A phage-based gene delivery system producing predefined protein in host cells

Abstract

The present invention relates to a phage-based gene delivery system targeting human cells or animal cells, including immune cells, other human cells and cancer cells. In particular, the present invention relates to novel phage particles and associated phagemid expression systems and their production for the delivery of transgenes, comprising DNA encoding pathogen antigens, to human immune cells, suitable for use in vaccines and vaccine development against infectious diseases and cancers.

Description

[0001] A PHAGE-BASED GENE DELIVERY SYSTEM PRODUCING PREDEFINED PROTEIN IN HOST CELLS

[0002] Field of the Invention

[0003] The present invention relates to a phage-based gene delivery system targeting human and animal cells, including immune cells and other cancer cells. In particular, the present invention relates to novel phage particles and associated phagemid expression systems and their production for the delivery of transgenes, comprising DNA encoding pathogen antigens or defined proteins, to human immune cells and cancer cells, suitable for use in vaccines, vaccine development against infectious diseases and cancers.

[0004] Background of the Invention

[0005] Vaccines are generally considered as one of the most important tools in the fight against infectious diseases and cancers. In light of the COVID- 19 pandemic, there has been a concerted effort globally to create vaccines and antiviral technologies that may help combat the spread of future disease outbreaks. Furthermore, it has been applied to the cancer vaccines. Current vaccine technologies are typically categorized into several types including whole virus (e.g., inactivated or live attenuated viral vaccines), viral vector vaccines (e.g., adenovirus), nucleic acid-based vaccines (e.g., mRNA and DNA), and protein-based (e.g., protein subunit, virus-like particle) vaccines. While useful, there are limitations associated with these vaccine platforms, which do not make them the most ideal for use especially in a global emergency situations. For example, whole virus vaccines are expensive to produce and do not scale up well due to the requirement of whole virus. Viral vectors have a complex manufacturing process, and response may be dampened by preexisting immunity against vector. MRNA vaccines on the other hand involve high production cost and require very careful cold storage and transportation. Clearly, there is still an urgent need for new and adaptable vaccine technologies that are not only fast and cheap to produce, but also suitable for large-scale production and distribution.

[0006] Bacteriophages are viruses which infect bacteria. In recent times, there has been a growing interest in using engineered phage particles in gene delivery systems and vaccine development because recombinant phage particles are cheap and fast to produce, easy to modify genetically, can elicit a strong immune response and are stable under harsh conditions, thus making them a suitable candidate for use as a novel and effective vaccine platform. However, phage vectors often have poor transfection efficiency in mammalian cells, as the phage particles are not able to withstand the harsh cellular microenvironment (such as non-specific adsorption and antibodybased neutralization). In this regard, a hybrid adeno-associated virus / phage (AAVP) vector, was created from recombinant adeno-associated virus (rAAV) and filamentous bacteriophage (phage). While the resulting AAVP vectors possess favourable characteristics, challenges still remained with AA VP-based transgene delivery, especially with respect to nonspecific protein adsorption resulting in antibody-based neutralization and endo-lysosomal degradation. As such, AA VPbased systems still show relatively low gene transduction efficacy when compared to mammalian viruses.

[0007] Accordingly, there is a need to provide improved methods of gene delivery and vaccine technology that overcome or at least ameliorate, one or more of the drawbacks described above. Summary of the Invention

[0008] Disclosed herein is a recombinant phagemid particle which specifically targets immune cells and cancer cells, a recombinant phagemid particle vaccine comprising said particle, a method of vaccination and a system for producing said recombinant phagemid particle thereof, suitable for use in the treatment or prophylaxis of infectious diseases and cancers. It would be understood that the invention may be adapted for use in the therapeutic treatment of a variety of infectious diseases and cancers depending on the antigen-expressing transgene inserted into the phagemid vector expression cassette.

[0009] In one aspect, there is provided a recombinant phagemid particle, wherein the phagemid particle comprises at least one transgene expression cassette comprising a polynucleotide encoding at least one or more antigens operably linked to at least one promoter, wherein said recombinant phagemid particle comprises an immune cell-targeting peptide or cancer cell targeting peptide and an endosome escape peptide on its outer surface wherein, in use, the particle is capable of delivering the polynucleotide to the targeted immune cell or cancer cell, such that one or more antigens are expressed, wherein; i) the immune cell-targeting peptide or cancer cell-targeting peptide is expressed on phagemid pill minor coat proteins, and / or ii) the new developed endosome escape peptide is expressed on phagemid pVIII major coat proteins; and iii) the one or more antigens are selected from the group comprising pathogen antigens such as virus antigens, bacterial antigens, parasite antigens, fungal antigens and cancer cell antigens, or any combination thereof, and said one or more antigens are capable of stimulating an immune reaction in a subject when expressed and presented by the target immune cell.

[0010] In some embodiments, the immune cell-targeting peptide comprises an amino acid sequence selected from the group comprising KYSFKLILAEYGGGS (TLR4 ligand; SEQ ID NO: 2), YEQDPWGVKWWYGGGS (M2pep; SEQ ID NO: 4) and FHRWPTWPLPSPGGGS (MP12 peptide; SEQ ID NO: 6), or functional amino acid sequence variant thereof; and / or the endosome escape peptide comprises an amino acid sequence selected from the group comprising SFGLFLAIALFILGGWLGLILGWYG (L5WYG; SEQ ID NO: 8) and SFGLFKAIAKFIKGGWKGLIKGWYG (K5WYG; SEQ ID NO: 10), or functional amino acid sequence variant thereof.

[0011] In a second aspect, there is provided a recombinant phagemid particle vaccine, wherein the vaccine comprises a recombinant phagemid particle of the first aspect.

[0012] In a third aspect, there is provided a use of a recombinant phagemid particle of the first aspect or a vaccine of the second aspect in the manufacture of a medicament for the prophylaxis or treatment of an infectious disease and cancer.

