Gastrointestinal stable protein delivery using disulfide bond TES system

Abstract

The present invention relates to an engineered disulfide bond linked ferritin assembly comprising at least one modified ferritin subunit wherein the at least one modified ferritin subunit comprises the amino acid sequence as set forth in SEQ ID NO: 1 and comprises; i) a F116H substitution, and zero or more amino acid substitutions at one or more positions selected from the group comprising E65, E128, E131 and D138 of SEQ ID NO: 1, and ii) a Cys substitution at two or more positions selected from the group comprising G37, L53, R66, G67, A74, A117 and A152 of SEQ ID NO: 1. The invention also relates to the use and preparation thereof.

Description

Technical Field

[0001] The present invention relates to thermostable ferritin assemblies, in particular engineered disulfide-linked ferritin assemblies, which have enhanced stability to acidic pH and human digestive enzymes, thereby also stabilizing the encapsulated payload, and having increased intestinal permeability compared to reference ferritin assemblies without added disulfide linkages. The present invention also relates to in vitro methods for forming engineered disulfide-linked ferritin assemblies, methods for delivering engineered disulfide-linked ferritin assemblies into cells; and kits for encapsulating molecules within the engineered disulfide-linked ferritin assemblies. Background Art

[0002] Oral administration is the most preferred and convenient route of all drug administration routes and is the main mode of drug administration for commonly used drugs. Oral delivery provides the best patient compliance because the drug is self-administered in flexible doses according to the patient's needs [Sadeghi, S., et al., ACS nano (2022); Majoul, I., et al., FEBS Letters 401: 104-108 (1997); Okumura, M., et al., The FEBS Journal 279: 2283-2295 (2012)]. In addition, the gastrointestinal (GI) tract mucosa and epithelial cells have a 30–40 m 2 The large surface area of ​​the GI tract provides an opportunity for high-quality transfer and absorption. Goblet cells, endocrine cells, Paneth cells, and specialized M cells in the GI tract can also play a specific role in drug absorption, providing unique properties for oral drug development [Deshpande, S., et al., Nature communications 8: 1-8 (2017); Zhang, M., et al., International Journal of Biological Macromolecules 138: 636-647 (2019)]. Despite the existence of standards for small molecule drug delivery routes, due to the high acidity and unfavorable physicochemical properties of the GI tract, biocompatible polymers and lipids generally require delicate superstructures, so the field of nanomedicine has not yet found a material basis for sustained oral drug delivery. Ultimately, nanoparticle delivery technology still has poor bioavailability, and alternative materials that are stable throughout the GI tract are needed [Sadeghi, S., et al., ACS nano (2022); Martien, R., et al., Nanotechnology 19:045101 (2008); Wu, W., et al., Food Hydrocolloids 80:78-87 (2018)].

[0003] Proteins and peptides are widely used in clinical care, but only <5% are administered orally, primarily due to instability with pH changes and degradative enzymes in the stomach and proximal small intestine [Ezpeleta, I., et al., International Journal of Pharmaceutics 191:25-32 (1999)]. Nanoparticle systems have improved oral delivery of small molecule drugs, but progress with functionally active proteins or enzymes has been relatively limited, as the organic emulsions used to synthesize these systems are incompatible with the tertiary structure of the proteins required for activity. Thus, the combination of denaturation and low encapsulation efficiency has led to ongoing challenges [Bies, C., et al., Advanced drug delivery reviews 56:425-435 (2004); Minko, T. Advanced drug delivery reviews 56:491-509 (2004); Penalva, R., et al., Food hydrocolloids 44:399-406 (2015)]. Highly stable nanoparticles are a promising approach to achieve biocompatibility, biodegradability, non-immunogenicity, resistance to enzymatic degradation, and amenability to surface engineering genetic modification [Shapira, A., et al., Nanomedicine 6:119-126 (2010); Penalva, R., et al., Journal of Agricultural and Food Chemistry 63:5603-5611 (2015); Umamaheshwari, R., et al., Aaps Pharmscitech 5:60-68 (2004)]. One such approach is the self-assembling and monodisperse protein cage iron protein, engineered to enhance circulation half-life and reduce toxicity [Umamaheshwari, R., et al., Aaps Pharmscitech 5:60-68 (2004); Irache, J Mand Gonzalez-Navarro, CJ Future Medicine 12:1209-1211 (2017); Patel, AR., et al., Journal of Agricultural and Food Chemistry 58:12497-12503 (2010)].However, the current gastric pH causes these protein cages to disintegrate, exposing the therapeutic protein cargo and leading to loss of activity and degradation by digestive enzymes [Sadeghi, S., et al., ACS nano (2022); Hong, S., et al., Pharmaceutics 12: 604 (2020); Kianfar, E. Journal of Nanobiotechnology 19: 1-32 (2021); Vallerinteavide Mavelli, G. et al., Pharmaceutics 13: 1790 (2021)]. In addition, these cages must pass through the thick mucus layer on the GI tract to reach the lumen, limiting their application as oral delivery carriers [Wu, W., et al., Food Hydrocolloids 80: 78-87 (2018); Estrada, L Hand Champion, J., Biomaterials Science 3: 787-799 (2015)].

[0004] There is a need to create an oral delivery vehicle with improved stability in the gastrointestinal tract. Summary of the invention

[0005] Thermostable shell (tES) is an engineered protein nanoparticle for rapid encapsulation of therapeutic proteins / enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. In view of the importance of oral delivery as a preferred mode of drug administration, the inventors structurally improved the stability of tES through multiple inter-subunit disulfide bonds that were not originally present in the parent molecule. Compared with tES, disulfide-linked tES significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and the activity against major human digestive enzymes, pepsin and trypsin. In addition, using Caco2 cell assessment, disulfide-linked tES (DS-tES) showed significant intestinal permeability. In vivo bioluminescence assays showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to free enzymes. After 24h of oral administration, the feces of mice treated with DS-tES had ~100 times more active enzymes compared to the control (free enzyme), indicating strong intestinal stability. Taken together, the in vitro and in vivo results confirm the potential of DS-tES for intraluminal and systemic oral drug delivery applications.

