Insulin Fusion Proteins

Abstract

Compositions and methods are provided for treating diabetes in dogs or cats, in which a canine or feline insulin-serum albumin fusion protein is administered to the subject.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 315,296, filed March 1, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] (Sequence Listing) The sequence listing XML associated with this application is provided in XML file format and is incorporated herein by reference. The XML file containing the sequence listing XML is named SCTB_017_01WO_ST26.xml. The XML file is 40,960 bytes, was created on February 27, 2023, and was submitted electronically via the USPTO Patent Center.

[0003] FIELD OF THEINVENTION The present disclosure relates generally to compositions and methods for treating diabetes. [Background technology]

[0004] Diabetes mellitus is a syndrome associated with persistent 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 the result of insulin resistance, islet amyloidosis, or chronic lymphoplasmocytic pancreatitis. In humans, β cell loss is caused by an autoimmune response in type 1 diabetes and decreased insulin sensitivity in type 2 diabetes.

[0005] Insulin is an endogenous peptide hormone produced by the beta cells of the pancreatic islets and is considered the body's major anabolic hormone. Insulin is the mainstay of diabetes treatment in mammals. The current standard of care is twice-daily insulin injections, which are expensive, time-consuming, and inconvenient, involving frequent clinic visits and disposable diagnostics.

[0006] The present disclosure provides compositions and methods relating to insulin fusion proteins to provide a sustained half-life of insulin. Summary of the Invention

[0007] The present disclosure provides a fusion protein for the treatment of companion animals comprising proinsulin and serum albumin, where the proinsulin is canine proinsulin or feline proinsulin.

[0008] In some embodiments, the proinsulin is canine proinsulin.

[0009] 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.

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

[0011] 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.

[0012] 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.

[0013] In some embodiments, the fusion protein includes an N-terminal signal peptide.

[0014] In some embodiments, the signal peptide is a canine insulin signal peptide.

[0015] In some embodiments, the signal peptide comprises a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with MALWMRLLPLLALLALWAPAPTRA (SEQ ID NO:7).

[0016] 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 set forth in SEQ ID NO:10.

[0017] In some embodiments, the canine proinsulin comprises K53R, R55K, and L86R mutations compared to the reference polypeptide sequence set forth in SEQ ID NO:10.

[0018] 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 to SEQ ID NO:8.

[0019] In some embodiments, the polynucleotide encoding the fusion protein is operably linked to a promoter.

[0020] In some embodiments, the promoter is the cytomegalovirus enhancer / chicken b-actin promoter.

[0021] The present disclosure provides a pharmaceutical composition suitable for use in treating a metabolic disease in a dog or cat comprising a fusion protein of an embodiment of the present disclosure.

[0022] 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 with a metabolic disease, optionally diabetes.

[0023] 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 with a metabolic disease, optionally diabetes.

[0024] 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 fusion protein, and / or the fusion protein is administered intravenously.

[0025] In some embodiments, the fusion protein is administered in a dose of at least about 0.01 mg, at least about 0.05 mg, at least 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.

[0026] In some embodiments, the fusion protein is administered intravenously to a canine or feline subject.

[0027] The present disclosure provides a method of treating a canine or feline subject having a metabolic disease, comprising administering to the canine or feline subject an effective amount of a fusion protein of the present disclosure and / or a pharmaceutical composition of the present disclosure.

[0028] In some embodiments, the metabolic disease is diabetes.

[0029] In some embodiments, the diabetes is type 1 diabetes.

[0030] In some embodiments, the diabetes is type 2 diabetes.

[0031] In some embodiments, the therapeutically effective amount is administered intravenously.

[0032] In some embodiments, the therapeutically effective amount is between 0.01 mg / kg and 15 mg / kg of the fusion protein.

[0033] In some embodiments, the therapeutically effective amount is between 1 mg / kg and 15 mg / kg of the fusion protein.

[0034] In some embodiments, the method reduces fasting glucose in a subject by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0035] The present disclosure provides a polynucleotide encoding a fusion protein for the treatment of companion animals, the fusion protein comprising proinsulin and serum albumin, wherein the proinsulin is canine proinsulin or feline proinsulin.

[0036] 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 to SEQ ID NO:2.

[0037] 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]

