Viral vector genomes encoding insulin fusion proteins

Abstract

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

Description

[0001] References to Electronic Sequence Listings

[0002] 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. Technical Field

[0003] The present invention generally relates to compositions and methods for treating diabetes in dogs or cats. Background Art

[0004] Diabetes mellitus is a syndrome associated with prolonged hyperglycemia resulting from loss or dysfunction of insulin secretion from 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 a result of insulin resistance, islet amyloidosis, or chronic lymphoplasmacytic pancreatitis.

[0005] Insulin is an endogenous peptide hormone produced by the 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's caregiver, along with frequent veterinary visits and one-time diagnostics that are expensive, time-consuming, and inconvenient.

[0006] The present disclosure provides compositions and methods relating to viral particles engineered to provide sustained expression of insulin. Summary of the invention

[0007] In certain embodiments, a recombinant adeno-associated virus (rAAV) virion for treating a companion animal is provided. The rAAV virion comprises an AAV capsid and a vector genome, wherein the vector genome comprises an expression cassette comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises proinsulin and serum albumin, wherein the expression cassette is flanked by 5' inverted terminal repeats (ITRs) and 3'ITRs, wherein the proinsulin is canine proinsulin or feline proinsulin. In certain embodiments, the AAV capsid is selected because of its ability to be expressed in muscle cells. In certain embodiments, the capsid is an AAVrh91 capsid.

[0008] In certain embodiments, the proinsulin is canine proinsulin. In other embodiments, the proinsulin is feline proinsulin. Suitably, the canine proinsulin or the feline proinsulin comprises an N-terminal signal peptide. In certain embodiments, the N-terminal signal peptide of the canine insulin is a canine insulin signal peptide. In certain embodiments, the N-terminal signal peptide of the feline insulin is a feline insulin signal peptide.

[0009] In certain embodiments, the canine proinsulin is a canine proinsulin variant having a mutation at one or more cleavage sites compared to the reference polypeptide sequence as shown in SEQ ID NO: 10. In certain embodiments, the proinsulin is a 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 with SEQ ID NO: 2. In certain embodiments, the proinsulin includes K29R, R31K and L62R mutations compared to the reference polypeptide sequence as shown in SEQ ID NO: 10. In certain embodiments, the canine proinsulin-serum albumin fusion protein includes a linker that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 8.

[0010] In certain embodiments, the feline proinsulin is a variant having a mutation at one or more cleavage sites compared to the reference polypeptide sequence as shown in SEQ ID NO: 24. In certain embodiments, the proinsulin is feline proinsulin fused to feline serum albumin.

[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. Compared to the reference polypeptide sequence as shown in SEQ ID NO: 24, the feline proinsulin may include K29R, R31K and L62R mutations.

[0012] In certain embodiments, the cat 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. Compared to the reference polypeptide sequence as shown in SEQ ID NO:24, the cat proinsulin may include K29R, R31K and L62R mutations. The polynucleotide encoding the fusion protein is operably linked to a promoter. In certain embodiments, the promoter is a CB7 promoter element including a cytomegalovirus enhancer and a chicken b-actin promoter. In certain embodiments, the expression cassette includes a polynucleotide sequence encoding a homology-directed repair (HDR) template, which is configured to be inserted into the cleavage site.

[0013] In certain embodiments, a pharmaceutical composition suitable for treating a metabolic disease in a dog or cat is provided, the pharmaceutical composition comprising the rAAV virus particle. The rAAV virus particle or the pharmaceutical composition can be used in a method for treating a dog or cat subject with a metabolic disease, optionally diabetes.

[0014] Certain embodiments provide the use of rAAV viral particles or pharmaceutical compositions for the preparation of a medicament for treating a canine or feline subject with a metabolic disease, optionally diabetes. Certain embodiments provide that the rAAV viral particles in the composition will be formulated to have a density of 1x10 9 GC / kg to 3x10 13 A dose of GC / kg of rAAV is administered to the canine or feline subject, and / or wherein the rAAV is delivered intramuscularly.

[0015] In certain embodiments, a method for treating a canine or feline subject with a metabolic disease is provided, the method comprising administering an effective amount of the rAAV virion or the pharmaceutical composition to the canine or feline subject. In certain embodiments, the method is used to treat a metabolic disease (e.g., 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 1x10 9 GC / kg to 3x10 13 GC / kg of the rAAV virus particles. In certain embodiments, the effective amount is 1x10 10 GC / kg to 3x10 13GC / kg of the rAAV virus particles. In certain embodiments, the method allows the fusion protein to be expressed in the subject for at least one week, at least two weeks, at least four 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 certain embodiments, the method allows the fusion protein to be expressed in the subject at a therapeutically effective concentration for at least three months, at least six months, or at least twelve months. In certain embodiments, the method reduces the fasting blood glucose of the subject by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0016] Still further aspects and advantages of the present invention will be apparent from the following detailed disclosure of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1C Schematic representations of illustrative canine insulin proteins of the present disclosure are shown. All three proteins incorporate a native signal peptide (SP) and a modified furin site. Figure 1A A schematic diagram of an illustrative canine preproinsulin-serum albumin fusion protein (cINS-Alb) is shown. The caINS-Alb protein has a glycine / serine linker connecting the A chain of insulin to canine serum albumin. Figure 1B A schematic diagram of an illustrative canine preproinsulin-transferrin fusion protein (cINS-Tf) is shown. The caINS-Tf protein has a glycine / serine linker connecting the A chain of insulin to canine transferrin. Figure 1C Shown is a schematic diagram of an illustrative canine preproinsulin protein containing a furin site modification and serving as a control (cINS-2-1).

[0018] Figure 2 The in vitro insulin bioactivity of caINS-Alb and caINS-Tf compared to a control insulin standard is shown. 50 Values ​​are listed in the table below the figure. Ligand-induced insulin receptor activation in response to increasing concentrations of purified cINS-Alb and cINS-Tf. Reference insulin standards were used as controls. Relative potency is expressed as relative light units.

[0019] Figure 3ABlood glucose levels of streptozotocin (STZ) NOD-SCID mice administered with AAV virions containing canine insulin analogs or PBS controls are shown. In vivo activity of AAV-cINS analogs (cINS-Alb and cINS-Tf) in NOD-SCID diabetic mice was observed. After intramuscular (IM) administration of the AAV candidates on day 0, fasting blood glucose was monitored over the duration of the study. The data shown are the mean and standard deviation (SD) of the cohort (n=7-10 / group).

[0020] Figure 3B Body weights of STZ NOD-SCID mice administered AAV virions containing canine insulin analogs or PBS controls are shown. In vivo activity of AAV-cINS analogs (cINS-Alb and cINS-Tf) in NOD-SCID diabetic mice was observed. After intramuscular (IM) administration of the AAV candidates on day 0, body weights were monitored over the duration of the study. Data shown are the mean and standard deviation (SD) of the cohort (n=7-10 / group).

[0021] Figure 3C and 3D Ex vivo serum insulin activity of STZ NOD-SCID mice administered AAV virions containing canine insulin analogs or PBS control is shown. 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 insulin activity was increased on day 28 ( Figure 3C ) and Day 56 ( Figure 3D ) collect serum samples and measure insulin bioactivity.Relative insulin activity is expressed as relative light unit.Shown data are the mean value and difference of independent mice.At two time points, statistical difference (analyzed by one-way ANOVA) is shown between contrast cINS-2-1 and cINS-Alb. DETAILED DESCRIPTION

[0022] As described elsewhere herein, the present disclosure is based, at least in part, on the surprising discovery of the inventors that viral particles encoding insulin fusion proteins achieve sustained expression of insulin in dogs and cats. Methods of making and using such viral particles are provided.

[0023] Insulin fusion proteins engineered to overcome the short half-life of natural hormones by fusion with proteins (e.g., serum albumin) with longer half-lives are therapeutic advances for the treatment of diabetes. Long-acting insulin fusion protein expression constructs for canines and felines have been developed. The expression construct includes a secretion signal peptide and a fusion domain intended to extend the circulation time of the resulting fusion protein.

[0024] The expression construct is delivered to a subject in need thereof by transduction of viral particles (such as AAV particles) and expression of the encoded insulin fusion protein in vivo. Also provided are methods for using these constructs in a regimen for treating type 1 diabetes (T1DM), type 2 diabetes (T2DM) or metabolic syndrome in a veterinary subject and increasing the half-life of insulin in the subject.

[0025] The present invention encompasses insulin-albumin fusion proteins comprising therapeutic proteins with insulin activity. The present invention also encompasses polynucleotides comprising or alternatively consisting of nucleic acid molecules encoding therapeutic proteins with insulin activity, the therapeutic protein being fused to albumin or a fragment (portion) or variant of albumin. Albumin can be fused to the N-terminus, C-terminus, or both ends 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 nucleic acid molecules encoding proteins, the proteins comprising therapeutic proteins with insulin activity fused to albumin or a fragment (portion) or variant of albumin, the therapeutic protein being sufficient to prolong its activity in vivo.

[0026] Preamble sequence

[0027] In one embodiment, the insulin protein includes 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 includes a leader or signal sequence and proinsulin. In one embodiment, the leader sequence is a native sequence (canine or feline insulin) leader. In another embodiment, the leader sequence is a heterologous sequence, i.e., derived from another protein other than canine or feline insulin.

[0028] In one embodiment, the leader is a canine IL-2 sequence. In some embodiments, 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:12.

[0029] SEQ ID NO:12:MYKMQLLSCIALTLVLVANS

[0030] In another embodiment, the leader is a native canine insulin sequence.In one embodiment, the canine leader comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:7.

[0031] SEQ ID NO:7:MALWMRLLPLLALLALWAPAPTRA

[0032] In one embodiment, the leader sequence is a feline IL-2 sequence. In some embodiments, 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.

[0033] SEQ ID NO:13:MYKIQLLSCIALTLILVTNS

[0034] In another embodiment, the leader is a native feline insulin sequence.In one embodiment, the canine leader comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:9.

[0035] SEQ ID NO:9:MAPWTRLLPLLALLSLWIPAPTRA

[0036] The leader sequence can be derived from the same species that is ultimately intended to be used, i.e., canines or felines. As used herein, the term "origin" or "derived from" means that a sequence or protein is derived from a specific subject species or shares the same sequence with a protein or sequence derived from a specific subject species. For example, a leader sequence "derived from" a dog or cat shares the same sequence (or its variant, as defined herein) with the same leader sequence as expressed in a dog or cat. However, it is not actually necessary to derive a nucleic acid or amino acid from a dog or cat. Various techniques known in the art that can produce a desired sequence include mutagenesis of similar proteins (e.g., homologues) or artificial generation of nucleic acid or amino acid sequences. "Derived" nucleic acids or amino acids retain the function of the same nucleic acids or amino acids in their "derived" species, regardless of the actual source of the derived sequence.