[0006] The inventors developed a new protein cage "tES" by engineering the heat-stable Archaeoglobus fulgidus ferritin, which has a diameter of 12nm, an 8nm aqueous cavity, and a capacity that can theoretically accommodate a molecular weight of ~270nm. 3 One or more proteins. The shell contains four 4.5 nm surface pores that allow small molecule substrates to penetrate while blocking the interaction of macromolecules such as antibodies and proteases [He, J., et al., Journal of Controlled Release 311: 288-300 (2019)]. The advantages of tES are: (1) simple and rapid encapsulation of protein cargo using charge complementation and mild pH titration, (2) prevention of aggregation, helping to form the correct tertiary structure and maintain protein activity, and (3) providing a highly stable protective environment for internalized proteins, resisting denaturation and proteolysis, making it an ideal carrier for enzyme delivery. In addition, tES has been shown to function as an artificial chaperone to fold proteins in vitro [Khoshnejad, M., et al., Journal of Controlled Release 282:13-24 (2018)], stabilize labile proteins from freezing and drying stress during lyophilization [Sadeghi, S., et al., Nature Communications 12:1-15 (2021)], and serve as a multilayer catalytic center for bioorthogonal catalysis to treat solid tumors [Yin, S. et al., Particuology 64:65-84 (2022)].

[0007] Here, the inventors describe the structural refinement of tES by integrating multiple inter-subunit disulfide bonds to create disulfide-linked tES with practical advantages in oral delivery applications. By using molecularly precise disulfide bonds (i.e., DS-tES) engineered natural covalent cross-links, the inventors were able to protect the encapsulated enzyme from the adverse GI environment when compared to tES and / or enzymes alone. In addition, in an in vitro Caco2 assay, DS-tES showed permeability through intestinal epithelial cells, confirming enhanced absorption. Finally, the rluc encapsulated in DS-tES showed significant protein stability in all parts of the GI tract via the presence of bioluminescence in feces after oral administration in mice.

[0008] What follows is a description of exemplary, non-limiting embodiments of the invention.

[0009] In a first aspect of the present invention, there is provided an engineered disulfide-linked ferritin assembly comprising at least one modified ferritin subunit, wherein the at least one modified ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 1, and comprises;

[0010] i) F116H substitution, and zero or more amino acid substitutions at one or more positions selected from the group consisting of E65, E128, E131 and D138 of SEQ ID NO: 1, and

[0011] ii) Cys substitution at two or more positions selected from the group consisting of G37, L53, R66, G67, A74, A117 and A152 of SEQ ID NO: 1.

[0012] Cys substitution forms inter-subunit disulfide bonds, which has the following advantages:

[0013] 1. Covalently locked shell

[0014] 2. Disulfide bridges do not affect the overall morphology of tES

[0015] 3. Stability under extremely acidic conditions (pH 4.0–pH 5.0)

[0016] 4. Stability against proteolytic enzymes (e.g. pepsin, trypsin)

[0017] The free thiols on the surface of tES have mucosal adhesion and intestinal permeability.

[0018] The disulfide-linked ferritin assembly of the present invention can encapsulate and stabilize different molecules, proteins and enzymes, thereby providing a wide range of potential applications for drug delivery. The potential application of this disulfide-linked ferritin assembly is as a gastrointestinal stable oral delivery carrier because it can protect the encapsulated molecules from adverse gastrointestinal conditions (including pepsin degradation and low pH conditions).

[0019] In some embodiments, the disulfide-linked ferritin assembly has a net positive internal charge or a net neutral internal charge.

[0020] In certain embodiments, the engineered ferritin assembly encapsulating the polypeptide can be delivered to cells (e.g., in vivo delivery) as a composition or formulation comprising the engineered ferritin assembly and one or more pharmaceutically acceptable carriers or excipients. Suitable pharmaceutical carriers will typically contain inert ingredients that do not interact with the agent or nucleic acid. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, normal saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's lactate, and the like. The formulation may also include a small amount of substances (e.g., emulsifiers, solubilizers, pH buffers, wetting agents) that enhance the effectiveness of the active ingredient. Methods of encapsulating the composition (such as in a hard gelatin or cyclodextrin coating) are known in the art.

[0021] In some embodiments, the modified ferritin subunit comprises an amino acid substitution at any one or more positions selected from E65, E128, E131, and D138 corresponding to the positions in the peptide sequence shown in SEQ ID NO:1.

[0022] In some embodiments, the amino acid substitutions include:

[0023] i) substitutions of E65K, E128K, E131K and D138A, which result in the assembly having a net positive internal charge; or

[0024] ii) Substitutions E65Q and D138A, which result in an assembly with a net neutral internal charge.

[0025] In some embodiments, prior to the Cys substitution, the modified ferritin subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0026] In some embodiments, the modified ferritin subunit includes a cysteine ​​residue at a position selected from the group consisting of A152C-G67C-A117C-G37C and A152C-L53C-G67C-A117C-G37C corresponding to a position in the peptide sequence set forth in SEQ ID NO: 1.

[0027] In some embodiments, the modified ferritin subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0028] In some embodiments, the bioorthogonal catalytic center, polypeptide or nucleic acid, or any combination thereof, is encapsulated within a disulfide-linked ferritin assembly.

[0029] In a second aspect, the present invention provides an in vitro method of forming an engineered disulfide-linked ferritin assembly of the first aspect, the method comprising adjusting the pH of a sample comprising at least one modified ferritin subunit of the first aspect from an acidic pH to an alkaline pH, wherein the assembly is stable in the gastrointestinal tract and capable of oral drug delivery.

[0030] In some embodiments, the method further comprises a bioorthogonal catalytic center, polypeptide or nucleic acid, or any combination thereof, in the sample, whereby the bioorthogonal catalytic center, polypeptide or nucleic acid, or any combination thereof, is encapsulated by the engineered ferritin assembly at an alkaline pH of the sample.

