hepatitis e virus-like particles (vlp) derived from consensus sequence

Abstract

Virus-like particles derived from the subfamily of a parvovirus-like virus and the subfamily of a orthohepatovirus, in particular virus-like particles derived from the species of the virus Ballum hepatitis E virus, which can cause acute hepatitis in humans and a variety of mammals, as well as chronic disease in immunocompromised patients. Compositions of virus-like particles comprising viral capsid proteins that are capable of assembling in cultured cells, which can be purified, disassembled and reassembled in the presence of other molecules, are provided, which are suitable for use as therapeutic drug products to facilitate the targeting and delivery of cargo molecules to specific cells or tissues, or as antigenic agents aimed at stimulating a response to heterologous epitopes exposed on the surface of the virus-like particles. Functional capsids are provided comprising polypeptide sequences containing amino acid substitutions, insertions or deletions of amino acids encoded by the consensus sequence of the ORF2 gene, which are functionally similar or have enhanced properties compared to capsid polypeptides encoded by naturally occurring viruses obtained from clinical samples or prototype hepatitis E virus (HEV).

Description

[0001] Priority Statement This application claims priority to U.S. Provisional Application US 63 / 609,538, filed on December 13, 2023.

[0002] By referencing and incorporating into the sequence list The sequence list contained in the file “762_196_008_US_01_Sequence_Listing_2023_12_11.xml” was created on November 6, 2023, and modified on December 11, 2023. The file size is 32,173 bytes. This sequence list, as well as any original and modified sequence lists, are incorporated herein by reference in their entirety. Technical Field

[0003] The study discloses virus-like particles (VLPs) containing capsid proteins derived from Hepeviridae viruses. These VLPs can encapsulate or conjugate nucleic acids, peptides, or small molecules for use in various diagnostic and therapeutic applications. VLPs derived from the following subfamilies are also disclosed: one is Parahepevirinae (infecting trout and salmon), and the other is Orthohepevirinae (infecting mammals and birds), particularly those derived from Bareny hepatitis E virus (Hepatitis E virus). Paslahepevirus balayani This invention relates to virus-like particles containing viral capsid proteins capable of assembling in cultured cells, and to virus-like particles that can cause acute hepatitis in humans and many other mammals, as well as chronic disease in immunocompromised patients. A key aspect of the invention relates to compositions of virus-like particles comprising viral capsid proteins capable of assembly in cultured cells, and to virus-like particles that can be purified, disassembled, and reassembled in the presence of other molecules. These compositions are suitable for use as therapeutic pharmaceutical products to facilitate the targeting and delivery of cargo molecules to specific cells or tissues, or as antigens designed to stimulate a response to heterologous epitopes exposed on the surface of virus-like particles. A preferred aspect relates to functional capsids derived from polypeptide sequences comprising one or more amino acid substitutions, insertions, or deletions of amino acids encoded by a common sequence of the ORF2 gene, wherein said variant polypeptides are functionally similar to or have enhanced properties compared to capsid polypeptides encoded by naturally occurring viruses obtained from clinical samples or prototype hepatitis E virus (HEV). Background Technology

[0004] Designing and assembling nucleic acids containing one or more genetic elements in a desired sequence typically requires a variety of techniques, including cloning one or more isolated DNA sequences into a vector that can reproduce in bacteria, sequencing the cloned insert, introducing the vector into a suitable host cell, and expressing the peptide under the control of a promoter operatively linked to the insert sequence. Structural and functional analysis of the expressed peptides accelerates research and often facilitates the development and commercialization of therapeutic products (e.g., small and large molecules, vaccines, components of cell and gene therapy vector systems, and research tools), thereby benefiting institutions with broad and significant interests in the life sciences.

[0005] Virus-like particles derived from the viral capsid system Virus-like particles (VLPs) have been used as nanocarriers to encapsulate nucleic acid or small molecule drug products, or to display epitopes on their surfaces, thereby facilitating the targeting and delivery of molecules to cells for the detection and treatment of various diseases. This review provides a comprehensive overview of the discovery, development, and commercialization of commercial drug delivery technologies, including VLPs (Vargason et al. (2021)).

[0006] Figure BK-1: This figure shows a classification of treatment and delivery modes.

[0007] Hepatitis E virus-like particles (HEV-VLP) Hepatitis E virus (HEV) possesses a non-enveloped icosahedral capsid enclosing a 7.2 kb single-stranded RNA (ssRNA) genome. The major capsid proteins are encoded by a second open reading frame (ORF2), which is crucial for viral assembly, immunogenicity, and host-cell interactions (Purcell 1996; Tam et al. 1991; Xing et al. 2011; Schofield et al. 2000; Yu et al. 2010). HEV-VLP forms a hollow T=1 icosahedral capsid composed of 60 identical units (Xing et al. 1999), providing high stability under storage and harsh physiological conditions. HEV-VLP has surface-exposed spike (P) domains that are connected to a stable icosahedral base via flexible hinges. The P domains form surface-exposed spikes on the icosahedral base. The shell (S) and intermediate (M) domains can assemble into a stable icosahedral shell of the HEV-VLP, while the P domain protrudes to form surface spikes, the depth of which (the spikes they carry) determines the antigenicity of HEV (Yamashita et al. 2009; Xing et al. 2011). The modularity of these three domains facilitates the assembly of antigenic particles, while reducing the size of these particles from T3 icosahedral particles to T1 icosahedral particles (Xing et al. 1999), without interfering with the HEV-VLP assembly process regardless of whether the P domain sequence is modified (Niikura et al. 2002; Jariyapong et al. 2013).

[0008] Figure BK-2 shows small figures A and B. Small figure A shows the crystal structure of HEV VLP, and small figure B shows the banding diagram of the capsid subunit.

[0009] Recombinant capsid protein (PORF2), when expressed in insect cells, can self-assemble into virus-like particles. These recombinant capsid proteins have 111 amino acids missing from their N-terminus and 52 amino acids missing from their C-terminus (Li et al. 2005b; Li et al. 1997). PORF2 folds into a shell domain, an intermediate domain, and a protruding domain, corresponding to amino acids 118-317, 318-451, and 452-606, respectively (Guu et al. 2009; Xing, Li, Miyazaki et al. 2010; Yamashita et al. 2009). The flexible hinge allows modification of the P-domain by inserting exogenous peptides (Jariyapong et al. 2013) or by conjugating chemicals to residues in the P-domain without disrupting its basic icosahedral structure (Chen et al. 2016). The three variable loops and the C-terminus of the P domain can also be modified to contain conjugation sites for bioactive agents exposed on the surface of HEV-VLP (Cheng et al. 2015; Chen et al. 2016; Cheng 2017). Replacing other amino acids with cysteine ​​residues or adding new cysteine ​​residues to the P domain can reduce the response of previously existing antibodies to HEV-VLP, thereby compensating for the deficiencies of many other protein-based delivery vector systems (Chen et al. 2016).

[0010] HEV-VLP-based nanotherapeutic agents and diagnostic agents Nanotherapeutic agents have accelerated the delivery of therapeutic and diagnostic products to specific cell and tissue types (Ludwig and Wagner 2007). These systems rely on delivering virus-like particles conjugated with therapeutic components to modulate the activity of regulatory and structural molecules in compromised host cell systems (Galaway and Stockley 2013; Ma, Nolte, and Cornelissen 2012; Chen et al. 2016). Many of these systems suffer from problems such as passive cellular uptake, premature degradation, toxicity, and insufficient binding to therapeutic molecules. However, VLP-based systems overcome many of these obstacles by enhancing the display of exogenous epitopes on their surfaces or by delivering small molecules directly to cells (Ludwig and Wagner 2007).

[0011] Modular therapeutics and diagnostics derived from HEV virus-like particles have been proposed as platforms for vaccine, cancer targeting, diagnostic, and therapeutic applications (Chen et al. 2016; Cheng et al. 2017; Holla et al. 2017; Stark et al. 2017; Chen, Baikoghli, and Cheng 2018; Jariyapong et al. 2013; Niikura et al. 2002). Flexible domains in HEV-VLPs can be used to conjugate functional peptides or compounds to their surfaces (Chen et al. 2016; Stark et al. 2017; Niikura et al. 2002; Jariyapong et al. 2013), thereby enabling the encapsulation of small molecule drug products (Chen, Baikoghli, and Cheng 2018), DNA and RNA molecules (Takamura et al. 2004), and inorganic beads (Chen CC 2017).

[0012] Figure BK-3: This figure illustrates the surface finishing capabilities of the HEV-VLP.

[0013] HEV-VLPs retain many of the biophysical properties of natural virions, including structural stability, antigenicity, and cell-binding ability. These particles, like natural viruses, are stable in acidic environments (Zafrullah et al. 2004) and resistant to digestion by proteolytic enzymes (Jariyapong et al. 2013; Chen, Baikoghli, and Cheng 2018), thus allowing for oral delivery. Administration of chimeric HEV-VLPs displaying exogenous epitopes stimulates systemic and mucosal immune responses (Niikura et al. 2002) with undetectable tolerability, and provides protection against HEV in non-human primates (Li et al. 2004). Orally delivered HEV-VLPs can also deliver plasmid DNA to small intestinal epithelial cells, stimulating antibody and cytotoxic T lymphocyte responses against plasmid-encoded antigens (Takamura et al. 2004).

[0014] HEV-VLPs with modified surface-protruding domains can also be used as diagnostic agents or as targeted drug delivery mediators for the treatment of cancer and other diseases. HEV-VLPs conjugated to ligand peptides (e.g., LXY30) with high affinity for malignant breast tumor cells have bound to tumor cells in in vitro and in vivo assays, suggesting that the delivery of diagnostic or therapeutic agents to pathological lesions can be facilitated by modulating the administration pathway (Chen et al. 2016; Stark et al. 2017).

[0015] Negatively charged nucleic acids (e.g., DNA and mRNA) can also be encapsulated for use in vaccines (Takamura et al. 2004) or as carriers for gene therapy delivery systems (Panda et al. 2015). HEV-VLP has also been proposed as a carrier for the oral delivery of proteins (e.g., insulin) to treat diabetes (Chen, Baikoghli, and Cheng 2018). Magnetic particles (e.g., ferrites) can also be encapsulated and used as contrast agents in magnetic resonance imaging or for tumor-targeted hyperthermia applications (Roemer 1999; Chen CC 2017).

[0016] HEV-VLPs can also be used in cell-specific drug targeting and delivery systems (DDS) by combining surface modification and payload encapsulation capabilities to deliver epitope and DNA vaccines, MRI / PET imaging enhancers, and drug delivery systems for gene therapy, genome editing, and cancer. All of these applications require scalable, low-cost expression and purification of stable functional particles.

[0017] HEV-VLP has also been proposed as a nanoplatform for developing therapeutic agents, introducing reactive sites by replacing or adding amino acids such as cysteine, thereby facilitating the conjugation of ligands or small molecules (e.g., maleimide-linked peptides or molecules) to prominent domains. Structural analysis of HEV-VLP and well-studied immunogenic epitopes has identified residues within the flexible ring and the exposed C-terminal sites, which can be replaced by or allowed to be inserted into cysteine ​​residues as chemical conjugation sites (Xing, Li, Mayazaki, et al. 2010; Xing et al. 2011; Chen et al. 2016).

[0018] Therapeutic and diagnostic agents targeting cancer cells require targeting ligands to facilitate the binding of the agent to and uptake by malignant cells. HEV-VLP conjugated with LXY30 bound to breast tumor cells in both in vitro and in vivo assay systems, suggesting that HEV-VLP can be modulated in other ways to facilitate drug delivery to pathological lesions (Xiao et al. 2016; Chen et al. 2016). Accumulation of HEV-VLP in abdominal organs, including the liver, has also been observed (Chen et al. 2016), which may inspire the development of liver-specific positron emission tomography (PET) imaging agents chemically conjugated with radioactive gallium-68[68Ga] (Lambidis, Chen, Lumén, et al. 2022; Lambidis, Chen, Baikoghli, et al. 2022), thus supporting the development of HEV-VLP as a nanotherapeutic agent, particularly one affecting the liver and related cells and tissues.

[0019] In recent years, reports describing VLP-based targeted drug delivery systems have increased dramatically. While nanocarriers incorporating synthetic polymers, liposomes, or linear-dendritic block copolymers have been extensively studied over the past 20 years, many limitations remain to be addressed, including toxicity, inability to accumulate sufficient quantities of molecules in the cytoplasm, and lack of biodegradability (Sebestik, Niederhafner, and Jezek 2011; Jian et al. 2012). Liposome carriers (e.g., Doxil®) lack tumor-targeting capabilities but tend to accumulate in solid tumors due to the characteristics of tumor regions (e.g., larger pores in neovascularization (EPR effect)) (Shi et al. 2020). Drug leakage during circulation, leading to adverse side effects, is another concern (Russell, Hultz, and Searson 2018).

[0020] Modular diagnostic and therapeutic HEV-VLPs can also be designed to utilize both the chemically exposed portion inside the capsid and the portion exposed on the capsid surface. HEV-VLPs have been designed to encapsulate viral RNA to form highly stable, non-infectious capsids capable of reversible in vitro assembly mediated by cation exchange (Xing, Li, Mayazaki, et al. 2010), demonstrating that they can also switch back and forth in a self-assembly reaction via a series of steps involving chemical reduction and cation chelation, mediated by reducing agents and chelating agents such as dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and calcium or magnesium ions.

[0021] Figure BK-4: This figure illustrates the encapsulation capability of HEV-VLP.

[0022] While HEV capsids typically encapsulate ssRNA molecules primarily mediated by charge-based interactions, modified capsids should be able to encapsulate small proteins or molecules for therapeutic drug applications. HEV-VLPs have been used for oral delivery of plasmid DNA encoding antigens to small intestinal epithelial cells for transient expression (Takamura et al. 2004; Cheng and Xing 2014). Negatively charged mRNA can also be encapsulated within HEV-VLPs for gene delivery. HEV-VLPs have also been shown to encapsulate magnetic virus-like particles, such as citrate-coated ferrites, suitable for diagnostic purposes under MRI and for tumor-targeted hyperthermia induced by ultrasound or radiofrequency electromagnetic radiation (Chen CC 2017). HEV-VLPs containing surface-bound cancer cell-targeting ligands have potential applications in gene therapy (Stark and Cheng 2016).

