Polynucleotides encoding the alpha-1 chain of human type ii collagen and methods of their use

Abstract

The present disclosure relates to compositions, particularly nucleic acid molecules, e.g., polynucleotides encoding a codon-optimized alpha-1 chain of human type II collagen (hCOL2A1), for use in therapy.

Description

POLYNUCLEOTIDES ENCODING THE ALPHA-1 CHAIN OF HUMAN TYPE II COLLAGEN AND METHODS OF THEIR USE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional App. No. 63 / 269,513, filed March 17, 2022, which is hereby incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The content of the electronically submitted sequence listing (Name: 4776_011PC02_Seqlisting_ST26; Size: 156,848 bytes; and Date of Creation: March 15, 2023) is herein incorporated by reference in its entirety. FIELD

[0003] The present disclosure relates to compositions, particularly nucleic acid molecules, e.g., polynucleotides encoding a codon-optimized alpha-1 chain of human type II collagen (hCOL2A1), for use in therapy, especially in gene therapy, more particularly for the treatment of type II collagen disorders. In some aspects polynucleotides may be encoded by or contained within a lentiviral vector. BACKGROUND

[0004] Type II collagen disorders are a group of autosomal dominant rare diseases caused by mutations in the COL2A1 gene encoding for the α1 chain of collagen type II. COL2A1 is a large homotrimeric protein and the main component of extracellular matrix of articular cartilage during bone growth (growth plates), also expressed in the cornea and the vitreous of the eye, the inner ear and the nucleus pulposus of the spine (Kosher et al. J Cell Biol.1986; 102(4):1151-115). Collagen type II plays a fundamental role on the endochondral bone formation and growth. COL2A1 mutations result in a diverse group of clinical phenotypes with a spectrum of severity ranging from severe perinatal lethal disorders to milder conditions presenting in adolescence or adulthood, with prematureosteoarthritis or degenerative joint disease as the primary feature (Gregersen PA, Savarirayan R. Type II Collagen Disorders Overview.2019 Apr 25).

[0005] Type II collagen disorders, or type II collagenopathies, encompass a diverse group of clinical phenotypes which are characterized by skeletal dysplasia, ocular manifestations, hearing impairment and orofacial features (Gregersen et al., 2019). Clinical entities caused by mutations in the COL2A1 gene include Achondrogenesis II, Hypochondrogenesis, Platyspondylic lethal skeletal dysplasias, spondyloepiphyseal dysplasia congenita (SEDc), Stickler syndrome, Kniest dysplasia, Spondyloepimetaphyseal dysplasia (SEMD) Strudwick type, Sponsyloperipheral dyplasia, Spondyloepiphyseal Dysplasia with metatarsal shortening Czech and mild spondyloepiphyseal dysplasia with premature onset arthrosis.

[0006] Achondrogenesis II, hypochondrogenesis, and platyspondylic lethal skeletal dysplasias have lethal presentation perinatally and are characterized by severely under ossified skeleton. Other collagenopathies have a neonatal or childhood presentation and is clinically ranked, severe / moderately severe, intermediate or mild. Spondyloepiphyseal dysplasia congenita (SEDc) is the most severe non-lethal type II collagenopathy. SEDc is a rare disease with a prevalence of 3.4 / 1,000,000, with a prevalence of 1 / 50,000 live births and an autosomal dominant transmission (Nenna R, et al., Appl Clin Genet.2019 Dec 4;12:235-238.; Terhal PA, et al., Am J Med Genet A.2015 Mar; 167A(3):461-75; Terhal PA et al., Am J Med Genet C Semin Med Genet.2012 Aug 15; 160C(3):205-16).

[0007] SEDc is characterized by an abnormality in the development of collagen type II tissues and has a neonatal presentation of severely short stature, hypertelorism, flat profile, myopia and other eye abnormalities. Hearing loss has also been reported. There is an increased risk for cervical instability and spinal cord compression and individuals with SEDc have a greater risk for tracheolaryngomalacia and related respiratory conditions.

[0008] The COL2A1 gene is specifically transcribed in a limited number of tissues including articular cartilage, the cornea and the vitreous of the eye, the inner ear and the nucleus polposus of the spine (Kosher RA, et al., J Cell Biol.1986 Apr; 102(4):1151-6). It encodes the α1 chain of procollagen type II. Three α1 chains are folded together on a triple-helical structure to form the procollagen homotrimer, characterized by Gly-X-Y triplets' repetition. Procollagen chains are first synthesized as pro-peptides containing N- and C-terminal portions. After secretion into the extracellular matrix, the terminal portions are cleaved forming the mature type II collagen that can form collagen fibrils.Collagen type II is the major fibrillar protein of hyaline cartilage and facilitates the specific arrangement of chondrocytes providing an architectural plan for the elongation of bones (Gentili C, Cancedda R. Curr Pharm Des.2009; 15(12):1334-48).

[0009] Several mutations in the COL2A1 gene have been identified in SEDc patients and are directly responsible for the clinical features. The most common mutations are substitutions of a glycine in cysteine or serine in the Gly-X-Y triplets leading to the absence or abnormal fibrils resulting in premature degradation of the cartilage matrix (Nishimura G, et al., Hum Mutat.2005 Jul; 26(1):36-43). It is important to notice that all other enzymes necessary for the complex maturation of collagen are normal. To better understand the mechanisms of said pathologies, some authors tried to develop animal or cellular models.

[0010] Zhang et al. have generated an animal model, a swine model of COL2A1 deficiency, which recapitulates the early skeletal developmental defects in humans (Zhang B, et al. Bone.2020 Aug; 137:115450). The COL2A1sedc / sedcdisease mouse model (B6(Cg)-Col2a1sedc / GrsrJ) is a spontaneous mutation (R1417C) described by the Jackson Laboratory (Donahue et al., J Bone Miner Res.18(9):1612-1621 (2003)), which, within 3 to 6 weeks from birth, recapitulates the key characteristics of the clinical, radiographic, histologic, and functional complications observed in SEDc pediatric patients. Okada et al. have generated new cellular models by converting patient fibroblasts from patients with achondrogenesis into induced chondrocytes or by generating Induced pluripotent stem (iPS) cells (Okada M, et al., Hum Mol Genet.2015 Jan 15;24(2):299-313). In this study, they attempted to decrease endoplasmic reticulum (ER) stress or to enhance other cartilage signaling pathways such as BMP signaling, showing some potential benefit, but most importantly they developed a cellular model that will be useful for pathophysiological studies and drug screening.

[0011] These phenotypes cause life-debilitating skeletal problems (severe disproportionate short stature) associated with joint pain and affect respiratory function, mobility, fatigue, ear and eye impairments, and severe impact on health-related quality of life.

[0012] There is no disease modifying treatment available. Currently, treatment or standard of care consists of surgical procedures to reduce the risk of paralysis (e.g., cervicomedullary decompression or spinal fusion and laminectomy) and utilization of long-term modalities of care (e.g., brace therapy) to reduce pain and restore function.Surgeries are potentially life-threatening or life-debilitating (Savarirayan R, et al., Genet Med.2019 Sep; 21(9):2070-2080) with children having multiple and very complex surgeries during infancy and early childhood, but also well into adulthood. Limb lengthening is not an option as COL2A1 are primarily disorders with short trunk and growth hormone is ineffective (Kanazawa et al. J Bone Miner Metab.21(5):307-310 (2003)).

[0013] Nevertheless, there still exists a serious and unfulfilled need for a curative treatment of type II collagenopathies. Furthermore, there exists a need for tools to treat this genetic disorder. The present disclosure aims at the treatment of type II collagenopathies utilizing gene therapy. BRIEF SUMMARY

[0014] Certain aspects of the disclosure or directed to a polynucleotide comprising a nucleic acid sequence of SEQ ID NOs: 1-13, wherein the polynucleotide encodes the alpha-1 chain of human type II collagen (hCOL2A1).

[0015] In some aspects, the polynucleotide further comprises a promoter element operably linked to the polynucleotide encoding the hCOL2A1 polypeptide.

[0016] In some aspects, the promoter element is a hybrid promoter comprising a fragment of the hEF1α promoter and a fragment of the hCOL2A1 promoter.

[0017] In some aspects, the promoter element comprises the nucleic acid sequence of SEQ ID NOs: 14-33.

[0018] In some aspects, the polynucleotide further comprises a nucleic acid sequence selected from SEQ ID NOs: 38-44, and combinations thereof.

[0019] In some aspects, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 7.

[0020] In some aspects, the promoter element comprises the nucleic acid sequence of SEQ ID NO: 14.

[0021] Certain aspects of the disclosure or directed to a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 36.

[0022] Certain aspects of the disclosure or directed to a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 37.

[0023] Certain aspects of the disclosure or directed to a vector comprising any of the polynucleotides disclosed herein.

[0024] In some aspects, the vector is an adenoviral, retroviral, lentiviral, or adeno- associated viral vector.

[0025] In some aspects, the vector is a lentiviral vector.

[0026] In some aspects, the vector further comprises a nucleic acid sequence of SEQ ID NO: 38-44.

[0027] Certain aspects of the disclosure or directed to a viral particle comprising any of the polynucleotide disclosed herein.

[0028] In some aspects, the particle is a lentiviral particle.

[0029] Certain aspects of the disclosure or directed to a host cell comprising any of the polynucleotides disclosed herein or any of the viral particles disclosed herein.

[0030] Certain aspects of the disclosure or directed to a method for producing a lentiviral particle comprising introducing any of the polynucleotides disclosed herein into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell.

[0031] Certain aspects of the disclosure or directed to a method for transducing a host cell comprising contacting the host cell with any of the lentiviral particle disclosed herein.

[0032] Certain aspects of the disclosure or directed to a pharmaceutical composition comprising any of the polynucleotides disclosed herein, any of the vectors disclosed herein, or any of the viral particles disclosed herein.

[0033] In some aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0034] In some aspects, the pharmaceutically acceptable excipient or carrier is histidine.

[0035] In some aspects, the pharmaceutical composition comprises between about 10 mM to about 30 mM of histidine.

[0036] In some aspects, the pharmaceutically acceptable excipient or carrier is sodium chloride (NaCl).

[0037] In some aspects, the pharmaceutical composition comprises between about 50 mM and about 150 mM of NaCl.

[0038] In some aspects, the pharmaceutically acceptable excipient or carrier is sucrose.

[0039] In some aspects, the pharmaceutical composition comprises between about 2% and about 10% of sucrose.

[0040] In some aspects, the pharmaceutically acceptable excipient or carrier is poloxamer 188.

[0041] In some aspects, the pharmaceutical composition comprises between about 0.0001% and about 0.015% of poloxamer 188.

[0042] In some aspects, the pharmaceutical composition has a pH between about 6 and about 9.

[0043] In some aspects, the pharmaceutical composition comprises about 20 mM of histidine, about 100 mM of NaCl, about 5% of sucrose, and about 0.005% of poloxamer 188.

[0044] In some aspects, the pharmaceutical composition has a pH of about 7.

[0045] Certain aspects of the disclosure or directed to a method of treating a skeletal dysplasia or a type II collagenopathy comprising administering to a subject in need thereof any of the lentiviral particles disclosed herein or any of the pharmaceutical compositions disclosed herein.

[0046] In some aspects, the lentiviral particle is administered subcutaneously, intravenously, intraperitoneally, orally, nasally, intramuscularly, sublingually, intrathecally or intradermally.

[0047] In some aspects, the method further comprises administering about 1E7 transduction units ("TU") / kg, about 1E8 TU / kg, about 1E9 TU / kg, or about 1E10 TU / kg of lentiviral particles to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate certain aspects of the invention, and together with the general description given above and the detailed description of the various aspects given below, serve to explain the principles of the invention.

[0049] Figure 1 (FIG.1) shows a schematic of the INS-101 plasmid construct comprising a hybrid promoter and a codon optimized hCOL2A1 of the present disclosure.

[0050] Figure 2 (FIG.2) shows micro-CT imaging of deformities of sternum forming a pectus in SEDc mice (middle panel, top row) and histology of the 3rdsterbena showing cartilage architecture organization in vehicle treated SEDc mice (middle panel, bottomrow), as compared to vehicle treat wild-type mice (left panel, bottom row) and INS-101 treated SEDc mice (right panel, bottom row).

[0051] Figures 3A-3C (FIGs.3A-3C) show micro-CT imaging and histology of cartilage architecture organization in vehicle treated wild-type mice (FIG.3A), vehicle treated SEDc mice (FIG.3B), and INS-101 treated SEDc mice (FIG.3C).

[0052] Figure 4 (FIG.4) shows GFP expression in ATDC5 (murine chondrogenic cell line) v. BAF cells (B-cell derived murine Baf3 cell) normalized to EF1a promoter expression for hybrid promoters.

[0053] Figure 5 (FIG.5) shows total body radiographic representation of one animal from WT / PBS, SEDc / PBS and SEDc / 1E7 groups (Left Panel); Total body length (mm) of animals that survived at PND22 are indicated as mean ± SEM (number of animals / group are indicated above each bar) (Middle Panel); and survival of animals over time is represented as a Kaplan-Meyer curve (Right Panel). Statistical analysis was performed by ANOVA with * P<0.05, **** P<0.0001, ns: not significant.

[0054] Figure 6 (FIG.6) shows the distance (d) in millimeters (mm) between the occipital edge and the foramen magnum at PND22 (number of mice per group is indicated above each bar). Statistical analysis was performed by ANOVA with ** P<0.005, **** P<0.0001, ns: not significant (note that data for 1E9 TU / kg dose in SEDc mice correspond to only n=1), stat N / A: statistical analysis not available.

[0055] Figure 7 (FIG.7) shows skeletal deformities at PDN22 by measuring the iliac angle (angle in degree between the purple lines on the left panel) and the lumbar 6 (L6) medial width (mm). Number of animals per group is indicated above each bar.

[0056] Figure 8 (FIG.8) shows measurement of pectus excavatum at PND22 as the sternum xyphoid process angle (degree). Number of animals per group is indicated above each bar.

[0057] Figure 9 (FIG.9) shows extracellular matrix content (ECM) as measured by Alcian blue staining (left panel). Histologic architecture of the chondrocyte proliferative region, arranged in column, in mice injected at 1E7 TU / kg was restored (circle). COL2A1 expression was measured by IF (green signal, right panel).

[0058] Figure 10 (FIG.10) shows radiographic imaging and histology of renal and retinal cells after treatment with PBS (top row), repeat dose of 1 x 3E9 INS-101 analog (middle row) or single dose of 1 x 3E9 of INS-101 analog (bottom row).

