EP4731646A2 - Novel insulin analogs to disrupt insulin fibrillation - Google Patents

Abstract

Provided herein are engineered insulin polypeptide that exhibit disrupted cross-β fibrillation and pharmaceutical compositions comprising the polypeptides. The disclosure also provides are methods for making the insulin polypeptide. Also included are methods of using the pharmaceutical compositions to treat diabetes and hyperglycemia.

Description

NOVEL INSULIN ANALOGS TO DISRUPT INSULIN FIBRILLATION

I. CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/510,024 filed on 23 June 2023, which is incorporated by reference herein in its entirety.

IL STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under GM142937, DK063608, DK132710, GM130289, and GM136976 awarded by the National Institutes of Health. The government has certain rights in the invention.

III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted 21 June 2024 as an XML file named ‘ 106546-805672- 4235_Sequence Listing’, created on 21 June 2024, and having a size of 68 KB is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

IV. FIELD

[0004] The present invention relates to insulin polypeptides and pharmaceutical compositions comprising these polypeptides. The current disclosure also encompasses methods of making and using the disclosed compositions.

V. BACKGROUND

[0005] Type 1 diabetes or insulin dependent diabetes is an autoimmune disease that results from destruction of beta cells by the immune system. Currently, about 37.3 million people (11.3% of the US population) have diabetes, while 96 million people aged 18 years or older have prediabetes. If left untreated, diabetes can cause several complications including rapid onset complications like diabetic ketoacidosis and nonketotic hyperosmolar coma. Long-term complications include heart disease, stroke, kidney failure, foot ulcers and damage to the eyes. There is currently no known way to prevent type 1 diabetes and treatment with insulin is required for survival. [0006] Insulin is produced by pancreatic P-cells. Insulin activates the insulin receptor (IR) and regulates glucose levels in animals. The mature form of insulin contains A- and B-chains that are linked by two inter-molecular disulfide bonds, while chain A contains an additional intramolecular disulfide bond. The binding of multiple insulin molecules to two distinct sites of IR synergistically promotes IR activation, thus triggering downstream signaling cascades, and promoting glucose uptake into the cells. Failure to produce sufficient insulin from pancreatic P- cells causes type 1 diabetes. Therefore, recombinant insulins have been widely used to treat type 1 diabetes.

[0007] Insulin, however, tends to undergo an alpha-to-beta conformational transition and ultimately forms amyloid fibrils under conditions commonly used for storage (i.e., low pH and high concentrations). Insulin in the fibrilized form cannot bind or activate IR, resulting in reduced therapeutic efficacy in treating type 1 diabetes. Furthermore, repeated injections of insulin can lead to insulin-derived amyloidosis, where insulin fibrils accumulate under the skin. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, patients with diabetes mellitus optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for such patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus, and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable fluctuations in blood glucose levels or even dangerous hyperglycemia.

[0008] Nevertheless, the molecular details of insulin fibrillation remain unclear, hindering efforts to prevent the fibrillation process. There is therefore an unmet need to develop insulin analogs with reduced fibrillation, to improve storage and patient outcomes.

VI. BRIEF SUMMARY

[0009] In some aspects, the current disclosure encompasses an engineered insulin polypeptide, comprising at least one mutation in the engineered insulin polypeptide, wherein the engineered insulin polypeptide exhibits disrupted cross-P fibrillation. In some aspects, the at least one mutation is in an A-chain polypeptide. In some aspects, the at least one mutation is in an amino acid in hydrophobic core of cross-P fold of insulin protomer. In some aspects, the at least one mutation is ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, IleAlOGlu, AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, AsnA18Gln or any combination thereof.

[0010] 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.

[0011] 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.

[0012] In an aspect, the at least one mutation is IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, IleAlOGlu, IleAlOAla, IleAlOAsp, IleAlOAsn, IleAlOCys, IleAlOGly, IleAlOHis, IleAlOLeu, IleAlOMet, IleAlOPro, IleAlOSer, IleAlOThr, or IleAlOVal.

[0013] 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 AsnAl 8Val.

[0014] In some aspects, the A-chain polypeptide comprises an amino acid sequence at least about 80% identical to a sequence as provided in SEQ ID NOS: 1-76, or a functional derivative thereof. In some aspects, the engineered insulin polypeptide further comprises one or more additional mutations.

[0015] In some aspects, the current disclosure also encompasses a composition comprising an engineered insulin polypeptide as disclosed herein, and at least a pharmaceutically acceptable excipient. In some aspects, the pharmaceutically acceptable excipient is a liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, taste-masking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a coloring agent, solvent or encapsulating material or any combination thereof. In some aspects, the composition further comprises an additional active agent. In some aspects, the additional active agent is glucagon or GLP-1. In some aspects, the composition is formulated to provide at least about 5U/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 some aspects, the composition is formulated for multiple dose administration or single dose administration. In some aspects, the composition is formulated for delivery using a injector, a closed loop system, an insulin pen or an insulin pump.

[0016] In some aspects, the current disclosure also encompasses a polynucleotide sequence encoding the engineered insulin polypeptide as disclosed herein. In some aspects, the polynucleotide sequence is a plasmid vector, a viral vector, a transposon, a gene edited polynucleotide sequence, or a virus.

[0017] In some aspects, the current disclosure also encompasses a host cell comprising the engineered insulin polypeptide or a polynucleotide encoding the engineered insulin polypeptide or both. In some aspects, the host cell is a bacterial, fungal, or a mammalian cell.

[0018] In some aspects, the current disclosure also encompasses a method for treating a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a composition disclosed herein. In some aspects, the subject is pre-diabetic or who is suffering from or diagnosed with type 1 diabetes, type 2 diabetes, or hyperglycemia. In some aspects, the administering is by any one of parenteral, oral, intraarterial, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, intravascular, liposomal, local, mucosal, subcutaneous, sublingual, topical, trans buccal, and transdermal route. In some aspects, the administration is on a chronic basis.

[0019] In some aspects, the current disclosure also encompasses a method in which, following the administration step, (i) the risk of unpredictable fluctuations of blood glucose levels or hyperglycemia may be prevented and/or decreased, (ii) the survival of the subject may be prolonged, (iii) the subject's quality of life may be enhanced and/or improved, (iv) normal metabolism of one or more organ systems in the subject may be improved and/or restored, (viii) one or more aspects of cellular homeostasis and/or cellular functionality, and/or metabolic dysregulation may be restored and/or improved, or (ix) any combination thereof.

[0020] In an aspect, the subject is a human subject.

[0021] In some aspects, the current disclosure also encompasses a kit comprising the composition as disclosed herein and instructions for use. In some aspects, the kit further comprises a means for administering the compositions disclosed herein.

VII. BRIEF DESCRIPTION OF THE FIGURES

[0022] The accompanying Figures and Examples are provided by way of illustration and not by way of limitation.

[0023] FIG. 1A shows a representative cryo-EM micrograph of insulin fibril sample. [0024] FIG. IB provides 2D class averages of type I, II and III insulin fibrils. [0025] FIG. 1C provides cryo-EM maps of type I, II, and III insulin fibrils. Type I fibril is comprised of one protofilament, while type II and III fibrils are comprised of two protofilaments, which are arranged antiparallel and parallel, respectively. Only the cryo-EM of type II fibril was resolved at sufficient resolution for accurate model building.

