High-throughput screening of gene variants associated with short stature

Abstract

This disclosure generally relates to the use of high-throughput screening methods for identifying gene mutations in genes associated with CNP dysfunction and short stature, such as the natriuretic peptide receptor 2 (NPR2) gene, and to methods for treating short stature. The high-throughput characterization of natriuretic peptide receptor 2 (NPR2) variants described herein may enable better prediction of novel NPR2 variants associated with CNP function, and for more commonly occurring variants, may be beneficial for the diagnosis and clinical trial enrollment of eligible patients with short stature.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 471,634, filed on June 7, 2023, U.S. Provisional Patent Application No. 63 / 540,792, filed on September 27, 2023, and U.S. Provisional Patent Application No. 63 / 564,071, filed on March 12, 2024, and the entire disclosures of which are incorporated herein by reference.

[0002] Incorporation by Reference of Sequence Listing This application includes a sequence listing electronically submitted in a file entitled 58818_Seqlisting.xml, created on June 3, 2024, and having a size of 56,620 bytes, which is incorporated herein by reference.

[0003] Field of the Disclosure The present disclosure generally relates to the use of high - throughput screening methods to identify gene mutations within genes related to CNP dysfunction and short stature disorders, such as the NPR2 gene.

Background Art

[0004] Methods for measuring the function of thousands of candidate regulatory sequences (CRSs) can be performed using a massively parallel reporter assay (MPRA) (Gordon et al., Nat Protoc. 2020 15(8):2387 - 2412).

[0005] Many different genes are potentially associated with skeletal disorders such as achondroplasia and short stature. C - type natriuretic peptide analogue (CNP) is currently approved by the FDA for achondroplasia, a common form of short stature. The binding of CNP to its receptor, natriuretic peptide receptor 2 (NPR2), induces endochondral and skeletal growth via cGMP production. Loss - of - function mutations in NPR2 cause dwarfism in mice and lack of intracellular cGMP response to CNP in cultured chondrocytes. NPR2 is a bidirectional therapeutic target associated with various forms of short stature and tall stature. NPR2 possesses guanylyl cyclase activity that leads to the synthesis of cyclic guanosine monophosphate (cGMP), and downregulation of this pathway is responsible for the short stature phenotype. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gordon et al., Nat Protoc.2020 15(8):2387-2412 [Overview of the project]

[0007] The high-throughput characterization of natriuretic peptide receptor 2 (NPR2) variants described herein may enable better prediction of novel NPR2 variants associated with CNP function and, for more commonly occurring variants, may be beneficial for the diagnosis and clinical trial enrollment of eligible patients with short stature.

[0008] This disclosure relates to a method for identifying variant genes associated with short stature that are gain-of-function (GoF) or loss-of-function (LoF) variants, - Transfect cells containing a cGMP-GFP reporter expression construct with a lentiviral vector expressing a polynucleotide encoding a variant protein associated with short stature, operably linked to one or more unique barcode sequences. - Contacting cells in a culture with C-type natriuretic peptide (CNP) or a variant thereof having CNP activity, -Selecting cells from the culture based on the level of GFP expression produced by the cells, - A method is provided for identifying variant proteins associated with short stature as GoF variants or LoF variants, wherein GoF variants have a higher level of cGMP production compared to a control, and LoF variants have a lower level of cGMP production compared to a control.

[0009] In various embodiments, variant genes associated with short stature include collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), DTL, PAPPA2, or a combination thereof. In various embodiments, variant genes associated with short stature are selected from the group consisting of natriuretic peptide receptor 2 (NPR2), natriuretic peptide precursor C (NPPC), fibroblast growth factor receptor 3 (FGFR3), or a combination thereof. In various embodiments, the variant gene associated with short stature is NPR2.

[0010] A method for identifying NPR2 variants as gain-of-function (GoF) or loss-of-function (LoF) variants, - Transfect cells containing a cGMP-GFP reporter expression construct with a lentiviral vector expressing a polynucleotide encoding an NPR2 variant protein operably linked to one or more unique barcode sequences, - Contacting cells in a culture with C-type natriuretic peptide (CNP) or a variant thereof having CNP activity, -Selecting cells from the culture based on the level of GFP expression produced by the cells, Methods are also contemplated herein that include identifying an NPR2 variant as a GoF variant or a LoF variant, wherein the GoF variant has a higher level of cGMP production compared to the control, and the LoF variant has a lower level of cGMP production compared to the control.

[0011] In various embodiments, the cells are mammalian cell lines. In specific embodiments, the cells are HEK293 cells.

[0012] In various embodiments, cells are sorted by flow cytometry.

[0013] In various embodiments, the lentiviral vector comprises a polynucleotide encoding a variant gene associated with short stature, a polynucleotide encoding a puromycin resistance gene, and a polynucleotide encoding a T2A-BFP promoter.

[0014] In various embodiments, the lentiviral vector further comprises 20 to 60 barcode sequences. In various embodiments, the barcode sequences are 15 to 30 base pairs long.

[0015] In various embodiments, the barcode sequence is located at the 3' end of the variant gene polynucleotide.

[0016] In various embodiments, the expression construct comprises a polynucleotide encoding a GFP protein operably linked to a cGMP-binding domain. In various embodiments, the cGMP-binding domain is derived from mouse or human phosphodiesterase.

[0017] In various embodiments, the expression construct further comprises a CMV promoter operably linked to cGull and a PGK promoter operably linked to a blastosidine resistance gene.

[0018] In various embodiments, the CNP variant 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)(Sequence ID 8), VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-50)(Sequence ID 9), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-49)(Sequence ID 10), TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-48)(Sequence ID 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47)(Sequence ID 12), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-46)(Sequence ID 13), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-45)(Sequence ID 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), LLQEHPNA RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(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), HPNARKYKGANKKGLSKGCFGLKLDRIGSMSG LGC(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), KYKGANKKGLSKGCFGLKLDRIGS MSGLGC(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), PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49), GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 50), GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 51), GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 52), and The selection is made from the group consisting of GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 53).

[0019] In various embodiments, CNPs are contacted with cells in doses of about 1 to about 100 nM. In various embodiments, CNPs are contacted with cells in doses of about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nM.

[0020] In various embodiments, lentiviruses are transfected into cells with an infection multiplicity of approximately 0.1 to approximately 0.5 MOI.

[0021] In various embodiments, the Disclosure provides a lentiviral vector comprising a polynucleotide encoding a variant gene associated with short stature, a polynucleotide encoding a puromycin resistance gene, and a polynucleotide encoding a T2A-BFP promoter.

[0022] In various embodiments, the variant gene associated with short stature is selected from the group consisting of natriuretic peptide receptor 2 (NPR2), natriuretic peptide precursor C (NPPC), fibroblast growth factor receptor 3 (FGFR3), or a combination thereof. In various embodiments, the variant gene associated with short stature is NPR2.

[0023] Further envisioned lentiviral vectors include a polynucleotide encoding the NPR2 variant, a polynucleotide encoding the puromycin resistance gene, and a polynucleotide encoding the T2A-BFP promoter. In various embodiments, the lentiviral vector further comprises 20 to 60 barcode sequences. In various embodiments, the barcode sequences are 15 to 30 base pairs long. In various embodiments, the barcode sequences are located at the 3' end of the variant gene polynucleotide.

[0024] In various embodiments, the present disclosure relates to a method for constructing a lentiviral library containing variant genes associated with short stature, - Amplifying variant genes associated with short stature from mammalian genomes or genomic DNA databases, - Cloning amplified variants into lentiviral vectors, wherein the lentiviral vectors contain 20 to 60 unique barcodes per vector. - Determining the sequence of variants related to barcodes within a vector, - Aligning variant sequences with control gene sequences to generate read structures, - Extracting barcodes from variant lead structures, - Identifying the barcode of each variant, - A method is provided which includes isolating a lentiviral vector expressing a variant gene.

[0025] In various embodiments, variant genes associated with short stature are selected from the group consisting of NPR2, NPPC, FGFR3, or combinations thereof.

[0026] A method for creating an NPR2 variant trentivirus library, - Amplifying NPR2 variants from mammalian genomes or genomic DNA databases, - Cloning amplified NPR2 variants into lentiviral vectors, wherein the lentiviral vectors contain 20 to 60 unique barcodes per vector. - Determining the sequence of NPR2 variants associated with barcodes in a vector, - Aligning the NPR2 variant sequence with the control NPR2 gene sequence to generate a read structure, - Extracting barcodes from NPR2 variant read structures, - Identify the barcode for each NPR2 variant, The present invention also provides a method comprising isolating a lentiviral vector expressing the -NPR2 variant.

[0027] This disclosure also contemplates a method for treating subjects with short stature disorder, comprising administering a CNP variant to a subject identified to have a loss-of-function variant of a gene associated with short stature, identified using the method described herein.

[0028] In various embodiments, the Disclosure provides a method for improving and / or maintaining bone strength in subjects who require improvement and / or maintenance of bone strength, comprising administering a C-type natriuretic peptide (CNP) to the subjects. In various embodiments, the subjects have short stature.

[0029] In various embodiments, the short stature disorder is achondroplasia, achondroplasia, short stature, idiopathic short stature, dwarfism, osteochondrodysplasia, thanatophoric osteodysplasia, osteogenesis congenita, achondroplasia, homozygous achondroplasia, chondrodysplasia congenita congenita), congenital limb dysplasia, congenital lethal hypophosphatasia, perinatal lethal type congenital osteogenesis imperfecta, short rib polydactyly syndrome, rhizomelic type of chondrodysplasia proximal limb type congenita), Jansen type metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia Congenital dysplasia, atelosteogenesis, twisting dysplasia, congenital short femur, Langer type mesomelic dysplasia, Nievergelt type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, microlimia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Leri syndrome) The group is selected from those consisting of disorders related to Weill, PTPN11 mutation (Noonan syndrome), and IGF1R mutation.

[0030] This disclosure also intends to provide a population PK model for administering a CNP variant to a subject. In various embodiments, this disclosure provides a method for treating a CNP-responsive bone-related disorder, skeletal dysplasia, or short stature disorder, comprising administering a CNP variant to a subject in need thereof, wherein the CNP variant is administered according to a weight-band administration regimen. i) Subjects weighing 10-11 kg received approximately 22-24 μg / kg of the CNP variant. ii) Subjects weighing 12-16 kg receive approximately 18-23 μg / kg of the CNP variant. iii) Subjects weighing 17-21 kg received administration of approximately 15-19 μg / kg of the CNP variant. iv) Subjects weighing 22-32 kg received administration of approximately 13-18 μg / kg of the CNP variant. v) Subjects weighing 33-43 kg received administration of approximately 12-15 μg / kg of the CNP variant. vi) Subjects weighing 44-59 kg received administration of approximately 10-14 μg / kg of the CNP variant. vii) Subjects weighing 60-89 kg who receive approximately 8-12 μg / kg of CNP variant, viii) A method is provided for subjects with a body weight of ≥90 kg to receive an administration of a CNP variant of approximately ≤9 μg / kg.

[0031] In various embodiments, i) Subjects weighing 10-11 kg received approximately 0.24 mg of the CNP variant. ii) Subjects weighing 12-16 kg received approximately 0.28 mg of the CNP variant. iii) Subjects weighing 17-21 kg received approximately 0.32 mg of the CNP variant. iv) Subjects weighing 22-32 kg received approximately 0.40 mg of the CNP variant. v) Subjects weighing 33-43 kg received approximately 0.50 mg of the CNP variant. vi) Subjects weighing 44-59 kg received approximately 0.60 mg of the CNP variant. vii) Subjects weighing 60-89 kg who receive approximately 0.7 mg of the CNP variant, viii) Subjects with a body weight of ≥90 kg receive approximately 0.80 mg of the CNP variant.

[0032] In various embodiments, CNP-responsive bone-related disorders, skeletal dysplasia, or short stature disorders include achondroplasia, hypochondrodysplasia, short stature, idiopathic short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, congenital osteogenesis imperfecta, achondroplasia, homozygous achondroplasia, congenital chondrodysplasia, flexor limb dysplasia, congenital lethal hypophosphatasia, perinatal lethal congenital osteogenesis imperfecta, short-rib polydactyly syndrome, proximal limb congenital chondrodysplasia, Jansen type metaphyseal dysplasia, and congenital vertebral epiphyseal dysplasia. The group consists of disorders related to osteogenesis imperfecta, torsional dysplasia, congenital femoral shortening, Langer type intermediate limb dysplasia, Nievergelt type intermediate limb dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysplasia, peripheral dysplasia, Kniest dysplasia, fibrous chondrodysplasia, Roberts syndrome, distal intermediate limb dysplasia, microlimia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepiphyseal metaphysical dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Leri Weill), PTPN11 mutation (Noonan syndrome), and IGF1R mutation.

[0033] In various embodiments, CNP variants are described in SEQ ID NOs: 1 to 53. In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP37)(SEQ ID NO: 1), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37)(SEQ ID NO: 2), or LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 21).

[0034] In various embodiments, the CNP variant further includes a hydrophilic portion. In various embodiments, the hydrophilic portion is PEG.

