Neoepitope vaccine delivery vehicle and method for making same

Abstract

Disclosed herein is mannan nanogel as a novel vaccine delivery platform, as well as a novel method for producing self-assembling mannan nanogel for in vivo delivery of therapeutic agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 287,176, filed December 8, 2021. The entire disclosure of U.S. Provisional Patent Application No. 63 / 287,176 is incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ST.26 XML format. The text file named "PAT005304_Sequence_Listing.xml" has a size of 7000 bytes and was recorded on December 6, 2022. The information contained in the text file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5). [Background technology]

[0003] Significant efforts have been put into the design of colloidal drug carriers to improve drug localization and bioavailability. Ideally, actively targeted particulate drug carriers would increase the therapeutic efficacy of drugs by delivery to the affected area while simultaneously reducing drug-associated side effects. Achieving this goal would significantly advance the treatment of diseases (e.g., cancer) where the toxic effects of systemically administered therapeutic agents may outweigh their benefits. To date, many types of delivery vehicles, including inorganic nanoparticles, polyelectrolyte complexes, liposomes, block copolymer micelles, and polymeric nanoparticles, have been explored for in vitro and in vivo drug delivery.

[0004] Several nanoparticle vaccine delivery platforms are under development, requiring the fastest possible delivery of the prime dose. Synthetic nanoscale excipients offer many advantages, such as bottom-up functional design, in vivo protection for sensitive bioactive cargoes such as peptides, and allow for scalable and reproducible manufacturing.

[0005] Disclosed herein is a novel nanogel vaccine platform produced by induced self-assembly of the polysaccharide mannan. The gel nanoparticles are modified with mannan chains (to attract the particles to the CD206 receptor) and cleavable neoepitope peptides. These nanoparticles can be rapidly produced according to the methods disclosed herein. Summary of the Invention [Means for solving the problem]

[0006] Disclosed herein is a method for producing self-assembling mannan nanogels for in vivo delivery of therapeutic agents, comprising: oxidizing mannan with periodate (NaIO4); purifying the oxidized mannan; adding aniline to the purified oxidized mannan to produce a mannan derivative having hydrophobic phenylimine groups covalently attached to the mannan; and sonicating the mannan derivative.

[0007] In one embodiment, a dihydrazide (DH) crosslinker is introduced into the self-assembled mannan nanogel, the method comprising reacting the mannan nanogel with succinic dihydrazide (SDH) and 3,3'-dithiobis(propanoic dihydrazide) (DPDH).

[0008] In one embodiment, dihydrazide cross-linked mannan nanogels were prepared for loading with thiol-containing cargo by a method that includes reducing the nanogel disulfide bridges with (tris(2-carboxyethyl)phosphine) (TCEP); reducing the nanogel imines and residual aldehydes with borohydride (NaBH4); and activating the nanogel thiols with 2,2-dithiopyridine (DTP).

[0009] In yet another embodiment, a thiol-containing cargo is loaded onto the DTP-activated nanogel, the cargo consisting of one or more peptides and, optionally, glutathione (GSH).

[0010] In yet another embodiment, the dihydrazide cross-linked nanogel is coated with NaIO4 oxidized mannan.

[0011] In one embodiment, a diamine (DA) crosslinker is introduced into mannan nanogels, the method comprising sequentially reacting the mannan nanogels with cystamine and ethylenediamine dihydrochloride (EDA) and then with sodium cyanoborohydride (NaCNBH3).

[0012] In one embodiment, diamine cross-linked mannan nanogels were prepared for loading with thiol-containing cargo by a method that involves reducing the nanogel disulfide bridges with (tris(2-carboxyethyl)phosphine) (TCEP) and activating the nanogel thiols with 2,2-dithiopyridine (DTP).

[0013] In one embodiment, a thiol-containing cargo is loaded onto the DTP-activated nanogel, the cargo consisting of one or more peptides and, optionally, glutathione (GSH).

[0014] In one embodiment, diamine crosslinked nanogels are coated with NaIO4 oxidized mannan and then reacted with sodium cyanoborohydride (NaCNBH3).

[0015] Also disclosed herein is a method for loading thiol-modified RNA onto DTP-activated dihydrazide- or diamine-crosslinked mannan nanogels, comprising 1) sequential reaction of the oxidized RNA with cystamine and TCEP, reductive amination of the oxidized RNA, 2) purification, and 3) addition to the DTP-activated nanogel.

[0016] Also disclosed herein is a method for loading RNA onto DTP-activated diamine-crosslinked mannan nanogels, comprising adding unmodified RNA to the DTP-activated nanogels.

[0017] Also disclosed herein is a composition comprising CD206-expressing 293T cells, wherein the 293T cells are genetically engineered to stably express a gene having the sequence of SEQ ID NO:1.

[0018] Further disclosed herein is a method for quantifying cellular uptake of mannan nanogel or cargo-loaded mannan nanogel, comprising treating CD206-expressing 293T cells with mannan nanogel, the 293T cells being genetically engineered to stably express a gene having the sequence of SEQ ID NO:1, and cellular uptake of the nanogel or cellular expression of the cargo being quantified.

[0019] In one embodiment of the method of quantifying cellular uptake, quantification is by fluorescence, luminescence, viability, apoptosis, cell size, cell proliferation, spheroid formation, cell surface expression, or intracellular localization.

