Hybrid promoters and their use in osteolysis syndrome

Abstract

The present invention relates to compositions comprising nucleic acid molecules, e.g., polynucleotides encoding a codon-optimized MAF BZIP Transcription Factor B (MafB), and / or hybrid promoters with a specific design, comprising fragments of the MafB promoter and fragments of the hEF1α promoter, which may be of interest in therapy, particularly in gene therapy. These compositions are useful for treating osteolysis syndromes, including MCTO.

Description

HYBRID PROMOTERS AND THEIR USE IN OSTEOLYSIS SYNDROMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional App. No. 63 / 269,514, filed March 17, 2022, and U.S. Provisional App. No. 63 / 269,517, 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_013PC02_Seqlisting_ST26; Size: 28,997 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 comprising hybrid promoters and / or polynucleotides encoding a codon-optimized MAF BZIP Transcription Factor B (MafB), for use in therapy, especially in gene therapy, more particularly for treating osteolysis syndromes.BACKGROUND OF THE INVENTION

[0004] Osteolysis syndromes are progressive conditions where bone tissue is destroyed.In this process, bones lose minerals (mostly calcium), soften, degenerate and become weaker. Osteolysis occurs when cells in the bone called osteoclasts increase their activity and break down the surrounding minerals. There are different types of osteolysis, and each has specific mechanisms that lead to this increase in osteoclast activity and the resulting condition of demineralization.

[0005] Idiopathic osteolysis, or vanishing bone disease, represents a group of rare diseases characterized by destruction and resorption of affected bones with subsequent skeletal deformities and functional impairment. Hardegger et al (J Bone Joint Surg Br. 1985) classified this disorder according to the phenotypes and genotypes as follows: 1)type 1, hereditary multicentric osteolysis with dominant transmission; 2) type 2, hereditary multicentric osteolysis with recessive transmission; 3) type 3, nonhereditary multicentric osteolysis with nephropathy; 4) type 4, Gorham-Stout syndrome; and 5) type 5, Winchester syndrome, defined as a monocentric disease with autosomal recessive inheritance.

[0006] Multicentric carpotarsal osteolysis syndrome (MCTO) is a rare osteolysis syndrome that is an autosomal dominant condition. MCTO presents as gradual loss of the small bones in the carpus and tarsus with and without nephropathy. This typically leads to joint subluxation and instability. This condition is caused by mutations in the transcription factor MafB, or V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), gene. This gene encodes a basic leucine zipper (bZIP) transcription factor. Optimal treatment for this condition is unclear.

[0007] Clinically, MCTO is characterized by aggressive osteolysis, which mainly affects the carpal tarsal bones, and is frequently associated with progressive nephropathy. Since the painful swelling and motion limitation on the wrists and / or ankles of MCTO mimics those of juvenile idiopathic arthritis (JIA), very often, MCTO is misdiagnosed as JIA.

[0008] Heterozygous missense mutations in the transcription factor MafB are found in patients with MCTO. MafB is reported to negatively regulate osteoclastogenesis in vitro. However, the in vivo function of MAFB and its relation to MCTO remains unknown.

[0009] The bZip transcription factor MafB is a member of the large Maf transcription factor family. It is expressed in multiple tissues including the spinal cord, retina and hematopoietic cells, and plays diverse functions in tissue development and cellular differentiation. MafB is highly expressed in monocytes and macrophages and is important for macrophage differentiation. It has also been demonstrated that MafB regulates osteoclastogenesis.

[0010] Osteoclasts are bone-resorbing cells that are derived from the hematopoietic monocyte-macrophage lineage. They are located at or near the bone surface and degrade the bone in a specialized extracellular compartment called Howship's lacunae by secreting acid and lytic enzymes, like tartrate-resistant acid phosphatase (TRAcP) and Cathepsin K(CTSK). Osteoclast cells are indispensable for normal bone development and remodeling.