[0013] In a fourth aspect, there is provided a method of vaccinating a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a recombinant phagemid particle of the first aspect or a recombinant phagemid particle vaccine of the second aspect.

[0014] In a fifth aspect, there is provided a system for producing a recombinant phagemid particle from a prokaryotic host, the system comprising: a) a first vector configured to persist inside a prokaryotic host, and comprising at least one transgene expression cassette comprising a polynucleotide encoding one or more antigens operably linked to at least one promoter, and a packaging signal for enabling replication of the vector into single- stranded DNA; i) the one or more antigens are selected from the group comprising pathogen antigens such as virus antigens, bacterial antigens, parasite antigens, fungal antigens and cancer cell antigens, or any combination thereof; and b) a second vector comprising a polynucleotide encoding structural proteins required for packaging the single-stranded DNA from (a), nucleic acid encoding a targeting peptide on its outer surface to target the phagemid particle to an immune cell or cancer cell, and nucleic acid encoding an endosome escape peptide on its outer surface, resulting in the formation and extrusion of a recombinant phagemid particle from the prokaryotic host which can target expression of the one or more antigens to within immune cells and cancer cells, wherein; i) the immune cell-targeting peptide or cancer cell-targeting peptide is expressed on phagemid pill minor coat proteins, and / or ii) the endosome escape peptide is expressed on phagemid pVIII major coat proteins.

[0015] In some embodiments, the immune cell-targeting peptide of the system comprises an amino acid sequence selected from the group comprising KYSFKLILAEYGGGS (TLR4 ligand; SEQ ID NO: 2), YEQDPWGVKWWYGGGS (M2pep; SEQ ID NO: 4) and FHRWPTWPLPSPGGGS (MP12 peptide; SEQ ID NO: 6), or functional amino acid sequence variant thereof; and / or the endosome escape peptide comprises an amino acid sequence selected from the group comprising SFGLFLAIALFILGGWLGLILGWYG (E5WYG; SEQ ID NO: 8) and SFGEFKAIAKFIKGGWKGEIKGWYG (K5WYG; SEQ ID NO: 10), or functional amino acid sequence variant thereof.

[0016] In a sixth aspect, there is provided a method of prophylaxis or treatment of an infectious disease in a subject, the method comprising administering to a subject in need of such treatment, a therapeutically effective amount of the recombinant phagemid particle according to the first aspect or the vaccine of the second aspect.

[0017] Advantageously, the engineered phagemid particles of the present disclosure not only display an improved transfection efficiency, they also are able to directly target immune cells, thereby stimulating strong immune responses. More advantageously, the phagemid particles and the phagemid particle expression system of the present disclosure are also cheaper and faster to produce, making them suitably adapted for rapid, large scale immunization. These and other advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description.

[0018] Brief Description of the Drawings

[0019] The accompanying drawings illustrate disclosed embodiments and serve to explain the principles of the disclosed embodiments. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0020] FIG. 1 shows an example of a structural DNA plasmid with restriction enzyme mapping that encodes a predefined protein (in this case, Nucleocapsid of SARs-CoV2).

[0021] FIG. 2 depicts an example of a helper phage DNA plasmid with restriction enzyme mapping that encodes envelop proteins and predefined protein for targeting.

[0022] FIG. 3 depicts a modified bacteriophage genome of one embodiment of the invention, for delivering gene sequences to eukaryotic cells by using AAV3B ITR gene cassette combined with CMV early enhancer fused to a modified chicken P-actin promoter.

[0023] FIG. 4 illustrates a modified phage of one embodiment of the invention. The M13 phage vector is modified to express the MP12 ligand on the pill minor coat protein as a guide ligand, which specifically binds with CD 14 on immune cells. The vector is also modified to express the endosome escape peptide (namely K5WYG) on the recombinant pVIII coat proteins to enhance transduction efficiency.

[0024] FIG. 5 depicts a plasmid vector map of modified vector pAAV3B-CBh-BEu2S.

[0025] FIG. 6 shows a genetic map of pAAV3B-CBh-BEu2S transgene cassette. FIG. 7 depicts a plasmid vector map of modified vector pAAV2-BLu2S.

[0026] FIG. 8 shows a genetic map of pAAV2- BLu2S transgene cassette.

[0027] FIG. 9 illustrates the pAAV3B-CBh-BLu2S construction scheme, showing the restriction enzyme sites used in construction.

[0028] FIG. 10 depicts a plasmid map of modified vector pM13KE MP12GGGS K5WYG.

[0029] FIG. 11 depicts the pM13KE MP12GGGS K5WYG construction scheme.

[0030] FIG. 12 shows the results of transfection with PAAV3 Luc in HEK293 cells. High luciferase activity was detected in PAAV3 Luc group only, at both 48 and 72 hours. Neither the CTRL (only culture media) nor FuGENE6 (only FuGENE6 reagent) groups showed any luciferase activity.

[0031] FIG. 13 shows the results of transfection with pAAV3 eGFP in HEK293 cells. Expression of the enhanced green fluorescent protein was only detected in the pAAV3 eGFP group but not in the CTRL and FuGENE6 groups, at both day 3 and 6 after transfection.

[0032] FIG. 14 shows the results of transduction with pAAV3-C / zB-eGFP-MP12 in (A) HEK293 cells and (B) THP1 cells. (A) The percentage of HEK293 cells that are transduced with pAAV3- ChB-eGFP-MP12 (Phage) containing the fluorescent intensity is low and is not sufficiently different from the non-transduced control. (CTRL). (B) The percentage of THP-1 cells that are transduced with phage pAAV3-C / zB-eGFP-MP12 (Phage) containing the fluorescent intensity is higher than the control (CTRL).

[0033] FIG. 15 Depicts the comparative results of transfection with pAAV2 and pAAV3, using Luc2 (luciferase) as reporter gene in HEK293 cells, after 3 days. The fluorescence signal due to transgene expression in the pAAV3 -transfected cells was greater than that for the pAAV2- transfected cells.