[0031] In some embodiments, the acidic pH in the sample is at least about 4.0 and / or the alkaline pH in the sample is about 8.0. In some embodiments, the acidic pH in the sample is at least 4.0 and / or the alkaline pH in the sample is 8.0.

[0032] In a third aspect, the present invention provides a method of delivering an engineered disulfide-linked ferritin assembly into a cell, comprising contacting the cell with the disulfide-linked ferritin assembly of the first aspect.

[0033] In some embodiments, the disulfide-linked ferritin assembly further comprises a cell targeting moiety, for example, on the surface of the engineered ferritin assembly. In certain embodiments, the cell targeting moiety binds to a molecule (e.g., a receptor or ligand) on a target cell (which may be a diseased cell). In some embodiments, the target cell is a cancer cell. Since the disulfide-linked ferritin assembly is very advantageous for oral therapeutic applications, surface modification thereof with these peptides may help to better target tumors in the gastrointestinal region. Examples of molecules to which the cell targeting moiety binds include, for example, EGFR and cell adhesion molecules (e.g., integrins and organ level selectivity, such as enhanced permeability and retention (EPR) effect).

[0034] Those skilled in the art will appreciate that a variety of molecules (e.g., peptides, receptors, ligands, and fragments thereof) can be used as targeting moieties to target and bind to molecules on diseased cells. In certain embodiments, the diseased cells are cancer cells, and the cell targeting moiety is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 (YHWYGYTPQNVI) or SEQ ID NO: 12 (LARLLT).

[0035] In some embodiments, the targeting moiety is cross-linked to the surface of the disulfide-linked ferritin assembly. Methods for cross-linking molecules (e.g., peptides) to other molecules are known in the art. For example, as described herein, a cross-linking agent (e.g., sulfo-SMCC) can be used to attach the targeting moiety to the surface of the engineered disulfide-linked ferritin assembly.

[0036] In some embodiments, the engineered ferritin assembly encapsulating the polypeptide can be delivered to cells (e.g., in vivo delivery) as a composition or formulation comprising the engineered ferritin assembly and one or more pharmaceutically acceptable carriers or excipients. Suitable pharmaceutical carriers will typically contain inert ingredients that do not interact with the agent or nucleic acid. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, normal saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's lactate, and the like. The formulation may also include a small amount of substances (e.g., emulsifiers, solubilizers, pH buffers, wetting agents) that enhance the effectiveness of the active ingredient. Methods of encapsulating the composition (such as in a hard gelatin or cyclodextrin coating) are known in the art.

[0037] In a fourth aspect, the present invention provides an isolated plasmid or vector polynucleic acid comprising a sequence encoding a modified ferritin subunit, wherein the modified ferritin subunit is as defined in the first aspect.

[0038] In a fifth aspect, the present invention provides a composition comprising at least one engineered disulfide-linked ferritin assembly for preventing or treating a disease in a subject.

[0039] In some embodiments, the at least one engineered disulfide-linked ferritin assembly has encapsulated:

[0040] i) enzymes; or

[0041] ii) an enzyme and one or more additional therapeutic agents.

[0042] In some embodiments, the composition is capable of delivering the drug via the oral route.

[0043] In a sixth aspect, the present invention provides a method of treatment or prevention, comprising administering an effective amount of the composition of the fifth aspect to a subject in need of such treatment or prevention.

[0044] In some embodiments, the composition is administered sequentially or simultaneously with another therapeutic agent.

[0045] When administered in combination therapy, the engineered ferritin assembly of the encapsulated polypeptide can be administered before, after, or in parallel with the other therapy. When co-administered simultaneously (e.g., in parallel), the engineered ferritin assembly of the encapsulated polypeptide and the other therapy can be separate preparations or the same preparation. Alternatively, the engineered ferritin assembly of the encapsulated polypeptide and the other therapy can be administered sequentially as separate compositions within an appropriate time range determined by a skilled clinician (e.g., a time sufficient to allow the drug effects of the therapy to overlap). In certain embodiments, the engineered ferritin assembly described herein is included in a composition or combination for treating a subject's disease. In certain embodiments, the disease is cancer.

[0046] In some embodiments, the composition comprises a bioorthogonal catalytic center encapsulated within an engineered disulfide-linked ferritin assembly, and the other therapeutic agent is a substrate for the catalytic center.

[0047] In a seventh aspect, the present invention provides use of the composition of the fifth aspect in the preparation of a medicament for treating a disease in a subject.

[0048] In some embodiments, the disease is cancer.

[0049] In an eighth aspect, the present invention provides a kit for encapsulating a bioorthogonal catalytic center, a polypeptide or a nucleic acid, or any combination thereof, within an engineered disulfide-linked ferritin assembly, the kit comprising at least one modified ferritin subunit according to the first aspect.

[0050] The present invention also provides a kit for encapsulating a bioorthogonal catalytic center, a polypeptide or a polynucleic acid or any combination thereof in an engineered disulfide-linked ferritin assembly, the kit comprising the plasmid or vector polynucleic acid of the fourth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 (a) Schematic diagram of sequence engineering of DS-tES is shown. Non-reducing SDS-PAGE of selected mutants, whereby lane 1 has mutant A152C; lane 2 has mutant A52C-L53C; lane 3 has mutant A152C-G67C; lane 4 has mutant A152C-L53C-G67C; lane 5 has mutant A152C-L53C-G67C-A117C-G37C (DS-tES). (b) Schematic diagram of DS-tES constructs with five point mutations G37C-L53C-G67C-A117C-A152C and restriction sites. (c) PyMOL diagram of DS-tES with all point mutations (black balls represent cysteine ​​residues).

[0052] Figure 2Shown are (ad) non-reducing SDS-PAGE photographs of designed cysteine ​​residue replacements. Lane 1tES, Lane 2tES R151C, Lane 3tES A152C, Lane 4tES A152C-L53C, Lane 5tES A152C-A74C, Lane 6tESA152C-G76C, Lane 7tES A152C-R66C, Lane 8tES A152C-L53C-R66C, Lane 9tES A152C-A74C-R66C, Lane 10tES A152C-G67C, Lane 11tES A152C-L53C-G67C, Lane 12tES A152C-A74C-G67C, Lane 13tES A152C-G67C-A117C-G37C, Lane 14tES A152C-L53C-G67C-A117C-G37C. M = size marker.