[0020] In one embodiment of the method for quantifying cellular uptake, mannan nanogels are doped with fluorescently labeled dextran. In one embodiment, the fluorescent label is fluorescein isothiocyanate (FITC). [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows a schematic diagram of the novel self-assembly process of uniform mannan nanogels. [Figure 2-1] Figure 2A shows that the nanogel sizes are highly monodisperse, and Figure 2B shows that the nanogels exhibit a negative surface charge. [Figure 2-2] Ultraviolet (UV) absorbance is shown to confirm the presence of aniline in the nanogels. [Diagram 3] A schematic representation of the process whereby nanogels are DTP-activated and loaded with peptide cargo is shown. [Figure 4-1]Figure 4A shows a schematic of how peptide cargo can be loaded onto nanogels via disulfide formation. Figure 4B shows the structure of FITC-RP426C. Figure 4C shows the structure of TCEP. [Figure 4-2] The fraction of thiol (-SH) thiol sites with FITC peptide loaded and released is shown. [Diagram 5] The modified cytomegalovirus (CMV) antigenic peptide relative to the disulfide bond is shown. The last box in the structure represents the "C" in QGQC. [Figure 6] 6 shows nanogel antigen delivery and presentation established using ELISPOT. Figure 6A is a schematic. Figure 6B shows the results for NLV-mod peptide and Figure 6C shows the results for DiHy and DiAm. [Figure 7-1] A stable 293T cell line was developed that expresses CD206. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8] Schematic diagrams of nanogels made with high or low oxidation mannan are shown. [Figure 9] Figure 9 shows that low oxidized mannan results in differential nanogel uptake by cell lines. The degree of oxidation (OD or OX) of the mannan determines the prevalence of reactive aldehydes. The fewer aldehydes, the fewer downstream surface groups susceptible to non-specific binding (e.g., charged, highly polar or hydrophobic). Differential uptake of low OD mannan gels by CD206-bearing cells may indicate improved receptor affinity and reduced non-specific binding or combination. Low OD mannans also produce less stable gels with lower cargo-holding capacity. Figure 9A shows the results with high Ox mannan; Figure 9B shows the results for low Ox mannan; and Figure 9C shows the fold change between the two. [Figure 10]A schematic diagram of secondary mannan assembly is shown. Dangling crosslinkers remain reactive towards aldehydes. Low OD mannans retain enough aldehydes to react with the coagel, but have few other sites for non-specific binding. Secondary core-shell assembly using low OD mannans should provide a l0w binding surface. [Figure 11] Secondary mannan reduces non-specific uptake and confers CD206 specificity. Figure 11A shows the results after 1 hour. Figure 11B shows the time course over 20 hours. [Figure 12] Figure 12 shows the oxidation level of secondary mannans optimized for CD206 specific uptake. Figure 12A shows the % positive versus oxidation level for 293T-Neg and 293T-CD206. Figure 12B shows the fold change in score versus oxidation level. [Figure 13] FIG. 13A shows that the charge of the nanogel surface can be controlled by the cargo mixture. NLV-mod carries a net + charge. GSH carries a net -1 charge. Peptides mixed in various ratios (NLV-mod:GSH) were loaded onto the nanogels. The observed zeta potential reflects the expected change in surface charge. The NVL peptide is an HLA-A2 restricted peptide from cytomegalovirus (CMV). It is used as a control peptide for CD8 T cell stimulation. FIG. 13B shows the structure of the NLV-mod antigen peptide. FIG. 13C shows the structure of glutathione (GSH). [Figure 14-1] Uptake and ELISPOT with GSH mixed cargo are shown. Figure 14A shows the results at 1:0; Figure 14B shows the results at 1:1 and Figure 14C shows the results at 1:9. [Figure 14-2] FIG. 14D shows interferon-gamma (INF-g) ELISPOT, NLP03. [Figure 15] Shown is the intracellular colocalization of nanogels and peptides in CD206 positive 293T cells at 1 hour (FIG. 15A) and overnight (FIG. 15B). [Figure 16]Schematic representation of RNA as nanogel cargo is shown. Figure 16A shows that the 3' end of mRNA can be selectively thiolated by periodate oxidation followed by reductive aminatation with cysteine. Figure 16B shows that cationic (DA, also called DiAm) nanogels can also form complexes with mRNA by electrostatics alone. [Figure 17] DiAm and DiHy nanogels deliver mCherry mRNA. Figure 17A shows gel uptake. Figure 17B shows mCherry expression. [Figure 18] A comparison of DiHY (Figure 18A) and DiAM (Figure 18B) mCherry RNA gels is shown. [Figure 19] 1 shows the structure of mannan from S. cerevisiae. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Unless otherwise defined herein, scientific and technical terms used in this application have the meanings commonly understood by those of ordinary skill in the art. In general, nomenclature used in connection with and in the arts of chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry is that well known and commonly used in the art.

[0023] All publications, patents, and published patent applications referenced in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0024] Mannan nanogel as a novel vaccine delivery platform, as well as a novel method of producing self-assembled mannan nanogel for in vivo delivery of therapeutics, are disclosed herein. Mannan targets dendritic cells (DC) via C-type lectin. Mannose receptor (CD206) is highly expressed on the DC cell surface. Targeting of mannan nanogel to DC has been confirmed by the inventors using a stable CD206-expressing cell line. Therapeutic agents, such as but not limited to peptides, can be linked by disulfide via cysteine. The method includes oxidizing mannan with periodate (NaIO4); purifying the oxidized mannan; adding aniline to the purified oxidized mannan to generate a mannan derivative with a hydrophobic phenylimine group covalently attached to the mannan; and sonicating the mannan derivative.

[0025] CD206 expression is restricted to DCs, macrophages, and a subpopulation of endothelial cells in humans. CD206 activation mediates endocytosis, which is ideal for processing antigen cargo. Thus, peptides bound to mannan nanogels are targeted to CD206-expressing dendritic cells, where they are taken up, processed, and presented by dendritic cells, activating T cells specific for the bound peptide.

[0026] In one embodiment, a crosslinker is introduced into the self-assembled mannan nanogel. Crosslinking transforms the hydrophobic assembly into a covalent network. As disclosed herein, the crosslinker can be a diamine or dihydrazide, which replaces aniline by transamination to provide stable interchain bonds and introduce disulfide linkage sites. Dihydrazide (DH) crosslinkers require organic solvents and reduce imines to hydrazides, and these DH crosslinkers do not introduce charge into the network at physiological pH. Diamine (DA) crosslinkers are water-soluble and reduce imines to secondary amines, and these DA crosslinkers introduce cationic (+) charge into the network.

[0027] In one embodiment, a DH crosslinker is introduced into the self-assembled mannan nanogel, the method comprising reacting the mannan nanogel with succinic dihydrazide (SDH) and 3,3'-dithiobis(propanoic dihydrazide) (DPDH).