[0034] FEG. 16 Showing the gene expression by phage particle containing AFP-DNA (alpha fetoprotein), and specific targeting the immune cells, including THP-1 (macrophage), RAW264.7 (macrophage) and PBMCs (peripheral blood mononuclear cells) which represent the white blood cells using the MP12 binding peptide.

[0035] Detailed Description of the Invention

[0036] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference but their mention in the specification does not imply that they form part of the common general knowledge.

[0037] Definitions

[0038] For convenience, certain terms employed in the specification, examples and appended claims are collected here.

[0039] In general, technical, scientific and medical terminologies used herein has the same meaning as understood by those skilled in the art to which this invention belongs. Further, the following technical comments and definitions are provided. These definitions should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

[0040] As used herein, “a” or “an” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. The inventors found that the more enzyme used the faster the reaction proceeded.

[0041] As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of’. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.

[0042] As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond. Whereas peptides are considered to be short amino acid chains, polypeptides are long amino acid chains and proteins tend to have a stable structure and may comprise modifications (e.g., glycosylation or phosphorylation). The term “protein” may encompass a naturally-occurring as well as artificial (e.g., engineered or variant) full-length protein as well as a functional fragment of the protein.

[0043] As used herein, the term “endosomal escape peptide” refers to a peptide which is expressed on the surface of a vector (for example, a phage particle of the present invention) which may facilitate endosomal release and / or its escape from the endocytic pathway. Used in the present context, the endosomal escape peptide may enhance the phage particle of the present invention’s ability to escape the endosome after endocytosis and facilitates its release into the cell cytosol, instead of for example, getting localised in the endosome and degraded by lysosomal action. In this regard, the endosome escape peptide may enhance the phage particle’s endurance / resistance from intracellular biodegradation upon entry into the targeted immune cell.

[0044] As used herein, the term “immune cell” may refer to any cell derived in the bone marrow that is a part of the immune system and help the body fight diseases and infections. Examples of immune cells may include, but are not limited to, granulocytes (such as eosinophils, neutrophils and basophils), B cells (such as memory B cells and plasma cells), natural killer cells, T cells (such as memory T cells, helper T cells and cytotoxic T cells), monocytes and monocyte -derived cells such as macrophages and dendritic cells. When used in context of the present invention, the “immune cells” may refer in particular, to TLR4 ligand positive cells such as monocyte-derivatives and dendritic cells.

[0045] As used herein, the term “phagemid particle” refers to a hybrid phagemid genome encapsulated by phage-derived coat proteins. As used in the present context, the phagemid particle may refers to a hybrid phagemid genome that has been encapsulated by phage proteins derived from a helper phage.

[0046] As used herein, the term "variant", or functional amino acid sequence variant, refers to an amino acid sequence that is altered by one or more amino acids of the non-variant reference peptide sequence, but retains the function of the non-variant reference peptide. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNASTAR® software (DNASTAR, Inc. Madison, Wisconsin, USA). For example, the immune cell-targeting peptide of the invention may comprise an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. The endosome escape peptide may comprise an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 10.

[0047] A description of exemplary, non-limiting embodiments of the invention follows.

[0048] The present disclosure is based, in part, on the development of a recombinant phagemid particle that has been engineered to advantageously target immune cells, such as monocytes, and provide an improved transduction efficiency. In this regard, the inventors have successfully modified a phagemid particle, referred to as Phagemid / Adeno-associated Virion (i.e. pAAV), to express an immune cell-targeting peptide and a modified endosomal escape peptide on its minor and major coat proteins, respectively.

[0049] As described herein, the immune cell-targeting peptide expressed on the recombinant phagemid particle of the present invention is displayed on the phagemid particle coat protein and serves as a homing mechanism to deliver directly said phagemid particle comprising a transgene of interest to the immune cells, thereby providing a more precise, targeted gene delivery system.

[0050] To this end, provided in one aspect of the present disclosure is a recombinant phagemid particle, wherein the phagemid particle comprises at least one transgene expression cassette comprising a polynucleotide encoding at least one or more antigens operably linked to at least one promoter, wherein said recombinant phagemid particle comprises an immune cell-targeting peptide and an endosome escape peptide on its outer surface wherein, in use, the particle is capable of delivering the polynucleotide to the targeted immune cell, such that one or more antigens are expressed, wherein; i) the immune cell-targeting peptide is expressed on phagemid pill minor coat proteins, and / or ii) the endosome escape peptide is expressed on phagemid pVIII major coat proteins; and iii) the one or more antigens are selected from the group comprising pathogen antigens such as virus antigens, bacterial antigens, parasite antigens, fungal antigens and cancer cell antigens, or any combination thereof, and said one or more antigens are capable of stimulating an immune reaction in a subject when expressed and presented by the target immune cell.

[0051] The targeting peptide as herein disclosed are preferably peptides that are capable of targeting immune cells. In particular, the targeting peptides disclosed herein may bind to receptors that are primarily expressed on immune cells, e.g., monocytes and monocyte-derived immune cells such as macrophages and dendritic cells. For example, in some embodiments, the targeting peptide may be MP 12 ligand (which binds to CD 14), TLR4 ligand (which binds to TLR4 receptor), or M2pep ligand (which binds to M2 macrophage).

[0052] In some embodiments, the immune cell-targeting peptide may comprise an amino acid sequence selected from the group comprising KYSFKLILAEYGGGS (TLR4 ligand; SEQ ID NO: 2), YEQDPWGVKWWYGGGS (M2pep; SEQ ID NO: 4) and FHRWPTWPLPSPGGGS (MP12 peptide; SEQ ID NO: 6), or a functional amino acid sequence variant thereof.