[0023] Therapeutic and diagnostic agents derived from HEV-VLP should be able to encapsulate a variety of molecules, including other types of inorganic nanoparticles, including metal nanoparticles, such as gold nanoparticles, and the aforementioned ferrite (Fe3O4) nanoparticles.

[0024] Key issues to be addressed in designing and optimizing methods for the disassembly and reassembly of VLPs include the concentration and ratio of chelating agents and calcium or magnesium ions, the concentration, charge, and other properties of the payload molecules. Application-related issues also need to be addressed, including the dosage of the encapsulated molecules, delivery and release of molecules at the target site, and cost-effectiveness, all of which are crucial for producing larger quantities of GMP-quality materials required for clinical trials.

[0025] HEV-VLP-based immunomodulators Atopic diseases (such as asthma, allergic rhinitis, and atopic dermatitis) are caused by environmental and genetic factors. Exposure to hepatitis A virus (HAV), poor sanitation, large family sizes, and enrollment in daycare facilities are all negatively associated with atopy. Individuals infected with HAV may be protected from atopy if they carry a variant of the gene encoding TIM-1 (the cell surface receptor used by HAV to infect human cells) (named HAVcr-1) (McIntire et al. 2003). These results suggest that the interaction between HAV and cells expressing the TIM-1 variant is somehow involved in the etiology of atopic diseases, thus supporting the sanitation hypothesis.

[0026] Hepatitis E virus transmitted orally is also considered a pathogen associated with poor sanitation. HEV-VLP can also be used to induce the expression of many genes in THP-1 cells derived from mononuclear cells of acute leukemia patients, and it is commonly used in studies of immune system disorders, immunology, and toxicology. The in vivo effects of HEV VLP as an oral immunomodulator can also be studied in various animal models (e.g., chickens vaccinated against avian influenza) by measuring immune response and weight gain. Summary of the Invention

[0027] A key aspect of the present invention relates to nucleotide sequences encoding polypeptide sequences derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming functional virus-like particles, wherein the virus-like particles encapsulate or conjugate one or more molecules selected from the group consisting of nucleic acids, polypeptides, inorganic nanoparticles, and small molecule pharmaceutical products.

[0028] A key aspect of the invention also relates to vectors comprising the aforementioned nucleotide sequences encoding functional virus-like particles, including vectors selected from the group consisting of cloning vectors and expression vectors, including expression vectors comprising nucleotide sequences encoding functional virus-like particles, the nucleotide sequences being operatively linked to a promoter at their 5' end and optionally operatively linked to a transcription termination signal at their 3' end, more preferably wherein the expression vector is a baculovirus expression vector, and most preferably wherein the baculovirus expression vector comprises a baculovirus promoter operatively linked to the nucleotide sequence encoding functional virus-like particles, for example, a vector comprising nucleotide sequences represented by SEQ ID NO: 01, 03, 05, 07, 09, 11 and 13.

[0029] The main aspect of this invention relates to prokaryotic or eukaryotic cells containing the aforementioned carrier, preferably eukaryotic cells, insect cells, lepidopteran insect cells, and most preferably lepidopteran insect cells selected from the group consisting of: fall armyworm (… Spodoptera frugiperda ), Powdered Noctuid moth ( Trichoplusia ni ) and silkworm ( Bombyx mori ), and other eukaryotic cell lines, including fungal cells (such as yeast cells) and prokaryotic cells (such as Escherichia coli). Escherichia coli )cell).

[0030] The main aspect of the present invention relates to a method for isolating functional virus-like particles derived from a common sequence of capsid proteins of the Hepatitis E Viridae family, comprising one or more amino acid substitutions, insertions, or deletions, the method comprising the steps of: (a) infecting susceptible insect cells with a baculovirus expression vector containing a nucleotide sequence encoding a functional virus-like particle under the control of a polyhedrosis protein promoter; (2) monitoring capsid protein expression over time; (3) concentrating, binding, and eluting the purified capsid protein; and (4) forming functional virus-like particles.

[0031] A key aspect of this invention relates to polypeptide sequences derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming functional virus-like particles, wherein the virus-like particles encapsulate or conjugate molecules selected from the group consisting of nucleic acids, polypeptides, inorganic nanoparticles, and small molecule pharmaceutical products. Related aspects include polypeptide sequences wherein the common sequence of hepatitis E virus family capsid proteins is derived from the hepatitis E virus subfamily, including those derived from the Bareni hepatitis E virus species within the hepatitis E virus subfamily.

[0032] A key aspect of the present invention is a functional virus-like particle derived from a common sequence of a capsid protein of the Hepatitis E Viridae family, comprising one or more amino acid substitutions, insertions, or deletions, wherein the virus-like particle encapsulates or conjugates a molecule selected from the group consisting of nucleic acids, peptides, or small molecule pharmaceutical products.

[0033] A better understanding of the invention will be obtained from the following detailed description and accompanying drawings, which illustrate illustrative embodiments and demonstrate various ways in which the principles of the invention are applied. Attached Figure Description

[0034] Statement regarding color illustrations This patent or application document contains the text of the specification and at least one color drawing. The Patent Office will provide a copy of the publication of this patent or application with one or more color drawings upon request and payment of the necessary fees.

[0035] Statement regarding aspects of the invention as understood with reference to the accompanying drawings. The foregoing aspects and numerous incidental advantages of the invention will become more readily understood as they become more apparent when taken in conjunction with the accompanying drawings and the following detailed description, wherein: Figure 1 The illustration shows a classification of treatment and delivery modes.

[0036] Figure 2 Figure A and Figure B are shown. Figure A shows the crystal structure of the HEV VLP, and Figure B shows the banding diagram of the capsid subunit.

[0037] Figure 3 The illustration shows the surface finishing capabilities of the HEV-VLP.

[0038] Figure 4 The illustration shown is titled "Encapsulation Capabilities of HEV-VLP".

[0039] Figure 5 The illustration shows the title "Pretreatment of expression cells before purification of HEV-VLP not secreted from cultured cells".

[0040] Figure 6 The illustration shows a purification process with the title "A capture / purification step via a pH gradient-based ion exchange method".

[0041] Figure 7 The illustration shows a purification process with the title "A capture / purification step via a salt gradient-based ion exchange method".

[0042] Figure 8The illustration shown is titled "A Universal Encapsulation Process for HEV-VLP".

[0043] Figure 9 The illustration, titled "Frequency of amino acid substitutions in the associated HEV ORF2 capsid sequence used to generate a common capsid sequence," is based on 124 different HEV capsid proteins, 93 of which are described as Barney hepatitis E virus, with the remainder derived from other sources (including pigs).

[0044] Figure 10 The illustration shows the predicted secondary structure of the common sequence derived from the associated HEV ORF2 capsid protein through multiple sequence alignment.

[0045] Figure 11 The diagram shows the distances of the number of amino acid variations across 124 sequences (lower green, higher red).

[0046] Figure 12 The graph shows the frequency of amino acid variations across 124 sequences (lower green, higher red).

[0047] Figure 2-13 The illustration shows a classification of treatment and delivery modes.

[0048] Figure 14 The figure shown is titled "Analysis of Peptide Hydrophobicity / Hydrophilicity".

[0049] Figure 15 The figure shown is titled "Hydrophobicity / hydrophilicity analysis of peptide NS5A (1-31)".

[0050] Figure 16 The illustration shown is titled "NCT-002: HEV-NS5A-VLP".

[0051] Figure 17 The illustration shows the title "PDB 5GGS (120 aa) (484 aa) (VHH Pembrolizumab 1-120 aa) inserted after 364 aa P".

[0052] Figure 18 The illustration shown is titled "Schematic diagram of the secondary structure interaction of pembrolizumab VHH based on the X-ray crystal structure of pembrolizumab (PDB: 5GGS)".

[0053] Figure 19 The illustration shows the P domain of the N / C-terminal deletion variant of the VHH substitution common sequence of mAb.

[0054] Figure 20 The illustration shows the title “NCT-006: VHH of mAb plus additional linker replaces the P domain of the proposed common sequence with N / C terminal missing version”.

[0055] Abbreviations, terms and their definitions The following is a list of abbreviations, terms, and their definitions used in the main text of the specification, figures, sequence list, supplementary data sheets (if any), and claims: Table 1 - List of Abbreviations Common abbreviations and their corresponding meanings include: aa or AA = amino acid; mg = milligram; ml or mL = milliliter; mm = millimeter; mM = millimole; nmol = nanomolar; pmol = picomolar; ppm = parts per million; RT = room temperature; U = unit; ug, µg = microgram; ul, µl = microliter; uM, µM = micromolar.

[0056] Specific abbreviations and their corresponding meanings include: The term “cell (cell and cells)” (meaning inclusive) refers to one or more types of cells that can be in isolation or culture (such as in a cell line containing homogeneous or heterogeneous cell populations), or in a tissue sample, or as part of an organism (such as a transgenic animal).

[0057] Unless otherwise stated, the term "amino acid" encompasses both naturally occurring and non-naturally occurring amino acids.

[0058] The term "pharmacokinetics" (PK) refers to the study of how organisms affect drug products, such as the absorption of drug products by the body, their conversion into other products as substrate organisms, the distribution of drug products and their metabolites in tissues, and the elimination of drugs and their metabolites from the body over a period of time.

[0059] The term "pharmacodynamics" (PD) refers to the study of how a drug product affects an organism.

[0060] Data from both PK and PD model systems influence decisions regarding the dosage, benefits, and adverse reactions of compositions containing one or more drug products. Detailed Implementation

[0061] This invention relates to virus-like particles derived from the following subfamilies: one is the Hepatitis E Virinae subfamily (infecting trout and salmon), and the other is the Hepatitis E Virinae subfamily (infecting mammals and birds), particularly virus-like particles derived from the Bareni Hepatitis E Virus species, which can cause acute hepatitis in humans and many other mammals, as well as chronic disease in immunocompromised patients.

[0062] The main aspects of the present invention relate to compositions of virus-like particles comprising viral capsid proteins capable of assembly in cultured cells, wherein the virus-like particles can be purified, disassembled, and reassembled in the presence of other molecules, and wherein the compositions are suitable for use as therapeutic pharmaceutical products to facilitate the targeting and delivery of cargo molecules to specific cells or tissues, or as antigens designed to stimulate a response to heterotopes exposed on the surface of virus-like particles.

[0063] Preferred aspects involve functional capsids comprising polypeptide sequences containing one or more amino acid substitutions, insertions, or deletions encoded by a common sequence of the ORF2 gene, wherein the variant polypeptide is functionally similar to or has enhanced properties compared to a capsid polypeptide encoded by a naturally occurring virus obtained from a clinical sample or prototype hepatitis E virus (HEV).

[0064] Other aspects include the design and assembly of modified vectors to facilitate basic and applied research, thereby developing and commercializing novel drug products, and serving as a tool to advance the interests of institutions involved in animal and human healthcare.

[0065] A nucleotide sequence encoding a polypeptide sequence derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming a functional virus-like particle (VLP), wherein the VLP encapsulates or conjugates one or more molecules, the one or more molecules being selected from the group consisting of nucleic acids, polypeptides, inorganic nanoparticles, and small molecule pharmaceutical products.

[0066] A key aspect of the present invention relates to nucleotide sequences encoding polypeptide sequences derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming functional virus-like particles, wherein the virus-like particles encapsulate or conjugate one or more molecules selected from the group consisting of nucleic acids, polypeptides, and small molecule pharmaceutical products.

[0067] The preferred aspect relates to a nucleotide sequence wherein the common sequence of the hepatitis E virus family capsid protein is derived from the hepatitis E virus subfamily, and a nucleotide sequence wherein the common sequence of the hepatitis E virus family capsid protein is derived from the Bareni hepatitis E virus species of the hepatitis E virus subfamily.

[0068] Other preferred aspects relate to nucleotide sequences, wherein the polypeptide sequence contains one or more conserved amino acid substitutions in a variable or invariant domain of a common sequence, the nucleotide sequence including nucleotide sequences in which the polypeptide sequence contains one or more conserved amino acid substitutions in a variable domain of a common sequence, or nucleotide sequences in which the polypeptide sequence contains one or more conserved amino acid substitutions in an invariant domain of a common sequence.

[0069] Other preferred aspects relate to nucleotide sequences, wherein the polypeptide sequence comprises one or more amino acid deletions, including wherein the polypeptide sequence comprises a deleted nucleotide sequence at the amino terminus, carboxyl terminus, or both amino terminus and carboxyl terminus of the common sequence, or wherein the polypeptide sequence comprises one or more deleted nucleotide sequences in a variable domain of the common sequence.

[0070] Other preferred aspects relate to nucleotide sequences, wherein the polypeptide sequence comprises one or more amino acid insertions, the nucleotide sequence including either a polypeptide sequence containing one or more inserted nucleotide sequences in a variable domain of a common sequence, or a polypeptide sequence containing one or more inserted nucleotide sequences in an invariant domain of a common sequence.

[0071] Other preferred aspects relate to nucleotide sequences, wherein the polypeptide sequence contains one or more conserved amino acid substitutions, and one or more insertions or deletions in a variable or invariant domain of a common sequence, or wherein all said conserved amino acid substitutions, insertions or deletions are located in a variable domain of the common sequence.

[0072] Other preferred aspects involve the aforementioned nucleotide sequences, which further comprise one or more insertions of DNA fragments encoding heterologous polypeptides selected from the group consisting of: nucleic acid binding domains, hydrophobic binding domains, polypeptide binding domains, and antibody binding domains, preferably wherein the domain is a nucleic acid binding domain, a hydrophobic binding domain, a hydrophilic binding domain, a polypeptide binding domain, or an antibody binding domain.

