CNP therapy

Abstract

The present disclosure relates generally to measures of efficacy in patients undergoing C-type natriuretic peptide (CNP) therapy to treat skeletal dysplasia, short stature, or bone-related disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 286,829, filed December 7, 2021, and U.S. Provisional Patent Application No. 63 / 380,509, filed October 21, 2022, which are incorporated by reference in their entireties.

[0002] Sequence Listing Reference This application contains a Sequence Listing that was submitted electronically in file entitled 56861_Seqlisting.xml, created on November 30, 2022, and having a size of 55,905 bytes, and is incorporated herein by reference.

[0003] The present disclosure relates generally to c-type natriuretic (CNP) therapy for treating skeletal dysplasia and measures of efficacy of the treatment. [Background technology]

[0004] C-type natriuretic peptide (CNP) is a paracrine growth factor that is widely expressed across multiple tissues (Prickett et al., Peptides 2020;132:170-363) and has diverse functions including regulation of endochondral bone growth, blood flow and pressure in the microcirculation, anti-inflammatory effects, gamete maturation and neurogenesis and connectivity (Kuhn M., Physiol Rev 2016;96:751-804). The most well-defined of these factors in humans is the hormone's critical role in skeletal growth, promoting the expansion of the growth plate.

[0005] Studies in experimental animals show that it is the local production of CNP acting through its specific receptor NPR2 in growth plate tissue that determines physiological endochondral bone growth (Nakao et al., Sci Rep 2015;5:10554). Study of the dynamic role of CNP in pediatric growth is difficult due to its rapid clearance and very low concentration in plasma. However, the inactive part of the synthetic product (proCNP) in tissues, i.e., amino-terminal proCNP (NTproCNP), does not undergo clearance or rapid degradation. Its levels in plasma reflect the variation of linear growth rate throughout growth in both children and experimental animals (Espiner et al., Horm Res Paediatr 2018;90:345-357). In particular, in subjects with genetic disorders of skeletal growth that affect CNP pathway activity, plasma NTproCNP concentrations are increased when intracellular CNP pathway activity is decreased (Olney et al., J Clin Endocrinol Metab 2015;100:E355-359; Wang et al., Hum Mutat 2015;36:474-481) and decreased when intracellular activity is enhanced (Hannema et al., J Clin Endocrin Metab 2013;98:E1988-1998; Boudin et al., Am J Hum Genet 2018;103:288-295; Miura et al. PloS one 2012;7:e42180). In achondroplasia, the normal reciprocal antagonism between FGFR3 pathway activity (inhibitory to endochondral bone growth) and CNP signaling (stimulating bone growth) (Ozasa et al., Bone 2005 36:1056-1064) is abrogated by gain of function mutations in FGR3 (Yasoda et al. Nature Medicine 2004 10:80-86), reducing intracellular CNP activity and associated with modestly elevated concentrations of CNP products in plasma (Olney et al., J Clin Endocrinol Metab 2015 100:E355-359). Nothing is known about the dynamics or significance of feedback regulation of CNP during periods of active long bone growth. Moreover, it is unclear whether such feedback is direct or time-dependent on cell-to-cell growth responses in skeletal tissues (indirect feedback). Direct feedback results from the action of a cell's own products on CNP production, whereas indirect feedback involves a longer loop mediated by cells other than the cells secreting the peptide. However, in a recent report addressing these important questions in rodent pups, exogenous CNP administered at high concentrations for 3 consecutive days inhibited CNP gene expression, but only in tissues containing the growth plate (Ueda et al., PLoS One 2020,15:e0240023). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Prickett et al.,Peptides2020;132:170363 [Non-Patent Document 2] Kuhn M.,Physiol Rev 2016;96:751-804 [Non-Patent Document 3] Nakao et al.,Sci Rep 2015;5:10554 [Non-Patent Document 4] Espiner et al., Horm Res Paediatr 2018;90:345-357 [Non-Patent Document 5] Olney et al.,J Clin Endocrinol Metab 2015;100:E355-359 [Non-Patent Document 6] Wang et al., Hum Mutat 2015;36:474-481 [Non-Patent Document 7] Hannema et al.,J Clin Endocrin Metab 2013;98:E1988-1998 [Non-Patent Document 8] Boudin et al.,Am J Hum Genet 2018;103:288-295 [Non-Patent Document 9] Miura et al.PloS one 2012;7:e42180 [Non-Patent Document 10] Ozasa et al.,Bone 2005 36:1056-1064 [Non-Patent Document 11] Yasoda et al.Nature Medicine 2004 10:80-86 [Non-Patent Document 12] Ueda et al.,PLoS One 2020,15:e0240023 Summary of the Invention

[0007] The present disclosure relates to real-time observations of the effects of exogenous CNP analogs (e.g., vosoritide) on endogenous CNP production in children with achondroplasia (Ach) during daily treatment for 5 years. Analysis of endogenous CNP levels in response to CNP treatment varies depending on the dose of exogenous CNP administered and the growth status of the subject. The present disclosure shows that levels of NTproCNP (representing endogenous CNP levels) and N-terminal fragment of collagen X (CXM) are useful as markers of growth rate and efficacy of endogenous CNP therapy in children with short stature or skeletal dysplasia, such as achondroplasia. Further measures of efficacy in young children include analysis of skull and brain morphology over time.

[0008] Provided herein is a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature and receiving C-type natriuretic peptide (CNP) therapy, comprising: i) administering CNP therapy to the subject; ii) obtaining a sample from the subject; iii) measuring levels of NTproCNP and / or N-terminal fragment of collagen X (CXM) in the sample obtained from the subject in (ii); and iv) altering or varying the dose of CNP to bring the NTproCNP level within + / - 2 SDS of the mean NTproCNP in a population.

[0009] In various embodiments, the CNP therapy dose level or frequency is increased when the level of NTproCNP increases, or the CNP therapy dose level is decreased when the level of NTproCNP decreases.

[0010] Also provided is a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature and receiving C-type natriuretic peptide (CNP) therapy, comprising: i) administering CNP therapy to the subject; ii) obtaining a sample from the subject; iii) measuring the level of N-terminal fragment of collagen X (CXM) in the sample obtained from the subject in (ii); and iv) increasing the dose level or frequency of the CNP therapy if the level of collagen X decreases.

[0011] In various embodiments, increasing the CNP therapy dose increases the average growth velocity (AGV) in the subject, hi various embodiments, the average growth velocity (AGV) in the subject increases over six months, over one year, or over two years, or more.

[0012] In various embodiments, increasing the CNP therapy dose includes increasing the frequency of administration or increasing the dosage.

[0013] In various embodiments, increasing CNP therapy dose levels and decreasing NTproCNP levels correlate with improved annualized growth velocity (AGV) in a subject.

[0014] In various embodiments, increasing CNP therapy dose levels and decreasing NTproCNP levels increases the duration of growth plate activity in a subject.

[0015] In various embodiments, the level of NTproCNP is maintained within 2 standard deviations of the mean NTproCNP level based on population analysis. In various embodiments, the level of NTproCNP is maintained within + / - 2 SDS of the mean NTproCNP level of the population. In various embodiments, NTproCNP is ±0.5, ±1.0, ±1.5 or ±2.0 standard deviations (SDS) of the mean NTproCNP level of the population into which the subjects are grouped.

[0016] In various embodiments, the CNP therapy is titrated toward zero NTproCNP SDS if the NTproCNP SDS falls below the average. In various embodiments, the CNP therapy is titrated until the NTproCNP SDS is zero. In various embodiments, the CNP therapy is titrated until a +0.5, +1.0, +1.5 or +2.0 NTproCNP SDS is achieved for the treated population. In various embodiments, a zero NTproCNP SDS predicts an optimal effect size. The optimal effect size is a measure of the expected average normal growth rate of a subject based on population norms.

[0017] In various embodiments, the sample is blood, urine, plasma, saliva, or tissue.

[0018] In various embodiments, the subject suffers from a bone-related disorder, skeletal dysplasia, or short stature. In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature is selected from the group consisting of achondroplasia, osteoarthropathy, hypophosphatemic rickets, hypochondrosis, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, ankylosing limb dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, rhizometaphyseal chondrodysplasia punctata, Jansen type metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, ... congenita, atelosteogenesis, warping dysplasia, congenital short femurs, Langer mesomelic dysostosis, Nievergelt mesomelic dysostosis, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromidextracellular dysostosis, micromelia, Morquio syndrome, Kniest syndrome, complex metatrophic dysplasia, spondyloepimetaphyseal dysplasia, NPR2 mutations, SHOX mutations (Turner syndrome / Leri Weill), PTPN11 mutations (Noonan syndrome), IGF1R mutation-associated disorders.

[0019] CNP therapy for treating a subject with a bone-related disorder, skeletal dysplasia, or short stature is contemplated to include administration of a CNP variant, conjugate, salt, or prodrug thereof.

[0020] In various embodiments, CNP variants are useful as supplements or substitutes for growth hormone for the treatment of idiopathic short stature and other skeletal dysplasias.

[0021] In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature disorder is due to an NPR2 mutation, a SHOX mutation (Turner syndrome / Leri Weill), or a PTPN11 mutation (Noonan syndrome).

[0022] In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature disorder is due to an NPR2 mutation, a SHOX mutation (Turner syndrome / Leri Weill), a PTPN11 mutation (Noonan syndrome), or an insulin growth factor 1 receptor (IGF1R).

[0023] In various embodiments, CNP variants are useful for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature, also known as dominantly inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is the result of a mutation in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0024] In various embodiments, the gene associated with skeletal dysplasia or short stature is selected from the group consisting of NPR2, SHOX, PTPN11, COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), NPPC, FGFR3, IGF1R, DTL, and pregnancy associated plasma protein A2 (PAPPA2), or a combination thereof.

[0025] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0026] In various embodiments, the bone-related disorder, skeletal dysplasia, or short stature disorder is caused by a RAS disease. In various embodiments, the RAS disease is Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome, neurofibromatosis type 1, or LEOPARD syndrome. In one embodiment, the RAS disease is hereditary gingival fibromatosis type 1.

[0027] In various embodiments, the CNP variants are useful for treating short subjects having a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, with at least one parent having a height SDS of less than SDS-1.0, -1.5, -2.0, or -2.5, and optionally the other parent's height is within the normal range. In various embodiments, the CNP variants are useful for treating short subjects having a height SDS of -2.0 to -3.0. In various embodiments, the CNP variants are useful for treating short subjects having a height SDS of -2.0 to -2.5. In various embodiments, the short stature is associated with one or more mutations in genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, the gene associated with skeletal dysplasia or short stature is selected from the group consisting of NPR2, SHOX, PTPN11, COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), NPPC, FGFR3, IGF1R, DTL, and pregnancy associated plasma protein A2 (PAPPA2), or combinations thereof. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0028] In various embodiments, the short stature is the result of mutations in multiple genes as determined by a polygenic risk score (PRS). In various embodiments, the subject has a mutation in NPR2 and has a low PRS. In various embodiments, the subject has a mutation in FGFR3 and has a low PRS. In various embodiments, the subject has a mutation in NPR2 and has a low PRS. In various embodiments, the subject has a mutation in IGF1R and has a low PRS. In various embodiments, the subject has a mutation in NPPC and has a low PRS. In various embodiments, the subject has a mutation in SHOX and has a low PRS. In various embodiments, the subject has one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX and has 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.

[0029] In various embodiments, CNP is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37) (SEQ ID NO: 1), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37) (SEQ ID NO: 2), GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP53) (SEQ ID NO: 3), PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP53) (SEQ ID NO: 4), MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP53) (SEQ ID NO: 5), DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53 (M48N)] (SEQ ID NO: 6), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 7), RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 8), VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 9), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-49) (SEQ ID NO: 10), TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 12), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-46) (SEQ ID NO: 13), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 14), AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 15), AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 16), WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 17), ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 18), RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 19), LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 20), LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 22), EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 23), HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 24), PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 25), NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 26), ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 27), RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 28), KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 29), YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 30), KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 31), GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 32), ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 33), NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 34), KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 35), KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 36), LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 37), SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 38), KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 39), GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 40), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37 (M32N)] (SEQ ID NO: 41), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37) (SEQ ID NO: 42), MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37) (SEQ ID NO: 43), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N)] (SEQ ID NO: 44), MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37) (SEQ ID NO: 45), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1).

[0030] In various embodiments, the variant peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus.

[0031] In various embodiments, the CNP variant composition is a sustained release composition. In various embodiments, the composition is a sustained release composition. In various embodiments, the sustained or extended release composition comprises a CNP variant prodrug.

[0032] In various embodiments, the 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.

[0033] In various embodiments, the conjugate moiety comprises one or more acid moieties linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu). In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) or OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) linked to one or more OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the acid moiety is a fatty acid. Exemplary fatty acids include short, medium, or long chain fatty acids, or dicarboxylic fatty acids. In various embodiments, the fatty acids are saturated or unsaturated. C-6 to C-20 fatty acids, saturated or unsaturated, are contemplated, including but not limited to C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acids. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids thereof.

[0034] In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-γGlu-C18DA.

[0035] In various embodiments, the variants include one or more linker groups. In various embodiments, the linker is on a residue in the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0036] In various embodiments, the linker is a hydrolyzable linker.

[0037] In various embodiments, the conjugate moiety comprises a synthetic polymer group. In various embodiments, the variant comprises a synthetic polymer group attached to the variant via a hydrolyzable linker. In various embodiments, the synthetic polymer group comprises a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG). In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG) having a chain length of 6 to 20 atoms.

[0038] 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) (sequence number 21).

[0039] In various embodiments, the variant has the structure: PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 46), or Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 46).

[0040] In various embodiments, the variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 46), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 47), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 48), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 48), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 46), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 49), and Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 49).

[0041] It is further contemplated that CNP variants include conjugates, salts, or prodrugs of the CNP variants described herein.

[0042] In various embodiments, levels of NTproCNP or CXM are measured in plasma samples, for example, before and after administration of a CNP variant.

[0043] In various embodiments, the subject is receiving 7.5 μg / kg to 30 μg / kg of CNP therapy. In various embodiments, the subject is receiving 15 μg / kg or 30 μg / kg of CNP therapy. In various embodiments, the dose may be increased to 30 μg / kg or 60 μg / kg.

[0044] In various embodiments, NTproCNP and / or CXM are measured at least 4 hours after administration. In various embodiments, the levels of NTproCNP and / or CXM are measured at least 3 months or 6 months after initiation of CNP therapy. In various embodiments, the levels of NTproCNP and / or CXM are measured at least every 3 months, every 6 months, or every year after initiation of CNP therapy. In various embodiments, the levels of NTproCNP and / or CXM are measured for at least 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, or until puberty / closure of the growth plates after initiation of CNP therapy.

[0045] In various embodiments, the level of NTproCNP in the sample is compared to a baseline measurement taken prior to the initiation of CNP therapy, hi various embodiments, the level of NTproCNP in the sample is compared to the average level in normal control patients.

[0046] In various embodiments, the dosage or frequency of CNP treatment is increased when a decrease in NTproCNP indicates an increase in AGV in the subject.

[0047] In various embodiments, the level of CXM in the sample is compared to a baseline measurement taken prior to the initiation of CNP therapy, hi various embodiments, the level of CXM in the sample is compared to the average level in normal control patients.

[0048] In various embodiments, an increase in CXM indicates increased bone growth, and the frequency or level of administration of CNP is increased if there is an increase in CXM which enhances AGV.