[0011] Bisphosphonates and denosumab can be of use for treating bone lesions, but there exists a need in the art for treatment of osteolysis syndromes, for example MCTO.BRIEF SUMMARY OF THE INVENTION

[0012] Certain aspects of the present disclosure are directed to a promoter comprising the polynucleotide of any one of SEQ ID NOs: 5-18. In some aspects, the promoter is contained in a vector. In some aspects, the vector is viral or non-viral. In some aspects, the promoter is operably linked to a nucleic acid sequence of interest. In some aspects, the promoter controls the expression of the MafB gene. In some aspects, the vector is an adenoviral, retroviral, lentiviral, or adeno-associated viral vector. In some aspects, the vector is a lentiviral vector.

[0013] Certain aspects of the disclosure are directed to a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 19, wherein the polynucleotide encodes MAF BZIP Transcription Factor B (MafB).

[0014] In some aspects, the polynucleotide further comprises a promoter element operably linked to the polynucleotide encoding the MafB polypeptide.

[0015] In some aspects, the promoter element is a hybrid promoter comprising a fragment of the hEFla promoter and a fragment of the MafB promoter.

[0016] In some aspects, the promoter element comprises the sequence of any one of SEQ ID NOs: 5-18.

[0017] Certain aspects of the disclosure are directed to a promoter comprising the polynucleotide of any one of SEQ ID NOs: 5-18.

[0018] Certain aspects of the disclosure are directed to a vector comprising the any of the polynucleotides disclosed herein, and / or any of the promoters disclosed herein.

[0019] In some aspects, the vector is viral or non-viral.

[0020] In some aspects, the promoter is operably linked to a nucleic acid sequence of interest.

[0021] In some aspects, the promoter controls the expression of the MafB gene.

[0022] In some aspects, the vector is an adenoviral, retroviral, lentiviral, or adeno- associated viral vector.

[0023] In some aspects, the vector is a lentiviral vector.

[0024] Certain aspects of the disclosure are directed to a viral particle comprising any of the vectors disclosed herein.

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

[0026] Certain aspects of the disclosure are directed to a host cell infected with any of the viral particles disclosed herein.

[0027] Certain aspects of the disclosure are directed to a method of treating osteolysis syndromes in a subject, comprising administering a therapeutic amount of any of the polynucleotides disclosed herein, any of the promoters disclosed herein, any of the vectors disclosed herein, or any of the viral particles disclosed herein to the subject.

[0028] In some aspects, the osteolysis syndrome is multicentric carpotarsal osteolysis syndrome (MCTO).

[0029] Certain aspects of the disclosure are 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.

[0030] Certain aspects of the disclosure are directed to a method for transducing a host cell comprising contacting the host cell with any of the lentiviral particles disclosed herein.

[0031] Certain aspects of the disclosure are directed to a method of treating a skeletal dysplasia or a disease of the musculoskeletal system in a subject, comprising administering a therapeutic amount of any of the polynucleotides disclosed herein, any of the promoters disclosed herein, any of the vectors disclosed herein, or any of the viral particles disclosed herein to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 shows a schematic representation of the MafB and EFla promoters. (E) - enhancer core element; (GC) - GC box; (CCAAT) - CAAT box; (TATA) - TATA box; (EFP1) and (EFP2) - EFP1 and EFP2 elements known to regulate EFla expression.

[0034] Figures 2A-2B show a schematic representation of the evaluated hybrid promoters.

[0035] Figure 3 shows a schematic representation of an exemplary plasmid comprising a promoter of the present disclosure.

[0036] Figures 4A-4B show GFP expression levels of HEK cells transfected with plasmids comprising the promoters of the present disclosure.

[0037] Figures 5A-5D show GFP expression levels of Jurkat cells transduced with lentiviral particles comprising the mla, m8a, or mlOa promoter of the present disclosure after 3 days (Figures 5A-5B) or 8 days (Figures 5C-5D).

[0038] Figure 5E shows average GFP expression intensity in Jurkat cells transduced with lentiviral particles comprising the mla, m8a, or mlOa promoter of the present disclosure relative to GFP expression driven by lentiviral particles comprising an EFla promoter.

