CNP variants and their conjugates

Abstract

The present disclosure relates generally to stable variants of C-type natriuretic peptide (CNP) and their use for treating bone-related disorders. The present disclosure relates to novel variants of C-type natriuretic peptide (CNP) with increased circulating half-life and stability in aqueous media, pharmaceutical compositions containing such CNP variants, and methods of using such CNP variants to treat disorders responsive to CNP, including, but not limited to, bone-related disorders such as achondroplasia.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 901,093, filed September 16, 2019, U.S. Provisional Patent Application No. 62 / 935,050, filed November 13, 2019, U.S. Provisional Patent Application No. 62 / 963,350, filed January 20, 2020, U.S. Provisional Patent Application No. 62 / 964,852, filed January 23, 2020, and U.S. Provisional Patent Application No. 63 / 038,667, filed June 12, 2020, which are incorporated by reference in their entireties.

[0002] Incorporation by Reference of Electronically Submitted Materials A sequence listing, which is part of this disclosure, is submitted as a text file concurrently with the specification. The name of the text file containing the sequence listing is "54736_Seqlisting.txt," which was created on August 6, 2020, and is 54,429 bytes in size. The contents of the sequence listing are incorporated herein by reference.

[0003] The present disclosure relates generally to variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants, and methods of use. CNP variants are useful as therapeutic agents for the treatment of diseases that respond to CNP, including, but not limited to, bone-related disorders, such as skeletal dysplasia (e.g., achondroplasia). [Background technology]

[0004] C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168:863-870 (1990) (GenBank accession number NP_077720 for CNP precursor protein, NPPC) (J. Hypertens., 10:907-912 (1992)) is a 17-amino acid loop structure (Levin et al. CNP is a small, single-chain peptide in a family of peptides (ANP, BNP, CNP) with a natriuretic peptide receptor-B (NPR-B, GC-B) and plays important roles in multiple biological processes (J. Hypertens., 10:1111-1114 (1992)). CNP is widely expressed in the central nervous system, reproductive tract, bone, and vascular endothelium (Hypertension, 49:419-426 (2007)).

[0005] In humans, CNP is initially produced as a single 126-amino acid prepropolypeptide from the natriuretic peptide precursor C (NPPC) gene (Biochem. Biophys. Res. Commun., 168:863-870 (1990)). Removal of the signal peptide generates proCNP, which is further cleaved by the endoprotease furin to generate the active 53-amino acid peptide (CNP-53), which is secreted and cleaved again by an unknown enzyme to produce the mature 22-amino acid peptide (CNP-22) (Wu, J. Biol. Chem. 278:25847-852 (2003)). CNP-53 and CNP-22 differ in their distribution, with CNP-53 predominating in tissues, while CNP-22 is found primarily in plasma and cerebrospinal fluid (J. Alfonzo, Recept. Signal. Transduct. Res., 26:269-297 (2006)). Both CNP-53 and CNP-22 bind similarly to NPR-B.

[0006] The downstream signal transduction mediated by cGMP production affects a wide variety of biological processes, including endochondral ossification. For example, knockout of either CNP or NPR-B in mouse models results in animals with a dwarf phenotype, with shorter long bones and vertebrae. Mutations in human NPR-B that block proper CNP signal transduction have been identified, causing dwarfism (Olney, et al., J. Clin. Endocrinol. Metab. 91(4):1229-1232(2006); Bartels, et al., Am. J. Hum. Genet. 75:27-34(2004)). In contrast, mice engineered to produce high levels of CNP exhibit elongated long bones and vertebrae. The therapeutic use of CNP (CNP22) is limited by its short plasma half-life, which has been shown to be 2.6 minutes in vivo in humans (J Clin. Endocrinol. Metab., 78:1428-35 (1994)). CNP variants with longer in vivo serum half-lives and exhibiting similar or improved activity to wild-type CNP are important for sustainable therapeutic strategies. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Biochem.Biophys.Res.Commun.,168:863-870(1990) [Non-patent document 2] J. Hypertens., 10:907-912(1992) [Non-patent document 3] Levin et al., N. Engl. J. Med., 339:863-870 (1998) [Non-patent document 4] J.Hypertens.,10:1111-1114(1992) [Non-patent document 5] Hypertension, 49: 419-426 (2007) Summary of the Invention [Means for solving the problem]

[0008] The present disclosure relates to novel variants of C-type natriuretic peptide (CNP) with increased circulating half-life and stability in aqueous media, pharmaceutical compositions comprising such CNP variants, and methods of using such CNP variants to treat disorders responsive to CNP, including, but not limited to, bone-related disorders such as achondroplasia.

[0009] In various embodiments, the present disclosure provides a variant of C-type natriuretic peptide (CNP) selected from the group consisting of PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6), and PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5).

[0010] In various embodiments, the present disclosure provides a variant of C-type natriuretic peptide (CNP) selected from the group consisting of PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0011] In various embodiments, the variant peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH group at the C-terminus.

[0012] In various embodiments, the variant peptide comprises a conjugate moiety. In various embodiments, the conjugate moiety is on a residue in the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the conjugate moiety is on a lysine residue. In various embodiments, the conjugate moiety comprises one or more acid moieties. In various embodiments, the acid moiety is a hydrophobic acid.

[0013] In various embodiments, the conjugate moiety comprises one or more acid moieties linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu). In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) or OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) linked to one or more OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the acid moiety is a fatty acid. Exemplary fatty acids include short-, medium-, or long-chain fatty acids, or dicarboxylic fatty acids. In various embodiments, the fatty acids are saturated or unsaturated. C-6 to C-20 fatty acids, saturated or unsaturated, are contemplated, including, but not limited to, C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acids. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or a diacid thereof.

[0014] In various embodiments, the acid moiety and hydrophilic spacer have the structure AEEA-AEEA-γGlu-C18DA. In various embodiments, the acid moiety and hydrophilic spacer have the structure: [ka] , in the formula, `` [ka] " to CNP variants join In various embodiments, " [ka] " is a linker to a hydrolyzable linker join represents a point where a hydrolyzable linker is attached to the CNP variant. In various embodiments, the hydrolyzable linker is capable of releasing the intact CNP variant.

[0015] In various embodiments, the CNP variant having a conjugate moiety is a component of a modified-release composition. In various embodiments, the modified-release composition is an extended-release composition. In various embodiments, the CNP variant comprising a conjugate moiety and a hydrolyzable linker is capable of releasing the CNP variant such that (i) less than about 20% of the CNP variant is released by day 1, and (ii) at pH 7-7.6, about 90% of the CNP variant is released weekly, or about 90% of the CNP variant is released every two weeks, or about 90% of the CNP variant is released monthly.

[0016] In various embodiments, (i) at pH 7.0-7.6, less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released by day 1; and (ii) at pH 7-7.6, about 90% of the peptide is released weekly, or about 90% of the peptide is released every two weeks, or about 90% of the peptide is released monthly. Further, (i) at pH 7.0-7.6, less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released by day 1; (ii) at pH 7-7.6, about 70%, about 80%, or about 90% of the peptide is released weekly, or about 70%, about 80%, or about 90% of the peptide is released every two weeks, or about 70%, about 80%, or about 90% of the peptide is released every three weeks, or about 70%, about 80%, or about 90% of the peptide is released every three weeks. or alternatively, ii) at pH 7-7.6, about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released weekly, or about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released every two weeks, or about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released every three weeks, or about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released monthly.

[0017] In various embodiments, the variant has the structure: PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 5), or Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8).

[0018] In various embodiments, the variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13), and Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0019] In various embodiments, the variants include one or more linker groups. In various embodiments, the linker is on a residue in the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0020] In various embodiments, the linker is a hydrolyzable linker.

[0021] In various embodiments, the CNP variant is attached to the conjugate moiety via a linker. In various embodiments, the linker is attached to the conjugate moiety via a hydrophilic spacer of the conjugate moiety. In various embodiments, the linker is aminoethoxy-2-ethoxyacetic acid (AEEA). In various embodiments, the linker is a bicine-type or peptoid linker, which refers to a linker that has a cleavable mechanism similar to bicine (bis-2-hydroxyethylglycinamide), but instead cleaves via an asymmetric N-alkyl peptide, i.e., a peptoid. In various embodiments, the linker is an electronic linker based on non-enzymatic beta-elimination. In various embodiments, the electronic linker includes an SO2 moiety. Examples of linkers shown in CNP conjugates are shown in Figure 1. See also Santi, et al., Proc Natl Acad Sci USA 109:6211-6216, 2012.

[0022] In various embodiments, the conjugate moiety is a synthetic polymer group. In various embodiments, the variant comprises a synthetic polymer group attached to the variant via a hydrolyzable linker. In various embodiments, the synthetic polymer group comprises a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG). In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG) having a chain length of 6 to 20 atoms.

[0023] In various embodiments, the variant peptide is synthetically produced.

[0024] In various embodiments, the variant peptides are stable for 10 days at about 37°C and pH 7.0-7.6. In various embodiments, the variant peptides are stable for at least 10 days at about 37°C and pH 7.0-7.4. In various embodiments, the variant peptides are stable for at least 10 days at about 37°C and pH 7.2-7.6.

[0025] In various embodiments, the variant peptide is stable to deamidation. In various embodiments, the variant peptide is stable to oxidation. In various embodiments, the variant peptide is stable to deamidation and / or oxidation, or a combination thereof. In various embodiments, methionine is replaced by norleucine. In various embodiments, there is little or no detectable deamidation after 10 days.

[0026] In various embodiments, the variant peptide has a half-life of about 10 days at about 37°C, pH 7.0-7.6. In various embodiments, the variant peptide has a half-life of about 10 days at about 37°C, pH 7.0-7.4. In various embodiments, the variant peptide has a half-life of about 10 days at about 37°C, pH 7.2-7.6. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37°C, pH 7.0-7.6. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37°C, pH 7.0-7.4. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37°C, pH 7.2-7.6. In various embodiments, the half-life is at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more days.

[0027] In various embodiments, the variant peptide has an EC50 of 0.1 to 10 nM in the cGMP assay. In various embodiments, the variant peptide has an EC50 of 0.1 to 25 nM in the cGMP assay.

[0028] In various embodiments, greater than 45% of the variant peptides are detectable after 10 days in an aqueous medium at physiological conditions, e.g., about 37° C., pH 7.0-7.6. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides are detectable after 10 days in an aqueous medium at physiological conditions, e.g., about 37° C., pH 7.0-7.6.

[0029] In various embodiments, greater than 45% of the variant peptides are detectable after 10 days in aqueous medium at pH 7.4 at 37° C. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides are detectable after 10 days in aqueous medium at pH 7.4 at 37° C.

[0030] In various embodiments, greater than 45% of the variant peptides are detectable after 10 days in physiological conditions, e.g., plasma at about 37° C. and pH 7.0-7.6. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides are detectable after 10 days in physiological conditions, e.g., plasma at about 37° C. and pH 7.0-7.6.

[0031] In various embodiments, greater than 45% of the variant peptides are detectable in plasma at pH 7.4 at 37° C. after 10 days. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides are detectable in plasma at pH 7.4 at 37° C. after 10 days.

[0032] In various embodiments, the variant peptide is conjugated to a lipid, a fatty acid, a hydrophilic spacer, or a linker, or optionally a combination thereof. In various embodiments, the linker is a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety is a synthetic hydrophilic polymer moiety.

[0033] In various embodiments, the variant peptide has a longer half-life compared to Pro-Gly-CNP37. In various embodiments, the variant peptide has a longer half-life compared to CNP-22. In various embodiments, the variant peptide has a longer half-life compared to Pro-Gly-CNP37 and / or CNP-22. In various embodiments, the variant peptide has a longer half-life in vitro and / or in vivo compared to Pro-Gly-CNP37 and / or CNP-22.

[0034] The present disclosure further provides pharmaceutical compositions comprising a CNP variant described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0035] In various embodiments, the composition is a lyophilized formulation prepared from a formulation comprising a citric acid / citrate buffer or an acetic acid / acetate buffer having a pH of about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a formulation further comprising an isotonicity adjuster or bulking agent selected from the group consisting of mannitol, sucrose, sorbitol, trehalose, polysorbate 80, and combinations thereof. In various embodiments, the lyophilized formulation is prepared from a formulation further comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, salt forms of ascorbic acid, thioglycerol, and combinations thereof. In various embodiments, the CNP variant composition is supplied as a lyophilized powder for reconstitution in amounts of 0.8 mg to 10 mg. In various embodiments, the CNP variant composition is supplied as a lyophilized, preservative-free powder for reconstitution in amounts of 0.8 mg or 2 mg.

[0036] In various embodiments, the composition is an extended release composition.

[0037] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37)(BMN111) (SEQ ID NO: 1).

[0038] Also provided are methods for treating a bone-related disorder or skeletal dysplasia in a subject in need thereof, comprising administering to the subject a composition comprising a CNP variant described herein.

[0039] In various embodiments, the bone-related disorder or skeletal dysplasia is selected from the group consisting of osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, dwarfism, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, achondrogenesis imperfecta, chondrodysplasia punctata, homozygous achondroplasia, chondrodysplasia punctata, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, imperfecta, short rib polydactyly syndrome, hypochondroplasia, rhizometaphyseal chondrodysplasia punctata, Janssen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, growth imperfect osteogenesis imperfecta, osseous dysplasia, short femora congenita, Langer metapeduncular dysplasia, Niebergelt metapeduncular dysplasia, Robinnow syndrome, Reinhardt syndrome, acrosopharyngeal dysplasia, peripheral dysplasia, Niest dysplasia, fibrochondrogenesis imperfecta, Roberts syndrome, distal mesopodial dysplasia, brachymetaphyseal dysplasia, Morquio syndrome, Niest syndrome, complex organotrophic dysplasia, and spondyloepiphyseal dysplasia.

[0040] In various embodiments, CNP variants are useful as supplements or alternatives to growth hormone for treating idiopathic short stature and other skeletal dysplasias.

[0041] In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature disorder is due to an NPR2 mutation, a SHOX mutation (Turner syndrome / Lelly-Weill), or a PTPN11 mutation (Noonan syndrome).