[0059] Figure 11 (FIG.11) shows matrix production as assessed by histology (Alcian blue staining and Sirius red staining (imaging with polarized light) after 3 weeks of 3-D culture from control (C1C5) or SEDc patient (D96C44) with or without treatment by INS-101. Bottom graph: INS-101 vector copy number (VCN) per cell after escalating dose (MOI) of INS-101 as measured by ddPCR using a probe / primer set targeting the INS-101 transgene cDNA.

[0060] Figure 12 (FIG.12) shows a graph comparing Viral Copy Number ("VCN") per cell from the high dose 1E9TU / kg at the time point 7 days between IV and IP administration. The median is shown, and each dot represents one animal.

[0061] Figure 13 (FIG.13) shows a comparison of INS-101 analog distribution (VCN / cell) in tissues, 1 week and 3 weeks after administration by IP or IV.

[0062] Figure 14 (FIG.14) shows Biodistribution (VCN / cell) of LV-13a-eGFP in minipigs, 1 week after IV or IP administration.

[0063] Figure 15 (FIG.15) shows dose response in SEDc mouse showing distance between occipital edge to foramen magnum, iliac angle and xiphoid process. Number of animals per group is indicated above each bar. DETAILED DESCRIPTION

[0064] Certain aspects of the disclosure or directed to a polynucleotide comprising a nucleic acid sequence of SEQ ID NOs: 1-13, wherein the polynucleotide encodes the alpha-1 chain of human type II collagen (hCOL2A1).

[0065] Certain aspects of the disclosure or directed to a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 36.

[0066] Certain aspects of the disclosure or directed to a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 37.

[0067] Certain aspects of the disclosure or directed to a vector comprising any of the polynucleotides disclosed herein.

[0068] Certain aspects of the disclosure or directed to a viral particle comprising any of the polynucleotide disclosed herein.

[0069] Certain aspects of the disclosure or directed to a host cell comprising any of the polynucleotides disclosed herein or any of the viral particles disclosed herein.

[0070] Certain aspects of the disclosure or directed to a method for producing a lentiviral particle comprising introducing any of the polynucleotides disclosed herein into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell.

[0071] Certain aspects of the disclosure or directed to a method for transducing a host cell comprising contacting the host cell with any of the lentiviral particle disclosed herein.

[0072] Certain aspects of the disclosure or directed to a pharmaceutical composition comprising any of the polynucleotides disclosed herein, any of the vectors disclosed herein, or any of the viral particles disclosed herein.

[0073] Certain aspects of the disclosure or directed to a method of treating a skeletal dysplasia or a type II collagenopathy comprising administering to a subject in need thereof any of the lentiviral particles disclosed herein or any of the pharmaceutical compositions disclosed herein. I. Definitions

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0075] Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

[0076] In order to further define this disclosure, the following terms and definitions are provided.

[0077] The singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or"an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple."

[0078] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0079] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Numeric ranges recited are inclusive of the numbers defining the range and include each integer within the defined range.

[0080] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0081] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure.Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0082] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0083] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0084] As used herein, the term "hCOL2A1" refers to the Homo sapiens α1 chain of human type II collagen (hCOL2A1) cDNA shown in SEQ ID NOs: 34-35 (accession numbers NM_001844.5 and NM_033150.3, that is the reference sequence for the CDS of the mRNA for COL2A1 human; MIM reference 120140), and variants thereof (e.g., codon optimized SEQ ID NOs: 1-13).

[0085] The phrase "operably linked" means that the nucleic acid sequence of interest is linked downstream of the promoter so that said nucleic acid sequence of interest is transcribed according to the promoter activity. The nucleic acid sequence of interest is a gene or a fragment thereof coding for a collagen chain. In some aspects, it is a human gene or fragment thereof coding for a collagen chain wherein said gene or fragment thereof may be native or recombinant.

[0086] The term "codon optimized" means that a codon that expresses a bias for human (i.e. is common in human genes but uncommon in other mammalian genes or non- mammalian genes) is changed to a synonymous codon (a codon that codes for the same amino acid) that does not express a bias for human. Thus, the change in codon does not result in any amino acid change in the encoded protein. Codon optimization allows forhigher levels of mRNA expression by improving mRNA stability, processivity for translation, removal of acceptor / donor sites, etc.

[0087] "Identical" refers to the sequence identity between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are identical at that position. The percent of identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10 of the positions in two sequences are matched then the two sequences are 60% identical. Generally, a comparison is made when two sequences are aligned to give maximum identity. Various bioinformatic tools known to the one skilled in the art might be used to align nucleic acid sequences such as BLAST or FASTA.

[0088] The term "decreased immunogenicity" as applied to the codon-optimized hCOL2A1 coding sequence or to the modified intron of the disclosure means that this codon-optimized gene or modified intron comprises a decreased number of potential alternative open reading frames (or ARFs) in either the intron, or the coding sequence, or both, thereby limiting the number of potential translation protein by-products, in particular from the coded mRNA, as compared to the wild-type cDNA or other hCOL2A1 cDNA variants. In particular, decreased ARFs are those whose length spans over 50 bp and have a stop codon in frame with a start codon. It will be appreciated that the nucleic acid of the disclosure may include one or more polyadenylation signals, typically located at the 3′-end of the molecule.

[0089] The term "promoter" refers to a nucleic acid sequence located upstream or 5' to a translational start codon of an open reading frame (or protein-coding region) of a gene and that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0090] A "hybrid promoter" is a non-native promoter that is functional in host cells, particularly in mammal cells such as human cells. Indeed, the hybrid promoter of the disclosure comprises a part of the sequence of the promoter of hCOL2A1 gene, but also at least the TATA box of the hEF1α promoter (See PCT application WO 2020 / 127533 which is incorporated by reference herein in its entirety). In some aspects, the hybrid promoter comprises a polynucleotide of SEQ ID NOs: 14-33.

[0091] As used herein, the term "vector" refers to a polynucleotide which is capable of being introduced or of introducing the nucleic acid sequence of interest into a cell. In the context of the present disclosure, the nucleic acid sequence of interest is expressed within the cell upon introduction of the vector. Both viral and non-viral vector systems can be employed to deliver a gene to a subject's cells.

[0092] Suitable vectors are known in the art and include, for example, plasmids, artificial chromosomes (e.g. bacterial, mammalian such as human, or yeast artificial), virus or phage vectors provided with an origin of replication, and optionally a regulator of the promoter. Vectors may be used to transfect or transform a host cell, for example, a mammalian host cell, e.g., for the production of the nucleic acid sequence of interest encoded by the vector. The vectors may also be adapted to be used in vivo, for example, in a method of vaccination or gene therapy.

[0093] Examples of suitable viral vectors include retroviral, lentiviral, adenoviral, adeno- associated viral, herpes viral, including herpes simplex viral, alpha-viral, pox viral, such as Canarypox and vaccinia-viral based systems. Gene transfer techniques using these viruses are known in the art. Retroviral vectors, for example, may be used to stably integrate the nucleic acid sequence of interest into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. Vectors capable of driving expression in insect cells (e.g., baculovirus vectors), in human cells, yeast, or in bacteria may be employed in order to produce quantities of the nucleic acid sequence of interest, for example, for use in subunit vaccines or in immunoassays. In an additional example, a replication-deficient simian adenovirus vector may be used as a live vector. These viruses contain an E1 deletion and can be grown on cell lines that are transformed with an E1 gene. These vectors can be manipulated to insert a nucleic acid sequence of interest, such that the encoded protein may be expressed.

[0094] As used herein, the term "subject" or "patient" denotes a human or non-human mammal, such as a rodent, a feline, a canine, an equine, or a primate. In certain aspects, the subject is a human being. Indeed, the codon-optimized sequence of human COL2A, hybrid promoter and vector according to the disclosure are able to penetrate into the cartilaginous matrix of the growth plate, and / or in cells which are annex to cartilage such as perichondrium.

[0095] As used herein, "treat", "treating", or "treatment" of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b)limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s).

[0096] The term "skeletal dysplasia" or "osteochondrodysplasia", as used herein, refers to a heritable group of more than 450 well delineated disorders that affect primarily bone and cartilage, but can also have significant effects on muscle, tendons and ligaments resulting in abnormal shape and size of the skeleton and disproportion of the long bones, spine, and head. By definition, skeletal dysplasias are heritable diseases that have generalized abnormalities in cartilage and bone.

[0097] The term "type II collagenopathy", as used herein, refers to a group of genetic disorders that affect connective tissue, and which are caused by defects in type II collagen. Type II collagenopathies are a subtype of skeletal dysplasia. For example, type II collagenopathies may include spondyloepiphyseal dysplasia congenita (SEDc), Kniest dysplasia, Stickler syndrome, achondrogenesis type 2, hypochondrogenesis, Czech dysplasia, Legg-Calve-Perthes disease, platyspondylic lethal skeletal dysplasia of Torrance type or spondyloepimetaphyseal dysplasia of Strudwick type.

[0098] The term "disease of the musculoskeletal system", as used herein, refers to a skeletal disease which affects joints. For example, the articular pathologies include arthritis, and in particular osteoarthritis, rheumatoid arthritis, gout, septic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis or Still's disease.

[0099] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

[0100] As used herein, the term "transduction unit" or "TU" refers to the number of functional viral particles in a solution that are capable of transducing a cell and expressing a transgene of interest (e.g., codon-optimized hCOL2A1).II. Polynucleotides

[0101] In some aspects, the disclosure relates to a polynucleotide comprising a nucleic acid sequence of SEQ ID NOs: 1-13, in which the polynucleotide encodes a codon optimized sequence of α1 chain of human type II collagen (hCOL2A1). COL2A1 is the gene coding for the α1 chain of procollagen type II (which is also called the pro-α1 (II) chain of type II collagen). The sequence of said human α1 chain of procollagen type II protein may be found in Uniprot under accession number P02458.

[0102] In some aspects, the disclosure results in increased expression of a nucleic acid sequence of interest. The "nucleic acid sequence of interest" may be an RNA or DNA sequence (including cDNA sequences), including that which encodes a gene. The nucleic acid sequence of interest is typically operably linked to the hybrid promoter (See PCT application WO 2020 / 127533 which is incorporated by reference herein in its entirety).

[0103] The collagen chain may be of any one of types I to XXVIII. The collagen chain can be a human collagen chain. In a particular aspect, the collagen chain is the pro-α 1 (II) chain of type II collagen, including the human one (encoded by hCOL2A1 gene).

[0104] To construct type II collagen, three pro-α 1 (II) chains twist together to form a triple- stranded, rope-like procollagen molecule. Procollagen molecules are then processed by enzymes in the cell. Once processed, the molecules leave the cell and arrange themselves into long, thin fibrils that link to one another (cross-link) in the spaces around cells. The cross-linkages result in the formation of very strong, mature type II collagen fibers.

[0105] The present disclosure also relates to a codon optimized hCOL2A1.

[0106] The sequences shown in SEQ ID NOs:1-13 are aspects of the codon optimized nucleic acid sequence of the disclosure.

[0107] The present disclosure also encompasses a nucleic acid sequence of the disclosure encoding a codon optimized human COL2A1 protein that is "substantially identical", i.e. about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 97%, 98% or even 99% identical to the sequence SEQ ID NOs:1-13.

[0108] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 1-13.

[0109] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-13.

[0110] In some aspects, the polynucleotide comprises any one of the nucleic acid sequences described in Table 1. Table 1 – Codon Optimized COL2A1 SE 123456789101112133435

[0111] Advantageously, the codon optimized nucleic acid of the disclosure provides for decreased immunogenicity with a subject.

[0112] The inventors have surprisingly developed a gene therapy to restore bone growth, particularly in the case of type II collagenopathies such as SEDc through the use of thepresent disclosure in gene therapy. In some aspects, this gene therapy approach is allowed thanks to designed hybrid promoters.

[0113] In some aspects, the hybrid promoters used the disclosure allow for the significant expression of type II collagen in specific cells and or tissues that naturally express type II collagen. Such an approach is highly beneficial, because the complex synthesis of type II collagen must be performed at the site of the disease (i.e. in the chondrocytes). COL2A1 gene is generally expressed in the articular cartilage, the cornea and the vitreous of the eye, the inner ear and the nucleus polposus of the spine.

[0114] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 14-33.

[0115] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 14-33.

[0116] In some aspects, the polynucleotide comprises any one of the nucleic acid sequences described in Table 2. Table 2 – Hybrid Promoters SE 14 15 1617 18 19 20 2122 23 2425 26 27 2829 30 31 3233

[0117] More particularly, this tissue specific expression would prove highly advantageous for gene therapy of type II collagenopathies.

[0118] In the context of the present disclosure gene therapy means the use of nucleic acids as a pharmaceutical agent to treat a disease. It derives its name from the idea that nucleic acids can be used to supplement or alter the expression of a gene within an individual's cells as a therapy to treat a disease. The most common form of gene therapy involves using nucleic acids that encode a functional, therapeutic protein in order to replace a mutated gene. Other forms involve direct correction of a mutation, or using nucleic acids that encode a therapeutic protein drug to provide treatment.

[0119] In gene therapy approaches, typically DNA is used even though RNA is also known in recent developments. Importantly, in all these gene therapy approaches mRNA functions as messenger for the sequence information of the encoded protein, irrespectively if DNA, viral RNA or mRNA is used. Delivery of the gene is generally achieved using a delivery vehicle, also known as a vector.

[0120] In some aspects, the vector for delivering the nucleic acid of the disclosure is a viral vector, for example, a retroviral vector, for example an AAV vector, or a non- pathogenic parvovirus, in some aspects a lentiviral vector.

[0121] In some aspects, the vector comprises a codon-optimized sequence of hCOL2A, and the vector encodes a hybrid promoter which controls the expression of the COL2A gene or a fragment thereof coding for a collagen chain, for example the pro-α1 (II) chain of type II collagen.

[0122] The vectors of the present disclosure provide long-term gene expression resulting in long-term expression of the therapeutic protein. As described herein, the phrases long term expression, permanent expression and persistent expression are used interchangeably. Long-term expression according to the present disclosure means the expression of a therapeutic gene and / or protein, at therapeutic levels, for at least 45 days, at least 60 days, at least 90 days, at least 120 days, at least 180 days at least 250 days, at least 360 days, at least 450 days, at least 730 days or more. Long-term expression means expression for at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days or more typically at least 360 days, at least 450 days, at least 720 days or more. This long term expression can be achieved through repeated doses or a single dose.

[0123] Repeated doses can be given twice a day, daily, twice a week, weekly, monthly, every two months, every three months, every four months, every six months, annually, every two years or more. The dosing can be continued as long as required, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years or more, up to all the lifetime of the patient to be treated.

