Insulin fusion protein

Abstract

The present disclosure provides compositions and methods for treating diabetes in dogs or cats. A dog or cat insulin-serum albumin fusion protein is administered to a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 315,296, filed on March 1, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] The sequence listing XML associated with this application is provided in XML file format and is incorporated into this specification by reference. The name of the XML file containing the sequence listing XML is SCTB_017_01WO_ST26.xml. The XML file is 40,960 bytes, created on February 27, 2023, and submitted electronically via the USPTO Patent Center. Technical Field

[0005] The present disclosure generally relates to compositions and methods for treating diabetes. Background Art

[0006] Diabetes is a syndrome associated with prolonged hyperglycemia due to loss or dysfunction of insulin secretion by pancreatic β cells, decreased insulin sensitivity in tissues, or both. In dogs, β cell loss tends to be rapid and progressive, and is usually due to immune-mediated destruction, vacuolar degeneration, or pancreatitis. In cats, β cell loss or dysfunction is a result of insulin resistance, islet amyloidosis, or chronic lymphoplasmacytic pancreatitis. In humans, β cell loss is caused by autoimmune responses in type 1 diabetes and decreased insulin sensitivity in type 2 diabetes.

[0007] Insulin is an endogenous peptide hormone produced by the beta cells of the pancreatic islets, and it is considered the body's primary anabolic hormone. Insulin is the primary means of treating diabetes in mammals. The current standard of care is twice-daily insulin injections and frequent medical visits and one-time diagnostics, which is expensive, time-consuming, and inconvenient.

[0008] The present disclosure provides compositions and methods related to insulin fusion proteins to provide sustained insulin half-life. Summary of the invention

[0009] The present disclosure provides a fusion protein for treating a companion animal, the fusion protein comprising proinsulin and serum albumin, wherein the proinsulin is canine proinsulin or feline proinsulin.

[0010] In some embodiments, the proinsulin is canine proinsulin.

[0011] In some embodiments, the canine proinsulin sequence shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:12.

[0012] In some embodiments, the proinsulin is feline proinsulin.

[0013] In some embodiments, the feline proinsulin sequence shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:15.

[0014] In some embodiments, the fusion protein comprises a polypeptide that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:1.

[0015] In some embodiments, the fusion protein comprises an N-terminal signal peptide.

[0016] In some embodiments, the signal peptide is canine insulin signal peptide.

[0017] In some embodiments, the signal peptide comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with MALWMRLLPLLALLALWAPAPTRA (SEQ ID NO: 7).

[0018] In some embodiments, the canine proinsulin is a canine proinsulin variant having a mutation at one or more cleavage sites compared to the reference polypeptide sequence shown in SEQ ID NO:10.

[0019] In some embodiments, canine proinsulin comprises K53R, R55K, and L86R mutations compared to the reference polypeptide sequence shown in SEQ ID NO:10.

[0020] In some embodiments, the canine proinsulin-serum albumin fusion protein comprises a linker that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:8.

[0021] In some embodiments, the polynucleotide encoding the fusion protein is operably linked to a promoter.

[0022] In some embodiments, the promoter is the cytomegalovirus enhancer / chicken b-actin promoter.

[0023] The present disclosure provides a pharmaceutical composition suitable for use in treating metabolic diseases in dogs or cats, comprising a fusion protein of an embodiment of the present disclosure.

[0024] In some embodiments, the fusion proteins and / or pharmaceutical compositions of the present disclosure are used in methods for treating a canine or feline subject suffering from a metabolic disease, optionally diabetes.

[0025] In some embodiments, the fusion proteins and / or pharmaceutical compositions of the present disclosure are used in the manufacture of a medicament for treating a canine or feline subject suffering from a metabolic disease, optionally diabetes.

[0026] In some embodiments, the fusion protein is formulated at a concentration of at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, or at least about 15 mg / kg of the fusion protein and / or wherein the fusion protein is administered intravenously.

[0027] In some embodiments, the fusion protein is administered at a dose of at least about 0.01 mg, at least about 0.05 mg, at least about 0.1 mg, at least about 1 mg, at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least about 15 mg.

[0028] In some embodiments, the fusion protein is administered intravenously to a canine or feline subject.

[0029] The present disclosure provides a method for treating a canine or feline subject suffering from a metabolic disease, the method comprising administering an effective amount of the fusion protein of the present disclosure and / or the pharmaceutical composition of the present disclosure to the canine or feline subject.

[0030] In some embodiments, the metabolic disease is diabetes.

[0031] In some embodiments, the diabetes is type 1 diabetes.

[0032] In some embodiments, the diabetes is type 2 diabetes.

[0033] In some embodiments, the therapeutically effective amount is administered intravenously.

[0034] In some embodiments, the therapeutically effective amount is between 0.01 mg / kg and 15 mg / kg of the fusion protein.

[0035] In some embodiments, the therapeutically effective amount is between 1 mg / kg and 15 mg / kg of the fusion protein.

[0036] In some embodiments, the method reduces the subject's fasting blood glucose by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0037] The present disclosure provides a polynucleotide encoding a fusion protein for treating a companion animal, the fusion protein comprising proinsulin and serum albumin, wherein the proinsulin is canine proinsulin or feline proinsulin.

[0038] In some embodiments, the canine proinsulin-serum albumin fusion polynucleotide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:2.

[0039] In some embodiments, the feline proinsulin-serum albumin fusion polynucleotide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:19. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A to 1C Schematic diagrams showing exemplary canine insulin proteins of the present disclosure. All three proteins contain a native signal peptide (SP) and a modified furin site. Figure 1A A schematic diagram showing an exemplary canine preproinsulin-serum albumin fusion protein (cINS-Alb). The caINS-Alb protein has a glycine / serine linker connecting the A chain of insulin to canine serum albumin. Figure 1B A schematic diagram showing an exemplary canine preproinsulin-transferrin fusion protein (cINS-Tf). The caINS-Tf protein has a glycine / serine linker connecting the A chain of insulin to canine transferrin. Figure 1C A schematic diagram of an exemplary canine preproinsulin protein containing a modification of the furin site is shown and used as a control (cINS-2-1).

[0041] Figure 2 The in vitro insulin bioactivity of caINS-Alb and caINS-Tf compared to a control insulin standard is shown. 50 Values ​​are listed in the table below the figure. Ligand-induced insulin receptor activation in response to increased concentrations of purified cINS-Alb and cINS-Tf. Reference insulin standards were used as controls. Relative potency is expressed as relative light units. DETAILED DESCRIPTION

[0042] As described elsewhere herein, the present disclosure is based, at least in part, on the surprising discovery by the inventors that insulin fusion proteins achieve sustained biological activity of insulin in dogs and cats. Methods of making and using the fusion proteins are provided.