[0038] [Figure 1A]Schematic diagrams of exemplary canine insulin proteins of the present disclosure. All three proteins incorporate a native signal peptide (SP) and modified furin site. FIG. 1A shows a schematic diagram of an exemplary canine preproinsulin-serum albumin fusion protein (cINS-Alb). The caINS-Alb protein has a glycine / serine linker linking the A chain of insulin to canine serum albumin. FIG. 1B shows a schematic diagram of an exemplary canine preproinsulin-transferrin fusion protein (cINS-Tf). The calNS-Tf protein has a glycine / serine linker linking the A chain of insulin to canine transferrin. FIG. 1C shows a schematic diagram of an exemplary canine preproinsulin protein (cINS-2-1) that contains a furin site modification and serves as a control. [Figure 1B] Schematic diagrams of exemplary canine insulin proteins of the present disclosure. All three proteins incorporate a native signal peptide (SP) and modified furin site. FIG. 1A shows a schematic diagram of an exemplary canine preproinsulin-serum albumin fusion protein (cINS-Alb). The caINS-Alb protein has a glycine / serine linker linking the A chain of insulin to canine serum albumin. FIG. 1B shows a schematic diagram of an exemplary canine preproinsulin-transferrin fusion protein (cINS-Tf). The calNS-Tf protein has a glycine / serine linker linking the A chain of insulin to canine transferrin. FIG. 1C shows a schematic diagram of an exemplary canine preproinsulin protein (cINS-2-1) that contains a furin site modification and serves as a control. [Figure 1C]Schematic diagrams of exemplary canine insulin proteins of the present disclosure. All three proteins incorporate a native signal peptide (SP) and modified furin site. FIG. 1A shows a schematic diagram of an exemplary canine preproinsulin-serum albumin fusion protein (cINS-Alb). The caINS-Alb protein has a glycine / serine linker linking the A chain of insulin to canine serum albumin. FIG. 1B shows a schematic diagram of an exemplary canine preproinsulin-transferrin fusion protein (cINS-Tf). The calNS-Tf protein has a glycine / serine linker linking the A chain of insulin to canine transferrin. FIG. 1C shows a schematic diagram of an exemplary canine preproinsulin protein (cINS-2-1) that contains a furin site modification and serves as a control. [Diagram 2] Figure 1 shows the in vitro insulin bioactivity of calNS-Alb and calNS-Tf compared to a control insulin standard. EC50 values ​​are listed in the table below the graph. Ligand-induced activation of the insulin receptor in response to increasing concentrations of purified cINS-Alb and cINS-Tf. A reference insulin standard was used as a control. Relative potency is expressed as relative luminescence units. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] As described elsewhere herein, the present disclosure is based, at least in part, on the inventors' surprising discovery that insulin fusion proteins achieve sustained biological activity of insulin in dogs and cats. Methods of making and using the fusion proteins are provided.

[0040] Insulin fusion proteins, engineered to overcome the short half-life of the native hormone by fusion to proteins with longer half-lives (e.g., serum albumin), are a therapeutic advance for the treatment of diabetes. Long-acting insulin fusion protein expression constructs have been developed for use in dogs and cats. The expression constructs contain a secretory signal peptide and a fusion domain that is intended to extend the time of circulation of the resulting fusion protein.

[0041] The expression constructs are administered to a subject in need thereof. Also provided are methods of using these fusion proteins in regimens for treating type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), or metabolic syndrome in a veterinary subject to increase the half-life of insulin in the subject.

[0042] The present disclosure encompasses insulin-albumin fusion proteins comprising a therapeutic protein with insulin activity. The present disclosure also encompasses polynucleotides comprising, or alternatively consisting of, a nucleic acid molecule encoding a therapeutic protein with insulin activity fused to albumin or a fragment (portion) or variant of albumin. Albumin may be fused to the N-terminus, C-terminus, or both termini of a 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 proteins, including a therapeutic protein with insulin activity fused to albumin or a fragment (portion) or variant of albumin, sufficient to prolong its activity in vivo.

[0043] Leader sequence In one embodiment, the insulin protein comprises a leader sequence, which may include 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 or signal sequence and proinsulin. The leader sequence, in one embodiment, is the native sequence (canine or feline insulin) leader. In another embodiment, the leader sequence is a heterologous sequence, i.e., derived from another protein other than canine or feline insulin.

[0044] 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.

[0045] SEQ ID NO:21: MYKMQLLSCIALTLVLVANS 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.

[0046] SEQ ID NO: 7: MALWMRLLPLLALLALWAPAPTRA 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.

[0047] SEQ ID NO: 13: MYKIQLLSCIALTLILVTNS 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.

[0048] SEQ ID NO: 9: MAPWTRLLPLLALLSLWIPAPTRA The leader sequence may be derived from the same species to which administration is ultimately intended, i.e., canine or feline. As used herein, the term "derived" or "derived from" means that the sequence or protein is sourced from a specific subject species or shares the same sequence as a protein or sequence sourced from a specific subject species. For example, a leader sequence "derived from" a canine or feline shares the same sequence (or a variant thereof as defined herein) as the same leader sequence expressed in a canine or feline. However, a particular nucleic acid or amino acid need not actually be sourced from a canine or feline. A variety of techniques are known in the art that can produce a desired sequence, including mutagenesis of similar proteins (e.g., homologs) or artificial production of a nucleic acid or amino acid sequence. A "derived" nucleic acid or amino acid retains the same nucleic acid or amino acid function in the species from which it is "derived," regardless of the actual source of the derived sequence.

[0049] Insulin Insulin is involved in regulating glucose utilization in the body. The inability of the body to synthesize insulin or cells resistant to insulin leads to diabetes mellitus, characterized by chronic hyperglycemia. Preproinsulin is transcribed as a 110 amino acid chain. Removal of the signal peptide from its N-terminus produces proinsulin. Formation of disulfide bonds between the A and B chain components and removal of the intervening C chain produces the biologically active insulin molecule, which is less than half the size of the original translation product and contains 51 amino acids.

[0050] As used herein, the term "insulin" refers to insulin or functional fragments thereof, including proinsulin and preproinsulin, as well as amino acid sequence variants of insulin or functional fragments thereof. The present disclosure provides proteins including 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 secretory 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 native canine proinsulin is set forth in SEQ ID NO: 10.

[0051] 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.