[0073] A key aspect of the invention relates to vectors comprising the aforementioned nucleotide sequences encoding functional virus-like particles, including vectors selected from the group consisting of cloning vectors and expression vectors, including expression vectors comprising nucleotide sequences encoding functional virus-like particles, the nucleotide sequences being operatively linked to a promoter at their 5' end and optionally operatively linked to a transcription termination signal at their 3' end, more preferably wherein the expression vector is a baculovirus expression vector, and most preferably wherein the baculovirus expression vector comprises a baculovirus promoter, such as a polyhedrosis protein or p10 promoter, operatively linked to the nucleotide sequence encoding functional virus-like particles. Other aspects relate to bacterial expression vectors, mammalian expression vectors, and yeast expression vectors. Preferred aspects relate to any of the aforementioned vectors comprising nucleotide sequences represented by SEQ ID NO: 01, 03, 05, 07, 09, 11, and 13, which encode polypeptides represented by SEQ ID NO: 02, 04, 06, 08, 10, 12, and 14, respectively.

[0074] The main aspects of this invention relate to prokaryotic or eukaryotic cells containing the aforementioned carriers, preferably eukaryotic cells, insect cells, lepidopteran insect cells, and most preferably lepidopteran insect cells selected from the group consisting of: fall armyworm, white armyworm, and silkworm. Other aspects relate to mammalian cells (including primate cells and human cells), fungal cells (including filamentous fungi and non-filamentous fungi, including yeast cells), and prokaryotic cells (including Escherichia coli cells).

[0075] A key aspect of this invention relates to polypeptide sequences derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming functional virus-like particles, wherein the virus-like particles encapsulate or conjugate one or more molecules selected from the group consisting of nucleic acids, polypeptides, inorganic nanoparticles, and small molecule pharmaceutical products. The relevant aspect includes polypeptide sequences wherein the common sequence of the hepatitis E virus family capsid protein is derived from the hepatitis E virus subfamily, including those derived from the Bareni hepatitis E virus species within the hepatitis E virus subfamily.

[0076] The relevant aspect includes a polypeptide sequence that contains one or more conserved amino acid substitutions in a variable or invariant domain of a common sequence, wherein the polypeptide sequence contains one or more conserved amino acid substitutions in a variable domain of a common sequence, or wherein the polypeptide sequence contains one or more conserved amino acid substitutions in an invariant domain of a common sequence.

[0077] The relevant aspect includes a polypeptide sequence containing one or more amino acid deletions, including the polypeptide sequence containing deletions at the amino terminus, carboxyl terminus, or both amino terminus and carboxyl terminus of the common sequence, or the polypeptide sequence containing one or more deletions in the variable domain of the common sequence.

[0078] The relevant aspect includes a polypeptide sequence containing one or more amino acid insertions, including the polypeptide sequence containing one or more insertions in a variable domain of a common sequence, or the polypeptide sequence containing one or more insertions in a constant domain of a common sequence.

[0079] The relevant aspect includes a polypeptide sequence that contains one or more conserved amino acid substitutions, and one or more insertions or deletions in a variable or invariant domain of a common sequence, including wherein all said conserved amino acid substitutions, insertions or deletions are located in a variable domain of the common sequence.

[0080] Other relevant aspects include the aforementioned polypeptide sequence, which further comprises one or more insertions of a DNA fragment encoding a heterologous polypeptide selected from the group consisting of: nucleic acid binding domains, hydrophobic binding domains, polypeptide binding domains, and antibody binding domains, including the polypeptide sequence, wherein the domain is a nucleic acid binding domain, a hydrophobic binding domain, a hydrophilic binding domain, a polypeptide binding domain, or an antibody binding domain.

[0081] A key aspect of the present invention is a functional virus-like particle derived from a common sequence of a capsid protein of the Hepatitis E Viridae family, comprising one or more amino acid substitutions, insertions, or deletions, wherein the virus-like particle encapsulates or conjugates one or more molecules selected from the group consisting of nucleic acids, peptides, or small molecule pharmaceutical products.

[0082] Related aspects include functional virus-like particles, wherein the virus-like particles encapsulate one or more molecules selected from the group consisting of: nucleic acids (including single-stranded RNA (ssRNA) molecules, double-stranded RNA (dsRNA) molecules, single-stranded DNA (ssDNA) molecules, and double-stranded DNA (dsDNA) molecules), peptides, inorganic nanoparticles, and small molecule drug products.

[0083] Other relevant aspects include functional virus-like particles, wherein the virus-like particles encapsulate one or more molecules selected from the group consisting of nucleic acids, peptides, and inorganic nanoparticles.

[0084] The main aspect of the present invention relates to a method for isolating functional virus-like particles derived from a common sequence of capsid proteins of the Hepatitis E Viridae family, comprising one or more amino acid substitutions, insertions, or deletions, the method comprising the steps of: (a) infecting susceptible insect cells with a baculovirus expression vector containing a nucleotide sequence encoding a functional virus-like particle under the control of a baculovirus promoter; (2) monitoring capsid protein expression over time; (3) concentrating, binding, and eluting the purified capsid protein; and (4) forming functional virus-like particles.

[0085] Example The foregoing discussion can be better understood in conjunction with the following representative examples, which are provided to illustrate the main methods and compositions of the invention and not to limit it. Various other examples will be apparent to those skilled in the art upon reading this disclosure without departing from the spirit and scope of the invention. All such other examples are intended to be included within the scope of the appended claims.

[0086] General Materials and Methods Unless otherwise stated, all portions are by weight (e.g., % w / w), and temperatures are in degrees Celsius (°C).

[0087] The table below provides an overview of the nucleotide and amino acid sequences described in this application. Even-numbered sequence identifier numbers (SEQ ID NO) generally indicate polypeptide sequences, while odd-numbered SEQ ID NOs indicate nucleotide sequences encoding the corresponding polypeptide sequences, typically obtained by reverse translating the polypeptide sequence into a nucleotide sequence.

[0088] Table 2 - Summary Table of Serial ID Numbers Expression and assembly of HEV VLP in baculovirus expression vector system Baculovirus expression vector systems (BEVS) have been widely used to promote the expression of heterologous proteins in cultured insect cells, most commonly based on alfalfa silver-striped armyworm nucleopolyhedrovirus (AcNPV) or silkworm nucleopolyhedrovirus (BmNPV), which are used to infect fall armyworm, cabbage white armyworm, or silkworm (silkworm) cells [O'Reilly, Miller and Luckow, 1992].

[0089] Several methods have been used to promote the generation of recombinant baculoviruses. The earliest method was based on homologous recombination between a transfer vector and a wild-type or modified baculovirus DNA sample transfected into cultured insect cells, replacing the polyhedrosis protein gene to generate a virus that forms inclusion-free plaques at a frequency of 0.1% to 1% on cultured cell moss under a low-melting-point agarose layer [O'Reilly, Miller, and Luckow, 1992]. When linearized parental viruses were used, the frequency increased to about 30%, and later, with the development of improved parental virus variants, the frequency increased to 100% [Kitts et al., 1990; Roy et al., 2012; Possee et al., 2019]. Linearized viral backbones are now available from a variety of sources.

[0090] The development of the baculovirus shuttle vector (bacmid) system has further improved the efficiency of the system. It involves inserting a miniature transposon containing one or more target genes (under the control of a baculovirus promoter derived from the donor vector) into a target vector containing a gene fusion with transposon attachment sites, using a component of the site-specific bacterial transposon Tn7, in the presence of an assistant vector containing four transposase genes. This target vector is then propagated in *Escherichia coli* as a very low copy number plasmid (named bacmid, or more generally, baculovirus shuttle vector) [Luckow et al. 1993]. DNA samples isolated from bacterial cells containing composite shuttle vectors containing transposon-derived cargo DNA segments are infectious when transfected into susceptible insect cells, thus avoiding the need for purifying recombinant viruses using cumbersome traditional methods or faster methods based on plaque assays of homologous recombination between the transfer vector and the circular or linearized parent viral DNA genome. Donor, auxiliary vector and target vector are available from Thermo Fisher Scientific as components of its Bac-To-Bac™ system.

[0091] Thousands of proteins have been expressed in baculovirus-infected insect cells for a variety of applications, including therapeutic proteins, components of vaccines and gene therapy vector systems (including viral capsid systems), for basic and applied research and development, and as biopharmaceutical products reviewed and approved by the FDA and similar regulatory agencies worldwide [Luckow et al. 1993; Chen et al. 2016; Possee 2019].

[0092] Fall armyworm and white armyworm cells are commonly used as substrates for expressing proteins encoded by target genes on recombinant baculoviruses derived from AcNPV. These include Sf9, Sf21, Tn368, and High Five (white armyworm) cell lines, which are available from biomaterials collections (e.g., ATCC (Manassas, Virginia)) and various commercial sources. Products intended for use in healthcare applications need to be expressed in cells free of foreign agents or purification process byproducts that could potentially impair the activity or efficacy of the product in its intended application, for example, potentially more significantly for patients receiving therapeutic proteins, vaccines, or gene therapy vectors than for products used for diagnostic assays or basic structure / function assays. Fall armyworm and white armyworm cell lines free of rhabdovirus and Nodamura virus are now available from various commercial sources.

[0093] VLP expression in baculovirus-infected insect cell lines While baculovirus expression vector systems have been used for many years to facilitate the large-scale expression and purification of virus-like particles used in human and animal vaccines (Kaba et al. 2004), there is growing interest in using these systems to express HEV-VLP at high levels, followed by efficient concentration and purification of the particles (Kawano et al. 2011). Because methods involving ultracentrifugation are labor-intensive and not easily scalable, alternative methods using cation exchange liquid chromatography (LC) and size exclusion columns have been developed to produce GMP-quality materials. Non-infectious HEV-VLP with a T=1 virus-like structure has been produced by baculovirus expression of the N-terminal and C-terminal truncated hepatitis E capsid protein PORF2 (Xing et al. 1999; Xing, Li, Mayazaki et al. 2010; Yamashita et al. 2009).

[0094] Construction of recombinant baculovirus containing HEV-VLP sequence Recombinant baculoviruses containing genes encoding HEV-VLP variant capsid proteins can be constructed using two basic methods: (1) inserting the capsid variant gene into a transfer vector and then transfecting it along with a circular or linearized viral backbone into cultured insect cells, where they recombine to generate an infectious virus capable of expressing a heterologous capsid protein (typically under the control of a polyhedrosis protein promoter); or (2) inserting the capsid variant gene into a donor vector transformed into Escherichia coli DH10Bac cells containing a shuttle vector (e.g., bMON14272) and an helper vector (e.g., pMON7124), and screening for cells containing a composite shuttle vector generated by transposing a cargo fragment of the donor to a target site within a disruptive lacZα gene fusion body on the shuttle vector, thereby generating a composite target vector that can be directly transfected as a pure virus into cultured insect cell lines.

[0095] Both methods require the synthesis of DNA segments encoding the desired gene. These DNA segments can be codon-optimized and flanked by recognition sites for restriction enzymes or regulatory sequences. Various commercial suppliers offer self-synthesized DNA fragments and vectors. In the first method, the DNA fragment is cloned into a transfer vector (e.g., pVL1392 or pVL1393), validated by sequencing, and transfected into insect cells with a linearized viral backbone (e.g., BestBac 2.0 (Δv-cath / chiA) (Expression Systems, Davis, CA)) to generate a series of viruses named AcBestBacORF2. In the second method, the DNA fragment is cloned into a donor vector (e.g., pFastBac1 (Thermo Fisher Scientific)) and transformed into competent Escherichia coli DH10Bac cells. After selection, a composite DNA sample is transfected into insect cells to generate the AcBacToBacORF2 viral vector. Stock solutions of AcBactoBac-ORF2 and AcBestBacORF2 can be prepared by repeated passages in Sf9 cells grown at 27°C in serum-free ESF921 medium (Expression Systems, Inc.). Viral titers can be measured using antibodies against gp64 (Mulvania, Hayes, and Hedin 2004).

[0096] General parameters for inoculation (via AcBactoBacORF2 or AcBestBacORF2) and harvesting of cell culture supernatant are described below. Day 1: High Five cell cultures were inoculated in ESF921 at 1 million cells / ml; Day 2: Cell cultures were infected with MOI = 0.1-5.0 and incubated with shaking for two hours; viable cell counts and viability percentages were measured daily, and cells were stained for gp64 expression on post-infection (pi) days 1 and 2. Days 3-7: Cell pelleting was performed, and the pellets of each culture were stored at -80°C, while virus supernatant samples were stored at 4°C. After each purification step, the yield and quality of HEV-VLP under each inoculation condition were analyzed and compared.

[0097] A stock of pure virus at a known titer can be used for subsequent studies to determine the optimal conditions for expression under various conditions. For example, High Five cells at 1-2 million cells / ml can be infected with a titrated viral sample to determine the time course of expression over a series of days, and cell samples and clarified supernatants can be collected for analysis to determine the optimal time to harvest cells, thereby ensuring that the desired protein is expressed at high levels without significant degradation or disassembly into inactive fragments.

[0098] Purification of HEV-VLP from baculovirus-infected insect cells The expression levels of heterologous proteins in baculovirus-infected insect cell lines will vary, typically starting around 48 hpi for genes controlled by strong polyhedromic protein promoters. The nature of the expressed protein will also affect its intracellular location, either as an active secretion by intact cells or as an accumulation of proteins or complex virus-like particles, or as an accumulation of proteins or complex virus-like particles exposed to the culture medium during late infection (48–72 hpi) when cells begin to lyse. Cell fractionation studies can be performed, using various assays (including immunoblotting or dye-binding properties of proteins separated by acrylamide gel electrophoresis) to determine the relative amount and activity of the target protein.

[0099] If HEV-VLP is secreted from infected cells, the culture medium and cells can be separated by low-speed centrifugation (e.g., at 1500-3000 rpm for 15 min at 4°C). The supernatant can be pretreated with a washing agent (e.g., 0.5%-1% Nonidet P-40) and gently shaken at room temperature for >2 h, then benzonatase at 5-20 U / mL in 1 mM MgCl2 can be added, followed by treatment at 37°C for 2.5 h to degrade endogenous DNA.

[0100] If HEV-VLP is not secreted from infected cells, the culture medium and cells can be separated by low-speed centrifugation (e.g., at 3,000 rpm for 15 min at 4°C). The precipitated cells can be pretreated with denaturing buffer (e.g., 50 mM sodium borate, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 5% 2-mercaptoethanol) and gently shaken at room temperature for 2 h. The lysate is diluted with cell culture medium before purification.