[0043] Insulin fusion proteins engineered to overcome the short half-life of the natural hormone by fusion with a protein with a longer half-life (e.g., serum albumin) are therapeutic advances for the treatment of diabetes. Long-acting insulin fusion protein expression constructs for canines and felines have been developed. The expression construct comprises a secretion signal peptide and a fusion domain intended to extend the circulation time of the resulting fusion protein.

[0044] The expression construct is administered to a subject in need thereof. Also provided are methods of using these fusion proteins in a regimen for treating type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), or metabolic syndrome in a veterinary subject and increasing the half-life of insulin in the subject.

[0045] The present disclosure encompasses insulin-albumin fusion proteins comprising a therapeutic protein with insulin activity. The present disclosure also encompasses polynucleotides comprising nucleic acid molecules encoding a therapeutic protein with insulin activity fused to albumin or a fragment (portion) or variant of albumin, or alternatively consisting thereof. Albumin can be fused to the N-terminus, C-terminus, or both ends of the therapeutic protein with insulin activity. In some embodiments, albumin is fused to the C-terminus of proinsulin. The present disclosure also encompasses polynucleotides comprising nucleic acid molecules encoding a protein comprising a therapeutic protein with insulin activity fused to albumin or a fragment (portion) or variant of albumin sufficient to prolong its activity in vivo.

[0046] Preamble sequence

[0047] In one embodiment, the insulin protein comprises a leader sequence, which may comprise a secretory signal peptide. As used herein, the term "leader sequence" refers to any N-terminal sequence of a polypeptide. In one embodiment, the canine or feline insulin protein described herein comprises a leader sequence or signal sequence and proinsulin. In one embodiment, the leader sequence is a native sequence (canine or feline insulin) leader region. In another embodiment, the leader sequence is a heterologous sequence, i.e., derived from another protein other than canine or feline insulin.

[0048] In one embodiment, the leader is a canine IL-2 sequence.In one embodiment, the IL-2 leader comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:21.

[0049] SEQ ID NO:21:MYKMQLLSCIALTLVLVANS

[0050] In another embodiment, the leader is a native canine insulin sequence.In one embodiment, the canine leader comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:7.

[0051] SEQ ID NO:7:MALWMRLLPLLALLALWAPAPTRA

[0052] In one embodiment, the leader sequence is a feline IL-2 sequence.In one embodiment, the IL-2 leader comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:13.

[0053] SEQ ID NO:13:MYKIQLLSCIALTLILVTNS

[0054] In another embodiment, the leader is a native feline insulin sequence.In one embodiment, the canine leader comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:9.

[0055] SEQ ID NO:9:MAPWTRLLPLLALLSLWIPAPTRA

[0056] The leader sequence can be derived from the same species that is ultimately intended to be administered, i.e., canines or felines. As used herein, the term "derived" or "derived from" means that the sequence or protein is derived from a specific test species or shares the same sequence with a protein or sequence derived from a specific test species. For example, a leader sequence "derived from" a dog or cat shares the same sequence (or a variant thereof, as defined herein) with the same leader sequence expressed in a dog or cat. However, the specified nucleic acid or amino acid does not actually have to be derived from a dog or cat. A variety of techniques known in the art that can produce the desired sequence include mutagenesis of similar proteins (e.g., homologues) or artificial generation of nucleic acid or amino acid sequences. "Derived" nucleic acids or amino acids retain the function of the same nucleic acids or amino acids in the species from which they are "derived", regardless of the actual source of the derived sequence.

[0057] insulin

[0058] Insulin is involved in the regulation of glucose utilization in the body. The inability of the body to synthesize insulin or the resistance of cells to insulin leads to diabetes characterized by chronic hyperglycemia. Preproinsulin is transcribed as a chain of 110 amino acids. Removal of the signal peptide from its N-terminus produces proinsulin. Formation of a disulfide bond between the A and B chain components and removal of the middle C chain produce a biologically active insulin molecule containing 51 amino acids, which is less than half the size of the original translation product.

[0059] As used herein, the term "insulin" refers to insulin or its functional fragments, including proinsulin and preproinsulin, and amino acid sequence variants of insulin or its functional fragments. The present disclosure provides proteins comprising canine insulin or feline insulin, as well as polynucleotides and expression vectors encoding such proteins. In some embodiments, the insulin protein comprises a polynucleotide sequence encoding a polypeptide comprising (a) a secretion signal peptide and (b) a proinsulin polypeptide. In one embodiment, the protein comprises a canine IL2 signal peptide and canine proinsulin. In another embodiment, the protein comprises a canine insulin signal peptide and canine proinsulin. The amino acid sequence of natural canine proinsulin is shown in SEQ ID NO: 10.

[0060] In one embodiment, the protein comprises a feline IL2 signal peptide and a feline proinsulin. In another embodiment, the protein comprises a feline insulin signal peptide and a feline proinsulin. The amino acid sequence of native feline proinsulin is shown in SEQ ID NO: 11.

[0061] In some embodiments, canine or feline insulin includes variants, which may include up to about 10% variation of an insulin nucleic acid or amino acid sequence as described herein or known in the art, which retains the function of the wild-type sequence. As used herein, "retaining function" means that the nucleic acid or amino acid works in the same manner as the wild-type sequence, although not necessarily at the same expression or activity level. For example, in one embodiment, compared with the wild-type sequence, the functional variant has increased expression or activity. In another embodiment, compared with the wild-type sequence, the functional variant has reduced expression or activity. In one embodiment, compared with the wild-type sequence (SEQ ID NO: 11), the functional variant has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase or reduction in expression or activity.

[0062] In one embodiment, compared with the native sequence, the canine proinsulin sequence contains one or more mutations. In some embodiments, these mutations are located at the cleavage site between the B / C chain and the C / A chain. In one embodiment, one or more cleavage sites are mutated to incorporate at least one furin cleavage site at the existing protease cleavage site. In one embodiment, the proinsulin sequence has a K53R mutation. In another embodiment, the proinsulin sequence has a R55K mutation. In another embodiment, the proinsulin sequence has a L86R mutation. In another embodiment, the proinsulin sequence has both K53R and R55K mutations. In another embodiment, the proinsulin sequence has both K53R and L86R mutations. In another embodiment, the proinsulin sequence has both R55K and L86R mutations. In another embodiment, the proinsulin sequence has K53R, R55K and L86R mutations.

[0063] In one embodiment, the canine proinsulin sequence is a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:12.

[0064] In one embodiment, the feline proinsulin sequence is a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:15.