[0037] insulin

[0038] Insulin is involved in regulating the utilization of glucose in the body. The body's inability to synthesize insulin or cells that are resistant to insulin lead to diabetes, which is 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. The formation of disulfide bonds between the A and B chain components, and the removal of the intermediate C chain produce a biologically active insulin molecule consisting of 51 amino acids, which is less than half the size of the original translation product.

[0039] Unless otherwise indicated, the term "insulin" refers to a biologically active insulin molecule or a functional fragment thereof that provides the desired insulin-like activity, as well as amino acid sequence variants of these molecules. 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 includes a polynucleotide sequence encoding a polypeptide including (a) a secretion signal peptide, (b) a proinsulin polypeptide, (c) an optional linker, and (d) an optional fusion partner.

[0040] In one embodiment, the protein includes canine IL2 signal peptide and canine proinsulin. In another embodiment, the protein includes canine insulin signal peptide and canine proinsulin. The amino acid sequence of native canine proinsulin is shown in SEQ ID NO:10. In another embodiment, the protein includes cat IL2 signal peptide and cat proinsulin. In another embodiment, the protein includes cat insulin signal peptide and cat proinsulin. The amino acid sequence of native cat proinsulin is shown in SEQ ID NO:24.

[0041] In some embodiments, canine or feline insulin comprises variants, and the variants may comprise up to about 10% changes from insulin nucleic acid or amino acid sequences described herein or known in the art, and the variants retain the function of the wild-type sequence. As used herein, "retaining function" means that the nucleic acid or amino acid works in the same manner as the wild-type sequence, although not necessarily at the same expression or activity level. For example, in one embodiment, compared with the wild-type sequence, the functional variant has increased expression or activity. In another embodiment, compared with the wild-type sequence, the functional variant has reduced expression or activity. In one embodiment, compared with the wild-type sequence [SEQ ID NO:24], the expression or activity of the functional variant increases or decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0042] In one embodiment, compared with the native sequence, the canine proinsulin sequence contains one or more mutations. In certain embodiments, these mutations are located in the cleavage site between the B / C chain and the C / A chain. In one embodiment, one or more cleavage sites in the cleavage site mutate to incorporate at least one furin cleavage site at the existing protease cleavage site. In one embodiment, the proinsulin sequence has a K29R mutation. In another embodiment, the proinsulin sequence has an R31K mutation. In another embodiment, the proinsulin sequence has an 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.

[0043] In one embodiment, the canine proinsulin sequence is a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 1, 3, 10 or 14.

[0044] In one embodiment, the feline proinsulin sequence is a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:15, 25 or 33.

[0045] When variants or fragments of the proinsulin sequence are desired, site-directed mutagenesis of the wild-type nucleic acid sequence can be used to generate the coding sequences of these peptides. Alternatively or in addition, 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 of EMBOSS; Gene Infinity; and / or ExPasy. In one embodiment, the RNA and / or cDNA coding sequence is designed to be optimally expressed in the subject species (i.e., dog or cat) that is ultimately intended to be administered.

[0046] Fusion domain

[0047] The present disclosure provides a fusion protein including a fusion domain. By fusing insulin with a fusion domain having a longer half-life, the insulin fusion protein overcomes the short half-life of the natural hormone. In some embodiments, the fusion domain includes any one of: (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 includes canine serum albumin.

[0048] In some embodiments, the fusion domain comprises any one of the following: (i) feline serum albumin or a functional variant thereof, (ii) feline IgG Fc or a functional variant thereof, or (iii) feline transferrin or a functional variant thereof. In some embodiments, the fusion domain comprises feline serum albumin.

[0049] In some embodiments, the fusion domain is canine serum albumin comprising a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:16.

[0050] In some embodiments, the fusion domain is canine transferrin comprising a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:17.

[0051] In some embodiments, the fusion domain is feline serum albumin comprising a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO:18.

[0052] Insulin fusion protein

[0053] The present disclosure provides fusion proteins including one or more copies of proinsulin, and polynucleotides and expression vectors encoding such fusion proteins. In certain embodiments, the fusion protein includes a polynucleotide sequence encoding the fusion protein, and the fusion protein includes any of the following fusion domains: (a) a leader sequence including a secretion signal peptide, (b) proinsulin and (c) including (i) IgG Fc or its functional variant, (ii) albumin or its functional variant or (iii) transferrin or its functional variant. In one embodiment, the fusion protein includes a thrombin leader sequence, proinsulin and IgG Fc or its functional variant. In another embodiment, the fusion protein includes a thrombin leader sequence, proinsulin and albumin or its functional variant.

[0054] In some embodiments, the fusion protein includes a canine insulin leader sequence, canine proinsulin (K29R, R31K and L62R relative to the numbering of SEQ ID NO: 10), a glycine / serine linker and canine serum albumin. In one embodiment, the fusion protein includes a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 1.

[0055] In some embodiments, the fusion protein includes a canine insulin leader sequence, canine proinsulin (K29R, R31K and L62R relative to the numbering of SEQ ID NO: 10), a glycine / serine linker and canine transferrin. In one embodiment, the fusion protein includes a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 3.

[0056] In some embodiments, the fusion protein includes a canine insulin leader sequence and canine proinsulin (K29R, R31K and L62R relative to the numbering of SEQ ID NO: 10). In one embodiment, the fusion protein includes a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO: 5.

[0057] In some embodiments, the fusion protein includes a feline insulin leader sequence, a feline proinsulin (K29R, R31K and L62R relative to the numbering of SEQ ID NO: 24), a glycine / serine linker and a feline serum albumin. In one embodiment, the fusion protein includes 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: 24 or 25.

[0058] In one embodiment, the fusion protein comprises the insulin leader sequence, proinsulin and albumin or a functional variant thereof. In one embodiment, the fusion protein comprises the insulin leader sequence, proinsulin and transferrin or a functional variant thereof.

[0059] In one embodiment, the fusion protein comprises the IL2 leader sequence, proinsulin and albumin or a functional variant thereof. In one embodiment, the fusion protein comprises the IL2 leader sequence, proinsulin and transferrin or a functional variant thereof.

[0060] In addition to the leader sequences, proinsulin and insulin polypeptides provided herein, nucleic acid sequences encoding these polypeptides (used interchangeably with "polynucleotides") are also provided. In one embodiment, nucleic acid sequences encoding the proinsulin-serum albumin fusion polypeptides described herein are provided. In some embodiments, the nucleic acid sequence encoding canine proinsulin-serum albumin fusions includes a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:2.

[0061] In some embodiments, the nucleic acid sequence encoding canine proinsulin-transferrin fusion comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:4.

[0062] In vivo function and stability of fusion protein of the present disclosure can be optimized by adding small peptide linkers, for example, to prevent potentially unwanted domain interactions or for other reasons. Further, glycine-rich linkers can provide some structural flexibility so that proinsulin part can effectively interact with the insulin receptor on target cells. Therefore, in one embodiment, the C-terminal of proinsulin and the N-terminal of the fusion domain of fusion protein are fused by a linker. In certain embodiments, the linker comprises 1, 2, 3 or n repeats of a G-rich peptide linker with sequence (GGGGS)n. In one embodiment, the linker comprises 1, 1.5 or 2 repeats of a G-rich peptide linker with sequence GGGGSGGGGSGGGGS (SEQ ID NO:19). In one embodiment, the linker comprises the repeats of a G-rich peptide linker with sequence GGGGSGGGGSGGGS (SEQ ID NO:8).

[0063] Vector genome

[0064] The vector genome may include a monocistronic or polycistronic transcript. In some embodiments, the viral particles of the present disclosure include a polynucleotide sequence encoding the following from 5' to 3':

[0065] (a) CB7 promoter element, which includes cytomegalovirus (CMV) enhancer and chicken b-actin promoter;

[0066] (b) canine insulin signal peptide;

[0067] (c) canine proinsulin (K29R, R31K, and L62R);

[0068] (d) a Gly / Ser linker; and

[0069] (e) Canine serum albumin.

[0070] In certain embodiments, the recombinant AAV vector (particle) comprises an AAV capsid and a vector genome, the vector genome comprising: 5'AAV ITR, a promoter, an enhancer, optional introns, an open reading frame, the open reading frame being a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a canine insulin signal peptide, canine proinsulin, a linker and canine serum albumin, polyA and 3'AAV ITR. In certain embodiments, canine proinsulin is engineered canine proinsulin (K29R, R31K and L62R).

[0071] In certain embodiments, the recombinant AAV vector (particle) comprises an AAV capsid and a vector genome, the vector genome comprising: 5'AAV ITR, a promoter, an enhancer, optional introns, an open reading frame, the open reading frame being a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a feline insulin signal peptide, a feline proinsulin, a linker and feline serum albumin, polyA and 3'AAV ITR. In certain embodiments, the feline proinsulin is an engineered feline proinsulin (K29R, R31K and L62R).

[0072] 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 with SEQ ID NO:26 (CB7.CI.cINS2-1_3xGSsp.ALB.rBG) or 28 (CB7.CI.cINS2-1_3xGSsp.Tf.rBG).

[0073] In certain embodiments, the vector genome of the production plasmid comprises a shortened 5' and / or 3' AAV ITR of 130 base pairs, wherein the external A element is missing. In certain embodiments, during the amplification of the vector DNA using the internal A element as a template, the shortened ITR is restored to a wild-type length of 145 base pairs. Therefore, the final viral particles produced by the production plasmid including the shortened ITR contain a full-length 145 ITR (by adding 15 nucleotides to the most 3' end of the vector genome and / or adding 15 nucleotides to the most 5' end). In other embodiments, full-length AAV 5' ITR and 3' ITR are used. ITR is selected so that it is trans-complemented by the expressed rep protein during production (e.g., in a trans plasmid). In the case where the source of ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting rAAV can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0074] Adeno-associated virus (AAV)

[0075] On the one hand, a virion comprising a polynucleotide encoding a leader sequence, proinsulin and insulin polypeptide as described herein is provided. In certain embodiments of the virion 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 a naturally occurring adeno-associated virus, an adeno-associated virus obtainable by a person skilled in the art and / or obtainable in view of the compositions and methods described herein, and an artificial AAV. Adeno-associated virus (AAV) virions are AAV DNA enzyme-resistant virus particles with an AAV protein capsid, wherein an expression cassette for delivery to a target cell is packaged with a flanking AAV inverted terminal repeat sequence (ITR) (collectively referred to as a "vector genome").