[0039] Figure 6 shows a Western Blot measuring MafB expression in HEK lysate or supernatant after transduction with lentiviral particles comprising the m8a promoter of the present disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0040] Certain aspects of the present disclosure are directed to a promoter comprising the polynucleotide of any one of SEQ ID NOs: 5-18. In some aspects, the promoter is contained in a vector. In some aspects, the vector is viral or non-viral. In some aspects, the promoter is operably linked to a nucleic acid sequence of interest. In some aspects, the promoter controls the expression of the MafB gene. In some aspects, the vector is an adenoviral, retroviral, lentiviral, or adeno-associated viral vector. In some aspects, the vector is a lentiviral vector.

[0041] Certain aspects of the disclosure are directed to a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 19, wherein the polynucleotide encodes MAF BZIP Transcription Factor B (MafB).

[0042] Certain aspects of the disclosure are directed to a promoter comprising the polynucleotide of any one of SEQ ID NOs: 5-18.

[0043] Certain aspects of the disclosure are directed to a vector comprising the any of the polynucleotides disclosed herein, and / or any of the promoters disclosed herein.

[0044] Certain aspects of the disclosure are directed to a viral particle comprising any of the vectors disclosed herein.

[0045] Certain aspects of the disclosure are directed to a host cell infected with any of the viral particles disclosed herein.

[0046] Certain aspects of the disclosure are directed to a method of treating osteolysis syndromes in a subject, comprising administering a therapeutic amount of any of the polynucleotides disclosed herein, any of the promoters disclosed herein, any of the vectors disclosed herein, or any of the viral particles disclosed herein to the subject.

[0047] Certain aspects of the disclosure are 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.

[0048] Certain aspects of the disclosure are directed to a method for transducing a host cell comprising contacting the host cell with any of the lentiviral particles disclosed herein.

[0049] Certain aspects of the disclosure are directed to a method of treating a skeletal dysplasia or a disease of the musculoskeletal system in a subject, comprising administering a therapeutic amount of any of the polynucleotides disclosed herein, any of the promoters disclosed herein, any of the vectors disclosed herein, or any of the viral particles disclosed herein to the subject.I. Definitions

[0050] 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.

[0051] 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.

[0052] In order to further define this disclosure, the following terms and definitions are provided.

[0053] 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."

[0054] 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).

[0055] 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.

[0056] 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. Forexample, 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.

[0057] 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.

[0058] 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).

[0059] 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.

[0060] As used herein, the term "hMafB" refers to the Homo Sapiens MAF bZIP transcription factor B cDNA (accession number NM_005461.5), and variants thereof (e.g. codon optimized SEQ ID NO: 19).

[0061] 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. 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 promoters of the invention comprise a part of the sequence of the promoter of the hMafB gene, but also at least theTATA box of the hEFla promoter. In some aspects, the hybrid promoter comprises a polynucleotide of any one of SEQ ID NOs: 5-18.

[0062] As used herein, the term "osteolysis syndromes" refers to skeletal disorders that include, but are not limited to multicentric carpotarsal osteolysis syndrome (MCTO) with and without nephropathy, idiopathic mulitcentric osteolysis (IMO), Hajdu-Cheney syndrome, Torg-Winchester syndrome, acro-osteolysis syndrome, progeria, familial expansile osteolysis, mandibuloacral dysaplsia types A and B. The most common types of osteolysis, which are generally unrelated to one other, include: distal clavicular osteolysis - commonly called "weight lifter's shoulder"; periprosthetic osteolysis - which affects some people who have had joint replacement surgeries; and acro-osteolysis (rare) - in which bone in the distal phalanges (fingers or toes) of the hand or feet erode and degenerate.

[0063] 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. In some aspects, the subject is a child (i.e., in terms of the present invention a human that is still growing, particularly in height).

[0064] 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).

[0065] The term "MafB" or "MAFB" refers to Homo sapiens MAF bZIP transcription factor B. Wild-type MafB mRNA is described at accession number NM_005461.5.