[0065] When variants or fragments of the proinsulin sequence are needed, site-directed mutagenesis of the wild-type nucleic acid sequence can be used to generate the coding sequences of these peptides. Alternatively or additionally, web-based or commercially available computer programs and service-based companies can be used to reverse-translate the amino acid sequence into nucleic acid coding sequences, including both RNA and / or cDNA. See, for example, backtranseq of EMBOSS: Gene Infinity; and / or ExPasy. In one embodiment, the RNA and / or cDNA coding sequences are designed to be optimally expressed in the test species, i.e., dog or cat, that is, the final intended administration.

[0066] Fusion domain

[0067] The present disclosure provides fusion proteins comprising fusion domains. By fusing insulin to a fusion domain having a longer half-life, insulin fusion proteins overcome the short half-life of natural hormones. In some embodiments, the fusion domain comprises (i) canine serum albumin or a functional variant thereof, (ii) canine IgG Fc or a functional variant thereof, or (iii) canine transferrin or a functional variant thereof. In some embodiments, the fusion domain comprises canine serum albumin.

[0068] In some embodiments, the fusion domain comprises (i) feline serum albumin or a functional variant thereof, (ii) feline IgG Fc or a functional variant thereof, or (iii) feline transferrin or a functional variant thereof. In some embodiments, the fusion domain comprises feline serum albumin.

[0069] In some embodiments, the fusion domain is canine serum albumin comprising a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:16.

[0070] In some embodiments, the fusion domain is canine transferrin, which comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:17.

[0071] In some embodiments, the fusion domain is feline serum albumin comprising a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:18.

[0072] Insulin fusion protein

[0073] The present disclosure provides a fusion protein comprising one or more copies of proinsulin, and polynucleotides and expression vectors encoding such fusion proteins. In some embodiments, the fusion protein comprises a polynucleotide sequence encoding the fusion protein, and the fusion protein comprises (a) a leader sequence comprising a secretion signal peptide, (b) proinsulin, and (c) a fusion domain comprising (i) IgG Fc or its functional variant, (ii) albumin or its functional variant, or (iii) transferrins or its functional variant. In one embodiment, the fusion protein comprises a thrombin leader sequence, proinsulin and IgG Fc or its functional variant. In another embodiment, the fusion protein comprises a thrombin leader sequence, proinsulin and albumin or its functional variant.

[0074] In some embodiments, the fusion protein comprises a canine insulin leader sequence, canine proinsulin (K53R, R55K and L86R), a glycine / serine linker and canine serum albumin. In embodiments, the fusion protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 1.

[0075] In some embodiments, the fusion protein comprises a canine insulin leader sequence, canine proinsulin (K53R, R55K and L86R), a glycine / serine linker and canine transferrin. In embodiments, the fusion protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 3.

[0076] In some embodiments, the fusion protein comprises a canine insulin leader sequence and canine proinsulin (K53R, R55K and L86R). In embodiments, the fusion protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:5.

[0077] In some embodiments, the fusion protein comprises a feline insulin leader sequence, feline proinsulin (K53R, R55K and L86R), a glycine / serine linker and a feline serum albumin. In embodiments, the fusion protein comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO:6.

[0078] In one embodiment, the fusion protein comprises the insulin leader sequence, proinsulin and albumin or a functional variant thereof. In one embodiment, the fusion protein comprises the insulin leader sequence, proinsulin and transferrin or a functional variant thereof.

[0079] In one embodiment, the fusion protein comprises the IL2 leader sequence, proinsulin and albumin or a functional variant thereof. In one embodiment, the fusion protein comprises the IL2 leader sequence, proinsulin and transferrin or a functional variant thereof.

[0080] In addition to the leader sequences, proinsulin and insulin polypeptides provided herein, nucleic acid sequences encoding these polypeptides (interchangeably used with "polynucleotides") are also provided. In one embodiment, a nucleic acid sequence encoding a proinsulin-serum albumin fusion polypeptide as described herein is provided. In some embodiments, the nucleic acid sequence encoding a canine proinsulin-serum albumin fusion comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 2.

[0081] In some embodiments, the nucleic acid sequence encoding the canine proinsulin-transferrin fusion comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:4.

[0082] In some embodiments, the nucleic acid sequence encoding the feline proinsulin-serum albumin fusion comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:19.

[0083] The in vivo function and stability of the fusion proteins disclosed herein can be optimized by adding small peptide linkers, for example, to prevent potentially undesirable domain interactions or for other reasons. In addition, glycine-rich linkers can provide some structural flexibility so that the proinsulin portion can effectively interact with the insulin receptor on the target cell. Therefore, in one embodiment, the C-terminus of the proinsulin of the fusion protein and the N-terminus of the fusion domain are fused via a linker. In some embodiments, the linker includes 1, 2, 3, or n repeats of the sequence (GGGGS) n In one embodiment, the linker comprises 1, 1.5 or 2 repeats of a G-rich peptide linker having the sequence GGGGSGGGSGGGGS (SEQ ID NO: 14). In one embodiment, the linker comprises repeats of a G-rich peptide linker having the sequence GGGGSGGGSGGGS (SEQ ID NO: 8). In one embodiment, the linker comprises repeats of a G-rich peptide linker having the sequence GGGGSGGGGS (SEQ ID NO: 20).

[0084] In some embodiments, the fusion protein of the present disclosure comprises from 5' to 3':

[0085] (a) Canine insulin signal peptide;

[0086] (b) canine proinsulin (K53R, R55K, and L86R);

[0087] (c) a Gly / Ser linker; and

[0088] (d) Canine serum albumin.

[0089] In some embodiments, the expression cassette comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:2.

[0090] In some embodiments, the fusion protein of the present disclosure comprises from 5' to 3':

[0091] (a) Feline insulin signal peptide;

[0092] (b) feline proinsulin (K53R, R55K, and L86R);

[0093] (c) a Gly / Ser linker; and

[0094] (d) Feline serum albumin

[0095] In some embodiments, the expression cassette comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:19.

[0096] Expression cassette

[0097] In some embodiments, expression cassette refers to a nucleic acid molecule comprising a proinsulin fusion construct coding sequence, a promoter, and may include other regulatory sequences thereof. The expression cassette can be engineered into a genetic element (e.g., a plasmid) for delivery into cells and purification of the fusion protein for therapeutic administration.

[0098] The expression cassette typically contains a promoter sequence as part of the expression control sequence. In one embodiment, a constitutive promoter is used. In the plasmids and expression vectors described herein, the CB7 promoter can be used. CB7 is a chicken B-actin promoter with a cytomegalovirus enhancer element. Other promoters that can be used in the expression vectors described herein are viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO 2011 / 126808 and WO2013 / 04943) or promoters that respond to physiological signals.

