WO2023175583A1 - Methods of treating bone fragility syndromes - Google Patents

Abstract

The present invention relates to methods of modulating delivery of a polynucleotide of interest by altering the route of administration. Additionally, the present invention relates to methods of treating bone fragility syndromes, for example osteogenesis imperfect. More particularly, the present invention relates to targeting gene therapy compositions to bone by providing the composition by intraperitoneal administration.

Description

METHODS OF TREATING BONE FRAGILITY SYNDROMES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional App. No. 63/269,519, filed March 17, 2022, and U.S. Provisional App. No. 63/269,520, filed March 17, 2022, each of 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_009PC02_Seqlisting_ST26; Size: 312,407 bytes; and Date of Creation: March 15, 2023) is herein incorporated by reference in its entirety.

FIELD

[0003] The present invention relates to methods of treating bone fragility syndromes. More particularly, the present invention relates to gene therapy approaches for treating bone fragility syndromes such as ostogenesis imperfecta.

BACKGROUND OF THE INVENTION

[0004] Formation of the skeletal system is one of the hallmarks that distinguish vertebrates from invertebrates. Patterning of the early skeletal system is controlled by several major signaling pathways that also regulate other pattern formation processes. These signaling pathways are mediated by morphogens including Wnts, Hedgehogs (Hhs), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Notch/Delta. Understanding skeletal development is indispensable for understanding pathological mechanisms of skeletal diseases, finding therapeutic targets, promoting consistent cartilage or bone repair in vivo.

[0005] Bone strength is determined by a number of important factors, including bone mass and bone shape. A reduction in bone strength is clearly related to fracture. Bone fragility results from a reduction in bone mass and density. If there is a reduction in the connectivity of bone and impact from a mechanical load occurs, bone will fracture. Rather than considering bone fragility as being the result of a reduced amount of bone, it is recognized that bone fragility is the result of changes in the material and structural properties of bone. A better understanding of the contribution of each component of the material composition and structure, and how these interact to maintain whole bone strength is obtained by the study of metabolic bone diseases. Disorders of collagen, of mineral content composition and distribution, disorders of remodeling and other diseases produce abnormalities in the material composition and structure that lead to bone fragility.

[0006] Genetic disorders arise via heritable or de novo mutations occurring in gene coding regions of the genome. In some cases, such genetic disorders are treated by administration of a protein encoded by the gene mutated in the individual having the genetic disorder. Such treatment has challenges however, as administration of the protein does not always result in the protein reaching the organs, cells, or organelle where it is needed. Furthermore, this treatment also often requires biweekly infusions, which are not needed with gene therapy, where a single treatment can offer lasting relief. Therefore, gene therapy has the potential to offer improved results over currently available treatments for genetic disorders related to bone fragility syndromes and bone growth.

BRIEF SUMMARY OF THE INVENTION

[0007] Certain aspects of the disclosure are directed to a method of targeting delivery of a polynucleotide of interest to bone, cartilage and/or bone growth plate comprising providing the polynucleotide of interest to a subject in need thereof via intraperitoneal administration.

[0008] Certain aspects of the disclosure are directed to a method of decreasing delivery of a polynucleotide of interest to the liver and/or spleen and/or the hematolymphoid system in a subject, comprising providing the polynucleotide of interest to the subject via intraperitoneal administration; wherein the amount of polynucleotide in the liver and/or spleen and/or the hematolymphoid system is decreased relative to the amount of polynucleotide in the liver and/or spleen and/or the hematolymphoid system following intravenous administration. [0009] In some aspects, the hematolymphoid system comprises the bone marrow, thymus, lymph nodes and/or mucosal lymphoid tissues.

[0010] Certain aspects of the disclosure are directed to a method of treating a bone disorder in a subject in need thereof, comprising providing a polynucleotide of interest to the subject via intraperitoneal administration, wherein the intraperitoneal administration of the polynucleotide of interest increases delivery to bone, cartilage, and/or the bone growth plate.

[0011] In some aspects, the intraperitoneal administration of the polynucleotide of interest reduces delivery to non-targeted tissues and increases the therapeutic window for treatment.

[0012] In some aspects, the non-targeted tissues are liver, spleen, and/or cells of the hematolymphoid system.

[0013] In some aspects, the polynucleotide of interest is delivered to the bone, the growth plate, the cartilage and/or the bone marrow.

[0014] In some aspects, the polynucleotide of interest comprises a nucleotide sequence encoding the alpha-1 chain of human type II collagen (hCOL2Al).

[0015] In some aspects, the polynucleotide comprises a nucleotide sequence of SEQ ID NOs: 1-13.

[0016] In some aspects, the polynucleotide of interest further comprises a promoter element.

[0017] In some aspects, the promoter element is a hybrid promoter comprising a fragment of the hEFla promoter and a fragment of the hCOL2Al promoter; wherein the hybrid promoter is operably linked to a nucleotide sequence encoding hCOL2Al.

[0018] In some aspects, the promoter element comprises the sequence of SEQ ID NOs:

14-33. In some aspects, the promoter element comprises the sequence of SEQ ID NO: 14.

[0019] In some aspects, the polynucleotide of interest is contained in a viral vector.

[0020] In some aspects, the vector is a lentiviral vector.

[0021] In some aspects, delivery of the polynucleotide results in at least about a 2-fold reduction of vector copy number in liver and/or spleen and/or hematolymphoid system compared to the copy number of the polynucleotide of interest following intravenous delivery.

[0022] In some aspects, the polynucleotide of interest is contained in a lentiviral particle. [0023] In some aspects, the polynucleotide of interest is present in the bone at least 3 weeks following administration.

[0024] In some aspects, the subject has a type II collagen disorder.

[0025] In some aspects, the subject has spondyloepiphyseal dysplasia congenital (SEDc).