[0028] As disclosed herein, DH cross-linked mannan nanogels can be prepared for loading thiol-containing cargo. The loading method includes reducing nanogel disulfide bridges with (Tris(2-carboxyethyl)phosphine) (TCEP), reducing nanogel imine and residual aldehydes with borohydride (NaBH4), and activating nanogel thiols with 2,2-dithiopyridine (DTP). Then, thiol-containing cargo is loaded onto the DTP-activated nanogel. The cargo includes one or more peptides and optionally glutathione (GSH). In one embodiment, the DH cross-linked nanogel can be further coated with NaIO4 oxidized mannan.

[0029] Also disclosed herein is a DA crosslinker that can be introduced into mannan nanogel. The method includes sequentially reacting mannan nanogel with cystamine and ethylenediamine dihydrochloride (EDA) and then with sodium cyanoborohydride (NaCNBH3). In one embodiment, DA crosslinked mannan nanogel is prepared for loading thiol-containing cargo, the method includes reducing nanogel disulfide bridges with TCEP and activating nanogel thiols with DTP. Then, thiol-containing cargo is loaded onto the DTP-activated nanogel, the cargo being composed of one or more peptides and, optionally, GSH. In one embodiment, DA crosslinked nanogel is coated with NaIO4 oxidized mannan and then reacted with sodium cyanoborohydride (NaCNBH3).

[0030] A further embodiment disclosed herein is a method for loading thiol-modified RNA onto DTP-activated, DH- or DA-crosslinked, mannan nanogels, which involves sequential reaction of the oxidized RNA with cystamine and TCEP, reductive amination of the oxidized RNA, followed by purification and addition to the DTP-activated nanogels.

[0031] Also contemplated is a method of loading RNA onto a DTP-activated, DA-crosslinked mannan nanogel, comprising adding unmodified RNA to the DTP-activated nanogel.

[0032] A further embodiment disclosed herein is a method for quantifying cellular uptake of mannan nanogel or cargo-loaded mannan nanogel. The method comprises treating CD206-expressing 293T cells with mannan nanogel, the 293T cells being genetically engineered to stably express a gene having the sequence of SEQ ID NO:1, and quantifying cellular uptake of the nanogel or cellular expression of the cargo. Quantification of cellular uptake can be by fluorescence, luminescence, viability, apoptosis, cell size, cell proliferation, spheroid formation, cell surface expression, or intracellular localization. In one aspect, the mannan nanogel is doped with fluorescently labeled dextran. In one aspect, the fluorescent label is FITC.

[0033] A further embodiment is a composition comprising CD206-expressing 293T cells. The 293T may be genetically engineered to stably express a gene having the sequence of SEQ ID NO:1.

[0034] As disclosed herein, nanogels are nanoparticles composed of hydrophilic polymer chains and hydrogels that are highly crosslinked physically or chemically. Nanogels can hold a large amount of water due to the presence of hydrophilic functional groups. Nanogels can swell in a good solvent while maintaining their internal structure. The term "nanogel" can refer to crosslinked polymer particles that can absorb liquid and retain at least a portion of the liquid to form swollen crosslinked polymer particles. Nanogels come in many sizes, which represent the nanogel in the solvent swollen state. Nanogel size can be optimized to remain in the bloodstream and can also traverse fenestrated tumor vasculature.

[0035] A nanogel-based delivery system comprises an active agent or cargo substantially contained within the nanogel, where the active agent is covalently or non-covalently bound to the nanogel. As used herein, the term "active agent" or "cargo" can refer to one or more moieties, such as a pharmacological moiety, a therapeutic moiety, a diagnostic moiety, a drug moiety, a biological moiety, etc.

[0036] Thus, terms such as "active agent," "cargo," "drug," "therapeutic," "diagnostic," "pharmaceutical," etc. may be used interchangeably throughout this disclosure. An active agent may also include one or more pharmaceutical additives such as, but not limited to, solubilizers, emulsifiers, buffers, preservatives, carriers, suspending agents, thickening agents, stabilizers, inactive ingredients, etc.

[0037] As used herein, the term "agent" includes, but is not limited to, biological or chemical compounds, such as single or complex organic or inorganic molecules, peptides, peptidomimetics, proteins (e.g., antibodies, growth factors), antigens or immunogens, mRNA, small interfering RNA (siRNA), or polynucleotides, viruses, or therapeutic agents. Organic or inorganic molecules include, but are not limited to, pharmaceuticals, radioisotopes, crude or purified plant extracts, and / or entities that alter, inhibit, activate, or otherwise affect biological or biochemical events, such as classes of molecules typically found in cells and tissues (e.g., proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, saccharides, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, growth factors, chemoattractants, cytokines, chemokines, etc.).

[0038] For example, when mRNA is the cargo or agent, the 3' end of the mRNA can be selectively thiolated by periodate oxidation followed by reductive aminatation with cysteine. We show that cationic DA nanogels can also form electrostatic complexes with mRNA alone.

[0039] Examples of such agents include, but are not limited to, gene therapy agents; analgesics; anti-arthritic agents; anti-asthma agents; anti-cancer agents; anticholinergic agents; anticonvulsants; antidepressants; anti-diabetic agents; anesthetics; antibiotics; antigens; antihistamines; anti-infective agents; anti-inflammatory agents; antibacterial agents; antifungal agents, anti-Parkinson's agents; antispasmodics; antipruritics; antipsychotics; antipyretics; antivirals; nucleic acids; DNA; RNA; siRNA; polynucleotides; nucleosides; nucleotides; amino acids; peptides; proteins; carbohydrates; lectins; lipids. id);lipids (fat);fatty acids;viruses;immunogens;antibodies and their fragments;serum;immuno-enhancing drugs;immunosuppressants;cardiovascular agents;channel blockers (e.g. potassium channel blockers, calcium channel blockers, beta blockers, alpha blockers);antiarrhythmic drugs;antihypertensive drugs;inhibitors of DNA, RNA, or protein synthesis;neurotoxins;vasodilators;vasoconstrictors;gases, growth factors;growth inhibitors;hormones;steroids;steroidal and non-steroidal anti-inflammatory agents;corticosteroids;angiogenic agents agents); angiogenesis inhibitors; hypnotics; muscle relaxants; muscle contractants; sedatives; tranquilizers; ionizing and non-ionizing agents; metals; small molecules; drugs; blood therapeutics; wound healing agents; indicators of changes in the biological environment; enzymes; enzyme inhibitors; nutrients; vitamins; minerals; clotting factors; anticoagulants; antithrombotic agents; neurochemicals (e.g., neurotransmitters); cell receptors; radioactive materials; imaging agents (e.g., fluorescent, magnetic, radioactive); nanoparticles (e.g., magnetic, semiconductor, dielectric, or metallic); vaccines; cell proliferation modifiers; cell adhesion modifiers; cell response modifiers; cells; chemicals or biological materials or compounds that induce a desired biological or pharmacological effect; and combinations thereof.

