Novel insulin analogue capable of blocking insulin fibrosis

Abstract

Provided herein are engineered insulin polypeptides that exhibit blocked cross-beta fibrosis, and pharmaceutical compositions comprising the polypeptides. The present disclosure also provides methods of making insulin polypeptides, and methods of treating diabetes and hyperglycemia using the pharmaceutical compositions.

Description

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 510,024, filed June 23, 2023, the entirety of which is incorporated by reference herein.

[0003] II. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under project numbers GM142937, DK063608, DK132710, GM130289, and GM136976, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] III. REFERENCE TO SEQUENCE LISTING The sequence listing (XML file name: 106546-805672-4235_SequenceListing, created on June 21, 2024, having a file size of 68 KB) filed on June 21, 2024, is incorporated by reference herein pursuant to 37 C.F.R. § 1.52(e)(5).

[0005] IV. TECHNICAL FIELD The present invention relates to insulin polypeptides and pharmaceutical compositions comprising these polypeptides. The disclosure also encompasses methods of making and using the disclosed compositions.

[0006] V. BACKGROUND Type 1 diabetes mellitus or insulin-dependent diabetes mellitus is an autoimmune disease caused by the immune system destroying beta cells. Currently, about 37.3 million people (11.3% of the total U.S. population) have diabetes, and another 96 million people aged 18 and older are pre-diabetic. If not treated promptly, diabetes can cause a variety of complications, including acute complications such as diabetic ketoacidosis and non-ketotic hyperosmolar coma. Long-term complications include heart disease, stroke, kidney failure, foot ulcers, and eye damage. There is currently no effective method to prevent type 1 diabetes, and insulin therapy is necessary to maintain survival.

[0007] Insulin is produced by pancreatic beta cells. Insulin activates the insulin receptor (IR) and regulates glucose levels in the body of an animal. Mature insulin comprises an A chain and a B chain connected by two intermolecular disulfide bonds, and the A chain further contains an additional intramolecular disulfide bond. Multiple insulin molecules bind cooperatively to two different sites of the insulin receptor, promoting receptor activation, and thus triggering downstream signaling cascades that promote glucose entry into cells. Failure of pancreatic beta cells to produce sufficient insulin leads to type 1 diabetes. Recombinant insulin has therefore been widely used to treat type 1 diabetes.

[0008] However, insulin is prone to undergo an alpha to beta conformational transition under regular storage conditions (low pH and high concentration), ultimately forming amyloid fibrils. The fibrillar form of insulin is unable to bind to or activate the IR, resulting in reduced efficacy in treating type 1 diabetes. Furthermore, repeated injection of insulin can induce insulin-derived amyloidosis, i.e., deposition of insulin fibrils in subcutaneous tissue. Current US Pharmacopoeia requires that insulin be discarded when the degree of fibrillation reaches 1% and above. Because high temperature accelerates the fibrillation process, it is recommended that insulin be stored cold before use by a diabetic patient. Fibrillation of insulin or insulin analogs is of particular concern for patients using an external insulin pump, which injects small amounts of insulin or insulin analogs at regular intervals into the patient. Under this mode of use, the insulin or insulin analogs cannot be kept cold in the pump device, and fibrillation of the insulin can cause blockage of the catheter used to inject the insulin or insulin analogs into the body, which can induce unpredictable fluctuations in blood glucose levels and even dangerous hyperglycemia.

[0009] However, the molecular details of insulin fibrillation are not well understood, hindering efforts to prevent the fibrillation process. There is therefore a need to develop insulin analogs with reduced degree of fibrillation to improve storage and patient outcomes.

[0010] VI. SUMMARY In certain aspects, the disclosure encompasses an engineered insulin polypeptide comprising at least one mutation in the engineered insulin polypeptide, wherein the engineered insulin polypeptide exhibits blocked cross-beta fibrillation. In certain aspects, the at least one mutation is in the A-chain polypeptide. In certain aspects, the at least one mutation is in a hydrophobic core amino acid of the cross-beta fold of the protomer. In certain aspects, the at least one mutation is ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, IleA10Arg, IleA10Lys, IleA10Phe, IleA10Tyr, IleA10Trp, IleA10Gln, IleA10Glu, AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, AsnA18Gln, or any combination thereof.

[0011] In an aspect, the at least one mutation is ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, ThrA8Ala, ThrA8Asp, ThrA8Asn, ThrA8Cys, ThrA8Gly, ThrA8His, ThrA8Ile, ThrA8Leu, ThrA8Met, ThrA8Pro, ThrA8Ser, or ThrA8Val.

[0012] In an aspect, the at least one mutation is SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, SerA9Ala, SerA9Asp, SerA9Asn, SerA9Cys, SerA9Gly, SerA9His, SerA9Ile, SerA9Leu, SerA9Met, SerA9Pro, SerA9Thr, or SerA9Val.

[0013] In an aspect, the at least one mutation is IleA10Arg, IleA10Lys, IleA10Phe, IleA10Tyr, IleA10Trp, IleA10Gln, IleA10Glu, IleA10Ala, IleA10Asp, IleA10Asn, IleA10Cys, IleA10Gly, IleA10His, IleA10Leu, IleA10Met, IleA10Pro, IleA10Ser, IleA10Thr, or IleA10Val.

[0014] In an aspect, the at least one mutation is AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, AsnA18Gln, AsnA18Ala, AsnA18Asp, AsnA18Cys, AsnA18Gly, AsnA18His, AsnA18Ile, AsnA18Leu, AsnA18Met, AsnA18Pro, AsnA18Ser, AsnA18Thr, or AsnA18Val.

[0015] In certain aspects, the A-chain polypeptide comprises an amino acid sequence that is at least about 80% identical to a sequence provided in SEQ ID NOs: 1-76, or a functional derivative thereof. In certain aspects, the engineered insulin polypeptide further comprises one or more additional mutations.

[0016] In certain aspects, the disclosure also encompasses a composition comprising an engineered insulin polypeptide disclosed herein, and at least one pharmaceutically acceptable excipient. In certain aspects, the pharmaceutically acceptable excipient is a liquid or solid filler, diluent, binding agent, buffer, pH adjusting agent, disintegrant, dispersant, preservative, lubricant or wetting agent, taste masking agent, antioxidant, carrier, adjuvant, stabilizer, emulsifying agent, solubility enhancer, salt, solubilizer, antifoaming agent, surfactant, flavoring agent, coloring agent, solvent, or encapsulating material, or any combination thereof. In certain aspects, the composition further comprises an additional active agent. In certain aspects, the additional active agent is glucagon or GLP-1. In certain aspects, the composition is formulated to provide at least about 5 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 150 U / mL, 200 U / mL, 250 U / mL, 300 U / mL, 350 U / mL, 400 U / mL, 450 U / mL, or 500 U / mL of insulin. In certain aspects, the composition is formulated for multiple dosing or single dosing. In certain aspects, the composition is formulated for delivery by a syringe, closed loop system, insulin pen, or insulin pump.

[0017] In certain aspects, the disclosure also encompasses a polynucleotide sequence encoding an engineered insulin polypeptide disclosed herein. In certain aspects, the polynucleotide sequence is a plasmid vector, viral vector, transposon, gene edited polynucleotide sequence, or virus.

[0018] In certain aspects, the disclosure also encompasses a host cell comprising the engineered insulin polypeptide or a polynucleotide encoding the engineered insulin polypeptide, or both. In certain aspects, the host cell is a bacterial, fungal, or mammalian cell.

[0019] In certain aspects, the disclosure also encompasses a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition disclosed herein. In certain aspects, the subject is pre-diabetic, or has or is diagnosed with type 1 diabetes, type 2 diabetes, or hyperglycemia. In certain aspects, the administration is by any of parenteral, oral, intra-arterial, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, intravascular, liposomal, topical, mucosal, subcutaneous, sublingual, topical, transbuccal, or transdermal routes. In certain aspects, the administration is chronic administration.

[0020] In certain aspects, the present disclosure also encompasses a method: (i) can prevent and / or reduce the risk of unpredictable fluctuations in blood glucose levels or hyperglycemia, (ii) can prolong the survival of the subject, (iii) can enhance and / or improve the quality of life of the subject, (iv) can improve and / or restore normal metabolic function of one or more organ systems of the subject, (viii) can restore and / or improve one or more aspects of cellular homeostasis and / or cellular functionality, and / or a metabolic disorder, or (ix) any combination thereof, after the administering step.

[0021] In one embodiment, the subject is a human subject.

[0022] In certain aspects, the present disclosure also encompasses a kit comprising the compositions and instructions for use disclosed herein. In certain aspects, the kit further comprises means for administering the compositions disclosed herein.

[0023] VII. BRIEF DESCRIPTION OF DRAWINGS The accompanying drawings and examples are intended to be illustrative only and not limiting.

[0024] Figure 1A Representative cryo-EM micrographs of insulin fibril samples are shown.

[0025] Figure 1B 2D class averages of Type I, Type II, and Type III insulin fibrils are provided.

[0026] Figure 1C Cryo-EM maps of Type I, Type II, and Type III insulin fibrils are provided. Type I fibrils are composed of one protofilament, while Type II and Type III fibrils are composed of two protofilaments arranged in an anti-parallel and parallel fashion, respectively. Only the Type II fibril was resolved to sufficient resolution to build an accurate model.

[0027] Figure 1D FSC curves (left) of the two half cryo-EM maps of Type II insulin fibril and the model optimized based on the cryo-EM maps (right) are shown.

[0028] Figure 1E Slices of the cryo-EM map of Type II insulin fibril are provided.

[0029] Figure 1F A local resolution map of Type II insulin fibril is provided.

[0030] Figure 1G The Type II insulin fibril model is shown in two perspectives. The Type II insulin fibril is composed of two similar protofilaments, with the insulin A and B chains labeled in blue and green, respectively.

