WO2025094056A1 - Gene therapy constructs and methods of use therefor - Google Patents

Abstract

The present invention is directed to polynucleotides, and to vectors comprising the polynucleotides, that are useful for the expression of a CTNNB1 transgene in cells and for use in gene therapy of CTNNB1-related disorders. In one aspect of the invention there is provided a nucleic acid construct comprising: a promoter operably linked to a nucleotide sequence encoding a functional CTNNB1 polypeptide; a 5' untranslated region (UTR) of a human CTNNB1 gene or a fragment of said 5' UTR; a microRNA (miRNA) targeting region; and a polyadenylation signal.

Description

GENE THERAPY CONSTRUCTS AND METHODS OF USE THEREFOR Field of Invention [0001] The present invention is directed to genetic constructs and vectors for the expression of a catenin beta 1 (CTNNB1) transgene in a host cell. The present invention is also directed to gene therapy applications for the treatment of disorders associated with loss of function mutations in the CTNNB1 gene, using the genetic constructs and vectors described herein. Background [0002] The CTNNB1 gene encodes β-catenin, a component of the Wnt signaling pathway involved in regulating cell proliferation and differentiation. Loss of function mutations in CTNNB1 are associated with intellectual disability. Specifically, CTNNB1 mutations are responsible for a group of CTNNB1-related disorders, characterised by mild to severe cognitive impairment. The disorders are also commonly associated with developmental delays, speech impairment, abnormal muscle tone and peripheral spasticity, ophthalmological defects, facial dimorphism, microcephaly and behavioral problems. The most common CTNNB1-related disorders are CTNNB1 neurodevelopmental disorder (CTNNB1 syndrome), familial exudative vitreoretinopathy (FEVR) and neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV). [0003] While genetic screening, including prenatal screening, has greatly improved the diagnosis of CTNNB1-related neurodevelopmental disorders, there is currently no curative treatment. Treatment is typically limited to supportive care and therapies to improve quality of life of sufferers, including for example, behavioral therapy, speech therapy, physiotherapy and ophthalmological therapies. [0004] There is a need for the development of approaches to the treatment and cure of CTNNB1-related neurodevelopmental disorders. [0005] Gene therapy has been used both experimentally and in the clinic to treat a range of conditions and diseases, including liver disease, heart disease, diabetes, cancer, immunodeficiencies, arthritis, cystic fibrosis, hemophilia, muscular dystrophy, sickle cell anemia, and retinal degenerative conditions. Gene therapy can be performed using viral vectors or using non-viral methods, such as transfection of naked DNA or formulation in microparticles and nanoparticles (e.g. liposomes) to transfer the transgene into the target cell. [0006] To facilitate expression of the transgene in the host cell, the transgene is typically contained in a construct that also contains various regulatory elements necessary to express the transgene. These elements can include, for example, promoters, enhancers, initiation signals, termination signals, introns and other regulatory elements, which must function together to facilitate not only stable expression of the transgene in the target cell, but also expression at levels that are sufficient to effect therapy. The promoter and other regulatory elements, as well as the vector or delivery characteristics, determine cell type specificity, transduction efficacy, and level and duration of expression. Stable and robust expression of a transgene in a target cell can be difficult to achieve. Summary of the Invention [0007] The present invention is directed to polynucleotides, and to vectors comprising the polynucleotides, that are useful for the expression of a CTNNB1 transgene in cells and for use in gene therapy of CTNNB1-related disorders. [0008] In one aspect of the invention there is provided a nucleic acid construct comprising: a promoter operably linked to a nucleotide sequence encoding a functional CTNNB1 polypeptide; a 5' untranslated region (UTR) of a human CTNNB1 gene or a fragment of said 5' UTR; a microRNA (miRNA) targeting region; and a polyadenylation signal. [0009] Typically, the CTNNB1 nucleotide sequence encodes a human CTNNB1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence having at least or about 80% identity thereto. The nucleotide sequence may comprise a CTNNB1 cDNA sequence comprising the sequence set forth in SEQ ID NO:2 or a sequence having at least or about 80% identity thereto. The nucleotide sequence encoding CTNNB1 may be codon optimized for expression in humans, optionally comprising the sequence set forth in SEQ ID NO:3. [0010] The CTNNB1 nucleotide sequence may comprise one or more intronic sequences. Optionally, the intronic sequences comprise: at least about 50 nucleotides derived from the 5' end of the human CTNNB1 intron 2, comprising the sequence set forth in SEQ ID NO:5 or a sequence at least or about 80% identical thereto; and at least about 50 nucleotides derived from the 3' end of the human CTNNB1 intron 2, comprising the sequence set forth in SEQ ID NO:6 or a sequence at least or about 80% identical thereto. In exemplary embodiments the intronic sequences comprise the sequence set forth in SEQ ID NO:7 or a sequence at least or about 80% identical thereto, located at a position corresponding to the position between nucleotides 13 and 14 of the nucleotide sequence set forth in SEQ ID NO:2 or 3. [0011] In particular embodiments the promoter is a hybrid form of the chicken β-actin promoter, preferably the CBh promoter. In particular embodiments, the CBh promoter comprises a nucleotide sequence set forth in SEQ ID NO:8, or a sequence having at least or about 80% sequence identity thereto. [0012] In particular embodiments, the 5' UTR sequence comprises a nucleotide sequence set forth in SEQ ID NO:9, or a sequence having at least or about 80% sequence identity thereto. Typically the 5'UTR is located between the promoter and CTNNB1 nucleotide sequence. [0013] The miRNA targeting region comprises one or more targeting sequences for one or more miRNAs, optionally selected from miR122, miR183 and/or miR199. The miR122 targeting sequence may comprise the sequence set forth in SEQ ID NO:11 or a sequence having at least or about 80% sequence identity thereto. The miR183 targeting sequence may comprise the sequence set forth in SEQ ID NO:12 or a sequence having at least or about 80% sequence identity thereto. The miR199 targeting sequence may comprise the sequence set forth in SEQ ID NO:13 or a sequence having at least or about 80% sequence identity thereto. [0014] The miRNA targeting region may comprise one or more targeting sequences for miR122, miR183 and miR199 in any order. Optionally the miRNA targeting region comprises one or more copies of the sequence set forth in SEQ ID NO:14 or a sequence having at least or about 80% sequence identity thereto. In exemplary embodiments the miRNA targeting region comprises three copies of the sequence set forth in SEQ ID NO:14 or a sequence having at least or about 80% sequence identity thereto. For example, the miRNA targeting region may comprise the sequence set forth in SEQ ID NO:15 or a sequence having at least or about 80% sequence identity thereto. [0015] The polyadenylation signal may be a bovine growth hormone (BGH) poly(A) signal, optionally comprising the sequence set forth in SEQ ID NO:16 or a sequence having at least or about 80% sequence identity thereto. Alternatively, the polyadenylation signal may be the endogenous polyadenylation signal residing within the 3' UTR of the human CTNNB1 gene. Optionally therefore the construct comprises a CTNNB13' UTR or fragment thereof comprising the endogenous polyadenylation signal. In an exemplary embodiment, the CTNNB13' UTR fragment comprises the sequence set forth in SEQ ID NO:10 or a sequence having at least or about 80% sequence identity thereto. [0016] In one aspect, the invention provides