[0066] 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.

[0067] 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 nonmammalian 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.

[0068] 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 x 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.

[0069] 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 some aspects, 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. 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. 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 virusescontain 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.

[0070] As used herein, the term "skeletal dysplasia" or "osteochondrodysplasia" 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. By definition, skeletal dysplasias are heritable diseases that have generalized abnormalities in cartilage and bone.

[0071] 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.

[0072] In some aspects, provided herein is a method for the treatment of a skeletal dysplasia, such as notably spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, Stickler syndrome or Jeune syndrome.

[0073] As used herein, the term "type II collagenopathy" 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.

[0074] In some aspects, provided herein is a method for the treatment of a type II collagenopathy, and preferably spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia or Stickler syndrome.

[0075] As used herein, the term "disease of the musculoskeletal system" 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.II. Promoters

[0076] In some aspects, the promoter of the invention comprise a part of the sequence of the promoter of the hMafB gene. In some aspects, the promoter further comprises at least the TATA box of the hEFla promoter. In some aspects, the promoter comprises a polynucleotide of any one of SEQ ID NOs: 5-18.

[0077] In some aspects, the promoter comprises a polynucleotide that is about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 97%, about 98% or about 99% identical to any one of SEQ ID NOs: 5-18.

[0078] In some aspects, the promoter comprises a polynucleotide that is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 5-18.

[0079] In some aspects, the promoter comprises a polynucleotide that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 97%, at least about 98% or at least about 99% identical to any one of SEQ ID NOs: 5-18.

[0080] In some aspects, the promoter comprises a nucleic acid sequence comprising SEQ ID NO: 2. In some aspects, the promoter comprises a nucleic acid sequence comprising SEQ ID NO: 3.

[0081] In some aspects, the promoter comprises any one of the sequences disclosed in Table 1.Table 1 - Promoters

[0082] In some aspects, the disclosure provides hMafB hybrid promoter mla, in which the TATA box is replaced by hEfla.

[0083] In some aspects, the disclosure provides hMafB hybrid promoter m2a, in which hEFla replaces sequence after the CCAAT box and replaces the TATA box.

[0084] In some aspects, the disclosure provides hMafB hybrid promoter m3a, in which hEFla is added upstream of the E box, and the TATA box is from hEFla.

[0085] In some aspects, the disclosure provides hMafB hybrid promoter m4a, in which hEFla replaces sequence upstream of the E box, and the TATA box is from hEFla.

[0086] In some aspects, the disclosure provides hMafB hybrid promoter m5a, in which hEFla replaces sequence containing the E box, GC box, GC box (60% homology), and the TATA box is from hEFla.

[0087] In some aspects, the disclosure provides hMafB hybrid promoter m6a, in which hEFla is added between the two GC boxes, and the TATA box is from hEFla.

[0088] In some aspects, the disclosure provides hMafB hybrid promoter m7a, in which hEFla replaces sequence between the two GC boxes, and the TATA box is from hEFla.

[0089] In some aspects, the disclosure provides hMafB hybrid promoter m8a, in which hEFla is added between the GC box and CCAAT box, and the TATA box is from hEFla.

[0090] In some aspects, the disclosure provides hMafB hybrid promoter m9a, in which hEFla replaces sequence between the GC box and CCAAT box, and the TATA box is from hEFla.

[0091] In some aspects, the disclosure provides hMafB hybrid promoter mlOa, in which hEFla is added between the E box and GC box, and the TATA box is from hEFla.

[0092] In some aspects, the disclosure provides hMafB hybrid promoter ml la, in which hEFla replaces the GC box, and the TATA box is from hEFla.

[0093] In some aspects, the disclosure provides hMafB hybrid promoter ml2a, in which hEFla replaces the GC box and sequence upstream of the CCAAT box, and the TATA box is from hEFla.

