WO2023087090A1 - Protéines recombinantes - Google Patents

Protéines recombinantes Download PDF

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Publication number
WO2023087090A1
WO2023087090A1 PCT/CA2021/051631 CA2021051631W WO2023087090A1 WO 2023087090 A1 WO2023087090 A1 WO 2023087090A1 CA 2021051631 W CA2021051631 W CA 2021051631W WO 2023087090 A1 WO2023087090 A1 WO 2023087090A1
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Prior art keywords
mbl
cell
recombinant protein
expression vector
acid sequence
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PCT/CA2021/051631
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English (en)
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Francis V A FERNANDES
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Magellan Therapeutics Inc.
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Priority to CA3238612A priority Critical patent/CA3238612A1/fr
Priority to PCT/CA2021/051631 priority patent/WO2023087090A1/fr
Publication of WO2023087090A1 publication Critical patent/WO2023087090A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K14/4726—Lectins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6408—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a recombinant protein comprising amino acid sequences of at least three MBL-2 proteins, fragment or variants thereof.
  • the invention also relates to a nucleic acid encoding such a recombinant protein, expression vector comprising said nucleic acid, as well as an exosome comprising the recombinant protein, nucleic acid, and/or expression vector.
  • the invention also relates to a cell comprising said recombinant protein, nucleic acid, expression vector, and/or exosome.
  • compositions comprising said recombinant protein, nucleic acid sequence, expression vector exosome, and/or cell.
  • the invention relates to the use of said recombinant protein, nucleic acid, expression vector, exosome, and/or composition, as a medicament, and/or for use in the treatment of a disease or disorder associated with MBL-2 deficiency.
  • the invention relates to methods of treating a disease or disorder associated with MBL-2 deficiency.
  • the complement system is a set of blood proteins that form a proteolytic enzyme cascade to help clear pathogens from the body.
  • Mannan Binding Lectin also known as mannosebinding lectin or mannan-binding protein (MBP)
  • MBL binds to carbohydrates, specifically D-mannose and L-fructose residues, commonly found on the surface of pathogens like bacteria, viruses, protozoa and fungi.
  • MBL binds to these antigens on the surfaces of foreign cells and thereby “labels” them for destruction by other components of the immune system. Only the correctly oligomerized forms of MBL are functional and capable of binding efficiently to microbial carbohydrates by associating with the Mannose-binding lectin (MBL)-associated serine proteases MASPs.
  • MBL Mannose-binding lectin
  • MASP Mannan-binding lectin serine protease
  • MASP-2 Mannosebinding lectin-associated serine protease-2
  • MASP-2 cleaves C4 releasing C4a and generating C4b, which attaches covalently to the pathogen surface upon exposure of its reactive thioester.
  • C2 binds to C4b and is also cleaved by MASP-2 to form the C3 convertase (C4b2a).
  • the interactions between MASP-2, C4, C2, and their activation fragments and MASP-2-catalyzed cleavage of C4b2 and C2 show that C2 binds tightly to C4b but not to C4, implying that C4 and C2 do not circulate as preformed complexes but that C2 is recruited only after prior activation of C4.
  • C4b Following cleavage of C4, C4b still binds to MASP-2 (Sepp, A., Dodds, A. W., Anderson, M. J., Campbell, R. D., Willis, A. C., and Law, S. K. (1993) Protein Sci. 2, 706-716). It has been shown that the C4b MASP-2 interaction favours attachment of C4b near to the activating MBL protein. It is proposed that the MASP complex on the bacterial surface, following recruitment of C2, with the proximity of enzyme and substrate (C4b2) drives the formation of the C3 convertase, promoting complement activation.
  • opsonins - proteins of the innate and adaptive immune system that facilitate phagocytosis and cell lysis by “marking” an antigen.
  • Opsonization is the modification of antigens by opsonins to make them more accessible to phagocytic cells and other immune cells.
  • MBL deficiency characterised by low levels of MBL in their blood.
  • Normal human plasma contains MBL concentrations ranging from 450ng/mL to 5,000 ng/ml. Those patients with an MBL serum concentration of less than 450ng/mL can lead to increased susceptibility to recurrent pathogen infections.
  • MBL deficiency is the most common human immunodeficiency identified to date, and it increases the susceptibility to and the severity of infections or inflammatory diseases, including HIV, hepatitis, cystic fibrosis and cancer.
  • Reduced MBL levels in serum are connected to an increased susceptibility for infections with regard to bacteria, fungi, and yeasts (in particular Candida albicans), due to the associated defect in the functioning of the complement system.
  • Defective MBL may furthermore cause decreased or delayed viral clearances (such as Herpes genitalis - HSV-2).
  • MBL deficiencies which are usually clinically objectively observable (Serum MBL ⁇ 50 ng/ml)
  • heterozygous mutation carriers most often get detected in context with other underlying diseases (during radio-, chemo-, or immunosuppressive therapy, alongside chronic infections).
  • Characteristic clinical pictures are recurrent candidiasis or bacterial infections, such as aggressive forms of pneumococcal infections or chronic recurrent respiratory infections.
  • the present invention is based on the inventor’s development of novel recombinant proteins which may be particularly effective in treating diseases or disorders associated with reduced levels of MBL-2 and/or MASP-2.
  • the inventor has synthesised DNA sequences encoding the recombinant proteins of the invention as plasmids which were incorporated into a vector and subsequently replicated in cell culture.
  • Furthemore the inventor has developed episomal minicircle DNA vectors which express MBL-2 and MASP2 proteins in cells such as mesenchymal stem cells and fibroblasts. These cells may have a natural tendency to migrate to the site of infection and express the recombinant protein in the desired location. However, other cells may also be used. Additionally, the inventor has shown that there may be more than one way to introduce the DNA encoding the recombinant protein of the invention into a recipient cell.
  • minicircle DNA This includes transfection of cells with exosomes containing the vector or natural uptake of exosomes by the target or recipient cells. Transfection of exosomes with minicircle DNA is a reproducible and established protocol. Moreover, exosomes are easily loaded in skin fibroblasts or mammalian cells in vitro without any treatment for cellular uptake rendering them especially usefully in a therapeutic context. Use of a Minicircle DNA also may have a number of advantages over other techniques used to deliver DNA to a recipient cell. Firstly, minicircle DNA is stable and can lead to episomal expression of the gene product sustained over a number of weeks. In addition, these DNA sequences contain no bacterial DNA sequences and as a result are unlikely to trigger an immune response themselves. Moreover, minicircle DNA can accommodate the insertion of a gene of interest of almost any size.
  • the DNA sequences encoding for recombinant proteins of the invention have been optimised by the inventor for high expression of the recombinant proteins. Additionally, the inventor has shown that the delivery of cells transfected with expression vectors (such as a minicircle DNA) comprising nucleic acid sequences encoding the recombinant protein of the invention or the delivery to cells of exosomes containing the vector may increase or restore expression of functional MBL-2- MASP2 complexes over an extended period of time without the risk of triggering an immune response.
  • expression vectors such as a minicircle DNA
  • the present invention provides a recombinant protein comprising amino acid sequences of at least three MBL-2 proteins, or fragments or variants thereof.
  • the recombinant protein may comprise amino acid sequences of at least four MBL-2 proteins, or fragments or variants thereof.
  • the recombinant protein may comprise an amino acid sequence of at least one MASP-2 protein, or a fragment or variant thereof.
  • the recombinant protein may comprise amino acid sequences of at least two MASP- 2 proteins, or fragments or variants thereof.
  • the recombinant protein may comprise one or more linkers.
  • the one or more linkers may comprise three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more glycine residues, optionally wherein some or all of the glycine residues are consecutive.
