Field of Invention

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. The invention also relates to compositions comprising said recombinant protein, nucleic acid sequence, expression vector exosome, and/or cell. Further, 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. Finally, the invention relates to methods of treating a disease or disorder associated with MBL-2 deficiency.

Background

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 (MBL), also known as mannosebinding lectin or mannan-binding protein (MBP), is an important factor in this system. 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.

Activation of component C3 is central to the pathways of complement and leads directly to neutralization of pathogens and stimulation of adaptive immune responses. This process is mediated by another protein called MASP (Mannan-binding lectin serine protease). Mannosebinding lectin (MBL)-associated serine protease-2 (MASP- 2) is an indispensable enzyme for the activation of the lectin pathway of complement system. The convertases that catalyse this reaction assemble from fragments of complement components via multistep reactions. In the lectin pathway, mannose-binding lectin (MBL) and ficolins bind to pathogens and activate MBL-associated serine protease-2 (MASP-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. 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.

Activation of the complement system results in the phagocytosis of the antigen. This process is mediated by opsonins - proteins of the innate and adaptive immune system that facilitate phagocytosis and cell lysis by “marking” an antigen. Opsonization, then, is the modification of antigens by opsonins to make them more accessible to phagocytic cells and other immune cells. Researchers estimate that between 5 and 30% of people have some level of 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. Mutations in the MBL2 DNA sequence are responsible for the chronic nature of several disease like AIDS, herpes and cancer. Further, when the condition is combined with chronic inflammatory or immune system dysfunction, then MBL deficiency becomes a much larger problem. 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). Unlike 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. There remains a need in the art for methods of treating diseases and disorders associated with MBL deficiency. The present invention aims to at least in part address this need by providing recombinant proteins that correctly oligomerize in order to be functional.

Summary of Invention

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.

As shown in the Examples section below, 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. 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 (such as for example the recombinant protein of SEQ ID NO: 1) 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. Furthemore, through a Sars-Cov-2 Pseudovirus replication inhibition assay, the inventor has confirmed that the recombinant proteins form functional complexes, which suggests that they have the ability to correctly oligomerize. The ability to cause the production of MBL-2 and MASP-2 proteins in vivo may be a useful tool in treating diseases or disorders associated with a decrease in MBL-2 or MASP-2 production, giving rise to the present invention as described hereinbelow.

In one aspect, the present invention provides a recombinant protein comprising amino acid sequences of at least three MBL-2 proteins, or fragments or variants thereof.

Suitably, the recombinant protein may comprise amino acid sequences of at least four MBL-2 proteins, or fragments or variants thereof.

Suitably, the recombinant protein may comprise an amino acid sequence of at least one MASP-2 protein, or a fragment or variant thereof.

Suitably, the recombinant protein may comprise amino acid sequences of at least two MASP- 2 proteins, or fragments or variants thereof.

Suitably, the recombinant protein may comprise one or more linkers.

Suitably, 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.

Suitably, 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 - MBL- 2); and SEQ ID NO: 8 (MBL-2 - L - MBL-2 - L - MBL-2 - L - MBL-2).

In one aspect, the present invention provides a nucleic acid sequence encoding the recombinant protein of the invention.

In one aspect, the present invention provides an expression vector comprising the nucleic acid sequence of the invention.

Suitably, the expression vector may be a minicircle DNA.

Suitably, the expression vector may comprise a promoter operably linked to the nucleic acid sequence of the invention. Suitably, the promoter may be a Human elongation factor-1 alpha (EF-1 alpha) promoter.

Suitably, the expression vector may further comprise an IRES.

In one aspect, the present invention provides an exosome comprising the recombinant protein, the nucleic acid sequence, and/or the expression vector of the invention.

In one aspect, the present invention provides a cell comprising the recombinant protein, the nucleic acid sequence, the expression vector, and/or the exosome of the invention.

Suitably, the cell may be an isolated cell, optionally a human isolated cell.

Suitably, the cell may localise to an infection site.

Suitably, the cell may be a mesenchymal stem cell, fibroblast or hepatocyte.

In one aspect, 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.

In one aspect, 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.

Suitably, 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.

Suitably, the mutation may be homozygous or heterozygous.

Suitably, 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.

Suitably, 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).

Suitably, the infection may be: a bacterial, viral, protozoal, and/or yeast infection; and/or a persistent, a recurrent, and/or a severe infection. Suitably, the subject with the infection may have cancer.

Suitably, a disease or disorder associated with MBL-2 deficiency may result in MBL-2 deficiency. As explained in more detail elsewhere in the present specification, a deficiency of human mannose-binding lectin (MBL) may result in increased risk and severity of infections and autoimmunity.

In one aspect, 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.

Except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to the recombinant protein, nucleic acid sequence, expression vector, cell, and/or exosome in accordance with the invention, and the uses thereof.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

Brief Description of the Drawings

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: 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 2927 to 3670); First MASP2 - nucleic acids 3671 to 5728; and Second MASP - nucleic acids 5729 to 7786.

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.

Figure 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.

Figure 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. On day 8, 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 recombinant proteins bind to pathogen particles in the blood and lymph circulation of patients with septicaemia.

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. In figure 7A MBL2 peaks can be seen at 41 KDa, 50KDa, 63KDa, 125KDa and 250KDa to 260KDa. In figure 7B, MASP2 peaks can be seen at 42KDa, 50KDa, 64KDa, 230KDa to 250KDa to 260KDa. The sizes of the peaks between 250KDa and 260KDa confirm that the recombinant proteins have oligomerized, which is required for function. 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-

2 x 3 - Homotrimer structural polypeptide 76 KDa; [MBL-2 x 3] x 4 - Tetramer comprised of

3 homotrimers 305 KDa; and [MBL-2 x 3] x 3 - Trimer comprised of 3 homotrimers 228 KDa. Monomeric MASP2 is 76kDa. Monomeric MBL-2 is 26KDa. Glycosylation may contribute to the KDa molecular weight of the bands observed in the Western blots.

Figure 8 A Cartoon representation of Western Blots - SDS-PAGE gradient electrophoresis from individuals with different MBL2 genotypes. B Table summary of peaks observed on the Western Blot Electropherogram. 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.

Figure 9 Schematic diagram showing performed experiments

Detailed Description

Recombinant proteins and nucleic acid sequences of the invention

In one aspect, the present invention provides a recombinant protein comprising amino acid sequences of at least three MBL-2 proteins, or fragments or variants thereof.

The term “recombinant protein” as used herein 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.

The terms “MBL-2” or “MBL2” as used herein refer to the protein Mannose-binding protein C encoded by the gene MBL2. Suitably 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). In humans 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. Thus 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). These homotrimers may further form bigger oligomeric structures, comprising of three, four, or more homotrimers. In humans, 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.

In the context of the present disclosure 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. Accordingly, 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.

Suitably, 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. Suitably, 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. Merely by way of example the recombinant protein may comprise an amino acid sequence of at least three MBL-2 proteins and at least one MASP-2 protein. Suitably, the recombinant protein may comprise an amino acid sequence of at least four MBL-2 proteins and at least one MASP-2 protein. Suitably the recombinant protein may comprise an amino acid sequence of at least three MBL-2 proteins and at least two MASP-2 protein. Suitably, the recombinant protein may comprise an amino acid sequence of at least four MBL-2 proteins and at least two MASP-2 protein.