[0052] In some embodiments, the dog or feline insulin comprises a variant that retains the function of the wild-type sequence and may include up to about 10% variation from the insulin nucleic acid or amino acid sequence described herein or known in the art. As used herein, "retains function" means that the nucleic acid or amino acid functions similarly to the wild-type sequence, although not necessarily at the same level of expression or activity. For example, in one embodiment, the functional variant has increased expression or activity compared to the wild-type sequence. In another embodiment, the functional variant has decreased expression or activity compared to the wild-type sequence. In one embodiment, the functional variant has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase or decrease in expression or activity compared to the wild-type sequence (SEQ ID NO: 11).

[0053] The canine proinsulin sequence, in one embodiment, comprises one or more mutations compared to the native sequence. These mutations, in some embodiments, are at the cleavage site between the B / C and C / A chains. In one embodiment, one or more of the cleavage sites are mutated to incorporate at least one furin cleavage site into an 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.

[0054] In one embodiment the canine proinsulin sequence is a sequence which shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:12.

[0055] 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.

[0056] If variants or fragments of the proinsulin sequence are desired, the coding sequences of these peptides can be generated using site-directed mutagenesis of the wild-type nucleic acid sequence. Alternatively, or in addition, web-based or commercially available computer programs, as well as service-based companies, can be used to back-translate the amino acid sequence into a nucleic acid coding sequence, including both RNA and / or cDNA. See, for example, backtranseq: Gene Infinity by EMBOSS, and / or ExPasy. In one embodiment, the RNA and / or cDNA coding sequence is designed for optimal expression in the target species for which administration is ultimately intended, i.e., dog or cat.

[0057] Fusion domains The present disclosure provides a fusion protein comprising a fusion domain. By fusing insulin to a fusion domain with a longer half-life, the insulin fusion protein overcomes the short half-life of the native hormone. In some embodiments, the fusion domain comprises either (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.

[0058] In some embodiments, the fusion domain comprises either (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, hi some embodiments, the fusion domain comprises feline serum albumin.

[0059] In some embodiments, the fusion domain is canine serum albumin comprising a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:16.

[0060] In some embodiments, the fusion domain is canine transferrin comprising a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:17.

[0061] In some embodiments, the fusion domain is feline serum albumin comprising a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:18.

[0062] Insulin Fusion Proteins The present disclosure provides fusion proteins comprising one or more copies of proinsulin, as well as polynucleotides and expression vectors encoding such fusion proteins. In some embodiments, the fusion protein comprises a polynucleotide sequence encoding a fusion protein comprising (a) a leader sequence comprising a secretory signal peptide, (b) proinsulin, and (c) a fusion domain comprising either (i) IgG Fc or a functional variant thereof, (ii) albumin or a functional variant thereof, or (iii) transferrin or a functional variant thereof. In one embodiment, the fusion protein comprises a thrombin leader sequence, proinsulin, and IgG Fc or a functional variant thereof. In another embodiment, the fusion protein comprises a thrombin leader sequence, proinsulin, and albumin or a functional variant thereof.

[0063] 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.

[0064] 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 that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:3.

[0065] 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.

[0066] In some embodiments, the fusion protein comprises a feline insulin leader sequence, feline proinsulin (K53R, R55K, and L86R), a glycine / serine linker, and feline 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:6.

[0067] In one embodiment, the fusion protein comprises an insulin leader sequence, proinsulin, and albumin or a functional variant thereof.In one embodiment, the fusion protein comprises an insulin leader sequence, proinsulin, and transferrin or a functional variant thereof.

[0068] In one embodiment, the fusion protein comprises an IL2 leader sequence, proinsulin, and albumin or a functional variant thereof.In one embodiment, the fusion protein comprises an IL2 leader sequence, proinsulin, and transferrin or a functional variant thereof.

[0069] In addition to the leader sequences, proinsulin, and insulin polypeptides provided herein, nucleic acid sequences (used interchangeably with "polynucleotides") encoding these polypeptides are provided. In one embodiment, a nucleic acid sequence encoding a proinsulin-serum albumin fusion polypeptide described herein is provided. In some embodiments, a 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.

[0070] 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.

[0071] 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.

[0072] The in vivo function and stability of the fusion proteins of the present disclosure may be optimized by adding small peptide linkers, for example, to prevent potentially unwanted domain interactions or for other reasons. Furthermore, a glycine-rich linker may provide some structural flexibility so that the proinsulin portion can productively interact with the insulin receptor on the target cell. Thus, the C-terminus of the proinsulin and the N-terminus of the fusion domain of the fusion protein are fused, in one embodiment, via a linker. In some embodiments, the linker has the sequence (GGGGS) nIn one embodiment, the linker comprises 1, 2, 3, or n repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 14). In one embodiment, the linker comprises 1, 1.5, or 2 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).

[0073] In some embodiments, the fusion protein of the present disclosure comprises, 5' to 3', (a) canine insulin signal peptide, (b) canine proinsulin (K53R, R55K, and L86R); (c) a Gly / Ser linker, and (d) Contains canine serum albumin.

[0074] 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.

[0075] In some embodiments, the fusion protein of the present disclosure comprises, 5' to 3', (a) feline insulin signal peptide, (b) feline proinsulin (K53R, R55K, and L86R); (c) a Gly / Ser linker, and (d) Contains feline serum albumin.