[0031] Figure 1H Cryo-electron microscopy images of type II insulin fibers and their corresponding models are provided.

[0032] Figure 1I This is a schematic diagram of a single profilament in type II insulin fibers.

[0033] Figure 2A The fibrosis of wild-type insulin and the designed mutant was shown and characterized by ThT detection.

[0034] Figure 2B The images show negatively stained electron micrographs of wild-type insulin and mutants designed to have reduced fibroblast-forming ability.

[0035] Figure 3A For Western blot analysis, IR autophosphorylation (pY IR) induced by treatment with a specified concentration of wild-type insulin or insulin mutant for 10 minutes was shown in 293FT cells expressing IR-WT.

[0036] Figure 3B Provided Figure 3A Quantification of Western blot data is shown. All data points are from 4 independent experiments (n=4). Data are expressed as mean ± SEM. pY IR levels were normalized to total IR levels and are shown as relative intensity values ​​of IR relative to the intensity of IR in cells treated with 10 nM wild-type insulin.

[0037] Figure 3C For Western blot analysis, IR autophosphorylation (pY IR) induced by 10 nM wild-type insulin or insulin mutant at specified time points was shown in 293FT cells expressing IR-WT.

[0038] Figure 3D Provided Figure 3C Quantification of Western blot data is shown. IleA10R 10 min, n=3; all other data points were performed 4 times independently (n=4). Mean ± SEM. pY IR levels were normalized to total IR levels and are shown as relative intensity values ​​of IR relative to the intensity of IR in cells treated with 10 nM wild-type insulin.

[0039] Figure 3E For Western blot analysis, IR autophosphorylation (pY IR) induced by treatment with 100 nM wild-type insulin or insulin mutant for 10 min was shown in IR-WT-expressing 293FT cells. Insulin stock solution was incubated at room temperature for 3 days.

[0040] Figure 3F Provided Figure 3EQuantification of the shown Western blot data. All data points are from 6 independent experiments (n=6). Data are presented as mean ± SEM. pY IR levels were normalized to total IR levels and shown as relative intensity values to the intensity of IR in 100 nM wild-type insulin treated cells.

[0041] Figures 4A to 4C Mutant AsnA18Gln vs. native insulin are shown in comparison for their stability after 16 hours of high temperature exposure at 70°C. The insulin receptor autophosphorylation levels ( Figure 4A ), AKT active phosphorylation levels ( Figure 4B ), and ERK active phosphorylation levels ( Figure 4C ) were determined using quantitative Western blot analysis. n=3. Mean ± SEM.

[0042] Figure 5 Mutant AsnA18Gln vs. native insulin are shown in comparison for their performance in an insulin tolerance test after 14 days of incubation at 37°C. n=7 per group. Mean ± SEM.

[0043] Figure 6 Comparison of the B-chain peptide segment in the crystal structure and the cryo-EM structure is shown. The insulin B-chain residues B11-B17 (LVEALYL) adopt a similar beta-strand conformation in the truncated peptide crystal structure and the full-length insulin fiber cryo-EM structure. However, this peptide segment forms a homotypic staggered stack in the crystal structure, rather than interacting with the insulin A-chain as seen in the cryo-EM structure.

[0044] VIII. DETAILED DESCRIPTION The following detailed description refers to the accompanying drawings that show various embodiments of the present disclosure. The drawings and description are intended to fully describe various aspects and embodiments of the present disclosure to enable one of ordinary skill in the art to practice the present disclosure. Other components can be used and modifications can be made without departing from the scope of the present disclosure. Accordingly, the following description should not be construed as limiting.

[0045] Insulin is a hormone that maintains normal blood glucose levels by activating the insulin receptor (IR) and is the main drug used to treat diabetes. Mature insulin comprises an A chain and a B chain, which are connected by two intermolecular disulfide bonds, and the A chain also contains an additional intramolecular disulfide bond. However, insulin is prone to unfolding and forms cross-β fibers through an α to β conformational transition. This fibration complicates the production, storage, and use of insulin. As used herein, the terms "fibration," "cross-β fibration," or "amyloid fibration" or "cross-β fiber" are used interchangeably and refer to a fibrillar protein aggregate formed through self-assembly, characterized by a fiber morphology of 7-13 nm in diameter, typically exhibiting a β-sheet secondary structure (i.e., a cross-β conformation), and can be stained by specific dyes (e.g., Congo red). In the current context, the formation of cross-β fibers in insulin is associated with loss of insulin function. The molecular details of insulin fibration are not well understood, which hinders efforts to prevent this fibration.

[0046] In some aspects, the present disclosure is based on the characterization of the insulin fiber structures disclosed herein by cryo-electron microscopy (Cryo-EM), which shows multiple forms built from different arrangements of protofilaments containing insulin A and B chains connected by disulfide bonds. The cryo-EM structures of the insulin fibers disclosed herein are the first amyloid fiber structures to clearly resolve the disulfide bonds. The structures reveal highly distorted β-sheets of insulin chains and their interactions as the basis for fibration. Constrained by both intra- and intermolecular disulfide bonds, the insulin fiber protofilaments exhibit highly distorted cross-β sheets. Structural characterization shows that full-length insulin forms diverse fiber forms containing different numbers of protofilaments. In the cryo-EM structure, two protofilaments are stacked in an antiparallel and asymmetric manner. In addition, another insulin fiber form containing two parallel stacked protofilaments is also observed.

[0047] Based on the structures disclosed herein, a series of insulin mutants that do not fibrate but retain wild-type IR signaling activity when activating the insulin receptor (IR) were conceived, generated, and successfully validated. In some aspects, the present disclosure encompasses engineered insulin polypeptides or designed insulin analogs, as well as pharmaceutical compositions comprising these polypeptides that can be effective for treating type 1 diabetes and related conditions. In certain aspects, the present disclosure also encompasses methods of making and using the compositions disclosed herein. These compositions can have multiple advantages over existing products, including: a) ease of production due to reduced propensity for insulin aggregation; b) ease of storage and extended shelf life; c) storage at room temperature; d) suitability for use with insulin pumps; e) improved dosing efficacy due to low aggregation propensity.

[0048] The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. For example, the use of "including" "comprising" "having" "containing" "involving" "characterized by" "characterized into" and variations thereof herein, is meant to encompass the item listed thereafter, and equivalents thereof as well as additional items. Further, the use of relational terms, such as "top," "bottom," "left," "right," "upper," "lower," "upward," "downward," "vertical," "horizontal," and the like are used for clarity in only the description of the aspects and are not limiting of the scope of the disclosure or the appended claims. For example, "upper" can be understood as "lower" and "lower" can be understood as "upper" and "left" can be understood as "right" and "right" can be understood as "left".

[0049] Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and instrumentalities shown and described. Any one of the features of the disclosure can be employed alone, or in combination with other features in various aspects of the disclosure. Descriptions in the specification of terms, such as "aspect," "aspects," and / or the like, refer to aspects of the description that are only described in one aspect. Separate references to "aspect," "aspects," and / or the like in this description do not necessarily refer to the same aspect, nor do they necessarily exclude possible incorporations of features of the described aspects in other aspects. For example, features described in one aspect can also be included in other aspects, although they are not necessarily included. Accordingly, various combinations and / or integrations of the described aspects are also contemplated. Further, not all aspects of the disclosure are necessarily required to practice the disclosure as described herein. Likewise, other systems, methods, features, and advantages of the disclosure will be apparent from the foregoing description, and from the accompanying drawings and claims. All of such additional systems, methods, features and advantages are intended to be included within the description that follows and are contemplated as falling within the scope of the disclosure. It is also noted that the specific

[0050] Any degree terms used in the specification, such as but not limited to "substantially", should be understood to encompass exact configurations or similar but not exact configurations. For example, "substantially planar surface" refers to both exact planar surfaces and similar but not exact planar surfaces. Similarly, terms such as "about" or "approximately" used in the specification and claims should be understood to encompass the recited numerical values, or values that are three times the recited numerical values, or values that are one third of the recited numerical values. For example, about 3 mm encompasses all values from 1 mm to 9 mm, and about 50 degrees encompasses all values from 16.6 degrees to 150 degrees. For example, they can refer to a range of less than or equal to ±5%, such as less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.2%, less than or equal to ±0.1%, less than or equal to ±0.05%.

[0051] In the disclosure, the terms "comprise", "include" and "have" are used interchangeably. The terms "comprise", "include" and "have" mean including, but not necessarily limited to, the described content.

[0052] As used herein, the terms "or" and "and / or" shall be construed to encompass both the inclusive and exclusive OR, unless otherwise indicated herein or by context or by understanding thereof within the field. Accordingly, "A, B, or C" or "A, B, and / or C" shall mean any of the following: "A," "B," or "C;" "A and B;" "A and C;" "B and C;" "A, B, and C." This definition applies regardless of the manner in which the word "or" or the phrase "and / or" is used.

[0053] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein. A "nucleic acid encoding for" or "nucleic acid molecule encoding for" shall be understood to refer to a nucleotide sequence that encodes a polypeptide.

[0054] A polynucleotide described herein can comprise one or more nucleic acids, each nucleic acid encoding a polypeptide and operably linked to one or more regulatory sequences (i.e., in functional relationship with one or more regulatory sequences), such as a promoter. Such a polynucleotide can also be referred to herein as a "nucleic acid construct" or "construct." As used herein, the term "operably linked" refers to functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or gene (or transgene) coding sequence such that the promoter, etc., is capable of initiating, assisting, influencing, eliciting, and / or facilitating transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in a particular tissue, developmental stage, and / or condition.