The term “MASP-2 protein” as used herein refers to the protein Mannan-binding lectin serine protease 2 encoded by the gene MASP2. Suitably the polypeptide is encoded by human gene MASP2, and has the following amino acid sequence: MRLLTLLGLL CGSVATPLGP KWPEPVFGRL ASPGFPGEYA NDQERRWTLT APPGYRLRLY FTHFDLELSH

LCEYDFVKLS SGAKVLATLC GQESTDTERA PGKDTFYSLG SSLDITFRSD YSNEKPFTGF

EAFYAAEDID ECQVAPGEAP TCDHHCHNHL GGFYCSCRAG YVLHRNKRTC

SALCSGQVFT QRSGELSSPE YPRPYPKLSS CTYSISLEEG FSVILDFVES FDVETHPETL

CPYDFLKIQT DREEHGPFCG KTLPHRIETK SNTVTITFVT DESGDHTGWK IHYTSTAQPC

PYPMAPPNGH VSPVQAKYIL KDSFSIFCET GYELLQGHLP LKSFTAVCQK

DGSWDRPMPA CSIVDCGPPD DLPSGRVEYI TGPG TTYKA VIQYSCEETF

YTMKVNDGKY VCEADGFWTS SKGEKSLPVC EPVCGLSART TGGRIYGGQK

AKPGDFPWQV LILGGTTAAG ALLYDNWVLT AAHAVYEQKH DASALDIRMG

TLKRLSPHYT QAWSEAVFIH EGYTHDAGFD NDIALIKLNN KWINSNITP ICLPRKEAES

FMRTDDIGTA SGWGLTQRGF LARNLMYVDI PIVDHQKCTA AYEKPPYPRG SVTANMLCAG LESGGKDSCR GDSGGALVFL DSETERWFVG GIVSWGSMNC GEAGQYGVYT KVINYIPWIE NIISDF (SEQ ID NO: 11).

The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a 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. As used herein, 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. In the context of the present specification, 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 . Suitably 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. In the context of MBL-2, 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. In the context of MASP2, 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. The term “fragment” as used herein 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 475 contiguous amino acid residues, at least 500 contiguous amino acid residues, at least 525 contiguous amino acid residues, at least 550 contiguous amino acid residues, at least 575 contiguous amino acid residues, at least 600 contiguous amino acid residues, at least 625 contiguous amino acid residues, at least 650 contiguous amino acid residues, or at least 675 contiguous amino acid residues. For example in the context of MBL- 2 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. For example, in the context of MASP-2 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.

The term "variant" as used herein 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). In one embodiment, 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. In another embodiment, 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. In another embodiment, 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).

As used herein, "sequence identity" or "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. 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. When 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.

It will be appreciated that in the context of the present disclosure, some or all of the domains of the recombinant proteins (for example each one of the three MBL-2 proteins, or fragments or variants thereof, and optionally each one of the two MASP-2 proteins or fragments or variants thereof, being a domain of the recombinant protein) may be the same or different. Thus, merely by way of example, the recombinant protein may comprise for example two full length MBL-2 proteins, and one MBL-2 fragment or variant, or vice versa. By the same taken when 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.

Suitably, the recombinant protein may comprise one or more linker. The term “linker” as used herein 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. In the present disclosure, the linker may be abbreviated as “L”. Thus, reference to, for example MBL-2 - L - MBL-2 refers to a MBL-2 domain followed by a linker followed by a second MBL-2 domain. For avoidance of doubt, 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. Choosing a suitable linker is within the capabilities of those having ordinary skill in the art. Suitably, 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. In a recombinant protein of the invention comprising of more than one linker, some or all of the linkers may be the same or different.

Suitably, 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 - MBL-2 - MBL-2); and SEQ ID NO: 8 (MBL-2 - L - MBL-2 - L - MBL-2 - L - MBL-2). In each of these configurations, it will be appreciated that one or more of the domains may be a fragment or variant of the parent or reference polypeptide.

Suitably, 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.

In a suitable embodiment 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.

Whilst SEQ ID NO: 1 shown hereinbelow contains 12 glycine residue linkers between the first and second, and second and third domains, it will be appreciated that different linkers may be used, the location of the linkers may different, or indeed the linkers may be not present at all. Merely by way of example, in the context of SEQ ID NO: 1 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.

Suitably, 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. Suitably, 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. In one aspect, the present invention provides a nucleic acid sequence encoding the recombinant protein of the invention.

The term “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. Unless otherwise limited “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. The term 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. The term 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.

Suitably, 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.

Expression Vectors

Suitably, 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. Herein the terms “vector” and “expression vector” are used interchangeably.

Suitably, the vector may be any vector capable of transferring the nucleic acid sequence (for example DNA) encoding the recombinant protein to a cell. Suitably, 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. In this context, the term “infection” is used to mean the process by which a virus transfers genetic material to its host or target cell. Suitably, 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. In such cases, the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques. Generally, any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.

Other 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. Such integrating and episomal vectors are well known to those skilled in the art and are fully described in the body of literature well known to those skilled in the art.

More suitably, 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. First of all, minicircle DNA is typically 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 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. In addition, minicircle DNA sequences tend to be smaller and easier for host cells to replicate. Suitably, the minicircle DNA may have a sequence according to SEQ ID NO: 9.

Suitably, 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).

As explained elsewhere in the present specification, the invention also provides a cell transformed or transfected with the vector of the present invention. The cell may be any mammalian cell. Suitably, the cell may be a rodent or human cell. Suitably, the cell may be an isolated cell. Exemplary cells are described hereinbelow.

Numerous techniques are known and are useful according to the invention for delivering the vectors described herein to cells, including 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).

Suitably, the expression vector may comprise a promoter operably linked to the nucleic acid sequence of the invention. Suitably, the promoter may be any promoter that allows the expression of the recombinant protein of the invention. The terms "promoter" as used herein, 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. Suitably, in the present invention the promoter may be Human elongation factor- 1 alpha (EF-1 alpha) promoter.

Suitably, 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.

Exosome

In one aspect, the present invention provides an exosome comprising the recombinant protein, the nucleic acid sequence, and/or the expression vector of the invention. Not all therapeutic delivery vehicles need to be cells (which are discussed hereinbelow). In some embodiments 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. As a result, they 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. Suitably, the expression vector may be a minicircle DNA. Thus suitably, the exosomes of the invention may comprise an expression vector (such as a minicircle DNA) of the present invention.

Cells

In one aspect, the present invention provides a cell comprising the recombinant protein, the nucleic acid sequence, the expression vector, and/or the exosome of the invention. Suitably, the cell may be an isolated cell. Suitably, the cell may be a mammalian cell, for example a human or rodent isolated cell. Suitably, 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. 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. Suitably, 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. Bone marrow-derived mesenchymal stem stromal cells (BM-MSCs) 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).

Researchers have been taking advantage of 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. For example, 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. 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.

In the context of the present disclosure, 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.

In one example, isolated cells (comprising for example the expression vector as disclosed herein and/or loaded with exosomes transferred with the expression vector) may be delivered to the subject in need of the recombinant protein of the invention. In such an example 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).

In another example, 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. In such an example the expressed recombinant protein and/or exosome may be purified (or otherwise prepared) for delivery to the subject. In such an example the cells may function as an expression chamber for the recombinant protein.

In such an embodiment, it is not necessary for the cells to localise to an infection site. Suitably, the cells may be ones that are capable of highly expressing the recombinant protein or releasing exosomes. Suitably, the cells may be hepatocytes.

Treatments

In one aspect, 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. As used herein, the term “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.

In one aspect, 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.

Suitably, a disease or disorder associated with MBL-deficiency may be primary MBL-2 deficiency.

Suitably, the primary MBL-2 deficiency may be caused by a mutation in the MBL-2 gene and/or MASP2 gene. Suitably, the mutation may be homozygous or heterozygous. Suitably, the mutation may be in the coding or non-coding region of MBL-2 or MASP2.

In another embodiment, 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. It will be appreciated that 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). Suitably, the infection may be: a bacterial, viral, protozoal, and/or yeast infection; and/or a persistent, recurrent, and/or severe infection. Suitably, the subject with the infection may have cancer.