[0026] Certain aspects of the disclosure are directed to a method of treating a bone fragility syndrome in a subject in need thereof, comprising providing at least one polynucleotide of interest to the subject via intraperitoneal administration.

[0027] In some aspects, the subject has osteogenesis imperfecta, osteoporosis, osteomalacia, or Paget's disease.

[0028] In some aspects, at least one polynucleotide of interest is delivered by intraperitoneal administration.

[0029] In some aspects, at least one polynucleotide of interest is preferentially delivered to the bone growth plate and/or bone marrow.

[0030] In some aspects, the polynucleotide of interest comprises a nucleotide sequence encoding the alpha- 1 or alpha-2 chain of human type I collagen, or the alpha- 1 chain of human type II collagen.

[0031] In some aspects, the polynucleotide comprises a nucleotide sequence of SEQ ID NOs: 34-63.

[0032] In some aspects, the administration of the polynucleotide of interest results in bone growth and/or a decrease in bone fragility.

[0033] In some aspects, the polynucleotide of interest further comprises a promoter element.

[0034] In some aspects, the promoter element is a hybrid promoter comprising a fragment of the hEFla promoter and a fragment of the hCOL2Al promoter; wherein the hybrid promoter is operably linked to a nucleotide sequence encoding hCOL2Al, hCOLl Al or hCOLlA2.

[0035] In some aspects, the promoter element comprises the sequence of SEQ ID NOs:

14-33. In some aspects, the promoter element comprises the sequence of SEQ ID NO: 14.

[0036] In some aspects, the polynucleotide of interest is contained in a viral vector.

[0037] In some aspects, the vector is a lentiviral vector.

[0038] In some aspects, the polynucleotide of interest is contained in a lentiviral particle.

[0039] In some aspects, the polynucleotide of interest is delivered ex vivo. [0040] In some aspects, the cells are delivered to mesenchymal stem cells.

[0041] In some aspects, the polynucleotide of interest is administered in combination with at least one therapeutic agent.

[0042] In some aspects, the polynucleotide of interest and the therapeutic agent are administered sequentially, and in any order.

[0043] In some aspects, the agent is selected from the group consisting of a bisphosphonate, a parathyroid hormone, a parathyroid hormone analog, calcitonin, and a selective estrogen receptor modulator.

[0044] In some aspects, the polynucleotide of interest is administered in combination with alendronate, pamidronate, zoledronate, or risedronate.

[0045] In some aspects, the polynucleotide of interest is administered in combination with teriparatide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] 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.

[0047] Figure 1 shows the increase in vector copy number (VCN) per diploid genome in the tibial growth plate when INS-101, a polynucleotide that restores COL2A1 function in bone growth plates, is delivered via intraperitoneal versus intravenous administration.

[0048] Figure 2 shows sustained INS-101 distribution in mouse growth plates following intraperitoneal administration in neonate SEDc mice upon single dose as compared to repeated administration of INS-101.

[0049] Figure 3 shows increased RNA expression in the same mouse growth plates following intraperitoneal administration in neonate SEDc mice upon single dose as compared to repeated administration of INS-101.

[0050] Figure 4 shows long-term 6-month INS-101 gene delivery in mice following intraperitoneal administration in neonate mice.

[0051] Figure 5 shows a comparison of INS-101 biodistribution in tissues when administered by intravenous versus intraperitoneal administration in 2-weeks old Gottingen minipigs. DETAILED DESCRIPTION OF THE INVENTION

[0052] Certain aspects of the disclosure are directed to a method of targeting delivery of a polynucleotide of interest to bone, cartilage and/or bone growth plate comprising providing the polynucleotide of interest to a subject in need thereof via intraperitoneal administration.

[0053] Certain aspects of the disclosure are directed to a method of decreasing delivery of a polynucleotide of interest to the liver and/or spleen and/or the hematolymphoid system in a subject, comprising providing the polynucleotide of interest to the subject via intraperitoneal administration; wherein the amount of polynucleotide in the liver and/or spleen and/or the hematolymphoid system is decreased relative to the amount of polynucleotide in the liver and/or spleen and/or the hematolymphoid system following intravenous administration.

[0054] Certain aspects of the disclosure are directed to a method of treating a bone disorder in a subject in need thereof, comprising providing a polynucleotide of interest to the subject via intraperitoneal administration, wherein the intraperitoneal administration of the polynucleotide of interest increases delivery to bone, cartilage, and/or the bone growth plate.

[0055] Certain aspects of the disclosure are directed to a method of treating a bone fragility syndrome in a subject in need thereof, comprising providing at least one polynucleotide of interest to the subject via intraperitoneal administration.

I. Definitions

[0056] 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.

[0057] 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.

[0058] In order to further define this disclosure, the following terms and definitions are provided.

[0059] 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."

[0060] 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).

[0061] 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.

[0062] Units, prefixes, and symbols are denoted in their Systeme 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] As used herein "bone disease" or "bone disorder" includes any disease or disorder which affects bone strength, function, and/or integrity, such as decreasing bone tensile strength and modulus. Examples of bone diseases include, but are not limited to, diseases of bone fragility, such as all types of osteoporosis according to etiology and pathogenesis, all types of metabolic bone diseases according to pathogenesis and etiology, osteonecrosis, and genetic diseases which result in abnormal bone formation. [0067] As used herein, 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 collagen disorders are a subtype of skeletal dysplasia. For example, type II collagen disorders 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. Accordingly, the disclosure provides for the treatment of a type II collagenopathy, and spondyloepiphyseal dysplasia congenita (SEDc), Kniest dysplasia or Stickler syndrome.

[0068] As used herein, 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.

[0069] As used herein a "bone defect" includes any defect which affects bone strength, function, and/or integrity, such as those resulting from injury, or a defect brought about during the course of surgery, infection, malignancy, or developmental malformation. Examples of bone defects include, but are not limited to, fractures (such as a critical defect or non-union fracture), dental or facial defects (such as cleft palate or facial or dental injuries or malformations).