[0053] Figure 3 Shown are (a) SDS-PAGE analysis of DS-tES and tES in the absence or presence of tri(2-carboxyethyl)phosphine (TCEP), a reducing agent that disrupts disulfide bonds. (b) Size exclusion chromatography of tES and DS-tES with similar elution profiles. (c) Hydrodynamic diameter measurements of tES and DS-tES using dynamic light scattering studies.

[0054] Figure 4 Shown are (ag) non-reducing SDS-PAGE pictures to increase disulfide bond formation in constructs 9, 10 and 11.

[0055] Figure 5 (a) Size exclusion chromatography of tES, A152C-G67C-A117C-G37C mutant tES, and DS-tES at pH 4.0 is shown. (b) Pepsin assay under different pH conditions. DS-tES protects encapsulated HRP from simulated gastric conditions. (c) Trypsin assay, tES and DS-tES protect HRP from trypsin. (d) Permeability assay in Caco2 monolayer shows that both tES and DS-tES can penetrate the intestinal epithelium. (bd: Data are shown as mean ± SEM, n = 3. ***P < 0.001.)

[0056] Figure 6 The Ellman assay standard curve is shown.

[0057] Figure 7Shown are (a) Schematic representation of IP injection of viviRen substrate after oral administration of DS-tES-rluc / rluc. (b) Bioluminescence emitted by mice within 3 and 24 h of administration of DS-tES-rluc / rluc. (c) Luciferase activity measurement in mouse fecal material up to 1 week. (b and c) Data are shown as mean ± SEM, n = 4. ***P < 0.001, **P < 0.01, *P < 0.05. )

[0058] Figure 8 Permeability assays in Caco2 monolayers demonstrate that both tES and DS-tES can penetrate the intestinal epithelium. (Data are shown as mean ± SEM, n = 3. ***P < 0.001. and **P < 0.01)

[0059] Fig. 9 (a, b) show the bioluminescence emitted by mice within 3 hours of administration of tES-rluc / rluc. DETAILED DESCRIPTION

[0060] For convenience, bibliographic references mentioned in this specification are listed in the form of a reference list and added to the end of the examples. The entire contents of such bibliographic references are incorporated herein by reference. Any discussion of the prior art does not constitute an admission that the prior art is part of the common general knowledge in the field of the present invention.

[0061] definition

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. For convenience, certain terms employed in the specification, examples, and appended claims are collected herein.

[0063] As used herein, "a" or "an" may mean one or more than one, unless stated otherwise or the context dictates otherwise.

[0064] As used herein, "comprising" or "including" should be interpreted as specifying the presence of stated features, integers, steps or components, but does not exclude the presence or addition of one or more features, integers, steps or components or groups thereof. However, in the context of the present disclosure, the term "comprising" or "including" also includes "consisting of". Variations of the word "comprising", such as "comprise" and "comprises", and variations of "including", such as "include" and "includes", have correspondingly varied meanings.

[0065] As used herein, the term "disulfide bond" or "disulfide bridge" refers to the inter-subunit covalent disulfide bond formed between cysteine ​​residues. The cysteine ​​residue can be a modified residue located at the tES dimerization intersection, such as arginine 151 (R151), alanine 152 (A152), leucine 53 (L53), alanine 74 (A74), glycine 76 (G76), arginine 66 (R66), glycine 67 (G67), glutamine 50 (Q50), glycine 37 (G37) and alanine 117 (A117).

[0066] As used herein, "ferritin assembly" is used synonymously with ferritin, which is a structure known in the art that includes 24 subunits (ferritin subunits), each subunit having a defined size (e.g., ˜20 kDa for AfFtn) and a structural motif (e.g., a four-helix bundle structural motif for AfFtn). The native ferritin assembly has a defined outer diameter (e.g., 12 nm for AfFtn) and a defined internal cage (e.g., 8 nm for AfFtn).

[0067] As used herein, a ferritin subunit refers to one of the 24 subunits that form the ferritin assembly as described above. The subunits of the ferritin assembly have a defined structure, including a four-helix bundle motif. In AfFtn, each subunit is ˜20 kDa and contains a four-helix bundle structural motif. The AfFtn wild-type subunit has the amino acid sequence shown in SEQ ID NO: 1 (Table 1).

[0068] Table 1. Amino acid sequences of AfFtn subunits

[0069]

[0070]

[0071]

[0072] As used herein, a "modified ferritin subunit" refers to a ferritin subunit that lacks the unstructured carboxyl terminal sequence possessed by a wild-type ferritin subunit. That is, the modified ferritin subunit includes a deletion of amino acids 165-173 of SEQ ID NO: 1. Table 1 summarizes the sequences described herein. For example, the subunit sequence is truncated at position 164 of SEQ ID NO: 1 and includes a F116H substitution, as shown in SEQ ID NO: 2 (Table 1).

[0073] As used herein, "engineered ferritin assembly" refers to a ferritin assembly formed by at least one modified ferritin subunit, as described herein with reference to Deshpande, S., et al., Nat. Commun. 8, 1-8 (2017). Engineered ferritin assemblies are sometimes referred to as "nanoshells" or "nanocages" or "nanoencapsulations", "NEs" or "thermostable shells" or "tES". An example of a modified ferritin subunit comprising residues 1-164 is shown in SEQ ID NO: 2. As known to those skilled in the art, there may be residues in the modified ferritin subunit other than the last residue generated by, for example, a cloning site, which residues do not affect the function of the engineered ferritin assembly. For example, as shown in Table 1, the AfFtn subunit with a truncated unstructured C-terminus includes residues "TS" generated by a cloning site (restriction site) in a cloning plasmid. Removal of the unstructured carboxyl terminal sequence not only frees up space inside the engineered ferritin assembly, but also confers stability to the assembly at low salt concentrations (e.g., less than 30 mM NaCl).