[0076] 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.

[0077] Expression cassette In some embodiments, an expression cassette refers to a nucleic acid molecule that includes a proinsulin fusion construct coding sequence, a promoter, and may include other regulatory sequences therefor. The expression cassette may be engineered into a genetic element (e.g., a plasmid) for delivery into a cell and purification of the fusion protein for therapeutic administration.

[0078] An expression cassette typically includes a promoter sequence as part of the expression control sequence. In one embodiment, a constitutive promoter is used. The CB7 promoter may be used in the plasmids and expression vectors described herein. CB7 is a chicken B-actin promoter with a cytomegalovirus enhancer element. Other promoters, such as viral promoters, constitutive promoters, regulatable promoters (see, for example, WO 2011 / 126808 and WO 2013 / 04943), or promoters that respond to physiological cues, may be used and may be utilized in the expression vectors described herein.

[0079] In some embodiments of the plasmids and expression vectors described herein, the CMV enhancer, chicken beta-Actin promoter and rabbit beta-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.

[0080] In addition to a promoter, the expression cassette and / or expression vector may contain other appropriate 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, if desired, sequences that enhance secretion of the encoded product. Illustrative examples of suitable polyA sequences include, for example, rabbit β-globin, SV40, bovine growth hormone (bGH), and TK polyA.

[0081] Illustrative examples of suitable enhancers include, among others, for example, alphatoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha1-microglobulin / bikunin enhancer). In one embodiment, the polyA is rabbit globin polyA.

[0082] 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 adjacent to a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0083] In one embodiment, an expression cassette is provided that includes a CB7 promoter, a chimeric intron, a coding sequence for the protein encoded by SEQ ID NO:2, and rabbit beta globin polyA.

[0084] In one embodiment, an expression cassette is provided that includes a CB7 promoter, a chimeric intron, a coding sequence for the protein encoded by SEQ ID NO:4, and rabbit beta globin polyA.

[0085] In one embodiment, an expression cassette is provided that includes a CB7 promoter, a chimeric intron, a coding sequence for the protein encoded by SEQ ID NO:19, and rabbit beta globin polyA.

[0086] The methods used to construct any embodiment of the present disclosure are known to those of skill in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques.

[0087] In some embodiments, the fusion protein of the present disclosure comprises, 5' to 3', (a) cytomegalovirus (CMV) enhancer / chicken b-actin (CAG) promoter; (b) canine insulin signal peptide, (c) canine proinsulin (K53R, R55K, and L86R); (d) a Gly / Ser linker, and (e) an expression cassette comprising canine serum albumin.

[0088] 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.

[0089] In some embodiments, the fusion protein of the present disclosure comprises, 5' to 3', (a) Cytomegalovirus (CMV) enhancer / chicken b-actin (CAG) promoter; (b) feline insulin signal peptide, (c) feline proinsulin (K53R, R55K, and L86R); (d) a Gly / Ser linker, and (e) comprises an expression cassette comprising feline serum albumin.

[0090] 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.

[0091] In one embodiment, the nucleic acid sequence encoding the proinsulin fusion construct described herein is engineered into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, recombinant AAV, etc., that transfers the proinsulin fusion sequence carried thereon into a host cell for generating nanoparticles carrying DNA or RNA that are virions in a packaging host cell, and / or for delivery to a host cell in a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0092] In some embodiments, the proinsulin fusion constructs described herein may be delivered via virions, including but not limited to recombinant AAV (rAAV). Such other virions may include any virus suitable for gene therapy, including but not limited to adenovirus, herpes virus, lentivirus, retrovirus, etc. Preferably, when one of these other virions is produced, it is produced as a replication-deficient virion.

[0093] cell culture 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 medium. Such cells are typically obtained from mammals or are cell lines derived from mammals, and are capable of growing and surviving when placed in either monolayer or suspension culture in media containing appropriate nutrients and / or growth factors. The growth factors and nutrients required for the growth and maintenance of a particular cell culture can be readily determined empirically by one of skill in the art, as described, for example, in Barnes and Sato, (1980, Cell, 22:649); in Mammalian Cell Culture, Ed. JP Mather, Plenum Press, NY, 1984, and U.S. Patent No. 5,721,121.

[0094] In addition, cell culture conditions typically include, in addition to temperature, pH, for example, about 6.5 to about 7.5, dissolved oxygen (O 2 ), e.g., about 5 to 90% air saturation and carbon dioxide (CO 2 ), agitation, and humidity are employed for batch, fed-batch, or continuous culture of cells and are well known.

[0095] Cell line cells are typically animal or mammalian cells that are capable of expressing and secreting, or can be molecularly engineered to express and secrete, large amounts of a particular protein into the culture medium. It will be understood that the protein of interest produced by the host cell can be endogenous or homologous to the host cell. In one embodiment, the protein of interest is produced and secreted by Chinese hamster ovary (CHO) host cells. In some embodiments, the protein of interest is a proinsulin-serum albumin fusion protein.

[0096] 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).

[0097] Cells suitable for culturing in the processes of the present disclosure may contain an expression vector (construct), such as a plasmid, carrying a coding sequence or a portion thereof encoding a protein for expression and production in the culturing process, introduced, for example, via transformation, transfection, infection, or injection. Such expression vectors contain the necessary elements for transcription and translation of the inserted coding sequence.