[0070] As used herein, "Osteogenesis" refers to the formation or production of bone. Bone-forming cells and mineral forming cells are cells having osteogenic potential. Examples include, but are not limited to: bone marrow stromal cells, osteoblasts, osteocytes, and dental pulp cells.

[0071] As used herein, the terms "Intraperitoneal" injection or "i.p." injection refer to the injection of a substance into the peritoneum (body cavity). Intraperitoneal injection typically results in high absorption due to the available large surface area.

[0072] As used herein, the terms "Intravenous" or "i.v." injection refer to the injection of a substance into a vein and directly into the bloodstream.

[0073] As used herein, the term "operably linked" means that the polynucleotide 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.

[0074] As used herein, the term "hCOL2Al" refers to the Homo sapiens al chain of human type II collagen (hCOL2Al) cDNA shown in SEQ ID NOs: 69-70 (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).

[0075] As used herein, the term "hCOLlAl" refers to the Homo sapiens al chain of human type I collagen (hCOLl Al) cDNA shown as SEQ ID NOs: 64-67 (accession numbers NM_000088.4, XM_011524341.2 (isoform XI), XM_005257058.5 (isoform X2), and XM_005257059.5 (isoform X3), that are the reference sequences for the CDS of the mRNA for COL1A1 human; MIM reference 120150), and variants thereof (e.g., codon optimized SEQ ID NOs: 34-57).

[0076] As used herein, the term "hCOLl A2" refers to the Homo sapiens a2 chain of human type I collagen (hCOLl A2) cDNA shown as SEQ ID NO: 68 (accession number NM_000089.4, that is the reference sequences for the CDS of the mRNA for COL1 A2 human; MIM reference 120160), and variants thereof (e.g., codon optimized SEQ ID NOs: 58-63).

[0077] As used herein, 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.

[0078] A "hybrid promoter" is a non-native promoter that is functional in host cells, particularly in mammal cells such as human cells. In some aspects, a hybrid promoter of the invention comprises a part of the sequence of the promoter of hCOL2Al gene, but also at least the TATA box of the hEFla promoter (See PCT application WO 2020/127533 which is incorporated by reference herein in its entirety). In some aspects, the hybrid promoters comprise any one of SEQ ID NOs: 14-33.

[0079] 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 some aspects, the subject is a human being. [0080] 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).

II. Bone Diseases

[0081] Bone as an organ is made up of the cartilaginous joints, the calcified cartilage in the growth plate (in developing individuals), the marrow space, and the cortical and cancellous mineralized structures. There are three cell types in bone tissue: bone-forming osteoblasts, osteocytes, and bone-destroying osteoclasts. The preponderance of bone is mineral and extracellular matrix (ECM) including collagen and noncollagenous proteins.

[0082] Skeletal dysplasia is 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. 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 bone manifestations; 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.

[0083] Accordingly, the disclosure provides for the treatment of any bone disease by delivering a therapeutic of interest by intraperitoneal administration. In some aspects, the disease is a skeletal dysplasia, such as spondyloepiphyseal dysplasia congenita (SEDc), Kniest dysplasia, Stickler syndrome or Jeune syndrome.

[0084] The cellular activities of bone modelling and remodeling determine the material composition and structure of bone. Bone modelling refers to the deposition of new bone without prior bone resorption. Bone remodeling is characterized by the appearance of focally and temporally distinct regions of resorption followed by bone formation that constitutes the basic multicellular units (BMUs). The purpose of bone modelling and remodeling during growth is to build peak bone strength. After the completion of growth, bone modelling continues in adulthood modestly to increase bone size further, whereas bone remodeling maintains bone strength by removal of microdamage.

[0085] The bone remodeling is initiated on a bone surface usually covered by a very thin layer of unmineralized matrix and lining cells. These cells may respond to stimuli (hormones, cytokines) that initiate the remodeling. After the resorption phase, osteoblasts secrete the bone matrix, which refills the resorption lacunae. Under normal conditions, the remodeling process of resorption followed by formation is closely coupled in BMU and results in no change of bone mass when the amounts of resorbed and newly formed bone are similar. The coupling between resorption and formation is controlled by several factors that are poorly defined. The absence of coupling formation occurring without prior resorption, is observed only under pathological conditions such as bone metastasis. The frequency of initiation of a new remodeling sequence characterized the bone turnover. Abnormalities in the rate and balance of bone remodeling play a pivotal role in the pathogenesis of bone loss and structural decay. A high remodeling rate contributes to bone fragility by reducing the time available for secondary mineralization; bone is removed and replaced with new, less densely mineralized bone, which reduces its material stiffness. High bone remodeling itself also alters collagen composition by impairing isomerization, maturation, and cross linking. The high remodeling rate produces stress concentrators excavated regions of bone that concentrate stress predisposing to microdamage.

[0086] In presence of a negative BMU balance produced by an increase in the volume of bone resorbed, a decrease in the volume of bone formed or both, each remodeling event during the high remodeling rate after menopause, and in disease states, accelerates bone loss and structural decay producing trabecular thinning, tunneling in the trabeculae, cortical thinning, and porosity. The resorptive phase of the remodeling cycle is responsible for the removal of microdamages, whereas the formation phase replaces bone and restores its material composition and structure.

[0087] Osteogenesis imperfecta (01) is an inherited disorder characterized by increased bone fragility with recurrent fractures that leads to skeletal deformities in severe cases. The phenotypic expression is heterogeneous with the most severe forms being fatal in the perinatal period to mild forms diagnosed only in adulthood. OI is characterized by a low bone mass, a reduced trabecular thickness and number and a decreased bone formation at the cellular level. The bone turnover is increased in children but decreased in adults.