[0049] In various embodiments, the subject is a pediatric subject with open growth plates and receives a dose of 15 or 30 μg / kg per day. In various embodiments, the subject is an early adolescent and receives a dose escalation to 30 μg / kg per day or 60 μg / kg per day. In various embodiments, the subject is a toddler and receives a dose escalation to 30 μg / kg per day.

[0050] The disclosure also provides a method of selecting initiation of CNP therapy in a subject comprising: i) measuring the subject's NTproCNP at multiple time points to establish a baseline NTproCNP level; ii) determining whether the NTproCNP level exhibits an SDS within ±2 of the mean NTproCNP level; and iii) initiating treatment with CNP therapy when the subject has an NTproCNP level below the mean NTproCNP SDS. In various embodiments, the subject has an NTproCNP SDS of about -2.5, -2.0, -1.5, -1.0, or -0.5. In various embodiments, the CNP therapy is adjusted such that the subject's NTproCNP SDS is about -0.4, -0.3, -0.2, -0.1, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, .5, .16, 1.7, 1.8, 1.9, or 2.0 after adjusting the dosage level and / or frequency of the CNP therapy.

[0051] In certain embodiments, the present disclosure contemplates a method of selecting initiation of CNP therapy in a subject with achondroplasia, the method comprising: i) measuring NTproCNP in the subject at multiple time points to establish a baseline NTproCNP level; ii) determining whether the NTproCNP level exhibits an SDS of zero, less than zero, or greater than zero; and iii) initiating treatment with CNP therapy if the subject has an NTproCNP level that exceeds an SDS of zero.

[0052] In various embodiments, NTproCNP is measured 2 weeks, 1 month, 3 months, and 6 months prior to CNP therapy to establish baseline NTproCNP levels, hi various embodiments, NTproCNP is measured by radioimmunoassay.

[0053] Also provided is a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature, comprising: i) identifying whether a subject has a loss-of-function (LoF) or gain-of-function (GoF) variant of a gene associated with short stature; ii) calculating a polygenic risk score (PRS) for the subject; and iii) determining whether the subject has a LoF variant and a PRS in the bottom 20%; iv) if the subject has a LoF variant and the PRS is in the bottom 20%, treating the subject with a CNP variant.

[0054] In various embodiments, the subject has a PRS in the bottom 20%, 19%, 18%, 17.5%, 17%, 16.5%, 16%, 15.5%, 15%, 14.5%, 14%, 13.5%, 13%, 12.5%, 12%, 11%, 10%, 9%, 8%, 7.5%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, or 1%. In various embodiments, steps iii) and iv) determine that the subject has a CNP variant if the subject has a LoF variant and a PRS in the bottom 12.5%.

[0055] In various embodiments, the gene associated with skeletal dysplasia or short stature is selected from the group consisting of NPR2, SHOX, PTPN11, COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), NPPC, FGFR3, IGF1R, DTL, and pregnancy associated plasma protein A2 (PAPPA2), or a combination thereof.

[0056] Also contemplated is a method of treating a subject having a bone-related disorder, a skeletal dysplasia, or short stature, i) identifying whether a subject has an NPR2 loss-of-function (LoF) or gain-of-function (GoF) variant; ii) calculating a polygenic risk score (PRS) for the subject; and iii) determining whether the subject has an NPR2 LoF variant and a PRS in the bottom 20%; iv) if the subject has an NPR2 LoF variant and the PRS is in the bottom 20%, treating the subject with a CNP variant.

[0057] In various embodiments, the subject has a PRS in the bottom 20%, 19%, 18%, 17.5%, 17%, 16.5%, 16%, 15.5%, 15%, 14.5%, 14%, 13.5%, 13%, 12.5%, 12%, 11%, 10%, 9%, 8%, 7.5%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, or 1%. In various embodiments, steps iii) and iv) determine that the subject has a CNP variant if the subject has a LoF variant and a PRS in the bottom 12.5%.

[0058] In various embodiments, the LoF or GoF variants are determined by biological activity assays. In various embodiments, the LoF or GoF variants may be predicted based on biological activity and mapping to the predicted 3D structure of the protein, for example using AlphaForm 3D mapping or other protein mapping tools.

[0059] In various embodiments, the PRS is calculated by genome-wide association studies (GWAS) of height. The PRS is an aggregate genetic score consisting of many common mutation effects of each small effect summed across the genome (Choi et al. Nat Protoc, 2020). To calculate the height PRS, genome-wide association studies (GWAS) association statistics are obtained to indicate the strength of each variation of association with height. These effect sizes are then applied to an independent sample, here a clinical population, by weighting the number of each height-associated allele (0, 1, or 2) by the GWAS effect size and summing this weighted number across the genome for each clinical sample. The PRS can be interpreted such that individuals with a low PRS carry a lower number of height-increasing genetic variants than average, and individuals with a high PRS carry a higher number of height-increasing variants than average.

[0060] In another embodiment, provided herein is a method for increasing facial volume, facial sinus volume, and foramen magnum area in a subject aged 6 months or less with bone-related disorder, skeletal dysplasia, or short stature, comprising administering a CNP variant, its conjugate, salt, or prodrug at a dose of at least 30 μg / kg.Also provided is a method for reducing the incidence of sudden infant death, sleep-disordered breathing, and the need for neurosurgical decompression of the foramen magnum in a subject aged 6 months or less with bone-related disorder, skeletal dysplasia, or short stature, comprising administering a CNP variant, its conjugate, salt, or prodrug at a dose of at least 30 μg / kg.

[0061] In various embodiments, the increase in facial volume, facial sinus volume, and foramen magnum area is measured by magnetic resonance imaging (MRI). In various embodiments, the changes in facial volume, facial sinus volume, and foramen magnum area are compared to baseline levels, healthy control subjects, or untreated control subjects.

[0062] In various embodiments, the CNP variant is administered subcutaneously. In various embodiments, the CNP variant is administered daily, weekly, every two weeks, monthly, or less frequently. In various embodiments, the CNP variant is administered at a dose of 30 μg / kg for 3 months, 6 months, 1 year or more. In various embodiments, the dose of the CNP variant is reduced to 15 μg / kg when the subject is about 2 years old.

[0063] It is understood that each feature or embodiment, or combination, described herein is intended to be a non-limiting, illustrative example of any of the aspects of the invention, and thus combinable with any other feature or embodiment, or combination, described herein. For example, when a feature is described with words such as "one embodiment," "some embodiments," "particular embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, without the need to recite every possible combination.

[0064] Any such feature or combination of features may be applied to any of the aspects of the invention. When example values ​​falling within ranges are disclosed, any of these examples are contemplated as possible endpoints for a range, and any and all values ​​between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned. [Brief description of the drawings]

[0065] [Figure 1] Annualized growth velocity (AGV) (Figure 1A), plasma NTproCNP concentrations (Figure 1B), and NTproCNP SDS (adjusted for age and sex) (Figure 1C) by cohort across the study. Values ​​are mean ± SE. Cohort 1 (six subjects, age range 6-10 years at screening) received 2.5 μg / kg / day for up to 10 months (until approximately day 300), followed by 7.5 μg / kg / day for approximately 2 months (until approximately day 360), then 15 μg / kg / day until study completion. Cohort 2 (six subjects, age range 5-10 years) received 7.5 μg / kg / day for the first 6-8 months (180-240 days), then titrated to 15 μg / kg / day. Cohort 3 (8 subjects, age range 6-11 years) and Cohort 4 (8 subjects, age range 5-8 years) received 15 μg / kg / day and 30 μg / kg / day, respectively, throughout the study. [Diagram 2]Changes in NTproCNP concentrations over time by cohort. Individuals within each cohort are depicted. The letter P indicates the time of the visit at which the individual was determined to have reached Tanner stage 2. Cohort 1 (Figure 2A) (6 subjects, age range 6-10 years at screening) received 2.5 μg / kg / day for up to 10 months (until approximately day 300), followed by 7.5 μg / kg / day for approximately 2 months (until approximately day 360), then 15 μg / kg / day until study completion. Cohort 2 (Figure 2B) (6 subjects, age range 5-10 years) received 7.5 μg / kg / day for the first 6-8 months (180-240 days), then titrated to 15 μg / kg / day. Cohort 3 ( Fig. 2C ) (8 subjects, age range 6–11 years) and cohort 4 ( Fig. 2D ) (8 subjects, age range 5–8 years) received 15 μg / kg / day and 30 μg / kg / day, respectively, throughout the study. [Diagram 3] Fold change from baseline (screening) in bone turnover markers (bALP, PINP) and plasma NTproCNP in three Cohort 4 subjects over years 3-4 of treatment. Each panel shows simultaneous analyte concentrations in a single subject. [Figure 4] Relationship between the change (delta) in plasma NTproCNP concentration 4 hours after injection on day 183 and the NTproCNP SDS before injection on the same day. [Figure 5A] Growth velocity and biomarker results over time for Phase 2 study 111-202. Annualized growth velocity (AGV) (Figure 5A), serum collagen X biomarker (CXM) (Figure 5B), and serum bone specific alkaline phosphatase (BSAP) (Figure 5C) were measured in subjects (cohorts 1, 2, 3, and 4) receiving 2.5, 7.5, 15, or 30ug / kg / day vosoritide. After 6 months of treatment, cohorts 1 and 2 were dose escalated to 15ug / kg / day. Change in AGV or biomarker from baseline is shown on the y-axis and days of vosoritide treatment are shown on the x-axis. Lines represent the mean for each cohort and error bars represent standard error of the mean. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A] Growth velocity and biomarker results over time for natural history study 111-901 and phase 3 trial 111-301. Annualized growth velocity (AGV) (Figure 6A), serum collagen X biomarker (CXM) (Figure 6B), and serum bone specific alkaline phosphatase (BSAP) (Figure 6C) were measured in untreated subjects in study 111-901 and in the same subjects receiving either 15ug / kg / day vosoritide or placebo in study 1110301. AGV or serum biomarker concentrations are shown on the y-axis and months from the start of study 111-301 (vosoritide or placebo treatment) are shown on the x-axis. Lines represent the mean for each group and error bars represent standard error of the mean. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7] 1 shows examples of CNP variant proteins that include conjugate moieties. [Figure 8A] FIG. 1 shows the CatchPoint assay, a competition-based ELISA assay used to measure cGMP (Molecular Devices). [Figure 8B] Normalized cGMP values ​​of various NPR2 LoF and GoF variants are shown. [Figure 9A] Figure 9A) Breakdown of variant activity levels based on protein prediction outcomes. Figure 9B) Breakdown of missense variant prediction outcomes based on Combined Annotation Dependent Depletion (CADD) scores. Figure 9C) Comparison of measured functional activity of NPR2 variants with their average effect on height of individuals carrying them. X-axis shows log base 2 activity levels relative to wild type NPR2 (0=wt, -1=½wt, -2=¼wt, etc.). Y-axis shows estimated effect size of variant on adult height. One unit of beta corresponds to one standard deviation (approximately 2.5 inches in males and 2.2 inches in females). [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 10A]Predicting idiopathic short stature (ISS) based on genetics. Figure 10A shows the probability of ISS based on a polygenic score of height alone. The first panel shows how the predictive power of the polygenic score changes in the context of NPR2 loss-of-function variants. Figure 10B shows the prediction of ISS by a polygenic risk score combined with the presence of NPR2 loss-of-function variants. [Figure 10B] Same as above. [Figure 11A] Figure 11A. 3D model of NPR2 protein, AF model, low confidence regions indicated (yellow and red). Figure 11B. Depiction of the different domains of NPR2 (dimer) as described in Hannema et al (J Clin Endocrinol Metab. 98:E1988-98, 2013). Figure 11C. A list of variants located in the ligand binding domain were mapped onto the 3D model. Figure 11D. Summary of all variants (LoF=red, GoF=green) and their potential impact on NPR2 function. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D-1] Same as above. [Figure 11D-2] Same as above. [Figure 12A] Magnetic resonance imaging at baseline and week 52 showing changes in (FIG. 12A) face volume, (FIG. 12B) sinus volume, and (FIG. 12C) foramen magnum area. Changes in face volume (upper panel), sinus volume (middle panel), and foramen magnum area (lower panel) on magnetic resonance imaging. The figure shows changes from baseline to week 52 in MRI results assessing the effect of vosoritide on skull morphology. Box plots display the 25th and 75th percentiles (box edges), median (midline), mean (open square symbols), and 2.5th and 97.5th percentiles (whiskers). Dots represent outliers. [Figure 12B] Same as above. [Figure 12C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] This application relates to the discovery that bone growth biomarkers are useful for assessing the improvement of bone growth and annual growth rate of CNP therapy in subjects with skeletal dysplasia. Biomarkers can be used to determine dose effectiveness and modify the timing and / or frequency of CNP administration based on the levels of biomarkers such as NTproCNP and CXM. Further measures of efficacy are provided, including analysis of infant skull and brain morphology over time.

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

[0068] The term "about" or "approximately" means within an acceptable error range 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 specified value or range. Whenever the term "about" or "approximately" appears before the first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to each of the numbers in the series.

[0069] The term "C-type natriuretic peptide" or "CNP" refers to a small single-chain peptide with a 17 amino acid loop structure at the C-terminus (GenBank accession number NP_077720 for CNP precursor protein, NPPC) and its variants. The 17-mer CNP loop structure is also called CNP17, CNP ring, or CNP cyclic domain. CNP includes an active 53 amino acid peptide (CNP-53) and a mature 22 amino acid peptide (CNP-22), as well as peptides with different lengths between the two peptides.

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

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

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

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

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

[0075] A "prophylactic" treatment is a treatment administered to a subject who does not show signs of a disease or who shows only early signs of a disease, for the purpose of reducing the risk of developing a condition. A compound or composition of the present disclosure may be given as a prophylactic measure to reduce the likelihood of developing a condition, or to minimize its severity if the condition does develop.

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

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

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

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

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

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

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

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

[0084] By "physiological pH" or "pH in the 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).

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

[0086] C-type natriuretic peptide C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168:863-870 (1990) (GenBank accession number NP_077720 for the CNP precursor protein, NPPC) (J. Hypertens., 10:907-912 (1992)) is a 17 amino acid loop structure (Levin et al. al., N. Engl. J. Med., 339: 863-870 (1998)) and has important roles in multiple biological processes. CNP interacts with the natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP) (J. Hypertens., 10: 1111-1114 (1992)). CNP is more widely expressed in the central nervous system, reproductive tract, bone, and vascular endothelium (Gardner et al., Hypertension, 49: 419-426 (2007)).

[0087] In humans, CNP is initially synthesized as a single 126 amino acid fragment from the natriuretic peptide precursor C (NPPC) gene (Sudoh et al., Biochem. Biophys. Res. Commun., 168:863-870 (1990)). Removal of the signal peptide results in proCNP, 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, whereas CNP-22 is found primarily in plasma and cerebrospinal fluid (J. Alfonzo, Recept. Signal. Transduct. Res., 26:269-297 (2006)). Both CNP-53 and CNP-22 bind similarly to NPR-B.

[0088] Downstream signaling mediated by cGMP production affects a wide variety of biological processes, including endochondral ossification. For example, knockout of either CNP or NPR-B in mouse models results in animals with a dwarfed phenotype with shorter long bones and vertebrae. Mutations in human NPR-B that block proper CNP signaling have been identified, causing dwarfism (Olney, et al., J. Clin. Endocrinol. Metab. 91(4):1229-1232 (2006); Bartels, et al., Am. J. Hum. 27-34 (2004)). In contrast, mice engineered to produce high levels of CNP show elongated long bones and vertebrae.