  • the recombinant protein may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 (MBL-2 - L - MBL-2 - L - MBL-2 - MBL-2 - MASP2 - MASP2); SEQ ID NO: 2 (MBL-2 - L - MBL-2 - L - MBL-2); SEQ ID NO: 3 (MBL-2 - L - MBL- 2 - L - MBL-2 - L - MASP-2 - L - MASP2); SEQ ID NO: 4 (MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP2); SEQ ID NO: 5 (MASP2 - L - MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP2); SEQ ID NO: 6 (MBL-2 - MBL-2 - MBL-2 - MBL-2); SEQ ID NO: 7 (MBL-2 - MBL-2
  • the present invention provides a nucleic acid sequence encoding the recombinant protein of the invention.
  • the present invention provides an expression vector comprising the nucleic acid sequence of the invention.
  • the expression vector may be a minicircle DNA.
  • the expression vector may comprise a promoter operably linked to the nucleic acid sequence of the invention.
  • the promoter may be a Human elongation factor-1 alpha (EF-1 alpha) promoter.
  • the expression vector may further comprise an IRES.
  • the present invention provides an exosome comprising the recombinant protein, the nucleic acid sequence, and/or the expression vector of the invention.
  • the present invention provides a cell comprising the recombinant protein, the nucleic acid sequence, the expression vector, and/or the exosome of the invention.
  • the cell may be an isolated cell, optionally a human isolated cell.
  • the cell may localise to an infection site.
  • the cell may be a mesenchymal stem cell, fibroblast or hepatocyte.
  • the present invention provides a composition comprising the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, and/or the cell and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • the present invention provides the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, the cell, and/or the composition of the invention for use in the treatment of a subject having or suspected of having a disease or disorder associated with MBL-2 deficiency.
  • the disease or disorder associated with MBL-2 deficiency may be a primary MBL-2 deficiency, optionally caused by a mutation in the MBL-2 gene and/or MASP2 gene.
  • the mutation may be homozygous or heterozygous.
  • a disease or disorder associated with MBL-2 deficiency may be a disease or disorder that is caused and/or exacerbated by a primary MBL-2 deficiency.
  • a disease or disorder that is caused and/or exacerbated by a primary MBL-2 deficiency may be selected from the group consisting of an infection, immunodeficiency, HIV, sepsis, malaria, respiratory failure, cystic fibrosis, cancer, or genetic factors (for example MBL- 2 gene polymorphisms which may cause defects in the polymerization of MBL-2 protein and result in a functional deficiency of lectin and/or low serum levels of lectin).
  • the infection may be: a bacterial, viral, protozoal, and/or yeast infection; and/or a persistent, a recurrent, and/or a severe infection.
  • the subject with the infection may have cancer.
  • a disease or disorder associated with MBL-2 deficiency may result in MBL-2 deficiency.
  • a deficiency of human mannose-binding lectin (MBL) may result in increased risk and severity of infections and autoimmunity.
  • the present invention provides a method of treating a subject the method comprising providing the subject in need thereof with a therapeutically effective amount of the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, the cell, and/or the composition of the invention.
  • Figure 1 is a graphic representation of an example of the parental plasmid coding for the minicircle DNA as described herein.
  • the sequence of the plasmid is shown in SEQ ID NO: 9.
  • the regions of the plasmid are as follow: SV40 ⁇ EEL ⁇ poly(A)s - nucleic acids 10130 to 10261 ; attB - nucleic acids 1 to 34 encode; attP - nucleic acids 10459 to 10497; EF1 promoter - nucleic acids 53 to 598; copGFP - nucleic acids 8386 to 9141 ; MCS (multiple cloning site) - nucleic acids 599 to 616 and 7790 to 7795; IRES - nucleic acids 7796 to 8373 and 8385 to 8385 4 MBL-2 proteins - nucleic acids 623 to 7789 (specifically 623 to 1366 is the location of first MBL-2 protein, followed by linker at position 1367 to 1402, followed by second MBL-2 protein at position 1403 to 2146, followed by linker at position 2147 to 2182, followed by third MBL-2 protein at position 2183 to 2926, followed by fourth MBL2 protein at position
  • Figure 2 is an illustration of the process of making the minicircle DNA from the parental plasmid.
  • Figure 3 is a graphic representation of the gene product coded for by the DNA sequence of invention.
  • FIG. 4 shows the expression levels of MBL2 and MASP2 in Huh7 Liver Cancer cell Line transfected with metafectene. Increased expression of MBL2 and MASP2 proteins in cancer cell line Huh7 transfected with minicircle DNA can be seen.
  • FIG. 5 shows the expression of green fluorescent protein (GFP) reporter protein in the minicircle gene construct in dermal fibroblast cells treated with exosomes transfected with minicircle DNA.
  • GFP green fluorescent protein
  • FIG. 5 shows the expression of green fluorescent protein (GFP) reporter protein in the minicircle gene construct in dermal fibroblast cells treated with exosomes transfected with minicircle DNA.
  • GFP fluorescence is visible in fibroblast cells.
  • Expression of GFP under the control of the human elongation factor 1 alpha promoter confirms the successful switching on of expression of the transgene and high efficiency of transfection of exosomes and subsequent loading of exosomes transfected with minicircle DNA into the skin fibroblast cells.
  • the expression of the genes of interest with linkers were detected by qPCR.
  • Skin fibroblasts will express the GOI while in the peripheral blood circulation for as long as the skin fibroblasts circulate in the blood which could be a couple of weeks. This could aid in expressed recomb
  • Figure 6 shows the results of an assay determining replication inhibition of Sars-Cov-2 Pseudovirus in vitro by a protein extract of a mouse humanized liver expressing the recombinant protein engineered in the minicircle DNA. It can be seen that the level of inhibition is proportional to amount of sample.
  • Figure 7 shows the results of a Western Blot from the humanized mouse liver extract.
  • MBL2 peaks can be seen at 41 KDa, 50KDa, 63KDa, 125KDa and 250KDa to 260KDa.
  • MASP2 peaks can be seen at 42KDa, 50KDa, 64KDa, 230KDa to 250KDa to 260KDa.
  • the theoretical size of recombinant protein of SEQ ID NO: 1 is 257.29KDa (including the linkers, wherein each of the two linkers (L) is 0.702KDa).
  • Natural oligomers formed in human liver have the following the sizes: MBL-
  • FIG. 8 A Cartoon representation of Western Blots - SDS-PAGE gradient electrophoresis from individuals with different MBL2 genotypes.
  • the peaks observed in the Western Blot Electropherogram are MBL2 proteins that interact with N-glycans of SARS-COVID-2 RBD & Mannan proteins respectively.
  • MASP2 proteins bind MBL2 proteins and so present peaks at nearly the same KDa as MBL2 proteins.
  • the present invention provides a recombinant protein comprising amino acid sequences of at least three MBL-2 proteins, or fragments or variants thereof.
  • recombinant protein refers to a protein produced by an artificial gene and/or process (e.g., genetic engineering). Such a recombinant protein comprises amino acid sequences of at least three MBL-2 proteins, or fragment or variants thereof. Suitably the recombinant proteins comprises amino acid sequences of at least three or at least four MBL- 2 proteins, or fragments or variants thereof. In the context of the present disclosure, each of the three MBL-2 proteins, or fragment or variants thereof may be referred to as a domain of the recombinant protein, or an MBL-2 domain of the recombinant protein.
  • MBL-2 or “MBL2” as used herein refer to the protein Mannose-binding protein C encoded by the gene MBL2.
  • the polypeptide is encoded by human gene MBL2, and has the following sequence: MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI (SEQ ID NO: 10).
  • MBL-2 has an oligomeric structure (400KDa to greater than 1000 KDa), built of subunits that contain three presumably identical peptide chains of about 26kDa each.
  • MBL-2 proteins assembles into a homotrimer.
  • the three polypeptides each form a N-terminal cysteine-rich region, a collagen-like region, an a-helical neck region, and a C-terminal carbohydrate recognition domain (CRD).
  • CCD C-terminal carbohydrate recognition domain
  • These homotrimers may further form bigger oligomeric structures, comprising of three, four, or more homotrimers.
  • MBL-2 is naturally produced primarily in the liver.