Animal studies and studies in human subjects suggest that skeletal fragility in OI is due to the defect in collagen synthesis, whereas the abnormalities in bone turnover and mineral are inconsistent.

[0088] Most forms of OI result from mutations in the genes that encode either the proalphal or proalpha2 polypeptide chains that comprise type I collagen molecules. Mutations in several genes can lead to OI. About 80%-90% of OI cases are caused by autosomal dominant mutations in the type 1 collagen genes, COL1 Al and COL1 A2. Mutations in one or the other of these genes cause the body to make either abnormally formed collagen or too little collagen. Mutations in these genes cause OI Types I through IV.

[0089] The remaining cases of OI (types VI-XI) are caused by autosomal recessive mutations in any of six genes (SERPINF1, CRTAP, LEPRE1, PPIB, SERPINH1, and FKBP10) that code for proteins that help make collagen. These mutations also cause the body to make too little collagen or abnormally formed collagen.

[0090] Osteomalacia is characterized by a defect of mineralization due to calcium and phosphate deficiencies that mainly result from a poor gut absorption due to vitamin D deficiency. Most common is vitamin D deficiency due to lack of sunlight exposure or intestinal malabsorption, but disorders of the vitamin D metabolism contribute (defect of hydroxylation, increased renal excretion, and increased catabolism by anticonvulsants). Osteomalacia may be drug-induced (fluoride or etidronate) or the result of aluminum exposure in parenteral nutrition or hemodialysis. Clinical features are pain, fissures, and fractures, which may occur after minimal trauma. The consequence of vitamin D deficiency is a fall of blood calcium concentration that induces a secondary hyperparathyroidism. The effect of hypocalcemia is a defect of bone mineralization, and the effect of secondary hyperparathyroidism is an increase in bone turnover. In contrast, in phosphate deficiency, parathyroid level and vitamin D status are normal, and the consequence of hypophosphatemia is osteomalacia. The reduced mechanical properties of osteomalacic bone results from the delayed primary mineralization, which is the cause of the small amount of mineralized tissue. Bone tissue is characterized by an accumulation of osteoid and a decrease in mineralization rate with a prolongation of the mineralization lag time the delay between the deposition of the matrix and the onset of the mineralization. In hypophosphatemic osteomalacia, bone tissue mass may be increased but the mineralized bone volume is decreased.

[0091] Postmenopausal osteoporosis. Osteoporosis is defined by a BMD lower than 2.5 SD from the young adult mean. This decreased BMD variably reflects the contributions of growth and age-related deficits in bone size, tissue mass, and the degree of mineralization of the bone. Fragility fractures occur in up to 50% of postmenopausal women, but half of the fractures occur in persons without osteoporosis, confirming that bone density is not the only determinant of bone strength in postmenopausal osteoporosis. Bone fragility in postmenopausal osteoporosis is the result of a decrease in bone mass and architectural decay in cortical and trabecular bone.

[0092] Primary hyperparathyroidism is common and usually asymptomatic. The skeletal manifestations are variable and include bone pain and fractures at several sites including vertebral, distal radius, and pelvis. Osteopenia at various degrees may be observed and localized on the cortical bone, trabecular bone, or both. The major consequence of primary hyperparathyroidism is an increase in the rate of bone remodeling. Increased bone resorption is shown by the extended resorption surfaces and increased osteoclast number. The augmentation of formation associates an increase in the osteoid surfaces, osteoblast number, and mineral apposition rate, which is the rate of the primary mineralization. Despite this accelerated bone remodeling, cancellous bone volume is maintained with a thinning of trabeculae but a preservation of connectivity. In contrast to postmenopausal osteoporosis, the coupling between resorption and formation remains balanced in primary hyperparathyroidism with an augmentation of the osteoblastic activity and lifespan or a decreased erosion depth that results in a normal or increased balance at the BMU level. In contrast, cortices are thinner and more porous.

[0093] Fragility fractures also occur in men. The incidence of fractures is higher in men than women from adolescence through middle life as a result of more severe trauma, but bone fragility may also contribute to the fracture risk. After 50 years, the incidence of fractures increases with aging in men. The age-adjusted incidence of both hip and vertebral fractures in men is about half of that in women. In addition, several other factors may contribute, including nutritional deficiencies, inactivity, hypogonadism, or alcoholism. Men with vertebral or hip fractures have reduced bone size. As in women, osteoporosis in men is characterized by decreased bone mass with a similar magnitude associated with a reduced cortical thickness and an increased porosity. Modifications of bone microarchitecture have been reported in osteoporotic men with vertebral fracture independently of BMD when compared with osteoporotic men without fracture. The bone microarchitecture is characterized by a lower trabeculae number and an increased trabecular separation. In contrast, in another study performed in younger men, no significant difference in trabecular architecture was observed between men with crush fractures and controls, except for a trend in decreasing number of free-ends. The reduced bone size may be due to reduced periosteal apposition during growth, aging, or both. Bone loss results mainly from a decreased bone formation. In addition, increased bone resorption contributes.

[0094] Corticosteroid-induced osteoporosis. Bone loss and fracture risk are related to the dose and duration of glucocorticoid exposure. Bone loss is rapid during the first 12 months, with a significant decrease of lumbar spine BMD since the third month of treatment, observed even with a low dose (10 mg/d) of prednisone. The fracture risk increases rapidly: the vertebral fracture incidence has been reported to be 2-fold higher in a large cohort of corticosteroid-treated patients compared with controls but decreases after cessation of therapy. Fracture risk is greater at predominantly trabecular sites such as the vertebrae and ribs, and the risk of hip fracture is also doubled in glucocorticoid- treated patients. However, the prevalence of fractures in corticosteroid-induced osteoporosis is higher than expected from the decreased BMD, suggesting that the low bone strength induced by glucocorticoid may be partly independent of the changes of BMD. Glucocorticoids induce apoptosis of osteocytes in animals as in humans. The mechanism by which osteocytes contribute to bone strength is still unknown, but osteocytes have been hypothesized to play a major role in the targeted remodeling, acting as mechanosensor and transducer in bone, and to be involved in the detection and repair of microcracks.