[0089] The native CNP gene and polypeptide have been previously described. U.S. Patent No. 5,352,770 discloses CNP-22 isolated and purified from pig brain with sequence identical 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. Mature CNP is a 22-amino acid peptide (CNP-22). Certain CNP variants are disclosed in U.S. Patent No. 8,198,242, which is incorporated herein by reference.

[0090] In various embodiments, the CNPs of the present disclosure range from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53), including truncated CNPs having wild-type amino acid sequences derived from hCNP-53 and variants thereof. Such truncated CNP peptides include: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37) (SEQ ID NO: 1), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37) (SEQ ID NO: 2), GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP53) (SEQ ID NO: 3), PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP53) (SEQ ID NO: 4), MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP53) (SEQ ID NO: 5), DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53 (M48N)] (SEQ ID NO: 6), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 7), RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 8), VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 9), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-49) (SEQ ID NO: 10), TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 12), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-46) (SEQ ID NO: 13), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 14), AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 15), AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 16), WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 17), ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 18), RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 19), LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 20), LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 22), EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 23), HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 24), PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 25), NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 26), ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 27), RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 28), KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 29), YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 30), KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 31), GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 32), ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 33), NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 34), KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 35), KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 36), LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 37), SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 38), KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 39), GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 40), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37 (M32N)] (SEQ ID NO: 41), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37) (SEQ ID NO: 42), MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37) (SEQ ID NO: 43), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N)] (SEQ ID NO: 44), MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37) (SEQ ID NO: 45), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (sequence number).

[0091] In various embodiments, the CNP variant peptide is a modified CNP-37 or CNP-38 peptide, optionally having a mutation / substitution at the furin cleavage site 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: 41] MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 43), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 42), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 44] PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO: 1), MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 45), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 2) [ka]

[0092] In various embodiments, the CNP variant of the disclosure is PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), or PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (sequence number).

[0093] The variant peptide may further comprise 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 NH2 group at the C-terminus.

[0094] The variant peptide may include 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 includes one or more acid moieties. In various embodiments, the acid moiety is a hydrophobic acid.

[0095] In various embodiments, the variant has the structure: PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 46), or Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 46).

[0096] In various embodiments, the variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 46), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 47), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 48), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 48), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 46), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 49), and Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 49).

[0097] In various embodiments, the CNP variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 46). In various embodiments, the CNP variant is Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 47). In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 47).

[0098] It is further contemplated that the CNP variants are conjugated or complexed with moieties, such as conjugating moieties, that provide increased stability or half-life. In various embodiments, the conjugating moieties are complexed through non-covalent bonds or attached by covalent bonds. The moieties can be non-covalently attached to the peptide through electrostatic interactions. Alternatively, the moieties can be covalently associated with the peptide through one or more linker moieties. The linkers can be cleavable and non-cleavable linkers. The cleavable linkers can be cleaved via enzymes, nucleophilic / basic reagents, reducing agents, light irradiation, electrophilic / acidic reagents, organometallic and metallic reagents, or oxidizing reagents. The linkers can also be self-immolative linkers. Exemplary linkers include N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as triene 2,6-diisocyanates), and the like. esters, 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-ethoxyacetic acid (AEEA), 5 aminovaleric acid (AVA), 6-aminocaproic acid (Abx), vicinal diol cleavable linkers, trimethyl lock lactonization, p-alkoxyphenyl carbamates, bicine, peptoid, or bicine type linkers, and electronic linkers as described herein.

[0099] It is contemplated that 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.

[0100] In various embodiments, the CNP variant is attached to the conjugate moiety via a linker. In various embodiments, the linker is attached to the conjugate moiety via a hydrophilic spacer of the conjugate moiety.

[0101] In various embodiments, the linker is a hydrolyzable linker.

[0102] 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 a SO2 moiety. Exemplary linkers are shown in Figure 7. It is further contemplated that the linkers in Figure 7 may be modified by substitution of the R group. For example, bicine-type linkers include the structures shown below: [ka]

[0103] In various embodiments, the moiety conjugated to the peptide is a synthetic polymer such as polyethylene glycol, a linker, a lipid moiety, or a fatty acid, or a combination thereof. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a spacer, and a linker. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a polyethylene glycol spacer, or a polyethylene glycol derivative spacer, and a linker. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a spacer, and a linker, where the spacer comprises a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, where the carbon atoms of the alkyl chain can be replaced by one or more of O, NH, N(C-1 to C-6 alkyl), or a carbonyl group.

[0104] In various embodiments, the CNP variant is conjugated to a fatty acid. It is hypothesized that lipid technology will increase the serum half-life of the CNP variant, allowing for less frequent injections and / or improved oral delivery. In various embodiments, the fatty acid is a short-chain, medium-chain, long-chain fatty acid, or a dicarboxylic acid fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. In various embodiments, the fatty acid is a C-6 to C-20 fatty acid. In various embodiments, the fatty acid is a C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acid. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids thereof. In various embodiments, the fatty acid is conjugated to a lysine residue.

[0105] In various embodiments, it is contemplated that the CNP variants described herein include 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 includes one or more acid moieties. In various embodiments, the acid moiety is a fatty acid. Exemplary CNP variants and peptide conjugates are described in International Patent Application Nos. PCT / US2020 / 051100 and USSN 17 / 642,150, which are incorporated herein by reference in their entireties. Variants, conjugates, and salts of CNP are disclosed in USSN 17 / 634,034, which are incorporated herein by reference.

[0106] In various embodiments, the conjugate moiety comprises an acid moiety linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, where the carbon atoms of the alkyl chain can be replaced by one or more of O, NH, N(C-1 to C-6 alkyl), or a carbonyl group. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu). In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 to C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 alkyl chain. In various embodiments, the hydrophilic spacer is one or more OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is one or two OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic 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.

[0107] In various embodiments, the present disclosure contemplates the use of CNP variants that include hydrophilic or water-soluble polymers (e.g., oxygenated alkyl chains in which carbon atoms can be replaced with one or more oxygen atoms, such as polyethylene glycol (PEG) or polyethylene oxide (PEO). In various embodiments, the water-soluble polymers can vary in type (e.g., homopolymer or copolymer; random, alternating, or block copolymer; linear or branched; monodisperse or polydisperse), linkage (e.g., hydrolyzable or stable linkages, such as amide, imine, aminal, alkylene, or ester bonds), conjugation site (e.g., N-terminal, internal, and / or C-terminal), and length (e.g., from about 0.2, 0.4, or 0.6 kDa to about 2, 5, 10, 25, 50, or 100 kDa). Hydrophilic or water-soluble polymers can be conjugated to the CNP variants by N-hydroxysuccinimide (NHS)-based or aldehyde-based chemistry or other chemistries, as known in the art. In various embodiments, negatively charged PEG-CNP variants can be designed for reduced renal clearance, including but not limited to the use of carboxylated, sulfated, and phosphorylated compounds (Caliceti, Adv. Drug Deliv. Rev., 55:1261-77 (2003); Perlman, J. Clin. Endo. Metab., 88:3227-35 (2003); Pitkin, Antimicrob. Ag. Chemo., 29:440-444 (1986); Vehaskari, Kidney Int'l, 22:127-135 (1982)). In one embodiment, the PEG (or PEO) moiety contains a carboxyl group, a sulfate group, and / or a phosphate group.

[0108] In another embodiment, the hydrophilic polymer (e.g., PEG or PEO) moieties conjugated to the N-terminus, C-terminus, and / or internal sites of the CNP variants described herein contain one or more functional groups that are positively charged under physiological conditions. Such moieties are designed to, among other things, improve the distribution of such conjugated CNP variants to cartilage tissue. In one embodiment, the PEG moiety contains one or more primary, secondary, or tertiary amino groups, quaternary ammonium groups, and / or other amine-containing (e.g., urea) groups.

[0109] How to Use CNP Variants Achondroplasia is the result of an autosomal dominant mutation in the gene for fibroblast growth factor receptor 3 (FGFR-3), which causes abnormalities in cartilage formation. FGFR-3 normally has a negative regulatory effect on chondrocyte growth and thus bone growth. In achondroplasia, mutant forms of FGFR-3 are constitutively active, which leads to severe shortening of bones. In humans, activating mutations in FGFR-3 are the main cause of genetic dwarfism. Mice with activated FGFR-3 serve as a model for achondroplasia, the most common form of skeletal dysplasia, and overexpression of CNP rescues these animals from dwarfism. Thus, functional variants of CNP are potential therapeutics for the treatment of various skeletal dysplasias.

[0110] By stimulating chondrocyte matrix production, proliferation, and differentiation and increasing long bone growth, the CNP variants of the present disclosure are useful for treating mammals, including humans, suffering from bone-related disorders such as skeletal dysplasia or short stature. Non-limiting examples of CNP-responsive bone-related disorders skeletal dysplasia or short stature disorders include achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta congenita, achondroplasia, chondrodysplasia congenita, homozygous achondroplasia, achondroplasia congenita, ankle dysplasia, lethal hypophosphatasia congenita, lethal osteogenesis imperfecta congenita, short rib polydactyly syndrome, hypochondroplasia, rhizometaphyseal congenital chondrodysplasia, Jansen type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, growth retardation, dysplasia of the skeletal dysplasia ... These include disorders associated with osteogenesis imperfecta, varicose dysplasia, congenital short femurs, Langer mesomelic dysplasia, Nievergelt mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondromatosis, Roberts syndrome, acromidial shortening dysplasia, micromelia, Morquio syndrome, Kniest syndrome, complex organotrophic dysplasia, spondyloepiphyseal dysplasia, NPR2 mutations, SHOX mutations (Turner syndrome / Leri Weill), PTPN11 mutations (Noonan syndrome), and IGF1R mutations.

[0111] Additional short stature and growth plate disorders contemplated by this method include disorders associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0112] Additionally, CNP variants are useful as supplements or substitutes for growth hormone for the treatment of idiopathic short stature and other skeletal dysplasias.

[0113] Growth plate disorders include disorders that result in short stature or abnormal bone growth and may be the result of genetic mutations in genes involved in bone growth, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RAS disease. In various embodiments, the subject with the 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, also known as dominantly inherited short stature, or idiopathic short stature. See, e.g., Plachy et al., J Clin Endocrinol Metab 104:4273-4281, 2019.

[0114] Mutations in ACAN can cause familial osteochondritis dissecans and short stature, ultimately osteoarthritis characterized by areas of bone damage (or lesions) caused by detachment of cartilage and sometimes bone from the ends of the bones at the joints. It has been suggested that a disorganized cartilage network in growing bones impairs their growth, leading to short stature. Mutations associated with ACAN and short stature include Val2303Met. See Stattin et al., Am J Hum Genet 86(2):126-37, 2010. It is contemplated that patients with mutations in ACAN that result in short stature will benefit from treatment with CNP, as administration may be able to increase the height of these patients through the known interaction of CNP with FGFR3.

[0115] The natriuretic peptide system, including the receptor NPR2, has been shown to be involved in the 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 Maroteaux-type distal midlimb dysplasia (AMDM), a skeletal dysplasia associated with very short stature (Vasquez et al., 2014, supra). Although reports have suggested heterozygous loss-of-function (e.g., dominant-negative) NPR2 mutations as the cause of short stature, gain-of-function NPR2 heterozygous mutations have been found to cause tall stature (Vasquez et al., 2014, supra). Given the interaction of CNP with NPR2 to stimulate cGMP production, increasing cGMP levels would be desirable in these conditions and would have therapeutic benefit in managing complications from these diseases and conditions.

[0116] Heterozygous mutations in NPR2 are believed to result in idiopathic short stature and other forms of short stature. Mutations in the NPR2 gene are 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, which are incorporated herein by reference. Subjects with short stature treated with the CNP variants described herein have a height SDS of less than -1.0, -1.5, -2.0, -2.5 or -3.0, and at least one parent has a height SDS of less than -1.0, -1.5, -2.0 or -2.5, and optionally the height of the other parent is within the normal range. In various embodiments, the CNP variants are useful for treating short subjects with a height SDS of -2.0 to -3.0. In various embodiments, the CNP variants are useful for treating short subjects with a height SDS of -2.0 to -2.5. However, because de novo mutations in NPR2 can result in short stature as defined as a height SDS less than -1.5, -2.0, -2.5, or -3.0, treatment of heterozygous carriers of deleterious mutations in NPR2 in whom neither parent has short stature is also contemplated. Also contemplated is treatment of individuals heterozygous for deleterious mutations in other growth plate genes with CNP to improve stature and / or enhance bone growth.

[0117] Exemplary mutations of NPR2 are disclosed in International Patent Publication No. WO2021 / 055497, which is incorporated by reference herein.

[0118] The role of NPPC in skeletal growth is well documented (Hisado-Oliva et al., Genetics Medicine 20:91-97, 2018). NPPC knockout mice showed severe disproportionate forms of dwarfism, including shortened limbs and endochondral ossification (Hisado-Oliva et al., 2018, supra). Human genome-wide studies have shown a relationship between NPPC and stature (Hisado-Oliva et al., 2018, supra). CNP haploinsufficiency is thought to be the cause of short stature in humans, but recent studies have identified heterozygous mutations in families with short stature and microhandia (Hisado-Oliva et al., 2018, supra). In these studies, a significant reduction in cGMP production was observed when measured in the heterozygous state (Hisado-Oliva et al., 2018, supra). NPPC mutations include the 355G>T missense mutation causing a Gly119Cys change and the 349C>G missense mutation causing an Arg117Gly change. CNP variants that rescue CGMP production may offer therapeutic benefit in managing the disorder in patients with heterozygous loss-of-function NPPC mutations.

[0119] Leri-Weill chondro-osseous dysplasia (LWD) is a rare genetic disorder characterized by shortened forearms and legs, abnormal wrist misalignment (Madelung deformity of the wrist), and associated short stature. LWD is caused by heterozygous mutations in the short stature homeobox-containing (SHOX) gene or its regulatory elements located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes. (See Rare Disease Database and Carmona et al., Hum Mol Genet 20:1547-1559, 2011). Langer type metapodial dysplasia occurs when there are two SHOX mutations, which can result from mutations on each chromosome, either homozygous or compound heterozygous mutations. A subset of SHOX mutations causes idiopathic short stature. Turner syndrome occurs due to a deletion of the X chromosome, which may include the SHOX gene. SHOX has been identified as being involved in the regulation of FGFR3 transcription and contributing to the control of bone growth (Marchini et al., Endocr Rev.37:417-448,2016). Deficiency of SHOX leads to increased FGFR3 signaling, and some evidence supports that SHOX also directly interacts with CNP / NPR2 (Marchini, supra). Given the association of SHOX with FGFR3 and bone growth, it is contemplated that subjects with homozygous or heterozygous SHOX mutations will benefit from treatment with the CNP variants described herein.

[0120] RASopathies are a group of rare genetic conditions caused by mutations in genes in the Ras / mitogen-activated protein kinase (MAPK) pathway. RASopathies are a group of disorders characterized by increased signaling through the RAS / MAPK pathway. This pathway leads to downstream activation of the RAF / MEK / ERK pathway. Short stature is a feature of certain RASopathies. For example, CNP signaling inhibits RAF, decreasing MEK and ERK activation.

[0121] The treatment of RASopathy is contemplated herein. RASopathy associated with short stature includes Noonan syndrome, Costello syndrome, cardiofacial cutaneous 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, cardiofacial cutaneous 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).

