Viral vector genome encoding insulin fusion protein

Abstract

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

Description

[Technical field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (UPN-22-10022.PCT.xml, size: 111 kb, and creation date: March 1, 2023) are incorporated herein by reference in their entirety.

[0002] The present invention relates generally to compositions and methods for treating diabetes in dogs or cats. [Background technology]

[0003] Diabetes mellitus is a syndrome associated with prolonged hyperglycemia due to loss or dysfunction of insulin secretion by pancreatic beta cells, decreased insulin sensitivity in tissues, or both. In dogs, beta cell loss tends to be rapid and progressive and is usually due to immune-mediated destruction, vacuolar degeneration, or pancreatitis. In cats, beta cell loss or dysfunction is the result of insulin resistance, islet amyloidosis, or chronic lymphoplasmocytic pancreatitis.

[0004] Insulin is an endogenous peptide hormone produced by beta cells of the pancreatic islets and is considered the body's primary anabolic hormone. Insulin is the mainstay of therapy for diabetic dogs and cats. The current standard of care is twice-daily insulin injections by the dog or cat caregiver along with frequent veterinary visits and diagnostics, which are expensive, time-consuming, and inconvenient.

[0005] The present disclosure provides compositions and methods relating to virions engineered to provide sustained expression of insulin. Summary of the Invention

[0006] In certain embodiments, recombinant adeno-associated virus (rAAV) virions are provided for the treatment of companion animals. The rAAV comprises an AAV capsid and a vector genome comprising an expression cassette comprising a polynucleotide encoding a fusion protein comprising proinsulin and serum albumin, the expression cassette being flanked by a 5' inverted terminal repeat (ITR) and a 3' ITR, and the proinsulin being canine proinsulin or feline proinsulin. In certain embodiments, the AAV capsid is selected for its ability to express in muscle cells. In certain embodiments, the capsid is an AAVrh91 capsid.

[0007] In certain embodiments, the proinsulin is canine proinsulin. In other embodiments, the proinsulin is feline proinsulin. Suitably, the canine or feline proinsulin comprises an N-terminal signal peptide. In certain embodiments, the N-terminal signal peptide of canine insulin is canine insulin signal peptide. In certain embodiments, the N-terminal signal peptide of feline insulin is feline signal peptide.

[0008] In certain embodiments, the canine proinsulin is a canine proinsulin variant having mutations at one or more cleavage sites compared to the reference polypeptide sequence set forth in SEQ ID NO: 10. In certain embodiments, the proinsulin is canine proinsulin fused to canine serum albumin. In certain embodiments, the 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. In certain embodiments, the proinsulin comprises K29R, R31K, and L62R mutations compared to the reference polypeptide sequence set forth in SEQ ID NO: 10. In certain 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.

[0009] In certain embodiments, the feline proinsulin is a variant having one or more cleavage site mutations compared to the reference polypeptide sequence set forth in SEQ ID NO: 24. In certain embodiments, the proinsulin is feline proinsulin fused to feline serum albumin.

[0010] In certain embodiments, the feline proinsulin-feline 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: 33. The feline proinsulin may contain K29R, R31K, and L62R mutations compared to the reference polypeptide sequence set forth in SEQ ID NO: 24.

[0011] In certain embodiments, the feline proinsulin-feline 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: 33. The feline proinsulin may include K29R, R31K, and L62R mutations compared to the reference polypeptide sequence set forth in SEQ ID NO: 24. The polynucleotide encoding the fusion protein is operably linked to a promoter. In certain embodiments, the promoter is a CB7 promoter element comprising a cytomegalovirus enhancer and / or chicken b-actin promoter. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding a homology-directed repair (HDR) template configured for insertion into the cleavage site.

[0012] In certain embodiments, a pharmaceutical composition suitable for use in treating a metabolic disease in a dog or cat is provided, comprising a rAAV virion. The rAAV virion or pharmaceutical composition may be used in a method for treating a dog or cat subject with a metabolic disease, optionally diabetes.

[0013] Certain embodiments provide for the use of rAAV virions or pharmaceutical compositions in the manufacture of a medicament for treating a canine or feline subject with a metabolic disease, optionally diabetes. Certain embodiments provide for the use of rAAV virions or pharmaceutical compositions in the manufacture of a medicament for treating a canine or feline subject with a metabolic disease, optionally diabetes. 9 GC / kg~3×10 13 The rAAV is provided in a composition that is formulated to be administered to a canine or feline subject at a dose of GC / kg and / or the rAAV is delivered intramuscularly.

[0014] In certain embodiments, a method of treating a canine or feline subject with a metabolic disease is provided, comprising administering to the canine or feline subject an effective amount of a rAAV virion or pharmaceutical composition. In certain embodiments, the method is for treating a metabolic disease, such as diabetes. In certain embodiments, the diabetes is type 1 diabetes. In other embodiments, the diabetes is type 2 diabetes. In certain embodiments, the effective amount is administered intramuscularly. In certain embodiments, the effective amount is greater than 1×10 9 GC / kg~3×10 13 GC / kg of rAAV virions. In certain embodiments, the effective amount is 1×10 10 GC / kg~3×10 13 In certain embodiments, the method comprises administering to the patient a rAAV virion at least for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, at least 60 weeks, at least 80 weeks, at least 90 weeks, at least 100 weeks, at least 120 weeks, at least 140 weeks, at least 160 weeks, at least 18 ... In certain embodiments, the method results in expression of the fusion protein in the subject at a therapeutically effective concentration for at least 3 months, at least 6 months, or at least 12 months. In certain embodiments, the method reduces fasting blood glucose in the subject by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0015] Still further aspects and advantages of the present invention will become apparent from the following detailed disclosure of the invention. [Brief description of the drawings]

[0016] [Figure 1A] 1 shows a schematic diagram of an exemplary canine insulin protein of the present disclosure. All three proteins incorporate a native signal peptide (SP) and a modified furin site. A schematic diagram of an exemplary canine preproinsulin-serum albumin fusion protein (cINS-Alb) is shown. The caINS-Alb protein has a glycine / serine linker linking the A chain of insulin to canine serum albumin. [Figure 1B] Schematic diagrams of exemplary canine insulin proteins of the present disclosure are shown. All three proteins incorporate a native signal peptide (SP) and a modified furin site. Schematic diagram of an exemplary canine preproinsulin-transferrin fusion protein (cINS-Tf) is shown. The caINS-Tf protein has a glycine / serine linker linking the A chain of insulin to canine transferrin. [Figure 1C] Schematic diagrams of exemplary canine insulin proteins of the present disclosure are shown. All three proteins incorporate a native signal peptide (SP) and a modified furin site. Schematic diagrams of an exemplary canine preproinsulin protein (cINS-2-1) containing a furin site modification and serving as a control are shown. [Diagram 2]Figure 1 shows the in vitro insulin bioactivity of caINS-Alb and caINS-Tf compared to control insulin standard. The EC50 values ​​are listed in the table of graphs below. Ligand-induced activation of insulin receptor in response to increasing concentrations of purified cINS-Alb and cINS-Tf. Reference insulin standard was used as control. Relative potency is expressed as relative light units. [Figure 3A] Blood glucose levels of streptozotocin (STZ)-induced NOD-SCID mice administered AAV virions containing canine insulin analogs or PBS control. In vivo activity of AAV-cINS analogs (cINS-Alb and cINS-Tf) in NOD-SCID diabetic mice was observed. After intramuscular (IM) administration of AAV candidates on day 0, fasting blood glucose was monitored for the duration of the study. Data shown are the mean and standard deviation (SD) of the cohort (n=7-10 / group). [Figure 3B] Figure 1 shows the body weight of STZ NOD-SCID mice administered AAV virions containing canine insulin analogs or PBS control. The in vivo activity of AAV-cINS analogs (cINS-Alb and cINS-Tf) in NOD-SCID diabetic mice was observed. After intramuscular (IM) administration of AAV candidates on day 0, body weight was monitored for the duration of the study. Data shown are the mean and standard deviation (SD) of the cohort (n=7-10 / group). [Figure 3C] Figure 1 shows ex vivo serum insulin activity of STZ NOD-SCID mice administered AAV virions containing canine insulin analogs or PBS control. The in vivo activity of AAV-cINS analogs (cINS-Alb and cINS-Tf) in NOD-SCID diabetic mice was observed. After intramuscular (IM) administration of AAV candidates on day 0, serum samples were taken on day 28 and assayed for insulin bioactivity. Relative insulin activity is expressed as relative light units. Data shown are the average and spread of individual mice. Statistical differences between control cINS-2-1 and cINS-Alb at both time points are shown (analyzed by one-way ANOVA). [Figure 3D]Figure 1 shows ex vivo serum insulin activity of STZ NOD-SCID mice administered AAV virions containing canine insulin analogs or PBS control. The in vivo activity of AAV-cINS analogs (cINS-Alb and cINS-Tf) in NOD-SCID diabetic mice was observed. After intramuscular (IM) administration of AAV candidates on day 0, serum samples were taken on day 56 and assayed for insulin bioactivity. Relative insulin activity is expressed as relative light units. Data shown are the average and spread of individual mice. Statistical differences between control cINS-2-1 and cINS-Alb at both time points are shown (analyzed by one-way ANOVA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] As described elsewhere herein, the present disclosure is based, at least in part, on the inventors' surprising discovery that virions encoding insulin fusion proteins achieve sustained expression of insulin in dogs and cats. Methods of making and using such virions are also provided.