[0076] The AAV capsid is composed of 60 capsid protein subunits VP1, VP2 and VP3, which are arranged in icosahedral symmetry, with a ratio of about 1:1:10 to 1:1:20, depending on the selected AAV. Various AAVs can be selected as the source of the capsid of the AAV virus particles as identified above. In one embodiment, the AAV capsid is an AAVrh91 capsid or a variant thereof. See, for example, WO2020 / 223232 (on the amino acid sequence predicted by AAVrh91 and the deamidation pattern of the assembled AAVrh91 capsid) and WO2022 / 036220. In certain embodiments, the capsid protein is specified by a number or a combination of numbers and letters after the term "AAV" in the name of the rAAV virus particle. Unless otherwise indicated, the AAV capsids, ITRs and other selected AAV components described herein can be readily selected from any AAV, including but not limited to AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVhu37, AAVrh32.33, AAV Anc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu37, AAVrh64R1 and AAVhu68.

[0077] Illustrative 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, EP 1310571, WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7790449, U.S. Pat. No. 7282199 (AAV8), WO 2005 / 033321, US 7,906,111 (AAV9), WO 2006 / 110689, WO 2003 / 042397 (rh10), WO 2005 / 033321, WO 2018 / 160582 (AAVhu68), which are incorporated herein by reference.

[0078] Other suitable AAVs may include but are not limited to AAVrh90, AAVrh91, AAVrh92, AAVrh93, and AAVrh91.93. Other suitable AAVs include the AAV3B variants described in International Patent Application No. PCT / US20 / 56511 filed on October 20, 2020, which describes 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, all of which are incorporated herein by reference. These documents also describe other AAV capsids that can be selected for production of rAAV and are incorporated by reference.

[0079] As used herein, with respect to AAV, the term "variant" means any AAV sequence derived from a known AAV sequence, the known AAV sequence comprising an AAV sequence with conservative amino acid substitutions and an AAV sequence sharing at least 90%, at least 95%, at least 97%, at least 99% or higher sequence identity with an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant, which may comprise a change of up to about 10% with 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 with the AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the identity percentage of the AAV capsid, any variable protein (e.g., vp1, vp2 or vp3) can be compared.

[0080] In one embodiment, the virion is a rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the virion is a rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the virion is a rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the virion is a rAAV having a capsid selected from the group consisting of AAV9, AAVrh64R1, AAVhu37 or AAVrh10. In one embodiment, the virion is a rAAV having a capsid of AAVhu68 or a functional variant thereof.

[0081] In certain embodiments, the viral vector has an AAVrh91 capsid. A nucleic acid sequence encoding an AAVrh91 capsid is provided in SEQ ID NO: 21, and an encoded amino acid sequence is provided in SEQ ID NO: 22. A rAAV is provided herein, 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 an AAVrh91 amino acid sequence is provided in SEQ ID NO: 22, and an 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 N-terminal and / or C-terminal truncations (e.g., truncations of about 1 to about 10 amino acids).

[0082] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:22, vp1 proteins produced by SEQ ID NO:21, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:21 encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:22, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:22, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:21, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:21 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:22. NO:21, a vp2 protein that is the product of a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2208 of SEQ ID NO:21, a heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: vp3 proteins 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:22, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2208 of SEQ ID NO:21, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2208 of SEQ ID NO:21 encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:22; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:22, and a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22. A heterogeneous population of vp3 proteins that are products of a nucleic acid sequence of at least amino acids 203 to 736 of SEQ ID NO:22, wherein: the vp1, vp2, and vp3 proteins contain a subpopulation having amino acid modifications that include at least two highly deamidated asparagines (N) in an asparagine-glycine pair in SEQ ID NO:22, and optionally further include 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 sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0083] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) AAVrh91 capsid proteins comprising: a heterogeneous population of AAVrh91 vp1 proteins selected from the group consisting of vp1 proteins produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:22, vp1 proteins produced by SEQ ID NO:21, or vp1 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:21 encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:22, a heterogeneous population of AAVrh91 vp2 proteins selected from the group consisting of vp2 proteins produced by expression of a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:22, vp2 proteins produced by a sequence comprising at least nucleotides 412 to 2208 of SEQ ID NO:21, or vp2 proteins produced by a nucleic acid sequence at least 70% identical to SEQ ID NO:21 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:22. NO:21, a vp2 protein that is the product of a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2208 of SEQ ID NO:21, a heterogeneous population of AAVrh91 vp3 proteins selected from the group consisting of: vp3 proteins 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:22, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2208 of SEQ ID NO:21, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2208 of SEQ ID NO:21 encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:22; and / or (2) a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:22, and a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22. A heterogeneous population of vp3 proteins that are products of a nucleic acid sequence of at least amino acids 203 to 736 of SEQ ID NO: 22, wherein: the vp1, vp2, and vp3 proteins contain a subpopulation with amino acid modifications that include at least two highly deamidated asparagines (N) in an asparagine-glycine pair in SEQ ID NO: 22, and optionally further include a subpopulation that includes 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 sequence and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence directing expression of the product in a host cell.;

[0084] In certain embodiments, the AAVrh91 vp1, vp2, and vp3 proteins contain a subpopulation with amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pairs in SEQ ID NO: 22, and optionally further include a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. Relative to the numbering of SEQ ID NO: 22, high levels of deamidation are observed at NG pairs N57, N383, and / or N512. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 can have other deamidated residues, e.g., typically less than 10%, and / or can have other modifications comprising phosphorylation (e.g., where present, in the range of about 2% to about 30%, or about 2% to about 20%, or about 2% to about 10%) (e.g., at S149) or oxidation (e.g., at one or more of about W22, about M211, W247, M403, M435, M471, W478, W503, about M537, about M541, about M559, about M599, M635, and / or W695). Optionally, W can be oxidized to kynurenine.

[0085] Table A - AAVrh91 deamidation

[0086] AAVrh91 deamidation based on VP1 numbering Deamidation% N57+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N94+ deamidation 2-15 or 2-5 N303+ deamidation 2-15 or 5-10 N383+deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 N497+deamidation 2-15 or 5-10 N512+ deamidation 65-90, 70-95, 80-95, 75-100, 80-100 or 90-100 About N691+deamidation 2-15, 2-10 or 5-10

[0087] In certain embodiments, the AAVrh91 capsid is modified in one or more of the positions identified in the foregoing table within the range provided as determined using mass spectrometry using trypsin. In certain embodiments, one or more of the positions are modified as described herein or the glycine after the N. The residue numbers are based on the AAVrh91 sequence provided herein. See, SEQ ID NO: 22.

[0088] In certain embodiments, the AAVrh91 capsid comprises: a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:22, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:22, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO:22.

[0089] In certain embodiments, the modified AAVrh91 nucleic acid sequence is used to generate a mutant rAAV having a capsid that is less deamidated than the native AAVrh91 capsid. Such mutant rAAV may have reduced immunogenicity and / or increased stability during storage, particularly when stored in suspension form.

[0090] In certain embodiments, the AAV68 capsid is further characterized by one or more of the following. AAV hu68 capsid proteins comprising: 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 encoding 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 at least about amino acids 138 to 736 of SEQ ID NO:37, a vp2 protein produced from a sequence including 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 encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:37, and / or an AAVhu68 vp3 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:37 AAVhu68 vp3 protein produced by expressing a nucleic acid sequence encoding at least about amino acids 203 to 736 of the predicted amino acid sequence of SEQ ID NO:37, a vp3 protein produced by a sequence including at least nucleotides 607 to 2211 of SEQ ID NO:35 or 36, or a vp3 protein produced by a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO:35 or 36 that encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:37.

[0091] Additionally or alternatively, an AAV capsid is provided that includes a heterogeneous population of vp1 proteins that optionally include valine at position 157, a heterogeneous population of vp2 proteins that optionally include valine at position 157, and a heterogeneous population of vp3 proteins, wherein based on the numbering of the vp1 capsid of SEQ ID NO:37, at least one subpopulation of vp1 and vp2 proteins includes valine at position 157 and optionally further includes glutamic acid at position 67. Additionally or alternatively, an AAVhu68 capsid is provided, comprising: a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:37, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 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 to 736 of SEQ ID NO:37, wherein: the vp1, vp2 and vp3 proteins contain subpopulations having amino acid modifications.

[0092] AAVhu68 vp1, vp2 and vp3 proteins are generally expressed as alternative splice variants encoded by the same nucleic acid sequence encoding the full-length vp1 amino acid sequence (amino acids 1 to 736) of SEQ ID NO: 37. Optionally, the vp1 coding sequence alone is used to express the vp1, vp2 and vp3 proteins. Alternatively, this sequence can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence (about aa 203 to 736) of SEQ ID NO:37 without 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 the complementary strands thereto, i.e., the corresponding mRNA or tRNA (about nt607 to about nt 2211 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 encoding aa 203 to 736 of SEQ ID NO:37. Additionally or alternatively, the vp1 encoding and / or vp2 encoding sequences can be co-expressed with one or more of the following: a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:37 (approximately aa 138 to 736) without the vp1 unique region (approximately aa 1 to approximately 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (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 encoding approximately aa 138 to 736 of SEQ ID NO:37.

[0093] 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, for example, encoding a vp3 protein that does not contain the unique regions of vp1 and / or vp2. rAAVhu68 produced from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1 proteins, vp2 proteins, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications of the predicted amino acid residues from SEQ ID NO:37. These subpopulations contain at least deamidated asparagine (N or Asn) residues. For example, the asparagine in the asparagine-glycine pair is highly deamidated.

[0094] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO:35 or 36 or a complementary strand thereto, such as a corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins can be expressed additionally or alternatively from a nucleic acid sequence different from vp1, such as to change the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO:37 (about aa 203 to 736) is also provided, which amino acid sequence lacks 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, i.e., the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO:35 or 36). In certain embodiments, nucleic acid sequences encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:37 (about aa 138 to 736) are also provided, wherein the amino acid sequence does not have the vp1 unique region (about aa 1 to about 137) or the complementary strand thereto, i.e., the corresponding mRNA or tRNA (nt 412 to 2211 of SEQ ID NO:35 or 36).

[0095] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 37 may be selected for use in generating rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 35 or 36, or 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 SEQ ID NO: 35 or 36 encoding SEQ ID NO: 37. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence 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 about nt 412 to about nt 2211 of SEQ ID NO: 35 or 36 encoding 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 of about nt 607 to about nt 2211 of SEQ ID NO:35 or 36, or a sequence that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to nt SEQ ID NO:35 or 36 encoding the vp3 capsid protein of SEQ ID NO:37 (about aa 203 to 736).

