Treatment of short stature with CNP variants

Abstract

The present disclosure relates, in general, to methods of treating short stature disorders in a subject who is non-responsive to growth hormone therapy using variants of C-type natriuretic peptide (CNP).

Description

TREATMENT OF SHORT STATURE WITH CNP VARIANTS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application No.63 / 678,712, filed August 2, 2024, U.S. Provisional Patent Application No. 63 / 689,001, filed August 30, 2024, U.S. Provisional Patent Application No.63 / 697,832, filed, September 23, 2024, U.S. Provisional Patent Application No.63 / 698,037, filed September 23, 2024, and U.S. Provisional Patent Application No.63 / 745,881, filed January 16, 2025, herein incorporated by reference in their entireties. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] The SequenceListing,whichisa part of the present disclosure,issubmittedconcurrently with the specification as a xml file. The name of the xm l file containing theSequence Listingis“70684_SeqListing.xml", which was createdonJuly 25, 2025, andis84,749 bytesinsize. The subject matter of the Sequence Listingisincorporated by reference herein in its entirety. FIELD OF THE DISCLOSURE

[0003] The present disclosure relates, in general, to variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants and methods of use to treat short stature disorders. BACKGROUND

[0004] C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168: 863-870 (1990) (GenBank Accession No. NP_077720, for the CNP precursor protein, NPPC) (J. Hypertens., 10: 907-912 (1992)) is a small, single chain peptide in a family of peptides (ANP, BNP, CNP) having a 17-amino acid loop structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and have important roles in multiple biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the generation of cyclic- guanosinemonophosphate (cGMP) (J. Hypertens., 10: 1111-1114 (1992)). CNP is expressed widely, including in the central nervous system, reproductive tract, bone and endothelium of blood vessels (Hypertension, 49: 419-426 (2007)).

[0005] In humans, CNP is initially produced from the natriuretic peptide precursor C (NPPC) gene as a single chain 126-amino acid pre-pro polypeptide (Biochem. Biophys. Res. Commun., 168: 863-870 (1990)). Removal of the signal peptide yields pro-CNP, and further cleavage by the endoprotease furin generates an 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 mainly found 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] Downstream signaling mediated by cGMP generation influences a diverse array of biological processes that include endochondral ossification. For example, knockout of either CNP or NPR-B in mouse models results in animals having a dwarfed phenotype with shorter long bones and vertebrae. Mutations in human NPR-B that block proper CNP signaling have been identified and result in 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 elevated levels of CNP display elongated long bones and vertebrae.

[0007] Therapeutic use of CNP (CNP22) has been 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)). A CNP variant having a longer in vivo serum half-life and exhibiting similar or improved activity to that of wild-type CNP is important for a sustainable therapeutic strategy.

[0008] Although Human Growth Hormone (hGH) is approved in many countries worldwide for the treatment of short stature associated with Turner syndrome, SHOX deficiency, and Noonan syndrome, and constitutes an effective therapeutic intervention in the early years of treatment, efficacy wanes over time (Blum et al., J Clin Endocrinol Metab.2013;98(8):E1383-E1392. correction appears in J Clin Endocrinol Metab.2013;98(12):4992; MacFarlane et al., J Clin Endocrinol Metab.2001;86(5):1953-1956.; Noonan et al., Horm Res Paediatr.2015;83(3):157- 166.; Ross et al., J Endocr Soc.2023;7(5):bvad026.; Wu et al., Mol Genet Genomic Med.2023;11(11):e2266). There are currently no approved second-line treatments available for such children and as such an unmet need for further treatment remains. SUMMARY

[0009] The present disclosure relates to methods of using CNP variants to treat short stature disorders.

[0010] Provided herein is a method of treating a short stature disorder in a subject in need thereof comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

[0011] Also provided is a method of elongating a bone or increasing long bone growth in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, and wherein the administering elongates a bone or increases long bone growth, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

[0012] Further contemplated is a method for increasing annualized growth velocity in a patient having a short stature disorder, comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the administering increases annualized growth velocity in the subject, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

[0013] The disclosure also provides a method for improving height SDS in a patient having a short stature disorder, comprising administering to the subject a composition comprising a C- type natriuretic peptide (CNP) variant, wherein the administering improves height SDS in the subject, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

[0014] In various embodiments, the subject that is unresponsive to growth hormone does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGVdetermined using median heights from CDC growth charts after a minimum one year of treatment.

[0015] In various embodiments, the subject does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from CDC growth charts after a minimum one year of treatment.

[0016] In various embodiments, the short stature disorder is selected from the group consisting of hypochondroplasia, NPPC mutation, NPR2 mutation, Short Stature Homeobox Containing Gene (SHOX) mutation, RASopathies, familial short stature, inherited short stature, and idiopathic short stature. In various embodiments, the short stature disorder results from a mutation in collagen (COL2A1, COL11A1, COL9A2, COL10), a mutation in aggrecan (ACAN), a mutation in Indian hedgehog (IHH), PTPN11 mutation, SHOX, insulin growth factor 1 receptor (IGF1R) mutation, or a mutation in FGFR3 that does not result in achondroplasia.

[0017] In various embodiments, the short stature disorder is hypochondroplasia. In various embodiments, the short stature disorder results from a mutation in NPR2 or NPPC.

[0018] In various embodiments, the short stature disorder is idiopathic short stature.

[0019] In various embodiments, the short stature disorder is associated with one or more mutations in a gene associated with a RASopathy. In various embodiments, the short stature disorder is a RASopathy. In various embodiments, the RASopathy is Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, or LEOPARD syndrome.

[0020] In one embodiment, the RASopathy is hereditary gingival fibromatosis type 1.

[0021] In various embodiments, the short stature disorder results from a mutation in SHOX. In various embodiments, the SHOX mutation results in Turner Syndrome or SHOX Deficiency.

[0022] In various embodiments, the short stature disorder is Turner syndrome, SHOX Deficiency, Noonan’s syndrome, or idiopathic short stature.

[0023] In various embodiments, the short stature is associated with one or more mutations in a gene associated with short stature, such as, collagen (COL2A1, COL11A1, COL9A2, COL10),aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, insulin growth factor 1 receptor (IGF1R), SHOX or combinations thereof.

[0024] In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of less than -1.0, -1.5, -2.0, -2.25, -2.5, -2.75 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, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -3.0. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5. In various embodiments, the subject having short stature has an SDS of -2.25 or less. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.25 or less. In various embodiments, the subject having short stature has an SDS of -2.00 or less. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.00 or less. In various embodiments, the subject having short stature has an SDS of -2.00 or less and does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from CDC growth charts after a minimum one year of treatment. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.00 or less and does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from CDC growth charts after a minimum one year of treatment.

[0025] In various embodiments, the short stature is a result of mutations in multiple genes as determined by 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 mutation 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 variousembodiments, the PRS is 2. Polygenic risk scores (PRS) were calculated for height as described in International Patent Publication WO 2021 / 055497. PRS 1 refers to the lowest height, PRS 5 the tallest height.

[0026] In various embodiments, the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.

[0027] In various embodiments, the CNP variant is administered at a dose of 7.5 to 30 µg / kg / day. In various embodiments, the CNP variant is administered at a dose of 7.5 to 22.5 µg / kg / day. In various embodiments, the CNP variant is administered at a dose of 7.5 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 22.5 µg / kg / day. In various embodiments, the CNP variant is administered at a dose of 30 µg / kg / day.

[0028] Also provided is a method of treating a short stature disorder in a subject in need thereof, a method of elongating a bone or increasing long bone growth in a subject, a method for increasing annualized growth velocity in a subject having short stature, or a method for improving height SDS in a subject having a short stature disorder, the method comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the CNP is administered at a dose of 7.5 µg / kg / day or a dose of 22.5 µg / kg / day.

[0029] Also provided is a method of treating a subject with mucopolysaccharidosis (MPS) IVA or MPS VI comprising administering a therapeutically effective amount of a CNP variant. In various embodiments, the subject with MPS IVA or MPS VI is receiving enzyme replacement therapy. In various embodiments, the enzyme replacement therapy is galactosamine-6-sulfate sulfatase. In various embodiments, the enzyme replacement therapy is N-acetylgalactosamine 4-sulfatase. In various embodiments, the subject is receiving about 15 µg / kg / day to about 30 µg / kg / day CNP variant. In various embodiments, the CNP variant is Pro-Gly CNP37 (SEQ ID NO: 1).

[0030] In various embodiments, administration of the CNP variant to a subject with MPS IVA or MPS VI improves one or more of height Z score, health and functional performance, pulmonary and cardiac systems, linear and segmental bone growth, biomarkers of growth, inflammation and bone metabolism, and self-reported tolerability and quality of life.

[0031] In various embodiments, the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.

[0032] In various embodiments, the CNP variant is selected from the group consisting of PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO:1); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); and GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55); 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); PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 1) 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); Ac- PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14). 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) (SEQ ID NO: 57)

[0033] In various embodiments, the CNP variant is selected from the group consisting of PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:1); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55); and LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO:2).

[0034] In various embodiments, the C-type natriuretic peptide (CNP) variant is selected from the group consisting of PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0035] In various embodiments, the CNP variant further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the CNP variant further comprises an OH or an NH2group at the C-terminus.

[0036] In various embodiments, the CNP variant peptide comprises a conjugate moiety. In various embodiments, the conjugate moiety is on a residue of 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.

[0037] 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 two or more OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the acid moiety is a fatty acid. Exemplary fatty acids include short chain, medium chain, or long chain fatty acids, or a dicarboxylic fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. Contemplated are C-6 to C-20 fatty acids, including but not limited to, C-6, C-8, C-10, C-12, C-14, C-16, C-18 or C-20 fatty acids, saturated or unsaturated. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids of the same.

[0038] In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-γGlu-C18DA.

[0039] 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).

[0040] In various embodiments, the variant comprises one or more linker groups. In various embodiments, the linker is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0041] In various embodiments, the linker is a hydrolysable linker.

[0042] In various embodiments, the conjugate moiety is a synthetic polymeric group. In various embodiments, the variant comprises a synthetic polymeric group coupled to the variant through a hydrolysable linker. In various embodiments, the synthetic polymeric 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 6 to 20 atom chain length.

[0043] In various embodiments, the CNP variant peptide is conjugated to a lipid, fatty acid, hydrophilic spacer, or linker, or optionally combinations thereof. In various embodiments, the linker is a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety is a synthetic hydrophilic polymer moiety.

[0044] The disclosure further provides a pharmaceutical composition comprising a CNP variant, and a pharmaceutically acceptable excipient, carrier or diluent for use in the methods.

[0045] In various embodiments, the composition is a lyophilized formulation prepared from a formulation that comprises a citric acid / citrate buffer or an acetic acid / acetate buffer having a pH from about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a formulation that further comprises an isotonicity-adjusting agent or a 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 that further comprises 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 from 0.8 mg to 10 mg. In various embodiments, the CNP variant composition is supplied as a 0.8-mg or 2-mg lyophilized, preservative-free powder for reconstitution.

[0046] In various embodiments, the composition is an extended release composition. In various embodiments, the composition is a sustained release composition. In various embodiments, the sustained or extended release compositions comprise a CNP pro-drug.

[0047] Further provided by the disclosure is a method of treating a short stature disorder in a subject in need thereof comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant selected from the group consisting of PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:1); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55); and LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO:2),

[0048] wherein the CNP variant comprises a conjugate moiety. In various embodiments, the conjugate moiety is a synthetic polymeric group. In various embodiments, the variant comprises a synthetic polymeric group coupled to the variant through a hydrolysable linker. In various embodiments, the synthetic polymeric 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 6 to 20 atom chain length.

[0049] In various embodiments, the variant comprises one or more linker groups. In various embodiments, the linker is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0050] It is further contemplated that the CNP variant includes a conjugate, salt or prodrug of the CNP variants described herein.

[0051] In various embodiments, the CNP variants are useful as an adjunct or alternative to growth hormone for treating idiopathic short stature and other short statures described herein. In various embodiments, the subject having short stature has an SDS of -2.00 or less and does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from CDC growth charts after a minimum one year of treatment. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.00 or less and does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from CDC growth charts after a minimum one year of treatment.

[0052] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37) (SEQ ID NO: 1); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37) (SEQ ID NO: 2); or LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21).

[0053] 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 an NH2 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 hydrolysable linker.

[0054] Further contemplated is a method of treating a CNP-responsive short stature disorder, comprising administering a CNP variant or composition as described herein to a subject, and monitoring the level of at least one bone- or cartilage-associated biomarker in the subject,wherein an increase in the level of the at least one bone- or cartilage-associated biomarker indicates a therapeutic effect of the CNP peptide or variant on the subject or the condition or disorder.

[0055] Further contemplated is a method of overcoming cell growth arrest induced by a constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3) in a subject with short stature comprising contacting a cell expressing the constitutively active FGFR-3 with a CNP variant or a composition as described herein.

[0056] In various embodiments, the method further comprises adjusting the amount or frequency of administration of the CNP variant as described herein, wherein i) the amount or frequency of administration of the CNP variant is increased if the level of the at least one bone- or cartilage-associated biomarker is below a target level; or ii) the amount or frequency of administration of the CNP variant is decreased if the level of the at least one bone- or cartilage- associated biomarker is above a target level.

[0057] In various embodiments, the at least one bone- or cartilage-associated biomarker is selected from the group consisting of CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, Collagen Type I C-Telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, and alkaline phosphatase, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N-propeptide (PINP), cross- linked C-telopeptide of type I collagen (CTx), cross-linked N-telopeptide of type I collagen (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, e.g., from PAXgene® RNA,, and CNP-variant bioactivity.

[0058] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. While the compositions, articles, and methods are susceptible of embodiments in various forms, the description hereafter includes specific embodiments with the understanding that the disclosure is illustrative, and is not intended to limit the invention to the specific embodiments described herein. For the compositions, articles, and methods described herein, optionalfeatures, including but not limited to components, compositional ranges thereof, substituents, conditions, and steps, are contemplated to be selected from the various aspects, embodiments, and examples provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 shows a schematic of a Phase 2 study protocol to treat subjects having short stature with CNP variant.

[0060] Figures 2A-2B show that 20nM vosoritide significantly reduced 0.3nM FGF18 ligand- induced ERK phosphorylation (Fig.2A) and increased chondrocyte growth (Fig.2B) in an ACH in vitro model with the FGFR3 G380R mutation.

[0061] Figures 3A-3B show that 20nM vosoritide significantly reduced 0.3nM FGF18 ligand- induced ERK phosphorylation (Fig.3A) and increased chondrocyte proliferation (Fig.3B) in an HCH in vitro model with the FGFR3 N540K mutation.

[0062] Figures 4A-4B show that 20nM vosoritide significantly reduced 0.3nM FGF18 ligand- induced ERK phosphorylation (Fig.4A) and increased chondrocyte proliferation (Fig.4B) in an NS in vitro model with the RAF1 L613V mutation.

[0063] Figures 5A-5B show that 20nM vosoritide significantly reduced ERK phosphorylation (Fig.5A) and increased chondrocyte proliferation (Fig.5B) in an ISS model represented in untreated RCS (wild-type) cells.

[0064] Figures 6A-6F show the effects of vosoritide on growth and skull morphology in in vivo Noonan Raf1L613V / + model. (Fig.6A) Raf1L613V / + showed reduced naso-anal length at baseline compared to control. (Fig.6B) Vosoritide significantly increased naso-anal length in Raf1L613V / + mice. (Fig.6C) in vivo micro-CT images of femur from vosoritide treated vs. vehicle mice. (Fig.6D) Vosoritide significantly increased femur length in Raf1 mice (Fig.6E) Vosoritide normalized inner canthal distance in Raf1 mice Fig.2A (Fig.6F). Vosoritide normalized skull width in Raf1 mice. (Fig.6G) Vosoritide increased skull length in Raf1 mice. (Fig.6H) Vosoritide normalized width / length ratio in Raf1 mice. White bar indicates Raf1+ / +, patterned bars indicate Raf1L613V / + mice.

[0065] Figures 7A-7H show that vosoritide treatment significantly increased growth and bone mass in wild-type (model of ISS) mice. (Fig.7A) Naso-anal length. (Fig.7B) in vivo micro-CT images of tibia from vosoritide treated vs. vehicle mice. (Fig.7C) Tibia length. (Fig.7D) Ex vivo micro-CT images of femur treated with vehicle, 40 µg / kg or 150 µg / kg vosoritide. (Fig.7E) 150 µg / kg significantly increased BV / TV and (Fig.7F) Tb. Th. (Fig.7G) 40 µg / kg and 150 µg / kg showed a trend to increase Tb. N. (Fig.7H) 40 µg / kg or 150 µg / kg did not affect Tb. Sp.

[0066] Figures 8A-8F show the evaluation of nasal-anal length, body weight, and skeletal growth in wild-type and L613V RAF1 HET mice treated with vosoritide. (Fig.8A) Nasal-Anal lengths, (Fig.8B) body weights, in vivo micro-CT images of femurs (Fig.8C, quantified in Fig. 8D) and tibias (Fig.8E, quantified in Fig.8F) from wild-type (WT) and L613V Raf1 Het mice treated with vosoritide (500 µg / kg, hatched bars) or vehicle (open bars). Results are expressed as biological replicates in each group (untreated groups, N=5; vosoritide-treated groups, N= 10) with the standard deviation included. In Figs.8A, 8B, 8D, and 8F, a one-way ANOVA with Tukey’s multiple comparison was performed, with statistical significance indicated.

[0067] Figures 9A-9C show the evaluation of FGF18 ligand-dependent FGFR3-RAS- MAPK / ERK and CNP-NPR2 signaling pathways in RCS cells. (Fig.9A) Endogenous pERK1 / 2 activation response in fgfr3+ / +or fgfr3- / -RCS cells treated with a dose range of FGF18 for 30 min. (Fig.9B) cGMP response in npr2+ / +RCS and npr2- / -RCS cells following treatment with a dose range of vosoritide. (Fig.9C) Evaluation of growth in parental RCS cell line over 72 hours in the absence or presence of drugs as indicated, with cells at each timepoint quantified by high content imaging of nuclei. For Figs.9A, 9B, and 9C, results are expressed as the mean of three biological replicates in each group with the standard deviation indicated as a bar. One way ANOVA with Tukey’s comparison was performed. * p < 0.1; ** p < 0.01; *** p < 0.0001.