[0040] Throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" refers to the inclusion of a specified integer (or component) or group of integers (or components) but not to the exclusion of any other integer (or component) or group of integers (or components).

[0041] The singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0042] "Including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0043] A pharma- ceutically acceptable carrier means a non-toxic carrier that may be administered to a patient together with a composition described herein and that does not destroy the pharmacological activity of the active agent within the composition. An "excipient" means an additive in a formulation or composition that is not a pharma- ceutically active ingredient.

[0044] A "pharmacologically effective amount" refers to an amount effective to treat a patient, for example, to produce a beneficial and / or desired change in the patient's overall health, suffering from a disease or condition, such as, but not limited to, cancer. Treatment includes, but is not limited to, killing cells, preventing the growth of new cells, improving the patient's functioning, enhancing the patient's well-being, reducing pain, improving appetite, improving the patient's weight, and any combination thereof. A "pharmacologically effective amount" also refers to the amount necessary to improve the patient's clinical symptoms.

[0045] "Peptide" and "polypeptide" are used interchangeably herein to refer to a polymer composed of amino acid residues. As used herein, "amino acid residue" refers to any naturally occurring amino acid (L or D form), a non-naturally occurring amino acid, or an amino acid mimetic (such as a peptide monomer).

[0046] "Identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences means two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a comparison window. For purposes of this disclosure, the degree of amino acid or nucleic acid sequence identity is determined using the BLAST algorithm described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. This algorithm identifies high scoring sequence pairs (HSPSs) by identifying short words of length W in a query sequence that match or meet some positive threshold score when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., (1990) J. Mol. Biol. 215:403-10). The initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended along each sequence in both directions for as far as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below 0 due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is not reached. For determining percent identity of amino acid sequences, the BLASTP settings are: word length (W), 3; expectation (E), 10; and the BLOSUM62 score matrix. For analysis of nucleic acid sequences, the BLASTN program settings are: word length (W), 11; expectation (E), 10; M=5; N=-4; and comparison of both strands.The BLASTN program (protein sequence against nucleotide sequence database query) uses a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0047] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-87). The smallest sum probability (P(N)) provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability is less than about 0.01 in a comparison of the test nucleic acid to the reference nucleic acid.

[0048] The "length" of a polypeptide is the number of amino acid residues linked end-to-end that make up the polypeptide, excluding any non-peptide linkers and / or modifications that the polypeptide may contain.

[0049] Hydrophobic amino acid residues are characterized by functional groups ("side chains") that are predominantly non-polar chemical nature. Such hydrophobic amino acid residues may be natural (L- or D-form) or non-natural. Alternatively, hydrophobic amino acid residues may be amino acid mimetics that are predominantly non-polar chemical nature. Conversely, hydrophilic amino acid residues are characterized by side chains that are predominantly polar (charged or uncharged) chemical nature. Such hydrophilic amino acid residues may be natural (L- or D-form) or non-natural. Alternatively, hydrophilic amino acid residues may be amino acid mimetics that are predominantly polar (charged or uncharged) chemical nature. Suitable non-natural amino acid residues and amino acid mimetics are known in the art. See, for example, Liang et al. (2013) PLoS ONE 8(7):e67844.

[0050] Although most amino acid residues are considered to be either hydrophobic or hydrophilic, some may behave as either hydrophobic or hydrophilic depending on the context. For example, the relatively weakly non-polar character of glycine, proline, and cysteine ​​allows them to sometimes function as hydrophilic amino acid residues, respectively. Conversely, the bulky, slightly hydrophobic side chains of histidine and arginine allow them to sometimes function as hydrophobic amino acid residues, respectively.

[0051] Unless otherwise specified, each embodiment disclosed herein may be used alone or in combination with one or the other embodiments herein.

[0052] "Transfection" refers to the introduction of foreign nucleic acid into a eukaryotic cell. Transfection can be accomplished by a variety of means known in the art, including electroporation, polymers (nanoparticles), calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, microinjection, liposome fusion, pofection, protoplast fusion, and biolistics.

[0053] "Stable transfection" or "stably transfected" refers to the introduction and integration of a foreign nucleic acid, DNA, into the genome of the transfected cell.

[0054] The term variant refers to a protein or fragment thereof having an amino acid sequence similar, but not identical, to a reference sequence (e.g., a SARS-CoV-2 protein sequence), where the activity of the variant protein is not significantly altered. These variations in sequence may be natural variations or may be engineered by using techniques known to those skilled in the art. Examples of suitable variations include, but are not limited to, amino acid deletions, insertions, substitutions, and combinations thereof.

[0055] Amino acids can be classified into groups based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants are those in which one amino acid is replaced with an amino acid from the same group. Such substitutions are called conservative substitutions.

[0056] Naturally occurring residues can be divided into the following classes based on common side chain properties: 1) Hydrophobic: Met, Ala, Val, Leu, Ile; 2) Neutral hydrophilic: Cys, Ser, Thr; 3) Acidic: Asp, Glu; 4) Basic: Asn, Gln, His, Lys, Arg; 5) Residues that affect chain orientation: Gly, Pro; and 6) Aromatics: Trp, Tyr, Phe.

[0057] Non-conservative substitutions may involve exchanging a member of one of these classes for a member from another class.

