Virus-like nanocapsids for oral delivery of insulin

Abstract

Hepatitis E virus (HEV)-based virus-like particles (VLPs) are provided that are constructed with a modified capsid protein containing at least a portion of the open reading frame 2 (ORF2) protein and an encapsulated insulin protein or insulin-encoding nucleic acid, as well as methods for targeted delivery of insulin using HEV VLPs.

Description

[Technical Field]

[0001] Related Applications This invention claims priority to U.S. Patent Application No. 62 / 642,356, filed March 13, 2018, the entire contents of which are incorporated herein by reference for all purposes.

[0002] STATEMENT REGARDING RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Contract Nos. AI095382, EB021230, and CA198880 awarded by the National Institutes of Health and the National Institute of Food and Agriculture under the U.S. Department of Agriculture. The government has certain rights in the invention. [Background technology]

[0003] Virus-like particles (VLPs) can serve as nanocarriers for targeted delivery of diagnostic and therapeutic regimens, such as DNA / RNA and various chemotherapeutics. Hepatitis E virus (HEV) is an enterally transmitted virus that causes acute hepatitis in humans. HEV virus-like particles (HEV VLPs) are icosahedral cage-shaped capsid proteins that can be produced by expressing the major capsid protein, HEV open reading frame 2 (ORF2), in a eukaryotic expression system. HEV VLPs are stable in acidic and proteolytic environments, a necessary feature for the natural infection pathway of HEV. Therefore, HEV VLPs are promising nanocarriers that can be utilized, for example, for the delivery of therapeutic agents, imaging agents, or vaccines.

[0004] One disease for which nanocarriers have been explored for treatment is diabetes, a highly prevalent disease, especially in developed countries. Despite the development of numerous other drugs to treat diabetes, insulin remains the first-line treatment for type 1 diabetes (T1D) and advanced type 2 diabetes (T2D). Although insulin has significantly reduced morbidity and mortality in diabetic patients, 60% of patients still fail to achieve long-term glucose control [1]. This is likely due to the discomfort and scarring associated with the typical needle-based insulin administration. Oral administration of insulin is considered a convenient, cost-effective, and preferred method of administration, with the highest patient compliance. In addition, the oral route mimics the endogenous insulin secretion pathway from the pancreas to the liver through the hepatic portal vein, achieving better glucose homeostasis [2-4]. Progress in oral insulin delivery has been hampered by the low bioavailability of insulin due to its degradation in the gastrointestinal (GI) tract as a protein and its low permeability through the intestinal epithelium [4, 5]. Oral delivery nevertheless remains an attractive alternative over needle injection, especially since the prospect of the once preferred pulmonary route has disappeared as a realistic possibility [6].

[0005] Several oral insulin delivery pharmacologies have been proposed using paracellular and / or transcellular transport through the ileum and colon via platforms such as tablets, capsules, intestinal patches, hydrogels, microparticles, and nanoparticles. The status of their oral insulin development and progress in different phases of clinical trials have been reviewed in several reviews [4, 7-10]. Among them, Oram Pharmaceuticals of Israel owns the patented Protein Oral Delivery (POD®) technology, which employs a three-pronged approach consisting of encapsulation, protease inhibitors, and chelators. Clinical trials are currently underway in both T1D and T2D patients. Novo Nordisk of Denmark conducted phase I and II clinical trials using an oral insulin tablet based on a microemulsion of a mixture of oils and surfactants or fatty acid derivatives within an enteric-coated gel capsule. Despite preliminary success in clinical trials, Novo Nordisk made the difficult decision to discontinue its oral insulin development program at the end of 2016 due to the system's low efficiency. Building on the technology and experience from these pioneering developments, the present inventors are addressing several cost-effective factors such as sufficient bioavailability and reproducible absorption of insulin for understanding the food-dependent absorption rate and mass production of an oral insulin delivery system.

[0006] Since the late 1970s, when the insulin gene was cloned and expressed in cultured cells, the development of gene therapy has also been proposed as a promising treatment for diabetes.

[11] Insulete, a startup company based in Madison, Wisconsin, USA, is commercializing a gene therapy that induces insulin production in patients' liver cells. They target the liver instead of the pancreas due to its regenerative potential. In preliminary animal studies, a single administration of naked insulin DNA plasmid resulted in glycemic control for up to six weeks.

[12] However, the system lacks specific tissue / cellular targeting, which remains to be addressed for effective treatment. Therefore, there is a clear need for the development of novel, effective insulin delivery methods for diabetes treatment. The present invention fulfills this and other related needs. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides HEV VLPs for the targeted delivery of insulin and methods for delivering insulin using such HEV VLPs.

[0008] In a first aspect, the present invention provides a composition comprising (a) a modified capsid protein comprising at least a portion of the Hepatitis E virus (HEV) open reading frame 2 (ORF2) protein and capable of forming an HEV virus-like particle (VLP), and (b) insulin in the form of a protein or polynucleotide encoding sequence encapsulated within the HEV VLP formed by the modified capsid protein. Typically, the modified ORF2 protein is less than the full-length of the wild-type protein (e.g., any one of SEQ ID NOS: 1-6). Specific modifications of the ORF2 protein may be those described in previous disclosures by the present inventors, such as U.S. Patent Nos. 8,906,862 and 8,906,863 and WO 2015 / 179321.

[0009] In some embodiments, the modified capsid protein is less than the full-length HEV ORF2 protein and includes a segment from residues 452 to 606 of the HEV ORF2 protein of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and includes a heterologous polypeptide sequence inserted into said portion of the HEV ORF2 protein within the segment from residues 483 to 490, 530 to 535, 554 to 561, 573 to 577, 582 to 593, or 601 to 603 of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the heterologous polypeptide sequence is inserted immediately after residue Y485 of SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the heterologous polypeptide may be involved in hepatocyte targeting for insulin delivery, such as RGD (Arg-Gly-Asp) peptide or cyclic RGD peptide [1], which are the most widely used homing peptides that exhibit strong affinity for integrins vb3 and vb5, or homing peptides that specifically target HCC and include TTPRDAY [2], FQHPSFI (HCBP1) [3], SFSIIHTPILPL (SP94) [4], RGWCRPLPKGEG (HC1) [5], AGKGTPSLETTP (A54) [6], KSLSRHDHIHHH (HCC79) [7], and AWYPLPP [8].

[0010] In some embodiments, the modified capsid protein is capable of forming acid- and proteolytically stable HEV VLPs and has at least one residue of Y485, T489, S533, N573, or T586 of SEQ ID NO: 1, 2, 3, 4, 5, or 6 substituted with a cysteine ​​or lysine, which may be chemically derivatized. In some embodiments, the cysteine ​​or lysine is alkylated, acylated, arylated, succinylated, oxidized, or conjugated with a detectable label or hepatocyte-targeting ligand. For example, the detectable label may comprise a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron-emitting isotope, or a cluster of an element from Groups 3-18 with an atomic number greater than 20. In some embodiments, the detectable label comprises a gold nanocluster. In another example, the hepatocyte targeting ligand is a heterologous polypeptide, which may be involved in hepatocyte targeting for insulin delivery, such as the most widely used homing peptides, RGD (Arg-Gly-Asp) peptide or cyclic RGD peptide [1], or homing peptides that specifically target HCC and include TTPRDAY [2], FQHPSFI (HCBP1) [3], SFSIIHTPILPL (SP94) [4], RGWCRPLPKGEG (HC1) [5], AGKGTPSLETTP (A54) [6], KSLSRHDHIHHH (HCC79) [7], and AWYPLPP [8].

[0011] In some embodiments, the composition may further comprise pharmaceutically acceptable non-medicinal excipients, or may be formulated for oral administration, for example, for the treatment of diabetic patients.

[0012] In a second aspect, the present invention provides a method for targeted delivery of insulin to hepatocytes, the method comprising contacting hepatocytes with any of the types of compositions described above and herein, in particular with a hepatocyte targeting ligand such as RGD (cyclic RGD) peptide [1].

[0013] In some embodiments, the hepatocytes are located in a patient, and the contacting step comprises administering to the patient a composition comprising an effective amount of HEV VLPs as described above and herein. In some embodiments, the administration is oral. In some embodiments, the modified capsid protein comprises a cysteine ​​or lysine bound to a gold nanocluster. In some embodiments, the patient is a patient diagnosed with diabetes. In some embodiments, the patient is an animal, particularly a mammal such as a primate, including a human. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows insulin-loaded HEVNPs (left panel) and the oral delivery route of insulin-loaded HEVNPs, which travel through the gastrointestinal tract and via the hepatic portal vein to the liver (right panel). [Figure 2] (A) TEM micrograph of insulin. (B) TEM micrograph of insulin-loaded HEVNPs. (C) Size distribution of insulin-loaded HEVNPs under TEM observation, with the majority of the HEVNPs measuring approximately 52 nm in size. (D) TEM image of insulin-loaded HEVNPs. The length of the bar is 100 nm. [Figure 3] (A) TEM micrograph of insulin-encapsulated HEVNPs as a control without pepsin treatment, (B) TEM micrograph of insulin-encapsulated HEVNPs after pepsin (38 U / ml) treatment at pH 3 and 37°C for 5 min, and (C) TEM micrograph of insulin-encapsulated HEVNPs after pepsin (38 U / ml) treatment at pH 4 and 37°C for 5 min. The bar length is 100 nm. [Figure 4] Insulin encapsulation in HEVNPs: Optimization of packaging conditions to increase the efficiency of insulin encapsulation in HEVNPs is shown. [Figure 5] Insulin encapsulation in HEVNPs: Optimization of packaging conditions to increase the efficiency of insulin encapsulation in HEVNPs, as tested by Bradford assay and ELISA. Increased loading capacity with ultrasound is shown (bottom panel). [Figure 6] Size exclusion column analysis: As shown in ELISA, there are clear overlapping peaks for insulin and HEVNP (indicated by the + symbols in conditions #16 to #32). [Figure 7] Insulin encapsulation in HEVNPs: Optimization of insulin packaging based on cryo-EM structures is followed by 3D modeling of insulin packaging and computational validation of the packaging mechanism, as well as serial tilt image data collection in electron microscope tomography to reconstruct a 3D depiction of HEVNP-insulin. [Figure 8] High Stability and Shelf Life: HEVNP-insulin samples were stored at 4°C for over a year and observed by cryo-electron microscopy. The micrographs show intact particles, indicating high stability to storage conditions. [Figure 9] Enhanced stability of HEVNPs with AuNCs: CryoArm 300kV microscopy and 3D image reconstruction of HEVNPs with enhanced stability due to clustered metal elements based on capsid surface modulation are shown. High-resolution structural determination is key to optimizing mucosal delivery of HEVNPs. DETAILED DESCRIPTION OF THE INVENTION

[0015] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0016] "Hepatitis E virus" or "HEV" refers to a virus, virus type, or virus class that i) causes waterborne infectious hepatitis, ii) is serologically distinct from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), or hepatitis D virus (HDV), and iii) contains a genetic region homologous to a 1.33 kb cDNA inserted into pTZKF1 (ET1.1), a plasmid introduced into the Escherichia coli strain deposited with the American Type Culture Collection (ATCC) under accession number 67717.