[0099] In some embodiments of the plasmids and expression vectors described herein, the CMV enhancer, chicken β-actin promoter, and rabbit β-globin splice acceptor site (CAG) promoter may be used. In some embodiments of the plasmids and expression vectors described herein, the elongation factor-1 alpha (EF1a) promoter may be used.

[0100] In addition to the promoter, the expression cassette and / or expression vector may also contain other suitable transcription initiation, termination, enhancer sequences, efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product. Illustrative examples of suitable polyA sequences include, for example, rabbit beta globulin, SV40, bovine growth hormone (bGH), and TK polyA.

[0101] Illustrative examples of suitable enhancers include, for example, the alpha fetoprotein enhancer, the TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha 1-microglobulin / bikunin enhancer), etc. In one embodiment, the polyA is rabbit globulin polyA.

[0102] These control sequences are "operably linked" to the proinsulin fusion construct sequence. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous to the gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest.

[0103] In one embodiment, an expression cassette is provided, which includes a CB7 promoter, a chimeric intron, a coding sequence for a protein encoded by SEQ ID NO: 2, and rabbit beta globin poly A.

[0104] In one embodiment, an expression cassette is provided, which includes a CB7 promoter, a chimeric intron, a coding sequence for a protein encoded by SEQ ID NO: 4, and rabbit beta globulin poly A.

[0105] In one embodiment, an expression cassette is provided, which includes a CB7 promoter, a chimeric intron, a coding sequence for a protein encoded by SEQ ID NO: 19, and rabbit beta globulin poly A.

[0106] Methods for constructing any embodiment of the present disclosure are known to those skilled in the art of nucleic acid manipulation, and include genetic engineering, recombinant engineering, and synthetic techniques.

[0107] In some embodiments, the fusion protein of the present disclosure comprises an expression cassette comprising, from 5' to 3':

[0108] (a) Cytomegalovirus (CMV) enhancer / chicken b-actin (CAG) promoter;

[0109] (b) canine insulin signal peptide;

[0110] (c) canine proinsulin (K53R, R55K, and L86R);

[0111] (d) a Gly / Ser linker; and

[0112] (e) Canine serum albumin.

[0113] In some embodiments, the expression cassette comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:2.

[0114] In some embodiments, the fusion protein of the present disclosure comprises an expression cassette comprising, from 5' to 3':

[0115] (a) Cytomegalovirus (CMV) enhancer / chicken b-actin (CAG) promoter;

[0116] (b) feline insulin signal peptide;

[0117] (c) feline proinsulin (K53R, R55K, and L86R);

[0118] (d) a Gly / Ser linker; and

[0119] (e) Feline serum albumin

[0120] In some embodiments, the expression cassette comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:19.

[0121] In one embodiment, the nucleic acid sequence encoding the proinsulin fusion construct described herein is engineered into any suitable genetic element, such as naked DNA, bacteriophage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, recombinant AAV, etc., and the proinsulin fusion sequence carried thereon is transferred to a host cell, such as for producing nanoparticles, virions carrying DNA or RNA in a packaging host cell and / or for delivery to a host cell of a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated mass, viral infection, and protoplast fusion. Methods for preparing such constructs are known to those skilled in the art of nucleic acid manipulation, and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0122] In some embodiments, the proinsulin fusion constructs described herein may be delivered via virosomes, including but not limited to recombinant AAV (rAAV). Such other virosomes may include any virus suitable for gene therapy that can be used, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus; etc. Suitably, when one of these other virosomes is produced, it is produced as a replication-defective virosome.

[0123] Cell culture

[0124] Animal cells, mammalian cells, cultured cells, animal or mammalian host cells, host cells, recombinant cells, recombinant host cells, manufacturing cell lines, etc. are all terms for cells that can be maintained in cell culture media. Such cells are typically cell lines obtained or derived from mammals and are able to grow and survive when placed in monolayer culture or suspension culture in a culture medium containing appropriate nutrients and / or growth factors. The growth factors and nutrients necessary for the growth and maintenance of a particular cell culture can be easily determined empirically by a person skilled in the art, such as Barnes and Sato, (1980, Cell, 22: 649); in Mammalian Cell Culture, ed. JPMather, Plenum Press, NY, 1984; and described in U.S. Pat. No. 5,721,121.

[0125] In addition, cell culture conditions are typically employed and are known for batch, fed-batch or continuous culture of cells, paying attention, in addition to temperature, to pH, e.g., about 6.5 to about 7.5, dissolved oxygen (O2), e.g., about 5% to 90% of air saturation and carbon dioxide (CO2), agitation and humidity.

[0126] Cell line cells are typically animal or mammalian cells that can express and secrete, or can be molecularly engineered to express and secrete large amounts of specific proteins into the culture medium. It should be understood that the target protein produced by the host cell can be endogenous or homologous to the host cell. In one embodiment, the target protein is produced and secreted by Chinese hamster ovary (CHO) host cells. In some embodiments, the target protein is a proinsulin-serum albumin fusion protein.

[0127] Non-limiting examples of animal or mammalian host cells suitable for harboring, expressing and producing proteins for subsequent isolation and / or purification include Chinese Hamster Ovary cells (CHO).

[0128] Cells suitable for culturing in the methods of the present disclosure may contain, for example, expression vectors (constructs) introduced via transformation, transfection, infection or injection, such as plasmids, etc., which carry coding sequences or portions thereof encoding proteins for expression and production during culture. Such expression vectors contain the necessary elements for transcription and translation of the inserted coding sequences.

[0129] In the culture methods encompassed by the present disclosure, proteins produced by the cells are typically collected, recovered, separated and / or purified or substantially purified as needed at the end of the total cell culture period using separation and purification methods known and practiced in the art. In some embodiments, the protein of interest is secreted from the cultured cells and separated from the culture medium or supernatant. In some embodiments, proteins can also be recovered from host cells (e.g., cell lysates) using methods known and practiced in the art.

[0130] Methods of treating a subject with the disclosed compositions

[0131] Also provided are compositions comprising fusion proteins as described herein. Pharmaceutical compositions as described herein are designed to be delivered to a dog or cat subject in need by any suitable route or combination of different routes. Direct delivery to the liver (optionally via intravenous, via the hepatic artery or by transplantation), direct delivery to the pancreas, oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal and other parenteral administration routes. Fusion proteins as described herein can be delivered in a single composition or multiple compositions.

[0132] In some embodiments, the pharmaceutical compositions described herein are designed for delivery to a canine or feline subject in need thereof via intramuscular administration.