[0074] The modified ferritin subunit may include one or more further modifications (e.g., amino acid substitutions) to its amino acid sequence. As described herein, at least one ferritin subunit may be modified to contribute to a net charge (e.g., positive, negative, or neutral) on the internal surface of the ferritin assembly. Methods for determining which residues to modify within a ferritin subunit to achieve a ferritin assembly having a net positive internal charge, a net negative internal charge, or a net neutral internal charge are known in the art, as described herein. An engineered ferritin assembly formed by truncating a natural AfFtn ferritin subunit has a net positive charge; examples of which are shown in SEQ ID NO: 3. An engineered ferritin assembly formed by truncating a natural AfFtn ferritin subunit has a net neutral charge; examples of which are shown in SEQ ID NO: 4. Mutants in which residues are substituted with cysteine ​​at different positions are shown as SEQ ID NOs: 5 to 10.

[0075] As used herein, "protein" and "polypeptide" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). The term "protein" encompasses full-length proteins, both naturally occurring and artificial (e.g., engineered or variant), as well as functional fragments of proteins.

[0076] The term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., biological activity or function, such as enzymatic activity) of a full-length protein, such as, for example, the ability to bind and / or interact with another protein or nucleic acid or to modulate the ability of another protein or nucleic acid. A functional fragment can be of any size as long as the fragment retains the ability to bind and interact with, for example, another protein or nucleic acid.

[0077] Preferably, the modified subunit of the thermostable ferritin assembly comprises the amino acid sequence shown in SEQ ID NO: 5 to 10, or a variant thereof that retains the assembly ability. The selection of the subunit sequence may depend on the required acid resistance and permeability.

[0078] As is well known to those skilled in the art, the biological function of a polypeptide / protein is closely related to its folding and structure. Therefore, as used herein, the term "reducing structural loss" refers to minimizing any changes in polypeptide structure / conformation, minimizing the degradation of its intended structure, enhancing its resistance to factors that may affect its intended structure, or a combination thereof, which results in improving its ability to maintain its intended structure over a period of time. Similarly, as used herein, the term "reducing functional loss" refers to minimizing any changes in the intended function of a polypeptide, minimizing the effectiveness of its intended function, enhancing its resistance to factors that may affect its intended function, or a combination thereof, which results in improving its ability to maintain its intended function over a period of time.

[0079] As used herein, the term "substrate" refers to any molecule that can be encapsulated by the tES of the present disclosure. Preferably, the substrate is a variable substrate. Preferably, the variable substrate is a variable molecule. As used herein, a "variable molecule" refers to any molecule that is sensitive to any one or more changes in its external environment, such as but not limited to temperature, concentration, ionic charge and pH, wherein the one or more changes in its external environment may affect its expected structure, conformation and / or function.

[0080] In some embodiments, the mutable molecule can be a polypeptide, a nucleic acid or a macromolecule. In some embodiments, the mutable molecule can have therapeutic applications. In some embodiments, the mutable molecule is a macromolecule. In some embodiments, the mutable molecule can be a macromolecule comprising an enzyme for converting a prodrug. In some embodiments, the mutable molecule is horseradish peroxidase (HRP).

[0081] As used herein, the term "polynucleic acid" refers to a polymer comprising a plurality of nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Polynucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Polynucleic acid molecules can be naturally occurring, recombinant or synthetic. In addition, polynucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In certain embodiments, polynucleic acid molecules can be modified. In the case of double-stranded polymers, "polynucleic acid" can refer to one or two chains of the molecule.

[0082] For in vivo delivery, the recombinant composition comprising the engineered ferritin assembly encapsulating the polypeptide can be delivered to a subject in need thereof by a variety of administration routes, including, for example, oral, dietary, topical, transdermal, or parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous, intradermal) administration routes. Administration can be local or systemic. The actual dosage and treatment regimen of the therapeutic polypeptide encapsulated by the engineered ferritin assembly can be determined by a skilled physician, taking into account the nature of the condition being treated and the characteristics of the patient.

[0083] Unless otherwise specified or obvious from the context and the understanding of those skilled in the art, the values ​​expressed in ranges can assume any specific value or sub-range within the range in various embodiments, accurate to one tenth of the unit of the lower limit of the range, unless otherwise clearly specified in the context. When referring to a numerical value, "about" generally refers to a range of values ​​within ±10%, ±5%, ±1%, and ±0.5% of the value, unless otherwise specified or the context clearly indicates otherwise. In any embodiment in which the numerical value is preceded by "about", an embodiment in which the exact numerical value is listed is provided. When an embodiment in which the numerical value is not preceded by "about", an embodiment in which the numerical value is preceded by "about" is also provided. When "about" is preceded by a range, an embodiment in which "about" applies to the lower and upper limits of the range or the lower or upper limits is provided, unless otherwise clearly specified in the context. When a phrase such as "at least", "up to", "not more than" or a similar phrase appears before a series of numbers, it should be understood that the phrase applies to each number in the list in various embodiments (it should be understood that, depending on the context, 100% of the value, for example, a value expressed as a percentage, may be an upper limit), unless otherwise clearly specified in the context. For example, "at least 1, 2, or 3" should be understood to mean "at least 1, at least 2, or at least 3" in various embodiments. It should also be understood that any and all reasonable lower and upper limits are expressly contemplated.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0085] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0086] Example

[0087] Example 1

[0088] Preparation of disulfide-linked tES (DS-tES)