[0094] In some aspects, the disclosure provides hMafB hybrid promoter ml3a, in which hEFla replaces the CCAAT box and sequence upstream to the GC box, and the TATA box is from hEFla.

[0095] In some aspects, the disclosure provides hMafB hybrid promoter ml4a, in which hEFla replaces the E box, and the TATA box is from hEFla.

[0096] The present invention also relates to a vector comprising a promoter disclosed herein.

[0097] In some aspects, the vector is a viral vector. In some aspects, the vector is a lentiviral vector or an adeno-associated virus (AAV).

[0098] In some aspects, vector comprises a hybrid promoter of the invention which controls the expression of a gene or a fragment thereof coding for a polypeptide of interest. In some aspects, the polypeptide of interest is MafB.III. Polynucleotides

[0099] Certain aspects of the disclosure relates to a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 19, in which the polynucleotide encodes a codon optimized sequence of MafB. The protein encoded by this gene is a basic leucine zipper(bZIP) transcription factor that plays an important role in the regulation of lineagespecific hematopoiesis. The encoded nuclear protein represses ETS1 -mediated transcription of erythroid-specific genes in myeloid cells. MafB contains no introns.

[0100] According to an aspect, 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. In some aspects, the hybrid promoter can be any one of SEQ ID NOs: 5-18.

[0101] The present disclosure also encompasses a nucleic acid sequence of the disclosure encoding a codon optimized human MAFB protein that is "substantially identical" to SEQ ID NO: 19.

[0102] In some aspects, the present disclosure comprises a nucleic acid sequence that is about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 97%, about 98% or about 99% identical to SEQ ID NO: 19.

[0103] In some aspects, the present disclosure comprises a nucleic acid sequence that is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19.

[0104] In some aspects, the present disclosure comprises a nucleic acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 19.

[0105] Advantageously, the codon optimized nucleic acid of the disclosure provides for decreased immunogenicity with a subject.

[0106] In some aspects, the polynucleotide comprises any one of the sequences disclosed in Table 2.Table 2 - Codon Optimized SequencesIV. Therapeutic uses

[0107] The present disclosure also relates to the use of a hybrid promoter disclosed herein, or to the use of a vector comprising a hybrid promoter disclosed herein in therapy. In some aspects, the therapy is gene therapy. In some aspects, the gene therapy is an ex vivo gene therapy. Ex vivo gene therapy consists of transfecting a vector comprising a hybrid promoter disclosed herein into isolated cells, and thereafter transplanting the transfected cells into the patient. Useful cells for ex vivo therapy are mesenchymal stem cells (MSC) cells or cells removed from the patient. Alternatively, said gene therapy may be an in vivo gene therapy. In vivo gene therapy comprises injecting a vector comprising a hybrid promoter disclosed herein directly into the patient.

[0108] In some aspects, the present disclosure relates to the use of a codon-optimized sequence of human MafB under the control of a hybrid promoter, or to the use of a vector comprising the codon-optimized sequence of human MafB and hybrid promoter according to the disclosure in therapy.

[0109] 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 toreplace a mutated gene. Other forms involve direct correction of a mutation, or using nucleic acids that encode a therapeutic protein drug to provide treatment.

[0110] 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.[OHl] In some aspects, provided herein is a method for the treatment of a skeletal dysplasia, such as notably spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, Stickler syndrome or Jeune syndrome.

[0112] In some aspects, provided herein is a method for the treatment of a type II collagenopathy, and preferably spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia or Stickler syndrome.

[0113] The present invention further relates to a method for treating a subject have osteolysis syndrome comprising administering to said subject a vector comprising any of the hybrid promoters or polynucleotides disclosed herein.

[0114] In some aspects, the subject has been diagnosed as suffering from an osteolysis syndrome. In some aspects, the subject has been diagnosed as suffering from MCTO.

[0115] In some aspects, a vector comprising a hybrid promoter according to the invention is preferably 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). Expression regulatory signals can be selected to be compatible with the host cell for which expression is designed.

[0116] 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.