[0068] Figures 10A-10D show the kinetics of FGF18 ligand-induced activation of RAS- MAPK / ERK signaling in RCS models of WT, N540K or G380R FGFR3 overexpression and correlation with growth. (Figs.10A-10D) Time course of untreated (open squares, solid line) or FGF18-ligand (2 nM, filled circles, solid line) induced activation of pERK1 / 2 signaling in fgfr3- / -RCS cells overexpressing GFP (Fig.10A), WT (Fig.10B), N540K (Fig.10C) or G380R FGFR3 (Fig.10D). Cells in Figs.10A-10D were also treated with 2 nM FGF18 ligand in combination with 20 nM vosoritide (open circles, broken line). (Fig.10E) Quantification of immunostaining with antihuman FGFR3 by high content imaging of fgfr3- / -RCS cells exogenously expressingWT (left 3 bars), N540K (middle 3 bars) or G380R FGFR3 (right 3 bars) left untreated or treated chronically with 2 nM FGF18 alone or in combination with 20 nM vosoritide for three days. Results are shown as the mean ± SD. One way ANOVA with Tukey’s multiple comparison was performed. ** p < 0.01; **** p < 0.0001

[0069] Figures 11A-11F shows the kinetics of FGF18 ligand-induced activation of RAS- MAPK / ERK signaling in RCS models of WT RAF1 overexpression or the L613V RAF1 variant associated with Noonan syndrome. (Figs.11A-11C) Time course of FGF18-ligand (0.3 nM) induced ERK1 / 2 signaling in parental RCS cells transduced with lentivirus encoding GFP (green solid line; Fig.11A), WT RAF1:T2A:GFP (black solid line; Fig.11B) or L613V RAF1:T2A:GFP (blue solid line; Fig.11C). An additional group of RCS cells in Fig.11A-11C were treated with 0.3 nM FGF18 ligand in the presence of 20 nM vosoritide (dashed line on each graph). (Fig.11D) ERK1 / 2 activation levels described in Figs.11A-11C plotted as histograms 1-hour post-ligand treatment. (Fig.11E) Quantification of GFP+RCS cells by high-content imaging at 72 hours post-lentivirus transduction. Results are shown as individual replicates, with significant differences indicated. One way ANOVA Turkey’s multiple comparison was performed. ** p < 0.01; *** p < 0.001; **** p< 0.0001

[0070] Figures 12A-12F show the kinetics of FGF18 ligand-induced activation of RAS- MAPK / ERK signaling in RCS models of WT KRAS overexpression or the P34R KRAS variant associated with Noonan syndrome. (Figs.12A-12C) Time course of FGF18-ligand (0.3 nM) induced ERK1 / 2 signaling in parental RCS cells transduced with lentivirus encoding GFP (solid line, Fig.12A), WT KRAS:T2A:GFP (solid line, Fig.12B) or the P34R KRAS:T2A:GFP variant associated with Noonan syndrome (solid line, Fig.12C). An additional group of RCS cells in Figs.12A-12C were treated with 0.3 nM FGF18 in the presence of 20 nM vosoritide (dashed line on each graph). (Fig.12D) ERK1 / 2 activation levels described in A, B, C, plotted as histograms 1-hour post-ligand treatment. (Fig.12E) Quantification of GFP+RCS cells described in E by high-content imaging at 72 hours post-lentivirus. Results are shown as individual replicates, with significant differences indicated. One way ANOVA with Turkey’s multiple comparison was performed. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0071] Figure 13A-13D show the evaluation of body weight and nasal-anal length in L613V Raf1 Het (HET) mice and wild-type (WT) littermate control mice, showing that L613V Het mice 18 33initially present with reduced body length at 3-weeks of age, which is no longer observed at 10- weeks of age. (Fig.13A-13B) Body weight measurements at 3 weeks of age (Fig.13A) and 10 weeks of age (Fig.13B) in wildtype (WT, black bars) and L613V Raf1 HET mice (blue bars). (Fig.13C-13D) Mouse body length measurements, as determined by measuring nasal-anal lengths at three weeks of age (Fig.13C) and at 10 weeks of age (Fig.13D). Biological repeats are indicated in each group with standard deviation indicated; WT, N = 16; Het, N = 20. Student T-test was performed, with statistically significant differences indicated.

[0072] Figure 14A-14D show the evaluation of skull morphology in WT mice and L613V RAF1 HET mice. (Fig.14A) Skull length. (Fig.14B) Skull width. (Fig.14C) Inner canthal distance. (Fig. 14D) Width / length ratio in wild-type (left bars) and L613V Raf1 Het (right bars) mice treated with vosoritide (500 µg / kg, hashed bars) or vehicle (open bars). Results are expressed as biological replicates in each group with standard deviation included. 1-way ANOVA Tukey’s multiple comparison was performed, with statistical significance indicated.

[0073] Figure 15 shows the change in annualized growth velocity (AGV) on vosoritide. Each line represents a subject and plots their baseline AGV (cm / y) compared to their 6-month AGV (cm / y). Subjects with prior GH exposure increased AGV by +4.9 and +1.57 cm / y over the AGV on GH treatment, while the GH naïve subject had an increased AGV +6.04 cm above the basline at the 6-month visit. AGV was calculated based on historical data closest to the 12 months prior to the screening visit.

[0074] Figure 16A-16C shows ANCOVA-adjusted differences in vosoritide versus placebo LSM change from baseline at week 52. (Fig. 16A) Interpedicular distance). (Fig 16B) Spinal canal width (inferior pedicle). (Fig.16C) Thoracolumbar kyphosis. DETAILED DESCRIPTION

[0075] The present disclosure relates to treating short stature disorders associated with C- type natriuretic peptide (CNP) signaling pathways with CNP variant peptides.

[0076] As used in the specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” include plural as well as singular referents unless the context clearly dictates otherwise.

[0077] The term “about” or “approximately” means an acceptable 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 a given value or range. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series.

[0078] The term “C-type natriuretic peptide” or “CNP” refers to a small, single chain peptide having a 17-amino acid loop structure at the C-terminal end (GenBank Accession No. NP_077720, for the CNP precursor protein, NPPC) and variants thereof. The 17-mer CNP loop structure, is also referred to as CNP 17, the CNP ring, or CNP cyclic domain. CNP includes the active 53-amino acid peptide (CNP-53) and the mature 22-amino acid peptide (CNP-22), and peptides of varying lengths between the two peptides.

[0079] In various embodiments, a “CNP variant” is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to the 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 the NPPC polypeptide. In one embodiment, a CNP variant may comprise a sequence of 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 acids derived from the NPPC polypeptide. CNP variant also includes conjugates, salts or prodrugs of CNP variants described herein.

[0080] The term “conjugate moiety” refers to a moiety that is conjugated to the variant peptide. Conjugate moieties include a lipid, fatty acid, hydrophilic spacer, synthetic polymer, linker, or optionally, combinations thereof.

[0081] The term “effective amount” refers to a dosage sufficient to produce a desired result on a health condition, pathology, or disease of a subject or for a diagnostic purpose. The desired result may comprise a subjective or objective improvement in the recipient of thedosage. "Therapeutically effective amount" refers to that amount of an agent effective to produce the intended beneficial effect on health. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experi- mentation. It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors, including the activity of the specific compound employed; the bioavailability, metabolic stability, rate of excretion and length of action of that compound; the mode and time of administration of the compound; the age, body weight, general health, sex, and diet of the patient; and the severity of the particular condition.

[0082] "Substantially pure" or "isolated" means an object species is the predominant species present (i.e., on a molar basis, more abundant than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition wherein the object 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 species of interest 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. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition. In an embodiment, the compounds of the disclosure are substantially pure or isolated. In another embodiment, the compounds of the 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 disclosure comprise a substantially pure or isolated CNP variant admixed with one or more pharmaceutically acceptable excipients, carriers or diluents, and optionally with another biologically active agent.

[0083] "Treatment" refers to prophylactic treatment or therapeutic treatment or diagnostic treatment. In certain embodiments, “treatment” refers to administration of a compound or composition to a subject for therapeutic, prophylactic or diagnostic purposes.

[0084] A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease, for the purpose ofdecreasing the risk of developing pathology. The compounds or compositions of the disclosure may be given as a prophylactic treatment to reduce the likelihood of developing a pathology or to minimize the severity of the pathology, if developed.

[0085] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology for the purpose of diminishing 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 disclosure may also be given as a therapeutic treatment or for diagnosis.

[0086] “Diagnostic" means identifying the presence, extent and / or nature of a pathologic condition. Diagnostic methods differ 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.

[0087] “Bone- or cartilage-associated biomarker” or “bone- or cartilage-associated marker” refers to a growth factor, enzyme, protein, or other detectable biological substance or moiety whose level is increased or decreased in association with, e.g., cartilage turnover, cartilage formation, cartilage growth, bone resorption, bone formation, bone growth, or combinations thereof. Such biomarkers may be measured before, during and / or after administration of a CNP variant as described herein. Exemplary bone- or cartilage-associated biomarkers include, but are not limited to, CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, propeptides of collagen type I and fragments thereof, collagen type I and fragments thereof, osteocalcin, proliferating cell nuclear antigen (PCNA), aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, alkaline phosphatase, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase (BSAP), cross-linked C-telopeptide of type I collagen, cross-linked N-telopeptide of type I collagen or tartrate-resistant acid phosphatase 5b (TRAP 5b), amino-terminal propeptide of type I collagen / procollagen type I N-propeptide (PINP), collagen type I C-telopeptide (CTx) collagen cross-linked N-telopeptide (NTx), transcriptomics readouts, e.g., from PAXgene® RNA, and CNP-variant bioactivity. Cartilage- and bone-associated biomarkers can be measured in any appropriate biological sample, including but not limited to tissues, blood, serum, plasma, cerebrospinal fluid, synovial fluid and urine. In some embodiments, the biomarkers aremeasured in blood, plasma, serum or urine from subjects undergoing efficacy / pharmacodynamic in vivo studies and / or from the conditioned media of ex vivo studies.

[0088] "Pharmaceutical composition" or "formulation" refers to a composition suitable for pharmaceutical use in subject animal, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of CNP variant, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier or diluent. In an embodiment, a pharmaceutical composition encompasses a composition comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the disclosure and a pharmaceutically acceptable excipient, carrier or diluent.

[0089] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and the like, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions (e.g., an oil / water or water / oil emulsion). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifying agents, wetting agents, lubricants, glidants, sweetening agents, 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 upon 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).

[0090] A "pharmaceutically acceptable salt" is a salt that can be formulated 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.

[0091] By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administeredto an individual without causing any undesirable biological effects or without interacting in a deleterious manner with any of the components of the composition in which it is contained or with any components present on or in the body of the individual.

[0092] “Physiological conditions” refer to conditions in the body of an animal (e.g., a human). Physiological conditions include, but are not limited to, body temperature and an aqueous environment of physiologic ionic strength, pH and enzymes. Physiological conditions also encompass conditions in the body of a particular subject which differ from the “normal” conditions present in the majority of subjects, e.g., which differ from the normal human body temperature of approximately 37 °C or differ from the normal human blood pH of approximately 7.4.

[0093] By “physiological pH” or a “pH in a physiological range” is meant a pH in the range of approximately 7.0 to 8.0 inclusive, more typically in the range of approximately 7.2 to 7.6 inclusive.

[0094] 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 apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or gender. In various embodiments, the subject is 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 older than 3, older than 2, older than 1, or older than 6 months in age. C-type natriuretic peptide variants

[0095] C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168: 863-870 (1990) (GenBank Accession No. NP_077720, for the CNP precursor protein, NPPC) (J. Hypertens., 10: 907-912 (1992)) is a small, single chain peptide in a family of peptides (ANP, BNP, CNP) having a 17-amino acid loop structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and have important roles in multiple biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the generation of cyclic- guanosinemonophosphate (cGMP) (J. Hypertens., 10: 1111-1114 (1992)). CNP is expressed more widely, including in the central nervous system, reproductive tract, bone and endothelium of blood vessels (Hypertension, 49: 419-426 (2007)).

[0096] Natural CNP gene and polypeptide have been previously described. U.S. Patent No. 5,352,770 discloses isolated and purified CNP-22 from porcine brain identical in sequence to human CNP and its use in treating cardiovascular indications. U.S. 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. The mature CNP is a 22-amino acid peptide (CNP-22). Certain CNP variants are disclosed in US Patent 8,198,242, incorporated by reference herein.

[0097] In various embodiments, CNP of the disclosure includes truncated CNP ranging from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53), and 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) (SEQ ID NO: 57).

[0098] In various embodiments, the CNP variant peptides are modified CNP-37 or CNP-38 peptides, optionally having mutation(s) / substitution(s) at the furin cleavage site (underlined), and / or containing glycine or proline-glycine at the N-terminus. Exemplary CNP-37 variants include but are not limited to:QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO:1); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); and GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55);

[0099] In various embodiments, CNP variants of the disclosure include PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); 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) .

[0100] 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.

[0101] In various embodiments, the peptide variant further comprises an OH or an NH2 group at the C-terminus.

[0102] In various embodiments, the CNP variants are selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8),PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 9), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 11), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 12), and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 7).

[0103] In various embodiments, the CNP variants are selected from the group consisting of: 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).

[0104] 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).

[0105] Variants, conjugates and salts of CNP are disclosed in USSN 17 / 642,150 and USSN 17 / 634,034, herein incorporated by reference.

[0106] In additional embodiments, for any of the CNP variants described herein that have asparagine (Asn / N) residue(s) and / or glutamine (Gln / Q) residue(s), whether they have a wild- type sequence or a non-natural amino acid sequence, any Asn residue(s) and / or any Gln residue(s) can independently be substituted with any other natural or unnatural amino acids,including conservative substitutions such as Asn to Gln. Such substitution(s) are designed in part to minimize or avoid any potential deamidation of asparagine and / or glutamine.

[0107] In additional embodiments, for any of the CNP variants described herein that have lysine (Lys / K) residue(s), whether they have a wild-type sequence or a non-natural amino acid sequence, any Lys residue(s) can independently be substituted with any other natural or unnatural amino acids, including substitutions such as Lys to Arg. In various embodiments, all lysine residues are independently substituted with any other natural or unnatural amino acids, including substitutions such as Lys to Arg, except the Lys residue in the CNP variant cyclic domain is not substituted with any other natural or unnatural amino acids.

[0108] In one embodiment, the CNP variants are cyclized via formation of a disulfide bond between Cys6and Cys22,as designated in the wtCNP22 peptide. Cys6can be a cysteine analog such as, e.g., homocysteine or penicillamine. In a further embodiment, the CNP variants 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 an embodiment, the covalent bond is formed between an amino acid at or toward the N-terminus and an amino acid at or toward the C-terminus of the peptide (referred to as “terminal” amino acids in this context). 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.

[0109] Head-to-tail cyclization of the terminal amine to the terminal carboxyl group can be carried out using a number of methods, e.g., using p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentafluorophenyl ester, the azide method, the mixed anhydride method, HATU, a carbodimide (e.g., DIC, EDC or DCC) with a catalyst such as HOBt, HONSu or HOAt, or on- resin cyclization.

[0110] In addition, the cyclic structure can be formed via a bridging group involving the side chains of amino acid residues of the CNP variant and / or the terminal amino acid residues. A bridging group is a chemical moiety that allows cyclization of two portions of the peptide. Non- limiting examples of bridging groups include amides, thioethers, thioesters, disulfides, ureas, carbamates, sulfonamides, and the like. A variety of methods are known in the art forincorporation of units having such bridging groups. For example, a lactam bridge (i.e., a cyclic amide) can be formed between the N-terminal amino group or an amino group on a side chain and the C-terminal carboxylic acid or a carboxyl group on a side chain, e.g., the side chain of lysine or ornithine and the side chain of glutamic acid or aspartic acid. A thioester can be formed between the C-terminal carboxyl group or a carboxyl group on a side chain and the thiol group on the side chain of cysteine or a cysteine analog.

[0111] Alternatively, a cross link can be formed by incorporating a lanthionine (thio-dialanine) residue to link alanine residues that are covalently bonded together by a thioether bond. In another method, a cross-linking agent, such as a dicarboxylic acid (e.g., suberic acid (octanedioic acid)), can link the functional groups of amino acid side chains, such as free amino, hydroxyl, and thiol groups.

[0112] Enzyme-catalyzed cyclization can also be used. For example, it has been reported that the thioesterase domain of tyrocidine synthetase can be used to cyclize a thioester precursor, a subtilisin mutant can be utilized to cyclize peptide glycolate phenylalanylamide esters, and the antibody ligase 16G3 can be employed to cyclize a p-nitrophenylester. For a review of peptide cyclization, see Davies, J. Peptide Sci., 9: 471-501 (2003), incorporated herein by reference in its entirety.

[0113] 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%. Peptide Conjugates

[0114] Peptide therapeutics are attractive biological therapeutic agents, but are often disadvantaged by low stability and short half-life in solution (Tang et al., Eur J Pharm Sci. 102:63-70, 2017). Attempts to improve efficacy of peptide therapeutics, by enhancing stability and / or increasing the half-life, include attempts to encapsulate hydrophilic peptides into biodegradable particles such as liposomes or polymer particles. However, this has been difficult due to the cationic nature of these peptides and their ability to electrostatically interact with liposomes of negatively charged polymers (Griesser et al., Int J Pharmaceutics 520:267-274, 2017). Generation of peptide conjugates has been one means used to enable better encapsulation of hydrophilic polymers into microparticles or liposomes (Lu et al., Mol. Pharmaceutics 15:216-225, 2018).

[0115] Peptides can be a string of amino acids from 5 to 100 amino acids. The peptide can have positively charged amino acids, negatively charged amino acids, or a mixture of both, such that the peptide is capable of interacting with charged moieties, e.g., a cation, anion or a combination thereof having charged species opposite to those in the peptide.

[0116] It is contemplated that the CNP variant is conjugated to or complexed to a moiety, e.g., a conjugate moiety, which confers increased stability or half-life. In various embodiments, the conjugate moiety is complexed via a non-covalent bond or is attached by a covalent bond. The moiety may be non-covalently attached with the peptide via electrostatic interactions. Alternatively, the moiety may be covalently associated to the peptide via one or more linker moieties. Linkers can be cleavable and non-cleavable linkers. Cleavable linkers may be cleaved via enzymes, nucleophilic / basic reagents, reducing agents, photo-irradiation, electrophilic / acidic reagents, organometallic and metal reagents, or oxidizing reagents. Linkers may also be self-immolative or traceless linkers. Exemplary linkers include, but are not limited to, 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 glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene), beta alanine, 4- aminobutyric acid (GABA), 2-aminoethoxy acid (AEA), aminoethoxy-2-ethoxy acetic acid (AEEA), 5 aminovaleric acid (AVA), 6-aminocaproic acid (Abx), a vicinal diol cleavable linker, Trimethyl Lock Lactonization, p-alkoxyphenyl carbamate, bicin, peptoid or bicin-type linkers, and electronic linkers as described herein.

[0117] 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.

[0118] In various embodiments, the CNP variant is attached to the conjugate moiety via the linker. In various embodiments, the linker is attached to the conjugate moiety via the hydrophilic spacer of the conjugate moiety.

[0119] In various embodiments, the linker is a hydrolysable linker.

[0120] 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 SO2moiety. Exemplary linkers are illustrated below. It is further contemplated that linkers are modified by substitution on the R groups. For example, bicin-type linkers include the structures as set out below: .

[0121] In various embodiments, the moiety conjugated to the peptide is a synthetic polymer such as polyethylene glycol, a linker, a lipid moiety or fatty acid, or a combination thereof. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a spacer and a linker. In various embodiments, the CNP variant is conjugated with 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 with 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, wherein 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 carbonyl groups.

[0122] In various embodiments, the CNP variant is conjugated with a fatty acid. It is hypothesized that the lipid technology increases 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, long chain fatty acid, or a dicarboxylic fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. In various embodiments, the fatty acidis 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 diacids of the same. In various embodiments, the fatty acid is conjugated to a lysine residue.

[0123] 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 on a residue of 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 fatty acid.

[0124] 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, wherein 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 carbonyl groups. 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.

[0125] In various embodiments, the CNP 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). 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 Asn to Glu variants of the above peptides.

[0126] In various embodiments, the disclosure contemplates use of hydrophilic or water soluble polymers (e.g., oxygenated alkyl chains, wherein the carbon atoms can be replaced with one or more oxygen atoms, such as polyethylene glycol (PEG) or polyethylene oxide (PEO) and the like). In various embodiments, the water soluble polymers can vary in type (e.g., homopolymer or copolymer; random, alternating or block copolymer; linear or branched; monodispersed or polydispersed), linkage (e.g., hydrolysable or stable linkage such as, e.g., amide, imine, aminal, alkylene, or ester bond), conjugation site (e.g., at the 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). The hydrophilic or water-soluble polymer can be conjugated to the CNP variant by means of N-hydroxy succinimide (NHS)- or aldehyde-based chemistry or other chemistry, as is known in the art. In various embodiments, negatively charged PEG-CNP variants can be designed for reduced renal clearance, including but not limited to 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 carboxyl group(s), sulfate group(s), and / or phosphate group(s).