[0133] In some embodiments, the pharmaceutical compositions described herein are designed for delivery to a canine or feline subject in need thereof by intravenous administration.

[0134] In some embodiments, the pharmaceutical compositions described herein are designed for delivery to a canine or feline subject in need thereof by subcutaneous administration.

[0135] In some embodiments, the treatment process may include repeated administration of the same fusion protein. Other combinations may also be selected using the fusion protein described herein. In some embodiments, the compositions described herein may be combined in a scheme involving other diabetes drugs or protein-based therapies (including, for example, insulin analogs, insulin, oral antihyperglycemic drugs, sulfonylureas, biguanides, thiazolidinediones, and alpha-glucosidase inhibitors). In some embodiments, the compositions described herein may be combined in a scheme involving a lifestyle change (including a diet and exercise regimen).

[0136] As used herein, the terms "proinsulin construct," "proinsulin expression construct," and synonyms include combinations of proinsulin sequences as described herein with a leader region, whether native or heterologous. The terms "proinsulin construct," "proinsulin expression construct," and synonyms may be used to refer to a nucleic acid sequence encoding a proinsulin fusion protein or its expression product.

[0137] In one aspect, the present disclosure relates to a method of treating a disease or condition in a subject in need thereof, the method comprising administering an effective amount of a proinsulin fusion protein, thereby ameliorating and / or treating one or more of the symptoms of type I diabetes, type II diabetes, or metabolic syndrome.

[0138] In some embodiments, the method comprises administering at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, or at least about 15 mg / kg of the fusion protein.

[0139] In some embodiments, a unit dose of proinsulin fusion protein comprises at least about 0.01 mg, at least about 0.05 mg, at least about 0.1 mg, at least about 1 mg, at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least about 15 mg.

[0140] In some embodiments, the fusion protein is administered intravenously. In some embodiments, the fusion protein is administered intramuscularly. In some embodiments, the fusion protein is administered subcutaneously. In some embodiments, the therapeutically effective amount of the proinsulin fusion protein is administered at a dosage of at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, or at least about 15 mg / kg.

[0141] In some embodiments, a therapeutically effective amount of the proinsulin fusion protein is administered in a unit dose of at least about 0.01 mg, at least about 0.05 mg, at least about 0.1 mg, at least about 1 mg, at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least about 15 mg.

[0142] In some embodiments, a therapeutically effective amount of the proinsulin fusion protein is administered in a unit dose of up to about 100 mg, up to about 90 mg, up to about 80 mg, up to about 70 mg, up to about 60 mg, up to about 50 mg, up to about 40 mg, up to about 30 mg, up to about 20 mg, or up to about 10 mg.

[0143] In some embodiments, the method comprises administering the dose of the proinsulin fusion protein once a day, once every 2 days, once every 3 days, once every 4 days, once every 7 days, once every 14 days, once every 21 days, or once every 28 days.

[0144] In some embodiments, the method comprises administering the dose of the proinsulin fusion protein for at least one, two, or four weeks.

[0145] In some embodiments, the method comprises administering the dose of the proinsulin fusion protein for a regimen of at least one month, two months, four months, six months, eight months, ten months, 12 months, 14 months, 16 months, or 18 months.

[0146] In some embodiments, the therapeutically effective amount of the proinsulin fusion protein is administered for a regimen of at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, eighteen months, two years, three years, four years, five years, six years, seven years, eight years, nine years, or ten years.

[0147] In some embodiments, the method comprises chronically administering the dosage regimen of the proinsulin fusion protein.

[0148] The fusion protein composition can be formulated into dosage units containing an amount of the fusion protein ranging from about 0.1 mg to about 15 mg.

[0149] In another aspect, a method for maintaining the biological activity of proinsulin fusion protein in a dog or cat subject is provided. The method includes applying a composition as described herein to a subject in need. In one embodiment, as described herein, the composition comprises a fusion protein containing proinsulin-serum albumin fusion protein. In some embodiments, compared with the native peptide, the proinsulin fusion protein described herein causes the half-life of insulin to be extended. In some embodiments, the fusion protein provided herein causes the half-life of proinsulin fusion protein in a subject to be at least 5 hours, at least 7 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours or at least 40 hours. In one embodiment, for veterinary subjects, the fusion protein can be delivered in a volume of 1 μL to about 100mL. See, for example, Diehl et al., J.Applied Toxicology, 21:15-23 (2001) for a discussion of good practices for administering substances to various veterinary animals. This document is incorporated herein by reference. As used herein, the term "dose" may refer to the total dose delivered to a subject during the course of treatment, or the amount delivered in a single (or multiple) administrations.

[0150] In one embodiment, the composition is administered in combination with an effective amount of insulin. Various commercially available insulin products are known in the art, including but not limited to protamine zinc recombinant human insulin Porcine Insulin Zinc Suspension and insulin glargine In some embodiments, compared with before the treatment with fusion protein, the combination of fusion protein as herein described and insulin reduces the insulin dosage requirement in the experimenter.Such dosage requirement can reduce 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more.The treatment physician can determine the correct dosage of insulin required for the experimenter.For example, insulin or other therapy treatment experimenter can be used, and the treatment physician can continue this treatment after using fusion protein.This insulin or other conjoint therapy can continue, reduce or interrupt as required subsequently.

[0151] In one embodiment, a therapeutically effective amount of a composition as described herein is delivered to a subject. As used herein, a "therapeutically effective amount" refers to an amount of a proinsulin-serum albumin fusion protein delivered in a target cell in an amount sufficient to achieve a therapeutic goal. In certain embodiments, the therapeutic goal is to improve or treat one or more symptoms of type I diabetes, type II diabetes, or metabolic syndrome. The therapeutically effective amount can be determined based on an animal model rather than a canine or feline subject. In another embodiment, the therapeutic goal is to alleviate a metabolic disease in a subject.

[0152] The above-mentioned fusion protein can be delivered to the host cell according to the disclosed method. The fusion protein, preferably suspended in a physiologically compatible carrier, can be administered to a desired subject including a dog. Taking into account the indications for which the transfer virus is directed, a person skilled in the art can easily select a suitable carrier. For example, a suitable carrier includes saline, which can be prepared with a variety of buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The selection of the carrier is not a limitation of the present disclosure.

[0153] In some embodiments, in addition to the fusion protein and / or variant and the carrier, the composition of the present disclosure may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol and p-chlorophenol. Exemplary chemical stabilizers include gelatin and albumin.

[0154] In some embodiments, the fusion protein of the present disclosure is formulated as a long-acting injection. Long-acting injection formulations deliver fusion proteins (e.g., proinsulin-serum albumin fusion proteins) at an adjustable, predetermined rate within the therapeutic range for a specified period of time. Release can occur directly at the site of action of local treatment or at the systemic level, thereby minimizing the adverse side effects of the fusion protein.