[0098] In the culture methods encompassed by the present disclosure, the protein produced by the cells is typically harvested, recovered, isolated, and / or purified, or substantially purified, as desired, at the end of the entire cell culture period using isolation and purification methods known and practiced in the art. In some embodiments, the protein of interest is secreted from the cultured cells and isolated from the culture medium or supernatant. In some embodiments, the protein can also be recovered from the host cells, e.g., from a cell lysate, using methods known and practiced in the art.

[0099] Methods of Treating Subjects with the Disclosed Compositions Also provided are compositions comprising the fusion proteins described herein.The pharmaceutical compositions described herein are designed for delivery to canine or feline subjects in need thereof by any suitable route or combination of different routes.Direct delivery to the liver (optionally via a vein, via the hepatic artery, or by transplantation), direct delivery to the pancreas, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration.The fusion proteins described herein can be delivered in a single composition or in multiple compositions.

[0100] In some embodiments, the pharmaceutical compositions described herein are designed for delivery to a canine or feline subject in need thereof by intramuscular administration.

[0101] In some embodiments, the pharmaceutical compositions described herein are designed for delivery to a canine or feline subject in need thereof by intravenous administration.

[0102] In some embodiments, the pharmaceutical compositions described herein are designed for delivery to a canine or feline subject in need thereof by subcutaneous administration.

[0103] In some embodiments, a course of treatment may involve repeated administration of the same fusion protein. Still other combinations may be selected using the fusion proteins described herein. In some embodiments, the compositions described herein may be combined in a regimen with other diabetes drugs or protein-based therapies, including, for example, insulin analogs, insulin, oral hypoglycemic agents, sulfonylureas, biguanides, thiazolidinediones, and α-glucosidase inhibitors. In some embodiments, the compositions described herein may be combined in a regimen with lifestyle changes, including diet and exercise regimens.

[0104] As used herein, the terms "proinsulin construct", "proinsulin expression construct" and synonyms include a proinsulin sequence as described herein in combination with a leader (whether native or heterologous). The terms "proinsulin construct", "proinsulin expression construct" and synonyms can be used to refer to a nucleic acid sequence that encodes a proinsulin fusion protein or its expression product.

[0105] In one aspect, the disclosure relates to a method of treating a disease or disorder in a subject in need thereof, including administering an effective amount of a proinsulin fusion protein, thereby ameliorating and / or treating one or more symptoms of Type I diabetes, Type II diabetes, or metabolic syndrome.

[0106] In some embodiments, the methods comprise 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.

[0107] In some embodiments, the 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.

[0108] 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, a therapeutically effective amount of a proinsulin fusion protein is administered at a dose 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.

[0109] 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.

[0110] In some embodiments, a therapeutically effective amount of a 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.

[0111] In some embodiments, the methods comprise administering a dose of proinsulin fusion protein once daily, 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.

[0112] In some embodiments, the methods comprise administering doses of the proinsulin fusion protein regimen for at least 1 week, 2 weeks, or 4 weeks.

[0113] In some embodiments, the methods include administering doses of the proinsulin fusion protein regimen for at least 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, or 18 months.

[0114] In some embodiments, a therapeutically effective amount of a proinsulin fusion protein regimen is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0115] In some embodiments, the methods comprise chronically administering doses of a proinsulin fusion protein regimen.

[0116] The fusion protein composition can be formulated in dosage units to contain an amount of fusion protein that is in the range of about 0.1 mg to about 15 mg.

[0117] In another aspect, a method of sustained biological activity of a proinsulin fusion protein in a canine or feline subject is provided. The method comprises administering a composition described herein to a subject in need thereof. In one embodiment, the composition comprises a fusion protein comprising a proinsulin-serum albumin fusion protein described herein. In some embodiments, the proinsulin fusion protein described herein provides an extended half-life of insulin compared to the native peptide. In some embodiments, the fusion protein provided herein provides a half-life of the proinsulin fusion protein in a subject of 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, the fusion protein can be delivered in a volume of 1 μL to about 100 mL for a veterinary subject. For a discussion of good practices for administration of substances to various veterinary animals, see, for example, Diehl et al, J. Applied Toxicology, 21:15-23 (2001), which is incorporated herein by reference. As used herein, the term "dose" can refer to the total dose delivered to a subject over the course of treatment or the amount delivered in a single dose (or doses).

[0118] 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 (ProZinc®), porcine insulin zinc suspension (Vetsulin®), and insulin glargine (Lantus®). In some embodiments, the combination of the fusion protein described herein with insulin reduces insulin dosage requirements in a subject compared to before treatment with the fusion protein. Such dosage requirements can be reduced by 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 treating physician can determine the correct dosage of insulin required by the subject. For example, the subject can be treated using insulin or other therapy, which the treating physician can continue upon administration of the fusion protein. Such insulin or other combination therapy can then be continued, reduced, or discontinued as needed.

[0119] In one embodiment, the subject is delivered a therapeutically effective amount of the composition described herein. As used herein, "therapeutically effective amount" refers to the amount of fusion protein that delivers an amount of proinsulin-serum albumin in target cells sufficient to reach a therapeutic goal. In certain embodiments, the therapeutic goal is to improve or treat one or more of the symptoms of type I diabetes, type II diabetes, or metabolic syndrome. The therapeutically effective amount may be determined based on an animal model rather than a canine or feline subject. In another embodiment, the therapeutic goal is the remission of metabolic disease in the subject.

