiment, the bacteria E. coli 0111: B4, bacterial concentration of about 10 ⁴ CFU / ml _o 4 experimental groups is provided as follows. (1) Bacterial control group: Mix ΙΟΟμΙ broth and ΙΟΟμΙ physiological saline, and bath at 37 °C for 1 hour. Apply the aliquot evenly to 4 plates and incubate at 37 °C for 16 hours. The colony counts are average. (2) Complement control group: The sputum sputum broth was mixed with ΙΟΟμΙ fresh guinea pig serum, and the water bath was incubated at 37 ° C for 1 hour; (3) Recombinant protein control group: Take ΙΟΟμΙ bacteria solution and mix with BPI-Fcyl recombinant protein of different dilutions of ΙΟΟμΙ, and water bath at 37 °C for 3 hours; (4) Recombinant protein plus complement experimental group: Mix ΙΟΟμΙ bacteria solution with ΙΟΟμΙ different dilution BPI-Fcyl recombinant protein, and mix with 豚μΙ 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.

4. 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 ΙΟΟμΙ bacteria solution and ΙΟΟμΙ 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. (2) White blood cell control group: Take ΙΟΟμΙ bacteria liquid and ΙΟΟμΙ human blood white blood cells (lxlO ⁵ white blood cells/ml) and mix, 37 ° C water bath for 1 hour; (3) Recombinant protein control group: The ΙΟΟμΙ broth was mixed with BPI-Fcyl recombinant protein of different dilutions of ΙΟΟμΙ, and water bath at 37 °C for 3 hours; (4) Recombinant protein plus leukocyte experimental group: Take ΙΟΟμΙ bacteria solution and mix with BPI-Fcyl recombinant protein of different dilutions of ΙΟΟμΙ, 37. After 3 hours of C water bath, each tube was added with ΙμΙ white blood cells (lxlO ⁵ white cells/ml), and mixed at 37 ° C for 1 hour; the same as the bacterial control group. The results are shown in Figure 9: BPI-Fcyl recombinant protein kills bacteria with the participation of white blood cells (phagocytic cells) With significantly enhanced, BPI-Fcyl recombinant protein was confirmed by adjusting ^a physical effect, enhance phagocytosis phagocytic killing effect on bacteria. Embodiment 4

AAV2-BPI-Fcyl recombinant virus mediates the expression of BPI-Fcyl gene in mice and its anti-infective effect in vivo

(a) Virus-mediated expression of the BPI-Fcyl gene in mice and its biological effects:

The AAV2-BPI-Fcyl recombinant virus (5χ10 ¹⁰ ν ./100μ1/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.

1. 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 in vivo transcription of BPI-Fcyl mRNA was detected by RT-PCR; the primers were P1 and P2, and after 45 minutes of reverse transcription at 48 °C, PCR reaction was carried out for 40 cycles, wherein the renaturation temperature was 51.5 Ό; condensed with 2% agarose The RT-PCR amplification product was identified by gel electrophoresis. The results are shown in Fig. 10: AAV2-BPI-Fcyl recombinant virus-infected mice showed an expected DNA amplification band of about 300b.

2. Immunohistochemistry was used to detect the expression of BPI-Fcyl recombinant protein in mouse muscle tissue:

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 (4-5μιη) 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 (8μιη), 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.

3. Detection of target gene products in mouse serum and its biological effects

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.

(1) The modified gene-linked immunosorbent assay is used to detect the target gene product BPI-Fc γ ΐ chimeric protein in mouse serum. The method is as follows: nitrocellulose with a diameter of 5 亳m The filter discs were placed in ΙΟΟμ 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. In the wells of the plate; then refer to the BPI ELISA kit (purchased from Hycult biotechnology.bV), add 100 L biotinylated anti-BPI antibody, and apply for 1 hour at room temperature; after washing 4 times, add ΙΟΟμί avidinized ^=艮Peroxidase, 1 hour at room temperature; After washing 4 times, add 100 L of color developer, react at room temperature for 20 minutes in the dark, add 100 L of stop solution, and measure OD ₄₅ immediately after mixing. Value (the same experiment was repeated three times). The results showed that the serum OD ₄₅₀ value of the control group [0.283+0.026 (n=3)] and the OD ₄₅₀ value of the albumin blank test group.

[0.290 ± 0.020 ( n=3 ) ] no difference ( t = 0.23, P = 0.832 ); AAV2-BPI-Fc

The recombinant virus serum OD V 1 ₄₅₀ experimental mice values [Soil 0.849 0.164 (n = 3)] OD serum and ₄₅ control mice. There was a significant difference in values (t = 5.92, P = 0.024). Above knot It was confirmed that the secreted target gene product was present in the serum of the AAV2-BPI-FC γ 1 recombinant virus experimental group mice.