[0124] The vectors of the present disclosure provide high levels of gene expression resulting in high levels (therapeutic levels) of the expression of a therapeutic protein. Expression can be measured by any suitable method (qualitative or quantitative), and concentrations can be reported in any appropriate unit of measurement, such as ng / ml. A high level of expression according to the present disclosure may mean expression of a therapeutic gene and / or protein at a concentration of at least 10 ng / ml, at least 20 ng / ml, at least 30 ng / ml, at least 40 ng / ml at least 50 ng / ml, at least 60 ng / ml, at least 70 ng / ml, at least 80 ng / ml, at least 90 ng / ml, at least 100 ng / ml, by at least 200 ng / ml, at least 300 ng / ml, at least 400 ng / ml, at least 500 ng / ml, at least 600 ng / ml, at least 700 ng / ml, atleast 800 ng / ml, at least 900 ng / ml, at least 1000 ng / ml, at least 2000 ng / ml, at least 3000 ng / ml, at least 4000 ng / ml, at least 5000 ng / ml, at least 10,000 ng / ml, at least 15,000 ng / ml, at least 20,000 ng / ml or more. Therapeutic expression can be set using the same values.

[0125] Lentiviral vectors of the present disclosure generally provide high levels of transgene expression when administered to a patient. The terms high expression and therapeutic expression are used interchangeably herein.

[0126] In some aspects, the polynucleotide of the present disclosure comprises the nucleic acid sequence corresponding to SEQ ID NOs: 38-44, or combinations thereof.

[0127] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 38-44.

[0128] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 38-44.

[0129] In some aspects, the polynucleotide comprises any one of the nucleic acid sequences described in Table 3 and combinations thereof. Table 3 – Lentiviral Plasmid Elements SE 38 39 40 41 42 43 44

[0130] Expression regulatory signals may be selected to be compatible with the host cell for which expression is designed. The introduced nucleic acid sequence of interest can be stably or transiently maintained in the host cell using the vector of the present disclosure. Stable maintenance typically requires that the introduced nucleic acid sequence of interest either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0131] In some aspects, the polynucleotide of the present disclosure comprises the nucleic acid sequence corresponding to SEQ ID NO: 36.

[0132] In some aspects, the polynucleotide of the present disclosure comprises the nucleic acid sequence corresponding to SEQ ID NO: 37.

[0133] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 36.

[0134] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 36.

[0135] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 37.

[0136] In some aspects, provided herein is a polynucleotide comprising a nucleic acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 37.

[0137] In some aspects, the polynucleotide comprises any one of the nucleic acid sequences described in Table 4.Table 4 – Exemplary Plasmid and Expression Construct SE 3637III. Pharmaceutical compositions and formulations

[0138] In some aspects, provided herein is a pharmaceutical composition comprising a polynucleotide of the present disclosure, and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject.

[0139] In some aspects, the pharmaceutical composition comprises about 20 mM of histidine, about 100 mM of NaCl, about 5% of sucrose, and about 0.005% of poloxamer 188. In some aspects, the pharmaceutical composition has a pH of about 7.

[0140] Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.

[0141] Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising the polynucleotide of the present disclosure or a pluralitythereof (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.18th ed. (1990)). The pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0142] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed.

[0143] In some aspects, the excipient is sucrose. In some aspects, the pharmaceutical composition comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of sucrose. In some aspects, the pharmaceutical composition comprises between about 2% and about 10% of sucrose.

[0144] In some aspects, the excipient is poloxamer 188. In some aspects, the pharmaceutical composition comprises about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, or about 0.015% of poloxamer 188. In some aspects, the pharmaceutical composition comprises between about .0001% and about 0.015% of poloxamer 188.

[0145] In some aspects, the pharmaceutical composition has a pH of about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9. In some aspects, the pharmaceutical composition has a pH between about 6 and about 9.

[0146] A composition of the present disclosure (e.g., including a vector comprising the codon-optimized nucleic acid sequence of interest and hybrid promoter according to the disclosure) can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals. In particular, the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon- optimized COL2A1, and the hybrid promoter, can be administered by any route known in the art, e.g., subcutaneous (e.g., by subcutaneous injection), intravenously, intraperitoneally, orally, nasally, intramuscularly, sublingually, intrathecally or intradermally. By way of example, pharmaceutical compositions of the disclosure can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gel cap, powder, gel, ointment, cream, nebulae, mist, atomized vapor, aerosol or phytosome.

[0147] Apart from the specific delivery systems embodied below in the examples, various delivery systems are known and can be used to administer the nucleic acid of thedisclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the codon-optimized nucleic acid of interest coding sequence, such as codon-optimized COL2A, receptor-mediated endocytosis, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of administration of the nucleic acid include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.

[0148] In some aspects, the nucleic acid sequence of the disclosure, whether vectorized or not, may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the disclosure into the liver of the subject by any suitable route. In addition naked DNA such as minicircles and transposons can be used for delivery or lentiviral vectors.

[0149] In some aspects, the nucleic acid of the disclosure can be delivered in a vesicle, in particular a liposome.

[0150] In some aspects, the nucleic acid of the disclosure can be delivered in a controlled release system.

[0151] Any amount of a pharmaceutical composition (including a vector comprising the codon-optimized nucleic acid sequence of interest and hybrid promoter according to the disclosure) can be administered to a patient, such as a patient with a skeletal dysplasia (e.g., a patient with a type II collagenopathy such as spondyloepiphyseal dysplasia congenita). The dosages will depend on many factors including the mode of administration and the age of the patient. Typically, the amount of the composition (e.g., including a vector according to the disclosure) can be an amount that is effective to treat a condition (e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita) as described herein without inducing significant toxicity.

[0152] In some aspects of the disclosure, at least one dose, of the composition comprising the vector of the disclosure, can be an amount that is effective to treat a condition (e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita) as described herein. In some aspects, the composition comprising the vector of the disclosure may be administered once a week.

[0153] Dosages of compositions comprising the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized COL2A1, and hybrid promoter according to the disclosure can be provided in either a single or multiple dosage regimens.

[0154] Alternatively, doses can be administered, e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, 10 times, 11 times or 12 times per 12 years.

[0155] The amount, frequency, and duration of dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Carriers / vehicles

[0156] The present disclosure also provides pharmaceutical compositions comprising a nucleic acid of the disclosure, or the vector of the disclosure, or the cell of the disclosure. Such compositions comprise a therapeutically effective amount of the therapeutic (the nucleic acid, vector or cell of the disclosure), and a pharmaceutically acceptable carrier.

[0157] Compositions comprising the vector of the disclosure may be provided to patients in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.

[0158] Intravenous or intraperitoneal vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991).

[0159] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the therapeutic, typically in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0160] The pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.

[0161] These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0162] In a particular aspect, the nucleic acid, vector or cell of the disclosure is formulated in a composition comprising phosphate-buffered saline and supplemented with 0.25% human serum albumin. In another particular aspect, the nucleic acid, vector or cell of the disclosure is formulated in a composition comprising ringer lactate and a non- ionic surfactant, such as pluronic F68 at a final concentration of 0.01-0.0001%, such as at a concentration of 0.001%, by weight of the total composition. The formulation may further comprise serum albumin, in particular human serum albumin, such as human serum albumin at 0.25%. Other appropriate formulations for either storage or administration are known in the art, in particular from WO 2005 / 118792 or Allay et al., 2011.

[0163] In some aspects, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration tohuman beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of the injection.

[0164] The amount of the therapeutic (i.e. a nucleic acid, vector or cell) of the disclosure which will be effective in the treatment can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays may optionally be employed to help predict optimal dosage ranges. As previously mentioned, the precise dose to be employed in the formulation will also depend on the route of administration and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. The dosage of the nucleic acid, the vector or the cell administered to the subject in need thereof will vary based on several factors including, without limitation, the route of administration, the specific disease treated, the subject's age or the level of expression necessary for the required therapeutic effect.

[0165] One skilled in the art can readily determine, based on their knowledge in this field, the dosage range required based on these factors and others.

[0166] In some aspects, the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized COL2A1, and hybrid promoter according to the disclosure can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0167] In some aspects, the pharmaceutical composition comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM of histidine. In some aspects, the pharmaceutical composition comprises between about 10 mM and about 30 mM of histidine.

[0168] In some aspects, the pharmaceutical composition comprises about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM,about 125 mM, about 130 mM, about 140 mM, or about 150 mM of NaCl. In some aspects, the pharmaceutical composition comprises between about 50 mM and about 150 mM of NaCl.

[0169] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be useful to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0170] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

[0171] For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of a specific fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the subject.

[0172] In some aspects, the pharmaceutical composition is stored in a vial or a syringe. In some aspects, the vial is suitable for cryostorage (i.e. a cryovial).

[0173] The invention is described by way of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention. IV. Therapeutic uses

[0174] The present disclosure also relates to the use of a codon-optimized sequence of human COL2A under the control of a hybrid promoter, to the use of a vector comprising the codon-optimized sequence of human COL2A and hybrid promoter according to the disclosure, or the use of the pharmaceutical composition of the present disclosure in therapy. In some aspects, the therapy is gene therapy. Said gene therapy may be an ex vivo gene therapy. Ex vivo gene therapy consists of transfecting a vector comprising the disclosure -optimized sequence of human COL2A and hybrid promoter according to the disclosure into isolated cells, and thereafter transplanting the transfected cells into the patient. Useful cells which may be of interest are mesenchymal stem cells (MSC) cells or cells removed from the patient himself. Alternatively, said gene therapy may be an in vivo gene therapy. In vivo gene therapy comprises injecting a vector comprising the hybrid promoter according to the disclosure directly into the patient.

[0175] The present disclosure further relates to a method for treating a subject in need thereof, which comprises administering to said subject a vector comprising the hybrid promoter according to the disclosure.

[0176] In some aspects, the virus is administered to a subject at a dose of about 1E6 transduction units ("TU") per kg, about 1E7 TU / kg, about 1E8 TU / kg, about 1E9 TU / kg, about 1E10 TU / kg, about 1E11 TU / kg, about 1E12 TU / kg, or about 1E13 TU / kg. In some aspects, the virus is administered to a subject at a dose of between about 1E6 TU / kg to about 1E13 TU / kg.

[0177] In some aspects, the subject has been diagnosed as suffering from a skeletal dysplasia as described herein.

[0178] In some aspects, the subject has been diagnosed as suffering from an asphyxiating syndrome as described herein.

[0179] In some aspects, the subject has been diagnosed as suffering from a type II collagenopathy as described herein.

[0180] In some aspects, the subject has been diagnosed as suffering from a disease of the musculoskeletal system as described herein.

[0181] The vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized COL2A1, and hybrid promoter according to the disclosure is delivered through gene therapy, where it is delivered to tissues of interest and expressed in vivo. Gene therapy methods are discussed, e.g., in Verme et al. (Nature 389:239-242, 1997), Yamamoto et al. (Molecular Therapy 17:S67-S68, 2009), and Yamamoto et al., (J. Bone Miner. Res.26: 135-142, 2011).

[0182] According to an aspect, the present disclosure further relates to the use of a codon- optimized human COL2A under the control of a hybrid promoter of the disclosure, or to the use of a vector comprising the codon-optimized human COL2A and hybrid promoter according to the disclosure, for treating a type II collagenopathy.

[0183] Examples of skeletal dysplasia include spondyloepiphyseal dysplasia congenita (SEDc), Kniest dysplasia, Stickler syndrome, Jeune syndrome (or asphyxiating thoracic dystrophy), achondroplasia, homozygous achondroplasia, heterozygous achondroplasia, achondrogenesis, acrodysostosis, acromesomelic dysplasia, atelosteogenesis, camptomelic dysplasia, chondrodysplasia punctata, rhizomelic type of chondrodysplasia punctata, cleidocranial dysostosis, congenital short femur, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), dactyly, brachydactyly, camptodactyly, Polydactyly, syndactyly, diastrophic dysplasia, dwarfism, dyssegmental dysplasia, enchondromatosis, fibrochondrogenesis, fibrous dysplasia, hereditary multiple exostoses, hypochondroplasia, hypophosphatasia, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Langer-type mesomelic dysplasia, Marfan syndrome, McCune-Albright syndrome, micromelia, metaphyseal dysplasia, Jansen-type metaphyseal dysplasia, metatrophic dysplasia, Morquio syndrome, Nievergelt-type mesomelic dysplasia, neurofibromatosis (e.g. type 1 , e.g., with bone manifestations or without bonemanifestations; type 2; schwannomatosis; or any described herein), osteochondrodysplasia, osteogenesis imperfecta, perinatal lethal type of osteogenesis imperfecta, osteopetrosis, osteopoikilosis, peripheral dysostosis, Reinhardt syndrome, Roberts syndrome, Robinow syndrome, short-rib Polydactyly syndromes, short stature, spondyloepimetaphyseal dysplasia, thanatophoric dysplasia, spondylocostal dysostosis, pseudorheumatoid dysplasia, all the aggrecanopathies, or Leri Weil dyschondrosteosis.

[0184] Accordingly, the disclosure provides for the treatment of a skeletal dysplasia, such as notably spondyloepiphyseal dysplasia congenita (SEDc), Kniest dysplasia, Stickler syndrome or Jeune syndrome.

[0185] Accordingly, the disclosure provides for the treatment of a type II collagenopathy, and spondyloepiphyseal dysplasia congenita (SEDc), Kniest dysplasia or Stickler syndrome.

[0186] Provided herein are notably methods for treating a skeletal dysplasia in a patient, such as a patient (like a human) having a type II collagenopathy such as spondyloepiphyseal dysplasia congenita. In particular, the patient may exhibit or may be likely to have one or more symptoms of a skeletal dysplasia (e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita). The method involves administering a vector comprising a codon-optimized nucleic acid sequence of interest, such as a sequence for a collagen chain (such as the pro-α 1 (II) chain of type II collagen encoded by COL2A1 gene), or a fragment thereof, operably linked to a hybrid promoter, or a fragment thereof, to the patient (e.g. a human).

[0187] For example, the patient exhibits signs or symptoms of a skeletal dysplasia, such as those described herein (e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita), e.g., prior to administration of the vector. Treatment with a vector of the disclosure can also occur after a patient (e.g., a human) has been diagnosed with a skeletal dysplasia, such as those described herein (e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita), or after a patient exhibits signs or symptoms of a skeletal dysplasia, such as those described herein (e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita).

[0188] Treatment with a vector comprising the codon-optimized nucleic acid of interest, such as human COL2A1, according to the disclosure can result in an improvement in a symptom of a skeletal dysplasia, e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita. The methods can be used to treat symptomsassociated with a skeletal dysplasia, e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita, such that there is reversal or a reduction in the severity of symptoms of the skeletal dysplasia, e.g., a type II collagenopathy such as spondyloepiphyseal dysplasia congenita.