[0035] Furthermore, expression constructs comprising polynucleotides encoding a GFP protein operably linked to a cGMP-binding domain as described herein, which are useful for the reporter assays described herein, are also intended. The cGMP-binding domain is derived from mouse or human phosphodiesterase. In various embodiments, the expression construct further comprises a CMV promoter operably linked to cGull and a PGK promoter operably linked to a blastosidine resistance gene.

[0036] Further embodiments and advantages will be apparent to those skilled in the art from the examination of the following detailed description in conjunction with the drawings. While compositions, articles, and methods can take various forms, the following description includes specific embodiments, with the understanding that this disclosure is illustrative and not intended to limit the invention to any particular embodiment described herein. Any features of the compositions, articles, and methods described herein, including but not limited to components, their compositional ranges, substituents, conditions, and processes, are to be selected from the various embodiments, examples, and examples provided herein. [Brief explanation of the drawing]

[0037] [Figure 1A-1D] A schematic diagram of a high-throughput screening process using lentiviruses expressing NPR2 and cGMP reporter constructs is shown. Figure 1A. Bidirectional lentivirus constructs expressing NPR2 and puro-T2A-BFP. Figure 1B. cGMP-responsive GFP fluorescent reporter reconstituted in HEK293 cells. Figure 1C. Barcode strategies for associating barcodes with variants, and PacBio (long-read) and Illumina (short-read) based methods. Figure 1D. GFP sorting-based screening strategy. [Figure 2] This document describes how to generate an NPR2 variant trentivirus library. [Figure 3A] The level of cGMP detected by the catchpoint assay (Figure 3A) or the cGMP-GFP reporter construct of this disclosure (Figure 3B) is shown. [Figure 3B] The level of cGMP detected by the catchpoint assay (Figure 3A) or the cGMP-GFP reporter construct of this disclosure (Figure 3B) is shown. [Figure 4A] Figure 4A shows the screening process of samples using PCR generation of NPR2 variants, screening for LoF or GoF functions with different levels of cGMP production (Figure 4B), and the results of cGMP-GFP reporter screening in GoF or LoF NPR2 variants (Figure 4C). [Figure 4B-4C] Figure 4A shows the screening process of samples using PCR generation of NPR2 variants, screening for LoF or GoF functions with different levels of cGMP production (Figure 4B), and the results of cGMP-GFP reporter screening in GoF or LoF NPR2 variants (Figure 4C). [Figure 5] This study demonstrates representative sample selection from flow cytometry analysis of high-GFP or low-GFP screening, showing that the screening is highly correlated with the function of NPR2 variants and cGMP-GFP expression. [Figure 6A] High-throughput screening using the cGMP-GFP construct (Figure 6A) correlates with previously published results for NPR2 variant function (Figure 6B). [Figure 6B] High-throughput screening using the cGMP-GFP construct (Figure 6A) correlates with previously published results for NPR2 variant function (Figure 6B). [Figure 7A]This shows variant functional activity due to genetic and phenotypic effects. Figure 7A. cGMP levels measured by the functional outcomes of variants. Figure 7B. Variance ER LOO prediction can partially distinguish missense variants that alter NPR2 function. Figure 7C. Comparison of library screening cGMP measurements with their effect on human adult height in the UK Biobank. [Figure 7B] This shows variant functional activity due to genetic and phenotypic effects. Figure 7A. cGMP levels measured by the functional outcomes of variants. Figure 7B. Variance ER LOO prediction can partially distinguish missense variants that alter NPR2 function. Figure 7C. Comparison of library screening cGMP measurements with their effect on human adult height in the UK Biobank. [Figure 7C] This shows variant functional activity due to genetic and phenotypic effects. Figure 7A. cGMP levels measured by the functional outcomes of variants. Figure 7B. Variance ER LOO prediction can partially distinguish missense variants that alter NPR2 function. Figure 7C. Comparison of library screening cGMP measurements with their effect on human adult height in the UK Biobank. [Figure 8A] Seven GoF variants were identified that showed low CNP stimulation and were not present in the high CNP screening (Figure 8A). In addition, constitutively active variants were observed within the CNP-free screening study (Figure 8B). [Figure 8B] Seven GoF variants were identified that showed low CNP stimulation and were not present in the high CNP screening (Figure 8A). In addition, constitutively active variants were observed within the CNP-free screening study (Figure 8B). [Figure 9A]This shows that polygenetic scores for height modify the effect of NPR2 variants. Figure 9A shows how adult height changes with polygenetic scores in individuals who reported being short at age 10. Figure 9B shows that a logistic regression model was trained to predict adult shortness of height in individuals who reported being short at age 10. The only independent variables were the presence of a reduced-activity NPR2 variant and the polygenetic score. This simple model can predict two-thirds of true positives while keeping the false-positive rate below 20%. [Figure 9B] This shows that polygenetic scores for height modify the effect of NPR2 variants. Figure 9A shows how adult height changes with polygenetic scores in individuals who reported being short at age 10. Figure 9B shows that a logistic regression model was trained to predict adult shortness of height in individuals who reported being short at age 10. The only independent variables were the presence of a reduced-activity NPR2 variant and the polygenetic score. This simple model can predict two-thirds of true positives while keeping the false-positive rate below 20%. [Figure 10] The sequence of the human NPR2 protein is shown. [Figure 11-1] A table of screened NPR2 variants is provided. [Figure 11-2] A table of screened NPR2 variants is provided. [Figure 12] The goodness-of-fit plot of the final PPK model is shown. [Figure 13] The dose-normalized VPC results for the PPK model are shown, representing the dose-normalized observed and simulated vosoritide concentrations as a function of time after the first dose. [Figure 14] The simulated vosolitide AUC values ​​are shown compared to the AUC values ​​observed at 15 μg / kg from experiments 111-301. [Figure 15] The simulated bosolitide Cmax values ​​are shown compared to the Cmax values ​​observed at 15 μg / kg from experiments 111-301. [Figure 16A] Figure 16A shows the metacarpal cortical area (mm²) at each time point in the procedure. Figure 16B shows the metacarpal rigidity (mm) at each time point in the procedure. [Figure 16B] Figure 16A shows the metacarpal cortical area (mm²) at each time point in the procedure. Figure 16B shows the metacarpal rigidity (mm) at each time point in the procedure. [Modes for carrying out the invention]

[0038] CNP targets the NPR2 receptor and simulates signaling that leads to signals stimulating bone growth. This high-throughput characterization of NPR2 variants described herein enables better prediction of novel variants. For these more commonly occurring mutations, this method may be beneficial for the diagnosis of eligible patients and clinical trial enrollment.

[0039] Conventional screening methods (e.g., catchpoint assays) required more than a year of effort to characterize approximately 160 variants. This library-based screening strategy offers several advantages, including higher throughput (e.g., simultaneous screening of >400-1000 variants in a few months) and higher data quality and reproducibility (e.g., increased replication).

[0040] As used in the specification and the attached claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context explicitly indicates otherwise.

[0041] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which depends in part on how that value is measured or determined. In certain embodiments, the terms “about” or “approximately” mean within one, two, three, or four standard deviations. In certain embodiments, the terms “about” or “approximately” mean within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the terms “about” or “approximately” precede the first number in a series of two or more numbers, it is understood that the terms “about” or “approximately” apply to each of those numbers.

[0042] "Amplification" refers to any means by which a polynucleotide sequence is copied and thus extended to a greater number of polynucleotide molecules, for example, by reverse transcription, polymerase chain reaction, and ligase chain reaction.

[0043] "cDNA" refers to DNA that is complementary to or identical to mRNA, in either single-stranded or double-stranded form.

[0044] Conventional notation is used herein to describe polynucleotide sequences, where the left end of a single-stranded polynucleotide sequence is called the 5' end, and the left-handed direction of a double-stranded polynucleotide sequence is called the 5' direction. The direction of nucleotide addition from 5' to 3' into a nascent RNA transcript is called the transcription direction. The DNA strand having the same sequence as mRNA is called the "coding strand," the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located at the 5' end of the RNA transcript is called the "upstream sequence," and the sequence on the DNA strand having the same sequence as RNA and located at the 3' end of the coding RNA transcript is called the "downstream sequence."

[0045] "Complementary" refers to the topological compatibility or agreement of the interacting surfaces of two polynucleotides. Therefore, two molecules can be described as complementary, and furthermore, their contact surface properties are also complementary. A first polynucleotide is complementary to a second polynucleotide if its nucleotide sequence is identical to the nucleotide sequence of its polynucleotide binding partner. Thus, a polynucleotide having the sequence 5'-TATAC-3' is complementary to a polynucleotide having the sequence 5'-GTATA-3'. A nucleotide sequence is "substantially complementary" to a reference nucleotide sequence if the sequence complementary to the nucleotide sequence in question is substantially identical to the reference nucleotide sequence.

[0046] "Code" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, which function as a template for the synthesis of any defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and other polymers and macromolecules in biological processes having the biological properties derived therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA produced by that gene produces a protein in a cell or other biological system. Both the coding strand, where the nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, used as a template for the transcription of a gene or cDNA, can be said to code for a protein or other product of that gene or cDNA. Unless otherwise specified, "nucleotide sequences that code for an amino acid sequence" include all nucleotide sequences that code for the same amino acid sequence, including degenerate versions of each other. Nucleotide sequences that code for proteins and RNA may contain introns.

[0047] A “regulatory expression sequence” refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and / or translation) of a nucleotide sequence operably linked to it. “Operatally linked” or “operably linked” refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in the action of the other part (e.g., transcription of a sequence). Regulatory expression sequences may include, but are not limited to, sequences of promoters (e.g., inductive or constitutive), enhancers, transcriptional terminators, start codons (i.e., ATGs), intron splicing signals, and stop codons.

[0048] As used herein, the term “promoter” refers to a region of DNA that functions to control the transcription of one or more DNA sequences and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site and other DNA sequences that interact to modulate promoter function. A promoter-enhancing fragment is a shortened or truncated promoter sequence that retains promoter activity. Promoter activity can be measured using any of the assays known in the art, for example, a reporter assay using luciferase or green fluorescent protein (GFP) as a reporter, or a commercially available reporter.

[0049] The term "vector" refers to any carrier of exogenous DNA or RNA that is useful for transferring exogenous DNA to a host cell for replication and / or proper expression of the exogenous DNA by the host cell. "Expression vector" refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by a host cell or an in vitro expression system. Expression vectors include all known in the art, such as viral vectors, cosmids, and plasmids (e.g., naked or contained in liposomes) that incorporate recombinant polynucleotides.

[0050] A "viral vector" refers to a vector that uses a viral skeleton to carry a polynucleotide expression cassette. Examples of viral vectors include lentiviral vectors, adenovirus vectors, or adeno-associated vectors (AAVs).

[0051] An "expression cassette" or "cassette" refers to a component of a vector or plasmid DNA that controls the expression of a gene or protein, and is interchangeable and can be easily inserted into or removed from a vector. An expression cassette often includes a promoter sequence, an open reading frame, and a 3' untranslated region containing polyadenylation sites.

[0052] A "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), including cDNA, as well as nucleic acid analogs. Nucleic acid analogs include those containing unnatural bases, nucleotides linked to other nucleotides by bonds other than natural phosphodiester bonds, or bases linked via bonds other than phosphodiester bonds. Therefore, nucleotide analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotryesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to a large polynucleotide. The term "oligonucleotide" typically refers to a short polynucleotide of about 50 nucleotides or less. If a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will also be understood that this includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0053] A "polypeptide" refers to a polymer composed of amino acid residues, associated naturally occurring structural variants, and non-naturally occurring analogs of its synthesis, linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using automated polypeptide synthesizers. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. Conventional notation is used herein to describe polypeptide sequences, where the left end of a polypeptide sequence is the amino terminus and the right end of a polypeptide sequence is the carboxyl terminus.

[0054] "Recombinant polynucleotides" refer to polynucleotides that have sequences not naturally occurring. Amplified or assembled recombinant polynucleotides may be included in a suitable vector, and a suitable host cell can be transformed using this vector. A host cell containing recombinant polynucleotides is called a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide." Recombinant polynucleotides may also perform non-coding functions (e.g., promoters, origins of replication, ribosome binding sites, etc.). Recombinant proteins refer to proteins encoded by recombinant polynucleotides.

[0055] The term "barcode" refers to a short nucleotide tag attached to a target polynucleotide sequence during the preparation of a DNA library, providing information about the specific polynucleotide to which the barcode is attached or the cells to which the target polynucleotide may be expressed. The barcode may be 10 to 30 base pairs (bp) in length, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs. Multiple barcodes can be attached to a target polynucleotide.

[0056] The term "polynucleotide library" or "library" refers to a set of polynucleotide fragments cloned into an expression vector for the purpose of identifying polynucleotide fragments and isolating one or more genes of interest. A polynucleotide library may be RNA or DNA, including genomic DNA or cDNA.