  • the homotrimers have the ability to bind to carbohydrates, for example D-mannose and L-fructose residues.
  • the recombinant protein and/or each of the MBL-2 domains forming the recombinant protein may have the ability to bind to carbohydrates, for example D-mannose and L-fructose residues.
  • the present invention also provides a recombinant protein having ability to bind to carbohydrates (for example D-mannose and L- fructose residues), wherein the recombinant protein comprises amino acid sequences of at least three proteins with such binding activity or capable for forming complexes with such binding activity, or fragments or variants thereof.
  • the recombinant protein may further comprise an amino acid sequence of at least one MASP-2 protein, or a fragment or variant thereof.
  • Such an additional MASP-2 protein, or a fragment or variant thereof, when present, is another domain of the recombinant protein.
  • This domain may be referred to herein as a MASP-2 domain.
  • the recombinant protein may comprise amino acid sequences of at least two, or more (for example three or four) MASP-2 proteins, or fragments or variants thereof.
  • the recombinant protein may comprise an amino acid sequence of at least three MBL-2 proteins and at least one MASP-2 protein.
  • the recombinant protein may comprise an amino acid sequence of at least four MBL-2 proteins and at least one MASP-2 protein.
  • the recombinant protein may comprise an amino acid sequence of at least three MBL-2 proteins and at least two MASP-2 protein.
  • the recombinant protein may comprise an amino acid sequence of at least four MBL-2 proteins and at least two MASP-2 protein.
  • MASP-2 protein refers to the protein Mannan-binding lectin serine protease 2 encoded by the gene MASP2.
  • the polypeptide is encoded by human gene MASP2, and has the following amino acid sequence: MRLLTLLGLL CGSVATPLGP KWPEPVFGRL ASPGFPGEYA NDQERRWTLT APPGYRLRLY FTHFDLELSH
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins.
  • the recombinant protein of the invention may comprise full lengths of the MBL-2 protein, and optionally MASP2 protein, or fragments or variants thereof.
  • the fragments or variants are described as compared to a parent or reference polypeptide. It will be appreciated that for a fragment or variant of MBL-2 the parent or reference protein may be one having an amino acid sequence according to SEQ ID NO: 10, whereas for a for a fragment or variant of MASP-2 the parent or reference protein may be one having an amino acid sequence according to SEQ ID NO: 11 .
  • the fragment or variants is a functional fragment or variant, meaning that it retains at least some of the biological activity of the parent or reference protein.
  • the fragment or variant may bind to carbohydrates, for example D-mannose and L-fructose residues, and/or may be able to form a complex that binds to carbohydrates, for example D-mannose and L-fructose residues.
  • the fragment or variant may cleave C2 and/or C4, and/or may be able to form a complex that may cleave C2 and/or C4.
  • Such biologically active fragments or variants of the reference or parent polypeptide may retain at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the biological activity of the parent or refence protein.
  • fragment refers to a polypeptide comprising an amino acid sequence of at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 225 contiguous amino acid residues, at least 250 contiguous amino acid residues, at least 275 contiguous amino acid residues, at least 300 contiguous amino acid residues, at least 325 contiguous amino acid residues, at least 350 contiguous amino acid residues, at least 375 contiguous amino acid residues, at least 400 contiguous amino acid residues, at least 425 contiguous amino acid residues, at least 450 contiguous amino acid residues, at least 4
  • the fragment may comprise at least 240, at least 241 , at least 242, at least 243, at least 244, at least 245, at least 246, at least or 247) contiguous amino acid residues of the parent or reference protein.
  • the fragment may comprise at least 680, at least 681 , at least 682, at least 683, at least 684, or at least 685 amino acid residues of the parent or reference protein.
  • variant refers to a polypeptide that comprises a polypeptide sequence that differs in one or more amino acid residues from the polypeptide sequence of a parent or reference polypeptide (such as, e.g., a wild-type (WT) polypeptide sequence, for example according to SEQ ID NO: 10 or 11).
  • a variant polypeptide may comprise a polypeptide sequence which differs from the parent or reference by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 20%, 30% 40%, 50% or more of the total number of residues of the parent or reference polypeptide sequence.
  • a variant polypeptide may comprise a polypeptide sequence that has at least about 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide sequence of a parent or reference polypeptide.
  • a variant polypeptide may comprise a polypeptide sequence that differs from the polypeptide sequence of a parent or reference polypeptide from 1 to 100 or more amino acid residues (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues).
  • a variant polypeptide may comprise a polypeptide sequence that differs from the polypeptide sequence of a parent or reference polypeptide by, e.g., the deletion, addition, or substitution of one or more amino acid residues (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues) of the parent or reference polypeptide, or any combination of such deletion(s), addition(s), and/or substitution(s).
  • the deletion, addition, or substitution of one or more amino acid residues e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues
  • sequence identity in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art.
  • the domains of the recombinant proteins may be the same or different.
  • the recombinant protein may comprise for example two full length MBL-2 proteins, and one MBL-2 fragment or variant, or vice versa.
  • the recombinant protein comprises two or more fragments or variants of MBL-2, some or all of these fragments or variants may be same or different.
  • the recombinant protein may comprise one or more linker.
  • linker refers to a short peptide (typically less than 30 amino acid residues in length, for example less than 20, or less than 15) which is located between two or more adjacent domains of the recombinant protein.
  • the linker may be abbreviated as “L”.
  • MBL-2 - L - MBL-2 refers to a MBL-2 domain followed by a linker followed by a second MBL-2 domain.
  • the domain may be full length MBL-2 protein, or a fragment or variant thereof.
  • a linker may enable or improve the folding of the recombinant protein to form a functional protein.
  • the linker may comprise three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more glycine residues, optionally wherein some or all of the glycine residues are consecutive.
  • some or all of the linkers may be the same or different.
  • the recombinant protein may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (MBL-2 - L - MBL-2 - L - MBL-2 - MBL-2 - MASP2 - MASP2); SEQ ID NO: 2 (MBL-2 - L - MBL-2 - L - MBL-2); SEQ ID NO: 3 (MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP-2 - L - MASP2); SEQ ID NO: 4 (MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP2); SEQ ID NO: 5 (MASP2 - L - MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP2); SEQ ID NO: 6 (MBL-2 - MBL-2 - MBL-2 - MBL-2); SEQ ID NO: 7 (MBL-2 - - M
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and/or SEQ ID NO: 8 are in the N- to C-terminal orientation.
  • the variant will have at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and/or SEQ ID NO: 8.
  • SEQ ID NO: 1 shown hereinbelow contains 12 glycine residue linkers between the first and second, and second and third domains
  • different linkers may be used, the location of the linkers may different, or indeed the linkers may be not present at all.
  • the linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in length. All, some, or none of the amino acids in the linker may be glycine residues. It will also be appreciated that some or all of the linkers may be the same or different. Therefore, merely by way of example, one linker may be comprise or consist of 12 glycine residues, while others may comprise or consist of less or more glycine residues.
  • the recombinant protein may have a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:1 , 2, 3, 4, 5, or 6.
  • the recombinant protein may have a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:1.
  • the present invention provides a nucleic acid sequence encoding the recombinant protein of the invention.
  • nucleic acid refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.
  • nucleic acids can include, in addition to the standard bases adenine, cytosine, guanine, thymine and uracil, various naturally occurring and synthetic bases (e.g., inosine), nucleotides and/or backbones.
  • nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; and mRNA.
  • cDNA complementary DNA
  • nucleic acid also encompasses any chemical modification thereof, such as by methylation and/or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole.
  • Such modifications may include base modifications such as 2- position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.
  • the nucleic acid sequence may be provided in an expression vector.
  • This embodiment gives rise to a further aspect of the invention, i.e. an expression vector comprising the nucleic acid sequence of the invention.
  • the nucleic acid sequence may be provided in an expression vector.
  • an expression vector comprising the nucleic acid sequence of the invention.
  • vector and “expression vector” are used interchangeably.