Suitably, 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.

MBL-2 deficiency as used herein 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. Thus, suitably, in the context of the present disclosure, 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). It the context of other mammals the skilled person will be easily able to determine is a subject has MBL-2 deficiency simply by comparing the levels of MBL-2 to a control sample or reference value. Suitably, 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.

In one aspect, 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. Suitably, 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.

The term “a therapeutically effective amount” as used herein 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.

It will be appreciated that 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.

A further aspect, provided herein is a composition 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.

As used herein, "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. The term “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.

EXAMPLES

1. Manufacture of the minicircle DNA construct with exemplary recombinant protein of the invention (SEQ ID NO: 1), and transfection of exosomes with minicircle DNA

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. In this example, 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. 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). Specifically, 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. Moreover, 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. However, one particular MSC for example will not act as an expression chamber for the therapeutic protein. Moreover, in this invention, 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. As shown in the diagram in Figure 3, 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. 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. In one experiment, stem cells were transfected with a commercially available transfection reagent made by System Biosciences Inc. In another experiment 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. As discussed above, MBL binds calcium-dependently to sugar residues mannose, fucose and N-acetyl glucosamine (GIcNAc) on the surface of pathogens thereby marking them for opsonization. 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. Once the minicircle DNA is generated, it has to be introduced into cells that express the gene product coded for by the DNA.

Commercially available transfection reagents can be used to transfect cells and exosomes with the engineered minicircles DNA. Polyplus, Metaphectene and Exo-Fect™ are examples of three transfection reagents tested for the transfection process.

Exo-Fect™ (SBI - Systems BioScience Inc.) was effective in the mini circle DNA transfection of exosomes. Metafectene (Bion Tex ) was effective in the minicircle DNA transfection of 2 cancer cell lines namely Huh7 and Hek293. Polyplus transfection agent was not efficient in the transfection of mini circle DNA of this invention in cells.

In the later experiments described herein, exosomes transfected with the minicircle DNA in accordance with manufacturers protocol for Exo-Fect™ 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.

2. Expression of recombinant protein according to SEQ ID NO: 1 in the minicircle DNA in Huh-7 human hepatocyte carcinoma (JCRB #0403) and 293T-hsACE2 cells (Integral Molecular C-HA102, HEK293T cells engineered to highly express Human ACE2).

Materials and methods

Minicircle plasmid DNA (“mcDNA”) was provided to Antibody Solutions for cell transfection studies. 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. Slowly and gently, 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.

A sample of conditioned supernatant was collected from each transfection condition at 24, 48, and 72 hours post-transfection. Using commercial ELISA kits for detection of MBL2 (BosterBio) and MASP2 (Biomatik), the supernatants were evaluated alongside standards and control medium to assess levels of protein secretion from the transfected cells. Briefly, serial dilutions of supernatants, standards, or controls were added to ELISA plates pre-coated with either MBL2 or MASP2 capture antibody. Plates were incubated for 1.5 hours at room temperature, then washed 4X, and either MBL2 or MASP2 specific HRP-conjugated detecting antibody was added. After another 1.5 hour incubation, plates were washed 4X and TMB substrate was added. After 15-20 minute incubation, the TMB reaction was stopped and absorbance read at 450 nm. “TFN Lib4” gave the highest detectable level of MBL2 secretion, however MASP2 was not detectable in any of the test samples.

Additionally, GFP expression was evaluated in the cells 72 hours post-transfection using an EMDMillipore Guava 8HT flow cytometer. TFN-Lib 4 and TFN-Lib5 exhibited higher levels of GFP expression at 72 hours compared to TFN-Lib 1 , 2, and 3.

Based on results of the pilot transfection experiment, 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. For the transfected Huh-7 cells, 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.

Results and Conclusion Cells were transfected with a minicircle DNA plasmid (mcDNA) containing genes for the expression of Human MBL2 and Human MASP2, as well as a GFP expression marker. 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.

3. Expression of recombinant protein (SEQ ID NO:1) in the minicircle DNA in humanized liver of mouse by tail vein injection.

Materials and methods

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.

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. Peripheral blood was collected on D-7, D4, D8 and at sacrifice (D14). Human 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.

Animals:

The study was carried out with female PIRF (IL-2R-gamma -/-, Rag2 -/-, Fah -/-, Por conditional KO) mice engrafted by injection of healthy adult hepatocytes in the spleen following TCS’s proprietary humanization protocol. Only mice with an amount of human albumin above 1 mg/mL were used. All procedures described in this study were reviewed and approved by the local ethic committee (CELEAG) and validated by the French Ministry of Research. Mice were hosted by groups of 3 individuals in TCS BSL-2 animal facility. Each mouse was uniquely identified. Animals were housed in a ventilated cage (type II (16x19x35 cm, floor area = 500 cm ² )) under the following controlled conditions:

• Room temperature (22±2°C)

• Hygrometry (55±10%)

• Photoperiod (12:12-hour light-dark cycle 7am:7pm)

• Water and food (Ref. 2018, Harlan France) available ad libitum

Mice were acclimated to the environment for 7 days prior to the beginning of the experiment.

At DO and D7, the 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:

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.

Blood collection:

Blood was collected from the retro-orbital sinus in EDTA-coated tubes at D-7 (100 pL), D4 (70 pL), D8 (70 pL). At sacrifice, maximum volume of blood was collected in sodium citrate-coated tubes. The obtained plasmas were stored 1 hour at -20 °C and then at -80 °C until analysis or shipment to Z-Biotech.

Organ collection:

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.

Date

Days post- boost D-7 D4 D8 D14

ELISA Mouse ID

Proteinl 434077 2.01 E+05 1.78E+05 2.10E+05 4.76E+05

(pg/mL) MBL2 434079 4.21 E+04 3.77E+04 5.08E+04 1.05E+05 434081 2.63E+04 4.91 E+04 8.64E+04 1.35E+05

Table 1 : Increased expression of MBL2 proteins in mouse with humanized liver transfected with minicircle DNA by hydrodynamic tail vein injection.

Results and Conclusions

Quantitative measurement of Human MBL2/MBP proteins was performed using ELISA assay on plasma samples on D-7 before hydrodynamic DNA minicircle injection, on D4, D8 and at sacrifice on D14. Following the first MN530A-4x MBL2-MASP2 minicircle DNA injection, the MBL2/MBP protein level was similar to the one detected at baseline. After the second DNA injection on D7, 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. Following the second injection on D7, 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.

The increased expression of MBL2 proteins[ELISA] in mice with humanized liver transfected with minicircle DNA by hydrodynamic tail vein injection was observed at Transcure mouse facility, France (Table 1).

An IV injection of the minicircle DNA in saline as a vehicle will be taken up by liver cells with no need of any lipid or other vehicle to enhance the biologic uptake by liver cells. Importantly no observable toxicity was observed in the three mice with humanized livers treated by hydrodynamic tail vein injection over 14 days. This data supports the claim that IV injection of the mcDNA in saline will express in human liver cells. There is no need for exosomes or fibroblasts or mesenchymal stem cells as delivery vehicles. Exosomes allow for mcDNA expression in most cells including liver that switch on the EF1 alpha promoter. Fibroblasts and mesenchymal stem cells are known to home in on diseased tissue and cancer cells.