[0053] It would be understood that the efficiency of gene delivery is affected by, to a certain degree, the efficiency of endosomal escape. Accordingly, to improve gene delivery efficiency, the phage particle of the present invention may express / display an endosome escape peptide on its surface. The endosome escape peptides as herein disclosed are preferably peptides that may facilitate the phage particle’s endosomal release into the cytosol (for example, via facilitating the rupturing of the endosomal membrane), thereby minimizing intracellular biodegradation upon entry into the target immune cell.

[0054] In some embodiments, the endosome escape peptide may be capable of reducing intracellular biodegradation upon entry into the targeted immune cell.

[0055] In some embodiments, the endosome escape peptide disclosed herein may be capable of disrupting endosomal membrane at a pH-dependent manner. In some embodiments, the endosome escape peptide may be a pH-dependent membrane-active peptide (PMAP).

[0056] In some embodiments, the endosome escape peptide disclosed herein may enhance endosomal escape by facilitating the rupturing of the endosomal membrane through a proton sponge effect. In this regard, it would be appreciated that peptides enriched with histidine show a proton sponge effect, and may disrupt the endosomal membrane. Accordingly in some embodiments, the endosome escape peptide as disclosed herein may be a peptide enriched with histidine residues and / or conjugated with one or more histidines.

[0057] In some embodiments, the endosome escape peptide may comprise one or more lysine residues. In some embodiments, the lysine-enriched peptide may be K5WYG, and / or may comprise an amino acid sequence set forth in SEQ ID NO: 10, or a functional amino acid sequence variant thereof.

[0058] In some embodiments, the endosome escape peptide may be the peptide K5WYG or L5WYG. Preferably, the endosome escape peptide may be selected from the group comprising peptides having an amino acid sequence comprising SFGLFLAIALFILGGWLGLILGWYG (E5WYG; SEQ ID NO: 8) and SFGEFKAIAKFIKGGWKGEIKGWYG (K5WYG; SEQ ID NO: 10), or a functional amino acid sequence variant thereof.

[0059] As those skilled in the art would appreciate, a protein’s function is directly related to its structure and sequence, and that there is a positive relationship between sequence identity and function similarity. In this regard, methods of determining a protein sequence identity are known in the art. Therefore, the sequences of the targeting peptides disclosed herein may be sufficiently varied so long as the targeting peptides maintain their functionality and can exhibit the required activity.

[0060] Accordingly in some embodiments, the targeting peptide may comprise an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. In some embodiments, the endosome escape peptide may comprise an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 10.

[0061] In contrast to the adeno-associated Virus / Phage (AAVP) vectors known in the art, the PAAV genome of the present disclosure lacks structural bacteriophage genes for the formation, packaging or extrusion of the phagemid particle from a prokaryotic host. Accordingly, a prokaryotic helper phage virus is needed to enable vector assembly in the host. The helper phage in this regard provides the capsid and other phage components, as well as the targeting and endosome escape peptides, to encapsulate the transgene expression cassette.

[0062] In some embodiments, the at least one transgene expression cassette may comprise an adeno-associated virus (AAV) transgene expression cassette or a lentivirus transgene expression cassette.

[0063] In some embodiments, the at least one expression cassette may comprise a bacteriophage fl origin and a drug resistance gene marker (such as Ampicillin resistance gene). It would be appreciated that the bacteriophage fl origin of replication may enable the phagemid particle of the present disclosure to replicate into single- stranded DNA while the drug resistance gene marker may allow for the phagemid genome to replicate efficiently inside the prokaryotic host by ensuring expression and providing selective pressure to prevent loss of phagemid genome.

[0064] In some embodiments, the at least one expression cassette further comprises a bacterial origin of replication. A bacterial origin of replication may enable replication of double-stranded vector inside a prokaryotic host.

[0065] In some embodiments, at least one of said expression cassettes comprises 5’ and 3’ Inverted Terminal Repeat (ITR) sequences and a poly A signal.

[0066] In some embodiments, at least one of said expression cassettes further comprises one or more components selected from the group comprising a AAV3B 5” ITR, a cytomegalovirus (CMV) enhancer, a chicken beta-actin promoter, a hybrid intron, a polynucleotide encoding one or more of said antigens, a poly A signal and a AAV3B 3” ITR.

[0067] In some embodiments, the AAV3B 5” ITR has the nucleotide sequence set forth in SEQ ID NO: 11, the CMV enhancer has the nucleotide sequence set forth in SEQ ID NO: 12, the chicken beta-actin promoter has the nucleotide sequence set forth in SEQ ID NO: 13, the hybrid intron has the nucleotide sequence set forth in SEQ ID NO: 14, the poly A signal has the nucleotide sequence set forth in SEQ ID NO: 15 and the AAV3B 3” ITR has the nucleotide sequence set forth in SEQ ID NO: 16, or a functional fragment or sequence variant thereof. It would be appreciated by a skilled artisan that the transgene expression cassette of the phagemid particle may be easily adapted to encode for any gene of interest. Preferably, the cassette may express an antigen from a pathogen, in order to elicit an immune response. For example in some embodiments, the pathogen antigen may be from a virus selected from the group comprising coronavirus, influenza virus, flavivirus, Bunyavirus, Filovirus, Herpes virus and Papilloma virus, or is from bacteria or fungi. In some embodiments, the virus is selected from the group comprising SARS-COV-2, Dengue, Zika, Rift Valley Fever, Ebola, hemorrhagic fever viruses, Hepatitis and serotypes thereof.

[0068] In some embodiments, the virus is SARS-COV-2.

[0069] In some embodiments, the pathogen antigen from SARS-COV-2 is a spike protein or immunogenic fragment or sequence variant thereof.

[0070] In some embodiments, the spike protein has the amino acid sequence set forth in SEQ ID NO: 18 or immunogenic fragment or sequence variant thereof.

[0071] In some embodiments, the genome of the recombinant phagemid particle comprises a packaging signal for enabling replication of the phagemid genome into single- stranded DNA, which can subsequently be packaged into the phagemid particle inside a prokaryotic host.