  • the vector may be any vector capable of transferring the nucleic acid sequence (for example DNA) encoding the recombinant protein to a cell.
  • the vector is an integrating vector or an episomal vector, more suitably the episomal vector is a minicircle DNA vector.
  • Suitable integrating vectors include recombinant retroviral vectors.
  • a recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells.
  • the term “infection” is used to mean the process by which a virus transfers genetic material to its host or target cell.
  • the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells.
  • the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques.
  • any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.
  • vectors useful in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirus vectors.
  • Adenovirus vectors are well known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain and skin (Hitt et al, 1997 Advances in Pharmacology 40: 137-206; Anderson, 1998 Nature 392: (6679 Suppl): 25-30) and to tumours (Mountain, 2000 Trends Biotechnol 78: 119-128).
  • a further suitable vector is the adeno-associated (AAV) vector.
  • AAV vectors are well known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid and haematopoietic stem cells for gene therapy applications (Philip et al, 1994 Mol Cell Biol 74: 2411-2418; Russell et a/, 1994 Proc Natl Acad Sci USA 91: 8915-8919; Flotte et al, 1993 Proc Natl Acad Sci USA 90: 10613- 10617; Walsh et al, 1994 Proc Natl Acad Sci USA 89 : 7257-7261 ; Miller et al, 1994 Proc Natl Acad Sci USA 97:10183-10187.; Emerson, 1996 Blood 87, 3082-3088).
  • International Patent Application WO 91/18088 describes specific AAV-based vectors.
  • Suitable episomal vectors include transient non-replicating episomal vectors and selfreplicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1.
  • viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1.
  • BK human papovavirus
  • the episomal vector is a minicircle DNA vector.
  • the inventor believes that a minicircle DNA has a number of advantages over some other techniques used to deliver DNA to a recipient cell.
  • minicircle DNA is typically stable and can lead to episomal expression of the gene product sustained over a number of weeks.
  • these DNA sequences contain no bacterial DNA sequences and as a result may be less likely to trigger an immune response themselves.
  • Typical transgene delivery methods involve plasmids which contain foreign DNA that can trigger an immune response.
  • Minicircle DNA can accommodate the insertion of a gene of interest of almost any size as well.
  • minicircle DNA sequences tend to be smaller and easier for host cells to replicate.
  • the minicircle DNA may have a sequence according to SEQ ID NO: 9.
  • the vector of the present invention may be a plasmid.
  • the plasmid may be a nonreplicating, non-integrating plasmid.
  • the term “plasmid” as used herein refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids.
  • the nucleic acid (DNA or RNA) may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
  • a non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell’s genome (i.e. does not integrate at high frequencies and does not integrate at specific sites).
  • Replicating plasmids can be identified using standard assays including the standard replication assay of Ustav et al (1991 EMBO J 70: 449-457).
  • the invention also provides a cell transformed or transfected with the vector of the present invention.
  • the cell may be any mammalian cell.
  • the cell may be a rodent or human cell.
  • the cell may be an isolated cell. Exemplary cells are described hereinbelow.
  • nucleic acid condensing agents include the use of nucleic acid condensing agents, electroporation, complexing with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984 Crit. Rev. Biochem 16’. 349- 379); Keown et a/ (1990 Methods Enzymol 785:527-37).
  • the expression vector may comprise a promoter operably linked to the nucleic acid sequence of the invention.
  • the promoter may be any promoter that allows the expression of the recombinant protein of the invention.
  • promoter refers to a DNA sequence that is in vicinity to the DNA sequence functions as a switch, activating the expression of a gene of interest. It will be appreciated that in the context of the present disclosure the gene of interest may be the DNA that encodes the recombinant protein of the invention. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene.
  • the promoter therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
  • the promoter may be Human elongation factor- 1 alpha (EF-1 alpha) promoter.
  • the expression vector may further comprise a sequence encoding an internal ribosome entry site (IRES).
  • IRES is an RNA element that allows for translation initiation in capindependent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex.
  • the present invention provides an exosome comprising the recombinant protein, the nucleic acid sequence, and/or the expression vector of the invention.
  • therapeutic delivery vehicles need to be cells (which are discussed hereinbelow).
  • they may be exosomes.
  • Exosomes are a class of round-shaped lipid bilayer vesicles with a diameter of ranging from about 30 to about 500 nm, more suitably from about 30 nm to about 150 nm.
  • Exosomes can be naturally secreted by a variety of cells, such as T and B lymphocytes, epithelial cells, endothelial cells, dendritic cells, mesenchymal stem cells, platelets and tumor cells.
  • Exosomes may be secreted in cells that are in vivo or in vitro. Exosomes contain multiple proteins, lipids, and nucleic acids. Exosomes can be used to stably carry drugs (in the context of the present disclosure the drug may be the recombinant protein of the invention) to avoid enzymatic degradation of drugs and thereby prolong the half-life of drugs during delivery. Exosomes may also have extremely high bioavailability, extremely low immunogenicity and toxicity, can spread in tumor tissues, and can pass through the human blood brain barrier. Exosomes generally interact with cell receptors on their target or recipient cells and when taken into cells, internalized exosomes may release their contents.
  • the recombinant protein of the present invention are good vehicles for delivering therapeutics to cells, including the recombinant protein of the present invention, where the recombinant protein may be delivered in the form of a nucleic acid sequence encoding said protein provided in an expression vector, or in the form of a recombinant protein.
  • the expression vector may be a minicircle DNA.
  • the exosomes of the invention may comprise an expression vector (such as a minicircle DNA) of the present invention.
  • the present invention provides a cell comprising the recombinant protein, the nucleic acid sequence, the expression vector, and/or the exosome of the invention.
  • the cell may be an isolated cell.
  • the cell may be a mammalian cell, for example a human or rodent isolated cell.
  • the cell is one that may localise to an infection site (for example by migrating to infection and/or injury site).
  • Cells that localise to an infection site include mesenchymal stem cells (MSCs) and fibroblasts.
  • MSCs mesenchymal stem cells
  • Other cells useful in the context of the present disclosure may be those that have the natural ability to promote MBL-2 and/or MASP proteins.
  • such cells are hepatocytes.
  • MSCs may migrate to sites of injury and then differentiate into functional cells, or they can fuse with compromised cells to regenerate damaged tissues. MSCs produce a number of molecules that act to modulate and/or suppress certain portions of the immune system. Human MSCs stimulated by inflammatory cytokines have been found to direct antibacterial activity. MSCs have been implicated in suppressing T-cell activity, inhibiting apoptosis, reduction of inflammatory cytokine levels, etc. Bone marrow-derived mesenchymal stem stromal cells (BM-MSCs) could have therapeutic potential for numerous conditions, including ischemia-related injury. MSCs have a from those interested in cell therapy and regenerative medicine.
  • BM-MSCs bone marrow-derived mesenchymal stem stromal cells
  • Bone marrow-derived mesenchymal stem stromal cells could have therapeutic potential for numerous conditions, including ischemia-related injury. MSCs have a high capacity for adhesion and as a result sometimes aggregate leading to undesirable outcomes such as clogging of capillaries especially in the lungs. Suspending MSCs in heparin before injecting a patient intravenously (i.v.) avoids a lot of these problems.
  • Human Mesenchymal Stromal Cells including hBM-MSCs are resistant to SARS-CoV-2 infection under steady-state, inflammatory conditions and in the presence of SARS-CoV-2- Infected cells (D0l:doi.org/10.1016/j.stemcr.2020.09.003).
  • MSCs innate ability to regulate immune system components and their anti-bacterial properties by introducing MSCs into tissues exhibiting certain illnesses. These stem cells naturally migrate to sites of inflammation. As a result, researchers have used modified MSCs to produce anti-cancer therapeutics since the cells naturally migrate to tumor microenvironments. Other researchers have coated MSCs with antigens in an attempt to create vaccines.
  • U.S. Patent Publication number 2019/0282694 describes immunoprotective primary mesenchymal stem cells (IP-MSC) that express multiple immunoreactive polypeptides targeted to a particular pathogen.