[0095] Paget's disease of bone is a localized disease characterized by increased bone remodeling, bone hypertrophy, and abnormal bone structure. The illness occurs in 2-3% of individuals over age 60. The consequences are pain, bone deformities, fractures of long bones or vertebrae, secondary osteoarthritis from deformity of bone near joints, and neurological complications. Paget's disease may affect only one bone or may involve several bones. Bone fragility in Paget's disease probably results from the accelerated bone turnover and the consequent disorganization of the matrix. Fragility occurs despite an increase in bone density/size at most skeletal sites. In affected bone, resorption is dramatically increased with abnormal osteoclasts containing numerous nuclei. Bone formation is also increased with numerous osteoblasts actively synthesizing bone matrix, which is rapidly mineralized. The accelerated bone turnover leads to the formation of abnormal woven bone with an irregular arrangement of collagen fibers that are not deposited in a lamellar fashion. The excessive bone formation results in the bone hypertrophy and osteosclerosis with thick and numerous trabeculae. The woven bone is not specific for Paget's disease but reflects an extremely high rate of bone turnover. The alteration of the bone texture due to abnormal turnover is likely to impair bone strength.

III. Polynucleotides

[0096] The "polynucleotide sequence of interest" may be an RNA or DNA sequence (including cDNA sequences), which encodes a gene. In some aspects, the polynucleotide of interest encodes a protein involved in a bone disorder or defect. The nucleic acid sequence of interest is typically operably linked to a hybrid promoter (See PCT application WO 2020/127533 which is incorporated by reference herein in its entirety). In some aspects, the polynucleotide of interest comprises any one of SEQ ID NOs: 1-13 or 34-63. [0097] The collagen chain can be of any one of types I to XXVIII. In some aspects, the collagen chain is of type I (i.e. encoded by COL1 Al or COL1 A2 genes), or of type II (i.e. encoded by COL2A1 gene). In some aspects, the collagen chain is of type II. In some aspects, the collagen chain is of type I. In some aspects, the collagen chain can be a human collagen chain. In some aspects, the collagen chain is the pro-a 1 chain of type II collagen. In some aspects, the collagen chain is the pro-a 1 or pro-a 2 chain of type I collagen.

[0098] To construct type II collagen, three pro-a 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.

[0099] In some aspects, the invention relates to the use of a polynucleotide comprising a nucleotide sequence of SEQ ID NOs: 1-13, in which the polynucleotide encodes a codon optimized sequence of al chain of human type II collagen (hCOL2Al), to treat a skeletal dysplasia.

[0100] In some aspects, the invention relates to the use of a polynucleotide comprising a nucleotide sequence of SEQ ID NOs: 34-57, in which the polynucleotide encodes a codon optimized sequence of al chain of human type I collagen (hCOLl Al), to treat a bone fragility syndrome.

[0101] In some aspects, the invention relates to the use of a polynucleotide comprising a nucleotide sequence of SEQ ID NOs: 58-63, in which the polynucleotide encodes a codon optimized sequence of a2 chain of human type I collagen (hCOLl A2), to treat a bone fragility syndrome.

IV. Hybrid Promoters

[0102] In some aspects, the invention provides for sufficient bone expression due to the presence of hybrid promoters linked to the polynucleotide of interest. In some aspects, the hybrid promoter comprises a nucleotide sequence selected from any one of SEQ ID NOs: 14-33.

[0103] In some aspects, the hybrid promoters used allow for the significant expression of the polynucleotide of interest in specific cells and or tissues that naturally express the polynucleotide. Such an approach is highly beneficial, because the complex synthesis of polynucleotide must be performed at the site of the disease (i.e. in the chondrocytes). In some aspects, hCOLlAl is the polynucleotide of interest. In some aspects, hCOLlA2 is the polynucleotide of interest. In some aspects, hCOL2Al is the polynucleotide of interest. hCOL2Al 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.

V. Administration

[0104] Methods of administration of the polynucleotide of interest include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.

[0105] In some aspects, intraperitoneal administration results in increased delivery/expression of a polynucleotide sequence of interest in bone, cartilage, and/or the bone growth plate.

[0106] In some aspects, the bone fragility syndrome is treated by administering one or more polynucleotides encoding the alpha 1 chain of collagen type 2.

[0107] In some aspects, the invention is directed to a gene therapy to restore bone growth, particularly in the case of type II collagen disorders, such as SEDc, through the targeting of a polynucleotide of interest to bone by intraperitoneal administration.

[0108] In the context of the present invention 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.

[0109] 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.

[0110] 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 invention, the nucleic acid sequence of interest is expressed within the cell upon introduction of the vector.

[0111] 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 El deletion and can be grown on cell lines that are transformed with an El gene. These vectors can be manipulated to insert a nucleic acid sequence of interest, such that the encoded protein may be expressed.

[0112] According to some aspects, the vector for delivering the nucleic acid of the invention 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.

[0113] In some aspects, the vector comprises a bone-specific polynucleotide of interest. In some aspects, the polynucleotide of interest is a codon-optimized sequence of hCOL2Al, and the vector encodes a hybrid promoter which controls the expression of the COL2A1 gene or a fragment thereof coding for a collagen chain. In some aspects, the polynucleotide of interest is a codon-optimized sequence of hCOLl Al or hCOLl A2, and the vector encodes a hybrid promoter which controls the expression of the hCOLl Al or hCOLl A2 gene or a fragment thereof coding for a collagen chain.