a nucleic acid construct comprising, from 5' to 3': a CBh promoter; a CTNNB15' UTR; a CTNNB1 coding sequence operably linked to the CBh promoter; a miRNA targeting region comprising one or more copies of targeting sequences for, sequentially, miR122, miR183 and miR199; and a BGH poly(A) signal. [0017] In a particular embodiment of the above aspect, the nucleic acid construct comprises the CBh promoter of SEQ ID NO:8, the CTNNB15' UTR sequence of SEQ ID NO:9, the CTNNB1 coding sequence of SEQ ID NO:2 or 3, the miRNA targeting region of SEQ ID NO:15, and the BGH poly(A) signal of SEQ ID NO:16. [0018] In another aspect, the invention provides a nucleic acid construct comprising, from 5' to 3': a CBh promoter; a CTNNB15' UTR; a CTNNB1 coding sequence operably linked to the CBh promoter, wherein the coding sequence comprises one or more intronic sequences; a miRNA targeting region comprising one or more copies of targeting sequences for, sequentially, miR122, miR183 and miR199, and a BGH poly(A) signal. [0019] In a particular embodiment of the above aspect, the nucleic acid construct comprises the CBh promoter of SEQ ID NO:8, the CTNNB15' UTR sequence of SEQ ID NO:9, the CTNNB1 coding sequence of SEQ ID NO:2 or 3 comprising the intronic sequences of SEQ ID NO:7 located between positions 13-14 of SEQ ID NO:2 or 3, the miRNA targeting region of SEQ ID NO:15, and the BGH poly(A) signal of SEQ ID NO:16. [0020] In another aspect, the invention provides a nucleic acid construct comprising, from 5' to 3': a CBh promoter; a CTNNB15' UTR; a CTNNB1 coding sequence operably linked to the CBh promoter; a miRNA targeting region comprising one or more copies of targeting sequences for, sequentially, miR122, miR183 and miR199, and a CTNNB13' UTR sequence comprising a poly(A) signal. [0021] In a particular embodiment of the above aspect, the nucleic acid construct comprises the CBh promoter of SEQ ID NO:8, the CTNNB15' UTR sequence of SEQ ID NO:9, the CTNNB1 coding sequence of SEQ ID NO:2 or 3, the miRNA targeting region of SEQ ID NO:15, and the 3' UTR sequence of SEQ ID NO:10. [0022] The nucleic acid constructs of the present invention may further comprise an adeno-associated virus (AAV) inverted terminal repeat (ITR) 5' of the promoter and an AAV ITR 3' of the poly(A) signal. For example, the AAV ITRs may be derived from an AAV serotype selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13. In a particular example, the AAV ITR 5' of the promoter comprises a sequence set forth in SEQ ID NO:17 and the AAV ITR 3' of the poly(A) signal comprises a sequence set forth in SEQ ID NO:18. [0023] In particular exemplary embodiments, there are provided nucleic acid constructs comprising the sequence of SEQ ID NO:19 or a sequence having at least or about 80% sequence identity thereto, the sequence of SEQ ID NO:20 or a sequence having at least or about 80% sequence identity thereto, or the sequence of SEQ ID NO:21, or a sequence having at least or about 80% sequence identity thereto. [0024] The nucleic acid constructs of the present invention are typically expression constructs designed for the expression of CTNNB1 in a suitable host cell. Optionally the constructs are in the form of, or are located within vectors. [0025] Accordingly, another aspect of the invention provides a vector comprising a nucleic acid construct described above and herein. The vector may, for example, be a polynucleotide vector (e.g. a plasmid, cosmid or transposon) or a viral vector (e.g. an AAV, lentiviral, retroviral, adenoviral, herpesviral or hepatitis viral vector). In particular embodiments, the vector is an AAV vector. [0026] Another aspect of the invention provides a host cell, comprising or transduced with a nucleic acid construct or vector described above and herein. [0027] By way of example, the cell may be a stem cell, for example a pluripotent or multipotent stem cell, optionally for example an induced pluripotent stem cell or a mesenchymal stem cell. [0028] In another aspect, provided is a method for the expression of CTNNB1, comprising introducing a nucleic acid construct or vector described above and herein into a host cell to facilitate expression of the CTNNB1 coding sequence present in the nucleic acid construct or vector in the host cell. [0029] In a further aspect there is provided a method for the treatment of a CTNNB1- related disorder or inhibiting or ameliorating at least one symptom thereof, wherein the disorder is characterised by, or associated with, a deficiency or mutation in CTNNB1 in a subject, comprising administering to the subject a nucleic acid construct, a vector or a cell as described above and herein. [0030] Optionally, the CTNNB1-related disorder is CTNNB1 neurodevelopmental disorder (CTNNB1 syndrome), familial exudative vitreoretinopathy (FEVR) or neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV). [0031] In an embodiment, the method further comprises the step, prior to administration, of identifying the subject as one that would benefit from receiving a therapy to treat a CTNNB1-related disorder or to inhibit or ameliorate at least one symptom thereof. [0032] A further aspect of the present invention provides the use of a nucleic acid construct, a vector or a cell as described above and herein for the manufacture of a medicament for the treatment of a CTNNB1-related disorder or inhibiting or ameliorating at least one symptom thereof, wherein the disorder is characterised by, or associated with, a deficiency or mutation in CTNNB1. Brief Description of the Drawings [0033] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings. [0034] Figure 1 - schematic representations of CTNNB1 expression cassettes as described herein. A, Construct #1 ("BCAT"), wild-type CTNNB1 coding sequence under the control of CBh promoter, further including a miRNA targeting region comprising a mixture of microRNA targeting sequences (miRTS) and the bovine Growth Hormone polyA signal (pA). B, Construct #2 ("5U"), wild-type CTNNB1 coding sequence including the 5’ untranslated region (5’UTR) under the control of CBh promoter, further including a miRNA targeting region comprising a mixture of microRNA targeting sequences (miRTS) and the bovine Growth Hormone polyA signal (pA). C, Construct #3 ("CTD"), truncated version of wild-type CTNNB1 coding sequence (∆CTD) including the 5’ untranslated region (5’UTR) under the control of CBh promoter, further including a miRNA targeting region comprising a mixture of microRNA targeting sequences (miRTS) and the bovine Growth Hormone polyA signal (pA). D, Construct #4 ("Intron"), wild-type CTNNB1 coding sequence including the 5’ untranslated region (5’UTR) and part of intron 2 under the control of CBh promoter, further including a miRNA targeting region comprising a mixture of microRNA targeting sequences (miRTS) and the bovine Growth Hormone polyA signal (pA). E, Construct #5 ("Opt"), codon-optimized version of CTNNB1 coding sequence under the control of CBh promoter, further including a miRNA targeting region comprising a mixture of microRNA targeting sequences (miRTS) and the bovine Growth Hormone polyA signal (pA). F, Construct #6 ("5U3U"), wild-type CTNNB1 coding sequence including the 5’ untranslated region (5’UTR) under the control of CBh promoter, further including a miRNA targeting region comprising a mixture of microRNA targeting sequences (miRTS) and a truncated endogenous CTNNB1 3’UTR containing the polyA signal (3’UTR). [0035] Figure 2 – A, Neuro-progenitor cells derived from induced pluripotent stem cells (iPSC) generated from cells sourced from a healthy donor. B, Neuro-progenitor cells derived from induced pluripotent stem cells (iPSC) generated from cells sourced from a patient. C, Relative expression levels of catenin beta 1 messenger RNA (mRNA), normalised with GAPDH (as a housekeeping gene), in healthy- (WT) and patient-derived (CTNNB1^mut) neuro- progenitor cells. D, Relative expression levels of vector-derived (exogenous) catenin beta 1 messenger RNA (mRNA), normalised with GAPDH (as a housekeeping gene), in healthy- (WT) and patient-derived (CTNNB1^mut) neuro-progenitor cells treated or not with AAV vectors containing constructs #1 to #6 at a multiplicity of infection (MOI) of 10,000 viral genomes/cell (vg/cell). In D: 1, Construct #1 (see Figure 1A); 2, Construct #2 (see Figure 1B); 3, Construct #3 (see Figure 1C); 4, Construct #4 (see Figure 1D); 5, Construct #5 (see Figure 1E); 6, Construct #6 (see Figure 1F). [0036] Figure 3 - Relative expression levels of catenin beta 1 messenger RNA (mRNA), normalised with GAPDH (as a housekeeping gene), in healthy- (WT) and patient-derived (CTNNB1^mut) neuro-progenitor cells treated or not with AAV vectors containing constructs #2, #4, or #6 at two different MOIs: LD= 5,000 vg/cell, HD= 10,000 vg/cell. LD, low dose; HD, high dose. 2, Construct #2 (see Figure 1B); 4, Construct #4 (see Figure 1D); 6, Construct #6 (see Figure 1F). [0037] Figure 4 - Relative expression levels of catenin beta 1 messenger RNA (mRNA), normalised with GAPDH (as a housekeeping gene), in 37-day-old healthy- (WT) and patient-derived (CTNNB1^mut) cortical brain organoids treated or not with AAV vectors containing constructs #2, #4, or #6 at an MOI of 10,000 vg/organoid. Organoids were transduced at day 23 of differentiation and harvested 2 weeks post-transduction. 