  • IP-MSC immunoprotective primary mesenchymal stem cells
  • Skin fibroblast cells are another type of cell that naturally migrates to sites of inflammation and/or injury.
  • Fibroblasts comprise the main cell type of connective tissue, whose function is believed to produce extracellular matrix responsible for maintaining structural integrity of tissue. Fibroblasts also play an important role in wound healing, resulting in deposition of extracellular matrix. Skin fibroblasts are known to home in on skin wounds. Recent research advances have established skin fibroblasts as a promising vehicle for therapeutic delivery. Skin fibroblasts have not induced host immunoreactivity upon local transplantation during systemic administrations. Intravenously injected human fibroblasts migrate to skin wounds, deliver type vii collagen, and promote wound healing in mice. Fibroblasts provide an alternative to mesenchymal stem cells with successful treatment and immune modulation in a Lewis rat model for Experimental autoimmune encephalomyelitis (EAE) of multiple sclerosis. Human skin fibroblasts are a safe carrier to deliver genes of interest (for example encoding the recombinant protein of the invention) into the tissues of interest for gene therapy applications.
  • EAE Experimental autoimmune encephalomyelitis
  • cells comprising the recombinant protein, the nucleic acid sequence, the expression vector, and/or the exosome of the invention may have at least two different uses.
  • isolated cells may be delivered to the subject in need of the recombinant protein of the invention.
  • the cells may function as an expression chamber for the recombinant protein and/or therapeutic delivery vehicle (the therapeutic being the recombinant protein of the invention).
  • the isolated cells may be used for producing (expressing) the recombinant protein of the invention or exosomes comprising the recombinant protein, nucleic acid or expression vector of the invention.
  • the expressed recombinant protein and/or exosome may be purified (or otherwise prepared) for delivery to the subject.
  • the cells may function as an expression chamber for the recombinant protein.
  • the cells may be ones that are capable of highly expressing the recombinant protein or releasing exosomes.
  • the cells may be hepatocytes.
  • the present invention provides the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, and/or the cell of the invention for use as a medicament.
  • medicament means an agent used to treat (e.g. fight, ameliorate, prevent, and/or slow progression of) an unwanted disease or disorder in a subject, and/or symptoms associated with such an unwanted disease or disorder.
  • the present invention provides the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, the cell, and/or the composition of the invention for use in the treatment of a subject having or suspected of having a disease or disorder associated with MBL-2 deficiency.
  • a disease or disorder associated with MBL-deficiency may be primary MBL-2 deficiency.
  • the primary MBL-2 deficiency may be caused by a mutation in the MBL-2 gene and/or MASP2 gene.
  • the mutation may be homozygous or heterozygous.
  • the mutation may be in the coding or non-coding region of MBL-2 or MASP2.
  • the disease or disorder associated with MBL-2 deficiency may be selected from the group consisting of infection; sepsis; immunodeficiency; immunosuppression; cancer; cystic fibrosis; autoimmune disease; and recurrent spontaneous abortions; optionally wherein the autoimmune disease is systemic lupus erythematosus or rheumatoid arthritis.
  • a disease or disorder associated with MBL-2 deficiency may be a disease or disorder that is caused and/or exacerbate by a primary MBL- 2 deficiency (such as for example a recurrent infection, immunodeficiency, respiratory failure, sepsis, cystic fibrosis, HIV, malaria, cancer, or genetic factors such as MBL-2 gene polymorphisms which may cause defects in the polymerization of MBL-2 protein and result in a functional deficiency of lectin and/or low serum levels of lectin).
  • the infection may be: a bacterial, viral, protozoal, and/or yeast infection; and/or a persistent, recurrent, and/or severe infection.
  • the subject with the infection may have cancer.
  • the subject may be any mammal that expresses MBL-2 and/or MASP2 protein. More suitably, the subject is human, monkey, rodent (for example rat or mouse), cat, dog, horse, cow, or pig.
  • rodent for example rat or mouse
  • MBL-2 deficiency refers to, in the context of human subjects, blood serum levels of MBL-2 that are less than 450 ng/ml, for example less than 400ng/ml, less than 350 ng/ml, less than 300 ng/ml, less than 250 ng/ml, or less.
  • the subject may have MBL-2 serum levels that are less than about 450 ng/ml, less than about 400ng/ml, less than about 350 ng/ml, less than about 300 ng/ml, less than 250 ng/ml, or less (for example about 200 ng/ml, about 150 ng/ml, about 100 ng/ml, or about 50 ng/ml).
  • a subject has MBL-2 deficiency simply by comparing the levels of MBL-2 to a control sample or reference value.
  • a subject may be determined to have MBL-2 deficiency of the subject has about 10%, about 20%, about 30%, about 40%, about 50% or lower MBL-2 levels that a control sample or reference value.
  • the present invention provides a method of treating a subject the method comprising providing the subject in need thereof with a therapeutically effective amount of the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, the cell, and/or the composition of the invention.
  • the subject in need thereof may be a subject that has or is suspected of having a primary MBL-2 deficiency, and/or has or is suspected of having a disease or disorder associated with MBL-2 deficiency. It will be appreciated that the subject in need thereof may be when the subject has MBL-2 serum levels below about 450 ng/ml.
  • a therapeutically effective amount refers to an amount of the recombinant protein, nucleic acid, expression vector, exosome, cell and/or composition of the invention sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder associated with MBL-2 deficiency, or to delay or minimize one or more symptoms associated with a disease or disorder associated with MBL-2 deficiency.
  • Symptoms associated with MBL-2 deficiency will be well known to those skilled in the art. Merely by way of example, the symptoms may include common and/or recurrent infections.
  • the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, and/or the cell of the invention may be formulated as a composition for use as a medicament, for example for use in treating a disease or disorder associated with MBL-2 deficiency.
  • compositions comprising the recombinant protein, the nucleic acid sequence, the expression vector, the exosome, and/or the cell of the invention, together with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected binding protein without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. an expression cassette, plasmid or virion), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.
  • Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
  • Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
  • Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
  • Figure 1 shows an example of the parental plasmid coding for the minicircle DNA. More specifically, Figure 1 shows a commercially available construct that has been modified to produce the desired minicircle DNA construct encoding recombinant protein of the invention.
  • the pMC.EF1a-MCS-IRES-GFP-SV40polyA Parental Minicircle Cloning Vector currently sold by SBI System Biosciences (cat # MN530A-1) has been modified to include an excisable strand of DNA that includes a promoter, such as EF1a (the naturally occurring MBL2 or MASP-2 promoters) and the polynucleotide sequences devised by the inventor.
  • EF1a the naturally occurring MBL2 or MASP-2 promoters
  • the cloning vector sold by the SBI contains other regulatory sequences such as SV40-poly-A and the parental plasmid contains two target sites or multiple cloning sites (MCSs) to allow for insertion of the Gene of Interest (GOI) (in this case the GOI encodes the recombinant protein of the invention according to SEQ ID NO: 1).
  • MCSs Multiple cloning sites
  • the parental plasmid uses the 385 bp attP site of A phage and 31 bp E. coli minimal attB sequence as MCSs.
  • the parental plasmid also includes components that allow for the replication and selection of the parental plasmid including a kanamycin-resistance gene and a replicon, in this case pUC ORI.
  • the parental plasmid also includes a plurality of l-Scel sites to facilitate degradation of the parental plasmid after the minicircle vector has been excised therefrom.
  • special features of the SBI cloning vector include the parental plasmid replication using ZYCY10P3S2T E. coli (hereinafter” the Minicircle Producer Strain”).
  • Figure 2 shows the process of creating the minicircle DNA from the parental plasmid.
  • the parental plasmid is transfected into this cell line that harbours an arabinose-inducible system to express the PhiC31 integrase and the l-Scel endonuclease simultaneously.