[0114] The vectors of the disclosure provide long-term gene expression resulting in longterm expression of a therapeutic protein. As described herein, the phrases long term expression, permanent expression and persistent expression are used interchangeably. Long-term expression results in expression of a therapeutic gene and/or protein, 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. This long term expression can be achieved through repeated doses or a single dose. [0115] Repeated doses can be given multiple times per day, daily, multiple times per 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.

[0116] Lentiviral vectors generally provide high levels of transgene expression when administered to a patient. The terms high expression and therapeutic expression are used interchangeably herein.

[0117] 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 invention. 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.

[0118] In some aspects, the polynucleotides of interest are provided ex vivo. In some aspects, the polynucleotides of interest are incorporated into mesenchymal stem cells. Mesenchymal stem cells (MSCs) are multipotent stromal cells that exist in bone marrow, fat, and so many other tissues, and can differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes, as well as myocytes and neurons. Moreover, they have great capacity for self-renewal while maintaining their multipotency. Their capacity for proliferation and differentiation, in addition to their immunomodulatory activity, makes them very promising candidates for cell-based regenerative medicine. Moreover, MSCs have the ability of mobilization to the site of damage; therefore, they can automatically migrate to the site of injury via their chemokine receptors following intravenous transplantation. In this respect, they can be applied for MSC-based gene therapy. In some aspects of the disclosure, genes of interest are introduced into MSCs via viral and non-viral-based methods that lead to transgene expression in the cells.

VI. Therapeutic uses

[0119] The present invention also relates to the use of polynucleotides of interest, under the control of a hybrid promoter, or to the use of a vector comprising the polynucleotide of interest and hybrid promoter according to the invention in therapy. In some aspects, the therapy is gene therapy. In some aspects, the polynucleotide of interest is a codon- optimized sequence of human C0L2A1 (e.g., SEQ ID NOs: 1-13). In some aspects, the polynucleotide of interest is a codon-optimized sequence of human COL1 Al (e.g., SEQ ID NOs: 34-57). In some aspects, the polynucleotide of interest is a codon-optimized sequence of human C0L1A2 (e.g., SEQ ID NOs: 58-63).

[0120] In some aspects, the subject has been diagnosed as suffering from a bone disease. In some aspects, the bone disease is a skeletal dysplasia. In some aspects, the bone disease is a bone fragility syndrome. In some aspects, the subject has been diagnosed as suffering from a disease of the musculoskeletal system.

[0121] The vector comprising the polynucleotide of interest, such as codon-optimized COL2A1, COL1 Al, or COL1 A2, and hybrid promoter according to the invention 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 1 7:S67-S68, 2009), and Yamamoto et al., (J. Bone Miner. Res. 26: 135-142, 2011 ).

[0122] Provided herein are 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-a 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).

[0123] 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 invention 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).

[0124] Despite the wide range of clinical manifestations observed in bone fragility syndromes, most cases of 01 result from mutations that affect the genes that encode the proal and proa2 polypeptide chains that comprise the type I collagen molecules. Type I collagen is the most abundant type of collagen and is widely distributed in almost all connective tissues with the exception of hyaline cartilage. It is the major protein in bone, skin, tendon, ligament, sclera, cornea and blood vessels. Mutations in COL1 Al and COL1 A2 genes that affect the amino-acid sequence in the helical domain of one of the proa chains, resulting in a complete lack of type I collagen production, or a substitution of the conserved glycine residue with an amino acid with a bulky side chain, result in 01. The genotype-phenotype relationship is not clearly understood, but what is clear is that bone fragility may result from either insufficient matrix synthesis or the accumulation of defective collagen molecules in the extracellular matrix.

[0125] The clinical expression of 01 traverses a broad spectrum from mild osteoporosis to prenatal death. Musculoskeletal abnormalities include long bone deformities with anterior bowing of the humerus, tibia and fibula and lateral bowing of the femur, radius and ulna. The hallmark of 01 is bone fragility with fractures occurring with minimal to moderate trauma. The number of fractures varies according to the severity of the disease. In general, the earlier the fractures occur in life, the more severe the disease. The lower limbs are more commonly involved, as they are more susceptible to trauma. Femural fractures are the most common fractures of long bones, with the fracture located usually at the convexity of the bone. They are usually transverse and minimally displaced. Multiple fractures within the same bone often occur as a result of the severe angulation in which it heals and because of disuse atrophy, both of which make it more susceptible to a second fracture. Bowing of the long bones results from the multiple transverse fractures and the pulling of strong muscles. Cranial deformity is also common. There is flattening of the posterior cranium with a bulging calvarium and a triangular-shaped face. In some 01 patients, the teeth are also severely affected; they are extremely brittle, breaking easily and susceptible to caries. When the teeth are affected in 01 patients, this is referred to as dentigenesis imperfecta and has been used by Sillence to subclassify Type I and Type III 01. Extraskeletal findings include blue sclera in some 01 patients and it is a hallmark characteristic of 01 Type I. Deafness occurs in approximately 40% of Type I 01 patients and in a lower percentage of Type IV 01.

[0126] Treatment with a vector comprising the polynucleotide of interest can result in an improvement in a symptom of a bone disease, e.g., a skeletal dysplasia or bone fragility syndrome, due to increased delivery of the polynucleotide of interest to bone, cartilage, and/or the bone growth plate and decreased delivery to liver, spleen, and/or the hematolymphoid system. The methods can be used to treat symptoms associated 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.

[0127] Any skeletal dysplasia that is a type II collagenopathy (e.g., caused by or associated with a mutation in C0L2A1 gene) can be treated by administering a vector comprising a hybrid promoter of the invention operably linked to a codon-optimized sequence coding for a collagen chain, such as the pro-a 1 (II) chain of type II collagen encoded by C0L2A1 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.