[0189] Any skeletal dysplasia that is a type II collagenopathy (e.g., caused by or associated with a mutation in COL2A1 gene) can be treated by administering a vector comprising a hybrid promoter of the disclosure operably linked to a codon-optimized sequence coding for a collagen chain, such as the pro-α 1 (II) chain of type II collagen encoded by COL2A1 gene, as described herein to a patient (e.g., a human). For example, such a vector can be administered to treat a type II collagenopathy such as spondyloepiphyseal dysplasia congenita.

[0190] Administration of such a vector can be used to treat the type II collagen disorders resulting from mutations of the pro-alpha 1 (II) chain of type II collagen encoded by COL2A1 gene. These mutations include, but are not limited to p.R275C, p.G504S, p.G546S, p.R437P, p.R740C, p.R789C, p.G207E, p.G246R, p.G333E, p.G351V, p.G354D, p.G354R, p.G369D, p.G372R, p.G375D, p.G384S, p.G393S, p.G396V, p.G408D, p.G411R, p.G420E, p.G429D, p.G438S, p.G441C, p.G444D, p.G447A, p.G450A, p.G456A, p.G474S, p.G477V, p.G483R, p.G483E, p.G486D, p.G495E, p.G504A, p.G522V, p.G537D, p.G537S, p.G561S, p.G594E, p.G609V, p.G612A, p.G621R, p.G621E, p.G624D, p.G654S, p.G672S, p.G675D, p.G684R, p.G687R, p.G687D, p.G687S, p.G690R, p.G705S, p.G725S, p.G726D, p.G759D, p.G759V, p.G771V, p.G801S, p.G813R, p.G822C, p.G822S, p.G831R, p.G846E, p.G849S, p.G870E, p.G873R, p.G897R, p.G903S, p.G909S, p.G912D, p.G918S, p.G921R, p.G1014V, p.G1062A, p.G1062C, p.G1083D, p.G1083V, p.G1086V, p.G1089V, p.G1092S, p.G1095S, p.G1101R, p.G1110S, p.G1152D, p.G1155V, p.G1158S, p.G1173R, p.G1173V, p.G1176V, p.G1179R, p.G1188A, p.G1197S mutations of the pro- alpha 1 (II) chain of type II collagen encoded by COL2A1 gene, as well as any one of the mutations of COL2A1 gene causing Stickler syndrome, such as the C57Y, G216D, G219R, G222V, G240D, G267D, G270R, G282D, del302-308, G453A, G492D, G501R, R565C, R904C, G1131A or G1158A mutations, or any other mutations of the COL2A1 gene. For example, 36% of SEDc mutations include: c.2965C>T p.(Arg989Cys) 8%; c.1510G>A p.(Gly504Ser) 5%; c.3589G>A p.(Gly1197Ser) 5%; c.4349T>C p.(Ile1450Thr) 5%; c.1781G>A p.(Gly594Glu) 4%; c.1214G>A p.(Gly405Asp) 3%;c.3283G>A p.(Gly1095Ser) 3%; c.3455G>A p.(Gly1152Asp) 3%. Interestingly, G susbstitution in D, S or V leads to severe type II collagen disorder (achondrogenesis, hypochondrogensis or SEDc). Especially, the type II collagenopathy may be spondyloepiphyseal dysplasia congenita (SEDc). In some aspects of the disclosure, the type II collagenopathy may be due to a glycine substitution.

[0191] In some aspects, the mutation is selected from the group consisting of c.74G>C; c.431G>T; c.611G>C; c.611G>T; c.619G>A; c.620G>A; c.629G>A; c.638G>A; c.638G>T; c.647G>A; c.655G>A; c.655G>C; c.656G>A; c.665G>T; c.692G>A; c.719G>A; c.718G>T; c.736G>C; c.737G>A; c.800G>A; c.808G>C; c.827G>A; c.845G>A; c.863G>C; c.881G>A; c.908G>A; c.926G>T; c.944G>T; c.952G>A; c.980G>A; c.998G>A; c.1016G>A; c.1034G>A; c.1043G>A; c.1043G>T; c.1052G>T; c.1060G>A; c.1070G>C; c.1106G>A; c.1114G>A; c.1115G>A; c.1123G>C; c.1124G>A; c.1132G>A; c.1133G>A; c.1141G>C; c.1142G>A; c.1148G>A; c.1150G>A; c.1151G>T; c.1177G>A; c.1187G>T; c.1195G>T; c.1196G>T; c.1205G>A; c.1214G>A; c.1223G>A; c.1223G>T; c.1231G>A; c.1259G>A; c.1276G>C; c.1286G>A; c.1312G>A; c.1321G>T; c.1331G>A; c.1340G>C; c.1340G>A; c.1339G>A; c.1349G>C; c.1349G>A; c.1348G>A; c.1358G>C; c.1358G>A; c.1357G>A; c.1358G>T; c.1367G>C; c.1366G>C; c.1376G>A; c.1385G>A; c.1385G>T; c.1394G>C; c.1393G>A; c.1403G>A; c.1402G>A; c.1403G>T; c.1420G>A; c.1430G>T; c.1447G>A; c.1448G>A; c.1457G>A; c.1466G>A; c.1475G>A; c.1475G>T; c.1484G>A; c.1493G>T; c.1501G>A; c.1501G>C; c.1511G>C; c.1510G>A; c.1511G>T; c.1519G>A; c.1529G>A; c.1537G>A; c.1547G>A; c.1555G>A; c.1565G>T; c.1574G>A; c.1610G>A; c.1609G>A; c.1628G>A; c.1636G>A; c.1637G>T; c.1654G>A; c.1681G>A; c.1717G>A; c.1736G>A; c.1744G>A; c.1772G>A; c.1781G>A; c.1789G>C; c.1799G>A; c.1799G>T; c.1808G>T; c.1817G>A; c.1826G>T; c.1835G>C; c.1861G>A; c.1862G>A; c.1871G>A; c.1879G>C; c.1888G>A; c.1960G>A; c.1969G>C; c.2014G>T; c.2014G>A; c.2024G>A; c.2032G>A; c.2033G>A; c.2050G>C; c.2051G>T; c.2059G>C; c.2060G>A; c.2059G>A; c.2068G>A; c.2104G>A; c.2113G>A; c.2131G>C; c.2141G>T; c.2149G>A; c.2150G>T; c.2175G>A; c.2177G>A; c.2213G>A; c.2230G>A; c.2249G>T; c.2258G>A; c.2276G>A; c.2275G>A; c.2276G>T; c.2303G>A; c.2312G>C; c.2312G>A; c.2312G>T; c.2320G>A; c.2338G>A; c.2357G>A; c.2383G>C; c.2384G>A; c.2401G>A; c.2411G>C; c.2419G>A; c.2437G>A; c.2465G>A; c.2464G>T; c.2464G>A;c.2491G>A; c.2537G>A; c.2546G>A; c.2545G>A; c.2564G>T; c.2574C>T; c.2582G>A; c.2582G>T; c.2600G>A; c.2609G>A; c.2617G>A; c.2618G>T; c.2662G>A; c.2671G>C; c.2681G>A; c.2689G>A; c.2707G>A; c.2725G>T; c.2725G>A; c.2735G>A; c.2752G>T; c.2752G>A; c.2761G>C; c.2789G>A; c.2807G>A; c.2806G>A; c.2833G>A; c.2843G>A; c.2860G>A; c.2897G>C; c.2905G>A; c.2906G>T; c.2941G>A; c.2950G>C; c.2950G>A; c.3013G>A; c.3023G>T; c.3041G>T; c.3050G>T; c.3121G>A; c.3158G>A; c.3158G>T; c.3185G>C; c.3184G>T; c.3194G>T; c.3238G>C; c.3248G>A; c.3248G>T; c.3257G>T; c.3265G>A; c.3266G>T; c.3275G>A; c.3274G>A; c.3283G>A; c.3293G>A; c.3301G>A; c.3328G>T; c.3328G>A; c.3329G>T; c.3337G>T; c.3355G>A; c.3364G>A; c.3383G>T; c.3392G>C; c.3419G>C; c.3419G>A; c.3427G>T; c.3427G>A; c.3437G>A; c.3455G>A; c.3464G>T; c.3473G>C; c.3472G>A; c.3490G>T; c.3490G>A; c.3508G>A; c.3517G>C; c.3517G>A; c.3518G>A; c.3518G>T; c.3527G>A; c.3526G>A; c.3527G>T; c.3535G>A; c.3545G>A; c.3563G>C; c.3562G>C; c.3563G>A; c.3571G>A; c.3590G>C; c.3589G>A; c.3598G>T; c.3598G>A; c.3608G>A; c.3644G>C; c.3674G>C; c.3914G>A; c.4327G>A; c.4433G>A; c.3G>T; c.1A>G; c.17C>A; c.170G>A; c.398C>A; c.409C>T; c.410G>A; c.434C>T; c.823C>T; c.905C>T; c.985C>T; c.1018G>A; c.1300C>T; c.1310G>C; c.1309C>T; c.1364C>T; c.1373C>T; c.1480C>T; c.1543C>T; c.1648C>T; c.1676C>G; c.1693C>T; c.1999C>T; c.2155C>T; c.2290A>G; c.2710C>T; c.2819G>A; c.2957C>T; c.2965C>T; c.2965C>G; c.2974A>G; c.3106C>G; c.3136C>T; c.3397C>T; c.3655G>C; c.3662C>T; c.3713A>G; c.3826C>T; c.3853G>T; c.3892T>A; c.3903C>A; c.3928G>A; c.3965C>A; c.3965C>T; c.3969C>G; c.3968G>A; c.3977C>A; c.4016G>T; c.4082A>G; c.4135C>T; c.4148C>T; c.4169C>A; c.4172A>G; c.4385T>G; c.4196A>G; c.4201G>C; c.4228G>C; c.4316C>T; c.4342A>C; c.4349T>A; c.4349T>C; c.4375C>T; c.4405G>C; c.4405G>T; c.4436T>G; c.4453T>C; c.4453T>G; c.70C>T; c.85C>T; c.115C>T; c.123T>A; c.140G>A; c.141G>A; c.192C>A; c.258C>A; c.556G>T; c.598C>T; c.625C>T; c.1030C>T; c.1522G>T; c.1597C>T; c.1714C>T; c.1777C>T; c.1957C>T; c.1963G>T; c.1993C>T; c.2047C>T; c.2101C>T; c.2116G>T; c.2129C>T; c.2353C>T; c.2467G>T; c.2473G>T; c.2653G>T; c.2659C>T; c.2794C>T; c.2818C>T; c.2839C>T; c.2869G>T; c.3106C>T; c.3280C>T; c.3574C>T; c.3709C>T; c.3714C>A; c.3878G>A; c.4011G>A; c.4314C>A; c.4335G>A; c.4339A>T; c.4423C>T; c.4462T>C; c.86- 50C>G; c.293-1G>A; c.292+258A>G; c.342+1G>A; c.430-1G>C; c.655-1G>C; c.654+1G>A; c.709-2A>G; c.709-1G>C; c.709-1G>T; c.763-2A>G; c.762+3G>C;c.762+5G>A; c.817-9G>A; c.817-1G>C; c.816+1G>A; c.816+6C>T; c.870+1G>A; c.870+5G>A; c.870+5G>T; c.925-1G>A; c.1024-2A>C; c.1023+1G>A; c.1023+1G>T; c.1023+2T>A; c.1023+2T>G; c.1023+6T>A; c.1069-2A>G; c.1068+1G>A; c.1123- 2A>G; c.1123-1G>A; c.1222-2A>G; c.1221+1G>A; c.1221+1G>C; c.1221+3A>C; c.1267-2A>G; c.1266+1G>C; c.1266+1G>T; c.1365+1G>A; c.1366-2A>T; c.1366- 1G>C; c.1375G>C; c.1365+1G>C; c.1420-2A>C; c.1420-2A>G; c.1420-1G>C; c.1419+5G>A; c.1419+5G>T; c.1528-2A>G; c.1527+1G>C; c.1527+4A>T; c.1527+104T>G; c.1527+135G>A; c.1582-2A>G; c.1581+1G>A; c.1680+1G>A; c.1681-1G>C; c.1735-1G>A; c.1734+5G>A; c.1833+1G>A; c.1888-2A>G; c.1888- 1G>C; c.1887+1G>A; c.1942-2A>G; c.1962C>T; p.Gly654Gly; c.1941+1G>A; c.1996- 9G>A; c.1996-1G>A; c.1995+1G>A; c.2050-1G>C; c.2049+1G>A; c.2095-1G>A; c.2094+1G>A; c.2194-1G>A; c.2193+2T>C; c.2302-1G>A; c.2301+1G>A; c.2356- 2A>G; c.2356-1G>A; c.2355+1G>A; c.2355+4A>C; c.2355+5G>A; c.2409+1G>T; c.2518-2A>G; c.2518-1G>A; c.2517+1G>C; c.2517+2T>G; c.2626-2A>G; c.2625+1G>T; c.2679+1G>A; c.2679+5G>C; c.2734-1G>T; c.2896-1G>A; c.2862C>T; p.Gly954Gly); c.3003+1G>A; c.3003+1G>T; c.3003+5G>A; c.3112-1G>A; c.3111+1G>A; c.3111+1G>T; c.3111+2T>C; c.3165+1G>T; c.3274-2A>G; c.3327+1G>C; c.3435+18C>A; c.3435+79A>T; c.3435+83C>G; c.3597+1G>A; c.3597+1G>C; c.3597+2T>G; c.3887-2A>G; c.3887-1G>T; c.3886+1G>A; c.3886+1G>T; c.3886+2T>C; c.4075-2A>G; c.4074+1G>T; c.4074+2_4074+3delTG; c.4317+2T>C; c.146delC; c.166_167delGT; c.171_172delTG; c.184delG; c.221delA; c.237delG; c.251_252delAG; c.266_278del13; c.294delG; c.311delG; c.492delT; c.510delT; c.572delC; c.724delC; c.744delT; c.793delG; c.816+1delG; c.883delC; c.895_898delAAGG; c.905_906delCG; c.924+1delG; c.931_934delAGTG; c.933delT; c.941delC; c.986delC; c.996_997delAG; c.1023+3_1023+6delGAGT; c.1032delA; c.1095delT; c.1172delC; c.1219_1221+4delTCCGTAA; c.1221+1delG; c.1266delT; c.1266+1delG; c.1279_1296del18; c.1313delG; c.1326delT; c.1416delA; c.1419+3_1419+6delGAGT; c.1435delC; c.1420_1430del11; c.1566_1570delTCTGG; c.1657_1675del19; c.1674delA; c.1732delT; c.1821delC; c.1828delG; c.1908delT; c.1931delC; c.1970delG; c.2077_2078delGG; c.2096_2105del10; c.2259_2266delCGAGAGGG; c.2263_2264delAG; c.2342delA; c.2344_2355+5del17; c.2381delC; c.2382delT; c.2463+4_2463+7delAGTG; c.2471delG; c.2478_2479delGA; c.2539delG; c.2588_2604del17; c.2678delC; c.2751delT; c.2760delT;c.2763_2771delTCCTTCTGG; c.2787delA; c.2789delG; c.2813delC; c.2814delT; c.2858delC; c.2948_2950delTCG; c.2976_2977delAG; c.2999delC; c.3012delC; c.3023delG; c.3050delG; c.3062_3079del18; c.3056_3058delCTG; c.3137delC; c.3138delT; c.3198_3206delTCCTGGAGC; c.3228delT; c.3261_3264delCAGA; c.3264_3265delAG; c.3266_3268delGAG; c.3325delC; c.3356delG; c.3401delG; c.3488delG; c.3495delT; c.3623delC; c.3624delT; c.3627_3644del18; c.3641delC; c.3778_3779delAA; c.3864_3865delCT; c.3872_3873delCT; c.3906delC; c.3957delT; c.3978delC; c.4088delA; c.4152delA; c.4161_4165delGAACA; c.4167delC; c.4231delC; c.4300delC; c.4311delC; c.4332delG; c.4337delG; c.4357delC; c.4413_4416delAGGG; c.4441_4452del12; c.4458_4460delCTT; SEDT; c.158_161dupGGAT; c.406dupG; c.491dupC; c.509dupC; c.583dupC; c.635dupC; c.756dupT; c.1137dupC; c.1399_1400dupCA; c.1428_1429insTGGC; c.1602dupG; c.1658_1675dup18; c.1783dupG; c.1836_1843dupTGAGCCTG; c.1943dupG; c.2219dupC; c.2381dupC; c.2436dupA; c.2490dupC; c.2493dupA; c.2678dupC; c.2715dupT; c.2723dupC; c.2750dupC; c.2813dupC; c.2908_2909dupCC; c.3023dupG; c.3046dupA; c.3137dupC; c.3464dupG; c.3469_3486dup18; c.3596dupC; c.3641dupC; c.3891_3898dupCTACTGGA; c.4134dupA; c.4287_4291dupCACGT; c.4384_4387dupATCA; c.25_26delACinsTA; c.1250_1256delGAGCCAAins17; c.1311_1313delGGGinsCA; c.1468_1475delAAGAGAGGinsT; c.1509_1510delCGinsTT; c.1680+1_1680+2delinsAA; c.2147_2151delAGGGTinsC; c.2631_2634delins14; c.3081_3087delGACGGGTinsCCTGG; c.3446_3447delGAinsTC; c.3447delAinsTCCTGCTG; c.3574_3576delCGAinsTGC; c.3619_3620delCCinsA; deletion of <700 kb incl. entire gene; deletion of 21 bp I17E18+1 to I17E18+21; deletion of 25 bp nt.25715 cd.731; deletion of 28 bp E12I12-19 to E12I12+9; deletion of 390 bp incl. ex.48; deletion of c.1491_1527+11del48; deletion of c.2745_3221del; deletion of c.2974_3003+5del36; deletion of c.85+1360_1023+555del; deletion of entire gene, c.(?_181)_(*442_?)del; deletion of Exons 2-18; duplication of 45 bp cd.970-984; duplication of c.211_233dup23; duplication of entire gene; p.Gly5Arg; p.Gly5Ala; p.Gly32Ser; p.Gly32Gly; p.Gly38Arg; p.Gly60Arg; p.Gly83Arg; p.Glu84Glu; p.Gly101Val; p.Gly104Arg; p.Gly117Arg; p.Gly117Ala; p.Gly162Ala; p.Gly168Asp; p.Gly171Arg; p.Gly171Cys; p.Gly171Val; p.Gly177Glu; p.Gly186Arg; p.Gly201Glu; p.Gly324Ala; p.Gly345Ser; p.Gly360Ser; p.Gly381Ala; p.Gln467Arg; p.Gly609Ala; p.Gly618Ala; p.Gly630Cys; p.Gly636Ser; p.Gly666Glu; p.Gly684Cys; p.Gly732Ser;p.Gly738Ala; p.Gly768Ser; p.Gly936Ser; p.Gly942Ala; p.Gly945Ser; p.Gly1014Ala; p.Gly1044Ala; p.Gly1215Ala; p.Gly1227Ser; p.Gly1232Ser; p.Gly1246Ser; p.Gly1274Arg; p.Gly1296Arg; p.Gly1296Ala; p.Gly1305Ala; p.Gly1355Asp; p.Gly1385Asp; p.Gly1385Ala; p.Gly1405Ser; p.Gly1471Arg; p.Gly1478Ser; p.Gly120Aspfs*79; p.Arg533*; p.Asn545Thrfs*84; p.Gly795TrpfsTer6; p.Gly939TrpfsTer5; p.Gly1101*; and combinations thereof.