2, Construct #2 (see Figure 1B); 4, Construct #4 (see Figure 1D); 6, Construct #6 (see Figure 1F). *, p<0.05. [0038] Figure 5 - Relative expression levels of catenin beta 1 messenger RNA (mRNA), normalised with GAPDH (as a housekeeping gene), in 141-day-old healthy- (WT) and patient- derived (CTNNB1^mut) cortical brain organoids treated or not with AAV vectors containing constructs #2, #4, or #6 at an MOI of 10,000 vg/organoid. Organoids were transduced at day 127 of differentiation and harvested 2 weeks post-transduction.2, Construct #2 (see Figure 1B); 4, Construct #4 (see Figure 1D); 6, Construct #6 (see Figure 1F). *, p<0.05. Detailed Description Definitions [0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information. [0040] As used herein, the singular forms "a", "an" and "the" also include plural aspects (i.e. at least one or more than one) unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a single polypeptide, as well as two or more polypeptides. [0041] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result. [0042] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or). [0043] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. [0044] The term “nucleic acid construct” refers to a genetic (nucleic acid) molecule including one or more polynucleotide (nucleic acid) sequences from one or more sources, from which CTNNB1 RNA and subsequently a functional CTNNB1 polypeptide or protein can be expressed. Thus, constructs include recombinant or chimeric molecules in which two or more polynucleotide sequences of different origin are assembled into a single nucleic acid molecule. Nucleic acid constructs defined herein typically include regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences). Nucleic acid constructs may be in the form of, or located in, a vector. Exemplary constructs include plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecules, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules are operably linked. For the practice of the methods of the present disclosure, conventional compositions and methods for preparing and using constructs are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3^rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000. [0045] A "transgene" as used herein refers to an exogenous DNA or cDNA sequence present in a polynucleotide, vector or host cell that encodes a functional CTNNB1 polypeptide or protein. The transgene may be foreign to the host cell into which it is introduced, or may represent a gene whose expression is otherwise absent or reduced in the host cell in the absence of the introduction and expression of the transgene. [0046] As used herein, the term "promoter" refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In some embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found about 70 to about 80 bases upstream from the start of transcription, e.g. a CNCAAT region where N may be any nucleotide. [0047] As used herein, the term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, enhancer or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence. Sequences may be in operable linkage regardless of the distance between the sequences or the relative orientations of the sequences in the construct. [0048] The term "enhancer" is used herein in its ordinary sense to refer to a nucleotide region comprising a sequence capable of increasing the level of transcription of a transgene from a promoter as compared to the level of transcription of the transgene from the promoter in the absence of the enhancer. Enhancers may be cis-acting or trans-acting, and may be located upstream or downstream of the transgene sequence, in either forward or reverse orientation with respect to the transgene sequence. An enhancer may be, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 base pairs, or any number of base pairs in a range defined by any two of the aforementioned values. [0049] As used herein, a "vector" includes reference to both polynucleotide vectors and viral vectors, each of which are capable of delivering a transgene contained within the vector into a host cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication-deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, cosmids and transposons. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpesviral and hepatitis viral vectors. [0050] As used herein, "adeno-associated viral vector" or AAV vector refers to a vector derived from an adeno-associated virus, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, or using synthetic or modified AAV capsid proteins. An AAV vector may also be referred to herein as "recombinant AAV", "rAAV", "recombinant AAV virion", and "rAAV virion," terms which are used interchangeably and refer to a replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. The AAV vector may be a chimeric vector comprising inverted terminal repeat regions (ITRs) derived from an AAV of one serotype wirth a capsid protein derived from an AAV of a different serotype. Examples include AAV2/8 and AAV2/5 vectors. [0051] The term "ITR" refers to an inverted terminal repeat at either end of the AAV genome. This sequence can form hairpin structures and is involved in AAV DNA replication and rescue, or excision, from prokaryotic plasmids. ITRs for use in the present invention need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging. Furthermore, the ITRs may be of any serotype or may be synthetic, and may be the same or different. [0052] As used herein, the term "host cell" refers to a cell into which a polynucleotide construct or vector of the invention is to be, or has been, introduced (e.g. transfected or transduced). In some embodiments, the host cells are mammalian cells in which the construct or vector can be introduced. Suitable mammalian host cells include, but are not limited to, human cells, murine cells, and non-human primate cells. The term includes the progeny of the original cell into which the construct or vector has been introduced. [0053] As used herein, the terms "transduce" or "transduction" refer to the delivery of a gene(s) using a viral or retroviral vector by means of infection rather than by transfection. Thus, a "transduced gene" is a gene that has been introduced into the cell via viral vector infection and provirus integration. Viral vectors (e.g., "transducing vectors") transduce genes into "target cells" or host cells. [0054] As used herein, the terms “treatment”, “treating”, and the like, refer to obtaining a desired pharmacologic and/or physiologic effect in a subject in need of treatment, that is, a subject who has a disease or disorder. By "treatment" is meant ameliorating or preventing one or more symptoms or effects (e.g. consequences) of a disease or disorder. Reference to “treatment”, “treat” or “treating” does not necessarily mean to reverse or prevent any or all symptoms or effects of a disease or disorder. For example, the subject may ultimately suffer one or more symptoms or effects, but the number and/or severity of the symptoms or effects is reduced and/or the quality of life is improved compared to prior to treatment. [0055] The term "CTNNB1-related disorder" as used herein refers to any disorder, for example a disorder characterised by or associated with mild to severe cognitive impairment, which is caused by or is associated with a deficiency or loss of function mutation in the CTNNB1 gene in one or more cells. [0056] The terms "subject", “patient” and “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, (e.g. human). [0057] By “effective amount”, in the context of treating a disease or condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the age, health and physical condition of the individual to be treated and whether symptoms of disease are apparent, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject. Optimum dosages may vary depending on the relative potency in an individual subject, and can generally be estimated based on EC50 values found to be effective in in vitro and in vivo models. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. [0058] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting. [0059] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise. Table 1. Brief Description of the Sequences SEQ ID Description NO. 1 Human CTNNB1 polypeptide 2 Human CTNNB1 coding sequence 3 Codon optimized CTNNB1 coding sequence 4 CTD domain of human CTNNB1 coding sequence of SEQ ID NO:2 5 5' end of human CTNNB1 intron 2 6 3' end of human CTNNB1 intron 2 7 5' and 3' ends of human CTNNB1 intron 2 8 CBh promoter 9 CTNNB15' UTR 10 Truncated CTNNB13' UTR with endogenous poly(A) signal 11 miR122 targeting sequence (A) 12 miR183 targeting sequence (B) 13 miR199 targeting sequence (C) 14 miRNA targeting region – (A+B+C)₁ 15 miRNA targeting region – (A+B+C)₃ 16 Bovine growth hormone poly(A) signal 17 5' inverted terminal repeat (5' ITR) 18 3' inverted terminal repeat (3' ITR) 19 Exemplary CTNNB1 expression construct #2 20 Exemplary CTNNB1 expression construct #4 21 Exemplary CTNNB1 expression construct #6 Nucleic acid constructs [0060] Provided are nucleic acid constructs that can be used for the expression of a CTNNB1 transgene, such as for gene therapy. The nucleic acid constructs therefore include a promoter operably linked to a CTNNB1 transgene. The nucleic acid constructs further include a 5' untranslated region of the CTNNB1 gene, a polyadenylation signal, one or more enhancer elements, and one or more miRNA targeting sequences. In particular embodiments, the nucleic acid construct also comprises viral elements to facilitate packaging of the polynucleotide into a viral vector. For example, some constructs of the present invention contain AAV inverted terminal repeat regions (ITRs) flanking the transgene and associated regulatory elements to facilitate packaging of the nucleic acid construct in an AAV vector. [0061] The various nucleic acid constructs described herein may be recombinant or synthetic and may be obtained by purification from a suitable source or produced by standard synthetic or recombinant DNA techniques such as those well known to persons skilled in the art, and described in, for example, Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press. [0062] As described herein, the selection or design of the various elements in the nucleic acid constructs, and the particular combination of the elements, is dictated at least in part by the requirements associated with the system used for delivery and/or expression of CTNNB1 and the gene therapy application intended. For example, where a viral vector is used, the constructs will contain the requisite viral elements to facilitate packaging, etc. Furthermore, viral vectors have limitations on the size of the genome that can be packaged, which in turn can dictate the size of each element in the genome. For example, AAV can package a genome slightly larger than the size of a wild-type genome, which is approximately 4.7 kb. Optimal packaging is achieved with genomes having a size of about 4.1-4.9 kb and packaging efficiencies can be adversely affected with genomes smaller or larger than this. For example, packaging may be significantly reduced when very large genomes are packaged. Packaging and transduction efficiency may also be adversely affected when smaller genomes are used. Without being bound by theory, there is the potential for additional DNA to be packaged in these circumstances, which in turn can result in errors in virus titration and dosage, and/or transductions efficiency. Thus, in particular embodiments of the present invention where the polynucleotides are designed for use in AAV vectors and thus represent the AAV genome (i.e. contain 5' and 3' AAV ITRs flanking the CTNNB1 coding sequence and regulatory elements), the size of the polynucleotide may be about or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the size of a wild-type AAV genome. [0063] Nucleic acid constructs of the present invention comprise a transgene expression cassette comprising a polynucleotide encoding a functional CTNNB1 polypeptide, operably linked to a promoter. Typically the CTNNB1 polypeptide is a human CTNNB1 polypeptide and the CTNNB1 polypeptide encoded by the polynucleotide is functional in that it produces a CTNNB1 polypeptide that shares qualitative and/or quantitative activity in common with the wild-type CTNNB1 protein, suitable to restore or provide cellular CTNNB1 levels and activity suitable to overcome or alleviate at least one symptom characteristic of a CTNNB1- related disorder. [0064] In exemplary embodiments, the CTNNB1 polypeptide encoded by the transgene is the wild-type human CTNNB1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, or a variant polypeptide comprising at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1, wherein the variant polypeptide retains activity of the wild-type CTNNB1 polypeptide. Typically, the variant polypeptide will retain at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the activity of the wild-type CTNNB1. CTNNB1 activity can be assessed by any method known in the art. Typically the CTNNB1 retains the carboxy terminal domain (CTD), encoded by the nucleotide sequence of SEQ ID NO:4. [0065] The CTNNB1 nucleotide sequence is typically a human CTNNB1 sequence, optionally a cDNA sequence. In exemplary embodiments, the CTNNB1 transgene comprises the nucleotide sequence set forth in SEQ ID NO:2, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, wherein the encoded CTNNB1 polypeptide retains at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the activity of the wild-type CTNNB1 protein. [0066] The CTNNB1 nucleotide sequence may be codon-optimized for expression in a suitable host cell. The term "codon optimized" as used herein has its plain meaning understood by those skilled in the art, referring to a polynucleotide encoding a polypeptide in which codon usage in the polynucleotide is altered to accommodate codon bias in a host cell and thereby optimize translational efficiency in the host cell. Codon optimization can be performed using algorithms known to those skilled in the art so as to create synthetic genetic transcripts optimized for high mRNA and protein yield in humans. Programs containing algorithms for codon optimization in humans are readily available. Such programs can include, for example, OptimumGene™ or GeneGPS® algorithms. Additionally human codon optimized sequences can be obtained commercially. [0067] In a particular embodiment the CTNNB1 nucleotide sequence is human codon- optimized and comprises the nucleotide sequence set forth in SEQ ID NO:3, or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, wherein the encoded CTNNB1 polypeptide retains at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the activity of the wild-type CTNNB1 protein. [0068] The inclusion of intronic sequences can promote the transcription of transgenes. Accordingly, the CTNNB1 transgene in constructs of the present invention may comprise one or more intronic sequences derived from the CTNNB1 gene. Optionally the transgene comprises sequences of the CTNNB1 intron 2, typically comprising sufficient sequences