[0026] FIG. ID shows FSC curve for two half cryo-EM maps of type II insulin fibril (left) and the refined model against the cryo-EM map (right).

[0027] FIG. IE provides the slice of the cryo-EM map of type II insulin fibril.

[0028] FIG. IF provides a local resolution map of type II insulin fibril.

[0029] FIG. 1G is a model of type II insulin fibril, shown in two views. Type II insulin fibril is made of two similar protofilaments. The A- and B-chains of insulin are colored in blue and green, respectively.

[0030] FIG. 1H provides the cryo-EM map of type II insulin fibril and the corresponding model fitted into the cryo-EM map.

[0031] FIG. II is a schematic of one protofilament from the type II insulin fibril.

[0032] FIG. 2A shows the fibrillation of insulin WT and designed mutants, characterized by ThT assay.

[0033] FIG. 2B shows negative stain EM images of insulin WT and the mutants designed to have reduced ability in forming fibril.

[0034] FIG. 3 A is a Western blot analysis showing IR autophosphorylation (pY IR) induced by the indicated concentration of insulin WT or insulin mutants for 10 min in 293FT cells expressing IR-WT.

[0035] FIG. 3B provides quantification of the Western blot data shown in FIG. 3 A. n=4 independent experiments for all data points. Mean ± SEM. Levels of pY IR were normalized to total IR levels and shown as intensities relative to that of IR in 10 nM insulin WT -treated cells.

[0036] FIG. 3C is a Western blot analysis showing IR autophosphorylation (pY IR) induced with 10 nM insulin WT or insulin mutants for the indicated time points in 293FT cells expressing IR- WT.

[0037] FIG. 3D provides quantification of the Western blot data shown in FIG. 3C. IleAlOR 10 min, n=3; all other data points, n=4 independent experiments. Mean ± SEM. Levels of pY IR were normalized to total IR levels and shown as intensities relative to that of IR in 10 nM insulin WT-treated cells.

[0038] FIG. 3E is a Western blot analysis showing IR autophosphorylation (pY IR) induced by 100 nM insulin WT or insulin mutants for 10 min in 293FT cells expressing IR-WT. The insulin stocks are incubated at room temperature for 3 days. [0039] FIG. 3F provides quantification of the Western blot data shown in FIG. 3E. n=6 independent experiments for all data points. Mean ± SD. Levels of pY IR were normalized to total IR levels and shown as intensities relative to that of IR in 100 nM insulin WT-treated cells. [0040] FIGS. 4A to 4C show stability of mutant AsnA18Gln compared to native insulin when exposed to high temperature (70 °C) for 16 hrs. Quantitative western blot analysis was used to determine the levels of insulin receptor autophosphorylation (FIG. 4A), active phosphorylation of AKT (FIG. 4B), and active phosphorylation at ERK (FIG. 4C). n=3. Mean ± SEM.

[0041] FIG. 5 shows mutant AsnA18Gln compared to native insulin in insulin tolerance tests following incubation at 37 °C for 14 days. n=7 per group. Mean ± SEM.

[0042] FIG. 6 shows a comparison of the B-chain peptide as seen in the crystal and cryo-EM structures. Insulin B-chain residues Bl 1 - B17 (LVEALYL) adopt a similar P-strand conformation in the crystal structure of the truncated peptide and the cryo-EM structure of full- length insulin fibril. However, this peptide in the crystal structure forms homotypic interdigitated packing, rather than interacting with insulin A-chain as seen in our cryo-EM structure.

VIII. DETAILED DESCRIPTION

[0043] The following detailed description references the accompanying drawings that illustrate various embodiments of the present disclosure. The drawings and description are intended to describe aspects and embodiments of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0044] Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. The mature form of insulin contains A- and B-chains that are linked by two inter-molecular disulfide bonds, while chain A contains an additional intra-molecular disulfide bond. However, insulin is prone to undergo unfolding to form cross-P fibers through alpha-to-beta conformational transition. The fibrillation complicates the manufacture, storage and therapeutic use of insulin. As used herein, the term “fibrillation” or “cross-P fibrillation” or “amyloid fibrillation” or “cross-P fibrils” are used interchangeably to encompass self-assembled fibrous protein aggregates characterized by a fibrillar morphology of 7-13 nm in diameter, usually with a P-sheet secondary structure (known as cross-P) and ability to be stained by particular dyes, such as Congo red. In the current context, cross-P fibrils formation in insulin is associated with non-functional insulin. The molecular details of insulin fibrillation remain unclear, hindering efforts towards preventing the fibrillation process.

[0045] In an aspect, the current disclosure is based on the characterization of Cryo-EM structures of insulin fibrils as disclosed herein, showing multiple forms that are built by different arrangements of one protofilament containing both the A- and B-chains of insulin linked by disulfide bonds. The cryo-EM structure of insulin fibril disclosed here is the first amyloid fibril structure with well resolved disulfide bonds. The structure revealed highly distorted P-sheets of the insulin chains and their interactions that underlie fibrillation. The protofilament of insulin fibrils adopts a highly distorted cross-P fold, due to the restraints imposed by the intra- and inter- molecular disulfide bonds. Structure characterization showed that full-length insulin forms diverse fibrillar morphologies containing different numbers of protofilaments. In the cryo-EM structure, the two protofilaments pack anti-parallelly and asymmetrically. Additionally, a different form of insulin fibrils containing two protofilaments packing in parallel was observed. [0046] Based on the structure disclosed herein, a series of insulin mutants that do not undergo fibrillation but retain wild-type IR signaling activity in activating insulin receptor (IR) were contemplated, generated and successfully tested. In some aspects, the current disclosure encompasses engineered insulin polypeptide or designed insulin analogs, and pharmaceutical compositions comprising these polypeptides for effective use as therapeutics for type 1 diabetes and associated medical conditions. In some aspects, the current disclosure also encompasses methods of making and using the compositions as disclosed herein. These compositions may provide several advantages over currently available products, including a) ease of manufacturing as the insulin has a lower tendency to aggregate, b) ease of storage and longer shelflife, c) room temperature storage, d) easier to use in insulin pump, e) greater efficacy on administration due to low propensity to aggregate.

[0047] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0048] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and/or the like in the description mean that the feature and/or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and/or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and/or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and/or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0049] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0050] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0051] The terms “or” and “and/or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and/or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive. [0052] The terms ‘’nucleic acid”, ‘’nucleic acid molecule”, and ‘’polynucleotide” are used interchangeably herein. The terms “nucleic acid encoding . . or “nucleic acid molecule encoding . . . ” should be understood as referring to the sequence of nucleotides which encodes a polypeptide.

[0053] A polynucleotide described herein may comprise one or more nucleic acids each encoding a polypeptide, operably linked to (i.e., in a functional relationship with) one or more regulatory sequences, such as a promoter. Such a polynucleotide may alternatively be referred to herein as a ‘’nucleic acid construct” or ‘’construct”. As used herein, the term “operably linked” refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter, etc., operates to initiate, assist, affect, cause, and/or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue(s), developmental stage(s) and/or condi tion(s).

[0054] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0055] Within the context of the application a protein is represented by an amino acid sequence and correspondingly a nucleic acid molecule or a polynucleotide represented by a nucleic acid sequence. Identity and similarity between sequences: throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.