[0057] The term "C-type natriuretic peptide" or "CNP" refers to a small single-chain peptide with a 17-amino acid loop structure at its C-terminus (CNP precursor protein, GenBank accession number NP_077720 for NPPC), and its variants. The 17-mer CNP loop structure is also called CNP17, CNP ring, or CNP cyclic domain. CNP includes the active 53-amino acid peptide (CNP-53) and the mature 22-amino acid peptide (CNP-22), as well as peptides with different lengths between the two peptides.

[0058] In various embodiments, the "CNP variant peptide" is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to wild-type NPPC with respect to the same number of amino acid residues. It is further intended that the CNP variant peptide may contain about 1 to about 53, or 1 to about 39, or 1 to about 38, or 1 to about 37, or 1 to about 35, or 1 to about 34, or 1 to 31, or 1 to about 27, or 1 to about 22, or 10 to about 35, or about 15 to about 37 residues of the NPPC polypeptide. In one embodiment, the CNP variant comprises amino acid sequences 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 derived from an NPPC polypeptide. The CNP variant also comprises a conjugate, salt, or prodrug of the CNP variant described herein. "CNP therapy" refers to the administration of a CNP variant to treat a subject having bone-related disorders, skeletal dysplasia, or short stature as described herein.

[0059] The term "conjugate moiety" refers to the portion that is conjugated to the variant peptide. The conjugate moiety may include lipids, fatty acids, hydrophilic spacers, synthetic polymers, linkers, or optionally, combinations thereof.

[0060] "Treatment" refers to preventive, therapeutic, or diagnostic measures. In specific embodiments, "treatment" refers to the administration of a compound or composition to a subject for therapeutic, preventive, or diagnostic purposes.

[0061] "Prophylactic" treatment is a treatment administered to subjects who show no signs of disease or only early signs of disease, with the aim of reducing the risk of developing a disease. The compounds or compositions of this disclosure may be given as prophylactic measures to reduce the likelihood of developing a disease or to minimize the severity of a disease if it does develop.

[0062] "Therapeutic" treatment is a treatment administered to an object exhibiting signs or symptoms of a disease, with the aim of reducing or eliminating those signs or symptoms. Signs or symptoms may be biochemical, cellular, histological, functional, or physical, subjective, or objective. The compounds of this disclosure may also be administered as therapeutic treatment or for diagnostic purposes.

[0063] "Diagnosis" means identifying the presence, degree, and / or nature of a pathological condition. Diagnostic methods differ in their specificity and selectivity. Certain diagnostic methods may not provide a definitive diagnosis of a condition, but it is sufficient if the method provides positive signs that aid in diagnosis.

[0064] "Pharmaceutical composition" or "formulation" refers to a composition suitable for pharmaceutical use in target animals, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of a CNP variant, optionally another biologically active agent, and optionally pharmaceutically acceptable excipients, carriers, or diluents. In one embodiment, a pharmaceutical composition includes an active ingredient, an inactive component constituting a carrier, and any product arising directly or indirectly from the dissociation of any two or more components, or one or more components, or from combinations, complex formation, or aggregation of one or more components from other types of reactions or interactions.

[0065] Accordingly, the pharmaceutical compositions of this disclosure include any compositions prepared by mixing the compounds of this disclosure with pharmaceutically acceptable excipients, carriers, or diluents.

[0066] "Pharmacologically acceptable carriers" refer to any of the standard pharmaceutical carriers and buffers, such as phosphate-buffered saline, a 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, flow enhancers, sweeteners, flavoring agents, and colorants. Preferred pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). The preferred pharmaceutical carrier depends 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).

[0067] 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.

[0068] "Pharmacologically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, that is, a material that can be administered to an individual without causing any undesirable biological effect, or without harmful interaction with any of the components of a composition containing it or any component present on or in the individual's body.

[0069] "Physiological state" refers to the internal state of an animal (e.g., a human). Physiological state includes, but is not limited to, body temperature, physiological ionic strength, pH, and the aqueous environment of enzymes. Physiological state also includes the physical state of a particular subject, which differs from the "normal" state present in most subjects, for example, from the normal human body temperature of approximately 37°C or from the normal human blood pH of approximately 7.4.

[0070] "Physiological pH" or "physiological pH range" refers to a pH range of approximately 7.0 to 8.0 (including both extreme values), or more typically, approximately 7.2 to 7.6 (including both extreme values). C-type natriuretic peptide variant

[0071] C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168:863-870 (1990) (GenBank accession number NP_077720 for CNP precursor protein, NPPC) (J. Hypertens., 10:907-912 (1992)) is a small single-chain peptide belonging to the peptide family (ANP, BNP, CNP) that has a 17-amino acid loop structure (Levin et al.) (al., N.Engl.J.Med., 339:863-870 (1998)) CNP plays an important role in multiple biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B, NPR2) to stimulate the production of cyclic guanosine monophosphate (cGMP) (J.Hypertens., 10:1111-1114 (1992)). CNP is expressed more broadly in the central nervous system, reproductive organs, bone, and vascular endothelium (Hypertension, 49:419-426 (2007)).

[0072] In humans, CNP is initially derived from the natriuretic peptide precursor C (NPPC) gene as a single-stranded 126-aminobutyric peptide (Sudoh et al., Biochem. Biophys. Res. Commun., 168:863-870 (1990)). Removal of the signal peptide results in pro-CNP, and further cleavage by the endoprotease furin produces an active 53-amino acid peptide (CNP-53), which is secreted and cleaved again by an unknown enzyme to produce a mature 22-amino acid peptide (CNP-22) (Wu, J. Biol. Chem. 278:25847-852 (2003)). CNP-53 and CNP-22 have different distributions; CNP-53 is dominant in tissues, while CNP-22 is mainly found in plasma and cerebrospinal fluid (J. Alfonzo, Recept. Signal. Transduct. Res., 26:269-297 (2006)). Both CNP-53 and CNP-22 bind to NPR-B in the same way.

[0073] 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 dwarf phenotype characterized by shorter long bones and vertebrae. Mutations in human NPR-B that block appropriate CNP signaling have been identified and cause dwarfism (Olney, et al., J. Clin. Endocrinol. Metab. 91(4):1229-1232 (2006), Bartels, et al., Am. J. Hum. Genet. 75:27-34 (2004)). In contrast, mice engineered to produce high levels of CNP exhibit elongated long bones and vertebrae.

[0074] Natural CNP genes and polypeptides have been previously described. U.S. Patent No. 5,352,770 discloses CNP-22 isolated and purified from porcine brain with the same sequence as human CNP, and its use in the treatment of cardiovascular indications. U.S. Patent No. 6,034,231 discloses the human gene and polypeptide of pre-proCNP (126 amino acids), as well as the human CNP-53 gene and polypeptide. Mature CNP is the 22-amino acid peptide (CNP-22). Specific CNP variants are disclosed in U.S. Patent No. 8,198,242, which is incorporated herein by reference.

[0075] In various embodiments, the CNPs of this disclosure include truncated CNPs having wild-type amino acid sequences derived from hCNP-53 and its variants, ranging from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53). 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)(Sequence ID 8), VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-50)(Sequence ID 9), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-49)(Sequence ID 10), TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-48)(Sequence ID 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47)(Sequence ID 12), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-46)(Sequence ID 13), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-45)(Sequence ID 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), LLQEHPNA RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(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), HPNARKYKGANKKGLSKGCFGLKLDRIGSMSG LGC(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), KYKGANKKGLSKGCFGLKLDRIGS MSGLGC(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)(Sequence ID 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)](Sequence ID 41), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37)(SEQ ID NO: 42), MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37)(SEQ ID NO: 43), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N)](Sequence ID 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: 49).

[0076] In various embodiments, the CNP variant peptide is a modified CNP-37 or CNP-38 peptide, optionally having mutations / substitutions at the furin cleavage site and / or containing glycine or proline-glycine at the N-terminus. Exemplary CNP-37 variants are, but are not limited to, QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N); Sequence ID 41], MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37; Sequence ID 43), PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37; Sequence ID 42), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N); Sequence ID 44], PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37; Sequence ID 1), MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37; Sequence ID 45), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37:Sequence ID 2) [ka]

[0077] In various embodiments, the CNP variants of the present disclosure include PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), or PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49).

[0078] The variant peptide may further contain an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the peptide further contains an OH or NH2 group at the C-terminus.

[0079] The variant peptide may contain a conjugate moiety. In various embodiments, the conjugate moiety is located 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 located on a lysine residue. In various embodiments, the conjugate moiety contains one or more acidic moieties. In various embodiments, the acidic moieties are hydrophobic acids.

[0080] In various embodiments, the variant comprises the following structures: PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 46), or Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 46).

[0081] In various embodiments, the variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (Sequence ID 46), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 47), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 48) Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 48), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 46), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 49), and Selected from the group consisting of Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 49).

[0082] 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).

[0083] The CNP variant is further intended to be conjugated or complexed with a moiety that provides increased stability or half-life, such as a conjugate moiety. In various embodiments, the conjugate moiety is complexed via non-covalent bonds or attached via covalent bonds. The moiety may be attached non-covalently to the peptide via electrostatic interactions. Alternatively, the moiety may be covalently associated to the peptide via one or more linker moieties. The linkers may be cleavable or non-cleavable linkers. Cleavable linkers may be cleaved via enzymes, nucleophilic / basic reagents, reducing agents, photoirradiation, electrophilic / acidic reagents, organometallic and metallic reagents, or oxidizing reagents. The linkers may also be self-destructive linkers. Examples of linkers include N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (such as dimethyladipymidate HCl), active esters (such as disuccinimidylsberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), and diisocyanates (such as triene 2,6-diisocyanate). Examples include, but are not limited to, bis-active fluorine compounds (such as nates), 1,5-difluoro-2,4-dinitrobenzene, beta-alanine, 4-aminobutyric acid (GABA), 2-aminoethoxylic acid (AEA), aminoethoxy-2-ethoxyacetic acid (AEEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Abx), bicinaldiol cleavable linkers, trimethylloclactonization, p-alkoxyphenylcarbamates, bicines, peptoids, or bicines-type linkers, and electron linkers as described herein.

[0084] The linker is intended to attach to a residue of the CNP variant either within the CNP cyclic domain or at a site outside the CNP cyclic domain. In various embodiments, the linker attaches to a lysine residue. In various embodiments, the linker attaches to a lysine residue within the CNP cyclic domain.

[0085] In various embodiments, the CNP variant is attached to the conjugate portion via a linker. In various embodiments, the linker is attached to the conjugate portion via a hydrophilic spacer on the conjugate portion.

[0086] In various embodiments, the linker is a hydrolyzable linker.

[0087] In various embodiments, the linker is a peptoid or an 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 includes an SO2 moiety. An exemplary linker is shown in Figure 7. The linker in Figure 7 is further intended to be modified by substitution of R groups. For example, a bisin-type linker includes the structure shown below: [ka]

[0088] In various embodiments, the portion conjugated to the peptide is a synthetic polymer such as polyethylene glycol, a linker, a lipid portion or a fatty acid, or a combination thereof. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a spacer, and a linker. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a polyethylene glycol spacer or a polyethylene glycol derivative spacer, and a linker. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a spacer, and a linker, the spacer comprising 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 carbonyl groups.

[0089] In various embodiments, the CNP variant is conjugated with a fatty acid. Lipid technology is assumed to increase the serum half-life of the CNP variant, enabling less frequent injection 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 their diacides. In various embodiments, the fatty acid is conjugated to a lysine residue.

[0090] In various embodiments, the CNP variants described herein are intended to include a conjugate moiety as described herein. The conjugate moiety is intended to be located 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 located on a lysine residue. In various embodiments, the conjugate moiety includes one or more acidic moieties. In various embodiments, the acidic moieties are fatty acids. Exemplary CNP variants and peptide conjugates are described in International Patent Application No. PCT / US2020 / 051100 and USSN No. 17 / 642,150, which are incorporated herein by reference in their entirety. Variants, conjugates, and salts of CNP are disclosed in USSN No. 17 / 634,034, which is incorporated herein by reference.

[0091] In various embodiments, the conjugate portion includes an acid portion linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain, 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 carbonyl groups. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (γGlu). In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 to C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 alkyl chain. In various embodiments, the hydrophilic spacer is one or more OEG(8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is one or two OEG(8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is OEG(8-amino-3,6-dioxaoctanoic acid). In various embodiments, the 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 acidic portion and the hydrophilic spacer have the structure AEEA-AEEA-γGlu-C18DA.

[0092] In various embodiments, the Disclosure intends to use CNP variants comprising hydrophilic or water-soluble polymers (e.g., oxygenated alkyl chains in which carbon atoms can be replaced by one or more oxygen atoms, e.g., polyethylene glycol (PEG) or polyethylene oxide (PEO)). In various embodiments, the water-soluble polymers may 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 links), conjugation site (e.g., N-terminus, internal, and / or C-terminus), and length (e.g., about 0.2, 0.4, or 0.6 kDa to about 2, 5, 10, 25, 50, or 100 kDa). The hydrophilic or water-soluble polymers can be conjugated to the CNP variant by N-hydroxysuccinimide (NHS)-based or aldehyde-based chemistry or other chemistry, as is known in the Art. In various embodiments, negatively charged PEG-CNP variants can be designed to reduce 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 base, and / or a phosphate base.