[0122] 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 faciocutaneous skeletal (FCS) syndrome, is caused by activating mutations in the H-Ras gene. Hereditary gingival fibromatosis type I (HGF) is caused by dominant mutations in the SOS1 gene (Son of Sevenless Homolog 1), which encodes a guanine nucleotide exchange factor (SOS) 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, SOS1, K-Ras, and Raf-1.

[0123] CNP has been demonstrated to be an effective therapy in RAS disease models. Ono et al. generated mice lacking Nf1 in type II collagen producing cells (Ono et al., Hum. Mol. Genet. 2013;22(15):3048-62). These mice showed constitutive ERK1 / 2 activation and reduced chondrocyte proliferation and maturation. Daily injection of CNP into these mice reduced ERK phosphorylation and corrected their short stature. A mouse model of cardio-facio-cutaneous syndrome using the Braf mutation (p.Q241R) (Inoue et al. Hum. Mol. Genet. 2019, 28(1):74-83) showed reduced body length and reduced growth plate width, smaller proliferative and hypertrophic zones compared to wild type, and CNP administration increased the body length of these animals.

[0124] Mutations in multiple genes can cause Noonan syndrome, which is characterized by short stature, heart defects, bleeding problems, and skeletal deformities. Mutations in the PTPN11 gene cause about half of all cases of Noonan syndrome. Mutations in the SOS1 gene cause another 10-15%, and the RAF1 and RIT1 genes each account for about 5% of cases. Mutations in other genes each account for a small number of cases. The cause of Noonan syndrome is unknown in 15-20 percent of people with the disorder.

[0125] The PTPN11, SOS1, RAF1, and RIT1 genes all code for important proteins in the RAS / MAPK cell signaling pathway, which is necessary for cell division and growth (proliferation), differentiation, and cell migration. Many of the mutations in the genes associated with Noonan syndrome turn on the resulting proteins, and this prolonged activation alters normal RAS / MAPK signaling, disrupting the regulation of cell growth and division, resulting in the characteristics of Noonan syndrome. See, for example, Chen et al., Proc Natl Acad Sci US A. 111(31):11473-8, 2014; Romano et al., Pediatrics. 126(4):746-59, 2010; and Milosavljevic et al., Am J Med Genet 170(7):1874-80, 2016. It is believed that subjects with mutations that activate MAPK pathway will benefit from treatment with CNP variants as described herein to improve bone growth and short stature.It is also contemplated that subjects with mutations that activate MAPK pathway will benefit from treatment with CNP variants as described herein to improve other comorbidities associated with overactive MAPK pathway in other cells throughout the body that express NPR2 receptor on their surface.

[0126] Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, cause disorders characterized by short stature, such as Noonan syndrome (Musente et al., Eur J Hum Genet 11:201-206 (2003). Musente (supra) has identified numerous mutations in the PTPN11 gene that lead to short stature. Gain of function mutations cause hyperactive signaling through SHP2, inhibiting growth hormone-induced IGF-1 release, thereby contributing to reduced bone growth (Rocca Serra-Nedelec, PNAS 109:4257-4262, 2012). Subjects with homozygous or heterozygous PTPN11 mutations are believed to benefit from treatment with CNP variants as described herein to improve bone growth and short stature.

[0127] Mutations in the Indian Hedgehog (IHH) gene, which is associated with the regulation of endochondral ossification, are also associated with short stature syndrome (Vasques et al., J Clin Endocrinol Metab.103:604-614,2018). Many identified IHH mutations segregate with short stature in a dominant inheritance pattern. Given the association of IHH with bone growth and ossification, it is contemplated that subjects with homozygous or heterozygous IHH mutations will benefit from treatment with the CNP variants described herein.

[0128] Mutations in FGFR3, including N540K and K650N, cause short stature and hypochondroplasia.

[0129] Insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (α2β2) transmembrane glycoprotein with intrinsic kinase activity. IGF1R has been shown to play a role in prenatal and postnatal growth. Heterozygous mutations in IGF1R have been identified in individuals with small for gestational age (SGA) and familial short stature (Kawashima et al., Endocrine J. 59:179-185, 2012). IGF1R mutations associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, supra).

[0130] Height is a highly heritable trait and can be influenced by the combined effects of hundreds or thousands of genes (Wood et al, 2014, Nature Genetics, 46:1173-1189). An individual's short stature may be the result of the combined effects of these genes, rather than a single gene being the primary cause. It is contemplated that such individuals with short stature, as defined by a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, may be beneficially treated with CNP variants, given the ability of CNP to increase the length of normal animals, e.g., to improve bone growth and bone length.

[0131] In various embodiments, the CNP variants are useful for treating short subjects having a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, with at least one parent having a height SDS of less than SDS-1.0, -1.5, -2.0, or -2.5, and optionally the other parent's height is within the normal range. In various embodiments, the CNP variants are useful for treating short subjects having a height SDS of -2.0 to -3.0. In various embodiments, the CNP variants are useful for treating short subjects having a height SDS of -2.0 to -2.5. In various embodiments, the short stature is associated with one or more mutations in genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), DTL, PAPPA2, or combinations thereof.

[0132] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in genes associated with RASopathy.

[0133] In various embodiments, the short stature is the result of mutations in multiple genes as determined by a polygenic risk score (PRS). The polygenic risk score (PRS) is calculated for height using the largest published genome-wide association study (GWAS) meta-analysis of height without samples from the UK Biobank Project described in WO2021 / 055497. The cohort may be divided into five PRS quintiles (PRS1 being the shortest height and PRS5 being the longest height). In various embodiments, the subject has a mutation in NPR2 and has a low PRS. In various embodiments, the subject has a mutation in FGFR3 and has a low PRS. In various embodiments, the subject has a mutation in NPR2 and has a low PRS. In various embodiments, the subject has a mutation in IGF1R and has a low PRS. In various embodiments, the subject has a mutation in NPPC and has a low PRS. In various embodiments, the subject has a mutation in SHOX and has a low PRS. In various embodiments, the subject has one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX, and has 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.

[0134] In addition, CNP variants are useful for the treatment of other bone-related conditions and disorders, such as rickets, hypophosphatemic rickets (including X-linked hypophosphatemic rickets (also called vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets), and osteomalacia (including tumor-induced osteomalacia (also called carcinogenic osteomalacia or carcinogenic hypophosphatemic osteomalacia)).

[0135] Provided herein is a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature, comprising: i) identifying whether a subject has a loss-of-function (LoF) or gain-of-function (GoF) variant of a gene associated with short stature; ii) calculating a polygenic risk score (PRS) for the subject; and iii) determining whether the subject has a LoF variant and a PRS in the bottom 20%; iv) if the subject has a LoF variant and the PRS is in the bottom 20%, treating the subject with a CNP variant.

[0136] In various embodiments, the subject has a PRS in the bottom 20%, 19%, 18%, 17.5%, 17%, 16.5%, 16%, 15.5%, 15%, 14.5%, 14%, 13.5%, 13%, 12.5%, 12%, 11%, 10%, 9%, 8%, 7.5%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, or 1%. In various embodiments, steps iii) and iv) determine that the subject has a CNP variant if the subject has a LoF variant and a PRS in the bottom 12.5%.

[0137] Exemplary genes associated with skeletal dysplasia or short stature include, but are not limited to, NPR2, SHOX, PTPN11, COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), NPPC, FGFR3, IGF1R, DTL, and pregnancy associated plasma protein A2 (PAPPA2).

[0138] The presence of LoF or GoF variants in genes associated with short stature is determined by biological activity assays. In various embodiments, LoF or GoF variants may be predicted based on mapping to the predicted 3D structure and activity domains of the protein encoded by the gene, for example, using AlphaForm 3D mapping or other protein mapping tools.

[0139] In an exemplary method, the PRS is calculated from a genome-wide association study (GWAS) of height.

[0140] In certain embodiments, the disclosed CNP variants and compositions and formulations comprising same are useful for improving one or more symptoms or physiological outcomes of skeletal dysplasia, where the improvement can be increased absolute growth, increased growth velocity, increased qualitative computed tomography (QCT) bone mineral density, improved growth plate morphology, increased long bone growth, improved spine morphology, improved elbow range of motion, and / or reduced sleep apnea. In this regard, it should be noted that the terms "improved," "improvement," "increase," "reduction," and their grammatical equivalents, when used in connection with a symptom or physiological outcome of a disease condition, are all relative terms referring to the state of the symptom or physiological outcome of the disease after treatment with a CNP variant of the invention (or a composition or formulation comprising same) compared to the same symptom or physiological outcome of the disease prior to treatment with a CNP variant of the invention (or a composition or formulation comprising same) (i.e., compared to the "baseline"). As noted above, a "baseline" condition can be determined either by measuring a subject's condition before treatment (which can then be compared with the condition of the same subject after treatment), or by measuring that condition in a population of subjects suffering from the same affliction who share the same or similar characteristics (e.g., age, sex and / or disease state or progression).

[0141] Also provided is a method of overcoming cell growth arrest induced by constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3), comprising contacting a cell expressing a constitutively active FGFR-3 with a CNP variant or composition described herein.

[0142] In yet another embodiment, the disclosure provides a CNP variant that stimulates the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP levels produced by the same concentration of wtCNP22 (e.g., 1 uM) in vitro or in vivo. In yet a further embodiment, the CNP variant of the disclosure stimulates the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP levels produced by the same concentration of wtCNP22 (e.g., 1 uM) in vitro or in vivo.

[0143] The present disclosure also contemplates that modulating treatment with CNP as described herein enhances or increases growth within a range of 25% to 50% change from baseline in a subject. In one embodiment, the enhanced or increased growth rate is an increase in annual growth rate of at least about 25% change from baseline in a subject, more preferably at least about 40% change.

[0144] It is contemplated that any of the CNP variants described herein, including conjugates, salts, or prodrugs thereof, are useful in this method.

[0145] 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 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, for example, a hydrolyzable linker as described herein.

[0146] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37 (M32N); SEQ ID NO: 41] MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 43), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 42), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 44] MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 45), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 2) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:50), GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:51), GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52), GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53), and LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), wherein the CNP variant comprises a conjugate moiety. In various embodiments, the conjugate moiety comprises a synthetic polymer group.

[0147] In various embodiments, the variant comprises a synthetic polymer group attached to the variant via a hydrolyzable linker. In various embodiments, the synthetic polymer group comprises a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG). In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG) having a chain length of 6 to 20 atoms. In various embodiments, the conjugate moiety comprises one or more acid moieties linked to a hydrophilic spacer as described herein.

[0148] In various embodiments, the conjugate moiety comprises one or more acid moieties linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu). In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) or OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu) linked to one or more OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the acid moiety is a fatty acid. Exemplary fatty acids include short, medium, or long chain fatty acids, or dicarboxylic fatty acids. In various embodiments, the fatty acids are saturated or unsaturated. C-6 to C-20 fatty acids, saturated or unsaturated, are contemplated, including but not limited to C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acids. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids thereof.

[0149] In various embodiments, the variants include one or more linker groups. In various embodiments, the linker is on a residue in the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0150] The efficacy of treatment is measured by various parameters. In various embodiments, efficacy is evaluated as the change in annual growth velocity from baseline period to intervention period. Efficacy is also evaluated as the change in height SDS from baseline to the end of treatment, when measured using CDC growth curves, and growth velocity SDS will be based on childhood bone mineral density studies (Kelly et al., J.Clin.Endocrinol.Metab.2014;99(6):2104-2112).

[0151] Efficacy can also be measured using analysis of skull and brain morphology, for example, using magnetic resonance imaging (MRI). At birth, children with achondroplasia have abnormalities of the skull base and the border of the foramen magnum due to defective endochondral ossification, resulting in stenosis of the foramen magnum and compression of important neural and vascular structures passing through it. Foramen magnum stenosis has been implicated as the main underlying cause of the increased risk of sudden death observed in children under 5 years of age with achondroplasia (Pauli et al., J Pediatr 1984;104:342-8; Hashmi et al., Am J Med Genet A 2018;176:2359-64). Skull and brain morphology analysis includes measurements of improvements in facial volume, sinus volume, and foramen magnum area in patients treated with CNP variants, for example, in young patients under 6 months of age.

[0152] Provided herein is a method for increasing facial volume, facial sinus volume, and foramen magnum area in a subject aged 6 months or less with bone-related disorder, skeletal dysplasia, or short stature, comprising administering a CNP variant, a conjugate, a salt, or a prodrug thereof at a dose of at least 30 μg / kg. Also provided is a method for reducing the incidence of sudden infant death, sleep-disordered breathing, and the need for neurosurgical decompression of the foramen magnum in a subject aged 6 months or less with bone-related disorder, skeletal dysplasia, or short stature, comprising administering a CNP variant, a conjugate, a salt, or a prodrug thereof at a dose of at least 30 μg / kg. In various embodiments, the CNP variant is administered at a dose of 30 μg / kg for 3 months, 6 months, 1 year, or more. In various embodiments, the dose of the CNP variant is reduced to 15 μg / kg when the subject is about 2 years old.

[0153] Changes in facial volume, facial sinus volume, and foramen magnum area can be measured by magnetic resonance imaging (MRI) and compared to baseline levels, healthy control subjects, or untreated control subjects.

[0154] The QoLISSY, Quality of Life in Short Stature Youth, was assessed as indicated (Quality of Life in Short Stature Youth-The QoLISSY Questionnaire User's Manual. Lengerich: Pabst Science Publishers; 2013).

[0155] Biomarkers A biomarker refers to a detectable biological substance or moiety whose level increases or decreases in association with a particular disease state or treatment regimen. In the present disclosure, biomarkers can be measured before, during, and / or after administration of the CNP variants described herein. Exemplary bone or cartilage-related biomarkers include, but are not limited to, NTproCNP, N-terminal fragment of collagen X (CXM), CNP, cGMP, propeptide and fragments of collagen type II, collagen type II and fragments thereof, propeptide and fragments of collagen type I, collagen type I and fragments thereof, osteocalcin, proliferating cell nuclear antigen (PCNA), aggrecan chondroitin sulfate, collagen X, and alkaline phosphatase. Cartilage and bone-related biomarkers can be measured in any suitable biological sample, including, but not limited to, tissue, blood, serum, plasma, cerebrospinal fluid, synovial fluid, and urine. In some embodiments, biomarkers are measured in blood, plasma, or serum from animals undergoing efficacy / pharmacodynamic in vivo testing and / or from conditioned medium of ex vivo testing.

[0156] NTproCNP is the amino-terminal propeptide of CNP (NTproCNP) that is released from cells in equimolar ratios with CNP. The biologically active form of CNP is found in plasma at low concentrations due to the rapid clearance rate of this peptide. NTproCNP is not cleared via the same mechanism and is found in the circulation at 20-50 times higher concentrations (Olney et al., Clin Endocrinol (Oxf). 2012, 77:416-422).