[0101] Figure 5 The illustration shows the title "Pretreatment of cells before purification of HEV-VLP not secreted from cultured cells".

[0102] HEV-VLP recovered by any method can be further purified through several additional steps: (1) clarification to remove cell debris and large aggregates; (2) concentration to minimize investment in equipment and consumables; (3) purification of the desired molecules; and (4) refining to remove host cell proteins and nucleic acids required to reach the acceptable threshold for clinical-grade biomaterials.

[0103] Figure 6 The illustration shows a purification process with the title "A capture / purification step via a pH gradient-based ion exchange method".

[0104] Figure 7 The illustration shows a purification process with the title "A capture / purification step via a salt gradient-based ion exchange method".

[0105] Other methods can also be used to purify HEV-VLP in a large-scale (scalable), high-quality (purity), high-titer (potency), and GMP-compliant manner. These methods include depth filtration, tangential flow ultrafiltration (TFF), cation exchange chromatography (IEX, FPLC) or cation exchange membrane chromatography and size exclusion (SEC, FPLC), replacing clarification, concentration, capture / purification, and refining steps, respectively. Methods relying on separating components from the mixture by ultracentrifugation can also be used (Xing et al. 1999; Niikura et al. 2002; Li et al. 2005a; Xing et al. 2011; Chen et al. 2018).

[0106] General Encapsulation Solution The DNA encapsulation process described in earlier studies (Barr, Keck, and Aposhian 1979; Nilsson et al. 2005) can be extended to encapsulate different payloads, including inorganic nanoparticles, DNA, mRNA, and peptides, into HEV-VLPs. The disassembly and reassembly of recombinant HEV-VLPs can be performed according to a procedure (with slight modifications) as described (Takamura et al. 2004). Briefly, purified HEV-VLPs are diluted to a final concentration of 0.4–1.2 mg / mL and incubated at room temperature in a buffer containing 1–10 mM EGTA and 10–20 mM DTT for 1–2 hours. The disassembly and reassembly of HEV-VLPs at approximately 10 nm can be measured and confirmed by dynamic light scattering (DLS) using a Malvern Antonpar Litesizer 500 device.

[0107] Figure 8 The illustration shown is titled "A Universal Encapsulation Process for HEV-VLP".

[0108] The payload can be added to the disassembled HEV-VLP at a specified ratio and mixed for >15 min. The mixture of disassembled HEV-VLP and payload can be reassembled by adding 1 / 4 volume of 4-10 mM CaCl2 or 5-10 mM MgCl2 four times per hour and incubating overnight at 4°C. The size of the encapsulated / reassembled HEV-VLP after the reaction can be measured by DLS and confirmed by TEM.

[0109] Negatively charged inorganic nanoparticles, including metallic nanoparticles such as gold nanoparticles and ferrite (Fe3O4) nanoparticles, such as citric acid-treated ferrite nanoparticles, can be encapsulated by HEV-VLPs regardless of their size. Unlike the formation of approximately 27 nm T=1 HEV-VLP particles, the particle size should vary depending on the size of the ferrite virus-like particles (NPs) (Chen CC 2017). The encapsulation of ferrite NPs by the capsid of the disassembled HEV-VLP is primarily due to the interaction between the negatively charged ferrite particles and the positively charged residues within the capsid particles (Takamura et al. 2004; Cheng and Xing 2014).

[0110] Examples of alternative encapsulation processes: Example 1: The encapsulation process can be performed at 37°C to reduce processing time and improve encapsulation efficiency. Generally, the payload (e.g., DNA plasmids and mRNA) can be added to the disassembled HEV-VLP at a specified ratio and mixed for >15 min. The mixture of disassembled HEV-VLP and payload can be reassembled by adding CaCl2 or MgCl2 to a final concentration of 5-10 mM, incubating at 37°C for one hour, and then incubating overnight at 4°C. The size of the HEV-VLP after the encapsulation / reassembly reaction can be measured by DLS and confirmed by TEM.

[0111] Example 2: Encapsulation can be performed using microfluidics, a process widely used for lipid nanoparticles (LNPs) (Ward 2015) to achieve automation and GMP compliance. Generally, the payload (e.g., DNA plasmids, mRNA, etc.) can be mixed with the disassembled HEV-VLP in a microfluidic device at a specified ratio. Similarly, the mixture of disassembled HEV-VLP and payload can be reassembled in the microfluidic device by adding CaCl2 or MgCl2 at 25°C–37°C to a final concentration of 2–10 mM. The size of the HEV-VLP after the encapsulation / reassembly reaction can be measured by DLS and confirmed by TEM.

[0112] Payload condensation and encapsulation procedures The payload typically needs to be condensed to a preferred size of approximately 10 nm to improve the efficiency of the encapsulation process. DNA condensation and charge reversal usually occur in multivalent counterionic solutions. The organic monovalent ion of tetraphenylarsine chloride (Ph4As+) can induce DNA compression even at low Ph4As+ concentrations of approximately 1 μM (Xia et al. 2017). When the Ph4As+ concentration is increased to 1 mM, the step pattern in the pull curve disappears, and spherical structures can be observed in the corresponding AFM images (Xia et al. 2017), indicating that plasmid payloads can be condensed to approximately 10 nm using approximately 1–2 mM Ph4As+ (e.g., 2 mM tetraphenylarsine chloride) (incubated overnight at 4°C). The size of the condensed plasmid payload can be measured by DLS.

[0113] Universal insulin, with its small size (5.8 kDa) and negative surface charge, is an attractive payload for use in HEV-VLP systems compared to long-chain linked insulin that may be extruded from assembled particles (Chen, Baikoghli, and Cheng 2018). Universal insulin prepared without an aggregation step cannot be encapsulated into HEV-VLPs, despite their size < 10 nm and negative surface charge, suggesting that payload aggregation is crucial for successful HEV-VLP encapsulation. Structural data confirm that the hexamer conformational change of insulin can be induced by Zn+2 (Olsen et al. 2003), indicating that 0.2–1.0 mg / mL universal insulin can aggregate in 2 mM ZnCl2 to reduce the payload size to approximately 10 nm prior to encapsulation. Insulin-encapsulated HEV-VLPs will be analyzed by DLS and confirmed by TEM. Insulin content can be analyzed by ELISA.

[0114] Evaluation of DNA samples in HEV-VLP HEV-VLPs can be considered nucleic acid-binding proteins that can form DNA / protein or RNA / protein complexes upon mixing. The resulting sample will contain a mixture of components, including empty HEV-VLPs, HEV-VLPs containing DNA, and unincorporated DNA and capsid protein molecules. DNA-encapsulated HEV-VLPs need to be effectively separated from the empty shell. Plasmid DNA can be agglomerated with >1 mM Ph4As+ before encapsulation. However, reagents like Ph4As+ are considered biohazardous. Size exclusion columns (SEC, with a cutoff size of 50–400 kDa) can be used to separate HEV-VLPs carrying DNA payloads to reduce the concentrations of Ph4As+ and other solutes to safe levels.

[0115] Encapsulation of plasmid DNA can be performed using the following general procedure. The encapsulated plasmid in HEV-VLP can be concentrated by filtration through a 100 kDa centrifuge filter (Amicon Ultra-0.5 centrifuge filter). The concentrated sample is then applied to a gravity-size exclusion column (SEC) (e.g., GE Sephacryl S-500 resin or TOSOH TSK gel G6000 SEC column). The collected fractions can be measured spectrophotometrically, by DLS, and confirmed by TEM. The encapsulated DNA content can be estimated by comparing fragment amplification through semi-quantitative PCR analysis of serial dilutions of unbound plasmid DNA samples separated on a 1% agarose gel.

[0116] Encapsulation of HEV-VLP intracellular RNA samples Small RNA molecules can be encapsulated into HEV-VLPs using a similar protocol to that used for plasmid DNA, without the need for a nucleic acid aggregation step. CleanCap™ eGFP mRNA (996 nt) (TriLink Biotechnology, San Diego, CA, USA) can be used as a control for encapsulation into HEV-VLPs. Free mRNA can be purified using size exclusion columns (SEC) (e.g., Capto™ Core 400 multimode column (Cytiva)). The collected fractions can be analyzed using UV spectrophotometry, DLS (Anton Parr Litesizer 500), and TEM.

[0117] The HEV-VLPs containing encapsulated RNA were also treated with RNase to confirm that the RNA was protected and packaged within the virus-like particles. The reaction could be terminated by adding an RNase inhibitor, and the sample was precipitated to remove the RNase and inhibitor. TEM analysis and UV spectrophotometry were also used to confirm that the treatment did not destroy the RNA molecules encapsulated within the HEV-VLPs. The HEV-VLPs containing RNA molecules could be disassembled, resulting in an increased nucleic acid concentration, which could be detected as bands on an agarose gel.

[0118] Encapsulation of a mixture containing one or more nucleic acids or peptides within an HEV-VLP The aforementioned disassembly, assembly, and encapsulation strategies can also be used to prepare HEV-VLPs containing one or more molecules that do not covalently associate with the capsid protein. The mixture may contain nucleic acids (e.g., single-stranded RNA, double-stranded RNA, single-stranded DNA, double-stranded DNA), as well as short peptides, longer polypeptides, and complexes of one or more peptides or polypeptides with single-stranded or double-stranded RNA or DNA molecules, including components of CRISPR-based systems, such as Cas proteins that guide RNA molecule complexation.

[0119] In vitro power assay using DNA and mRNA payloads from encapsulated HEV-VLPs seeded into mammalian cells. PC-3 cells monolayered in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 IU / ml penicillin, and 100 ug / ml streptomycin can be seeded into 12-well plates (Nunc, Life Technologies) and allowed to grow to 80% confluence (approximately 4 × 10⁻⁶ cells / well). 5Cells per well. Cells can be washed with PBS. A sample of HEV-VLP encapsulated with DNA diluted in 100 μL PBS can be added to the wells and incubated at 37°C for 1 h. Following this step, 1 mL of DMEM supplemented with 10% FBS is added, and the cells are incubated at 37°C for 48 and 72 h. GFP expression and activity levels are measured by comparing the number of fluorescent and non-fluorescent cells using a Cellometometer device.

[0120] Transmission electron microscopy (TEM) The purified VLP can be loaded onto a glow discharge, carbon-coated EM grid and stained with 2% uranyl acetate. The formation of HEV-VLP can be examined under transmission electron microscopy (TEM) at magnifications of 10,000x to 30,000x.

[0121] Example 1: Common sequences derived from related hepatitis E virus ORF2 capsid sequences A variety of methods can be used to design and assemble viral capsids based on known sequences stored in publicly accessible nucleotide and amino acid databases, such as GenBank. In many cases, basic research uses a limited set of vectors containing nucleotide sequences encoding a target peptide or polypeptide and its variants to facilitate the characterization of direct relationships between molecular structural and functional features, such as the binding of repressors or activators to DNA, mRNA stability, and the folding, assembly, and catalytic activity of oligomeric complexes of monomeric proteins or subunits encoded by the same or different genes. When resources are unrestricted, some strategies, such as saturation mutagenesis across target nucleotide sequence segments, are more likely to occur in applied research settings to generate large amounts of data that are invaluable for understanding the structural and functional relationships of molecules involved in chronic and acute diseases, including cancer, infectious diseases, and immunodeficiency. This is essential for facilitating the discovery, development, and commercialization of novel drug products, including compositions containing components of therapeutic agents, vaccines, and cell and gene therapy vector systems.

[0122] More than 2,200 nucleotide sequences have been deposited in GenBank. These sequences are described as components encoding hepatitis E virus, many of which are based on clinical samples collected from patients or veterinarians in hospitals around the world. Deciding where to begin and which sequences to use is often challenging because the prototype virus, despite being studied for decades, does not appear to be clinically relevant or suitable for mutagenesis, expression, and proliferation in cultured cells, making it difficult to characterize key components for vaccine development, or as virus-like particles that can encapsulate nucleic acids, proteins, or small molecules, or as molecular conjugates that can be used in other diagnostic procedures or therapeutic products.

[0123] This application discloses methods for designing and characterizing novel viral capsids derived from one or more common sequences obtained by comparing the arrangement of numerous related HEV ORF2 capsid polypeptide sequences. In the examples below, the amino acid frequencies of 660 or 674 amino acids derived from ORF2 capsid proteins from 124 related HEV genomes were determined at each position. These strategies should lead to the discovery and development of novel capsids, such as virus-like particles, that function in a variety of human cells and can efficiently deliver a variety of other molecules encapsulated or conjugated to particles (which bind to and are absorbed by specific types of host cells). Virus-like particles based on common sequences derived from HEVs or variants thereof do not appear to be disclosed or mentioned in publicly available sequence databases, journal articles, or patent documents.

[0124] By summarizing tables and graphs of the frequencies of variants derived from nucleotide or polypeptide sequence arrangements, you can identify segments containing a “sea” of highly conserved residues, interspersed with “islands” of two or more residues, while considering their biochemical properties, such as the position of the nucleotide in the codon or the characteristics of the amino acid side chains observed at any given position. If a segment of one or more amino acids contains a mixture of aliphatic, hydrophobic, positively charged, or negatively charged residues, then the segment can tolerate many kinds of amino acids that are not necessarily observed in the frequency table. For example, if they are all hydrophobic or negatively charged, then the tolerance to changes may be lower, and only biochemically similar amino acids can be substituted.

[0125] Site-specific mutagenesis or directed evolution experiments can also be performed, in which one or more amino acids are substituted, inserted, or deleted at specific positions and evaluated to determine whether the capsid properties are altered in the desired manner. The gene sequence encoding a variant capsid protein can be identified and combined with one or more other variants to generate other desired sequences that may not exist in nature but have similar or unexpected properties compared to those disclosed in GenBank or other publicly available nucleotide or amino acid sequence databases.

[0126] Thousands of HEV or HEV-like virus sequences have been deposited in GenBank, and comparing multiple sequence alignments of related sequences helps to determine the location of identical, highly conserved or variable amino acid segments in the full length of the capsid protein.