[0192] In some aspects, the mutation is a glycine missense mutation (e.g., a mutation as described in Al Kaissi (2013) Orthopade 42, 963; Almatrafi (2019) Genet Test Mol Biomarkers 23: 310; Bae (2016) Genet Med 18, 563; Barat-Houari (2016) Eur J Hum Genet 24, 992; Barat-Houari (2016) Hum Mutat 37, 7; Bertoli-Avella (2021) Eur J Hum Genet 29, 141; Bogaert (1992) J Biol Chem 267, 22522; Bonaventure (1995) Biochem J 307, 823; Boucher (2020) Proc Natl Acad Sci U S A 117, 31278; Cao (2012) Genet Mol Res 11: 4130; Carss (2014) Hum Mol Genet 23, 3269; Chan (1991) J Biol Chem 266, 12487; Chan (1995) J Biol Chem 270, 1747; Chandler (2018) Genet Med 20, 1430; Chen (2017) BMC Pediatr 17: 175; Chen (2020) Eur J Obstet Gynecol Reprod Biol 251, 119; Chitty (2006) Prenat Diagn 26, 861; Choy (2019) Front Genet 10, 761; Codsi (2015) Case Rep Obstet Gynecol 2015: 935204; Comstock (2010) Am J Med Genet A 152A, 1822; Cui (2012) Orphanet J Rare Dis 7, 55; Dasa (2019) Am J Med Genet A 179, 534; Deden (2020) Prenat Diagn 40, 972; Dikaiakou (2018) Mol Syndromol 9: 241; Dogan (2019) Balkan J Med Genet 22, 89; Downie (2020) Eur J Hum Genet 28, 587; Faivre (2004) Am J Med Genet 126A, 308; Fan (2021) J Genet Genomics epub, epub; Forzano (2007) Am J Med Genet A 143A, 2815; Freisinger (1994) J Biol Chem 269, 13663; Güneş (2018) Mol Syndromol 9134; Han (2020) Prenat Diagn 40: 577; Hauer (2018) Genet Med 20, 630; Heinrich (2015) Cytogenet Genome Res 145: 25; Higuchi (2017) J Med Case Rep 11, 237; Homma (2019 J Pediatr 215:192 PMID 31630891; Hoornaert (2010) Eur J Hum Genet 18, 872; Horton (1992) Proc Natl Acad Sci U S A 89, 4583; Hu (2018) Genet Med 20, 1045; Huang (2015) PLoS One 10, 0127529; Huang (2018) Cell Physiol Biochem 49: 295; Huang (2020) Genes (Basel) 11, 882; Jackson (2012) Hum Mutat 33, 144; Jakkula (2005) Osteoarthritis Cartilage 13, 497; Ji (2019) Cold Spring Harb Mol Case Stud 5, ; Jung (2004) J Pediatr Orthop B 13, 63; Jurgens (2015) Hum Mutat 36, 1004; Kannu (2010) Arthritis Rheum 62, 1421; Kannu (2011) Clin Orthop Relat Res 469, 1785; Karen collection; Kawano (2015) Am J Med Genet A 167, 1578; Kim (2021) Front Genet 12:670608 PMID 34122524; Kishiya (2014) J Med Case Rep 8, 276; Klee (2021) GenetMed 23, 498; Korkko (1993) Am J Hum Genet 53, 55; Korkko (2000) Am J Med Genet 92, 95; Kuivaniemi (1997) Hum Mutat 9, 300; Lee (2011) J Genet Med 8125; Lefebvre (2021) J Med Genet 58, 400; Li (2014) Joint Bone Spine 81, 86; Li (2014) PLoS One 9, e100505; Li (2015) Eur Spine J 24: 1813; Li (2021) Int J Gen Med 14: 2569; Liu (2005) N Engl J Med 352, 2294; Liu (2015) PLoS One 10, e0133636; Liu (2016) Eur Spine J 25, 2967; Liu (2018) Mol Med Rep 18:254 PMID 29750297; Liu (2019) Diagn Pathol 14, 76; Liu (2019) Mol Genet Genomic Med 7, ; Machol (2017) Am J Med Genet A 173: 733; Maddirevula (2018) Genet Med 20, 12; Maddox (1997) Matrix Biol 16: 93; Marinakis (2021) Am J Med Genet A 185: 2561; Matsubayashi (2013) Mol Syndromol 4, 148; Meng (2017) JAMA Pediatr 171, 173438; Meredith (2007) Br J Ophthalmol 91, 1148; Merrick (2015) Am J Med Genet A 167A, 3103; Miyamoto (2007) Hum Genet 121: 625; Modi (2017) PLoS One 12: e0174356; Morrison (2020) Am J Med Genet A 182, 2191; Mortier (2000) J Med Genet 37, 263; Mundlos (1996) Am J Med Genet 63, 129; Nagendran (2012) Am J Med Genet A 158A, 1204; Nakane (2011) Mol Syndromol 1, 21; Nakane (2011) Mol Syndromol 2, 21; Nakashima (2016) Hum Genome Var 3, 16007; Nishimura (2005) Hum Mutat 26, 36; Okamoto (2012) Am J Med Genet A 158A, 1953; Peng (2021) Front Genet 12, 728544; Petrovski (2019) Lancet 393: 758; Polla (2015) PLoS One 10, e0138314; Powis (2018) Genet Med 20, 1468; Quaio (2020) Am J Med Genet C Semin Med Genet 184, 955; Retterer (2016) Genet Med 18, 696; Richards (2002) J Med Genet 39, 661; Richards (2005) Invest Ophthalmol Vis Sci 46, 663; Richards (2007) Hum Mutat 28, 639; Richards (2008) Eye (Lond) 22, 1243; Richards (2010) Hum Mutat 31, E1461; Ritvaniemi (1994) Hum Mutat 3, 261; Rolvien (2020) Osteoarthritis Cartilage 28: 334; Ruault (2020) Arthritis Rheumatol 72:1689; Rukavina (2014) Semin Arthritis Rheum 44, 101; Sangsin (2016) Genet Mol Res 15, 017624; Savasta (2015) Eur J Pediatr 174;697 PMID 2580973; Scocchia (2019) NPJ Genom Med 4: 5; Sellick (2006) Clin Dysmorphol 15, 197; Sergouniotis (2015) Eye (Lond) 29: 475; Sobetzko (2000) Am J Med Genet 90, 239; Stranneheim (2021) Genome Med 13, 40; Su (2010) Int Orthop 34: 1333; Sun (2015) Invest Ophthalmol Vis Sci 56, 8365; Takagi (2016) Am J Med Genet A 170, 795; Tang (2020) Clin Chim Acta 507, 187; Terhal (2012) Am J Med Genet C Semin Med Genet 160C, 205; Terhal (2015) Am J Med Genet A 167, 461; Tiller (1995) Nat Genet 11, 87; Tolusso (2021) Genet Med 23:909 PMID 33442022; Travessa (2020) Am J Med Genet A 182: 2715; Trujillano (2017) Eur J Hum Genet 25, 176; Turner (2019) Am J Hum Genet 105: 1274; Tysoe (2003) QJM 96, 663;van der Sluijs (2019) Genet Med 21, 1074; Vikkula (1993) Genomics 16, 282; Vissing (1989) J Biol Chem 264, 18265; Walter (2007) Am J Med Genet A 143A, 161; Wang (2014) Mol Med Rep 10;1835 PMID 25050885; Wang (2019) Case Rep Obstet Gynecol 2019, 7981767; Wang (2019) Zhonghua Yi Xue Yi Chuan Xue Za Zhi 36, 694; Wang (2021) Hum Mutat 42, 434; Weis (1998) J Biol Chem 273, 4761; Wilkin (1994) Hum Mol Genet 3, 1999; Williams (1995) Hum Mol Genet 4, 309; Winterpacht (1994) Hum Mutat 4, 257; Winterpacht (1995) Hum Genet 95, 437; Wright (2019) Nat Commun 10, 2985; Wu (2021) BMC Med Genetomics 14:170 PMID 34182999; Wubben (2018) Ophthalmic Genet 39: 615; Xia (2007) Clin Chim Acta 382: 148; Xiong (2015) Science 347: 1254806; Xiong (2018) Hum Genome Var 5, 17059; Xu (2014) Eur Spine J 23S2: 271; Xu (2020) Int J Biol Sci 16, 859; Yan (2020) Prenat Diagn 40: 1459; Zanolli (2020) Ophthalmic Genet 41, 36; Zechi-Ceide (2008) Clin Dysmorphol 17, 225; Zhang (2011) Biochem Biophys Res Commun 413, 504; Zhang (2015) J Hum Genet 60, 769; Zhang (2019) Zhonghua Fu Chan Ke Za Zhi 54, 221; Zhang (2021) BMC Med Genomics 14, 147; Zhang (2021) Clin Genet 100, 219; Zheng (2020) Mol Genet Genomic Med 8, ; Zhou (2018) Exp Eye Res 171, 76; Zhou (2021) J Clin Lab Anal 35: e23728).