[0127] In another embodiment, the hydrophilic polymer (e.g., PEG or PEO) moieties conjugated to the N-terminus, C-terminus and / or internal site(s) of CNP variants described herein contain one or more functional groups that are positively charged under physiological conditions. Such moieties are designed, inter alia, to improve distribution of such conjugated CNP variants to cartilage tissues. In one embodiment, PEG moieties contain one or moreprimary, secondary or tertiary amino groups, quaternary ammonium groups, and / or other amine- containing (e.g., urea) groups. Methods of Use

[0128] Children with short stature often present to the pediatric endocrinologist for medical evaluation. After elimination of an underlying chronic medical illness or hormonal deficiency, many of the patients’ growth impairments are attributed to an underlying genetic predisposition to short stature. In recent years, advances in genomic technologies have allowed us to dissect the genetic underpinnings of short stature (Jee et al., Endocrinol. Metab. Clin. North Am. 46(2):259–281, 2017; Dauber et al., J. Clin. Endocrinol. Metab.99(9):3080–3092, 2014). Many of the genetic etiologies are centered on defects in the growth plate which is the primary site of action responsible for linear growth in humans (Baron et al., Nat. Rev. Endocrinol.11(12):735– 746, 2015). In a comprehensive study examining the yield of genetic diagnosis in short stature, Hauer et al. found that heterozygous carriers of recessive skeletal dysplasia genes represented 3.5% of all patients presenting for evaluation with short stature (Hauer et al., Genet. Med. 20(6):630–638, 2018). In the group of patients who underwent exome sequencing, 2.5% had mutations in ACAN (the gene for Aggrecan, a proteoglycan found in the extracellular matrix of the growth plate) and 1.5% had mutations in NPR2, the gene encoding the NPR-B receptor for C-type natriuretic peptide (CNP) (Hauer, supra). In a recent study from the Czech Republic, 33 patients with familial short stature who were being treated with growth hormone were studied to identify a genetic etiology (Plachy et al., J. Clin. Endocrinol. Metab.104(10):4273–4281, 2019). Seventeen of the 33 (52%) had a likely genetic etiology identified with 9 of 17 (53%) being attributed to variants in growth plate related genes (Plachy, supra). Current therapeutic options to treat patients with genetic growth disorders are quite limited. Growth hormone has been approved for a limited number of indications but response to growth hormone is quite variable. Therefore, there is a need to explore novel therapeutic approaches to genetic causes of short stature.

[0129] A recently approved drug vosoritide is a modified recombinant CNP that is administered as a daily subcutaneous injection. Vosoritide has initially been studied in patients with achondroplasia, a condition due to an activating mutation in FGFR3 which leads to severe perturbations in growth plate physiology. Achondroplasia was targeted due to the clear interaction between the CNP / NPR-B signaling pathway and the FGFR3 pathway. Activatingmutations in FGFR3 lead to increased downstream signaling via SOS > RAS > RAF-1 ultimately increasing signaling in the MEK / ERK pathway. Increased phosphorylation of ERK1 / 2 is an important marker of increased activity of this pathway and ultimately leads to decreased chondrocyte proliferation and hypertrophy. CNP binds to its receptor, NPR-B, and generates cGMP, which activates a number of different signaling mediators including cGMP-dependent protein kinases. One of these kinases, protein kinase G, inhibits the activation of RAF-1, and ultimately leads to decreased activation of ERK1 / 2. Thus, this inhibitory effect of CNP on the FGFR3 pathway leads to an increase in chondrocyte proliferation and differentiation and an increase in cartilage matrix synthesis.

[0130] It is hypothesized herein that patients with selected genetic causes of short stature, in particular those with evidence suggesting that they affect the same signaling pathways, will also respond to vosoritide treatment with an increase in growth velocity. Furthermore, it is plausible that these patients will have more robust growth responses than those with achondroplasia as achondroplasia is an extremely severe perturbation of chondrocyte physiology.

[0131] By stimulating matrix production, proliferation and differentiation of chondrocytes and increasing long bone growth, the CNP variants of the disclosure are useful for treating mammals, including humans, suffering from a bone-related disorder, such as a skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasias include achondroplasia, hypochondroplasia, idiopathic short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis congenita, achondrogenesis, chondrodysplasia congenita, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis congenita, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia congenita, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenital, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia / Kniest syndrome, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia.

[0132] Short stature or growth plate disorders contemplated herein include idiopathic short stature, disorders related to NPR2 mutation, SHOX mutation (e.g., Turner’s syndrome / Leri Weill Dyschondrosteosis, SHOX Deficiency), PTPN11 mutations (e.g., Noonan’s syndrome), IGF1Rmutation, mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), NPPC, in FGFR3 that does not result in achondroplasia, or combinations thereof.

[0133] Further, the CNP variants are useful as an adjunct or alternative to growth hormone for treating idiopathic short stature and other growth plate disorders. Although hGH is approved in many countries worldwide for the treatment of short stature associated with Turner syndrome, SHOX deficiency, and Noonan syndrome, and constitutes an effective therapeutic intervention in the early years of treatment, efficacy wanes over time. This waning of response may relate to the fact that the pathophysiology underlying impaired growth in these conditions does not involve a disruption of the GH axis, but rather disruption of growth plate biology. There are currently no approved second-line treatments available for such children and as such an unmet need for further treatments remains. A participant’s response to hGH treatment is defined as inadequate if their annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from Centers for Disease Control and Prevention (CDC) growth charts after a minimum 1 year of treatment.

[0134] According to clinical guidelines, idiopathic short stature (ISS) is defined as short stature in the absence of i) a causative systemic disease, ii) growth hormone deficiency or other endocrine disorder, or iii) known chromosomal imbalance or genetic variant associated with short stature (Grimberg et al., Horm Res Paediatr 2016;86:361-397). The level of growth impairment, quantified as height Z-score, used to define ISS is variable in the literature and in clinical practice; however, a height Z-score ≤ -2.25 standard deviations (SDs) is often taken to describe a severely impacted population. While ISS is, by definition, considered to be idiopathic, height is known to be associated with many genetic variants across the genome (Wood et al., Nat Genet.2014; 46:1173-1186). The impact of such variants, alone or in aggregate, may determine final adult height in such populations.

[0135] Individuals with ISS may experience lower quality of life (QoL) both in childhood and adulthood compared to those of average stature (Backeljauw et al., Growth Horm IGF Res 2021;57-58:101392). In childhood, individuals may experience functional and psychosocial challenges such as low self-esteem, self- consciousness, and body image issues (Lipman et al., Pediatr Endocrinol Rev 2017;14:472-477). Further, they may suffer bullying, leading to psychological distress, anxiety, and depression. There is also an association between lowheight Z-score and suicidal ideation among Korean adolescents (Song et al., Front Psychiatry 2023;14:1172940). In adulthood, ISS may be associated with lower health status, including increased adiposity (Bosy-Westphal et al., Br J Nutr 2009;102:453461), increased prevalence of type 2 diabetes (Wittenbecher et al., Diabetologia 2019; 62:2211-2221), and increased rates of coronary heart disease (Paajanen et al., Eur Heart J 2010;31:1802–1809). Concern around psychosocial issues is a key reason families seek access to growth promoting therapies for their children.

[0136] Growth plate disorders include disorders that result in short stature or abnormal bone growth and that may be the result of a genetic mutation in a gene involved in bone growth, including collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), SHOX, PTPN11, NPR2, NPPC, FGFR3 or IGF1R. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy. In various embodiments, a 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, the mutation is a gain-of-function mutation. Growth plate disorders include, but are not limited to, familial short stature, dominant familial short stature which is also known as dominant inherited short stature, or idiopathic short stature. See, e.g., Plachy et al., J Clin Endocrinol Metab 104: 4273-4281, 2019.

[0137] Mutations in ACAN can give rise to familial osteochondritis dissecans and short stature and eventually osteoarthritis, characterized by areas of bone damage (or lesions) caused by the detachment of cartilage and sometimes bone from the end of the bone at a joint. It has been suggested that the disorganized cartilage network in growing bones impairs their growth, leading to short stature. A mutation associated with ACAN and short stature includes Val2303Met. See Stattin et al., Am J Hum Genet 86(2):126-37, 2010. It is contemplated that patients with a mutation in ACAN resulting in short stature would benefit from treatment with CNP as administration may be able to increase height in these patients by the known interaction of CNP with FGFR3.

[0138] The natriuretic peptide system, including receptor NPR2, has been shown to be involved in regulation of endochondral bone growth (Vasques et al., Horm Res Pediat 82:222- 229, 2014). Studies have shown that homozygous or compound heterozygous loss-of-function mutations in NPR2 cause acromesomelic dysplasia type Maroteaux (AMDM), which is a skeletaldysplasia having extremely short stature (Vasquez et al., 2014, supra). There are reports implicating heterozygous loss-of-function (such as dominant negative) NPR2 mutations as a cause of short stature, whereas gain-of-function NPR2 heterozygous mutations have been found to be responsible for tall stature (Vasquez et al., 2014, supra). In view of CNP’s interaction with NPR2 to stimulate cGMP generation, increasing cGMP levels is desirable in these conditions and would have therapeutic benefit in the management of the complications from these diseases and conditions. It was shown in Estrada et al (Nat Commun.12(1):2224, 2021) and in International Patent Publication WO 2021 / 055497 (herein incorporated by reference in their entireties) that rat chondrosarcoma cell lines in which NPR2 was either completely knocked out or was haploinsufficient (i.e. mimicking a heterozygous loss-of-function mutation), homozygous knock out lines had absolutely no response to CNP. However, the heterozygous knock outs were able to respond to CNP, and at a concentration >0.163nM were able to generate a level of cGMP sufficient to activate downstream protein kinase G signaling. Taken together, this data support the hypothesis that vosoritide will be able to increase signaling via the CNP / NPR-B pathway in patients with heterozygous NPR2 mutations resulting in an ultimate increase in height.

[0139] Heterozygous mutations of NPR2 are believed to result in idiopathic short stature and other forms of short stature. Mutations in the NPR2 gene are set out below and 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, hereby incorporated by reference. It is contemplated that a subject having short stature to be treated with a CNP variant as described herein has a height SDS of less than -1.0, -1.5, - 2.0, -2.25, -2.5, -2.75 or -3.0, and has at least one parent with a height SDS of less than -1.0, - 1.5, -2.0 or -2.5, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -3.0. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.25 or less. 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, -2.25, -2.5, -2.75 or -3.0, treatment of individuals who are heterozygous carriers of a deleterious mutation in NPR2 with neither parenthaving short stature is also contemplated. Further contemplated is treatment of individuals who are heterozygous for deleterious mutations in other growth plate genes with CNP to improve stature and / or enhance bone growth.

[0140] Exemplary NPR2 mutations in patients that may be treated with a CNP variant include: Amino acid Disease Nucleotide Mutation change9:35793980:T:A Tyr250Ter 9:35807085:C:T Thr861Ile 9:35793906:A:G Ile226Val 9:35808558:G:A Arg921Gln 9:35802741:G:A Glu609Lys 9:35802594:G:A Arg601His 9:35808663:T:A Leu956Gln 9:35808545:G:C Gly917Arg

[0141] NPPC’s role in skeletal growth is well documented (Hisado-Oliva et al., Genetics Medicine 20:91-97, 2018). The NPPC knock out mouse showed severe disproportionate form of dwarfism including shortening of limbs and endochondral ossification (Hisado-Oliva et al., 2018, supra). Human genome wide studies have shown a link between NPPC and height (Hisado-Oliva et al., 2018, supra). Although CNP haploinsufficiency has been believed to be a cause of short stature in humans, a recent study identified heterozygous mutations in families with short stature and smaller hands, presenting with dominantly inherited short stature with heights ranging from -4.3 to -2.3 SD (Hisado-Oliva et al., 2018, supra). These studies observed significant reduction in cGMP production as measured in heterozygous state (Hisado-Oliva et al., 2018, supra). Mutations in NPPC include a 355G>T missense mutation causing a Gly119Cys change and a 349C>G missense mutation causing a Arg117Gly change. In vitro studies showed that these missense variants led to decreased cGMP production upon stimulation of the NPR-B receptor in both the homozygous and heterozygous state. A CNP variant rescuing CGMP production may provide therapeutic benefit in the management of a disorder in patients having heterozygous loss-of-function NPPC mutations.

[0142] Turner syndrome, short stature homeobox-containing gene (SHOX) deficiency disorders, and Noonan syndrome are all rare genetic conditions that share short stature as a key element of their phenotype (Karlberg et al., Acta Paediatr Scand.1991;80(12):1158-1165; Karlberg, Excerpta Med ICS 1991;924:89–94; Rongen-Westerlaken et al., Swedish Study Group GH Treat. Acta Paediatr.1997;86(9):937–942; Binder and Rappold, SHOX Deficiency Disorders. In: GeneReviews®. University of Washington, Seattle, Seattle (WA), 2018; Fukami et al., Mol Syndromol.2016;7(1):3-11; Jorge et al., Pediatr Endocrinol Rev.2010;8(2):79-85; Bhambhani et al., Am Fam Physician.2014;89(1):37-43; Noonan et al., Am J Medical Genetics -Part A.2003;123A(1):68-71). Further, all 3 share a common pathophysiologic mechanism underlying short stature, disruption of the RAS-mitogen-activated protein kinase (RAS-MAPK) pathway (Roberts et al., Lancet.2013;381; 333-342).

[0143] SHOX is another gene implicated at the growth plate presenting with short stature. It encodes a transcription factor expressed throughout the growth plate with many functions including influencing the NPR-B and FGFR3 pathways. SHOX mutations have been estimated to account for approximately 2-10% of patients with idiopathic short stature (Rappold et al., J. Clin. Endocrinol. Metab.87:1402–1406, 2002; Marchini et al., Endocr Rev.37: 417–448, 2016). The wide range of mechanistic effects of SHOX were extensively reviewed by Marchini et al. (Marchini et al., Endocr Rev.37: 417–448, 2016). Relevant to the current protocol, SHOX has been shown to be a repressor of FGFR3 transcription. Decker et al. used chromatin immunoprecipitation to demonstrate that SHOX directly binds to multiple upstream regulatory elements around FGFR3 (Decker et al., Hum. Mol. Genet.20:1524–35, 2011). They then employed an in vitro model system for chondrogenesis and endochondral ossification using micromass cultures of chicken mesenchymal cells. Injecting a construct that expressed SHOX led to significant decreases in Fgfr3 expression (Decker, supra). Thus, there is a possible link in the pathophysiology of SHOX deficiency and the FGFR3 pathway. SHOX deficiency leads to increased FGFR3 signaling, similar to what is seen in achondroplasia. Additionally, there is some evidence to support that SHOX has direct interactions with CNP / NPR2 as well (Marchini, supra).

[0144] Isolated SHOX deficiency is one of the more prevalent monogenic causes of short stature (birth prevalence approximately 10 per 100,000 [Marchini et al., Endocr Rev.37: 417– 448, 2016]) (Genoni et al., Pediatr Res.2018;83(2):438-444). The SHOX gene, which is located on both the X and Y chromosomes, encodes for a transcription factor expressed throughout the growth plate with functions including influencing the natriuretic peptide receptor-type B (NPR-B) and fibroblast growth factor receptor 3 (FGFR3) pathways. The SHOX gene has been shown to be a repressor of FGFR3 transcription, and SHOX deficiency leads to increased FGFR3 signaling (Marchini, supra). Several pathogenic variants in SHOX are known to give rise to short stature. Individuals with pathogenic variants causing SHOX deficiency present with growth failure in the first year of life, height that often remains below -2.00 SDs compared to CDCpopulation norms, and no development of a pubertal growth spurt accentuating final statural impairment (Binder 2018, supra; Fukami 2016, supra; Jorge 2010, supra).

[0145] Turner syndrome is a rare chromosomal disorder (birth prevalence 32 per 100,000 females [16 per 100,000 total population] [Martin-Giacalone et al. Am J Med Genet A. 2023;191(5):1339-1349]) causing short stature and other phenotypic features in girls. It is caused by a structurally abnormal, or a partial or complete absence of 1 X chromosome which can in turn result in a loss of one copy of the SHOX gene (located on the X-chromosome). The absence of 1 copy of the SHOX gene results in disordered growth and skeletal anomalies (Gravholt et al., Nat Rev Endocrinol.2019;15(10):601-614). Girls with Turner syndrome exhibit growth rate slowing after their first 3 years of life, with adult height deficits further impacted by an absent pubertal growth spurt. Individuals have final heights significantly shorter than average stature women, with a mean height deficit of 20 cm from that predicted by mid-parental height (Karlberg 1991, supra; Rongen-Westerlaken et al., Swedish Study Group GH Treat. Acta Paediatr.1997;86(9):937–942). Given the association of SHOX with FGFR3 and bone growth, it is contemplated that a subject having a homozygous or heterozygous SHOX mutation would benefit from treatment with CNP variants as described herein.

[0146] Leri-Weill dyschondrosteosis (LWD) is a rare genetic disorder characterized by shortening of the forearms and lower legs, abnormal misalignment of the wrist (Madelung deformity of the wrist), and associated short stature. LWD is caused by a heterozygous mutation in the short stature homeobox-containing (SHOX) gene or its regulatory elements located on the pseudoautosomal region 1 (PAR1) of the sex chromosomes. (See the Rare Disease Database and Carmona et al., Hum Mol Genet 20:1547-1559, 2011). The disorder Langer mesomelic dysplasia arises when there are two SHOX mutations, and may result from a mutation on each chromosome, either a homozygous or compound heterozygous mutations. A subset of SHOX mutations give rise to idiopathic short stature. Turner syndrome results due to a deletion on the X chromosome that can include the SHOX gene. SHOX has been identified as involved in the regulation of FGFR3 transcription and contributes to control of bone growth (Marchini et al., Endocr Rev.37: 417–448, 2016). SHOX deficiency leads to increased FGFR3 signaling, and there is some evidence to support that SHOX has direct interactions with CNP / NPR2 as well (Marchini, supra). Given the association of SHOX with FGFR3 and bone growth, it iscontemplated that a subject having a homozygous or heterozygous SHOX mutation would benefit from treatment with CNP variants as described herein.

[0147] RASopathies are a group of rare genetic conditions caused by mutations in genes of the Ras / mitogen-activated protein kinase (MAPK) pathway. RASopathies are a group of disorders characterized by increased signaling through RAS / MAPK pathway. This pathway leads to downstream activation of the RAF / MEK / ERK pathway. Short stature is a characteristic feature of certain RASopathies. For example, CNP signaling inhibits RAF and leads to decreased MEK and ERK activation.

[0148] Treatment of RASopathies is provided herein. RASopathies associated with short stature include Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, and LEOPARD syndrome. Hereditary gingival fibromatosis type 1 is also a RASopathy contemplated herein. RASopathy patients (including Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, LEOPARD syndrome, 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).

[0149] 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 called faciocutaneoskeletal (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) that acts on 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, and SOS1, as well as K-Ras and Raf-1.

[0150] CNP has been demonstrated to be an effective therapy in RASopathy models. Ono et al. generated mice deficient in Nf1 in type II collagen producing cells (Ono et al., Hum. Mol. Genet.2013;22(15):3048–62). These mice demonstrated constitutive ERK1 / 2 activation, and decreased chondrocyte proliferation, and maturation. Daily injections of CNP in these mice ledto decreased ERK phosphorylation and corrected the short stature. A mouse model of Cardiofaciocutaneous syndrome using a Braf mutation (p.Q241R) (Inoue et al., Hum. Mol. Genet.2019;28(1):74–83) exhibited decreased body length and reduced growth plate width with smaller proliferative and hypertrophic zones compared to wild type, and CNP administration led to increases in body length in these animals.

[0151] Mutations in multiple genes can cause Noonan syndrome, which is characterized by short stature, heart defects, bleeding problems, and skeletal malformations. Mutations in the PTPN11 gene cause about half of all cases of Noonan’s syndrome. SOS1 gene mutations cause an additional 10 to 15 percent, and RAF1 and RIT1 genes each account for about 5 percent of cases. Mutations in other genes each account for a small number of cases. The cause of Noonan syndrome in 15 to 20 percent of people with this disorder is unknown.