4. Expression of recombinant protein (SEQ ID NO: 1) in the minicircle DNA in skin fibroblast.

Materials and Methods Human Dermal Fibroblast (Lot#201491) was treated with or without Exosome (see item 1 of Example section for details on exosome transfection with minicircle DNA) twice at Day 0 and Day 3. Cell pellet was collected on Day 8 post treatment and total RNA was extracted using Zymo RNA Miniprep kit according to the instructions. 1 pg RNA was used for the cDNA synthesis by Transcriptor Universal cDNA master (Sigma). Gene expression was measured by qPCR using Lightcycler 96. The expression of MBL2, MASP2 and MBL2-Trimer was measured by qPCR using 2 sets of primers separately. GAPDH serves as internal control. The expression of each gene detected by 2 set of primers were compared to their own GAPDH internal control firstly, followed by normalization to the untreated sample 1 . # indicates each set of primer targeted the individual gene.

Table 2 - primers used for qPCR

Gene Primers Amplicon size (bp)

MBL2- 5’-CCCTGTTTCCATCACTCCCTC-3’ (SEQ ID NO: 12) 92

Internal 5’-GCAGGTCTTTTGGGCATCC-3’ (SEQ ID NO: 13)

5’-TAGTAGCCTGGCTGCCTCAGAA-3’ (SEQ ID NO: 14) 119

5’-CACCATTGGTCAGGAAGAACTTG-3’ (SEQ ID NO: 15)

MASP2- 5’-TCCAGGGGAGTATGCCAATGA-3’ (SEQ ID NO: 16) 96

Internal 5’-TCCAGGTCGAAGTGGGTGAA-3’ (SEQ ID NO: 17) 5’-CTGGCAAGGACACTTTCTACTC-3’ (SEQ ID NO: 18) 81

5’-TGAACGGCTTCTCGTTGGAG-3’ (SEQ ID NO: 19)

MBL2-Cross- 5’-CAGGCAATAGACTGACCTACAC-3’ (SEQ ID NO: 20) 101 liner 5’-GAAGAACGGCCAGTGGAAT-3’ (SEQ ID NO: 21)

5’-AGGCAATCGCCTGACTTATAC-3’ (SEQ ID NO: 22) 102

5’-GAAGAATGGACAGTGGAACGA-3’ (SEQ ID NO: 23)

GAPDH 5’-GTCTCCTCTGACTTCAACAGCG-3’ (SEQ ID NO: 24) 5’-GTCTCCTCTGACTTCAACAGCG-3’ (SEQ ID NO: 25)_

Results

- QPCR result using 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.

5. Inhibition study of Covid by Hu liver extract expressing recombinant protein (SEQ ID NO: 1) of minicircle DNA administered by tail vein injection.

Materials and methods

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.

Reagents:

Cells expressing ACE2 receptor - A549

Cells not expressing ACE2 receptor (available upon request) - NIH3T3

Cell culturing - DMEM complete growth medium with 10% FBS and 2% penicillin-streptomycin, Trypsin, Cell counter hemacytometer, U shape 96-well cell culture plate, 37C incubator with 5% CO2, Microscope, Pipettes, syringes, & tubes, Sterile serological pipettes (5 ml, 10 ml, and 25 ml), Micropipettes, Multichannel micropipettes, Micropipette tips (10 pl, 200 pl, and 1000 pl), Plastic syringe (1 ml), Polypropylene sterile conical tubes (15 ml and 50 ml), 96-well cell culture plate (white plate with flat, clear bottom)

Pseudovirus - Supernatant containing the pseudoviruses rVSV-AG-pCAGGS/Spike- luciferase

Detection - Firefly luciferase substrate; Luminometer (BioTek synergy HT)

Other: 70% Ethanol, Millex GV Filter Unit (Hydrophilic Durapore membrane) and sample(s) Procedure:

Day 0: Culture cells-of-interest

Day 1 : Seed cells-of-interest - 20,000 cells per well are seeded in a 96-well white cell culture plate with flat, clear bottom for 24 hours at 37°C I 5% CO2.

Day 2: Cell infection with generated rVSV pseudoviruses

Tissue lysate preparation: sample dilution: 1 , 5, 10, 100, 500, 1000 pg/ml

1) Combined protein extracts from six liver lobes (Sample ID: 434079-CL, CL2, LM1 , LM2, RM1 , and RM2) into one liver lysate sample.

2) Measured sample concentration (100 mg/ml) using the BCA kit.

3) 1000 pg/ml sample: Mixed 14.4 pL stock solution with 705.6 pL DMEM. Added 60 ul of diluted sample to the each well of 96-well plate (sample now 2000 pg/ml). Added 60 pL pseudovirus to the plate (sample now 1000 pg/ml).

4) 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).

5) 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).

6) 10 pg/ml sample: Mixed 0.29 pL stock solution with 719.71 pL DMEM. Added 60 ul of diluted sample to the each well of 96-well plate (sample now 20 pg/ml).

Added 60 pL pseudovirus to the plate (sample now 10 pg/ml).

7) 5 pg/ml sample: Mixed 0.15 pL stock solution with 719.85 pL DMEM. Added 60 ul of diluted sample to the each well of 96-well plate (sample now 10 pg/ml).

Added 60 pL pseudovirus to the plate (sample now 5 pg/ml).

8) 1 pg/ml sample: Mixed 0.03 pL stock solution with 719.97 pL DMEM. Added 60 ul of diluted sample to the each well of 96-well plate (sample now 2 pg/ml).

Added 60 pL pseudovirus to the plate (sample now 1 pg/ml).

9) Incubated the plate for 1 hour at 37°C / 5% CO2.

10) Removed media from cells prepared on Day 1. Added 100 pL of diluted compound samples and pseudovirus mixture to the 96-well plate seeded with cells.

11 ) Incubated the plate for 1.5 hours at 37°C / 5% CO2.

12) Added 100 pl pre-warmed DMEM complete growth medium. 13) Incubated the plate for 24 hours at 37°C / 5% CO2.

Sample Preparation:

If serum or plasma, 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 (mock infection): No pseudovirus was added to account for background noise.

Day 3: Luciferase assay

- Removed 200 pl of supernatants from each well.

- Added 50 pl of luciferase substrate to each well. Shaked the plate for 3 minutes at 300-600 rpm to lyse cells and equilibrate samples.

- Measured luminescence in a luminometer with an integration time of 0.5-1 second and recorded the results.

Day 4: Data analysis

Background subtraction: The average optical density (OD) reading of the “mock infection” negative control replicates is subtracted from all samples’ OD readings.

Inhibition rate (%):

Inhibition rate = [(“No Sample” positive control OD - sample OD)/ “No Sample” positive control OD] x 100%

Results

This experiment shows the therapeutics promise of the present invention when treating Covid- 19. Specifically, 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. As described above 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.

As described above 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. In another aspect 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.

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. 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.

6. Enrichment of liver tissue proteins that interact with N-glycans ofSARS-COVID-2 RBD & M proteins

Background:

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.

Materials:

1 . Liver extract (47.77 mg/ml), two tubes (~800 ul each), used one tube

2. COVID-19 Microplate Spike + M Proteins (XpressBio, SP866C), the strips with black mark is Tissue Control (TC) antigen 3. PNGase F (NEB P0709L, 75000 units)

4. RIPA Lysis and Extraction Buffer (Thermo Fisher 89900)

5. Glycan Array Assay Buffer (GAAB): TBST with 2 mM MgCI ₂ and 2mM CaCI ₂ Methods:

1. Prepared liver extract samples (15 mg/ml) for 16 wells of RBD/M. Used GAAB buffer as dilution buffer to prepare the sample solution.

2. Added 10Oul of above prepared sample solution to each well and incubate at room temperature for 4 hours; rotating the well at 120 rpm to make sure the sample is sufficient interact with immobilized RBD/M.

3. After incubation, washed the well with 10Oul GAAB /well, 4 times, 4-5 minutes each at 120 rpm rotation.

4. After washing by GAAB, washed one more time with 100 ul RIPA buffer, 4-5 minutes at 120 rpm.

5. Prepared PNGase releasing and enrichment solution by diluting PNGase F in the RIPA buffer.

PNGaseF stock = 500 U/uL

Prepare as 500 U/30uL, namely, 16.67 U/uL

Prepare total 540uL of solution: adding 18 uL of PNGaseF in 540uL of RIPA buffer.