[0042] In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature disorder is due to an NPR2 mutation, a SHOX mutation (Turner syndrome / Lelly-Weill), or a PTPN11 mutation (Noonan syndrome), or an insulin growth factor 1 receptor (IGF1R).

[0043] In various embodiments, CNP variants are useful for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature, also known as dominantly inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is the result of a mutation in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0044] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0045] In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature disorder is caused by a RASopathy. In various embodiments, the RASopathy is Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome, neurofibromatosis type 1, or Leopard syndrome.

[0046] In one embodiment, the RAS disease is hereditary gingival fibromatosis type 1.

[0047] In various embodiments, CNP variants are useful for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature, also known as dominantly inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is the result of a mutation in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R).

[0048] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0049] In various embodiments, CNP variants are useful for treating short subjects with a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and having at least one parent with a height SDS of less than -1.0, -1.5, -2.0, or -2.5, and optionally, the height of a second parent is within the normal range. In various embodiments, CNP variants are useful for treating short subjects with a height SDS of -2.0 to -3.0. In various embodiments, CNP variants are useful for treating short subjects with a height SDS of -2.0 to -2.5. In various embodiments, the short stature is associated with one or more mutations in genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), or a combination thereof. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0050] In various embodiments, short stature is the result of mutations in multiple genes as determined by a polygenic risk score (PRS). In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in FGFR3 and a low PRS. In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in IGF1R and a low PRS. In various embodiments, the subject has a mutation in NPPC and a low PRS. In various embodiments, the subject has a mutation in SHOX and a low PRS. In various embodiments, the subject has one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX and a low PRS. In various embodiments, the PRS is 1 or 2. In various embodiments, the PRS is 1. In various embodiments, the PRS is 2. A polygenic risk score (PRS) for height was calculated as described in Example 4. PRS1 refers to the shortest height, and PRS5 refers to the longest height.

[0051] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker.

[0052] In various embodiments, the present disclosure provides methods of lengthening bones or increasing long bone growth in a subject in need thereof, comprising administering to the subject a composition comprising a CNP variant described herein, wherein administering lengthens the bones or increases long bone growth.

[0053] In various embodiments, the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.

[0054] In various embodiments, the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0055] In various embodiments, the composition is an extended release composition.

[0056] Further contemplated are methods for treating a CNP-responsive condition or disorder, comprising administering a CNP variant or composition described herein to a subject and monitoring the level of at least one bone- or cartilage-related biomarker in the subject, wherein an increase in the level of the at least one bone- or cartilage-related biomarker indicates a therapeutic effect of the CNP peptide or variant on the subject or condition or disorder.

[0057] Further contemplated is a method of overcoming cell growth arrest induced by constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3), comprising contacting a cell expressing constitutively active FGFR-3 with a CNP variant or composition described herein.

[0058] Further contemplated is a method of stimulating cGMP production in a cell expressing natriuretic peptide receptor B (NPR-B), comprising contacting the cell expressing NPR-B with a CNP variant or composition described herein.

[0059] In various embodiments, the method further comprises adjusting the amount or frequency of administration of the CNP peptide or variant described herein, i) increasing the amount or frequency of administration of the CNP peptide or variant if the level of at least one bone- or cartilage-related biomarker is below a target level, or ii) decreasing the amount or frequency of administration of the CNP peptide or variant if the level of at least one bone- or cartilage-related biomarker is above a target level.

[0060] In various embodiments, the at least one bone or cartilage-related biomarker is selected from the group consisting of CNP, cGMP, propeptide of type II collagen and fragments thereof, type II collagen and fragments thereof, type I collagen C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptide of type I procollagen (PINP) and fragments thereof, type I collagen and fragments thereof, aggrecan chondroitin sulfate, collagen X, and alkaline phosphatase.

[0061] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the acetyl group is on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker.

[0062] Also provided is a method of making the CNP variants described herein, comprising synthesizing the peptide on a solid phase resin using Fmoc amino acids.

[0063] In various embodiments, the method includes acetylating the peptide by reacting the resin with NMP / Ac2O / DIEA (10:1:0.1, v / v / v).

[0064] In various embodiments, the method includes conjugating the peptide to a conjugate moiety, optionally on a lysine residue, hi various embodiments, the method includes cleaving the protected amino group on the lysine, reacting the peptide with 2xFmoc-aminoPEG(2), followed by reaction with an amino acid, followed by conjugation of a lipid or fatty acid moiety. In an embodiment of the present invention, for example, the following items are provided: (Item 1) PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6), and A variant of C-type natriuretic peptide selected from the group consisting of: PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7). (Item 2) 2. The variant of claim 1, wherein the peptide further comprises an acetyl group. (Item 3) 3. The variant according to item 1 or 2, wherein the acetyl group is on the N-terminus of the peptide. (Item 4) The peptide has an OH or NH 2 The variant of any one of the preceding items, further comprising a group. (Item 5) 10. The variant of any one of the preceding items, comprising a conjugate moiety. (Item 6) 6. The variant of claim 5, wherein the conjugate moiety is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. (Item 7) 7. The variant of item 5 or 6, wherein the conjugate moiety is on a lysine residue. (Item 8) 8. The variant according to any one of items 5 to 7, wherein the conjugate moiety comprises an acid moiety. (Item 9) 9. The variant of any one of items 5 to 8, wherein the conjugate moiety comprises an acid moiety linked to a hydrophilic spacer. (Item 10) 10. The variant of any one of items 8 or 9, wherein the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-γGlu-C18DA. (Item 11) The peptide is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH 2 (SEQ ID NO: 9), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH 2 (SEQ ID NO: 11), and Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH 2 (SEQ ID NO: 12). (Item 12) 12. The variant of any one of items 10 or 11, wherein the CNP variant is Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1) or PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1). (Item 13) The variant of any one of the preceding items, comprising a linker. (Item 14) 14. The variant of item 13, wherein the linker is located on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. (Item 15) 15. The variant of any one of items 13 or 14, wherein the linker is a hydrolyzable linker. (Item 16) 16. The variant according to any one of items 13 to 15, wherein the linker is on a lysine residue. (Item 17) 17. The variant of any one of items 5 to 16, wherein the CNP variant is linked to the conjugate moiety via the linker. (Item 18) 18. The variant of claim 17, wherein the linker is attached to the conjugate moiety via the hydrophilic spacer of the conjugate moiety. (Item 19) 19. The variant of any one of items 13 to 18, wherein the linker is a peptoid linker or an electronic linker. (Item 20) The variant of any one of the preceding items, wherein the peptide is synthetically produced. (Item 21) 8. The variant of any one of the preceding items, wherein the peptide is stable at 37° C. and pH 7-7.4 for 10 days. (Item 22) The variant of any one of the preceding items, wherein the peptide has an EC50 of 0.1 to 10 nM in a cGMP assay. (Item 23) Item 10. The variant of any one of the preceding items, wherein more than 45% of the peptides are detected after 10 days in aqueous medium at 37°C and pH 7.4. (Item 24) The variant of any one of the preceding items, wherein the peptide is conjugated to one or more lipids, fatty acids, hydrophilic spacer moieties, or optionally combinations thereof. (Item 25) The peptide is Pro-Gly-CNP37, CNP-22, or Pro-Gly The variant of any one of the preceding items, which has a longer half-life compared to both CNP and CNP-22. (Item 26) 26. A pharmaceutical composition comprising the CNP variant according to any one of items 1 to 25 and a pharmaceutically acceptable excipient, carrier, or diluent. (Item 27) 27. The composition according to item 26, which is a lyophilized formulation prepared from a formulation comprising a citric acid / citrate buffer or an acetic acid / acetate buffer having a pH of about 4 to about 6. (Item 28) 28. The composition of claim 27, wherein the lyophilized formulation is prepared from a formulation further comprising an isotonicity adjusting agent or bulking agent selected from the group consisting of mannitol, sucrose, sorbitol, and combinations thereof. (Item 29) 29. The composition of any one of items 27 to 28, wherein the lyophilized formulation is prepared from a formulation further comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, a salt form of ascorbic acid, thioglycerol, and combinations thereof. (Item 30) 30. A method of treating a bone-related disorder or skeletal dysplasia in a subject in need thereof, comprising administering to the subject a composition comprising the CNP variant or composition of any one of items 1 to 29. (Item 31) The bone-related disorder or skeletal dysplasia is selected from the group consisting of osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, dwarfism, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chondrodysplasia punctata, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, hypochondroplasia, rhizometaphyseal chondrodysplasia punctata, Janssen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, osseous dysplasia, osseous dysplasia congenita, osseous dysplasia varus ... 31. The method of claim 30, wherein the dysplasia is selected from the group consisting of midfoot dysplasia, Niebergelt midfoot dysplasia, Robinnow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral dysplasia, Niest dysplasia, fibrochondrodysplasia, Roberts syndrome, distal intermedius dysplasia, brachymetaphyseal dysplasia, Morquio syndrome, Niest syndrome, complex organotrophic dysplasia, and spondyloepiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Lelly-Weill), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation, and idiopathic short stature. (Item 32) 30. A method of lengthening bones or increasing long bone growth in a subject in need thereof, comprising administering to said subject a composition comprising the CNP variant or composition of any one of paragraphs 1-29, wherein said administering lengthens bones or increases long bone growth. (Item 33) 33. The method of any one of items 30 to 32, wherein the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly. (Item 34) 34. The method of any one of items 30 to 33, wherein the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months. (Item 35) 35. The method according to any one of items 30 to 34, wherein the composition is an extended release composition. (Item 36) 1. A method of treating a CNP-responsive condition or disorder, comprising: Administering the CNP variant or composition according to any one of items 1 to 29 to a subject; monitoring the level of at least one bone- or cartilage-related biomarker in the subject; The method, wherein an increase in the level of said at least one bone- or cartilage-related biomarker is indicative of a therapeutic effect of said CNP variant on said subject or said condition or disorder. (Item 37) further comprising adjusting the amount or frequency of administration of said CNP variant; i) increasing the amount or frequency of administration of the CNP variant if the level of the at least one bone- or cartilage-related biomarker falls below a target level; or ii) the amount or frequency of administration of the CNP variant is reduced if the level of the at least one bone- or cartilage-related biomarker is above a target level. (Item 38) 38. The method of item 36 or 37, wherein the at least one bone or cartilage-related biomarker is selected from the group consisting of CNP, cGMP, type II collagen propeptide and fragments thereof, type II collagen and fragments thereof, type I collagen C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), type I procollagen propeptide (PINP) and fragments thereof, type I collagen and fragments thereof, aggrecan chondroitin sulfate, collagen X, and alkaline phosphatase. (Item 39) 26. A method for producing a CNP variant according to any one of items 1 to 25, comprising synthesizing the peptide on a solid-phase resin using Fmoc amino acids. (Item 40) The peptide was dissolved in NMP / Ac. 2 39. The method according to item 38, wherein the acetylated cellulose is acetylated by reacting with O / DIEA (10:1:0.1, v / v / v). (Item 41) 41. The method of claim 39 or 40, wherein the peptide is conjugated to a lipid moiety, optionally on a lysine residue. (Item 42) 42. The method of claim 41, comprising cleaving the protected amino group on the lysine, reacting the peptide with 2xFmoc-aminoPEG(2), followed by reaction with an amino acid, followed by conjugation of a lipid or fatty acid moiety. [Brief explanation of the drawings]

[0065] [Figure 1] 1 illustrates the use of peptoid or electronic linkers in the CNP conjugates described herein. [Figure 2A] 1 shows the stability of CNP variants in human plasma over a 24 hour period. [Figure 2B] 1 shows the stability of CNP variants in human plasma over a 24 hour period. [Figure 3] 1 shows the stability of CNP variants under different culture conditions. [Figure 4] 1 shows the effect of a CNP variant (Pro-Gly-CNP) on cells harboring either NPR2 homozygous or heterozygous mutations, as measured by cGMP stimulation. [Figure 5-1] The nucleotide and predicted protein sequences of the first exon in NPR2 mutant clones transfected into RCS cells are shown. [Figure 5-2] The nucleotide and predicted protein sequences of the first exon in NPR2 mutant clones transfected into RCS cells are shown. [Figure 5-3] The nucleotide and predicted protein sequences of the first exon in NPR2 mutant clones transfected into RCS cells are shown. [Figure 6] 1 shows exemplary NPR2 mutations analyzed for response to CNP. [Figure 7-1] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-2] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-3] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-4] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-5] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-6] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-7] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-8] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-9] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 7-10] Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown. [Figure 8] Figure 8A shows the combined effect of PRS and rare coding variants on height. Figure 8A shows the effect on height as a quantitative trait. Samples were divided into five groups based on their PRS. The violin plot is shown with horizontal lines representing the 25th, 50th, and 75th percentiles of height. Samples were grouped by having missense, loss-of-function, or none status in any of the five core genes. Figure 8B shows the effect reflected in the odds ratios for "idiopathic short stature" or ISS. Odds of ISS using PRS=3 as the reference versus other PRS groups. Figure 8C shows the odds of ISS having missense and / or loss-of-function variants in core genes versus ISS using PRS=1 as the reference. Figure 8D shows the odds of ISS having missense and / or loss-of-function variants in core genes versus ISS using PRS=1 noncarriers as the reference. Figure 8E shows the odds of ISS having missense and / or loss-of-function variants in core genes versus ISS using PRS=2 noncarriers as the reference. Figure 8F. Odds of ISS carrying a missense and / or loss-of-function variant in a core gene versus ISS using PRS=3 non-carriers as reference. G. Odds of ISS carrying a missense and / or loss-of-function variant in a core gene versus ISS using PRS=4 non-carriers as reference. DETAILED DESCRIPTION OF THE INVENTION

[0066] The present disclosure relates to stable CNP variants useful for treating skeletal dysplasias and bone growth disorders.

[0067] As used in the specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly dictates otherwise.

[0068] The term "about" or "approximately" means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of the specified value or range. Whenever the term "about" or "approximately" appears before the first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to each of the numbers in the series.