[0152] Noonan syndrome (birth prevalence 40 per 100,000 [NORD 2019]) is the most common RASopathy, a clinically defined group of disorders caused by a germline mutation in one of the genes encoding components of the RAS-MAPK pathway, typically resulting in increased signaling through this pathway. The RAS-MAPK pathway leads to downstream activation of RAF / MEK / extracellular-signal-regulated kinase (ERK). C-type natriuretic peptide (CNP) signaling intersects with this pathway by inhibiting RAF, leading to decreased MEK and ERK activation. Noonan syndrome primarily results from gain of function (GoF) mutations in genes encoding components of the RAS-MAPK pathway. Noonan syndrome is characterized by short stature in 50-70% of those affected (Bhambhani 2014, supra), with typical facial features and cardiac defects in more than 80% of patients (Noonan JA, Progress in Pediatric Cardiology. 2005;20(2):177-185), and multi-system involvement in older children (Allanson et al., Cassidy and Allanson's Management of Genetic Syndromes.2021:651-669; Breilyn et al., Pediatr Endocrinol Rev.2019;16(Suppl 2):428-434). Growth failure occurs in the first year of life and the height of the child often remains below -2.00 SDs until puberty, when the growth is further affected due to an attenuated pubertal growth spurt (Carcavilla et al., Anales De Pediatría (English Edition) 2020;93(1):61.e1-61.e14.). Approximately 50% of adults with Noonan syndrome have significantly reduced height (Noonan et al., Am J Medical Genetics - Part A. 2003;123A(1):68-7).

[0153] The PTPN11, SOS1, RAF1, and RIT1 genes all encode for proteins that are important in the RAS / MAPK cell signaling pathway, which is needed for cell division and growth(proliferation), differentiation, and cell migration. Many of the mutations in the genes associated with Noonan syndrome cause the resulting protein to be turned on (active) and this prolonged activation alters normal RAS / MAPK signaling, which disrupts the regulation of cell growth and division, leading to the characteristic features of Noonan syndrome. See, e.g., Chen et al., Proc Natl Acad Sci U S A.111(31):11473-8, 2014, Romano et al., Pediatrics.126(4):746-59, 2010, and Milosavljević et al., Am J Med Genet 170(7):1874-80, 2016. It is contemplated that a subject having mutations that activate the MAPK pathway would benefit from treatment with CNP variants as described herein to improve bone growth and short stature. It is also contemplated that a subject having mutations that activate the MAPK pathway would benefit from treatment with CNP variants as described herein to improve other comorbidities associated with an overactive MAPK pathway in other cells throughout the body where the NPR2 receptor is expressed on its surface.

[0154] Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, lead to disorders characterized by short stature such as Noonan’s Syndrome (Musente et al., Eur J Hum Genet 11:201–206 (2003). Musente (supra) identifies numerous mutations in the PTPN11 gene that lead to short stature. Gain of function mutations lead to overactive signaling through SHP2 and inhibit Growth Hormone-induced IGF-1 release, thereby contributing to a decrease in bone growth (Rocca Serra-Nédélec, PNAS 109:4257- 4262, 2012). It is contemplated that a subject having a homozygous or heterozygous PTPN11 mutation would benefit from treatment with CNP variants as described herein to improve bone growth and short stature.

[0155] Mutations in the Indian hedgehog (IHH) gene, which is related to regulation of endochondral ossification, have also been associated with short stature syndromes (Vasques et al., J Clin Endocrinol Metab.103:604-614, 2018). Many IHH mutations identified segregate with short stature in a dominant inheritance pattern. Given the association of IHH with bone growth and ossification, it is contemplated that subjects having a homozygous or heterozygous IHH mutation will benefit from treatment with a CNP variant as described herein.

[0156] Hypochondroplasia is due to milder mutations in the FGFR3 gene with the p.Asn540Lys mutation being by far the most common cause (Bober et al., Hypochondroplasia. In GeneReviews®, University of Washington, Seattle; 1993–2021.1999 Jul 15). Mutations in FGFR3, including N540K and K650N, lead to short stature and hypochondroplasia. Therationale for using vosoritide in hypochondroplasia is primarily an extension of the positive clinic trial results in patients with achondroplasia, a more severe perturbation of the same gene. In vitro studies have shown that the p.Asn540Lys mutation causing hypochondroplasia leads to increased pERK (a marker of activation of the FGFR3 pathway) and decreased cell proliferation (Krejci et al., PLoS One 3(12), 2008). In certain embodiments, the subjects having hypochondroplasia caused by a mutation in FGFR3 are in an FGFR3 variant subgroup, e.g., having GFR3 mutations c.1620C>G, c.1620C>A resulting in Asn540Lys change.

[0157] Others changes in FGRF3 that can confirm diagnosis of hypochondroplasia include Leu326Trp, Leu324Val, Gly268Cys, Thr264Met, Ser348Cys, Ser351Phe, Ser351Cys, Lys650Thr, Gly342Cys, Ser84Leu, Tyr278Cys, Ser279Cys, Lys650Gln, Lys650Asn, Asn540Thr, Pro250Arg

[0158] Insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (α2β2) transmembrane glycoprotein with an intrinsic kinase activity. IGF1R has been shown to have a role in prenatal and postnatal growth. Heterozygous mutations in IGF1R have been identified in Small for gestational age children (SGA) and individuals with familial short stature (Kawashima et al., Endocrine J.59:179-185, 2012). Mutations in IGF1R associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, supra).

[0159] Height is a highly heritable trait that can be influenced by the combined effect of hundreds or thousands of genes (Wood et al, 2014, Nature Genetics, 46:1173-1189. Short stature in an individual can be the result of the combined effect of these genes, without a single gene being the primary contributor. It is contemplated that such individuals with short stature defined by a height SDS of less than -1.0, -1.5, -2.0, -2.25, -2.5, -2.75 or -3.0, can be beneficially treated with a CNP variant given the ability of CNP to increase the length of normal animals, for example, enhance bone growth and length of bones.

[0160] In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of less than -1.0, -1.5, -2.0, -2.25, -2.5, -2.75 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, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -3.0. In various embodiments, the CNP variants are useful to treat a subject with short stature having aheight SDS of between -2.0 to -2.5. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.25 or less. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.00 or less. In various embodiments, the short stature is associated with one or more mutations in a gene associated with short stature, such as, collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, SHOX, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), or combinations thereof.

[0161] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.

[0162] In various embodiments, the short stature is a result of mutations in multiple genes as determined by polygenic risk score (PRS). Polygenic risk scores (PRS) were calculated for height using the largest published GWAS meta-analysis for height that did not include any samples from the UK Biobank project as described in International Patent Publication WO 2021 / 055497. The cohort was divided into five PRS quintiles (PRS 1 being the lowest height, PRS 5 the tallest 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 mutation 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.

[0163] In certain embodiments, the CNP variants and compositions and formulations comprising the same of the present disclosure are useful for improving one or more of the symptom(s) or physiological consequences of a short stature disorder, wherein the improvement may be increased absolute growth, increased growth velocity, increased qualitative computed tomography (QCT) bone mineral density, improvement in growth plate morphology, increased long bone growth, improvement in spinal morphology, improved elbow joint range of motion and / or decreased sleep apnea.

[0164] In various embodiments, the CNP variants and compositions and formulations comprising the same of the present disclosure are useful for improving one or more of the symptom(s) or physiological consequences of a short stature disorder, wherein the improvement may be increased absolute growth, increased growth velocity, increased qualitative computed tomography (QCT) bone mineral density, improvement in growth plate morphology, increased long bone growth, improvement in spinal morphology, improved tibial bowing, improved elbow joint range of motion and / or decreased sleep apnea.

[0165] The disclosure also provides methods of treating growth deficiencies or short stature associated with MPS IVA, (Morquio Syndrome) or MPS VI (Maroteaux-Lamy Syndrome) comprising administering a therapeutically effective amount of a CNP variant as described herein. Assessments of efficacy of the treatment include improvement in one or more of height Z score, evaluation of health and functional performance, pulmonary and cardiac systems, linear and segmental bone growth, biomarkers of growth, inflammation and bone metabolism, and self-reported tolerability and quality of life.

[0166] In this regard, it is noted that the terms "improved", "improvement", "increase", "decrease" and grammatical equivalents thereof are all relative terms that when used in relation to a symptom or physiological consequence of a disease state, refer to the state of the symptom or physiological consequence of the disease after treatment with a CNP variant (or composition or formulation comprising the same) of the present invention as compared to the same symptom or physiological consequence of the disease before treatment with a CNP variant (or composition or formulation comprising the same) of the present invention (i.e., as compared to "baseline"). As described above, a "baseline" state can be determined either through measurement of the state in the subject prior to treatment (which can subsequently be compared to the state in the same subject after treatment), or through measurement of that state in a population of subjects suffering from the same affliction that share the same or similar characteristics (e.g., age, sex and / or disease state or progression).

[0167] Also provided is a method of overcoming cell growth arrest induced by a constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3) comprising contacting a cell expressing the constitutively active FGFR-3 with a CNP variant or a composition as described herein.

[0168] 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 a composition as described herein.

[0169] In yet another embodiment, the disclosure provides for use of CNP variants that in vitro or in vivo stimulate the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP level produced under the same concentration of wtCNP22 (e.g., 1 uM). In a still further embodiment, the CNP variants described herein in vitro or in vivo stimulate the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP level produced under the same concentration of wtCNP22 (e.g., 1 uM).

[0170] It is contemplated that any of the CNP variants including conjugates, salts or prodrugs thereof, described herein are useful in the methods.

[0171] 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 an NH2 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 hydrolysable linker.

[0172] In various embodiments, the CNP variant is selected from the group consisting of PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:1); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO:MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55); and LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO:2), wherein the CNP variant comprises a conjugate moiety. In various embodiments, the conjugate moiety is a synthetic polymeric group.

[0173] In various embodiments, the variant comprises a synthetic polymeric group coupled to the variant through a hydrolysable linker. In various embodiments, the synthetic polymeric 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 6 to 20 atom chain length. For purposes of this conjugate, the synthetic polymeric group is not a peptide.

[0174] In various embodiments, the conjugate moiety comprises one or more acid moieties linked to a hydrophilic spacer as described herein. 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 two or more OEG (8-amino- 3,6-dioxaoctanoic acid). In various embodiments, the acid moiety is a fatty acid. Exemplary fatty acids include short chain, medium chain, or long chain fatty acids, or a dicarboxylic fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. Contemplated are C-6 to C-20 fatty acids, including but not limited to, C-6, C-8, C-10, C-12, C-14, C-16, C-18 or C-20 fatty acids, saturated or unsaturated. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids of the same.

[0175] In various embodiments, the variant comprises one or more linker groups. In various embodiments, the linker is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0176] Efficacy of treatment is measured by various parameters. In various embodiments, efficacy is assessed as the change in annualized growth velocity from the baseline period to the intervention period. Efficacy will also be assessed as the change in height SDS from baseline to end of treatment as measured using the CDC growth curves, and growth velocity SDS will be based on the Bone Mineral Density in Childhood Study (Kelly et al., J. Clin. Endocrinol. Metab. 2014;99(6):2104–2112).

[0177] Health Related Quality of Life (HRQoL) indices are also measured.

[0178] QoLISSY, the Quality of Life in Short Stature Youth, is assessed as directed (Quality of Life in Short Stature Youth - The QoLISSY Questionnaire User’s Manual. Lengerich: Pabst Science Publishers; 2013). The QoLISSY questionnaire is a disease-specific clinical outcome assessment designed for short stature youth, which has both child self-report and parent-report versions and consists of questions across 8 domains, physical, social, emotional, coping, treatment, belief, future, and effects on parents. Domain total scores are then linearly transformed to standard scores on a scale of 0 to 100: min score = 0 (worst QoL), max score = 100 (best QoL).

[0179] The PedsQL™ is a robust generic pediatric quality of life clinical outcome assessment that is applicable broadly for any condition and includes domains (Physical Function, Emotional Function, School Function, Social Function) assessing HRQoL impacts in children and adolescents, that has both child self-report and parent-report versions and consists of questions across 4 domains: physical functioning, emotional functioning, social functioning, and school functioning. Scores are linearly transformed to a standard score on a scale of 0 to 100: min score = 0 (worst QoL), max score = 100 (best QoL).

[0180] The Patient and Caregiver Global Impression of Severity (PGI-S; CaGI-S) are standard, single items that have been widely used across a broad range of disease areas and are typically included in clinical studies as anchor measures to help establish meaningful change in other clinical outcome assessments. The PGI-S and CaGI-S are single global items used to rate the overall condition of the patient, as reported by the patient (PGI-S) and observedby the caregiver (CaGI-S); the items include a 5-point Likert scale from “None” to “Very severe”. Both global and domain-specific items will be administered in this study and patient-reported items will be administered only to those who are at the age of ≥ 8 years where they can reliably and validly self-report.

[0181] The Patient and Caregiver Global Impression of Change (PGI-C; CaGI-C) are standard single items that have been widely used across a broad range of disease areas and are typically included in clinical studies as anchor measures to help establish meaningful change in other clinical outcome assessments. The PGI-C and CaGI-C are single global items used to assess if there has been an improvement or change in clinical status; the items include a 5-point Likert scale from “Much better” to “Much worse”. Both global and domain-specific items will be administered in this study and patient-reported items will be administered only to those who are at the age of ≥ 8 years where they can reliably and validly self-report.

[0182] Also measured Is the Kaufman Assessment Battery for Children-II (KABC-II) to monitor cognitive function using a neurocognitive battery of tests to monitor cognitive decline. The KABC-II (Kaufman and Kaufman 1983) is a standardized clinical instrument (psychological diagnostic test) for assessing cognitive development in children aged 3 to 18 years. The KABC- II measures a child’s cognitive ability and processing skills, and measures these abilities in a way that reduces score differences between ethnic and cultural groups, providing confidence in the assessment of children and adolescent from a variety of backgrounds. Using a single battery of 18 core (and supplementary) subtests, the KABC-II supports interpretation through three index scores: Mental Processing Index, Fluid-Crystallized Index, and Nonverbal Index. The time for administration can be up to 70 minutes, depending on the index selected, the age of the child and the corresponding subtests administered. Further information on the administration of subtests and scoring is outlined in the KABC-II Manual.

[0183] Respiratory complications in HCH include multiple otitis media, sleep apnea and conductive hearing loss. Seizures are also known complications of hypochondroplasia. Patients are measured for any change in incidence of otitis media or frequency or severity of seizures as a result of treatment described herein. Pharmaceutical Compositions

[0184] The 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 the clearance receptor NPR-C).

[0185] The disclosure provides for modified release compositions comprising a conjugate moiety as described herein. Modified-release compositions include those that deliver a drug with a delay after its administration (delayed-release dosage) or for a prolonged period of time (extended-release dosage). Various embodiments of a CNP peptide conjugate provided herein include modified-release compositions, such as extended release, sustained or controlled release, and delayed release. The term “extended release composition” refers to a composition formulated in a manner in order to make the active ingredient / drug available over an extended period of time following administration (US Pharmacopeia). Extended-release dosage include sustained-release (SR) or controlled-release (CR) forms in which. Sustained release maintains drug release over a sustained period but not necessarily at a constant rate, while CR maintains drug release over a sustained period at a nearly constant rate (Pharmaceutics: Drug Delivery and Targeting, Yvonne Perrie, Thomas Rades, Pharmaceutical Press, 2009). Delayed-release compositions or products are modified to delay release of the drug substance for some period of time after initial administration.

[0186] In various embodiments, the modified release composition is an extended release composition.

[0187] 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.

[0188] Non-limiting examples of excipients, carriers and diluents include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and so on. The compositions can contain liquids (e.g., water, ethanol); diluents of various buffer content (e.g., Tris-HCl, phosphate, acetate buffers, citrate buffers), pH and ionic strength; detergents and solubilizing agents (e.g., Polysorbate 20,Polysorbate 80); anti-oxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., Thimerosol, 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, e.g., Remington's Pharmaceutical Sciences, 18thEdition, pages 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0189] For example, carriers include without limitation diluents, vehicles and adjuvants, as well as implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials that do not react with the active ingredient(s). Non-limiting examples of carriers include phosphate buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). A carrier can be a solvent or dispersing medium containing, e.g., ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), a vegetable oil, and mixtures thereof.

[0190] In some embodiments, the compositions are liquid formulations. In certain embodiments, the formulations comprise a CNP variant in a concentration range 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 in 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.

[0191] In further embodiments, the compositions comprise a buffer solution or buffering agent to maintain the pH of a CNP-containing solution or suspension within a desired range. Non- limiting examples of buffer solutions include phosphate buffered saline, Tris buffered saline, and Hank's buffered saline. Buffering agents include without limitation 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 or citric acid / citrate. The amount of buffering agent suitable in a 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 a composition is fromabout pH 3 to about pH 9, or from about pH 3 to about pH 7.5, or from about pH 3.5 to about pH 7, or from about pH 3.5 to about pH 6.5, or from about pH 4 to about pH 6, or from about pH 4 to about pH 5, or is at 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.

[0192] In other embodiments, the compositions contain an isotonicity-adjusting agent to render the solution or suspension isotonic and more compatible 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, NaCl is in 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.

[0193] In yet other embodiments, the compositions comprise a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In certain embodiments, the preservative is in a concentration of about 0.4% ± 0.2%, or about 1% ± 0.5%, or about 1.5% ± 0.5%, or about 2.0% ± 0.5%.

[0194] In still other embodiments, the compositions contain an anti-adsorbent (e.g., to mitigate adsorption of a CNP variant to glass or plastic). Anti-adsorbents include without limitation benzyl alcohol, Polysorbate 20, and Polysorbate 80. In certain embodiments, the anti- adsorbent is in a concentration from about 0.001% to about 0.5%, or from about 0.01% to about 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about 1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, or from about 1% to about 2%.

[0195] In additional embodiments, the compositions comprise a stabilizer. Non-limiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and salts thereof. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or a salt thereof, the stabilizer is in a concentration from about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer is in a concentration from about 0.01% to about 0.5%, or from about 0.01% to about 0.2%. In still other embodiments, when the stabilizer is glycerin, the stabilizer is in a concentration from about 5% to about 100% (neat).

[0196] In further embodiments, the compositions contain an antioxidant. Exemplary antioxidants include without limitation methionine and ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP is from about 0.1:1 to about 15:1, or from about 1:1 toabout 15:1, or from about 0.5:1 to about 10:1, or from about 1:1 to about 10:1 or from about 3:1 to about 10:1.

[0197] Pharmaceutically acceptable salts can be used in the compositions, including without limitation mineral 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 thorough discussion of pharmaceutically acceptable salts is found in Remington's Pharmaceutical Sciences, 18thEdition, Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0198] Pharmaceutical compositions can be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage forms of the compositions can be tailored to the desired mode of administration of the compositions. For oral administration, the compositions can take the form of, e.g., a tablet or capsule (including softgel capsule), or can be, e.g., an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules for oral administration can include 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 lubricating agents (e.g., magnesium stearate, sodium stearyl fumarate). If desired, flavoring, coloring and / or sweetening agents can be added to the solid and liquid formulations. Other optional ingredients for oral formulations include without limitation preservatives, suspending agents, and thickening agents. Oral formulations can also have an enteric coating to protect the CNP variant from the acidic environment of the stomach. Methods of preparing solid and liquid dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington's Pharmaceutical Sciences, referenced above).

[0199] Formulations for parenteral administration can be prepared, e.g., as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in a liquid medium 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., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution), dispersing agents, wetting agents, emulsifying agents, suspending agents, and the like. In addition, sterile fixed oils, fatty esters, polyols and / or other inactive ingredients can be used. As further examples, formulations for parenteral administration include aqueous sterile injectable solutions,which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can contain suspending agents and thickening agents.

[0200] Compositions comprising a CNP variant can also be lyophilized formulations. In certain embodiments, the lyophilized formulations comprise a buffer and bulking agent, and optionally an antioxidant. Exemplary buffers include without limitation acetate buffers and citrate buffers. Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, mannitol is in an amount from about 3% to about 10%, or from about 4% to about 8%, or from about 4% to about 6%. In certain embodiments, sucrose is in an amount from about 6% to about 20%, or from about 6% to about 15%, or from about 8% to about 12%. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid.

[0201] In various embodiments, the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).