[0155] In some embodiments, the proinsulin-serum albumin fusion proteins of the present disclosure are formulated as long-acting injections.

[0156] The recombinant fusion proteins described herein can be used to prepare a medicament for delivering the proinsulin fusion protein to a subject in need thereof, providing the subject with insulin with an increased half-life, and / or for treating type I diabetes, type II diabetes, or metabolic syndrome in a subject.

[0157] In one aspect, a method of treating diabetes is provided. The method comprises administering a composition as described herein to a canine or feline subject in need thereof. In one embodiment, the composition comprises a proinsulin fusion as described herein.

[0158] In another embodiment, a method for treating type 2 diabetes in dogs or cats is provided. The method comprises administering a proinsulin fusion protein as described herein.

[0159] In another embodiment, a method for treating type 1 diabetes in dogs or cats is provided. The method comprises administering a proinsulin fusion protein as described herein.

[0160] In another aspect, a method for treating a metabolic disease in a dog or cat is provided. The method comprises administering a composition as described herein to a dog or cat subject in need. In one embodiment, the composition comprises a proinsulin fusion protein as described herein. In one embodiment, the metabolic disease is type I diabetes. In one embodiment, the metabolic disease is type II diabetes. In one embodiment, the metabolic disease is metabolic syndrome.

[0161] In another embodiment, a method for treating diabetes in a dog or cat is provided. The method comprises administering a proinsulin-serum albumin fusion protein as described herein, wherein the fusion protein is administered after administration of insulin to the subject.

[0162] In another embodiment, a method for preventing cataract formation in a diabetic dog or cat is provided. The method comprises administering a proinsulin-serum albumin fusion protein as described herein.

[0163] In another embodiment, a method for reducing blood glucose concentration in a diabetic dog or cat is provided. The method comprises administering a proinsulin-serum albumin fusion protein as described herein.

[0164] As used herein, the term "treatment" or "treating" is defined to encompass the administration of one or more compounds or compositions described herein to a subject for the purpose of improving one or more symptoms of type I diabetes, type II diabetes (T2DM), or metabolic syndrome. Thus, "treatment" may include one or more of reducing the progression of type I diabetes, type II diabetes, or metabolic syndrome, reducing the severity of symptoms, eliminating disease symptoms, delaying the progression of the disease, or increasing the efficacy of a therapy in a given subject.

[0165] As used herein, the term "remission" refers to the ability to discontinue insulin therapy when a cat or dog no longer exhibits clinical signs of diabetes and has normal blood glucose levels.

[0166] In another embodiment, a method for treating T2DM in a cat or dog is provided. The method comprises administering a fusion protein as described herein.

[0167] In another aspect, a method for treating a metabolic disease in a cat or dog is provided. The method comprises administering a composition as described herein to a cat or dog subject in need thereof. In one embodiment, the composition comprises a proinsulin fusion protein as described herein.

[0168] In another aspect, a method for reducing fasting blood glucose in a canine or feline subject is provided. The method comprises administering a composition as described herein to a subject in need thereof. In one embodiment, the composition comprises a proinsulin-serum albumin fusion protein as described herein. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by about 20%. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by about 30%. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by about 40%.

[0169] definition

[0170] Unless otherwise defined, the terms used herein (including technical and scientific terms) have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. It should also be understood that those terms such as those defined in common dictionaries should be interpreted as having the same meanings as their meanings in the context of the present application and the related art, and should not be interpreted with idealized or overly formal meanings, unless clearly defined in this article. The terms used in this specification are only used to describe the purpose of a specific embodiment and are not intended to be limited. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a term conflict, this specification shall prevail.

[0171] Reference to "one embodiment" or "another embodiment" when describing an embodiment does not mean that the referenced embodiment is mutually exclusive of another embodiment (eg, an embodiment described before the referenced embodiment) unless explicitly specified otherwise.

[0172] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides, i.e., when compared and aligned over a comparison window or a specified region to obtain maximum correspondence, there is at least about 80% identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, over a specified region with a reference sequence, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are referred to as "substantially identical". This definition also refers to the complementary sequence of a test sequence. In some embodiments, the identity is present over a region of at least about 25 amino acids or nucleotides in length, e.g., over a region of 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the full length of a reference sequence.

[0173] For sequence comparison, usually a sequence serves as a reference sequence, and a test sequence is compared thereto. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then based on the program parameters, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence. In some embodiments, BLAST and BLAST 2.0 algorithms and default parameters are used.

[0174] In the context of amino acid sequences, the terms "percent (%) identity", "sequence identity", "percent sequence identity" or "percent identical" refer to the residues in two sequences that are identical when aligned for correspondence. The percent identity of an amino acid sequence over the full length of a protein, polypeptide, about 70 amino acids to about 100 amino acids, or a peptide fragment thereof, or the corresponding nucleic acid sequence encoding the sequence, can be readily determined. Suitable amino acid fragments can be at least about 8 amino acids in length, and can be up to about 150 amino acids. Typically, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to the "aligned" sequence.

[0175] "Aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences, typically containing corrections for missing or extra bases or amino acids. Alignment is performed using any of a variety of disclosed or commercially available multiple sequence alignment programs. Sequence alignment programs can be used for amino acid sequences, such as "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used under default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art may utilize another algorithm or computer program that provides at least the identity or alignment level provided by the cited algorithms and programs.

[0176] The term "amino acid substitution" and its synonyms are intended to cover the modification of an amino acid sequence by replacing an amino acid with another substituted amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. When referring to two amino acids, the term conservative is intended to mean that these amino acids have a common property recognized by those skilled in the art. For example, an amino acid with a hydrophobic non-acidic side chain, an amino acid with a hydrophobic acidic side chain, an amino acid with a hydrophilic non-acidic side chain, an amino acid with a hydrophilic acidic side chain, and an amino acid with a hydrophilic basic side chain. The common property may also be an amino acid with a hydrophobic side chain, an amino acid with an aliphatic hydrophobic side chain, an amino acid with an aromatic hydrophobic side chain, an amino acid with a polar neutral side chain, an amino acid with a charged side chain, an amino acid with a charged acidic side chain, and an amino acid with a charged basic side chain. Naturally occurring and non-naturally occurring amino acids are all known in the art and may be used as substituted amino acids in embodiments. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. Reference herein to "one or more" is intended to encompass, for example, 1, 2, 3, 4, 5, 6 or more separate embodiments.