from the 5' and 3' ends of the intron as are required for splicing. For example, the intronic sequences may comprise at least about 50 nucleotides, preferably at least about 100 nucleotides, from the 5' end of intron 2 as set forth in SEQ ID NO:5, or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and at least about 50 nucleotides, preferably at least about 100 nucleotides, from the 3' end of intron 2 as set forth in SEQ ID NO:6, or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [0069] In exemplary embodiments, the CTNNB1 transgene comprises the intronic sequences set forth in SEQ ID NO:7, or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, located at a position corresponding to between nucleotides 13 and 14 of the CTNNB1 nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3. [0070] In constructs of the invention the CTNNB1 transgene is operably linked to a heterologous promoter. Typically the promoter facilitates the expression of the CTNNB1 transgene in one or more suitable cell types in which it is beneficial to express CTNNB1 for the treatment or alleviation of symptoms of CTNNB1-related disorders. The promoter may drive constitutive, conditional or inducible expression of the CTNNB1 transgene to which it is operably linked. In particular embodiments, of the present invention the promoter is a hybrid chicken β-actin (CBA) promoter, preferably the CBh promoter (see, for example, Gray et al., 2011, Hum Gene Ther 22:1143-1153) comprising the sequence set forth in SEQ ID NO:8, or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The CBh promoter may be a functional fragment of the promoter sequence set forth in SEQ ID NO:8 or sequence at least or about 80% identical thereto, wherein the functional fragment retains the ability to drive transcription of an operably linked CTNNB1 transgene. An exemplary functional fragment may comprise at least or about 300, 350, 400, 450. 500, 550, 600, 650, 700, or 750 consecutive nucleotides of the sequence set forth in SEQ ID NO:8, or sequence at least or about 80% identical thereto. [0071] The CTNNB15' UTR present in constructs of the present invention typically comprises the sequence set forth in SEQ ID NO:9, or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The 5' UTR is typically located 5' to the CTNNB1 transgene and 3' to the promoter. [0072] Constructs of the present invention comprise a miRNA targeting region. The miRNA targeting region is typically located 3' to the CTNNB1 transgene. The miRNA targeting region is typically designed to repress or inhibit expression of the transgene in undesired cell types or tissues, such as, for example, the liver and dorsal root ganglia. The miRNA targeting region as used herein comprises one or more copies of targeting sequences directed to one or more miRNAs. Exemplary miRNAs which may be targeted are miR-122, miR-183 and miR-199. Thus, embodiments of the invention contemplate miRNA targeting regions comprising one or more copies of miRNA targeting sequences (miRTSs) against one or more of miR-122, miR-183 and miR-199. Each miR-122 miRTS may comprise the sequence set forth in SEQ ID NO:11 or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Each miR- 183 miRTS may comprise the sequence set forth in SEQ ID NO:12 or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Each miR-199 miRTS may comprise the sequence set forth in SEQ ID NO:13 or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [0073] Where the miRNA targeting region comprises more than one miRTS, the miRTS may be in any order. In exemplary embodiments, the miRNA targeting region comprises an miR-122 miRTS, an miR-183 miRTS and an miR-199 miRTS in the order (miR-122 miRTS / miR-183 miRTS / miR-199 miRTS), as set forth in SEQ ID NO:14. Optionally the miRNA targeting region comprises two, three or more copies of (miR-122 miRTS / miR-183 miRTS / miR-199 miRTS). In particular exemplary embodiments, the miRNA targeting region comprises three copies of (miR-122 miRTS / miR-183 miRTS / miR-199 miRTS), with a sequence set forth in SEQ ID NO:15 or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [0074] Constructs of the invention further comprise a polyadenylation signal 3' to the CTNNB1 transgene, and typically 3' to the miRNA targeting region. The polyadelylation signal may be, for example, a bovine growth hormone (BGH) poly(A), rabbit β-globin (RBG) poly(A), SV40 poly(A), thymidine kinase (TK) poly(A), and any variants thereof. In exemplary embodiments, the polyadenylation signal is a BGH poly(A), for example comprising the sequence set forth in SEQ ID NO:16 or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In other exemplary embodiments, the polyadenylation signal is an endogenous CTNNB1 poly(A) signal residing in the CTNNB13' UTR. Thus, the construct may comprise the CTNNB1 3' UTR or a truncated form thereof containing the poly(A) sequences. For example, the truncated 3' UTR may comprise the sequence set forth in SEQ ID NO:10 or a sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [0075] Constructs described herein may further comprise one or more additional post- transcriptional regulatory elements that can function to increase expression of the transgene, such as for example a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), an RNA transport element (RTE), one or more additional translational initiation enhancers, such as a Kozac sequence, and one or more regulatory sequences modulating transduction of a vector comprising the construct, such as one or more polypuyrine tracts (PPTs). Delivery vehicles [0076] The present disclosure also contemplates delivery vehicles for delivering the nucleic acid constructs of the invention to a cell. Suitable delivery vehicles for nucleic acid molecules are well known in the art and may, for example, be viral or non-viral vectors. Suitable viral vectors for delivery of nucleic acid constructs include, but are not limited to adeno-associated viral vectors (AAV), lentiviral vectors, adenovirus vectors and herpes simplex viral vectors. [0077] For the purposes of embodiments of the present disclosure, a viral vector is a vector which comprises nucleic acid that includes at least one component part derivable from a virus, such as, for example, an AAV, an adenovirus, or a lentiviurus. That component part may be involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated. Thus, viral vectors include nucleic acid molecules such as plasmids, and virus particles. [0078] In particular embodiments described herein, the vector is an AAV vector. Embodiments of the present disclosure are described below in the context of an AAV vector, however the person skilled in the art will recognise these as exemplary embodiments only and will appreciate that the scope of the present disclosure is not limited thereto. [0079] The rep coding region of the AAV genome encodes the replication proteins Rep 78, Rep 68, Rep 52, and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other exogenous) promoters. The Rep expression products are collectively required for replicating the AAV genome. The AAV cap coding region of the AAV genome encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. Recombinant AAV-based vectors have the rep and cap viral genes that account for 96% of the viral genome removed, leaving the two flanking 145-basepair (bp) inverted terminal repeats (ITRs), which are used to initiate viral DNA replication, packaging and integration. In the absence of helper virus, wild-type AAV integrates into the human host-cell genome or may be maintained episomally. A single AAV particle can accommodate up to 5 kb of ssDNA, therefore leaving about 4.5 kb for a transgene and regulatory elements, which is typically sufficient. However, trans-splicing systems as described, for example, in U.S. Pat. No. 6,544,785, may nearly double this limit. [0080] In vectors of the present invention the nucleic acid construct is typically flanked at the 5' and 3' region with functional AAV inverted terminal repeat sequences (ITRs). The nucleotide sequences of AAV ITR regions are known. The ITR sequences for AAV2 are described, for example, by Kotin et al. Human Gene