[0055] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a compound comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must comprise at least two amino acids, and there is no maximum number of amino acids in a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids linked to one another by peptide bonds. As used herein, the term refers to both short chains (often referred to in the art as peptides, oligopeptides, and oligomers, among others) as well as longer chains (often referred to in the art as proteins). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide can comprise a natural peptide, a recombinant peptide, or a combination thereof.

[0056] In the context of the present application, a protein is represented by an amino acid sequence, and correspondingly, a nucleic acid molecule or polynucleotide is represented by a nucleic acid sequence. Identity and similarity between sequences: Throughout the present application, each time a specific amino acid sequence SEQ ID NO (as an example SEQ ID NO: Y) is mentioned, it can be replaced by: a polypeptide represented by an amino acid sequence comprising a sequence having at least 60% sequence identity or similarity with the amino acid sequence SEQ ID NO: Y. Another preferred sequence identity or similarity level is 65%. Another preferred sequence identity or similarity level is 70%. Another preferred sequence identity or similarity level is 75%. Another preferred sequence identity or similarity level is 80%. Another preferred sequence identity or similarity level is 85%. Another preferred sequence identity or similarity level is 90%. Another preferred sequence identity or similarity level is 95%. Another preferred sequence identity or similarity level is 98%. Another preferred sequence identity or similarity level is 99%.

[0057] Each of the amino acid sequences described herein, based on their percentage of identity or similarity to a particular amino acid sequence, has in more preferred aspects at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, respectively. The terms "homology", "sequence identity" and the like are used interchangeably herein. Sequence identity as described herein refers to the relatedness between two or more amino acid (polypeptide or protein) sequences or between two or more nucleic acid (polynucleotide) sequences as determined by comparing the sequences. In preferred aspects, sequence identity is calculated over the full length of the two given SEQ ID NOs or a portion thereof. A portion thereof preferably refers to at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the two SEQ ID NO sequences. In the art, "identity" also refers to the degree of sequence correlation between amino acid or nucleic acid sequences (as the case can be) which is determined by the degree of matching between such sequence strings. The degree of sequence identity can be determined, for example, by comparing two sequences using a computer program commonly used for this purpose, such as a global or local alignment algorithm. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or other suitable methods or algorithms. The Needleman and Wunsch global alignment algorithm can be used to align the full length or a portion (a portion can refer to at least 50%, 60%, 70%, 80%, or 90% of the length of the sequence) of two sequences to maximize the number of matches and minimize the number of gaps. Default settings can be employed, with preferred programs being: Needle for pairwise alignment (in one aspect: using EMBOSS Needle version 6.6.0.0, gap opening penalty: 10, gap extension penalty: 0.5, end gap penalty: off (false), end gap opening penalty: 10, end gap extension penalty: 0.5); MAFFT for multiple sequence alignment.(In one aspect, MAFFT v7 default values were used: BLOSUM62 [bl62], Gaps Open: 1.53, Gaps Extend: 0.123, Alignment: Aligned, Tree Rebuilds: 2, Guide Tree Output: On [True], Max Iterations 2, FFTS Execution: Off (none)).

[0058] As used herein, the term "treatment" refers to reversing, alleviating, inhibiting the progress of, or preventing the disease or condition to which such term applies, or ameliorating one or more symptoms of the condition or disease. "Treatment" or "therapy" of a subject refers to any type of intervention performed on the subject in an effort to alter the disease's progression, or to improve the disease's symptoms, complications, conditions, or biochemical indicia, either physically, physiologically, or both. In certain aspects, the disease is a cancer and / or tumor as described herein.

[0059] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness, and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0060] An "effective amount" of a compound refers to an amount necessary or sufficient to treat or prevent cancer or any other disease or condition associated with ISR. In one embodiment, an effective amount of a compound described herein is an amount sufficient to treat leukemia; in one embodiment, an effective amount of a compound described herein is an amount sufficient to treat colon cancer. The effective amount can vary depending on the size and weight of the subject, the type of disease, or the particular class of compound, among other factors. For example, the selection of a compound described herein will affect what constitutes an "effective amount." One of ordinary skill in the art can determine an effective amount of a compound described herein without undue experimentation, by utilizing the factors noted herein.

[0061] The term "pharmaceutically acceptable excipient" includes any pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, binding agent, buffer, pH adjusting agent, disintegrating agent, dispersing agent, preservative, lubricant or wetting agent, taste modifying agent, antioxidant, carrier, adjuvant, stabilizer, emulsifying agent, solubility enhancing agent, salt, solubilizer, antifoaming agent, surfactant, flavoring agent, coloring agent, solvent, or encapsulating material, or any combination thereof, which is suitable for use in administering a compound described herein to a mammal. Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The nature and quantity of excipients used in the formation of a pharmaceutical composition can be selected according to principles known in the pharmaceutical sciences. In each aspect described herein, the compositions of the present disclosure can optionally comprise one or more additional pharmaceutical or therapeutically active agents in addition to at least one of the factors disclosed herein. Thus, a subject can be provided with other therapies known to be effective for a disease, disorder, or condition in addition to the therapies described herein.

[0062] A. Compositions In certain aspects, the compositions of the present disclosure encompass engineered polypeptides and pharmaceutical compositions comprising the polypeptides. In another aspect, the compositions of the present disclosure encompass engineered polynucleotides encoding the polypeptides described herein, and / or host cells comprising a polynucleotide encoding an engineered polypeptide described herein and / or comprising an engineered polypeptide described herein.

[0063] In one aspect, the present disclosure encompasses an engineered insulin polypeptide or functional derivative or analog thereof comprising at least one mutation in the insulin polypeptide, wherein the polypeptide exhibits blocked cross-β fiberization. In certain aspects, the at least one mutation is in the A chain of the polypeptide. In certain aspects, the at least one mutation is in a hydrophobic core amino acid of the cross-β fold of the proinsulin body. In certain aspects, the mutation is in a mutable residue (e.g., Thr A8, Ser A9, lie A10, or Asn A18).

[0064] In various aspects, the at least one mutation is selected from the group consisting of ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, IleA10Arg, IleA10Lys, IleA10Phe, IleA10Tyr, IleA10Trp, IleA10Gln, IleA10Glu, AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, and AsnA18Gln, wherein the first three letters correspond to the amino acid to be mutated, the fourth letter plus number corresponds to the chain and residue number (e.g., A18 corresponds to the 18th residue of the A chain), and the last three letters correspond to the new amino acid.

[0065] In certain aspects, the mutation occurs at a residue, such as ThrA8, SerA9, IleA10, or AsnA18, which can be mutated to any of the remaining 19 naturally occurring amino acids, thereby effectively reducing the fibrillation of insulin. In one aspect, the at least one mutation is selected from the group consisting of ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, ThrA8Ala, ThrA8Asp, ThrA8Asn, ThrA8Cys, ThrA8Gly, ThrA8His, ThrA8Ile, ThrA8Leu, ThrA8Met, ThrA8Pro, ThrA8Ser, and ThrA8Val.

[0066] In another aspect, the at least one mutation is selected from the group consisting of SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, SerA9Ala, SerA9Asp, SerA9Asn, SerA9Cys, SerA9Gly, SerA9His, SerA9Ile, SerA9Leu, SerA9Met, SerA9Pro, SerA9Thr, and SerA9Val.

[0067] In another aspect, the at least one mutation is Ile A10Arg, Ile A10Lys, Ile A10Phe, Ile A10Tyr, Ile A10Trp, Ile A10Gln, Ile A10Glu, Ile A10Ala, Ile A10Asp, Ile A10Asn, Ile A10Cys, Ile A10Gly, Ile A10His, Ile A10Leu, Ile A10Met, Ile A10Pro, Ile A10Ser, Ile A10Thr, and Ile A10Val.

[0068] In other aspects, the at least one mutation is selected from the group consisting of: Asn A18Arg, Asn A18Lys, Asn A18Phe, Asn A18Tyr, Asn A18Trp, Asn A18Glu, Asn A18Gln, Asn A18Ala, Asn A18Asp, Asn A18Cys, Asn A18Gly, Asn A18His, Asn A18Ile, Asn A18Leu, Asn A18Met, Asn A18Pro, Asn A18Ser, Asn A18Thr, and Asn A18Val.

[0069] In certain aspects, the engineered insulin polypeptide can comprise at least 1, or at least 2, or at least 3, or at least 4, or more mutations in the A chain of the insulin polypeptide. In one aspect, the at least 1, or at least 2, or at least 3, or at least 4, or more mutations are in the hydrophobic core amino acids of the cross-beta fold of the proinsulin body. In one aspect, the at least 1, or at least 2, or at least 3, or at least 4, or more mutations are in the residues, for example, but not limited to, Thr A8 and Ser A9, Ile A10, or Asn A18.

[0070] In one aspect, the mutations can be located at Thr A8 and Ser A9, Thr A8 and Ile A10, Thr A8 and Asn A18, Ser A9 and Ile A10, Ser A9 and Asn A18, Ile A10 and Asn A18.

[0071] In one aspect, the mutations can be located at Thr A8, Ser A9, and Ile A10. In one aspect, the mutations can be located at Thr A8, Ser A9, and Asn A18. In one aspect, the mutations can be located at Thr A8, Ile A10, and Asn A18. In one aspect, the mutations can be located at Ser A9, Ile A10, and Asn A18.