[0089] PyMOL software was used to identify the amino acid at the tES dimerization intersection based on the interatomic distance for nucleotide replacement. The PRSF plasmid carrying tES gene 19 was purified using OMEGAE.ZNA plasmid DNA small amount kit I (D6922-02). Primers for nucleotide replacement were designed using QuikChange Primer Design, and PCR reactions were performed using QuikChange lightening Site-Directed Mutagenesis kit (210519). The PCR product was transformed into competent cells provided by the kit. Colonies were selected using kanamycin (25 μg mL-1; ThermoFisher, Singapore) resistant LB agar (Axil Scientific, USA) plates, and then sequenced for positive confirmation. The positive construct was transformed into BL21 (DE3) Escherichia coli (E. coli) competent cells. An overnight starter culture (25 ml) prepared with a single positive colony was inoculated with LB broth (1000 mL) maintained at 37 ° C and supplemented with kanamycin. Bacterial cultures were grown until the absorbance (OD600) reached ~0.7. Protein expression was induced using isopropyl β-D-1-thiogalactoside (IPTG, 0.4 mM; Axil Scientific), and the cultures were allowed to grow for another 5 h at 37 °C. The bacteria were pelleted by centrifugation at 14,000 × g for 15 min. The pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM BME, and 0.1% Triton-X 100), incubated on ice for 15 min, and then sonicated and centrifuged. The supernatant obtained was purified using a two-step chromatographic procedure. The lysate samples were subjected to hydrophobic interaction chromatography (HIC) using a HiPrepTM Phenyl FF (low substitution) 16 / 10 column (Cytivia), followed by size exclusion chromatography (SEC) using a Superdex S-200 10 / 300GL column (Cytivia). The purity of the protein was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stored under reducing conditions for long-term storage.

[0090] Characterization of DS-tES

[0091] The particle size of tES and DS-tES was measured by dynamic light scattering (DLS) study (Nanobrook Omni, Brookhaven). The purified tES and DS-tES were divided into cuvettes of suitable size and measured at a scattering angle of 90 °. The formation of disulfide bonds was evaluated using non-reducing SDS-PAGE. In addition, the concentration of free thiols present in DS-tES was determined using the Elman assay. In brief, 5,5'-dithio-bis-2-(nitrobenzoic acid) or DTNB prepared at a concentration of 4mg / ml in 0.1M sodium phosphate buffer was mixed with protein samples, and the generation of yellow adducts was measured at 412nm. The standard graph was determined using L-cysteine ​​of known concentration, and free thiols were quantified. In addition, according to user instructions, the presence of endotoxins in purified protein samples was determined using an endotoxin kit (Thermo Fisher Scientific, Waltham, MA, USA).

[0092] Preparation of DS-tES-HRP / rluc

[0093] DS-tES was acidified to pH 5.8 in the presence of a reducing agent to obtain subunits. The subunits were mixed with a 10-fold molar excess of HRP (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 4°C for 30 minutes. The pH of the mixture was raised to 8.0, and the assembled DS-tES encapsulated HRP (DS-tES-HRP) was separated from free HRP on SEC using a non-reducing buffer. HRP encapsulation was analyzed using SDS-PAGE.

[0094] In vitro experiments

[0095] Pepsin assay

[0096] DS-tES-HRP / tES-HRP / HRP were incubated for 2 h in the presence of 1 mg / ml pepsin. The molar ratio of HRP to pepsin used in this study was 1:100 (for DS-tES-HRP and tES-HRP, HRP activity equivalent to free HRP was used), and different pH values ​​ranged from 2 to 8 using 3 M HCl, and the pH was restored to 8 with NaOH to inactivate pepsin. After 2 h, HRP activity was determined using 3,3',5,5'-tetramethylbenzidine (TMB) substrate in a 96-well clear polystyrene plate (Greiner Bio-One). Absorbance was measured at 450 nm using a Perkin plate reader.

[0097] Trypsin assay

[0098] DS-tES-HRP / tES-HRP / HRP were incubated in different concentrations of trypsin (0.25 and 0.4%) for 2 h. After 2 h, HRP activity was measured via 3,3',5,5'-tetramethylbenzidine (TMB) substrate in a 96-well clear polystyrene plate (Griener Bio-one).

[0099] Cell permeability

[0100] DS-tES penetration into the intestinal epithelium was performed in the human colon cancer cell line Caco-2. Briefly, 500,000 Caco2 cells were seeded on a transwell plate and incubated at 37°C in a 10% CO2 incubator for 21 days. After the culture medium was replaced with HBSS, DS-tES-HRP was added to the apical layer. The plate was incubated at 37°C for 3h, and the HRP activity of the culture medium from the basolateral region was evaluated.

[0101] In vivo experiments

[0102] To investigate the in vivo stability of DS-tES, Balb / c mice were orally administered with 0.8 mM DS-tES-rluc or free rluc of comparable activity. Then, ViviRen was intraperitoneally injected within 3 and 24 h. TM In vivo substrate (0.4 mM). The distribution of bioluminescence was evaluated using IVIS spectroscopy. In addition, mouse feces were collected 5, 24, 48, 72 h and 1 week after oral administration of DS-tES-rluc to evaluate the presence of enzymes in the gastrointestinal tract.