[0093] In another embodiment, a hydrophilic polymer (e.g., PEG or PEO) moiety conjugated to the N-terminus, C-terminus, and / or internal portion of the CNP variant described herein contains one or more functional groups that are positively charged under physiological conditions. Such moieties are, among other things, designed to improve the distribution of such conjugated CNP variant into cartilage tissue. In one embodiment, the PEG moiety contains one or more primary, secondary, or tertiary amino groups, quaternary ammonium groups, and / or other amine-containing (e.g., urea) groups.

[0094] In additional embodiments, for any of the CNP variants described herein having a lysine (Lys / K) residue, regardless of whether they have a wild-type sequence or a non-natural amino acid sequence, any Lys residue can be independently substituted with any other natural or non-natural amino acid, including Lys to Arg substitutions. In various embodiments, all lysine residues are independently substituted with any other natural or non-natural amino acid, including Lys to Arg substitutions, except that the Lys residue in the cyclic domain of the CNP variant is not substituted with any other natural or non-natural amino acid.

[0095] C-type natriuretic peptide variant This specification describes a method for constructing a library of variant genes associated with short stature using lentiviral vector expression constructs. Large-scale parallel reporter assays (MPRAs) have been previously disclosed (Gordon et al., Nat Protoc. 2020 15(8):2387-2412). These assays utilized libraries of regulatory genes (e.g., promoters and enhancers) to determine the effect of gene mutations on the ability of regulatory regions to perform their regulatory functions. Another assay generated a library of T cell open reading frames to identify genes that positively regulate T cell activity (Daniloski, Nature 2022 603(7902):1-8).

[0096] However, these parallel reporter assays, when coupled with functional reporter systems, have not been used to identify variants in unregulated genes. This disclosure provides a method for constructing a lentiviral library of cell surface receptor variants, which is then introduced into cells expressing reporter constructs having readouts correlated with cell surface receptor function.

[0097] Lentiviral vectors are well-known in the field of genetic engineering. Commercially available lentiviral vectors can be adapted as needed to express variant genes associated with short stature or wild-type proteins. For example, lentiviral vectors can be derived from human immunodeficiency virus, feline immunodeficiency virus, equine immunodeficiency virus, pseudotyped lentivirus, VSVg pseudotyped lentiviral vector, pLS-SceI vector (ADDGENE), and other commercially available customizable vectors (e.g., VectorBuilder Inc.).

[0098] Other commercially available viral vectors are also being considered.

[0099] In various embodiments, this method is useful for creating libraries of variant genes associated with short stature. In various embodiments, variant genes associated with short stature include 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. In various embodiments, the variant gene associated with short stature is NPR2, NPPC, or FGFR3. In various embodiments, the variant gene associated with short stature is NPR2.

[0100] In various embodiments, the lentiviral vector contains an NPR2 variant. NPR2 protein mutation variants were identified in a UK Biobank study and described in Estrada et al. (Nat Commun. 2021, 12(1):2224) and International Patent Publication WO2021 / 055497, which are incorporated herein by reference. The sequence of NPR2 is shown in Figure 10, and NPR2 variants identified for screening in the library include those shown in Figure 11. Approximately 160 NPR2 gene variants were recently phenotypically characterized using the aforementioned cGMP quantification “catchpoint” assay (Estrada et al., above). The preparation and screening of this library are improved compared to previous catchpoint assays.

[0101] In one embodiment, the lentiviral vector includes a promoter region. In various embodiments, the promoter is the CMV promoter, EFGR promoter, MND promoter, CAG promoter, PGK promoter, or EF1A promoter.

[0102] In various embodiments, the lentiviral vector includes a select gene. In various embodiments, the select gene includes puromycin, kanamycin, blastocydin G418, or neomycin.

[0103] In one embodiment, a bidirectional lentiviral construct expressing NPR2 and puromycin-T2A-BFP was used. In one embodiment, a lentiviral NPR2 expression construct was constructed by subcloning a bidirectional lentiviral vector expressing puro-T2A-BFP in the opposite direction (Robinson et al., PLoS One 2021 Apr9;16(4):e0249117).

[0104] In various embodiments, the lentiviral vector further includes unique barcodes associated with the variant gene. In various embodiments, the variant in the vector includes 10 to 60 barcodes. In various embodiments, the variant in the vector includes 20 to 50 barcodes, 20 to 40 barcodes, or 30 to 45 barcodes.

[0105] In various embodiments, the barcode is 15–30 base pairs long. In various embodiments, the barcode is 18–25 base pairs long. In various embodiments, the barcode is 20 base pairs long. An exemplary barcode library contains approximately 20 base-pair randommer sequences with complexity >10^9. The barcode library is constructed, for example, as described by Azenta (Burlington, MA), and cloned into a lentiviral vector containing the variant gene of interest. Primers and next-generation sequencing pipelines for sequencing the barcodes were designed and executed by Cellecta using a similar protocol described by the manufacturer (manuals.cellecta.com / ngs-prep-kit-for-sgrna-shrna-dna-barcode).

[0106] Screening method The presence of LoF or GoF variants in genes associated with short stature is determined by a biological activity assay. In various embodiments, the biological assay is a cGMP reporter assay.

[0107] In various embodiments, the reporter assay uses an expression construct containing a cGMP-binding domain linked to a reporter construct. In various embodiments, the reporter is a green fluorescent protein, a red fluorescent protein, a luciferase, a beta-galactosidase, etc. In various embodiments, the cGMP-binding domain is derived from a human or mouse phosphodiesterase.

[0108] In various embodiments, the expression construct is a lentiviral vector.

[0109] In various embodiments, the lentiviral vector includes a promoter region. In various embodiments, the promoter is a CMV promoter, an EFGR promoter, an MND promoter, a CAG promoter, a PGK promoter, or an EF1A promoter.

[0110] In various embodiments, the lentiviral vector includes a select gene. In various embodiments, the select gene includes puromycin, kanamycin, blastocydin G418, or neomycin.

[0111] In various embodiments, the expression construct is transfected or transduced into a host cell. In various embodiments, the host cell is a mammalian cell. Exemplary mammalian cell lines include, but are not limited to, HEK293, CHO, MDCK, BHK, NIH / 3T3, COS, A549, MEF, or HeLa cells.

[0112] In various embodiments, the expression construct comprises a cGMP-binding domain and a GFP reporter. In various embodiments, the expression construct comprises a CMV promoter operably linked to the cGMP-GFP reporter and a PGK promoter operably linked to a blastosidine resistance gene. In various embodiments, the expression construct is transfected into HEK293 cells.

[0113] In various embodiments, cells transfected with the cGMP expression constructs described herein are brought into contact with CNPs to induce cGMP production. In various embodiments, cells are brought into contact with CNPs in concentrations of approximately 1–60 nM. In various embodiments, cells are brought into contact with CNPs in concentrations of approximately 1–5 mM, approximately 5–50 mM, approximately 10–40 mM, or approximately 15–50 mM.

[0114] In various embodiments, the level of the reporter expressed by the expression construct is measured by ELISA, flow cytometry, enzyme substrate assay, or other suitable assay for the selected reporter.

[0115] In various embodiments, LoF or GoF variants may be predicted, for example, using AlphaForm 3D mapping or other protein mapping tools, based on the mapping of changes in the variant sequence to the predicted 3D structure and active domain of the gene-encoded protein.

[0116] Genes related to short stature Achondroplasia is the result of an autosomal dominant mutation in the fibroblast growth factor receptor 3 (FGFR-3) gene, causing abnormalities in chondrogenesis. FGFR-3 normally has a negative regulatory effect on chondrocyte growth, and therefore bone growth. In achondroplasia, the mutant FGFR-3 is constitutively active, leading to significant bone shortening. In humans, activation of FGFR-3 mutations is the primary cause of hereditary dwarfism. Mice with activated FGFR-3 serve as a model for achondroplasia, the most common form of skeletal dysplasia, and CNP overexpression rescues these animals from dwarfism. Therefore, functional variants of CNP are a potential therapeutic agent for the treatment of various skeletal dysplasias.

[0117] By stimulating chondrocyte matrix production, proliferation, and differentiation, and increasing long bone growth, the CNP variants of this disclosure are useful for treating mammals, including humans, that suffer from bone-related disorders such as skeletal dysplasia or short stature. CNP-responsive bone-related disorders Non-limited examples of skeletal dysplasia and short stature disorders include achondroplasia, hypochondrodysplasia, short stature, idiopathic short stature, dwarfism, osteochondrodysplasia, thanatophoric ossification, congenital osteogenesis imperfecta, achondroplasia, homozygous achondroplasia, congenital chondrodysplasia, flexor limb dysplasia, congenital lethal hypophosphatasia, perinatal lethal congenital osteogenesis imperfecta, short-rib polydactyly syndrome, proximal limb congenital chondrodysplasia, Jansen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteogenesis imperfecta, Disorders associated with torsional dysplasia, congenital femoral shortening, Langer type intermediate limb dysplasia, Nievergelt type intermediate limb dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysplasia, peripheral dysplasia, Kniest dysplasia, fibrous chondrodysplasia, Roberts syndrome, distal intermediate limb dysplasia, microlimia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepiphyseal metaphysical dysplasia, as well as disorders associated with NPR2 mutations, SHOX mutations (Turner syndrome / Leri Weill), PTPN11 mutations (Noonan syndrome), and IGF1R mutations.

[0118] Additional short stature and growth plate disorders targeted 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. In various embodiments, the short stature gene is NPR2, NPPC, or FGFR3.

[0119] Growth plate disorders include disorders resulting 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, growth plate disorders or short stature are associated with mutations in one or more genes related to RAS disease. In various embodiments, subjects with growth plate disorders are heterozygous for mutations in growth plate genes. In various embodiments, the mutations are loss-of-function mutations. In various embodiments, the mutations are gain-of-function mutations. Growth plate disorders include, but are not limited to, familial short stature, dominant familial short stature (also known as dominant inherited short stature), or idiopathic short stature. See, for example, Plachy et al., J Clin Endocrinol Metab 104:4273-4281, 2019.

[0120] Mutations in ACAN can cause familial osteochondritis dissecans and short stature, and osteoarthritis characterized by areas of bone damage (or lesions) ultimately caused by detachment of cartilage and sometimes bone from the ends of the joint bones. A disordered cartilage network in growing bones is suggested to impair their growth and lead to short stature. Mutations associated with ACAN and short stature include Val2303Met. See Stattin et al., Am J Hum Genet 86(2):126-37, 2010. Patients with ACAN mutations resulting in short stature are expected to benefit from treatment with CNP, as administration may increase the height of these patients due to known interactions between CNP and FGFR3.

[0121] The natriuretic peptide system, including the receptor NPR2, has been shown to be involved in regulating endochondral bone growth (Vasquez et al., Horm Res Pediat 82:222-229, 2014). Studies have shown that homozygous or compound heterozygous loss-of-function mutations in NPR2 cause Maroteaux's distal intermediate limb dysplasia (AMDM), a skeletal dysplasia characterized by very short stature (Vasquez et al., 2014, see above). While some reports suggest heterozygous loss-of-function (dominant-negative, etc.) NPR2 mutations as a cause of short stature, gain-of-function heterozygous NPR2 mutations are known to cause tall stature (Vasquez et al., 2014, see above). Considering the interaction of CNP with NPR2 to stimulate cGMP production, increasing cGMP levels would be desirable in these conditions and would have therapeutic benefits in managing complications from these diseases and conditions.

[0122] Heterozygous mutations in NPR2 are thought 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 less than -1.0, -1.5, -2.0, -2.5, or -3.0, and it is intended that at least one parent has a height SDS less than -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 subjects with short stature having a height SDS between -2.0 and -3.0. In various embodiments, the CNP variants are useful for treating subjects with short stature having a height SDS between -2.0 and -2.5. However, since de novo mutations in NPR2 can result in short stature as defined by a height SDS less than -1.5, -2.0, -2.5, or -3.0, treatment of heterozygous carriers of harmful mutations in NPR2 where neither parent has short stature is also conceivable. Furthermore, the aim is to treat individuals heterozygous for harmful mutations in other growth plate genes with CNP in order to improve height and / or enhance bone growth.

[0123] Exemplary variants of NPR2 are disclosed in International Patent Publication No. 2021 / 055497, which is incorporated herein by reference.

[0124] The role of NPPC in skeletal growth is well documented (Hisado-Oliva et al., Genetics Medicine 20:91-97, 2018). NPPC knockout mice exhibited severe dwarfism with disproportionate morphological features, including limb shortening and endochondral ossification (Hisado-Oliva et al., 2018, see above). Studies of the entire human genome have shown a relationship between NPPC and height (Hisado-Oliva et al., 2018, see above). CNP haploinsufficiency is thought to be a cause of short stature in humans, and recent studies have identified heterozygous mutations in families with short stature and microhandia (Hisado-Oliva et al., 2018, see above). These studies observed a significant reduction in cGMP production when measured in a heterozygous state (Hisado-Oliva et al., 2018, see above). NPPC mutations include the 355G>T missense mutation, which causes changes in Gly119Cys, and the 349C>G missense mutation, which causes changes in Arg117Gly. CNP variants that rescue cGMP production may offer therapeutic benefits in managing disability in patients with heterozygous loss-of-function NPPC mutations.