[0128] 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.Gl lOIR, p.Gl l lOS, p.G1152D, p.G1155V, p.G1158S, р.G1173R, p.G1173V, p.G1176V, p.G1179R, p.G1188A, p.G1197S mutations of the proalpha 1 (II) chain of type II collagen encoded by C0L2A1 gene, as well as any one of the mutations of C0L2A1 gene causing Stickler syndrome, such as the C57Y, G216D, G219R, G222V, G240D, G267D, G270R, G282D, del302-308, G453A, G492D, G501R, R565C, R904C, G1131 A or G1158A mutations, or any other mutations of the C0L2A1 gene. For example, 36% of SEDc mutations include: C.2965OT p.(Arg989Cys) 8%; с, 1510G>A p.(Gly504Ser) 5%; c.3589G>Ap.(Glyl 197Ser) 5%; c.4349T>C р.(Ilel450Thr) 5%; c,1781G>A p.(Gly594Glu) 4%; c,1214G>A p.(Gly405Asp) 3%; с.3283G>A p.(GlylO95Ser) 3%; c.3455G>A p.(Glyl 152Asp) 3%. Interestingly, G substitution 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 invention, the type II collagenopathy may be due to a glycine substitution.

[0129] Genetic abnormalities, for example any of the aforementioned mutations in the COL2A1 and COL1 A2 genes, can be detected in a sample from the patient prior to or after treatment. 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.

[0130] Administration of a vector as disclosed herein can result in an improvement in symptoms including, but not limited to, fractures, 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.

[0131] Symptoms of bone disease 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 invention. 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 invention 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 and/or skull width but also cervical instability, respiratory complications and/or neurological 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 a polynucleotide of interest. 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 polynucleotide of interest, and hybrid promoter according to the invention. For example, body weight and/or skull size are measured in response to administration of the vector of the present invention.

[0132] 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.

[0133] Measurements can be taken by any known or self-devised standardized method. For a human subject, the measurement of skull circumference is performed. 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) is performed.

[0134] 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 performed. 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.

[0135] 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.

VII. Pharmaceutical compositions and formulations

[0136] 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.

[0137] 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.

[0138] Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising the polynucleotide of the present disclosure or a plurality thereof (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.

[0139] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed.

[0140] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.

[0141] A composition of the present invention (e.g., including a vector comprising the polynucleotide sequence of interest) is administered via intraperitoneal administration. 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 the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the polynucleotide of interest, receptor-mediated endocytosis, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.

[0142] In some aspects, the nucleic acid of the invention can be delivered in a vesicle, in particular a liposome. In yet another aspect, the nucleic acid of the invention can be delivered in a controlled release system.

[0143] Any amount of a pharmaceutical composition (including a vector comprising the polynucleotide of interest according to the invention) 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). In some aspects, the patient has a bone fragility syndrome. The dosages will depend on many factors including the age of the patient. Typically, the amount of the composition (e.g., including a vector according to the invention) 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.

[0144] 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.

EXAMPLES

[0145] The following examples are illustrative and do not limit the scope of the claimed aspects.

Example 1. In vivo administration of INS-101

[0146] To examine whether the route of administration influenced gene delivery, 6-8 week old wild-type C57B1/6 mice were injected with INS-101 at a dosage of IxlO⁹ transducing units (TU)/kg either by intravenous or intraperitoneal administration (N=6 per group, 3 males and 3 females). Biodistribution was analyzed one week postadministration in genomic DNA extracted from the liver, spleen, bone marrow and tibial growth plates. Vector copy number (VCN) per diploid genome was determined by quantitative polymerase chain reaction (qPCR). As shown in Figure 1, both intraperitoneal and intravenous administration resulted in higher levels of INS-101 gene delivery in the liver (red square) and spleen (green round). VCN per diploid genome were not significantly different between the two routes of administrations in these tissues. By contrast, INS-101 gene delivery was significantly increased in the tibial growth plates after intraperitoneal administration compared to intravenous gene delivery after one week (Figure 1, p< 0.005 by Mann-Whitney test).

[0147] The sustained high-level gene delivery by intraperitoneal route was reproduced in neonate homozygous COL2Al^sedc/sedc mice at 3 weeks-old after single dosing at Day 5 or repeated dosing of INS-101 at day 1, 8 and 15 days after birth (Figure 2). The COL2Al^sedc/sedc disease mouse model (B6(Cg)-Col2alsedc/GrsrJ) is a spontaneous mutation (R1417C) described by the Jackson Laboratory (Donahue et al., 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. As shown in Figure 2, SEDc mice dosed intraperitonealy demonstrated similar biodistribution levels in liver, spleen and sternal growth plate tissues after either 3 doses of IxlO⁹ TU/kg INS-101 on Day 1, 8, and 15 (repeat dose) or a single-dose of 3xl0⁹ TU/kg on Day 5 only (single dose), supporting that both repeat and single intraperitoneal dose result in similar and sustained high level biodistribution of INS-101 in the bone growth plates of neonatal SEDc mice despite an expected turn-over of chondrocytes as part of the biology of the bone growth plate during rapid skeletal growth from birth to 3 weeks of age in mice. These results were confirmed by the high level of RNA expression in the sternum growth plates as measured by RT-qPCR from the same tissues (Figure 3). Finally, long-term high-level gene delivery was confirmed in the bone growth plate after intraperitoneal administration in both wild-type and heterozygote COL2Al^sedc/+ mice injected intraperitoneally with 3 doses of IxlO⁹ TU/kg INS-101 on Day 1, 8, and 15 after birth and sacrificed at +6 months (Figure 4).

[0148] The tissue targeting effects of intraperitoneal administration were confirmed using a minipig model. Two week old minipigs were dosed with IxlO⁹ INS-101 by intravenous or intraperitoneal administration. Biodistribution in tissue was analyzed by qPCR one week post-administration. As shown in Figure 5, VCN per diploid genome was increased in the tibial growth plate. However, a two log reduction in VCN was also seen in the liver and spleen. These results were confirmed using both a EFla and a hCOL2Al hybrid promoter in N=6 minipig with males and females.