Therapy, 5:793-01 (1994); Fields & Knipe, Fundamental Virology, “Parvoviridae and their Replication” (2d ed. 1986). The skilled artisan will appreciate that AAV ITR's can be modified using standard molecular biology techniques (e.g., Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)). Accordingly, AAV ITRs used in the vectors of the present disclosure need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, and the like. Furthermore, the 5' and 3' ITRs, which flank a selected nucleotide sequence in an AAV expression vector, need not necessarily be identical or derived from the same AAV serotype or isolate, so long as the ITR's function as intended, i.e., to allow for excision and replication of the bounded nucleotide sequence of interest when AAV rep gene products are present in the cell. Exemplary AAV ITRs useful for the nucleic acid constructs described herein include those, for example, set forth in SEQ ID NOs:17 and 18. [0081] AAV vectors can be constructed using known techniques. An AAV vector harboring a nucleic acid construct of the invention flanked by AAV ITRs, can be constructed by directly inserting the sequence(s) of interest into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome can also be deleted, as long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. These constructs can be designed using techniques well known in the art. (See, e.g., Lebkowski et al., Molecular & Cellular Biology, 8:3988-96 (1988); Vincent et al., Vaccines 90 (Cold Spring Harbor Laboratory Press, 1990); Carter, Current Opinion Biotechnology, 3:533-39 (1992); Muzyczka, Current Topics Microbiology & Immunology, 158:97-29 (1992); Kotin, Human Gene Therapy, 5:793-01(1994); Shelling et al., Gene Therapy, 1:165-69 (1994); and Zhou et al., J Experimental Medicine, 179:1867- 75 (1994)). Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected polynucleotide construct that is present in another vector using standard ligation techniques, such as those described in Green & Sambrook (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)). [0082] In order to produce recombinant AAV particles, AAV cis- and trans-acting plamids can be introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art (See, e.g., Graham et al., Virology, 52:456 (1973); Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012); Davis et al., Basic Methods Molecular Biology, (Elsevier, 1986); and Chu et al., Gene, 13:197 (1981)). Exemplary transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles. [0083] Suitable host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an exogenous nucleic acid molecule. The host cell includes any eukaryotic cell or cell line so long as the cell or cell line is not incompatible with the protein to be expressed, the selection system chosen or the fermentation system employed. In some embodiments, the host cells are cells from the stable human cell line, 293 (readily available through, e.g., the ATCC under Accession No. ATCC CRL 1573), which is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments, and expresses the adenoviral E1a and E1 b genes. The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce AAV virions. Host cells containing the above-described AAV vectors must be rendered capable of providing AAV helper functions in order to replicate and encapsidate the expression cassette flanked by the AAV ITRs to produce recombinant AAV particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV vectors. Thus. AAV helper functions include one, or both of the major AAV open reading frames (ORFs), namely the rep and cap coding regions, or functional homologues thereof. [0084] AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV vector comprising the expression construct. AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and plM29+45 which encode both Rep and Cap expression products (See, e.g., Samulski et al., J. Virology, 63:3822-28 (1989); McCarty et al., J. Virology, 65:2936-45 (1991)). A number of other vectors have been described which encode Rep and/or Cap expression products (See. e.g., U.S. Pat. No.5,139,941, incorporated by reference). [0085] As a consequence of the transfection of the host cell with a helper plasmids, the AAV Rep and/or Cap proteins are produced in trans. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, and the AAV genome is packaged into the capsids. This results the AAV being packaged into recombinant AAV particles comprising the expression cassette. Following recombinant AAV replication, recombinant AAV particles can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. The resulting recombinant AAV particles are then ready for use for gene delivery to various cell types. [0086] In some embodiments, the number of viral vector and/or virion particles administered to a subject may be on the order ranging from 10³ to 10¹⁵ particles/mL, or any values therebetween, such as for example, about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ particles/mL. In some embodiments, vector and/or virion particles of higher than 10¹³ particles/mL are administered. Volumes between 1 µL and 10 mL may be administered such that the subject receives between 10² and 10¹⁶ total vector and/or virion particles. Thus, in some embodiments, about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ or 10¹⁶ vector and/or virion particles are administered. [0087] In accordance with the present invention, an AAV of any serotype can be used. The serotype of the viral vector used in certain embodiments of the invention is selected from the group consisting from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13 (see, e.g., Gao et al., 2002, PNAS 99:11854-11859; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Other serotypes besides those listed herein can be used. Furthermore, pseudotyped AAV vectors may also be utilized in the methods described herein. Pseudotyped AAV vectors are those which contain the ITRs of one AAV serotype in the capsid of a second AAV serotype. [0088] As will be appreciated by a skilled artisan, any method suitable for purifying AAV can be used in the embodiments described herein to purify AAV vectors comprising a polynucleotide construct of the invention, and such methods are well known in the art. For example, the recombinant AAV can be isolated and purified from packaging cells and/or the supernatant of the packaging cells. In some embodiments, the AAV is purified by separation method using a CsCl gradient. In other embodiments, AAV is purified as described in US20020136710 using a solid support that includes a matrix to which an artificial receptor or receptor-like molecule that mediates AAV attachment is immobilized. [0089] A vector of the present invention can be a viral vector other than an AAV vector as described above. For example, replication defective retroviruses, adenoviruses, herpes simplex viruses, and lentivirus can be used. Protocols for producing recombinant retroviruses and for transducing cells in vitro or in vivo with such vectors can be found in Ausubel et al., Current Protocols in Molecular Biology §§9.10-9.14 (Greene Publishing Associates, 1989) and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM, which are well known to those skilled in the art. Examples of suitable packaging virus lines include Crip, Cre, 2 and Am. The genome of adenovirus can be manipulated such that it encodes and expresses the protein of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (See e.g., Berkner et al., BioTechniques, 6:616-29 (1988); Rosenfeld et al., Science, 252:431-34 (1991); Rosenfeld et al., Cell 68:143-55 (1992)). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. [0090] The production of suitable lentiviral vectors is well known in the art (See, e.g., U.S. patent application Ser. No.13/893,920, incorporated by reference). A lentiviral vector according to the present disclosure may be derived from or may be derivable from any suitable lentivirus. A recombinant lentiviral particle is capable of transducing a target cell with a nucleotide of interest. Once within the cell, the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the genome of the target cell. A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. That component part may be involved in the biological mechanisms by which the vector transduces cells, expresses genes or is replicated. The basic structure of retrovirus and lentivirus genomes share many common features such as a 5' LTR and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components, which are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as the rev and rev response element (RRE) sequences, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. In the provirus, the viral genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. The LTRs themselves are identical sequences that can be divided into three elements, which are called “U3,” “R” and “U5.” U3 is derived from the sequence unique to the 3' end of the RNA, R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different viruses. [0091] Nucleic acid constructs of the invention can also be delivered using virus-like particles (VLPs) and virus-like nanoparticles (VLNPs). VLPs and VLNPs comprise one or more virus-derived structural proteins, but lacking viral genetic material, thereby forming self-assembling structures with the ability to be recognised and taken up by host cells (e.g. see Chung et al., Advanced Drug Delivery Rev, 156:214-235 (2020); Nooraei et al., J Nanobiotech, 19:59 (2021)). VLPs and VLNPs can be exploited as efficient vehicles for delivery of a range of compounds and molecules, including small molecules, peptides, proteinaceous molecules and nucleic acids, including both DNA and RNA, cells and subjects in vivo. As such, VLPs and VLNPs can be utilized for gene therapy applications, vaccine delivery, diagnostics and theranostics. In the context of the present disclosure, VLPs and VLNPs can be used for the delivery of DNA and RNA nucleic acid constructs of the disclosure. [0092] VLPs and VLNPs can be generated using structural proteins (e.g. capsid, envelope and/or core proteins) derived from any one or more suitable viruses, such as those hereinbefore described, and/or recombinant viral proteins, using methods known to those skilled in the art. It will also be understood by those skilled in the art that a range of symmetrical particles, with an organized underlying geometry, formed from non-viral and artificial proteins can also be considered VLPS and VLNPs (e.g. see Heddle et al., Curr Opin Struct Biol, 43:148-155 (2017)). The scope of the present disclosure is not considered to be limited by reference to any one form, type or size of VLP or VLNP. [0093] Nucleic acid constructs of the invention can also be delivered using a non-viral delivery system. Any delivery method or system for delivery of nucleic acid molecules known in the art can be used, including for example delivery to the desired tissues in colloidal dispersion systems that include, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Other delivery systems include exosomes, virosomes, nanoparticles (including gold or silica nanoparticles), polymers (e.g. dendrimers, polymeric nanogels, etc.) Suitable delivery reagents for nucleic acid include, but are not limited to, e.g., the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Research, 32:e109 (2004); Hanai et al. Annals N.Y. Acad. Sci., 1082:9-17 (2006); Kawata et al. Molecular Cancer Therapeutics, 7:2904- 12 (2008). [0094] In one example, a liposome is used as the delivery vehicle. Liposomes suitable for delivery of the nucleic acid constructs described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. Examples of suitable lipids liposomes production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Additional examples of lipids include, but are not limited to, polylysine, protamine., sulfate and 3.beta.-[N--(N',N'-dimethylaminoethane) carbamoyl] cholesterol. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al., Annual Rev. Biophysics & Bioengineering, 9:467-08 (1980); and U.S. Pat. Nos.4,235,871; 4,501,728; 4,837,028; and 5,019,369, which are herein incorporated by reference. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms, containing an aqueous solution in the core. Host cells [0095] The present disclosure also provides a host cell comprising, transformed, transfected or transduced with a nucleic acid construct or vector of the present invention. In some embodiments, the host cells are mammalian cells in which the polynucleotide construct can be expressed. Suitable mammalian host cells include, but are not limited to, human cells, murine cells, non-human primate cells (e.g. rhesus monkey cells), or human progenitor cells or stem cells. In certain embodiments, the host cell comprising a construct or vector of the disclosure is a pluripotent or multipotent progenitor/stem cell, such as induced pluripotent stem cell (iPSC) or a mesenchymal stem cell. The cell may be an iPSC-derived cell such as a neuro-progenitor cell. The cell may be, for example, a neuronal cell such as a motor neuron, or a muscle cell. Those skilled in the art would appreciate the conditions under which the construct or vector can be introduced into a host cell and the conditions that support or facilitate expression of the CTNNB1 transgene within the cell. Furthermore, the methods may be in vitro, ex vivo or in vivo. In some embodiments, transduction of host cells with vectors of the invention may be increased by contacting the host cell, in vitro, ex vivo, or in vivo, with a vector of the present invention and one or more compounds that increase transduction efficiency, as will be well known to those skilled in the art. [0096] In some embodiments, transduced host cells may be combined with a pharmaceutically acceptable carrier for subsequernt administration to a subject. In some embodiments, the transduced host cells are formulated with PLASMA-LYTE A (e.g. a sterile, nonpyrogenic isotonic solution for intravenous administration; where one liter of PLASMA-LYTE A has an ionic concentration of 140 mEq sodium, 5 mEq potassium, 3 mEq magnesium, 98 mEq chloride, 27 mEq acetate, and 23 mEq gluconate). In other embodiments, the host cells or transduced host cells are formulated in a solution of PLASMA-LYTE A, the solution comprising between about 8% and about 10% dimethyl sulfoxide (DMSO). In some embodiments, the less than about 2x10⁷ host cells/transduced host cells are present per mL of a formulation including PLASMA-LYTE A and DMSO. Pharmaceutical compositions [0097] The present disclosure also provides for compositions, including pharmaceutical compositions, comprising one or more constructs, vectors or cells as disclosed herein. In some embodiments, pharmaceutical compositions comprise an effective amount of a construct or vector as described herein and a pharmaceutically acceptable carrier. An effective amount can be readily determined by those skilled in the art based on factors such as body size, body weight, age, health, sex of the subject, ethnicity, and viral titers. [0098] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. For example, a vector may be formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. Methods for the formulation of compounds with pharmaceutical carriers are known in the art and are described in, for example, in Remington's Pharmaceutical Science, (17th ed. Mack Publishing Company, Easton, Pa. 1985); and Goodman & Gillman's: The Pharmacological Basis of Therapeutics (11th Edition, McGraw- Hill Professional, 2005); the disclosures of each of which are hereby incorporated herein by reference in their entirety. [0099] In some embodiments, the pharmaceutical compositions may comprise the construct or vector in an amount of from about 0.1% to about 99.9% by weight. Pharmaceutically acceptable carriers suitable for inclusion within any pharmaceutical composition include water, buffered water, saline solutions such as, for example, normal saline or balanced saline solutions such as Hank's or Earle's balanced solutions), glycine, hyaluronic acid etc. The pharmaceutical composition may be formulated for parenteral administration, such as intravenous, intramuscular or subcutaneous administration. Pharmaceutical compositions for parenteral administration may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and