[0017] In the context of HEV, the terms "capsid protein" and "modified capsid protein" refer to mature or modified (e.g., truncated, recombinantly mutated, or chemically derivatized) HEV open reading frame 2 (ORF2) polypeptides. As used herein, reference to such ORF2 polypeptides or ORF2 proteins is intended to encompass the full-length polypeptide and fragments thereof, as well as any substitutions, deletions, insertions, or other modifications to the ORF2 protein. The capsid protein must be capable of forming virus-like particles (VLPs). While capsid proteins can tolerate various additional substitutions, deletions, or insertions as long as they do not inhibit VLP formation, capsid proteins typically comprise at least residues 112-608 of HEV ORF2.

[0018] In one embodiment, the term "modified capsid protein" refers to a capsid protein or a portion thereof (i.e., less than the full-length capsid protein) that has modifications, such as one or more additions, deletions, or substitutions, but the resulting modified capsid protein maintains the ability to form a VLP. These modifications include those described in U.S. Patent Nos. 8,906,862 and 8,906,863 and WO 2015 / 179321. For example, a heterologous polypeptide may be inserted into a capsid protein or a portion thereof within the segment at positions 483-490, 530-535, 554-561, 573-577, 582-593, or 601-603, or immediately after residue Y485 (see U.S. Patent Nos. 8,906,862 and 8,906,863). As another example, WO 2015 / 179321 describes another example of a modified capsid protein in which the surface variable loop of the P domain of HEV ORF2 has been modified to incorporate one or more cysteines or lysines not present in the wild-type capsid protein sequence. Alternatively, or in addition, the term "modified capsid protein" refers to a capsid protein or portion thereof in which the C-terminus of HEV ORF2 (e.g., position 608) has been modified to incorporate one or more cysteines or lysines not present in the wild-type capsid protein sequence. Alternatively, or in addition, the term "modified capsid protein" refers to a capsid protein or portion thereof in which a cysteine ​​or lysine (e.g., a cysteine ​​or lysine in the surface variable loop of the P domain of HEV ORF2, or a cysteine / lysine introduced by recombinant technology at position 608) has been chemically derivatized, and the protein is covalently linked to at least one heterologous atom or molecule. Cysteines or lysines may be inserted to increase the length of the HEV ORF2 protein, substituting one or more residues in the surface variable loop and / or C-terminus of the P domain.

[0019] Generally, the modified capsid protein retains the ability to form HEV VLPs. In some cases, the one or more cysteines or lysines are conjugated to a bioactive agent (e.g., a cell-targeting ligand such as peptide LXY30). The surface variable loop of the P domain includes, for example, one or more of residues 475-493, 502-535, 539-569, 572-579, and 581-595 of HEV ORF2 (SEQ ID NO: 1, 2, 3, 4, 5, or 6). The surface variable loop of the P domain further comprises polypeptide residues that have at least about 80%, 85%, 90%, 95%, 99% or more identity to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, and that comprise an amino acid sequence corresponding to one or more of residues 475-493, residues 502-535, residues 539-569, residues 572-579, and residues 581-595 of SEQ ID NOs: 1, 2, 3, 4, 5, or 6.

[0020] As used herein, the term "virus-like particle" (VLP) refers to an icosahedral shell formed by capsid proteins (e.g., T1 or T3). VLPs lack the viral genome and are therefore not infectious. "VLP" refers to a non-replicating, icosahedral viral shell derived from the capsid protein HEV ORF2 of Hepatitis E virus or a portion thereof. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. In some embodiments, VLPs are formed from modified capsid proteins, for example, capsid proteins containing one or more cysteine / lysine residues in the surface variable loops of HEV ORF2 or a portion thereof. HEV VLPs can comprise a mixture of modified and / or unmodified HEV ORF2 proteins.

[0021] The term "acid- and proteolytically stable" in the context of HEV VLPs refers to HEV VLPs that are resistant to the acidic and proteolytic environment of the mammalian digestive system. Methods for assessing acid and proteolytic stability are described in Jariyapong et al. (2013), and include, but are not limited to, placing HEV VLPs in an acidic environment (e.g., pH 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2, or a pH of about 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2) and / or a proteolytic environment (e.g., trypsin and / or pepsin), observing the contacted HEV VLPs by electron microscopy, gel filtration chromatography, or other suitable method, and determining whether the quaternary structure of the HEV VLP (e.g., T=1, T=3, icosahedron, dodecahedron, etc.) is maintained. A population of HEV VLPs (e.g., modified or unmodified) can be incubated under acidic and / or proteolytic conditions for a suitable period of time (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, or 60 minutes, or at least about 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, or 60 minutes) and then tested to determine the extent of quaternary structure retention. In this context, acid- and proteolytically stable modified HEV VLPs refer to modified HEV VLPs in which at least 10%, 25%, 50%, 75%, 90%, 95%, 99%, or 100% of the population of VLPs retain quaternary structure when incubated as a population of VLPs under acidic and / or proteolytic conditions and assessed by electron microscopy as a percentage of the VLPs.

[0022] Alternatively, HEV VLPs can be delivered to a subject orally, and delivery efficiency can be assessed by detecting and / or quantifying a biological response resulting from (i) an immune response to an antigen within the HEV VLP, (ii) a detectable label bound to, recombinantly introduced into, or encapsulated in the HEV VLP, or (iii) delivery of a bioactive agent bound to (e.g., recombinantly introduced into, bound to, or encapsulated in) the HEV VLP to cells. In this context, a modified HEV VLP that is stable against acid and proteolysis refers to a modified HEV VLP that retains at least 10%, 25%, 50%, 75%, 90%, 95%, 99%, or 100% of the oral delivery efficiency and / or cell-penetrating activity of an unmodified HEV VLP.

[0023] The term "heterologous," when used in the context of describing the relative positions of two elements, refers to two elements, such as nucleic acids (e.g., promoters or protein-encoding sequences) or proteins (e.g., HEV ORF2 protein or portions thereof, or modified capsid proteins and other proteins), that are not found in the same relative positions in nature. Thus, a "heterologous promoter" of a gene refers to a promoter that is not operably linked to that gene in nature. Similarly, a "heterologous polypeptide" or "heterologous nucleic acid" in the context of an HEV VLP or HEV capsid protein refers to one that is derived from a non-HEV source.

[0024] Hepatitis E virus (HEV) is known to cause severe acute liver failure. HEV belongs to the genus Hepevirus in the family Hepeviridae. HEV contains a single-stranded, positive-sense RNA molecule of approximately 7.2 kb. The RNA is 3' polyadenylated and contains three open reading frames (ORFs). ORF1 encodes the viral nonstructural proteins and is located in the 5' half of the genome. ORF2 encodes the proteins that form the viral capsid and is located at the 3' end of the genome. ORF3 encodes a 13.5 kDa protein and overlaps with the C-terminus of ORF1 and the N-terminus of ORF2. ORF3 associates with membrane and cytoskeletal compartments.

[0025] As used herein, the term "encapsulation" or "encapsulated" refers to the inclusion of a heterologous substance, such as a heterologous nucleic acid or protein, a chemotherapeutic agent, an imaging agent, a ferrite nanoparticle, or the like, within a VLP as defined herein.

[0026] The term "bioactive agent" refers to any chemical, drug, compound, or mixture thereof that targets a specific biological location (targeting agent) and / or exerts some local or systemic physiological or pharmacological effect that can be demonstrated in vivo or in vitro. Non-limiting examples include drugs, hormones, vaccines, antibodies, antibody fragments, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA, snRNA, RNAi, DNA, cDNA, antisense constructs, ribozymes, etc.).

[0027] A "pharmaceutically acceptable" or "pharmacologically acceptable" substance is one that is not biologically harmful or undesirable, i.e., the substance can be administered to a human together with the capsid protein, HEV VLP, or composition of the invention without causing undesired biological effects. The substance does not interact in a deleterious manner with any of the components of the composition in which it is included.

[0028] The term "non-medicinal ingredient" refers to any essentially accessory substance that may be present in the final dosage form of the compositions of the present invention. For example, the term "non-medicinal ingredient" includes vehicles, binders, disintegrants, excipients (diluents), lubricants, glidants (glidants), compression aids, dyes, sweeteners, preservatives, suspending / dispersing agents, film-forming / coating agents, flavorings, and printing inks.

[0029] The term "adjuvant" refers to a compound that, when administered together with an antigen, enhances the immune response to the antigen, but does not produce an immune response to the antigen when administered alone. Adjuvants can enhance the immune response through various systems, including lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of macrophages.