[0056] Each amino acid sequence described herein by virtue of its identity or similarity percentage with a given amino acid sequence respectively has in a further preferred aspect an identity or a similarity of 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% with the given nucleotide or amino acid sequence, respectively. The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and preferred program is Needle for pairwise alignment (in an aspect, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment ( in an aspect, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned, Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2, Perform FFTS: none is used). [0057] The term “treating,” as used herein refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or ameliorating one or more symptoms of such condition or disorder. The term “treatment,” or “therapy” of a subject refers to any type of intervention, or the administration of a compound as disclosed herein, to a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. In some aspects, the disease is a cancer and/or a tumor as provided herein.

[0058] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce a toxic, 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. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0059] The language “effective amount” of the compound is that amount necessary or sufficient to treat or prevent cancer or any other disease or disorder that is linked to ISR. In an example, an effective amount of the compound described herein is the amount sufficient to treat leukemia. In an example, an effective amount of the compound described herein is the amount sufficient to treat a colorectal cancer. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular compound described herein. For example, the choice of the compound described herein can affect what constitutes an “effective amount.” One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the compounds described herein without undue experimentation.

[0060] The phrase “pharmaceutically acceptable excipient” includes any pharmaceutically acceptable material, composition, or vehicle, suitable for administering the compounds described herein to mammals. The excipient includes liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, taste-masking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a coloring agent, solvent or encapsulating material or any combination thereof. 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 amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science. In each of the aspects described herein, a composition of the disclosure may optionally comprise one or more additional drug or therapeutically active agent in addition to the at least one factor disclosed herein. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

A. Compositions

[0061] In some aspects, the compositions of the current disclosure encompass engineered polypeptides and pharmaceutical compositions comprising said polypeptides. In another aspect, the compositions of the current disclosure encompass engineered polynucleotides encoding the disclosed polypeptides disclosed herein and/or host cell comprising polynucleotides encoding the engineered polypeptide disclosed herein and/or comprising the engineered polypeptides disclosed herein.

[0062] In an aspect, the current disclosure encompasses an engineered insulin polypeptide or a functional derivative or analog thereof, comprising at least one mutation in the insulin polypeptide, wherein the polypeptide exhibits disrupted cross-P fibrillation. In some aspects, the at least one mutation is in the A-chain of the polypeptide. In some aspects, the at least one mutation is in an amino acid in the hydrophobic core of the cross-P fold of the insulin protomer. In some aspects, the mutation is in a residue, for example ThrA8, SerA9, IleAlO or AsnAl 8, that may be mutated.

[0063] In various aspects, the at least one mutation is selected from ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, UeAlOGlu, AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnAl 8Trp, AsnAl 8Glu, and AsnAl 8Gln, wherein the first 3 letters correspond to the amino acid to be mutated, the fourth letter followed by the number correspond to the chain and residue number (for example Al 8, corresponds to the 18th residue on the A chain), and the last three letters correspond to the new amino acid.

[0064] In some aspects, the mutation is in a residue, for example ThrA8, SerA9, IleAlO or AsnAl 8, that may be mutated to any of the 19 remaining naturally occurring amino acids to be effective in reducing fibrillation in insulin. In an aspect, the at least one mutation is selected from ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, ThrA8Ala, ThrA8Asp, ThrA8Asn, ThrA8Cys, ThrA8Gly, ThrA8His, ThrA8Ile, ThrA8Leu, ThrA8Met, ThrA8Pro, ThrA8Ser, and ThrA8Val. [0065] In another aspect, the at least one mutation is selected from SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, SerA9Ala, SerA9Asp, SerA9Asn, SerA9Cys, SerA9Gly, SerA9His, SerA9Ile, SerA9Leu, SerA9Met, SerA9Pro, SerA9Thr, or SerA9Val.

[0066] In still another aspect, the at least one mutation is IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, IleAlOGlu, IleAlOAla, IleAlOAsp, IleAlOAsn, IleAlOCys, IleAlOGly, IleAlOHis, IleAlOLeu, IleAlOMet, IleAlOPro, IleAlOSer, IleAlOThr, and IleAlOVal.

[0067] In a further aspect, the at least one mutation is selected from AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, AsnA18Gln, AsnA18Ala, AsnA18Asp, AsnA18Cys, AsnA18Gly, AsnA18His, AsnA18Ile, AsnA18Leu, AsnA18Met, AsnA18Pro, AsnA18Ser, AsnA18Thr, and AsnA18Val.

[0068] In some aspects, the engineered insulin polypeptide may 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 an aspect, the at least 1, or at least 2, or at least 3, or at least 4 or more mutation(s) is in an amino acid in the hydrophobic core of the cross-P fold of the insulin protomer. In an aspect, the at least 1, or at least 2, or at least 3, or at least 4 or more mutation(s) is in a residue, such as, but not limited to ThrA8 and SerA9, IleAlO, or AsnA18.

[0069] In an aspect, the mutations may be in ThrA8 and SerA9, ThrA8 and IleAlO, ThrA8 and AsnA18, SerA9 and IleAlO, SerA9 and AsnA18, IleAlO and AsnA18.

[0070] In an aspect, the mutations may be in ThrA8, SerA9, and IleAlO. In an aspect, the mutations may be in ThrA8, SerA9, and AsnA18. In an aspect, the mutations may be in ThrA8, IleAlO, and AsnA18. In an aspect, the mutations may be in SerA9, IleAlO, and AsnA18.

[0071] In some aspects, the engineered insulin polypeptide comprises an A chain comprising an amino acid sequence at least about 80% identical to one or more of SEQ ID NOS: 1-76 as provided in Table 1 or a functional derivative or analog thereof. In some aspects, the engineered insulin polypeptide comprises an A-chain comprising an amino acid sequence 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 SEQ ID NOS: 1-76 or a functional derivative or analog thereof. In some aspects, the B-chain of the engineered polypeptide comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 77 or a functional derivative thereof. In some 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 NOS: 77 or a functional derivative thereof. As used herein, a functional derivative or analog of insulin includes but are not restricted to acylated insulin, basic insulin, insulin degludec, isophane insulin, insulin aspart, insulin lispro, and insulin glulisine, desoctapeptide insulin, insulin-zinc complexes, higher molecular weight insulins, N- carboxyaroyl insulins, insulin glargine, detemir, insulin derivatives wherein a lipophilic side chain is attached to the B-chain, insulin comprising a long hydrocarbon group like palmitoyl, or recombinant insulin or combinations thereof. These derivatives have been designed in the art to provide various benefits to insulin production, delivery and/or use, and the mutation/s disclosed herein can be made in the context of these derivatives.

Table 1: [0072] In some aspects, the current disclosure also encompasses a composition comprising an engineered insulin polypeptide as disclosed, and at least a pharmaceutically acceptable excipient. In some aspects, the composition as disclosed herein is formulated with at least one pharmaceutically acceptable excipient for administration into a subject in need thereof. In some aspects, the composition can be formulated as a liquid, gel, or a dry powder formulation.

[0073] In some aspects, the pharmaceutical acceptable excipient may be a liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, taste-masking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a coloring agent, solvent or encapsulating material or any combination thereof.

[0074] In some aspects, for example wherein the composition is formulated as a liquid, the pharmaceutically acceptable excipient may comprise one or more of a buffering agent, an isotonic agent, a preservative, or combinations thereof. In some aspects, where in the composition is formulated as a dry powder, the formulation may further comprise bulking agents, carriers, additives, dispersants, lubricants, or disintegrants. The amount and types of excipients utilized to form the compositions may be selected according to known principles of pharmaceutical science.