[0072] In certain aspects, the engineered insulin polypeptide comprises an A chain comprising an amino acid sequence that is at least about 80% identical to one or more of the sequences provided in SEQ ID NOs: 1-76 of Table 1, or a functional derivative or analog thereof. In certain aspects, the engineered insulin polypeptide comprises an A chain comprising an amino acid sequence that is at least about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100% identical to one or more of the sequences provided in SEQ ID NOs: 1-76 of Table 1, or a functional derivative or analog thereof. In certain aspects, the B chain of the engineered polypeptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 77, or a functional derivative thereof. In certain aspects, the B chain is at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about, 99%, or about 100% identical to SEQ ID NO: 77, or a functional derivative thereof. As used herein, a functional derivative or analog of insulin includes, but is not limited to, acylated insulin, basic insulin, degludec insulin, insulin isophane, insulin aspart, insulin lispro, insulin glulisine, des (octapeptide) insulin, insulin-zinc complex, high molecular weight insulin, N-carboxyaryl insulin, insulin glargine, detemir insulin, insulin derivatives with lipophilic side chains attached to the B chain, insulin containing long hydrocarbon groups such as palmitoyl, recombinant insulin, or combinations thereof. These derivatives are designed in the art to improve the production, delivery, and / or use of insulin, and the mutations disclosed herein can be constructed in the context of these derivatives.

[0073] Table 1

[0074]

[0075] In certain aspects, the present disclosure also encompasses a composition comprising the disclosed engineered insulin polypeptide and at least one pharmaceutically acceptable excipient. In certain aspects, the composition of the present disclosure is formulated with at least one pharmaceutically acceptable excipient into a formulation for administration to a subject in need thereof. In certain aspects, the composition can be formulated into a liquid, gel, or dry powder formulation.

[0076] In certain aspects, the pharmaceutically acceptable excipient can be a liquid or solid filler, diluent, binder, buffer, pH adjusting agent, disintegrant, dispersant, preservative, lubricant or wetting agent, taste masking agent, antioxidant, carrier, adjuvant, stabilizer, emulsifying agent, solubility enhancer, salt, solubilizer, antifoaming agent, surfactant, flavoring agent, coloring agent, solvent, or encapsulating material, or any combination thereof.

[0077] In certain aspects, for example when the composition is formulated as a liquid, the pharmaceutically acceptable excipient can include one or more of a buffer, an isotonicity agent, a preservative, or a combination thereof. In certain aspects, when the composition is formulated as a dry powder, the formulation can further include a filler, a carrier, an additive, a dispersant, a lubricant, or a disintegrant. The class and amount of excipient used to form the composition can be selected according to principles known to those of ordinary skill in the pharmaceutical sciences.

[0078] In certain aspects, the at least one excipient can be a buffer. The buffer can be selected from the group consisting of: sodium acetate, sodium carbonate, citrate, glycine, lysine, arginine, disodium hydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, ADA (N-[2-acetamido]-2-iminodiacetic acid), ACES (N-[2-acetamido]-2-aminoethanesulfonic acid), BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), bicine (N,N-bis[2-hydroxyethyl]glycine), BIS-TRIS (bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane), DIPSO (3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), ethylenediamine dihydrochloride, glycinamide, HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), HEPPSO (N-[2-hydroxyethyl]piperazine-N'-[2-hydroxypropanesulfonic acid]), imidazole, MOBS (4-[N-morpholino]butanesulfonic acid), MOPS (3-[N-morpholino]propanesulfonic acid), PIPES (piperazine-N,N'-bis[2-ethanesulfonic acid]), TAPSO (3-[N-tris[hydroxymethyl]methylamino]-2-hydroxypropanesulfonic acid), THAM (tris[hydroxymethyl]aminomethane), TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), tricine (N-tris[hydroxymethyl]methylglycine), adipic acid, aspartic acid, glutaric acid, malic acid, malonic acid, succinic acid, and / or salts and / or mixtures thereof. In one aspect, the pH of the composition can be about 5.5 to about 8.5. In another aspect, the pH is about 5.8 to about 6.8, about 6.8 to about 7, or about 7.0 to about 8.5.

[0079] In a further aspect, the pharmaceutically acceptable excipient can comprise a pharmaceutically acceptable preservative, e.g., phenol, o-cresol, m-cresol, p-cresol, methyl paraben, propyl paraben, 2-phenoxyethanol, butyl paraben, 2-phenylethanol, benzyl alcohol, chlorobutanol, bronopol, benzoic acid, imidurea, chlorhexidine, sodium dehydroacetate, chlorocresol, ethyl paraben, benzalkonium chloride, chlorphenesin (3-(4-chlorophenoxy)propane-1,2-diol), or mixtures thereof. The use of preservatives in pharmaceutical compositions is well known to those skilled in the art. For ease of reference, see Remington: The Science and Practice of Pharmacy, 19thedition, 1995.

[0080] In one aspect, the pharmaceutically acceptable excipient can comprise an isotonic agent, e.g., a salt (such as sodium chloride), a sugar or sugar alcohol, an amino acid (such as 1-glycine, 1-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol, polyethylene glycol (such as PEG 400), or mixtures thereof. Any sugar, e.g., monosaccharide, disaccharide, or polysaccharide, or water-soluble dextran, e.g., fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. In one aspect, the sugar additive is sucrose. Sugar alcohols are defined as C4-C8hydrocarbons having at least one -OH group, including, e.g., mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabinitol. In one aspect, the sugar alcohol additive is mannitol. The above-mentioned sugars or sugar alcohols can be used individually or in combination. Each of the above-mentioned isotonic agents constitutes an alternative to the application. The use of isotonic agents in pharmaceutical compositions is well known to those skilled in the art. For ease of reference, see Remington: The Science and Practice of Pharmacy, 19thedition, 1995.

[0081] In further aspects, the formulation can include a surfactant. Examples of surfactants that have been disclosed for use in parenteral pharmaceutical compositions include: polysorbates, such as polysorbate 20 (TWEEN® 20); polyethylene glycols, such as PEG 400, PEG 3000; TRITON™ X-100); polyethylene glycols, such as polyoxyethylene (23) lauryl ether (CAS number: 9002-92-0, trade name BRIJ®), alkoxylated fatty acids (such as MYRJ™), polypropylene glycol, block copolymers (such as polyoxyethylene polyoxopropylene ether polymer 188 (CAS number 9003-11-6, trade name PLURONIC® F-68); alkoxylated fatty acids, such as MYRJ™; polypropylene glycol; block copolymers, such as poloxamer 188 (CAS number 9003-11-6, trade name PLURONIC® F-68) and poloxamer 407 (PLURONIC® F127); sorbitan alkyl esters (e.g., SPAN®); polyethoxylated castor oil (e.g., KOLLIPHOR®, CREMOPHOR®); and trehalose and its derivatives, such as trehalose laurate. In certain embodiments, the surfactant is selected from the group consisting of polyoxyethylene (23) lauryl ether, poloxamer 188, and trehalose laurate. Most preferred is poloxamer 188. In certain aspects, the composition is free of surfactants, as it is less prone to fibrosis.

[0082] Typical additives include monosaccharides, disaccharides, and polysaccharides; sugar alcohols and other polyols, such as lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitol, dipalmitoylphosphatidylcholine, or lecithin; or similar substances. In certain aspects, the pharmaceutically acceptable excipient can include ions, such as zinc.

[0083] In certain aspects, the composition can further include an excipient for stabilizing the insulin or derivative thereof, such as a buffer, a salt (e.g., sodium chloride, magnesium chloride), a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins that can be used to formulate insulin include albumin, protamine, and the like. Typical carbohydrates that can be used to formulate insulin include sucrose, mannitol, lactose, trehalose, glucose, and the like.

[0084] The weight fraction of the excipient or combination of excipients in the composition can be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less, of the total weight of the composition.

[0085] The compositions described herein can be formulated using one or more pharmaceutically acceptable excipients, by any of the conventional means, e.g., see A.R. Gennaro, ed., Remington: The Science and Practice of Pharmacy, 21sted., ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations will include a therapeutically effective amount of the biologically active factors described herein (which can be in purified form), as well as an appropriate amount of excipients to provide a dosage form suitable for administration to a subject.

[0086] In aspects described herein, the compositions of the disclosure can optionally include one or more additional pharmaceutical or therapeutically active agents in addition to at least one factor disclosed herein. Thus, a subject can be provided with known other therapies effective for a disease, disorder, or condition in addition to the therapies described herein. Examples of additional pharmaceuticals can include glucagon or glucagon-like peptide-1 (GLP-1).

[0087] In certain aspects, the compositions of the disclosure can be formulated in unit-dose or multi-dose form. In certain aspects, the compositions are formulated to provide at least about 5 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 150 U / mL, 200 U / mL, 250 U / mL, 300 U / mL, 350 U / mL, 400 U / mL, 450 U / mL, or 500 U / mL of insulin. In certain aspects, the compositions of the disclosure are formulated for delivery by a syringe, a closed loop system, an insulin pen, or an insulin pump.

[0088] In certain aspects, the disclosure also encompasses polynucleotide sequences comprising nucleic acid sequences encoding engineered insulin polypeptides comprising an A chain described herein or a B chain described herein or any combination thereof. In certain aspects, the polynucleotide can comprise a nucleic acid sequence encoding an insulin A chain having at least about 85% identity to any one of SEQ ID NOs: 1-76. In certain aspects, the polynucleotide can comprise a nucleic acid sequence encoding an insulin A chain comprising an amino acid sequence having at least about 80% identity to any one of SEQ ID NOs: 1-76 or a functional derivative thereof; and an insulin B chain comprising an amino acid sequence having at least about 90% identity to SEQ ID NO: 77 or a functional derivative thereof. The polynucleotide can comprise a nucleic acid sequence encoding an insulin polypeptide disclosed herein further comprising other mutations or modifications that can provide additional benefits related or unrelated to fibrosis. The nucleic acid sequence can encode a modified A chain or B chain sequence containing unrelated substitutions or extensions at other positions in the polypeptide or modified proinsulin analog. In certain aspects, the polynucleotide can be operably linked to appropriate terminators, polyadenylation signals, transcription enhancer sequences, and translation enhancer sequences as desired. In certain aspects, to direct the insulin peptide into the host cell secretory pathway, the polynucleotide can further encode a secretion signal sequence (also known as a leader sequence, pre-pro sequence, or pre sequence). The secretion signal sequence is linked in-frame to the nucleic acid sequence encoding the polypeptide. The secretion signal sequence is typically located 5' to the nucleic acid sequence encoding the polypeptide. The signal sequence can typically be a sequence associated with the peptide or can be from a gene encoding another secreted protein.