[0202] The disclosure also provides kits containing, e.g., bottles, vials, ampoules, tubes, cartridges and / or syringes that comprise a liquid (e.g., sterile injectable) formulation or a solid (e.g., lyophilized) formulation. The kits can also contain pharmaceutically acceptable vehicles or carriers (e.g., solvents, solutions and / or buffers) for reconstituting a solid (e.g., lyophilized) formulation into a solution or suspension for administration (e.g., by injection), including without limitation reconstituting a lyophilized formulation in a syringe for injection or for diluting concentrate to a lower concentration. Furthermore, extemporaneous injection solutions and suspensions can be prepared from, e.g., sterile powder, granules, or tablets comprising a CNP- containing composition. The kits can also include dispensing devices, such as aerosol or injection dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes, and / or needles.

[0203] As a non-limiting example, a kit can include syringes having a single chamber or dual chambers. For single-chamber syringes, the single chamber can contain a liquid CNP formulation ready for injection, or a solid (e.g., lyophilized) CNP formulation or a liquid formulation of a 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. For dual-chambersyringes, one chamber can contain a pharmaceutically acceptable vehicle or carrier (e.g., solvent system, solution or buffer), and the other chamber can contain a solid (e.g., lyophilized) CNP formulation or a 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, for injection.

[0204] As a further example, a kit can include one or more pen injector or autoinjector devices, and dual-chamber cartridges. One chamber of a cartridge can contain a pharmaceutically acceptable vehicle or carrier (e.g., solvent system, solution or buffer), and the other chamber can contain a solid (e.g., lyophilized) CNP formulation or a 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, for injection. A cartridge can comprise an amount of the CNP variant that is sufficient for dosing over a desired time period (e.g., 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen injector or autoinjector can be adjusted to administer a desired amount of the CNP formulation from a cartridge. Administration and Dosing

[0205] The CNP variants, or pharmaceutical compositions or formulations comprising them, can be administered to subjects in various ways such as, e.g., 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 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.

[0206] The CNP variants or compositions thereof can also be administered by implantation of a depot at the target site of action (e.g., an abnormal or degenerated joint or cartilage area). Alternatively, the CNP variant can be administered sublingually under the tongue (e.g., sublingual tablet) by transdermal delivery (e.g., by means of a patch on the skin) or orally in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamides, polylactide, polyglycolide, poly(lactide-glycolide)), microemulsions, and the like.

[0207] The CNP variant compositions described herein can be administered to patients in need thereof at therapeutically effective doses to treat, ameliorate or prevent bone-related disorders or short stature disorders (e.g., skeletal dysplasias, including achondroplasia, hypochondroplasia, idiopathic short stature, Turner syndrome, SHOX deficiency, Noonan’s syndrome, etc.). The safety and therapeutic efficacy of the CNP variant can be determined by standard pharmacological procedures in cell cultures or experimental animals, such as, for example, by determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Active agents exhibiting a large therapeutic index are normally preferred.

[0208] In certain embodiments, the CNP variant compositions described herein are administered at a dose in the range 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 compositions are 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 dose deemed appropriate by the treating physician. In other embodiments, the CNP variant compositions are administered at a dose 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, 700, 750, 800, 850, 900, 950 or 1000 µg / kg, or 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 other dose deemed appropriate by the treating physician. The doses of CNP or CNP variant described herein can be administered according to the dosing frequency / frequency of administration described herein, including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc. In various embodiments, the CNP or CNP variant is administered daily subcutaneously. In various embodiments, the CNP or CNP variant is administered weekly subcutaneously. In various embodiments, the CNP variant is administered at a dose of 2.5 µg / kg / day to 30 µg / kg / day, 7.5 µg / kg / day to 22.5 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 7.5 µg / kg / day. In variousembodiments, 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 22.5 µg / kg / day.

[0209] In various embodiments, the administered dose is 0.4 mg, 0.56 mg, or 1.2 mg lyophilized powder in a single-dose vial for reconstitution, e.g., at 0.8 mg / mL or 2 mg / mL.

[0210] The frequency of dosing / administration of a CNP variant for a particular subject may vary depending upon various factors, including the disorder being treated and the condition and response of the subject to the therapy. The CNP variant can be administered in a single dose or in multiple doses per dosing. In certain embodiments, the CNP variant composition is administered, in a single dose or in multiple doses, once daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 6 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.

[0211] In some embodiments, a CNP variant composition is administered so as to allow for periods of growth (e.g., chondrogenesis), followed by a recovery period (e.g., osteogenesis). For example, the CNP composition may be administered subcutaneously or by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, 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 per week) is for 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 a related embodiment, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain embodiments, the dosing regimen of the CNP variant compositions 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. Biomarkers

[0212] For treatment of bone-related disorders, indicators of growth can be measured, such as long bone growth measurements in utero and neonatal and measurements of bone growthbiomarkers such as CNP, cGMP, Collagen II, Collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), osteocalcin, Proliferating Cell Nuclear Antigen (PCNA) NTproCNP, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N-propeptide (PINP), cross-linked C-telopeptide of type I collagen (CTx), cross-linked N-telopeptide of type I collagen (NTx) or tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts such as PAXgene RNA, and CNP- variant bioactivity.

[0213] One CNP signaling marker is cGMP (guanosine 3’,5’ cyclic monophosphate). The level of this intracellular signaling molecule increases after CNP binds to and activates its cognate receptor NPR-B. Elevated levels of cGMP can be measured from cell culture extracts (in vitro) after CNP exposure, conditioned media from bone ex-plant studies (ex vivo) after CNP exposure, and in the plasma (in vivo) within minutes of CNP administration subcutaneously, intravenously, or via other routes of administration known in the art.

[0214] Cartilage and bone-specific analytes (or cartilage- and bone-associated markers) can also be measured to assess CNP efficacy. For example, fragments of cleaved collagen type II are a cartilage-specific marker for cartilage turnover. Type II collagen is the major organic constituent of cartilage and fragments of type II collagen (cleaved collagen) are released into circulation, and subsequently secreted into the urine, following cartilage turnover. Cartilage turnover precedes new bone formation.

[0215] NTproCNP is an amino-terminal propeptide (NTproCNP) of CNP that is released from cells at an equimolar ratio with CNP. The biologically active forms of CNP are found in plasma in low concentrations due to the quick clearance rate of the peptide. NTproCNP is not cleared via the same mechanism and it is found in the circulation at 20- to 50-fold higher concentration (Olney et al., Clin Endocrinol (Oxf).2012, 77:416–422).

[0216] Collagen type X biomarker (CXM) is a degradation fragment of collagen type X, comprising intact trimeric noncollagenous 1 (NC1) domain of type X collagen. CXM is released by active growth plates and decreases in samples as subjects age. CXM levels have been correlated with growth velocity in children (Coghlan et al., Sci Transl Med 2017, 9(419):eaan4669).

[0217] Bone-specific alkaline phosphatase (BSAP or BAP) is a bone growth biomarker produced by osteoblasts and osteoclasts in growth plates and mineralized bone. Changes in BSAP may reflect growth plate activity, bone growth, and / or bone remodeling activity.

[0218] A bone-specific biomarker for bone formation which can be measured is N-terminal propeptides of type I procollagen (PINP). The synthesis of type I collagen is an important step in bone formation, as type I collagen is the major organic component in bone matrix. During collagen synthesis, propeptides are released from the procollagen molecule and can be detected in serum. In addition, fragments of collagen type I can be measured as a marker for bone resorption.

[0219] Other potential biomarkers for cartilage and bone formation and growth include aggrecan chondroitin sulfate (cartilage-specific marker for cartilage turnover), propeptides of type II collagen (cartilage-specific marker for cartilage formation), collagen type I C-telopeptide (CTx), alkaline phosphatase (bone-specific) osteocalcin (bone-specific marker for bone formation), cross-linked N-telopeptide of type I collagen (NTx) or tartrate-resistant acid phosphatase 5b (TRAP-5b), and transcriptomics readouts, e.g., from PAXgene® RNA.

[0220] NTx detection in urine is useful to assess bone resorption in patients with metabolic bone disease and measuring osteoporotic bone loss. A decline in NTx during treatment is suggestive of effective therapy. TRAP 5b is a marker of bone resorption.

[0221] Cartilage- and bone-associated biomarkers can be measured, e.g., in urine, in serum from efficacy / pharmacodynamic in vivo studies and from the conditioned media of ex vivo studies, using commercially available kits.

[0222] In one embodiment, the level of at least one bone- or cartilage-associated biomarker is assayed or measured in a subject that has been administered a CNP variant or composition described herein in order to monitor the effects of the CNP composition on bone and cartilage formation and growth in vivo. For example, an increase in the level of at least one bone- or cartilage-associated biomarker may indicate that administration of a CNP variant or composition has a positive effect on bone growth and is a useful treatment for short stature disorder and other bone- or cartilage-related diseases or disorders associated with decreased CNP activity. Exemplary bone- or cartilage-associated biomarkers include, but are not limited to, CNP (e.g., endogenous levels of CNP), cGMP, propeptides of collagen type II and fragments thereof,collagen type II and fragments thereof, collagen type I C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen (PINP) and fragments thereof, collagen type I and fragments thereof, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), aggrecan chondroitin sulfate, NTproCNP, alkaline phosphatase, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, cross- linked C-telopeptide of type I collagen (CTx), cross-linked N-telopeptide of type I collagen (NTx) or tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, e.g., from PAXgene® RNA, and CNP-variant bioactivity.

[0223] In various embodiments, biomarkers are measured by obtaining a biological sample from a subject who will be administered, is being administered or has been administered a CNP variant. Biomarkers can be measured using techniques known in the art, including, but not limited to, Western Blot, enzyme linked immunosorbant assay (ELISA), enzymatic activity assay, and using nucleic acid assays, or transcriptomics readouts, e.g., from PAXgene® RNA. The biological sample can be blood, serum, urine, or other biological fluids.

[0224] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. EXAMPLES Example 1 – Phase 2 Study of CNP Variant in Children with Short Stature Disorders with Inadequate Response to Human Growth Hormone (hGH).

[0225] Multiple clinical studies using a CNP variant, vosoritide (Pro-Gly CNP37) have been completed. Wendt et al., (J Pharmacol Exp Ther.2015 Apr;353(1):132-49) describes an initial study of CNP variants to treat achondroplasia in children. Savarirayan et al. (Lancet.2020 Sep 5;396(10252):684-692) describes a phase 3 trial of vosoritide in children with achondroplasia. Savarirayan et al., Lancet Child Adolesc Health.2024 Jan;8(1):40-50, describes the results of a phase 2 trial using vosoritide in additional populations of achondroplasia patients, such as infants Qi et al., (Clin Pharmacokinet.2024 May;63(5):707-719) describes weight band dosing of vosoritide.

[0226] An ongoing investigator-led study (IND 145918), in pre-pubertal participants (Tanner Stage 1) who are aged ≥ 3 to < 10 years (females), and ≥ 3 to < 11 years (males) aims toassess the effect of vosoritide in children with selected genetic causes of short stature, including children with short stature attributed to variants in NPR2 and in ACAN. Children with short stature attributable to variants in FGFR3 (causing HCH), mutations in NPPC, mutations causing SHOX deficiency, and mutations in specific members of the RAS superfamily (RASopathies) are also eligible for inclusion in this study. Participants have a height deficit at Screening of ≤ -2.25 SDs compared to CDC average stature norms and are treated with daily subcutaneous (SC) vosoritide at a dose of 15 µg / kg, following a 6-month observation period to establish the baseline height velocity. Preliminary results suggest a clear growth promoting effect in children with selected mutations; results are provided in the current version of the vosoritide IB. Initial safety results from this study demonstrate an adverse event (AE) profile similar to that observed for ACH, and no cardiac electrophysiologic AEs have been reported. No treatment-limiting AEs were seen over up to 2 years of treatment. See Dauber et al., EClinicalMedicine.2024 Apr 11:71:102591.

[0227] Summary

[0228] Described below is a Phase 2 study to assess vosoritide (Pro-Gly CNP37) in children with Turner syndrome, short stature homeobox‑containing gene (SHOX) deficiency disorders, and Noonan syndrome who have an inadequate response to human growth hormone (hGH) treatment. The study will primarily assess the efficacy of daily vosoritide at 7.5 µg / kg, 15 µg / kg, and 22.5 µg / kg versus hGH (referred to as the Dose-Finding Phase). The long-term efficacy and safety of the selected therapeutic dose will be assessed up to final adult height (FAH) (referred to as the Long-Term Phase).

[0229] Pediatric patients currently receiving hGH treatment without an inadequate response were selected, given that there are currently no approved treatment options specifically addressing residual unmet need in this population. Such patients may have the potential to benefit from vosoritide, an analog of the master-growth-regulator C-type natriuretic peptide (CNP), as compared to further treatment with hGH since the pathophysiology underlying impaired growth in all these conditions involves disruption of growth plate biology rather than a disruption of the growth hormone (GH) axis.

[0230] Turner Syndrome: Participants must have a previously confirmed diagnosis of Turner syndrome based on a karyotype with a minimum of 30 cells or on a chromosomal microarray.Participants with Turner syndrome who showed mosaicism (such as a 46,XX / 45,X karyotype) will be required to have a minimum of 10% mosaicism of the 45,X cell line in order to participate in the study (Doğer et al., Reprod Biol Endocrinol.2015;13:59; Shankar et al., Ther Adv Endocrinol Metab.2018;9(1):33-40; Zhong et al., Fertil Steril.2012;98(4):775-779). Further, to be eligible participants must not have Y-chromosome material unless they have undergone gonadectomy and have fully external female genitalia.

[0231] SHOX Deficiency: Participants with SHOX deficiency will be required to have either at least 1 deletion in SHOX or its enhancer sequences or a heterozygous pathogenic or likely pathogenic variant as per ACMG guidelines. The deletion can cover all or part of the SHOX gene and can include single base pair deletions which result in a loss of function due to a frameshift (Binder and Rappold, SHOX Deficiency Disorders. In: GeneReviews®. University of Washington, Seattle, Seattle (WA);2018; Vannelli et al., Ital J Pediatr.2020;46(1):163).

[0232] Noonan Syndrome: Participants with Noonan syndrome will be required to have a heterozygous variant, defined as pathogenic or likely pathogenic as per ACMG guidelines, in one of the following genes: BRAF, KRAS, LZTR1, MAP2K1, MRAS, NRAS, PTPN11, RAF1, RIT1, RRAS2, SOS1, SOS2 (Roberts AE, GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993-2024).

[0233] Study Duration: A 6-month observational Baseline Growth Phase, a Dose-Finding Phase of at least 6 months, and a Long-Term Phase up to FAH.

[0234] Observations: This study will collect data on medical history, prior hGH treatment / dosing, anthropometric measurements (retrospective and prospective), health-related quality of life and functionality indicators, adverse events (AEs), clinical laboratory parameters, vital signs, anti-drug antibodies, biomarkers, genetics, dual X-ray absorptiometry (DXA) imaging and bone age assessment, lower extremity X-rays, pharmacokinetics, electrocardiograms (ECGs) and echocardiograms (ECHOs), and concomitant medications and medical procedures.

[0235] Number of Participants: Approximately 72 participants with genetically confirmed Turner syndrome (N ≥ 24), SHOX deficiency (N ≥ 16), or Noonan syndrome (N ≥ 24) are planned to be enrolled in the study (a minimum of 4 participants with SHOX deficiency and 6 participants each with Turner syndrome and with Noonan syndrome per study group). Patients with idiopathic short stature may also be enrolled. Eligible participants will be aged ≥ 3 yearsand < 10 years (females) or < 11 years (males) and have a height Z-score of ≤ -2.00 standard deviations (SDs). They must have been receiving continuous treatment with hGH for ≥ 1 year, and be receiving a dose of ≥ 0.35 mg / kg weekly with no dose changes in the last 6 months and no future dose changes planned. A participant’s response to hGH treatment will be defined as inadequate if their annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from Centers for Disease Control and Prevention (CDC) growth charts after a minimum 1 year of treatment.

[0236] Study Groups and Duration: At the Screening Visit, AGV will be assessed using height measurements obtained during that visit and historic height measurements obtained from their medical record (acquired in the period 5 to 12 months prior to the visit).

[0237] Eligible participants will enter a 6-month observational Baseline Growth Phase and will continue on their prior standard of care hGH regimen. At the end of the observational Baseline Growth Phase but within 28 days of randomization, a Pre-Randomization Assessment will be performed to re-check key eligibility criteria (height Z-score, Tanner stage, baseline AGV, and safety parameters). Participants who meet these key eligibility criteria will be stratified by condition and randomized in a 1:1:1:1 ratio to receive 1 of the following 4 study treatments: • vosoritide 7.5 µg / kg daily (n=18) • vosoritide 15 µg / kg daily (n=18) • vosoritide 22.5 µg / kg daily (n=18) • hGH daily provided by the Sponsor at an equivalent dose to their previous hGH treatment (n=18).

[0238] Participants will receive study treatment daily for at least 6 months in the Dose-Finding Phase. An interim analysis on dose response will be performed after at least 50% of participants have completed their 6-month follow-up visit in each treatment group. The final analysis to determine the therapeutic dose will be performed after all participants have either completed the 6-month follow-up visit or discontinued from the study.

[0239] All participants in the vosoritide groups will transition to the therapeutic dose of vosoritide for the Long-Term Phase. Participants randomized to the hGH group will receive open-label hGH until they have completed 24 months of hGH study treatment afterrandomization. Upon completion, participants will transition to vosoritide at the therapeutic dose for the remainder of the Long-Term Phase.

[0240] After 24 months of study treatment, an efficacy analysis will be performed to compare vosoritide at the therapeutic dose versus hGH. Treatment will continue until the participant reaches near-final adult height (NFAH), defined as: 1) decreased growth velocity (AGV < 1.5 cm / year) as assessed over a period of at least 6 months, and 2) growth plate fusion.

[0241] Participants who discontinue treatment should remain in the study until they either reach NFAH or are at least 16 years of age for females and 18 years of age for males, whichever comes later (i.e., until FAH).

[0242] Clinical Outcome Assessment

[0243] The impact of treatment on patients’ health-related quality of life (HRQoL) is an important and meaningful efficacy outcome measure. To understand the impact of short stature on well-being and functioning and to assess the impact of vosoritide on this important outcome, the following key clinical outcomes assessment (COA) measures will be studied: the Quality of Life in Short Stature Youth (QoLISSY), a validated measure in specific pediatric short stature populations (Bloemeke et al., J Pediatr Endocrinol Metab.2019;32(10):1089-1101; Bullinger et al., Health Qual Life Outcomes.2013;11:76), and the Pediatric Quality of Life Inventory (PedsQL), a reliable and valid generic measure used extensively to assess HRQoL in children (Varni et al., Med Care.2001;39(8):800-812). In order to generate additional content validity evidence to support the use of the QoLISSY in these specific populations and to help estimate and interpret meaningful change thresholds, interviews with a subset of participants and / or their caregivers will be conducted as part of this study. Additionally, the inclusion of global impression scales for patients and caregivers (PGI / CaGI-S, PGI / CaGI-C) will also help facilitate analyses aimed at establishing and interpreting meaningful change thresholds for the key aforementioned COAs.

[0244] Objectives and Endpoints

[0245] The primary and secondary objectives and endpoints are described in Table 1: Table 1Objectives Endpoints d A ity;AUC0-t, AUC from 0 to the time of last measurable concentration; BMC, bone mineral content; BMD, bone mineral density; CL / F, apparent clearance; Cmax, maximum observed plasma concentration; CXM, collagen X marker; DXA, dual X-ray absorptiometry; FAH, final adult height; hGH, human growth hormone; PD, pharmacodynamic; PK, pharmacokinetic; t1 / 2, elimination half-life; Tmax, time to reach maximum concentration; Vz / F, apparent volume of distribution.

[0246] Number of Participants: Approximately 72 participants with Turner syndrome (N ≥ 24), SHOX deficiency (N ≥ 16), or Noonan syndrome (N ≥ 24) will be randomized in 111-211 (a minimum of 4 participants with SHOX deficiency and 6 participants each with Turner syndrome and with Noonan syndrome per study group). An additional 10% more participants may be enrolled to achieve approximately 72 participants to be randomized.