[0072] In second aspect, there is provided a recombinant phagemid particle vaccine, wherein the vaccine comprises a recombinant phagemid particle of the first aspect.

[0073] In a third aspect, there is provided a use of a recombinant phagemid particle of the first aspect or a vaccine of the second aspect in the manufacture of a medicament for the prophylaxis or treatment of an infectious disease.

[0074] In a fourth aspect, there is provided a method of vaccinating a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a recombinant phagemid particle of the first aspect or a recombinant phagemid particle vaccine of the second aspect.

[0075] In some embodiments, the method of the fourth aspect is for use in treating or preventing disease.

[0076] For in vivo delivery, the phagemid particles and vaccines comprising the same may be delivered to a subject in need thereof by a variety of routes of administration including, for example, oral, dietary, topical, transdermal, or parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection) routes of administration. Administration can be local or systemic. The actual dose and treatment regimen of said phagemid particles and the vaccines herein can be determined by a skilled physician, taking into account the nature of the condition being treated, and patient characteristics. In some embodiments, the phagemid particles and the vaccine comprising the same as disclosed herein may preferably be formulated for topical application. Preferably, the topical formulation may be in the form of a liquid solution or mixture, dispersion, suspension, gel, lotion, emulsion, paste, cream, ointment, milk, pomade, spray or a medicated bandage, pad or mask. It would be appreciated that the methods to prepare topical formulations are known is the art and is based on standard principles and methods described in various pharmaceutical literature.

[0077] In a fifth aspect, there is provided a system for producing a recombinant phagemid particle from a prokaryotic host, the system comprising: a) a first vector configured to persist inside a prokaryotic host, and comprising at least one transgene expression cassette comprising a polynucleotide encoding one or more antigens operably linked to at least one promoter, and a packaging signal for enabling replication of the vector into single- stranded DNA; i) the one or more antigens are selected from the group comprising pathogen antigens such as virus antigens, bacterial antigens, parasite antigens, fungal antigens and cancer cell antigens, or any combination thereof; and b) a second vector comprising a polynucleotide encoding structural proteins required for packaging the single- stranded DNA from (a), nucleic acid encoding a targeting peptide on its outer surface to target the phagemid particle to an immune cell, and a nucleic acid encoding an endosome escape peptide on its outer surface, resulting in the formation and extrusion of a recombinant phagemid particle from the prokaryotic host which can target expression of the one or more antigens to within immune cells, wherein; i) the targeting peptide is expressed on phagemid pill minor coat proteins, and / or ii) the endosome escape peptide is expressed on phagemid pVIII major coat proteins.

[0078] In some embodiments, the endosome escape peptide of the system may comprise an amino acid sequence selected from the group comprising KYSFKLILAEYGGGS (TLR4 ligand; SEQ ID NO: 2), YEQDPWGVKWWYGGGS (M2pep; SEQ ID NO: 4) and FHRWPTWPLPSPGGGS (MP12 peptide; SEQ ID NO: 6), or functional amino acid sequence variant thereof; and / or the endosome escape peptide comprises an amino acid sequence selected from the group comprising SFGLFLAIALFILGGWLGLILGWYG (L5WYG; SEQ ID NO: 8) and SFGLFKAIAKFIKGGWKGLIKGWYG (K5WYG; SEQ ID NO: 10), or functional amino acid sequence variant thereof.

[0079] In some embodiments, said recombinant phagemid particle is defined according to the first aspect. In a sixth aspect, there is provided a method of prophylaxis or treatment of an infectious disease in a subject, the method comprising administering to a subject in need of such treatment, a therapeutically effective amount of the recombinant phagemid particle according to the first aspect or the vaccine of the second aspect.

[0080] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in various embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. “About” in reference to a numerical value generally refers to a range of values that fall within ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5% of the value unless otherwise stated or otherwise evident from the context. In any embodiment in which a numerical value is prefaced by “about”, an embodiment in which the exact value is recited is provided. Where an embodiment in which a numerical value is not prefaced by “about” is provided, an embodiment in which the value is prefaced by “about” is also provided. Where a range is preceded by “about”, embodiments are provided in which “about” applies to the lower limit and to the upper limit of the range or to either the lower or the upper limit, unless the context clearly dictates otherwise. Where a phrase such as “at least”, “up to”, “no more than”, or similar phrases, precedes a series of numbers, it is to be understood that the phrase applies to each number in the list in various embodiments (it being understood that, depending on the context, 100% of a value, e.g., a value expressed as a percentage, may be an upper limit), unless the context clearly dictates otherwise. For example, “at least 1, 2, or 3” should be understood to mean “at least 1, at least 2, or at least 3” in various embodiments. It will also be understood that any and all reasonable lower limits and upper limits are expressly contemplated.

[0081] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

[0082] EXAMPLES

[0083] Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0084] Table 1. DNA and peptide sequences of the present study

[0085]

[0086]

[0087] Example 1: Design and construction of recombinant phagemid vectors

[0088] Construction ofpAAV plasmid

[0089] The pAAV vector was constructed as a fusion of synthesized vectors from Vectorbuilder (VB221020-1033kcn) and Geneart (pAAV-CbhGFPImp-115166). The vectors were digested with the appropriate restriction enzymes and ligated to make a recombinant vector, as shown in FIGS.

[0090] 5, 6 and 9. pAAV-CBheGFP-115166 contained AAV3B ITR sequence. With digestion and ligation, VB 1033kcn-AAV3B was generated. This vector contained the AAV3B and eGFP genes. After that, BamHl and Spel restriction enzyme site from VB221020-1033kcn (BNuS) were added and this adapter was used for cloning Luciferase gene from VB220826- 1406asx. Luciferase gene of VB220826-1406asx was amplified by using specific primers 5'- TAAGCAGGATCCAATGGAAGATGCCAAAAACAT-3' (SEQ ID NO: 19) and 5'- TAAGCAACTAGTTTACACGGCGATCTTGCCGCCCTTCTTGG-3’ (SEQ ID NO: 20) as forward and reverse primers, respectively. The PCR product was ligated with AAV3B vector and resulted in pAAV3B-CBh-BLu2S (FIG. 5) Construction of helper vector plasmid

[0091] The M13 helper vector was constructed as a fusion of original M13KE vector (New England Biolabs) and synthesized gene fragments from Vectorbuilder and Geneart. The vector was digested with the appropriate restriction enzymes and ligated to make a recombinant vector, as shown in FIG. 10 - FIG. 11.