[0117] In some aspects, the vector comprises a codon-optimized sequence of hMafB, and the vector encodes a hybrid promoter which controls the expression of the MafB gene.

[0118] The introduced nucleic acid sequence of interest can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced nucleic acid sequence of interest either contains an origin of replication compatible withthe 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.

[0119] MCTO is a very rare condition characterized by progressive loss of some of the bones of the hand and feet, usually the carpal (bones of the wrist) and tarsal bones (bones of the ankle), as well as chronic kidney (renal) failure in many cases. The bone problems lead to deformity and disability. Some patients also have intellectual disability and facial abnormalities.

[0120] MCTO is caused by mutations in the MafB gene. Autosomal dominant inheritance has been described in many families but most cases are sporadic (Mumms S. Am J Med Genet A. September, 2014; 164A(9):2287-93). Mutations in MafB that reportedly cause MCTO include, but are not limited to c,161C>G, p.Ser54Trp; c,161C>T, p.Ser54Leu; c, 167C>T, p.Ser56Phe; c, 176C>T, p.Pro59Leu; c,184A>C, p.Thr62Pro; c,185C>T, р.Thr62Ile; c,188C>G, p.Pro63Arg; c,188C>T, p.Pro63Leu; c, 194G>T, p.Ser65Ile; с, 197C>G, p.Ser66Cys; c.206C>T, p.Ser69Leu; c.208T>G, p.Ser70Ala; c.209C>T, p.Ser70Leu; c.211C>T, p.Pro71Ser; and c.212C>T, p.Pro71Leu. Treatment is with longterm immunosuppressive therapy (Dolgikh VV, et al., Ter Arkh. 2015; 87(3):88-91).

[0121] Genetic abnormalities, for example any of the aforementioned mutations in the MafB gene, 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.

[0122] In some aspects, administration of a polynucleotide of interest decreases the gradual loss of the small bones in the carpus and tarsus. In some aspects, administration decreases joint subluxation and instability. In some aspects, craniofacial abnormalities including triangular facies, micrognathia, maxillary hypoplasia, and exophthalmos are decreased.

[0123] 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 meansexpression 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.

[0124] 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.

[0125] 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, at least 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.

[0126] 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.

[0127] 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.

[0128] Symptoms of osteolysis syndromes in patients (e.g., humans) may also be monitored prior to or after a patient is treated with a vector comprising the hybrid promoters and a 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 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.V. Pharmaceutical compositions and formulations

[0129] In some aspects, provided herein is a pharmaceutical composition comprising the hybrid promoters and / or the polynucleotide sequence of the present disclosure, and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject.

[0130] 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.

[0131] In some aspects, 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 MafB, 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.

[0132] 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 disclosure, 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 MafB, 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.

[0133] 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 mini circles and transposons can be used for delivery or lentiviral vectors.

[0134] In some aspects, the nucleic acid of the invention can be delivered in a vesicle, in particular a liposome. In some aspects, the nucleic acid of the invention can be delivered in a controlled release system.

[0135] 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 an osteolysis syndrome (e.g., a patient with MCTO). 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., osteolysis syndromes such as MCTO) as described herein without inducing significant toxicity.

[0136] 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 an osteolysis syndrome as described herein. In some aspects, the composition comprising the vector of the disclosure may be administered once a week.

[0137] Dosages of compositions comprising the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized MafB, and hybrid promoter according to the disclosure can be provided in either a single or multiple dosage regimens.

[0138] 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.

[0139] 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

[0140] 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.

[0141] 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.

[0142] In a specific aspect, 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.

[0143] 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).

[0144] 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.

[0145] The pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.

[0146] 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.

[0147] 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 nonionic surfactant, such as pluronic F68 at a final concentration of 0.01-0.0001%, such as ata 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.

[0148] In some aspects, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human 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.

[0149] 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.

[0150] 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.

[0151] In some aspects, the vector comprising the codon-optimized nucleic acid sequence of interest, such as codon-optimized MafB, 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.

[0152] 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.

[0153] 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.