[0018] Insulin fusion proteins engineered to overcome the short half-life of the native hormone by fusion to a protein with a longer half-life (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 canine and feline animals. The expression constructs contain a secretory signal peptide and a fusion domain intended to extend the circulation time of the resulting fusion protein.

[0019] The expression constructs are delivered to a subject in need thereof via transduction of a viral particle, such as an AAV particle, and in vivo expression of the encoded insulin fusion protein. Methods of using these constructs in regimens for treating type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), or metabolic syndrome in veterinary subjects, and methods of increasing the half-life of insulin in a subject are also provided.

[0020] The present invention encompasses insulin-albumin fusion proteins comprising a therapeutic protein with insulin activity. The present invention 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 invention also encompasses polynucleotides comprising a nucleic acid molecule encoding a protein comprising a therapeutic protein with insulin activity fused to albumin or a fragment (portion) or variant of albumin sufficient to prolong insulin activity in vivo.

[0021] 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 a protein other than canine or feline insulin.

[0022] In one embodiment, the leader is a canine IL-2 sequence. In one embodiment, the IL-2 leader is at least 80%, at least 85%, at least 90% identical to SEQ ID NO:12. %, at least 95%, at least 99%, or 100% identity. SEQ ID NO:12: MYKMQLLSCIALTLVLVANS

[0023] 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 to SEQ ID NO:7. SEQ ID NO: 7: MALWMRLLPLLALLALWAPAPTRA

[0024] 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. SEQ ID NO: 13: MYKIQLLSCIALTLILVTNS

[0025] 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 to SEQ ID NO:9. SEQ ID NO: 9: MAPWTRLLPLLALLSLWIPAPTRA

[0026] The leader sequence may be derived from the same species as that for which administration is ultimately intended, i.e., canine or feline animals. As used herein, the term "derived" or "derived from" means that the sequence or protein is sourced from or shares the same sequence as a protein or sequence sourced from a particular 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, the specified 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 nucleic acid or amino acid sequences. 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.

[0027] Insulin Insulin is involved in regulating glucose utilization in the body. The inability of the body to synthesize insulin or cells that are resistant to insulin leads to diabetes, characterized by chronic hyperglycemia. Preproinsulin is transcribed as a 110 amino acid chain with an N-terminal signal peptide. 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 a biologically active insulin molecule that is less than half the size of the original translation product and contains 51 amino acids.

[0028] Unless otherwise specified, the term "insulin" refers to biologically active insulin molecules or functional fragments thereof, and amino acid sequence variants of these molecules that provide the desired insulin-like activity. 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, (b) a proinsulin polypeptide, (c) an optional linker, and (d) an optional fusion partner.

[0029] 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. In another embodiment, the protein comprises a feline IL2 signal peptide and feline proinsulin. In another embodiment, the protein comprises a feline insulin signal peptide and feline proinsulin. The amino acid sequence of native feline proinsulin is set forth in SEQ ID NO: 24.

[0030] In some embodiments, the canine or feline insulin comprises a variant that may include up to about 10% variation from the insulin nucleic acid or amino acid sequence described herein or known in the art that retains the function of the wild-type sequence. As used herein, "retains function" means that the nucleic acid or amino acid functions in the same manner as 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 an increase or decrease in expression or activity of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the wild-type sequence [SEQ ID NO: 24].

[0031] The canine proinsulin sequence, in one embodiment, contains one or more mutations compared to the native sequence. These mutations are in some embodiments 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 K29R mutation. In another embodiment, the proinsulin sequence has a R31K mutation. In another embodiment, the proinsulin sequence has a L62R mutation. In another embodiment, the proinsulin sequence has both K29R and R31K mutations. In another embodiment, the proinsulin sequence has both K29R and L62R mutations. In another embodiment, the proinsulin sequence has both R31K and L62R mutations. In another embodiment, the proinsulin sequence has K29R, R31K, and L62R mutations.

[0032] In one embodiment the canine proinsulin sequence comprises 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: 1, 3, 10 or 14.

[0033] In one embodiment, the feline proinsulin sequence comprises 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: 15, 25, or 33.

[0034] 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 additionally, web-based or commercially available computer programs and service-based companies can be used to reverse translate the amino acid sequence into a nucleic acid coding sequence, including both RNA and / or cDNA. See, for example, backtranseq by EMBOSS, Gene Infinity, 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.

[0035] Fusion domains The present disclosure provides a fusion protein comprising a fusion domain. By fusing to a fusion domain with reduced half-life, insulin fusion proteins overcome 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.

[0036] 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.

[0037] 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 to SEQ ID NO:16.

[0038] 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.

[0039] 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 to SEQ ID NO:18.

[0040] Insulin Fusion Proteins The present disclosure provides fusion proteins comprising one or more copies of proinsulin, as well as polynucleotides and 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.

[0041] In some embodiments, the fusion protein comprises a canine insulin leader sequence, canine proinsulin (K29R, R31K, and L62R, with respect to the numbering of SEQ ID NO: 10), a glycine / serine linker, and canine serum albumin. In embodiments, the fusion protein comprises 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:1.

[0042] In some embodiments, the fusion protein comprises a canine insulin leader sequence, canine proinsulin (K29R, R31K, and L62R, with respect to the numbering of SEQ ID NO: 10), a glycine / serine linker, and canine transferrin. In embodiments, the fusion protein comprises 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:3.

[0043] In some embodiments, the fusion protein comprises a canine insulin leader sequence and canine proinsulin (K29R, R31K, and L62R, with respect to the numbering of SEQ ID NO:10). In embodiments, the fusion protein comprises 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:5.

[0044] In some embodiments, the fusion protein comprises a feline insulin leader sequence, feline proinsulin (K29R, R31K, and L62R, with respect to the numbering of SEQ ID NO: 24), a glycine / serine linker, and feline serum albumin. In embodiments, the fusion protein comprises 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: 24 or 25.

[0045] 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.

[0046] 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.

[0047] In addition to the leader sequences, proinsulin, and insulin polypeptides provided herein, nucleic acid sequences (used interchangeably with "polynucleotide") 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 sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:2.

[0048] In some embodiments, the nucleic acid sequence encoding the canine proinsulin-transferrin fusion comprises 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:4.

[0049] The in vivo function and stability of the fusion proteins of the present disclosure may be optimized by adding a small amount of peptide linker, for example, to prevent potentially undesirable domain interactions or for other reasons. Furthermore, the 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 comprises 1, 2, 3... or n repeats of a G-rich peptide linker having 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 GGGGSGGGGSGGGGS (SEQ ID NO: 19). In one embodiment, the linker comprises repeats of a G-rich peptide linker having the sequence GGGGSGGGGSGGGS (SEQ ID NO: 8).

[0050] Vector genome The vector genome may comprise a monocistronic or polycistronic transcript. In some embodiments, the viral particle of the present disclosure comprises, from 5' to 3', a polynucleotide sequence encoding: (a) Cytomegalovirus (CMV) enhancer and chicken b-actin promoter CB7 promoter elements, including the ter (b) canine insulin signal peptide, (c) canine proinsulin (K29R, R31K, and L62R); (d) a Gly / Ser linker, and (e) Canine serum albumin.

[0051] In certain embodiments, the recombinant AAV vector (particle) comprises an AAV capsid and a vector genome comprising a 5' AAV ITR, a promoter, an enhancer, an optional intron, an open reading frame that is a nucleic acid sequence encoding a fusion protein comprising canine insulin signal peptide, canine proinsulin, a linker, and canine serum albumin, polyA, and a 3' AAV ITR. In certain embodiments, the canine proinsulin is engineered canine proinsulin (K29R, R31K, and L62R).

[0052] In certain embodiments, the recombinant AAV vector (particle) comprises an AAV capsid and a vector genome comprising a 5' AAV ITR, a promoter, an enhancer, an optional intron, an open reading frame that is a nucleic acid sequence encoding a fusion protein comprising feline insulin signal peptide, feline proinsulin, a linker, and feline serum albumin, a polyA, and a 3' AAV ITR. In certain embodiments, the feline proinsulin is engineered feline proinsulin (K29R, R31K, and L62R).

[0053] In some embodiments, the viral particle 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 to SEQ ID NO: 26 (CB7.CI.cINS2-1_3xGSsp.ALB.rBG) or 28 (CB7.CI.cINS2-1_3xGSsp.Tf.rBG).

[0054] In certain embodiments, the vector genome of the production plasmid contains a shortened 5' and / or 3' AAV ITR of 130 base pairs, and the external A element is deleted. In certain embodiments, the shortened ITR is restored to the wild-type length of 145 base pairs during vector DNA amplification using the internal A element as a template. Thus, the final virus particles generated from the production plasmid containing the shortened ITR contain a full-length 145 ITR (by adding 15 nucleotides to the extreme 3' end and / or 15 nucleotides to the extreme 5' end of the vector genome). In other embodiments, full-length AAV 5' and 3' ITRs are used. The ITRs are selected such that they are transcomplemented by the rep protein expressed during production (e.g., in a transplasmid). If the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting rAAV may be referred to as pseudotyped. However, other configurations of these elements may also be suitable.