[0120] The above fusion protein can be delivered to host cells according to published methods. The fusion protein, preferably suspended in a physiologically compatible carrier, can be administered to a desired subject, including a dog. A suitable carrier can be easily selected by a person skilled in the art, taking into consideration the indication for which the transferred virus is intended. For example, one suitable carrier includes saline (e.g., phosphate buffered saline), which can be formulated with various buffers. 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.

[0121] In some embodiments, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the fusion protein and / or variant and carrier. Exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Exemplary chemical stabilizers include gelatin and albumin.

[0122] In some embodiments, the fusion protein of the present disclosure is formulated as a depot injection. The depot injection formulation delivers the fusion protein (e.g., proinsulin-serum albumin fusion protein) at a tunable, predetermined rate within the therapeutic range for a specific period of time. The release can occur directly at the site of action for localized treatment or at a systemic level, thus minimizing the adverse side effects of the fusion protein.

[0123] In some embodiments, the proinsulin-serum albumin fusion proteins of the present disclosure are formulated as a depot injection.

[0124] The recombinant fusion proteins described herein can be used in preparing a medicament for delivering a proinsulin fusion protein to a subject in need thereof, for providing a subject with insulin having an increased half-life, and / or for treating type I diabetes, type II diabetes, or metabolic syndrome in a subject.

[0125] In one aspect, a method of treating diabetes is provided. The method comprises administering a composition described herein to a canine or feline subject in need thereof. In one embodiment, the composition comprises a proinsulin fusion described herein.

[0126] In another embodiment, a method is provided for treating type 2 diabetes in a dog or cat, the method comprising administering a proinsulin fusion protein as described herein.

[0127] In another embodiment, a method is provided for treating type 1 diabetes in a dog or cat, the method comprising administering a proinsulin fusion protein as described herein.

[0128] In another aspect, a method of treating a metabolic disease in a dog or cat is provided. The method comprises administering a composition described herein to a dog or cat subject in need thereof. In one embodiment, the composition comprises a proinsulin fusion protein 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.

[0129] In another embodiment, a method is provided for treating diabetes in a dog or cat, the method comprising administering a proinsulin-serum albumin fusion protein described herein, where the fusion protein is administered after insulin has been administered to the subject.

[0130] In another embodiment, a method is provided for preventing cataract formation in a diabetic dog or cat comprising administering a proinsulin-serum albumin fusion protein as described herein.

[0131] In another embodiment, a method is provided for reducing blood glucose concentrations in a diabetic dog or cat, the method comprising administering a proinsulin-serum albumin fusion protein described herein.

[0132] As used herein, the term "treatment" or "treating" is defined to encompass administration of one or more compounds or compositions described herein to a subject for the purpose of ameliorating one or more symptoms of type I diabetes, type II diabetes (T2DM), or metabolic syndrome. Thus, "treatment" can include one or more of reducing the progression of type I diabetes, type II diabetes, or metabolic syndrome, reducing the severity of a symptom, eliminating disease symptoms, slowing disease progression, or increasing the effectiveness of a therapy in a given subject.

[0133] As used herein, the term "remission" refers to the ability to discontinue insulin treatment when a cat or dog no longer shows clinical signs of diabetes and has normal blood glucose levels.

[0134] In another embodiment, a method is provided for treating T2DM in a cat or dog, the method comprising administering a fusion protein as described herein.

[0135] In another aspect, a method of treating a metabolic disease in a cat or dog is provided. The method comprises administering a composition described herein to a feline or canine subject in need thereof. In one embodiment, the composition comprises a proinsulin fusion protein described herein.

[0136] In another aspect, a method of reducing fasting blood glucose in a canine or feline subject is provided. The method comprises administering a composition described herein to a subject in need thereof. In one embodiment, the composition comprises a proinsulin-serum albumin fusion protein described herein. In some embodiments, the method provided herein reduces fasting blood glucose in 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 fasting blood glucose in the subject by about 20%. In some embodiments, the method provided herein reduces fasting blood glucose in the subject by about 30%. In some embodiments, the method provided herein reduces fasting blood glucose in the subject by about 40%.

[0137] definition Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terms, the present specification shall control.

[0138] References to "one embodiment" or "another embodiment" when describing an embodiment do not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise.

[0139] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, i.e., share at least about 80% identity with a reference sequence over a specified region, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. In some embodiments, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, e.g., over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0140] For sequence comparison, typically one sequence acts as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.In some embodiments, BLAST and BLAST 2.0 algorithms and default parameters are used.

[0141] The terms "percent identity", "sequence identity", "percent sequence identity", or "percent identity" in the context of amino acid sequences refer to the residues in two sequences that are the same when aligned for correspondence. Percent identity can be readily determined for amino acid sequences spanning the entire length of a protein, polypeptide, from about 70 amino acids to about 100 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence coding sequence. Suitable amino acid fragments can be at least about 8 amino acids in length, and up to about 150 amino acids in length. In general, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to the "aligned" sequences.