[0096]

[0097]

[0098] In certain embodiments, the AAVhu68 capsid is characterized as 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 about 90% of the N residues 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) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized as having a population of proteins in which about 1% to about 20% of the N residues are deamidated at one or more of the following 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 includes at least a subset of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of the following 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, based on numbering of the amino acid sequence of SEQ ID NO: 37, or a combination thereof. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0099] In another embodiment, a recombinant adeno-associated virus (rAAV) is provided having an AAVhu68 capsid and a vector genome, 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, wherein the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins contain a subpopulation having an amino acid modification, the amino acid modification comprising SEQ ID NO: 1 as determined using mass spectrometry. NO:37 wherein at least two asparagines (N) in an asparagine-glycine pair in two or more of N57, N329, N452, N512 are deamidated to 50% to 100%, and optionally further comprises a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change wherein the deamidated asparagines are deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid / isoaspartic acid pair, or a combination thereof, wherein the AAVhu68 capsid further comprises a subpopulation having one or more of:

[0100] (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;

[0101] (b) at least 75% of the N in the asparagine-glycine pair at position N329 of the vp1, v2 and vp3 proteins, based on the residue numbering of the amino acid sequence of SEQ ID NO: 37, is deamidated,

[0102] (c) at least 50% of the N in the asparagine-glycine pair in position N452 of the vp1, v2 and vp3 proteins based on the residue numbering of the amino acid sequence of SEQ ID NO: 37 is deamidated; and / or

[0103] (d) at least 75% of the N in the asparagine-glycine pair in position N512 of the vp1, v2 and vp3 proteins based on the residue numbering of the amino acid sequence of SEQ ID NO: 37 is deamidated, and

[0104] The vector genome in the AAVhu68 capsid comprises a nucleic acid molecule comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a PTH fusion as described herein operably linked to a sequence that directs expression of the PTH fusion in a target cell.

[0105] In one aspect, a recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid from adeno-associated virus rh91 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a coding sequence of a canine proinsulin-serum albumin fusion of SEQ ID NO: 1, and a regulatory sequence directing the expression of the canine proinsulin fusion.

[0106] In another embodiment, rAAV comprises an AAV capsid from adeno-associated virus rh91 and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), a coding sequence for a canine proinsulin-transferrin fusion of SEQ ID NO: 3, and a regulatory sequence directing the expression of the canine proinsulin fusion.

[0107] In another embodiment, rAAV comprises an AAV capsid from adeno-associated virus rh91 and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), a coding sequence for a feline proinsulin-serum albumin fusion of SEQ ID NO: 32, and a regulatory sequence directing the expression of the feline proinsulin fusion.

[0108] In one aspect, a recombinant AAV (rAAV) is provided. The rAAV comprises an AAV capsid from adeno-associated virus hu68 and a vector genome packaged in the AAV capsid, wherein the vector genome comprises an AAV inverted terminal repeat (ITR), a coding sequence of a canine proinsulin-serum albumin fusion of SEQ ID NO: 1, and a regulatory sequence directing the expression of the canine proinsulin fusion.

[0109] In another embodiment, rAAV comprises an AAV capsid from adeno-associated virus hu68 and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), a coding sequence for canine proinsulin-transferrin fusion of SEQ ID NO: 3, and a regulatory sequence directing the expression of the canine proinsulin fusion.

[0110] In another embodiment, rAAV comprises an AAV capsid from adeno-associated virus hu68 and a vector genome packaged in the AAV capsid, wherein the vector genome includes an AAV inverted terminal repeat (ITR), a coding sequence for a feline proinsulin-serum albumin fusion of SEQ ID NO: 32, and a regulatory sequence directing the expression of the feline proinsulin fusion.

[0111] In one embodiment, the nucleic acid sequence encoding the proinsulin fusion construct described herein is engineered into any suitable genetic element, for example, naked DNA, bacteriophage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., and the proinsulin fusion sequence carried thereon is transferred to a host cell, for example, for producing nanoparticles, virions and / or for delivery to a host cell in a subject in a packaging host cell carrying DNA or RNA. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The method for preparing such a construct is known to nucleic acid manipulation technicians and includes genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0112] In some embodiments, the proinsulin fusion constructs described herein can be delivered by viral particles other than rAAV. Such other viral particles can include any virus suitable for gene therapy that can be used, including but not limited to adenovirus; herpes virus; lentivirus; retrovirus, etc. Suitably, when one of these other viral particles is produced, it is produced as a replication-defective viral particle.

[0113] "Replication-defective virus" or "virion" refers to a synthetic or artificial virus particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, the synthetic or artificial virus particle cannot produce progeny virus particles but retains the ability to infect target cells. In one embodiment, the genome of the virus particle does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"-containing only transgenes of interest flanking the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for gene therapy because replication and infection by progeny virus particles will not occur unless the viral enzymes required for replication are present.

[0114] Expression cassette

[0115] In certain embodiments, expression cassette refers to nucleic acid molecules, and the nucleic acid molecules include proinsulin fusion construct encoding sequence, promoter, and may include other regulatory sequences for it. Expression cassettes may be engineered into genetic elements and / or packaged into the capsid of virions (e.g., viral particles). Typically, such expression cassettes for producing virions contain proinsulin fusion constructs described herein, and the packaging signal and other expression control sequences of the proinsulin fusion construct flanking viral genome, such as sequences described herein. Any expression control sequence may be optimized for a particular species using techniques known in the art, and the technology may include, for example, codon optimization, such as described herein.

[0116] The expression cassette generally contains a promoter sequence as part of the expression control sequence. In one embodiment, a constitutive promoter is used. In the plasmids and expression vectors described herein, the CB7 promoter can be used. CB7 is a chicken B-actin promoter with a cytomegalovirus enhancer element. Alternatively, liver-specific promoters can be used, including but not limited to α1 antitrypsin (A1AT), human albumin (Miyatake et al., Journal of Virology (J.Virol.) 71:512432 (1997)) [humAlb]; Hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002 9 (1996)), TTR minimal enhancer / promoter, α-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 such as viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO 2011 / 126808 and WO 2013 / 04943), or promoters that are responsive to physiological cues can be used in the expression vectors described herein.

[0117] In one embodiment, an expression cassette is provided that includes a CB7 promoter, a chicken beta actin intron, a canine mutant proinsulin having a canine insulin leader fused to feline albumin via a linker, and a rabbit beta globin poly, such as in SEQ ID NO: 26. SEQ ID NO: 27 shows a vector genome that includes a shortened AAV 5′ ITR, a spacer sequence, SEQ ID NO: 27, a spacer sequence, and a shortened AAV 3′ ITR.

[0118] In another embodiment, an expression cassette is provided that includes a CB7 promoter, a chicken beta actin intron, a canine mutant proinsulin having a canine insulin leader fused to feline transferrin via a linker, and a rabbit beta globin poly, such as in SEQ ID NO: 28. SEQ ID NO: 29 shows a vector genome that includes a shortened AAV 5′ ITR, a spacer sequence, SEQ ID NO: 28, a spacer sequence, and a shortened AAV 3′ ITR.

[0119] In another embodiment, an inducible promoter is used. Examples of inducible promoters that can be used herein include inducible promoters described in International Patent Application No. PCT / US2021 / 043219, entitled "Canine and Feline Inducible Expression Constructs for Gene Therapy Applications," published on February 3, 2022, which is incorporated herein by reference. Briefly, the inducible promoter includes a promoter; an activation domain including a canine or feline transactivation domain and a canine or feline FKBP12-rapamycin-associated protein (FRAP) FKBP12-rapamycin binding (FRB) domain; a DNA binding domain including a zinc finger homology domain (ZFHD) and one, two or three FK506 binding protein domain (FKBP) subunit genes; and at least 8 copies of a ZFHD binding site (8XZFHD), followed by a minimal IL2 promoter. The presence of an effective amount of rapamycin or a rapamycin analog induces expression of a transgene in a host cell.

[0120] In addition to the promoter, the expression cassette and / or expression vector may also 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 sequences that enhance secretion of the encoded product when desired. Illustrative examples of suitable polyA sequences include, for example, rabbit beta globin, SV40, bovine growth hormone (bGH), and TK polyA.

[0121] Illustrative examples of suitable enhancers include, for example, α-fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / α1 microglobulin / bikunin enhancer), etc. In one embodiment, polyA is rabbit globin polyA. In one embodiment, polyA has a sequence of SEQ ID NO: 20.

[0122] These control sequences are "operably linked" to the proinsulin fusion construct sequence. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest.

[0123] In one embodiment, rAAV is provided, comprising 5'ITR, CB7 promoter, chicken β-actin intron, coding sequence of protein of SEQ ID NO: 1, rabbit globin poly A and 3'ITR. In one embodiment, rAAV is provided, comprising 5'ITR, CB7 promoter, chicken β-actin intron, coding sequence of protein of SEQ ID NO: 3, rabbit globin poly A and 3'ITR. In one embodiment, rAAV is provided, comprising 5'ITR, CB7 promoter, chicken β-actin intron, coding sequence of protein of SEQ ID NO: 3, rabbit globin poly A and 3'ITR.

[0124] The minimal sequences required to package the expression cassette into AAV viral particles are AAV 5' and 3' ITRs of the same AAV origin as the capsid or of a different AAV origin (to generate AAV pseudotypes).

[0125] In order to package the expression cassette into the AAV capsid to form virions (i.e., rAAV vectors or rAAV particles), ITR is the only AAV component that needs to be in cis in the construct identical to the gene. In one embodiment, the coding sequence for replication (rep) and / or capsid (cap) is removed from the AAV genome and supplied in trans or supplied by a packaging cell line to produce AAV virions. For example, as described above, pseudotype AAV can contain ITRs from sources different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid can be utilized. Still other AAV components can be selected. The source of such AAV sequences is described herein and can also be separated or obtained from academic, commercial or public sources (e.g., the American Type Culture Collection in Manassas, Virginia, VA). AAV sequences can be obtained by synthesis or other suitable means by reference to publicly disclosed sequences (e.g., in the literature or in databases such as GenBank, PubMed, etc.).