[0127] The following table and graphs were created based on queries from GenBank, sequences were extracted, and multiple sequence permutations were prepared using ClustalP (Sievers, 2011). The frequencies of variant amino acids at different positions throughout the protein were determined. The original permutations of the sequences were pasted into Excel, each group containing 60 to 100 amino acids, and tables were created to compare the frequency of amino acids in each alignment column of the 100 sequences in each group. Some amino acids appeared only once (e.g., starting methionine), while others had multiple amino acids; for the initial group of 124 highly similar sequences, there were up to 6 different amino acids. Using data generated from other types of search queries, even fewer or more sequences can be analyzed.

[0128] Table 3: Search results and sources of 124 HEV-related capsid proteins in GenBank Search strategy: (txid291484[organism:extended] and (capsid[all fields] and complete[all fields])) and "Bareny Hepatitis E Virus"[porgn] and ("651"[sequence length] : "675"[sequence length]). Records 116 to 124 also contain the same supplementary note: "Recommended name: full name = precursor secretory protein ORF2; alias: full name = protein ORF2; abbreviation = pORF2; contains: recommended name: full name = secretory protein ORF2; abbreviation = ORF2; label: precursor".

[0129] A graph was also created that sorts the number of different amino acids at each position, and then several algorithms were used to analyze it, comparing its biochemical properties based on hydrophobicity, charge, size and other characteristics. The values ​​of each algorithm were different.

[0130] If all amino acids in a column are identical, the position is highly conserved, indicating that the residue is essential for protein expression (amino-terminal Met), transport (signal peptide), catalysis, or other structural or functional features.

[0131] If the amino acids are different but have similar biochemical properties, then substitutions at those positions indicate greater tolerance to other amino acids of the same chemical class, but not necessarily those amino acids represented in the analyzed sequence set.

[0132] If amino acid changes but have drastically different biochemical properties, then a wide variety of substitutions, or even insertions and deletions, can be tolerated at or near that position. This could be because segments of that amino acid or nearby consecutive amino acids are exposed inside or outside the cell, or in disordered internal segments, such as highly ordered segments or spacers between polypeptide domains.

[0133] Figure 9 The illustration, titled "Frequency of amino acid substitutions in the associated HEV ORF2 capsid sequence used to generate a common capsid sequence," is based on 124 different HEV capsid proteins, 93 of which are described as Barney hepatitis E virus, with the remainder derived from other sources (including pigs).

[0134] Note the variable "islands" within the "sea" of highly conserved amino acids, particularly at the amino and carboxyl ends of 124 protein sequences. This group contains longer segments of highly conserved amino acids compared to the first group.

[0135] Figure 10 The illustration shows the predicted secondary structure of the common sequence derived from the associated HEV ORF2 capsid protein through multiple sequence alignment.

[0136] Figure 11 The diagram shows the distances of the number of amino acid variations across 124 sequences (lower green, higher red).

[0137] The image illustrates the fundamental differences between 124 sequences, with green diagonal lines indicating equivalence between identical sequences. Large blocks show comparisons of sequences with 660 AA each (both preceded by 15 dashes in the permutation) or sequences with 674 AA each. The color pattern of the differences resembles farmland.

[0138] Figure 12 The graph shows the frequency of amino acid variations across 124 sequences (lower green, higher red).

[0139] Sequence alignment 1: The common sequence NCT-660 (SEQ ID NO: 02) derived from 124 related HEV ORF2 capsid sequences. The explicit patterns of variability derived from these analyses are valuable in the design of vectors encoding common sequences derived from HEVs and their variants, where one or more amino acids are substituted, inserted, or deleted at specific positions, and evaluated to determine whether the capsid properties are altered in a desired manner. Genetic sequences encoding variant capsid proteins can be identified and combined with those sequences encoding other desired variants, which may not exist in nature and possess altered or improved properties, particularly for the development and commercialization of clinically significant therapeutic drug products, vaccines, and as components of cell and gene therapy vector systems, as described in other examples below.

[0140] Note that the description of variable and invariant sequences depends heavily on the query sequence submitted to the nucleotide or peptide sequence database and the results returned by the algorithm used to identify similar sequences. A query that is highly similar to many other sequences represented in the database will return a sequence group that can show many invariant, identical, or highly conserved regions and fewer variable regions compared to a query that is less similar to other sequences represented in the database. A query that returns more variable regions and fewer invariant, identical, or highly conserved sequence regions will return more variable regions and fewer invariant, identical, or highly conserved sequence regions.

[0141] In this paper, a capsid derived from HEV common sequences and their variants (containing one or more “functionally similar” amino acid substitutions, insertions, or deletions) refers to a polypeptide that can be expressed in prokaryotic or eukaryotic host cell systems that can assemble and form VLPs or virus-like particles with desired properties, such as the ability to form three-dimensional structures similar to wild-type viruses observed using electron microscopy and virus-like particle counting equipment, and the ability to encapsulate nucleic acids (e.g., RNA molecules encoding viral proteins) or heterologous polypeptides based on amino acid side chains within the capsid that can attract and bind to RNA molecules specifically or non-specifically.

[0142] Capsids derived from HEV common sequences and their variants (containing one or more amino acid substitutions, insertions, or deletions) have enhanced properties in this context. They also refer to capsids capable of encapsulating and binding dsDNA molecules, peptides, or being modified to contain residues on the outer or inner surface, thereby facilitating conjugation with other molecules, for use as vaccines, or as agents for targeting and delivering non-covalently associated nucleic acid, peptide, or small molecule therapeutic products to cells.

[0143] By identifying and displaying variable regions among a large number of conserved residues in the common sequence of the HEV ORF2 capsid protein, the discovery, development, and commercialization of a variety of products for use as therapeutics or diagnostics have been significantly accelerated, avoiding the need to design, express, and test hundreds of variants with properties required for specific applications through traditional methods.

[0144] Example 2: Development of HEV-VLP for Hydrophobic Drug Delivery Systems In recent years, the application of virus-like particle-based targeted drug delivery has increased significantly. Although many types of nanocarriers, such as synthetic polymers, liposomes, and linear-dendritic block copolymers, have been designed and evaluated over the past 20 years, limitations such as toxicity, inability to accumulate sufficient numbers of molecules in the cytoplasm, and lack of biodegradability still need to be addressed (Sebestik, Niederhafner et al. 2011; Jian, Zhang et al. 2012). Liposome carriers (e.g., Doxil) ® Drugs such as 5-fluorouracil do not have the ability to target specific cell types, but they tend to accumulate in solid tumors due to the characteristics of tumor regions (e.g., larger pores in neovascularization (EPR effect)) (Shi, van der Meel et al. 2020). Drug leakage during circulation, leading to undesirable side effects and stability issues, is a major concern (Russell, Hultz et al. 2018).

[0145] Lipid-based virus-like particle delivery systems, including micelles, liposomes, polymeric vesicles, polymeric nanospheres, and dendritic polymers, have been used as mediators for the targeted and controlled delivery of various agents, including small and large molecule therapeutics, gene and diagnostic imaging agents (Hughes, Misra et al. 2023). MRNA-containing vaccines have attracted significant attention due to their ability to protect against the SARS-CoV-2 virus. Compositions containing mRNA vaccines also incorporate lipid nanocarriers that not only encapsulate the mRNA but also protect it from in vivo degradation and precisely transport it to the cytoplasm (Zeng, Zhang et al. 2022). COVID vaccines containing protein-based virus-like particles (including those developed by Novavax) have also been evaluated in clinical trials (Anselmo and Mitragotri 2021).

[0146] Many diseases, such as cancer, can benefit from therapeutics that modulate the activity of intracellular targets. All FDA-approved intracellular targeted virus-like particle delivery systems utilize a parenteral administration route. Doxorubicin (DOX), an anthracycline chemotherapeutic agent first approved in 1995, is the most widely used doxorubicin formulation, formulated with a surface PEG coating (Anselmo and Mitragotri 2016). Other FDA-approved chemotherapeutic liposomal virus-like particle formulations include vincristine, irinotecan, mivaminectide, daunorubicin, cytarabine, and cisplatin (Anselmo and Mitragotri 2016).

[0147] Clinical trials and vaccinations of COVID-19 mRNA vaccines have demonstrated very high levels of protection and varying degrees of side effects. Lipid-based virus-like particles (LNPs), used in many preclinical studies, exhibit high inflammatory activity in mice. Intradermal injection of LNPs elicits a rapid and intense inflammatory response characterized by extensive neutrophil infiltration, activation of multiple inflammatory pathways, and the production of various inflammatory cytokines and chemokines. Intranasal delivery of the same dose of LNPs elicits a similar inflammatory response in the lungs but results in a higher mortality rate (Ndeupen, Qin et al. 2021). LNPs may not be the optimal delivery system for mRNA used in vaccines and other gene therapy applications. Similar safety concerns appear to have arisen with the application of lipid-based intracellular cancer therapeutic delivery systems.

[0148] Other strategies for delivering biomolecules into cells are needed, including strategies that use viral capsids alone as protein-based carriers. The inherent ability of viral capsids to encapsulate nucleic acids, small molecules, and proteins makes them ideal carriers for delivering therapeutic anticancer agents. Viral capsids can penetrate cells through active endocytosis and undergo proteolytic degradation after delivery.

[0149] Hepatitis E virus-like particles (HEV-VLPs) derived from HEV are non-infectious, self-assembled capsids capable of binding to and entering cells. HEV-VLPs appear to maintain their structural integrity in low pH environments (Zafrullah, Khursheed et al. 2004), which is advantageous for intratumoral penetration. The primary route of HEV infection is the fecal-oral route. HEV-VLPs are also resistant to proteolytic and acidic mucosal conditions, making them ideal mucosal delivery capsules (Jariyapong, Xing et al. 2013; Chen, Baikoghli et al. 2018; Shizuo G. Kamita 2019).

[0150] HEV-VLP can be expressed and purified in high quantities in High-Five insect cells (Kawano, Xing et al. 2011). The structure of HEV-VLP was determined by X-ray crystallography and cryo-electron microscopy (cryo-EM) (Xing, Kato et al. 1999; Xing, Li et al. 2010). The major capsid protein, open reading frame 2 (ORF2, approximately 500 amino acids), consists of three domains, with the N-terminal shell domain and the intermediate domain forming the base of the shell. The remaining approximately 150 amino acids of the C-terminal domain (the overhanging domain or P domain) constitute the major binding site for neutralizing antibodies and receptors, as well as the antibody site.

[0151] The P domain forms surface-exposed spikes at the icosahedral base, while the flexible hinge allows the P domain to be modified by inserting exogenous peptides through genetic engineering (Jariyapong, Xing et al. 2013) or chemical conjugation (Chen, Xing et al. 2016) without disrupting the icosahedral structure at the base. The P domain and three surface-variable loops at the C-terminus of the HEV capsid protein ORF2 have been designed and engineered as conjugation sites for at least one or more bioactive agents because they are well exposed on the surface of HEV-VLP (Cheng, Xing et al. 2015, Chen, Xing et al. 2016, R. Holl and Cheng 2017).

[0152] A major problem with many protein-based delivery systems is their non-reusable nature due to the immune response that arises during delivery. The antigenic determinants of HEV-VLP reside entirely within its surface P domain (Holla, Baikoghli et al. 2017, Baikoghli 2018). Surface modifications to the P domain (e.g., through point mutations, chemical conjugations, and amino acid insertions) can eliminate or significantly reduce the immune response to HEV-VLP from pre-existing HEV antibodies (Chen, Xing et al. 2016). The geometric confinement of the intermediate domain (M domain) provides a physical barrier for antibody binding, which may help HEV-VLP evade surveillance of the immune system by HEV-specific antibodies. Overall, the structural features of HEV-VLP make it a valuable component of reusable delivery platforms (Shizuo G. Kamita 2019).

[0153] HEV-VLP has the endogenous ability to deliver heterologous epitopes to and across the mucosal surface without requiring any potentially harmful exogenous enhancers (such as mucosal degrading enzymes, pH adjusters, or uptake cofactors).

[0154] Several characteristics make HEV-VLP an ideal medium for targeted and mucosal delivery of other molecules (Kawano, Xing et al. 2011; Shizuo G., Kamita 2019): (i) Surface plasticity. The surface of HEV-VLP can be genetically or chemically modified while maintaining the integrity of the core structure. Different sites on the P domain can be engineered for site-specific attachment or insertion of one or more epitopes. Chemical grafting of tumor-targeting ligands onto the surface of HEV-VLP enhances in vivo tumor-targeting properties (Chen, Xing et al. 2016). The P domain also carries antigenic sites of HEV-VLP, and modification of the P domain can neutralize the immunogenicity against HEV-VLP.

[0155] (ii) Signal amplification. Each HEV-VLP consists of 30 dimer structural units, in which antigen presentation can be amplified by up to 60 times.

[0156] (iii) Gastrointestinal (GI) stability. Surface-modified HEV-VLP remains stable under the harsh conditions of low pH and proteolytic enzymes present in the GI tract, and can be used for oral delivery of epitopes. HEV-VLP also has the potential to directly target the mucosa of the GI tract, small intestine, nasal cavity, lungs, and colon (Chen, Baikoghli et al. 2021).

[0157] (iv) Packaging capabilities. Modular therapeutic HEV-VLPs can involve utilizing internal properties beyond their functionalized surfaces. HEV-VLP sequences are optimized to not encapsulate viral RNA, forming highly stable, non-infectious capsids capable of reversible in vitro assembly via cation-mediated processes (Xing, Li et al. 2010).

[0158] Figure 13 A diagram titled "Classification of Treatment and Delivery Modes" is shown. HEV-VLP can switch back and forth between chemical reduction and cationic chelation in self-assembly. This property is illustrated by an in vitro encapsulation method for therapeutic nanomaterials. HEV-VLP can be disassembled in the presence of reducing agents and chelating agents (such as dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)), while the addition of calcium or magnesium ions initiates a reassembly reaction.

[0159] The encapsulation of HEV-VLPs is based on charge-force interactions, enabling the packaging of negatively charged nucleic acids, nanoscale proteins, and inorganic nanoparticles (including metallic nanoparticles, such as gold and ferrite (Fe3O4) nanoparticles) for therapeutic applications (Chen, Baikoghli et al. 2018, Shizuo G. Kamita 2019). The oral delivery capability of HEV-VLPs for exogenous genes can be used to deliver plasmid cDNA to small intestinal epithelial cells for transient expression of cDNA-encoded antigens (Takamura, Niikura et al. 2004, Cheng and Xing 2014). HEV-VLPs encapsulated with magnetic ferrite NPs can also be used in procedures such as MRI imaging and tumor-targeted hyperthermia induced by radiofrequency electromagnetic radiation (Roemer 1999).