[0092] M13KE was used as the core vector to be modified. MP12 gene (fragment synthesized from vector 21AAU2ID_3120896_RGDM13H5WSacIIMP12GG) was first inserted into gene III of M13KE and a modified M13KE comprising MP 12 gene was generated. After that, a copy of modified Gene VIII comprising K5WYG sequence was added within the KasI and Bglll restriction sites, and the M13KE MP12 K5WYG vector was generated.

[0093] Digestion of plasmid DNA with restriction enzymes

[0094] The plasmid DNA prepared as described above was digested with desirable restriction endonucleases based on the desired transformants. Approximately 1 pg of plasmid DNA was digested with restriction enzyme using 5 units of enzyme / pg plasmid DNA in lx reaction buffer and the total volume should contain <5% glycerol. The buffers for specific restriction enzymes were based on the commercial recommendation. The digestion reaction was incubated at 37 °C for each enzyme for 1 h. The digested plasmid DNAs were then analyzed on 1.0% agarose gel electrophoresis at 90 volts. The gel was stained with Redsafe dye (Invitrogen). The plasmid DNA patterns were visualized under UV transluminator, and the size of plasmid DNA fragments were estimated by comparing to the size of the plasmid DNA standard base pair marker. The plasmid DNA bands were cut and extracted from gel by using GeneAid gel / PCR mini kit and a ligation reaction was performed.

[0095] Ligation of plasmid vector

[0096] The ligation reaction was done based on T4 ligation kit (New England Biolabs) with the appropriate insert / vector ratio. 20 pl of mixture was set up by adding 50 ng of DNA vector and 5 unit of T4 DNA ligase into reaction mixture. Then, the ligation mixture was incubated at room temperature (25 °C) for 30 min, and was transformed directly into the competent E. coli cells. Preparation of E. coli competent cells

[0097] The competent E. coli cells were prepared by MgCh-CaCh treatment. A single colony of appropriate E. coli strain was inoculated into 5 ml Luria-Bertani (LB) broth without antibiotics for TGI strain at 37°C overnight (14-16 h). The overnight culture was used as inoculum (1% inoculum size) to inoculate 250 ml LB media with or without appropriate antibiotics. The culture was shaken at 37°C until the ODeoo reading reached about 0.5, and the cells were harvested by centrifugation at 3,500 rpm for 10 min at 4°C. After removing the supernatant, the cell pellet was gently resuspended in 1 / 5 original culture volume of ice-cold 100 mM MgCh. The cell suspension was kept on ice for 15-20 min before centrifugation at 3,500 rpm for 5 min at 4°C to pellet the cells. After removing the supernatant, the cell pellet was gently resuspended with 1 / 50 original culture volume of ice-cold 100 mM CaCh and was allowed to settle on ice for 1 h. Sterile glycerol was then added to the cell suspension at 15% final concentration with gentle swirling. The resulting competent cells were stored in aliquots in pre-chilled sterile microcentrifuge tubes, followed by freezing at -80 °C until use.

[0098] Bacterial cell transformation

[0099] The frozen MgCh / CaCh-treated E. coli cells prepared as described above were thawed on ice for 15 min and aliquots of 100 pl were dispensed into pre-chilled microcentrifuge tubes. The ligation reaction or purified plasmid DNA (50-100 ng) was gently mixed with 100 pl of competent E. coli cells and incubated on ice for 30 min. The mixture was heat shocked at 42°C for 1 min without agitation and then abruptly chilled on ice for additional 2 min. One mL of non-antibiotic LB broth was added and the transformed cells were then shaken at 37°C with 180 rpm for 1 h. The transformed cells were centrifuged at 2,500 rpm RT for 30 sec and plated onto LB agar supplemented with 100 pg / ml ampicillin plate. The plates were incubated at 37°C overnight (14- 16 hours). The isolated colony was picked randomly and grown in media for plasmid extraction and verified by using restriction enzymes.

[0100] Example 2: Production of the recombinant phagemid particle

[0101] For phagemid / AAV (PAAV) vector production, the transformed pAAV TGI was added to 2XYT medium with ampicillin (or carbenicillin) at 100 pg / ml at 37°C and 250 rpm for 16 - 18 h. The preculture TGI was regrown in 20 ml of 2XYT media with ampicillin (or carbenicillin) 100 pg / ml until ODeoo nm was between 0.3 and 0.6. The 10 - 15 pl of 1011TFU helper phage was then added to the culture and incubated without shaking for 1 h. Next, the culture was added to 1000 ml of 2XYT with ampicillin (or carbenicillin) at pg / ml and kanamycin at 50 pg / ml for 1 h and then IPTG was added with a final concentration of 100 mM. The culture was continuously shaken for 18 h. To get rid of bacteria from the culture supernatant, the overnight bacterial culture was then centrifuged twice at 6000x g. The vectors in the supernatant were precipitated twice using polyethylene glycol (PEG)ZNaCl at a final concentration of 30% (w / v). The precipitate phage particles were separated by centrifugation at 10,000x g for 30 min. After being dissolved in 2 mL of PBS and then filtered through a 0.45micrometer (Dm) low protein binding PVDF filter, a pellet of the PAAV vector was finally used. PAAV titers were calculated as functional bacterial transducing units (TU) using colony counting of the host bacteria.