[0058] The terms subject, patient, individual, etc. are not intended to be limiting and are generally interchangeable. That is, an individual described as a patient does not necessarily have a given disease, but may simply seek medical advice. As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The terms do not imply a particular age or sex. Thus, adult and neonatal subjects, regardless of male or female, are intended to be encompassed. As used herein, patient, individual, and subject may be used interchangeably, and these terms are not intended to be limiting. That is, an individual described as a patient does not necessarily have a given disease, but may simply seek medical advice. The terms patient or subject include human and veterinary subjects.

[0059] References to "therapeutic" and "prophylactic" herein should be considered in their broadest context. "Therapeutic" does not necessarily mean that a mammal is treated until complete recovery. Similarly, "prophylactic" does not necessarily mean that a subject will not eventually contract a disease state. The term "prophylaxis" may be considered as reducing the severity of a particular symptom onset. Treatment may also reduce the severity of an existing symptom or the frequency of acute attacks. As used herein, "treat," "treating," and similar words refer to stabilizing and / or reducing the symptoms of a disease or condition. In some embodiments, the compositions disclosed herein can prevent the onset of a disease or condition or cure a disease or condition, which is distinct from treatment.

[0060] The route and frequency of administration of the therapeutic compositions described herein, as well as the dosage, will vary from person to person and from disease to disease, and can be readily established using standard techniques. In general, pharmaceutical compositions can be administered by injection (e.g., intradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., by inhalation), in pill form (e.g., suppository for swallowing, vaginal, or rectal delivery).

[0061] According to the methods provided herein, the subject is administered an effective or therapeutically effective amount of one or more of the agents provided herein. The terms effective amount, therapeutically effective amount, and effective dosage are used interchangeably. The term effective amount is defined as the amount necessary to produce the desired physiological response (e.g., reduction of inflammation). The effective amount and the schedule for administering the agent can be empirically determined by those of skill in the art. The dosage range of administration is large enough to produce the desired effect (e.g., reduction or delay) in which one or more symptoms of the disease or disorder are affected. The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, etc. In general, dosage will vary depending on age, medical condition, sex, type of disease, extent of disease or disorder, route of administration, or whether other drugs are included in the administration regimen, and can be determined by those of skill in the art. Dosage can be adjusted by the individual physician in the event of contraindications. Dosage can vary and can be administered daily, in one or multiple doses, for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceuticals. For example, for a given parameter, an effective amount will exhibit at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least a 100% decrease or reduction. Efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or greater effect compared to a control. The exact dosage or formulation will vary depending on the purpose of treatment, and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Editor (2012), and Pickar, Dosage Calculations (1999)).

[0062] Compositions suitable for injectable use include sterile aqueous solutions (if water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0063] Injectable sterile solution can be prepared by incorporating active compound in the required amount in suitable solvent with various other components listed above, and then, if necessary, for example, sterilization by filtration or other suitable means.By incorporating various sterilized active components into sterilized vehicle, dispersion can be prepared, which contains basic dispersion medium and other components required from the components listed above.In the case of sterile powder for preparing injectable sterile solution, the preferred method of preparation includes vacuum drying and freeze-drying technology, which produces powder of active component + any additional desired components from the solution previously sterilized and filtered.

[0064] When the active ingredients are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0065] In combination with any of the above methods, the composition may be administered in conjunction with another drug. In each case, the composition may be administered before, simultaneously with, or after the administration of the other drug. In accordance with the methods described herein, the compounds or compositions may be co-administered with one or more other compounds, such as known chemotherapeutic agents, antiviral compounds or molecules, as well as antibiotics, chloroquine, hydroxychloroquine, known drugs for treating pneumonia, analgesics (such as lidocaine or paracetamol), anti-inflammatory agents (betamethasone, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, ibuprofen, naproxen), and / or other suitable drugs. The provided methods may further be combined with other tumor treatments, such as radiation therapy, surgery, hormone therapy, and / or immunotherapy. Thus, the provided methods may further comprise administering to the subject one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, analgesics, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antineoplastics, allosteric inhibitors, anabolic steroids, antirheumatics, psychotherapeutic agents, nerve blocks, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antivirals, dopaminergic agents, blood system agents, immune agents, muscarinic agents, protease inhibitors, vitamins, growth factors, and hormones. The choice of agent and dosage can be easily determined by those skilled in the art based on the given disease to be treated. Optionally, the additional therapeutic agent is octreotide acetate, interferon, pembrolizumab, glucopyranosyl lipid A, carboplatin, etoposide, or any combination thereof.

[0066] "Co-administered" means simultaneous administration in the same formulation or in two different formulations by the same or different route, or sequential administration by the same or different route. "Sequential" administration means a time difference of seconds, minutes, hours, or days between the administration of two or more separate compounds.

[0067] In some embodiments, it may be beneficial to include one or more excipients in the composition. Those skilled in the art will recognize that the selection of one excipient may affect the selection of the other excipient. For example, the selection of a particular excipient may preclude the use of one or more additional excipients, since the combination of excipients would cause undesirable effects. Those skilled in the art will be able to empirically determine which excipients, if any, to include in the formulations or compositions disclosed herein. Excipients include, but are not limited to, co-solvents, solubilizers, buffers, pH adjusters, bulking agents, surfactants, encapsulating agents, tonicity adjusters, stabilizers, protectants, and viscosity adjusters. In some embodiments, it may be beneficial to include a pharma- ceutical acceptable carrier.

[0068] In some embodiments, it may be beneficial to include a solubilizer. The solubilizer may be useful for enhancing the solubility of any of the components of the formulation or composition, such as the peptides or excipients disclosed herein. The solubilizers described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary solubilizers that may be used. In certain embodiments, the solubilizer includes, but is not limited to, ethyl alcohol, t-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, and pharma- ceutically acceptable salts thereof and / or combinations thereof.

[0069] The pH can be any pH that provides the composition with desirable properties, including, for example, peptide stability, increased peptide retention compared to compositions at other pHs, and improved filtration efficiency.

[0070] In some embodiments, it may be beneficial to include a tonicity adjuster. The tonicity of a liquid composition is an important consideration, for example, when administering the composition to a patient via parenteral administration. Thus, tonicity adjusters may be used to help make the composition suitable for administration. Tonicity adjusters are well known in the art. Thus, the tonicity adjusters described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary tonicity adjusters that may be used. Tonicity adjusters may be ionic or non-ionic, and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars include sugar alcohols, such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

[0071] In some embodiments, it may be beneficial to include a stabilizer, which helps to increase the stability of the peptide in the composition of the invention.