[0069] The term "C-type natriuretic peptide" or "CNP" refers to a small, single-chain peptide with a 17-amino acid loop structure at its C-terminus (GenBank accession number NP_077720 for the CNP precursor protein, NPPC), and its variants. The 17-mer CNP loop structure is also referred to as CNP-17, the CNP ring, or the CNP cyclic domain. CNP includes an active 53-amino acid peptide (CNP-53) and a mature 22-amino acid peptide (CNP-22), as well as peptides of varying lengths.

[0070] In various embodiments, a "CNP variant" is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to wild-type NPPC over the same number of amino acid residues. It is further contemplated that a CNP variant peptide may comprise from about 1 to about 53, or 1 to 39, or 1 to 38, or 1 to 37, or 1 to 35, or 1 to 34, or 1 to 31, or 1 to 27, or 1 to 22, or 10 to 35, or about 15 to about 37 residues of an NPPC polypeptide. In one embodiment, the CNP variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acid sequence derived from an NPPC polypeptide.

[0071] The term "conjugated moiety" refers to a moiety that is conjugated to a variant peptide. The conjugated moiety comprises a lipid, a fatty acid, a hydrophilic spacer, a synthetic polymer, a linker, or optionally a combination thereof.

[0072] The term "effective amount" refers to a dosage sufficient to produce a desired result with respect to a subject's health condition, medical condition, or disease, or for diagnostic purposes. The desired result may include a subjective or objective improvement in the recipient of the dosage. A "therapeutically effective amount" refers to an amount of an agent effective to produce an intended beneficial effect on health. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. It will be understood that the specific dose level and frequency of administration for a particular patient may vary and will depend on a variety of factors, including the activity of the particular compound used, the bioavailability, metabolic stability, excretion rate and length of action of that compound, the mode and time of administration of the compound, the patient's age, weight, general health, sex, and diet, and the severity of the particular condition.

[0073] "Substantially pure" or "isolated" means that the target species is the predominant species present (i.e., more abundant than any other individual macromolecular species in the composition, on a molar basis), and a substantially purified fraction means that the target species comprises at least about 50% (on a molar basis) of all macromolecular species present. In one embodiment, a substantially pure composition means that the target species comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of the macromolecular species present in the composition, on a molar or weight basis. A target species is purified to essential homogeneity (contaminating species in the composition cannot be detected by conventional detection methods) when the composition consists essentially of a single macromolecular species. For purposes of this definition, solvent species, small molecules (<500 daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species. In one embodiment, the compounds of the present disclosure are substantially pure or isolated. In another embodiment, the compounds of the present disclosure are substantially pure or isolated with respect to the macromolecular starting materials used in their production. In yet another embodiment, the pharmaceutical compositions of the present disclosure comprise a substantially pure or isolated CNP variant mixed with one or more pharmaceutically acceptable excipients, carriers, or diluents, and optionally another biologically active agent.

[0074] "Treatment" refers to prophylactic or therapeutic or diagnostic treatment. In certain embodiments, "treatment" refers to the administration of a compound or composition to a subject for therapeutic, prophylactic, or diagnostic purposes.

[0075] "Prophylactic" treatment is treatment administered to a subject who shows no signs of disease or who shows only early signs of disease, for the purpose of reducing the risk of developing a condition. The compounds or compositions of the present disclosure may be given as prophylactic treatment to reduce the likelihood of developing a condition or to minimize its severity if the condition does develop.

[0076] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of a medical condition with the intent of reducing or eliminating those signs or symptoms. The signs or symptoms may be biochemical, cellular, histological, functional or physical, subjective or objective. The compounds of the present disclosure may also be given as a therapeutic treatment or for diagnosis.

[0077] "Diagnosis" means identifying the presence, extent, and / or nature of a pathological condition. Diagnostic methods vary in their specificity and selectivity. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0078] "Bone- or cartilage-related biomarker" or "bone- or cartilage-related marker" refers to a growth factor, enzyme, protein, or other detectable biological substance or moiety, the level of which increases or decreases in association with, for example, cartilage turnover, chondrogenesis, cartilage growth, bone resorption, bone formation, bone growth, or a combination thereof. Such biomarkers can be measured before, during, and / or after administration of a CNP variant described herein. Exemplary bone- or cartilage-related biomarkers include, but are not limited to, CNP, cGMP, type II collagen propeptide and fragments thereof, type II collagen and fragments thereof, type I collagen propeptide and fragments thereof, type I collagen and fragments thereof, osteocalcin, proliferating cell nuclear antigen (PCNA), aggrecan chondroitin sulfate, collagen X, and alkaline phosphatase. Cartilage- and bone-related biomarkers can be measured in any suitable biological sample, including, but not limited to, tissue, blood, serum, plasma, cerebrospinal fluid, synovial fluid, and urine. In some embodiments, biomarkers are measured in blood, plasma, or serum from animals undergoing efficacy / pharmacodynamic in vivo studies and / or from conditioned medium from ex vivo studies.

[0079] A "pharmaceutical composition" or "formulation" refers to a composition suitable for pharmaceutical use in subjects, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of a CNP variant, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In one embodiment, a pharmaceutical composition encompasses compositions containing the active ingredient, the inactive ingredients that make up the carrier, and any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more components, or from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Thus, a pharmaceutical composition of the present disclosure encompasses any composition made by mixing a compound of the present disclosure with a pharmaceutically acceptable excipient, carrier, or diluent.

[0080] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, etc., such as phosphate-buffered saline solution, 5% dextrose in water, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection, or topical, transdermal, or transmucosal administration).

[0081] A "pharmaceutically acceptable salt" is a salt that can be incorporated into a compound for pharmaceutical use, including, but not limited to, metal salts (e.g., sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0082] "Pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained, or any components present on or in the individual's body.

[0083] "Physiological conditions" refers to the conditions within the body of an animal (e.g., a human). Physiological conditions include, but are not limited to, body temperature, physiological ionic strength, pH, and an aqueous environment for enzymes. Physiological conditions also include the physical conditions of a particular subject that differ from the "normal" conditions present in most subjects, for example, a normal human body temperature of approximately 37°C or a normal human blood pH of approximately 7.4.

[0084] "Physiological pH" or "pH in the physiological range" means a pH in the range of approximately 7.0 to 8.0, more typically in the range of approximately 7.2 to 7.6.

[0085] As used herein, the term "subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; livestock such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or sex. In various embodiments, the subject is a human. In various embodiments, the subject is a child or adolescent. In various embodiments, the subject is an infant. In various embodiments, the subject is over 3 years of age, over 2 years of age, over 1 year of age, or over 6 months of age.

[0086] C-type natriuretic peptide variants C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168:863-870 (1990) (GenBank accession number NP_077720 for CNP precursor protein, NPPC) (J. Hypertens., 10:907-912 (1992)) is a 17-amino acid loop structure (Levin et al. CNP is a small, single-chain peptide in a family of peptides (ANP, BNP, CNP) with a specific function (see, for example, NPR-B, GC-B) and plays an important role in multiple biological processes. CNP interacts with the natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP) (J. Hypertens., 10:1111-1114 (1992)). CNP is more widely expressed in the central nervous system, reproductive tract, bone, and vascular endothelium (Hypertension, 49:419-426 (2007)).

[0087] The native CNP gene and polypeptide have been previously described. US Patent No. 5,352,770 discloses CNP-22 isolated and purified from pig brain, which has the same sequence as human CNP, and its use in the treatment of cardiovascular indications. US Patent No. 6,034,231 discloses the human gene and polypeptide of pre-proCNP (126 amino acids), and the human CNP-53 gene and polypeptide. Mature CNP is a 22-amino acid peptide (CNP-22). Specific CNP variants are disclosed in US Patent No. 8,198,242, which is incorporated herein by reference.

[0088] In various embodiments, the CNPs of the present disclosure range from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53), including truncated CNPs having wild-type amino acid sequences derived from hCNP-53. Such truncated CNP peptides include: DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53) (SEQ ID NO: 56), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 15), RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 16), VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 17), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-49) (SEQ ID NO: 18) TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 19), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 20), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-46) (SEQ ID NO: 21), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 22), AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 23), AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 24), WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 25), ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 26), RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 27), LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 28), LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 3), EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 29), HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 30), PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 4), NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 31), ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 32), RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 33), KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 34), YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 35), KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 36), GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 37), ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 38), NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 39), KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 40), KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 41), GLSKGCFGLKLDRIGSMSGLGC (CNP-22) (SEQ ID NO: 68), LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 42), SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 43), KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 44), GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 45), and CFGLKLDRIGSMSGLGC(CNP-17) (sequence number 67).

[0089] In various embodiments, the CNP variant peptide is a modified CNP-37 or CNP-38 peptide, optionally with a mutation / substitution in the furin cleavage site (underlined) and / or containing a glycine or proline-glycine at the N-terminus. Exemplary CNP-37 variants include, but are not limited to: [ka] .

[0090] In various embodiments, the CNP variants of the disclosure are selected from the group consisting of PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7), PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 5), and PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 1).

[0091] It is further contemplated that any of the variants described herein having fewer than 53 amino acids can be extended by adding amino acids to the N-terminus of the peptide. For example, if a CNP variant is a 34-, 35-, 36-, 37-, 38-, or 39-mer with altered amino acids compared to wt CNP, such variants can be extended at the N-terminus with either wild-type or modified residues from CNP53 or from other peptides.

[0092] In various embodiments, the CNP variant further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus, C-terminus, or attached to an internal amino acid side group. In various embodiments, the acetyl group is on the N-terminus of the peptide.

[0093] In various embodiments, the peptide variant further comprises an OH or NH2 group at the C-terminus.

[0094] In various embodiments, the CNP variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11), and Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12).

[0095] In various embodiments, the CNP variant is PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11), and PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12) PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 7).

[0096] In various embodiments, the CNP variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13), and Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0097] In various embodiments, the CNP variant is selected from the group consisting of Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8). In various embodiments, the CNP variant is Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1). In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1).

[0098] In additional embodiments, for any of the CNP variants described herein having asparagine (Asn / N) and / or glutamine (Gln / Q) residues, whether they have a wild-type sequence or a non-natural amino acid sequence, any Asn residue and / or any Gln residue can be independently substituted with any other natural or non-natural amino acid, including conservative substitutions such as Asn to Gln. Such substitutions are designed, in part, to minimize or avoid potential deamidation of asparagine and / or glutamine.

[0099] In additional embodiments, for any of the CNP variants described herein that have lysine (Lys / K) residues, whether they have a wild-type sequence or a non-natural amino acid sequence, any Lys residue can be independently substituted with any other natural or non-natural amino acid, including substitutions such as Lys to Arg. In various embodiments, all lysine residues are independently substituted with any other natural or non-natural amino acid, including substitutions such as Lys to Arg, except that Lys residues in the CNP variant cyclic domain are not substituted with any other natural or non-natural amino acid.

[0100] In one embodiment, the CNP variant is a CNP variant designated Cys in the wtCNP22 peptide. 6 and Cys 22 It is cyclized via the formation of a disulfide bond between Cys 6 can be, for example, a cysteine ​​analog such as homocysteine ​​or penicillamine. In further embodiments, the CNP variant can be cyclized by a covalent bond formed head-to-tail, side chain-to-side chain, side chain-to-head, or side chain-to-tail. In one embodiment, the covalent bond is formed between an amino acid at or toward the N-terminus of the peptide and an amino acid at or toward the C-terminus of the peptide (referred to in this context as the "terminal" amino acid). In another embodiment, the covalent bond is formed between the side chains of the two terminal amino acids. In yet another embodiment, the covalent bond is formed between the side chain of one terminal amino acid and the terminal group of the other terminal amino acid, or between the terminal groups of the two terminal amino acids.

[0101] Head-to-tail cyclization of the terminal amine to the terminal carboxyl group can be accomplished using several methods, for example, using p-nitrophenyl esters, 2,4,5-trichlorophenyl esters, pentafluorophenyl esters, the azide method, the mixed anhydride method, HATU, carbodiimides (e.g., DIC, EDC, or DCC) with catalysts such as HOBt, HONSu, or HOAt, or on-resin cyclization.

[0102] Additionally, cyclic structures can be formed via bridging groups comprising the side chains of amino acid residues and / or terminal amino acid residues of CNP variants. Bridging groups are chemical moieties that allow for cyclization of two portions of a peptide. Non-limiting examples of bridging groups include amides, thioethers, thioesters, disulfides, ureas, carbamates, sulfonamides, and the like. Various methods for incorporating units bearing such bridging groups are known in the art. For example, lactam bridges (i.e., cyclic amides) can be formed between the N-terminal amino or amino group of a side chain and the C-terminal carboxylic or carboxyl group of side chains, such as those of lysine or ornithine and glutamic or aspartic acid. Thioesters can be formed between the C-terminal carboxyl or carboxyl group of a side chain and the thiol group of a side chain of cysteine ​​or a cysteine ​​analog.

[0103] Alternatively, crosslinks can be formed by incorporating lanthionine (thio-dialanine) residues to link alanine residues covalently attached by thioether bonds. Alternatively, crosslinkers such as dicarboxylic acids (e.g., suberic acid (octanedioic acid)) can link functional groups on amino acid side chains, such as free amino, hydroxyl, and thiol groups.

[0104] Enzyme-catalyzed cyclization can also be used.For example, it has been reported that the thioesterase domain of tyrocidine synthase can be used to cyclize thioester precursors, subtilisin mutants can be used to cyclize peptide glycolate phenylalanyl amide esters, and antibody ligase 16G3 can be used to cyclize p-nitrophenyl esters.For a general review of peptide cyclization, see Davies, J. Peptide Sci., 9:471-501 (2003), the entire contents of which are incorporated herein by reference.

[0105] In certain embodiments, the final product has a purity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99%.