* * *

[0149] 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.

[0150] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0151] Any examples provided herein are offered by way of illustration and not by way of limitation. SEQUENCES

Claims

WHAT IS CLAIMED IS: A method of targeting delivery of a polynucleotide of interest to bone, cartilage and/or bone growth plate comprising providing the polynucleotide of interest to a subject in need thereof via intraperitoneal administration. A method of decreasing delivery of a polynucleotide of interest to the liver, spleen and/or the hematolymphoid system in a subject, comprising providing the polynucleotide of interest to the subject via intraperitoneal administration; wherein the amount of polynucleotide in the liver, spleen and/or the hematolymphoid system is decreased relative to the amount of polynucleotide in the liver, spleen and/or the hematolymphoid system following intravenous administration. The method of claim 2, wherein the hematolymphoid system comprises the bone marrow, thymus, lymph nodes and/or mucosal lymphoid tissues. A method of treating a bone disorder in a subject in need thereof, comprising providing a polynucleotide of interest to the subject via intraperitoneal administration, wherein the intraperitoneal administration of the polynucleotide of interest increases delivery to bone, cartilage, and/or the bone growth plate. The method of claim 4, wherein the intraperitoneal administration of the polynucleotide of interest reduces delivery to non-targeted tissues and increases the therapeutic window for treatment. The method of claim 5, wherein the non-targeted tissues are liver, spleen, and/or cells of the hematolymphoid system. The method of any one of claims 1-6, wherein the polynucleotide of interest is delivered to the bone, the growth plate, the cartilage and/or the bone marrow. The method of any one of claims 1-7, wherein the polynucleotide of interest comprises a nucleotide sequence encoding the alpha-1 chain of human type II collagen (hCOL2Al). The method of claim 8, wherein the polynucleotide comprises a nucleotide sequence of SEQ ID NOs: 1-13. The method of any one of claims 1-9, wherein the polynucleotide of interest further comprises a promoter element. The method of claim 10, wherein the promoter element is a hybrid promoter comprising a fragment of the hEFla promoter and a fragment of the hCOL2Al promoter; wherein the hybrid promoter is operably linked to a nucleotide sequence encoding hCOL2Al. The method of claim 11, wherein the promoter element comprises the sequence of SEQ ID NOs: 14-33. The method of any one of claims 1-12, wherein the polynucleotide of interest is contained in a viral vector. The method of claim 13, wherein the vector is a lentiviral vector. The method of claim 14, wherein delivery of the polynucleotide results in at least about a 2-fold reduction of vector copy number in liver and/or spleen and/or hematolymphoid system compared to the copy number of the polynucleotide of interest following intravenous delivery. The method of any one of claims 1-15, wherein the polynucleotide of interest is contained in a lentiviral particle. The method of any one of claims 1-16, wherein the polynucleotide of interest is present in the bone at least 3 weeks following administration. The method of any one of claims 1-17, wherein the subject has a type II collagen disorder. The method of claim 18, wherein the subject has spondyloepiphyseal dysplasia congenital (SEDc). A method of treating a bone fragility syndrome in a subject in need thereof, comprising providing at least one polynucleotide of interest to the subject via intraperitoneal administration. The method of claim 20, wherein the subject has osteogenesis imperfecta, osteoporosis, osteomalacia, or Paget's disease. The method of claims 20 or 21, wherein at least one polynucleotide of interest is delivered by intraperitoneal administration. The method of any one of claims 20-22, wherein at least one polynucleotide of interest is preferentially delivered to the bone growth plate and/or bone marrow. The method of any one of claims 20-23, wherein the polynucleotide of interest comprises a nucleotide sequence encoding the alpha-1 or alpha-2 chain of human type I collagen, or the alpha-1 chain of human type II collagen. The method of claim 24, wherein the polynucleotide comprises a nucleotide sequence of SEQ ID NOs: 1-13 or 34-63. The method of claim 25, wherein administration of the polynucleotide of interest results in bone growth and/or a decrease in bone fragility. The method of any one of claims 20-26, wherein the polynucleotide of interest further comprises a promoter element. The method of claim 27, wherein the promoter element is a hybrid promoter comprising a fragment of the hEFla promoter and a fragment of the hCOL2Al promoter; wherein the hybrid promoter is operably linked to a nucleotide sequence encoding hCOL2Al, hCOLlAl or hCOLlA2. The method of claim 28, wherein the promoter element comprises the sequence of SEQ ID NOs: 14-33. The method of any one of claims 20-29, wherein the polynucleotide of interest is contained in a viral vector. The method of claim 30, wherein the vector is a lentiviral vector. The method of any one of claims 20-31, wherein the polynucleotide of interest is contained in a lentiviral particle. The method of any one of claims 20-32, wherein the polynucleotide of interest is delivered ex vivo. The method claim 33, wherein the cells are delivered to mesenchymal stem cells. The method of any one of claims 20-34, wherein the polynucleotide of interest is administered in combination with at least one therapeutic agent. The method of claim 35, wherein the polynucleotide of interest and the therapeutic agent are administered sequentially, and in any order. The method of claims 35 or 36, wherein the agent is selected from the group consisting of a bisphosphonate, a parathyroid hormone, a parathyroid hormone analog, calcitonin, and a selective estrogen receptor modulator. The method of claim 37, wherein the polynucleotide of interest is administered in combination with alendronate, pamidronate, zoledronate, or risedronate. The method of claim 37, wherein the polynucleotide of interest is administered in combination with teriparatide. The method of claim 12, wherein the promoter element comprises the sequence of SEQ ID NO: 14. The method of claim 29, wherein the promoter element comprises the sequence of SEQ ID NO: 14.