[0102] Example 3: Transfection in human HEK293 cells

[0103] Human embryonic kidney 293 (HEK293) cells were seeded into 96- well plate at 10,000 cells / well in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) for 24 hours. The cells were then transfected with PAAV3 Luc (0.2 pg DNA) using FuGENE® 6 Transfection Reagent (Promega, US), following the manufacturer’s protocol. In short, serum-free DMEM and FuGENE® 6 Transfection Reagent were incubated together for 5 minutes, 0.2 pg DNA was then added and incubated for 20 minutes. The mixture was added to HEK293 cells culture media. During transfection, cells were maintained in a humidified atmosphere at 37 °C and 5% CO2.

[0104] After 48 and 72 hours of transfection, the cells were processed for luciferase quantification using Luciferase Assay Systems (Promega, US). In brief, cell culture media was removed, then the cells were rinsed with phosphate buffered saline (PBS). After that, lysis reagent was added to the well then transferred to an opaque plate. Luciferase Assay Reagent was added, and the plate was measured for the light produced.

[0105] Results

[0106] HEK293 is a human cell line which is the most utilized human cell line for expression of recombinant proteins for various fields of research applications. In this study, HEK293 cells were used as a host eukaryotic cell for plasmid transfection to determine the capability of the PAAV3 Luc transgene cassette. Luciferase transgene was utilized as a reporter gene and the luciferase expression was detected as a synthesized cloned structural gene. In this regard, high activity of luciferase product was found only in PAAV3 Luc group, at both 48 and 72 hours (FIG. 12). Neither CTRL (only culture media) nor FuGENE6 (only FuGENE6 reagent) groups showed any luciferase activity. This phenomenon indicates that the PAAV3 Luc is capable of entering the host eukaryotic cells and synthesize the cloned structural gene.

[0107] Next, the capability of the present plasmid carrying transgene cassette was confirmed by changing the luciferase reporter gene to enhanced Green Fluorescent Protein (eGFP). The same transfection assay was carried out as in the prior experiment and the expression of the eGFP was then observed at day 3 and 6 after transfection.

[0108] Huge expression of eGFP was detected in the PAAV3 eGFP group only, but not in CTRL and FuGENE6 groups, both at day 3 and 6 after transfection (FIG. 13). This shares the same phenomenon as the previous experiment. Hence, the capability of the present plasmid for transgene delivery was confirmed. Example 4: Transduction into THP-1 cells (immune cells)

[0109] THP1 cells or HEK293 cells were seeded into 6-well plates at 300,000 cells / well in 10% FBS RPMI-1640 or 10% FBS DMEM, respectively, for 24 hours to reach 60%-70% confluence. Before transduction, culture media were replaced with serum- free media (RPMI-1640 for THP1, DMEM for HEK293) for 2 hours. pAAV3-C / zB-eGFP-MP12 was diluted in serum-free media as the transduction media. Both cells were transduced with pAAV3-C / zB-eGFP-MP12 at 2,000,000 TU / cell. During transduction, cells were maintained in a humidified atmosphere at 37 °C and 5% CO2.

[0110] At day 6 post-transduction, THP1 cells or HEK293 cells were harvested using Trypsin - EDTA (0.05%), phenol red (Gibco, US) and washed twice with phosphate-buffered saline (PBS). Cell numbers were adjusted to a concentration of 1 x 106cells / mL in cold Fluorescence-activated Cell Sorting (FACS) buffer and subjected to Flow cytometer (BD FACSAria™ III, Franklin Fakes, NJ, USA).

[0111] Results

[0112] THP1, which expresses some levels of CD14 on the cell surface at around 30% (Mittar et al., 2011), is a human monocyte cell line as antigen presenting cells. On the other hand, HEK293, which rarely expresses the CD14, is used as a negative control (Figuera et. al., 2012). FIG. 14 shows that pAAV3-C / zB-eGFP-MP12 was able to specifically bind to the THP1 and enter the cells. After that, the cassette gene expressed eGFP proteins which could be shown by the flow cytometry (48.2%; FIG. 14B). On the contrary, the fluorescence in the transduced HEK293 cells showed a very low amount of % positive cells (11.3%; FIG. 14A). These data demonstrate the positive capability of pAAV3-C / zB-eGFP-MP12, in terms of selective binding to CD14+ immune cells, gene delivery and transgene expression.

[0113] Example 5: Comparison between transfection and expression ofpAAV2 and pAAV3 using Luc2 as reporter sene

[0114] HEK293 cells were seeded into 96 well plate at 10,000 cells / well in DMEM with 10% FBS. The cells were then transfected with PAAV3 Euc (FIGS. 5 and 6) or PAAV2 Luc (FIGS. 7 and 8) (0.2 pg DNA) using FuGENE® 6 Transfection Reagent (Promega, US), following the manufacturer’s protocol. In brief, serum-free DMEM and FuGENE® 6 Transfection Reagent were incubated together for 5 minutes, 0.2 pg DNA (PAAV3 Luc or PAAV2 Luc) was then added and incubated for 20 minutes. The mixture was added to HEK293 cells culture media. During transfection, cells were maintained in a humidified atmosphere at 37 °C and 5% CO2.

[0115] At 2 and 3 days after transfection, the cells were processed for luciferase quantification using Luciferase Assay Systems (Promega, US). In detail, cell culture media was removed, then the cells were rinsed with phosphate buffered saline (PBS). Next, lysis reagent was added to the well then transferred to an opaque plate. Luciferase Assay Reagent was added, and the plate was measured the light produced.

[0116] Results There are 11 serotypes of AAV that have been identified. The best characterized and most commonly used is AAV2. In this study, we constructed the AAV3 transfer plasmid as a transgene cassette for gene delivery. Then, we compared the transduction efficiency between AAV3 and AAV2 transfer plasmid. We used HEK293 cell as a host eukaryotic cell for plasmid transfection and luciferase (Luc) transgene was utilized as a reporter gene. We detected the luciferase expression as a synthesized cloned structural gene. We found the high activity of luciferase product in both AAV2 and AAV3 group, at 48 and 72 hours (FIG. 15). Neither CTRL (only culture media) nor FuGENE6 (only FuGENE6 reagent) group showed any luciferase activity. Significantly, AAV3 group showed higher activity of luciferase product than AAV2 group at both observation days. Best Mode of the Invention

[0117] As described in Detailed Description of the Invention.