[0030] An "immunogenic response" to an antigen or composition is the development in a subject of a humoral and / or cellular immune response to the antigen present in the composition of interest. For purposes of this disclosure, "humoral immune response" refers to an immune response mediated by antibody molecules, and "cellular immune response" refers to one mediated by T lymphocytes and / or other white blood cells. One important aspect of cellular immunity involves antigen-specific responses by cytotoxic T cells ("CTLs"). CTLs have specificity for peptide antigens expressed on the cell surface, presented in association with proteins encoded by the major histocompatibility complex (MHC). CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves antigen-specific responses by helper T cells. Helper T cells help stimulate the function and focus the activity of nonspecific effector cells against cells presenting peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other white blood cells, including cells derived from CD4+ T cells and CD8+ T cells. Thus, an immune response may include one or more of the following effects: antibody production by B cells and / or activation of suppressor T cells and / or γΔT cells specifically directed against an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity and / or mediate antibody-complement or antibody-dependent cellular cytotoxicity (ADCC) to protect an immunized host. Such responses can be assessed using standard immunoassays and neutralization assays well known in the art.

[0031] A "label," "detectable label," or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32Detectable labels include P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens, and proteins that can be made detectable, for example, by incorporating a radioactive moiety into the peptide or can be used to detect antibodies specifically reactive with the peptide. Typically, a detectable label is a heterologous moiety attached to a probe or molecule with defined binding properties (e.g., a polypeptide or polynucleotide with known binding specificity) such that the presence of the probe / molecule (and therefore its binding target) is readily detectable. The heterologous nature of the label ensures that it is of a different origin from the probe or molecule it labels, so that a probe / molecule attached to a detectable label does not constitute a naturally occurring composition.

[0032] The terms "treating" or "treatment" as used in this application refer to an act that results in the elimination, reduction, alleviation, amelioration, prevention, or delay in the onset or recurrence of any symptoms of the associated disease. In other words, "treatment" of a disease includes both therapeutic and prophylactic interventions against the disease.

[0033] The term "effective amount" as used herein refers to a quantity of a given substance that is quantitatively sufficient to produce a desired effect. For example, an effective amount of insulin-encapsulated HEV nanoparticles (HEVNPs) is the amount of HEVNPs that achieves a detectable effect in a patient receiving the HEVNPs for therapeutic purposes, such as reducing, reversing, eliminating, preventing, or delaying the onset of the symptoms, severity, and / or likelihood of recurrence of the targeted disease (e.g., diabetes). An amount adequate to achieve this is defined as a "therapeutically effective dose." Dosage ranges vary depending on the nature of the therapeutic agent being administered, as well as other factors such as the route of administration and the severity of the patient's disease.

[0034] As used herein, the term "patient" refers to a vertebrate, e.g., an avian or mammalian species, particularly a mammal (e.g., bull / cow, pig, sheep / goat, horse, rabbit, rodent, dog, cat, fox, etc.), including primates such as chimpanzees, monkeys, or humans.

[0035] A. Introduction The present disclosure relates to a viral-derived nanocapsid for oral delivery of insulin that is chemically stable and resistant to enzymatic activity in the gastrointestinal tract. It is well known that certain limitations in the treatment of diabetes, including low patient compliance, are due to the discomfort and adverse effects associated with the common use of needle injections for insulin administration. Oral delivery is the most preferred route for insulin delivery, but the 5.8 kDa protein faces challenges, including degradation in the gastrointestinal tract by proteolytic enzymes and harsh, acidic physiological conditions, as well as post-absorption delivery efficiency and permeability through the intestinal epithelium. While several oral insulin delivery systems have been developed and approved for clinical trials, numerous cost-related factors remain to be addressed, including improving low bioavailability, achieving reproducible absorption, gaining an understanding of food-dependent absorption rates, and the need for mass production of orally administered insulin delivery systems.

[0036] Hepatitis E virus nanoparticles (HEVNPs) are derived from self-assembling, noninfectious nanocapsids. HEVNPs are stable in acidic environments and resistant to proteolytic digestion, making them advantageous as oral delivery vehicles. They can be administered orally and subsequently transported to the small intestine and ultimately the liver, following the natural HEV infection pathway. Their in vitro disassembly / reassembly capabilities allow HEVNPs to encapsulate drugs or nucleic acids and deliver them through the gastrointestinal digestive system. Specific targeting ligands (e.g., ligands targeting delivery to the liver) can be attached to the protruding domains of HEVNPs by genetic engineering or chemical conjugation. For improved bioavailability of orally delivered drugs (e.g., insulin), the HEVNP structure can be stabilized by conjugation with monodisperse gold nanoclusters (AuNCs)

[18] .

[0037] Certain embodiments of this disclosure and our previous publications (see, e.g., U.S. Pat. Nos. 8,906,862 and 8,906,863, and WO 2015 / 179321) outline the production of HEVNPs and methods and applications for surface modification, encapsulation for oral delivery of insulin to the liver, and mimicking the physiological secretory pathway from the pancreas to the liver.

[0038] The construction of stabilized HEVNPs as oral insulin delivery capsules offers the following benefits: (1) elimination of the need for needles and associated hazards and disposal; (2) insulin, either as a polypeptide or a polynucleotide encoding the sequence itself, can be easily encapsulated in the HEVNP structure in vitro and delivered to the liver without a targeting ligand; however, a therapeutic targeting ligand would enable and enhance delivery of insulin (e.g., the insulin gene) specifically to the pancreas; and (3) HEVNPs composed of capsid proteins pose few toxicological concerns and can be biodegraded through proteolytic pathways.

[0039] Combinations of various forms of insulin-loaded HEVNPs can be used as a multimodality treatment for better control of blood glucose levels in diabetic patients. Scaled-up production and expression of HEVNPs will be followed by animal studies for cost analysis of the treatment regimen.

[0040] B. Production and Purification of Modified Capsid Proteins and VLP Formation One aspect of the present invention relates to methods for producing and purifying capsid proteins and VLPs derived therefrom. (Expression and self-assembly of empty virus-like particles of hepatitis E virus. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T., J Virol. 1997 Oct;71(10):7207-13. Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus. Li TC, Takeda N, Miyamura T, Matsuura Y, Wang JC, Engvall H, Hammar L, Xing L, Cheng RH. J Virol. 2005 Oct;79(20):12999-3006. Niikura M et al., Chimeric recombinant hepatitis E virus-like particles as an oral vaccine vehicle presenting foreign epitopes. Virology 2002;293:273-280). In one embodiment, the capsid protein is a modified capsid protein, and the VLP derived therefrom is a cysteine / lysine modified HEV VLP. For example, the modified capsid protein contains one or more cysteine / lysine residues in the surface variable loop of HEV ORF2 or a portion thereof.

[0041] A variety of expression systems can be used to express the capsid proteins of the present invention. Examples of expression systems useful for producing the virus-like particles of the present invention include, but are not limited to, bacterial expression systems (e.g., E. coli), insect cells, yeast cells, and mammalian cells. A preferred expression system of the present invention is the baculovirus expression system using insect cells. For example, general methods for manipulating and preparing baculovirus vectors and baculovirus DNA, as well as procedures for culturing insect cells, are outlined in A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures.

[0042] The capsid proteins of the present invention can be cloned into a baculovirus vector and used to infect appropriate host cells (see, e.g., O'Reilly et al., "Baculovirus Expression Vectors: A Lab Manual," Freeman & Co., 1992). Insect cell lines (e.g., Sf9 or Tn5) can be transformed with a transfer vector containing a polynucleic acid encoding the capsid protein of the present invention. Transfer vectors include, for example, linearized baculovirus DNA and a plasmid containing the desired polynucleotide. To produce recombinant baculovirus, a host cell line can be co-transfected with the linearized baculovirus DNA and the plasmid.

[0043] Purification of the virus-like particles of the present invention can be carried out according to standard techniques in the art (see Li TC, et al., J. Virol. 1997 Oct; 71(10):7207-13; Li TC, et al., J. Virol. 2005 Oct; 79(20):12999-3006; Niikura M et al., Virology 2002; 293:273-280). The purified VLPs are then resuspended in an appropriate buffer.

[0044] In some embodiments, the modified capsid protein or VLP derived therefrom can be chemically conjugated to one or more bioactive agents. For example, one or more cysteine / lysine residues of the capsid protein can be acylated, alkylated, arylated, succinylated, or oxidized using methods well known in the art. In some cases, one or more cysteine / lysine residues can be conjugated to the thiol moiety of a cysteine ​​or lysine using a maleimide functional group that is covalently attached to the bioactive agent. In some cases, the bioactive agent can be modified to introduce a maleimide functional group using click chemistry. For example, an alkyne derivative of the bioactive agent can be contacted with maleimide azide in the presence of copper sulfate and ascorbic acid to produce a maleimide bioactive agent. This maleimide can then be contacted with one or more cysteine / lysine residues of the modified capsid protein to covalently link the two molecules. In some cases, conjugation is performed on capsid proteins that are not assembled into VLPs (e.g., in the presence of reducing agents such as EDTA, EGTA, and / or DTT or β-mercaptoethanol), and in some cases, conjugation is performed on capsid proteins that have assembled into VLPs.

[0045] C. Encapsulation of Bioactive Agents Another aspect of the present invention relates to the encapsulation of one or more bioactive agents into HEV virus-like particles (e.g., cysteine / lysine-modified HEV VLPs) (see, DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration, Gene Therapy 2004.11, 628-635. S Takamura, M Niikura, TC Li, N Takeda, S Kusagawa, Y Takebe, T Miyamura, and Y Yasutomi). Heterologous nucleic acids, proteins, polypeptides, chemotherapeutic agents, contrast agents, nanoparticles, and the like can be encapsulated into the VLPs of the present invention using standard techniques in the art. An exemplary bioactive agent is insulin in protein or nucleic acid form. A typical procedure involves (1) disassembling the VLPs formed by the capsid proteins of the present invention and (2) reconstituting the VLPs in the presence of a bioactive agent. Those skilled in the art will appreciate that it is preferable to purify the VLPs prior to the encapsulation procedure. It is particularly preferred to remove, or substantially remove, any undesired material (eg, nucleic acids) from the VLPs prior to the encapsulation procedure.