[0193] In some aspects, the mutation is a missense mutation (e.g., a mutation as described in Ala-Kokko (1990) Proc Natl Acad Sci U S A 87, 6565; Bae (2016) Genet Med 18, 563; Ballo (1998) Am J Med Genet 80, 6; Barat-Houari (2016) Am J Med Genet A 170A: 263; Barat-Houari (2016) Eur J Hum Genet 24, 992; Barat-Houari (2016) Hum Mutat 37, 7; Bhattacharya (2018) Genome Res 28, 423; Bleasel (1995) J Rheumatol 22: 255; Bleasel (1996) J Rheumatol 23: 1594; Capalbo (2019) PLoS Genet 15: e1008409; Chan (1993) J Biol Chem 268, 15238; Jensen (2011) Hum Mutat 32: 794; Chen (2020) Eur J Obstet Gynecol Reprod Biol 251, 119; Chung (2013) Am J Med Genet A 161: 918; Clark (2019) Sci Transl Med 11, ; Copiova (2020) Ann Hum Genet 84:380 PMID 32427345; Deden (2020) Prenat Diagn 40: 972; Fakhro (2019) Hum Mol Genet 28, 3970; Girisha (2020) Am J Med Genet A 182, 338; Goyal (2016) Case Rep Pediatr 2016: 3198597; Guo (2017) Hum Genome Var 4:17040 PMID 28983407; Hammarsjö (2016) Am J Med Genet A 170A, 266; Hoornaert (2006) J Med Genet 43, 406; Hoornaert (2007) Eur J Hum Genet 15: 1269; Huang (2018) Cell Physiol Biochem 49, 295; Huang (2018) Int J Mol Med 42, 1819; Huang (2020) Genes (Basel) 11:882; Kannu (2011) Am J Med Genet A 155, 1759; Kars (2021) Proc Natl Acad Sci U S A 118: e2026076118; Kausthubham (2021) Hum Mutat 42: e15-e61; Kosaki (2020) Am J Med Genet A 182, 1601; Kusano (2017) HumGenome Var 4, 17003; Liu (2020) Hum Mutat 41, 2167; Marinakis (2021) Am J Med Genet A 185, 2561; Mark (2011) Am J Med Genet A 155, 174; McAlinden (2008) Hum Mutat 29, 83; Mier (2001) Mol Genet Metab 74: 338; Miyagawa (2013) PLoS One 8, e71381; Modi (2017) PLoS One 12: e0174356; Morgan (2020) Genes (Basel) 11, 1237; Mortier (2000) J Med Genet 37, 263; Nagendran (2012) Am J Med Genet A 158A: 1204; Nguyen (2021) Am J Med Genet A 185, 2108; Nishimura (2004) J Med Genet 41, 75; Nishimura (2005) Hum Mutat 26, 36; Peng (2021) Front Genet 12, 728544; Plachy (2019) J Clin Endocrinol Metab 104, 4273; Plachy (2021) J Clin Endocrinol Metab 106: 1742; Richards (2000) Am J Hum Genet 67, 1083; Richards (2007) Hum Mutat 28, 639; Richards (2010) Hum Mutat 31, E1461; Ruault (2020) Arthritis Rheumatol 72:1689; Seo (2020) Clin Genet 98:562 PMID 32901917; Silveira (2015) Am J Med Genet 167A:894 PMID 25735649; Sulko (2005) Am J Med Genet 137A, 292; Sun (2015) Invest Ophthalmol Vis Sci 56: 8365; Terhal (2012) Am J Med Genet C Semin Med Genet 160C, 205; Terhal (2015) Am J Med Genet A 167, 461; Tham (2015) Clin Genet 87, 496; Tomckova (2018) Cesk Slov Oftalmol 74:108 PMID 30650974; Turner (2019) Am J Hum Genet 105, 1274; Unger (2001) Am J Med Genet 104, 140; Wang (2016) Mol Vis 22: 697; Williams (1993) Hum Genet 92, 499; Wu (2015) Wubben (2018) Ophthalmic Genet 39, 615; Xu (2020) In J Biol Sci 16:859 PMID 32071555; Yang (2018) BMC Med Gente 19:212 PMID 30541462; Zampaglione (2020) Genet Med 22:1079 PMID 32037395; Zankl (2005) Am J Med Genet 133A, 61; Zhang (2011) Biochem Biophys Res Commun 413: 504; Zhang (2021) BMC Med Genomics 14; Zhou (2018) Exp Eye Res 171:76 PMID 29453956).

[0194] In some aspects, the mutation is a nonsense mutation (e.g., a mutation as described in Ahmad (1991) Proc Natl Acad Sci U S A 88, 6624; Ahmad (1993) Am J Hum Genet 52, 39; Annunen (1999) Am J Hum Genet 65, 974; Barat-Houari (2016) Eur J Hum Genet 24, 992; Bedeschi (2011) Orphanet J Rare Dis 6, 7; Donoso (2002) Am J Ophthalmol 134, 720; Hoornaert (2010) Eur J Hum Genet 18, 872; Huang (2020) Genes (Basel) 11, 882; Khan (2021) Mol Genet Genomic Med 9: e1628; Kondo (2016) Hum Genome Var 3, 16018; Liberfarb (2003) Genet Med 5, 21; Maddirevula (2018) Genet Med 20, 12; Maddirevula (2018) Genet Med 20: 1609; McAlinden (2008) Hum Mutat 29, 83; Mehta (2017) Ophthalmic Surg Lasers Imaging Retina 48, 83; Nagendran (2012) Am J Med Genet A 158A: 1204; Nishimura (2005) Hum Mutat 26, 36; Plachy (2021) J Clin Endocrinol Metab 106, 1742; Richards (2000) Br J Ophthalmol 84, 364; Richards (2006)Hum Mutat 27, 696; Richards (2010) Hum Mutat 31, E1461; Riise (2018) BMC Med Genet 19: 155; Savasta (2015) Eur J Pediatr 174: 697; Stark (2017) Eur J Hum Genet 25: 1268; Stone (2017) Ophthalmology 124: 1314; Sun (2015) Invest Ophthalmol Vis Sci 56: 8365; Tompson (2017) Ophthalmic Genet 38, 43; Tran-Viet (2013) Mol Vis 19: 759; Wang (2016) Mol Vis 22, 697; Wang (2019) Acta Ophthalmol 98: e440; Xiong (2015) Science 347:1254806; Xu (2020) Int J Biol Sci 16, 859; Zankl (2004) Am J Med Genet 129A, 144; Zankl (2005) Am J Med Genet 133A, 61; Zechi-Ceide (2008) Eur J Med Genet 51, 183; Zhou (2018) Exp Eye Res 171:76; Zhou (2018) Mol Vis 24: 560.

[0195] In some aspects, the mutation is a splicing mutation (e.g., a mutation as described in Ala-Kokko (1990) Proc Natl Acad Sci U S A 87, 6565; Bae (2016) Genet Med 18, 563; Ballo (1998) Am J Med Genet 80, 6; Barat-Houari (2016) Am J Med Genet A 170A: 263; Barat-Houari (2016) Eur J Hum Genet 24: 992; Barat-Houari (2016) Hum Mutat 37:7; Bhattacharya (2018) Genome Res 28, 423; Bleasel (1995) J Rheumatol 22: 255; Bleasel (1996) J Rheumatol 23: 1594; Capalbo (2019) PLoS Genet 15: e1008409; Chan (1993) J Biol Chem 268, 15238; Jensen (2011) Hum Mutat 32: 794; Chen (2020) Eur J Obstet Gynecol Reprod Biol 251, 119; Chung (2013) Am J Med Genet A 161: 918; Clark (2019) Sci Transl Med 11; Copiova (2020) Ann Hum Genet 84:380 PMID 32427345; Deden (2020) Prenat Diagn 40: 972; Fakhro (2019) Hum Mol Genet 28, 3970; Girisha (2020) Am J Med Genet A 182, 338; Goyal (2016) Case Rep Pediatr 2016: 3198597; Guo (2017) Hum Genome Var 4:17040 PMID 28983407; Hammarsjö (2016) Am J Med Genet A 170A, 266; Hoornaert (2006) J Med Genet 43, 406; Hoornaert (2007) Eur J Hum Genet 15: 1269; Huang (2018) Cell Physiol Biochem 49, 295; Huang (2018) Int J Mol Med 42, 1819; Huang (2020) Genes (Basel) 11; Kannu (2011) Am J Med Genet A 155, 1759; Kars (2021) Proc Natl Acad Sci U S A 118: e2026076118; Kausthubham (2021) Hum Mutat 42: e15-e61; Kosaki (2020) Am J Med Genet A 182, 1601; Kusano (2017) Hum Genome Var 4, 17003; Liu (2020) Hum Mutat 41, 2167; Marinakis (2021) Am J Med Genet A 185, 2561; Mark (2011) Am J Med Genet A 155, 174; McAlinden (2008) Hum Mutat 29, 83; Mier (2001) Mol Genet Metab 74: 338; Miyagawa (2013) PLoS One 8: e71381; Modi (2017) PLoS One 12: e0174356; Morgan (2020) Genes (Basel) 11, 1237; Mortier (2000) J Med Genet 37, 263; Nagendran (2012) Am J Med Genet A 158A: 1204; Nguyen (2021) Am J Med Genet A 185, 2108; Nishimura (2004) J Med Genet 41, 75; Nishimura (2005) Hum Mutat 26, 36; Peng (2021) Front Genet 12, 728544; Plachy (2019) J Clin Endocrinol Metab 104, 4273; Plachy (2021) J Clin Endocrinol Metab 106: 1742; Richards (2000) AmJ Hum Genet 67, 1083; Richards (2007) Hum Mutat 28, 639; Richards (2010) Hum Mutat 31, E1461; Ruault (2020) Arthritis Rheumatol 72: 1689; Seo (2020) Clin Genet 98:562 PMID 32901917; Silveira (2015) Am J Med Genet 167A:894 PMID 25735649; Sulko (2005) Am J Med Genet 137A, 292; Sun (2015) Invest Ophthalmol Vis Sci 56: 8365; Terhal (2012) Am J Med Genet C Semin Med Genet 160C, 205; Terhal (2015) Am J Med Genet A 167, 461; Tham (2015) Clin Genet 87, 496; Tomckova (2018) Cesk Slov Oftalmol 74:108 PMID 30650974; Turner (2019) Am J Hum Genet 105, 1274; Unger (2001) Am J Med Genet 104, 140; Wang (2016) Mol Vis 22:697 PMID 4919091; Williams (1993) Hum Genet 92, 499; Wubben (2018) Ophthalmic Genet 39, 615; Xu (2020) In J Biol Sci 16:859 PMID 32071555; Yang (2018) BMC Med Gente 19:212 PMID 30541462; Zampaglione (2020) Genet Med 22:1079 PMID 32037395; Zankl (2005) Am J Med Genet 133A, 61; Zhang (2011) Biochem Biophys Res Commun 413: 504; Zhang (2021) BMC Med Genomics 14; Zhou (2018) Exp Eye Res 171:76 PMID 29453956).

[0196] In some aspects, the mutation is a small deletion (e.g., a deletion as described in Ahmad (1995) Arch Ophthalmol 113, 1454; Annunen (1999) Am J Hum Genet 65, 974; Bae (2016) Genet Med 18, 563; Baijens (2004) Int J Pediatr Otorhinolaryngol 68, 1573; Barat-Houari (2016) Eur J Hum Genet 24, 992; Barat-Houari (2016) Hum Mutat 37:7; Brown (1992) Arch Ophthalmol 110, 1589; Brown (1995) Hum Mol Genet 4, 141; Chem (2019) Clin exp Ophthalmol 48:486 PMID 31872526; Čopíková (2020) Ann Hum Genet 84, 380; Čopíková (2020) Ann Hum Genet 84: 380; Dai (2021) Arch Dis Child epub; Désir (2012) Am J Med Genet A 158A, 1948; Faber (2000) J Med Genet 37, 318; Gerth- Kahlert (2011) J AAPOS 15, 311; Gupta (2002) Am J Ophthalmol 133, 203; Hoornaert (2010) Eur J Hum Genet 18, 872; Huang (2020) Genes (Basel) 11, 882; Ideura (2019) Sci Rep 9, 11976; Kondo (2016) Hum Genome Var 3, 16018; Korkko (2000) Am J Med Genet 92, 95; Kosaki (2020) Am J Med Genet A 182: 1601; Kosmicki (2017) Nat Genet 49: 504; Liberfarb (2003) Genet Med 5, 21; Liu (2020) Exp Eye Res 197: 108118; Meredith (2007) Br J Ophthalmol 91, 1148; Mortier (2000) J Med Genet 37, 263; Nishimura (2004) J Med Genet 41, 75; Nishimura (2005) Hum Mutat 26, 36; Richards (2000) Br J Ophthalmol 84, 364; Richards (2006) Hum Mutat 27, 696; Richards (2010) Hum Mutat 31, E1461; Sangsin (2016) BMC Med Genet 17, 96; Smith (2010) Obstet Gynecol 115, 460; Spranger (1997) Am J Med Genet 69, 79; Stevenson (2012) Eur J Med Genet 55, 418; Stranneheim (2021) Genome Med 13: 40; Terhal (2012) Am J Med GenetC Semin Med Genet 160C, 205; Terhal (2015) Am J Med Genet A 167:461; Walter (2007) Am J Med Genet A 143A, 161; Wang (2016) Mol Vis 22, 697; Wang (2019) Acta Ophthalmol 98, e440; Wang (2019) Case Rep Obstet Gynecol 2019:7981767; Wilkin (1999) Am J Med Genet 85, 105; Winterpacht (1996) J Med Genet 33, 649; Wubben (2018) Ophthalmic Genet 39, 615; Yoshida (2006) Eye (Lond) 20, 743; Zankl (2004) Am J Med Genet 129A, 144; Zankl (2005) Am J Med Genet 133A, 61; Zechi-Ceide (2008) Eur J Med Genet 51, 183; Zhang (2013) Gene 522:107; Zhang (2015) J Hum Genet 60, 769; Zhou (2018) Exp Eye Res 171: 76; Zhou (2018) Mol Vis 24:560).

[0197] In some aspects, the mutation is a small insertion (e.g., an insertion as described in Barat-Houari (2016) Eur J Hum Genet 24:992; Barat-Houari (2016) Hum Mutat 37, 7; Clark (2019) Sci Transl Med 11; Hoornaert (2010) Eur J Hum Genet 18, 872; Huang (2020) Genes (Basel) 11, 882; Kingsmore (2019) Am J Hum Genet 105, 719; Nishimura (2005) Hum Mutat 26, 36; Richards (2000) Br J Ophthalmol 84, 364; Richards (2006) Hum Mutat 27, 696; Richards (2010) Hum Mutat 31, E1461; Stranneheim (2021) Genome Med 13, 40; Sun (2015) Invest Ophthalmol Vis Sci 56, 8365; Sun (2015) Invest Ophthalmol Vis Sci 56: 8365; Terhal (2012) Am J Med Genet C Semin Med Genet 160C, 205; Terhal (2015) Am J Med Genet A 167, 461; Vu (2003) Ophthalmology 110, 70; Wang (2021) Hum Mutat 42, 434; Yoon (2016) Ann Lab Med 36:166; Zabel (1996) Am J Med Genet 63, 123; Zechi-Ceide (2008) Eur J Med Genet 51, 183).

[0198] In some aspects, the mutation is an indel (e.g., an indel as described in Chung (2009) J Mol Biol 390, 306; Hoornaert (2010) Eur J Hum Genet 18, 872; Huang (2020) Genes (Basel) 11, 882; Liu (2016) Eur Spine J 25, 2967; Nishimura (2005) Hum Mutat 26, 36; Olavarrieta (2008) Clin Genet 73, 262; Richards (2006) Hum Mutat 27, 696; Tiller (1995) Nat Genet 11, 87; Zhou (2018) Exp Eye Res 171, 76).