[0103] Example 2

[0104] Design and screening of disulfide bond tES

[0105] Previously, we reported the application of tES in delivering iron-mediated catalytic centers for intratumoral bioorthogonal catalysis and solid tumor regression [Sadeghi, S., et al., ACS nano 16:10292-10301 (2022)]. To expand the potential application of tES to other delivery routes, we improved the stability of tES by introducing engineered disulfide bonds at the subunit interface. First, the tES subunit amino acid sequence was screened to find the interatomic distance of We found that arginine 151 (R151), alanine 152 (A152), leucine 53 (L53), alanine 74 (A74), glycine 76 (G76), arginine 66 (R66), glycine 67 (G67), glutamine 50 (Q50), glycine 37 (G37), and alanine 117 (A117) are located at the tES dimerization intersection, so mutagenic primers were designed to systematically replace them with cysteine. As observed in non-reducing SDS-PAGE, R151C did not form disulfide bonds between subunits, while A152C formed dimers ( Figure 2 a). The subsequent double mutants (A152C-L53C and A152C-A74C) showed a higher tendency to form intersubunit disulfide bonds compared to A152C-G76C ( Figure 2 b). Therefore, these two variants were selected for further mutagenesis to form double / triple mutants (A152C-R66C; A152C-G67C; A152C-L53C-R66C; A152C-A74C-R66C; A152C-L53C-G67C; and A152C-A74C-G67C) ( Figure 1 a. Figure 2 c). Among them, A152C-G67C and A152C-L53C-G67C improved stability on non-reducing SDS-PAGE, although no fully connected shells were observed ( Figure 2 c). Various disulfide bond induction methods were used, such as using dimethyl sulfoxide, glutathione system (GSH-GSSG) [Majoul, I., et al., FEBS Letters 401: 104-108 (1997)], methanol, hydrogen peroxide, iodine [Montemiglio, LC, et al., Nature Communications 10: 1-8 (2019)], BMOE crosslinker and air oxidation at different pH. However, none of these methods resulted in the formation of fully connected and stable tES ( Figure 4 ). Further screening revealed the significance of G37C and A117C in stabilizing tES in a non-reducing environment ( Figure 1 ac and Figure 2 d). Therefore, A152C-G67C and A152C-L53C-G67C tES were mutated to their corresponding constructs A152C-G67C-A117C-G37C and A152C-L53C-G67C-A117C-G37C and evaluated for their stability at pH 4.0. Interestingly, A152C-L53C-G67C-A117C-G37C (DS-tES) showed higher stability at acidic pH and was selected for further studies ( Figure 5a) All clones were sequence confirmed, mutants expressed, and proteins purified and evaluated in non-reducing SDS-PAGE.

[0106] Example 3

[0107] Purification and characterization of DS-tES

[0108] like Figure 1 As shown in a and b, mutagenesis at five target sites (A152C-L53C-G67C-A117C-G37C) resulted in a DS-tES with a hydrodynamic diameter of ∼13 nm and an elution volume of 10 ml in size exclusion chromatography ( Figure 3 b, c). Furthermore, reduction to single subunits using TCEP showed that DS-tES was more stable than tES in a non-reducing SDS-PAGE environment and remained largely intact even in the presence of denaturing agents ( Figure 2 , Figure 3 a). Compared with tES, which dissociates into monomers at pH 4.0, DS-tES remains fully assembled and folded at extremely acidic pH ( Figure 5 a). Thiolation is an effective method to tailor the mucoadhesive and mechanical properties of drug delivery systems, so the thiol group content in DS-tES was quantified using Ellman's reagent and determined to be 1.2 μmol per mg protein ( Figure 6 ). In addition, endotoxin levels in purified protein samples were estimated to be 0.018 ng / ml per mg protein, within previously reported limits. Overall, we found that the cage is covalently "locked" and has extremophile stability characteristics. This new property is the result of 60 rationally engineered disulfide bonds that stabilize inter-subunit interactions without affecting the overall morphology of tES. A comparison of the properties of DS-TES and tES is shown in Table 2.

[0109] Table 2. Comparative analysis of DS-tES and tES

[0110]

[0111] Example 4

[0112] DS-tES stabilizes HRP activity under simulated GI conditions

[0113] Acidic gastric fluid, together with enzyme barriers such as pepsin and trypsin, is biologically designed to degrade proteins, thus prohibiting the administration of most protein-based therapies. Therefore, the stability of HRP as a model peptide encapsulated within DS-tES (DS-tES-HRP) was evaluated in a simulated GI environment, and the activity was compared with free HRP or tES-HRP (non-disulfide-stabilized). In the presence of pepsin and pH 4.0, the enzymatic activity of DS-tES-HRP was ~5-fold and ~14-fold higher than that of tES-HRP or free HRP, respectively ( Figure 5 b). However, when incubated with trypsin at pH 7.0, both DS-tES and tES similarly protected encapsulated HRP from proteolysis ( Figure 5 c). The protective properties of tES for encapsulated protein cargo from trypsin hydrolysis have been previously reported [Deshpande, S. et al., Nature Communications 8: 1-8 (2017)]. Similarly, after tES was incubated with serum proteases for 8 days, its HRP activity was maintained at ~ 30%, but no quantifiable activity was observed when incubated with free HRP [Sadeghi, S., et al., ACS nano 16: 10292-10301 (2022)]. Therefore, DS-tES can protect internalized protein substrates from the two major GI proteases in both normal and acidic pH environments.

[0114] Example 5

[0115] DS-tES can penetrate the intestinal cell layer

[0116] To evaluate the transcytotic transport of DS-tES across the intestinal epithelium, permeability assays were performed in the human colon cancer cell line Caco2, as it represents the best available in vitro model of the small intestinal enterocytes. Figure 5d As can be seen, after encapsulation in DS-tES, the HRP signal was significantly higher at all evaluation points. After 3 hours, DS-tES-HRP exhibited 3 times more peroxidase activity compared to free HRP. In addition, a slight decrease in peroxidase activity from DS-tES to tES was observed, with the difference being attributed to the free thiols present on DS-tES. Similar thiol-mediated Caco2 monolayer permeability has been reported previously [Zhang, M., et al., International Journal of Biological Macromolecules 138: 636-647 (2019); Martien, R., et al., Nanotechnology 19: 045101 (2008)]. In summary, these observations suggest that DS-tES plays an important role in intestinal epithelial cell metastasis.

[0117] Example 6

[0118] Oral delivery of DS-tES

[0119] To further understand the in vivo capability of DS-tES as an oral delivery vehicle, a single dose of both rluc and rluc encapsulated in DS-tES (DS-tES-rluc) was orally administered to Balb / c mice. Then, Viviren substrate was injected intraperitoneally into the mice (3 and 24 h later). Figure 7 a). Figure 7 As can be seen in b, both rluc and DS-tES-rluc moved through the GI within 3 hours after administration. Over the next 24 hours, encapsulated luciferase activity was maintained in the GI tract, but the activity of free rluc was negligible. Thereafter, as rluc (whether free or encapsulated) was eliminated from the GI tract, the total signal decreased over time. In addition, rluc activity was measured from mouse feces. A strong and significant effect of DS-tES was observed (P<0.001), which protected the activity of rluc in the length of the GI tract for up to 24 hours. Then, luciferase activity decreased by ~6 times from 24 hours to 72 hours. In contrast, free luciferase activity decreased by more than 50 times after 24 hours, indicating that DS-tES can protect and maintain luciferase activity after oral administration ( Figure 7 c).