[0046] Disassembly of VLPs can be performed using standard techniques in the art. Reconstituted virus-like particles can be produced under physiological conditions (see U.S. Patent Application Publication No. 2008 / 0131928). Disassembly of virus-like particles often requires the use of reagents that disrupt VLP assembly, such as reducing or chelating agents (see U.S. Patent Application Publication No. 2004 / 0152181). Those skilled in the art will appreciate that factors and conditions that affect assembly and disassembly include, among others, pH, ionic strength, post-translational modifications of viral capsid proteins, disulfide bonds, and divalent cation binding. For example, the importance of cation binding, particularly calcium, in maintaining virion integrity has been demonstrated for polyomaviruses (Brady et al., J. Virol., 23:717-724, 1977) and rotoviruses (Gajardo et al., J. Virol., 71:2211-2216, 1997). Disulfide bonds appear to be important for the stabilization of polyomaviruses (Walter et al., Cold Spring Har Symp. Quant. Biol., 39:255-257, 1975; Brady et al., J. Virol., 23:717-724, 1977) and SV40 viruses (Christansen et al., J. Virol., 21:1079-1084, 1977). Factors such as pH and ionic strength are known to affect the stability of polyomavirus capsids, possibly by affecting electrostatic interactions (Brady et al., J. Virol., 23:717-724, 1977; Salunke et al., Cell., 46:895-904, 1986; Salunke et al., Biophys. J., 56:887-900, 1980). It is also known that post-translational modifications of several viral capsid proteins, such as glycosylation, phosphorylation, and acetylation, affect capsid stability and assembly (Garcea et al., Proc. Natl. Acad. Sci. USA, 80:3613-3617, 1983; Xi et al., J. Gen. Virol, 72:2981-2988, 1991).Thus, there are many interrelated factors that influence capsid stability, assembly, and disassembly.

[0047] Preferably, the VLPs of the present invention are disassembled by removal of calcium ions (see Touze A, Coursaget P. In vitro gene transfer using human papillomavirus-like particles. Nucleic Acids Res 1998;26:1317-1323; Takamura et al., DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration. Gene Therapy 2004;11:628-635). According to the present invention, VLPs are disassembled using a reducing agent or a chelating agent, or both. Various reducing agents can be used. A preferred embodiment of the reducing agent includes, but is not limited to, dithiothreitol (DTT). Various chelating agents can be used, such as ethylene glycol tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA). An example of conditions for VLP degradation includes, but is not limited to, disrupting purified VLPs by incubation for 30 minutes in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, and 20 mM dithiothreitol.

[0048] Those skilled in the art will understand that, although preferred, complete disassembly of the VLP is not required to encapsulate the bioactive agent. Those skilled in the art will also understand that in other cases, partial disassembly of the VLP may be preferred. According to the present invention, the conditions for partial disassembly of the VLP can be controlled so that the bioactive agent is still efficiently encapsulated. Partial disassembly of the VLP can be achieved by treating the VLP with a reducing agent alone (e.g., 20 mM DTT) (Sapp et al., J. Gen. Virol., 76:2407-2412, 1995). According to the present invention, complete or partial disassembly of the VLP allows for encapsulation of the bioactive agent by reconstitution of the VLP in the presence of the bioactive agent. In some cases, it is advantageous to use a bioactive agent with a net negative charge to enhance encapsulation. For example, nucleic acids have a net negative charge and may be preferentially encapsulated compared to positively or neutrally charged compounds.

[0049] In some embodiments of the present invention, VLP reassembly is achieved by replenishing the disintegrated VLPs with calcium ions. Alternatively, VLP reassembly is achieved by removing the reducing agent or chelating agent. Optionally, factors such as pH and ionic strength, as well as other factors described herein, can be adjusted to achieve efficient VLP reassembly and efficient encapsulation of the bioactive agent.

[0050] In some embodiments, encapsulation is performed as follows: After a 30-minute incubation at room temperature, a bioactive agent in 50 mM Tris-HCl buffer (pH 7.5) and 150 mM NaCl is added to the disassembled VLP preparation. The disassembled VLP preparation is then refolded by incubation for 1 hour in increasing concentrations of CaCl to a final concentration of 5 mM. The VLPs are pelleted by ultracentrifugation and resuspended in 10 mM potassium MES buffer (pH 6.2). To estimate the amount of encapsulated agent, the refolded, purified VLPs are purified from any unencapsulated bioactive agent and disrupted with EGTA (1 mM). The absorbance of the supernatant, or other suitable methods, can be used to detect the bioactive agent.

[0051] In some embodiments, the bioactive agent (e.g., insulin protein or a nucleic acid encoding insulin) or imaging agent to be encapsulated is linked to an encapsidation signal. For example, the RNA element corresponding to codons 35-59 of HEV open reading frame 1 is a strong encapsidation signal that enables specific interaction in vitro with HEV capsid proteins, including truncated and / or cysteine / lysine-modified HEV ORF2 VLPs described herein. To use VLPs as carriers of therapeutic or imaging agents, chemical linkers (e.g., LC-SPDP, aptamers, telodendrimers) can be used to tag drugs (e.g., chemotherapeutic agents) with HEV encapsidation signals, such as the aforementioned RNA elements, prior to capsid self-assembly.

[0052] In some embodiments, a detectable label (imaging agent) is encapsulated. A detectable label can be a moiety that makes the molecule to which it is attached detectable by various mechanisms, including chemical, enzymatic, immunological, or radiological means. Examples of detectable labels include fluorescent molecules (such as fluorescein, rhodamine, Texas Red, and phycoerythrin) and enzymatic molecules (such as horseradish peroxidase, alkaline phosphatase, and β-galactosidase) that allow detection based on fluorescence or the product of an enzyme-catalyzed chemical reaction. Detectable by any suitable method of recording radiation, such as autoradiography. 3 H, 125 I, 35 S, 14 C, or 32Radioactive labels, including various isotopes such as P, can also be attached to appropriate molecules. See, for example, Tijssen, "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 920. Label introduction, labeling procedures, and label detection can also be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd Ed., Springer Verlag, NY (1997); and in Haugland, Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue published by Molecular Probes, Inc. (1996). Other detectable labels include, but are not limited to, superparamagnetic labels (e.g., ferrites), contrast-enhancing agents (e.g., MRI contrast agents), atomic clusters (e.g., gold clusters), and the like. The attachment of monodisperse gold clusters onto modified capsid proteins, for example onto cysteine / lysine residues, including artificially introduced cysteine / lysine residues in the modified capsid proteins, can be carried out according to methods well known in the art and described in various publications

[18] .

[0053] In some embodiments, the bioactive agent is encapsulated. In some cases, the bioactive agent is a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, cytotoxic drugs. Examples of cytotoxic drugs that can be used in the present invention include alkylating agents such as cyclophosphamide, ifosfamide, chlorambucil, melphalan, busulfan, lomustine, carmustine, chlormethine (mustine), estramustine, threosulfan, thiotepa, and mitobronitol; cytotoxic antibiotics such as doxorubicin, epirubicin, aclarubicin, idarubicin, daunorubicin, mitoxantrone (mitoxantrone), bleomycin, dactinomycin, and mitomycin; methotrexate, capecitabine, cytarabine, fludarabine, cladribine, gemcitabine, fluorouracil, and raltitrexed (Tomudex). antimetabolites such as mercaptopurine, tegafur, and thioguanine; vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, and etoposide; other antineoplastic agents such as amsacrine, artetermine, crisantaspase, dacarbazine, and temozolomide, platinum compounds including hydroxycarbamide (hydroxyurea), pentostatin, carboplatin, cisplatin, and oxaliplatin, porfimer sodium, procarbazine, and razoxane; taxanes including docetaxel and paclitaxel; topoisomerase I inhibitors including inotecan and topotecan, trastuzumab, and tretinoin. In some cases, one or more of the aforementioned imaging agents and / or bioactive agents, or combinations thereof, can additionally or alternatively be conjugated via a thiol bond to a cysteine ​​or lysine (e.g., a recombinantly introduced cysteine ​​or lysine) in a surface variable loop or at the C-terminus of the P domain. In some cases, one or more of the aforementioned imaging agents and / or bioactive agents, or combinations thereof, can additionally or alternatively be conjugated via a thiol bond to a second cysteine ​​or lysine (e.g., a recombinantly introduced cysteine ​​or lysine) in a surface variable loop or at the C-terminus of the P domain.