[0125] Lelyweil chondrodysplasia (LWD) is a rare genetic disorder characterized by shortening of the forearms and lower limbs, abnormal wrist deformity (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 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 intermediate-limb dysplasia occurs when there are two SHOX mutations, which can result from homozygous or compound heterozygous mutations on each chromosome. A subset of SHOX mutations causes idiopathic short stature. Turner syndrome is caused by a deletion on the X chromosome that may contain the SHOX gene. SHOX has been shown to be involved in the regulation of FGFR3 transcription and contribute to the control of bone growth (Marchini et al., Endocr Rev.37:417-448, 2016). SHOX deficiency leads to increased FGFR3 signaling, and there is some evidence supporting that SHOX also directly interacts with CNP / NPR2 (Marchini, above). Given the association between SHOX, FGFR3, and bone growth, subjects with homozygous or heterozygous SHOX mutations are expected to benefit from treatment with the CNP variants described herein.

[0126] RAS disease is a group of rare genetic conditions caused by mutations in the Ras / mitogen-activated protein kinase (MAPK) pathway. RAS disease is a group of disorders characterized by increased signaling via the RAS / MAPK pathway. This pathway leads to downstream activation of the RAF / MEK / ERK pathway. Short stature is a characteristic feature of certain RAS diseases. For example, CNP signaling inhibits RAF and reduces the activation of MEK and ERK.

[0127] RAS diseases associated with short stature include Noonan syndrome, Costello syndrome, cardiac facial cutaneous syndrome, neurofibromatosis type 1, and LEOPARD syndrome. Hereditary gingival fibromatosis type 1 is also a RAS disease as intended herein. Patients with RAS disease (including Noonan syndrome, Costello syndrome, cardiac facial cutaneous syndrome, neurofibromatosis type 1, LEOPARD syndrome, and hereditary gingival fibromatosis type 1) include those 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).

[0128] CFC is caused by mutations in several genes in the Ras / MAPK signaling pathway, including K-Ras, B-Raf, Mek1, and Mek2. Costello syndrome, also known as facial dermatoskeletal (FCS) syndrome, is caused by the activation of mutations in the H-Ras gene. Hereditary gingival fibromatosis type 1 (HGF) is caused by a dominant mutation in the SOS1 gene (Son of Sevenless homolog 1), which encodes a guanine nucleotide exchange factor (SOS) that acts on the Ras subfamily of low molecular weight GTPases. Neurofibromatosis type 1 (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 a mutation in one of several genes, including PTPN11 which encodes SHP2, SOS1, K-Ras, and Raf-1.

[0129] CNP has been demonstrated as 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 short stature. A mouse model of cardiac-facial-cutaneous syndrome using the Braf mutation (p.Q241R) (Inoue et al. Hum.Mol.Genet. 2019, 28(1):74-83) showed reduced body length and growth plate width compared to the wild type, with smaller growth and hypertrophy zones, and CNP administration increased the body length of these animals.

[0130] Mutations in multiple genes can cause Noonan syndrome, characterized by short stature, heart defects, bleeding problems, and skeletal malformations. 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 explain a small number of cases. The cause of Noonan syndrome in 15–20 percent of people with this disorder is unknown.

[0131] The PTPN11, SOS1, RAF1, and RIT1 genes all encode proteins crucial to the RAS / MAPK cell signaling pathway, which is essential for cell division and growth (proliferation), differentiation, and migration. Many mutations in the genes associated with Noonan syndrome activate the resulting proteins, and this prolonged activation alters normal RAS / MAPK signaling, disrupting the regulation of cell growth and division and leading to the characteristics of Noonan syndrome. See, for example, Chen et al., Proc Natl Acad Sci USA. 111(31):11473-8, 2014; Romano et al., Pediatrics. 126(4):746-59, 2010; and Milosavljevic et al., Am J Med Genet 170(7):1874-80, 2016. Subjects with mutations that activate the MAPK pathway are expected to benefit from treatment with CNP variants as described herein to improve bone growth and short stature. Subjects with mutations that activate the MAPK pathway are also expected to benefit from treatment with CNP variants as described herein to improve other comorbidities associated with hyperactivated MAPK pathways in other cells throughout the body on which the NPR2 receptor is expressed on its surface.

[0132] 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 (above) has identified numerous mutations in the PTPN11 gene that lead to short stature. Gain-of-function mutations cause hyperactivation signaling via SHP2, inhibiting growth hormone-induced IGF-1 release and thereby contributing to reduced bone growth (Rocca Serra-Nedelec, PNAS 109:4257-4262, 2012). Individuals with homozygous or heterozygous PTPN11 mutations are thought to benefit from treatment with CNP variants to improve bone growth and short stature.

[0133] Mutations in the Indian Hedgehog (IHH) gene, which is associated with the regulation of endochondral ossification, are also linked to short stature syndrome (Vasques et al., J Clin Endocrinol Metab. 103:604-614, 2018). Many of the IHH mutations identified segregate in short stature in a dominant inheritance pattern. Given the association between IHH and bone growth and ossification, subjects with homozygous or heterozygous IHH mutations are expected to benefit from treatment with the CNP variants described herein.

[0134] Mutations in FGFR3, including N540K and K650N, cause short stature and achondroplasia.

[0135] The insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (α2β2) transmembrane glycoprotein with intrinsic kinase activity. IGF1R has been shown to play a role in prenatal and postnatal growth. Heterozygous mutations in IGF1R have been identified in gestational age (SGA) children and individuals with familial short stature (Kawashima et al., Endocrine J. 59: 179-185, 2012). Mutations in IGF1R associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, see above).

[0136] 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). Short stature in individuals may not be primarily caused by a single gene, but rather by the combined effects of these genes. Such individuals with short stature, defined by height SDS less than -1.0, -1.5, -2.0, -2.5, or -3.0, are intended to be beneficially treated with CNP variants, given the ability of CNP to increase length in normal animals, for example, to improve bone growth and bone length.

[0137] In various embodiments, the CNP variant is useful for treating subjects with short stature who have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0 and have at least one parent with a height SDS of less than -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 variant is useful for treating subjects with short stature who have a height SDS of -2.0 to -3.0. In various embodiments, the CNP variant is useful for treating subjects with short stature who have a height SDS of -2.0 to -2.5. In various embodiments, short stature is associated with mutations in one or more genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, insulin growth factor 1 receptor (IGF1R), DTL, PAPPA2, or combinations thereof.

[0138] In various embodiments, growth plate disorders or short stature are associated with mutations in one or more genes related to RAS disease.

[0139] In various embodiments, short stature is the result of mutations in multiple genes, such as those 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 on height, excluding samples from the UK Biobank Project described in WO2021 / 055497. The cohort can be divided into five PRS quintiles (PRS1 being the shortest height, and PRS5 being the longest height). In various embodiments, subjects have a mutation in NPR2 and have a low PRS. In various embodiments, subjects have a mutation in FGFR3 and have a low PRS. In various embodiments, subjects have a mutation in NPR2 and have a low PRS. In various embodiments, subjects have a mutation in IGF1R and have a low PRS. In various embodiments, subjects have a mutation in NPPC and have a low PRS. In various embodiments, subjects have a mutation in SHOX and have a low PRS. In various embodiments, the subjects have one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX, and have 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.

[0140] In addition, CNP variants are useful in treating other bone-related conditions and disorders such as rickets, hypophosphatemic rickets [including X-linked hypophosphatemic rickets (also known as vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets], and osteomalacia [including tumor-induced osteomalacia (also known as oncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)].

[0141] 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).

[0142] CNP is also reported herein to improve bone strength in subjects with achondroplasia receiving long-term CNP therapy. This specification provides a method for improving and / or maintaining bone strength in subjects requiring improvement and / or maintenance of bone strength, comprising administering C-type natriuretic peptide (CNP) to the subject. In various embodiments, the subject has a bone-related disorder, e.g., skeletal dysplasia or short stature. CNP-responsive bone-related disorder Non-limited examples of skeletal dysplasia and short stature disorders include achondroplasia, hypochondrodysplasia, short stature, idiopathic short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, congenital osteogenesis imperfecta, achondroplasia, homozygous achondroplasia, congenital chondrodysplasia, flexor limb dysplasia, congenital lethal hypophosphatasia, perinatal lethal congenital osteogenesis imperfecta, short-rib polydactyly syndrome, proximal limb congenital chondrodysplasia, Jansen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, and osteogenesis imperfecta. This includes all conditions, torsional dysplasia, congenital femoral shortening, Langer type intermediate limb dysplasia, Nievergelt type intermediate limb dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysplasia, peripheral dysplasia, Kniest dysplasia, fibrous chondrodysplasia, Roberts syndrome, distal intermediate limb dysplasia, microlimia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepiphyseal metaphysical dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Leri Weill), PTPN11 mutation (Noonan syndrome), disorders associated with IGF1R mutation, rickets, hypophosphatemic rickets [including X-linked hypophosphatemic rickets (also called vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets], and osteomalacia [including neoplastic osteomalacia (also called oncogenic osteomalacia or oncogenic phosphatemic osteomalacia)].

[0143] In certain embodiments, the CNP variants and compositions and formulations comprising the present disclosure are useful in improving one or more symptoms or physiological outcomes of skeletal dysplasia, which may include increased absolute growth, increased growth rate, increased qualitative computed tomography (QCT) bone mineral density, improved growth plate morphology, increased long bone growth, improved spinal morphology, improved elbow joint range of motion, and / or reduced sleep apnea. In this regard, it should be noted that the terms “improved,” “improved,” “increased,” “decreased,” and their grammatical equivalents are all relative terms referring to the state of symptoms or physiological outcomes of the disease after treatment with the CNP variant (or composition or formulation) of the present invention, compared to the same symptoms or physiological outcomes of the disease before treatment with the CNP variant (or composition or formulation) of the present invention (i.e., compared to “baseline”), when used in relation to the symptoms or physiological outcomes of the disease. As described above, the “baseline” state can be determined by either measuring the subject’s condition before treatment (which can then be compared to the same subject’s condition after treatment), or by measuring their condition in a group of subjects suffering from the same distress who share the same or similar characteristics (e.g., age, sex, and / or disease state or progression).

[0144] 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 level produced at the same concentration of wtCNP22 (e.g., 1 μM), either in vitro or in vivo.

[0145] Any of the CNP variants described herein is intended to be useful in this method.

[0146] Further aspects and details of this disclosure will be apparent from the following examples, which are intended to be illustrative rather than restrictive.

[0147] Further embodiments and advantages will be apparent to those skilled in the art from the examination of the following detailed description in conjunction with the drawings. While compositions, articles, and methods can take various forms, the following description includes specific embodiments, with the understanding that this disclosure is illustrative and not intended to limit the invention to any particular embodiment described herein. Any features of the compositions, articles, and methods described herein, including but not limited to components, their compositional ranges, substituents, conditions, and processes, are to be selected from the various embodiments, examples, and examples provided herein. [Examples]

[0148] Example 1 - Construction of a lentiviral construct expressing NPR2 Materials and methods Vector and cell line construction. The cGMP-GFP-on reporter was designed by subcloning the cGull insert from (Matsuda et al, ACS Sens 2017 2(1):46-51) and cloning it into a VectorBuilder (VectorBuilder Inc., Chicago, IL) custom lentiviral vector containing a CMV promoter driving cGull and a mouse PGK driving blastosidine resistance. cGull contains a cGMP-binding domain from mouse phosphodiesterase 5α. The HEK293 cGMP-GFP-on reporter line was constructed by transducing HEK293T p11 cells with a cGMP-GFP reporter virus with an MOI of 0.2 and performing blastosidine selection (10 μg / ml) for 4 days. Cells were tested by transfecting with a WT NPR expression vector and stimulating the cells with 50 nM CNP.

[0149] A custom bidirectional lentiviral construct expressing NPR2 and puro-T2A-BFP was used. The lentivirus NPR2 expression construct was constructed by subcloning (PspXI / NotI) into a custom bidirectional vector expressing puro-T2A-BFP in the opposite direction (Robinson et al., PLoS One 2021 Apr9;16(4):e0249117).