[0247] To identify and study genetic variants that may modify Turner syndrome, SHOX deficiency, or Noonan syndrome, or idiopathic short stature, or that may influence CNP signaling or vosoritide efficacy and safety profile, WGS data from a whole blood sample will be collected. Exploratory genetic research may consist of the analysis of 1 or more candidate genes or the analysis of genetic markers throughout the genome (as appropriate). Example 2 – CNP Effects on Short Stature Disease in In Vitro Models

[0248] Vosoritide is an FDA-approved recombinant CNP analog that increases linear growth velocity in children with Achondroplasia (ACH) Lorget et al. Am J Hum Genet.2012;91(6):1108- 1114; Savarirayan et al. Genet Med 23, 2443–2447 (2021). Individuals with ACH harbor autosomal dominant activating variants in Fibroblast Growth Factor Receptor 3 gene (FGFR3, >95% G380R) which result in elevated FGFR3 activity and therefore increased downstream MAPK pathway signaling. Similar to ACH, individuals with Hypochondroplasia (HCH) (Dauber et al. EClinicalMedicine.2024;71:102591. Published 2024 Apr 11) harbor activating variants in the FGFR3 gene (>70% N540K), which also activate the MAPK pathway. ACH and HCH are FGFR3-dependent short-stature conditions. Dysregulation of the MAPK pathway is associated with other growth disorders including Noonan Syndrome (NS) (Boucher et al., J Endocr Soc. 2022 Nov 1;6(Suppl 1):A618–9), where increased MAPK activity impairs chondrocyte proliferation and differentiation, leading to abnormal bone growth including short stature (Dauber et al., supra) NS is an FGFR3-independent short stature condition.

[0249] Based on CNP’s mechanism of action and human genetic evidence indicating the CNP / NPR2 pathway is a “master regulator” of skeletal growth, it was hypothesized that CNP should increase stature across cellular and rodent models dependent and independent of FGFR3. To test this, CNP effects in in vitro and in vivo models of HCH, NS, and Idiopathic Short Stature (ISS; represented by wild-type [WT] systems) were investigated.

[0250] For in vitro analysis G380R FGFR3 and N540K FGFR3 variants associated with ACH and HCH, respectively, or the L613V RAF1 variant associated with NS in rat chondrosarcoma (RCS) cells were treated with FGF18 and / or vosoritide (Pro-Gly-CNP37, SEQ ID NO: 1). RCS were transduced with lentivirus encoding human G380R FGFR3 variant associated with ACH, a N540K FGFR3 missense variant associated with HCH, or the L613V RAF1 gene variant associated with NS. Cells were treated with 0.3 mM FGF18 and with or without 20nM vosoritide for 3 days. Cells were then collected and AlphaLISA® was used to measure phospho-ERK and total ERK levels using p-ERK Thr202 / Tyr204 and total ERK1 / 2 antibodies. Cell count was measured by high-content imaging.

[0251] Vosoritide significantly reduced FGF18 ligand-induced ERK phosphorylation and increased chondrocyte growth (proliferation) in RCS cells expressing G380R or N540K mutation in FGFR3 or expressing L613V in RAF1 (Figures 2 and 3).

[0252] It was shown that vosoritide treatment can rescue the growth arrest phenotype in chondrocytes expressing the G380R FGFR3 variant associated with ACH by reducing FGFR3- ERK1 / 2 signaling and restore FGF18-ligand-induced growth arrest in RCS cells, which coincides with down-regulation of FGFR3-ERK1 / 2 signaling). The data also show vosoritide significantly reduces pERK and increases proliferation in the NS cellular model. Overall, the data demonstrate that CNP variant administration reduces pERK and increases proliferation in ACH, HCH, NS and WT (ISS) cellular models

[0253] To examine effects of vosoritide on ERK phosphorylation and chondrocyte proliferation in wild-type rat chondrosarcoma cells (RCS), model of idiopathic short stature (ISS), wild-type RCS were treated with 0.3 nM FGF18 and with or without 20nM Vosoritide for 3 days. Cells were collected and phosphor-ERK and total Erk levels were measured via AlphaLISA®. Cell count was measured by high-content imaging. Vosoritide significantly reduced FGF18-mediated increase ERK phosphorylation in the wild-type RCS cells compared to cells without Vosoritide treatment, and increases proliferation in wild-type (model of ISS) cellular model, which coincides with increased growth (Figures 4 and 5). Example 3: CNP Effects on Short Stature Disease In Vivo Models

[0254] To determine effects of vosoritide on skeletal defects in Noonan Syndrome (NS), male Raf1L613V S129 and female C57BL / 6 mice were used to generate 50:50 mix strain of Raf1 L613V / + heterozygous mice to recapitulate phenotypes of NS. 4-week-old Raf1 L613V / + and + / + animals received daily dosing of 500 µg / kg vosoritide for 6 weeks. Naso-anal length, femur length and skull morphology were measured at 10 weeks of age.3-week-old C57BL / 6J animals were treated daily with vehicle, 40 µg / kg or 150 µg / kg for 5 weeks. Naso-anal lengths measured weekly via in vivo micro-CT and right femurs were used for ex vivo high-resolution. Raf1 L613 / + animals showed significant reduction in naso-anal length (p = 0.0130) compared to control Raf1+ / + mice at baseline. Raf1 L613V / + mice treated with 500 µg / kg Vosoritide showed significant increase in naso-anal length (p<0.0001) and femur length (p=0.0006) (Figure 6). Vosoritide treatment also rescued skull morphology seen in Raf1 L613V / + animals, which exhibited reduced skull length and increased inner canthal distance, skull width and width / length ratio. Vosoritide administration resulted in increased femur length, skull length ,and normalized width / length ratio in Raf1 mouse model of Noonan’s. These data show vosoritide improves growth and craniofacial deficits in NS mice.

[0255] To examine effects of vosoritide on growth and bone mass in male wild-type (model of idiopathic short stature, ISS) mice, 3-week-old C57BL / 6 male mice were treated with vehicle, 40 µg / kg and 150 µg / kg of vosoritide daily for 5 weeks and they were scarified by the end of the study when they were 8 weeks of age. During in-life, these animals underwent weekly in vivo micro-CT imaging to measure naso-anal and tibia length. After sacrifice, right femurs were collected and ex vivo micro-CT imaging was used to examine bone volume fraction (BV / TV), trabecular thickness, trabecular number, and trabecular separation.

[0256] Both 40ug / kg and 150ug / kg vosoritide treatment showed significant increase in naso- anal and tibia lengths compared to vehicle (Figure 7). Significant naso-anal length increase was observed after a week of 150 µg / kg treatment. The 150 µg / kg treatment group also showed significant increase in bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and exhibited a trend to increase trabecular number (Tb.N) while not affecting trabecular separation (Tb. Sp). These data show vosoritide increases growth and bone mass in a wild-type (model of ISS) mice.

[0257] The data presented herein demonstrate across cellular and in vivo models that CNP can 1) reduce the effects of FGFR3-dependent MAPK activation, 2) dampen the aberrantlyactivated MAPK pathway in the growth plate, and 3) work in a FGFR3-independent manner, all leading to increased growth velocity. This supports the hypothesis that CNP works independent of the genetic cause of ACH, HCH, Noonan, and ISS – and suggests that CNP therapy will work across indications. Example 4: Evaluate the Safety and Efficacy of Vosoritide for the Treatment of Growth Deficits in MPS IVA and VI

[0258] Mucopolysaccharidoses (MPS) are a group of 11 rare genetic lysosomal storage diseases caused by deficiency in various enzymes responsible for the breakdown of glycosaminoglycans (GAGs). The progressive accumulations of GAGs in the tissues and organs of these patients are responsible for many adverse health outcomes, including severe growth deficits and growth-related decreased quality of life. Common manifestations include coarse facies, skeletal deformities, cardiac disease, upper airway obstruction, cognitive impairment (in some types), growth deficiency. Average height Z-score in fully mature patients with MPS IVA and VI naïve to enzyme replacement therapy (ERT) is reportedly between -8.0 and -9.0. Growth deficiencies are likely due to a variety of factors, including GAG accumulation at the growth plate, inflammation, osteoblast or chondrocyte dysfunction and / or hormone deficiencies. Despite overall health and functional improvements with ERT, growth deficits persist.

[0259] MPS IVA, (Morquio Syndrome) is characterized by deficiency in the enzyme galactosamine-6-sulfate sulfatase resulting in accumulation of the GAG heparan sulfate. MPS VI (Maroteaux-Lamy Syndrome) is characterized by a deficient in the enzyme N- acetylgalactosamine 4-sulfatase (also called arylsulfatase B) resulting in accumulation of dermatan sulfate.

[0260] MPS patients have severe growth deficits and growth-related decreased quality of life that persist despite ERT. Average height Z-score in adult MPS IV without ERT is between -7.7 and -8.0. Height Z-score only increased 0.1 from baseline in a phase III ERT trial (Tomatsu 2012; Hendriksz CJ J Inherit Metab Dis.37(6):979-90, 2014; Harmatz J Inherit Metab Dis. 2017). Pre-ERT adult height ranges from 110 to 140 cm, far below -2 SDS of normal age- adjusted population. Average height Z-score for MPS VI patients is -4.8±2.2 despite earlyintroduction of ERT (Quartel JIMD Rep.18:1-11, 2015; Miller J Inherit Metab Dis.46:695-704, 2023).

[0261] Growth Deficits in MPS IVA and MPS VI include structural skeletal changes such as joint contractures, scoliosis, kyphosis, genu valgum. Osteoblast and chondrocyte dysfunction leads to decreased endochondral ossification. Growth hormone deficiency treatments yield inconsistent results. GAG accumulation disrupts growth plate and causes inflammation.

[0262] While multiple clinical trials have aimed to improve health outcomes in MPS disorders, none have focused primarily on growth. A proposal focused on growth improvement and related quality of life is warranted. Vosoritide, a C-type natriuretic peptide (CNP) analog, has been shown to enhance growth velocity in Achondroplasia, a genetic form of dwarfism. Vosoritide may have similar growth-enhancing effects in MPS by reducing inflammation at the growth plate. In 2020, Yamashita et al. reported that vosoritide had a synergistic effect on growth in a mouse model of MPS VII when delivered with ERT. Given the many similarities in MPS disorders, the proposed mechanism, and the preliminary data, it seems feasible that vosoritide provided along with ERT may improve growth deficits in patients with MPS IVA and VI.

[0263] A phase I / II, single arm, open-label study was designed to deliver vosoritide subcutaneously at approximately 15 ug / kg / day for 48 weeks to 6 patients with MPS IVA or VI who are receiving enzyme replacement therapy. Prior to the start of intervention, subjects will be followed for a minimum of 24 weeks to observe baseline changes in health and function.

[0264] Measures of safety, tolerability, and early efficacy will be collected at baseline, the start of intervention, and at 24 and 48 weeks following intervention. Assessments will include evaluation of health and functional performance, pulmonary and cardiac systems, linear and segmental bone growth, biomarkers of growth, inflammation and bone metabolism, and self- reported tolerability and quality of life.

[0265] Results

[0266] Six subjects with either MPS IVA or MPS VI have enrolled. Baseline data on growth, inflammatory markers, and bone turnover biomarkers are provided in Table 2. Table 2 MPS IVA MPS VIGender Male Male Male Female Female Male Age at screening 87 71 83 88 56 74

[0267] These early data confirm the existence of inflammation and altered bone metabolism in these subjects with MPS, providing a basis for the treatment of growth deficits with vosoritide. Results from this study will provide preliminary data on the safety and tolerability of vosoritide in MPS. Example 5 – Vosoritide improves tibial bowing in infants and toddlers with achondroplasia

[0268] Vosoritide, a stimulator of endochondral bone growth, is approved for use in children with achondroplasia (ACH). As well as increasing linear growth, vosoritide positively impacts body proportionality and quality of life. This analysis assesses its impact on a key orthopedic feature of ACH, tibial bowing.

[0269] A randomized, placebo-controlled, phase 2 trial was conducted in children aged less than 5 years with ACH. Following an observational period, children (n=75) were enrolled in age-based cohorts and, with the exception of sentinels (n=11), randomized 1:1 to receivevosoritide (30·0 μg / kg for infants 0-23 months and 15·0 μg / kg for older children) or placebo for one year. Participants were then followed-up in an ongoing open-label extension study, with those receiving placebo transitioning to vosoritide. The degree of tibial bowing was measured, radiographically observed as of the linear intersections derived from the upper and lower physical plates and the mid-shaft of the tibia. Magnitude of change in tibial bowing is quantified as least square mean (LSM) change from baseline, applying an ANCOVA model, and is reported with units of degrees.

[0270] In the overall population the mean (SD) baseline values for tibial bowing angle in the left and right leg were 10.13 (7.26) (n=39) and 12.03 (6.67) (n=38) with vosoritide, and 12.00 (7.36) and 10.59 (5.96) with placebo (n=27). The LSM difference (95% CI and two-sided p- value) between vosoritide and placebo in change from baseline at year 1 was -2.96 (-5.85, - 0.07; p=0.045) in the left leg and -1.21 (-4.02, 1.59; p=0.39) in the right leg, in favor of vosoritide. In the open-label extension study, mean (SD) change from baseline in tibial bowing angle decreased over time in both the left leg (-0.97 (5.88), after 1 year (n=39); -4.07 (8.19) after 2 years (n=27) and -2.40 (7.79), after 3 years (n=41)) and the right leg (-2.34 (5.37) after 1 year (n=38); -0.75 (8.74), after 2 years (n=27) and -3.68 (6.99), after 3 years (n=41)).

[0271] Treatment with vosoritide improved tibial bowing, with results reaching statistical significance over the one-year placebo-controlled period. Improvement continued over later years. Tibial bowing can be an important cause of pain and impaired function, and a common reason for surgical intervention, in children with ACH. These results further point towards the potential for vosoritide to positively impact elements of ACH beyond height. Example 6 – Evaluation of vosoritide in preclinical short stature models of achondroplasia, hypochondroplasia and Noonan syndrome

[0272] Vosoritide is an analog of C-type natriuretic peptide (CNP), designed to improve growth velocity in children with achondroplasia (ACH) caused by FGFR3 gene variants. By binding NPR2 on chondrocytes within the growth plate, vosoritide promotes cGMP production and is thought to inhibit RAS-MAPK / ERK-driven growth arrest pathways to promote chondrocyte proliferation and differentiation. The present disclosure investigates whethervosoritide might also target other genetic segments associated with short stature via growth factor-mediated activation of the RAS-MAPK / ERK signaling pathway.

[0273] The L613V Raf1 HET mouse model of Noonan syndrome has previously been reported to exhibit reduced body length from 3-weeks of age and up to 20 weeks of age (Wu et al., J. Clin. Invest., 121:1009-1025). The model for genetic short stature tested here predicts that activating FGFR3, RAS and RAF gene variants, or loss-of-function NPR2 gene variants associated with short stature intersect with a common growth factor-dependent RAS-MAPK / ERK signaling pathway, which leads to premature engagement of chondrocyte growth arrest in the growth plate. Resulting in the proposal that pathological RAS-MAPK / ERK signaling associated with genetic short stature can be antagonized with CNP (vosoritide), which activates NPR2 and inhibits RAS-MAPK / ERK signaling at the level of Raf to downregulate ERK-driven chondrocyte growth arrest pathways and promote skeletal growth. In this model, it was predicted that the activation of RAS-MAPK / ERK signaling is potentiated by multiple growth factor ligands sequestered in the extracellular matrix of the growth plate, which will also activate the wild-type gene counterparts of FGFR3, RAS and RAF. While FGF18 ligand was used to test this model in the in vitro cell modeling studies of chondrocyte growth arrest, several additional FGF ligands and non-FGF ligands can potentially also activate the RAS-MAPK / ERK signaling pathway.

[0274] L613V Raf1 HET mice initially exhibited a lower body weight and reduced nasal-anal length, when compared to their wild-type siblings at three weeks of age (Fig.13A, Fig.13C). However, these differences in body weight and nasal-anal length between L613V Raf1 HET and wild-type groups are no longer observed at 10 weeks of age (Fig.13B, Fig.13D).

[0275] Daily dosing with vosoritide at a dose of 500 µg / kg over a period of 6 weeks in wild-type mice and L613V Raf1 HET mice, starting at 4 weeks of age, leads to significant increase in body length (Naso-Anal length; Fig.8A), when compared with their respective vehicle-treated controls (Fig.8A). Daily dosing with vosoritide over six weeks commencing at 4-weeks of age did not lead to any weight changes in any of the groups tested, when compared with their respective vehicle- treated controls (Fig.8B). Vosoritide treatment also coincides with increased lengthening of femur (Fig.8C, quantification in 8D), along with rescue of facial dysmorphia (Fig.14) in L613V Raf1 HET mice and in WT littermate controls. While tibia lengthening was observed in WT mice treated with vosoritide, this effect was not observed in L613V Raf1 HET mice treated with Vosoritide (Fig.8E, quantification in 8F). These results suggest that the increase in body length following daily dosingobserved with vosoritide for six weeks in L613V Raf1 HET and in WT mice (Fig.8A), is directly attributed to drug-mediated skeletal growth (Fig.8D, 8F, Fig 14). Moreover, vosoritide promotes a small but significant reduction in width / length ratio for skull measurements in WT mice (p=0.0325; Fig 14), which were not observed with wild-type mice treated with a MEK inhibition approach by Wu et al., (Wu et al., J Clin Invest., 121:1009-1025, 2012), suggesting that vosoritide influences skeletal growth both in conditions with and without pathogenic activation of MAPK signaling. Collectively, these results suggest that an ERK1 / 2 inhibition approach using vosoritide leads to skeletal growth in the L613V Raf1 HET mouse model of Noonan syndrome, as well as WT littermate control mice (Fig.8, Fig 14).

[0276] Cell-based modeling studies were employed to next understand at the signaling level how vosoritide promotes skeletal growth in L613V Raf1 HET mice and in wild-type mice (Fig.8, Fig 14). RCS cell-based models previously described by Krejci et al. (J Cell Sci., 118:5089-5100, 2005) were used to test the hypothesis that inherited or de novo gene variants associated with genetic short stature act through a common RAS-MAP / ERK signaling pathway to activate ERK- driven growth arrest in chondrocytes, which can be therapeutically targeted with vosoritide. A CRISPR-engineered fgfr3- / -rat chondrosarcoma (RCS) chondrocyte-like cell line was used to study FGF18 ligand-induced RAS-MAPK / ERK signaling associated with the N540K and G380R FGFR3 missense variants commonly diagnosed in HCH and ACH, respectively. Both endogenous rat fgfr3 alleles were knocked out, rendering a mutant fgfr3- / -RCS cell line significantly less responsive to FGF18, which predominantly acts as a ligand for FGFR3. FGF18- ligand-induced activation of RAS-MAPK / ERK1 / 2 signaling is reduced in the fgfr3- / -line when compared with fgfr3+ / +RCS chondrocyte-like cells (Fig.9A). Presumably, the lower level of FGF18-induced ERK1 / 2 activation that is observed in fgfr3- / -RCS cells is arising from FGF18- mediated activation of FGFR1 (Fig.9A, Murugaiyan et al., J Funct Biomater, 14:36, 2023). As previously observed in a npr2+ / -RCS clone (Estrada et al., Nat Commun.12:2224), disclosed here is in an additional npr2+ / -RCS clone a dose-dependent cGMP response following treatment with vosoritide (Vmax = 12.6 pmol; Fig.9B), which is augmented further in the RCS parental line (Vmax = 50.4 pmol; Fig.9B).