6. Added 30 uL of above solution into each well and incubate at room temperature for overnight (rotating at 80 rpm). Make sure there is a cover on the top to prevent evaporation.

7. After incubation, transferred solution to a 1 ,5mL tube and all the target proteins were enriched in the solution.

8. Measured protein concentration using BOA protein assay. The final protein concentration is 0.08 mg/ml.

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.

7. Enrichment of liver tissue proteins that interact with mannose of mannan

Background:

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.

Materials:

1 . Liver extract (47.77 mg/ml), two tubes (~800 ul each), used one tube

2. Streptavidin-coated plate (Thermo Fisher 15125)

3. Mannosidase (Sigma M7257)

4. RIPA Lysis and Extraction Buffer (Thermo Fisher 89900)

5. Glycan Array Assay Buffer (GAAB): TBST with 2 mM MgCI ₂ and 2mM CaCI ₂ Methods:

1 . Added 50 ul of Biotinylated Mannan (0.1 mg/ml) into each of 16 wells on the streptavidin-coated plate.

2. Incubated the plate at room temperature for 30 min then kept the plate in 4C refrigerator overnight.

3. Prepared enough liver extract samples (15 mg/ml) for 16 wells of RBD/M. Used GAAB buffer as dilution buffer to prepare the sample solution.

4. Added 10Oul of above prepared sample solution to each well and incubate at room temperature for 4 hours; rotating the well at 120 rpm to make sure the sample was sufficient interact with immobilized mannan.

5. After incubation, washed the well with 10Oul GAAB /well, 4 times, 4-5 minutes each at 120 rpm rotation.

6. After washing by GAAB, washed one more time with 100 ul RIPA buffer, 4-5 minutes at 120 rpm.

7. Prepare mannosidase releasing and enrichment solution by diluting mannosidase in the RIPA buffer (16 ul mannosidase in 480 ul RIPA).

8. Added 30 uL of above solution into each well and incubate at room temperature for one hour (rotating at 80 rpm). Make sure there is a cover on the top to prevent evaporation.

9. After incubation, transferred solution to a 1 ,5mL tube and all the target proteins were enriched in the solution. 10. Measured protein concentration using BCA protein assay. The final protein concentration is 0.003 mg/ml.

A 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 (below) indicates the presence of MBL2 and MASP2 proteins in this 0.003 mg/ml concentrate from step 10.

8. Western Blot -Electropherogram

Materials and Methods

Twelve lanes were run - six lanes for the detection of MBL2 with anti-MBL2 antibodies and six lanes for the detection of MASP2 with anti-MASP2 antibodies.

Table 3

Note: 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.

Humanized liver from one mouse was homogenized and the protein extract was used for this binding study.

Con: COVID-19 Microplate Spike + M Proteins (XpressBio, SP866C); and

Man: Biotinylated Mannan bound to Streptavidin-coated plate (Thermo Fisher 15125) were used for this study.

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.

Glycosylation will contribute to the KDa molecular weight of the bands observed in the Western Blots. Results

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 - Homotrimer structural polypeptide 76 KDa;

[MBL-2 x 3] x 4 - Tetramer comprised of 3 homotrimers 305 KDa; and

[MBL-2 x 3] x 3 - T rimer comprised of 3 homotrimers 228 KDa.

Post translational modifications are the reason for small variations in KDa molecular weights (DOI: 10.1111/sji.12441). A monomeric MASP2 is 76kDa. A monomeric subunit of MBL-2 is 26KDa.

In figure 7A MBL2 peaks can be seen at 41 KDa, 50KDa, 63KDa, 125KDa, 230KDa and 250KDa to 260KDa. In figure 7B, MASP2 peaks can be seen at 42KDa, 50KDa, 64KDa and 230KDa to 260KDa.

In Figure 8 bands detected can be compared with individuals with different MBL2 genotypes, doi: 10.1038/sj. gene.6364283. Of significance is the presence of a 50KDa band in all individuals with different MBL2 genotypes. The 50KDa band corresponds with the observations in Figures 7A and 7B. A 63KDa/64KDa band is not observed in all individuals with different MBL2 genotypes. A 63KDa peak is observed for MBL2. A 64KDa band observed for MASP2. This suggests a proteolytic cleavage product derived from a proportion of MBL2 - MASP2 complex of this invention.

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 Teillet 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. It can be concluded that these molecular entities do not bind effectively to the N-glycans of SARS-COVID-2 RBD & Mannan proteins respectively. A peak is observed corresponding with [MBL-2 x 3] x 3 - Trimer of 228 KDa comprising 3 homotrimers. This suggests that naturally occurring MBL2 trimers present in the humanized mouse liver extract have oligomerized, which is required for binding to carbohydrate moieties of N-glycans. Alternatively, naturally assembled [MBL-2 x 3] x 2 - Dimer of 152 KDa comprising 2 homotrimers and one MASP2 monomeric unit of 76 KDA, correspond with the peak observed at 228 KDa.

Without wishing to be bound by this hypothesis, the inventor hypothesises that all peaks less than 220 KDa are due to proteolytic cleavage of oligomerized MBL2 complexed with MASP2. The 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. However, of certainty is the 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. The implication of this binding in the pseudoviruses rVSV-AG-pCAGGS/Spike-luciferase experiment is very clear - Up to over 90% inhibition of viral replication at the highest liver fraction extract concentration. The data obtained in this in vitro experiment are significant as the inhibition was in human serum with whole liver extract expressing the genes of interest and not cell culture.

SEQUENCES

(MBL-2 - underlined; Linker (L) - italic ; MASP2 - bold)

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. MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKG E PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEM ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIK EE AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHL AVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSP GINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGK SPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQ A SVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDE D CVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSE TVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPG NPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFL TNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTG NRL TYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIMSLFPSLPLLLLSMV A ASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGP PGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLG KQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKT EG QFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIMRLLT LLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHF DLELSHLCEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNE K

PFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSA LC SGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFSVILDFVESFDVETHPETLCPYD FL KIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYPMAP P NGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKDGSWDRPMPACSIVDCG PPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSL PVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVY EQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKWINSN I TPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYEKPPYP R GSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQY GVYTKVINYIPWIENIISDFMRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYA ND

QERRWTLTAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQESTDTER APG KDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGG FYCSCRAGYVLHRNKRTCSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFS VILDFVESFDVETHPETLCPYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDE SGD HTGWKIHYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFT A VCQKDGSWDRPMPACSIVDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVN DGKYVCEADGFWTSSKGEKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGG TTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYT HDAGFDNDIALIKLNNKWINSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNL M YVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETER

WFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDF*

SEQ ID NO: 2 amino acid sequence of MBL-2 - L - MBL-2 - L - MBL-2

MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKG EKGE PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEM ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIK EE AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHL AVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSP GINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGK SPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQ A

SVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAG SDED CVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSE

TVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGP PG

NPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNK FFL

TNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVD LTGNRL TYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPI*

SEQ ID NO: 3 amino acid sequence of MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP-2 - L - MASP2

MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKG EKGE

PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTE M

ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQN LIKEE

AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCST SHL

AVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIAC SSP GINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGK

SPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCV KFQA

SVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAG SDED CVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSE

TVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGP PG

NPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNK FFL

TNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVD LTGNRL

TYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGG

MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRL RL

YFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDIT FRSD

YSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNK RT

CSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFSVILDFVESFDVETH PETLC

PYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQ PCPYP

MAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKDGSWDRPMPA CSI

VDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKG

EKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLT AA

HAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLN NKW

INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLMYVDIPIVDHQKCTA AYEKP

PYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGE A GQYGVYTKVINYIPWIENIISDFGGGGGGGGGGGGMRLLTLLGLLCGSVATPLGPKWPEP VFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAK VLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQV A PGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSALCSGQVFTQRSGELSSPEYPRP YPKLSSCTYSISLEEGFSVILDFVESFDVETHPETLCPYDFLKIQTDREEHGPFCGKTLP HRI ETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIF C ETGYELLQGHLPLKSFTAVCQKDGSWDRPMPACSIVDCGPPDDLPSGRVEYITGPGVTTY KAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVCGLSARTTGGRIYG GQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLS PHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKWINSNITPICLPRKEAESFMRTDDI GT ASGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDS CRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDF*

SEQ ID NO: 4 amino acid sequence of MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP2 MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKG E PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEM ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIK EE AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHL AVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSP GINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGK SPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQ A SVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDE D CVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSE TVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPG NPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFL TNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTG NRL TYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGG MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRL YFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRS D YSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRT CSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFSVILDFVESFDVETHPET LC PYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCP YP MAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKDGSWDRPMPACSI VDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKG EKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAA HAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKW INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYE KP PYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEA GQYGVYTKVINYIPWIENIISDF*

SEQ ID NO: 5 amino acid sequence of MASP2 - L - MBL-2 - L - MBL-2 - L - MBL-2 - L - MASP2

MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRL RL YFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRS D

YSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNK RT CSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFSVILDFVESFDVETHPET LC PYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCP YP MAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKDGSWDRPMPACSI

VDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTS SKG EKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAA HAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKW INSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYE KP PYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEA

GQYGVYTKVINYIPWIENIISDFGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSET VTCE DAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPS

GSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTN GEI MTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTY TN

WNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMSLF P SLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQG L

RGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIK KW LTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLG ITD

EKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCE FPI GGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPG

KDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDS SLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATP RN

AAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVL LLKN GQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMRLLTLLGLLCGSVATPLGPKWPEPV

FGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGA KVL ATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAP G EAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSALCSGQVFTQRSGELSSPEYPRPYP KLSSCTYSISLEEGFSVILDFVESFDVETHPETLCPYDFLKIQTDREEHGPFCGKTLPHR IET KSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIFCE T GYELLQGHLPLKSFTAVCQKDGSWDRPMPACSIVDCGPPDDLPSGRVEYITGPGVTTYK

AVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVCGLSARTTGGRIY GG QKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLSP HYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKWINSNITPICLPRKEAESFMRTDDIG TA SGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSC

RGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDF *

SEQ ID NO: 6 amino acid sequence of MBL-2 - MBL-2 - MBL2 - MBL-2

MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKG EKGE

PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTE M

ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQN LIKEE AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHL AVCEFPIMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRD GT KGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERK

ALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAE NGAIQ NLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVP CSTSHLAVCEFPIMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFP GKD GRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLA

ASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATP RNAA

ENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLL KNGQ

WNDVPCSTSHLAVCEFPIMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACS SPGIN GFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSP DGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQAS V

ATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSD EDCV LLLKNGQWNDVPCSTSHLAVCEFPI*

SEQ ID NO: 7 amino acid sequence of MBL-2 - MBL-2 - MBL-2

MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKG EKGE

PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTE M

ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQN LIKEE AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHL AVCEFPIMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRD GT KGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERK

ALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAE NGAIQ NLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVP CSTSHLAVCEFPIMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFP GKD GRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLA ASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNA A ENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNG Q WNDVPCSTSHLAVCEFPI*

SEQ ID NO: 8 amino acid sequence of MBL-2 - L - MBL-2 - L - MBL-2 - L - MBL-2 MSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKG E PGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEM ARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIK EE AFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHL AVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSP GINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGK SPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQ A SVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDE D CVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGMSLFPSLPLLLLSMVAASYSE TVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGEPGQGLRGLQGPPGKLGPPG NPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMARIKKWLTFSLGKQVGNKFFL TNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKEEAFLGITDEKTEGQFVDLTG NRL TYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAVCEFPIGGGGGGGGGGGGM SLFPSLPLLLLSMVAASYSETVTCEDAQKTCPAVIACSSPGINGFPGKDGRDGTKGEKGE P GQGLRGLQGPPGKLGPPGNPGPSGSPGPKGQKGDPGKSPDGDSSLAASERKALQTEMA RIKKWLTFSLGKQVGNKFFLTNGEIMTFEKVKALCVKFQASVATPRNAAENGAIQNLIKE EAF LGITDEKTEGQFVDLTGNRLTYTNWNEGEPNNAGSDEDCVLLLKNGQWNDVPCSTSHLAV