[0054] In some embodiments, insulin is the bioactive agent encapsulated in the HEV VLP constructs of the invention. While insulin may be used in the form of a biologically active polypeptide (which may optionally include post-translational modifications, such as glycosylation, PEGylation, or substitution of one or more artificial amino acid analogs, including D-amino acids), insulin may also be in the form of a polynucleotide sequence (e.g., cDNA) encoding insulin and / or proinsulin protein; for example, an insulin-encoding nucleic acid is a human insulin gene expression construct in a TA1m vector

[12] . The insulin protein may be recombinant or isolated from a natural source. It may be human insulin or derived from other animals, such as bovine, porcine, feline, or canine. It may also be proinsulin. Different forms of insulin can be used: rapid-acting (aspart (Novolog), glulisine, Apidra, lispro (Humalog)), short-acting (regular (Humulin), Humulin R, Novolin), intermediate-acting (NPH (Humulin N), Novolin N), intermediate-long-acting (detemir), and long-acting (e.g., glargine). Additionally, the bioactive agent can be an insulin analog, such as the commercial insulin analog sold under the trade name Levemir, or insulin glargine, a long-acting basal insulin analog sold under the trade name Lantus. The bioactive agent can also be a combination of insulin and a glucagon-like peptide (GLP-1) receptor agonist or other drug. Examples of GLP-1 receptor agonists include liraglutide (Victoza, Saxenda), lixisenatide (Lyxumia), albiglutide (Tanzeum), dulaglutide (Trulicity), and semaglutide (Ozempic).Suitable forms or combinations of insulin include insulin glargine, insulin lispro, insulin aspart, insulin detemir, insulin (human), insulin aspart + insulin aspart protamine, insulin glulisine, insulin (human) + isophane insulin [International Nonproprietary Name], insulin aspart + insulin degludec, insulin aspart + isophane insulin [International Nonproprietary Name], insulin degludec + liraglutide, insulin glargine + lixisenatide, human insulin + isophane insulin [International Nonproprietary Name], isophane insulin [International Nonproprietary Name] + neutral insulin, human isophane insulin [International Nonproprietary Name] + human insulin, insulin (bovine), insulin degludec, human insulin zinc, isophane insulin [International Nonproprietary Name], human isophane insulin [International Nonproprietary Name], neutral insulin, human insulin + human isophane insulin [International Nonproprietary Name], neutral insulin + isophane insulin [International Nonproprietary Name], insulin (porcine), insulin These include, but are not limited to, neutral, insulin protamine zinc, insulin, insulin tregopir [International Nonproprietary Name], human insulin + human proinsulin, insulin glargine + insulin lispro, human insulin + pramlintide acetate, dulaglutide, dulaglutide + insulin glargine, exenatide + insulin lispro, insulin glargine + liraglutide, insulin lispro + pramlintide, efpeglenatide [International Nonproprietary Name], human insulin + pramlintide, exenatide + human insulin, insulin lispro + insulin lispro protamine, clioquinol [International Nonproprietary Name] + human insulin, insulin glargine + insulin glulisine, and insulin I 131. Additionally, various peptidyl and non-peptidyl insulin analogs, such as those described in Nankar et al. (Drug Discovery Today, Volume 18, Issues 15-16, August 2013, Pages 748-755), may be used as bioactive agents for inclusion in HEV VLPs.

[0055] The size of the VLP can be altered by using capsid proteins with different configurations. For example, the N-terminal portion of the capsid protein can be adjusted to increase or decrease the size and encapsulation capacity of the VLP. In some embodiments of the present invention, when constructing an HEV VLP, a portion of the HEV ORF3 protein fused to the N-terminus of a portion of the HEV ORF2 protein is used to adjust the size of the VLP. Typically, HEV VLPs are formed from a portion of HEV ORF2 containing at least residues 112 to 608 of HEV ORF2.

[0056] D. Pharmaceutical Compositions, Formulations, and Administration The present invention also provides pharmaceutical or physiological compositions comprising HEV VLPs formed by modified capsid proteins encapsulating a bioactive agent, such as insulin, in the form of a protein or nucleic acid. Such pharmaceutical or physiological compositions may also include one or more pharmaceutically or physiologically acceptable non-medicinal ingredients or carriers. The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief description of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0057] The compositions of the present invention can be administered to a host along with non-medicinal ingredients, including, but not limited to, solvents, binders, disintegrants, excipients (diluents), lubricants, glidants (glidants), compression aids, dyes, sweeteners, preservatives, suspending / dispersing agents, film-forming / coating agents, flavorings, and printing inks.

[0058] One advantage of the present invention is that the compositions of the present invention are suitable for oral delivery. Because the HEV VLPs of the present invention can target hepatocytes, site-specific delivery of insulin can be achieved efficiently. Furthermore, as a result of the capsid protein modifications, the HEV VLPs of the present invention are stable in acidic environments and resistant to digestion in the gastrointestinal tract, making them suitable for oral delivery of insulin. Gold nanoclusters attached to cysteine ​​or lysine residues, particularly those surface-modified in some embodiments of the modified capsid protein of the present invention, further enhance the stability, bioavailability, and delivery efficiency of HEV VLPs. Thus, oral delivery of the compositions of the present invention can efficiently provide therapeutic benefits to patients suffering from insulin deficiency or insulin dysregulation disorders, such as type I or type II diabetes and related conditions. The HEV VLPs of the present invention may be formulated in solid (e.g., powder) or liquid form so that they can be used as dietary supplements in familiar foods or beverages for daily consumption.

[0059] The compositions of the present invention may also be formulated for mucosal delivery, such as delivery to the buccal or labial mucosa or mucosa of the respiratory tract, including the nasal mucosa.

[0060] The pharmaceutical compositions of the present invention can be administered by various routes, including, for example, oral, subcutaneous, transdermal, intradermal, intramuscular, intravenous, or intraperitoneal. A preferred route of administration of the pharmaceutical composition is oral delivery of a daily dose of about 0.01 to 5000 mg, preferably 5 to 500 mg, of HEV VLPs. Oral administration is the preferred method of administration, and an appropriate amount may be administered in the form of tablets, capsules, or as a dietary supplement in food and beverage products, as a single daily dose or in divided doses spaced at appropriate intervals, e.g., two, three, four, or more divided doses per day.

[0061] Inert and pharmaceutically acceptable carriers are used to prepare the pharmaceutical compositions of the present invention. Pharmaceutical carriers can be either solid or liquid. Solid preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Solid carriers can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents, and can also be encapsulating materials.

[0062] In powders, the carrier is generally a finely divided solid that is in admixture with the finely divided active ingredient, e.g., chimeric virus-like particles with encapsulated nucleic acids. In tablets, the active ingredient (chimeric virus-like particles with encapsulated nucleic acids) is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0063] For preparing pharmaceutical compositions in the form of suppositories, a low melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into suitable sized molds and allowed to cool and solidify.

[0064] The powders and tablets preferably contain about 5% to about 70% by weight of the active ingredient. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

[0065] The pharmaceutical composition can include a formulation of the active compound with an encapsulating material as a carrier to provide a capsule in which the active ingredient (with or without other carriers) is surrounded by the carrier, thereby binding the carrier to the compound. In a similar manner, cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

[0066] Liquid pharmaceutical compositions include, for example, solutions, suspensions, and emulsions suitable for oral or parenteral administration. Examples of liquid compositions suitable for parenteral administration include sterile aqueous solutions of the active ingredient (e.g., chimeric virus-like particles having encapsulated nucleic acids) or sterile solutions of the active ingredient in a solvent, such as water, buffered water, saline, PBS, ethanol, or propylene glycol. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters, buffers, tonicity adjusters, wetting agents, and detergents, as needed to approximate physiological conditions. It is also anticipated that HEV VLPs may be available commercially in the form of tablets / capsules in prepackaged powder form or in the form of a concentrated liquid. This may be added to food or beverages containing water by the patient before ingestion. Alternatively, HEV VLPs in liquid form may be ingested directly without further dilution.

[0067] Sterile solutions can be prepared by suspending the active ingredient (e.g., chimeric virus-like particles with encapsulated nucleic acid) in a desired solvent system and then passing the resulting solution through a membrane filter for sterilization, or by dissolving the sterilized compound in a previously sterilized solvent under sterile conditions. The resulting aqueous solution may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterilized aqueous carrier prior to administration. The pH of the preparation will typically be 3-9, more preferably 5-8, and most preferably 6-7.

[0068] The pharmaceutical compositions of the present invention can be administered for prophylactic and / or therapeutic therapy. In therapeutic applications, a sufficient amount of the composition is administered to a patient already suffering from the disease to prevent, treat, ameliorate, or at least partially slow or inhibit the symptoms of the disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective amount." Amounts effective for this use will depend on the severity of the illness or disease and the patient's weight and general condition, but will generally range from about 0.1 mg to about 2000 mg of the composition per day for a 70 kg patient, with dosages of about 5 mg to about 500 mg of the composition per day for a 70 kg patient being more commonly used.

[0069] In prophylactic applications, the pharmaceutical compositions of the present invention are administered to a patient susceptible to or at risk of developing a disease or disorder, such as diabetes, in an amount sufficient to delay or prevent the onset of symptoms. Such an amount is defined as a "prophylactically effective amount." Again, the precise amount of composition used in this application will depend on the patient's health and weight, but generally ranges from about 0.1 mg to about 2000 mg of inhibitor per day for a 70 kg patient, and more generally from about 5 mg to about 500 mg per day for a 70 kg patient.

[0070] Single or multiple administrations of the compositions can be carried out with the dose level and pattern being selected by the treating physician. In any event, the pharmaceutical formulation should provide a quantity of the composition of the invention sufficient to achieve the intended effect in the patient, either therapeutically or prophylactically. [Example]

[0071] The following examples are offered by way of illustration only, and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially the same or similar results.

[0072] Example 1: Oral insulin delivery by HEVNPs I. Background For the past 80 years, subcutaneous (SC) injection has been the primary route of insulin administration used to replace suboptimal insulin secretion as a treatment for diabetes. While this method is effective, SC injection is painful, inconvenient, and carries a high risk of infection, which reduces patient compliance. Insulin-encapsulated hepatitis E virus nanoparticles (HEVNPs), consisting of non-infectious hepatitis E virus capsids, hold promise for delivering insulin from the gastrointestinal (GI) tract to the liver after ingestion. HEVNPs may be the answer to the long-standing search for an effective and efficient means for oral administration of insulin, the most preferred route of drug delivery with the highest patient compliance.

[0073] II. Structurally stabilized HEVNPs for oral delivery of insulin From a physiological perspective, orally administered insulin has therapeutic advantages in managing hepatic glucose production due to its ability to mimic the endogenous insulin secretory pathway [4]. Following the route of natural HEV infection, insulin encapsulated in HEVNPs can travel through the gastrointestinal tract and enter the liver via the portal vein (Figure 1). In contrast, parenteral or inhaled insulin is absorbed directly into the peripheral circulation by bypassing hepatic extraction, thereby failing to restore the portal-peripheral insulin gradient and physiological hepatic insulin disposal. Furthermore, these routes expose peripheral targets to higher insulin concentrations compared to the liver, placing patients at higher risk of hypoglycemia and predisposing them to the adverse effects of hyperinsulinemia [4].