[0055] Adeno-associated virus (AAV) In one aspect, a virion is provided that comprises a polynucleotide encoding a leader sequence, proinsulin, and an insulin polypeptide as described herein. In certain embodiments of the virions described herein, the virion is an adeno-associated virus (AAV) or a recombinant AAV (rAAV). As used herein, the term "recombinant AAV" or "rAAV" refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to one of skill in the art and / or available in the context of the composition(s) and method(s) described herein, as well as artificial AAV. An adeno-associated virus (AAV) virion is an AAV DNase-resistant viral particle having an AAV protein capsid, in which: An expression cassette is packaged that is flanked by AAV inverted terminal repeats (ITRs) for delivery to target cells (together referred to as the "vector genome").

[0056] The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs can be selected as the source of capsids for the AAV viral particles identified above. In one embodiment, the AAV capsid is an AAVrh91 capsid or a variant thereof. See, for example, WO2020 / 223232 (for the AAVrh91 predicted amino acid sequence and deamidation pattern of assembled AAVrh91 capsids) and WO2022 / 036220. In certain embodiments, the capsid proteins are designated by numbers or combinations of numbers and letters following the term "AAV" in the name of the rAAV virion. Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, but not limited to, the AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh32.33, AAVAnc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu37, AAVrh64R1, and AAVhu68.

[0057] Exemplary AAV virions are described, for example, in U.S. Published Patent Application No. 2007-0036760-A1, U.S. Published Patent Application No. 2009-0197338-A1, EP1310571, WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449, U.S. Patent No. 7,282,199 (AAV8), WO2005 / 033321, US 7,906,111 (AAV9), WO2006 / 110689, WO2003 / 042397 (rh10), WO2005 / 033321, WO2018 / 160582 (AAVhu68), which are incorporated herein by reference.

[0058] Other suitable AAVs may include, but are not limited to, AAVrh90, AAVrh91, AAVrh92, AAVrh93, AAVrh91.93. Other suitable AAVs include the AAV3B variants described in International Patent Application No. PCT / US20 / 56511, filed October 20, 2020, including AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, or AAV3B.AR2.17, which are incorporated herein by reference. These documents also describe other AAV capsids that can be selected to generate rAAV, and are incorporated by reference.

[0059] As used herein, with respect to AAV, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including AAV sequences with conservative amino acid substitutions and AAV sequences that share at least 90%, at least 95%, at least 97%, at least 99% or more sequence identity across amino acid or nucleic acid sequences. In another embodiment, the AAV capsid includes a variant that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity to an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, The comparison can be made across any of the variable proteins (eg, vp1, vp2, or vp3).

[0060] In one embodiment, the virion is an rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the virion is an rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the virion is an rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the virion is an rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10. In one embodiment, the virion is an rAAV having a capsid of AAVhu68 or a functional variant thereof.

[0061] In certain embodiments, the viral vector has an AAVrh91 capsid. A nucleic acid sequence encoding the AAVrh91 capsid is provided in SEQ ID NO: 21, and the encoded amino acid sequence is provided in SEQ ID NO: 22. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 22). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91 (SEQ ID NO: 21). In yet another embodiment, a nucleic acid sequence encoding the AAVrh91 amino acid sequence is provided in SEQ ID NO: 22, and the encoded amino acid sequence is provided in SEQ ID NO: 22. Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVrh91eng (SEQ ID NO: 23). In certain embodiments, vp1, vp2, and / or vp3 are full-length capsid proteins of AAVrh91 (SEQ ID NO: 22). In other embodiments, vp1, vp2, and / or vp3 have an N-terminal and / or C-terminal truncation (e.g., a truncation of about 1 to about 10 amino acids).

[0062] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) a heterologous collection of AAVrh91 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 1-736 of SEQ ID NO:22, a vp1 protein produced from SEQ ID NO:21, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 1-736 of SEQ ID NO:22; a vp2 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138-736 of SEQ ID NO:22, a vp2 protein produced from a sequence that includes at least nucleotides 412-2208 of SEQ ID NO:21, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:22; a heterologous collection of vp2 proteins, a vp3 protein produced from expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:22, a vp3 protein produced from a sequence comprising at least nucleotides 607-2208 of SEQ ID NO:21, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:21 encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:22. and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:22, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:22, and a heterogeneous collection of vp3 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 203-736 of SEQ ID NO:22, wherein the vp1, vp2, and vp3 proteins are a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, a heterogeneous collection of vp3 proteins, each of which comprises a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in lysine pairs, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change, and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0063] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) a heterologous collection of AAVrh91 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 1-736 of SEQ ID NO:22, a vp1 protein produced from SEQ ID NO:21, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 1-736 of SEQ ID NO:22; a vp2 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138-736 of SEQ ID NO:22, a vp2 protein produced from a sequence that includes at least nucleotides 412-2208 of SEQ ID NO:21, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:22; a heterologous collection of vp2 proteins, a vp3 protein produced from expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:22, a vp3 protein produced from a sequence comprising at least nucleotides 607-2208 of SEQ ID NO:21, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:21 encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:22.and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:22, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:22, and a heterogeneous collection of vp3 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 203-736 of SEQ ID NO:22, wherein the vp1, vp2, and vp3 proteins are at least one of the asparagine-glycine pairs of SEQ ID NO:22. a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, a heterogeneous collection of vp3 proteins, each of which comprises a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N), and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change, and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0064] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh91 comprise a subpopulation with an amino acid modification comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:22, and optionally further comprising a subpopulation comprising other deamidated amino acids, where the deamidation results in an amino acid change. High levels of deamidation are observed at NG pairs N57, N383, and / or N512, relative to the numbers in SEQ ID NO:22. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 may have other residues that are deamidated, e.g., typically less than 10%, and / or are phosphorylated (e.g., in the range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%, if present) (e.g., at S149), or oxidized (e.g., at about W22, about M211, W247, M403, M435, M471, W478, W 503, about M537, about M541, about M559, about M599, M635, and / or W695. Optionally, W can be oxidized to kynurenine. [Table 1]

[0065] In certain embodiments, the AAVrh91 capsid is modified at one or more of the positions specified in the previous table to the extent provided, as determined using mass spectrometry with trypsin enzyme.In certain embodiments, one or more of the positions, or the glycine following N, is modified as described herein.The residue number is based on the AAVrh91 sequence provided herein.See SEQ ID NO:22.

[0066] In certain embodiments, the AAVrh91 capsid comprises a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 138-736 of SEQ ID NO:22, and a heterogeneous collection of vp3 proteins that are the product of a nucleic acid sequence encoding at least about amino acids 203-736 of SEQ ID NO:22.

[0067] In certain embodiments, modified AAVrh91 nucleic acid sequences can be used to generate mutant rAAVs having capsids that contain less deamidation than the native AAVrh91 capsid. Such mutant rAAVs can have reduced immunogenicity and / or increased stability upon storage, particularly in suspension form.

[0068] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following: The AAV hu68 capsid protein may be an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:37, a vp1 protein produced from SEQ ID NO:35 or 36, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:35 or 36, which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO:37; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:37, a vp2 protein produced from a sequence that includes at least nucleotides 412 to 2211 of SEQ ID NO:35 or 36, or a vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:35 or 36, which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:37, and / or an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:37. vp3 protein, a small amount of SEQ ID NO: 35 or 36 These include vp3 proteins produced from a sequence including at least nucleotides 607 to 2211, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO: 35 or 36, which encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO: 37.

[0069] Additionally or alternatively, AAV capsids are provided that optionally comprise a heterogeneous population of vp1 proteins that comprise a valine at position 157, optionally a heterogeneous population of vp2 proteins that comprise a valine at position 157, and a heterogeneous population of vp3 proteins, where at least a subpopulation of vp1 and vp2 proteins comprise a valine at position 157 and optionally further comprise a glutamic acid at position 67 based on the numbering of the vp1 capsid of SEQ ID NO: 37. Additionally or alternatively, AAVhu68 capsids are provided that comprise a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 37, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO: 37, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO: 37, where the vp1, vp2, and vp3 proteins include subpopulations with amino acid modifications.

[0070] The AAVhu68 vp1, vp2, and vp3 proteins are typically expressed as alternative splice variants encoded by the same nucleic acid sequence that encodes the full-length vp1 amino acid sequence (amino acids 1-736) of SEQ ID NO: 37. Optionally, the vp1 coding sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 37 (about aa203-736) without the vp1 unique region (about aa1 to about aa137) and / or the vp2 unique region (about aa1 to about aa202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 35 or 36), or a sequence at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 35 or 36 encoding aa203-736 of SEQ ID NO: 37. Additionally or alternatively, the vp1 and / or vp2 coding sequences can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 37 (about aa 1 to about 137) without the vp1 unique region (about aa 1 to about 137), or a complementary strand thereto, the corresponding mRNA or tRNA (e.g., nt 412 to 2212 of SEQ ID NO: 35 or 36), or a sequence that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 35 or 36, which encodes about aa 138 to 736 of SEQ ID NO: 37.