  • the ZYCY10P3S2T strain also contains a robust arabinose transporter LacY A177C gene. Adding arabinose to the media containing the minicircle producer strain turns on expression of the PhiC31 integrase and endonuclease genes, resulting in separation of the Parental Minicircle Plasmid into the individual minicircle and the leftover bacterial backbone (from the PhiC31 Integrase activity), and the degradation of the parental plasmid (from Sce-1 endonuclease activity).
  • An embodiment of this invention is the combination of three non-immunogenic entities namely, the minicircle DNA, exosomes and cells (for example MSCs or fibroblasts). Toxicity of the therapeutic is predicably minimized.
  • the minicircle DNA is transfected into exosomes with the transfection reagent.
  • the exosomes transfected with the minicircle DNA is then loaded into recipient cells (such as fibroblasts or MSCs). Skin fibroblasts and several MSCs support the switching on of the EFlalpha promoter which in turn leads to the expressed proteins if this invention.
  • MBL2 and MASP2 are expressed only in the liver under normal physiological conditions.
  • Minicircle DNA and exosomes biodistribution may vary each time a dose is administered for treatment of infected tissue.
  • the use of a particular MSC to home in on diseased tissue with the minicircle DNA payload maybe very efficient.
  • one particular MSC for example will not act as an expression chamber for the therapeutic protein.
  • the method may utilise skin fibroblasts as the episomal transient DNA expression chamber while circulating in the blood stream. T reatment of infections in the blood stream is thus made possible.
  • the minicircle contains a nucleic acid motif that functions as the protein translation initiation site called the Kozak consensus sequence (Kozak) that is present in most eukaryotic mRNA transcripts.
  • Kozak Kozak consensus sequence
  • the minicircle has four open reading frames coding for the MBL-2 protein and one open reading frame coding for the MASP-2 protein.
  • the minicircle DNA construct codes for a chain of four MBL-2 proteins linked end to end with one of them being linked to MASP-2 protein.
  • Production of the minicircle DNA starts by creating a parental plasmid, a plasmid derived from another organism like bacteria, with eukaryotic DNA inserted therein and exposing the plasmid to a site-specific recombinase such as arabinose.
  • the recombinase will excise the DNA at specific sites freeing the eukaryotic DNA from the parental plasmid and resulting in two products, the plasmid by itself and the minicircle DNA.
  • the minicircle DNA can then be recovered using gel electrophoresis and amplified using the RNA polymerase chain reaction (PCR).
  • the minicircle DNA is then introduced into the target cells via transfection or lipofection - processes that use vesicles capable of fusing with the membranes of the target cells allowing the DNA contained therein to be delivered to the target cells.
  • stem cells were transfected with a commercially available transfection reagent made by System Biosciences Inc.
  • Metafectene Bion Tex was effective in the minicircle DNA transfection of 2 cancer cell lines.
  • Skin fibroblasts, mesenchymal stem cells and other cells that naturally migrate to the site of inflammation can be used as delivery vehicles for minicircle DNA that are engineered to express the recombinant protein in vitro and/or in vivo and treat a variety of infections or other diseases or disorders caused by or associated with a deficiency of MBL.
  • In vitro may entail capture of the expressed recombinant protein and vialing for human IV administration.
  • In vivo may entail IV administration of the minicircle DNA or the exosome transfected DNA or the skin fibroblasts/MSCs for treatment of diseases in humans.
  • MSCs including hBM-MSCs are resistant to SARS-CoV-2 infection under steady-state, inflammatory conditions and in the presence of SARS-CoV-2- Infected cells Richard Schafer, et. al.
  • the disclosed method also uses skin fibroblasts that have been engineered to deliver episomal minicircle DNA that codes for the production of MBL and MASP-2 complexes to target inflammatory cells such that the production of the gene product causes the opsonization of pathogens.
  • MBL binds calcium-dependently to sugar residues mannose, fucose and N-acetyl glucosamine (GIcNAc) on the surface of pathogens thereby marking them for opsonization.
  • GIcNAc N-acetyl glucosamine
  • the transplanted skin fibroblasts will express the MBL and MASP-2 complexes while circulating in the peripheral blood system for as long as a couple of weeks allowing for continued expression of the recombinant protein. This could aid it mopping up of pathogen particles in the circulation in patients with septicaemia.
  • the minicircle DNA Once the minicircle DNA is generated, it has to be introduced into cells that express the gene product coded for by the DNA.
  • transfection reagents can be used to transfect cells and exosomes with the engineered minicircles DNA.
  • Polyplus, Metaphectene and Exo-FectTM are examples of three transfection reagents tested for the transfection process.
  • Exo-FectTM (SBI - Systems BioScience Inc.) was effective in the mini circle DNA transfection of exosomes.
  • Metafectene Bion Tex
  • Polyplus transfection agent was not efficient in the transfection of mini circle DNA of this invention in cells.
  • exosomes transfected with the minicircle DNA in accordance with manufacturers protocol for Exo-FectTM Exosome Transfection Reagent (SBI System BioScience) were used.
  • Minicircle DNA transfected exosomes loaded on skin fibroblasts or MSCs in the following concentrations were found to be effective in producing measurable and therapeutic levels of MBL-2 and/or MASP-2 protein in vitro: 25 pg minicircle DNA for 25 pg Exosomes; 1 pg minicircle DNA : 1 pg Exosomes; and 20 pg Exosomes for 10e 5 skin fibroblast cells or MSCs, as explained in more detail below.
  • mcDNA Minicircle plasmid DNA
  • the provided mcDNA vector contained genes for the expression of Human MBL2 (Mannan-binding lectin) and Human MASP2 (Mannan-binding lectin serine protease 2) (SEQ ID NO: 1) as well as a GFP expression marker.
  • Two cell lines were evaluated in the transfection studies - Huh-7 human hepatocyte carcinoma (JCRB #0403) as well as 293T- hsACE2 cells (Integral Molecular C-HA102, HEK293T cells engineered to highly express Human ACE2).
  • a pilot transfection of the MBL2-MASP2 mcDNA construct was performed in 293T-hsACE2 cells using two transfection reagents - jetOptimus (Polyplus) and Metafectene Pro (Biontex). Five transfection conditions were evaluated - three with jetOptimus and two with Metafectene Pro. The transfection conditions are summarized in the table below. In brief, the indicated amount of mcDNA plasmid was mixed with the respective volumes of transfection reagent and transfection medium, then incubated at ambient temperature for 10- 15 minutes. During this incubation, a suspension of the indicated number of cells was centrifuged gently (400g for 5 minutes) and the supernatant discarded.
  • each mcDNA I transfection reagent complex was added to the respective cell pellet over a period of 1 minute with continuous agitation.
  • Cells were incubated with the mcDNA mixture for 10-15 minutes at ambient temperature, with mild agitation every 3-5 minutes.
  • Each condition was then fully re-suspended with 6 mL transfection medium, transferred to a T-75 cell culture flask, and placed in a 37°C incubator for 5-6 hours. After 5-6 hours, the total culture medium volume was adjusted to 18 mL and the flasks were returned to the incubator.
  • TFN-Lib 4 and TFN-Lib5 exhibited higher levels of GFP expression at 72 hours compared to TFN-Lib 1 , 2, and 3.
  • Huh-7 cells were transfected with mcDNA and Metafectene Pro using the same conditions as “TFN Lib4” above, both with and without supplementation of CaCI2 (100 ug/mL) in the transfection and culture medium.
  • conditioned supernatant was collected at 72, 96, and 120 hours post-transfection for MBL2 and MASP2 ELISA testing. Both MBL2 and MASP2 exhibited detectable levels in each sample, with the level of each protein increasing over time posttransfection. Results are shown in Figure 4.
  • mcDNA minicircle DNA plasmid
  • mcDNA minicircle DNA plasmid
  • Multiple transfection conditions and reagents were initially evaluated in 293T-hsACE2 cells.
  • GPF expression in mcDNA transfected cells was confirmed by flow cytometry, and MBL2 and MASP2 protein secretion from the cells was evaluated by ELISA testing of conditioned culture supernatant.