Claims

AMENDED CLAIMS received by the International Bureau on 11 August 2025 (11 .08.2025)1. A recombinant phagemid particle comprising a transgene expression cassette, wherein the transgene expression cassette comprises:I) inverted terminal repeats (ITRs) comprising an AAV3B 5” ITR and an AAV3B 3” ITR, the amino acid sequences of which are set forth in SEQ ID NO: 11 and SEQ ID NO: 16;II) a CMV enhancer as set forth in SEQ ID NO: 12;III) a chicken P-actin promoter as set forth in SEQ ID NO: 13;IV) a predefined protein encoding DNA andV) a polyadenylation (poly A) signal as set forth in SEQ ID NO: 15; wherein the recombinant phagemid particle further comprises:I) an immune cell-targeting peptide as set forth in SEQ ID NO: 2 or SEQ ID NO: 6;II) an endosome escape peptide as set forth in SEQ ID NO: 10;III) a hybrid intron as set forth in SEQ ID NO: 14; andIV) one or more antigens.

2. The recombinant phagemid particle according to claim 1, wherein the expression cassette comprises a bacteriophage fl origin and a drug resistance gene marker.

3. The recombinant phagemid particle according to claim 1, wherein the expression cassette further comprises a bacterial origin of replication.

4. The recombinant phagemid particle according to claim 1, wherein the predefined encoding protein DNA derived from a microorganisms or mammalian cell.

5. The recombinant phagemid particle according to claim 4, wherein the mammalian cell is selected from the group consisting of normal epithelial cells, immortalized cell lines, pluripotent stem cells, cancer-derived cells, or other animal cells including but not limited to Human Embryonic Kidney cells, Baby Hamster Kidney cells, Vero cells, HeLa cells, MDCK cells, and mouse hepatocytes.

6. The recombinant phagemid particle according to claim 4, wherein the microorganisms are from a virus, bacteria or fungi.

7. The recombinant phagemid particle according to claim 6, wherein the virus is selected from the group comprising SARS-COV-2, Dengue, Zika, Rift Valley Fever, Ebola, hemorrhagic fever viruses, Hepatitis and serotypes thereof or virus disease.

8. The recombinant phagemid particle according to any one of claims 4 to 7, wherein the SARS-COV-2 is a spike protein or immunogenic fragment thereof.

9. The recombinant phagemid particle according to claim 8, wherein the spike protein has the amino acid sequence set forth in SEQ ID NO: 18 or immunogenic fragment thereof.

10. The recombinant phagemid particle according to any one of claims 1 to 9, wherein the genome of the recombinant phagemid particle comprises a packaging signal for enabling replication of the phagemid genome into single-stranded DNA, which can subsequently be packaged into the phagemid particle inside a prokaryotic host.

11. The recombinant phagemid particle according to any one of claims 1 to 10, wherein the particle is capable of providing a therapeutic or prophylactic effect against an infectious disease.

12. The recombinant phagemid particle according to claim 11, wherein the particle is incorporated into a pharmaceutical composition or a pharmaceutically acceptable carrier.

13. The recombinant phagemid particle according to claim 12, wherein the pharmaceutically composition is vaccine, tablet, capsule, liquid formulation, topical formulation or inhalation from nasal spray.

14. A system for producing a recombinant phagemid particle from a prokaryotic host, comprising: a) a first vector configured to persist inside a prokaryotic host, and comprising at least one transgene expression cassette comprising a polynucleotide encoding one or more antigens operably linked to at least one promoter, and a packaging signal for enabling replication of the vector into single-stranded DNA; wherein i) the one or more predefined encoding protein DNA are selected from the group comprising pathogenic microorganisms such as virus antigens, bacterial antigens, parasite antigens, fungal antigens and cancer cell antigens, or any combination thereof; a) a second vector comprising a polynucleotide encoding structural proteins required for packaging the single-stranded DNA from step a), a polynucleotide encoding a targeting peptide on its outer surface to target the phagemid particle to an immune cell, and a polynucleotide encoding an endosome escape peptide on its outer surface, resulting in the formation and extrusion of a recombinant phagemid particle from the prokaryotic host which can target expression of the one or more antigens to within immune cells; wherein i) the immune cell-targeting peptide is expressed on phagemid pill minor coat proteins, and / orii) the endosome escape peptide is expressed on phagemid pVIII major coat proteins.

15. The system according to claim 12, wherein the endosome escape peptide is a pH-dependent membrane-active peptide (PMAP).

16. The system according to claim 12 or 13, wherein the endosome escape peptide comprises one or more lysine residues.

17. The system according to any one of claims 12 to 14, wherein the immune cell -targeting peptide comprises an amino acid sequence selected from the group comprising KYSFKLILAEYGGGS (TLR4 ligand; SEQ ID NO: 2), YEQDPWGVKWWYGGGS (M2pep; SEQ ID NO: 4) and FHRWPTWPLPSPGGGS (MP12 peptide; SEQ ID NO: 6), or functional amino acid sequence variant thereof; and / or the endosome escape peptide comprises an amino acid sequence selected from the group comprising SFGLFLAIALFILGGWLGLILGWYG (L5WYG; SEQ ID NO: 8) and SFGLFKAIAKFIKGGWKGLIKGWYG (K5WYG; SEQ ID NO: 10) or functional amino acid sequence variant thereof.

18. The system according to any one of claims 12 to 15, wherein said recombinant phagemid particle is defined in any one of claims 1 to 11.

19. A method for the treatment or prophylaxis of an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant phagemid particle according to any one of claims 1 to 11.

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