[0126] Methods for producing and isolating AAV virions suitable for delivery to a subject are known in the art. See, e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199; WO 2003 / 042397; WO 2005 / 033321; WO2006 / 110689; and US 7,588,772. In one system, a production cell line is transiently transfected with a construct encoding a transgene flanking the ITRs and a construct encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanking the ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpes virus, thereby requiring separation of rAAV from contaminating viruses. Recently, systems have been developed that do not require infection with a helper virus to recover AAV - systems by which the desired helper functions (i.e., adenovirus E1, E2a, VA and E4 or herpesvirus UL5, UL8, UL52 and UL29, and herpesvirus polymerase) are also supplied in trans. In these systems, helper functions can be supplied by transiently transfecting cells with constructs encoding the desired helper functions, or the cells can be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In another system, transgenes flanking ITRs and rep / cap genes are introduced into insect cells by infection with baculovirus-based expression vectors. For a review of these production systems, see generally, 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 entirety. Methods for making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each of which are incorporated herein by reference in their entirety: U.S. Pat. Nos. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065.See generally, e.g., Grieger and Samulski, 2005, Adv. Biochem. 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. Methods for constructing any embodiment of the present invention are known to nucleic acid manipulation technicians and include genetic engineering, recombinant engineering, and synthetic techniques.

[0127] Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation of the present invention. See, for example, K. Fisher et al., (1993) Journal of Virology, 70:520-532 and U.S. Pat. No. 5,478,745.

[0128] 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 the polynucleotide sequence encoding an insulin protein to be inserted into the genome of a host cell.

[0129] Methods of treating a subject with the disclosed compositions

[0130] Compositions comprising virion constructs described herein are also provided. Pharmaceutical compositions described herein are designed to be delivered to a dog or cat subject in need by any suitable approach or combination of different approaches. Direct delivery to the liver (optionally intravenously, by the hepatic artery or by transplantation), direct delivery to the pancreas, oral administration routes, inhalation administration routes, intranasal administration routes, intratracheal administration routes, intraarterial administration routes, intraocular administration routes, intravenous administration routes, intramuscular administration routes, subcutaneous administration routes, intradermal administration routes, and other maternal administration routes. Virions described herein can be delivered in the form of a single composition or a variety of compositions. Optionally, two or more different AAVs, or a variety of viruses [see, for example, WO 2011 / 126808 and WO 2013 / 049493] can be delivered.

[0131] In some embodiments, the pharmaceutical compositions described herein are designed to be delivered to a canine or feline subject in need thereof by intramuscular administration.

[0132] In another embodiment, the plurality of viruses may contain different replication-defective viruses (e.g., AAV and adenovirus). In one embodiment, administration is performed intramuscularly. In another embodiment, administration is performed intravenously.

[0133] In some embodiments, the course of treatment may involve repeated administration of the same viral particles (e.g., AAVrh91 viral particles) or different viral particles (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the viral particles described herein. In some embodiments, the compositions described herein may be combined in regimens involving other diabetes drugs or protein-based therapies including, for example, insulin analogs, insulin, oral antihyperglycemic drugs (sulfonylureas, biguanides, thiazolidinediones, and alpha-glucosidase inhibitors). In some embodiments, the compositions described herein may be combined in regimens involving lifestyle changes including diet and exercise regimens.

[0134] As used herein, the term "proinsulin construct," "proinsulin expression construct," and synonyms include combinations of proinsulin sequences and leaders as described herein (whether native or heterologous). The term "proinsulin construct," "proinsulin expression construct," and synonyms may be used to refer to nucleic acid sequences encoding proinsulin fusion proteins or their expression products. In this specification, the proinsulin sequence with a leader is also referred to as "preproinsulin." In certain embodiments, a "proinsulin construct" may also encompass fusion proteins as described herein.

[0135] Replication-defective viruses can be formulated with physiologically acceptable carriers for 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 preparation. Any method known in the art can be used to determine the number of genome copies (GC) of the replication-defective virus composition of the present invention. A method for titrating the number of AAV GC is as follows: a purified AAV vector genome sample is first treated with DNA enzyme to eliminate unencapsidated AAV genomic 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 targeting a specific region of the viral genome (usually a poly A signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), particularly optimized qPCR or digital droplet PCR.

[0136] In addition, the replication-defective virus composition can be formulated in a dosage unit to contain about 1.0 x 109 GC to about 1.0x10 15 In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, for an average canine or feline subject of about 5-10 kg, the dose is about 1.0 x 10 10 GC to about 3.0x10 13 GC. In another embodiment, the dose is about 1x10 9 GC. For example, the dose of AAV virus can be about 1x10 10 GC, 1x10 11 GC, about 5x10 11 GC, about 1x10 12 GC, about 5x10 12 GC or about 1x10 13 GC. In another embodiment, for canine or feline subjects, the dose is about 1.0 x 10 9 GC / kg to about 3.0x10 13 GC / kg body mass. In another embodiment, the dose is about 1x10 9 GC / kg. In another embodiment, the dose is about 1x10 10 GC / kg. In another embodiment, the dose is about 1x10 11 GC / kg. For example, the dose of AAV virus can be about 1x10 10 GC / kg、1x10 11 GC / kg, about 5x10 11 GC / kg, about 1x10 12 GC / kg, about 5x10 12 GC / kg or about 1x10 13 GC / kg.

[0137] On the other hand, a method for sustained expression of proinsulin fusion protein in a canine or cat subject is provided. The method comprises administering a composition as described herein to a subject in need thereof. In one embodiment, the composition comprises a virion containing a proinsulin-serum albumin fusion protein expression cassette, as described herein. In some embodiments, the method provided herein allows the proinsulin fusion protein to be expressed in the subject for at least one week, at least two weeks, at least four 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 allows the proinsulin fusion protein to be expressed in the subject for at least eight weeks. In some embodiments, the method provided herein allows the proinsulin fusion protein to be expressed in the subject for at least one month, at least three months, at least six months, or at least twelve months at a therapeutically effective concentration.

[0138] In one embodiment, for veterinary subjects, the construct can be delivered in a volume of 1 μL to about 100 mL. See, for example, Diehl et al., J. Applied Toxicology, 21:15-23 (2001) for a discussion of good practices for administering substances to various veterinary animals. This document is incorporated herein by reference. As used herein, the term "dose" may refer to the total dose delivered to a subject during treatment or the amount delivered in a single (or multiple) administration.

[0139] In one embodiment, the composition is administered in combination with an effective amount of insulin. Various commercially available insulin products are known in the art, including but not limited to protamine zinc recombinant human insulin Porcine Insulin Zinc Suspension and insulin glargine In some embodiments, the combination of rAAV and insulin described herein reduces the insulin dosage requirement of the 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 dose of insulin required for the subject. For example, insulin or other therapies can be used to treat the subject, and the treating physician can continue treatment after administering the AAV virions. Such insulin or other co-therapies can be subsequently continued, reduced or interrupted as needed.

[0140] In one embodiment, a composition comprising an expression cassette, a vector genome, rAAV or other compositions described herein for gene therapy is delivered in a single dose / subject. In one embodiment, a therapeutically effective amount of a composition described herein is delivered to a subject. As used herein, a "therapeutically effective amount" refers to the amount of proinsulin-serum albumin delivered and expressed in a target cell by an expression cassette or a virion or a combination thereof to achieve a therapeutic goal. In certain embodiments, the therapeutic goal is to improve or treat one or more symptoms of type I diabetes, type II diabetes or metabolic syndrome. The therapeutically effective amount can be determined based on an animal model rather than a canine or feline subject. In another embodiment, the therapeutic goal is to alleviate a metabolic disease in a subject.

[0141] The recombinant AAV virions described above can be delivered to host cells according to the disclosed methods. The rAAV preferably suspended in a physiologically compatible carrier can be administered to a desired subject including a dog. In view of the indications for which the transfer virus is directed, a suitable carrier can be easily selected by those skilled in the art. For example, a suitable carrier comprises saline, which can be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The selection of the carrier is not a limitation of the present invention.

[0142] In some embodiments, in addition to rAAV and / or variants and carriers, the compositions of the invention may also contain other conventional pharmaceutical ingredients (such as preservatives or chemical stabilizers). Illustrative preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Illustrative chemical stabilizers include gelatin and albumin.

[0143] The recombinant viral particles and constructs described herein can be used to prepare a medicament for delivering a proinsulin fusion protein construct to a subject in need thereof, for supplying insulin with increased half-life to a subject, and / or for treating type I diabetes, type II diabetes, or metabolic syndrome in a subject.

[0144] In one aspect, a method for treating diabetes is provided. The method comprises administering a composition as described herein to a canine or feline subject in need thereof. In one embodiment, the composition comprises a viral particle containing a proinsulin fusion protein expression cassette, as described herein.

[0145] In another embodiment, a method for treating type 2 diabetes in a dog or cat is provided. The method comprises administering a viral particle comprising a nucleic acid molecule comprising a sequence encoding a proinsulin fusion protein as described herein.

[0146] In another embodiment, a method for treating type 1 diabetes in a dog or cat is provided. The method comprises administering a viral particle comprising a nucleic acid molecule comprising a sequence encoding a proinsulin fusion protein as described herein.

[0147] In another aspect, a method for treating a metabolic disease in a dog or cat is provided. The method comprises administering a composition as described herein to a dog or cat subject in need thereof. In one embodiment, the composition comprises a viral particle containing a proinsulin fusion protein expression cassette, as described herein. In one embodiment, the metabolic disease is type I diabetes. In one embodiment, the metabolic disease is type II diabetes. In one embodiment, the metabolic disease is metabolic syndrome.

[0148] In another embodiment, a method for treating diabetes in a dog or cat is provided. The method comprises administering a viral particle comprising a nucleic acid molecule comprising a sequence encoding a proinsulin-serum albumin fusion protein as described herein, wherein the viral particle is administered after insulin is administered to the subject. In some embodiments, the viral particle is administered at least 1 day, at least 3 days, at least 5 days, at least one week, at least 2 weeks, at least 3 weeks, or at least 4 weeks after insulin is administered to the subject.

[0149] In another embodiment, a method of preventing cataract formation in a diabetic dog or cat is provided. The method comprises administering a viral particle comprising a nucleic acid molecule comprising a sequence encoding a proinsulin-serum albumin fusion protein as described herein. In some embodiments, the viral particle is administered after insulin is administered to the subject.

[0150] In another embodiment, a method of reducing blood glucose concentration in a diabetic dog or cat is provided. The method comprises administering a viral particle comprising a nucleic acid molecule comprising a sequence encoding a proinsulin-serum albumin fusion protein as described herein.

[0151] As used herein, the term "treatment" or "treating" is defined as covering the administration of one or more compounds or compositions described herein to a subject for the purpose of improving one or more symptoms of type I diabetes, type II diabetes (T2DM) or metabolic syndrome. Therefore, "treatment" may include one or more of the following: reducing the progression of type I diabetes, type II diabetes or metabolic syndrome in a given subject, reducing the severity of symptoms in a given subject, removing disease symptoms in a given subject, delaying the progression of a given subject's disease, or increasing the efficacy of a given subject's therapy.

[0152] As used herein, the term "remission" refers to the ability to discontinue insulin therapy when a cat or dog no longer exhibits clinical signs of diabetes and has normal blood glucose levels.