(2), take 50pL of mouse serum and 50pL of non-pyrogenic aqueous solution with endotoxin content of 1.0EU/mL, and after 37min of water bath for 37min, refer to the operation instructions of sputum kit to detect the target gene product in mouse serum. Neutralization of toxins (repeated three times in the same experiment). The results showed that the serum OD ₅₄₅ of the AAV2-BPI-FC Y 1 recombinant virus experimental group was 0.173 ± 0·021 (n=3); the serum OD ₅₄₅ of the control group was 0.287 ± 0.021 (n=3); The OD _S4S value of the control group without heat source was 0.087 ± 0.005 (n=3). There was a significant difference in serum OD ₅₄₅ values between the AAV2-BPI-Fc γ 1 recombinant virus experimental group and the mouse control group (t=6.61, Ρ=0·003). The above results indicate that the target gene product present in the serum of the AAV2-BPI-FC γ 1 recombinant virus experimental group has a neutralizing effect on endotoxin.

(3), take the mouse serum ΙΟΟμΙ and ΙΟΟμΙ E. coli OH1.B4 bacterial solution (1 X 10 ³ CFU / mL), 4. After 15 minutes of C action, the bacteria were plated by a flat pour method, and two plates were equally divided into each sample to be tested, and cultured at 37 ° C for 16 hours, and the colony counts were averaged (the same test was repeated three times). The results showed that the serum colony counts of the AAV2-BPI-FC γ 1 recombinant virus experimental group and the control group were 22.5 soil 5.20 (n=3) and 33.00 soil 4.93 (n=3), respectively (t=3.88, P =0.018). The above results indicate that the target gene product present in the serum of the AAV2-BPI-FC γ 1 recombinant virus experimental group has a killing effect on E. coli.

(2), AAV2-BPI-Fcyl recombinant virus gene therapy for the protective effect of lethal dose of GNB-infected mice

E. coli (E, coli) 0111: B4 Intestinal infection of Balb/c mice with minimum lethal dose (MLD) determination: E. coli 0111:B4 was diluted with PBS containing 5% high active dry yeast. Different concentrations of bacterial solution, randomized group of 6-7 weeks old Balb/c mice were injected intraperitoneally (0.5ml/each), The death of the mice was examined; the minimum bacterial amount causing 80-100% of the mice to die within 72 hours was taken as MLD. The results are shown in Table 1. 2.5 x 10 ⁴ cfu was determined as intraperitoneal injection (0.5 ml/each) of MLD of Escherichia coli 0111:B4 in Balb/c mice. Table 1. Determination of minimum lethal dose (MLD) of E. coli 0111: B4 intraperitoneal infection of Balb/c mice

Randomized grouping of 5-6 week old Balb/c mice each in the right hind limb quadriceps injection

AAV2-BPI-Fcyl recombinant virus, each mouse was intraperitoneally injected with 1 MLD of E. coli 0111:B4 liquid (0.5 ml/each) for infection attack, and the following observation and detection were carried out; Group and AAV2-EGFP recombinant virus control group.

1. Protection against lethal bacterial infection in mice:

The mortality of the above mice was observed, and the results are shown in Table 2: 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

2. Counting blood and organ bacteria in mice with lethal bacterial infection

At different times after the bacterial infection attack, blood was collected and organs were taken for bacterial count as follows: (1), mouse eyeballs were collected, allowed to stand for 40 min, centrifuged at 1000 rpm/min for 10 min, and serum was diluted 1:10. Serum samples; (2), take the liver and spleen of the mice separately, rinse with sterile saline, add 3ml of sterile physiological saline, grind with sterile cell sieve, and make specimens of organ slurry. Take 50μ1 of each of the above serum samples and organ grouting specimens for bacterial plating (each specimen is repeatedly coated), culture at 37 ° C for 16 hours, and the colony counts are average. The results are shown in Figure 12: The bacterial content in the serum and organs of the AAV2-BPI-Fcyl recombinant virus gene therapy group was significantly lower than that of the PBS control group.

3. Detection of serum endotoxin levels in mice with lethal bacterial infection: Blood was collected from the eyeball at different times after the bacterial infection attack, and the serum obtained was free of heat source water.

After 1:10 dilution, follow the 鲎 kit instructions to detect changes in serum endotoxin levels. The results are shown in Figure 13: The serum endotoxin levels in the AAV2-BPI-Fcyl recombinant virus gene treatment group were significantly lower at each time point than in the PBS control group (using paired t-test, t = 9.29, p = 0.001).

4. Pathological examination of mice with lethal bacterial 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. Compared with normal mice, 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.

5. Dynamic changes of endotoxin and pro-inflammatory cytokines in serum of mice with lethal bacterial infection and their correlation

The 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).

The dynamic changes of endotoxin and pro-inflammatory cytokines (IL-1|5, TNF-α) in mouse serum are shown in Figure 15: After bacterial infection, AAV2-BPI-FC Y 1 recombinant virus experimental group mice serum The endotoxin, IL-Ιβ and TNF-ix were significantly lower than the control mice, and they reached their respective peaks at 12 hours after infection (experimental mice) and 18 hours (control mice). There is a correlation between endotoxin and pro-inflammatory cytokines (IL-Ιβ, TNF-o dynamic changes. Control group mouse serum The content of pro-inflammatory cytokines was significantly increased (peaking at 18 hours after infection), corresponding to a mortality rate of 92.5% after infection (initiation of death 18 hours after infection) (see Table 1).