[0157] It is contemplated that evaluation of the effect of the CNP therapy described herein on bone growth is measured relative to NTproCNP levels, e.g., NTproCNP levels are measured in a sample and the dose of CNP is altered or varied to bring the NTproCNP levels within ±2 SDS of the mean NTproCNP of the population. NTproCNP mean levels in different populations have been studied in the following publications, which are incorporated herein by reference: Olney et al. (2015). C-type natriuretic peptide plasma levels are elevated in subjects with achondroplasia, hypochondroplasia, and thanatophoric dysplasia. J Clin Endocrinol Metab, 100(2), E355-359; Prickett et al., (2013). Impact of age, phenotype and cardio-renal function on plasma C-type and B-type natriuretic peptide forms in an adult population. Clin Endocrinol(Oxf), 78(5), 783-789; Espiner et al. (2018). Plasma C-Type Natriuretic Peptide: Emerging Applications in Disorders of Skeletal Growth. Horm Res Paediatr, 90(6), 345-357; Olney et al. al.(2012).Amino-terminal propeptide of C-type natriuretic peptide(NTproCNP) predicts height velocity in healthy children.Clin Endocrinol(Oxf),77(3),416-422, Olney et al.,(2007).Amino-terminal propeptide of C-type natriuretic peptide and linear growth in children:effects of puberty,testosterone,and growth hormone.J Clin Endocrinol Metab,92(11),4294-4298 and Olney et al.(2016).Dynamic response of C-type natriuretic peptide and its aminoterminal propeptide (NTproCNP) to growth hormone treatment in children with short stature.Clin Endocrinol (Oxf), 85(4), 561-568. .

[0158] For example, Olney 2016 shows that children aged 6-10 years with idiopathic short stature can have a mean baseline NTproSDS of -0.6 with a range of -1.0 to 0.7. Olney 2012 reported NTproCNP levels in healthy children / adolescents at growth stages. NTproCNP SDS can be calculated based on the mean NTproCNP levels of different age groups, and therefore + / - 2 SDS can also be calculated from this mean. NTproCNP levels in subjects with achondroplasia or hypochondroplasia are described in Olney 2015, showing that children aged approximately 3-8 years have a NTproSDS mean of 1.4 with a range of 0.4 to 1.8, while subjects with hypochondroplasia (6.6-11 years) have a mean NTproCNP SDS of 1.9 with a range of 1.8 to 2.3. Methods for determining NTproSDS levels are described herein and in the publications mentioned above.

[0159] The collagen type X biomarker (CXM) is a degradation fragment of collagen type X that contains the intact trimeric noncollagen 1 (NC1) domain of collagen type X. CXM is released by active growth plates and decreases in samples with subject age. CXM levels correlate with pediatric growth velocity (Coghlan et al., Sci Transl Med 2017,9(419):eaan4669).

[0160] Bone-specific alkaline phosphatase (BSAP or BAP) is a bone growth biomarker produced by osteoblasts and osteoclasts in the growth plate and mineralized bone. Changes in BSAP may reflect growth plate activity, bone growth, and / or bone remodeling activity.

[0161] The N-terminal propeptide of type I procollagen (PINP) is a potential pharmacodynamic bone growth biomarker that is released during the production of type I collagen. Changes in PINP may reflect alterations in growth plate activity, bone growth, and / or bone remodeling.

[0162] Cross-linked C-telopeptide of type II collagen (CTXII) is a potential pharmacodynamic bone growth biomarker that is released during the degradation of type II collagen. Changes in CTXII may reflect changes in growth plate activity, bone growth, bone remodeling, and / or articular cartilage remodeling.

[0163] formulation The present disclosure provides pharmaceutical compositions, including modified release compositions, comprising a CNP variant described herein and one or more pharma- ceutically acceptable excipients, carriers, and / or diluents. In certain embodiments, the compositions further comprise one or more other biologically active agents (e.g., inhibitors of proteases, receptor tyrosine kinases, and / or clearance receptors NPR-C).

[0164] The present disclosure provides modified release compositions comprising conjugate moieties as described herein. Modified release compositions include those that deliver a drug for a delayed period (delayed release dosage) or extended period (extended release dosage) after administration. Various embodiments of the CNP peptide conjugates provided herein include modified release compositions such as sustained, extended or controlled release, and delayed release. The term "sustained release composition" refers to a composition formulated in a manner that makes the active ingredient / drug available for an extended period after administration (USP). Sustained release dosages include sustained release (SR) or controlled release (CR) forms. Sustained release maintains drug release over a sustained period, but not necessarily at a constant rate, while CR maintains drug release at an approximately constant rate over a sustained period (Pharmaceutics: Drug Delivery and Targeting, Yvonne Perrie, Thomas Rades, Pharmaceutical Press, 2009). Delayed release compositions or products are engineered to delay the release of the drug substance for a period of time after initial administration.

[0165] In various embodiments, the modified release composition is a sustained release composition. In various embodiments, the modified release composition is a sustained release composition. In various embodiments, the sustained or extended release composition comprises a CNP prodrug.

[0166] In various embodiments, the composition comprises an excipient, diluent, or carrier. In various embodiments, the sustained release composition comprises an excipient, diluent, or carrier. In various embodiments, the excipient, diluent, or carrier is a pharma- ceutically acceptable excipient, diluent, or carrier.

[0167] Non-limiting examples of excipients, carriers, and diluents include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and the like. The composition can include liquids (e.g., water, ethanol). Diluents of various buffer contents (e.g., Tris-HCl, phosphate, acetate buffer, citrate buffer), pH and ionic strength, detergents and solubilizers (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, m-cresol), and bulking substances (e.g., lactose, mannitol, sucrose). The use of excipients, diluents, and carriers in the formulation of pharmaceutical compositions is known in the art, and can be found, for example, in Remington's Pharmaceutical Sciences, 18, incorporated herein by reference in its entirety. th Edition, pages 1435-1712, Mack Publishing Co., Easton, Pennsylvania (1990).

[0168] For example, carriers include, but are not limited to, 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 active ingredients.Non-limiting examples of carriers include phosphate buffered saline, saline, water, and emulsions (e.g., oil / water emulsions).Carriers can be, for example, solvents or dispersion media, including ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, and mixtures thereof.

[0169] In some embodiments, the composition is a liquid formulation. In certain embodiments, the formulation comprises a CNP variant in a concentration range of about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 0.5 to 5 mg / ml, or about 0.5 to about 3 mg / ml, or about 1 mg / ml to about 10 mg / ml. In various embodiments, the CNP variant is at a concentration of 0.8 mg / ml to 2 mg / ml. In various embodiments, the CNP variant is at a concentration of 0.8 mg / ml. In various embodiments, the CNP variant is at a concentration of 2.0 mg / ml. In various embodiments, the CNP variant is reconstituted from a lyophilized powder.

[0170] In further embodiments, the composition includes a buffer or buffering agent to maintain the pH of the CNP-containing solution or suspension within a desired range. Non-limiting examples of buffers include phosphate buffered saline, Tris buffered saline, and Hank's buffered saline. Buffering agents include, 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 for the composition depends in part on the particular buffer used and the desired pH of the solution or suspension. In some embodiments, the buffering agent has a concentration of about 10 mM ± 5 mM. In certain embodiments, the pH of the composition is about pH 3 to about pH 9, or about pH 3 to about pH 7.5, or about pH 3.5 to about pH 7, or about pH 3.5 to about pH 6.5, or about pH 4 to about pH 6, or about pH 4 to about pH 5, or about pH 5.0 ± 1.0. In various embodiments, the pH is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In various embodiments, the pH is 5.5.

[0171] In other embodiments, the composition contains an isotonicity adjusting agent that makes 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, the NaCl is at a concentration of about 160±20 mM, or about 140 mM±20 mM, or about 120±20 mM, or about 100 mM±20 mM, or about 80 mM±20 mM, or about 60 mM±20 mM.

[0172] In yet other embodiments, the composition comprises a preservative, including but not limited to m-cresol and benzyl alcohol. In certain embodiments, the preservative is at 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%.

[0173] In yet other embodiments, the composition contains an anti-adsorption agent (e.g., to reduce adsorption of the CNP variant to glass or plastic). Anti-adsorption agents include, but are not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In certain embodiments, the anti-adsorption agent is at a concentration of about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%.

[0174] In additional embodiments, the composition includes a stabilizer. Non-limiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and their salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or their salts, the stabilizer is at a concentration of about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer is at a concentration of about 0.01% to about 0.5%, or about 0.01% to about 0.2%. In yet other embodiments, when the stabilizer is glycerin, the stabilizer is at a concentration of about 5% to about 100% (neat).

[0175] In further embodiments, the composition contains an antioxidant. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP is about 0.1:1 to about 15:1, or about 1:1 to about 15:1, or about 0.5:1 to about 10:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0176] Pharmaceutically acceptable salts can be used in the compositions, including, but not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate), and amine salts (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A complete discussion of pharma- ceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 18th Edition, pp. 111-115, 2002. th Edition, Mack Publishing Company, Easton, Pennsylvania (1990).

[0177] The pharmaceutical composition can be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage form of the composition can be adjusted to suit the desired mode of administration of the composition. For oral administration, the composition can take the form of, for example, a tablet or capsule (including soft gel capsules), or can be, for example, an aqueous or non-aqueous solution, a suspension, or a syrup. Tablets and capsules for oral administration can use one or more commonly used excipients, diluents, and carriers, such as mannitol, lactose, glucose, sucrose, starch, corn starch, sodium saccharin, talc, cellulose, magnesium carbonate, and lubricants (e.g., magnesium stearate, sodium stearyl fumarate). Flavorings, colorings, and / or sweeteners can be added to solid and liquid formulations as needed. Other optional components of oral formulations include, but are not limited to, preservatives, suspending agents, and thickening agents. Oral formulations can also be enteric coated to protect the CNP variant from the acidic environment of the stomach. Methods for preparing solid and liquid dosage forms are known, or will be apparent, to those skilled in the art (see, eg, Remington's Pharmaceutical Sciences, referenced above).

[0178] Preparations for parenteral administration can be prepared, for example, as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in liquid media prior to injection, or as emulsions. For example, sterile injectable solutions and suspensions can be formulated according to techniques known in the art using suitable diluents, carriers, solvents (e.g., aqueous buffer solutions, Ringer's solution, isotonic sodium chloride solution), dispersants, wetting agents, emulsifying agents, suspending agents, and the like. In addition, sterile fixed oils, fatty acid esters, polyols, and / or other inert ingredients can be used. By way of further example, preparations for parenteral administration include aqueous sterile injectable solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0179] Exemplary CNP formulations are described in US Patent Nos. 9,907,834 and 10,646,550. CNP formulations having a pH in the range of about 4 to about 6 are contemplated for use.

[0180] The composition comprising the CNP variant may also be a lyophilized formulation. In certain embodiments, the lyophilized formulation comprises a buffer and bulking agent, and optionally an antioxidant. Exemplary buffers include, but are not limited to, acetate buffer and citrate buffer. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, mannitol is in an amount of about 3% to about 10%, or about 4% to about 8%, or about 4% to about 6%. In certain embodiments, sucrose is in an amount of about 6% to about 20%, or about 6% to about 15%, or about 8% to about 12%. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid.

[0181] In various embodiments, the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).

[0182] The disclosure also provides kits that include, for example, bottles, vials, ampoules, tubes, cartridges, and / or syringes that contain liquid (e.g., sterile injectable) or solid (e.g., lyophilized) formulations. The kits can also include pharma- ceutically acceptable vehicles or carriers (e.g., solvents, solutions, and / or buffers) for reconstituting solid (e.g., lyophilized) formulations into solutions or suspensions for administration (e.g., by injection), including, but not limited to, reconstituting lyophilized formulations in syringes for injection or for diluting concentrates to lower concentrations. Additionally, extemporaneous injection solutions and suspensions can be prepared, for example, from sterile powders, granules, or tablets that contain the CNP-containing compositions. The kits can also include dispensing devices, such as aerosol or injection dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes, and / or needles.

[0183] As a non-limiting example, the kit can include a syringe with a single or dual chamber. In the case of a single chamber syringe, the single chamber can contain a liquid CNP formulation ready for injection, or a liquid formulation of a solid (e.g., lyophilized) CNP formulation or CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension for injection. In the case of a dual chamber syringe, one chamber can contain a pharma- ceutically acceptable vehicle or carrier (e.g., a solvent system, solution, or buffer) for injection, and the other chamber can contain a liquid formulation of a solid (e.g., lyophilized) CNP formulation or CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension using the vehicle or carrier from the first chamber.

[0184] As a further example, the kit can include one or more pen injectors or auto-injector devices and a dual-chamber cartridge. One chamber of the cartridge can contain a pharma- ceutically acceptable vehicle or carrier (e.g., a solvent system, solution, or buffer) for injection, and the other chamber can contain a solid (e.g., lyophilized) CNP formulation or liquid formulation of a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension using the vehicle or carrier from the first chamber. The cartridge can contain a sufficient amount of CNP variant to be dosed for a desired period of time (e.g., 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen injector or auto-injector can be adjusted to administer the desired amount of the CNP formulation from the cartridge.

[0185] In addition, pharmaceutical compositions containing CNP variants can be formulated as sustained-, controlled-, or sustained-release systems to maintain a relatively constant level of dosage over a desired period of time, such as 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. Sustained-, controlled-, and sustained-release formulations can be prepared, for example, using biodegradable polymeric systems {which can include, for example, hydrophilic polymers [e.g., polylactide, polyglycolide, poly(lactide-glycolide)]} and can take the form of, for example, microparticles, microspheres, or liposomes, as known in the art.

[0186] Administration and Dosing As used herein, the term "therapeutically effective amount" of an active agent (e.g., a CNP variant) refers to an amount that provides a therapeutic benefit to a patient. The amount may vary from individual to individual and may depend on many factors, including the overall physical condition of the patient. The therapeutically effective amount of a CNP variant can be readily ascertained by one of skill in the art using publicly available materials and procedures. For example, the amount of CNP variant used for treatment should provide an acceptable rate of reversal of cartilage degeneration or increase in cartilage growth.

[0187] The dosing frequency for a particular individual can vary depending on a variety of factors, including the disorder being treated and the individual's condition and response to therapy. In certain embodiments, a pharmaceutical composition containing a CNP variant is administered to a subject about once per day, once every two days, once every three days, or once per week, twice per week, three times per week, once every two weeks, or once per month.

[0188] The CNP variant compositions described herein can be administered to a patient in need thereof in a therapeutically effective dose to treat, ameliorate, or prevent bone-related disorders and short stature disorders (e.g., skeletal dysplasias, including achondroplasia, hypochondroplasia, and the like). The CNP variants contemplated for use herein can be administered to a patient in a therapeutically effective dose to treat, ameliorate, or prevent osteoarthritis and other conditions having osteoarthritis-related symptoms. The safety and therapeutic efficacy of the CNP variants can be determined by standard pharmacological procedures in cell cultures or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the ratio LD 50 / ED 50 Generally, active agents that exhibit large therapeutic indices are preferred.

[0189] In certain embodiments, the CNP variant compositions described herein are administered at a dose ranging from about 3, 4, 5, 6, 7, 8, 9, or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP composition is administered at a dose of about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, or 2000 nmol / kg, or other doses as deemed appropriate by the treating physician. In other embodiments, the CNP variant composition comprises 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, 7 The CNP or CNP variant may be administered at a dose of about 0.5, 0.8, 1.0, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / kg, or other doses deemed appropriate by the treating physician. Doses of CNP or CNP variants described herein may be administered according to dosing / administration frequencies described herein, including, without limitation, daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc. In various embodiments, the CNP or CNP variant is administered subcutaneously daily. In various embodiments, the CNP or CNP variant is administered subcutaneously weekly. In various embodiments, the CNP variant is administered at a dose of 2.5 μg / kg / day to 60 μg / kg / day, 10 μg / kg / day to 45 μg / kg / day, or 15 μg / kg / day to 30 μg / kg / day.In various embodiments, the CNP variant is administered at a dose of 15 μg / kg / day. In various embodiments, the CNP variant is administered at a dose of 30 μg / kg / day.