[0160] Encapsulating molecules with HEV-VLPs has the potential to impact many areas requiring drug and gene delivery systems (Stark and Cheng 2016). HEV-VLPs containing surface-binding ligands that target specific types of cancer cells also have the potential to cure or improve the condition of many types of cancer.

[0161] Next-generation therapeutic agents, including proteins and peptides, monoclonal antibodies (mAbs), nucleic acids, and live cells, offer novel therapeutic capabilities. HEV-VLPs have been proposed as drug delivery systems (DDS), including dsDNA vectors and RNA, for gene therapy applications. Many major classes of therapeutic agents (small molecule drugs) cannot be packaged into HEV-VLPs due to their hydrophobicity. In this example, we disclose a method to extend the ability of HEV-VLPs to encapsulate hydrophobic cancer drugs by modifying the N-terminus of HEV-VLPs.

[0162] NCT-002: A proposed common sequence derived from 160 sequences (inserting Cys between 490 aa and 491 aa). Hydrophobic peptides inserted into HEV-VLPs possess icosahedral structures with double, triple, and quintuple symmetry axes (Shizuo G. Kamita 2019). The capsid PORF2 of hepatitis E virus exhibits characteristics typical of secreted proteins: an N-terminal signal sequence and conserved glycosylation sites. The 111 N-terminal amino acids show the greatest sequence difference between HEV genotypes, and expression of the full-length ORF2 in insect cells typically results in proteolytic cleavage of this region. The virion possesses T=3 symmetry with 180 monomers, while the truncated pORF folds into a T=1 particle with 60 subunits and 30 protruding spikes, which has been promoted for use as a drug delivery system (Shizuo G. Kamita 2019). In structural studies, additional mass can be detected within the VLP, which may correspond to an exogenous peptide fused to the C-terminus of ORF2 (Wang, Miyazaki et al. 2008).

[0163] In this example, a hydrophobic peptide named NS5A(1-31) (shown in yellow) was selected for insertion into the C-terminus of ORF2 to generate HEV-NS5A-VLP, which is expected to facilitate more efficient encapsulation of hydrophobic drugs (Buehler, Marsden et al. 2014). Cysteine ​​residues inserted between amino acids N490 and P491 (underlined and shown in green) can be used for ligand conjugation to facilitate VLP targeting of the desired cell type. The expected size of the VLP is between 20 and 27 nm.

[0164] Figure 14 The figure shown is titled "Analysis of Peptide Hydrophobicity / Hydrophilicity".

[0165] Sequence alignment 2: Proposed AA sequence of NCT-002: HEV-NS5A-VLP (SEQ ID NO: 08) VLPs containing hydrophobic peptides at their amino terminus can be expressed in baculovirus-infected insect cells, followed by purification steps involving clarification, concentration, purification, and refining to produce materials suitable for a variety of structural and functional studies, including TEM for confirming the conformation of HEV-NS5A-VLP particles. The ability of HEV-NS5A-VLP to encapsulate hydrophobic drug products (such as doxorubicin) is demonstrated by reassembling functional virus-like particles by mixing the payload with disassembled HEV-NS5A-VLP and gradually adding calcium or magnesium ions.

[0166] Example 3: Development of a Therapeutic HEV-VLP for Medical Applications Hepatitis E virus-like particles (HEV-VLPs) derived from HEV are non-infectious, self-assembled capsids capable of binding to and entering cells. HEV-VLPs appear to maintain their structural integrity in low pH environments (Zafrullah, Khursheed et al., 2004), which is advantageous for intratumoral penetration. HEV infection primarily occurs via the fecal and oral routes. HEV-VLPs are also resistant to proteolytic and acidic mucosal conditions, making them ideal for use as mucosal delivery capsules (Jariyapong, Xing et al., 2013; Chen, Baikoghli et al., 2018). HEV-VLPs also possess surface-exposed protruding (P) domains connected to a stable icosahedral base via flexible hinges. Engineered HEV-VLPs form hollow nanoscale capsids composed of 60 identical units (Xing, Kato et al., 1999), exhibiting high stability during storage and under harsh physiological conditions. Surface engineering, i.e., replacing or adding cysteine ​​residues as chemical conjugation sites on the P domain, can reduce the response of HEV-VLP to pre-existing antibodies (Chen, Xing et al. 2016). Due to the presence of 60 repeating units, single-site specific modification on the P domain generates 60 symmetrical sites on the assembled capsid for conjugation and presentation of numerous exogenous molecules.

[0167] Cancer diagnosis and treatment require direct contact between drugs and pathological lesions. HEV-VLP-based diagnostic agents, when conjugated with LXY30, exhibit high affinity for human malignant breast tumor cells (Xiao, Li et al. 2016) and demonstrate specific targeting of breast tumor cells in vitro and in vivo, suggesting that HEV-VLP can be manipulated to facilitate targeted delivery of diagnostic or therapeutic agents to pathological lesions (Chen, Xing et al. 2016). HEV-VLP also accumulates in abdominal organs, including the liver (Chen, Xing et al. 2016), indicating their potential use as liver-specific positron emission tomography (PET) imaging agents conjugated with radioactive gallium-68 [68Ga] (Lambidis, Chen et al. 2022).

[0168] Modular therapeutic agents can be designed and expanded by modifying the internal cavities of HEV-VLPs. They can be reversibly disassembled and reassembled via chemical reduction and chelation, providing a method for encapsulating therapeutic nanomaterials in vitro. HEV-VLPs will disassemble in the presence of reducing agents and chelating agents such as dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Cation concentration can trigger VLP reassembly. HEV-VLPs can also encapsulate negatively charged insulin peptides for oral insulin administration (Chen, Baikoghli et al. 2018), as well as magnetic virus-like particles (e.g., ferrite virus-like particles) for MRI diagnostics and tumor-targeted hyperthermia induced by alternating magnetic fields (AMF) (Ito, Honda et al. 2006, Chen CC 2017).

[0169] Passive encapsulation of different payloads is typically accomplished by mixing the payload with disassembled HEV-VLPs and gradually adding calcium or magnesium ions to form virus-like particles. Non-toxic, biocompatible, and stable magnetic iron oxide virus-like particles can be synthesized in various shapes and sizes (Cassim, Giustini et al. 2011). According to several established physical theories, magnetic virus-like particles can generate heat when placed in an AMF (Wu, Zhuo et al. 2015; Attar and Haghpanahi 2016; Espinosa, Di Corato et al. 2016; Kaur, Aliru et al. 2016). Encapsulation of HEV-VLPs with such properties can enhance magnetic resonance imaging (MRI) by producing a darker signal on T2-weighted images than surrounding tissue sections (Serkova 2017).

[0170] Ferrite virus-like particles have been shown to possess the ability to be heated by an alternating magnetic field (AMF) (Ito, Shinkai et al. 2003; Ito, Honda et al. 2006). Magnetic virus-like particles injected into targeted cancer tissue rapidly heat up upon activation by an external alternating magnetic field (AMF). If the particle concentration and AMF amplitude are sufficient, necrosis occurs in the microenvironment. Tumor-specific hyperthermia induces necrotic cell death through the expression of heat shock proteins (HSPs), thereby inducing anti-tumor immunity (Ito, Shinkai et al. 2003).

[0171] HIFU (High-Intensity Focused Ultrasound) is a non-invasive method for treating solid tumors and metastatic diseases. HIFU has been effectively used in clinical practice to treat a variety of solid malignancies, including those of the pancreas, liver, prostate, breast, uterine fibroids, and soft tissue sarcomas. However, long procedure times and collateral damage remain challenges in HIFU medical procedures. Accidental injuries (such as skin burns and damage to covering tissues) have been reported during HIFU procedures due to the high energy intensity. The gastrointestinal tract (GI) is considered a heat-sensitive tissue and remains challenging for HIFU hyperthermia. One strategy to reduce the required acoustic intensity and the resulting side effects is the use of hyperthermia enhancers. Ferrites and gold virus-like particles have both reduced the required acoustic intensity and exposure time in HIFU thermal ablation procedures (Kosheleva, Lai et al. 2016; Devarakonda, Myers et al. 2017; Devarakonda, Myers et al. 2017; Devarakonda, Myers et al. 2018; Kaczmarek, Hornowski et al. 2018). Magnetic ferrite virus-like particles can also be used as magnetic resonance imaging (MRI) agents for tracking guidance and temperature monitoring. MRI-guided high-intensity focused ultrasound (MRg-HIFU) therapy has been extended to pre- and post-thermotherapy treatments to ensure safe and effective therapy (Kim 2015). Virus-like particles can only be injected into the tumor site to enhance heating intensity, partly because they lack cancer-targeting capabilities.

[0172] Encapsulation of HEV-VLPs is based on charge-based interactions, enabling the packaging of negatively charged nucleic acids, nanoscale proteins, and inorganic nanoparticles (including metal nanoparticles such as gold and ferrite (Fe3O4) nanoparticles) for therapeutic applications (Chen, Baikoghli et al. 2018; Shizuo G. Kamita 2019). In this example, we outline a strategy to enhance the ability of HEV-VLPs to encapsulate negatively charged payloads by modifying the N-terminus of HEV-VLPs with positively charged peptides.

[0173] NCT-001: A proposed common sequence derived from 160 sequences (99 a-608 aa, between 490 aa and 491 aa). N&C truncated version with Cys inserted in between The icosahedral viral structure of HEV-VLP exhibits double, triple, and quintuple symmetry axes (Shizuo G. Kamita 2019). The capsid PORF2 of hepatitis E virus possesses characteristics typical of secreted proteins: an N-terminal signal sequence and conserved glycosylation sites. The 111 N-terminal amino acids show the greatest sequence difference between HEV genotypes, and expression of the full-length ORF2 in insect cells typically results in proteolytic cleavage of this region. The virion exhibits T=3 symmetry with 180 monomers, while the truncated pORF folds into a T=1 particle with 60 subunits and 30 protruding spikes (Shizuo G. Kamita 2019). Additional mass can be detected within the VLP, which may correspond to an exogenous peptide fused to the C-terminus of ORF2 (Wang, Miyazaki et al. 2008).

[0174] Figure 15 The figure shown is titled "Hydrophobicity / hydrophilicity analysis of peptide NS5A (1-31)".

[0175] Select the negatively charged peptide (99-111 aa) of ORF2 (protein sequence: RRRSAPAGAAPLT The gene is inserted at the C-terminus of ORF2 to generate HEV-ORF2(99-608)-VLP. The insertion of a cysteine ​​residue after 490 aa on the P domain of HEV-VLP can serve as a chemical conjugation site for exogenous peptides / ligands for antigen or specific cell targeting. The 490 aa is located within one of three flexible loops in the P domain; these loops are non-structurally essential and can be genetically engineered without affecting VLP formation (Chen et al. 2016). The choice of Cys after 490 aa in HEV-VLP (HEV-Cys VLP) demonstrates the generation of modular therapeutic capsules. HEV-Cys VLP can utilize two different conjugation methods: thiol-selective conjugation between maleimide and cysteine, and amine-selective conjugation between an amino group and an NHS ester.

[0176] Sequence alignment 3: NCT-001 - ORF2 (99-608 aa) with N and C ends missing (SEQ ID NO: 10) Expected results: T=1, VLP conformation, 27 nm; positively charged peptide elongated at the N-terminus. This will help to more effectively encapsulate negatively charged drugs, DNA, RNA, and Fe3O4 NPs.

[0177] The ligand can be chemically conjugated to the Cys mutation site on the HEV-Cys VLP with a maleimide-linked peptide / ligand, and conjugation is achieved by reacting the HEV-Cys VLP (4 µM–40 µM) with the maleimide-linked peptide / ligand (20 µM–200 µM) overnight in 0.01 M PBS (pH 7.2) at 4°C. Unbound maleimide-peptide / ligand can then be removed using a 7,000 MWCO or similar desalting column (Zeba centrifugal desalting column, Thermo Scientific).

[0178] Example 4: Development of HEV-VLP for Cancer / Tissue-Targeted Drug Delivery Systems A fundamental challenge in developing gene-based therapeutic systems is ensuring the safety and efficacy of the delivery vector or agent. While many types of viruses used in gene therapy vector systems have been evaluated as highly effective in delivering genes to cells, their application is limited by concerns related to oncogenicity, immunogenicity, and the biomanufacturing of these vectors. Non-viral synthetic delivery vectors based on lipid virus-like particles have also been developed to enhance the delivery of therapeutic nucleic acids to their sites of action. Only a few synthetic delivery vector systems have been approved for clinical use, often facing obstacles such as lower delivery efficiency compared to viral vector systems. Although viruses have evolved to effectively target and deliver their genomes to mammalian cells, many synthetic vectors or delivery systems cannot efficiently transport their payloads across the multiple barriers they face.

[0179] Hepatitis E virus-like particles (HEV-VLPs) are non-infectious, self-assembled particles containing capsid proteins capable of binding and entering cells. HEV-VLPs also appear to maintain their structural integrity in low pH environments (Zafrullah, Khursheed et al. 2004), which is advantageous for intratumoral penetration. The primary route of HEV infection is the fecal-oral route. HEV-VLPs are resistant to proteolytic and acidic mucosal conditions, making them ideal mucosal delivery capsules (Jariyapong, Xing et al. 2013; Chen, Baikoghli et al. 2018; Shizuo G. Kamita 2019). HEV-VLPs can be purified in high quantities from *Spodoptera litura* insect cells infected with baculovirus expression vectors (Kawano, Xing et al. 2011).