[0089] The polynucleotides disclosed herein can be an isolated polynucleotide, an expression vector, a plasmid vector, a viral vector, a transposon, a gene editing polynucleotide sequence, or a virus, and can be inserted into a host cell, such as a prokaryotic host cell (e.g., an E. coli cell line), or a eukaryotic cell line (e.g., a fungal cell line, such as a S. cerevisiae or Pischia pastoris strain or cell line), or a mammalian cell line (e.g., BHK or CHO). In certain aspects, the polynucleotides can be used in vitro or in vivo to produce the engineered insulin polypeptides of the disclosure. In certain aspects, the host cells disclosed herein can be used to express and purify the disclosed polypeptides. Methods for using these polynucleotides and host expression systems are well known in the art.10 microns.

[0090] B. Methods of manufacture In certain aspects, the present disclosure also encompasses methods of making and using the engineered insulin polypeptides and compositions disclosed herein.

[0091] In certain aspects, the methods of making the engineered insulin polypeptides can employ standard techniques known in the art. For example, in one aspect, the method can comprise: a) cloning a polynucleotide sequence encoding an engineered insulin polypeptide disclosed herein into a suitable expression system (e.g., expression vector, expression cassette, plasmid vector, viral vector, transposon); b) transfecting or transforming the expression vector or cassette into a suitable host cell (e.g., prokaryotic or eukaryotic cell); c) expressing the engineered insulin polypeptide; d) purifying the polypeptide; e) formulating it into a suitable pharmaceutical composition. Methods of cloning and expressing engineered proteins are standard techniques in the art (see, e.g., Sambrook, J, Fritsch, EF and Maniatis, T Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). In one aspect, standard in vitro protein expression methods can be employed to produce the engineered insulin polypeptides in vitro.

[0092] In one aspect, the disclosed methods can restore and / or improve one or more aspects of cellular homeostasis and / or cellular function, and / or metabolic disorders.

[0093] C. Therapeutic methods and uses In certain aspects, the present disclosure encompasses a method for treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed engineered insulin polypeptide or a composition comprising a disclosed engineered insulin polypeptide and at least one excipient as disclosed herein.

[0094] In one aspect, the engineered insulin polypeptide comprises at least one mutation in a residue, e.g., Thr A8, Ser A9, Ile A10, or Asn A18. In certain aspects, the at least one mutation can be Thr A8Arg, Thr A8Lys, Thr A8Phe, Thr A8Tyr, Thr A8Trp, Thr A8Gln, Thr A8Glu, Ser A9Arg, Ser A9Lys, Ser A9Phe, Ser A9Tyr, Ser A9Trp, Ser A9Gln, Ser A9Glu, Ile A10Arg, Ile A10Lys, Ile A10Phe, Ile A10Tyr, Ile A10Trp, Ile A10Gln, Ile A10Glu, Asn A18Arg, Asn A18Lys, Asn A18Phe, Asn A18Tyr, Asn A18Trp, Asn A18Glu, or Asn A18Gln. In one aspect, the at least one mutation can be Thr A8Arg, Thr A8Lys, Thr A8Phe, Thr A8Tyr, Thr A8Trp, Thr A8Gln, Thr A8Glu, Thr A8Ala, Thr A8Asp, Thr A8Asn, Thr A8Cys, Thr A8Gly, Thr A8His, Thr A8Ile, Thr A8Leu, Thr A8Met, Thr A8Pro, Thr A8Ser, or Thr A8Val. In one aspect, the at least one mutation can be Ser A9Arg, Ser A9Lys, Ser A9Phe, Ser A9Tyr, Ser A9Trp, Ser A9Gln, Ser A9Glu, Ser A9Ala, Ser A9Asp, Ser A9Asn, Ser A9Cys, Ser A9Gly, Ser A9His, Ser A9Ile, Ser A9Leu, Ser A9Met, Ser A9Pro, Ser A9Thr, or Ser A9Val. In one aspect, the at least one mutation can be Ile A10Arg, Ile A10Lys, Ile A10Phe, Ile A10Tyr, Ile A10Trp, Ile A10Gln, Ile A10Glu, Ile A10Ala, Ile A10Asp, Ile A10Asn, Ile A10Cys, Ile A10Gly, Ile A10His, Ile A10Leu, Ile A10Met, Ile A10Pro, Ile A10Ser, Ile A10Thr, or Ile A10Val.In one aspect, the at least one mutation can be Asn A18Arg, Asn A18Lys, Asn A18Phe, Asn A18Tyr, Asn A18Trp, Asn A18Glu, Asn A18Gln, Asn A18Ala, Asn A18Asp, Asn A18Cys, Asn A18Gly, Asn A18His, Asn A18Ile, Asn A18Leu, Asn A18Met, Asn A18Pro, Asn A18Ser, Asn A18Thr, or Asn A18Val.

[0095] In certain aspects, the disclosure encompasses a method of reducing blood glucose levels in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered insulin polypeptide disclosed herein, or a composition comprising an engineered insulin polypeptide disclosed herein and at least one excipient disclosed herein. In certain aspects, the subject is suspected of having or is diagnosed with diabetes. In certain aspects, the subject is suspected of having or is diagnosed with type 1 diabetes, type 2 diabetes, prediabetes, or hyperglycemia.

[0096] As used herein, the term "subject" can include an animal or human that receives treatment according to the methods of the disclosure. More specifically, the term subject can include animals for detection, such as animals used in preclinical testing, including but not limited to mice, rats, monkeys, dogs, pigs, and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equines, avians, felines, bovids, murids, canids, and the like. The present disclosure contemplates both human and veterinary applications. The term includes, but is not limited to, birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents (e.g., mice and rats), rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep, chickens, and goats. In certain aspects, the subject is a human, a chicken, or a mouse. In certain aspects, the subject is a human. Both pediatric and adult subjects are contemplated. For example, in any of the methods described herein, the subject can be at least 6 months of age (e.g., 6 months of age and older, 12 months of age and older, 18 months of age and older, 2 years of age and older, 4 years of age and older, 6 years of age and older, 10 years of age and older, 13 years of age and older, 16 years of age and older, 18 years of age and older, 21 years of age and older, 25 years of age and older, 30 years of age and older, 35 years of age and older, 40 years of age and older, 45 years of age and older, 50 years of age and older, 60 years of age and older, 65 years of age and older, 70 years of age and older, 75 years of age and older, 80 years of age and older, 85 years of age and older, 90 years of age and older, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 18, 20, 21, 24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 years of age or older).

[0097] As described herein, the disclosed compositions can be administered to humans and other animals by any suitable route of administration, including: parenteral, oral, intraadiposal, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intra vascular, intravitreal, liposome, local, mucosal, subcutaneous, sublingual, topical, buccal, and transdermal routes. In certain particular aspects, the route and mode of administration of the disclosed compositions can be selected from self-subcutaneous injection (e.g., using a needle and syringe or pen device), or continuous subcutaneous insulin infusion therapy by an insulin pump device, although intravenous, intradermal, or intraperitoneal routes can also be employed. In certain aspects, the disclosure also provides articles of manufacture comprising or containing the disclosed compositions herein. In particular aspects, the article of manufacture is a multi-dose vial; in other aspects, the article of manufacture is a multi-dose prefilled cartridge; in other aspects, the article of manufacture is a reusable pen-type injector; in other aspects, the article of manufacture is a disposable pen device. In other aspects, the article of manufacture is a pump device for continuous subcutaneous insulin infusion therapy. In other aspects, the article of manufacture is a container closure system for a pump device for continuous subcutaneous insulin infusion therapy. Any of these articles of manufacture are understood to be administration tools, such as a needle and syringe.

[0098] Regardless of the route of administration selected, the formulations described herein, which can be used in a suitable hydrated form, and / or formulated by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the disclosed compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, compositions, and mode of administration, without being toxic to the subject. The selected dosage level will depend on a variety of factors including the activity of the particular insulin composition described herein employed, the route of administration, the time of administration, other drugs, compounds, and / or materials used in combination with the particular compositions employed, the age, sex, weight, health, medical history, and prior medical history of the subject being treated, and like factors well known in the medical arts. In certain aspects, insulin therapy for the treatment of diabetes is chronic therapy, which can require daily administration. If desired, the effective daily dose can be divided into two, three, four, five, six, or more sub-doses that are administered at appropriate intervals throughout the day, optionally in unit dosage forms.

[0099] Those of ordinary skill in the art of medicine or veterinary medicine can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian could start doses of the compounds of the pharmaceutical composition employed according to the disclosed methods at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0100] While the compounds described herein can be administered alone, they can also be used in combination with other active ingredients. The term "combination" is intended to encompass the use of fixed or unitary dosages of the individual agents in a single dosage unit form, as well as the administration of the individual agents in a kit of parts, where the kit contains separate dosages for each active ingredient intended for administration concomitantly. The compounds described herein can be administered simultaneously or sequentially with glucagon, GLP-1, anti-inflammatory agents, anti-proliferative agents, antibiotics, NSAIDs, analgesics, chemotherapeutic agents, immunosuppressive agents, other anti-cancer drugs, cytotoxic agents, or salts thereof.

[0101] In one aspect, the disclosed methods can comprise modifying one or more of the disclosed steps. For example, modifying one or more steps of the disclosed methods can comprise modifying or altering one or more features or aspects of one or more steps of the method. For example, in one aspect, the method can be adjusted by altering the amount of the other active ingredient used in the combination administration.