[0153] In another embodiment, a method for treating T2DM in a cat or dog is provided. The method comprises administering a viral particle comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein.

[0154] In another aspect, a method for treating a metabolic disease in a cat or dog is provided. The method comprises administering a composition as described herein to a cat or dog subject in need thereof. In one embodiment, the composition comprises a viral particle containing a proinsulin fusion protein expression cassette, as described herein.

[0155] In another aspect, a method for reducing fasting blood glucose in a canine or feline subject is provided. The method comprises administering a composition as described herein to a subject in need thereof. In one embodiment, the composition comprises a viral particle containing a proinsulin-serum albumin fusion protein expression cassette, as described herein. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by about 20%. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by about 30%. In some embodiments, the method provided herein reduces the fasting blood glucose of the subject by about 40%.

[0156] Unless otherwise defined, all terms used herein (including technical terms and scientific terms) have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. It will be further understood that terms, such as those defined in common dictionaries, should be interpreted as having the same meaning as that in the context of the present application and the related art, and unless clearly defined as such in this article, should not be interpreted as idealized or over-formal meaning. The terms used in the specification are only for the purpose of describing specific embodiments, and are not intended to be restrictive. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict of terms, this specification shall prevail.

[0157] Reference to "one embodiment" or "another embodiment" when describing an embodiment does not mean that the referenced embodiment is mutually exclusive of another embodiment (eg, an embodiment described before the referenced embodiment) unless explicitly stated otherwise.

[0158] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., when compared and aligned for maximum correspondence over a comparison window or designated region, sharing at least about 80% identity with a reference sequence over a designated region, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, as measured using the following sequence comparison algorithm or by manual alignment and visual inspection). Such sequences are then referred to as "substantially identical". This definition also refers to the complement of a test sequence. In some embodiments, identity is present over a region of at least about 25 amino acids or nucleotides in length, e.g., over a region of 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the full-length reference sequence.

[0159] In order to carry out sequence comparison, usually a sequence serves as a reference sequence compared with a test sequence. When using a sequence comparison algorithm, test sequence and reference sequence are input into a computer, coordinates are specified (if necessary) subsequently, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on program parameters. In certain embodiments, BLAST and BLAST 2.0 algorithms and default parameters are used.

[0160] In the context of amino acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "identical percent" refer to the residues in two sequences that are identical when aligned for correspondence. The percent identity of an amino acid sequence over a full-length protein, polypeptide, about 70 amino acids to about 100 amino acids, or a peptide fragment thereof, or a corresponding nucleic acid sequence encoding sequence can be readily determined. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 150 amino acids. Typically, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to the "aligned" sequence.

[0161] "Compared" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence. Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs can be used for amino acid sequences, for example, "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used with default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same identity or alignment level as provided by a reference algorithm and program.

[0162] The term "amino acid substitution" and its synonyms are intended to cover the modification of an amino acid sequence by replacing one amino acid with another substituted amino acid. The substitution may be a conservative substitution. The substitution may also be a non-conservative substitution. With respect to two amino acids, the term conservative is intended to mean that the amino acids share common properties recognized by those skilled 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 properties 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 amino acids and non-naturally occurring amino acids are known in the art and may be used as substituted amino acids in embodiments. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. Reference herein to "one or more" is intended to encompass, for example, 1, 2, 3, 4, 5, 6 or more separate embodiments.

[0163] Codon optimization can be used to design the coding sequence for optimal expression. Codon-optimized coding regions can be designed by various methods. This optimization can be performed using methods available online, disclosed methods, or companies providing codon optimization services. For example, a codon optimization method is described in International Patent Application Publication No. WO 2015 / 012924, which is incorporated herein by reference. In short, the nucleic acid sequence of the encoded product is modified with a synonymous codon sequence. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only the fragment of the ORF can be changed. By using a method in these methods, frequency can be applied to any given polypeptide sequence, and a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide is produced.

[0164] In one embodiment, the insulin-serum albumin fusion is caninized or felineized.

[0165] "Caninized" means that the fusion protein includes an amino acid sequence that is compatible with dogs, such that the amino acid sequence is less likely to be viewed as foreign by the immune system of a canine subject. In the present disclosure, polypeptide terms prefixed with "ca" refer to variants of human polypeptides in which the human fusion domain is replaced by a canine homolog of the fusion domain, and when proinsulin is a fragment or variant of a human protein, proinsulin is replaced by a canine homolog of the fragment or variant.

[0166] "Felinization" means that the fusion protein includes an amino acid sequence that is compatible with cats, making it less likely that the amino acid sequence will be considered foreign by the immune system of a feline subject. In the present disclosure, polypeptide terms prefixed with "fe" refer to variants of human polypeptides in which the human fusion domain is replaced by a feline homolog of the fusion domain, and when proinsulin is a fragment or variant of a human protein, proinsulin is replaced by a feline homolog of the fragment or variant.

[0167] As described elsewhere herein, the present disclosure extends to fusion proteins that are compatible with species other than canine or feline.In this context, a fusion protein may be referred to as "specific," referring to the target species to which the molecule will be administered.

[0168] In certain embodiments, compositions and methods described herein are intended for use in feline animals. The term cat (family Felidae) refers to any of 37 cat species, including cheetahs, cougars, jaguars, leopards, lions, lynxes, tigers, and house cats, etc. In one embodiment, the subject is a house cat. In certain embodiments, compositions and methods described herein are intended for use in canine animals. The term dog refers to any species in the Canidae family, including domestic dogs, wolves, and foxes, etc. In one embodiment, the subject is a domestic dog, also referred to as Canis lupus familiaris or Canis familiaris.

[0169] As used in the description of 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 indicates otherwise.

[0170] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative (or).

[0171] As used herein, the phrase "consisting essentially of refers to the genus or species of active agent recited in the method or composition, and may further include other agents that themselves are not substantially active for the recited indication or purpose.

[0172] The words "comprise", "comprises" and "comprising" are to be interpreted as inclusive rather than exclusive. The words "consist", "consisting" and variations thereof are to be interpreted as exclusive rather than inclusive. Although various embodiments in the specification are presented using "comprising" language, in other cases, it is also intended to use the language of "consisting of" or "consisting essentially of" to explain and describe the relevant embodiments.

[0173] As used herein, unless otherwise indicated, the term "about" means a variability of 10% relative to a given reference.

[0174] 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.

[0175] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state of a subject.

[0176] The terms "subject," "individual," and "patient" refer interchangeably to mammals, humans or non-human primates, domestic mammals (e.g., dogs or cats), laboratory mammals, and agricultural mammals. In various embodiments, the subject can be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a male child, a female child). In various embodiments, the subject is a companion animal. Illustrative companion animals include, but are not limited to, dogs, cats, horses, rabbits, ferrets, birds, and guinea pigs. In various embodiments, the subject is a dog. In various embodiments, the subject is a cat. In various embodiments, the subject is a mammal.

[0177] As used herein, the term "viral particle" or "virion" refers to a macromolecular complex capable of delivering foreign nucleic acid molecules to cells unrelated to another agent. Independent particles of a virus, "viral particles" or "virions", consist of genetic material (i.e., DNA or RNA encoding a transgenic) 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" can be used interchangeably with "recombinant adeno-associated virus (rAAV) vector" and indicates the presence of a nuclease-resistant AAV capsid, in which a replication-defective vector genome is packaged. Non-viral particles are limited to liposomes, nanoparticles, and other encapsulation systems for delivering polynucleotides to cells.

[0178] The term "transgenic" refers to the nucleic acid itself that is transferred. The transgenic can be a naked nucleic acid molecule (such as a plasmid) or RNA. The transgenic can include a polynucleotide encoding one or more polypeptides (e.g., an insulin fusion protein). The transgenic can include a polynucleotide encoding one or more heterologous proteins (e.g., an insulin fusion protein), one or more capsid proteins, and other proteins required for transducing the polynucleotide into the target cell.

[0179] The term "transduction" refers to the introduction of nucleic acid into a cell or host organism by particles (e.g., adeno-associated virus particles). Therefore, the introduction of a transgene into a cell by a viral particle can be referred to as the "transduction" of a 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, the transgene can be stably maintained in the cell. Alternatively, the introduced transgene can be present in the recipient cell or host organism extrachromosomally or only transiently. Therefore, "transduced cells" refer to cells into which a transgene is introduced by means of transduction. Therefore, "transduced" cells are cells introduced into a polynucleotide.

[0180] As used herein, the term "host cell" may refer to a packaging cell line, wherein virions (e.g., recombinant AAV or rAAV) are produced by a production plasmid. In an alternative, the term "host cell" may refer to any target cell in which a gene product described herein is desired to be expressed. Therefore, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, human cell, or insect cell), which contains exogenous or heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion). In certain embodiments herein, the term "host cell" refers to a culture of cells of various mammalian species for in vitro evaluation of compositions described herein. In other embodiments herein, the term "host cell" refers to a cell for producing and packaging virions or recombinant viruses. In another embodiment, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, a liver cell.

[0181] As used herein, the term "target cell" refers to any target cell in which it is desired to express a heterologous nucleic acid sequence or protein. In certain embodiments, the target cell is a liver cell. In certain embodiments, the target cell is a muscle cell.

[0182] As used herein, "expression cassette" or "vector genome" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably linked thereto, which 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 comprises both regulatory sequences (also referred to as elements) adjacent or non-adjacent to the nucleic acid sequence and regulatory sequences that function as trans or cis nucleic acid sequences. Such regulatory sequences typically include, for example, one or more of the following: a promoter, an enhancer, a transcription factor, a transcription terminator, an intron, a sequence that enhances translation efficiency (i.e., a Kozak consensus sequence), an effective RNA processing signal (e.g., a slicing and polyadenylation sequence), a sequence that stabilizes cytoplasmic mRNA (e.g., a woodchuck hepatitis virus (WHP) post-translational regulatory element (WPRE)), and a TATA signal. The expression cassette can contain the regulatory sequence of the upstream (5' place) of the gene sequence, for example, one or more and enhancer in promoter, enhancer, intron etc., or one or more in the regulatory sequence of the downstream (3' place) of the gene sequence, for example, 3' untranslated region (3'UTR) including polyadenylation site, and other elements. In certain embodiments, the regulatory sequence is operably connected to the nucleotide sequence of the gene product, wherein the regulatory sequence is separated from the nucleotide sequence of the gene product by the intermediate nucleic acid sequence, i.e. 5'-untranslated region (5'UTR). In certain embodiments, the expression cassette includes the nucleotide sequence of one or more gene products in the gene product. In certain embodiments, the expression cassette can be a monocistronic expression cassette or a bicistronic expression cassette. In other embodiments, the term "transgenic" refers to one or more DNA sequences inserted into the target cell from an exogenous source. Generally, such expression cassettes can be used to produce virions, and contain the coding sequence and other expression control sequences of the packaging signal of the flanking viral genome of the gene product described herein, such as the sequence described herein. In certain embodiments, the vector genome can contain two or more expression cassettes.