[0180] Modular therapeutic HEV-VLPs can bind to molecules encapsulated within them or to amino acids or other parts exposed on their surface. The HEV-VLP sequence is optimized to not encapsulate viral RNA, forming a highly stable, non-infectious capsid capable of reversible in vitro assembly via cation-mediated processes (Xing, Li et al. 2010). HEV-VLPs can switch back and forth between chemical reduction and cation chelation during self-assembly. This property provides a method for encapsulating therapeutic nanomaterials in vitro. HEV-VLPs can be disassembled in the presence of reducing agents and chelating agents (e.g., dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)), while reassembly is initiated by calcium or magnesium ions. Encapsulation of HEV-VLPs occurs through charge-based interactions, enabling the packaging of negatively charged nucleic acids, nanoscale proteins, and inorganic nanoparticles (including metallic nanoparticles, such as gold and ferrite (Fe3O4) nanoparticles) for therapeutic applications (Chen, Baikoghli et al. 2018, Shizuo G. Kamita 2019). Oral delivery of plasmid cDNA containing exogenous genes to small intestinal epithelial cells has been demonstrated by showing transient expression of antigens encoded by cDNA (Takamura, Niikura et al. 2004, Cheng and Xing 2014). HEV-VLPs encapsulating magnetic ferrite NPs are suitable for MRI imaging and tumor-targeted hyperthermia induced by radiofrequency electromagnetic radiation (Roemer 1999). Encapsulation of molecules with HEV-VLPs has shown a variety of applications in drug and gene delivery systems (Stark and Cheng 2016). Binding cancer cell targeting ligands to their surfaces may also have implications for many applications focused on cell and gene therapy vector systems.

[0181] Chemotherapy is generally recommended for patients with intermediate or advanced cancer. Conventional therapies are often limited by the non-specific delivery of chemotherapy agents. Drug delivery systems can effectively target and deliver cytotoxic agents to the tumor site, thereby enhancing efficacy and reducing unwanted side effects.

[0182] Cancer diagnosis and treatment require direct contact between drugs and pathological lesions, often necessitating the targeted delivery of anticancer agents via capsules carrying specific ligands. To evaluate targeting and diagnostic capabilities, the breast cancer cell-targeting ligand LXY30 was chemically added to the surface of HEV-VLP via cysteine-anchored melamine-alkyne. In vivo NIR optical imaging was used to investigate the distribution of HEV-VLP in nude mice with MDA-MB-231 breast cancer xenografts, serving as a standard for assessing tumor-targeting specificity. Cy5.5-labeled LXY30-HEV-VLP and Cy5.5-labeled wild-type VLP were injected intravenously via the tail vein into nude mice with MDA-MB-231 xenografts, respectively. Both Cy5.5-labeled wild-type VLP and LXY30-HEV-VLP were immediately distributed throughout the mouse body after intravenous injection and accumulated in the liver. Tumor uptake of LXY30-HEV-VLP was observed, but not of wild-type VLP. LXY30-HEV-VLP retention at the tumor site begins as early as one hour after injection and continues until six hours later. HEV, as an enterotransmitted hepatitis virus, preferentially enters hepatocytes in the liver. The observation of HEV-VLP accumulation in abdominal organs, including the liver, is not surprising.

[0183] Zwitterionic materials have attracted widespread attention for their effectiveness, robustness, and stability in resisting nonspecific protein uptake. Ultra-low-fouling surfaces can be achieved when the surface contains a nanoscale homogeneous mixture of balanced charge groups from zwitterionic moieties (such as polyEK peptides, where both positively and negatively charged portions are attached to the same group). Nonspecific binding of HEV-VLPs in the liver can be reduced by chemically conjugating the low-fouling peptide polyEK to the P-domain of HEV-VLPs or by inserting the polyEK gene into the fully exposed C-terminus of HEV-VLPs.

[0184] Hepatitis E virus-like particles (HEV-VLPs) derived from modified HEV capsid proteins are non-infectious, self-assembled capsids capable of binding and entering cells. Like the natural virus, HEV-VLPs are stable in acidic environments and resistant to proteolytic digestion, making them highly advantageous as oral delivery mediators. HEV-VLPs can be manufactured cost-effectively using systems already used to produce FDA-approved biomolecules. The major capsid protein, open reading frame 2 (ORF2, approximately 500 amino acids), consists of three domains: an N-terminal shell domain and a middle domain forming the base of the capsid. The remaining approximately 150 amino acids of the C-terminal domain (the overhanging domain or P domain) comprise the major binding sites for neutralizing antibodies and receptors, as well as antibody sites.

[0185] The P domain forms surface-exposed spikes at the icosahedral base, while the flexible hinge allows the P domain to be modified by inserting exogenous peptides through genetic engineering (Jariyapong, Xing et al. 2013) or chemical conjugation (Chen, Xing et al. 2016) without disrupting the icosahedral structure at the base. The P domain and three surface-variable loops at the C-terminus of the HEV capsid protein ORF2 have been designed, genetically engineered, and / or used as chemical conjugation sites for at least one or more bioactive agents because they are well exposed on the HEV-VLP surface (Cheng, Xing et al. 2015, Chen, Xing et al. 2016, R. Holl and Cheng 2017).

[0186] NCT-003: A proposed common sequence derived from 160 sequences (inserting Cys between 490 aa and 491 aa). The C-terminal fused RGD peptide of the N / C-terminal missing version The icosahedral viral structure of HEV-VLP exhibits double, triple, and quintuple symmetry axes (Shizuo G. Kamita 2019). The capsid PORF2 of hepatitis E virus possesses typical characteristics of secreted proteins: an N-terminal signal sequence and conserved glycosylation sites. Interestingly, the N-terminal 111 amino acids show the greatest sequence difference between HEV genotypes, and expression of the full-length ORF2 in insect cells typically leads to proteolytic cleavage of this region. The virion exhibits T=3 symmetry with 180 monomers, while the truncated pORF folds into a T=1 particle with 60 subunits and 30 protruding spikes; this is HEV-VLP, which has been promoted for use as a drug delivery system (Shizuo G. Kamita 2019). In previous structural studies (Wang et al. 2008), the C-terminus of ORF2 was exposed on the surface of HEV-VLP. In this example, the ORF2 with HSV linkers ( RGD peptide ( ) and aggregate EKEK It is fused with VLP to reduce liver uptake.

[0187] Sequence alignment 4: NCT-003: RGD peptide fused at the C-terminus of the N / C-terminal deleted version (SEQ ID NO:12) :99-111 aa contains RRR, which can improve the encapsulation efficiency of negatively charged payloads; Cys was inserted between 490aa N and 491aa as a chemical conjugation site; It fuses with the C-terminus for cancer targeting.

[0188] Expected results: T=1, VLP conformation, 27 nm; The additional HSV linker at the C-terminus, the EKEK-RGD peptide, will help bind to cancer cells; The EKEK peptide between the HSV linker and the RGD peptide may reduce liver binding; and The Cys insertion between positions 490 aa and 491 aa can be used as a chemical conjugation site for cell-targeting ligands.

[0189] Passive encapsulation of different payloads will be accomplished by mixing the payload with the disassembled HEV-RGD-VLP and then gradually adding calcium or magnesium ions to form virus-like particles.

[0190] Key issues to be addressed in developing HEV-RGD-VLP as a mucosal drug delivery agent include: (1) the optimal payload per virus-like particle; (2) the relative cost and efficiency of encapsulating different payloads; (3) the effective separation of unpackaged drug / payload from encapsulated HEV-VLP, which requires meeting GMP standards for products used in clinical trials; and (4) the optimization of HEV-RGD-VLP encapsulation procedures for different types of payloads.

[0191] Example 5: Development of a therapeutic HEV-VLP (T = 3 viral particles) for medical applications HEV consists of a non-enveloped icosahedral capsid and a 7.2 kb single-stranded positive-sense RNA genome (encoding three open reading frames (ORFs)) (Tam, Smith et al., 1991). The capsid protein encoded by ORF2 consists of 660 amino acids and is responsible for most capsid-related functions, such as virion assembly, host interaction, and immunogenicity. The N-terminal 111 amino acids show the greatest sequence difference between HEV genotypes, and expression of the full-length ORF2 in insect cells typically results in proteolytic cleavage of this region.

[0192] The virion exhibits T=3 symmetry and contains 180 monomers, while the truncated pORF2 (amino acids 112-608) folds into a T=1 particle with 60 subunits and 30 protruding spikes (Xing, Kato et al. 1999). Elements in the PORF2 protein essential for T=1 VLP assembly include amino acids 125-600 (Li, Takeda et al. 2005). VLPs containing amino acids 14-608, expressed and purified from insect cells, possess a T=3 VLP structure with a diameter of 410 Å and an inner radius of 170 Å, significantly larger than T=1 VLPs (Xing, Li et al. 2010).

[0193] Three linear domains form distinct structural elements: S, a continuous capsid; M, a triplet protrusion; and P, a double spike. The S domain (amino acids 118-317) employs a jelly-roll β-barrel fold, commonly observed in small RNA viruses. Both the M (amino acids 318-451) and P domains (amino acids 452-606) employ β-barrel folds. The P domain contains binding sites for neutralizing antibodies and receptors. The HEV-VLP with a (T = 1) structure has a surface-exposed protruding (P) domain connected to a stable icosahedral base via a flexible hinge.

[0194] Engineered HEV-VLP exhibited high affinity for human malignant breast tumor cells after conjugation with LXY30 and demonstrated specific targeting of breast tumor cells both in vitro and in vivo (Xiao, Li et al. 2016). These results suggest that HEV-VLP can be manipulated to facilitate targeted delivery of diagnostic or therapeutic agents to pathological lesions (Chen, Xing et al. 2016). HEV-(T=1)VLP also accumulates in abdominal organs, including the liver (Chen, Xing et al. 2016). Recently, HEV-(T=1)VLP has been proposed as a liver-specific positron emission tomography (PET) imaging agent by chemically conjugating it with radioactive gallium-68[68Ga] (Lambidis, Chen et al. 2022). This supports the continued development of HEV-(T=1)VLP as a diagnostic and therapeutic nanocarrier, particularly for therapies targeting hepatocytes.

[0195] In this example, we propose using a T=3 VLP with a diameter of 410 Å and an inner radius of 170 Å to improve the payload encapsulation capability of the HEV-VLP compared to a T=1 VLP with a diameter of 270 Å and an inner radius of 110 Å. The encapsulation capability of the HEV-(T=3)VLP will be enhanced through charge-based interactions, allowing negatively charged nucleic acids, nanoscale proteins, and inorganic nanoparticles (including metal nanoparticles, such as gold and ferrite (Fe3O4) nanoparticles) to be packaged for therapeutic applications (Chen, Baikoghli et al. 2018, Shizuo G. Kamita 2019). Cancer / tissue targeting capability will be enhanced by inserting additional cysteine ​​residues on its surface, such as NCT-001:HEV(T=1)VLP.

[0196] NCT-004: A proposed common sequence derived from 124 sequences (14 aa-660 aa, between 490 aa and 491 aa). The full-length N-terminal deletion of Cys) was 13 aa. HEV consists of a non-enveloped icosahedral capsid and a 7.2 kb single-stranded positive-sense RNA genome (encoding three open reading frames (ORFs)) (2). The capsid protein encoded by ORF2 consists of 660 amino acids and is responsible for most capsid-related functions, such as virion assembly, host interaction, and immunogenicity. In this example, NCT-004, containing amino acids 14-608 of ORF2, is disclosed, which generates a T = 3 VLP with a diameter of 410 Å and an inner radius of 170 Å, significantly larger than the previously reported T = 1 VLP (Xing, Li, Mayazaki, et al. 2010).

[0197] Figure 16 The illustration shown is titled "NCT-002: HEV-NS5A-VLP".

[0198] Sequence alignment 5: NCT-004: Near full-length ORF2 (SEQ ID NO: 04) Expected results: T = 3, VLP conformation, and 40 nm.

[0199] Other sizes may help encapsulate negatively charged drugs, DNA, RNA, insulin, and Fe3O4 NPs.

[0200] Insert between amino acid positions 490 and 491 The residues can be used as sites for chemically conjugating cell-targeting ligands.

[0201] Example 6: P-domain substitution: The VHH substitution of MAB is derived from the N / C-terminal missing version of the P-domain of the proposed common sequence. HEV consists of a non-enveloped icosahedral capsid and a 7.2 kb single-stranded positive-sense RNA genome (encoding three open reading frames (ORFs)) (Tam, Smith et al., 1991). The capsid protein encoded by ORF2 consists of 660 amino acids and is responsible for most capsid-related functions, such as virion assembly, host interaction, and immunogenicity. The N-terminal 111 amino acids show the greatest sequence difference between HEV genotypes, and expression of the full-length ORF2 in insect cells typically results in proteolytic cleavage of this region.

[0202] The virion exhibits T=3 symmetry and contains 180 monomers, while the truncated pORF2 (amino acids 112-608) folds into a T=1 particle with 60 subunits and 30 protruding spikes (Xing, Kato et al. 1999). Elements in the PORF2 protein essential for T=1 VLP assembly include amino acids 125-600 (Li, Takeda et al. 2005). When expressed and purified in baculovirus-infected insect cells, the capsid protein containing amino acids 14-608 produces a T=3 VLP with a diameter of 410 Å and an inner radius of 170 Å, significantly larger than a T=1 VLP (Xing, Li et al. 2010).

[0203] Three linear domains form distinct structural elements: S, a continuous capsid; M, a triple spike; and P, a double spike. The S domain (amino acids 118-317) employs a jelly-roll β-barrel fold, commonly observed in small RNA viruses. Both the M (amino acids 318-451) and P domains (amino acids 452-606) employ β-barrel folds. The P domain contains binding sites for neutralizing antibodies and receptors. The M domain of HEVNP interacts strongly with the S domain and is linked to the P domain via a proline-rich long hinge (amino acids 445-463), which appears to influence the formation of the dimer (the structural unit of HEVNP) (Xing, Li et al. 2010).

[0204] In this example, a portion of the P domain (amino acids 463-608) was replaced with a sequence substitution from a 120 aa nanobody derived from VHH, an FDA-approved anti-PD-1 drug product (Sun, Yan et al. 2018; Jeong, Lee et al. 2022), resulting in a chimeric nanobody – HEVNP. The deletion of approximately 145 amino acids in the capsid protein is similar in size to the added nanobody fragment.