[0277] A dose of 20 nM vosoritide, approximately 10-fold above the EC50 for the cGMP response detected in RCS cells (Fig.9B, EC50 = 2.3 nM), was chosen to evaluate its effect on chondrocyte growth in RCS cells. Chronic treatment of the parental RCS cell line with 20 nMvosoritide alone does not lead to any changes in their growth, when compared with untreated RCS cells (Fig.9C). In contrast, chronic treatment of RCS cells with 0.5 nM FGF18 for three days leads to reduced growth, which can be partially rescued by co-treatment with 20 nM vosoritide (Fig.9C). Collectively, these results suggest that in RCS cells the majority of FGF18 ligand-induced ERK1 / 2 signaling is dependent upon fgfr3 gene expression (Fig.9A), and most of the vosoritide-induced production of cGMP is npr2-dependent (Fig.9B). Furthermore, vosoritide mediates growth in RCS cells in a FGF-ligand-dependent manner. These results corroborate results obtained by the Krejci lab using FGF2 ligand in this model of chondrocyte growth arrest (Krejci et al., J Cell Sci., 118:5089-5100, 2005; Krejci et al., Bone, 47:102-10, 2010), inferring that a CNP approach with vosoritide safeguards RCS chondrocytes against upregulation of ERK1 / 2-driven growth arrest pathways by various FGF ligands, which coincides with a mitogenic response and partial rescue of the growth arrest phenotype (Fig.9, Krejci et al., J Cell Sci., 118:5089-5100, 2005). Previous results have attributed CNP-mediated rescue of growth arrest to be arising from attenuation of ERK1 / 2-driven senescence pathways (Krejci et al., Bone, 47:102-10, 2010).

[0278] It was then hypothesized that several genetic segments of short stature that intersect with the RAS-MAPK / ERK signaling pathway, will also be activated with growth factors, leading to prolonged engagement of ERK1 / 2 driven growth arrest pathways. In this model, the growth factor can potentially be any type of ligand sequestered in the extracellular matrix that activates a receptor tyrosine kinase (RTK), upstream of RAS. For in vitro modeling experiments, FGF18 was used to first test the hypothesis in RCS models of ACH and HCH, which are commonly associated with the G380R FGFR3 and N540K FGFR3 variants. Transduction of fgfr3- / -RCS cells with lentivirus encoding GFP, WT FGFR3, N540K FGFR3 or G380R FGFR3 and grown for three days results in the detection of GFP-encoded protein or FGFR3-encoded protein by Western blotting.

[0279] To permit comparison of the kinetics of FGF18 ligand-dependent activation of WT and mutant FGFR3 signaling in fgfr3- / -RCS cells over a three-hour period, ERK / pERK results were plotted for each receptor along with the GFP control on individual graphs using the same maximum value on the Y-axis (Fig.10A-10D). Following treatment with 2 nM FGF18, fgfr3- / -RCS cells transduced with lentivirus encoding the GFP reporter control fail to exhibit a robust phosphorylated ERK1 / 2 (pERK1 / 2) signaling response (Fig.10A). In fgfr3- / -RCS cells transduced with lentivirus encoding WT FGFR3 (Fig.10B) a higher level of pERK1 / 2 signaling was observed in response to treatment with 2nM FGF18 over 3 hours, when compared with cells expressing GFP (Fig.10A).The peak of pERK1 / 2 activation in fgfr3- / -RCS cells expressing WT FGFR3 is rapidly detected within 30 minutes post-ligand treatment, maintained at 1 hour-post ligand treatment and approaches basal levels at the 3-hour timepoint (Fig.10B). When compared with the WT receptor, fgfr3- / -RCS cells transduced with lentivirus encoding N540K FGFR3 reach their maximal activation at 30 minutes post-ligand treatment, which is then reduced, but still elevated above basal levels of ERK1 / 2 signaling at the 1-hour and 3-hour timepoints (Fig.10C). In RCS cells overexpressing the G380R FGFR3 variant, the highest level of pERK1 / 2 signaling is observed at 30-minutes post-treatment with FGF18-ligand (Fig.10D) and remains elevated throughout the experiment at the 1-hour and 3- hour timepoints (Fig.10D), when compared fgfr3- / -KO RCS chondrocytes over-expressing WT (Fig. 10B) or N540K FGFR3 (Fig.10C). Treatment of fgfr3- / -RCS cells over-expressing GFP (Fig.10A), WT (Fig.10B), N540K (Fig.10C) or G380R FGFR3 (Fig.10D) with 20 nM vosoritide reduces FGF18 ligand-dependent activation of pERK1 / 2 signaling, as indicated by ERK1 / 2 phosphorylation, particularly at the earlier timepoints tested (Fig.10A-10D).

[0280] Transduction of fgfr3- / -RCS cells with cDNA encoding WT or mutant FGFR3 leads to detection of FGFR3+RCS cells by immunofluorescence with an FGFR3-specific antibody, which was used for evaluation of their growth in high-content imaging experiments (N=3 biological repeats). These results show that the FGFR3 antibody employed in these studies does not react with endogenous rat fgfr3 in RCS cells and only reacts with exogenously expressed human WT and mutant receptors. Growth in fgfr3- / -RCS cells transduced with lentivirus encoding WT FGFR3 is significantly reduced in the presence of chronic exposure to 2 nM FGF18 for three days (Fig.10E). Lentiviral-mediated over-expression of fgfr3- / -RCS cells with the N540K FGFR3 variant associated with HCH leads to reduced growth over three days when compared with fgfr3- / - RCS cells transduced with lentivirus encoding the WT receptor (Fig.10E). This impaired growth associated with the N540K FGFR3 variant is reduced further following chronic exposure to 2 nM FGF18 (Fig.10E). Similarly, in fgfr3- / -RCS cells overexpressing the G380R FGFR3 variant associated with ACH, there is reduced growth when compared with fgfr3- / -RCS cells overexpressing the WT receptor, which can be reduced further following chronic treatment with 2 nM FGF18 (Fig.10E). The FGF18-dependent reduced growth associated with WT FGFR3 along with the N540K and G380R FGFR3 variants (Fig.10E) can all be partially rescued with vosoritide co-treatment over three days of culture (Fig.10E).

[0281] Collectively, these results suggest that increased RAS-MAPK / ERK signaling associated with the N540K FGFR3 and G380R FGFR3 variants commonly diagnosed in HCH and ACH occur due to overall increases in the strength and duration of ligand-evoked ERK1 / 2 signaling in chondrocytes, which leads to their reduced growth over time. Moreover, differences in the kinetics of ERK1 / 2 activation were noticed between the two variants, with faster activation of pERK1 / 2 signaling with the N540K FGFR3 variant associated with HCH (Fig.10C), when compared with more sustained activation of the G380R FGFR3 variant associated with ACH (Fig.10D), which may provide some insight into differential clinical presentation of these short stature indications. The attenuation of agonist-induced ERK1 / 2 signaling observed with vosoritide in fgfr3- / -RCS chondrocytes exogenously expressing N540K FGFR3 (Fig.10C) and G380R FGFR3 (Fig.10D), and subsequent increases in cell growth in this study (Fig.10E) agree with previous results observed in a mouse model of ACH, where vosoritide also increased skeletal growth (Lorget et al., Am J Hum Genet, 91:1108-1114, 2012).

[0282] It was next hypothesized that in addition to variants in the FGFR3 gene associated with ACH and HCH, additional gene variants in the RAS-MAPK / ERK signaling pathway may also be responsive to ligand-induced activation of ERK1 / 2 signaling. To test this hypothesis FGF18 ligand was used to activate endogenous rat FGFR3-RAS-MAPK / ERK signaling in the RCS parental cell line over-expressing the GFP reporter control (Fig.11A), WT RAF1:T2A:GFP (Fig.11B) or L613V RAF1:T2A:GFP associated with short stature in Noonan syndrome (Wu et al., Am J Hum Genet., 91:1108-1114, 2012; Fig.11C). Treatment of RCS cells expressing WT RAF1:T2A:GFP with 0.3 nM FGF18 ligand (Fig.11B) leads to increased ERK1 / 2 signaling over a 3-hour period, when compared with the GFP control (Fig.11A, quantification in 11D). FGF18 ligand-induced activation of ERK1 / 2 signaling is increased further in RCS cells expressing the L613V RAF1 variant at the earlier 30 and 1-hour timepoints (Fig.11C, quantification in 11D). Chronic treatment of RCS cells with vosoritide is sufficient to attenuate FGF18 ligand-dependent pERK1 / 2 signaling in RCS cells over-expressing GFP (Fig.11A, quantification in 11D), WT RAF1 (Fig.11B, quantification in 11D) and the L613V RAF1 variant (Fig.11C, quantification in 11D), which coincides with increased growth of GFP+RCS cells over three days (quantification in 11E). Collectively, these modeling studies of the L613V RAF1 variant in RCS cells (Fig.5) are in agreement with the findings in the L613V Raf1 HET mouse model (Fig.8, Fig.14), providing further support for the hypothesis herein that vosoritide can be used to antagonize growth factor-dependent activation of RAS-MAPK / ERK-driven growth arrest associated with genetic short stature in chondrocytes to mediate increased skeletal growth (Fig.8, Fig.14, Fig.11).

[0283] Along with the L613V RAF1 variant, the P34R KRAS variant associated with short stature in Noonan syndrome was also evaluated in the same RCS experiment described in Fig 11, with results summarized in Fig 12 using the same GFP results shown in Fig 11A but with a different Y-axis scale (Fig.12A). The kinetics of FGF18 ligand-induced activation of ERK1 / 2 signaling in RCS cells overexpressing WT KRAS is increased over a 3-hour period (Fig.12B), when compared with the GFP control (Fig.12A, quantification in 12D). The kinetics of FGF18 ligand-induced ERK1 / 2 signaling were increased further in RCS cells expressing the L613V RAF1 variant (Fig.12C, quantification in 12D). Chronic treatment with 20 nM vosoritide is sufficient to attenuate FGF18 ligand-dependent pERK1 / 2 signaling in RCS cells over-expressing WT KRAS (Fig.12B, quantification in 12D) and the P34R KRAS variant (Fig.12C, quantification in 12D), which coincides with increased growth of GFP+RCS cells over three days (quantification in 12E).

[0284] Discussion

[0285] These results demonstrated that variants commonly associated with several genetic segments of short stature are responsive to vosoritide using in vitro and in vivo approaches. Increased body length, femur lengthening, and improvement in skull morphology were demonstrated in response to vosoritide in the L613V Raf1 Het mouse model of Noonan syndrome (Fig, 8, Fig 14). Chondrocyte-like RCS cell models were also used to provide evidence for a pathological mechanism by which commonly diagnosed short stature variants in the RAS- MAPK / ERK signaling pathway genes FGFR3 (Fig.10), RAF1 (Fig.11) and KRAS (Fig.12) potentiate FGF ligand-dependent RAS-MAPK / ERK1 / 2 signaling, as measured by ERK1 / 2 phosphorylation in kinetic studies, when compared with their WT gene counterparts. It was demonstrated that an ERK1 / 2 inhibition approach with vosoritide is sufficient to attenuate FGF18 ligand-induced ERK1 / 2 signaling in these genetic models of ACH, HCH and Noonan syndrome, which coincide with increased proliferation of RCS cells. In support of these preclinical results in RCS cells and the L613V RAF1 mouse model, vosoritide has been approved for patients with ACH (Duggan, Drugs, 81:2057-2062, 2021) and recently been reported to improve growth in ongoing prospective clinical trials in patients with HCH and Noonan syndrome (Dauber et al., Horm Res Paediatr., 95(supp 1):137-138, 2022a; Dauber et al., Horm Res Paediatr.95(suppl 2):293-294,2022b; Dauber et al., Horm Res Paediatr., 95(suppl 2):30, 2022c; Dauber et al., EClinicalMedicine, 71:102591, 2024). In further support of the model proposed, it was demonstrated that vosoritide can upregulate cGMP production in an additional npr2+ / -RCS clone (Fig.9B), providing further support for an ERK-inhibition approach to treat short stature associated with variants in the NPR2 gene (Estrada et al., Nat Commun., 12:2224, 2021; Pejchalova et al., Mol Genet Metab.92:210-215, 2007).

[0286] The L613V Raf1 HET mouse models utilized a 50:50 mixed strain background of C57BL / 6 and 129S. Wu et al., (J Clin Invest, 121:1009-1025, 2011) have previously demonstrated that this mixed strain strategy is needed to achieve optimal phenotype penetrance, with the phenotype being lost following three backcrosses into 129S or C57BL / 6 backgrounds (Wu et al., 2011, supra). While the L613V Raf1 HET model was derived using a CRISPR knock-in genetic strategy, and a Cre recombinase approach was employed by Wu et al., 2011 (Wu et al., J Clin Invest, 121:1009-1025), both models exhibit growth defects and exhibit increased skeletal growth in response to pharmacological inhibition of RAS-MAPK signaling with the MEK inhibitor, PD0325901 (Wu et al.2011, supra) or with vosoritide. One notable difference is a more pronounced reduction in body length in the Wu et al study, commencing at 3 weeks of age and observed up to 20 weeks of age, whereas here a transient growth defect is observed at 3-weeks, which is no longer observed at 10-weeks of age

[0287] Mechanistically, growth was not observed in RCS cells treated with vosoritide alone (Fig. 9C). Instead, a mitotic effect was only observed with vosoritide in RCS cells following co-treatment with FGF18 (Fig.9C). These results suggest that vosoritide attenuates FGF18 ligand-induced activation of FGFR3-RAS-MAPK / ERK signaling in RCS cells over-expressing WT FGFR3 (Fig. 10B), WT RAF1 (Fig.11B) and WT KRAS (Fig.12B), which coincides with a mitotic effect (Figs. 10E, 11E, and 12E). As previously reported (Lorget et al., Am J Hum Genet, 91:1108-1114) and shown here (Fig.8), vosoritide also promotes increases in body length, femur length and tibia length in WT mice, when compared with vehicle-treated WT mice (Fig.8; Lorget, 2012, supra). Collectively, these results imply that CNP exerts a ligand-dependent mitotic effect in RCS cells and increases skeletal growth in mice with genetically unmodified MAPK signaling, and potentially through MAPK-independent pathways. In support of this, previous studies have demonstrated that CNP can upregulate proteoglycan matrix levels in RCS cells in the absenceof FGF ligand, an area that deserves further investigation (Krejci et al., J Cell Sci., 118:5089- 5100, 2005).

[0288] Daily administration of vosoritide over six weeks increased femur length and improved skull morphology in both wild-type mice and in a heterozygous L613V RAF1 mouse model of Noonan syndrome. In cellular models, fibroblast growth factor-18 (FGF18) increases RAS- MAPK / ERK signaling in rat chondrosarcoma (RCS) cells, which coincides with chondrocyte growth arrest. FGF 18-induced RAS-MAPK / Erk signaling increase further in RCS cells overexpressing wild-type FGFR3, wild-type RAF1, wild-type K-RAS, or variants associated with achondroplasia, hypochondroplasia and Noonan syndrome. Vosoritide reduces ERK signaling and promotes chondrocyte growth across all RCS models. These preclinical findings support ongoing clinical trials for using vosoritide to treat various segments of genetic short stature involving RAS-MAPK / ERK signaling pathway. Furthermore, these results in wild-type in vitro models and in wild-type mice suggest that vosoritide also activates chondrocyte proliferation and skeletal growth in systems without genetic variants activating the RAS-MAPK / ERK signaling pathway.

[0289] Together, this data illustrates the versatility of the CNP pathway to modulate skeletal growth in both WT models and models of dominantly inherited short stature driven by activation of the RAS-MAPK / ERK signaling pathway.

[0290] Experimental procedures:

[0291] Reagents. Fibroblast growth factor 18 (FGF18, #PHG0234) was acquired from Thermo Fisher Scientific (Waltham, M.A.). A modified variant of human CNP (vosoritide), resistant to proteolytic degradation by neutral endopeptidase, that retains the ability to stimulate signaling downstream of NPR2 has previously been described (Pejchalova et al., Mol Genet Metab., 92:210-215, 2007). Enolase antibody for western blotting was purchased from Santa Cruz Biotech (#sc-271384) and GFP antibody was purchased from Abcam (ab13970). FGFR3 antibody was used for detection of the human isoform of FGFR3 by western blotting and immunofluorescence and was purchased from Abcam (ab133644).

[0292] Animal Welfare. All mouse procedures conducted in this study were approved by the Institutional Animal Care and Use Committee at BioMarin Pharmaceutical Inc., and performed under the guidelines set by the Institute for Laboratory Animal Research (ILAR). Animals weregroup housed (no more than 5 per cage) in disposable rodent caging (Innovive, San Diego CA) on 1 / 8 inch pre-bedded sterile corn cob (M-BTM-C8, Innovive San Diego CA) and provided acidified pre-prepared water bottles (Innovive, San Diego CA) and pelleted rodent chow (Envigo Teklad 2918) in rooms using a 12:12 on:off light cycle. Additional environmental enrichment was also provided in the form of envirodri shredded paper (Shepherd Specialty Papers), Cotton nestlets (Ancare), and cardboard tubes (Newco). Efforts were employed to minimize pain and distress and adhere to 3R guidelines as outlined by Russel and Burch Principles of Humane Experimental Techniques (1959).

[0293] L613V Raf1 HET mouse model generation. The N1 generation of male WT or L613V Raf1 HET mice of 50% 129S1 / SvlmJ and 50% C57BL / 6J background was generated through crossing of male L613V Raf1 HET 129S1 / SvlmJ mice (model was custom made via CRISPR cas9 knock at Jackson Laboratories, Sacramento, California, USA) to female WT C57BL / 6J mice (JAX stock #000664, Jackson Laboratories, Sacramento, California, USA). Briefly, the silent guide blocking-mutation sequence used was AAAATC>AAGATT, followed by the PAM targeting sequence and remaining guide RNA sequence according to Ensembl ENSMUSE 00000693732. The guide RNA oligo was used in combination with a cas9 CRSPR in S129 embryos and male pups were genotyped for the Raf1 L613V mutation using PCR. Upon receipt, breeding pairs for male Raf1 S129S1 / SvlmJ and female C57BL / 6J mice were acclimated for at least 7 days prior to initiation of mating set ups. Pups were born in house, kept with their litter in their mothers’ home cage during genotyping, and weaned into new home cages for study enrollment between age postnatal days (PND) 21 to 23. Mice were genotyped via tail tip sample at 2 weeks old and received a neonatal tail tattoo per litter prior to study enrollment.

[0294] Animal Dosing and Enrolment. At three weeks of age, mice were weaned from their mothers and sorted by genotype into study cohorts and new home cages and received a study ID by ear notching. Baseline measurements of bodyweight and body length under anesthesia using a metric ruler were made at 3 weeks of age (Study Week -1). Cohort assignment was based on keeping the average mouse age and weight as similar as possible among the groups respective to genotype. The following week (Study week 1), when mice were 4 weeks old, daily bodyweight measurements and dosing of test articles commenced and continued for the 6-week study period. Animals receiving vosoritide or corresponding vehicle were dosed once daily each day between 9 AM-12 PM via subcutaneous (sc) injection in the subscapular region for the 6-week treatmentperiod. Animals were weighed daily to calculate appropriate dosing volume. Doses of vosoritide or corresponding vehicle were formulated to be delivered at a volume of 4mL / kg and a dose of 500 ug / kg.

[0295] Evaluation of body length. At Study Week -1 (mouse age ~3 weeks) and Study Week 6 (mouse age ~10 weeks) mice were put into a surgical plane of anesthesia by isoflurane inhalation (3-3.5% induction). Anesthetized mice were gently laid across a metric ruler in the dorsal position with the tip of the nose placed at 0 cm. The distance in centimeters from the tip of the nose to the anus, just before the start of the base of the tail (but not including the tail) was used to determine the body length.