[0102] In one aspect, the disclosed methods can further comprise monitoring the subject for adverse reactions.

[0103] In one aspect, the disclosed methods can improve and / or prolong the survivability of the subject, can improve the quality of life of the subject, can increase and / or prolong the life of the subject, or any combination thereof. In one aspect, the life expectancy of the subject can be compared to the life expectancy of a control (i.e., untreated). In one aspect, the control can be a subject that does not receive the disclosed composition. In one aspect, the control can be a collection of subjects that do not receive the disclosed pharmaceutical composition. In one aspect, the control is one or more subjects having the same type of condition as the subject. As used herein, "life expectancy" is defined as the point in time at which 50% of the subjects are alive and 50% of the subjects are dead.

[0104] In one aspect, the life expectancy of the subject can be increased indefinitely following treatment with the disclosed methods. In one aspect, the life expectancy of the subject can be increased by at least about 5% or more, at least about 10% or more to at least about 95% or more, at least about 20% or more to at least about 80% or more, at least about 40% or more to at least about 60% or more, as compared to an untreated subject having the same or nearly the same viral infection and having the same or nearly the same prognosis.

[0105] In one aspect, the disclosed methods can improve and / or enhance the quality of life of the subject, which can be assessed subjectively and / or objectively.

[0106] In one aspect, the disclosed methods can increase the treatment options for the subject.

[0107] In one aspect, the disclosed methods can provide personalized treatment for a subject.

[0108] In one aspect, the disclosed methods can improve and / or restore normal metabolism of one or more organ systems in a subject.

[0109] In one aspect, the disclosed methods can restore and / or improve one or more aspects of cellular homeostasis and / or cellular function, and / or metabolic disorders.

[0110] In certain aspects, the present disclosure also encompasses the use of the compositions disclosed herein for laboratory research and preclinical testing.

[0111] D. Kits In certain aspects, the present disclosure encompasses kits comprising the compositions disclosed herein and instructions for use. In certain aspects, the present disclosure provides kits for administering the compositions disclosed herein. Such kits can comprise tools for containing and / or administering the insulin compositions.

[0112] In certain aspects, the kits disclosed herein can have a medical container that contains the composition in a safe, stable, and durable manner. In certain embodiments, the kits disclosed herein can also comprise tools for administering the composition, such as a needle, a catheter, a spatula, or a combination thereof.

[0113] Any of the kits can further comprise an instruction manual that provides guidance for using the kit for treatment. The manual can be written with the physician or hepatology specialist as the intended reader.

[0114] It will be understood by those within the art that, in some aspects of the disclosure, relative terms are used to describe one aspect in terms of another aspect. However, this usage of relative terms is intended to convey a possible relationship between various aspects or elements in terms of how they can be arranged in relation to one another and is not intended to convey an absolute relationship between the aspects or elements, unless specifically stated.

[0115] In one aspect, the disclosed kits can be used to provide personalized treatment for a subject.

[0116] IX. Examples The following examples are intended to demonstrate preferred aspects of the disclosure. Those of skill in the art will understand that the technical solutions disclosed in the following examples represent techniques that the inventors have found to work well in practicing the disclosure, and thus can be considered preferred modes of its practice. However, those of skill in the art will understand, in light of the present disclosure, that numerous changes can be made to the specific aspects disclosed and yet still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0117] Materials and Methods Cryo-EM data collection: Wild-type insulin (I2643) purchased from Sigma-Aldrich was used to determine the fibril structure. Insulin was dissolved in pH 2 buffer at a concentration of 1 mg / ml and incubated at 65 °C for 7 days to promote fibril formation.

[0118] Cryo-EM grid preparation: 3 μl of insulin fibril sample was applied to glow-discharged Quantifoil R1.2 / 1.3 300 mesh gold hole carbon grids (Quantifoil, Micro Tools GmbH, Germany). After 4.0 s of blotting at 4 °C and 100% humidity, the grids were plunged into liquid ethane using a Mark IV Vitrobot. Microscopy images were collected using a Titan Krios microscope operated at 300 kV, equipped with a K3 direct electron detector (Gatan) and set up with a GIF-Quantum energy filter with a 20 eV slit width. Data collection was performed using SerialEM 3.8 software. Insulin fibril samples were imaged using a calibrated 60241x magnification, resulting in a pixel size of 0.83 Å for the sample. Defocus was set to 1.6 μm to 2.6 μm. Each microscopy image was dose fractionated into 30 frames, with a total dose of approximately 60 e- / Å 2 .

[0119] Image processing: After motion correction using MotionCor21.2, super-resolution movie frames were combined into single micrographs with a 2x binning factor. Contrast transfer function (CTF) parameters were estimated using GCTF 1.16. All subsequent image processing was performed using Relion 3.1. After manual picking of insulin fibrils, they were sorted by multiple rounds of reference-free 2D classification. Different widths of fibrils were clearly visible in the 2D class averages. Three types, Type I, Type II and Type III, with higher abundances were selected for further processing. Initial models were generated using previously described methods. The helical rise per subunit was set to 4.75 Å, and the helical twist angle was estimated from the cross- distance of the fibrils in the micrographs. Three-dimensional reconstruction and CTF optimization were performed using the standard Relion pipeline. The final map was sharpened using a soft-edged solvent mask, and the resolution was estimated based on the gold-standard Fourier shell correlation between half-maps.

[0120] Model building and optimization: The map quality of the type II fiber was sufficient for de novo modeling using Coot 0.98. The coordinates were refined under secondary structure constraints using ISOLDE 1.2 in Chimera X 1.2 in conjunction with Phenix 1.19. Model quality and its correlation with the map were assessed using the validation tools in Phenix. Molecular structure figures were rendered using Chimera and ChimeraX.

[0121] Expression and purification of insulin wild type and mutants: All wild type (WT) insulins and mutants were obtained by bacterial expression and refolding. The genetically engineered insulin precursor (comprising the amino acid sequence "B1...B29 Lys-Ala-8xHis-Ala-Lys-A1 Gly...A21") was integrated into the pET28a vector for recombinant expression. The precursor protein was first expressed and refolded to obtain the single-chain insulin precursor. Subsequently, the attached His-tag was cleaved off using the endopeptidase Lys-C, resulting in the mature insulin linked by a disulfide bridge of two polypeptide chains. All insulins were purified in the form of des-B30Thr. It is worth noting that this truncation of the insulin molecule has no effect on its biological activity.

[0122] The specific purification procedure is as follows: Cells containing the above vector were cultured at 37 °C and induced with 1 mM IPTG for 24 h before harvesting. The cells were lysed using the French press method, and the lysis buffer was pH 8.0, containing 50 mM Tris-HCl and 2 mM EDTA. The inclusion bodies in the lysate were collected by centrifugation and washed with a washing buffer containing 50 mM Tris-HCl, 2 mM EDTA, and 1% Triton X-100 at pH 8.0. The inclusion bodies were dissolved in a solubilization buffer containing 30 mM Tris, 8 M urea, 1 mM EDTA, and 5 mM DTT at pH 8 for 24 h. 10 ml of the solubilized inclusion bodies were dialyzed in 500 ml of a refolding buffer containing 0.5 M arginine and 0.6 mM oxidized glutathione at pH 8.0 for 24 h. The supernatant was concentrated and loaded onto a HisTrap excel column (Cytiva™), and the eluate was collected after elution with imidazole. Subsequently, 50 µL of Lys-C enzyme was added to digest and remove the His-tag. The final insulin sample was purified by size exclusion chromatography using a Superdex 75 increase 10 / 300 GL column (Cytiva™) for storage, and the eluate was a pH 2 HC1 solution containing 150 mM NaCl.

[0123] Thioflavin T (ThT) assay: Wild-type or mutant insulin samples were diluted to a concentration of 0.5 mg / ml with either pH 2 (pH 2 HC1, 150 mM NaCl) or pH 7 (pH 7 20 mM HEPS, 150 mM NaCl) buffer. Each 50 μΐ of protein sample was mixed with 50 μΜ ThT and aliquoted into a clear-bottom 384-well plate. All ThT assays were performed on a multi-functional microplate reader (FLUOstar OPTIMA) under pre-set test settings. Prior to the assay, a 3-minute double-orbit shaking was performed, followed by hourly scanning of the fluorescence signal with a 430 / 10 nm (center wavelength / bandwidth) excitation filter and a 475 / 30 nm (center wavelength / bandwidth) emission filter. All conditions were repeated in triplicate at 37 °C. The signal was normalized to the maximum signal of the wild-type and blank wells containing buffer and ThT were subtracted from the experimental values. Data were plotted by GraphPad Prism, with data points and connecting lines with error bars representing the mean and standard error of the mean.

[0124] Insulin receptor activation in cultured cells: Insulin receptor activation assays were performed as previously described with some modifications. 293FT cells (R70007, Invitrogen) were cultured in high glucose (4.5 g / L) DMEM supplemented with 10% (v / v) FBS, 2 mM L-glutamine and 1% penicillin / streptomycin. Cells were not mycoplasma-contaminated. Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen). One day later, cells were serum-starved for 14-16 hours. For dose-dependent experiments, insulin mutants were diluted to a concentration of 1 nM, 10 nM or 100 nM in serum-free high glucose DMEM and cells were treated for 10 minutes at 37 °C. For time-dependent experiments, insulin mutants were diluted to a concentration of 10 nM in serum-free high glucose DMEM and cells were treated for 10, 30 or 60 minutes at 37 °C. For incubation of the reagents at room temperature, each insulin mutant was aliquoted at a concentration of 77.2 μΜ in buffer (20 mM HEPES pH 7.4, 150 mM NaCl) and stored in the dark at room temperature for three days. At the end of the incubation, insulin mutants were diluted to a concentration of 100 nM in serum-free high glucose DMEM and cells were treated for 10 minutes at 37 °C.