[0190] The frequency of dosing / administration of the CNP variant for a particular subject may vary depending on a variety of factors, including the disorder being treated and the subject's condition and response to therapy. The CNP variant can be administered in a single dose or multiple doses per administration. In certain embodiments, the CNP variant composition is administered in a single dose or multiple doses once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months, or as deemed appropriate by the treating physician. In various embodiments, the CNP variant is administered for 3 months, 6 months, 12 months, or more.

[0191] In some embodiments, the CNP variant composition is administered to allow for a period of growth (e.g., cartilage formation), followed by a period of recovery (e.g., bone formation). For example, the CNP composition is administered subcutaneously, or otherwise, daily or multiple times per week for a period of time, followed by a period of no treatment, and then the cycle is repeated. In some embodiments, the initial period of treatment (e.g., administration of the CNP variant composition daily or multiple times per week) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In related embodiments, the period of no treatment lasts 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the dosing regimen of the CNP variant composition is daily for 3 days followed by 3 days off, or daily or multiple times per week for 1 week followed by 3 days or 1 week off, or daily or multiple times per week for 2 weeks followed by 1 week 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.

[0192] CNP variants, or pharmaceutical compositions comprising them, can be administered to a subject in a variety of ways, such as, for example, subcutaneous, intra-articular, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. In one embodiment, the CNP variant is administered by a single subcutaneous, intra-articular, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection.

[0193] The CNP variant can be administered by implantation of a depot at the target site of action (e.g., an abnormal or degenerative joint or cartilage region). Alternatively, the CNP variant can be administered sublingually under the tongue (e.g., a sublingual tablet), or by inhalation to the lungs (e.g., an inhaler or aerosol spray), by delivery to the nasal cavity (e.g., an intranasal spray), by delivery to the eye (e.g., eye drops), or by transdermal delivery (e.g., by a patch on the skin). The CNP variant can also be administered orally in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamide, polylactide, polyglycolide, poly(lactide-glycolide)), microemulsions, and the like.

[0194] A further method of administration is by osmotic pump (e.g., Alzet pump) or minipump (e.g., Alzet miniosmotic pump), which allows for controlled, continuous and / or sustained release delivery of the CNP variant or pharmaceutical composition over a defined period of time. The osmotic pump or minipump can be implanted subcutaneously or near the target site (e.g., long bones of the limbs, epiphysis, etc.).

[0195] It will be apparent to one of skill in the art that the CNP variant or composition thereof can also be administered by other modes. Determining the most effective mode of administration of the CNP variant or composition thereof is within the skill of one of ordinary skill in the art.

[0196] The CNP variants can be administered, for example, as pharmaceutical formulations suitable for oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous, intraarticular and intravenous) administration, or in a form suitable for administration by inhalation or inhalation. Depending on the intended mode of administration, the pharmaceutical formulations can be in the form of solid, semi-solid or liquid dosage forms such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, emulsions, creams, ointments, lotions, and the like. The formulations can be provided in unit dosage forms suitable for single administration of precise dosage amounts. The formulations comprise an effective amount of the CNP variant, as well as one or more pharma- ceutically acceptable excipients, carriers, and / or diluents, and optionally one or more other biologically active agents.

[0197] Further aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. EXAMPLES

[0198] Example 1 - Measurement of NTproCNP We studied 1) whether daily administration of vosoritide depending on dose and duration inhibits endogenous CNP secretion during the phase of increased growth rate (indirect feedback) and 2) whether endogenous CNP is unaffected by vosoritide administered 4 h earlier (direct feedback).

[0199] Briefly, 35 children (ages 5-14 years) were enrolled in four consecutive cohorts across nine investigational sites in this dose-finding and follow-on study. See, for example, Savarirayan, et al. N Engl J Med 2019,381:25-35. Measurement of plasma NTproCNP was available only in 28 of these subjects (age range 5-11 years, 12 boys, 16 girls), so all data presented here apply only to this subgroup. After screening at baseline, four separate cohorts (balanced by gender but differentiated by dose and timing of dose escalation) received daily subcutaneous injections of vosoritide for up to 5.5 years. Cohort 1 (six subjects, age range 6-10 years at screening) received 2.5 μg / kg / day for up to 10 months, followed by 7.5 μg / kg / day for approximately 2 months, then 15 μg / kg / day until study completion. Cohort 2 (six subjects, age range 5-10 years) received 7.5 μg / kg / day for the first 6-8 months, then titrated to 15 μg / kg / day. Cohort 3 (eight subjects, age range 6-11 years) and cohort 4 (eight subjects, age range 5-8 years) received 15 μg / kg / day and 30 μg / kg / day, respectively, throughout the study. At the completion of NTproCNP sampling, the mean ages were 13.2 years in cohort 1, 13.7 years in cohort 2, 13.6 years in cohort 3, and 11.4 years in cohort 4. Signs of pubertal development were observed 2 years after treatment in all but three subjects in cohort 2, one subject in cohort 3, and two subjects in cohort 4.

[0200] Serial (pre-injection) sampling was performed throughout the study to evaluate possible correlations with annual growth velocity (AGV) and / or pubertal changes. EDTA-anticoagulated plasma was collected at screening (baseline) and then at 12 different time points over a 5-year period. Anticipating a link between endogenous CNP and the early phase of growth acceleration (Olney et al., Clin Endocrinol (Oxf) 2016, 85:561-568), sampling was performed more frequently in all cohorts during the first 2 months of treatment. Measurements were also performed at day 85 in cohort 1 and in three subjects in cohort 2, but not in the other cohorts, and therefore are not included in the analysis of serial changes in AGV. Sampling was performed less frequently after 1 year (biannually at years 1, 2, and until the final sampling at 5.5 years).

[0201] To examine possible acute effects of vosoritide on plasma NTproCNP, samples were collected 4 hours after injection in all subjects on nine separate occasions over the first two years of the study. In subjects in cohorts 1 and 2, samples were collected both 4 and 8 hours after the first injection. All other samples were collected 4 hours after each morning injection.

[0202] All plasma NTproCNP measurements were performed in duplicate by the Christchurch Laboratory assay using their previously determined age- and sex-adjusted normal ranges (standard deviation scores, SDS) for normal children (Olney et al. Clin Endocrinol (Oxf) 2012, 77:416-422). There was no detectable cross-reactivity of CNP (1-37pro gly) in the NTproCNP assay. The assay has a detection limit of 1.5 pmol / l, with intra- and inter-assay coefficients of variation of 6% and 7%, respectively, at 18 pmol / l. In a previous study (unpublished) of six healthy adolescents, there was no evidence of diurnal variation or the influence of food intake between 0900 and 1500 hours (mean coefficient of variation 6.4%), and no significant variation in samples taken at close intervals on consecutive days. Because both age and sex affect plasma concentrations of NTproCNP in normal children (Prickett, et al. Pediatr Res 2005 58:334-340), all measured NTproCNP concentrations were converted to SDS using data from a reference range previously determined from 258 normal healthy children aged 2 months to 20 years (Olney et al. Clin Endocrinol (Oxf) 2012;77:416-422). Because SDS of plasma NTproCNP is not available for untreated Ach, and because the tempo of height velocity and pubertal timing in untreated Ach appear to be different from those of normal non-Ach children, the above approach was deemed to be the optimal comparator to use in these samples extracted from children of different ages and sexes (Merker et al. Am J Med Genet A 2018;176:1723-1734).

[0203] Baseline NTproCNP is elevated in subjects with achondroplasia compared to the general population. Baseline plasma NTproCNP values ​​at screening were elevated despite significantly lower AGV (mean 3.9 ± 0.3 cm / y) compared to the general population children in this age group, as indicated by AGV SDS (mean SDS 0.66 ± 0.17, P < 0.001) (Kelly et al. J Clin Endocrinol Metab 2014,99:2104-2112). Relevant baseline data for the four cohorts relating to age at screening, plasma NTproCNP, AGV and AGV SDS are shown in Table 1, along with the increase in AGV after 6 months of treatment. NTproCNP SDS was lower in cohort 1 and age was lower in cohort 4. Cohorts 3 and 4, which received high doses of vosoritide (15 μg / kg / day and 30 μg / kg / day, respectively), showed significant and similar increases in AGV assessed at 6 months (P<0.05 for both). [Table 1]

[0204] The sudden changes in growth velocity are associated with the suppression of NTproCNP. The dynamic changes in NTproCNP and SDS associated with the growth-promoting action of vosoritide are shown for each cohort in Figure 1. The individual changes per cohort are shown in Figure 2. The sharp inflection of AGV in cohorts 3 and 4 over the first 3 months was associated with a significant decline in NTproCNP SDS at 1 month (P = 0.04 and 0.004, cohorts 3 and 4, respectively). At time points after the first year of treatment in these two groups, when AGV was stable, the mean NTproCNP SDS was more variable but tended to be lower than baseline (Figure 1). In cohorts 1 and 2, when the dose was titrated to 15 μg / kg / day after 1 year, the increase in AGV was associated with a decline in NTproCNP (Figure 1), although the lack of frequent sampling early in the course of dose titration prevents a more detailed analysis of the time changes.

[0205] Abnormal increases in NTproCNP. Examination of Figure 1 shows relatively unchanged AGV from years 2 to 4 associated with unchanged NTproCNP, except for that in cohort 4. In cohorts 2 and 3 (both receiving a dose of 15 μg / kg), mean NTproCNP SDS was lower than at screening at all time points. In cohorts 1 and 4, some abnormal and unexplained elevations were observed, but all these were not associated with changes in AGV. After 3 to 4 years of treatment with 30 μg / kg / day (cohort 4), a notable increase in plasma levels was observed in three of the eight children (Figure 2). The increases in SDS in these children (3.1, 6.7; 1.4, 3.0; and 0.3, 1.9-pre and peak, respectively) persisted for at least 1 year and were not consistently associated with changes in either BALP or PINP (Figure 3). In two subjects, Tanner stage 2 pubertal development was documented, but in the subject with the highest NTproCNP (138 pmol / L, SDS 6.7), puberty did not develop until breast budding was documented 6 months after the last measurement. In other subjects, occasional spikes in plasma NTproCNP were observed, some of which were consistent with pubertal staging (Figure 2). In contrast, in one girl (cohort 1) aged 6.5 years, plasma levels suddenly increased approximately 6 months after starting the 2.5 μg / kg dose (Figure 2). SDS (0.16 at screening) increased to 2.6 at 6 months and remained elevated for the remaining 4.5 years, but was not affected by normal pubertal progression at 10.5 years of age. No consistent associations with BALP, PINP, or AGV were evident.

[0206] Sustained growth under exogenous CNP effects was associated with sustained suppression of NTproCNP. To evaluate the effect of age or duration of treatment over time on plasma NTproCNP, we compared SDS at year 1 with SDS at completion of the study. Values ​​were combined because cohorts 1-3 all received a dose of 15 μg / kg / day after 1 year. As shown in Table 2, during periods of relatively unchanged AGV, a significant decline in NTproCNP SDS was observed over time (F=14, p=0.002,) (Figure 3). [Table 2]

[0207] Acute exposure to exogenous CNP analogues inhibits the production of NTproCNP. The impact of the first injection of vosoritide was studied only in cohorts 1 and 2. In cohort 1, no change in NTproCNP 4 hours after injection was observed after 2.5 μg / kg / day, but not in any of the six studies performed in the subsequent 10 months of treatment. In cohort 2, a significant decline occurred from the initial 7.5 μg / k dose (43.3 ± 3.5 pre-injection, 35 ± 2.7 pmol / L post-injection, P = 0.013, n = 8), returning to pre-injection levels (44.3 ± 4.7 pmol / L) 8 hours after injection. In these same subjects, a significant suppression was observed 1 month after the start of treatment, at a time when AGV was unchanged (P = 0.02) (Table 1). Across all subjects, results from paired samples were available at up to eight different time points during the first 24 months of vosoritide treatment. Because there were no differences in response according to dose (F=0.8, P=0.5), results from any given time point were combined for statistical analysis. [Table 3]

[0208] As shown in Table 3, when the grouped data were analyzed, no significant changes were observed at 4 hours within the first 6 months of treatment (i.e., days 29, 43, and 127). However, at each subsequent time point, significant declines in plasma NTproCNP were observed at day 183 (P=0.003, n=30), month 12 (P=0.006, n=22), and month 24 (P=0.015, n=17) (see Table 3). When the association with concurrent NTproCNP SDS at the time of injection was examined, an association was confirmed between the fall in plasma NTproCNP(delta) and pre-injection plasma NTproCNP SDS at screening (r=-0.71, P<0.001), day 29 (r=-0.45, P=0.012), day 127 (r=-0.42, P=0.02), day 183 (r=-0.59, P<0.001, see Figure 4), and at 24 months (r=-0.36, P=0.10), regardless of the significance of the decrease at 4 hours. A higher pre-test SDS is strongly associated with a fall in plasma NTproCNP at 4 hours. Together, these results support a direct inhibitory effect of exogenous CNP on CNP production that is not observed during the first 6 months when skeletal growth accelerates.

[0209] Furthermore, the data suggest that NTproCNP SDS is significantly increased at baseline. Second, the significant decline in plasma NTproCNP during the initial acceleration of AGV with a growth-promoting dose of vosoritide provides evidence for a feedback regulation that is likely indirect. Third, the decrease in plasma NTproCNP 4 hours after vosoritide injection in proportion to plasma NTproCNP SDS immediately prior to injection is consistent with a direct feedback effect. These and other findings of a marked increase in NTproCNP in early adolescence in some subjects who received the higher dose are important new observations that require more detailed study.

[0210] Studies from rodent puppies, growing lambs, and kids support the view that circulating levels of CNP products (CNP and NTproCNP) in plasma are sourced primarily from the growth plate or closely related tissues (Espiner 2018, supra). Therefore, measurements of the possible effects of administered CNP1-37ProGly (which strongly cross-reacts with CNP but not with the NTproCNP assay) on endogenous proCNP production are only feasible using plasma concentrations of NTproCNP adjusted for age and sex (SDS). As previously reported (Olney et al., J Clin Endocrinol Metab 100:E355-359, (2015) we confirmed that levels in healthy children with Ach were significantly increased before treatment and remained so until the end of the study. Notably, at the doses that caused a rapid increase in AGV within 2-3 months (15 and 30 μg / kg / day in cohorts 3 and 4), NTproCNP SDS was significantly decreased, coinciding with the first signs of an increase in serum collagen X marker, a breakdown product of type X collagen. These changes were not observed in subjects who received lower doses (2.5 μg / kg / day and 7.5 μg / kg / day), which did not affect AGV at this time point. It is instructive to compare these dynamic changes in CNP with those seen in short non-Ach children of a similar age who start a daily dose of human growth hormone (HGH) (Olney, et al. Clin Endocrinol (Oxf) 85:561-568, 2016). In that setting, a similar refraction in AGV was associated with an increase in NTproCNP (mean 11 pmol / L, delta 22% on day 21) in contrast to a concomitant decline in NTproCNP on day 29 during 15-30 μg / kg / day vosoritide treatment in cohorts 3 and 4 (~6 pmol / L, range 3-11 pmol / L, delta -12%). The different responses are not surprising, since growth plate concentrations of CNP products are clearly increased by growth hormone but not by exogenous CNP.These kinetic responses may be related to the estimated time (approximately 20-22 days) (Sansone et al., J Pediatr Orthop 29:61-67, 2009) for recruited chondrocytes to traverse their respective regions of the proliferating growth plate and populate the primary corpus cavernosum. However, unlike the sustained elevation of NTproCNP above baseline in the first year of HGH treatment (Olney, 2016, supra), the initial marked decline in concentrations during vosoritide treatment was less sustained and may reflect the much shorter half-life of vosoritide (28 min) compared to the much longer duration of HGH activity (up to 12 h) (Olney, 2016, supra).