[0106] Peptide conjugates Peptide therapeutics are attractive biological therapeutics, but are often disadvantaged by poor stability and short half-lives in solution (Tang et al., Eur J Pharm Sci. 102:63-70, 2017). Attempts to improve the efficacy of peptide therapeutics by improving stability and / or increasing half-life include encapsulating hydrophilic peptides in biodegradable particles such as liposomes or polymer particles. However, this is difficult due to the cationic nature of these peptides and their ability to electrostatically interact with negatively charged polymeric liposomes (Griesser et al., Int J Pharmaceutics 520:267-274, 2017). The generation of peptide conjugates is one approach used to enable better encapsulation of hydrophilic polymers into microparticles or liposomes (Lu et al., Mol. Pharmaceutics 15:216-225, 2018).

[0107] A peptide can be a string of 5 to 100 amino acids. Peptides can have positively charged amino acids, negatively charged amino acids, or a mixture of both, so that the peptide can interact with charged moieties, such as cations, anions, or combinations thereof, that have species of opposite charge to those in the peptide.

[0108] It is contemplated that the peptide may be conjugated with a moiety that provides increased stability or half-life, such as a conjugate moiety. In various embodiments, the conjugate moiety is conjugated via a non-covalent bond or attached by a covalent bond. The moiety may be non-covalently attached to the peptide via electrostatic interactions. Alternatively, the moiety may be covalently associated with the peptide via one or more linker moieties. The linker may be cleavable or non-cleavable. Cleavable linkers may be cleaved via enzymes, nucleophilic / basic reagents, reducing agents, photoirradiation, electrophilic / acidic reagents, organometallic and metallic reagents, or oxidative reagents. The linker may also be a self-immolative linker. Exemplary linkers include N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and the like. linkers, such as bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene), beta-alanine, 4-aminobutyric acid (GABA), 2-aminoethoxy acid (AEA), aminoethoxy-2-ethoxyacetic acid (AEEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Abx), vicinal diol cleavable linkers, trimethyl lock lactonization, p-alkoxyphenylcarbamate, bicine, peptoid, or bicine-type linkers, and electronic linkers described herein.

[0109] In various embodiments, the linker is attached to a residue of the CNP variant within the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is attached to a lysine residue. In various embodiments, the linker is attached to a lysine residue in the CNP cyclic domain.

[0110] In various embodiments, the CNP variant is attached to the conjugate moiety via a linker. In various embodiments, the linker is attached to the conjugate moiety via a hydrophilic spacer of the conjugate moiety.

[0111] In various embodiments, the linker is a hydrolyzable linker.

[0112] In various embodiments, the linker is a peptoid or electronic linker. In various embodiments, the linker is a peptoid linker. In various embodiments, the linker is an electronic linker. In various embodiments, the linker comprises an SO2 moiety. Exemplary linkers are shown in Figure 1. It is further contemplated that the linkers in Figure 1 may be modified by substitution of the R group. For example, bicine-type linkers include the structures shown below: [ka] .

[0113] In various embodiments, the moiety conjugated to the peptide is a synthetic polymer such as polyethylene glycol, a linker, a lipid moiety, or a fatty acid, or a combination thereof. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a spacer, and a linker. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a polyethylene glycol spacer, or a polyethylene glycol derivative spacer, and a linker. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a spacer, and a linker, wherein the spacer comprises a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, and the carbon atoms of the alkyl chain can be replaced by one or more of O, NH, N(C-1 to C-6 alkyl), or a carbonyl group.

[0114] In various embodiments, the CNP variant is conjugated to a fatty acid. Lipid technology is hypothesized to increase the serum half-life of the CNP variant, allowing for less frequent injections and / or improved oral delivery. In various embodiments, the fatty acid is a short-chain, medium-chain, or long-chain fatty acid, or a dicarboxylic acid fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. In various embodiments, the fatty acid is a C-6 to C-20 fatty acid. In various embodiments, the fatty acid is a C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acid. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or a diacid thereof. In various embodiments, the fatty acid is conjugated to a lysine residue.

[0115] In various embodiments, it is contemplated that the CNP variants described herein comprise a conjugate moiety as described herein. It is contemplated that the conjugate moiety is located on a residue in the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the conjugate moiety is located on a lysine residue. In various embodiments, the conjugate moiety comprises one or more acid moieties. In various embodiments, the acid moiety is a fatty acid.

[0116] In various embodiments, the conjugate moiety comprises an acid moiety linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, where the carbon atoms of the alkyl chain can be replaced by one or more of O, NH, N(C-1 to C-6 alkyl), or a carbonyl group. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu). In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 to C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 alkyl chain. In various embodiments, the hydrophilic spacer is one or more OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is one or two OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the spacer is OEG (8-amino-3,6-dioxaoctanoic acid) or γGlu. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) linked to one or more OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) linked to one or two OEG (8-amino-3,6-dioxaoctanoic acid) groups (diEG). In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-γGlu-C18DA.

[0117] In various embodiments, the CNP variant has the following structure: PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 5), and

[0118] Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8). In various embodiments, the CNP variant has the structure PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC, Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1), or PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1). In various embodiments, the CNP variant comprises an Asn-to-Glu variant of the above peptide.

[0119] In various embodiments, the present disclosure contemplates the use of hydrophilic or water-soluble polymers (e.g., oxygenated alkyl chains in which carbon atoms can be replaced with one or more oxygen atoms, such as polyethylene glycol (PEG) or polyethylene oxide (PEO)). In various embodiments, the water-soluble polymers can vary in type (e.g., homopolymer or copolymer; random, alternating, or block copolymer; linear or branched; monodisperse or polydisperse), linkage (e.g., hydrolyzable or stable linkage, such as amide, imine, aminal, alkylene, or ester bond), conjugation site (e.g., N-terminus, internal, and / or C-terminus), and length (e.g., from about 0.2, 0.4, or 0.6 kDa to about 2, 5, 10, 25, 50, or 100 kDa). Hydrophilic or water-soluble polymers can be conjugated to CNP variants via N-hydroxysuccinimide (NHS)-based or aldehyde-based chemistry or other chemistries, as known in the art. In various embodiments, negatively charged PEG-CNP variants can be designed for reduced renal clearance, including, but not limited to, the use of carboxylated, sulfated, and phosphorylated compounds (Caliceti, Adv. Drug Deliv. Rev., 55:1261-77 (2003); Perlman, J. Clin. Endo. Metab., 88:3227-35 (2003); Pitkin, Antimicrob. Ag. Chemo., 29:440-444 (1986); Vehaskari, Kidney Int'l, 22:127-135 (1982)). In one embodiment, the PEG (or PEO) moiety contains a carboxyl group, a sulfate group, and / or a phosphate group.

[0120] In another embodiment, the hydrophilic polymer (e.g., PEG or PEO) moiety conjugated to the N-terminus, C-terminus, and / or internal site of the CNP variant described herein contains one or more functional groups that are positively charged under physiological conditions. Such moieties are designed, among other things, to improve the distribution of such conjugated CNP variants into cartilage tissue. In one embodiment, the PEG moiety contains one or more primary, secondary, or tertiary amino groups, quaternary ammonium groups, and / or other amine-containing (e.g., urea) groups.

[0121] Production method Also contemplated herein are methods of making compositions comprising a CNP variant as described herein and optionally a conjugate moiety.

[0122] In various embodiments, CNP variants are synthetically produced using standard protein synthesis chemistry. For example, peptides are synthesized stepwise using solid-phase resins and standard Fmoc chemistry. The peptides are cleaved from the resin using trifluoroacetic acid (TFA) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC).

[0123] In various embodiments, the method further comprises acetylating the peptide by reacting the resin with NMP / Ac2O / DIEA, optionally 10:1:0.1, v / v / v.

[0124] Further provided is a method in which a peptide is conjugated to a conjugate moiety, optionally on a lysine residue, by the steps of cleaving the protected amino group on the lysine, reacting the peptide with 2xFmoc-aminoPEG(2), followed by reaction with an amino acid, and then conjugating a lipid or fatty acid moiety. In various embodiments, the conjugate moiety comprises one or more lipids or fatty acids and a hydrophobic spacer.

[0125] The method further provides for cleaving the peptide from the resin by contact with trifluoroacetic acid and purifying the peptide by reverse phase HPLC.

[0126] In certain embodiments, the CNP variants described herein are produced by a recombinant process comprising culturing a host cell in a culture medium containing a first polynucleotide encoding a CNP variant polypeptide, optionally linked to a second polynucleotide encoding a cleavable peptide or protein, under conditions that result in expression of a fusion polypeptide encoded by the polynucleotide. In some embodiments, the host cell is transformed with an expression vector containing a polynucleotide encoding a CNP variant polypeptide, optionally linked to a polynucleotide encoding a cleavable peptide or protein. In certain embodiments, the fusion polypeptide is expressed as a soluble protein or as an inclusion body. The expressed fusion polypeptide can be isolated from the host cell or culture medium, and the isolated fusion polypeptide can be contacted with a cleavage agent to release the CNP variant.

[0127] Methods for making CNP variant peptides, including the use of host cells, expression vectors, cleavable peptides, and culture parameters, are disclosed in US Pat. No. 8,198,242, which is incorporated herein by reference.

[0128] How to use Achondroplasia is the result of an autosomal dominant mutation in the fibroblast growth factor receptor 3 (FGFR-3) gene, which causes abnormalities in cartilage formation. FGFR-3 normally has a negative regulatory effect on chondrocyte growth and, therefore, bone growth. In achondroplasia, mutant forms of FGFR-3 are constitutively active, resulting in severe bone shortening. In humans, activating mutations in FGFR-3 are the primary cause of hereditary dwarfism. FGFR-3-activated mice serve as a model of achondroplasia, the most common form of skeletal dysplasia, and overexpression of CNP rescues these animals from dwarfism. Therefore, functional variants of CNP are potential therapeutic agents for the treatment of various skeletal dysplasias.

[0129] By stimulating chondrocyte matrix production, proliferation, and differentiation and increasing long bone growth, the CNP variants of the present disclosure are useful for treating mammals, including humans, suffering from bone-related disorders such as skeletal dysplasias. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasias include achondroplasia, hypochondroplasia, dwarfism, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta congenita, achondroplasia, chondrodysplasia congenita, homozygous achondroplasia, chondrodysplasia congenita, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta congenita, chondrodysplasia congenita, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta congenita, kyphotic dysplasia ... congenita), short rib polydactyly syndrome, hypochondroplasia, rhizometachondrodysplasia congenita, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, growth imperfect osteogenesis imperfecta, flexor dysplasia, congenital short femurs, Langer metapeduncular dysplasia, Niebergelt metapeduncular dysplasia, Robinnow syndrome, Reinhardt syndrome, acrosophysia imperfecta, peripheral dysplasia, Niest dysplasia, fibrochondrogenesis imperfecta, Roberts syndrome, distal intermedullary dysplasia, brachymetaphyseal dysplasia, Morquio syndrome, Niest syndrome, metabolic dysplasia, and spondyloepiphyseal dysplasia. Short stature, growth plate disorders, bone-related disorders, or skeletal dysplasias contemplated herein include disorders associated with NPR2 mutations, SHOX mutations (Turner syndrome / Lelly-Weill syndrome), and PTPN11 mutations (Noonan syndrome).

[0130] By stimulating chondrocyte matrix production, proliferation, and differentiation and increasing long bone growth, the CNP variants of the present disclosure are useful for treating mammals, including humans, suffering from bone-related disorders such as skeletal dysplasias. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasias include achondroplasia, hypochondroplasia, dwarfism, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta congenita, achondroplasia, chondrodysplasia congenita, homozygous achondroplasia, chondrodysplasia congenita, kyphoplasia limbis, lethal hypophosphatasia congenita, perinatal lethal osteogenesis imperfecta congenita, short rib polydactyly syndrome, hypochondroplasia, rhizomelic chondrodysplasia congenita, Janssen syndrome, rhizomelic dysplasia ... Short stature, growth plate disorders, bone-related disorders, or skeletal dysplasias include spondyloepiphyseal dysplasia, congenital spondyloepiphyseal dysplasia, growth-imperfect osteogenesis imperfecta, skeletal dysplasia, congenital short femurs, Langer midcrus dysplasia, Niebergelt midcrus dysplasia, Robinnow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral dysplasia, Niest dysplasia, fibrochondrodysplasia, Roberts syndrome, distal intermedius dysplasia, brachymetaphyseal dysplasia, Morquio syndrome, Niest syndrome, metabolic dysplasia, and spondyloepiphyseal dysplasia. Short stature, growth plate disorders, bone-related disorders, or skeletal dysplasias contemplated herein include disorders associated with NPR2 mutations, SHOX mutations (Turner syndrome / Lelly-Weill syndrome), PTPN11 mutations (Noonan syndrome), and IGF1R mutations.

[0131] Additional short stature and growth plate disorders contemplated by this method include disorders associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0132] Additional short stature and growth plate disorders contemplated by this method include disorders associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0133] Additionally, CNP variants are useful as supplements or alternatives to growth hormone for treating idiopathic short stature and other skeletal dysplasias.

[0134] Growth plate disorders include disorders that result in short stature or abnormal bone growth and may be the result of a genetic mutation in a gene involved in bone growth, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3. In various embodiments, growth plate disorders include disorders that result in short stature or abnormal bone growth and may be the result of a genetic mutation in a gene involved in bone growth, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RAS-related diseases. In various embodiments, the subject with a growth plate disorder is heterozygous for a mutation in a growth plate gene. In various embodiments, the mutation is a loss-of-function mutation. In various embodiments, mutation is a gain-of-function mutation.Growth plate disorders include but are not limited to familial short stature, dominant familial short stature (also known as dominant inherited short stature) or idiopathic short stature.For example, see Plachy et al., J Clin Endocrinol Metab 104:4273-4281,2019.

[0135] Mutations in ACAN can cause familial osteochondritis dissecans and short stature, ultimately resulting in osteoarthritis characterized by areas of bone damage (or lesions) caused by detachment of cartilage and sometimes bone from the ends of bones at the joints. It has been suggested that a disorganized cartilage network in growing bones impairs their growth, leading to short stature. Mutations associated with ACAN and short stature include Val2303Met. See Stattin et al., Am J Hum Genet 86(2):126-37, 2010. Patients with ACAN mutations that result in short stature are contemplated to benefit from treatment with CNP, as administration may increase the height of these patients through the known interaction of CNP with FGFR3.