[0142] "Aligned" sequences or "alignment" often refers to multiple nucleic acid sequences or protein (amino acid) sequences, including corrections for missing or additional bases or amino acids compared to a reference sequence. Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used with default settings, but one of skill in the art can change these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as provided by the referenced algorithms and programs.

[0143] The term "amino acid substitution" and its synonyms are intended to encompass the modification of an amino acid sequence by replacing an amino acid with another replacement amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. The term conservative, when referring to two amino acids, is intended to mean that the amino acids share a common property recognized by those of skill in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. The common property may also be amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as replacement amino acids in embodiments. Methods for replacing amino acids are well known to those of skill in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. References herein to "one or more" are intended to encompass, for example, one, two, three, four, five, six or more individual embodiments.

[0144] Coding sequences can be designed for optimal expression using codon optimization. Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be performed using methods available online, published methods, or companies that provide codon optimization services. One codon optimization method is described, for example, in WO 2015 / 012924, which is incorporated herein by reference. Briefly, a nucleic acid sequence that codes for a product is modified with synonymous codon sequences. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only a fragment of the ORF may be modified. By using one of these methods, a frequency can be applied to any given polypeptide sequence to produce a nucleic acid fragment of a codon-optimized coding region that codes for a polypeptide.

[0145] In embodiments, the insulin-serum albumin fusion is humanized, canine, feline, or equine.

[0146] By "humanized" it is meant that the fusion protein contains an amino acid sequence that is compatible with humans, such that the amino acid sequence is unlikely to be regarded as foreign by the immune system of a human subject.

[0147] "Caninized" means that the fusion protein comprises an amino acid sequence that is canine compatible, such that the amino acid sequence is unlikely to be viewed as foreign by the immune system of a canine subject. In this disclosure, terms for polypeptides preceded by the prefix "ca" refer to variants of human polypeptides in which a human fusion domain is replaced with a canine homolog of that fusion domain, and in the case where proinsulin is a fragment or variant of a human protein, proinsulin is replaced with the canine homolog of that fragment or variant.

[0148] "Fenified" means that the fusion protein comprises an amino acid sequence that is feline compatible, such that the amino acid sequence is unlikely to be viewed as foreign by the immune system of a feline subject. In the present disclosure, terms for polypeptides preceded by the prefix "fe" refer to variants of human polypeptides in which the human fusion domain is replaced with a feline homolog of that fusion domain, and in the case where proinsulin is a fragment or variant of a human protein, the proinsulin is replaced with the feline homolog of that fragment or variant.

[0149] By "equineized" it is meant that the fusion protein contains an amino acid sequence that is compatible with horses, such that the amino acid sequence is unlikely to be viewed as foreign by the immune system of an equine subject.

[0150] As described elsewhere herein, the present disclosure extends to fusion proteins that are compatible with species other than humans, dogs, cats, and horses, and in this context, the fusion proteins may be referred to as "specialized" referring to the target species to which the molecule is administered.

[0151] In some embodiments, the compositions and methods described herein are intended for use with felines. The term cat (feline) refers to any of the 37 cat species, including cheetah, puma, jaguar, leopard, lion, lynx, tiger, and domestic cat, among others. In an embodiment, the subject is a domestic cat. In some embodiments, the compositions and methods described herein are intended for use with canines. The term dog refers to any of the species found in the canine family, including domestic dogs, wolves, and foxes, among others. In an embodiment, the subject is a domestic dog, also known as Canis lupus familiaris or Canis familiaris.

[0152] As used in the specification of this 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.

[0153] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0154] As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agents recited in a method or composition, and can further include other agents that do not alone have substantial activity for the recited indication or purpose.

[0155] The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and variations thereof are to be interpreted exclusively rather than inclusively. Although various embodiments herein are presented using the word "comprising", it is intended that, under other circumstances, the relevant embodiment also be construed and described using the word "consisting of" or "consisting essentially of".

[0156] As used herein, the term "about" means a variability of 10% from the cited reference, unless otherwise specified.

[0157] The term "modulate" or variations thereof as used herein refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0158] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal condition in a subject.

[0159] The terms "subject," "individual," and "patient" refer interchangeably to mammals, humans or non-human primates, domesticated 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. Illustrative 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.

[0160] 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 liver cell. In some embodiments, the target cell is a muscle cell.

[0161] As used herein, an "expression cassette" refers to a nucleic acid molecule that includes a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, an enzyme or other useful gene product, mRNA, etc.) and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, an "operably linked" sequence includes both regulatory sequences (also referred to as elements) that are adjacent or not adjacent to the nucleic acid sequence, and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include one or more of, for example, promoters, enhancers, transcription factors, transcription terminators, introns, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), efficient RNA processing signals such as slicing and polyadenylation sequences, sequences that stabilize cytoplasmic mRNA, such as the Woodchuck Hepatitis Virus (WHP) posttranslational Regulatory Element (WPRE), and a TATA signal. An expression cassette may include, among other elements, regulatory sequences upstream of the gene sequence (5' to the gene sequence), such as one or more of a promoter, enhancer, intron, etc., and enhancers, or regulatory sequences downstream of the gene sequence (3' to the gene sequence), such as one or more of a 3' untranslated region (3' UTR) including a polyadenylation site. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of the gene product, and the regulatory sequences are separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5'-untranslated region (5' UTR). In certain embodiments, the expression cassette includes one or more nucleic acid sequences of the gene products. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette.