[0072] In some embodiments, it may be beneficial to include a protective agent. A protective agent is an agent that protects a pharma- ceutical active ingredient (e.g., a peptide disclosed herein) from undesirable conditions (e.g., instability caused by freezing or lyophilization, or oxidation). Protective agents include, for example, cryoprotectants, lyoprotectants, and antioxidants. Cryoprotectants are useful for preventing loss of potency of an active pharmaceutical ingredient (e.g., a peptide disclosed herein) when the formulation is exposed to a temperature below its freezing point. For example, a cryoprotectant can be included in a reconstituted lyophilized formulation, such that the formulation can be frozen prior to intravenous (IV) administration. Cryoprotectants are well known in the art. Thus, the cryoprotectants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary cryoprotectants that may be used. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizers, viscosity modifiers, and combinations thereof. Cryoprotectants can include, for example, disaccharides (eg, sucrose, lactose, maltose, and trehalose), polyols (eg, glycerol, mannitol, sorbitol, and dulcitol), glycols (eg, ethylene glycol, polyethylene glycol, propylene glycol).

[0073] Lyoprotectants are useful for stabilizing the components of lyophilized formulations or compositions. For example, the peptides disclosed herein can be lyophilized using a lyoprotectant prior to reconstitution. Lyoprotectants are well known in the art. Thus, the lyoprotectants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary lyoprotectants that may be used. Lyoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizers, viscosity modifiers, and combinations thereof. Exemplary lyoprotectants can be, for example, sugars and polyols, trehalose, sucrose, dextran, with hydroxypropyl-β-cyclodextrin being a non-limiting example of a lyoprotectant.

[0074] Antioxidants are useful for preventing oxidation of the components of the composition. Oxidation can cause drug product aggregation or other adverse effects on drug product purity or its potency. Antioxidants are well known in the art. Thus, the antioxidants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary antioxidants that can be used. Antioxidants can be, for example, sodium ascorbate, sodium citrate, thiols, metabisulfites, and combinations thereof.

[0075] Variations, modifications, and other embodiments of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the disclosure, and therefore the following claims are not limited to only the foregoing illustrative description.

[0076] Each of the embodiments described herein may be combined individually or in conjunction with one or more other embodiments of the present invention.

[0077] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the compounds, compositions, and methods of use described herein, which are deemed to be within the scope of the compositions and methods disclosed herein.

[0078] The contents of all references, patents and published patent applications cited throughout this application, as well as any drawings accompanying it, are hereby incorporated by reference in their entirety. EXAMPLES

[0079] The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention or to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

[0080] Example 1: Mannan nanogel synthesis protocol (4 days): A. Oxidation and purification of mannan and dextran: Materials: Mannan; FITC dextran; 100 mM sodium acetate (NaOAc buffer, pH 5.5); PD10 desalting column; CENTRI-SEP™ column (Princeton Separations); sodium periodate (NaIO4); SPIN-X® 10k MWCO concentrator (Corning)

[0081] Preparation: For each mannan oxidation performed, prepare one PD10 desalting column by removing the top buffer, cutting off the bottom tip, and attaching it to a clamp stand. Prime the column by filling the NaOAc buffer to the top four times, allowing it to drain completely after each fill.

[0082] If fluorescent dextran is being oxidized, one CENTRI-SEP™ column is prepared for every 500 μg of oxidized dextran required by adding 800 μL of NaOAc buffer to each column and vortexing thoroughly.

[0083] Oxidation: Weigh out mannan (>15 mg / oxidation performed) directly into a 5 mL tube and dissolve in NaOAc buffer to a concentration of 10 mg / mL. Weigh out NaIO4 to a solution volume equal to the volume of mannan solution. The concentration of NaIO4 is 120 mM for oxidation of mannan for nanogel core formulation and 2 mM for oxidation of mannan for secondary mannan addition. NaIO4 is dissolved in NaOAc buffer.

[0084] For each oxidation performed, 1.5 mL of mannan solution is mixed with 1.5 mL of the appropriate NaIO4 solution in a 5 mL tube. Final NaIO4 concentration = 60 mM for the core formulation and 1 mM for the secondary mannan.

[0085] If oxidized fluorescent dextran is being prepared, a 10 mg / mL solution is prepared in NaOAc buffer and for every 500 μg of oxidized dextran required, 60 μL of the dextran solution is mixed with 60 uL of 120 mM NaIO4 solution in a tube.

[0086] The tube is wrapped in foil and allowed to react for 30 minutes with rotation.

[0087] purification: Each oxidized mannan (ox-mannan) reaction solution is added to its own PD10 desalting column and drained completely into waste. A SPIN-X® concentrator is placed under each column and the purified mannans are eluted into the concentrator by adding 3.5 mL of NaOAc buffer to the column and allowing it to drain completely. The concentrators are then spun at 4000×g for 13 minutes.

[0088] Transfer all of the mannan from the top of the concentrator to a 1.7 mL tube, measure the starting volume with a 1 mL pipette, and bring the total volume up to 1.25 mL by adding NaOAc buffer. Assuming zero losses, this is 10 mg / mL.

[0089] For dextran purification, drain the CENTRI-SEP™ column by removing the top and bottom caps and spin the column beds at 800×g for 2 minutes. Then add 100 uL of dextran reaction solution to each column bed stacked in a clean 1.7 mL tube and spin again at 800×g for 2 minutes. Approximately 90 μL is eluted, which means the concentration is approximately 5.6 mg / mL assuming zero loss.

[0090] If not used immediately, store the purified oxidized mannan or dextran at 4°C.

[0091] B. Mannan Self-Assembly: Material: Aniline Preparation of 500 μg nanogel (NG) batch: A total of 500ug (assuming zero losses) of oxidized mannan and dextran are added to 1.7mL tubes for each batch. 5uL of aniline is added to each tube, then the tubes are immediately shaken. All tubes are batch sonicated for approximately 10 seconds each, then left under foil overnight.