[0150] Barcode synthesis and barcode-seq NGS primers. A 20 bp randommer sequence with complexity >10^9 was constructed and cloned into the PmeI site of the NPR2 vector (Genewiz / Azenta). Primers and an NGS pipeline for barcode sequencing were designed and executed by Cellecta using a similar protocol described by the manufacturer (manuals.cellecta.com / ngs-prep-kit-for-sgrna-shrna-dna-barcode-libraries). [Table 1]

[0151] Cloning of NPR2 variants and PacBio association of barcodes to variants. 160 NPR2 variants (Table 2) were selected from UKBB, amplified as a pool, and cloned into barcoded NPR2 vectors via PspXI / NotI. 25 unique barcodes were selected for each variant, resulting in a library of approximately 4K elements. PacBio sequencing was performed using a non-amplified (HpaI / BstEII digestion) method (library-pre info), and sequencing with Smart-seq Sequel I yielded X-aligned reads. Long-read alignment was required for analysis to associate variants with barcodes. The reference sequence used for alignment consisted of a single fasta entry containing the NPR2 gene sequence, followed by a 54bp spacer sequence (AGCGGCCGCGTTGGTCAGGCTTGGATTTCTATAACTTCGTATAGCAGTTTAAAC) (SEQ ID NO: 59), a 20bp barcode sequence, and finally a 20bp 3' barcode adjacency sequence (GTTTAAACCGAGAGATGGGG) (SEQ ID NO: 60). To account for the semi-degenerate structure of the barcode, this portion of the reference was filled with N. Alignment was performed using the PacBio-specific implementation of minimap2, i.e., pbmm2 align, with all default parameters preserved. All reads that did not map to the reference sequence were excluded from further analysis. Read structure assembly and barcode extraction were performed using pysam, the samtools Python® API, which iterated through the aligned reads and extracted the aligned reference position, aligned bases of the read (excluding soft clipped* bases), and cigar* string. Reads that did not cover the start site and barcode sequence of the NPR2 gene were excluded from further analysis. The barcode sequence was extracted using a simple regular expression with the Python® regular expression library (regex library).To locate the barcode, the 5' barcode adjacency sequence, consisting of the last 20 bp of the aforementioned spacer sequence, was used. The regular expression allowed for a 1 bp error (insertion, deletion, or substitution) when searching this sequence. The 20 bp following the adjacency sequence were then assumed to be the barcode. After barcode extraction, the aligned reads were reconstructed using a cigar string, taking insertions and deletions into account. Where deletions were identified within the reads, a dash (-) was inserted, and the inserted base was removed. After the reads were reconstructed, known variant locations could be simply extracted by indexing. Barcode polishing was used to correct potential sequencing errors in the barcodes. Here, barcodes with identical sequences were grouped into clusters. If a particular barcode sequence was observed five or more times, it was assumed to be a genuine barcode. If it was observed five or fewer times, it was likely due to a sequencing error. Barcodes with potential sequencing errors were compared to all barcodes in the "authentic" list using the Levenstein distance* with the Python® library pylev. Barcodes with a Levenstein distance of 2 or less to an "authentic" barcode were placed in this cluster.

[0152] Library packaging, infection, and screening. HEK293T cells were seeded in 40 mL of medium (DMEM, 10% fetal bovine serum) at a rate of 65,000 cells per ccm in a T225 flask and incubated overnight at 37°C and 5% CO2. The following morning, 10.5 μg of sgRNA library plasmid, 12 μg of LV-MAX lentivirus packaging mix (ThermoFisher), and 22.5 μL of Lipofectamine 2000 (ThermoFisher) were mixed in 2 mL of serum-free OptiMEM (Gibco), vortexed, incubated at room temperature for 10 minutes, and then added to the cells. After 24 hours, 40 U of DNAseI (NEB) was added to each plate to remove non-transfected plasmids. 72 hours after transfection, the supernatant was collected, passed through a 0.45 μm filter (Millipore, Stericup), and aliquots were stored at -80°C. HEK293-cGMP-GFP reporter cells were infected with the library at MOI=0.2 to obtain an initial coverage of approximately 500X. Library-infected cells were puro-selected (approximately 2 ug / ml) for 4 days to maintain coverage above 1000X throughout the screening. This screening involved resuspending cells (approximately 150 M cells per replication) and stimulating them with 0, 1, or 50 nM CNP for 1 hour with gentle shaking. Replication sorting was performed using Aria2, and coverage of approximately 250x per replication (4M cells) was collected for both high-GFP (top 15%) and low-GFP (bottom 15%) cells. The cell pellets were frozen and then processed for barcode sequencing using the primer sequences described above. Mageck and Mann-Whitney analyses were performed comparing four high-GFP replication samples with four low-GFP sorted samples.

[0153] Example 2 - Screening Strategy It is anticipated that multiple rounds of screening may be required to achieve a high-throughput method. Library #1 is used to establish a screening strategy using a cGMP-responsive GFP-on reporter system to phenotypically characterize NPR2 gene variants. Library #2 (approximately 60 times larger) is useful as a similar screening strategy for phenotypicly characterizing variants on a larger scale (e.g., approximately 160 NPR2s) in parallel. Library #3 can phenotypically characterize approximately 450 NPR2 variants in parallel. Library generation requires initial long-read alignment of sequences, assembly of read structures, and barcode extraction using 5' and 3' flacking sequences. Identify the barcode for each variant and determine whether they are unique barcodes that identify the variant.

[0154] A large number of barcodes per variant are desired in this method (e.g., about 30) to enable increased replication numbers, increased statistical power of the method, control for incorporation, provision of cellular heterogeneity, and increased gene coverage. In the preparation of Library 1, approximately 20 barcodes per variant were observed, resulting in a 93.3% unique barcode association rate.

[0155] Example 3. Establishment of the wrench NPR2 and cGMP-GFP-on reporter. We developed a lentiviral reporter system to determine whether a lentiviral vector containing the NPR2 gene can be detected when transduced into cells.

[0156] Cells and vectors were generated as described in Example 1. Stimulation of HEK cells containing the lenti-NPR2 vector with cGMP resulted in detectable GFP levels within the cells. These levels were comparable to those obtained using catch-point assays previously used to characterize NPR2 variants (Figure 3).

[0157] Next, the variant library was screened for the effect of variants on NPR2 function using a cGMP-GFP reporter assay with HEK293 cells expressing a GFP reporter construct and transfected with the NPR2 variant library. Library-positive cells were isolated based on puromycin selectivity and then sorted based on high to low GFP expression. DNA was extracted from the cells, and the DNA ligated to the library barcode was amplified by next-generation sequencing. The sequenced variants were then associated with previously identified high or low GFP profiles, and the effect of the NPR2 variants on function, e.g., gain-of-function or loss-of-function, was evaluated. For example, in the screening, LoF would have a higher count in the low-GFP sample (compared to the WT) (Figure 4B).

[0158] Using controls with known functions, variants were screened in this assay, and it was observed that the selected samples correlated highly with function. In addition, the Wilcoxon-Mann-Whitney test can be used to analyze the data because it is a nonparametric test of the null hypothesis that for values ​​X and Y randomly selected from two populations, the probability that X is greater than Y is equal to the probability that Y is greater than X. This analysis is robust, provides phenotypic directionality, and successfully identifies positive controls and other significant variants.

[0159] Screening of Library 2, which contains approximately 160 variants, demonstrated that the method provides a highly correlated screening tool. Screening of NPR2 variants previously identified by Estrada et al. (above) using this method yielded results consistent with previously identified NPR2 phenotypes, identifying both LoF and GoF variants (Figure 6).

[0160] The functional activity of the variants was classified by genetic and phenotypic effects (Figures 7A-7B). cGMP levels were measured by the functional outcomes of the variants. In the case of missense variants, Variation ER LOO prediction can partially distinguish missense variants that alter NPR2 function. A comparison of the cGMP measurements from this library screening with the effect on human adult height determined by UKBiobank is shown in Figure 7C.

[0161] This method is also useful for identifying mischaracterized mutations. For example, the variant Gln744del was previously thought to be an in-frame 3-nucleotide deletion that does not result in alteration of NPR2 function. However, in this screening, the inconsistency was identified, and the variant was characterized as a 4-nucleotide deletion that results in a LoF variant rather than a neutral variant.

[0162] This method, which has higher sensitivity than previous methods, was hypothesized to potentially allow variant screening using low concentrations of CNP. Several GoF mutation responses (e.g., R562Q) have been shown to saturate at high doses of CNP. Under conditions where CNP concentrations are limited, identifying GoF mutations may be easier.

[0163] CNP stimulation curves were generated using a cGMP catchpoint assay with eight concentrations of CNP ranging from 50 nM to 0 nM Pro-Gly-CNP37. cGULL GFP ON was transiently transfected using Lipo3000 in 2 ug plasmids in 6-well plates. Quadruplicates were tested for better accuracy. This screening identified seven more GoFs at lower CNP concentrations (only three GoFs were identified from the first screening at higher CNP concentrations), yielding non-overlapping GoFs (Arg804) (Figure 8).

[0164] Furthermore, it was confirmed that screening at low CNP levels, such as 1 nM, does not impair the isolation of LoF variants.

[0165] Polygenetic scores for height, when used in conjunction with screening methods to correct for the effect of NPR2 variants on polygenetic risk scores for short stature, can help predict individuals at high risk of idiopathic short stature. Figure 9A shows how adult height changes with polygenetic scores for individuals who reported being short at age 10. Of these individuals, only 29% were short as adults (height Z score < -2.25 SD), while 98% were below average height. Polygenetic scores summarize the combined effect of thousands of common variants that have little impact on height. While these scores capture 43% of the population variability in adult human height, their ability to predict at both ends of the distribution is limited. Figure 9B shows a logistic regression model trained to predict adult short stature for individuals who reported being short at age 10. Independent variables included only the presence of a reduced-activity NPR2 variant and the polygenetic score. This simple model can predict two-thirds of true positives while maintaining a false-positive rate of less than 20%. A model using only polygene scores and not reflecting the presence of reduced active NPR2 variants had an AUC-ROC of 0.66. In this sample, 20% of individuals reported short stature at age 10. In clinics, children with short stature are in the bottom 3%, so a similar model would likely perform much better.

[0166] The results described herein demonstrate the development of a novel pooled NPR2 variant screening system using lentiviral expression of variant genes, a cGMP-GFP reporter, a barcoding system, and a selection-based screening strategy. This screening successfully identifies both LoF and GoF variants. The screening allowed for the reclassification of 160 missense variants into 31 LoF, 6 GoF, and 123 neutral-activity variants compared to WT NPR2. Functional data indicate that at least 34 out of 100,000 individuals carry low-activity NPR2 variants that reduce height. By incorporating multi-gene scoring, this method enables the prediction of adult short stature in short-stature children carrying these variants. High-throughput methods are advantageous because they offer scalability (screening a large number of variants), complexity (identifying a large number of replications), and certainty (providing higher reliability for variant phenotypes).

[0167] Example 4. CNP body weight-based dosing model The PPK model was developed using data from five clinical trials in children (0.95–15 years old) with achondroplasia who received a daily dose of vosolitide per kg. The model was used to simulate expected exposure levels in children using a refined weight-band dosing regimen. The appropriateness of the weight-band dosing regimen was evaluated by comparing the simulated exposure levels with those observed in pivotal clinical trials.

[0168] Sampling for pharmacokinetic (PK) analysis of bosolitide was conducted as part of three Phase II and two Phase III trials, including the use of a daily subcutaneous dose of 15 μg / kg. In two of these analyses (Chan et al., Clin Pharmacokinet. 2022 61:263-80), the mean peak plasma concentration (Cmax) after a single subcutaneous administration of 15 μg / kg of bosolitide ranged from 4750 to 7180 pg / mL, the area under the plasma concentration-time curve (AUC) from time 0 to the last measurable concentration ranged from 175,000 to 290,000 pg-min / mL, and the mean time to reach peak plasma concentration ranged from 13.8 to 16.8 minutes (Chan, above). PK data also showed a positive correlation between plasma exposure to vosolitide (AUC) and the body weight of patients treated daily with a dose of vosolitide per kg, suggesting that alternative body weight-based dosing methods with vosolitide may result in more consistent exposure across the patient body weight range (Chan, above).

[0169] This study was designed to develop a population PK (PPK) model of bosolitide in children with achondroplasia and to evaluate the impact of clinically relevant covariates on bosolitide PK to better understand the causes of post-subcutaneous administration variability. The original weight-based dosing regimen (15 μg / kg) used in clinical trials of bosolitide required multiple different dose levels for children weighing 10–83 kg. A weight-band dosing regimen for bosolitide was developed to account for the weight-characterized effect on bosolitide clearance and volume of distribution and to ensure more consistent drug exposure throughout the patient's treatment period. This regimen also allows for fewer required dose levels and fewer dose changes, as a new dose is only needed when the child progresses from one weight band to the next, thus potentially simplifying medication for children with achondroplasia and their caregivers. Simulations were performed using the PPK model to develop medication recommendations.

[0170] This PPK analysis included PK, laboratory, and demographic data from children with achondroplasia. Data were collected from five clinical trials: Trials 111-202 (NCT02055157), a Phase II, non-randomized, open-label, consecutive cohort, dose-finding study of vosolitide (2.5, 7.5, 15, or 30 μg / kg) administered for 24 months to 35 children (5-14 years) with achondroplasia. [(Chan, above), Savarirayan et al., N Engl J] [Med.2019;381:25-35]; Ongoing Phase II open-label extension study of vosolitide (15 or 30 μg / kg) administered to 30 children with achondroplasia who completed 24 months of treatment in Study 111-205 (NCT02724228) and Study 111-202 (Savarirayan, above); Phase III, randomized, double-blind, parallel-allocated efficacy and safety study of vosolitide (15 μg / kg) administered for 12 months in 121 children (5-18 years old) with achondroplasia (Chan, above); Study 111-302 (NCT03 424018), an ongoing Phase III, open-label extension, safety and efficacy study of vosolitide (15 μg / kg) administered to children (≥6 years) with achondroplasia who completed 12 months of treatment in clinical trial 111-301; and clinical trial 111-206 (NCT03583697), a Phase II, randomized, double-blind, parallel-allocated, safety and efficacy study of vosolitide (15 or 30 μg / kg) administered for 12 months to 75 children (≤5 years) with achondroplasia; at the time of PPK model construction, the trials were ongoing, so only interim data from sentinel patients were available for inclusion in model development.