[0136] The natriuretic peptide system, including the receptor NPR2, has been shown to be involved in regulating endochondral bone growth (Vasquez et al., Horm Res Pediat 82:222-229, 2014). Studies have shown that homozygous or compound heterozygous loss-of-function mutations in NPR2 cause distal mesomelic dysplasia type Maroteau (AMDM), a skeletal dysplasia associated with very short stature (Vasquez et al., 2014, supra). While some reports suggest heterozygous loss-of-function (e.g., dominant-negative) NPR2 mutations as the cause of short stature, heterozygous gain-of-function NPR2 mutations have been shown to cause tall stature (Vasquez et al., 2014, supra). Given CNP's interaction with NPR2 to stimulate cGMP production, increasing cGMP levels is desirable in these conditions and may have therapeutic benefit in managing these diseases and their associated complications.

[0137] Heterozygous mutation of NPR2 is believed to cause idiopathic short stature and other forms of short stature.Mutation of NPR2 gene is described in Amano et al., J Clin Endocrinol Metab 99:E713-718,2014, Hisado-Oliva et al., J Clin Endocrinol Metab 100:E1133-1142,2015 and Vasques et al., J Clin Endocrinol Metab 98:E1636-1644,2013, and is incorporated herein by reference. Subjects with short stature treated with the CNP variants described herein have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one parent has a height SDS of less than -1.0, -1.5, -2.0, or -2.5, and optionally, the height of a second parent is within the normal range. In various embodiments, CNP variants are useful for treating subjects with short stature who have a height SDS of -2.0 to -3.0. In various embodiments, CNP variants are useful for treating subjects with short stature who have a height SDS of -2.0 to -2.5. However, because de novo mutations in NPR2 can result in short stature, as defined by a height SDS of less than -1.5, -2.0, -2.5, or -3.0, treatment of heterozygous carriers of deleterious mutations in NPR2 in whom neither parent has short stature is also contemplated. Further contemplated is treating individuals heterozygous for deleterious mutations in other growth plate genes with CNP to improve stature and / or enhance bone growth.

[0138] Exemplary NPR2 mutations in patients who may be treated with CNP variants include: [Table 1] [Table 2]

[0139] The role of NPPC in skeletal growth is well documented (Hisado-Oliva et al., Genetics Medicine 20:91-97, 2018). NPPC knockout mice exhibited severe, disproportionate forms of dwarfism, including shortened limbs and endochondral ossification (Hisado-Oliva et al., 2018, supra). Human genome-wide studies have shown a relationship between NPPC and height (Hisado-Oliva et al., 2018, supra). CNP haploinsufficiency is thought to be the cause of short stature in humans, but recent studies have identified heterozygous mutations in families with short stature and hands (Hisado-Oliva et al., 2018, supra). These studies observed a significant reduction in cGMP production when measured in the heterozygous state (Hisado-Oliva et al., 2018, supra). NPPC mutations include the 355G>T missense mutation, which causes a Gly119Cys change, and the 349C>G missense mutation, which causes an Arg117Gly change. CNP variants that rescue CGMP production may offer therapeutic benefit in managing the disorder in patients with heterozygous loss-of-function NPPC mutations.

[0140] Leri-Weill chondro-osseous dysplasia (LWD) is a rare genetic disorder characterized by shortened forearms and lower limbs, abnormal wrist misalignment (Madelung's wrist deformity), and associated short stature. LWD is caused by heterozygous mutations in the short stature homeobox-containing (SHOX) gene or its regulatory elements, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes. (See Rare Disease Database and Carmona et al., Hum Mol Genet 20:1547-1559, 2011.) Langer's metapodial dysplasia occurs when there are two SHOX mutations, which can result from mutations on each chromosome, either homozygous or compound heterozygous mutations. A subset of SHOX mutations causes idiopathic short stature. Turner syndrome occurs due to deletion of the X chromosome, which may include the SHOX gene. SHOX has been identified as being involved in the regulation of FGFR3 transcription and contributing to the control of bone growth (Marchini et al., Endocr Rev. 37:417-448, 2016). Deficiency of SHOX leads to increased FGFR3 signaling, and some evidence supports that SHOX also directly interacts with CNP / NPR2 (Marchini, supra). Given the association of SHOX with FGFR3 and bone growth, it is contemplated that subjects with homozygous or heterozygous SHOX mutations will benefit from treatment with the CNP variants described herein.

[0141] RASopathies are a group of rare genetic conditions caused by mutations in genes in the Ras / mitogen-activated protein kinase (MAPK) pathway. RASopathies are a group of disorders characterized by increased signaling through the RAS / MAPK pathway. This pathway leads to downstream activation of the RAF / MEK / ERK pathway. Short stature is a hallmark of certain RASopathies. For example, CNP signaling inhibits RAF, reducing MEK and ERK activation.

[0142] The present invention contemplates the treatment of RAS diseases. RAS diseases associated with short stature include Noonan syndrome, Costello syndrome, cardiofacial cutaneous syndrome, neurofibromatosis type 1, and Leopard syndrome. Hereditary gingival fibromatosis type 1 is also a RAS disease contemplated herein. RAS disease patients (including Noonan syndrome, Costello syndrome, cardiofacial cutaneous syndrome, neurofibromatosis type 1, Leopard syndrome, and hereditary gingival fibromatosis type 1) include patients with heterozygous variants in one or more of the following genes: BRAF, CBL, HRAS, KRAS, LZTR1 MAP2K1, MAP2K2, MRAS, NF1, NRAS, PPP1CB, PTPN11, RAF1, RRAS, RIT1, SHOC2, SOS1, or SOS2 (Tajan et al. Endocr. Rev. 2018; 39 (5): 676-700).

[0143] CFC is caused by mutations in several genes in the Ras / MAPK signaling pathway, including K-Ras, B-Raf, Mek1, and Mek2. Costello syndrome, also known as faciocutaneous skeletal (FCS) syndrome, is caused by activating mutations in the H-Ras gene. Hereditary gingival fibromatosis type I (HGF) is caused by dominant mutations in the SOS1 gene (Son of Sevenless Homolog 1), which encodes a guanine nucleotide exchange factor (SOS) for the Ras subfamily of small GTPases. Neurofibromatosis type I (NF1) is caused by mutations in the neurofibromin 1 gene, which encodes a negative regulator of the Ras / MAPK signaling pathway. Noonan syndrome (NS) is caused by mutations in one of several genes, including PTPN11, which encodes SHP2, SOS1, K-Ras, and Raf-1.

[0144] CNP has been demonstrated to be an effective therapy in RAS disease models. Ono et al. generated mice lacking Nf1 in type II collagen-producing cells (Ono et al., Hum. Mol. Genet. 2013;22(15):3048-62). These mice exhibited constitutive ERK1 / 2 activation and reduced chondrocyte proliferation and maturation. Daily injection of CNP into these mice reduced ERK phosphorylation and corrected their short stature. A mouse model of cardiofacial cutaneous syndrome using the Braf mutation (p.Q241R) (Inoue et al., Hum. Mol. Genet. 2019;28(1):74-83) showed reduced body length and growth plate width, smaller areas of proliferation and hypertrophy, and increased body length compared to wild-type mice. CNP administration resulted in increased body length in these animals.

[0145] Mutations in multiple genes can cause Noonan syndrome, which is characterized by short stature, heart defects, bleeding problems, and skeletal deformities. Mutations in the PTPN11 gene cause approximately half of all cases of Noonan syndrome. Mutations in the SOS1 gene cause another 10-15%, and the RAF1 and RIT1 genes each account for approximately 5% of cases. Mutations in other genes each account for a small number of cases. The cause of Noonan syndrome is unknown in 15-20 percent of people with the disorder.

[0146] The PTPN11, SOS1, RAF1, and RIT1 genes all encode important proteins in the RAS / MAPK cell signaling pathway, which is necessary for cell division and growth (proliferation), differentiation, and cell migration. Many of the gene mutations associated with Noonan syndrome turn on (activate) the resulting proteins, and this prolonged activation alters normal RAS / MAPK signaling, disrupting the regulation of cell growth and division and resulting in the characteristics of Noonan syndrome. See, for example, Chen et al., Proc Natl Acad Sci US A. 111(31):11473-8, 2014; Romano et al., Pediatrics. 126(4):746-59, 2010; and Milosavljevic et al., Am J Med Genet 170(7):1874-80, 2016. Subjects with mutations that activate the MAPK pathway are likely to benefit from treatment with CNP variants as described herein to improve bone growth and short stature. It is also contemplated that subjects with mutations that activate the MAPK pathway will benefit from treatment with the CNP variants described herein to ameliorate other comorbidities associated with an overactive MAPK pathway in other cells throughout the body that express the NPR2 receptor on their surface.

[0147] Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, cause disorders characterized by short stature, such as Noonan syndrome (Musente et al., Eur J Hum Genet 11:201-206 (2003)). Musente (supra) has identified numerous mutations in the PTPN11 gene that lead to short stature. Gain of function mutations cause hyperactive signaling through SHP2, inhibiting growth hormone-induced IGF-1 release, thereby contributing to reduced bone growth (Rocca Serra-Nedelec, PNAS 109:4257-4262, 2012). Subjects with homozygous or heterozygous PTPN11 mutations are contemplated to benefit from treatment with CNP variants as described herein to improve bone growth and short stature.

[0148] Mutations in the Indian Hedgehog (IHH) gene, which regulates endochondral ossification, are also associated with short stature syndrome (Vasques et al., J Clin Endocrinol Metab. 103:604-614, 2018). Many identified IHH mutations segregate with short stature in a dominant inheritance pattern. Given the association of IHH with bone growth and ossification, subjects with homozygous or heterozygous IHH mutations may benefit from treatment with the CNP variants described herein.

[0149] Mutations in FGFR3, including N540K and K650N, cause short stature and hypochondroplasia.

[0150] The insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (α2β2) transmembrane glycoprotein with intrinsic kinase activity. IGF1R has been shown to play a role in prenatal and postnatal growth. Heterozygous mutations in IGF1R have been identified in individuals with small-for-gestational age (SGA) and familial short stature (Kawashima et al., Endocrine J. 59:179-185, 2012). IGF1R mutations associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, supra).

[0151] Height is a highly heritable trait and can be influenced by the combined effects of hundreds or even thousands of genes (Wood et al., 2014, Nature Genetics, 46:1173-1189). An individual's short stature may be the result of the combined effects of these genes, rather than a single gene being the primary cause. It is contemplated that such individuals with short stature, as defined by a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, could be beneficially treated with CNP variants, for example, to improve bone growth and bone length, given CNP's ability to increase the length of normal animals.

[0152] In various embodiments, CNP variants are useful for treating short subjects with a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and having at least one parent with a height SDS of less than -1.0, -1.5, -2.0, or -2.5, and optionally, the height of a second parent is within the normal range. In various embodiments, CNP variants are useful for treating short subjects with a height SDS of -2.0 to -3.0. In various embodiments, CNP variants are useful for treating short subjects with a height SDS of -2.0 to -2.5. In various embodiments, the short stature is associated with one or more mutations in genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), or a combination thereof.

[0153] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0154] In various embodiments, short stature is the result of mutations in multiple genes as determined by a polygenic risk score (PRS). The largest published GWAS meta-analysis of height, not including any samples from the UK Biobank Project, described in Example 4, was used to calculate the polygenic risk score (PRS) for height. The cohort was divided into five PRS quintiles (PRS1 being the shortest height and PRS5 being the longest height). In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in FGFR3 and a low PRS. In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in IGF1R and a low PRS. In various embodiments, the subject has a mutation in NPPC and a low PRS. In various embodiments, the subject has a mutation in SHOX and a low PRS. In various embodiments, the subject has one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX and a low PRS. In various embodiments, PRS is 1 or 2. In various embodiments, PRS is 1. In various embodiments, PRS is 2.

[0155] In addition, CNP variants are useful for the treatment of other bone-related conditions and disorders, such as rickets, hypophosphatemic rickets (including X-linked hypophosphatemic rickets (also called vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets), and osteomalacia (including tumor-induced osteomalacia (also called oncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)).

[0156] The CNP variants of the present disclosure can also be used to treat osteoarthritis. Osteoarthritis is a degenerative disease of articular cartilage that frequently occurs in the elderly. Osteoarthritis involves cartilage destruction due to degeneration of joint components and proliferative changes in bone and cartilage, which lead to secondary arthritis (e.g., synovitis). In osteoarthritis, extracellular matrix proteins, the functional entities of cartilage, are reduced, and the number of chondrocytes decreases (Arth. Rheum. 46(8):1986-1996(2002)). By promoting chondrocyte matrix production, growth, and differentiation, the CNP composition counters the undesirable effects of FGF-2 and increases matrix synthesis in subjects suffering from arthritis, including osteoarthritis, making it useful for treating arthritis, including osteoarthritis.

[0157] In certain embodiments, CNP variants and compositions and formulations comprising the same of the present disclosure are useful for improving one or more symptoms or physiological consequences of skeletal dysplasia, where the improvement can be increased absolute growth, increased growth velocity, increased qualitative computed tomography (QCT) bone mineral density, improved growth plate morphology, increased long bone growth, improved spine morphology, improved elbow range of motion, and / or reduced sleep apnea. In this regard, it should be noted that the terms "improved," "improvement," "increase," "reduction," and their grammatical equivalents, when used in connection with a symptom or physiological consequence of a disease state, are all relative terms referring to the state of the symptom or physiological consequence of the disease after treatment with a CNP variant of the invention (or a composition or formulation comprising same) compared to the same symptom or physiological consequence of the disease before treatment with a CNP variant of the invention (or a composition or formulation comprising same) (i.e., compared to the "baseline"). As noted above, the "baseline" condition can be determined either by measuring the subject's condition before treatment (which can then be compared with the condition of the same subject after treatment), or by measuring that condition in a population of subjects suffering from the same affliction who share the same or similar characteristics (e.g., age, sex and / or disease state or progression).