[0092] C. Nanogel Crosslinking Materials: Succinic acid dihydrazide (SDH); 3,3'-dithiobis(propanoic acid dihydrazide; DPDH); cystamine dihydrochloride ("cystamine"); ethylenediamine dihydrochloride (EDA); sodium cyanoborohydride (NaCNBH3); acetonitrile (ACN); 100 mM sodium acetate, pH 5.5 (NaOAc buffer); PBS (never DPBS); 1 normal (molar) sodium hydroxide (1N NaOH).

[0093] Dihydrazide (DH) crosslinking: Bath sonicate all tubes to ensure a good pellet on spin. Spin nanogels (NG) from section B above at 15000×g for 4 min. Pipette off supernatant, add 1 mL NaOAc buffer, and resuspend NG using a short pulse from a probe sonicator. Spin NG and resuspend in 1 mL 2:1 (vol) ACN:NaOAc containing 50 mM SDH and 50 mM DPDH. Leave to react overnight under foil with rotation.

[0094] Diamine (DA) crosslinking Spin nanogels (NG) from section B above, wash with 1 mL NaOAc buffer, then spin and resuspend in 1 mL PBS with 50 mM EDA and 50 mM cystamine. Allow to react for 1 hour under foil with rotation. Prepare a 5M solution of NaCNBH3 in 1N NaOH, then add 20 μL of this solution to each NG tube. Allow to react overnight under foil with rotation.

[0095] Storage for later use: After overnight reaction at room temperature, the D-H cross-linked NG can be stored as is at 4 °C for later use.

[0096] Prior to storage at 4° C., the DA-crosslinked NG should be washed once with 1 mL of PBS and then resuspended in 1 mL of PBS.

[0097] D. Secondary Mannan Treatment: Materials: 1 mM ox-mannan (10 mg / mL) from section A; aniline; NaOAc buffer (100 mM sodium acetate, pH 5.5); PBS; sodium cyanoborohydride (NaCNBH3)

[0098] Addition of secondary mannan to DH crosslinked NG: Bath sonicate all tubes to ensure a good pellet on spin. Spin NG (still in DH solution) and wash with 1 mL of 2:1 (vol) ACN:NaOAc. Resuspend each NG batch in 1 mL of NaOAc buffer. Then split each tube into two new tubes with 500 uL (250 ug ox-mannan) each. All samples prepared may be pooled before splitting. All subsequent steps are for this 250 μg half batch size.

[0099] Each new tube is spun and resuspended in 50 μL 1 mM ox-mannan + 350 μL NaOAc buffer and allowed to react overnight under foil.

[0100] Addition of secondary mannan to DA cross-linked NG: Bath sonicate all tubes to ensure a good pellet in the spin. Split the NG (already washed and in 1 mL PBS) into two new tubes with 500 μL (250 ug ox-mannan) each. All samples prepared may be pooled before splitting. All subsequent steps are for this 250 μg half batch size.

[0101] Spin and resuspend each new tube in 50 μL 1 mM ox-mannan + 350 μL PBS and leave to react under foil for 1 hour. Prepare a 5M solution of NaCNBH3 in 1N NaOH. Add 10 μL of this solution to each NG tube and leave to react overnight under foil.

[0102] E. Activation and cargo loading: Materials: PBS; tris(2-carboxyethyl)phosphine (TCEP); 18 MΩ deionized water (DI); 100% methanol (MeOH); sodium borohydride (NaBH4); 2,2-dithiopyridine (DTP); glacial acetic acid (AcOH); glutathione, reduced (GSH); dimethyl sulfoxide (DMSO); 10 normal (molar) sodium hydroxide (10 N NaOH); peptides.

[0103] Reduction of disulfide bridges to thiols with TCEP: Bath sonicate all tubes to ensure a good pellet on spin. Prepare a 100 mM solution of TCEP in PBS, then for every 10 mL, add 350 µL of 10 N NaOH to this solution. Ensure pH 6.9-7.5. Spin and resuspend NGs (DH and DANG treated similarly in subsequent steps) in 1 mL of TCEP solution and leave to react for 1 h under foil with rotation.

[0104] The imine and residual aldehydes are reduced with borohydride (this step can be omitted for DA-bridged NGs without damaging them): Wash the NG once with 1 mL of deionized water and once with 1 mL of MeOH. Resuspend the NG in 1 mL of 100 mM NaBH4 in MeOH and allow to react for 1 h.

[0105] Activating thiols with DTP: Wash NG with 1 mL MeOH (if above step is omitted, wash with 1 mL deionized water first) and resuspend in 100 mM DTP in MeOH:AcOH (160:1). Incubate under foil with rotation for 4 hours.

[0106] Thiol cargo load (cargo frequently changes between experiments): Cargo solution is prepared by dissolving GSH in deionized water to 200 mM, then diluting with DMSO to 20 mM. Dissolve peptide in DMSO to 20 mM. Dissolution of 200 mM in either deionized water or other solvent may be required for some peptides. NG is washed with 1 mL MeOH. A total of 100 μL of 20 mM cargo solution is added to each tube, then add 900 μL MeOH and resuspend. Allow to react overnight under foil with rotation.

[0107] Preparation for delivery: Wash the NG once with MeOH and once with deionized water, resuspend in either 200 uL (=1000x concentration) or 1 mL (200x concentration) of sterile PBS and sonicate in a bath sonicator (approximately 30 s to 1 min).

[0108] Example 2: Nanogel-RNA delivery by conjugation of thiol-modified RNA via disulfide bonds: A. Materials: Tris(2-carboxyethyl)phosphine (TCEP); 100 mM sodium acetate (NaOAc buffer, pH 5.5); CENTRI-SEP™ column (Princeton Separations); sodium periodate (NaIO4); sodium cyanoborohydride (NaCNBH3); cystamine dihydrochloride ("cystamine"); phosphate buffer saline (PBS, pH 7.4); 1 normal (molar) sodium hydroxide (1N NaOH).

[0109] B. Oxidation of RNA 3': Mix 50 μL of a 20 mM solution of NaIO4 in NaOAc buffer with 50 μL of 10 mg / mL RNA and leave to react under foil for 30 minutes. Purify the oxidized RNA using a CENTRI-SEP™ column in PBS.