[0071] As described herein, rAAVhu68 has rAAVhu68 capsids produced in a production system that expresses capsids from an AAVhu68 nucleic acid encoding the vp1 amino acid sequence of SEQ ID NO:37, and optionally, an additional nucleic acid sequence encoding, for example, a vp3 protein that does not include the vp1 and / or vp2 unique regions. rAAVhu68 resulting from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1, vp2, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO:37. These subpopulations contain, at a minimum, deamidated asparagine (N or Asn) residues. For example, the asparagine in an asparagine-glycine pair is highly deamidated.

[0072] In one embodiment, the AAVhu68 vp1 nucleic acid sequence is the sequence of SEQ ID NO: 35 or 36. AAVhu68 vp3 amino acid sequence of SEQ ID NO: 37 (about aa 203-736) is also provided that does not include the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 35 or 36). In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 37 (about aa 138-736), without the vp1 unique region (about aa 1 to about 137), or the complementary strand thereto, the corresponding mRNA or tRNA (nt 412-2211 of SEQ ID NO: 35 or 36), is also provided.

[0073] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 37 may be selected for use in producing rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO: 35 or 36, or SEQ ID NO: 35 or 36 encoding SEQ ID NO: 37. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO: 35 or 36, or a sequence from about nt 412 to about nt 2211 of SEQ ID NO: 35 or 36, and encodes the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 37. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence from about nt 607 to about nt 2211 of SEQ ID NO: 35 or 36, or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to nt SEQ ID NO: 35 or 36 and encodes the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO: 37. [Table 2-1] [Table 2-2]

[0074] In certain embodiments, an AAVhu68 capsid is characterized in having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 37. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues are deamidated at one or more of these NG positions (i.e., N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 37). In these and other embodiments, the AAVhu68 capsid is further characterized in that it has a population of proteins in which about 1% to about 20% of N residues have deamidation at one or more of positions N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO:37. In certain embodiments, AAVhu68 comprises a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or combinations thereof, based on the numbering of the amino acid sequence of SEQ ID NO: 37. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0075] In another embodiment, a recombinant adeno-associated virus (rAAV) having an AAVhu68 capsid and a vector genome is provided, wherein (a) the AAV hu68 capsid comprises a heterogeneous population of AAVhu68 vp1 proteins, a heterogeneous population of AAVhu68 vp2 proteins, and a heterogeneous population of AAVhu68 vp3 proteins, and wherein the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 the vp3 protein comprises a subpopulation having amino acid modifications comprising 50% to 100% deamidation of at least two asparagines (N) in asparagine-glycine pairs at two or more of N57, N329, N452, N512 of SEQ ID NO: 37, as determined using mass spectrometry, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change where the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof; and the AAVhu68 capsid comprises (a) at least 65% of the asparagine (N) in the asparagine-glycine pair located at position N57 of the vp1 protein, based on the numbering of SEQ ID NO:37, is deamidated; (b) at least 75% of the N in the asparagine-glycine pair at position N329 of the vp1, v2, and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:37; (c) at least 50% of the N in the asparagine-glycine pair at position N452 of the vp1, v2, and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO: 37; and / or (d) at least 75% of the N in the asparagine-glycine pair at position N512 of the vp1, v2, and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:37;

[0076] The vector genome further comprises an AAVhu68 capsid, the vector genome comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a PTH fusion described herein operably linked to a sequence that directs expression of the PTH fusion in a target cell.

[0077] In one aspect, a recombinant AAV (rAAV) is provided. The present invention relates to an AAV capsid derived from Canine rh91, and a vector genome packaged in the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for the canine proinsulin serum albumin fusion of SEQ ID NO:1, and regulatory sequences directing expression of the canine proinsulin fusion.

[0078] In another embodiment, the rAAV comprises an AAV capsid derived from the adeno-associated virus rh91 and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for the canine proinsulin-transferrin fusion of SEQ ID NO:3, and regulatory sequences that direct expression of the canine proinsulin fusion.

[0079] In another embodiment, the rAAV comprises an AAV capsid derived from the adeno-associated virus rh91 and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for a feline proinsulin-serum albumin fusion of SEQ ID NO:32, and regulatory sequences directing expression of the feline proinsulin fusion.

[0080] In one aspect, a recombinant AAV (rAAV) is provided that comprises an AAV capsid derived from the adeno-associated virus hu68 and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for a canine proinsulin serum albumin fusion of SEQ ID NO:1, and regulatory sequences that direct expression of the canine proinsulin fusion.

[0081] In another embodiment, the rAAV comprises an AAV capsid derived from the adeno-associated virus hu68 and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for the canine proinsulin-transferrin fusion of SEQ ID NO:3, and regulatory sequences that direct expression of the canine proinsulin fusion.

[0082] In another embodiment, the rAAV comprises an AAV capsid derived from the adeno-associated virus hu68 and a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for a feline proinsulin-serum albumin fusion of SEQ ID NO:32, and regulatory sequences directing expression of the feline proinsulin fusion.

[0083] 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, etc., which introduces the proinsulin fusion sequence carried thereon into a host cell, e.g., for generating nanoparticles carrying DNA or RNA, 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).

[0084] In some embodiments, the proinsulin fusion constructs described herein may be delivered via virions other than rAAV. Such other virions may include any virus suitable for gene therapy that may be used, including, but not limited to, adenovirus, herpesvirus, lentivirus, retrovirus, etc. Preferably, when one of these other virions is produced, it is produced as a replication-deficient virion.

[0085] "Replication-defective virus" or "virion" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, and any viral genomic sequences packaged within the viral capsid or envelope are also replication-defective, i.e., they are unable to generate progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the virion does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless", containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0086] Expression cassette In some embodiments, 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. An expression cassette may be engineered into a genetic element and / or packaged into a virion capsid (e.g., a viral particle). Typically, such an expression cassette for producing virions contains a proinsulin fusion construct sequence as described herein adjacent to a packaging signal of the viral genome, and other expression control sequences, such as those described herein. Any of the expression control sequences may be optimized for a particular species using techniques known in the art, including codon optimization, for example, as described herein.

[0087] Expression cassettes typically contain 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 may be used. CB7 is a chicken B-actin promoter with a cytomegalovirus enhancer element. Alternatively, liver-specific promoters may be used, including, but not limited to, alpha 1 antitrypsin (A1AT), human albumin (Miyatake et al., J. Virol. 71:5124 32 (1997)) [humAlb], Hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002 9 (1996)), TTR minimal enhancer / promoter, alpha-antitrypsin promoter, or liver-specific promoter (LSP) (Wu et al. Mol Ther. 16:280-289 (2008)). In one embodiment, the liver-specific promoter thyroxine-binding globulin (TBG) is used. Other promoters may be used in the vectors described herein, such as viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), or promoters that respond to physiological cues.

[0088] In one embodiment, the expression cassette comprises a CB7 promoter, a chicken beta actin intron, a canine mutant pre-insulin with a canine insulin leader fused to feline albumin via a linker, and a rabbit beta globin polynucleotide, e.g., as provided in SEQ ID NO: 26. SEQ ID NO: 27 illustrates a vector genome comprising a truncated AAV 5'ITR, a spacer sequence, SEQ ID NO: 27, a spacer sequence, and a truncated AAV 3'ITR. Shows.

[0089] In another embodiment, the expression cassette comprises a CB7 promoter, a chicken beta actin intron, a canine mutant pre-insulin with a canine insulin leader fused via a linker to feline transferrin, and a rabbit beta globin poly, e.g., as provided in SEQ ID NO: 28. SEQ ID NO: 29 shows a vector genome comprising a truncated AAV 5'ITR, a spacer sequence, SEQ ID NO: 28, a spacer sequence, and a truncated AAV 3'ITR.

[0090] In another embodiment, an inducible promoter is used. Examples of inducible promoters useful herein include those described in "Canine and Feline Inducible Expression Constructs for Examples include those described in International Patent Application No. PCT / US2021 / 043219, entitled "Inducible Gene Therapy Applications," which is incorporated herein by reference. Briefly, the inducible promoter comprises a promoter, an activation domain comprising a canine or feline transactivation domain and an FKBP12-rapamycin binding (FRB) domain of a canine or feline FKBP12-rapamycin related protein (FRAP), a DNA binding domain comprising a zinc finger homeodomain (ZFHD) and one, two, or three FK506 binding protein domain (FKBP) subunit genes, and at least eight copies of binding sites for ZFHD (8XZFHD), followed by a minimal IL2 promoter. The presence of an effective amount of rapamycin or a rapalog induces expression of the transgene in the host cell.

[0091] In addition to the 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, optionally, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, for example, rabbit beta globin, SV40, bovine growth hormone (bGH), and TK polyA.

[0092] Examples of suitable enhancers include, for example, alpha fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha 1-microglobulin / bikunin enhancer), among others. In one embodiment, the polyA is rabbit globin polyA. In one embodiment, the polyA has the sequence of SEQ ID NO:20.