[0075] In some aspects, the at least one excipient may be a buffering agent. The buffer may be selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycine, lysine, arginine, sodium dihydrogen 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 -hydroxy ethyl]-2-aminoethanesulfonic acid), bicine (N,N-bis-[2- hydroxyethyl]glycine), BIS-TRIS (bis[2 -hydroxy ethyl]iminotris[hydroxymethyl]-methane), DIPSO (3 [N,N-bis(2-hydroxethyl]amin]-2 -hydroxypropanesulfonic acid), ethylenediamine dihydrochloride, glycylglycine, HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), HEPPSO(N-[2 -hydroxy ethyl]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]- aminomethan), 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 thereof and/or mixtures thereof. In one aspect the composition may have a pH between about 5.5 to about 8.5. In another aspect the pH is from about 5.8 to about 6.8, about 6.8 to about 7, or about 7.0 to about 8.5.

[0076] In a further aspect, the pharmaceutically acceptable excipient may comprise a pharmaceutically acceptable preservative for example, phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl phydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3-(4-chlorophenoxy)propane-l,2-diol) or mixtures thereof. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0077] In one aspect, the pharmaceutically acceptable excipient may comprise an isotonic agent for example, a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. 1 -glycine, 1 -histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), 1,2-propan ediol (propyleneglycol), 1,3 -propanediol, 1,3 -butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxy ethyl starch and carboxymethylcellulose-Na may be used. In one aspect the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one — OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one aspect the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. Each one of these specific isotonic agents constitutes an alternative aspect of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0078] In a further aspect, formulation may comprise a surfactant. Examples of surfactants 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, sold under trade name BRIJ®), alkoxylated fatty acids, such as MYRJ™, polypropylene glycols, block copolymers such as poloxamer 188 (CAS Number 9003-11-6, sold under trade name PLURONIC® F-68) and poloxamer 407 (PLURONIC® F127), sorbitan alkyl esters (e.g., SPAN®), poly ethoxylated castor oil (e.g., KOLLIPHOR®, CREMOPHOR®) and trehalose and derivatives thereof, such as trehalose laurate ester. In certain embodiments, the surfactant is selected from the group consisting of polyoxyethylene (23) lauryl ether, poloxamer 188 and trehalose laurate ester. Most preferred is poloxamer 188. In some aspects, the composition does not include a surfactant, as it is not prone to fibrillation.

[0079] Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. In some aspects, the pharmaceutically acceptable excipient may comprise an ion, for example zinc.

[0080] In some aspects, the compositions can also include an excipient for stabilization of the insulin or a derivative thereof, such as a buffer, salts (for example: sodium chloride, magnesium chloride), a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating insulin include albumin, protamine, or the like. Typical carbohydrates useful in formulating insulin include sucrose, mannitol, lactose, trehalose, glucose, or the like.

[0081] The weight fraction of the excipient or combination of excipients in the composition may 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.

[0082] The compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active factor described herein, which can be in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the subj ect.

[0083] In each of the aspects described herein, a composition of the disclosure may optionally comprise one or more additional drug or therapeutically active agent in addition to the at least one factor disclosed herein. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition. Examples of additional drugs may include glucagon or glucagon like peptide - 1 (GLP-1). [0084] In some aspects, the compositions as disclosed herein is formulated in a unit dosage form, or a multiple dosage form. In some 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 some aspects, the composition as disclosed herein is formulated for delivery using an injector, a closed loop system, an insulin pen or an insulin pump.

[0085] In some aspects, the current disclosure also encompasses a polynucleotide sequence comprising a nucleic acid sequence that encodes an engineered insulin polypeptide comprising an A-chain as disclosed herein, or a B-chain as disclosed herein or any combinations thereof. In some aspects, the polynucleotide may comprise a nucleic acid sequence encoding an insulin A- chain at least about 85% identical to any one of SEQ ID NOS: 1-76. In some aspects, the polynucleotide may comprise a nucleic acid sequence encoding an insulin A-chain comprising an amino acid sequence at least about 80% identical to any one of SEQ ID NOS: 1-76 or a functional derivative thereof and encoding an insulin B-chain comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 77, or functional derivatives thereof. The polynucleotide may comprise a nucleic acid sequence that encodes insulin polypeptides as disclosed herein which further comprise other mutations or modifications that provide additional benefits unrelated or related to fibrillation. The nucleic acid sequence may encode a modified A- or B- chain sequence containing an unrelated substitution or extension elsewhere in the polypeptide or modified proinsulin analogues. In some aspects the polynucleotide may also, if necessary, be operably linked to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. In some aspects, to direct the insulin peptide into the secretory pathway of the host cells, the polynucleotide may further encode a secretory signal sequence (also known as a leader sequence, pre-pro sequence or pre sequence). The secretory signal sequence is joined to the nucleic acid sequence encoding the polypeptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the nucleic acid sequence encoding the polypeptide. The secretory signal sequence may be that which is normally associated with the peptide or may be from a gene encoding another secreted protein.

[0086] The polynucleotide, as disclosed herein may be an isolated polynucleotide, an expression vector, a plasmid vector, a viral vector, a transposon, a gene editing polynucleotide sequence, or a virus and may be inserted into a host cell such as a prokaryotic host cell like an E. coli cell line, or an eukaryotic cell line, for example a fungal cell line such as S. cerevisiae or Pischia pastoris strain or cell line, or a mammalian cell line, for example BHK or CHO. In some aspects, the polynucleotide can be used in vitro or in vivo to produce the engineered insulin polypeptides of the current disclosure. In some aspects, the host cells as disclosed herein may be used for the expression and purification of the disclosed polypeptides. Methods of using these polynucleotide and host expression systems are well known in the art.10 micrometers.

B. Methods of Making

[0087] In some aspects, the current disclosure also encompasses methods of making and using the engineered insulin polypeptide and compositions as disclosed herein.

[0088] In some aspects, the method of making the engineered insulin polypeptide may involve standard techniques known in the field. For example, in one aspect, the method may involve a) cloning a polynucleotide sequence encoding the engineered insulin polypeptide disclosed herein into a suitable expression system, for example an expression vector, expression cassette, a plasmid vector, a viral vector, a transposon, b) transfecting or transforming the expression vector or expression cassette into a suitable host cell, for example a prokaryotic or a eukaryotic cell, c) expressing the engineered insulin polypeptide, d) purifying the polypeptide, and e) formulating it into suitable a pharmaceutical composition. Methods of cloning and expression of engineered protein are standard in the field (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). In one aspect, the engineered insulin polypeptide may be produced in vitro using a standard method of in vitro protein expression.

[0089] In an aspect, a disclosed method can restore and/or improve one or more aspects of cellular homeostasis and/or cellular functionality, and/or metabolic dysregulation.

C. Methods of Treatment and Use

[0090] In some aspects, the current 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.