[0183] As used herein, "administration" refers to local and systemic administration, for example, including enteral administration, parenteral administration, pulmonary administration and local / transdermal administration. The route of administration of the drug component (e.g., virion) used in the methods described herein includes, for example, oral (oral (PO)) administration, nasal administration or inhalation administration, administration in the form of suppositories, local contact, transdermal delivery (e.g., by transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration or subcutaneous ("sc") administration, or sustained release devices (e.g., micro-osmotic pumps, reservoir preparations, etc.) are implanted in the subject. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ion introduction and intracranial.

[0184] The terms "systemic administration" and "systemically administered" refer to a method of administering a pharmaceutical ingredient or composition to a mammal so that the pharmaceutical ingredient or composition is delivered to a site in the body (including a target site of drug action) via the circulatory system. Systemic administration includes, but is not limited to, oral administration, intranasal administration, rectal administration, and parenteral administration (e.g., administration other than through the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous).

[0185] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or dosage regimen of one or more pharmaceutical ingredients (eg, viral particles) required to produce the desired result.

[0186] As used herein, the terms "treating" and "treatment" refer to delaying the onset of the disease or condition to which the term applies, or one or more symptoms of such disease or condition, slowing or reversing the progression of the disease or condition or its symptoms, reducing the severity of the disease or condition or its symptoms, or alleviating or preventing the disease or condition or its symptoms. The terms "treating" and "treatment" also encompass preventing, alleviating, ameliorating, reducing, inhibiting, eliminating and / or reversing one or more symptoms of a disease or condition.

[0187] The term "alleviate" refers to reducing or eliminating one or more symptoms of a pathology or disease, and / or reducing the incidence or delaying the onset of one or more symptoms of a pathology or disease or reducing its severity and / or preventing the pathology or disease. In some embodiments, the reduction or elimination of one or more symptoms of a pathology or disease can include, for example, a measurable and sustained reduction in fasting blood glucose.

[0188] ****

[0189] All publications and patents mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control. However, reference to any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be taken as an admission or any form of suggestion that it constitutes valid prior art or forms part of the common general knowledge in any country in the world.

[0190] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0191] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

[0192] Examples

[0193] The following examples are presented in order to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated, and are intended to be purely illustrative of the invention and are not intended to be limiting as to the scope of the invention.

[0194] Example 1: Production of canine insulin-serum albumin fusions with extended half-life and evaluation of in vitro potency of canine insulin fusion proteins

[0195] A canine insulin-serum albumin fusion protein designed to be administered via AAV virions has been developed to manage hyperglycemia and hyperglycemia-related clinical signs of diabetes in dogs.

[0196] Canine preproinsulin-serum albumin fusion protein (cINS-Alb) was produced by constructing a fusion polypeptide containing the following elements: Figure 1A ) (SEQ ID NO: 1):

[0197] Natural canine insulin signal peptide (SP)

[0198] Canine proinsulin in which the native sequence was modified at 3 amino acid positions (K29R, R31K and L62R) to incorporate 2 furin cleavage sites at existing protease cleavage sites (B chain, C peptide, A chain)

[0199] · 14aa Gly / Ser linker comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 8)

[0200] Canine serum albumin

[0201] Canine preproinsulin fusion protein ( Figure 1B )(SEQ ID NO:3). The expression cassette for the albumin fusion is provided in SEQ ID NO:26, and the vector genome for the production plasmid is in SEQ ID NO:27. In the control (cINS-2-1) ( Figure 1C A control canine preproinsulin sequence (no fusion) with three amino acid modifications (K29R, R31K and L62R) was also produced in AAV virions containing SEQ ID NO:5; the expression cassette for the control is in SEQ ID NO:30, and the vector genome is in SEQ ID NO:31. The vector genomes of the production plasmids all contained shortened AAV 5' and 3' ITRs. SEQ ID NO:28 provides an expression cassette for a canine proinsulin-transferrin fusion protein. The vector genome is provided in SEQ ID NO:29.

[0202] A study was performed to evaluate the in vitro potency of two fusion proteins (cINS-Alb and cINS-Tf). C-terminally histidine-tagged versions of each protein were produced in mammalian cells following transient transfection of expression plasmids containing cDNA encoding the corresponding proteins. The proteins were purified from cell supernatants by nickel affinity chromatography and used according to the kit instructions. Insulin bioassay kit (Eurofins TM DiscoverX Products Ltd. (Eurofins TM DiscoverX Products, LLC)) to determine the bioactivity of insulin. The standard insulin provided in the kit was used as a reference control. Both cINS-Alb and cINS-Tf showed bioactivity in this assay ( Figure 2 ). If through EC 50 Modifications of the insulin molecule in both fusion proteins resulted in a slight loss of potency, as indicated by the change in the values.

[0203] Example 2: Production of AAV Viral Particles Including Canine Insulin Fusion Protein Transgene and Evaluation of In Vivo Efficacy

[0204] To produce AAV virions, a codon-optimized transgene encoding a control (AAVrh91.cIns.2-1) with three amino acid modifications but without a fusion partner and two insulin fusion proteins (SEQ ID NOs: 1, 3, and 5) was cloned into a plasmid containing CB7 promoter elements (including a hybrid cytomegalovirus enhancer and a chicken b-actin promoter). The expression construct was flanked by shortened 130 bp 5′ and 3′ AAV inverted terminal repeats (ITRs) in the plasmid and packaged in AAV serotype rh91 capsid (AAVrh91) virions by triple transfection with a trans plasmid expressing AAVrh91 capsid and a helper plasmid expressing adenoviral helper functions required for replication and production. HEK 293 packaging cells expressing E1 adenoviral functions did not provide adenoviral helper functions. During replication and packaging, the 5′ and 3′ ITRs were restored to the full wild-type 145 bp ITR length. The expression construct was then expressed by Taqman TM AAV virions were purified by quantitative PCR titration and iodixanol gradient purification.

[0205] A study was conducted to evaluate the in vivo efficacy of three AAV-delivered canine insulin fusion proteins in a diabetic mouse model. NOD-SCID mice that had been treated with streptozotocin to induce diabetes were assigned to one of the four groups shown in Table 1 below. Non-diabetic NOD-SCID mice included in Group 5 served as controls.

[0206] Table 1: Study groups

[0207] Group quantity STZ-induced diabetes? Vector genome Dosage (GC / mouse) 1 7 yes PBS - 2 8 yes AAVrh91.cIns-Alb <![CDATA[1x10 11 ]]> 3 8 yes AAVrh91.cIns-Tf <![CDATA[1x10 11 ]]> 4 8 yes AAVrh91.cIns.2-1 (control) <![CDATA[1x10 11 ]]> 5 10 no PBS -

[0208] Virus particles (1x10 11 GC / mouse). Food was removed from the cage twice a week 6 hours before fasting blood glucose sampling. Two mice from Group 2 were euthanized due to hypoglycemia (on 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 a glucometer with an upper limit of 600 mg / dL.

[0209] Animals that received AAV virions containing the control cINS-2-1 (Group 4) and cINS-Alb (Group 2) transgenes had significantly lower blood glucose levels compared to control group 1 ( Figure 3A). Compared with AAV containing control cINS-2-1 (Group 4), mice receiving AAV containing cINS-Alb fusion (Group 2) showed a more obvious and faster decline in fasting blood glucose levels. By the 32nd day, blood glucose levels in Group 2 had normalized and were similar to those observed in the non-diabetic mouse cohort (Group 5). In contrast to the significant reduction in blood glucose levels observed in animals receiving AAV containing cINS-Alb fusion (Group 2), no reduction in blood glucose was observed in the animal cohort (Group 3) administered with AAV containing cINS-Tf fusion.

[0210] The weight changes of mice Figure 3B Mice administered AAV containing the cINS-Alb fusion tended to gain more weight than diabetic animals in the other groups, although this was not statistically significant.

[0211] To examine the relative biological potency of the expressed canine insulin proteins, as described in detail above, Sera from mice collected on days 28 and 56 were tested in the insulin bioassay kit. Figure 3C As shown in Figure 2, at the 28th day and the 56th day, the circulating potent insulin level of the animal using the AAV containing cINS-Alb fusion was significantly higher than the circulating potent insulin level of the animal in other queues. Surprisingly, although the cINS-Tf protein shows moderate activity in vitro assays, the activity or efficacy of insulin-transferrin fusion protein is not observed in vivo. These data show that cINS-Alb shows a higher biological activity than cINS-Tf, and the AAV containing cINS-All fusions shows an in vivo efficacy higher than the AAV containing cINS-Tf fusions.

[0212] Example 3: Evaluation of AAV-caINS-Alb in Healthy Canine Subjects

[0213] A study will be conducted to evaluate the 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 10kg to 15kg will be administered 1x10 11 or 1x10 12 AAV-caINS-Alb was administered intramuscularly at a dose of GC / kg. Before administration of AAV-caINS-Alb, a series of health screening measures including veterinary examination, serum fructosamine analysis, and hematology, clinical chemistry analysis, and urinalysis will be performed. Serum samples were then collected weekly for up to 8 weeks to determine the level of circulating caINS-Alb protein using a sandwich ELISA protocol as described below.

[0214] The potential immunogenicity of caINS-Alb will be determined by measuring anti-caINS-Alb antibodies in a bridging assay. Blood glucose levels will be measured twice daily and monitored using a continuous glucose monitoring system (FreeStyle Libre TM Interstitial glucose concentrations were recorded at regular intervals by a RT-PCR system (Abbott). Animals were observed daily for signs of irritability, lethargy, and hypoglycemia.

[0215] In order to measure the serum concentration of caINS-Alb, sandwich ELISA was used. The ELISA plate was coated with 0.2 μg / ml of mouse anti-human insulin (clone 7F8, Life Technologies) and blocked with PBS / 0.05% Tween 20 / 1% casein. The coated wells were incubated at room temperature for 1 hour with 100 μL of appropriately diluted serum samples, which were diluted in PBS / 0.05% Tween 20 / 1% casein. The purified caINS-Alb standard within the range of 200 ng / ml to 0.19 ng / ml was used to establish a standard curve. After washing, the plate was incubated with 1 / 2000 diluted goat anti-dog albumin (Bethyl Laboratories) in PBS / 0.05% Tween 20 / 1% casein for 1 hour. The plate was then washed five times with PBS / 0.05% Tween 20, and developed by adding tetramethylbenzidine (TMB) substrate. Development was stopped by adding 2M H2SO4 and the absorbance was read at 450 nm.