[0093] 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 with a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0094] In one embodiment, an rAAV is provided that includes a 5'ITR, a CB7 promoter, a chicken beta-actin intron, a coding sequence for a protein of SEQ ID NO:1, a rabbit globin polyA, and a 3'ITR. In one embodiment, an rAAV is provided that includes a 5'ITR, a CB7 promoter, a chicken beta-actin intron, a coding sequence for a protein of SEQ ID NO:3, a rabbit globin polyA, and a 3'ITR. In one embodiment, an rAAV is provided that includes a 5'ITR, a CB7 promoter, a chicken beta-actin intron, a coding sequence for a protein of SEQ ID NO:3, a rabbit globin polyA, and a 3'ITR.

[0095] The minimal sequences required for packaging an expression cassette into an AAV viral particle are the AAV 5' and 3' ITRs, which can be of the same AAV origin as the capsid or of a different AAV origin (to produce AAV pseudotypes).

[0096] The ITRs are the only AAV components required in cis in the same construct as the genes for packaging the expression cassette into the AAV capsid to form virions (i.e., rAAV vectors or rAAV particles). In one embodiment, the coding sequences for replication (rep) and / or capsid (cap) are removed from the AAV genome and supplied in trans or by a packaging cell line to generate AAV virions. For example, as described above, pseudotyped AAV may include ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid may be utilized. Still other AAV components may be selected. Sources of such AAV sequences are described herein and may be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). AAV sequences may be obtained via synthesis or other suitable means, for example, by reference to published sequences as available in the literature or databases such as GenBank, PubMed, etc.

[0097] Methods for producing and isolating AAV virions suitable for delivery to a subject are known in the art.See, for example, US Patent No. 7,790,449, US Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7,588,772.In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap.In the second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs.In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, and rAAV needs to be separated from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to restore AAV, and the necessary helper functions (i.e., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied by the system in trans. In these systems, the helper functions can be supplied by transient transfection of cells with constructs encoding the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In another system, the transgene flanked by ITRs and the rep / cap genes are introduced into insect cells by infection with a baculovirus-based expression vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties.Methods of making and using these and other AAV production systems are also described in the following United States patents, the contents of each of which are incorporated herein by reference in their entirety: US 5,139,941, US 5,741,683, US 6,057,152, US 6,204,059, US 6,268,213, US 6,491,907, US 6,660,514, US 6,951,753, US 7,094,604, US 7,172,893, US 7,201,898, US 7,229,823, and US 7,439,065. See generally, e.g., Grieger & Samulski, 2005, Adv. Bi. ochem.Engin / Biotechnol.99:119-145, Buning et al., 2008, J. Gene Med. 10:717-733, and the references cited below, each of which is incorporated herein by reference in its entirety. The methods used to construct any embodiment of the present invention are known to those of skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques.

[0098] Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limitation of the present invention, see, e.g., K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0099] The rAAV described herein comprises a selected capsid having a vector genome packaged inside. The vector genome (or rAAV genome) comprises 5' and 3' AAV inverted terminal repeats (ITRs), a polynucleotide sequence encoding an insulin protein, and regulatory sequences that direct insertion of the polynucleotide sequence encoding the insulin protein into the genome of a host cell.

[0100] Methods of Treating Subjects with the Disclosed Compositions Compositions comprising the virion constructs described herein are also provided. The pharmaceutical compositions described herein are designed to be delivered to a canine or feline subject in need thereof by any suitable route or combination of different routes. Direct delivery to the liver (optionally via intravenous, via 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 virions described herein can be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, can be delivered [see, for example, WO2011 / 126808 and WO2013 / 049493].

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

[0102] In another embodiment, the multiple viruses can include different replication-deficient viruses (e.g., AAV and adenovirus). In one embodiment, administration is intramuscular. In another embodiment, administration is intravenous.

[0103] In some embodiments, a course of treatment may include repeated administration of the same virion (e.g., AAVrh91 virion) or different virions (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the virions described herein. In some embodiments, the compositions described herein may be combined in regimens that include other diabetes drugs or protein-based therapies (e.g., insulin analogs, insulin, oral antihyperglycemic agents, including sulfonylureas, biguanides, thiazolidinediones, and alpha-glucosidase inhibitors). In some embodiments, the compositions described herein may be combined in regimens that involve lifestyle changes, including dietary 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 may be used to refer to a nucleic acid sequence encoding a proinsulin fusion protein or its expression product. A proinsulin sequence with a leader is also referred to herein as a "preproinsulin". In certain embodiments, "Proinsulin constructs" can also include fusion proteins as described herein.

[0105] Replication-defective viruses can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications. In the case of AAV virions, quantification of genome copies ("GC" or "gc") can be used as a measure of the dose contained in the formulation. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective virus composition of the present invention. One method for performing titration of AAV GC number is as follows: A purified AAV vector genome sample is first treated with DNase to eliminate non-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The nuclease-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set that targets a specific region of the viral genome (usually the polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), particularly optimized qPCR or digital droplet PCR.

[0106] In addition, the replication-deficient virus composition contains approximately 1.0×10 9 GC~approx. 1.0×10 15The virus can be formulated into a dosage unit containing an amount of the replication-defective virus in the range of GC. In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, the dose is about 1.0×10 for an average canine or feline subject of about 5-10 kg. 10 GC~approx.3.0×10 13 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC, 1×10 11 GC, approx. 5×10 11 GC, approx. 1×10 12 GC, approx. 5×10 12 GC, or approximately 1 × 10 13 In another embodiment, the dosage is about 1.0×10 9 GC / kg ~ approx. 3.0×10 13 In another embodiment, the dose is about 1×10 9 In another embodiment, the dose is about 1×10 10 In another embodiment, the dose is about 1×10 11 For example, the dose of AAV virus is about 1×10 10 GC / kg, 1×10 11 GC / kg, approx. 5×10 11 GC / kg, approx. 1×10 12 GC / kg, approx. 5×10 12 GC / kg, or approximately 1 x 10 13 It can be GC / kg.

[0107] In another aspect, a method for sustained expression 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 virions comprising a proinsulin-serum albumin fusion protein expression cassette described herein. In some embodiments, the method provided herein results in expression of a proinsulin fusion protein in the subject for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, or at least 60 weeks. In some embodiments, the method provided herein results in expression of a proinsulin fusion protein in the subject for at least 8 weeks. In some embodiments, the method results in expression of a proinsulin fusion protein in the subject at therapeutically effective concentrations for at least 1 month, at least 3 months, at least 6 months, or at least 12 months.

[0108] In one embodiment, the constructs may be delivered to a veterinary subject in a volume of 1 μL to about 100 mL. For a discussion of good practices for administering substances to various veterinary animals, see, e.g., Diehl et al, J. Applied Toxicology, 21:15-23 (2001), which is incorporated herein by reference. As used herein, the term "dosage" may refer to the total dose delivered to a subject over the course of treatment, or the amount delivered in a single dose (of multiple doses).

[0109] 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 rAAV and insulin described herein reduces insulin dosage requirements in a subject compared to before treatment with AAV virions. 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 may be being treated with insulin or other therapy that the treating physician may continue upon administration of the AAV virions. Such insulin or other combination therapy may then be continued, reduced, or discontinued, as necessary.

[0110] In one embodiment, the expression cassette, vector genome, rAAV-containing composition described herein for gene therapy, or other composition is delivered as a single dose per subject. 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 expression cassette or virion, or combination thereof, that delivers and expresses a sufficient amount of proinsulin serum albumin in target cells to achieve a therapeutic goal. In certain embodiments, the therapeutic goal is to alleviate or treat one or more symptoms of type I diabetes, type II diabetes, or metabolic syndrome. The therapeutically effective amount may be determined based on animal models other than canine or feline subjects. In another embodiment, the therapeutic goal is the remission of metabolic disease in the subject.

[0111] The above recombinant AAV virions can be delivered to host cells according to published methods. Preferably, rAAV 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 in view of the indication for which the introduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various 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 invention.

[0112] In some embodiments, the compositions of the invention may contain, in addition to the rAAV and / or variant and carrier(s), 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, glycerin, phenol, and parachlorophenol. Exemplary chemical stabilizers include gelatin and albumin.

[0113] The recombinant virions and constructs described herein can be used to deliver a proinsulin fusion protein construct to a subject in need thereof, to provide a subject with insulin having an increased half-life, and / or to prepare a medicament for treating type I diabetes, type II diabetes, or metabolic syndrome in a subject.

[0114] 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 virions comprising a proinsulin fusion protein expression cassette described herein.

[0115] In another embodiment, a method is provided for treating type 2 diabetes in a dog or cat, the method comprising administering a virion comprising a nucleic acid molecule comprising a sequence encoding a proinsulin fusion protein as described herein.

[0116] In another embodiment, a method is provided for treating type 1 diabetes in a dog or cat, the method comprising administering a virion comprising a nucleic acid molecule comprising a sequence encoding a proinsulin fusion protein as described herein.

[0117] 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 virion comprising a proinsulin fusion protein expression cassette 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.