[0199] In some aspects, the mutation is a gross deletion (e.g., a gross deletion as described in Annunen (1999) Am J Hum Genet 65, 974; Barat-Houari (2016) Eur J Hum Genet 24, 992; Kondo (2016) Hum Genome Var 3, 16018; Lee (1989) Science 244, 978; Richards (2010) Hum Mutat 31, E1461; van der Hout (2002) Hum Mutat 20, 236; Wilkin (1999) Am J Med Genet 85:105; Winterpacht (1993) Nat Genet 3, 323).

[0200] In some aspects, the mutation is a gross duplication (e.g., a gross duplication as described in Tiller (1990) Proc Natl Acad Sci U S A 87, 3889; Richards (2010) Hum Mutat 31, E1461).

[0201] Thus, a patient treated with the vector described herein may have any of the mutations in the COL2A1 gene as described above.

[0202] Any of the aforementioned mutations in the COL2A1 gene can be detected in a sample from the patient (e.g., a human with a type II collagenopathy such as spondyloepiphyseal dysplasia congenita) prior to or after treatment (e.g., treatment with the vector described herein). Additionally, the parents and siblings of the patient and / or fetal samples (e.g., fetal nucleic acid obtained from maternal blood, placental, or fetal samples) may be tested by methods known in the art for the mutation.

[0203] Administration of a vector as disclosed herein may result in an improvement in symptoms including, but not limited to, growth retardation, skull deformities, orthodontic defects, cervical cord compression (with risk of death, e.g., from central apnea or seizures), spinal stenosis (e.g., leg and lower back pain), hydrocephalus (e.g., requiring cerebral shunt surgery), hearing loss due to chronic otitis, cardiovascular disease, neurological disease, respiratory problems, fatigue, pain, numbness in the lower back and / or spin, and obesity.

[0204] Patients treated using the vector described herein may include, e.g., infants, children, and adults with a skeletal dysplasia or disease of the musculoskeletal system. Infants are often diagnosed with type II collagenopathies at birth, and thus, treatment with a vector as described herein, may begin as early as possible in the patient's life, e.g., shortly after birth, or prior to birth (in utero).

[0205] Symptoms of skeletal dysplasia (e.g., type II collagenopathy) or disease of the musculoskeletal system in patients (e.g., humans) may also be monitored prior to or after a patient is treated with a vector comprising the codon-optimized nucleic acid sequence of interest according to the disclosure. For instance, the symptoms may be monitored prior to treatment to assess the severity of the disease and condition of the patient prior to performing the methods. The methods of the disclosure may include diagnosis of the disease in a patient and monitoring the patient for changes in the symptoms of the disease, such as changes in body weight, body height, sitting height, skull shape, skull length, growth velocity and / or skull width but also cervical instability, respiratory complications of the patient based on changes monitored over a period of time, e.g., 1 , 2, 3, 4 or more times per month or per year or approximately every 1, 2, 3, 4, 5, 6, 7, 8, 12 or 16 weeks over the course of treatment with the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized COL2A1, and hybrid promoter of thepresent disclosure. Body weight and / or skull size of the patient or changes thereof can also be determined at treatment specific events, e.g. before and / or after administration of the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized COL2A1, and hybrid promoter according to the disclosure. For example, body weight and / or skull size are measured in response to administration of the vector of the present disclosure.

[0206] Body weight can be measured simply be weighing the subject on a scale, in a standardized manner, e.g. with the same (in particular for humans) or no clothes or at a certain time of the day, typically in a fasting state (for example in the morning before breakfast is taken, or after at least 1, 2, 3, 4, 5 or more hours of fasting). Skull size is represented by length, height, width and / or circumference.

[0207] Measurements can be taken by any known or self-devised standardized method. For a human subject, the measurement of skull circumference is done. It is usually taken with a flexible and non-stretchable material such as a tape, which is wrapped around the widest possible circumference of the head (though not around the ears or the facial area below and including the eyebrows), e.g. from the most prominent part of the forehead around to the widest part of the back of the head. Another measurement for a human subject can determine the height of the skull, for example from the underside of the chin to the uppermost point of the head. For a rodent subject, the measurement of the length of the skull (e.g. tip of the nasal bone to back of the occipital bone).

[0208] Alternatively, also the width of the skull (e.g. widest points of the parietal bone), the height of the skull (e.g. lowest point of the angular process of lower jaw to frontal bone) or the volume of the skull are measured. Any measurement is taken more than once, e.g. at least 3 times, and the largest number is taken as the length, height, width and / or circumference.

[0209] In the case of human subjects, the symptoms of the disease to be considered are typically changes in body weight, body height, sitting height, skull shape, long bone length (such as tibia length, humerus length and / or femur length), cervical instability, respiratory complications and / or neurological complications. EXAMPLE Example 1 – Codon Optimization of hCOL2A1

[0210] The codon optimization for hCOL2A1 was adapted to the codon bias of Homo sapiens genes with regions of very high (> 80 %) or very low (< 30 %) GC content avoided whenever possible. Cis-acting sequence motifs were avoided where applicable, including, but not limited to, internal TATA-boxes, chi-sites and ribosomal entry sites, AT-rich or GC-rich sequence stretches, RNA instability motifs, repeat sequences and RNA secondary structures and (cryptic) splice donor and acceptor sites in higher eukaryotes. Examples of Codon Optimized hCOL2A1 can be found in Table 1 (i.e. SEQ ID NOs.1-13). Example 2 – Design and Development of INS-101 Description and composition of the pharmaceutical product

[0211] INS-101 was developed an investigational third generation LV gene therapy product (Milone et al., Leukemia 32(7):1529-1541 (2018)) consisting in a replication defective, self-inactivating, Human Immunodeficiency Virus (HIV-1)-based LV pseudotyped with the Vesicular Stomatitis Virus glycoprotein G (VSV-G) envelope protein and carrying the wild-type human α1 chain of procollagen type II (COL2A1) therapeutic gene under the control of a chimeric hybrid promoter (HP) for enhanced expression in chondrocytes.

[0212] The drug product (DP) INS-101 was developed as a sterile suspension for injection of the DS formulated in 20 mM Histidine, 100 mM NaCl, 5% (w / v) Sucrose, 0.005% (w / v) Poloxamer 188, pH = 7.0 and aseptically filled in cryovials. LV Vector Construct

[0213] The vector is composed of two main elements: 1) A linear dimeric single-stranded ribonucleic acid (RNA) genome containing a hybrid promoter (HP), derived from the endogenous human COL2 promoter (13a COL2 Hybrid) followed by a codon-optimized wild-type human cDNA of Collagen Type II Alpha 1 Chain (COL2A1) and the mutated Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE4) and 2) The structural and functional elements of the HIV capsid and cell membrane, pseudo-typed with VSV-G. See Figure 1.

[0214] The description of INS-101 plasmid elements is provided below in Table 5.Table 5 - Description of the INS-101 genome ReHI ProThe tran WPDesign and Selection of the Promoter

[0215] The selected promoter is "13a COL2 Hybrid". This promoter contains the DNA sequence of the human COL2 endogenous promoter (nucleotides -1 to -716 from the transcription start site) and has been modified on its 3' end to include ~80 nucleotides of the EF1a promoter, with the EFP1, EFP2 and TATA box sequences, to increase COL2A1 expression yet maintaining tissue specificity and a similar pattern of expression as for the endogenous COL2A1 in tissues (www.proteinatlas.org / ENSG00000139219- COL2A1 / tissue).

[0216] This hybrid promoter has been tested in a murine chondrogenic cell line (ATDC5) and selected based on an increased expression of a GFP reporter gene compared to the endogenous human COL2 promoter and based on a similar or lower expression than the ubiquitous EF1a promoter (Figure 4). This hybrid promoter showed: i) no or marginal expression in B-cell derived murine Baf3 cell line, as a safety marker for no promoter activity in lymphoblastoid cells, as expected from endogenous COL2A1 expression; ii) similar activity as the endogenous human COL2 promoter in murine chondrocytes cell line(ATDC5); and iii) the ability to express the therapeutic COL2A1 transgene in SEDc and healthy induced Pluripotent Stem Cells (iPSC)-derived human chondrocytes. Formulation Development

[0217] A new formulation buffer was developed to optimize the stability of INS-101 DP. The formulation buffer contains the following excipients: 20 mM Histidine, 100 mM NaCl, 5% (w / v) Sucrose, 0.005% (w / v) Poloxamer 188, and has a pH = 7.0.

[0218] The new buffer was introduced through diafiltration performed during the TFF step.

[0219] All excipients were GMP grade and comply with European and United States Pharmacopeias quality standards. Col2a1sedc / sedcmouse model

[0220] The homozygote Col2a1sedc / sedcmouse model (B6(Cg)-Col2a1sedc / GrsrJ) is a spontaneous strain expressing a mutated COL2A1(R1417C) protein and was described by the Jackson Laboratory (Donahue et al., J Bone Miner Res.18(9):1612-1621 (2003)). This model recapitulates key characteristics of the clinical, radiographic, histologic, and functional complications observed in SEDc pediatric and related disorders patients i.e., disproportionate short stature, pectus excavatum, respiratory dysfunction, atlanto-axial instability, and hip deformity. This model is used to test the efficacy of INS-101.

[0221] A milder type-2 collagen disorder phenotype, with apparent clinical manifestations at 6-12 weeks of age, is also observed in the Col2a1sedc / +heterozygote mouse. The phenotype includes a significant reduction of cartilage femoral head cap and increased osteoarthritis lesions (data not shown). Gottingen Minipig

[0222] The pig is an established large-animal model for toxicology studies for small molecules (Bode et al. J Pharmacol Toxicol Methods.62(3):196-220 (2010)), immunogenicity (Mair et al. Dev Comp Immunol.45(2):321-343 (2014)) as well as biodistribution, safety or efficacy in gene therapy of cardiovascular and inflammatory diseases (Ishikawa et al., Methods Mol Biol.1521:3-14 (2017); Hulot et al., Eur Heart J. 37(21):1651-1658 (2016); and Hinkel et al., J. Am. Coll. Cardiol.66(2):154-165 (2015)). Skeletal growth, anatomical, physiological, and developmental similarities between theGottingen mini-pig and humans are documented for pediatric research (Ayuso et al. Pharmaceutics.13(1):44 (2020)). The pig is increasingly used to study the musculoskeletal system, including joints, bone, cartilage, and ligaments; in particular, to evaluate the role of skeletal growth on the biomechanics and engineered replacements of these joints and tissues. The growth curve of mini-pigs has been described: mini-pigs reach an adult mature body weight of 20 kg about 12 months after birth; epiphyseal lines are closed at 20 months, whereas they close at 15–17 years in humans (Tohyama et al. Cell Transplant.28(2):224-228 (2019)). Furthermore, the mini-pig skeletal macrostructure, physiology and lamellar micro-structure closely resemble those of human bones (Behrends et al. J Funct Biomater.6(2):407-421 (2015)).

[0223] For these reasons, the wild-type Gottingen minipig was chosen to assess INS-101 biodistribution and toxicity in a formal pharmacology and toxicology (Pharm / Tox) GLP study. Conservation of the transgene and promotor activities between species

[0224] The fragments of the native COL2 core promoter used in the 13a hybrid promoter are highly conserved between species (Niebler and Bosserhoff, FEBS J.280(6):1397- 1408 (2013)) and are thus expected to produce similar levels of expression in the different species.

[0225] The transgene is a functional protein and has no enzymatic activity. The protein sequence similarity is 99.87% between NHP and human, 98.86% between minipig and human, and 97.38% between mouse and human. It is thus supposed to be equally functional in the different species. Age equivalence between selected animals and humans

[0226] The target population of the FIH is children aged between 6 months and 12 years. The table below compares age equivalence between mouse, minipig, NHP and human (www.jax.org / research-and-faculty / research-labs / the-harrison-lab / gerontology / life-span- as-a-biomarker). Table 6 - Age equivalence between species6 8 32 4 6 2 4 6 9

[0227] The mouse proof-of-concept studies have been conducted in mice at PND1, 8 and 15, corresponding to a 5 / 6-month-, 3 / 4-year- and 6 / 7-year-old child, respectively. Because murine growth plates are not fused at sexual maturity and are accessible to LV transduction, additional biodistribution studies were conducted in adult mice (INBD003- 21 & INDB004-21).

[0228] In the selection of the age of animals, limitations and differences in the skeletal development between mice and humans have been considered, including: 1 / the absence of fusion of secondary ossification centers in long bones, leading to reduced but maintained growth throughout the life of rodents, 2 / rapid bone growth in mice, particularly during the first month after birth compared to humans, 3 / the absence of a growth spurt in mice and 4 / the dominance and sex bias appearing after weaning (PND22) that further affects the SEDc mouse phenotype. In the ongoing studies, a cohort at PND5 was added in addition to PND1, 8 and 15, to cover the younger age group. The mini-pig biodistribution studies have been conducted in wild-type animals aged 2 or 6 weeks, which corresponds to a 6-month-old or 2-year-old child, respectively. The GLP biodistribution and toxicology study will be performed in 2-week-old wild-type mini- pigs.

[0229] The NHP biodistribution studies utilized 2 / 3-year-old animals, corresponding to an 8 / 10-year-old child.

[0230] In conclusion, the sponsor believes that the selected animal models as well as their ages were relevant, provided a significant coverage (6 months to 10 years) of the therapeutic window and are sufficient to assess the biodistribution and toxicity that can be extrapolated to the target population in the clinical trial. In vitro model using chondrocytes and MSCs derived from iPS cells from SEDc and related disorders patients

[0231] During embryonic development, skeletal elements including bone and cartilage were derived from the mesenchymal lineage. To evaluate treatment effect on patient cells, mesenchymal stem cells (MSCs) were obtained from the differentiation of induced Pluripotent Stem Cells (iPSCs). iPSCs were generated from peripheral blood mononuclear cells (PBMCs) obtained from SEDc patients and two unrelated healthy individuals as controls. After isolation, the cells were reprogrammed using Sendai virus containing the reprogramming factor OCT4, SOX2, KLF4 and c-MYC and characterized for self-renewal, expression of embryonic markers and pluripotency.

[0232] Differentiation of SEDc and healthy iPSCs into MSCs was confirmed by the expression of the typical MSC surface markers CD90, CD105 and CD73.