[0177] Codon optimization can be used to design coding sequences for optimal expression. Codon optimized coding regions can be designed by various methods. The optimization can be carried out using methods obtained online, disclosed methods or companies providing codon optimization services. A codon optimization method is described in, for example, International Patent Application Publication No. WO 2015 / 012924, which is incorporated herein by reference. In short, the nucleic acid sequence of the encoded product is modified with a synonymous codon sequence. Suitably, the entire length of the open reading frame (ORF) of the modified product. However, in some embodiments, only the fragment of the ORF can be changed. By using one of these methods, frequency can be applied to any given polypeptide sequence, and a nucleic acid fragment of the codon optimized coding region encoding the polypeptide is produced.

[0178] In embodiments, the insulin-serum albumin fusion is humanized, caninized, feline or equinized.

[0179] By "humanized" is meant that the fusion protein comprises an amino acid sequence that is compatible with humans, such that the amino acid sequence is less likely to be viewed as foreign by the immune system of a human subject.

[0180] "Caninization" means that the fusion protein comprises an amino acid sequence that is compatible with dogs, such that the amino acid sequence is less likely to be viewed as foreign by the immune system of a canine subject. In the present disclosure, the term "canine (ca)" prefixed polypeptide refers to a variant of a human polypeptide in which the human fusion domain is replaced by a canine homolog of the fusion domain, and when proinsulin is a fragment or variant of a human protein, proinsulin is replaced by a canine homolog of the fragment or variant.

[0181] "Felinization" means that the fusion protein comprises an amino acid sequence that is compatible with cats, making it less likely that the amino acid sequence will be considered foreign by the immune system of a feline subject. In the present disclosure, the term "feline (fe)" prefixed polypeptide refers to a variant of a human polypeptide in which a human fusion domain is replaced by a feline homolog of the fusion domain, and when proinsulin is a fragment or variant of a human protein, proinsulin is replaced by a feline homolog of the fragment or variant.

[0182] By "equined" is meant that the fusion protein comprises an amino acid sequence that is compatible with horses, such that the amino acid sequence is less likely to be viewed as foreign by the immune system of an equine subject.

[0183] As described elsewhere herein, the present disclosure extends to fusion proteins that are compatible with species other than humans, dogs, cats, and horses. In this context, the fusion protein may be referred to as "speciesized," referring to the target species to which the molecule will be administered.

[0184] In some embodiments, compositions and methods described herein are intended for use in feline animals. The term cat (Felidae) refers to any of 37 cat species, including cheetahs, cougars, jaguars, jaguars, lions, lynxes, tigers, and house cats. In embodiments, the subject is a house cat. In some embodiments, compositions and methods described herein are intended for use in canine animals. The term dog refers to any species found in the canine family, including domestic dogs, wolves, and foxes. In embodiments, the subject is a domestic dog (domestic dog, also referred to as Canis lupus familiaris or Canis familiaris).

[0185] As used in the description of the present disclosure and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0186] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative (or).

[0187] As used herein, the phrase "consisting essentially of refers to the genus or species of active agent recited in the method or composition, and may also include other agents that themselves are not substantially active for the recited indication or purpose.

[0188] The words "comprise / comprises / comprising" are to be interpreted as inclusive rather than exclusive. The words "consist / consisting" and variations thereof are to be interpreted exclusively rather than inclusively. Although various embodiments in the specification are presented using "comprising" language, in other cases, the relevant embodiments are also intended to be interpreted and described using "consisting of" or "consisting essentially of" language.

[0189] As used herein, unless otherwise indicated, the term "about" means a variability of 10% from a given reference.

[0190] As used herein, the term "modulate" or variations thereof refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0191] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

[0192] The terms "subject", "individual" and "patient" refer interchangeably to mammals, humans or non-human primates, domestic mammals (e.g., dogs or cats), laboratory mammals, and agricultural mammals. In various embodiments, the subject can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a boy, a girl). In various embodiments, the subject is a companion animal. Exemplary companion animals include, but are not limited to, dogs, cats, horses, rabbits, ferrets, birds, and guinea pigs. In various embodiments, the subject is a dog. In various embodiments, the subject is a cat. In various embodiments, the subject is a mammal.

[0193] As used herein, the term "target cell" refers to any target cell in which expression of a protein is desired. In certain embodiments, the target cell is a hepatocyte. In some embodiments, the target cell is a muscle cell.

[0194] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and a regulatory sequence that is operably linked thereto to direct or regulate the transcription, translation and / or expression of the nucleic acid sequence and its gene product. As used herein, "operably linked" sequences include both regulatory sequences (also referred to as elements) that are continuous or discontinuous with the nucleic acid sequence and regulatory sequences that function in a trans or cis nucleic acid sequence. Such regulatory sequences typically include, for example, promoters, enhancers, transcription factors, transcription terminators, introns, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), effective RNA processing signals such as slicing and polyadenylation sequences, sequences that stabilize cytoplasmic mRNA (e.g., woodchuck hepatitis virus (WHP) post-translational regulatory elements (WPRE)) and TATA signals. The expression cassette may contain a regulatory sequence upstream (5') of the gene sequence, for example, one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or a regulatory sequence downstream (3') of the gene sequence, for example, a 3' untranslated region (3'UTR) including a polyadenylation site, and other elements. In certain embodiments, the regulatory sequence is operably connected to the nucleic acid sequence of the gene product, wherein the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, that is, the 5'-untranslated region (5'UTR). In certain embodiments, the expression cassette comprises the nucleic acid sequence of one or more gene products. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette.

[0195] As used herein, "administration" refers to local and systemic administration, for example, including enteral, parenteral, pulmonary and topical / transdermal administration. The administration routes of the drug components that can be used in the methods described herein include, for example, oral (oral (PO)) administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration or subcutaneous ("sc") administration, or implantation of a sustained-release device (e.g., mini-osmotic pump, long-acting preparation, etc.) to a subject. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ion introduction and intracranial.

[0196] The terms "systemic administration" and "systemically administered" refer to a method of administering a pharmaceutical ingredient or composition to a mammal so that the pharmaceutical ingredient or composition is delivered to a site in the body (including a targeted site of drug action) via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., other than through the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0197] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or dosage regimen of one or more pharmaceutical ingredients (eg, fusion protein) necessary to produce the desired result.

[0198] As used herein, the terms "treating" or "treatment" refer to delaying the onset of, slowing or reversing the progression of, reducing the severity of, or alleviating or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or condition. The term "treating" also includes preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of a disease or condition.

[0199] The term "alleviate" refers to a reduction or elimination of one or more symptoms of the pathology or disease, and / or a reduction in the rate or delay of onset or severity of one or more symptoms of the pathology or disease, and / or the prevention of the pathology or disease. In some embodiments, the reduction or elimination of one or more symptoms of a pathology or disease may include, for example, a measurable and sustained reduction in fasting blood glucose.