[0091] In an aspect, the engineered insulin polypeptide comprises at least one mutation in a residue, for example ThrA8, SerA9, IleAlO or AsnA18. In some aspects, the at least one mutation may be ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, IleAlOGlu, AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, or AsnA18Gln. In an aspect, the at least one mutation may be ThrA8Arg, ThrA8Lys, ThrA8Phe, ThrA8Tyr, ThrA8Trp, ThrA8Gln, ThrA8Glu, ThrA8Ala, ThrA8Asp, ThrA8Asn, ThrA8Cys, ThrA8Gly, ThrA8His, ThrA8Ile, ThrA8Leu, ThrA8Met, ThrA8Pro, ThrA8Ser, or ThrA8Val. In an aspect, the at least one mutation may be SerA9Arg, SerA9Lys, SerA9Phe, SerA9Tyr, SerA9Trp, SerA9Gln, SerA9Glu, SerA9Ala, SerA9Asp, SerA9Asn, SerA9Cys, SerA9Gly, SerA9His, SerA9Ile, SerA9Leu, SerA9Met, SerA9Pro, SerA9Thr, or SerA9Val. In an aspect, the at least one mutation may be IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, IleAlOGlu, IleAlOAla, IleAlOAsp, IleAlOAsn, IleAlOCys, IleAlOGly, IleAlOHis, IleAlOLeu, IleAlOMet, IleAlOPro, IleAlOSer, IleAlOThr, or IleAlOVal. In an aspect, the at least one mutation may be AsnA18Arg, AsnA18Lys, AsnA18Phe, AsnA18Tyr, AsnA18Trp, AsnA18Glu, AsnA18Gln, AsnA18Ala, AsnA18Asp, AsnA18Cys, AsnA18Gly, AsnA18His, AsnA18Ile, AsnA18Leu, AsnA18Met, AsnA18Pro, AsnA18Ser, AsnA18Thr, or AsnA18Val.

[0092] In some aspects, the current disclosure encompasses a method of reducing the blood glucose level of a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of the engineered insulin polypeptide as disclosed herein or a composition comprising the engineered insulin polypeptide as disclosed herein and at least one excipient as disclosed herein. In some aspects, the subject is suspected of having, or diagnosed to have diabetes. In some aspects, the subject is suspected of having, or diagnosed to have type 1 diabetes, type 2 diabetes, pre-diabetes or hyperglycemia.

[0093] As used herein, the term "subject" may include an animal, or human, to whom treatment according to the methods of the present disclosure is provided. More particularly, the term subject can include animals used in assays such as those 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, equine, poultry, felines, bovines, murines, canines, and the like. Human and veterinary applications are anticipated by the present disclosure. The term includes but is not limited to birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents, such as mice and rats, rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep, chickens and goats. In some aspects, the subjects are humans, chickens, or mice. In some aspects, the subject is a human. Both pediatric and adult subjects are included. For example, in any of the methods described herein, the subject can be at least 6 months old (e.g., 6 months or older, 12 months or older, 18 months or older, 2 years or older, 4 years or older, 6 years or older, 10 years or older, 13 years or older, 16 years or older, 18 years or older, 21 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or 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, or more years old). [0094] As provided herein, the composition as disclosed herein may 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, intraprostatical, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposomal, local, mucosal, subcutaneous, sublingual, topical, trans buccal, and transdermal route. In some particular aspects, the route and means of administration for the compositions of the present disclosure may be selected from self-administered subcutaneous injection, e.g., by use of a needle and syringe or a pen device, or by continuous subcutaneous insulin infusion therapy with an insulin pump device, though intravenous, intradermal, or intraperitoneal routes may also be used. In some aspects, the present disclosure also provides an article of manufacture comprising or containing the composition as disclosed herein. In certain aspects, the article of manufacture is a multi-use vial. In other aspects, the article of manufacture is a multi-use pre- filled cartridge. In other aspects, the article of manufacture is a re-usable pen 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 use in a pump device for continuous subcutaneous insulin infusion therapy. Any of these articles of manufacture are to be understood as means of administration, as is a needle and syringe.

[0095] Regardless of the route of administration selected, the compositions described herein, which may be used in a suitable hydrated form and/or are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the compositions described herein may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of factors including the activity of the particular insulin composition described herein, the route of administration, the time of administration, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. In some aspects, when used in the treatment of diabetes, insulin therapy is chronic and may require daily dosing. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. [0096] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition 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.

[0097] While it is possible for a compound described herein to be administered alone, it may be administered in combination with other active composition. By the term “combination” is meant either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a composition described herein and a combination partner may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a cooperative, e.g., synergistic, effect, or any combination thereof. The compounds described herein may be administered, simultaneously or sequentially, with glucagon, GLP-1, an anti-inflammatory, antiproliferative, antibiotics, NSAIDs, painkillers, chemotherapeutic agent, immunosuppressant, other anti-cancer drugs, cytotoxic agent or salt thereof.

[0098] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of the other active composition administered in combination.

[0099] In an aspect, a disclosed method can further comprise monitoring the subject for adverse effects.

[0100] In an aspect, a disclosed method can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof. In an aspect, the subject’s life expectancy can be compared to the life expectancy of a control (i.e., no treatment). In an aspect, a control can be a subject not receiving a disclosed composition. In an aspect, a control can be a pooled number of subjects not receiving a disclosed pharmaceutical composition. In an aspect, a control is one or more subjects having the same type of condition as the subject. As used herein, “life expectancy” is defined as the time at which 50 percent of subjects are alive and 50 percent have passed away.

[0101] In an aspect, subject life expectancy can be indefinite following treatment with a disclosed method. In an aspect, subject life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with the identical or near identical viral infection and the identical or near identical predicted outcome.

[0102] In an aspect, a disclosed method can improve and/or enhance the subject’s quality of life, which can be measured subjective and/or objectively.

[0103] In an aspect, a disclosed method can increase treatment options of a subject.

[0104] In an aspect, a disclosed method can provide a personalized treatment of the subject.

[0105] In an aspect, a disclosed method can improve and/or restore normal metabolism of one or more organ systems in the subject.

[0106] In an aspect, a disclosed method can restore and/or improve one or more aspects of cellular homeostasis and/or cellular functionality, and/or metabolic dysregulation.

[0107] In some aspects, the current disclosure also encompasses use of the compositions disclosed herein for laboratory studies and preclinical testing.

D. Kits

[0108] In some aspects, the current disclosure encompasses a kit comprising the composition as disclosed herein and instructions for use. In some aspects, the present disclosure provides a kit for administering a composition as disclosed herein. Such a kit may comprise a means for holding and/or administering insulin compositions.

[0109] In some aspects, kits disclosed herein can have a medical container, which holds the composition in a safe, stable and durable way. In some examples, kits disclosed herein may also comprise a means to administer the composition, such as a needle, tube, a spatula or combinations thereof.

[0110] Any of the kits may further comprise an instruction manual providing guidance for using the kit for treatment. The manual may be written with the physician or the liver specialist as the intended reader.

[oni] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.

[0112] In an aspect, a disclosed kit can be used to provide a personalized treatment of the subject.