CEFPI

SEQ ID NO: 9 minicircle DNA sequence gccagggcgt gccct tgggc tccccgggcg cgactagtga at tgatacta gtaaggatct 60 gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 120 tggggggagg ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg gtaaactggg 180 aaagtgatgt cgtgtactgg ctccgcct t t t tcccgaggg tgggggagaa ccgtatataa 240 gtgcagtagt cgccgtgaac gt tct t t t tc gcaacgggt t tgccgccaga acacagctga 300 agct tcgagg ggctcgcatc tctcct tcac gcgcccgccg ccctacctga ggccgccatc 360 cacgccggt t gagtcgcgt t ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg 420 ccgtctaggt aagt t taaag ctcaggtcga gaccgggcct t tgtccggcg ctccct tgga 480 gcctacctag actcagccgg ctctccacgc t t tgcctgac cctgct tgct caactctacg 540 tct t tgt t tc gt t t tctgt t ctgcgccgt t acagatccaa gctgtgaccg gcgcctactc 600 tagagctagc gaat tcgcca ccatgtccct gt tcccatct ctgccactgc tgctgctgag 660 catggtggca gccagctact ccgagaccgt gacatgcgag gacgcacaga agacctgccc 720 tgccgtgatc gcctgtagct cccctggcat caacggct t t ccaggcaagg acggccggga 780 tggcacaaag ggagagaagg gagagccagg acagggactg agaggeetge agggaccacc 840 tggcaagctg ggaccaccag gaaatccagg accatctggc agccctggac caaagggaca 900 gaagggcgat cccggcaagt cccctgacgg egat tetage ctggcagcat etgagaggaa 960 ggccctgcag accgagatgg ccagaatcaa gaagtggctg acat tcagcc tgggcaagca 1020 agtgggcaac aagt tct t tc tgaccaatgg cgagatcatg acat tcgaga aggtgaaggc 1080 cctgtgcgtg aagt t tcagg catccgtggc aaccccaagg aacgcagccg agaatggege 1140 catccagaac ctgatcaagg aggaggeet t cctgggcatc accgacgaga agacagaggg 1200 ccagt t tgtg gatctgacag gcaatagact gacctacaca aactggaatg agggegagee 1260 taacaatgcc ggcagcgacg aggat tgegt getgetgetg aagaaeggee agtggaatga 1320 cgtgccatgc tccacctctc acctggccgt gtgcgagt tc cccatcggag geggeggegg 1380 cggcggaggc gggggcggcg gcatgagcct gt t tcct tcc ctgcctctgc tgctgctgtc 1440 catggtggcc gccagctat t ccgagaccgt gacatgtgag gatgcacaga agacctgccc 1500 agccgtgatc gcatgt tcct ctccaggcat caacggct tc cctggcaagg acggcaggga 1560 tggcacaaag ggcgagaagg gagageetgg acagggactg aggggactgc agggccctcc 1620 aggcaagctg ggaccacctg gaaatccagg acct tctggc agcccaggcc ctaagggaca 1680 gaagggcgac ccaggcaaga gcccagacgg cgatagctcc ctggcagcat ccgaaagaaa 1740 ggccctgcag actgaaatgg cccgcatcaa gaagtggctg acct tcagct taggcaagca 1800 agtcggaaac aagt tct t tc tgactaacgg agaaat tatg acat tcgaga aagteaagge 1860 cctgtgcgtg aaat tccagg ccagcgtcgc cactcctaga aatgccgctg aaaaeggage 1920 tat tcagaac ctgatcaagg aagaageet t cctgggaat t aetgatgaaa aaaccgaagg 1980 acagt tcgtc gatctgacag gcaatcgcct gact tatacc aat tggaacg aaggagaacc 2040 aaataacgct ggcagcgatg aagaetgegt getgetgetg aagaatggac agtggaacga 2100 cgtgcct tgt tccacctctc acctggccgt gtgcgagt tc ccgat tggcg ggggcggggg 2160 cggcggcggc gggggaggag gcatgtctct gt t tcccagt ctgcccctgc tgctgctgtc 2220 tatggtggcc get tcctata gegaaaetgt cacatgtgag gatgcccaga aaact tgccc 2280 agccgtgatc gcctgt tcta gcccaggcat caacggat tc ccaggcaagg aeggaaggga 2340 tggaaccaaa ggegaaaaag gcgaacctgg ccagggcctg agaggeetge agggcccacc 2400 cggcaagctg ggccctccag gaaatcctgg accatctggc agcccaggac ccaaaggaca 2460 gaagggcgac cctggcaagt ctccagacgg cgat tcctct ctggcagcaa gcgaacgcaa 2520 ggccctgcag aetgagatgg ctagaatcaa gaagtggctg acct tcagcc t tggcaagca 2580 agtcgggaac aagt tct t tc tgacaaacgg agaaatcatg acat tcgaga aagt taaggc 2640 cctgtgcgtg aagt tccagg ccagcgtcgc tactcctaga aatgccgcag aaaaeggage 2700 tatacaaaac ctgatcaagg aagaggeet t cctgggaat t acagatgaaa aaaccgaagg 2760 gcagt tcgtg gatetgaeeg gaaacaggct gact tatacc aactggaacg aaggagaacc 2820 caataacgct ggcagcgacg aagaetgegt getgetgetg aagaaeggte agtggaatga 2880 cgtgccctgc tccacctctc acctggccgt gtgcgagt tc cctatcatgt ccctgt t tcc 2940 tagct tacct t tat tactgc tgagcatggt cgccgct tcc tacagcgaaa ctgtgacctg 3000 tgaggatgcc cagaagact t gtcccgccgt gateget tge tccagcccag gcatcaacgg 3060 gt t tcctggc aaggatggaa gagacggaac aaagggcgag aagggcgaac ctggccaggg 3120 gctgagggga ctgcaggggc ctcccggcaa gctgggcccc cctggcaacc ccggccct tc 3180 cggcagcccc ggccccaaag gccagaaggg cgaccccggc aagagcccag atggegat te 3240 ctccctggcc gcctccgaaa ggaaggccct gcaaactgaa atggetegea tcaagaagtg 3300 gctgacat tc aget taggea agcaagtagg taacaagt tc t t tetgaega aeggagagat 3360 tatgacat tc gagaaggtea aggccctgtg cgtgaagt t t caagccagcg tcgccacccc 3420 aaggaacgca gcagaaaacg gagctatcca aaacctgatc aaggaggaag cct tcctggg 3480 aatcactgat gaaaaaaeeg agggacagt t cgtggatctg actggcaatc gcctgact ta 3540 caccaat tgg aacgagggcg agcccaataa cgcaggcagc gatgaagat t gegtgetget 3600 gctgaagaac ggacagtgga atgaegtgee t tgctccacc tctcacctgg ccgtgtgcga 3660 gt tcccaatc atgaggctgc tgaccctgct gggactgctg tgcggctctg tggcaacacc 3720 tctgggacca aagtggccag agcccgtgt t cggcagactg gcaagcccag gat t tcctgg 3780 agagtacgcc aacgatcagg agcggagatg gaccctgaca gcacctccag gatacaggct 3840 gagactgtat t tcacccact t tgacctgga gctgagccac ctgtgcgagt acgat t t tgt 3900 gaagctgtcc tctggagcaa aggtgctggc caccctgtgc ggacaggagt ctaccgacac 3960 agagcgggcc ccaggcaagg atacct tcta tagcctgggc agctccctgg acatcacct t 4020 ccggagcgat tacagcaacg agaagccct t caccggct t t gaggcct tct atgccgccga 4080 ggacatcgat gagtgccagg tggcaccagg agaggcacca acatgcgacc accactgtca 4140 caaccacctg ggcggct tct actgctct tg tagggccggc tatgtgctgc accggaataa 4200 gagaacctgc agcgccctgt gctccggaca ggtgt t taca cagcggagcg gagagctgtc 4260 tagcccagag tacccaagac cctatcctaa gctgtcctct tgtacctaca gcatctccct 4320 ggaggagggc t tctccgtga tcctggact t cgtggagtct t t tgatgtgg agacccaccc 4380 cgagacactg tgccct tatg act tcctgaa gatccagacc gatcgggagg agcacggccc 4440 ct t t tgtggc aagaccctgc ctcacagaat cgagacaaag tctaacaccg tgacaatcac 4500 ct t tgtgacc gacgagagcg gcgatcacac aggctggaag atccactaca catccaccgc 4560 ccagccatgc ccctatccta tggcaccacc taatggacac gtgagcccag tgcaggccaa 4620 gtacatcctg aaggactct t t tagcatct t ctgcgagacc ggctatgagc tgctgcaggg 4680 ccacctgcct ctgaagtcct tcacagccgt gtgccagaag gacggcagct gggataggcc 4740 aatgccagca tgctccatcg tggat tgtgg cccacccgac gatctgcct t ccggcagagt 4800 ggagtacatc accggcccag gcgtgaccac atacaaggcc gtgatccagt at tct tgcga 4860 ggagacct tc tacacaatga aggtgaacga cggcaagtac gtgtgcgagg ccgatggct t 4920 t tggaccagc tccaagggag agaagagcct gcccgtgtgc gagcccgtgt gcggactgtc 4980 cgcccggacc acaggcggca gaatctatgg aggacagaag gcaaagccag gcgact tccc 5040 t tggcaggtg ctgatcctgg gaggaaccac agcagcaggc gccctgctgt acgataactg 5100 ggtgctgacc gcagcacacg ccgtgtatga gcagaagcac gacgccagcg ccctggatat 5160 ccggatgggc accctgaaga gactgagccc