  • An optimal condition (“TFN Lib 4”) was identified and subsequently used for the transfection of Huh-7 cells, which exhibited detectable levels of MASP2 and MBL2 in conditioned culture supernatant after 72 hours.
  • the aim of this study was to determine the protein expression of human MBL2/MBP proteins in humanized liver PIRF mouse model after minicircle DNA hydrodynamic injection.
  • mice Three mice were boosted with MN530A-4x MBL2-MASP2 (SEQ ID NO: 1) minicircle DNA by hydrodynamic tail vein injection at day (D) 0 and D7. Body weight was monitored three times per week and global clinical score were monitored weekly. No strong adverse reaction was observed after the hydrodynamic injection of MN530A-4x MBL2-MASP2 minicircle DNA. All mice were sacrificed on D14. Whole livers were harvested and weighted. Each lobe was split in two parts and each part was weighted. Liver lobes and plasma collected at sacrifice were stored for 1 hour at -20°C and then stored at -80 °C until the shipment to RayBiotech, and Z- Biotech CROs for further analysis.
  • SEQ ID NO: 1 Three mice were boosted with MN530A-4x MBL2-MASP2 (SEQ ID NO: 1) minicircle DNA by hydrodynamic tail vein injection at day (D) 0 and D7. Body weight was monitored three times per week and global clinical score were monitored weekly. No strong adverse reaction was
  • MBL2/MPB ELISA tests were performed on plasma. Following the first MN530A-4x MBL2-MASP2 minicircle DNA hydrodynamic injection, the level of MBL2/MBP protein detected in plasma was similar on D7 and D4. Nevertheless, after the second hydrodynamic injection on D7, a significant increase of MBL2/MBP level for all mice was observed at D14 (day of sacrifice) by comparison to the baseline level. In conclusion, increased MBL2 expression was observed in PIRF humanized liver after hydrodynamic injection of minicircle DNA encoding for MBL2 and MASP2 protein.
  • mice were acclimated to the environment for 7 days prior to the beginning of the experiment.
  • mice were treated by a tail vein hydrodynamic injection of 25 pg of MN530A- 4x MBL2MASP2 (Cat# CS950MC-1 Lot# 200901-005) minicircle DNA. Ringer lactate solution was used as vehicle.
  • ELISA were performed for the quantitative measurement of MBL2/MBP proteins in plasma samples using Human LBK2/MPBboster (EK0805) kits according to the manufacturer’s instructions. Blood and plasma were collected at different time points: D-7 for the baseline, D4, D8 and at sacrifice on D14.
  • liver The five lobes of the liver were harvested and split in two parts. Liver parts were stored 1 hour at -20 °C and then at -80 °C until shipment to RayBiotech as well as Z-biotech for further analysis.
  • Table 1 Increased expression of MBL2 proteins in mouse with humanized liver transfected with minicircle DNA by hydrodynamic tail vein injection.
  • MBL2/MBP protein level was similar to the one detected at baseline.
  • the MBL2/MBP protein level showed a slight increased for each mouse on D8 and kept increased on D14 to reach a significant 3-fold increase compared to the baseline (Table 1).
  • the aim of this study was to evaluate MBL2/MBP protein expression in liver of humanized PIRF mouse model following hydrodynamic injection of MN530A-4x MBL2-MASP2 minicircle DNA.
  • the first injection of the MN530A-4x MBL2-MASP2 minicircle DNA on DO did not lead to any increase of the MBL2/MBP protein level in plasma.
  • the MBL2/MBP protein level increased for each mouse and kept increasing until sacrifice allowing at least 3-fold average increase for MBL2 level protein in plasma.
  • MBL2-Cross- 5’-CAGGCAATAGACTGACCTACAC-3’ (SEQ ID NO: 20) 101 liner 5’-GAAGAACGGCCAGTGGAAT-3’ (SEQ ID NO: 21)
  • MBL2-internal primer set 1 and 2 indicates that endogenous MBL2 expression remains similar; however, MBL2 primer set 2 may work more specific compared to primer set 1 ;
  • MASP2-internal primer set 2 may work more specific for endogenous gene expression compared to primer set 1 ;
  • MBL2-Cross-linker can be examined by both set of primers
  • Positive control for MBL2-Cross-linker primer may help to verify the better primer set in the future experiment.
  • Figure 5A and B shows qPCR results of skin fibroblasts treated with minicircle DNA transfected exosomes versus untreated skin fibroblast cells.
  • the qPCR probes were run specifically to detect the MBL-2 - Linker L - MBL-2 - Linker L - MBL-2 trimer region in duplicate runs.
  • the pseudovirus expresses the SARS-CoV-2 Spike protein on the surface while a plasmid encoding for luciferase is contained inside the particle. No other nucleic acid is present inside the pseudovirus. After the Spike protein binds to the ACE2 receptor on the host cell, the plasmid is released into the cell where luciferase is expressed. In the presence of a luciferase substrate, cells that have been successfully infected with the pseudovirus will luminescence. Spike-ACE2 inhibitors will decrease the level of luminescence.
  • Tissue lysate preparation sample dilution: 1 , 5, 10, 100, 500, 1000 pg/ml
  • 500 pg/ml sample Mixed 7.2 pL stock solution with 712.8 pL DMEM. Added 60 ul of diluted sample to the each well of 96-well plate (sample now 1000 pg/ml). Added 60 pL pseudovirus to the plate (sample now 500 pg/ml).
  • sample 100 pg/ml sample: Mixed 2.88 pL stock solution with 717.12 pL DMEM. Added 60 ul of diluted sample to the each well of 96-well plate (sample now 200 pg/ml). Added 60 pL pseudovirus to the plate (sample now 100 pg/ml).
  • samples were heat inactivated at 56°C for 30 minutes and filtered. If the sample was antibodies, the sample was filtered. Since the samples were neither serum/plasma nor antibodies, no heat inactivation or filtering was performed.
  • Negative control No pseudovirus was added to account for background noise.
  • Inhibition rate [(“No Sample” positive control OD - sample OD)/ “No Sample” positive control OD] x 100%
  • This experiment shows the therapeutics promise of the present invention when treating Covid- 19.
  • the protein extract from humanized mouse liver cells expressing the recombinant protein engineered in the minicircle DNA when treated with RBD + M Microplate showed binding to the membrane protein of the Covid-19 strain as evidenced from further PNGaseF treatment that produced 300pl, 0.08 mg/mL protein concentration corresponding N- Glycan binding proteins.
  • the inventor has also demonstrated that proteins from a humanized mouse liver lead to replication inhibition of a SARS-COV-2 pseudo virus in vitro. Moreover, the protein extract from the humanized mouse liver with a concentration of 500 pg/mL and up to 1000 pg/mL caused an inhibition of SARS-COV-2 pseudo virus replication up to 80% compared to a positive control.
  • the humanized mouse liver expressed 409.35 ng/mL of human MBL-2 protein.
  • the inhibitory replication effects observed with the SARS-COV-2 pseudo virus can be used according to the invention for the general treatment for all pathogenic infections as the MBL-2 protein and its complexes are selective for binding mannose residues present on the glycocalyx of the membranes of pathogens. The results of this experiment are in Figure 6.
  • the inhibitory replication effects observed with the SARS-COV-2 pseudovirus can be used according to the invention forthe general treatment for all pathogenic infections as the MBL-2 protein and its complexes are selective for binding mannose residues present on the glycocalyx of the membranes of pathogens.
  • the amount of protein in the liver extract that inhibited the Covid replication in vitro is between 500 ug/mL and up to 1000 ug/mL.
  • the corresponding mouse blood plasma levels of the MBL-2 was in the 100ng/mL to 500ng/mL range in the three minicircle DNA treated mice with humanized livers on Day 14.
  • SARS-CoV-2 The entry of SARS-CoV-2 into human host cell is mediated by the Spike (S) protein, found on the surface of the virus. This interacts with the angiotensin converting enzyme 2 (ACE2) on the host cell.