[0205] The β-barrel folding of nanobodies is expected to form structural units with dicyclic structures capable of forming T = 1 VLPs. The 3D structure of pembrolizumab (PDB: 5GGS) indicates that the active domain HCD3, after fusion with the S and M domains of ORF2, will be oriented inward. Even if VHH fusion with ORF2 can form VLPs in the chimeric molecule, its expected tertiary structure may hinder the presentation of HCD3 by pembrolizumab.

[0206] Figure 17 The illustration shows the title "PDB 5GGS (120 aa) (484 aa) (VHH Pembrolizumab 1-120 aa) inserted after 364 aa P".

[0207] Figure 18 The illustration shown is titled "Schematic diagram of the secondary structure interaction of pembrolizumab VHH based on the X-ray crystal structure of pembrolizumab (PDB: 5GGS)".

[0208] Sequence alignment 6: NCT-005-ORF2 (99-452 aa) fused with VHH-pembrolizumab 1-120 aa (PDB: 5GGS) (1-120 aa), with a total length of 474 aa (ORF2 99-608 aa, 510 aa) (SEQ ID NO: 14). AA sequence: :99-111 aa contains RRR, which can improve the encapsulation efficiency of negatively charged payloads; : The β-fold structure obtained by predicting the secondary structure using PredictProtein.

[0209] PDB: 5GGS (120 aa) (484 aa) inserted after 364 aa P (VHH Pembrolizumab 1-120 aa) Secondary structure features generated by the Predict Protein program indicate that The region will have a β-fold structure as shown in the PDB:5GGS obtained through X-ray analysis, and The region will have an α-helical structure as shown in PDB, and 5GGS was analyzed by X-ray.

[0210] Figure EX-6-19: Illustration of the P domain of the N / C-terminal deletion variant of the VHH substitution concordant sequence in NCT-005: mAb Figure EX-6-20: This figure illustrates the title "NCT-006: VHH of mAb with additional linker replaces the P domain of the proposed shared sequence with N / C-terminal missing version". Sequence alignment 7: NCT-006-PORF2 (99-452 aa) fused with VHH-pembrolizumab 1-120 aa (PDB: 5GGS), with a linker (22 aa) and a total length of 496 Gret:aa (ORF2 99-608 aa, 510 aa) (SEQ ID NO: 16) AA sequence: Amino acid 99-111 contains RRR, which can improve the encapsulation efficiency of negatively charged payloads; underline Connector I and Connector II : The β-fold structure obtained by secondary structure prediction using PredictProtein. Connectors I and II: (forming another β-fold (I) and hairpin (II), so that the HCDR3 of VHH faces outward): RVRRRV (formation) ); and CARRDYRFDMGFDYWG (for forming hairpins, HCDR-3).

[0211] Connectors I and II were inserted after aa4(L) in PDB: 5GGS (120 aa, VHH pembrolizumab); 120 + 22 = 142 aa . underline : Connector I and connector II; light gray: PDB analyzed by X-ray: β-fold structure shown in 5GGS; The α-helical structure shown in the PDB, and analyzed by 5GGS using X-rays.

[0212] After inserting additional peptides that form the β-sheet of a stable nanobody structure, the HCDR3 region targeting PD-1 can be outward.

[0213] The ability to bind chimeric nanobodies to HEVNPs and deliver them to specific target sites on different cell types, including breast cancer cells expressing the HER2 antigen, will be evaluated using a variety of established in vitro and in vivo assay systems. The ability to encapsulate different types of payloads (e.g., DNA, mRNA, tRNA, RNP, and inorganic molecules) within virus-like particles will be assessed using established protocols. Routes of administration will be evaluated in animals, including intranasal or oral delivery to mucosal sites, or intravenous injection.

[0214] Statements regarding specific aspects, various modifications, and alternatives are intended to illustrate, rather than limit, the scope of the invention. While specific aspects of the invention have been described in detail, those skilled in the art will understand that various modifications and alternatives can be made to these details in light of the general teachings of this disclosure. It should be recognized that numerous changes can be made to the invention as presently described without departing from the scope and spirit of the invention or impairing any of its advantages. These include replacing different genetic elements, particularly those used to improve the expression of heterologous genes in host cells, including genetic elements used for expression in eukaryotic cells, rather than those specified. Therefore, the specific arrangements disclosed are intended to be illustrative only and not to limit the scope of the invention, which will be given by the full scope of the appended claims and any equivalents thereof.

[0215] Bibliography Statement regarding the inclusion of journal articles and patent documents through citation All references, patents or applications cited in this article are incorporated in their entirety as written in this article.

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Claims

1. A nucleotide sequence encoding a polypeptide sequence derived from a common sequence of capsid proteins of the Hepatitis E Viridae family, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming a functional virus-like particle (VLP), wherein the VLP encapsulates or conjugates one or more molecules, the one or more molecules being selected from the group consisting of nucleic acids, polypeptides, inorganic nanoparticles, or small molecule pharmaceutical products.

2. The nucleotide sequence of claim 1, wherein the common sequence of the hepatitis E virus family capsid protein is derived from the hepatitis E virus subfamily.

3. The nucleotide sequence of claim 2, wherein the common sequence of the hepatitis E virus family capsid protein is derived from the Bareni hepatitis E virus species of the Hepatitis E Virinae subfamily.

4. The nucleotide sequence of claim 1, wherein the polypeptide sequence comprises one or more conserved amino acid substitutions in the variable or invariant domain of the common sequence.

5. The nucleotide sequence of claim 1, wherein the polypeptide sequence contains one or more conserved amino acid substitutions in the variable domain of the common sequence.

6. The nucleotide sequence of claim 1, wherein the polypeptide sequence contains one or more conserved amino acid substitutions in the invariant domain of the common sequence.

7. The nucleotide sequence of claim 1, wherein the polypeptide sequence comprises one or more amino acid deletions.

8. The nucleotide sequence of claim 7, wherein the polypeptide sequence contains a deletion at the amino terminus, carboxyl terminus, or both amino terminus and carboxyl terminus of the common sequence.

9. The nucleotide sequence of claim 7, wherein the polypeptide sequence contains one or more deletions in the variable domain of the common sequence.

10. The nucleotide sequence of claim 1, wherein the polypeptide sequence comprises one or more amino acid insertions.

11. The nucleotide sequence of claim 10, wherein the polypeptide sequence contains one or more insertions in the variable domain of the common sequence.

12. The nucleotide sequence of claim 10, wherein the polypeptide sequence contains one or more insertions in the invariant domain of the common sequence.

13. The nucleotide sequence of claim 1, wherein the polypeptide sequence comprises one or more conserved amino acid substitutions, and one or more insertions or deletions, in the variable or invariant domain of the common sequence.

14. The nucleotide sequence of claim 13, wherein all the conserved amino acid substitutions, insertions or deletions are located in the variable domains of the common sequence.

15. The nucleotide sequence of any one of claims 1-14, wherein the nucleotide sequence further comprises one or more insertions of a DNA fragment encoding a heterologous polypeptide selected from the group consisting of: nucleic acid binding domains, hydrophobic binding domains, hydrophilic binding domains, polypeptide binding domains, and antibody binding domains.

16. The nucleotide sequence of claim 15, wherein the domain is a nucleic acid binding domain.

17. The nucleotide sequence of claim 15, wherein the domain is a hydrophobic binding domain or a hydrophilic binding domain.

18. The nucleotide sequence of claim 15, wherein the domain is a polypeptide-binding domain.

19. The nucleotide sequence of claim 15, wherein the domain is an antibody-binding domain.

20. A vector comprising a nucleotide sequence as described in any one of claims 1-20, the nucleotide sequence encoding a functional virus-like particle.

21. The vector of claim 21, wherein the vector is selected from the group consisting of cloning vectors and expression vectors.

22. The vector of claim 22, wherein the vector is an expression vector comprising a nucleotide sequence encoding a functional virus-like particle, the nucleotide sequence being operatively linked to a promoter at its 5' end and optionally operatively linked to a transcription termination signal at its 3' end.

23. The vector of claim 23, wherein the expression vector is a baculovirus expression vector.

24. The vector of claim 24, wherein the baculovirus expression vector comprises a baculovirus promoter operatively linked to a nucleotide sequence encoding a functional virus-like particle.

25. The vector of claim 25, wherein the promoter is a polyhedral protein promoter.

26. The carrier of claim 25, wherein the promoter is a P10 promoter.

27. The vector of claim 23, wherein the expression vector is a bacterial expression vector.

28. The vector of claim 23, wherein the expression vector is a mammalian expression vector.

29. The vector according to any one of claims 21-29, wherein the vector comprises a nucleotide sequence represented by SEQ ID NO: 01, 03, 05, 07, 09, 11 and 12.

30. A prokaryotic or eukaryotic cell containing the carrier as described in claim 21.

31. The cell of claim 31, wherein the cell is a eukaryotic cell.

32. The cell of claim 32, wherein the cell is an insect cell.

33. The cell of claim 33, wherein the cell is a lepidopteran insect cell.

34. The cell of claim 34, wherein the lepidopteran insect cell is selected from the group consisting of: fall armyworm, white armyworm, and silkworm.

35. The cell of claim 32, wherein the cell is a mammalian cell.

36. The cell of claim 33, wherein the cell is a human cell.

37. The cell of claim 32, wherein the cell is a yeast cell.

38. The cell of claim 31, wherein the cell is a prokaryotic cell.

39. The cell of claim 33, wherein the cell is an Escherichia coli cell.

40. A polypeptide sequence derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, capable of forming a functional virus-like particle, wherein the virus-like particle encapsulates or conjugates one or more molecules, the one or more molecules being selected from the group consisting of nucleic acids, polypeptides, inorganic nanoparticles, and small molecule pharmaceutical products.

41. The polypeptide sequence of claim 41, wherein the common sequence of the hepatitis E virus family capsid protein is derived from the hepatitis E virus subfamily.

42. The polypeptide sequence of claim 42, wherein the common sequence of the hepatitis E virus family capsid protein is derived from the Bareni hepatitis E virus species of the Hepatitis E Virinae subfamily.

43. The polypeptide sequence of claim 41, wherein the polypeptide sequence comprises one or more conserved amino acid substitutions in the variable or invariant domains of the common sequence.

44. The polypeptide sequence of claim 44, wherein the polypeptide sequence contains one or more conserved amino acid substitutions in the variable domain of the common sequence.

45. The polypeptide sequence of claim 44, wherein the polypeptide sequence contains one or more conserved amino acid substitutions in the invariant domain of the common sequence.

46. ​​The polypeptide sequence of claim 41, wherein the polypeptide sequence comprises one or more amino acid deletions.

47. The polypeptide sequence of claim 47, wherein the polypeptide sequence contains a deletion at the amino terminus, carboxyl terminus, or both amino terminus and carboxyl terminus of the common sequence.

48. The polypeptide sequence of claim 47, wherein the polypeptide sequence contains one or more deletions in the variable structural domain of the common sequence.

49. The polypeptide sequence of claim 41, wherein the polypeptide sequence comprises one or more amino acid insertions.

50. The polypeptide sequence of claim 50, wherein the polypeptide sequence contains one or more insertions in the variable structural domain of the common sequence.

51. The polypeptide sequence of claim 50, wherein the polypeptide sequence contains one or more insertions in the invariant domain of the common sequence.

52. The polypeptide sequence of claim 41, wherein the polypeptide sequence comprises one or more conserved amino acid substitutions, and one or more insertions or deletions in the variable or invariant domains of the common sequence.

53. The polypeptide sequence of claim 53, wherein all the conserved amino acid substitutions, insertions or deletions are located in the variable domains of the common sequence.

54. The polypeptide sequence of any one of claims 41-54, wherein the polypeptide sequence further comprises one or more insertions of a DNA fragment encoding a heterologous polypeptide, the heterologous polypeptide being selected from the group consisting of one or more nucleic acid binding domains, hydrophobic binding domains, polypeptide binding domains, and antibody binding domains.

55. The polypeptide sequence of claim 55, wherein the domain is a nucleic acid binding domain.

56. The polypeptide sequence of claim 55, wherein the domain is a hydrophobic binding domain.

57. The polypeptide sequence of claim 55, wherein the domain is a hydrophilic binding domain.

58. The polypeptide sequence of claim 55, wherein the domain is a polypeptide-binding domain.

59. The polypeptide sequence of claim 55, wherein the domain is an antibody-binding domain.

60. A functional virus-like particle, said functional virus-like particle being derived from a common sequence of a capsid protein of the Hepatitis E Viridae family, comprising one or more amino acid substitutions, insertions, or deletions, wherein said virus-like particle encapsulates or conjugates one or more molecules, said one or more molecules being selected from the group consisting of: nucleic acids, peptides, inorganic nanoparticles, and small molecule pharmaceutical products.

61. The functional virus-like particle of claim 61, wherein the virus-like particle encapsulates nucleic acid.

62. The functional virus-like particle of claim 62, wherein the virus-like particle encapsulates a single-stranded RNA (ssRNA) molecule.

63. The functional virus-like particle of claim 62, wherein the virus-like particle encapsulates a double-stranded RNA (dsRNA) molecule.

64. The functional virus-like particle of claim 62, wherein the virus-like particle encapsulates a single-stranded DNA (ssDNA) molecule.

65. The functional virus-like particle of claim 62, wherein the virus-like particle encapsulates a double-stranded DNA (dsDNA) molecule.

66. The functional virus-like particle of claim 61, wherein the virus-like particle encapsulates a polypeptide.

67. The functional virus-like particle of claim 61, wherein the virus-like particle encapsulates one or more molecules selected from the group consisting of nucleic acids, peptides, and inorganic nanoparticles.

68. The functional virus-like particle of claim 61, wherein the virus-like particle encapsulates a small molecule drug product.

69. A method for isolating functional virus-like particles, said functional virus-like particles being derived from a common sequence of hepatitis E virus family capsid proteins, comprising one or more amino acid substitutions, insertions, or deletions, said method comprising the following steps: (a) Infecting susceptible insect cells with a baculovirus expression vector containing a nucleotide sequence encoding a functional virus-like particle under the control of a baculovirus promoter; (2) Monitoring the expression of capsid proteins over time; (3) Concentrating, binding, and eluting the purified capsid proteins; and (4) Forming functional virus-like particles.

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