ccactacaca caggcctggt ccgaggccgt 5220 gt tcatccac gagggctata cccacgacgc cggct t tgac aatgatatcg ccctgatcaa 5280 gctgaacaat aaggtggtca tcaactccaa tatcacacca atctgcctgc cccggaagga 5340 ggccgagtct t tcatgagaa ccgacgatat cggcacagca agcggatggg gactgaccca 5400 gaggggct t t ctggcacgca acctgatgta cgtggacatc cctatcgtgg atcaccagaa 5460 gtgtaccgcc gcctacgaga agcctccata tccacggggc tctgtgacag ccaatatgct 5520 gtgcgcagga ctggagtccg gaggcaagga ctct tgtagg ggcgatagcg gaggcgccct 5580 ggtgt tcctg gacagcgaga ccgagagatg gt t tgtgggc ggcatcgtgt cctggggctc 5640 tatgaactgc ggagaggcag gacagtacgg cgtgtataca aaagtgatca actacatccc 5700 ctggatcgag aatatcatca gcgact tcat gagactgctg accctgctgg gcctgctgtg 5760 cggctccgtg gcaacaccac tgggaccaaa gtggcctgag ccagtgt tcg gccgcctggc 5820 aagccctgga t t tccaggcg agtatgccaa tgatcaggag aggcgctgga cactgaccgc 5880 accacctgga tacaggctga ggctgtact t cacccact tc gacctggagc tgtctcacct 5940 gtgcgaatat gact t tgtga agctgtctag cggcgccaaa gtcctggcca ccctgtgcgg 6000 ccaggagtcc acagacaccg agagggcccc tggcaaggac acct tctat t ctctgggctc 6060 ctctctggac atcacat t tc gcagcgat ta ctctaatgaa aaaccct tca ccggct tcga 6120 ggcct t t tat gctgctgaag atat tgacga gtgccaggtg gcacctggcg aagcccctac 6180 ctgtgatcat cat tgccata atcacctggg aggct tctac tgctcctgta gagctggata 6240 tgtgctgcac aggaataaga ggact tgtag cgccctgtgc agtggccagg tgt t tacaca 6300 gaggtctgga gagctgagct ccccagagta cccaaggcct tatccaaagc tgtctagctg 6360 tacctactcc atctctctgg aggagggct t ctctgtgatc ctggat t t tg tggagagct t 6420 tgatgtggaa actcacccag agacactgtg cccctacgac t tcctgaaaa tccagaccga 6480 tagggaggag cacggcccat tctgcggcaa gaccctgccc caccgcat tg aaacaaagtc 6540 taacacagtg accatcacat ttgtgactga tgaatccggc gatcataccg gatggaaaat 6600 ccactacacc tccacagccc agccctgccc t tatccaatg gccccaccca atggccacgt 6660 gtcccctgtc caggctaaat acatcctgaa ggacagct tc tccatctttt gegaaaetgg 6720 atacgaactg ctgcagggac acctgcccct gaagtettte acagccgtgt gccaaaagga 6780 cggctcttgg gataggccca tgcctgcctg cagcatcgtg gattgtggac ctccagacga 6840 tctgccaagc ggaagggtgg agtacatcac cggacctgga gtcactacct ataaageegt 6900 gatccagtat teetgtgaag agaccttcta caccatgaaa gtcaatgatg gcaagtacgt 6960 gtgcgaagct gacggctttt ggacctcctc taagggagag aagtctctgc ccgtgtgcga 7020 acccgtgtgc ggactgtctg ccagaactac cggaggcaga atctatggcg gccagaaggc 7080 caagccaggc gacttcccat ggcaggtcct gatcctgggc ggcactaccg ccgccggcgc 7140 cctgctgtat gacaattggg tcctgactgc cgcccatgcc gtgtatgaac agaaacatga 7200 tgctagcgcc ctggacatca ggatgggcac cctgaagcgc ctgtccccac attatacaca 7260 ggcctggtct gaageegtgt tcatccatga aggatacact catgatgctg gat tegataa 7320 cgacatcgcc ctgatcaagc tgaacaataa ggtggtcatc aacagcaata tcacacctat 7380 ctgcctgcca aggaaggagg ccgagagctt catgcgcact gatgatateg gcacagcatc 7440 tggctggggc ctgactcaga gaggetttet ggccagaaac ctgatgtatg tggacatccc 7500 cat tgtcgac catcagaaat gcactgctgc etaegagaag ccccct tatc ctagaggcag 7560 cgtcaccgct aacatgctgt gcgccggcct ggaaagegge ggcaaggatt cctgcagagg 7620 cgat tccggc ggcgccctgg tgttcctgga t tccgagacc gagcggtggt tegteggegg 7680 catcgtgtct tggggcagca tgaattgtgg cgaagccggc cagtatggcg tgtataccaa 7740 agtgatcaac tatat tcct t ggattgagaa tatcatcagc gatttetgag gatccgcggc 7800 cgcgcccctc tccctccccc ccccctaacg ttactggccg aageeget tg gaataaggee 7860 ggtgtgcgtt tgtctatatg ttattttcca ccatat tgee gtcttttggc aatgtgaggg 7920 cccggaaacc tggccctgtc ttettgaega gcattcctag gggtctttcc cctctcgcca 7980 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gettettgaa 8040 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt 8100 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggeggea caaccccagt 8160 gccacgttgt gagttggata gttgtggaaa gagteaaatg gctctcctca agegtat tea 8220 acaaggggct gaaggatgee cagaaggtac cccat tgtat gggatetgat ctggggcctc 8280 ggtgcacatg ct t tacatgt gtttagtcga ggt taaaaaa aegtetagge cccccgaacc 8340 acggggacgt ggttttcctt tgaaaaacac gataatacct ccggaatgga gagegaegag 8400 agcggcctgc ccgccatgga gategagtge cgcatcaccg gcaccctgaa eggegtggag 8460 ttcgagctgg tgggcggcgg agagggcacc cccaagcagg gccgcatgac caacaagatg 8520 aagagcacca aaggcgccct gacct tcagc ccctacctgc tgagccacgt gatgggctac 8580 ggct tctacc act tcggcac ctaccccagc ggetaegaga acccct tcct gcacgccatc 8640 aacaacggcg gctacaccaa cacccgcatc gagaagtacg aggaeggegg cgtgctgcac 8700 gtgagcttca gctaccgcta cgaggccggc egegtgateg gcgact tcaa ggtggtgggc 8760 accggct tcc ccgaggacag egtgatet tc accgacaaga tcatccgcag caacgccacc 8820 gtggagcacc tgcaccccat gggcgataac gtgctggtgg gcagct tege ccgcacct tc 8880 agcctgcgcg aeggeggeta ctacagct tc gtggtggaca gccacatgca cttcaagagc 8940 gccatccacc ccagcatcct gcagaacggg ggccccatgt tcgccttccg ccgcgtggag 9000 gagctgcaca gcaacaccga gctgggcatc gtggagtacc agcacgcctt caagaccccc 9060 atcgcct teg ccagatcccg cgctcagtcg tccaat tetg ccgtggacgg caccgccgga 9120 cccggctcca ccggatctcg etagagetga atetaagteg acaatcaacc tctggattac 9180 aaaatttgtg aaagattgac tggtattett aactatgt tg ctccttttac gctatgtgga 9240 taegetgett taatgeettt gtatcatgct attgcttccc gtatggcttt cattttctcc 9300 tcct tgtata aatcctggt t getgtetett tatgaggagt tgtggcccgt tgtcaggcaa 9360 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cat tgccacc 9420 acctgtcagc tcctttccgg gact t tcgct t tccccctcc ctattgccac ggcggaactc 9480 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaat tee 9540 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg 9600 attctgcgcg ggacgtcctt ctgctacgtc cct tcggccc tcaatccagc ggaccttcct 9660 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 9720 agtcggatct ccctttgggc cgcctccccg cctggtacct ttaagaccaa tgacttacaa 9780 ggcagctgta gatct tagcc actttttaaa agaaaagggg ggactggaag ggetaat tea 9840 ctcccaacga agataagatc tgctttttgc ttgtactggg tctctctggt tagaccagat 9900 ctgagcctgg gagctctctg gctaactagg gaacccactg ct taagcctc aataaaget t 9960 gccttgagtg ct tcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 10020 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagt teat gteatet tat 10080 tat tcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat 10140 tgcagct tat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 10200 tttttcactg cat tctagt t gtggtttgtc caaactcatc aatgtatctt atcatgtctg 10260 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 10320 cat tctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 10380 gcctctgagc tat tccagaa gtagtgagga ggcttttttg gaggcctaga cttttgcaga 10440 tcgacccatg ggggcccgcc ccaactgggg taacctttga gt tctctcag ttggggg 10497