[0154] 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 for intravenous, 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 in1 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.EXAMPLES

[0155] The following examples are illustrative and do not limit the scope of the claimed aspects.Example 1 - Codon Optimized MafB

[0156] The codon optimization for MafB 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.Example 2 - MafB promoters

[0157] MafB promoter and EFla promoter (Figure 1) were hybridized to produce hybrid promoters (Figures 2A-2B).

[0158] HEK293 cells plated in a 24-well plate were transfected with 1 pg per well of plasmids comprising the hybrid promoters of Figures 2A-2B (e.g., Figure 3). GFP expression was measured by FACS 40 hours after transfection (see Figures 4A-4B). All 14 constructs showed expression of GFP. However, the mla, m8a, and mlOa constructs unexpectedly showed the strongest expression.

[0159] Next, Jurkat cells (immortalized human T lymphocyte cell line) were transduced with lentivirus comprising the mla construct, the m8a construct, the mlOa construct, or an EFla-GFP construct (positive control) to determine specificity of the constructs. GFP expression was measured via FACs 3 days after transduction (or 2 days for the positive control) and 8 days after transduction. All 3 constructs showed expression in the Jurkat cells after 3 days (Figures 5A-5B) and 8 days (Figures 5C-5D). Average of GFP intensity on live cells was measured, and the mla construct was the most specific (11% of EFl Aintensity compared to 17.9% for the m8a construct and 13.7% for the mlOa construct) (Figure 5E).

[0160] Additionally, HEK cells were transduced with the m8a construct for 3 days, media was replaced, and the cells were incubated overnight prior to protein extraction. This experiment showed that there is no MafB secretion into the supernatant after the overnight incubation (Figure 6).* * *

[0161] 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.

[0162] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0163] Any examples provided herein are offered by way of illustration and not by way of limitation.

Claims

WHAT IS CLAIMED IS: A polynucleotide comprising a nucleotide sequence of SEQ ID NO: 19, wherein the polynucleotide encodes MAF BZIP Transcription Factor B (MafB). The polynucleotide of claim 1, further comprising a promoter element operably linked to the polynucleotide encoding the MafB polypeptide. The polynucleotide of claim 2, wherein the promoter element is a hybrid promoter comprising a fragment of the hEFla promoter and a fragment of the MafB promoter. The polynucleotide of claim 3, wherein the promoter element comprises the sequence of any one of SEQ ID NOs: 5-18. A promoter comprising the polynucleotide of any one of SEQ ID NOs: 5-18. A vector comprising the polynucleotide of any one of claims 1-4, and / or the promoter of claim 5. The vector of claim 6, which is viral or non-viral. The vector of claims 6 or 7, wherein the promoter is operably linked to a nucleic acid sequence of interest. The vector of any one of claims 6-8, wherein the promoter controls the expression of the MafB gene. The vector of any one of claims 6-9, wherein the vector is an adenoviral, retroviral, lentiviral, or adeno-associated viral vector. The vector of claim 10, wherein the vector is a lentiviral vector. A viral particle comprising the vector of claim 10. The viral particle of claim 12, wherein the particle is a lentiviral particle.A host cell infected with the viral particle of claim 12. A method of treating osteolysis syndromes in a subject, comprising administering a therapeutic amount of the polynucleotide of any one of claims 1-4, the promoter of claim 5, the vector of any one of claims 6-11, or the viral particle of claims 12 or 13 to the subject. The method of claim 15, wherein the osteolysis syndrome is multicentric carpotarsal osteolysis syndrome (MCTO). A method for producing a lentiviral particle comprising introducing a polynucleotide of claim 1 into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell. A method for transducing a host cell comprising contacting the host cell with the lentiviral particle of claim 13. A method of treating a skeletal dysplasia or a disease of the musculoskeletal system in a subject, comprising administering a therapeutic amount of the polynucleotide of any one of claims 1-4, the promoter of claim 5, the vector of any one of claims 6-11, or the viral particle of claims 12 or 13 to the subject.

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