[0118] In another embodiment, a method for treating diabetes in a dog or cat is provided. The method comprises administering a virion comprising a nucleic acid molecule comprising a sequence encoding a proinsulin-serum albumin fusion protein described herein, wherein the virion is administered after insulin is administered to the subject. In some embodiments, the virion is administered at least 1 day, at least 3 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks after insulin is administered to the subject.

[0119] In another embodiment, a method is provided for preventing cataract formation in a diabetic dog or cat. The method comprises administering a virion comprising a nucleic acid molecule comprising a sequence encoding a proinsulin-serum albumin fusion protein described herein. In some embodiments, the virion is administered after insulin has been administered to the subject.

[0120] In another embodiment, a method is provided for reducing blood glucose levels in a diabetic dog or cat, the method comprising administering a virion comprising a nucleic acid molecule comprising a sequence encoding a proinsulin-serum albumin fusion protein as described herein.

[0121] As used herein, the term "treatment" or "treating" is defined to include administering to a subject one or more compounds or compositions described herein for the purpose of alleviating one or more symptoms of type I diabetes, type II diabetes mellitus (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 condition, eliminating a symptom of the disease, slowing the progression of the disease, or increasing the effectiveness of a therapy in a given subject.

[0122] As used herein, the term "remission" refers to the ability to discontinue insulin treatment when the cat or dog no longer exhibits clinical signs of diabetes and has normal blood glucose levels.

[0123] In another embodiment, a method is provided for treating T2DM in a cat or dog, the method comprising administering a virion comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein.

[0124] In another aspect, a method of treating a metabolic disorder in a cat or dog is provided, the method comprising administering a composition described herein to a feline or canine subject in need thereof. In one embodiment, the composition comprises virions comprising a proinsulin fusion protein expression cassette described herein.

[0125] 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 virion comprising a proinsulin-serum albumin fusion protein expression cassette 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%.

[0126] 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 as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and it will be further understood that they should not be interpreted in an ideal or overly formal sense unless expressly defined herein. The terms used in the description are for the purpose of describing specific embodiments only 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 will control.

[0127] 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.

[0128] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, 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, 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 over a particular region (i.e., two or more sequences or subsequences that share at least about 80% identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity). Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. In some embodiments, 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.

[0129] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as 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 for the test sequence relative to the reference sequence based on the program parameters. In some embodiments, the BLAST and BLAST 2.0 algorithms and default parameters are used.

[0130] The terms "percent identity", "sequence identity", "percent sequence identity" or "percent identical" in the context of amino acid sequences refer to the residues in two sequences that are the same when aligned accordingly. Percent identity can be readily determined for the corresponding nucleic acid sequence encoding a full-length polypeptide of a protein, a polypeptide, about 70 amino acids to about 100 amino acids, or a peptide fragment thereof or sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and up to about 150 amino acids. Generally, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to the "aligned" sequences.

[0131] "Aligned" sequences or "alignment" refers to multiple nucleic acid sequences or protein (amino acid) sequences, often 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 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 same level of identity or alignment as that provided by the referenced algorithm and program.

[0132] 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 alternative 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 substituting 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, 1, 2, 3, 4, 5, 6 or more individual embodiments.

[0133] 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 International Patent Application Publication No. 2015 / 012924, which is incorporated herein by reference. Briefly, a nucleic acid sequence encoding a product is modified with synonymous codon sequences. Preferably, the entire length of the product's open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF may be altered. 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 encodes a polypeptide.

[0134] In embodiments, the insulin-serum albumin fusion is caninized or felinized.

[0135] "Caninized" means that the fusion protein comprises an amino acid sequence that is compatible with dogs such that the amino acid sequence is unlikely to be regarded as foreign by the immune system of a canine subject. In this disclosure, the term polypeptide preceded by the prefix "ca" refers to a variant of a human polypeptide in which the 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 a canine homolog of that fragment or variant.

[0136] "Fenitized" means that the fusion protein comprises a feline-compatible amino acid sequence such that the amino acid sequence is less likely to be considered foreign by the immune system of a feline subject. In the present disclosure, the term polypeptide preceded by the prefix "fe" refers to a variant of a human polypeptide 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, proinsulin is replaced with a feline homolog of that fragment or variant.

[0137] As noted elsewhere herein, the present disclosure extends to fusion proteins that are compatible with species other than dogs or cats, in this context the fusion protein may be referred to as "speciesized," referring to the target species to which the molecule is administered.

[0138] In some embodiments, the compositions and methods described herein are intended for use in feline animals. The term cat (feline) refers to any of the 37 species of cats, including cheetahs, pumas, jaguars, leopards, lions, lynxes, tigers, and domestic cats, among others. In an embodiment, the subject is a domestic cat. In some embodiments, the compositions and methods described herein are intended for use in canine animals. The term dog refers to any of the species found in the Canidae 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.

[0139] As used in describing the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0140] 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.

[0141] As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical ingredients listed in the method or composition, and may further include other agents that do not themselves have substantial activity for the listed indication or purpose.

[0142] The terms "comprise", "comprises", and "comprising" should be interpreted inclusively rather than exclusively. The terms "consist", "consisting", and variations thereof should be interpreted exclusively rather than inclusively. Although various embodiments herein are expressed using the word "comprising", it is also intended that under other circumstances the relevant embodiment should be interpreted and described using the word "consisting of" or "consisting essentially of".

[0143] As used herein, the term "about" means a 10% variability from a given reference, unless otherwise specified.

[0144] 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.

[0145] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal condition in a subject.

[0146] 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., adult male, adult female, adolescent male, adolescent female, male child, female child). 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.

[0147] The term "viral particle" or "virion" as used herein refers to a macromolecular complex capable of delivering a foreign nucleic acid molecule to a cell independent of another agent. An independent particle of a virus, a "viral particle" or "virion", consists of genetic material (i.e., DNA or RNA encoding a transgene) and a capsid. The particle can be a viral particle or a non-viral particle. Viral particles include retroviral particles, lentiviral particles, and adeno-associated viral particles. The term "adeno-associated viral particle" may be used interchangeably with "recombinant adeno-associated viral (rAAV) vector" to indicate the presence of a nuclease-resistant AAV capsid with a replication-defective vector genome packaged therein. Non-viral particles are limited to liposomes, nanoparticles, and other encapsulation systems for the delivery of polynucleotides to cells.

[0148] The term "transgene" refers to the transferred nucleic acid itself. A transgene can be a naked nucleic acid molecule (such as a plasmid) or RNA. A transgene can include a polynucleotide that encodes one or more polypeptides (e.g., an insulin fusion protein). A transgene can include a polynucleotide that encodes one or more heterologous proteins (e.g., an insulin fusion protein), one or more capsid proteins, and other proteins required for transduction of the polynucleotide into a target cell.

[0149] The term "transducing" refers to the introduction of a nucleic acid into a cell or host organism by a particle (e.g., an adeno-associated virus particle). Thus, the introduction of a transgene into a cell by a viral particle may be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it may be stably maintained in that cell. Alternatively, the introduced transgene may be present extrachromosomally or only transiently in the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a polynucleotide has been introduced.

[0150] As used herein, the term "host cell" may refer to a packaging cell line in which virions (e.g., recombinant AAV or rAAV) are produced from a production plasmid. Alternatively, the term "host cell" may refer to a cell line in which expression of the gene products described herein is desired. It may refer to any target cell. Thus, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial, human, or insect cell) that contains exogenous or heterologous DNA introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to cultures of cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to cells used to produce and package virions or recombinant viruses. In further embodiments, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, or a liver cell.

[0151] As used herein, the term "target cell" refers to any target cell in which expression of a heterologous nucleic acid sequence or protein is desired. In certain embodiments, the target cell is a liver cell. In some embodiments, the target cell is a muscle cell.

[0152] As used herein, an "expression cassette" or "vector genome" refers to a nucleic acid molecule that includes 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 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 contiguous or non-contiguous with 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, e.g., Woodchuck Hepatitis Virus (WHP) post-translational regulatory element (WPRE), and TATA signal. An expression cassette may contain, among other elements, regulatory sequences upstream (5') of a gene sequence, such as one or more of a promoter, enhancer, intron, etc., and enhancers, or gene sequences downstream (3') of the regulatory sequences, such as one or more 3' untranslated regions (3'UTRs) including polyadenylation sites. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of a 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, an expression cassette comprises one or more nucleic acid sequences of a gene product. In some embodiments, the expression cassette may be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell. Typically, such expression cassettes may be used to generate virions and include coding sequences of gene products described herein adjacent to a packaging signal of the viral genome, and other expression control sequences, such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

[0153] As used herein, "administering" refers to local and systemic administration, including, for example, enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration for pharmaceutical ingredients (e.g., virions) that find use in the methods described herein include, for example, oral (per os (PO)) administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT), intravenous ("iv"), intraperitoneal ("ip"), intramuscular ("im"), intralesional, or subcutaneous ("sc") administration, or implantation of a delayed release device, such as a mini-osmotic pump, 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, or other methods. These include intracranial and intracranial.

[0154] 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 via the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0155] The term "effective amount" or "pharmacologically effective amount" refers to the amount and / or dosage and / or dosage regimen of one or more pharmaceutical components (e.g., virions) necessary to produce a desired result.