IX. EXAMPLES [0113] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Materials and Methods

[0114] Cryo-EM data acquisition: WT insulin purchased from Sigma- Aldrich (12643) was used for the structural determination of fibrils. The insulin was dissolved in a pH 2 buffer solution at 1 mg/ml. These solutions were incubated at 65 °C for 7 days to promote the fibril formation. [0115] The cryo-EM grid was prepared by applying 3 pl of the insulin fibril samples to glow- discharged Quantifoil Rl.2/1.3 300-mesh gold holey carbon grids (Quantifoil, Micro Tools GmbH, Germany). Grids were blotted for 4.0 seconds under 100% humidity at 4 °C before being plunged into the liquid ethane using a Mark IV Vitrobot. Micrographs were acquired on a Titan Krios microscope operated at 300 kV with a K3 direct electron detector (Gatan), using a slit width of 20 eV on a GIF -Quantum energy filter. SerialEM 3.8 was used for the data collection. A calibrated magnification of 60,241 was used for imaging of insulin fibril sample, yielding a pixel size of 0.83 A on specimen. The defocus range was set from 1.6 pm to 2.6 pm. Each micrograph was dose-fractionated to 30 frames with a total dose of about 60 e-/A2.

[0116] Image processing: Super-solution movie frames were binned by a factor of 2 and summed into single micrographs after motion correction using MotionCor2 1.2. GCTF 1.16 was used for estimating the contrast transfer function (CTF) parameters. All subsequent image processing steps were carried out using Relion 3.1. Insulin filaments were picked manually and cleaned up by multiple-round reference-free 2D classification. Filaments of different width could be readily seen in the 2D class averages. Three of the abundant types, types I, II and III, were chosen for further processing. Initial models were generated by using the method described before. The helical rises per subunit were set to 4.75 A, while the helical twists were estimated from the cross-over distances of filaments in the micrographs. The subsequent 3D reconstruction and CTF refinement were performed with standard procedures in Relion. The final maps were sharpened with a soft-edged solvent mask, and the resolution was estimated based on the gold- standard Fourier shell correlation between the two half maps. [0117] Model building and refinement: The quality of the map of type II filaments were sufficient for de novo model building using Coot 0.98. Coordinates were refined by using the combination of ISOLDE 1.2 within Chimerax 1.2 and Phenix 1.19, with secondary structure restraints. The model quality and its correlation with the map were assessed with the validation tools in Phenix. Molecular figures were rendered with Chimera and ChimeraX.

[0118] Expression and purification of insulin WT and mutants: All insulin wild-type (WT) and mutants were obtained by bacterial expression and refolding. The genetically engineered insulin precursor, comprising the amino acid sequence "Bl...B29Lys-Ala-8xHis-Ala-Lys-AlGly...A21," was incorporated into the pET28a vector to facilitate its recombinant expression. First, the precursor protein underwent expression and refolding to yield the single-chain insulin precursor. Following this, the connecting His-tag was enzymatically cleaved using lysyl endopeptidase (Lys-C), thereby generating the mature insulin consisting of two polypeptide chains joined by disulfide bonds. All insulins were purified in the des-B30Thr form. Notably, this truncation of the insulin molecule shows no effects on its biological activity.

[0119] The detailed purification process is described as follows. The cells containing the vector described above were cultured at 37 °C, induced with 1 mM IPTG for 24 hours, and subsequently harvested. The cells were lysed by a French press method with a lysis buffer of 50 mM Tris-HCl and 2 mM EDTA at a pH of 8.0. Then, the inclusion body was collected through centrifugation of previous lysate and washed with a wash buffer containing 50 mM Tris-HCl, 2 mM EDTA, and 1% Triton X-100 at a pH of 8.0. The inclusion body was then solubilized in a solubilize buffer containing 30 mM Tris, 8 M Urea, 1 mM EDTA, and 5 mM DTT at a pH of 8 for 24 hours. A 10 ml portion of solubilized inclusion body was dialyzed in a 500 ml refolding buffer comprising 0.5 M Arg and 0.6 mM oxidized glutathione at a pH of 8.0 for 24 hours. The supernatant was concentrated and loaded onto a HisTrap excel column (Cytiva™), where the elution solution was collected after washing with imidazole. Then, the sample was digested using 50 pL Lys-C enzyme to cleave the His-tag. The final insulin sample was purified using size exclusion chromatography by Superdex 75 increase 10/300 GL columns (Cytiva™) under a pH 2 solution of HC1 and 150 mM NaCl for storage.

[0120] Thioflavin T (ThT) assay: The wild-type or mutant insulin samples were prepared by diluting them to a concentration of 0.5 mg/ml in either pH 2 (pH2 HC1, 150 mM NaCl) or pH 7 (pH 7 20 mM HEPS, 150 mM NaCl) buffer. 50 pL of each protein sample, mixed with 50 pM of ThT, was then aliquoted into a clear-bottomed 384-well plate. All ThT assays were performed using a multifunctional microplate reader (FLUOstar OPTIMA) with a predetermined test setup. The fluorescence signals were scanned every hour with excitation filters at 430/10 nm (center wavelength/b and width) and emission filter at 475/30 nm (center wavelength/bandwidth) and were preceded by a 3 -minute double orbital shaking. All conditions were done in triplicates at 37 °C. The signals were normalized to the maximum signal produced by wild-type and blank wells containing buffer and ThT were subtracted from the experimental values. The data were then plotted using GraphPad Prism, with the mean and standard error of the mean displayed as points and connecting lines with error bars.

[0121] Insulin receptor activation in cultured cells: The insulin receptor activation assay were performed as described earlier 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 free from mycoplasma contamination. Plasmid transfection was performed with Lipofectamine 2000 (Invitrogen). After 1 day, the cells were serum starved for 14-16 hours. For dose-dependent experiments, serum-starved cells were treated with insulin mutants diluted to 1, 10, or 100 nM in high-glucose DMEM without serum for 10 minutes at 37 °C. For time-dependent experiments, serum-starved cells were treated with insulin mutants diluted to 10 nM in high-glucose DMEM without serum for 10, 30, or 60 minutes at 37 °C. For incubation of reagents at room temperature, aliquots of each insulin mutant were prepared at 77.2 pM using buffer (20 mM HEPES pH 7.4, 150 mM NaCl) and stored protected from light for three days at room temperature. After the incubation period, serum-starved cells were treated with insulin mutants diluted to 100 nM in high-glucose DMEM without serum for 10 minutes at 37 °C.

[0122] Cells were incubated with 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 phenylmethyl sulfonyl fluoride (PMSF)] supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche) and PhosSTOP™ (Roche) on ice for 1 hour. After centrifugation at 18,213 g at 4 °C for 20 minutes, samples of cell lysates were analyzed by SDS-PAGE and Western blotting. Anti-IR-pYl 150/1151 (1 : 1000, 19H7, Cell signaling; labeled as pY IR) and anti-Myc (1 : 1000; 9E10, Roche; labeled as IR) were used as primary antibodies. For quantitative Western blots, anti-rabbit immunoglobulin G (IgG) (H+L) (Dylight 800 conjugates) and anti-mouse IgG (H+L) (Dylight 680 conjugates) (Cell signaling) were used as secondary antibodies. The membranes were scanned with the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). Levels of pY- IR were normalized to total IR levels and shown as intensities relative to that of IR treated with 10 nM WT insulin for 10 minutes. [0123] Statistical analysis: Prism 9 was used for the generation of graphs and for statistical analyses. Results are presented as mean ± s.d. or mean ± s.e.m. Two-tailed unpaired t tests were used for pairwise significance analysis for FIG. 3. Power analysis for sample sizes, and randomization and blinding methods were not used, and data were analyzed after the completion of all data collection in each experiment.