[0162] As used herein, "administering" refers to local and systemic administration, including, for example, enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration of pharmaceutical ingredients used in the methods described herein include, for example, oral (per os (PO)), nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT), intravenous (intravenous, "iv"), intraperitoneal (intraperitoneal, "ip"), intramuscular (intramuscular, "im"), intralesional, or subcutaneous (subcutaneous, "sc") administration, or implantation of a sustained release device, such as a mini-osmotic pump, a depot formulation, or the like, into a subject. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, and intracranial administration.

[0163] The terms "systemic administration" and "administered systemically" refer to a method of administering a pharmaceutical ingredient or composition to a mammal such that the pharmaceutical ingredient or composition is delivered via the circulatory system to a site within the body, including the target site of pharmaceutical action. 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).

[0164] The term "effective amount" or "pharmacologically effective amount" refers to the amounts and / or dosages and / or dosing regimen of one or more pharmaceutical components (e.g., fusion protein) necessary to bring about a desired result.

[0165] As used herein, the terms "treating" and "treatment" refer to delaying the onset of, hindering the progression of, or reversing, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. The terms "treating" and "treatment" also include preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of a disease or condition.

[0166] The term "alleviating" refers to the reduction or elimination of one or more symptoms of the pathology or disease, and / or the reduction or delay in the rate 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 the pathology or disease can include, for example, a measurable and sustained reduction in fasting blood glucose. *****

[0167] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, this application, including any definitions herein, will control. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited in this specification is not, and should not be construed as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0168] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0169] While illustrative embodiments have been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the disclosure. EXAMPLES

[0170] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be merely illustrative of the disclosure and are not intended to limit the scope of what is regarded as the disclosure.

[0171] Example 1: Generation of half-life extended canine insulin-serum albumin fusions and evaluation of the in vitro potency of canine insulin fusion proteins A canine insulin-serum albumin fusion protein has been developed for the management of hyperglycemia and hyperglycemia-related clinical signs of diabetes mellitus in dogs.

[0172] Canine preproinsulin-serum albumin fusion protein (cINS-Alb) was generated by constructing a fusion polypeptide (FIG. 1A) (SEQ ID NO:1) containing the following elements: Natural canine insulin signal peptide (SP) Canine proinsulin, the native sequence of which has been modified at three amino acid positions (K53R, R55K, and L86R) to incorporate two furin cleavage sites into the existing protease cleavage sites (B chain, C peptide, A chain). a Gly / Ser linker comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO:8) ·Canine serum albumin

[0173] A canine preproinsulin fusion protein was made using the same elements as above, except that the canine serum albumin sequence was replaced with canine transferrin (cINS-Tf) (FIG. 1B) (SEQ ID NO: 3). A control canine preproinsulin sequence with three amino acid modifications (K53R, R55K, and L86R) was also generated as a control (cINS-2-1) (FIG. 1C) (SEQ ID NO: 5).

[0174] A study was conducted to evaluate the in vitro potency of the two fusion proteins (cINS-Alb and cINS-Tf), where a C-terminal histidine-tagged version of each of the proteins was produced in mammalian cells after transient transfection of an expression plasmid containing the cDNA encoding the respective protein. The proteins were purified from cell supernatants via nickel affinity chromatography and assayed for insulin bioactivity using the PathHunterO™ Insulin Bioassay Kit (Eurofins™ DiscoverX Products, LLC) according to the kit's instructions. 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). The modifications made to the insulin molecule in both fusion proteins were consistent with the EC 50 This resulted in a slight loss of potency as indicated by a change in values.

Claims

1. A fusion protein comprising proinsulin and serum albumin, wherein the proinsulin is canine proinsulin that shares at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 12 or feline proinsulin that shares at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:

15.

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

1.

3. The fusion protein of claim 1 , wherein the fusion protein comprises an N-terminal signal peptide.

4. The fusion protein of claim 3, wherein the N-terminal signal peptide is a canine insulin signal peptide comprising a sequence that shares at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:

7.

5. 2. The fusion protein of claim 1, wherein the canine proinsulin comprises K53R, R55K and L86R mutations compared to the reference polypeptide sequence shown in SEQ ID NO:

10.

6. 2. The fusion protein of claim 1, 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.

7. A polynucleotide encoding the fusion protein of claim 1, wherein the polynucleotide shares at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 2 or SEQ ID NO:

19.

8. The polynucleotide of claim 7, wherein the polynucleotide is operably linked to a promoter, preferably linked to the cytomegalovirus enhancer / chicken b-actin promoter.

9. A pharmaceutical composition comprising the fusion protein of claim 1.

10. A pharmaceutical composition for treating a canine or feline subject with a metabolic disease, comprising the fusion protein of claim 1.

11. The pharmaceutical composition described in claim 10, wherein the metabolic disease is diabetes.

12. 11. The pharmaceutical composition of claim 10, wherein the fusion protein is formulated to be administered at a concentration of the fusion protein 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.

13. 11. The pharmaceutical composition of claim 10, wherein the fusion protein is administered in 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.

14. 11. The pharmaceutical composition of claim 10, wherein the fusion protein or pharmaceutical composition is administered intravenously to the canine or feline subject.

Images