[0110] C. Reductive amination of RNA 3' aldehyde: 10 μL of 100 mM cystamine in PBS is added to the oxidized RNA and allowed to react for 1 hour. Then, μL of a 5 M solution of NaCNBH3 in 1 N NaOH is added and allowed to react for 2 hours. The oxidized RNA is purified using a CENTRI-SEP™ column in PBS.

[0111] D. Reduction with TCEP to expose the RNA 3' thiol: 10 μL of 100 mM TCEP in PBS (pH approx. 7) is added to the cystamine modified RNA and allowed to react for 1 hour. The oxidized RNA is purified using a CENTRI-SEP™ column in PBS.

[0112] E. RNA-nanogel linking / complex formation: A suspension of mannan nanogel in PBS is added to a solution of 2 μg of thiol-modified RNA in the DTP-activated state (see nanogel protocol in Example 1) to a total volume of 200 μL (the amount of nanogel used can be varied to achieve different nanogel:RNA ratios; the highest expression was achieved with a weight:weight ratio of approximately 25). The mixture is then vortexed on high for approximately 30 seconds and allowed to react overnight under foil.

[0113] Example 3: Nanogel-RNA delivery by electrostatic complexation of RNA with nanogels: RNA-nanogel complex formation: A suspension of DiAm (cationic) mannan nanogel in PBS in the DTP-activated state (see nanogel protocol in Example 1) is added to a solution of 2 μg of unmodified RNA for a total volume of 200 μL (the amount of nanogel used can be varied to achieve different nanogel:RNA ratios; the highest expression was achieved with a weight:weight ratio of approximately 25). The mixture is then vortexed on high for approximately 30 seconds and allowed to react for 15 minutes.

[0114] Example 4: Methods for CD206-expressing 293T cells: A CMV promoter-driven CD206 expression plasmid was constructed with a codon-optimized CD206 sequence and a puromycin resistance gene. 293T cells were transfected with the CD206 expression plasmid using Lipofectamine2000 according to the manufacturer's recommended conditions and placed under puromycin selection for 2 weeks. Single CD206 positive cells were cultured in 96-well plates using a cell sorter, and 293T clones showing CD206 expression were selected.

Claims

1. 1. A method for making a self-assembling mannan nanogel for in vivo delivery of a therapeutic agent, comprising: a. Oxidizing mannan with periodate (NaIO4); b. purifying the oxidized mannans; c. adding aniline to the purified oxidized mannans to produce a mannan derivative having a hydrophobic phenylimine group covalently attached to the mannans; and d. Sonicating the mannan derivative A method comprising:

2. 2. The method of claim 1, comprising introducing a dihydrazide (DH) crosslinker to the self-assembled mannan nanogel and reacting the mannan nanogel with succinic dihydrazide (SDH) and 3,3'-dithiobis(propanoic dihydrazide) (DPDH).

3. Dihydrazide-crosslinked mannan nanogels were prepared for loading with thiol-containing cargo by reducing the nanogel disulfide bridges with tris(2-carboxyethyl)phosphine (TCEP) and catalyzing the nanogel imines and residual aldehydes with borohydride (NaBH ４ 3. The method of claim 2, comprising reducing the nanogel thiol with 2,2-dithiopyridine (DTP) and activating the nanogel thiol with 2,2-dithiopyridine (DTP).

4. The method of claim 3, wherein the thiol-containing cargo is loaded onto the DTP-activated nanogel, the cargo consisting of one or more peptides and, optionally, glutathione (GSH).

5. The method of claim 2, wherein the dihydrazide cross-linked nanogel is coated with NaIO4 oxidized mannan.

6. A diamine (DA) crosslinker was introduced into the mannan nanogel, which was then crosslinked with cystamine and ethylenediamine dihydrochloride (EDA) and then sodium cyanoborohydride (NaCNBH ３ 2. The method of claim 1 , comprising sequentially reacting

7. 7. The method of claim 6, wherein the diamine cross-linked mannan nanogels are prepared for loading with thiol-containing cargo, comprising reducing the nanogel disulfide bridges with (tris(2-carboxyethyl)phosphine) (TCEP) and activating the nanogel thiols with 2,2-dithiopyridine (DTP).

8. The method of claim 7, wherein the thiol-containing cargo is loaded onto the DTP-activated nanogel, the cargo consisting of one or more peptides and, optionally, glutathione (GSH).

9. The diamine cross-linked nanogels were coated with NaIO4 oxidized mannan and then sodium cyanoborohydride (NaCNBH ３ 7. The method of claim 6, wherein the reactant is a carboxylic acid.

10. 4. A method for loading thiol-modified RNA onto a DTP-activated mannan nanogel according to claim 3, comprising: a) reductive amination of oxidized RNA by sequentially reacting the oxidized RNA with cystamine and TCEP; b) purification; and c) Addition to DTP-activated nanogels The method includes:

11. 8. A method for loading thiol-modified RNA onto a DTP-activated mannan nanogel according to claim 7, comprising: a) reductive amination of oxidized RNA by sequentially reacting the oxidized RNA with cystamine and TCEP; b) purification; and c) Addition to DTP-activated nanogels The method includes:

12. A method for loading RNA onto a DTP-activated mannan nanogel as described in claim 7, comprising adding unmodified RNA to the DTP-activated nanogel.

13. 1. A composition comprising CD206-expressing 293T cells, wherein the 293T cells are genetically engineered to stably express a gene having the sequence of SEQ ID NO:

1.

14. A method for quantifying cellular uptake of mannan nanogel or cargo-loaded mannan nanogel, comprising treating CD206-expressing 293T cells with mannan nanogel, the 293T cells being genetically engineered to stably express a gene having the sequence of SEQ ID NO:1, and cellular uptake of the nanogel or intracellular expression of the cargo being quantified.

15. 15. The method of claim 14, wherein the quantification is by fluorescence, luminescence, viability, apoptosis, cell size, cell proliferation, spheroid formation, cell surface expression, or subcellular localization.

16. The method of claim 14, wherein the mannan nanogel is doped with fluorescently labeled dextran.

17. 17. The method of claim 16, wherein the fluorescent label is fluorescein isothiocyanate (FITC).

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