[0074] Hepatitis E virus nanoparticles (HEVNPs), derived from an engineered form of the hepatitis E virus (HEV) capsid protein, are noninfectious, self-assembling capsids lacking the viral genome and capable of binding and penetrating cells. Because HEV evolved for oral mucosal delivery, the assembled capsid protein is equally stable to proteolytic degradation and acidic mucosal conditions

[13] . High-yield HEVNP production has been achieved using a baculovirus-vectored insect cell expression system. Due to its proteolytic stability, self-assembled HEVNPs can be extracted and purified directly from cell supernatants, thereby substantially reducing the number of required purification steps. Furthermore, HEVNPs possess a surface-exposed protruding domain (P domain) that is connected to a stable icosahedral base via a flexible hinge. The P domain can be modified by inserting foreign peptides via genetic engineering

[13] or chemical conjugation

[14] without disrupting the underlying icosahedral structure. Three well-exposed surface variable loops on the P domain, encoded by open reading frame 2 (ORF2), and the C-terminus of the HEV capsid protein (CP) are designed as genetic engineering and / or chemical conjugation sites for at least one or more bioactive agents [ 14 , 15 ].

[0075] Targeted drug delivery to specific organs and cellular compartments has been proposed to reduce nonspecific organ / cellular side effects. HEVNP has been proposed as a cell-targeting delivery system because its surface-exposed cysteine ​​or lysine residues allow the use of synthetic ligands for tissue targeting [14, 15]. Its ability to orally deliver genes has been demonstrated in previous studies where HEVNP orally delivered plasmid cDNA to small intestinal epithelial cells for transient expression of insulin and / or proinsulin [16, 17]. In vivo biodistribution assays of HEVNP in a mouse model using far-infrared (FIR) imaging (data not shown) demonstrated that orally delivered HEVNP accumulated in the liver, even without specific liver-targeting ligands.

[0076] Encapsulation by HEVNPs is based on charge interactions, allowing negatively charged nucleic acids and nanosized proteins / small molecules to be packaged for therapeutic applications. HEVNPs can encapsulate the commercial insulin analog, i.e., insulin detemir from Levemir (website: levemir.com), which is approximately 52 nm in size (Figure 3). Considering the drug toxicology of HEVNPs, they consist of a single copy of the capsid protein ORF2 and are biodegradable. In addition, HEVNPs can encapsulate insulin or proinsulin cDNA for oral gene delivery. The ability to target pancreatic β cells and / or the liver can be added by inserting specific cell-targeting ligands into the protruding domain of HEVNPs via overnight chemical conjugation or time-consuming but cost-effective genetic engineering. The tissue-targeting ability of HEVNPs makes them a convenient oral delivery vehicle for transporting insulin genes to the pancreas and / or liver, enabling in situ insulin expression.

[0077] The concept of using HEVNPs as oral delivery vehicles was not only unproven by the previous studies mentioned above, but also unsupported by in vitro stability studies. In vitro stability evaluations (unpublished data) using different pH and pepsin digestion tests showed that insulin-encapsulated HEVNPs could survive 5 minutes of pepsin digestion at pH 3 (Figure 2). HEVNPs contain a modified ORF2 capsid protein with one or more modifications described in WO 2015 / 179321, U.S. Patent No. 8,906,862, and U.S. Patent No. 8,906,863. The bioavailability of encapsulated insulin can be further ensured by taking them before meals to avoid the harsh digestive environment in the stomach. Furthermore, bioavailability can be stabilized by chemically attaching monodisperse gold nanoclusters (AuNCs) to the five-fold symmetric region of HEVNPs

[18] . Furthermore, AuNCs have been proposed as an in vivo imaging agent due to their FIR-detectable signal, which can penetrate deep tissues.

[19] The combination of HEVNPs' functions, including insulin encapsulation, insulin / proinsulin cDNA encapsulation, and tissue / cell targeting through surface binding ability, makes them an ideal oral delivery system for insulin itself or insulin genes to treat diabetes. The delivery system improves patient compliance by eliminating the use of needles.

[0078] The present invention resides in a HEVNP platform having (1) surface-bound tissue / cell targeting ligands (particularly ligands capable of specifically directing HEVNPs to hepatocytes) for enhanced absorption, and (2) encapsulated insulin (either in the form of insulin polypeptide or a polynucleotide sequence encoding insulin) for drug / gene delivery. The HEVNPs are constructed in accordance with prior disclosures by the inventors, including U.S. Patent No. 8,906,862, U.S. Patent No. 8,906,863, and WO 2015 / 179321.

[0079] III. Overview HEVNPs, nanocapsules derived from HEV that lack viral infectivity, retain essential characteristics of HEV, including gastrointestinal stability, target cell binding, and cell entry. Combined with their in vitro degradation / reassembly capabilities, HEVNPs have been proposed as attractive nanocapsules for oral delivery via ingestion. Encapsulation by HEVNPs relies on electrostatic interactions between the payload and capsid proteins, allowing negatively charged nucleic acids and nanosized proteins / small molecules to be packaged for therapeutic applications. In addition to encapsulating insulin for oral delivery to the liver via the gastrointestinal tract, insulin genes can also be encapsulated. If necessary, targeting of pancreatic β cells and / or the liver can be achieved by inserting specific cell-targeting ligands into the protruding domain of HEVNPs via overnight chemical conjugation or time-consuming but cost-effective genetic engineering. Thus, HEVNPs have the potential to serve as cell-targeting gene delivery vehicles capable of delivering insulin genes to the pancreas and transiently expressing insulin in situ. Insulin-loaded HEVNPs are expected to deliver insulin from the gastrointestinal tract to the liver via oral administration, which is the preferred route of drug administration.

[0080] In a multimodality treatment plan, diabetic patients are treated with two or more diabetes therapies to improve blood glucose control. Multiple therapies for diabetes treatment can be provided by HEVNPs by exchanging the payload between insulin in the form of insulin / proinsulin polypeptide and insulin in the form of insulin / proinsulin cDNA to achieve different in vivo kinetics of the delivered insulin. Other levels of therapy are provided by binding of different tissue / cell targeting ligands on the protruding domains of HEVNPs. By orally delivering HEVNPs containing insulin and / or insulin / proinsulin cDNA encapsulated in HEVNPs, these diverse therapeutic combinations can provide an alternative to needle injections for diabetes treatment.

[0081] IV. Materials and Methods 1. HEVNP encapsulation of insulin 1.1. Decomposition of HEVNP 1.1.1. Decompose HEVNP in 20 mM DTT, 10 mM EDTA at 4°C overnight. 1.1.2. Dialyze the degraded HEVNP against 50 mM Tris (pH 7.5), 150 mM NaCl at room temperature for at least 1 hour. 1.1.3. Examination by TEM, protein concentration determination by spectrometry.

[0082] 1.2. Encapsulation of insulin into HEVNPs 1.2.1. Mix the degraded HEVNP with insulin in 50 mM Tris (pH 7.5), 150 mM CaCl2, and add CaCl2 to a final concentration of 2-5 mM. Incubate overnight at 4°C. 1.2.2. Pass through a size exclusion column to remove free insulin. 1.2.3. Collect fractions and measure protein concentration by spectrophotometry. 1.2.4. Examine insulin-encapsulated HEVNPs by TEM.

[0083] 2. Characterization of HEVNPs 2.1. Using a spectrophotometer, record the absorbance at 280 nm and the ratio of absorbance at 260 nm to absorbance at 280 nm. The molar extinction coefficient of HEVNP ORF2 is 60280, which corresponds to 1.019 times the absorbance value of the protein at 280 nm. Since this is close to 1:1, the concentration of HEVNP can be expressed by measuring the protein concentration of absorbance at 280 nm using a spectrophotometer. Considering that the molecular weight of ORF2, a component of HEVNP, is 53.318 kDa, the following is obtained:

number

[0084] 2.2. Instruction Manual 14 Prepare a 1.0 mm, 17-well 4-12% SDS-PAGE Bis-Tris protein gel according to the protocol. 2.2.1. Add 2 μL of 4x loading buffer to 6 μL of protein sample. Incubate the sample mixture in a heat block at 100°C for 10 minutes to denature the protein. Load the protein sample onto a NuPAGE gel setup. 2.2.2. Run the SDS-PAGE with the DC power supply set at 100V for 10 minutes, then 150V for 45 minutes until the sample reaches approximately 1 cm above the bottom of the gel. 2.2.3. Stain the SDS-PAGE gel with Coomassie Blue (0.25% (w / v) Coomassie Brilliant Blue R250, 30% (v / v) methanol, 10% (v / v) acetic acid) for 1 hour. 2.2.4. After the staining procedure, remove the Coomassie blue stain and apply destaining buffer (30% (v / v) methanol, 10% (v / v) acetic acid) to the protein gel for at least 12 hours at room temperature. 2.2.5. Record the gel under white light to confirm the presence of HEVNP ORF2 in the 52 kDa band.

[0085] 2.3. Observation of HEVNPs using TEM 2.3.1. Prepare or dilute HEVNP samples to 0.5–2 mg / mL with 10 mM MES (pH 6.2) for imaging with TEM. 2.3.2. Ionize the carbon-coated grid with a 40 mA glow discharge for 30 seconds to create a hydrophilic carbon surface. The glow discharge device may be an EMS glow discharger. The hydrophilic carbon surface of the grid lasts for only 30 minutes after the glow discharge treatment. 2.3.3. Hold the grid with tweezers and add 2 μL of HEVNP sample to it, wait 15-30 seconds, and wipe it off with filter paper. 2.3.4. Immediately wash the grid with deionized distilled water and wipe with filter paper. Immediately add 2 μL of 2% uranyl acetate to the grid, wait 15 seconds, then wipe with filter paper. Dry the sample grid overnight by placing it in a microwave drying cabinet. 2.3.6. Transfer the grid to a transmission electron microscope (TEM) and image at magnifications of 10K to 80K. Due to the absence of viral RNA, HEVNP appears in the TEM as an empty icosahedron of protein approximately 27 nm in diameter.