Example 1 Cryo-EM structures of insulin fibrils

[0124] To promote insulin fibrillation, human insulin was dissolved in a buffer of pH 2 and incubated at 65 °C for 7 days. Cryo-EM images of the resulting samples showed that most insulin molecules under this condition formed remarkedly long fibrils with a variety of widths and morphologies (FIG. 1 A). Filaments belonging to three abundant types were manually selected for 2D and 3D classifications (named type I, II, and III fibrils) (FIGs. 1 A-1C). A 3.6 A resolution 3D reconstruction of type II fibril was obtained, with the helical rise of 4.75 A and the left-handed twist of 1.27°, which allowed building of an atomic model (FIG. 1D-1F). The fibrils in type II contain two protofilaments packed in an asymmetric and anti-parallel manner (FIG. 1G). A 3D reconstruction of type III fibril reached lower resolution, for which an atomic model was not built (FIG. 1C). However, the density map clearly showed that this fibril contained two copies of the same protofilament as type II, but they were packed in parallel (FIG. 1C). A high- resolution 3D reconstruction of type I fibril could not be obtained, while the 2D class averages and low-resolution reconstruction indicated that type I fibril is comprised of a single protofilament (FIGs. 1 A-C). Without being bound by theory, it is hypothesized that the one intrachain disulfide bond in chain A (CysA6-CysAl 1) and two inter-chain disulfide bonds (CysA7- CysB7 and CysA20-CysB19) likely impose strong conformational restraints on the individual protomers, resulting in a single defined conformation of the protofilament that is formed by sequential stacking of the protomers along the long axis of the filament. In the three types of fibrils analyzed here, different numbers and arrangements of proto filaments determine the fibril form (FIGs. 1 A-C). In addition, wider fibrils composed of more than two protofilaments were also found in the sample, although they are less abundant.

[0125] The cross-B fold of the insulin protomer: Native insulin is comprised of mostly helical structural elements that are packed into a compact global domain. In contrast, insulin protomers in the fibril switch to an extended and flat overall shape, allowing them to stack sequentially to form the cross-P protofilament (FIG. 1G). The N-terminal portion of chain A adopts an S-shape, with the first half stapled by the intra-chain disulfide bond (CysA6-CysAl 1), which is followed by a P-strand formed by the C-terminal portion (FIGs. 1H and II). The middle portion of chain B forms a relatively long P-strand, which is tethered to chain A by the two inter-chain disulfide bonds (CysA7-CysB7 and CysA20-CysB19) (FIGs. 1H and II). The C-terminal portion of chain B extends away and contributes to the interaction between two protofilaments (FIGs. 1G and 1H).

[0126] In addition to the two inter-chain disulfides, the A- and B-chains of insulin interact through two clusters of hydrophobic residues (FIGs. 1H and II). Particularly, IleAlO and LeuA16 make direct interaction with LeuB15 and LeuB17, meanwhile LeuB6 and LeuBl 1 contact CysA7-CysB7 disulfide bond (FIGs. 1H and II). Moreover, several polar residues are trapped between the A- and B-chains in the protofilament. In particular, ThrA8 and SerA9 both make contact with GluB13 (FIGs. 1H and II). The involvement of GluB13 in the core of the protofilament provides an explanation for the requirement of low pH for inducing fibrillation, as it helps protonate the carboxyl group of GluB13 and neutralize the negative charge which would otherwise be unfavorable to be trapped in the hydrophobic core of the protofilament.

[0127] It is worth noting that, in the cross-P fold of monomeric insulin, the C-terminus of insulin B-chain is located ~50 A away from the N-terminus of insulin A-chain (FIG. II). This means that, to connect these two termini without disturbing the cross-P fold, a loop longer than 20 residues is required. Without being bound by theory, this may explain why the single-chain insulin analogs, which normally contain a short inter-chains loop, cannot form stable amyloid fibrils.

[0128] Subunit stacking: Insulin protomers along the protofilament axis form regular backbone hydrogen bonds that contribute to their interaction (FIG. 1G). In addition, the subunit stacking in each protofilament is driven by the 7t-stacking of sidechains of aromatic residues, such as TyrA14, TyrA19 and TyrB16, as well as the hydrogen-bonded ladders formed by sidechains of asparagine and glutamine residues in the A-chain of insulin, including GlnA5, GlnA15, AsnA18 and AsnA21 (FIG. 1H). Furthermore, due to the out-of-plane tilting of the disulfide bonded loop in the A-chain of insulin (FIG. 1G), ThrA8 forms a hydrogen bond with the GluB13 residue that is from the neighboring subunit along the length of the protofilament. Such non-planar subunit conformation further stabilizes the protofilament structure by providing additional subunitsubunit contact along the protofilament axis (FIG. 1G). These structural observations indicate that both inter- and intra-molecular disulfide bonds are critical for the structural stability of insulin fibril, which supports previous results that insulins in the presence of a reducing agent, such as tris (20-carboxy ethyl) phosphine (TCEP), tend to assemble into highly flexible filaments. In addition, ArgB22 and the C-terminal carboxyl group of A-chain of insulin form an alternating positive-negative charge electrostatic interaction, which contributes to the protofilament stacking (FIG. 1H).

[0129] Inter-protofilament assembly: In the type II insulin fibril, the two protofilaments pack against each other anti-parallelly (FIGs. 1C and 1G). The packing between the two protofilaments is predominately mediated by hydrophobic interactions involving the side chains of PheB24 and TyrB26 in the C-terminus of chain B from one protofilament and the side chains of SerA12 and TyrA14 on the outer surface of the ‘S’-shaped insulin A-chain from the second protofilament (FIG. 1H). In addition, the electrostatic interactions between ArgB22 in the first protofilament and GluA4 in the second protofilament further contribute to the inter-protofilament packing in the type II fibril (FIG. 1H).

[0130] Different from the type II fibril, the C-terminus of insulin B-chain plays an exclusive role in mediating the parallel packing of the two protofilaments in the type III fibril through forming a homotypic interface (FIG. 1C). Without being bound by theory, these structural observations indicate that the C-terminus of insulin B-chain is crucial for the supra-protofilament organization of insulin fibril, although it is not involved in the fold of each subunit. Furthermore, in one protofilament of both type II and III fibrils, the C-terminus of the insulin B-chain adopts a 90° bent conformation (FIGs. 1H and II). This bending makes the aromatic residues in C-terminus of insulin B-chain more exposed, which facilitates the lateral protofilament interaction and thus promotes the formation of the larger supra-fil ament bundles.

Example 2 Designing insulin mutants resistant to fibriiiization

[0131] Based on the structure of type II insulin fiber described here and the structure of the IR/insulin complex previously published, specific mutants of insulin were designed to reduce fibriiiization, while retaining its binding to IR. It was hypothesized that mutating small or hydrophobic residues in the hydrophobic core of the cross-P fold of the insulin protomer to large polar residues would reduce the formation of the cross-P structure of insulin and thus render its resistant to fibriiiization. Three mutants were made, ThrA8Arg, IleAlOArg, and AsnA18Gln. Importantly, none of these residues participates in the insulin binding to IR. Fibril formation of these three mutants was first evaluated using the well-established Thioflavin T (ThT) assay for amyloid formation. Incubation of insulin WT in the pH 2 buffer at 65 °C for approximately 2 days resulted in a strong ThT signal, indicating robust insulin fibril formation (Fig. 2A). Strikingly, no significant ThT signal was observed for the three mutants, indicating that these mutations successfully prevented insulin fibrilization (Fig. 2A). A negative stain EM analysis confirmed that none of the three insulin mutants formed any long fibrils after incubation under the same condition for 7 days (Fig. 2B). These results validate the structural model of insulin fibril and demonstrate that the fibril forming ability of insulin could be abolished by a point mutation.