[0156] As used herein, the terms "treating" and "treatment" refer to delaying the onset of, hindering or reversing the progression of, 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.

[0157] The term "alleviating" refers to the reduction or elimination of one or more symptoms of the condition or disease, and / or the slowing of the rate or delaying of the onset or severity of one or more symptoms of the condition or disease, and / or the prevention of the condition or disease. In some embodiments, the reduction or elimination of one or more symptoms of the condition or disease can include, for example, a measurable and sustained decrease in fasting blood glucose.

[0158] 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 considered as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0159] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0160] While exemplary 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 invention. EXAMPLES

[0161] 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 purely illustrative of the invention and are not intended to limit the scope of what is claimed.

[0162] 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 designed to be administered via AAV virions has been developed for the management of diabetic hyperglycemia and hyperglycemia-related clinical signs in dogs.

[0163] Canine preproinsulin-serum albumin fusion protein (cINS-Alb) was generated by constructing a fusion polypeptide containing the following elements ( FIG. 1A ) (SEQ ID NO:1): Natural canine insulin signal peptide (SP) Canine proinsulin with modifications to the native sequence at three amino acid positions (K29R, R31K, and L62R) and the incorporation of two furin cleavage sites into the existing protease cleavage sites (B-chain, C-peptide, A-chain) A 14 aa Gly / Ser linker containing the sequence GGGGSGGGGSGGGS (SEQ ID NO:8) ·Canine serum albumin

[0164] A canine preproinsulin fusion protein was made with 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). The expression cassette for the albumin fusion is provided in SEQ ID NO: 26, and the vector genome of the production plasmid is in SEQ ID NO: 27. A control canine preproinsulin sequence (no fusion) with three amino acid modifications (K29R, R31K, and L62R) was also produced in AAV virions as a control (cINS-2-1) (FIG. 1C) (SEQ ID NO: 5, the control expression cassette is in SEQ ID NO: 30, and the vector genome is in SEQ ID NO: 31). All vector genomes of the production plasmids contain shortened AAV 5' and 3' ITRs. SEQ ID NO: 28 provides the expression cassette for the canine proinsulin-transferrin fusion protein. The vector genome is provided in SEQ ID NO: 29.

[0165] Studies were conducted to evaluate the in vitro potency of the two fusion proteins (cINS-Alb and cINS-Tf), where C-terminal histidine-tagged versions of each of the proteins were produced in mammalian cells after transient transfection of expression plasmids containing the cDNAs encoding the respective proteins. 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 the shift in values.

[0166] Example 2: Generation of AAV viruses containing a canine insulin fusion protein transgene and evaluation of efficacy in vivo To generate AAV virions, codon-optimized transgenes encoding a control with three amino acid modifications without a fusion partner (AAVrh91.cIns.2-1) and two insulin fusion proteins (SEQ ID NOs: 1, 3, and 5) were cloned into a plasmid containing a CB7 promoter element (comprising a hybrid cytomegalovirus enhancer and chicken b-actin promoter). The expression constructs were flanked by truncated 130 bp 5' and 3' AAV inverted terminal repeats (ITRs) in the plasmid and packaged into AAV serotype rh91 capsid (AAVrh91) virions by triple transfection with a trans plasmid expressing the AAVrh91 capsid and a helper plasmid expressing the adenoviral helper functions necessary for replication and production that are not provided by HEK293 packaging cells expressing E1 adenoviral functions. During replication and packaging, the 5' and 3' ITRs revert to the full wild-type 145 bp ITR length. AAV virions were subsequently purified by iodixanol gradient purification titrated by Taqman™ quantitative PCR.

[0167] A study was conducted to evaluate the in vivo efficacy of three AAV-delivered canine insulin fusion proteins in a mouse model of diabetes. NOD-SCID mice treated with streptozotocin to induce diabetes were assigned to one of four groups, as shown in Table 1 below. A fifth group of non-diabetic NOD-SCID mice was included as a control. [Table 3]

[0168] Mice were administered virions (1 × 10 11Group 1 mice were administered 100 mg / kg / mouse (100 mg / kg / mouse). Food was removed from cages 6 hours before sampling for fasting blood glucose twice a week. Two mice from group 2 were euthanized due to hypoglycemia (days 45 and 52, respectively). One mouse from group 3 was euthanized on day 28 due to poor body condition score and high blood glucose. Fasting blood glucose was measured by glucometer with an upper limit of 600 mg / dL.

[0169] Animals receiving AAV virions containing the control cINS-2-1 (group 4) and cINS-Alb (group 2) transgenes had a significant reduction in blood glucose levels compared to control group 1 (Figure 3A). Mice receiving AAV containing the cINS-Alb fusion (group 2) showed a more pronounced and more rapid reduction in fasting blood glucose levels than AAV containing the control cINS-2-1 (group 4). By day 32, blood glucose levels in group 2 were normalized and were similar to those observed in the nondiabetic cohort of mice (group 5). In contrast to the significant reduction in blood glucose levels observed in animals receiving AAV containing the cINS-Alb fusion (group 2), no reduction in blood glucose was observed in the cohort of animals administered AAV containing the cINS-Tf fusion (group 3).

[0170] The changes in mouse body weight are shown in Figure 3B. Mice administered AAV containing the cINS-Alb fusion tended to gain more weight than diabetic animals in other groups, although this was not statistically significant.

[0171] To examine the relative biological potency of the expressed canine insulin proteins, serum from mice collected on days 28 and 56 was tested with the PathHunterO™ Insulin Bioassay Kit, as described above. As shown in FIG. 3C, on both days 28 and 56, animals administered AAV containing cINS-Alb fusions had significantly higher levels of circulating potent insulin than those in other cohorts. Surprisingly, while cINS-Tf protein showed moderate activity in in vitro assays, no activity or evidence of efficacy of insulin-transferrin fusion protein was observed in vivo. This data indicates that cINS-Alb showed higher biological activity than cINS-Tf, and AAV containing cINS-Alb fusions showed significantly higher in vivo efficacy than AAV containing cINS-Tf fusions.

[0172] Example 3: Evaluation of AAV-caINS-Alb in healthy canine subjects A study will be conducted to evaluate expression, immunogenicity, and effects on blood glucose following administration of AAVrh91 virions expressing caINS-Alb in healthy dogs. Two groups of healthy dogs weighing approximately 10 kg to 15 kg will be administered 1 × 10 11 Or 1×10 12 An intramuscular dose of AAV-caINS-Alb is administered at a dose of 1000 GC / kg. Prior to administration of AAV-caINS-Alb, a series of health screening measurements are performed, including a veterinary examination, serum fructosamine analysis, and hematology, clinical chemistry, and urinalysis. Serum samples are then collected weekly for up to 8 weeks to determine the levels of circulating caINS-Alb protein using a sandwich ELISA protocol, as described below.

[0173] Potential immunogenicity to caINS-Alb is determined by measuring anti-caINS-Alb antibodies in a bridging assay. Blood glucose levels are measured twice daily, and interstitial glucose concentrations are recorded at regular intervals using a continuous glucose monitoring system (FreeStyle Libre™, Abbott). Animals are observed daily for signs of anaphylaxis, lethargy, and hypoglycemia.

[0174] To measure serum concentrations of caINS-Alb, a sandwich ELISA is used. ELISA plates are coated with 0.2 μg / ml mouse anti-human insulin (clone 7F8, Life Technologies) and blocked with PBS / 0.05% Tween 20 / 1% casein. Coated wells are incubated for 1 h at room temperature with 100 μL of appropriately diluted serum samples diluted in PBS / 0.05% Tween 20 / 1% casein. A standard curve is established using purified caINS-Alb standards ranging from 200 ng / ml to 0.19 ng / ml. After washing, plates are incubated for 1 h with a 1 / 2000 dilution of goat anti-canine albumin (Bethyl Laboratories) in PBS / 0.05% Tween 20 / 1% casein. Plates are then washed 5 times with PBS / 0.05% Tween 20 and developed by addition of tetramethylbenzidine (TMB) substrate. Development is stopped by addition of 2M H2SO4 and absorbance is read at 450 nm.

[0175] Example 4: Evaluation of the efficacy of AAV-caINS-Alb in a canine streptozotocin (STZ)-induced diabetes model The efficacy of a single administration of AAVrh91 virions expressing caINS-Alb to adequately maintain blood glucose levels in diabetic dogs is evaluated in a non-randomized, open-label, laboratory study of six Beagle dogs with chemically induced diabetes receiving a stable diabetic treatment regimen.

[0176] Low dose (1×1011 gc / kg, N = 3) of AAVrh91 virions expressing caINS-Alb, and a high dose (1 × 10 12 AAVrh91 virions expressing caINS-Alb will be evaluated at 100 mg / kg (N=3). Prior to administration of AAV-caINS-Alb, a battery of health screening measurements will be performed, including a veterinary exam, serum fructosamine analysis, and hematology, clinical chemistry, and urinalysis. Three times daily blood glucose measurements via glucometer and twice daily clinical observations will be performed throughout the study. On day -1, subjects will be acclimated to placement of a continuous glucose monitoring device and interstitial glucose concentrations will be collected three times daily.