[0158] Also provided are methods for overcoming cell growth arrest induced by constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3), comprising contacting a cell expressing constitutively active FGFR-3 with a CNP variant or composition described herein.

[0159] Further provided is a method of stimulating cGMP production in a cell expressing natriuretic peptide receptor B (NPR-B), comprising contacting the cell expressing NPR-B with a CNP variant or composition described herein.

[0160] In yet another embodiment, the present disclosure provides a CNP variant that stimulates in vitro or in vivo the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP levels produced by the same concentration of wtCNP22 (e.g., 1 uM). In yet a further embodiment, the CNP variant of the present disclosure stimulates in vitro or in vivo the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP levels produced by the same concentration of wtCNP22 (e.g., 1 uM).

[0161] It is contemplated that any of the CNP variants described herein are useful in this method.

[0162] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker.

[0163] The effectiveness of treatment is measured by various parameters. In various embodiments, effectiveness is evaluated as the change in annual growth velocity from the baseline period to the intervention period. Effectiveness is also evaluated as the change in height SDS from baseline to the end of treatment, measured using CDC growth curves, and growth velocity SDS is based on childhood bone mineral density studies (Kelly et al., J.Clin.Endocrinol.Metab.2014;99(6):2104-2112).

[0164] QoLISSY, Quality of Life in Short Stature Youth, will be assessed as instructed (Quality of Life in Short Stature Youth-The QoLISSY Questionnaire User's Manual. Lengerich: Pabst Science Publishers; 2013).

[0165] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions, including modified-release compositions, comprising a CNP variant described herein and one or more pharmaceutically acceptable excipients, carriers, and / or diluents. In certain embodiments, the compositions further comprise one or more other biologically active agents (e.g., inhibitors of proteases, receptor tyrosine kinases, and / or clearance receptors NPR-C).

[0166] The present disclosure provides modified-release compositions comprising the conjugate moieties described herein. Modified-release compositions include those that deliver a drug for a delayed period (delayed-release dosage) or an extended period (extended-release dosage) after administration. Various embodiments of the CNP peptide conjugates provided herein include modified-release compositions, such as extended-release, sustained-release, or controlled-release, and delayed-release. The term "extended-release composition" refers to a composition formulated in a manner that makes the active ingredient / drug available for an extended period after administration (USP). Extended-release dosages include sustained-release (SR) or controlled-release (CR) forms. Sustained release maintains drug release over a sustained period, but not necessarily at a constant rate, while CR maintains drug release at a nearly constant rate over a sustained period (Pharmaceutics: Drug Delivery and Targeting, Yvonne Perrie, Thomas Rades, Pharmaceutical Press, 2009). Delayed-release compositions or products are engineered to delay the release of the active drug substance for a period of time after initial administration.

[0167] In various embodiments, the modified release composition is an extended release composition.

[0168] In various embodiments, for an extended release composition, at pH 7-7.6, (i) less than about 20% of the peptide is released by day 1, and (ii) about 90% of the peptide is released weekly, or about 90% of the peptide is released every other week, or about 90% of the peptide is released monthly.

[0169] In various embodiments, at pH 7-7.6, less than about 20% of the peptide is released by day 1. In various embodiments, at pH 7-7.6, less than about 10% of the peptide is released by day 1. Furthermore, it is contemplated that (i) at pH 7.0-7.6, less than about 30%, or about 40%, or about 50% of the peptide is released by day 1, and (ii) at pH 7-7.6, about 90% of the peptide is released weekly, or about 90% of the peptide is released every two weeks, or about 90% of the peptide is released monthly. It is further contemplated that (i) at pH 7.0-7.6, about 30%, or about 40%, or about 50%, or less than about 60% of the peptide is released by day 1; and (ii) at pH 7.0-7.6, about 70%, about 80%, or about 90% of the peptide is released weekly, or about 70%, about 80%, or about 90% of the peptide is released every two weeks, or about 70%, about 80%, or about 90% of the peptide is released every three weeks, or about 70%, about 80%, or about 90% of the peptide is released monthly. In various embodiments, at pH 7-7.6, about 90% of the peptide is released weekly. In various embodiments, at pH 7-7.6, about 90% of the peptide is released every two weeks. In various embodiments, at pH 7-7.6, about 90% of the peptide is released monthly. It is further contemplated that release can be at a pH of pH 7.0-7.6, pH 7.1-7.5, pH 7.2-7.4, pH 7.2-7.6, or pH 7.0-7.4.

[0170] In various embodiments, (i) at pH 7.0-7.6, less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released by day 1; and (ii) at pH 7-7.6, about 90% of the peptide is released weekly, or about 90% of the peptide is released every two weeks, or about 90% of the peptide is released monthly. Further, (i) at pH 7.0-7.6, less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released by day 1; (ii) at pH 7-7.6, about 70%, about 80%, or about 90% of the peptide is released weekly, or about 70%, about 80%, or about 90% of the peptide is released every two weeks, or about 70%, about 80%, or about 90% of the peptide is released every three weeks, or about 70%, about 80%, or about 90% of the peptide is released every three weeks. or alternatively, ii) at pH 7-7.6, about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released weekly, or about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released every two weeks, or about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released every three weeks, or about 70%, about 75%, about 80%, about 85%, or about 90% of the peptide is released monthly.

[0171] In various embodiments, the composition comprises an excipient, diluent, or carrier. In various embodiments, the extended release composition comprises an excipient, diluent, or carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0172] Non-limiting examples of excipients, carriers, and diluents include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, etc. The composition can include liquids (e.g., water, ethanol). Diluents with various buffer contents (e.g., Tris-HCl, phosphate, acetate buffer, citrate buffer), pH and ionic strength, detergents and solubilizers (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, m-cresol), and bulking substances (e.g., lactose, mannitol, sucrose). The use of excipients, diluents, and carriers in the formulation of pharmaceutical compositions is known in the art, see, for example, Remington's Pharmaceutical Sciences, 18, incorporated herein by reference in its entirety. th Edition, pages 1435-1712, Mack Publishing Co., Easton, Pennsylvania (1990).

[0173] For example, carriers include, but are not limited to, diluents, vehicles and adjuvants, and implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials that do not react with active ingredients.Non-limiting examples of carriers include phosphate buffered saline, saline, water, and emulsions (e.g., oil / water emulsions).Carriers can be, for example, solvents or dispersion media, including ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, and their mixtures.

[0174] In some embodiments, the composition is a liquid formulation. In certain embodiments, the formulation comprises a CNP variant at a concentration ranging from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 0.5 to 5 mg / ml, or from about 0.5 to 3 mg / ml, or from about 1 mg / ml to about 10 mg / ml. In various embodiments, the CNP variant is at a concentration of 0.8 mg / ml to 2 mg / ml. In various embodiments, the CNP variant is at a concentration of 0.8 mg / ml. In various embodiments, the CNP variant is at a concentration of 2.0 mg / ml. In various embodiments, the CNP variant is reconstituted from a lyophilized powder.

[0175] In further embodiments, the composition includes a buffer or buffering agent to maintain the pH of the CNP-containing solution or suspension within a desired range. Non-limiting examples of buffers include phosphate-buffered saline, Tris-buffered saline, and Hank's buffered saline. Buffering agents include, but are not limited to, sodium acetate, sodium phosphate, and sodium citrate. Mixtures of buffering agents can also be used. In certain embodiments, the buffering agent is acetic acid / acetate salt or citric acid / citrate salt. The amount of buffering agent suitable for the composition depends in part on the particular buffer used and the desired pH of the solution or suspension. In some embodiments, the buffering agent has a concentration of about 10 mM ± 5 mM. In certain embodiments, the pH of the composition is about pH 3 to about pH 9, or about pH 3 to about pH 7.5, or about pH 3.5 to about pH 7, or about pH 3.5 to about pH 6.5, or about pH 4 to about pH 6, or about pH 4 to about pH 5, or about pH 5.0 ± 1.0. In various embodiments, the pH is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In various embodiments, the pH is 5.5.

[0176] In other embodiments, the composition contains an isotonicity adjusting agent to make the solution or suspension isotonic and more suitable for administration.Non-limiting examples of isotonicity agents include NaCl, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol.In certain embodiments, the isotonicity agent is NaCl.In certain embodiments, the NaCl is at a concentration of about 160±20 mM, or about 140 mM±20 mM, or about 120±20 mM, or about 100 mM±20 mM, or about 80 mM±20 mM, or about 60 mM±20 mM.

[0177] In yet other embodiments, the composition contains a preservative, including but not limited to m-cresol and benzyl alcohol. In certain embodiments, the preservative is present at a concentration of about 0.4%±0.2%, about 1%±0.5%, about 1.5%±0.5%, or about 2.0%±0.5%.

[0178] In yet other embodiments, the composition contains an anti-adsorption agent (e.g., to reduce adsorption of the CNP variant to glass or plastic). Anti-adsorption agents include, but are not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In certain embodiments, the anti-adsorption agent is at a concentration of about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%.

[0179] In additional embodiments, the composition includes a stabilizer. Non-limiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and their salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or a salt thereof, the stabilizer is present at a concentration of about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer is present at a concentration of about 0.01% to about 0.5%, or about 0.01% to about 0.2%. In yet other embodiments, when the stabilizer is glycerin, the stabilizer is present at a concentration of about 5% to about 100% (neat).

[0180] In further embodiments, the composition includes an antioxidant. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP is about 0.1:1 to about 15:1, or about 1:1 to about 15:1, or about 0.5:1 to about 10:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0181] Pharmaceutically acceptable salts can be used in the compositions, including, but not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), salts of organic acids (e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate), and salts of amines (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A complete discussion of pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Company, Easton, Pennsylvania (1990).

[0182] Pharmaceutical compositions can be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage form of the composition can be adjusted to suit the desired mode of administration of the composition. For oral administration, the composition can take the form of, for example, a tablet or capsule (including softgel capsules), or can be, for example, an aqueous or non-aqueous solution, suspension, or syrup. Tablets and capsules for oral administration can contain one or more commonly used excipients, diluents, and carriers, such as mannitol, lactose, glucose, sucrose, starch, corn starch, sodium saccharin, talc, cellulose, magnesium carbonate, and lubricants (e.g., magnesium stearate, sodium stearyl fumarate). Flavorings, colorings, and / or sweeteners can be added to solid and liquid formulations as needed. Other optional components of oral formulations include, but are not limited to, preservatives, suspending agents, and thickeners. Oral formulations can also be enteric coated to protect the CNP variant from the acidic environment of the stomach. Methods for preparing solid and liquid dosage forms are known, or will be apparent, to those skilled in the art (see, eg, Remington's Pharmaceutical Sciences, referenced above).

[0183] Preparations for parenteral administration can be prepared, for example, as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in liquid media prior to injection, or as emulsions. For example, sterile injectable solutions and suspensions can be formulated according to techniques known in the art using suitable diluents, carriers, solvents (e.g., aqueous buffer solutions, Ringer's solution, isotonic sodium chloride solution), dispersants, wetting agents, emulsifiers, suspending agents, etc. In addition, sterile fixed oils, fatty acid esters, polyols, and / or other inactive ingredients can be used. For further example, preparations for parenteral administration include aqueous sterile injectable solutions and aqueous and non-aqueous sterile suspensions, which can contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient, and can include suspending agents and thickening agents.

[0184] Compositions containing CNP variants may also be lyophilized formulations. In certain embodiments, lyophilized formulations contain a buffer and a bulking agent, and optionally, an antioxidant. Exemplary buffers include, but are not limited to, acetate buffer and citrate buffer. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, mannitol is present in an amount of about 3% to about 10%, or about 4% to about 8%, or about 4% to about 6%. In certain embodiments, sucrose is present in an amount of about 6% to about 20%, or about 6% to about 15%, or about 8% to about 12%. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid.

[0185] In various embodiments, the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).

[0186] The present disclosure also provides kits comprising, for example, bottles, vials, ampoules, tubes, cartridges, and / or syringes containing liquid (e.g., sterile injectable) or solid (e.g., lyophilized) formulations. The kits may also include pharmaceutically acceptable vehicles or carriers (e.g., solvents, solutions, and / or buffers) for reconstituting solid (e.g., lyophilized) formulations into solutions or suspensions for administration (e.g., by injection), including, but not limited to, reconstituting lyophilized formulations in syringes for injection or for diluting concentrates to lower concentrations. Furthermore, extemporaneous injection solutions and suspensions can be prepared, for example, from sterile powders, granules, or tablets containing the CNP-containing composition. The kits may also include dispensing devices, such as aerosol or injection dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes, and / or needles.

[0187] As a non-limiting example, the kit can include a syringe with a single or dual chamber. In the case of a single-chamber syringe, the single chamber can contain a liquid CNP formulation ready for injection, or a liquid formulation of a solid (e.g., lyophilized) CNP formulation or CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension for injection. In the case of a dual-chamber syringe, one chamber can contain a pharmaceutically acceptable vehicle or carrier (e.g., a solvent system, solution, or buffer) for injection, and the other chamber can contain a liquid formulation of a solid (e.g., lyophilized) CNP formulation or CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension using the vehicle or carrier from the first chamber.

[0188] As a further example, the kit can include one or more pen injector or auto-injector devices and a dual-chamber cartridge. For injection, one chamber of the cartridge can contain a pharmaceutically acceptable vehicle or carrier (e.g., a solvent system, solution, or buffer), and the other chamber can contain a solid (e.g., lyophilized) CNP formulation or liquid formulation of a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin), which can be reconstituted into a solution or suspension using the vehicle or carrier from the first chamber. The cartridge can contain a sufficient amount of CNP variant for dosing over a desired period of time (e.g., 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen injector or auto-injector can be adjusted to administer the desired amount of CNP formulation from the cartridge.

[0189] Administration and Dosage CNP variants, or pharmaceutical compositions or formulations comprising them, can be administered to a subject in a variety of ways, such as subcutaneously, intraarticularly, intraperitoneally, intramuscularly, intradermally, or orally, In one embodiment, the CNP variant composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0190] The CNP variant or a composition thereof can also be administered by implantation of a depot at the target site of action (e.g., an abnormal or degenerative joint or cartilage region). Alternatively, the CNP variant can be administered sublingually (e.g., a sublingual tablet), by transdermal delivery (e.g., by a patch on the skin), or orally in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamide, polylactide, polyglycolide, poly(lactide-glycolide)), microemulsions, etc.