[0086] 3. Chemical conjugation of HEVNP with biotin, tissue / cell targeting ligands, and fluorophores 3.1. One-step conjugation of HEVNP with maleimide-conjugated biotin 3.1.1 Buffer exchange: Place the HEVNP in a mini dialysis unit and dialyze it against 0.01 M PBS (pH 7.4) at room temperature for 1 hour according to the manufacturer's instructions (Zeba Spin Desalting Column, 40K MWCO, 0.5 mL). Transfer the HEVNP to a 1.5 mL tube and measure the protein concentration at 280 nm using a spectrophotometer. 3.1.2. HEVNP at 1 mg / mL, corresponding to 18.8 μM cysteine ​​reactive sites (see step 2.2.4 for details), is mixed with an equal volume of maleimide-biotin (100 μM) in 0.01 M PBS (pH 7.4) at a molar ratio of 1:5 and reacted overnight at 4°C. Unbound maleimide-biotin is removed using a 40K MWCO Spin Desalting Column (Zeba Spin Desalting Column, 40K MWCO, 0.5 mL) according to the manufacturer's protocol. 3.1.3. Analyze samples using standard reducing SDS-PAGE (step 3.1). 3.1.4 Prepare a Western blot using HRP-conjugated streptavidin chemiluminescence. Capture the chemiluminescent signal on X-ray film (Figure 2).

[0087] 3.2. Two-step conjugation of tissue-targeting ligands (RGD peptides) to exposed cysteines on the HEVNP surface 3.2.1 Buffer exchange: HEVNP was applied to a mini dialysis unit and dialyzed against 0.01 M PBS (pH 7.4) at room temperature for 1 hour. HEVNP was transferred to a 1.5 mL tube and the protein concentration was measured at 280 nm using a spectrophotometer. 3.2.2. Add 650 μM maleimide azide and 650 μM alkyned Ligand X to 0.01 M PBS (pH 7.4) containing 200 μM CuSO and 1 mM ascorbic acid to form 650 μM maleimide-linked Ligand X (Mal-LigandX). Incubate the mixture overnight at 4°C. 3.2.3. HEVNP at 1 mg / mL, corresponding to 18.8 μM cysteine ​​reactive sites (see step 2.2.4 for details), is mixed with Mal-LigandX (650 μM) in approximately 10% volume of 0.01 M PBS (pH 7.4) at a molar ratio of 1:3 and reacted overnight at 4 °C. Due to the relatively high concentration of maleimide-conjugated LXY30, the final concentration of reactants such as CuSO4 is reduced by approximately 10-fold after mixing to avoid damage to HEVNP. Alternatively, copper-free conjugation methods are available. 15 . 3.2.4. Remove unbound maleimide click ligand X using a 40K MWCO Spin desalting column according to the manufacturer's procedure (Table of Materials). Keep the LXY30-conjugated HEVNP (LXY30-HEVNP) at 4 °C.

[0088] 3.3. One-step conjugation of LXY30-conjugated HEVNP (LigandX-HEVNP) with Cy5.5 NHS ester (NHS-Cy5.5) 3.3.1. 1 mg / mL of Ligand X-linked HEVNP (LigandX-VLP), corresponding to 18.8 μM of cysteine ​​reactive sites (see step 2.2.4 for details), is mixed with an equal volume of Cy5.5 NHS ester (NHS-Cy5.5 100 μM) in 0.01 M PBS (pH 7.4) at a molar ratio of 1:5, and the mixture is incubated overnight at 4°C. 3.3.2. Remove unbound Cy5.5-NHS using a 40K MWCO Spin Desalting Column (Zeba Spin Desalting Column, 40K MWCO, 0.5 mL) according to the manufacturer's instructions. Keep the RGD-Cy5.5-conjugated HEVNP (RGD-HEVNP-Cy5.5) at 4°C.

[0089] Example 2: In vivo studies I. HEVNP Encapsulation Design In formulations, HEVNP can be formulated as tablets, capsules, sprinkle powder, or liquid for inclusion in beverages. Subcomponents of HEVNP have been proven to be safe vaccines for humans and animals. In contrast to other proposed enhancers of oral insulin administration, HEVNP capsules can be used as a mucosally focused delivery system with enhanced bioavailability for protein payloads such as insulin via the oral route. Quaternary structure-based payloads are designed to take advantage of macromolecular properties to extend usable retention time.

[0090] To optimize insulin encapsulation efficiency, multiple evaluations were performed to determine the optimal conditions. As shown in Figure 4, insulin encapsulation in HEVNPs exhibited the highest stability and structural uniformity during and after encapsulation in Tris buffer. The optimal encapsulation conditions were narrowed down to 10–50 mM Tris and 0–150 mM NaCl in the neutral pH range. While PBS buffer produced a high degree of precipitation, MES buffer provided the least favorable conditions for encapsulation of the payload. The highest encapsulation yield was further identified as being in Tris buffer, as Tris buffer provided HEVNPs with stable and monodisperse protein payloads in solution.

[0091] For encapsulation, HEVNP subunits are incubated with a corresponding molar ratio of a protein payload, such as insulin, to gradually assemble the capsules with CaCl2 added to the system. The efficiency of insulin encapsulation was measured and evaluated as follows.

[0092] 1. Cesium chloride density gradient separation; coexistence of HEVNP and insulin is demonstrated by ELISA (for HEV and insulin) (Figure 5) 2. Size exclusion column separation; coexistence of HEVNP and insulin is demonstrated by ELISA (for HEV and insulin) (Figure 6).

[0093] II. HEVNP Encapsulation Using Density Assessment Upon buffer optimization, the cesium chloride gradient clearly showed the coexistence of insulin and HEVNPs within a single peak in the ELISA readings, which explained the efficiency of insulin encapsulation in HEVNPs. "+" indicates a positive reading from the ELISA and the coexistence of HEV and insulin in fractions 6–13.

[0094] Size assessment identifies a novel structure of HEVNPs with insulin cargo.

[0095] As shown by ELISA, SEC shows distinct overlapping peaks for insulin and HEVNP (indicated by + signs in fractions #16 to #32).

[0096] Further evidence for identifying the coexistence of insulin and HEVNP capsules, as shown in the first peak (red peak), was verified by ELISA evaluation according to the specificity of anti-insulin and anti-HEVNP antibodies, respectively. To further identify the new morphology of HEVNP (lower panel of Figure 5), i.e., the optimization of ultrasound-mediated loading into a single peak (excluding the fraction of outliers over 35), further encapsulation was systematically measured and shown as a unified peak with both insulin and HEV (verified by ELISA, absorbance reading at 492 nm).

[0097] III. Prolonged storage period of HEVNP For an effective drug delivery system, high product stability and shelf life are crucial. HEVNP-insulin samples were stored at 4°C for over a year and observed by cryo-electron microscopy. Micrographs show intact particles, indicating high stability under storage conditions. Cryo-electron microscopy was used to observe HEVNP particles with encapsulated insulin detemir, as shown in Figure 8.

[0098] IV. Structural characterization of HEVNP-insulin Electron microscopy has yielded results demonstrating insulin encapsulation. However, the two-dimensional distribution and three-dimensional structural features of these nanoparticles have not yet been fully characterized. Using a combination of in-house procedures and commercially available image processing packages, large datasets have been collected and analyzed to 1) statistically analyze particle distributions and 2) determine the high-resolution 3D structure of insulin-loaded HEVNPs.

[0099] Evaluation of TEM images reveals a novel conformation of HEVNP-insulin fabricated with a diameter of approximately 45 nm, approximately two-fold larger than the diameter of our previously filed first-generation HEVNPs (27 nm). Within these HEVNPs, the novel shape and size appear optimal for carrying insulin payloads with protruding strands of hexameric nodes that are easily visualized. To achieve structure-based optimization of insulin packaging efficiency, further 3D volumetric characterization was performed using cryo-electron microscopy. This new generation HEVNP conformation was realized through computational modeling to perfect preloaded packaging. Electron 3D tomography was performed using sequential tilt data collected to reconstruct a 3D representation of HEVNP-insulin, along with digital segmentation. The packaging system was analyzed using a 200 kV electron microscope (JEOL 2100F) from -60° to +60° in 1° increments. In Figure 7, 3D reconstruction was performed using a simultaneous iterative reconstruction method, clearly demonstrating the segmented strands of insulin protruding from the HEVNPs.

[0100] V. HEVNP Encapsulation Validated in Large and Small Animal Models Mice were randomly assigned to one of two treatment groups and subjected to insulin tolerance testing as follows. A. Oral administration of 0.1 U of insulin (encapsulated in HEVNP) per mouse. B. Oral administration of 1 U of insulin (encapsulated in HEVNP) per mouse.

[0101] Assuming an average 25% reduction in blood glucose concentrations after oral insulin administration and a 50% reduction in blood glucose concentrations after intraperitoneal insulin administration, a standard deviation of 15%, a desired alpha error of 5%, and a power of 80% were used to subgroup 10% of the mice to detect significant differences between groups.

[0102] Oral delivery is performed by force using light isoflurane anesthesia and a flexible gavage needle. A 26-gauge needle is used for intraperitoneal injection. Insulin and / or HEVNP are dissolved in 0.9% saline. When oral insulin formulations are absorbed through the mucous membranes, the expected decrease in blood glucose levels is achieved.

[0103] In addition, 8 to 10 diabetic disease model dogs were tested as "patients" for glucose monitoring measurements.