[0120] Summarize

[0121] The transport of protein nanoparticles through the GI tract requires exposure to high pH gradients (pH 2.0-8.0), digestive enzymes (pepsin, trypsin, etc.), and mucosal and cell barriers, all of which will change the structural function of the loaded therapeutic protein or peptide. The present invention illustrates that disulfide-bonded tES (DS-tES) protects the encapsulated substrate from this adverse gastrointestinal (GI) environment. The high abundance of cysteine ​​and disulfide bonds that stabilize the quaternary structure are one of the most significant structural characteristics of DS-tES. Free thiols may play an additional role in cellular absorption, especially through transferrin receptor-mediated endocytosis. We also note that when taken orally, DS-tES encapsulating Renilla luciferase will stay in the GI tract for a long period of time before being removed from the system. Since DS-tES uses a simple and rapid encapsulation technology without affecting the properties of the encapsulated substrate, it may have a significant impact on the nutritional health products and pharmaceutical industries.

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Claims

1. An engineered disulfide-linked ferritin assembly comprising at least one modified ferritin subunit, wherein: The at least one modified ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 1, and comprises; i) F116H substitution, and zero or more amino acid substitutions at one or more positions selected from the group consisting of E65, E128, E131 and D138 of SEQ ID NO: 1, and ii) Cys substitution at two or more positions selected from the group consisting of G37, L53, R66, G67, A74, A117 and A152 of SEQ ID NO:

1.

2. The engineered disulfide-bonded ferritin assembly according to claim 1, wherein: The assembly has a net positive internal charge or a net neutral internal charge.

3. The engineered disulfide-bonded ferritin assembly according to claim 2, wherein: The modified ferritin subunit comprises an amino acid substitution at any one or more positions selected from the group consisting of E65, E128, E131 and D138.

4. The engineered disulfide-bonded ferritin assembly according to claim 3, wherein: The amino acid substitutions include: i) substitutions of E65K, E128K, E131K and D138A, which result in the assembly having a net positive internal charge; or ii) Substitutions E65Q and D138A, which result in the assembly having a net neutral internal charge.

5. The engineered disulfide-linked ferritin assembly according to any one of claims 1 to 4, wherein: Prior to the Cys substitution, the modified ferritin subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

4.

6. The engineered disulfide-linked ferritin assembly according to any one of claims 1 to 5, wherein: The modified ferritin subunit includes a cysteine ​​residue at a position selected from the group consisting of A152C-G67C-A117C-G37C and A152C-L53C-G67C-A117C-G37C.

7. The engineered disulfide-bonded ferritin assembly according to claim 6, wherein: The modified ferritin subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:

10.

8. The engineered disulfide-linked ferritin assembly according to any one of claims 1 to 7, wherein: A bioorthogonal catalytic center, a polypeptide or a nucleic acid or any combination thereof is encapsulated within the ferritin assembly.

9. The engineered disulfide-linked ferritin assembly according to any one of claims 1 to 8, wherein: The disulfide-linked ferritin assembly further comprises a cell targeting moiety.

10. An in vitro method for forming an engineered disulfide-linked ferritin assembly, the method comprising adjusting the pH of a sample comprising at least one modified ferritin subunit according to any one of claims 1 to 9 from an acidic pH to an alkaline pH, wherein: The assemblies are stable in the gastrointestinal tract and enable oral drug delivery.

11. The method of claim 10, further comprising a bioorthogonal catalytic center, a polypeptide or a nucleic acid or any combination thereof in the sample, wherein: The bioorthogonal catalytic center, polypeptide or nucleic acid or any combination thereof is encapsulated by the engineered ferritin assembly at the alkaline pH of the sample.

12. The method according to claim 10 or 11, wherein: The acidic pH in the sample is at least about 4.0 and / or the alkaline pH in the sample is about 8.

0.

13. A method of delivering an engineered disulfide-linked ferritin assembly into a cell, comprising contacting the cell with the disulfide-linked ferritin assembly according to any one of claims 1 to 9.

14. An isolated plasmid or vector polynucleic acid comprising a sequence encoding a modified ferritin subunit, wherein: The modified ferritin subunit is as defined in any one of claims 1 to 9.

15. A composition comprising at least one engineered disulfide-linked ferritin assembly according to any one of claims 1 to 9, for use in preventing or treating a disease in a subject.

16. The composition according to claim 15, wherein The at least one engineered disulfide-linked ferritin assembly encapsulates: i) enzymes; or ii) an enzyme and one or more additional therapeutic agents.

17. The composition according to claim 15 or 16, which can be delivered via oral route.

18. A method of treatment or prevention comprising administering to a subject in need of such treatment or prevention an effective amount of a composition according to any one of claims 15 to 17.

19. The method according to claim 18, wherein: The composition is administered sequentially or simultaneously with other therapeutic agents.

20. The method according to claim 19, wherein: The composition includes a bioorthogonal catalytic center encapsulated within the engineered disulfide-linked ferritin assembly, and the other therapeutic agent is a substrate for the catalytic center.

21. Use of a composition according to any one of claims 15 to 17 in the preparation of a medicament for treating a disease in a subject.

22. The use according to claim 19, wherein The disease is cancer.

23. A kit for encapsulating a bioorthogonal catalytic center, a polypeptide or a nucleic acid, or any combination thereof, in an engineered disulfide-linked ferritin assembly, the kit comprising at least one modified ferritin subunit according to any one of claims 1 to 9.

24. A kit for encapsulating a bioorthogonal catalytic center, a polypeptide or a nucleic acid or any combination thereof in an engineered disulfide-linked ferritin assembly, the kit comprising the plasmid or vector nucleic acid of claim 14.

Images