[0171] The data was pooled into a single Nonlinear Mixed Effects Modeling (NONMEM®, version 7.4.4, ICON Development Solutions, Dublin, Ireland) database. For bosolitide PPK modeling, a logarithmic transformation approach on both sides and stochastic approximate expectation maximization (SAEM), followed by importance sampling (IMP), were used.

[0172] The PPK model was developed in a series of steps. A base model was created without considering covariate effects and was used to explain the structural and stochastic components of the model and to perform a graph evaluation of the covariates. Using single-covariate models, pre-specified covariate-parameter relationships were graphically validated using covariates known to affect bosolitide PK or that were physiologically valid. Once all single-covariate evaluations were complete, a complete model was constructed using all single-covariate models that were statistically significant (p<0.01) and well-estimated. Then, backpassing was performed on the complete covariate model, removing one covariate at a time. Covariates with increased objective function (p<0.001) were retained in the final model.

[0173] The final PPK model parameters were used in the simulation without considering parameter precision. 500 replications were run using the PPK model to generate intensive concentration-time profiles (at 5-minute intervals) over the first 5 hours after subcutaneous administration of various stratified doses of vosolitide in pediatric patients weighing 10–90 kg. The simulation was performed using doses from the smallest stock-keeping unit (0.8 mg / mL [0.5 mL], 0.8 mg / mL [0.70 mL], 2 mg / mL [0.60 mL]). The highest withdrawal doses (0.32, 0.48, 1, and 1.2 mg) were included in the simulation. PK non-compartmental analysis (PKNCA) to calculate AUC and Cmax values ​​was performed on the simulation data using the PKNCA package in R, version 0.9.3. The simulated exposures were compared to the exposure data observed from trial 111-301. The median, 5th percentile, and 95th percentile values ​​of PK parameters from simulations were compared with PK parameters calculated from observational data evaluated at a daily dose of 15 μg / kg.

[0174] The final database used included 4,741 observations from 158 patients aged 0.95–15 years, with a mean age of 8.43 years. Patient weights ranged from 9–74.5 kg, with a mean baseline weight of 23.8 kg. Actual doses administered during the study to patients whose data were included in the PPK model included 2.5 μg / kg / day (6 patients), 7.5 μg / kg / day (12 patients), 15 μg / kg / day (151 patients), and 30 μg / kg / day (11 patients).

[0175] The final PPK model consisted of a one-compartment system with primary elimination and change-point primary absorption, allowing for time-dependent changes in the absorption rate coefficient. Body weight was found to be a predictor of drug CL / F and V / F (Table 2). [Table 2]

[0176] Furthermore, the 0.2 mg / mL SOLNC dose, used only in trials 111–202, and the duration of treatment were found to predict the relative bioavailability (F) of vosolitide. Separate residual errors were estimated for the two assay types. The model also considered CL / F, V / F, and IIV at the transition point. An additional secondary study identification number (SIDN) was used to represent the clinical trial in which each patient was enrolled, taking into account the fact that a patient may be enrolled in >1 trial. The effect of SIDN on IIV for CL / F and V / F was modeled by additional hierarchical levels (StudyCL and StudyV). Typical values ​​of the parameter estimates for the PPK model and parameter precision (standard error %) are presented in Table 3. Parameter precision was less than 30%, except for the terms IIV StudyCL and IIV StudyV, as there were only three SIDN values ​​in this database. The estimated typical parameter values ​​were consistent with the median of the bootstrap parameter estimates, the confidence intervals were reliably narrow, and no nulls were included. [Table 3]

[0177] The performance of the PPK model for bosolitide was evaluated using diagnostic goodness-of-fit plots and VPC (Figures 12 and 13). These demonstrated that the model was able to accurately describe the data. The model's predicted distribution of median concentrations across various time intervals was compared to the observed medians using VPC (Figure 13). The lower and upper bounds of the observed and simulated data were generally similar, and the observed concentrations fell within the 5th and 95th percentile ranges of the final model's predicted distribution across the time intervals.

[0178] Using a PPK model of vosolitide, improved recommended dosages for pediatric patients with achondroplasia were developed. Simulations were performed for various weight groups. Four initial regimens were identified for four weight bands: 0.32 mg for 10–19 kg, 0.48 mg for 20–34 kg, 0.7 mg for 35–64 kg, and 1 mg for ≥65 kg. The simulated AUC for patients <65 kg was within or slightly above the upper limit of the AUC observed in trials 111-301, 111-202, and 111-205. However, the simulated AUC for patients ≥65 kg was above the upper limit. Considering the results obtained with the initial regimens for the four weight bands, revised dosing regimens were tested. The new dosing regimens included more weight bands (8 compared to 4) to produce simulated exposures that better fit observed exposures. The best identified weight band dosing regimens were 0.24 mg for weights of 10–11 kg, 0.28 mg for weights of 12–16 kg, 0.32 mg for weights of 17–21 kg, 0.40 mg for weights of 22–32 kg, 0.50 mg for weights of 33–43 kg, 0.60 mg for weights of 44–59 kg, 0.70 mg for weights of 60–89 kg, and 0.80 mg for weights of ≥90 kg (Table 4). [Table 4]

[0179] The proposed regimen includes doses of <15 μg / kg for patients with a body weight of ≥44 kg and doses of >15 μg / kg for patients with a body weight of 10–16 kg. The new body weight band dosing regimen was found to result in more consistent exposure across the body weight range. The 5th–95th percentiles of the simulated AUC were within the range of the AUC observed at 15 μg / kg, and the median of the simulated AUC was distributed around the median of the observed AUC (Figure 14). In addition, the median of the simulated Cmax was in general agreement with the Cmax observed at 15 μg / kg, but the 5th and 95th percentiles of the simulated Cmax were lower than the 5th and 95th percentiles of the observed Cmax (Figure 15). This discrepancy may be due to the model underestimating Cmax, as shown in the VPC plot, or it may be a result of the simulation being performed with only one SIDN instead of the three SIDNs present in the model.

[0180] Using the PPK model, drug concentrations and exposures were simulated with the aim of developing more refined weight-band dosing regimens. While four weight-band regimens were initially proposed, greater consistency between simulated and observed exposures was achieved with eight weight-band regimens. The weight-band regimens provided more consistent drug exposures across the entire weight range. Specifically, for patients with a weight of >44 kg, a dose of <15 μg / kg was proposed to account for the observed non-linear correlation between exposure and patient weight. Similarly, for children aged 2–5 years and / or patients weighing 10–16 kg, a dose of >15 μg / kg was proposed to avoid suboptimal exposure and to allow for extrapolation approaches. The 30 μg / kg dose was tested in Phase II trials (trials 111–202, 111–205 [(Chan, above), Savarirayan above], and 111–206) and demonstrated a similar safety profile to the 15 μg / kg dose.

[0181] The simulated exposures from the eight proposed weight-band dosing regimens fall within the range demonstrated by good tolerability and efficacy in previous studies (Chan, above). Importantly, the proposed weight-band regimens ensure more consistent vosolitide exposure both within the patient population and throughout the entire treatment period for individual patients, while simplifying administration for children with achondroplasia and their caregivers.

[0182] Example 5: Improvement of bone strength using CNP The main objective of this study was to determine, using measurements of the second metacarpal bone, whether the CNP variant affects both bone strength length and development in children with achondroplasia.

[0183] This study included 103 anonymized AP hand / wrist radiographs from 30 children with achondroplasia (13 males, 17 females, aged 7.8–16 years). Proprietary data included hand films collected at four time points after the start of treatment: 104 weeks (rollover to the Phase II extension trial), 156 weeks, 208 weeks, and 260 weeks. The length and width of the second metacarpal bone, cortical thickness, robustness (total area / length), and cortical area (correlated with strength) were measured. The measurements were compared to 378 radiographs from 114 mean height controls (61 males, 53 females, aged 6–16 years) via a nonparametric Kruskal-Wallis test (p<0.05).

[0184] At weeks 208 and 260 of treatment, children with achondroplasia showed longer metacarpals with increased cortical area and thickness compared to week 104 (all p<0.05). There were no significant changes in RCA and robustness throughout the treatment time points (Table 5). Furthermore, there were no differences between males and females with achondroplasia at any time point. Compared to controls, children with achondroplasia had higher robustness and cortical area throughout the treatment (p<0.001). [Table 5]

[0185] Additional measurements were taken at weeks 208 and 260. The results are shown in Table 6 and Figures 16A-B. [Table 6]

[0186] An additional 2-3 years of bosolitide treatment was observed to be associated with increased bone length and increased metacarpal cortical area, which correlates with strength, compared to the initial time point (104 weeks). This study suggests that this bone lengthening treatment did not adversely affect bone strength in children with achondroplasia. The lack of a significant difference in robustness after treatment indicates that periosteal expansion continued outward at a pace that maintained robustness, allowing the bone to maintain strength even as it lengthened. Further studies comparing treated and untreated children with achondroplasia are needed to more rigorously confirm whether the treatment did not adversely affect bone strength development. Overall, this study may have important clinical implications for treatment choices in children with achondroplasia.

[0187] As illustrated in the above examples, numerous modifications and variations in this disclosure are expected to occur for those skilled in the art. Therefore, only such limitations as those found in the appended claims should be imposed on this disclosure.

Claims

1. A method for identifying variant genes associated with short stature that are gain-of-function (GoF) or loss-of-function (LoF) variants, - Transfecting cells containing a cGMP-GFP reporter expression construct with a lentiviral vector expressing a polynucleotide encoding a variant protein associated with short stature, which is operablely linked to one or more unique barcode sequences, - Contacting the cells in the culture with C-type natriuretic peptide (CNP) or a variant thereof having CNP activity, - Selecting the cells from the culture based on the level of GFP expression produced by the cells, A method comprising: identifying the variant protein associated with short stature as a GoF variant or a LoF variant, wherein the GoF variant has a higher level of cGMP production compared to a control, and the LoF variant has a lower level of cGMP production compared to a control.

2. The method according to claim 1, wherein the variant gene associated with short stature is selected from the group consisting of natriuretic peptide receptor 2 (NPR2), natriuretic peptide precursor C (NPPC), fibroblast growth factor receptor 3 (FGFR3), or a combination thereof.

3. A method for identifying NPR2 variants as gain-of-function (GoF) or loss-of-function (LoF) variants, - Transfecting cells containing a cGMP-GFP reporter expression construct with a lentiviral vector expressing a polynucleotide encoding an NPR2 variant protein operably linked to one or more unique barcode sequences, - Contacting the cells in the culture with C-type natriuretic peptide (CNP) or a variant thereof having CNP activity, - Selecting the cells from the culture based on the level of GFP expression produced by the cells, A method comprising: identifying the NPR2 variant as a GoF variant or a LoF variant, wherein the GoF variant has a higher level of cGMP production compared to a control, and the LoF variant has a lower level of cGMP production compared to a control.

4. The method according to any one of claims 1 to 3, wherein the cells are a mammalian cell line.

5. The method according to any one of claims 1 to 4, wherein the cells are HEK293 cells.

6. The method according to any one of claims 1 to 5, wherein the cells are sorted by flow cytometry.

7. The method according to any one of claims 1 to 6, wherein the lentiviral vector comprises a polynucleotide encoding a variant gene associated with short stature, a polynucleotide encoding a puromycin resistance gene, and a polynucleotide encoding a T2A-BFP promoter.

8. The method according to any one of claims 1 to 7, wherein the lentiviral vector further comprises 20 to 60 barcode sequences.

9. The method according to claim 8, wherein the barcode sequence is 15 to 30 base pairs.

10. The method according to claim 8 or 9, wherein the barcode sequence is located at the 3' end of the variant gene polynucleotide.

11. The method according to any one of claims 1 to 10, wherein the expression construct comprises a polynucleotide encoding a GFP protein operably linked to a cGMP-binding domain.

12. The method according to claim 11, wherein the cGMP-binding domain is derived from mouse phosphodiesterase 5α.

13. The method according to claim 11 or 12, wherein the expression construct further comprises a CMV promoter operably linked to cGull and a PGK promoter operably linked to a blastosidine resistance gene.