[0125] Cells were incubated in cell lysis buffer [50 mM HEPES pH 7.4, 150 mM NaCl, 10% (v / v) glycerol, 1% (v / v) Triton X-100, 1 mM EDTA, 10 mM sodium fluoride, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.5 mM dithiothreitol (DTT), 2 mM phenylmethylsulfonyl fluoride (PMSF)] supplemented with cOmplete™ protease inhibitor cocktail (Roche) and PhosSTOP™ (Roche) for 1 hour on ice. After centrifugation at 18,213 g for 20 minutes at 4 °C, samples of the cell lysate were taken for SDS-PAGE and Western blot analysis. Anti-IR-pY1150 / 1151 (1:1000, 19H7, Cell Signaling; labelled pYIR) and anti-Myc (1:1000; 9E10, Roche; labelled IR) were used as primary antibodies. Quantitative Western blotting used anti-rabbit immunoglobulin (IgG) (H+L) (Dylight 800 conjugate) and anti-mouse IgG (H+L) (Dylight 680 conjugate) (Cell Signaling) as secondary antibodies. Membrane bands were scanned using an Odyssey infrared imaging system (LI-COR, Lincoln, NE). pY-IR levels were normalised to total IR levels and expressed as intensity relative to the intensity of IR after 10 minutes of treatment with 10 nM wild-type insulin.

[0126] Statistical analysis: Prism 9 software was used to generate graphs and perform statistical analysis. Results are expressed as mean ± standard deviation (s.d.) or mean ± standard error (s.e.m.). Pairwise significance analysis for Figure 3 used a two-tailed unpaired t-test. Sample size power analysis, randomisation and blinding were not performed and all experiments were uniformly analysed statistically after data collection was complete.

[0127] Example 1 cryo-EM structure of insulin fibrils To promote insulin fibrillation, human insulin was dissolved in a buffer at pH 2 and incubated at 65 °C for 7 days. Cryo-EM images of the resulting sample showed that most insulin molecules formed fibrils of significant length under these conditions, with varying widths and morphologies Figure 1A Three types of fibrils (named Type I, Type II and Type III fibrils) with higher abundance were manually picked out for 2D and 3D classification Figures 1A to 1C A 3.6 Å resolution 3D reconstruction model of Type II fibrils was obtained, with a helical rise of 4.75 Å and a left-handed twist angle of 1.27°, allowing the construction of an atomic model Figures 1D to 1F Type II fibrils contain two protofilaments folded in an asymmetric and antiparallel mannerFigure 1G ). The 3D reconstruction of type III fibers had a lower resolution and failed to build an atomic model ( Figure 1C ). However, the density map clearly showed that it contained two protofilaments identical to type II, but folded parallel to the ground ( Figure 1C ). High-resolution 3D reconstruction of type I fibers failed, but 2D class averages and low-resolution reconstruction suggested that it was composed of a single protofilament ( Figure 1A - Figure C). Without being bound by theory, it was speculated that one intra-chain disulfide bond (CysA6-CysA11) and two inter-chain disulfide bonds (CysA7-CysB7 and CysA20-CysB19) in chain A might impose strong conformational constraints on the individual protomers, resulting in a conformationally unique protofilament formed by the sequential stacking of protomers along the long axis of the fiber. In the three fibers analyzed in this study, the number and arrangement of protofilaments determined the fiber form ( Figures 1A to 1C ). In addition, wider fibers composed of more than two protofilaments were also found in the sample, but their abundance was low.

[0128] Cross-β fold of insulin protomers: Native insulin mainly contains helical structural elements that are stacked into compact global domains. In contrast, insulin protomers in the fiber are transformed into an extended and flattened global shape, enabling them to sequentially stack into cross-β protofilaments ( Figure 1G ). The N-terminal of chain A has an S-shape, with the first half pinned by an intra-chain disulfide bond (CysA6-CysA11), followed by a β-strand formed by the C-terminal part ( Figure 1H and Figure 1I ). The middle section of the B chain forms a relatively long β-strand, connected to the A chain by two inter-chain disulfide bonds (CysA7-CysB7 and CysA20-CysB19) ( Figure 1H and Figure 1I ). The C-terminal part of the B chain extends and participates in the interaction between the two protofilaments ( Figure 1G and Figure 1H ).

[0129] In addition to the two inter-chain disulfide bonds, the A and B chains of insulin also interact through two clusters of hydrophobic residues ( Figure 1H and Figure 1I ). In particular, IleA10 and LeuA16 directly interact with LeuB15 and LeuB17, while LeuB6 and LeuB11 are in contact with the CysA7-CysB7 disulfide bond ( Figure 1H and Figure 1I ). In addition, multiple polar residues in the protofilament are buried between the A and B chains. Among them, ThrA8 and SerA9 both contact GluB13 ( Figure 1H and Figure 1IGluB13 participates in the conformation of the protofilament core, explaining why low pH is required to induce fibrillation: it promotes the protonation of the GluB13 carboxyl groups, neutralizing their negative charge, which would otherwise hinder embedding into the hydrophobic core of the protofilament.

[0130] It is worth noting that in the cross-β-sheet of monomeric insulin, the C-terminus of the insulin B chain is approximately 50 Å away from the N-terminus of the A chain. Figure 1I This means that a loop longer than 20 residues is required to connect the two ends without disrupting the cross-β-sheet. Without being theoretically constrained, this might explain why single-chain insulin analogs, which typically contain short interchain loops, cannot form stable amyloid fibrils.

[0131] Subunit stacking: Insulinogenomeres along the protonematrix axis form regular skeletal hydrogen bonds that promote interactions between subunits. Figure 1G Furthermore, the subunit stacking in each protofilament is driven by the π-stacking of aromatic residues (TyrA14, TyrA19, and TyrB16) side chains and the hydrogen-bonded ladder structure formed by the asparagine and glutamine residues (GlnA5, GlnA15, AsnA18, and AsnA21) side chains in the insulin A chain. Figure 1H Furthermore, due to the out-of-plane tilt of the disulfide ring in the insulin A chain (…), Figure 1G ThrA8 forms hydrogen bonds with GluB13 residues from adjacent subunits along the length of the protonemafur. This non-planar subunit conformation further stabilizes the protonemafur structure by providing additional inter-subunit contacts along the protonemafur axis. Figure 1G These structural observations indicate that intermolecular and intramolecular disulfide bonds are crucial for the structural stability of insulin fibers, supporting previous findings that insulin tends to assemble into highly flexible fibers in the presence of reducing agents, such as tris(20-carboxyethyl)phosphine (TCEP). Furthermore, ArgB22 forms alternating positive and negative charge electrostatic interactions with the C-terminal carboxyl group of the insulin A chain, promoting protofilament stacking. Figure 1H ).

[0132] Protoplast assembly: In type II insulin fibers, two protoplasts stack on each other in an antiparallel manner. Figure 1C and Figure 1G The stacking between the two protonemata is primarily mediated by hydrophobic interactions, involving the PheB24 and TyrB26 side chains from the C-terminus of the B chain of the first protonemata, and the SerA12 and TyrA14 side chains from the outer surface of the "S"-shaped insulin A chain of the second protonemata. Figure 1H Furthermore, the electrostatic interaction between ArgB22 of the first precursor and GluA4 of the second precursor further promotes the stacking between the two precursors in type II fibers. Figure 1H ).

[0133] Unlike type II fibrils, the C-terminus of the insulin B-chain plays a unique role in mediating the parallel stacking of two protofilaments in type III fibrils by forming a homointerface Figure 1C Without being bound by theory, these structural observations suggest that, although the C-terminus of the insulin B-chain does not participate in the folding of the individual subunits, it is critical for the assembly of the supra-protofilament of insulin fibrils. Furthermore, in both type II and type III fibrils, the C-terminus of the insulin B-chain adopts a 90° bent conformation in the single protofilament Figure 1H and Figure 1I This bend exposes the aromatic residues of the C-terminus of the insulin B-chain more readily for lateral inter-protofilament interactions, which in turn facilitates the formation of larger supra-filament bundles.

[0134] Example 2 Designing Anti-fibrillogenic Insulin Mutants Based on the type II insulin fibril structure described herein and previously published IR / insulin complex structures, specific insulin mutants were designed to reduce fibrillogenicity while retaining their ability to bind to the IR. It was hypothesized that mutating small or hydrophobic residues in the core of the insulin proto-cross-β sheet to large polar residues would reduce the formation of the insulin cross-β sheet, rendering it anti-fibrillogenic. Three mutants were constructed: ThrA8Arg, IleA10Arg, and AsnA18Gln. Importantly, none of these residues participate in insulin binding to the IR. First, the three mutants were evaluated for fibril formation using the well-established thioflavin T (ThT) assay. Wild-type insulin, when incubated in pH 2 buffer at 65°C for about 2 days, exhibited a strong ThT signal, indicating that insulin formed robust fibrils Figure 2A Remarkably, none of the three mutants exhibited a significant ThT signal, confirming that these mutations successfully prevented insulin fibrillogenicity Figure 2A Negatively stained electron microscopy analysis confirmed that none of the three insulin mutants formed any long fibrils after incubation for 7 days under the same conditions Figure 2B These results validated the structural model of insulin fibrils and demonstrated that single-point mutations can abolish the fibril-forming ability of insulin.

[0135] The ability of these anti-insulin fibrillogenic mutants (ThrA8Arg, IleA10Arg, and AsnA18Gln) to induce IR activation was subsequently tested. Consistent with the structural model, all of the insulin fibril mutants were able to fully activate the IR at a wide range of concentrations and at multiple time points Figures 3A to 3D Notably, all three insulin mutants were able to slightly enhance IR autophosphorylation compared to wild-type insulin Figure 3A andFigure 3B ).