[0296] Micro Computed Tomography 3D Imaging and skeletal growth measurements. At Study Week 6 (mouse age ~10-weeks), mice underwent whole-body Micro Computed Tomography (Micro-CT) scanning via Qantum GX2 (Perkin Elmer, now Revvity) to assess femur and tibia length. Mice were anesthetized in an induction chamber with 3.2% inhaled isoflurane then transferred to a nosecone within the micro-CT scanning chamber that delivered 2.5% isoflurane and oxygen. Whole body Micro-CT images were collected in both prone and supine positions. Femur length was measured from the proximal end of the femoral head to the distal end of the medial condyle, and tibia length was measured from the proximal medial plateau to the medial malleolus on the distal end. Mice were imaged in a ventral position then turned over to the dorsal position for a 2ndfull body scan. For skull length measurements, distance from the tip of the nasal bone to occipital condyle was measured. For skull width, distance between the frontal bone was measured. For inner canthal distance, the distance between the right anterolateral corner of the frontal bone to the left anterolateral corner of the frontal bone was measured. Skull W / L ratio was the ratio between the width over the length.

[0297] RCS cell culture and maintenance. Rat chondrosarcoma (RCS) cells (Krejci et al., J Cell Sci., 118:5089-5100, 2005) were a kind gift from Pavel Krejci, Masaryk University, Czech Republic. RCS cells were grown and passaged in DMEM (Thermo Fisher Scientific, #10566) supplemented with 10% fetal bovine serum (FBS; 10% v / v), antibiotics and Glutamax (Gibco). RCS cells were routinely tested and shown to be negative for mycoplasma contamination.

[0298] Knockdown of FGFR3 in RCS cells. The RCS cells line (Mukhopadhyay et al, 1995) was cultured with DMEM containing 10% fetal bovine serum, penicillin / streptomycin, andGlutamax (Gibco) at 37°C with 5% CO2. RCS cells were harvested with TrypLE Express Enzyme (Life Technologies) and transfected with RNPs formed with guide RNA containing ATTO 550 labelled tracrRNA (IDT 1075928) and crRNA targeting FGFR3, with PAM in bold:

[0299] gRNA15'- CCTGGGTGCAGGCCCCCAACAGG-3' (SEQ ID NO: 58)

[0300] GTTGGGGGCCTGCACCCAGG (SEQ ID NO: 59)

[0301] Briefly, equimolar ratios of crRNA and tracrRNA were annealed and 12 pmol was complexed with 104pmol HiFi Cas9 (IDT 1081061) according to the manufacturer’s protocol. 2E5 cells were transfected with 500 ng of GFP mRNA (TriLink Biotechnologies, cat# L-7201) or RNP using a 96-well Shuttle nucleofector (Lonza) with Amaxa solution SF (Lonza) and program CA-150. Post transfection, each well was split and seeded into two wells of a 96-well plate. Three days after transfection, single cells were sorted into 96-well plates containing 200 μl of media using a FACSMelody cell sorter (BD Biosciences). The plates were monitored biweekly by imaging (Cell Metric, Solentim). When confluent, cells were expanded into 6-well plates and genotyped.

[0302] Genomic DNA was extracted using Quanta Extracta solution (Quanta Biosciences, cat #95091). DNA amplicons covering the gRNA target region were amplified and analyzed by amplicon sequencing following the manufacturers protocols (Genewiz, Amplicon-EZ). Amplicon libraries were prepared using primers with universal sequences and the locus specific primers FW 5'-CCTGTCACCGTGGCCGTGAA-3' (SEQ ID NO: 60) and REV 5'- ATGCCGTGAGACCCTGGAGC-3' (SEQ ID NO: 61). To analyze NGS FASTQ files from amplicon sequencing, Geneious Prime 2019.0.4 software was used for quality assessment, quality filtering, reference alignment, and calculating indels. The sequence of exon 11 of the FGFR3 KO RCS clone has a 4bp deletion on one allele and allele 2 has a 1bp insertion leading to an out of frame coding sequence and premature stop codon.

[0303] FGFR3 KO RCS clone, Allele 1:

[0304] ATGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGA A AATGATTGGCAAGCACAAGAACATCATTAACCTGTTGCCTGCACCCAGGGTG (SEQ ID NO: 62)

[0305] FGFR3 KO RCS clone, Allele 2:

[0306] ATGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGA A AATGATTGGCAAGCACAAGAACATCATTAACCTGTTGGGGGGCCTGCACCCAGGGTG. (SEQ ID NO: 63)

[0307] Lentivirus constructs. LV-EF1α-eGFP, LV-EF1α-WT FGFR3, LV-EF1α-N540K FGFR3, LV-EF1α-G380R FGFR3, LV-UBC-GFP, LV-UBC-WT-RAF1:T2A:eGFP, LV-UBC- L613V-RAF1:T2A:eGFP, LV-UBC-WT-KRAS:T2A:GFP, LV-UBC-P34R-KRAS:T2A:GFP plasmids were designed, constructed, purified, packaged for large scale lentiviral production, and ultra-purified to produce lentivirus particles by VectorBuilder (Chicago, I.L.). Virus was stored in 100 µL aliquots at -80º C, with a titer of 109transduction units per mL, as determined by VectorBuilder. RCS cells were treated with 1x collagenase (Thermo Fisher Scientific, #17101015) for four hours, then plated into 96-well plates at 2500-5000 cells per well in the presence of lentivirus containing 7.5 micrograms / mL polybrene.

[0308] Ligand Dependent ERK1 / 2 Signaling. Activation of ERK1 / 2 signaling in RCS cells with FGF-18 ligand was evaluated alone or in combination with vosoritide. For kinetic time- course experiments, replicate plates were taken down at 0, 15, 60, and 180 minutes of FGF-18 ligand treatment. Activation of ERK1 / 2 signaling was quantified using pPERK and total ERK1 / 2 alphaLISA kits (Revvity, #ALSU-TPERK-A500 and #ALSU-TERK-A500 respectively) according to manufacturer protocol, which detect phosphorylation of ERK1 / 2 at Thr202 / Tyr204, and Total ERK1 / 2, respectively.

[0309] cGMP Production & Measurement. RCS cells were stimulated for cGMP production by pre-incubation with 0.75 mM 3-isobutyl-1-methylxanthine (IBMX; Enzo life sciences, #89161- 340) for 15 minutes at 37°C in serum-free media followed by treatment with vosoritide for 15 minutes at 37°C in serum-free media. cGMP production was measured using kits from Molecular Devices (#R8075) according to manufacturer protocol.

[0310] Evaluation of cell growth in RCS cells expressing GFP, WT FGFR3 and N540K FGFR3. RCS were seeded into 96-well black imaging plates (Greiner, #655090) at 2500 cells per well and treated with collagenase prior to transduction with lentivirus in the presence of polybrene. RCS cell growth was then evaluated by high-content imaging. Briefly, RCS cells transduced with lentivirus were fixed (4% PFA in PBS) and permeabilized using normal goat serum (Thermo Fisher Scientific, #50062Z) with 0.01% Triton X-100. RCS cells were then blocked in normal goatserum and stained with an anti-FGFR3 antibody (Abcam, #ab133644; diluted 1:200 in normal goat serum) overnight at 4°C. Cells were imaged on the ImageXpress high content microscope FGFR3+cells were quantified using MetaMorph software. In some instances, lentivirus constructs also expressed GFP, interrupted by the 2A self-cleaving peptide T2A, to permit quantification of GFP+RCS cells by high content imaging.

[0311] For evaluation of ligand-dependent RCS cell growth, RCS cells were typically treated with FGF18 ligand, which predominantly acts as a ligand for FGFR3. RCS cells were incubated for 24 hours after plating and drug was administered daily. For instances where RCS were treated with FGF18 and vosoritide, cells were pre-treated with vosoritide for 30 minutes prior to addition of FGF18 and vosoritide. For time course experiments, one plate was taken down at each timepoint as indicated and fixed with 4% PFA in PBS (Santa Cruz Biotech, #sc-281692) with Hoescht (Thermo Fisher Scientific, #H3570) diluted 1:1000 for 15 minutes at room temperature. Stained RCS cells were imaged on the high content microscope at 2x and 20x objectives, acquiring 4 sites per well. Number of nuclei was analyzed using MetaMorph software to generate growth curves.

[0312] Statistical Analysis. All statistics shown in RCS cells were calculated by running student t-tests or one way ANOVA using GraphPad prism software. Example 7 – Preliminary Assessment of Vosoritide for Short Stature in Turner Syndrome

[0313] Short-stature homeobox (SHOX) haploinsufficiency associated with increased extracellular signal-regulated kinase (ERK) activity is one of the mechanisms of short stature in Turner syndrome (TS). Vosoritide, a C-type natriuretic peptide (CNP) analog promotes endochondral bone growth by reducing ERK activity leading to the hypothesis that vosoritide may increase height in girls with TS.

[0314] During endochondral bone development, FGF9 and FGF18, derived from the perichondrium and surrounding tissue, signal to chondrocytes. Activity is mediated in part by regulated diffusion through the extracellular matrix through affinity for HS and potentially other sulfated glycosaminoglycans. Activation of FGR3 in proliferating chondrocytes activates the STAT1 and MAPK signaling pathways. FGFR3 signaling results in increased expression of Snail1, which in turn is required for STAT1 and MAPK signaling. FGFR3 signaling can alsoactivate PP2a. Activation of downstream signals, p107, p21Waf1 / Cip1, and Sox9 regulates chondrocyte proliferation and differentiation to hypertrophic chondrocyte proliferation and differentiation to hypertrophic chondrocytes. C-type natriuretic peptide (CNP) signals through the natriuretic peptide receptor 2 (NPR2), a guanylyl cyclase. cGMP activates cyclic GMP- protein kinase II (cGKII), which, through p38 MAPK activation, functionally antagonizes RAF1 activation of MEK. SHOX increases CNP signaling while simultaneously decreasing FGRF3 signaling.

[0315] A prospective single center Phase II open label clinical trial of Vosoritide was conducted in pre-pubertal girls with TS (ages 3-11). Karyotyping confirmed TS in participating pre-pubertal girls who placed in the less than fifth percentile for height. Participants either were naïve to growth hormone (GH) or on GH for over a year with a 6m annualized growth velocity (AGV) less than the fiftieth percentile of US girls for age and sex. AGV referring to the AGV after 1 year of treatment with 1SD on the National Cooperative Growth Study response curves.

[0316] The study design consisted of an initial screen visit with approximately 1 month and 2 weeks for review of screening labs. The subjects then started Vosoritide on the Day 1 / Day 2 visit followed by a telephone follow-up. The next two visits then consisted of a 6-month visit and 12 months visit which included a telephone follow-up in between.

[0317] Subjects received daily subcutaneous vosoritide injections (using weight-based dose banding approved for achondroplasia) for 12 months, after discontinuation of GH. The primary outcomes were change in AGV and height SD from baseline, as well as rate of adverse events (AEs). Table 3 Age 50-cell Growth Baseline Baseline 6m Vosoritide Other TS- edel(9)(q12) / 46,X,del(X / p s, e

[0318] Of the first 4 subjects enrolled in the trial, one failed screen due to new onset celiac disease and 3 completed 6 months of treatment. Subjects with prior GH exposure increased AGV by +4.9 and +1.57 cm / y over the AGV on GH treatment, while the GH naïve subject had an increased AGV +6.04 cm above the baseline at the 6-month visit (Fig.15).

[0319] Vosoritide was generally well tolerated, with transient injection site erythema / swell (Grade 1-2). No subjects discontinued participation due to AEs. There were no serious AEs related to treatment.

[0320] This is the first clinical trial of vosoritide therapy in TS and shows promise in increasing AGV in both GH naïve as well as previously GH-treated girls at 6-months. Example 8: Effect of Vosoritide on Spine Morphology in Young Children with Achondroplasia

[0321] Spinal stenosis is a serious complication of achondroplasia (ACH) caused by the narrowing of the spinal canal and exacerbated by the presence of thoracolumbar kyphosis (TLK). Clinical evidence of the effects of vosoritide on spinal morphology is limited. Thepresent example reports spinal morphological parameters in young children with ACH treated with vosoritide vs placebo for over 52 weeks in a clinical study.

[0322] A placebo-controlled, randomized phase 2 trial was conducted evaluating the safety and efficacy of vosoritide in children with ACH aged 0 to <5 years. Participants were randomized to daily subcutaneous placebo or vosoritide (weight-based dose of 15 or 30 µg / kg / day). Spinal morphology parameters assessed by lateral or anterior / posterior X-ray images at baseline and week 52 included interpedicular distance ([IPD]; distance [mm] between medial aspects of pedicles) and spinal canal width (sagittal width [mm] at inferior level of pedicle) at lumbar vertebrae (L) 1–5. Additionally, TLK angle ([degrees], top of thoracic vertebrae 11 to top of L3) was assessed across age subgroups 0 to <0.5, ≥0.5 to <2, and ≥2 to <5 y. Efficacy of vosoritide vs placebo was determined by an ANCOVA-adjusted difference least squares mean (LSM) change from baseline that included treatment, sex, age, baseline age, baseline Z-score, baseline AGV, and baseline IPD as model terms. Vosoritide (N=43) led to measurable improvements in each parameter compared with placebo (N=32) at all lumbar vertebrae at week 52 (Fig.16). LSM change from baseline was equal to or higher than placebo with vosoritide at all lumbar vertebrae for IPD (L1: 1.3 vs 1.1; L2: 1.2 vs 0.8; L3: 0.8 vs 0.8; L4: 1.0 vs 0.5; L5: 1.0 vs 0.5) and sagittal canal width (L1: 0.7 vs 0.1; L2: 0.5 vs 0.1; L3: 0.8 vs 0.4; L4: 1.6 vs 0.2; L5: 1.4 vs 0.5). Lower increases in TLK angle in children aged 0 to <0.5 y and higher decreases in children aged ≥0.5 to <5 y also indicated a positive difference in favor of vosoritide. Fewer children treated with vosoritide (33%) compared with placebo (57%) had TLK angle ≥20° at week 52.

[0323] Vosoritide improved spinal morphology, including spinal canal width and TLK angle, in young children with ACH vs placebo after 52 weeks of treatment.

[0324] It is understood that every embodiment of the disclosure described herein may optionally be combined with any one or more of the other embodiments described herein. Every patent literature and every non-patent literature cited herein are incorporated herein by reference in their entirety.

[0325] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description, and / or shownin the attached drawings. Consequently only such limitations as appear in the appended claims should be placed on the disclosure.

Claims

WHAT IS CLAIMED:

1. A method of treating a short stature disorder in a subject in need thereof comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

2. A method of elongating a bone or increasing long bone growth in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C- type natriuretic peptide (CNP) variant, and wherein the administering elongates a bone or increases long bone growth, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

3. A method for increasing annualized growth velocity in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the administering increases annualized growth velocity in the subject, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

4. A method for improving height SDS in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the administering improves height SDS in the subject, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment.

5. The method of any one of claims 1-4, wherein the subject does not have an adequate response to Human Growth Hormone treatment, wherein the annualized growth velocity (AGV) is less than that of age- and sex-matched average stature AGV determined using median heights from CDC growth charts after a minimum one year of treatment.

6. A method of treating a short stature disorder in a subject in need thereof comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the CNP is administered at a dose of 7.5 µg / kg / day or a dose of 22.5 µg / kg / day.

7. A method of elongating a bone or increasing long bone growth in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C- type natriuretic peptide (CNP) variant, and wherein the administering elongates a bone or increases long bone growth, wherein the CNP is administered at a dose of 7.5 µg / kg / day or a dose of 22.5 µg / kg / day.

8. A method for increasing annualized growth velocity in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the administering increases annualized growth velocity in the subject, wherein the CNP is administered at a dose of 7.5 µg / kg / day or a dose of 22.5 µg / kg / day.

9. A method for improving height SDS in a subject having a short stature disorder, comprising administering to the subject a composition comprising a C-type natriuretic peptide (CNP) variant, wherein the administering improves height SDS in the subject, wherein the CNP is administered at a dose of 7.5 µg / kg / day or a dose of 22.5 µg / kg / day.

10. The method of any one of claims 1-9, wherein the short stature disorder is selected from the group consisting of hypochondroplasia, NPPC mutation, NPR2 mutation, SHOX mutation, RASopathies, familial short stature, inherited short stature, and idiopathic short stature.

11. The method of any one of claims 1 to 10, wherein the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.

12. The method of any one of claims 1 to 11, wherein the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.

13. A method of treating a C-type natriuretic peptide (CNP)-responsive short stature condition or disorder, wherein the subject has previously received treatment with growth hormone and is unresponsive to growth hormone treatment, comprising administering a CNP variant to a subject, and monitoring the level of at least one bone- or cartilage-associated biomarker in the subject, wherein an increase in the level of the at least one bone- or cartilage-associated biomarker indicates a therapeutic effect of the CNP variant on the subject or the condition or disorder.

14. The method of claim 13, further comprising adjusting the amount or frequency of administration of the CNP variant, wherein i) the amount or frequency of administration of the CNP variant is increased if the level of the at least one bone- or cartilage-associated biomarker is below a target level; or ii) the amount or frequency of administration of the CNP variant is decreased if the level of the at least one bone- or cartilage-associated biomarker is above a target level.

15. The method of claim 13 or 14, wherein the at least one bone- or cartilage-associated biomarker is selected from the group consisting of CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, collagen type I C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, alkaline phosphatase, N-terminal collagen type I pro-peptide (PINP), bone-specific alkaline phosphatase (BSAP), cross-linked C-telopeptide of type I collagen (CTx), cross-linked N-telopeptide of type I collagen (NTx), tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, and CNP-variant bioactivity.

16. The method of any one of claims 1 to 15, wherein the subject having short stature has a height Z-score of -2.00 standard deviations (SDs) or less.

17. The method of any one of claims 1 to 4 and 9 to 16, wherein the CNP variant is administered at a dose of 7.5 µg / kg / day, 15 µg / kg / day or a dose of 22.5 µg / kg / day.

18. The method of any one of claims 1 to 17, wherein the CNP variant is administered at a dose of 7.5 µg / kg / day.

19. The method of any one of claims 1 to 17, wherein the CNP variant is administered at a dose of 22.5 µg / kg / day.

20. The method of any one of claims 1 to 19, wherein the short stature disorder is idiopathic short stature.

21. The method of any one of claims 1 to 20, wherein the short stature disorder is a RASopathy.

22. The method of claim 21, wherein the RASopathy is Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, or LEOPARD syndrome.

23. The method of any one of claims 1 to 19, wherein the short stature disorder results from a mutation in NPR2 or NPPC.

24. The method of any one of claims 1 to 19, wherein the short stature disorder results from a mutation in SHOX.

25. The method of claim 24, wherein the SHOX mutation is Turner Syndrome or SHOX Deficiency.

26. The method of any one of claims 1 to 25, wherein the CNP variant is selected from the group consisting of PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO:1); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46];MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); and GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55); 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); PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 1) 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); Ac- PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14). 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) (SEQ ID NO: 57).

27. The method of any one of claims 1-26, wherein the CNP variant is selected from the group consisting of PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:1); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7) QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:54); and GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55).

28. The method of claim 26 or 27, wherein the CNP variant further comprises an acetyl group. 29 The method of claim 28, wherein the acetyl group is on the N-terminus of the peptide.

30. The method of any one of claims 26-29, wherein the CNP variant further comprises an OH or an NH2 group at the C-terminus.

31. The method of any one of claims 26-30, wherein the CNP variant comprises a conjugate moiety.

32. The method of claim 31, wherein the conjugate moiety is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain.

33. The method of claim 31 or 32, wherein the conjugate moiety is on a lysine residue.

34. The method of any one of claims 26 to 33, wherein the CNP variant comprises a linker.

35. The method of claim 34, wherein the linker is a hydrolysable linker.

36. The method of claim 34 or 35, wherein the linker is on a lysine residue.

37. The method of any one of claims 34 to 36, wherein the CNP variant is linked to the conjugate moiety via the linker.

38. The method of any one of claims 1 to 37, wherein the composition further comprises a pharmaceutically acceptable excipient, carrier or diluent.

39. The method of claim 38, wherein the composition is a lyophilized formulation prepared from a formulation that comprises a citric acid / citrate buffer or an acetic acid / acetate buffer having a pH from about 4 to about 6.

40. The method of claim 39, wherein the lyophilized formulation is prepared from a formulation that further comprises an isotonicity-adjusting agent or a bulking agent selected from the group consisting of mannitol, sucrose, sorbitol, and combinations thereof.

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