[0191] The CNP variant compositions described herein can be administered to a patient in need thereof at a therapeutically effective dose to treat, ameliorate, or prevent bone-related disorders (e.g., skeletal dysplasias, including achondroplasia). The safety and therapeutic efficacy of the CNP variants can be determined by standard pharmacological procedures in cell cultures or experimental animals, e.g., LD 50 (a dose lethal to 50% of the population) and ED 50 The LD can be determined by determining the therapeutically effective dose in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Generally, active agents that exhibit large therapeutic indices are preferred.

[0192] In certain embodiments, the CNP variant compositions described herein are administered at a dose ranging from about 3, 4, 5, 6, 7, 8, 9, or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP composition is administered at a dose of about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, or 2000 nmol / kg, or other doses deemed appropriate by the treating physician. In other embodiments, the CNP variant composition comprises a CNP variant having a saturation of about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 70 The CNP or CNP variant may be administered at a dose of 0, 750, 800, 850, 900, 950, or 1000 μg / kg, or at a dose of about 0.5, 0.8, 1.0, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / kg, or at other doses deemed appropriate by the treating physician. The dose of CNP or CNP variant described herein may be administered according to the dosing / administration frequencies described herein, including, but not limited to, daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc. In various embodiments, the CNP or CNP variant is administered subcutaneously daily. In various embodiments, the CNP or CNP variant is administered subcutaneously weekly. In various embodiments, the CNP variant is administered at a dose of 2.5 μg / kg / day to 60 μg / kg / day, 10 μg / kg / day to 45 μg / kg / day, or 15 μg / kg / day to 30 μg / kg / day.In various embodiments, the CNP variant is administered at a dose of 15 μg / kg / day. In various embodiments, the CNP variant is administered at a dose of 30 μg / kg / day.

[0193] The frequency of dosing / administration of a CNP variant to a particular subject can vary depending on various factors, including the disorder being treated and the subject's condition and response to therapy. The CNP variant can be administered in a single dose or multiple doses per dose. In certain embodiments, the CNP variant composition is administered in a single dose or multiple doses once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months, or as deemed appropriate by the treating physician. In various embodiments, the CNP variant is administered for 3 months, 6 months, 12 months or more.

[0194] In some embodiments, the CNP variant composition is administered to allow for a period of growth (e.g., chondrogenesis), followed by a period of recovery (e.g., bone formation). For example, the CNP composition is administered subcutaneously or otherwise daily or multiple times weekly for a period of time, followed by a period of no treatment, and then the cycle is repeated. In some embodiments, the initial period of treatment (e.g., administration of the CNP variant composition daily or multiple times weekly) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In related embodiments, the period of no treatment lasts 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the dosing regimen for the CNP variant composition is daily for 3 days followed by 3 days off, or daily or multiple times per week for 1 week followed by 3 days or 1 week off, or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off, or daily or multiple times per week for 3 weeks followed by 1, 2, or 3 weeks off, or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks followed by 1, 2, 3, or 4 weeks off.

[0195] Biomarkers In the treatment of bone-related disorders, indicators of growth can be measured, including intrauterine and neonatal long bone growth measurements and bone growth biomarkers such as CNP, cGMP, collagen II, collagen X, osteocalcin, and proliferating cell nuclear antigen (PCNA).

[0196] One CNP signaling marker is cGMP (guanosine 3',5' cyclic monophosphate). Levels of this intracellular signaling molecule increase after CNP binds to and activates its cognate receptor, NPR-B. Elevated levels of cGMP can be measured in cell culture extracts (in vitro) after CNP exposure, in conditioned medium from bone explant studies (ex vivo) after CNP exposure, and in plasma (in vitro) within minutes of CNP administration via subcutaneous, intravenous, or other routes of administration known in the art.

[0197] Cartilage- and bone-specific analytes (or cartilage- and bone-related markers) can also be measured to assess the efficacy of CNP. For example, cleaved collagen type II fragments are a cartilage-specific marker of cartilage turnover. Type II collagen is the major organic component of cartilage, and type II collagen fragments (cleaved collagen) are released into the circulation and secreted into the urine following cartilage turnover. Cartilage turnover precedes new bone formation.

[0198] A measurable bone-specific biomarker of bone formation is the N-terminal propeptide of type I procollagen (PINP). Because type I collagen is the major organic component of the bone matrix, its synthesis is a critical step in bone formation. During collagen synthesis, the propeptide is released from the procollagen molecule and can be detected in serum. Furthermore, fragments of type I collagen can be measured as a marker of bone resorption.

[0199] Other potential biomarkers for cartilage and bone formation and growth include aggrecan chondroitin sulfate (a cartilage-specific marker for cartilage turnover), type II collagen propeptide (a cartilage-specific marker for chondrogenesis), type I collagen C-telopeptide (CTx), alkaline phosphatase (bone-specific), and osteocalcin (a bone-specific marker for bone formation). Cartilage- and bone-related biomarkers can be measured, for example, in serum from efficacy / pharmacodynamic in vivo studies and from conditioned medium from ex vivo studies using commercially available kits.

[0200] In one embodiment, to monitor the effect of the CNP composition on bone and cartilage formation and growth in vivo, the level of at least one bone- or cartilage-related biomarker is assayed or measured in subjects administered the CNP variant or composition described herein. For example, an increase in the level of at least one bone- or cartilage-related biomarker may indicate that the administration of the CNP variant or composition has a positive effect on bone growth and is a useful treatment for skeletal dysplasia and other bone- or cartilage-related diseases or disorders associated with decreased CNP activity. Exemplary bone- or cartilage-related biomarkers include, but are not limited to, CNP (e.g., endogenous levels of CNP), cGMP, type II collagen propeptide and its fragments, type II collagen and its fragments, type I collagen C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), type I procollagen propeptide (PINP) and its fragments, type I collagen and its fragments, collagen X, aggrecan chondroitin sulfate, and alkaline phosphatase.

[0201] In various embodiments, biomarkers are measured by obtaining biological samples from subjects who are administered, are being administered, or have been administered CNP variants. Biomarkers can be measured using techniques known in the art, including but not limited to Western blot, enzyme-linked immunosorbent assay (ELISA), and enzyme activity assay. Biological samples can be blood, serum, urine, or other bodily fluids.

[0202] Further aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. [Example]

[0203] Example 1: Synthesis of CNP variants CNP variant peptides were synthesized on a solid phase using a resin leaving a C-terminal COOH on a Symphony / Prelude (Protein Technologies Inc., USA), Voyager (CEM GmbH, Germany), or SyroII (MultiSyntech, Germany) synthesizer.

[0204] All Fmoc-amino acids were purchased from Biosolve (Netherlands) or Bachem GmbH (Germany) and had side chain functional groups protected with Nt-Boc (KW), Ot-Bu (DESTY), N-Trt (HNQ), S-Trt (C), or N-Pbf (R) groups. All amino acid coupling steps used a 5-fold excess of HBTU / HOBt / amino acid / DIPEA (1:1:1:2) in NMP with a 20 min activation time using double coupling.

[0205] Acetylation (Ac) of the peptide was carried out by reacting the resin with NMP / Ac2O / DIEA (10:1:0.1, v / v / v) for 30 min at room temperature.

[0206] For moiety conjugation, the protected amino group on the lysine was cleaved to create a reactive group. Standard Fmoc synthesis was used to react 2xFmoc-aminoPEG (2), followed by glutamic acid, followed by the C18-diacid.

[0207] The completed peptide was cleaved from the resin by reaction with TFA (40 mL / mmol resin) for 2 h at room temperature. The crude peptide was filtered and precipitated with ice-cold EtO, followed by lyophilization and final purification by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The final product and purity were confirmed by mass spectrometry.

[0208] CNP variants were generated based on the sequence of Pro-Gly CNP-37 (PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1)), optionally with different amino acid residue changes and / or acetylation at the N-terminus and / or other modifications, including the following (amino acid changes are underlined): [ka] .

[0209] The fully reduced peptide was dissolved in 0.1 M Tris buffer (pH 8.0) containing 1 mM cysteine ​​(SS form) and 8 mM cysteine ​​(SH form) at a final concentration of 0.1 mg / mL, with or without guanidine HCl, and stirred at room temperature. Disulfide formation was monitored by HPLC analysis until no further peak changes were observed. The mixture was loaded onto a preparative RP-HPLC for purification.

[0210] Example 2: Characterization of synthesized CNP variants CNP variants synthesized as in Example 1 were analyzed by mass spectrometry and UV spectroscopy to determine purity and stability after 10 days.

[0211] Stability was measured using RP-HPLC analysis of purity over time periods T0 to T10. Briefly, CNP variants were diluted 1:5 with buffer: 5 mM citrate, 6% sucrose, 1.5% mannitol, 0.7 mg / ml methionine, and 0.005% Tween® 80, pH 5.6. A 10 μL injection volume was injected onto a Phenomenex Aeris XB-C18 RP column (2 μm, 100A, 2.1 x 250 mm, p / n 00G-4505-AN). HPLC conditions were: Mobile phase A: HO / 0.05% TFA, pH 3 w / NH4OH (approximately 3 mM final); Mobile phase B: CH3CN / 0.05% TFA in 70% HO, pH 3 w / NH4OH (approximately 3 mM final); and a flow rate of 0.25 mL / min. Measurements were performed at a column temperature of 55°C and UV: 214 nm (below 4 nm), ref: 360 nm (below 20 nm), UV: 280 nm (below 4 nm), ref: 360 nm (below 20 nm). Stability measurements were obtained by storing CNP variants in PBS, pH 7.4, at 37°C for 10 days. The results of the stability measurements are shown in Table 1 below as % variant detected at T0 (day 0) or T10 (day 10). [Table 3]

[0212] CNP variants were tested for activity by cGMP stimulation assay using the CatchPoint Cyclic-GMP Fluorescent Assay Bulk Kit (Molecular Devices, R8075). Briefly, NIH3T3 cells (ATCC, CRL-1658) and HEK293 cells were seeded at 60,000 cells / well in 96-well plates (96-well black imaging plates, Grenier, #655090). The media were as follows: NIH3T3 medium: DMEM high glucose, pyruvate (Thermo, 11995-073) + 10% FBS + 1x PenStrep (abbreviated as P / S, Thermo, catalog number 15140122). NIH3T3 was the control system for the cGMP assay. HEK293 medium: EMEM + 10% FBS + 1x P / S + 1x GMAX. Serum-free NIH3T3 medium: DMEM + 1x P / S for treating cells with IBMX (CAS28822-58-4), serum-free NIH3T3 medium with BSA: DMEM + 1x P / S + 0.5mg / mL BSA (Thermo, A9418-100G) for treating cells with CNP.

[0213] The cells were incubated at 37°C, 5% CO2 for 24 hours. For cells treated with CNP variants, the plates were pretreated with IBMX (Enzo Life Sciences, 89161-340, 1 g) 15 minutes before use. IBMX is a potent, nonspecific inhibitor of phosphodiesterases. An 800 mM stock solution of IBMX was diluted to a 0.75 mM working stock in IBMX dilution medium (serum-free medium (DMEM + 1x PBS mixed 1:1 with 1x PBS)).

[0214] CNP variants were prepared as follows: 10 mg / ml CNP solution was diluted 1:1000 in CNP dilution medium (DMEM + 1x P / S + 0.5 mg / mL BSA). This solution was further diluted to obtain a CNP starting solution seeded with 100 nM CNP / well. This 100 nM CNP solution was further serially diluted 1:5 into six dilutions to obtain a lower concentration of 0.0064 nM CNP / well. This provided a 7-point dose curve for analysis.

[0215] For cell treatment, cells were removed from the incubator, growth medium was removed from the cells, and cells were treated with IBMX. 80 μL of 0.75 mM IBMX was added to each well, and the cells were returned to the 37°C incubator for 15 minutes. After 15 minutes, CNP (40 μL / well) was added to each test well, and the cells were returned to the 37°C incubator for 15 minutes. The plate was mixed by gentle tapping. The plate was imaged with a Solentim cell meter to visualize the cells and determine if they had lifted, and then returned to the 37°C incubator.

[0216] The reaction was stopped and 40 μL of lysis buffer (from the cGMP kit) was added to lyse the cells. The plate was placed on a shaker for 5 minutes to complete the lysis. The cell lysate was used in the cGMP assay.

[0217] The cGMP assay was performed using a cGMP calibrator, rabbit anti-cGMP antibody, and HRP-cGMP prepared according to the manufacturer's protocol. 40 μL of the calibrator was added to wells of a plate coated with anti-cGMP antibody, and 40 μL of the lysate to be analyzed was added to the appropriate wells. 40 μL of reconstituted rabbit anti-cGMP antibody was added to all wells, and the plate was placed on a shaker for 5 minutes to mix. 40 μL of reconstituted HRP-cGMP was added to each well and incubated for 2 hours at room temperature. The plate was manually aspirated and washed four times with 300 μL of wash buffer. 100 μL of Stoplight Red substrate was added to each well, and the plate was covered, protected from light, and left at room temperature for at least 10 minutes. The plate was read for fluorescence intensity at excitation 530 nm and emission 590 nm on a Spectramax M or similar instrument.

[0218] Table 1 demonstrates that CNP variants stimulate cGMP production, indicating that the stable variants described herein are useful as therapeutic agents for treating bone-related disorders.

[0219] Example 2A: Stability of CNP variants or conjugates in plasma The stability of different CNP variants or CNP conjugates was tested in human plasma over a 24-hour period. Briefly, two OEG spacers were used to fuse a C18 fatty acid to gamma glutamic acid (see, for example, Lau et al., J. Med. Chem 58:7370-7380, 2015). For chromatography, a Waters UPLC H-class was connected to a BEH C18 1.7um, 2.1x150mm column, with mobile phase A: 1% DMSO 0.1% formic acid in water, and mobile phase B: 1% DMSO 0.1% formic acid in CAN. Mass spectrometry was performed using an AB Sciex QTRAP.