14. The aforementioned CNP variant is PGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP37) (Sequence ID 1), GQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37) (Sequence ID 2), GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP53) (Sequence No. 3), PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP53) (Sequence No. 4), MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLS KGCFGLKLDRIGSMSGLGC (Met-CNP53) (Sequence No. 5), DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53 (M48N)] (Sequence No. 6), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (Sequence No. 7), RVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-51) (Sequence ID 8), VDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-50) (Sequence No. 9), DTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-49) (Sequence ID 10), TKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-48) (Sequence ID 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47) (Sequence ID 12), SRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-46) (Sequence ID 13), RAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-45) (Sequence ID 14), AAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-44) (Sequence ID 15), AWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 16), WARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 17), ARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 18), RLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 19), LLQEHPNA RKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 20), LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 22), EHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 23), HPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC LGC (CNP-35) (SEQ ID NO: 24), PNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 25), NARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 26), ARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 27), RKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 28), KYKGANKGLSKGCFGLKLDRIGS MSGLGC (CNP-30) (SEQ ID NO: 29), YKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 30), KGANKGLSKGCFGLKLDRIGSMSGLGC (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) (Sequence ID 35), KGLSKGCFGLKLDRIGSMSGLGC(CNP-23) (Sequence ID 36), LSKGCFGLKLDRIGSMSGLGC(CNP-21) (Sequence ID 37), SKGCFGLKLDRIGSMSGLGC(CNP-20) (Sequence ID 38), KGCFGLKLDRIGSMSGLGC(CNP-19) (Sequence ID 39), GCFGLKLDRIGSMSGLGC(CNP-18) (Sequence ID 40), QEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N)](Sequence ID 41), PQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37)(Sequence ID 42), MQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37) (Sequence ID 43), GQEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N)](Sequence ID 44), MGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37) (Sequence ID 45), PGQEHPQARRYRGAQRRRRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNAARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNAARRYRGANKRRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49), 【Transformation 3】 A method according to any one of the prior claims, selected from the group consisting of the following.

15. The method according to any one of the prior claims, wherein the CNP is brought into contact with the cells in a dose of 1 to 100 nM.

16. The method according to any one of the prior claims, wherein the lentivirus is transfected into the cells with an infection multiplicity of about 0.1 to about 0.5 MOI.

17. A lentiviral vector comprising a polynucleotide encoding a variant gene associated with short stature, a polynucleotide encoding a puromycin resistance gene, and a polynucleotide encoding a T2A-BFP promoter.

18. The lentiviral vector according to claim 17, wherein the variant gene associated with short stature is selected from the group consisting of natriuretic peptide receptor 2 (NPR2), natriuretic peptide precursor C (NPPC), fibroblast growth factor receptor 3 (FGFR3), or a combination thereof.

19. A lentiviral vector comprising a polynucleotide encoding an NPR2 variant, a polynucleotide encoding a puromycin resistance gene, and a polynucleotide encoding a T2A-BFP promoter.

20. A lentiviral vector according to any one of claims 17 to 19, further comprising 20 to 60 barcode sequences.

21. The lentiviral vector according to claim 20, wherein the barcode sequence is 15 to 30 base pairs.

22. The lentiviral vector according to claim 20 or 21, wherein the barcode sequence is located at the 3' end of the variant gene polynucleotide.

23. A method for creating a lentiviral library containing variant genes associated with short stature, - Amplifying variant genes associated with short stature from mammalian genomes or genomic DNA databases. - Cloning an amplified variant into a lentiviral vector, wherein the lentiviral vector contains 20 to 60 unique barcodes per vector. - Determining the arrangement of the variants related to the barcode in the vector, - Aligning the variant sequence with the control gene sequence to generate a read structure, - Extracting the barcode from the variant lead structure, - Identifying the barcode of each variant, A method comprising isolating a lentiviral vector that expresses a variant gene.

24. The method according to claim 23, wherein the variant gene associated with short stature is selected from the group consisting of NPR2, NPPC, FGFR3, or a combination thereof.

25. A method for creating an NPR2 variant trentivirus library, - Amplifying NPR2 variants from mammalian genomes or genomic DNA databases. - Cloning an amplified NPR2 variant into a lentiviral vector, wherein the lentiviral vector contains 20 to 60 unique barcodes per vector. - Determining the sequence of the NPR2 variants related to the barcode in the vector, - Aligning the NPR2 variant sequence with the control NPR2 gene sequence to generate a read structure. - Extracting the barcode from the NPR2 variant lead structure, - Identifying the barcode of each NPR2 variant, A method comprising isolating a lentiviral vector expressing an NPR2 variant.

26. A method for treating a subject with short stature, comprising administering a CNP variant to a subject identified as having a loss-of-function variant of a gene associated with short stature, identified using the method according to any one of claims 1 to 16.

27. The short stature disorder includes achondroplasia, achondroplasia, short stature, idiopathic short stature, dwarfism, osteochondrodysplasia, thanatophoric osteodysplasia, osteogenesis congenita, achondroplasia, and congenital chondrodysplasia. congenita), homozygous achondroplasia, congenital chondrodysplasia, flexural limb dysplasia, congenital lethal hypophosphatasia hypophosphatasia), perinatal lethal type congenital osteogenesis imperfecta, short rib polydactyly syndrome, proximal limb type chondrodysplasia congener (rhizomeric type of chondrodysplasia congenit), Jansen type metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia congenital, atelosteogenesis, torsional dysplasia, congenital short femur femur), Langer mesomelic dysplasia, Nievergelt mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis The method according to claim 26, selected from the group consisting of disorders associated with dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomellic dysplasia, microlimia, Morquio syndrome, Kniest syndrome, metatropic dysplasia, and spinyloepimetaphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Leri Weill), PTPN11 mutation (Noonan syndrome), and IGF1R mutation.

28. The aforementioned CNP variant is PGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP37) (Sequence ID 1), GQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37) (Sequence ID 2), GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP53) (Sequence No. 3), PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP53) (Sequence No. 4), MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLS KGCFGLKLDRIGSMSGLGC (Met-CNP53) (Sequence No. 5), DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53 (M48N)] (Sequence No. 6), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (Sequence No. 7), RVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-51) (Sequence ID 8), VDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-50) (Sequence No. 9), DTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-49) (Sequence ID 10), TKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-48) (Sequence ID 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47) (Sequence ID 12), SRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-46) (Sequence ID 13), RAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-45) (Sequence ID 14), AAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-44) (Sequence ID 15), AWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 16), WARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 17), ARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 18), RLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 19), LLQEHPNA RKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 20), LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 22), EHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 23), HPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC LGC (CNP-35) (SEQ ID NO: 24), PNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 25), NARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 26), ARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 27), RKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 28), KYKGANKGLSKGCFGLKLDRIGS MSGLGC (CNP-30) (SEQ ID NO: 29), YKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 30), KGANKGLSKGCFGLKLDRIGSMSGLGC (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) (Sequence ID 35), KGLSKGCFGLKLDRIGSMSGLGC(CNP-23) (Sequence ID 36), LSKGCFGLKLDRIGSMSGLGC(CNP-21) (Sequence ID 37), SKGCFGLKLDRIGSMSGLGC(CNP-20) (Sequence ID 38), KGCFGLKLDRIGSMSGLGC(CNP-19) (Sequence ID 39), GCFGLKLDRIGSMSGLGC(CNP-18) (Sequence ID 40), QEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N)](Sequence ID 41), PQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37)(Sequence ID 42), MQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37) (Sequence ID 43), GQEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N)](Sequence ID 44), MGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37) (Sequence ID 45), PGQEHPQARRYRGAQRRRRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNAARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNAARRYRGANKRRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49), 【Chemistry 4】 The method according to claim 26 or 27, selected from the group consisting of the following.

29. The method according to any one of claims 26 to 28, wherein the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP37) (SEQ ID NO: 1), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37) (SEQ ID NO: 2), or LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (SEQ ID NO: 21).

30. The method according to any one of claims 26 to 29, wherein the CNP variant includes a hydrophilic portion.

31. The method according to claim 30, wherein the hydrophilic portion is PEG.

32. A method for treating CNP-responsive bone-related disorders, skeletal dysplasia, or short stature disorders, comprising administering a CNP variant to a subject in need thereof, wherein the CNP variant is administered according to a weight-banding regimen. i) Subjects weighing 10-11 kg receive administration of approximately 22-24 μg / kg of CNP variant. ii) Subjects weighing 12-16 kg received administration of approximately 18-23 μg / kg of the CNP variant. iii) Subjects weighing 17-21 kg received administration of approximately 15-19 μg / kg of CNP variant. iv) Subjects weighing 22-32 kg received administration of approximately 13-18 μg / kg of the CNP variant. v) Subjects weighing 33-43 kg received administration of approximately 12-15 μg / kg of CNP variant. vi) Subjects weighing 44-59 kg received administration of approximately 10-14 μg / kg of the CNP variant. vii) Subjects weighing 60-89 kg who receive approximately 8-12 μg / kg of CNP variant, viiii) A method in which subjects with a body weight of ≥90 kg receive an administration of a CNP variant of approximately ≤9 μg / kg.

33. i) Subjects weighing 10-11 kg received approximately 0.24 mg of the CNP variant. ii) Subjects weighing 12-16 kg received approximately 0.28 mg of the CNP variant. iii) Subjects weighing 17-21 kg received approximately 0.32 mg of the CNP variant. iv) Subjects weighing 22-32 kg received approximately 0.40 mg of the CNP variant. v) Subjects weighing 33-43 kg received approximately 0.50 mg of the CNP variant. vi) Subjects weighing 44-59 kg received approximately 0.60 mg of the CNP variant. vii) Subjects weighing 60-89 kg who receive approximately 0.7 mg of CNP variant, The method according to claim 32, wherein a subject with a body weight of ≥ 90 kg receives an administration of approximately 0.80 mg of a CNP variant.

34. The short stature disorder includes achondroplasia, achondroplasia, short stature, idiopathic short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, congenital osteogenesis imperfecta, achondroplasia, congenital achondroplasia, homozygous achondroplasia, and congenital achondroplasia. Dysplasia, flexural limb dysplasia, congenital fatal hypophosphatasia, perinatal fatal congenital osteogenesis imperfecta, short rib polydactyly syndrome, proximal limb congenital chondrodysplasia, Jansen type metaphyseal dysplasia, congenital spondyloephyseal dysplasia, osteogenesis imperfecta, torsion The method according to claim 32 or 33, selected from the group consisting of disorders associated with ossicular dysplasia, congenital femoral shortening, Langer type intermediate limb dysplasia, Nievergelt type intermediate limb dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysplasia, peripheral dysplasia, Kniest dysplasia, fibrous chondrodysplasia, Roberts syndrome, distal intermediate limb dysplasia, microlimia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepiphyseal metaphysis dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Leri Weill), PTPN11 mutation (Noonan syndrome), and IGF1R mutation.

35. The aforementioned CNP variant is PGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP37) (Sequence ID 1), GQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37) (Sequence ID 2), GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP53) (Sequence No. 3), PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP53) (Sequence No. 4), MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLS KGCFGLKLDRIGSMSGLGC (Met-CNP53) (Sequence No. 5), DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-53 (M48N)] (Sequence No. 6), LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (Sequence No. 7), RVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-51) (Sequence ID 8), VDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-50) (Sequence No. 9), DTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-49) (Sequence ID 10), TKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-48) (Sequence ID 11), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47) (Sequence ID 12), SRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-46) (Sequence ID 13), RAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-45) (Sequence ID 14), AAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-44) (Sequence ID 15), AWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-43) (SEQ ID NO: 16), WARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 17), ARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 18), RLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 19), LLQEHPNA RKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 20), LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 21), QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 22), EHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 23), HPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC LGC (CNP-35) (SEQ ID NO: 24), PNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 25), NARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 26), ARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 27), RKYKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 28), KYKGANKGLSKGCFGLKLDRIGS MSGLGC (CNP-30) (SEQ ID NO: 29), YKGANKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 30), KGANKGLSKGCFGLKLDRIGSMSGLGC (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) (Sequence ID 35), KGLSKGCFGLKLDRIGSMSGLGC(CNP-23) (Sequence ID 36), LSKGCFGLKLDRIGSMSGLGC(CNP-21) (Sequence ID 37), SKGCFGLKLDRIGSMSGLGC(CNP-20) (Sequence ID 38), KGCFGLKLDRIGSMSGLGC(CNP-19) (Sequence ID 39), GCFGLKLDRIGSMSGLGC(CNP-18) (Sequence ID 40), QEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N)](Sequence ID 41), PQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37)(Sequence ID 42), MQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37) (Sequence ID 43), GQEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N)](Sequence ID 44), MGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37) (Sequence ID 45), PGQEHPQARRYRGAQRRRRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 46), PGQEHPNAARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 47), PGQEHPNAARRYRGANKRRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 48), PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 49), 【Transformation 5】 The method according to any one of claims 32 to 34, selected from the group consisting of the following.

36. The method according to any one of claims 32 to 35, wherein the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP37) (SEQ ID NO: 1), GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37) (SEQ ID NO: 2), or LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (SEQ ID NO: 21).

37. The method according to any one of claims 32 to 36, wherein the CNP variant includes a hydrophilic portion.

38. The method according to claim 37, wherein the hydrophilic portion is PEG.

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