[0211] Taken together, the current findings are consistent with an indirect feedback mechanism in which factors (or bone-like tissue) generated by accelerated growth plate activity reduce local NPPC expression or secretion of proCNP into the extracellular fluid, consistent with the indirect negative feedback from exogenous CNP observed in 4-week-old rodent pups.10 However, in that study, high-dose continuous intravenous infusion of CNP53 for 3 days, while significantly reducing lumbar NPPC expression, did not reduce plasma NTproCNP in male rats (n=6) and was associated with a slight reduction in females (n=6). As no skeletal indicators of growth plate activity in this brief exposure to CNP53 have been reported, the possible association of accelerated endochondral bone growth with reduced plasma NTproCNP in this setting remains to be investigated. Further studies of larger subject groups and more appropriately timed sampling points, especially within the first 3 months of initiating exogenous CNP therapy, are expected to advance our understanding of these dynamic changes associated with altered bone growth in children and may provide clinical applications. For example, targeting NTproCNP decline at 1 month, or NTproCNP SDS zero at Ach, can be used to predict optimal effect size, duration of effect on growth plate activity, and selection of injection dose and frequency. Optimal effect size refers to a measure of the average normal growth rate expected based on population norms.

[0212] Surprisingly, multiple studies conducted at different times over the first 2 years of treatment revealed strong evidence of acute feedback, but only after 6 months of exogenous treatment. This time constraint may be related to the early growth spurt observed in many of the children tested. Patients in cohorts 3 and 4 showed early rapid refractions with AGV-reducing plasma NTproCNP drawn 24 hours after injection during this period. This may limit any decline at 4 hours from injection administration. Although neither group was tested before 1 month, the inhibitory effect from either the 15 μg or 30 μg doses was not seen at days 29, 43, 127, or 183 (when AVG was accelerating), but was clearly observed at later time points. AGV during the first 6 months was not significantly affected in either cohort 1 or cohort 2. In the former, the first dose was insufficient to affect bioactivity (urinary cGMP) on day 1 (Savarirayan, et al., N Engl J Med 381:25-35 2019) or consistently increase plasma CNP39 within 2 hours (Yasoda, 2004, supra), without affecting AGV. Thus, the lack of suppression at 4 hours is not surprising in any of the eight studies performed. On the other hand, after 7.5 μg / kg / day (cohort 2), a significant increase in urinary cGMP and peak CNP39 was observed on day 1, and a significant suppression of NTproCNP at 4 hours was observed - also on day 29 in this cohort. Unfortunately, during the period of dose escalation in these two cohorts, sampling frequency was insufficient to assess the effect of accelerated AGV on NTproCNP, or the effect this may have on the response at 4 hours. Although other factors such as increased body weight, pubertal age, dose, and bioavailability (much greater in cohorts 3 and 4) (Savarirayan, supra), as well as the small number of subjects studied, must be taken into account, the results suggest that an interaction between the indirect and direct feedback systems may explain the reduced effect of vosoritide at 4 h during the first 6 months.Combining all groups, highly significant reductions in NTproCNP were found at 4 hours post-dose on days 183, 365 and 730 (Table 3). Notably, assessing response across all trials according to NTproCNP SDS shows that acute falls (effect size) are strongly dependent on the SDS at dosing. This finding suggests that by restoring intracellular CNP activity via a functional receptor (NPR2), CNP production is reduced in proportion to the general tolerance level. Possibly, increasing the expression of the clearance receptor NPR3, reducing CNP and increasing NTproCNP (the antithesis of loss of function in NPR3) (Boudin et al., Am J Hum Genet 103:288-295, 2018) could explain the observed findings. However, both CNP and NTproCNP are similarly increased in untreated children with Ach (Olney, supra), and the ratio of NTproCNP / CNP is normal, making upregulation of NPR3 unlikely. Of note, no direct feedback was seen in wild-type rodent pups (Ueda, supra), raising the possibility that the findings of inhibition (not sex-dependent) are specific to Ach, where circulating CNP products are elevated above normal. In a clinical (safety) study (BMN111-101) conducted in optimally healthy normal adult males, no significant changes in plasma NTproCNP from baseline were observed 4 hours after dosing in the range of 2.5-15 μg / kg / day. This suggests that direct inhibition characterizes the immature skeleton, but whether it is limited to Ach, as well as Ach, where SDS is close to zero, such as in children without genetic disorders, requires further testing. Despite these findings, given the very high concentrations associated with significant inhibition (peak >350 pmol / L) and levels seen in pathophysiology (2-8 pmol / L), it is unlikely that the observed direct feedback contributes to CNP regulation in vivo (Olney et al., J Clin Endocrinol Metab 100:E355-359, 2015).

[0213] Example 2 - Measurement of CXM and other biomarkers Vosoritide acts on growth plate chondrocytes via the B-type natriuretic peptide receptor to stimulate increased endochondral bone growth, leading to increased growth rate in treated subjects. In clinical trials, subjects' blood and urine samples were analyzed to monitor putative bone growth biomarkers, including cross-linked C-terminal telopeptide of collagen II (CTxII), bone-specific alkaline phosphatase (BSAP), N-terminal propeptide of collagen I (PINP), and N-terminal fragment of collagen X (CXM). Changes in the biomarkers over time were analyzed in relation to observed changes in growth rate in subjects receiving vosoritide.

[0214] Collagen type X biomarker (CXM; Coghlan 2017) is a degradation fragment of collagen type X released by active growth plates. A relative quantitative biomarker ECLA was developed and validated at BioMarin to measure CXM. 96-well Meso Scale Discovery (MSD) streptavidin plates were blocked with StartingBlock PBS with Tween®-20 (ThermoFisher Scientific, Waltham, MA, USA). After decanting the blocking buffer, biotinylated anti-human collagen NC1 domain capture SOMAmer was incubated on the plate. Standard stocks (recombinant human collagen type X NC1 domain in assay diluent [AD]) were serially diluted in AD, while serum quality control samples (QCs) and serum test samples were diluted 1:100 in AD. After washing the assay plates, diluted calibrators and samples were incubated on the plates. After a second wash, a ruthenium-labeled mouse monoclonal anti-collagen type X NC1 domain IgG detection antibody was incubated on the plate. The plate was then washed, MSD Read Buffer T with detergent was added, and the plate was read on the MSD Quickplex instrument. The raw signal from each well was proportional to the collagen type X concentration in each sample. The concentration of collagen type X in each unknown sample was determined by interpolating the raw assay signal using the standard calibrator curve. Standard regression performed by Watson LIMS was calculated as 1 / Y 2 A four-parameter logistic (4-PL) Marquardt model was used with weighting coefficients of 0.01 pg / mL CXM in human serum.

[0215] An enzyme immunoassay for measuring bone-specific alkaline phosphatase in human serum was validated at ICON Labs (Farmingdale, NY, USA; validation N08-024VR-1,4). The assay used monoclonal anti-BAP antibody-coated wells to capture BAP in samples. Enzyme activity of captured BAP was detected using p-nitrophenyl phosphate substrate. Raw assay signal was read using a SpectraMax spectrophotometer (Molecular Devices, San Jose, CA, USA). The concentration of BAP in each sample was determined by interpolation using a standard calibrator curve with a linear curve fit. The assay limit of quantification was 2 U / L neat.

[0216] A quantitative competitive format radioimmunoassay (RIA) method based on the UniQ PINP RIA assay kit (Orion Diagnostica, Espoo, Finland) was validated by ICON Labs (Farmingdale, NY, USA; validation N06-016VR). 125 I-labeled PINP and an unknown amount of unlabeled PINP competed for a limited number of high affinity binding sites on a polyclonal rabbit anti-PINP IgG antibody. Antibody-bound PINP was separated from matrix components using a secondary anti-rabbit IgG antibody coated on solid kaolin particles. Bound 125 The radioactivity of I-PINP was measured using a WIZARD automated gamma counter (Perkin Elmer, Waltham, MA, USA). The amount of radioactivity in each tube was inversely proportional to the concentration of PINP in each sample. The concentration of PINP in each sample was determined by interpolation using the standard calibrator curve and linear regression curve fitting. The lower limit of quantification was 5 μg / PINP in neat human serum.

[0217] A quantitative competitive format ELISA for the measurement of CTXII in human urine using the CartiLaps ELISA kit from ImmunoDiagnostic Systems (East Boldon, UK) was validated at ICON Labs to support test 111-202 / 205 (validation N06-114VR). The assay was based on competitive binding of mouse monoclonal anti-CTXII antibodies to urinary fragments of collagen type II or biotinylated synthetic peptides bound to the surface of a streptavidin-coated microtiter plate. First, biotinylated synthetic peptides were bound to the surface of the streptavidin-coated wells of the microtiter plate. After washing, standards, controls, and urine samples containing unlabeled CTXII were pipetted into the wells, followed by the addition of a solution of mouse monoclonal anti-CTXII IgG. The wells were washed and a solution of peroxidase-conjugated rabbit anti-mouse IgG was added to the wells. After a second washing step, tetramethylbenzidine (TMB) chromogenic substrate was added to all wells. The yellow color development was stopped with sulfuric acid, and the absorbance at 450 nm was read on a SpectraMax Plus spectrophotometer (Molecular Devices, San Jose, CA, USA). The raw signal in each well was inversely correlated with the concentration of CTXII in each sample. The concentration of CTXII in each sample was determined by interpolation using standard calibrator curves and linear curve fitting. The lower limit of quantification was 0.60 ng / mL CTXII in neat urine.

[0218] In studies 111-202, pediatric subjects with achondroplasia aged 5-15 years were administered vosoritide at 2.5 μg / kg / day, 7.5 μg / kg / day, 15 μg / kg / kg / day, or 30 μg / kg / kg / day (cohorts 1, 2, 3, and 4, respectively) for the first 6 months. After 6 months, a significant increase in annualized growth velocity (AGV) was observed in subjects receiving vosoritide at 15 μg / kg / day or 30 μg / kg / day, but not for subjects receiving 2.5 μg / kg / day or 7.5 μg / kg / day (Figure 5). After 6 months, the dose concentration was increased to 15 μg / kg / day for subjects in cohorts 1 and 2, resulting in an increase in AGV.

[0219] All biomarkers analyzed in 111-202 were variable, but CXM showed a dose-dependent increase, BSAP increased slightly over time during treatment at all dose levels, and there were no clear trends or dose-dependent responses for CTxII or P1NP. Based on these results, BSAP and CXM were incorporated into the placebo-controlled Phase III vosoritide clinical trial 111-301 and the pretreatment natural history study 111-901.

[0220] Prior to entry into Study 111-301, pediatric subjects with achondroplasia aged 5-15 years were monitored without treatment for at least 6 months and up to 15 months prior to treatment initiation in Study 111-901. AGV, CXM, and BSAP were measured during the natural history study 111-901 and during the placebo-controlled, double-blind, Phase 3 study 111-301. AGV was relatively stable prior to treatment initiation and in subjects who received placebo. In contrast, AGV increased dramatically over 3 months in subjects who received vosoritide (Figure 6). Similarly, CXM levels increased dramatically in treated subjects in Study 111-301, but not in subjects who received placebo. Serum BSAP levels increased in treated subjects, but to a lesser extent in subjects who received placebo.

[0221] The data suggest that CXM is superior to CTxII, PINP, and BSAP for monitoring changes in endochondral bone growth. Data from the Phase 3 study clearly demonstrated an increase in serum CXM levels associated with an increase in AGV in subjects treated with vosoritide but not placebo. There appeared to be some increase in BSAP in vosoritide-treated subjects over that observed in placebo-treated subjects, but this increase was relatively small compared to that observed in CXM. The study data indicated that serum CXM is a useful growth plate biomarker associated with changes in AGV.

[0222] Example 3 - High-throughput characterization of NPR2 variants associated with genetic causes of short stature High-throughput characterization of NPR2 variants allows those skilled in the art to better predict novel variants and, for more commonly occurring variants, can improve diagnosis and clinical trial enrollment of eligible patients. The methods herein predict benign vs. pathogenic classification of short stature genetic variants, and it is hypothesized that NPR2 variant activity predicts total body length. Achondroplasia is defined as height <2SD from the mean.

[0223] Described herein is an in vitro assay (cGMP) to assess the activity of over 260 NPR2 variants. NPR2 protein alteration variants were identified in a UK Biobank study and described in Estrada et al. (Nat Commun. 2021 12(1):2224). NPR2 expression constructs were transfected into HEK293 cells, and after 3 days, cells were treated with 0.4 nM IBMX and 20 nM CNP in serum-free DMEM before measuring cGMP levels. cGMP was measured using a cGMP catchpoint ELISA competition assay (Molecular Devices) according to the manufacturer's recommendations. RedLuc was used as a transfection control (Figure 8A). Figure 8B shows the normalized cGMP values ​​of the various LoF and GoF variants.

[0224] Figure 9A shows a breakdown of variant activity levels based on predicted protein outcomes. Protein truncation variants (stop-gains and frameshifts) have activity levels close to zero, while synonymous mutations have activity levels close to wild type. Missense and in-frame deletions span a wide range of activity levels. Figure 9B shows a breakdown of predicted outcomes for missense variants based on the Combined Annotation Dependent Depletion (CADD) score (Kircher et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014, 46(3):310-5), which incorporates evolutionary conservation and over 60 other annotations. Figure 9C is a comparison of the measured functional activity of NPR2 variants and their average effect on the height of individuals carrying them. These results show that NPR2 variant activity data predicts height effect size.

[0225] Polygenic risk scores (PRS) can be used with phenotypic data to identify patient populations. Idiopathic short stature (ISS) can be predicted based on genetics. Figure 10A shows the probability of ISS based on a polygenic score for height alone. Polygenic scores summarize the combined effect of thousands of common variants with small effects on height. These scores capture 43% of the population variation in adult human height, but have limited predictive power at the extreme ends of the distribution. Figure 10B shows how the predictive power of the polygenic score changes with respect to NPR2 loss-of-function variants. Individuals with NPR2 LoF variants and polygenic scores in the bottom 12.5% ​​have a nearly 100% chance of ISS as adults. Approximately 1 in 800 people have a NPR2 variant that has been characterized and confirmed to have low activity levels, so at least 1 in 6,600 people can be predicted to have idiopathic short stature based on genetics alone. Similar procedures could be applied to other genes with large effects on height (ACAN, SHOX, DTL, PAPPA, etc.).

[0226] Mapping selected variants to the 3D structure will reveal potential mechanisms of disruption. Approximately 160 NPR2 gene variants have been recently phenotypically characterized using the previously described cGMP quantification "catchpoint" assay (Estrada et al., supra). 3D modeling of high-confidence loss-of-function (LoF) and gain-of-function (GoF) variants may provide mechanistic insight into how specific amino acids are altered and how the variants may regulate the activity of NPR2. The goal was to map approximately 35 high-confidence (LoF and GoF) NPR2 variants to known functional domains within NPR2 (2D mapping) and then map these variants to the alpha-fold 3D structure (3D mapping).