[0104] VI. Whole Animal Imaging to Track Encapsulated Payloads In vivo optical imaging of mice using cyanine 5.5 (Cy5.5)-labeled HEVNPs was previously demonstrated in Chen et al., "Chemically activatable viral capsid functionalized for cancer targeting," Nanomedicine 11, no. 4 (2016): 377-390, in which a breast cancer targeting molecule (LXY30) was conjugated to Cy5.5 linked to engineered cysteine ​​arms and exposed lysine residues. Whole-animal imaging demonstrated that LXY30-bearing HEVNPs accumulated at tumor sites. Here, the surface of insulin-encapsulated HEVNPs was modified with Cy5.5 NHS ester (Lumiprobe) at a molar ratio of 300:1 (Cy5.5:HEVNP) in 0.01 M PBS (pH 7.2) buffer for 2 hours at room temperature, followed by overnight incubation at 4°C. Next, free Cy5.5 NHS ester is removed by a 7000 MWCO desalting column (Zeba Spin Desalting Column, Thermo Scientific). Cy5.5 has an excitation maximum at 682 nm, an emission maximum at 702 nm, and a peak at 250,000 cm. -1 M -1 It has a molar extinction coefficient of

[0105] Next, to track the distribution of HEVNP-insulin, whole-animal imaging was performed using IVIS Spectrum for optical imaging (resolution of approximately 20 μm to 5 mm) and MicroXCT-200 for high-resolution CT (resolution of approximately 1 to 20 μm). Oral insulin delivery involves passage through the stomach, the mucosal lining of the gastrointestinal tract, and the hepatic portal vein to the liver. Therefore, nanoparticles accumulate in the liver, where they release insulin.

[0106] VII. Molecular Features Illustrated by Electron Microscopy To study HEVNP distribution at the cellular level, liver biopsies were performed using high-pressure freezing and cryofixation to embed the tissue. The extracted tissue was weakly fixed in formaldehyde and then placed in a specimen holder. The frozen tissue was then fixed in a resin block, sectioned using an ultramicrotome, and examined using transmission electron microscopy (TEM). HEVNP was identified by adding contrast with either gold atomic clusters or 10 nm ferrite oxide particles. Electron-dense HEVNP particles provided sufficient contrast for identification by TEM.

[0107] High-pressure freezing and TEM preparation allow for high-resolution 3D imaging of cellular ultrastructure using a JEM2100F electron microscope, as described, for example, in Paavolainen et al., "Compensation of missing wedge effects with sequential statistical reconstruction in electron tomography," PloS one 9, no. 10 (2014): e108978; Soonsawad et al., "Permeability changes of integrin-containing multivesicular structures triggered by picornavirus entry," PloS one 9, no. 10 (2014): e108948; and Soonsawad et al., "Structural evidence of glycoprotein assembly in cellular membrane compartments prior to alphavirus budding," Journal of Virology 84, no. 21 (2010): 11145-11151.

[0108] All patents, patent applications, and other publications, including GenBank accession numbers, cited in this application are incorporated by reference in their entirety for all purposes.

[0109] reference 1.Saaddine JB,Cadwell B,Gregg EW,Et Al.Improvements in diabetes processes of care and intermediate outcomes:United states,1988-2002.Annals of Internal Medicine 144(7),465-474(2006). 2.Hoffman A,Ziv E.Pharmacokinetic considerations of new insulin formulations and routes of administration.Clinical pharmacokinetics 33(4),285-301(1997). 3.Owens DR.New horizons--alternative routes for insulin therapy.Nature reviews.Drug discovery 1(7),529-540(2002). 4.Arbit E,Kidron M.Oral Insulin Delivery in a Physiologic Context:Review.J Diabetes Sci Technol 11(4),825-832(2017). 5.Carino GP,Mathiowitz E.Oral insulin delivery1Abbreviations:GI,gastrointestinal;IDDM,insulin-dependent diabetes mellitus;IU,international units;NIDDM,non-insulin-dependent diabetes mellitus;PIN,phase inversion nanoencapsulation;ZOT,zona occludens toxin.1.Advanced drug delivery reviews 35(2),249-257(1999). 6.Heinemann L.New ways of insulin delivery. International journal of clinical practice.Supplement doi:10.1111 / j.1742-1241.2010.02577.x(170),31-46(2011). 7.Fonte P,Araujo F,Reis S,Sarmento B.Oral insulin delivery:how far are we? J Diabetes Sci Technol 7(2),520-531(2013). 8.Zijlstra E,Heinemann L,Plum-Morschel L.Oral insulin reloaded:a structured approach. J Diabetes Sci Technol 8(3),458-465(2014). 9.Zaykov AN,Mayer JP,Dimarchi RD.Pursuit of a perfect insulin.Nature reviews.Drug discovery 15(6),425-439(2016). 10.Wong CY,Martinez J,Dass CR.Oral delivery of insulin for treatment of diabetes:status quo,challenges and opportunities. The Journal of pharmacy and pharmacology 68(9),1093-1108(2016). 11.Samson SL,Chan L.Gene therapy for diabetes:reinventing the islet.Trends Endocrinol Metab 17(3),92-100(2006). 12.Alam T,Wai P,Held D,Vakili ST,Forsberg E,Sollinger H.Correction of Diabetic Hyperglycemia and Amelioration of Metabolic Anomalies by Minicircle DNA Mediated Glucose-Dependent Hepatic Insulin Production.PloS one 8(6),e67515(2013). 13.Jariyapong P,Xing L,Van Houten NE et al.Chimeric hepatitis E virus-like particle as a carrier for oral-delivery.Vaccine 31(2),417-424(2013). 14.Chen CC,Xing L,Stark M et al.Chemically activatable viral capsid functionalized for cancer targeting.Nanomedicine(Lond)11(4),377-390(2016). 15.Cheng RH,Xing L,Chen CC,Stark MC:WO / 2015 / 179321(2015). 16.Takamura S,Niikura M,Li TC et al.DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.Gene therapy 11(7),628-635(2004). 17.Cheng RH,Xing L:US8906863(2014). 18. Stark MC, Baikoghli MA, Lahtinen T et al. Structural characterization of site-modified nanocapsid with monodispersed gold clusters. Scientific reports 7(1), 17048(2017). 19. Li W, Chen X. Gold nanoparticles for photoacoustic imaging. Nanomedicine 10(2), 299 - 320(2015).

[0110] [Table 1]

[0111] [Table 2]

[0112] [Table 3]

[0113] [Table 4] Aspects of the present invention include the following. ​​​​​​​​​​​​​​​​​​​​​​​​ <6> the cysteine ​​or lysine is alkylated, acylated, arylated, succinylated, oxidized, or conjugated with a detectable label or a hepatocyte-targeting ligand; <5> The composition described in <7> the detectable label comprises a fluorescent dye molecule, a superparamagnetic label, an MRI contrast agent, a positron-emitting isotope, or a cluster of an element from Groups 3 to 18 with an atomic number greater than 20; <6> The composition described in <8> the detectable label comprises a gold nanocluster; <7> The composition described in <9> the hepatocyte targeting ligand is an RGD peptide or a cyclic RGD peptide; <6> The composition described in <10> Further comprising a pharmaceutically acceptable non-medicinal excipient, <9> The composition described in <11> Formulated for oral administration, <9> The composition described in <12> Hepatocytes <1> ~ <11> A method for targeted delivery of insulin, comprising contacting a subject with a composition described in any one of the above. <13> The hepatocytes are present in a patient, and the contacting step <1> administering to the patient a composition according to claim 1, <12> The method described below. <14> The administration is oral administration. <12> The method described below. <15> the modified capsid protein comprises a cysteine ​​or lysine bound to a gold nanocluster; <13> The method described below. <16> The patient is a patient diagnosed with diabetes. <13> The method described below.

Claims

1. (a) a hepatitis E virus (HEV) virus-like particle (VLP) formed from a modified capsid protein comprising at least a segment of bases 452-606 of the hepatitis E virus (HEV) open reading frame 2 (ORF2) protein of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and a heterologous polypeptide sequence inserted into said portion of the HEV ORF2 protein within a segment of bases 483-490, 530-535, 554-561, 573-577, 582-593, or 601-603 of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and (b) a bioactive agent encapsulated within the HEV VLP. Including, the heterologous polypeptide sequence is an RGD peptide or a cyclic RGD peptide, and the bioactive agent is an insulin protein or a nucleic acid encoding an insulin protein; composition.

2. 2. The composition of claim 1, wherein the heterologous polypeptide sequence is inserted immediately after residue Y485 of SEQ ID NO: 1, 2, 3, 4, 5, or 6.

3. 2. The composition of claim 1, wherein the HEV VLP is an acid- and proteolytically stable HEV VLP, and the modified capsid protein has at least one of residues Y485, T489, S533, N573, and T586 of SEQ ID NO: 1, 2, 3, 4, 5, or 6 substituted with a cysteine ​​or lysine that may be chemically derivatized.

4. The cysteine ​​or lysine is alkylated, acylated, arylated, succinylated, oxidized, or detectably labeled. or the composition of claim 3, which is conjugated to a hepatocyte targeting ligand.

5. 5. The composition of claim 4, wherein the detectable label comprises a fluorescent dye molecule, a superparamagnetic label, an MRI contrast agent, a positron-emitting isotope, or a cluster of an element from Groups 3-18 with an atomic number greater than 20.

6. The composition of claim 5 , wherein the detectable label comprises a gold nanocluster.

7. The composition of claim 4 , wherein the hepatocyte targeting ligand is an RGD peptide or a cyclic RGD peptide.

8. 8. The composition of claim 7, further comprising a pharmaceutically acceptable non-medicinal excipient.

9. 9. The composition of claim 7 or claim 8, formulated for oral administration.

10. A composition according to any one of claims 1 to 9 for use in targeted delivery of insulin to liver cells.

11. The composition of claim 10 , wherein the hepatocytes are in a patient and the targeted delivery of insulin comprises administering the composition to the patient.

12. The composition of claim 11 , wherein the administration is oral administration.

13. 12. The composition of claim 11, wherein the modified capsid protein comprises a cysteine ​​or lysine bound to a gold nanocluster.

14. The composition of claim 11 , wherein the patient is a patient diagnosed with diabetes.

Images