[0132] Next, the ability of these insulin fibrillizati on-resistant mutants (ThrA8Arg, IleAlOArg, and AsnA18Gln) to induce IR activation was tested. Consistent with the structural models, all the insulin fibril mutants were able to fully activate IR over a wide range of concentrations and at multiple time points (FIGs. 3 A-D). Strikingly, all the three insulin mutants stimulated slightly increased IR autophosphorylation in comparison to insulin WT (FIGs. 3 A and 3B).

[0133] To further investigate whether these mutations could protect against the disruption of insulin function caused by fibril formation, insulin stocks were incubated at room temperature for three days to promote insulin fibril formation and the effect of insulin on the activation of IR was examined. Under this condition, all the three mutants drove small but statistically significant increased IR autophosphorylation compared to insulin WT (FIGs. 3E and 3F). These data indicate that these insulin mutations prevent insulin fibril formation, thus preserving insulin activity on IR activation over a longer period of time.

[0134] Next, the high temperature and long-term stability of one of the mutants, AsnA18Gln, compared to native insulin (WT) was tested. First, the activity of insulin WT and AsnA18Gln mutant was evaluated after incubation at a high temperature (70 °C) for 16 hrs. with 1000 rpm agitation. Quantitative western blot analysis was used to determine the levels of insulin receptor autophosphorylation (FIG. 4 A), active phosphorylation of AKT (FIG. 4B), and active phosphorylation at ERK (FIG. 4C). The higher activity of the AsnA18Gln mutant is indicative of higher stability.

[0135] Next, referring to FIG. 5, insulin WT and the AsnA18Gln mutant were incubated for 14 days at 37 °C with 250 rpm agitation to conduct in vivo insulin tolerance tests on mice (B6, male, 2-moth-old). Prior to testing, the mice were fasted for 4 hours. The mice were injected with 1.5 U/kg of inulin WT or AsnA18Gln mutant and glucose levels were measured at various time points. The increased effectiveness at lowering blood glucose levels over native insulin indicates that the AsnA18Gln mutant has increased thermal stability at physiological temperature.

Conclusion

[0136] The studies described above show that insulin fibril exhibits a high degree of structural heterogeneity, which is referred to as amyloid polymorphism. The cryo-EM dataset showed at least three distinct types of insulin fibrils. In both type II and III, the protofilaments assume quite similar conformations, indicating that the polymorphs of insulin fibril result from the different arrangements of similar protofilaments. In both type II and III insulin fibrils that comprise of two protofilaments, the C-terminus of insulin B-chain plays a role in the lateral interaction between protofilaments. Notably, GluB13 is trapped inside the hydrophobic core of the protofilament.

The packing of the GluB13 in the hydrophobic environment is favored at low pH when the negative charge of GluB13 is neutralized. This explains why low pH promotes the process of insulin fibrilization.

[0137] Disulfide bonds may affect the subunit assembly at various stages of amyloid fibrillation and the eventual fibril morphology. The cryo-EM structure showed that both intra- and inter- molecular disulfide bonds of insulin participate in stabilization of the cross-P fold of each subunit and maintaining the close packing of A- and B-chains of insulin within each subunit. In addition, the disulfide bonded loop in the insulin A-chain simultaneously contacts two adjacent subunits along the protofilament axis, thereby contributing to the subunits stacking in each protofilament, leading to increased rigidity of the fibrils.

[0138] Insulin B-chain residues Bl 1 - B17 (LVEALYL) adopt a similar P-strand conformation in the crystal structure of the truncated peptide and the cryo-EM structure of full-length insulin fibril (FIG. 6). However, this peptide in the crystal structure forms homotypic interdigitated packing, rather than interacting with insulin A-chain as seen in our cryo-EM structure. This segment of insulin B-chain may form a P-strand and cross-P amyloids, even in the absence of A- chain. In the context of full-length insulin, the presence of both A- and B-chains and the disulfide bonds strongly constrain the conformations of both chains and their interactions, resulting in one well-defined protofilament, which can further pack through the more flexible C- terminal tail of B-chain into different types of fibrils.

[0139] In conclusion, the structural work disclosed herein provides valuable information for defining the molecular mechanisms underlying the formation of full-length insulin fibrils. Understanding the structural basis of insulin fibrillation can enable designing insulin analogs, such as those exemplified above, to treat patients with type-1 diabetes more effectively.

Claims

X. CLAIMS What is claimed is:

1. An engineered insulin polypeptide, comprising at least one mutation in the engineered insulin polypeptide, wherein the engineered insulin polypeptide exhibits disrupted cross-P fibrillation.

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

3. The engineered insulin polypeptide of any one of claims 1 or 2, wherein the at least one mutation is in an amino acid in hydrophobic core of cross-P fold of insulin protomer.

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, IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, UeAlOGlu, 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 IleAlOArg, IleAlOLys, IleAlOPhe, IleAlOTyr, IleAlOTrp, IleAlOGln, IleAlOGlu, IleAlOAla, IleAlOAsp, IleAlOAsn, IleAlOCys, IleAlOGly, IleAlOHis, IleAlOLeu, IleAlOMet, IleAlOPro, IleAlOSer, IleAlOThr, or lleAlOVal.

8. The engineered insulin polypeptide of any one of claims 1-3, wherein 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.

9. The engineered insulin polypeptide of any one of claims 1-8, wherein the A-chain polypeptide comprises an amino acid sequence at least about 80% identical to a sequence as provided in 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 an engineered insulin polypeptide of any one of claims 1-10, and at least a pharmaceutically acceptable excipient.

12. The composition of claim 11, wherein the pharmaceutically acceptable excipient is a liquid or solid filler, a diluent, a binder, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant or wetting agent, taste-masking agent, an antioxidant, carrier, adjuvant, stabilizing agent, emulsifying agent, solution promoter, salt, solubilizer, antifoaming agent, surfactant, a flavoring agent, a 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 claim 11-14, wherein the composition is formulated to provide at least about 5U/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 dose administration or single dose administration.

17. The composition of any one of claims 11-16, wherein the composition is formulated for delivery using an injector, a closed loop system, an insulin pen or an 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 a 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 a 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 pre-diabetic or who is suffering from type 1 diabetes, type 2 diabetes, or hyperglycemia.

24. The method of any one of claims 22 or 23, wherein the administering is by any one of parenteral, oral, intraarterial, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, intravascular, liposomal, local, mucosal, subcutaneous, sublingual, topical, trans buccal, and transdermal route.

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

26. The method of any one of claims 22-25, wherein following the administration step, (i) the risk of unpredictable fluctuations of blood glucose levels or hyperglycemia is prevented and/or decreased, (ii) the survival of the subject is prolonged, (iii) the subject's quality of life is enhanced and/or improved, (iv) normal metabolism of one or more organ systems in the subject are improved and/or restored, (viii) one or more aspects of cellular homeostasis and/or cellular functionality, and/or metabolic dysregulation 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 as disclosed in any one of claims 11-17;

(B) instructions for use.

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