[0177] Over the course of the study, in addition to daily procedures beginning at baseline, blood draws for fructosamine analysis, hematology, and clinical chemistry will be performed every 14 days until day 42 and again on day 63. Circulating caINS-Alb protein and anti-caINS-Alb Blood samples for antibody determination will be collected periodically throughout the study and body weights will be measured every 7 days. Required replacement insulin dose levels, weight gain, and glycemic control will be used to assess efficacy of the test product at the two doses tested in individual subjects.

[0178] Example 5: Evaluation of the efficacy of AAV-caINS-Alb in a canine diabetes model A study will be conducted to evaluate the efficacy of a single administration of AAVrh91 virions expressing caINS-Alb to adequately maintain blood glucose levels in diabetic dogs.

[0179] Dogs previously diagnosed with diabetes, on stable insulin therapy, and with moderate to good glycemic control are treated with either placebo or AAV-caINS-Alb (intramuscular administration). Dogs newly diagnosed with diabetes are treated with either placebo or AAV-caINS-Alb (intramuscular administration). Prior to administration of AAV-caINS-Alb, a series of health screening measurements are performed, including a veterinary exam, serum fructosamine analysis, and hematology, clinical chemistry analysis, and urinalysis. Glycemic control is assessed prior to AAV administration and throughout the study by measuring interstitial glucose concentrations either continuously with a continuous glucose monitor or twice daily using a glucometer. Dogs are fed a constant diet and weight is monitored throughout the study. Over the course of the study, blood draws for fructosamine analysis, hematology, clinical chemistry, and circulating caINS-Alb protein are analyzed periodically, in addition to blood glucose monitoring. Serum levels of caINS-Alb are measured using a custom sandwich ELISA. Improvement in replacement insulin requirements, insulin dose, body weight, and glycemic control, as well as clinical signs, are used to assess the efficacy of the test product.

[0180] Example 6: Evaluation of the efficacy of AAV-feINS-Alb in a feline diabetes model A study will be conducted to evaluate the efficacy of a single administration of AAVrh91 virions expressing feINS-Alb to adequately maintain blood glucose levels in diabetic cats.

[0181] Cats previously diagnosed with diabetes, on stable insulin therapy, and with moderate to good glycemic control are treated with either placebo or AAV-feINS-Alb (administered intramuscularly). Cats newly diagnosed with diabetes are treated with either placebo or AAV-feINS-Alb (administered intramuscularly). Prior to administration of AAV-feINS-Alb, a series of health screening measurements are performed, including a veterinary exam, serum fructosamine analysis, and hematology, clinical chemistry analysis, and urinalysis. Glycemic control is assessed prior to administration and throughout the study by measuring interstitial glucose concentrations either continuously with a continuous glucose monitor or twice daily using a glucometer. Cats are fed a constant diet and weight monitored throughout the study. Over the course of the study, blood draws for fructosamine analysis, hematology, clinical chemistry, and circulating feINS-Alb protein are analyzed periodically, in addition to blood glucose monitoring. Serum levels of feINS-Alb are measured using a custom sandwich ELISA. Improvement in replacement insulin requirements, insulin dose, body weight, and glycemic control, as well as clinical signs and remission rates and times to remission are used to assess efficacy of the test product.

[0182] To measure serum concentrations of feINS-Alb, a custom sandwich ELISA is used as follows: ELISA plates are coated with 0.2 μg / ml mouse anti-human insulin (clone 7F8, Life Technologies) and blocked with PBS / 0.05% Tween 20 / 1% casein. Coated wells are incubated for 1 h at room temperature with 100 μL of appropriately diluted serum samples diluted in PBS / 0.05% Tween 20 / 1% casein. A standard curve is established using purified feINS-Alb standards ranging from 200ng / ml to 0.19ng / ml. After washing, the plates are incubated for 1 hour with a 1 / 2000 dilution of goat anti-feline albumin (Bethyl Laboratories) in PBS / 0.05% Tween 20 / 1% casein. The plates are then washed 5 times with PBS / 0.05% Tween 20 and developed by the addition of TMB substrate. Development is stopped by the addition of 2M H2SO4 and absorbance is read at 450nm.

[0183] All publications cited herein are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 315,252, filed March 1, 2022, is incorporated herein by reference in its entirety. Although the present invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A recombinant adeno-associated virus (rAAV) virion for the treatment of companion animals, comprising: an AAV capsid; and a vector genome comprising an expression cassette comprising a polynucleotide encoding a fusion protein comprising proinsulin and serum albumin, said expression cassette being flanked by a 5' inverted terminal repeat (ITR) and a 3' ITR, and said proinsulin being canine proinsulin or feline proinsulin.

2. the AAV capsid comprises an AAVrh91 capsid, and optionally 2. The rAAV virion of claim 1, wherein the AAVrh91 capsid comprises 60 AAVrh91 capsid proteins that comprise at least 99%, or at least 100%, identity to amino acids 203-736 of SEQ ID NO:

22.

3. The rAAV virion of claim 1, wherein the proinsulin is canine proinsulin, and optionally, the canine proinsulin sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

14.

4. The rAAV virion of claim 1, wherein the proinsulin is feline proinsulin, and optionally the feline proinsulin sequence shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:

15.

5. 3. The rAAV virion of claim 1 or 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 to SEQ ID NO:

1.

6. The rAAV virion of any one of claims 1 to 4, wherein the fusion protein comprises an N-terminal signal peptide.

7. 4. The rAAV virion of any one of claims 1 to 3, wherein the signal peptide is a canine insulin signal peptide, and optionally 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 to MALWMRLLPLLALLALWAPAPTRA (SEQ ID NO: 7).

8. the canine proinsulin is a canine proinsulin variant having one or more cleavage site mutations compared to the reference polypeptide sequence set forth in SEQ ID NO: 10, and / or the proinsulin is canine proinsulin fused to canine serum albumin, and optionally 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; and / or the canine proinsulin comprises the mutations K29R, R31K, and L62R compared to the reference polypeptide sequence set forth in SEQ ID NO: 10, and / or 4. The rAAV virion of any one of claims 1 to 3, 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 to SEQ ID NO:

8.

9. 5. The rAAV virion of claim 1, 2, or 4, wherein the fusion protein comprises a feline N-terminal signal peptide, and optionally 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 to SEQ ID NO:

9.

10. The feline proinsulin is a feline proinsulin variant having one or more cleavage site mutations compared to the reference polypeptide sequence set forth in SEQ ID NO: 24, and / or the proinsulin is feline proinsulin fused to feline serum albumin, and optionally the feline proinsulin-feline 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: 33; and / or 5. The rAAV virion of claim 1, wherein the feline proinsulin comprises K29R, R31K, and L62R mutations compared to the reference polypeptide sequence set forth in SEQ ID NO:

24.

11. the polynucleotide encoding the fusion protein is operably linked to a promoter, optionally the promoter being a CB7 promoter element comprising a cytomegalovirus enhancer and a chicken b-actin promoter; and / or 5. The rAAV virion of any one of claims 1 to 4, wherein the expression cassette comprises a polynucleotide sequence encoding a homology-directed repair (HDR) template configured for insertion into the cleavage site.

12. A pharmaceutical composition for use in the treatment of metabolic diseases in dogs or cats, comprising an rAAV virion according to any one of claims 1 to 4.

13. Virion composition is 1 x 10 9 GC / kg ~ 3 x 10 13 5. The rAAV virion of any one of claims 1 to 4, wherein the rAAV is formulated to be administered to the canine or feline subject at a dose of GC / kg of the rAAV, and / or the rAAV is delivered intramuscularly.

14. 5. The rAAV virion of any one of claims 1 to 4, for use in 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 rAAV virion, optionally wherein the metabolic disease is diabetes, and optionally wherein the diabetes is type 1 diabetes or type 2 diabetes.

15. The effective amount is administered intramuscularly, and optionally the effective amount is 1×10 9 GC / kg to 3×10 13 GC / kg of the rAAV virions; or the effective amount is 1×10 10 GC / kg to 3×10 13 GC / kg of the rAAV virions; or the method results in expression of the fusion protein in the subject for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, or at least 60 weeks; or the method results in expression of the fusion protein in the subject at a therapeutically effective concentration for at least 3 months, at least 6 months, or at least 12 months; or 15. The rAAV of claim 14, wherein the method reduces fasting blood glucose in the subject by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

16. 13. The pharmaceutical composition of claim 12, for use in a method for 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 pharmaceutical composition, optionally wherein the metabolic disease is diabetes, and optionally wherein the diabetes is type 1 diabetes or type 2 diabetes.

17. The effective amount is administered intramuscularly, and optionally the effective amount is 1×10 9 GC / kg to 3×10 13 GC / kg of the rAAV virions; or the effective amount is 1×10 10 GC / kg to 3×10 13 GC / kg of the rAAV virions; or the method results in expression of the fusion protein in the subject for at least 1 week, at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks, or at least 60 weeks; or the method results in expression of the fusion protein in the subject at a therapeutically effective concentration for at least 3 months, at least 6 months, or at least 12 months; or 17. The pharmaceutical composition of claim 16, wherein the method reduces fasting blood glucose in the subject by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

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