[0227] The NPR2 alpha-fold structure was obtained from the recent alpha-fold publication (Jumper et al., Nature 596:583-589 (2021)). A list of phenotypically characterized "high confidence" variants was generated (Table 4). Phenotypes are calculated by determining the levels of cGMP using a standard curve. This value is normalized to RedLuciferase (transfection control). This value is further normalized by setting WT to 1. Mean values ​​are calculated from at least three replicate experiments with four replicates each. [Table 4-1] [Table 4-2]

[0228] The variants were separated based on their localization to different protein domains (ECD (extracellular domain): ligand (CNP) binding, KHD (kinase homology domain) (binds ATP, a negative regulator of GC function), or GCD (guanylyl cyclase domain: produces cGMP). We mapped these variants onto the alphafold 3D structure (Figure 11A) and provided conclusions regarding possible mechanisms by which some variants may alter NPR2 activity (Figures 11C-11D). For example, many GoF mutations appear to be in the KHD region.

[0229] The data herein suggest that the presence of NPR LoF variants and polygenic risk scores (PRS) in the bottom 12.5% ​​can accurately predict adult idiopathic short stature. Our 3D modeling of high-confidence LoF and GoF variants provides mechanistic insight into these variant-specific effects of NPR2 activity.

[0230] Example 4 - Skull and brain morphology as a measure of efficacy of CNP therapy Additional markers of the effectiveness of CNP therapy include increases in skull and brain morphology such as facial volume, sinus volume, and foramen magnum area.

[0231] In an ongoing Phase II study of CNP therapy in children, a group of patients aged 3-6 months (Cohort 3) were administered CNP (BMN111) subcutaneously at a dose of 30 μg / kg daily and annual growth mass, change in height, and change in skull morphology were measured. See also PCT Application No. PCT / US22 / 73605.

[0232] Pharmacokinetic studies showed that participants in cohorts 2 and 3 who received 30 μg of vosoritide per kg had higher mean exposure than participants in cohort 1 who received 15 μg of vosoritide per kg. [Table 5]

[0233] Magnetic resonance imaging (MRI) was used to confirm possible therapeutic effects of vosoritide on brain and skull morphology, including dimensions of the foramen magnum, ventricles, and brain parenchyma. It was also used to confirm the eligibility of each patient entering the study based on exclusion criteria indicating the presence of clinically significant corticomedullary or spinal cord injury (based on evidence of cervical spinal cord stenosis based on brain MRI obtained during the screening period, or the presence of lesions or anatomical abnormalities). Scan parameters were standardized across all clinical sites and are detailed in Table 6 below. [Table 6]

[0234] T1-weighted and T2-weighted MRI examinations (1.2 mm slice thickness, from skull vertex to C2) were performed on study subjects under anesthesia using standardized acquisition techniques at baseline (screening period days −30 to 1) and after 52 weeks of treatment (+ / − days 7) or at early termination visits to ensure consistency over time and between sites.

[0235] In the youngest cohort (cohort 3, children aged 3 to 6 months), participants treated with vosoritide showed greater increases in facial volume, facial sinus volume, and foramen magnum area at 52 weeks, as assessed by MRI.

[0236] The MRI changes observed in participants in cohort 3 (3-6 months) are noteworthy because abnormal craniofacial and skull base endochondral ossification leading to midfacial hypoplasia and foramen magnum stenosis, by causing sleep-disordered breathing (observed in one participant in this study) and brainstem compression (Hoover-Fong et al.), are thought to be a major contributing factor to the reported excess of sudden death in children under 5 years of age with achondroplasia (Hecht et al., Am J Med Genet 1985;20:355-60; Hoover-Fong et al., Bone 2021;146:115-872). Given that vosoritide improves craniofacial skeletal and foramen magnum abnormalities in mouse models of achondroplasia (Lorget et al., Am J Hum Genet 2012;91:1108-14) and accelerates endochondral ossification in children with achondroplasia (Savarirayan et al., The Lancet 2020;396:684-92; Savarirayan et al., N Engl J Med 2019;381:25-35), it is plausible that these MRI changes reflect a direct effect of vosoritide on craniofacial and foramen magnum growth. Whether these observed MRI changes translate into reduced incidence of sudden infant death, sleep-disordered breathing, and the need for neurosurgical decompression of the foramen magnum in these infants will be evaluated during long-term follow-up (Study 208, ClinicalTrials.gov number, NCT03989947).

[0237] Changes in skull / brain morphology may be a better measure of efficacy in younger patients, as the treatment effect of vosoritide on annual growth velocity in this group was not as high as in children aged 5 years or older. Explanations for this discrepancy include the high variability and rapid decline in growth velocity in very young children with achondroplasia, and the practical challenges in consistently and accurately measuring body length in these infants. The treatment effect on growth velocity observed in the youngest participants from cohort 3 reflected this, with measurements showing wide variability and large confidence intervals.

[0238] Another ongoing trial comparing current standard of care versus CNP variant (e.g., vosoritide) treatment in infants under 1 year of age with achondroplasia at risk for requiring surgical decompression of the foramen magnum also directly addresses this issue (ClinicalTrials.gov number, NCT04554940) (Savarirayan et al., Sci Prog 2021;104:368504211003782). These MRI changes were observed only in participants from cohort 3, consistent with the fact that growth of the foramen magnum, especially in the transverse plane, is negligible after 6 months of age (Hecht et al., Am J Med Genet 1989;32:528-35).

[0239] Subcutaneous administration of vosoritide once daily in children aged 3 to 60 months was associated with an overall adverse event profile that appeared to be generally mild and resulted in an increase in height Z score. Treatment in children aged 3 to 6 months resulted in an increase in facial and sinus volumes, as well as an increase in the area of ​​the foramen magnum.

[0240] It is understood that all embodiments of the present disclosure described herein may be optionally combined with any one or more of the other embodiments described herein. All patent and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0241] It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications that are within the spirit and scope of the invention as defined by the appended claims, the above description, and / or the accompanying drawings. Accordingly, only such limitations as appear in the claims should be placed on the invention.

Claims

1. 1. A composition for use in a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature, comprising C-type natriuretic peptide (CNP), said method comprising: i) administering said composition to said subject; ii) obtaining a sample from said subject; iii) measuring the level of NTproCNP and / or N-terminal fragment of collagen X (CXM) in the sample obtained from the subject in (ii); iv) modifying or varying the dose of CNP therapy in said composition to bring NTproCNP levels within + / - 2 SDS of the mean NTproCNP in the population.

2. The composition described in claim 1, wherein the dose level of CNP therapy in the composition or the frequency of administration of the composition increases when the level of NTproCNP increases, or the dose level of CNP therapy in the composition decreases when the level of NTproCNP decreases.

3. 1. A composition for use in a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature, comprising C-type natriuretic peptide (CNP), said method comprising: i) administering said composition to said subject; ii) obtaining a sample from said subject; iii) measuring the level of N-terminal fragment of collagen X (CXM) in the sample obtained from the subject in (ii); iv) increasing the dosage level of CNP therapy in the composition or the frequency of administration of the composition if the level of collagen X decreases.

4. A composition described in any one of claims 1 to 3, wherein increasing the CNP therapeutic dose in the composition increases the average growth velocity (AGV) in the subject.

5. 5. The composition of claim 4, wherein the average growth velocity (AGV) in the subject is increased over six months, one year, or two years or more.

6. A composition described in any one of claims 1 to 3, wherein increasing the CNP therapeutic dose in the composition comprises increasing the administration frequency and / or increasing the dosage of the composition.

7. A composition described in any one of claims 1 to 3, wherein an increase in CNP therapeutic dose level and a decrease in NTproCNP level in the composition correlates with an improvement in annual growth velocity (AGV) in the subject.

8. A composition described in any one of claims 1 to 3, wherein increasing the CNP therapeutic dose level and decreasing the NTproCNP level in the composition extends the duration of growth plate activity in the subject.

9. The composition of any one of claims 1 to 3, wherein the level of NTproCNP is maintained between + / - 2 SDS of the mean NTproCNP level of the population.

10. 4. The composition of any one of claims 1 to 3, wherein the composition is titrated toward zero NTproCNP SDS if the NTproCNP SDS is below the average.

11. The composition of claim 10 , wherein the zero NTproCNP SDS predicts an optimal effect size.

12. The composition of any one of claims 1 to 3, wherein the sample is blood, urine, plasma, saliva, or tissue.

13. The subject has achondroplasia, osteoarthropathy, hypophosphatemic rickets, hypochondroplasia, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chiropteran dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, rhizometaphyseal chondrodysplasia punctata, Jansen's metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia congenita), atelosteogenesis, torsion dysplasia, congenital short femur, Langer's mesomelic dysplasia, Nievergelt mesolimb dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis 4. The composition of claim 1, wherein the patient is suffering from a bone-related disorder, skeletal dysplasia or short stature selected from the group consisting of disorders associated with: dysplasia, Kniest's dysplasia, fibrochondrogenesis, Roberts' syndrome, acromembranous short limb dysplasia, micromelia, Morquio syndrome, Kniest's syndrome, complex metatrophic dysplasia, spondyloepimetaphyseal dysplasia, NPR2 mutations, SHOX mutations (Turner syndrome / Leri-Weill), PTPN11 mutations (Noonan syndrome), IGF1R mutations.

14. The CNP therapy is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37) (SEQ ID NO: 1), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37) (SEQ ID NO: 2), GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP53) (SEQ ID NO: 3), PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP53) (SEQ ID NO: 4), MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP53) (SEQ ID NO: 5), DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53 (M48N)] (SEQ ID NO: 6), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 7), RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 8), VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 9), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-49) (SEQ ID NO: 10), TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 12), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-46) (SEQ ID NO: 13), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 14), AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 15), AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 16), WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 17), ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 18), RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 19), LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 20), LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 22), EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 23), HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 24), PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 25), NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 26), ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 27), RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 28), KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 29), YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 30), KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 31), GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 32), ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 33), NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 34), KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 35), KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 36), LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 37), SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 38), KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 39), GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 40), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37 (M32N)] (SEQ ID NO: 41), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37) (SEQ ID NO: 42), MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37) (SEQ ID NO: 43), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N)] (SEQ ID NO: 44), MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37) (SEQ ID NO: 45), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), and The composition of any one of claims 1 to 3, wherein the CNP variant is selected from the group consisting of PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49).

15. the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP37) (SEQ ID NO: 1); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37) (SEQ ID NO: 2), or The composition of claim 14, wherein the amino acid sequence is LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21).

16. The composition of any one of claims 1 to 3, wherein the level of NTproCNP or CXM is measured in a plasma sample.

17. The composition according to any one of claims 1 to 3, wherein the subject is administered the composition at a CNP regimen of 7.5 μg / kg to 30 μg / kg.

18. A composition described in any one of claims 1 to 3, wherein the dose of the CNP therapy in the composition is increased to 30 μg / kg.

19. The composition according to any one of claims 1 to 3, wherein the NTproCNP and / or CXM are measured at least 4 hours after administration.

20. The composition according to any one of claims 1 to 3, wherein the levels of NTproCNP and / or CXM are measured at least 6 months after the start of administration of the composition.

21. The composition of any one of claims 1 to 3, wherein the level of NTproCNP in the sample is compared to a baseline measurement taken before the start of administration of the composition.

22. A composition described in any one of claims 1 to 3, wherein the dose of CNP therapy in the composition or the frequency of administration of the composition is increased if a decrease in NTproCNP indicates an increase in AGV in the subject.

23. The composition of any one of claims 1 to 3, wherein the level of CXM in the sample is compared to a baseline measurement taken before the start of administration of the composition.

24. The composition of any one of claims 1 to 3, wherein an increase in CXM indicates increased bone growth, and the frequency of administration of CNP therapy or the dose level of CNP therapy in the composition is increased if there is an increase in CXM that enhances AGV.

25. The composition according to any one of claims 1 to 3, wherein the subject is a pediatric subject with open growth plates, and the composition is administered at a CNP therapy dose of 15 or 30 μg / kg per day.

26. 1. A method for obtaining NTproCNP levels as an indicator for selecting initiation of CNP therapy in a subject, the method comprising: i) measuring NTproCNP in said subject at multiple time points to establish a baseline NTproCNP level; ii) determining whether the NTproCNP level exhibits an SDS of zero, less than zero, or greater than zero; wherein the subject having an NTproCNP level of + / - 2 SDS indicates that treatment with CNP therapy should be initiated.

27. 1. A method for obtaining NTproCNP levels as an indicator for selecting initiation of CNP therapy in a subject with achondroplasia, comprising: i) measuring NTproCNP in said subject at multiple time points to establish a baseline NTproCNP level; ii) determining whether the NTproCNP level exhibits zero or greater than zero SDS; wherein the subject having an NTproCNP level of SDS greater than zero indicates that treatment with CNP therapy should be initiated.

28. 28. The method of claim 27, wherein NTproCNP is measured at 2 weeks, 1 month, 3 months, and 6 months to establish baseline NTproCNP levels.

29. The composition according to any one of claims 1 to 3, wherein NTproCNP is measured by radioimmunoassay.

30. 1. A composition for use in a method of treating a subject having a bone-related disorder, skeletal dysplasia, or short stature, comprising a CNP variant, said method comprising: [i) identifying whether a subject has a loss-of-function (LoF) or gain-of-function (GoF) variant in a gene associated with a bone-related disorder, skeletal dysplasia, or short stature; ii) calculating a polygenic risk score (PRS) for height of said subject; iii) determining whether the subject has a LoF variant and PRS in the bottom 20%; iv) if the subject has a LoF variant and PRS is in the bottom 20%, treating the subject with the composition.

31. 31. The composition of claim 30, wherein the gene associated with bone-related disorders, skeletal dysplasia, or short stature is selected from the group consisting of NPR2, SHOX, PTPN11, COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), NPPC, FGFR3, IGF1R, DTL, and pregnancy-associated plasma protein A2 (PAPPA2), or a combination thereof.

32. 32. The composition of claim 30 or 31, wherein the gene associated with bone-related disorders, skeletal dysplasia, or short stature is NPR2.

33. A composition for increasing facial volume, facial sinus volume, and foramen magnum area in a subject aged 6 months or less who has a bone-related disorder, skeletal dysplasia, or short stature, the composition comprising a CNP variant, wherein the composition is administered to the subject at a dose of at least 30 μg / kg of the CNP variant.

34. A composition for reducing the incidence of sudden infant death, sleep-disordered breathing, and neurosurgical decompression of the foramen magnum in a subject aged 6 months or younger with a bone-related disorder, skeletal dysplasia, or short stature, the composition comprising a CNP variant, wherein the composition is administered to the subject at a dose of at least 30 μg / kg of the CNP variant.

35. 35. The composition of claim 33 or 34, wherein the increase in facial volume, facial sinus volume, and foramen magnum area is measured by magnetic resonance imaging (MRI).

36. 36. The composition of claim 35, wherein changes in facial volume, facial sinus volume, and foramen magnum area are compared to baseline levels, healthy control subjects, or untreated control subjects.

37. 35. The composition of claim 33 or claim 34, wherein the composition is administered subcutaneously.

38. The composition of claim 33 or claim 34, wherein the composition is administered daily, weekly, every two weeks, monthly, or less frequently.

39. The composition described in claim 33 or claim 34, characterized in that the composition is administered at a dose of 30 μg / kg of the CNP variant for 3 months, 6 months, 1 year or more.

40. The composition described in claim 33 or claim 34, wherein the dose of the composition is reduced to 15 μg / kg of the CNP variant when the subject is approximately 2 years old.

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