[0220] Each CNP variant was prepared at 200 nM in human plasma Li-heparin, and samples were incubated at 37°C and 5% CO. Reactions were quenched with 0.5 M sodium citrate pH 4 for 0, 1, 2, 4, 8, and 24 hours. Plasma proteins were precipitated with 0.2% formic acid in MeOH, and samples were prepared using a WCX 96-well uElution plate and analyzed by LC-MS / MS 6500.

[0221] The variant CNP-R refers to a CNP37 variant in which a K residue not in the ring portion is changed to an R residue, and CNP Q / R refers to a CNP37 variant in which an N residue is changed to a Q and a K residue not in the ring portion is changed to an R.

[0222] Figure 1 shows CNP conjugates with different linker structures, and Figure 2 shows that lipidated conjugates have improved stability in plasma compared to PEGylated or unconjugated peptides in plasma.

[0223] The stability of the different conjugates was also analyzed under varying conditions. Briefly, the same protocol as above was used at 37°C (control), 37°C + 0.5 M NaCl, 37°C + protease inhibitors, or 4°C. Figure 3 shows that PG-CNP37 and variants are susceptible to proteolysis in human plasma, with PG-CNP37 exhibiting greater stability compared to the other variants.

[0224] Example 3: Heterozygous NPR2 mutations respond to CNP treatment To determine the effects of CNP on subjects with short stature due to NPR2 mutations, a cellular model of NPR2 mutations was developed. Exemplary NPR2 mutations analyzed are shown in Figure 6. Rat chondrosarcoma (RCS) cells harboring knockout or heterozygous loss-of-function mutations in the NPR2 gene were generated by RNP transfection into RCS cells using 125 ng of NPR2 variant or wild-type NPR2 plasmid DNA transfected into RCS or HEK293 cells. Single-cell clones were seeded and genotyped by Sanger sequencing. The cellular model is able to recapitulate the published cGMP phenotypes of various mutations.

[0225] NPR2 clones were created by creating insertions and deletions in the first exon of NPR2 in RCS cells. The sequence of the first exon of NPR2 was confirmed by next-generation sequencing and is shown in Figure 5. NPR2 mutant cells were treated with 6 nM Pro-Gly-CNP37 and then tested for activity in response to CNP administration by cGMP stimulation assay using the CatchPoint cyclic-GMP fluorescent assay. RCS (rat chondrosarcoma) cells were seeded at 40,000 cells / well in RCS medium: DMEM + 10% FBS + 1x Pen-Strep. Figure 4 shows that adding exogenous Pro-Gly-CNP37 variant rescues cGMP upregulation in the NPR2+ / - rat chondrosarcoma cell model.

[0226] Previous activation data have reported cGMP EC50 values ​​in the range of 40-360 nM for PRKG2 activation (Campbell et al., ACS Chem Biol 12, 2388-2398, 2017; Vaandrager et al., J Biol Chem 272, 11816-23, 1997; Pohler et al., FEBS Lett 374, 419-25, 1995). In heterozygous NPR2 knockout cells, a CNP dose of >0.163 nM can achieve intracellular concentrations above the EC50 range for PRKG2 activation (Figure 4). In contrast, in wild-type cells, the same cGMP concentration can be achieved with a CNP dose of 0.040 nM. These results indicate that CNP supplementation can achieve the cGMP levels required for PRKG2 activation and proliferation in cells with loss-of-function mutations in NPR2.

[0227] These results also suggest that administration of CNP variants may be useful in restoring bone growth in short subjects with reduced NPR2 activity. Furthermore, treatment with CNP variants may be beneficial in subjects with mutations in other growth plate genes that may impair cGMP signaling.

[0228] Example 4: Identification of mutations associated with short stature It is hypothesized that genes that show clear evidence of genetically driven bidirectional effects are likely to represent therapeutic targets that can be effectively modulated across a wide range of patient populations. To identify genes that are core regulators of growth, we analyzed the intersection of five gene lists, including a list of genes from genome-wide association studies (GWAS). Core growth regulators would most likely contain rare coding variants with bidirectional effects (i.e., short stature or skeletal dysplasia and tall stature or overgrowth).

[0229] Databases queried included: GWAS: 2,067 non-recurrent nearest genes were extracted for each of 3,290 independent genetic variants reported by a large-scale GWAS meta-analysis of height using approximately 700,000 individuals. HGMD: The "allmut" table in HGMD version v2019_2 was queried to find all pathogenic variants labeled "DM" with either "short stature" and "tall stature or overgrowth" in the same gene. OMIM: A list of OMIM genes associated with growth disorders was previously described and created using the keywords short stature, overgrowth, skeletal dysplasia, and brachydactyly.

[0230] First, the Human Gene Mutation Database (HGMD version v2019_2) was queried for genes associated with short or tall stature (Stenson et al., Hum Genet 136:665-677, 2017). There were 47 genes annotated with at least one pathogenic variant reported in the literature as causing "short stature." Only 20 genes were annotated as tall or overgrowth genes. Second, a manually curated list of 258 OMIM genes (248 short stature, 20 tall stature) created using the keywords: short stature, overgrowth, skeletal dysplasia, and brachydactyly was used (Wood et al., Nat Genet 46:1173-86, 2014). Third, the intersection of these lists was compared with the gene lists from GWAS studies. The intersection of these lists included three genes known to be associated with height (IGF1R, NPPC, and NPR2), and two additional genes (FGFR3 and SHOX) were identified.

[0231] Additional analyses generated a new group of five core genes that were significantly associated with decreased height (β = -0.20, 95% CI [-0.26 to -0.14], p = 4.04 x 10-11) and significantly increased risk of idiopathic short stature (ISS) (OR = 2.75, 95% CI [1.92 to 3.96]). Each of the core five genes (FGFR3, IGF1R, NPPC, NPR2, and SHOX) was associated with height when considered individually and also with short stature when combined with other mutations. Examples of mutations in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown in Figure 7.

[0232] The combination of loss-of-function (LoF) and missense variants in NPR2 and IGF1R was also associated with an increased risk of ISS (OR=3.31, P=0.001; OR=2.85, P=0.002, respectively). Whole-gene deletions and / or mutations causing loss of protein function in SHOX, IGF1R, NPPC, and NPR2 have been reported in familial short stature of varying degrees of severity.

[0233] Analysis indicates that carriers of variants in any of the five core genes have an approximately three-fold increased risk of ISS, accounting for 6.7% of the total ISS population. Furthermore, dose-dependent rescue of NPR2 signaling in a cellular model of NPR2 haploinsufficiency after the addition of exogenous CNP was demonstrated.

[0234] According to the omnigenic model (Liu, et al., Cell 177:1022-1034 e6 (2019), Boyle et al., Cell 169:1177-1186 (2017)), if these genes are core human growth genes, their effects should be modulated by multiple weaker common genetic variants driving a regulatory network. To indirectly test this hypothesis, we calculated a polygenic risk score (PRS) for height using the largest published GWAS meta-analysis on height without samples from the UK Biobank project. The cohort was divided into five equal-sized (n = 6,824) PRS quintiles (PRS1 = lowest height, PRS5 = highest height). There was a dose-dependent relationship between increasing PRS score and mean height (β = 0.30 per increasing PRS quintile) (Figure 8A). Carriers of LoF variants in the five core genes were consistently shorter than carriers across five different PRS backgrounds. See Figure 8. The data suggest that the combined effects of PRS and rare protein variants are consistent with an additive model: polygenic effects modulated height in both carriers and non-carriers.

[0235] The risk of ISS across PRS groups was calculated using PRS = 3 as the reference. The lowest PRS group was associated with an increased risk of ISS, while the highest PRS group was associated with a decreased risk (OR = 5.43, P = 8.58x10-34, and OR = 0.22, P = 4.49x10-7 for PRS1 and PRS5, respectively). The effect of rare coding variants in the five core genes was evaluated on ISS stratified by PRS group. Carriers of any of the five core genes had a higher risk of ISS in the first three quintiles (OR = 2.64, P = 3.09x10-5, OR = 2.17, P = 0.04, OR = 5.29, P = 1.58x10-5, OR = 2.72, P = 0.09, Figure 8C-F). A consistent direction of effect was observed for carriers of each individual core gene on ISS risk stratified by PRS (Figure 8C-F).

[0236] Furthermore, the additive effects of PRS, primarily derived from multiple common genetic variants with small individual effects, predicted 20.1% of the variance in height in the dataset. These additive effects of PRS appeared to be similar in magnitude for carriers and noncarriers of rare coding variants in core genes. This observation indicates that PRS may significantly contribute to differential penetrance of rare pathogenic variants, particularly in haploinsufficiency models such as those described here. Supporting this idea, we observed that two of eight NPR2 variant carriers with low NPR2 activity had short, normal height. This data suggests that most ISS individuals with mutations in NPR2 may have a polygenic background that predisposes them to the pathogenic effects of losing NPR2 activity.

[0237] These results support the idea that CNP-based therapy may be effective in NPR2 haploinsufficient patient populations. Furthermore, our results demonstrating a significant bidirectional (LoF and GoF) correlation between cGMP levels and height in NPR2 carriers from the general population suggest that targeting this receptor with CNP analogs may be an effective therapy for all individuals with ISS.

[0238] It is understood that all embodiments of the present disclosure described herein may be optionally combined with any one or more of the other embodiments described herein. All patent and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0239] It is understood, therefore, that the present invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications that are within the spirit and scope of the invention as defined by the appended claims, the above description, and / or the accompanying drawings. Accordingly, only such limitations as appear in the claims should be placed on the invention.

Claims

1. A variant of C-type natriuretic peptide (CNP), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6), and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7); and Conjugate moiety containing the AEEA-AEEA-γGlu-C18DA structure wherein the AEEA-AEEA-γGlu-C18DA structure is conjugated to a lysine residue (K) of the variant within the CNP cyclic domain. variant.

2. The variant of claim 1, wherein the lysine residue is at position 27 of the amino acid sequences of SEQ ID NOs: 1 and 5-7.

3. The peptide is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1) 3. The variant of claim 1 or 2, wherein:

4. The variant according to any one of claims 1 to 3, wherein the AEEA-AEEA-γGlu-C18DA structure is conjugated to the lysine residue with a linker.

5. The variant of claim 4, wherein the linker is a hydrolyzable linker.

6. The variant of claim 4 or 5, wherein the linker is a peptoid linker.

7. The AEEA-AEEA-γGlu-C18DA structure together with the peptoid linker gives the following structure: 【Chemistry 10】 wherein 【Chemistry 11】 The variant of claim 6, wherein represents the attachment point of the variant to the lysine residue within the CNP cyclic domain.

8. The variant according to any one of claims 4 to 5, wherein the linker is an electronic linker.

9. The AEEA-AEEA-γGlu-C18DA structure together with the electronic linker forms the following structure: 【Chemistry 12】 wherein 【Chemistry 13】 The variant of claim 8, wherein represents the attachment point of the variant to the lysine residue within the CNP cyclic domain.

10. The variant of any one of claims 1 to 9, wherein the variant is synthetically produced.

11. A pharmaceutical composition comprising a variant according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient, carrier or diluent.

12. 12. The pharmaceutical composition of claim 11, which is a lyophilized formulation prepared from a formulation comprising an isotonicity adjusting agent or bulking agent selected from the group consisting of mannitol, sucrose, sorbitol, and combinations thereof, and / or the lyophilized formulation is prepared from a formulation further comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, salt forms of ascorbic acid, thioglycerol, and combinations thereof.

13. 13. A composition comprising the variant of any one of claims 1 to 10 or the composition of any one of claims 11 to 12 for use in the treatment of a bone-related disorder or skeletal dysplasia, or for lengthening bones or increasing long bone growth in a subject.

14. The bone-related disorder or skeletal dysplasia is osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, dwarfism, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chondrodysplasia punctata, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, rhizometaphyseal chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, osseous dysplasia, short femora congenita, Langer mid-foot dysplasia 14. The composition of claim 13, wherein the cause is selected from the group consisting of: pediatric dysplasia, Niebergelt midfoot dysplasia, Robinnow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral dysplasia, Niest dysplasia, fibrochondrodysplasia, Roberts syndrome, distal mesomelic dysplasia, brachymetaphyseal dysplasia, Morquio syndrome, Niest syndrome, complex organotrophic dysplasia, and spondyloepiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Lelly-Weill syndrome), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation, and idiopathic short stature.

15. The composition according to any one of claims 13 to 14, characterized in that the composition is administered subcutaneously, intradermally, intraarticularly, orally or intramuscularly.

16. The composition of any one of claims 13 to 15, wherein the composition is an extended release composition.

17. 13. A composition comprising a variant according to any one of claims 1 to 10 or a composition according to claim 11 or 12 for use in a method of treating a CNP-responsive condition or disorder, said method comprising: administering the composition to a subject; monitoring the level of at least one bone- or cartilage-related biomarker in the subject; A composition wherein an increase in the level of said at least one bone- or cartilage-related biomarker is indicative of a therapeutic effect of said variant on said subject or said condition or disorder.

18. The method further comprises adjusting the amount or frequency of administration of the variant; i) if the level of said at least one bone- or cartilage-related biomarker falls below a target level, the amount or frequency of administration of said variant is increased, or ii) if the level of the at least one bone or cartilage related biomarker is above a target level, the amount or frequency of administration of the variant is reduced.

19. 19. The composition of claim 17 or 18, wherein the at least one bone or cartilage-related biomarker is selected from the group consisting of CNP, cGMP, type II collagen propeptide and fragments thereof, type II collagen and fragments thereof, type I collagen C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), type I procollagen propeptide (PINP) and fragments thereof, type I collagen and fragments thereof, aggrecan chondroitin sulfate, collagen X, and alkaline phosphatase.

20. A method for producing a variant according to any one of claims 1 to 10, comprising synthesizing the peptide on a solid phase resin using Fmoc amino acids.

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