[0136] To further investigate whether these mutations could prevent the disruption of insulin function caused by fibril formation, insulin stock solution was incubated at room temperature for three days to promote insulin fibril formation, and the effect of insulin on IR activation was tested. Under this condition, all three mutants were able to drive a slight but statistically significant enhancement of insulin receptor autophosphorylation compared to wild-type insulin ( Figure 3E and 3F ). These data suggest that insulin mutations can inhibit fibril formation, thus maintaining the effect of insulin on IR activation for a longer period of time.

[0137] Next, one of the mutants, AsnA18Gln, was tested for its high-temperature and long-term stability relative to native insulin (wild-type). First, the activity of wild-type insulin and the AsnA18Gln mutant was evaluated after incubation at 70 °C for 16 hours with stirring at 1000 rpm. The level of insulin receptor autophosphorylation ( Figure 4A ), AKT active phosphorylation ( Figure 4B ), and ERK active phosphorylation ( Figure 4C ) were determined by quantitative western blot analysis. The AsnA18Gln mutant exhibited higher activity, indicating that it has superior stability.

[0138] Subsequently, referring to Figure 5 , wild-type insulin and the AsnA18Gln mutant were incubated at 37 °C with stirring at 250 rpm for 14 days for in vivo insulin tolerance testing in mice (B6 strain, male, 2 months old). The mice were fasted for 4 hours before testing. After the mice were injected with a dose of 1.5 U / kg of either wild-type insulin or the AsnA18Gln mutant, blood glucose levels were measured at different time points. The mutant showed a stronger hypoglycemic effect compared to native insulin, indicating that the AsnA18Gln mutant has higher thermal stability at physiological temperatures compared to wild-type insulin.

[0139] CONCLUSION The above studies show that insulin fibrils have a high degree of structural heterogeneity, a phenomenon known as amyloid polymorphism. Cryo-EM data sets reveal at least three different types of insulin fibrils. In Type II and Type III, protofilaments exhibit a highly similar conformation, suggesting that insulin fibril polymorphism arises from different arrangements of similar protofilaments. In Type II and Type III insulin fibrils, which are composed of two protofilaments, the C-terminal end of the insulin B chain plays a key role in the lateral interactions between protofilaments. Notably, GluB13 is buried within the hydrophobic core of the protofilament. When the negative charge of GluB13 is neutralized in a low-pH environment, its stacking in a hydrophobic environment is more favorable. This explains why low-pH conditions promote the process of insulin fibrillation.

[0140] Disulfide bonds can affect subunit assembly at different stages of amyloid fibrillation and ultimately determine the fibril morphology. Cryo-EM structures show that both intra- and intermolecular disulfide bonds contribute to stabilizing the cross-β sheet of each subunit and maintaining the close stacking of the A and B chains within each subunit. In addition, the disulfide bond loop in the insulin A chain simultaneously contacts two adjacent subunits along the protofilament axis, thereby promoting the stacking of subunits in the protofilament and leading to increased fibril rigidity.

[0141] The insulin B chain residues B11-B17 (LVEALYL) exhibit a similar β-strand conformation in both the crystal structure of the truncated peptide and the cryo-EM structure of the full-length insulin fibril ( Figure 6 ). However, this peptide segment forms a homologous, staggered stack in the crystal structure, rather than interacting with the insulin A chain as shown in the cryo-EM structure. This segment of the insulin B chain can form a β-strand and a cross-β amyloid even in the absence of the A chain. In full-length insulin, the presence of the A and B chains and disulfide bonds strongly constrain the two-chain conformation and its interactions, ultimately forming a single, well-defined protofilament. This protofilament can be further stacked through the more flexible C-terminal tail of the B chain to form different types of fibrils.

[0142] In summary, the structural studies disclosed herein provide important information for elucidating the molecular mechanisms underlying the formation of full-length insulin fibrils. Understanding the structural basis of insulin fibrillation will aid in the design of insulin analogs (such as the examples listed above) to more effectively treat patients with type 1 diabetes.

Claims

1. An engineered insulin polypeptide comprising at least one mutation in the engineered insulin polypeptide, wherein, The engineered insulin polypeptide exhibits blocked cross-β fibrilization.

2. The engineered insulin polypeptide of claim 1, wherein the at least one mutation is in the A-chain polypeptide.

3. The engineered insulin polypeptide of claim 1 or 2, wherein the at least one mutation is in an amino acid of the hydrophobic core of the cross-β fold of the proinsulin body.

4. The engineered insulin polypeptide of any one of claims 1-3, wherein the at least one mutation is ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, IleA10Arg, IleA10Lys, IleA10Phe, IleA10Tyr, IleA10Trp, IleA10Gln, IleA10Glu, AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, AsnA18Gln, or any combination thereof.

5. The engineered insulin polypeptide of any one of claims 1-3, wherein the at least one mutation is ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, ThrA8Ala, ThrA8Asp, ThrA8Asn, ThrA8Cys, ThrA8Gly, ThrA8His, ThrA8Ile, ThrA8Leu, ThrA8Met, ThrA8Pro, ThrA8Ser, or ThrA8Val.

6. The engineered insulin polypeptide of any one of claims 1-3, wherein the at least one mutation is SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, SerA9Ala, SerA9Asp, SerA9Asn, SerA9Cys, SerA9Gly, SerA9His, SerA9Ile, SerA9Leu, SerA9Met, SerA9Pro, SerA9Thr, or SerA9Val.

7. The engineered insulin polypeptide of any one of claims 1-3, wherein the at least one mutation is: Ile A10Arg, Ile A10Lys, Ile A10Phe, Ile A10Tyr, Ile A10Trp, Ile A10Gln, Ile A10Glu, Ile A10Ala, Ile A10Asp, Ile A10Asn, Ile A10Cys, Ile A10Gly, Ile A10His, Ile A10Leu, Ile A10Met, Ile A10Pro, Ile A10Ser, Ile A10Thr, or Ile A10Val.

8. The engineered insulin polypeptide of any one of claims 1-3, wherein the at least one mutation is: Asn A18Arg, Asn A18Lys, Asn A18Phe, Asn A18Tyr, Asn A18Trp, Asn A18Glu, Asn A18Gln, Asn A18Ala, Asn A18Asp, Asn A18Cys, Asn A18Gly, Asn A18His, Asn A18Ile, Asn A18Leu, Asn A18Met, Asn A18Pro, Asn A18Ser, Asn A18Thr, or Asn A18Val.

9. The engineered insulin polypeptide of any one of claims 1-8, wherein the A chain polypeptide comprises an amino acid sequence that is at least about 80% identical to one of SEQ ID NOs: 1-76, or a functional derivative thereof.

10. The engineered insulin polypeptide of any one of claims 1-9, wherein the engineered insulin polypeptide further comprises one or more additional mutations.

11. A composition comprising the engineered insulin polypeptide of any one of claims 1-10, and at least one pharmaceutically acceptable excipient.

12. The composition of claim 11, wherein the pharmaceutically acceptable excipient is a liquid or solid filler, diluent, binder, buffering agent, pH adjusting agent, disintegrating agent, dispersing agent, preservative, lubricant or wetting agent, taste masking agent, antioxidant, carrier, adjuvant, stabilizer, emulsifying agent, solubility enhancer, salt, solubilizer, antifoaming agent, surfactant, flavoring agent, coloring agent, solvent or encapsulating material, or any combination thereof.

13. The composition of claim 11 or 12, further comprising an additional active agent.

14. The composition of claim 13, wherein the additional active agent is glucagon or GLP-1.

15. The composition of any one of claims 11-14, wherein the composition is formulated to provide at least about 5 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 150 U / mL, 200 U / mL, 250 U / mL, 300 U / mL, 350 U / mL, 400 U / mL, 450 U / mL, or 500 U / mL of insulin.

16. The composition of any one of claims 11-15, wherein the composition is formulated for multiple doses or dose administration.

17. The composition of any one of claims 11-16, wherein the composition is formulated for delivery by a syringe, closed loop system, insulin pen, or insulin pump.

18. A polynucleotide sequence encoding the engineered insulin polypeptide of any one of claims 1-10.

19. The polynucleotide sequence of claim 18, wherein the polynucleotide sequence is a plasmid vector, a viral vector, a transposon, a gene edited polynucleotide sequence, or a virus.

20. A host cell comprising the engineered insulin polypeptide of any one of claims 1-10 or the polynucleotide of any one of claims 18-19, or both.

21. The host cell of claim 20, wherein the host cell is a bacterial, fungal, or mammalian cell.

22. A method for treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 11-17.

23. The method of claim 22, wherein the subject is in a pre-diabetic state or has type 1 diabetes, type 2 diabetes, or hyperglycemia.

24. The method of claim 22 or 23, wherein the administration is by any one of parenteral, oral, intra-arterial, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, intravascular, liposomal, topical, mucosal, subcutaneous, sublingual, topical, buccal, or transdermal routes.

25. The method of any one of claims 22-24, wherein the administration is chronic administration.

26. The method of any one of claims 22-25, wherein after the step of administering: (i) the risk of unpredictable fluctuations in blood glucose levels or hyperglycemia is reduced, (ii) the survival of the subject is prolonged, (iii) the quality of life of the subject is improved and / or enhanced, (iv) normal metabolism of one or more organ systems of the subject is improved and / or restored, (viii) one or more aspects of cellular homeostasis and / or cellular functionality, and / or metabolic disorders are restored and / or improved, or (ix) any combination thereof.

27. The method of any one of claims 22-26, wherein the subject is a human.

28. A kit comprising: (A) the composition of any one of claims 11-17; (B) instructions for use.

29. The kit of claim 2, further comprising means for administering (A).

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