[0216] Example 4: Evaluation of the efficacy of AAV-caINS-Alb in canine streptozotocin (STZ)-induced diabetes model

[0217] In a non-randomized, unblinded laboratory study, six beagle dogs with chemically induced diabetes on a stable diabetes management regimen will be evaluated for the efficacy of a single administration of AAVrh91 virions expressing caINS-Alb to adequately maintain blood glucose levels in diabetic dogs.

[0218] Low doses (1x10 11 gc / kg, N=3) AAVrh91 virions expressing caINS-Alb and high dose (1x10 12gc / kg, N=3) AAVrh91 virions expressing caINS-Alb. Prior to administration of AAV-caINS-Alb, a battery of health screening measures will be performed including veterinary examination, serum fructosamine analysis, and hematology, clinical chemistry analysis, and urinalysis. Throughout the study, blood glucose measurements will be performed 3 times a day by glucometer and clinical observations will be performed 2 times a day. On Day -1, subjects will be acclimated to the placement of a continuous glucose monitor and interstitial glucose concentrations will be collected 3 times a day.

[0219] During the study, in addition to the daily procedures initiated at baseline, blood collections for fructosamine analysis, hematology, and clinical chemistry will be performed every 14 days until Day 42 and again on Day 63. Blood collections for determination of circulating caINS-Alb protein and anti-caINS-Alb antibodies will be collected regularly throughout the study, and body weight will be measured every 7 days. The required supplemental insulin dose level, weight gain, and glycemic control will be used to evaluate the efficacy of the test product at the two doses tested in individual subjects.

[0220] Example 5: Evaluation of the efficacy of AAV-caINS-Alb in a canine diabetes model

[0221] 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.

[0222] Dogs previously diagnosed with diabetes, receiving stable insulin treatment and with moderate to good glycemic control will be treated with placebo or AAV-caINS-Alb (intramuscular administration). Dogs newly diagnosed with diabetes will be treated with placebo or AAV-caINS-Alb (intramuscular administration). Before the administration of AAV-caINS-Alb, a series of health screening measures including veterinary examination, serum fructosamine analysis, and hematology, clinical chemistry analysis, and urinalysis will be performed. Before AAV administration and throughout the study, glycemic control will be assessed by continuously measuring interstitial glucose concentration using a continuous glucose monitor or measuring interstitial glucose concentration twice a day using a glucometer. Throughout the study, dogs were fed a consistent diet and their body weight was monitored. During the study, in addition to blood glucose monitoring, blood collection for fructosamine analysis, hematology, clinical chemistry, and circulating caINS-Alb protein will be regularly analyzed. Serum levels of caINS-Alb are measured using a customized sandwich ELISA. Requirements for insulin supplementation, insulin dosage, body weight and glycemic control, and improvement of clinical signs will be used to evaluate the efficacy of the test product.

[0223] Example 6: Evaluation of the efficacy of AAV-feINS-Alb in a feline diabetes model

[0224] 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.

[0225] Cats previously diagnosed with diabetes, receiving stable insulin treatment and with moderate to good glycemic control will be treated with placebo or AAV-feINS-Alb (administered intramuscularly). Cats newly diagnosed with diabetes will be treated with placebo or AAV-feINS-Alb (administered intramuscularly). Before the administration of AAV-feINS-Alb, a series of health screening measures including veterinary examination, serum fructosamine analysis, and hematology, clinical chemistry analysis, and urinalysis will be performed. Before administration and throughout the study, glycemic control will be assessed by continuously measuring interstitial glucose concentrations using a continuous glucose monitor or measuring interstitial glucose concentrations twice a day using a glucometer. Throughout the study, cats will be fed a consistent diet and body weight will be monitored. During the study, in addition to blood glucose monitoring, blood collection for fructosamine analysis, hematology, clinical chemistry, and circulating feINS-Alb protein will be analyzed regularly. Serum levels of feINS-Alb will be measured using a customized sandwich ELISA. Requirements for supplemental insulin, insulin dose, body weight, and glycemic control, as well as clinical sign improvement and remission rate and remission time will be used to evaluate the efficacy of the test product.

[0226] To measure the serum concentration of feINS-Alb, a customized sandwich ELISA as shown below will be used. The ELISA plate is coated with 0.2 μg / ml of mouse anti-human insulin (clone 7F8, Life Technologies) and blocked with PBS / 0.05% Tween 20 / 1% casein. The coated wells are incubated at room temperature for 1 hour with 100 μL of appropriately diluted serum samples, which are diluted in PBS / 0.05% Tween 20 / 1% casein. Purified feINS-Alb standards within the range of 200ng / ml to 0.19ng / ml are used to establish a standard curve. After washing, the plate is incubated with 1 / 2000 diluted goat anti-cat albumin (Bethel Laboratories) in PBS / 0.05% Tween 20 / 1% casein for 1 hour. The plate is then washed five times with PBS / 0.05% Tween 20, and developed by adding TMB substrate. Development was stopped by adding 2M H2SO4 and the absorbance was read at 450 nm.

[0227] All publications cited in this specification are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 315,252, filed on March 1, 2022, is incorporated herein by reference in its entirety. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the present invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

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

2. The rAAV virion of claim 1, wherein the AAV capsid comprises an AAVrh91 capsid.

3. The rAAV virus particle of claim 2, wherein the AAVrh91 capsid comprises 60 AAVrh91 capsid proteins, and the AAVrh91 capsid proteins have at least 99% or at least 100% identity with amino acids 203 to 736 of SEQ ID NO:

22.

4. The rAAV virion of claim 1, wherein the proinsulin is canine proinsulin.

5. The rAAV virion of claim 4, wherein 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.

6. The rAAV virion of claim 1, wherein the proinsulin is feline proinsulin.

7. The rAAV virion of claim 6, wherein the feline proinsulin sequence shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

15.

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

1.

9. The rAAV virion of any one of claims 1 to 8, wherein the fusion protein comprises an N-terminal signal peptide.

10. The rAAV virion of any one of claims 1 to 4, wherein the signal peptide is a canine insulin signal peptide.

11. The rAAV virion of claim 9 or claim 10, wherein the signal peptide comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with MALWMRLLPLLALLALWAPAPTRA (SEQ ID NO: 7).

12. The rAAV virion according to any one of claims 1 to 5, wherein the canine proinsulin is a canine proinsulin variant having a mutation at one or more cleavage sites compared to the reference polypeptide sequence as shown in SEQ ID NO:

10.

13. The rAAV virion of any one of claims 1 to 5, 11 or 12, wherein the proinsulin is canine proinsulin fused to canine serum albumin.

14. The rAAV virion of any one of claims 1 to 5 or 8 to 13, wherein the canine proinsulin-serum albumin fusion polynucleotide shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

2.

15. The rAAV virion of any one of claims 1 to 5 or 8 to 14, wherein the canine proinsulin comprises K29R, R31K and L62R mutations compared to the reference polypeptide sequence as shown in SEQ ID NO:

10.

16. The rAAV virion of any one of claims 1 to 5 or 8 to 15, wherein the canine proinsulin-serum albumin fusion protein comprises a linker that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

8.

17. The rAAV virion of any one of claims 1 to 3, 6 or 7, wherein the fusion protein comprises a feline N-terminal signal peptide.

18. The rAAV virion of claim 17, wherein the signal peptide comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

9.

19. The rAAV virion of any one of claims 1 to 3, 6, 7, 17 or 18, wherein the feline proinsulin is a feline proinsulin variant having a mutation at one or more cleavage sites compared to the reference polypeptide sequence as shown in SEQ ID NO:

24.

20. The rAAV virion of any one of claims 1 to 3, 6, 7 or 18, wherein the proinsulin is feline proinsulin fused to feline serum albumin.

21. The rAAV virion of any one of claims 1 to 3, 6, 7, 17 to 20, wherein 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.

22. The rAAV virion of any one of claims 1 to 5 or 8 to 134, wherein the feline proinsulin comprises K29R, R31K and L62R mutations compared to the reference polypeptide sequence as shown in SEQ ID NO:

24.

23. The rAAV virion of any one of claims 1 to 22, wherein the polynucleotide encoding the fusion protein is operably linked to a promoter.

24. The rAAV virion of claim 23, wherein the promoter is a CB7 promoter element comprising a cytomegalovirus enhancer and a chicken b-actin promoter.

25. The rAAV virion of any one of claims 1 to 24, wherein the expression cassette comprises a polynucleotide sequence encoding a homology directed repair (HDR) template configured to insert into the cleavage site.

26. A pharmaceutical composition suitable for treating metabolic diseases in dogs or cats, the pharmaceutical composition comprising the rAAV virus particle according to any one of claims 1 to 25.

27. The rAAV virion of any one of claims 1 to 25 or the pharmaceutical composition of claim 26 for use in a method for treating a canine or feline subject suffering from a metabolic disease, optionally diabetes.

28. Use of the rAAV virion according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26 for the preparation of a medicament for treating a canine or feline subject suffering from a metabolic disease, optionally diabetes.

29. The rAAV virion according to any one of claims 1 to 26, wherein the virion composition is formulated to have a concentration of 1 x 10 9 GC / kg to 3x 10 13 A dose of GC / kg of rAAV is administered to the canine or feline subject, and / or wherein the rAAV is delivered intramuscularly.

30. 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 of any one of claims 1 to 25 or the pharmaceutical composition of claim 26.

31. The method of claim 30, wherein the metabolic disease is diabetes.

32. The method of claim 31 , wherein the diabetes is type 1 diabetes.

33. The method of claim 31, wherein the diabetes is type 2 diabetes.

34. The method of any one of claims 30 to 33, wherein the effective amount is administered intramuscularly.

35. The method according to any one of claims 30 to 34, wherein the effective amount is 1 x 10 9 GC / kg to 3x10 13 GC / kg of the rAAV virus particles.

36. The method according to any one of claims 30 to 34, wherein the effective amount is 1 x 10 10 GC / kg to 3x10 13 GC / kg of the rAAV virus particles.

37. according to the method described in any one of claims 30 to 34, wherein said method makes said fusion protein be expressed in the subject for at least one week, at least two weeks, at least four 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.

38. The method of any one of claims 30 to 34, wherein the method results in expression of the fusion protein at a therapeutically effective concentration in the subject for at least three months, at least six months, or at least twelve months.

39. The method of any one of claims 30 to 34, wherein the method reduces the subject's fasting blood glucose by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

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