The above experimental results show that 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.

(3:), AAV2-BPI-Fcyl recombinant virus combined with antibiotics for synergistic protection of lethal dose of GNB-infected mice

Balb/c 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 ((μΐ only). The results showed that: within 72 hours after the injection of antibiotics, the first group of mice died; the third and fourth groups of mice all survived; the second group of mice had a mortality rate of 91.4% ± 3.5% (η = 4), so The dose of cefuroxime sodium used in this group of mice (250 g/mL x ΙΟΟμΙ^ only = 25 μ _§ / 100 μΐν only) was determined as a synergistic dose.

The mice were divided into the following four groups: PBS group, cefuroxime sodium group (25μ ₈ /100μΙ7), AAV2-BPI-Fcyl recombinant virus group, AAV2-BPI-Fcyl recombinant virus and cefuroxime sodium (25μ^100μΐ 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). The results showed that the survival rate of AAV2-BPI-Fcyl recombinant virus combined with cefuroxime sodium was 60~70%, which was significantly higher than that of cefuroxime sodium group (survival rate 0~10%) and AAV ² _BPI-Fcyl. Recombinant virus group (survival rate ³⁰ %) mice. The above test results show that the combination of AAV2-BPI-Fcyl recombinant virus and antibiotic has a synergistic antibacterial effect, which can significantly enhance the 4th anti-effect of mice on the minimum lethal dose of E.coli infection. Applicant or agent file number IEC050023PCT Description of microbial deposit

(Rule 13 bis)

Filled in by the International Bureau

□ International Bureau receives authorized officials on the date of this page

Claims

A recombinant virus comprising a viral vector, and a genetic construct selected from the group consisting of:

2) 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 linked to the 3' terminus of the human BPI gene or a functional fragment thereof gene or Its allele gene.

2. The recombinant virus according to claim 1, wherein the viral vector is selected from the group consisting of an adenovirus vector, a herpes simplex virus vector, and a retroviral vector, or is selected from a virus-free viral vector, including an adeno-associated virus vector, an adenovirus. Microcarriers (Mini-Ad) and HSV amplicon vectors.

The recombinant virus according to claim 2, wherein the adeno-associated virus vector is an AAV vector based on AAV1 - AAV8 serotype adeno-associated virus, and currently includes an AAV2, AAV1, AAV3, AAV5 type adeno-associated virus vector and The hybrid vector is preferably an AAV2-type adeno-associated virus vector.

The recombinant virus according to claim 1, wherein the functional fragment gene of the human BPI gene is

1) A polynucleotide sequence encoding a BPIW99 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 SEQ ID NO: 1 has an amino acid at position 132, optionally Replaced with Ala or Ser, or

2) A polynucleotide sequence encoding a BPI _{1 193} functional fragment of the C-terminus of the amino acid fragment of 1) that is truncated by 6 amino acids.

The recombinant virus according to claim 1, wherein the human immunoglobulin heavy chain constant region Fc gene is one selected from the group consisting of Cyl, Cy2, Cy3, Cod and Ca2 genes.

The recombinant virus according to claim 1, which is an AAV2 type recombinant adeno-associated virus containing a BPI-Fcyl chimeric gene, wherein the amino acid sequence encoded by the BPI-Fcyl chimeric gene is as shown in SEQ ID NO: 2. The human BPIW99 functional fragment gene and the human Fcyl gene are chimeric, and the 5th and 3' ends thereof are ligated to the CMV promoter and the SV40 poly A expression control element, respectively.

7. Use of a recombinant virus according to claim 1 for the preparation of a pharmaceutical composition for gene therapy against a mammal, in particular a human GNB and/or a GNB-like pathogen, such as Chlamydia, Rickettsia and spirochete, an infectious disease .

A pharmaceutical composition for gene therapy of GNB and/or GNB-like pathogen-infected diseases, comprising a therapeutically effective amount of the recombinant virus of claim 1, and a pharmaceutically acceptable carrier.

A pharmaceutical composition for gene therapy of GNB and/or GNB-like pathogen-infected diseases, comprising a pharmaceutically acceptable non-viral vector, and a genetic construct selected from the group consisting of:

10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable non-viral vector is a liposome.

11. A method of gene therapy for GNB and/or GNB-like pathogen infection, comprising: administering a therapeutically effective amount of the recombinant virus of claim 1 or the pharmaceutical composition of claim 9 or 10 to a patient.

12. The method of claim 11, further comprising administering a known antibiotic compound in combination before, simultaneously or after administering a therapeutically effective amount of the recombinant virus of claim 1 or the pharmaceutical composition of claim 9 or 10 Patient steps.