[0233] Finally, MSCs were differentiated into chondrocytes (De Kinderen et al. J Bone Miner Res.37(3):397-410 (2022)). Characterization of the obtained 3D chondrocyte cultures showed that SEDc patient's cells have impaired extracellular matrix production compared to healthy control cells, showing that these patient iPSC-derived chondrocytes are a relevant model to assess the pharmacodynamic of INS-101. Example 3 – Pharmacological Analysis

[0234] Treatment effects of LV-13a-COL2A1-ires-GFP in the SEDc mouse model (Col2a1sedc / sedc): single and repeat dosing Repeat injections in Col2a1sedc / sedcpups

[0235] Proof-of-concept studies (A005 and A006) were performed in Col2a1sedc / sedc mice with intraperitoneal (IP) administration of an INS-101 surrogate (LV-13a-COL2A1- ires-GFP) expressing both human COL2A1 and the eGFP reporter tracer under the HP promoter. The LV vector was injected once a week for 3 weeks (at PND1, PND8 and PND15) in wild-type (WT), heterozygote Col2a1sedc / + mice or homozygoteCol2a1sedc / sedc (SEDc) mice. Doses ranged from 1E6 to 1E9 TU / kg. Survival, restoration of skeletal growth and prevention of the main complications were assessed by measuring specific radiographic criteria relevant to SEDc and by histology. Controls were injected with phosphate buffered saline (PBS).

[0236] Following repeat injections of the INS-101 surrogate at 1E7 TU / kg, survival of SEDc mice improved from 55 to 86% compared to saline-treated controls (Figure 5). SEDc mice treated at doses ranging from 1E7 to 1E9 TU / kg showed a dose-dependent, long-term correction of histologic, radiographic, and clinical anomalies.

[0237] Figure 2 shows micro-CT imaging of thoracic cage deformities in vehicle treated SEDc mice that is restored by INS-101 treatment. Figures 3A-3C show CT imaging and histology of hip deformities in mice with and without INS-101 treatment. Figure 6 shows INS-101 analog corrected the distance between occipital edge to foramen magnum in SEDc mice. Figure 7 shows INS-101 analog corrected hip and vertebral deformities in SEDc mice. Figure 8 shows INS-101 analog prevented pectus excavatum and kyphosis in SEDc mice. Figure 9 shows INS-101 analog restored bone growth histologic architecture in SEDc mice.

[0238] Specifically, the Sponsor demonstrated i) Expression of the COL2A1 transgene in the chondrocytes of bone growth plates; ii) Restoration of columnar architecture and chondrocyte differentiation; iii) Radiographic correction of sternal, hip, long bone measurements, and total body length; iv) Restoration of the thoracic cage volume and correction of the cranio-cervical instability and vertebrae maturation; and v) Prevention of spine deformities and, on observation, improved locomotor function. Single injection in Col2a1sedc / sedcpups

[0239] Study INEF-003-21 (proof of concept study, biodistribution and single and repeat dose) was performed using the INS-101 analog (LV-13a-COL2A1-ires-GFP) to assess biodistribution in Col2a1sedc / sedcmice treated with three doses (1E9 TU / kg injected at PND1, PND8 and PND15, which is equivalent of 5-6 months, 3-4 years and 6-7 years old children respectively) versus mice treated with a single dose of 3E9 TU / kg (at PND5). Controls included mice treated with phosphate buffered saline (PBS).

[0240] Radiographic imaging (Figure 10) showed that INS-101 analog corrected the rib cage deformities both with single and repeat dosing. Histology showed that INS-101 restored sternum growth plate architecture and prevented retinal detachment. Altogether,these results showed that a single administration can improve SEDc-associated complications.

[0241] In vitro Proof of concept study in patient cells: Treatment effects of INS-101 and analogs on MSCs differentiation and chondrocyte matrix production from SEDc patient cells.

[0242] To evaluate treatment effect on patient cells, induced Pluripotent Stem Cells (iPSCs) from healthy controls and SEDc patients were generated and differentiated into chondrocytes - with a mesenchymal stem cells (MSCs) intermediate stage. The selected SEDc iPSCs carry the following missense mutation: D96 (NM_001844.4: c.2689G>A; p.(Gly897Arg)) or D919 (NM_001844.4:c.3589G>A; p.(Gly1197Ser)).

[0243] Matrix production was evaluated in a 3D chondrocyte cell culture system in patient cells (D96C44 and D919C27) and controls cells (C1C5 and C1C36) by histology, 3-weeks after treatment with increasing doses of INS-101. INS-101 treatment restored a functional matrix production and a fibrillar organization in SEDc iPSC-derived chondrocytes (Figure 11). No abnormal extracellular matrix deposition was observed in the treated healthy controls, suggesting that INS-101 does not lead to deleterious overproduction of COL2A1 in this condition. Nonclinical studies comparing IP and IV RoAs in mice and minipigs.

[0244] Because of the age and size of mice at the time of injection, the proof-of-concept studies were conducted using the IP RoA. Biodistribution in the adult mice:

[0245] Nonclinical biodistribution studies using wild-type mice were conducted to compare IP and IV RoAs. Mice were IP- or IV-injected with LV-13a-COL2A1-ires-GFP at 6-8 weeks of age, with doses of 1E8 or 1E9 TU / kg. Animals were euthanized at either 1 or 3 weeks after injection, and tissues were collected for histological and molecular analyses. Data at 3 weeks were compared to that at 1 week to assess the kinetic of the COL2A1 transgene expression.

[0246] In this study, no significant difference in transduction (VCN) of the COL2A1 transgene was observed in the long bone growth plate, sternum, liver and spleen between IP and IV administrations (Figure 12). Transgene mRNA expression correlated with VCN(data not shown). The LV vector did not distribute to other off-target tissues like thymus, pancreas, muscle, lung, kidney, and heart.

[0247] Figure 13 shows persistence of the VCN detection in the sternum and tibial growth plates from 1 week to 3 weeks. In most tissues, a peak of transduction at 1 week was seen when compared to 3 weeks. Conversely, there is a significant reduction of VCNs in the liver and spleen, demonstrating a more rapid loss of the transgene in these off-target tissues. Transgene mRNA expression as measured by qRT-PCR correlated with the VCN data (data not shown). Biodistribution in the minipig:

[0248] Nonclinical studies using the wild-type Gottingen minipig were also conducted to compare IP and IV RoAs.2-week- and 6-week-old mini-pigs were IV- or IP-injected with a LV construct expressing the eGFP reported gene (LV-13a-eGFP) at a dose of 1E9 TU / kg. Animals were euthanized 1-week post-injection, and tissues were collected for molecular analyses.

[0249] VCN data obtained with both 2-week- and 6-week-old animals showed that LV- 13a-eGFP could efficiently transduce growth plates in long bones and sternum of a large animal (Figure 14). Interestingly, the IP RoA led to reduced off-target liver and spleen exposure compared to IV (Figure 14). This suggests that the IP administration increased the safety profile of INS-101 in large species. Conclusion on the proof-of-concept studies

[0250] In vitro and in vivo proof-of-concept studies suggested that systemic delivery of INS-101 can efficiently target chondrocytes of the skeletal bone growth plates and prevent the development of severe complications related to SEDc and related disorders.

[0251] Data in the mouse disease model of SEDc demonstrated that INS-101 mediated high expression of wild-type COL2A1 in resting and proliferating chondrocytes during bone growth and restored a normal cartilage matrix and thereby partially or completely corrected the phenotypic, functional and radiographic anomalies. A dose-dependent response from 1E7 to 1E9 TU / kg was observed. The administration with one dose every week for 3 consecutive weeks (PND1, PND8 and PND15) using an INS-101 surrogate shows: reduction of mortality and restoration of bone growth, correction of the distance between the occipital edge to foramen magnum and correction of atlanto-axial instability,correction of the thoracic cage deformity, prevention of pectus excavatum and respiratory dysfunction, correction of hips and vertebral deformities, prevention of scoliosis and kyphosis, restoration of bone growth histologic architecture.

[0252] Studies assessing a single dose vs multiple dosing demonstrates the potential efficacy of a single dose to treat the clinical complications in SEDc and related disorders.

[0253] The IP was a preferred route of administration compared to IV for delivery in the target tissue and in reducing distribution in off-target tissues such as the liver or spleen.

[0254] In the non-clinical studies conducted to date, no in-life adverse events were seen and off-target distribution seemed to be low Example 4 – Dose Selection and Dose Escalation in Mice

[0255] In the SEDc mouse model, doses from 1E6 TU / kg to 1E9 TU / kg have been tested. Increased survival, skeletal growth and prevention of the main complications have been observed with a dose starting at 1E7 TU / kg (Figure 15). Figure 15 also shows dose response in SEDc mouse showing distance between occipital edge to foramen magnum, iliac angle and xiphoid process.

[0256] The minimum effective dose (MED) will be defined in the future studies:

[0257] The doses to be administered will range from 3.3E7 - 1.0E9 TU / kg. These dose escalation studies will lead to the identification of the minimal effective dose that provides a significant and broad prevention of bone deformities at most of the ages that were selected for injection (PND1 to PND15). Specifically, we expect INS-101 treatment to increase the rib cage volume, as well as prevent spine and pelvis deformities, which are three relevant symptoms associated with SEDc and related.

[0258] The dose range finding study will be performed to confirm the MED using an INS-101 LV preparation generated using the same process as the one for the clinic. It will also include higher doses to determine the NOAEL and the safety margin in mice. We propose using one age group (either PND5 or PND8) to limit the number of mice as data can be extrapolated from the previous studies. Juvenile 5 or 8-day-old COL2A1sedc / sedcmice will be enrolled into the vehicle control group or one of four INS-101 treatment groups, administered as a single dose of 1E7, 1E8, 1E9, or 1E10 TU / kg, with additional controls including WT healthy mice. Tissues will be analyzed to correlate the efficacy with the biodistribution and the toxicology profile.

[0259] Projection: Based on the accumulated data from the proof-of-concept studies, the sponsor projects a MED between 1E7 and 1E8 TU / kg in mice. Consequently, a lowclinical dose of 1E8 TU / kg and a high clinical dose of 1E9 TU / kg are considered, assuming the translatability of the MED between mice to humans is within a log difference. These projections will be confirmed in the studies, combined with Minipig and NHP biodistribution and toxicology studies that will determine the translatability and safety margin (NOAEL). Translation of data from animal studies into human

[0260] Our translational plan is based on biodistribution data obtained in mice to extrapolate the MED to larger non-rodent species and humans. This will be achieved in two steps:

[0261] 1 – To identify the MED in the SEDc mouse model, as discussed above, and to determine the corresponding biodistribution (VCN / cell; mRNA expression; number of transduced cells) in target tissues (planned studies).

[0262] 2 – To perform dose escalation in minipigs (planned study) to identify the dose providing the same biodistribution in target tissues as the one defined in SEDc mice with the MED. Because minipigs are good models based on bone physiology and growth, a corrective parameter will be established and will lead to the definition of the lower clinical dose. For instance: if a dose (TU / kg) leads to the same biodistribution in mice and minipigs, the low clinical dose will be the same as the ME; if there is a log difference between mice and minipigs to achieve the same biodistribution, the lower clinical dose will be a log higher than the mouse MED. Table 7 - INBD-MP-003 biodistribution study Stu Stu* * *

[0263] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

[0264] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0265] Any examples provided herein are offered by way of illustration and not by way of limitation.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide comprising a nucleic acid sequence of SEQ ID NOs: 1-13, wherein the polynucleotide encodes the alpha-1 chain of human type II collagen (hCOL2A1).

2. The polynucleotide of claim 1, further comprising a promoter element operably linked to the polynucleotide encoding the hCOL2A1 polypeptide.

3. The polynucleotide of claim 2, wherein the promoter element is a hybrid promoter comprising a fragment of the hEF1α promoter and a fragment of the hCOL2A1 promoter.

4. The polynucleotide of claim 3, wherein the promoter element comprises the nucleic acid sequence of SEQ ID NOs: 14-33.

5. The polynucleotide of claim 1, further comprising a nucleic acid sequence selected from SEQ ID NOs: 38-44, and combinations thereof.

6. The polynucleotide of any one of claims 1-5, comprising the nucleic acid sequence of SEQ ID NO:

7.

7. The polynucleotide of claim 6, wherein the promoter element comprises the nucleic acid sequence of SEQ ID NO:

14.

8. A polynucleotide comprising a nucleic acid sequence of SEQ ID NO:

36.

9. A polynucleotide comprising a nucleic acid sequence of SEQ ID NO:

37.

10. A vector comprising a polynucleotide of any one of claims 1-9.

11. The vector of claim 10, wherein the vector is an adenoviral, retroviral, lentiviral, or adeno-associated viral vector.

12. The vector of claim 11, wherein the vector is a lentiviral vector.

13. The vector of claim 12, further comprising a nucleic acid sequence of SEQ ID NO: 38-44.

14. A viral particle comprising the polynucleotide of any one of claims 1-8.

15. The viral particle of claim 14, wherein the particle is a lentiviral particle.

16. A host cell comprising the polynucleotide of any one of claims 1-9 or the viral particle of claims 14-15.

17. A method for producing a lentiviral particle comprising introducing a polynucleotide of any one of claims 1-9 into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell.

18. A method for transducing a host cell comprising contacting the host cell with the lentiviral particle of claim 15.

19. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-9, the vector of any one of claims 10-13, or the viral particle of any one of claims 14-15.

20. The pharmaceutical composition of claim 19, further comprising a pharmaceutically acceptable excipient or carrier.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutically acceptable excipient or carrier is histidine.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises between about 10 mM to about 30 mM of histidine.

23. The pharmaceutical composition of claim 20, wherein the pharmaceutically acceptable excipient or carrier is sodium chloride (NaCl).

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition comprises between about 50 mM and about 150 mM of NaCl.

25. The pharmaceutical composition of claim 20, wherein the pharmaceutically acceptable excipient or carrier is sucrose.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition comprises between about 2% and about 10% of sucrose.

27. The pharmaceutical composition of claim 20, wherein the pharmaceutically acceptable excipient or carrier is poloxamer 188.

28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition comprises between about 0.0001% and about 0.015% of poloxamer 188.

29. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition has a pH between about 6 and about 9.

30. The pharmaceutical composition of any one of claims 19-29, wherein the pharmaceutical composition comprises about 20 mM of histidine, about 100 mM of NaCl, about 5% of sucrose, and about 0.005% of poloxamer 188.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition has a pH of about 7.

32. A method of treating a skeletal dysplasia or a type II collagenopathy comprising administering to a subject in need thereof the lentiviral particle of claim 15 or the pharmaceutical composition of any one of claims 19-3133. The method of claim 32, wherein the lentiviral particle is administered subcutaneously, intravenously, intraperitoneally, orally, nasally, intramuscularly, sublingually, intrathecally or intradermally.

34. The method of claim 32 or 33, comprising administering about 1E7 transduction units ("TU") / kg, about 1E8 TU / kg, about 1E9 TU / kg, or about 1E10 TU / kg of lentiviral particles to the subject.

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