[0200] ****

[0201] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference. In the event of a conflict, the present application (including any definition herein) shall prevail. However, the reference to any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be regarded as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge of any country in the world.

[0202] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0203] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.

[0204] Example

[0205] The following examples are presented to provide one of ordinary skill in the art with a description of how to use, prepare, and evaluate the compositions and methods described herein and are intended to be purely exemplary of the present disclosure and are not intended to limit the scope of the present disclosure.

[0206] Example 1: Production of canine insulin-serum albumin fusions with extended half-life and canine insulin fusion proteins Evaluation of in vitro potency

[0207] Canine insulin-serum albumin fusion protein has been developed for use in the control of hyperglycemia and clinical symptoms of diabetes associated with hyperglycemia in dogs.

[0208] Canine preproinsulin-serum albumin fusion protein (cINS-Alb) was produced by constructing a fusion polypeptide containing the following elements: Figure 1A ) (SEQ ID NO: 1):

[0209] Natural canine insulin signal peptide (SP)

[0210] Canine proinsulin in which the native sequence has been modified at three amino acid positions (K53R, R55K and L86R) to incorporate two furin cleavage sites at existing protease cleavage sites (B chain, C peptide, A chain)

[0211] A Gly / Ser linker comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 8)

[0212] Canine serum albumin

[0213] Canine preproinsulin fusion protein was prepared using the same components as above, except that canine transferrin (cINS-Tf) ( Figure 1B )(SEQ ID NO:3) to replace the canine serum albumin sequence. A control canine preproinsulin sequence with three amino acid modifications (K53R, R55K and L86R) was also generated as a control (cINS-2-1) ( Figure 1C )(SEQ ID NO:5).

[0214] A study was performed to evaluate the in vitro potency of two fusion proteins (cINS-Alb and cINS-Tf). A C-terminally histidine-tagged version of each protein was produced in mammalian cells following transient transfection of expression plasmids containing cDNA encoding the corresponding proteins. The proteins were purified from cell supernatants by nickel affinity chromatography and expressed using Insulin bioassay kit (Eurofins TM Insulin bioactivity was determined by the manufacturer's manufacturer (DiscoverX Products, LLC) according to the kit instructions. The standard insulin provided in the kit was used as a reference control. Both cINS-Alb and cINS-Tf showed bioactivity in this assay ( Figure 2 ). Modifications made to the insulin molecules in the two fusion proteins resulted in a small loss of potency, such as EC 50 Indicated by the change in value.

Claims

1. A fusion protein for treating a companion animal, the fusion protein comprising proinsulin and serum albumin, wherein the proinsulin is canine proinsulin or feline proinsulin.

2. The fusion protein according to claim 1, wherein the proinsulin is canine proinsulin.

3. The fusion protein of claim 2, wherein the canine proinsulin sequence shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

12.

4. The fusion protein of claim 1, wherein the proinsulin is feline proinsulin.

5. The fusion protein of claim 4, wherein the feline proinsulin sequence shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

15.

6. The fusion protein of claim 1 or claim 2, wherein the fusion protein comprises a polypeptide that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

1.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein comprises an N-terminal signal peptide. The fusion protein according to claim 7 , wherein the signal peptide is canine insulin signal peptide.

9. The fusion protein of claim 7 or claim 8, wherein the signal peptide comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with MALWMRLLPLLALLALWAPAPTRA (SEQ ID NO: 7).

10. The fusion protein according to any one of claims 1 to 3 or 6 to 9, wherein the canine proinsulin is a canine proinsulin variant having a mutation at one or more cleavage sites compared to the reference polypeptide sequence shown in SEQ ID NO:

10.

11. The fusion protein according to any one of claims 1 to 3 or 6 to 10, wherein the canine proinsulin comprises K53R, R55K and L86R mutations compared to the reference polypeptide sequence shown in SEQ ID NO:

10.

12. The fusion protein of any one of claims 1 to 3 or 6 to 11, wherein the canine proinsulin-serum albumin fusion protein comprises a linker that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

8.

13. The fusion protein according to any one of claims 1 to 12, wherein the polynucleotide encoding the fusion protein is operably linked to a promoter. The fusion protein according to claim 13 , wherein the promoter is a cytomegalovirus enhancer / chicken b-actin promoter.

15. A pharmaceutical composition suitable for use in treating metabolic diseases in dogs or cats, comprising the fusion protein according to any one of claims 1 to 14.

16. The fusion protein according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 for use in a method for treating a canine or feline subject suffering from a metabolic disease, optionally diabetes.

17. Use of the fusion protein according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 in the manufacture of a medicament for treating a canine or feline subject suffering from a metabolic disease, optionally diabetes.

18. The fusion protein of any one of claims 1 to 14, wherein the fusion protein is formulated to be administered at a concentration of at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, or at least about 15 mg / kg of the fusion protein, and / or wherein the fusion protein is administered intravenously.

19. The fusion protein of any one of claims 1 to 14, wherein the fusion protein is administered at a dose of at least about 0.01 mg, at least about 0.05 mg, at least about 0.1 mg, at least about 1 mg, at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least about 15 mg.

20. The fusion protein of any one of claims 1 to 19, wherein the fusion protein is administered intravenously to the canine or feline subject.

21. A method of treating a canine or feline subject having a metabolic disease, the method comprising administering to the canine or feline subject an effective amount of the fusion protein of any one of claims 1 to 14 or the pharmaceutical composition of claim 15.

22. The method of claim 21, wherein the metabolic disease is diabetes.

23. The method of claim 22, wherein the diabetes is type 1 diabetes.

24. The method of claim 22, wherein the diabetes is type 2 diabetes.

25. The method of any one of claims 21 to 24, wherein the therapeutically effective amount is administered intravenously.

26. The method of any one of claims 21 to 24, wherein the therapeutically effective amount is between 0.01 mg / kg and 15 mg / kg of the fusion protein.

27. The method of any one of claims 21 to 26, wherein the therapeutically effective amount is between 1 mg / kg and 15 mg / kg of the fusion protein.

28. The method of any one of claims 21 to 27, wherein the method reduces the subject's fasting blood glucose by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

29. A polynucleotide encoding a fusion protein for treating a companion animal, the fusion protein comprising proinsulin and serum albumin, wherein the proinsulin is canine proinsulin or feline proinsulin.

30. The polynucleotide of claim 29, wherein the canine proinsulin-serum albumin fusion polynucleotide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

2.

31. The polynucleotide of claim 29, wherein the feline proinsulin-serum albumin fusion polynucleotide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 19.

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