  • ACE2 angiotensin converting enzyme 2
  • the data clearly shows replication inhibition by the liver protein extract binding to the spike(S) protein on the covid pseudo virus. Moreover, the liver protein extract binds to other glycoproteins on the covid pseudovirus.
  • the goal is to enrich proteins from liver tissue that potentially interact with N-glycans decorated on the SARS-COVID-2 RBD or M proteins.
  • Liver tissue lysate was incubated with COVID-19 microplate, in which RBD and M protein are immobilized. Proteins potentially interacted with RBD/M or N-glycans of RBD/M were retained after incubation.
  • a PNGase F was used to specifically release the N-glycan binding proteins from RBD/M.
  • RBD/M N-glycan specific binding proteins could be enriched for downstream analysis.
  • Glycan Array Assay Buffer (GAAB): TBST with 2 mM MgCI 2 and 2mM CaCI 2 Methods:
  • Peptide -N-Glycosidase F also known as PNGase F, is an amidase that cleaves between the innermost GIcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. PNGase F was used to specifically release the N-glycan binding proteins from RBD/M.
  • the final protein concentration obtained in step 8. of 0.08 mg/ml indicates that proteins from the mouse liver bound to the COVID-19 Microplate Spike + M Proteins decorated with N-glycans.
  • the Western Blot experiment to follow indicates the presence of MBL2 and MASP2 proteins in this 0.08 mg/ml concentrate.
  • the goal is to enrich proteins from liver tissue that potentially interact with mannan in the biotinylated mannan.
  • the biotinylated mannan was coated on the streptavidin-coated plate. Then the liver tissue lysate was incubated with the microplate, in which mannan is immobilized. Proteins potentially interact with mannan was retained after incubation.
  • a mannosidase was used to specifically release the mannose-binding proteins. Mannose specific binding proteins could be enriched for downstream analysis.
  • Glycan Array Assay Buffer (GAAB): TBST with 2 mM MgCI 2 and 2mM CaCI 2 Methods:
  • Mannosidase was used to specifically release the mannose-binding proteins.
  • Mannose specific binding proteins from the mouse liver namely MBL2 and MASP2 complexes were predicted to be bound to mannan.
  • the Western Blot experiment indicates the presence of MBL2 and MASP2 proteins in this 0.003 mg/ml concentrate from step 10.
  • Origene LC434596 MASP2 Human Over-expression Lysate protein standard shows a strong peak at 120KDa which may be interpreted as complexed with other molecules from the lysate.
  • Abeam ab151947 Recombinant MBL2 protein standard shows a strong peak at 39KDa which may be interpreted as complexed with other molecules from the cell lysate.
  • the glycocalyx binding ability to COVID-19 Microplate Spike + M Proteins electropherogram view depicts several minor and major peaks for both MBL2 and MASP2 proteins respectively.
  • the theoretical size of the recombinant protein of SEQ ID NO: 1 is 256 KDa.
  • Natural oligomers formed in human liver have the following the sizes:
  • [MBL-2 x 3] x 4 - Tetramer comprised of 3 homotrimers 305 KDa;
  • [MBL-2 x 3] x 3 - T rimer comprised of 3 homotrimers 228 KDa.
  • a monomeric MASP2 is 76kDa.
  • a monomeric subunit of MBL-2 is 26KDa.
  • MBL2 peaks can be seen at 41 KDa, 50KDa, 63KDa, 125KDa, 230KDa and 250KDa to 260KDa.
  • MASP2 peaks can be seen at 42KDa, 50KDa, 64KDa and 230KDa to 260KDa.
  • Peaks 41 KDa, 50KDa, 63KDa, 125KDa, 230KDa can be interpreted as deriving from natural proteins of MBL2 and MASP2 assembled in the liver. Peaks 250KDa to 260KDa can be concluded as covalently linked complexes derived from the engineered MBL2-MASP2 recombinant protein of the invention. Natural oligomers of MBL2 and MASP2 complexed together do not exist in human serum in the 250KDa to 260KDa range as measured by mass spectroscopy, which is explained in Sectionlet et al., 2005 (doi.org/10.4049/jimmunol.174.5.2870).
  • the peaks observed in the Western Blot are MBL2 proteins that interact with N-glycans of SARS-COVID-2 RBD & Mannan proteins respectively.
  • MASP2 proteins bind MBL2 proteins and so present peaks at nearly the same KDa as MBL2 proteins in the Western Blot Electropherogram. This observation indicates presence of MBL2 - MASP2 complex formation.
  • PNGase F was used to specifically release the N-glycan binding proteins from RBD/M.
  • a mannosidase was used to specifically release the mannose-binding proteins.
  • a monomeric MASP2 peak of 76kDa was not detected.
  • a monomeric subunit MBL-2 peak of 26KDa was not detected. Neither was a homotrimer of MBL2 of 76KDa detected.
  • MBL2 - MASP2 complexes are interpreted to be from natural humanized mouse liver expression and also from the covalently linked MBL2 - MASP2 complex of this invention.
  • MBL2 and MASP2 entities are effective binding to the N-glycans of SARS-COVID-2 RBD & Mannan proteins respectively by MBL2 and MASP2 entities from natural oligomers in liver as well as the minicircle expressed proteins of this invention.
  • SEQ ID NO: 1 Exemplary recombinant protein of the invention with the following domain configuration: MBL-2 - L - MBL-2 - L - MBL-2 - MBL-2 - MASP2 - MASP2.

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Abstract

L'invention concerne des protéines recombinantes utiles dans le traitement de maladies ou de troubles associés à une déficience en MBL-2. L'invention concerne également des acides nucléiques codant pour de telles protéines recombinantes, un vecteur d'expression comprenant lesdits acides nucléiques, ainsi que des exosomes comprenant les protéines recombinantes, les acides nucléiques et/ou les vecteurs d'expression. L'invention concerne en outre des cellules comprenant lesdites protéines recombinantes, des acides nucléiques, des vecteurs d'expression et/ou des exosomes. L'invention concerne également des méthodes de traitement de maladies ou de troubles associés à une déficience en MBL-2.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000070043A1 (fr) * 1999-05-14 2000-11-23 Steffen Thiel Lectine humaine de recombinaison se fixant a la mannane
WO2003090774A1 (fr) * 2002-04-24 2003-11-06 The Council Of The Queensland Institute Of Medical Research Lectine de fixation du mannose et utilisations correspondantes
US7060267B2 (en) * 1997-04-03 2006-06-13 Jensenius Jens Chr MASP-2, a complement-fixing enzyme, and uses for it

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Publication number Priority date Publication date Assignee Title
US7060267B2 (en) * 1997-04-03 2006-06-13 Jensenius Jens Chr MASP-2, a complement-fixing enzyme, and uses for it
WO2000070043A1 (fr) * 1999-05-14 2000-11-23 Steffen Thiel Lectine humaine de recombinaison se fixant a la mannane
WO2003090774A1 (fr) * 2002-04-24 2003-11-06 The Council Of The Queensland Institute Of Medical Research Lectine de fixation du mannose et utilisations correspondantes

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GASPAR VÍTOR, MELO-DIOGO DUARTE DE, COSTA ELISABETE, MOREIRA ANDRÉ, QUEIROZ JOÃO, PICHON CHANTAL, CORREIA ILÍDIO, SOUSA FANI: "Minicircle DNA vectors for gene therapy: advances and applications", EXPERT OPINION ON BIOLOGICAL THERAPY, INFORMA HEALTHCARE, vol. 15, no. 3, 4 March 2015 (2015-03-04), pages 353 - 379, XP093069435, ISSN: 1471-2598, DOI: 10.1517/14712598.2015.996544 *
HWANG HYUN-JU, HAN JIN-WOO, JEON HANCHEOL, HAN JONG: "Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin", BIOMOLECULES, vol. 8, no. 4, pages 146, XP093069434, DOI: 10.3390/biom8040146 *
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