mixtures thereof, vegetable oils (such as olive oil), injectable organic esters (e.g. ethyl oleate). [00100] The pharmaceutical composition may be formulated for any suitable route of administration, such as intravenous, subcutaneous, oral, intramuscular, intraperitoneal, pulmonary, intracranial, intraosseous, buccal, or nasal administration. [00101] The pharmaceutical compositions may comprise a construct or vector disclosed herein in an encapsulated form. For example, the construct or vector may be encapsulated within a nanocapsule, such as a nanocapsule comprising one or more biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). In some embodiments, the vectors are encapsulated within polymeric nanocapsules. In other embodiments, the vectors are encapsulated within biodegradable and/or erodible polymeric nanocapsules. In some embodiments, the polymeric nanocapsules are comprised of two different positively charged monomers, at least one neutral monomer, and a crosslinker. In some embodiments, the nanocapsules further comprise at least one targeting moiety, such as an antibody. Methods of treatment [00102] A nucleic acid construct or vector of the present invention, or a cell comprising a nucleic acid or vector of the invention can be administered to a subject in need thereof to restore or provide a normal level of expression of CTNNB1 in cells that normally express CTNNB1, to thereby treat a CTNNB1-related disorder, or to inhibit or alleviate one or more symptoms characteristic of, or associated with a CTNNB1-related disorder. Typically, treatment with the constructs, vectors or cells described herein genetically corrects or alleviates one or more of the symptoms or pathologies associated with a deficiency or loss- of-function mutation in CTNNB1. [00103] Accordingly, provided herein is a method for the treatment of a CTNNB1-related disorder or inhibiting or ameliorating at least one symptom thereof, wherein the disorder is characterised by, or associated with, a deficiency or mutation in CTNNB1 in a subject, comprising administering to the subject a nucleic acid construct, a vector or a cell as described herein. [00104] The CTNNB1-related disorder may be, for example, CTNNB1 neurodevelopmental disorder (CTNNB1 syndrome), familial exudative vitreoretinopathy (FEVR) or neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV). [00105] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples. [00106] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Examples Example 1. Nucleic acid constructs [00107] Nucleic acid backbones containing nucleotide sequences of the CBh promoter of SEQ ID NO:8, the miRNA targeting region of SEQ ID NO:15, the poly(A) signal of SEQ ID NO:10 or16, the 5' ITR of SEQ ID NO:17, and the 3' ITR of SEQ ID NO:18 as described herein were generated by PCR according to known methods, with size confirmed by agarose gel electrophoresis. PCR products were cleaned up using QIAquick® PCR Purification Kit (QIAGEN). PCR products (1.5 ug) were digested with XbaI and NotI, and desired bands purified from agarose gel. Inserts containing the CTNNB1 nucleotide sequences of SEQ ID NOs:2, 3 and 7 were isolated from plasmids by PCR reaction with Q5® High-Fidelity 2X Master Mix (New England Biolabs). PCR products were run in an agarose gel and the desired bands purified. Inserts were introduced into linearized backbone by Gibson assembly followed by transformation, colony PCR, and sequencing. [00108] Schematic representations of constructs described herein are shown in Figure 1. The locations of specific elements within exemplary construct #2 (see Figure 1B), exemplary construct #4 (Figure 1D) and exemplary construct #6 (Figure 1F) are shown in Table 2. Table 2. Constructs – locations of elements within sequences Element Construct #2 Construct #4 Construct #6 (SEQ ID NO:19) (SEQ ID NO:20) (SEQ ID NO:21) 5' ITR 1-145 1-145 1-145 (SEQ ID NO:17) CBh promoter 181-1019 181-1019 181-1019 (SEQ ID NO:8) 5' UTR 1026-1239 1026-1239 1026-1239 (SEQ ID NO:9) CTNNB1 coding 1240-3585 1240-3785 1240-3585 sequence (SEQ ID NO:2) Intron 2 sequences - 1253-1452 - (SEQ ID NO:7) miRTS 3598-3798 3798-3998 3598-3798 (SEQ ID NO:15) 3' UTR - - 3825-4695 (SEQ ID NO:10) BGH polyA 3805-4029 4005-4229 - (SEQ ID NO:16) 3' ITR 4107-4251 4307-4451 4775-4919 (SEQ ID NO:18) Example 2. AAV vector production and validation [00109] AAV vector production required pAd5 helper plasmids. For studies that aimed to establish the transduction efficiency in patient-derived neuro-progenitor cells and cortical brain organoids, AAV-7m8 was used for packaging the nucleic acid constructs (pTransgene). 10 × 15 cm dishes of HEK293T cells were transfected using PEI (polyethylenimine; PolyPlus) with a 1:1:3 ratio of pCap7m8:pTransgene:pAd5. All aforementioned constructs were harvested three days post-transfection and the AAVs were recovered by iodixanol gradient purification as described in Strobel et al., 2015, Hu. Gene Ther Methods 26: 147-157. A final buffer exchange (phosphate-buffered saline (PBS; Gibco), 50 mM NaCl (Sigma-Aldrich), 0.001%, Pluronic F68 (v/v, Gibco)) and concentration step were performed using Amicon Ultra-4 Centrifuge Filter Units with Ultracel-100 kDa membrane (EMD Millipore). Iodixanol-purified AAVs were quantified using droplet digital PCR (ddPCR; Bio-Rad) using QX200 ddPCR EvaGreen Supermix (Bio-Rad). Example 3. Neural models for CTNNB1 syndrome [00110] The inventors generated patient-derived neuro-progenitor cells and cortical brain organoids as models of CTNNB1 syndrome for the evaluation of AAV vector constructs described above. Reprogramming of patient-derived (CTNNB1^mut) PBMC (Peripheral Blood Mononuclear Cells) into iPSC (induced Pluripotent Stem Cells) [00111] Peripheral blood mononuclear cells from a patient with CTNNB1 syndrome were expanded with cytokines and nucleofected (electroporated using Amaxa 4D) with Yamanaka plasmids expressing L-MYC/LIN28, OCT3/4 and shRNA against p53 and plated in IPSC matrix. After 2 days, media was changed to iPSC media and single iPSCs were visible and after approximately 3 weeks, colonies were big enough to isolate by mechanical excision. Each colony was dissociated by mechanical means into single well in iPSC conditions and subsequently maintained and passaged till passage 10 and characterized for pluripotency marker expression. Differentiation of iPSC into Neuro-Progenitor Cells (NPCs) [00112] Wells of a 6-well plate were coated with 1.5 mL of 10 μg/mL laminin (LAM) in PBS -/- (no calcium, no magnesium) ensuring the whole well was covered by gently tapping the side of the plate. Parafilm and incubated overnight at 4^oC. The wells rinsed once with PBS. Approximately 20,000-30,000 cells were seeded per LAM-coated wellwith StemFlex. After 24 hours media was changed to N2/B27 freshly supplemented with 10 μM SB 431542 and 500 ng/mL Noggin for 7 days to generate NPCs. Freshly supplemented media was changed every 2 days. NPCs were maintained in media containing 20 ng/mL FGF2 and 20 ng/mL EGF. Differentiation of iPSCs into cortical brain organoids [00113] iPSCs were pre-treated with 1% DMSO at Day -2. At Day -1, 3D spheroids were generated as follows: • Pre-warm E8 medium, Accutase, and DMEM/F-12 at RT. Supplement E8 medium with the ROCK inhibitor Y-27632 to a final concentration of 10 μM. • Prepare the AggreWell plate adding 1 ml per well of E8 supplemented with Y-27632 and centrifuge at 2,000 x g for 5 minutes in a swinging bucket rotor. Check under the microscope to ensure bubbles have been removed from microwells. Set the plate in an incubator while preparing the single cell suspensions of iPSC. • Aspirate maintenance medium from the iPSC 100 mm plates and rinse cells twice with PBS -/-. • Add 4 ml of Accutase per 100 mm culture plate and incubate for 7 minutes at 37°C, i^{n a 5% CO} ₂ ^incubator. • Add pre-warmed E8 medium up to 10 ml volume and centrifuge the cell suspension at 200 x g for 4 minutes. Resuspend the pellet with E8 medium and count cell number. • Centrifuge the cell suspension at 200 x g for 4 minutes. Resuspend the pellet with pre-warmed EB medium supplemented with Y-27632 to obtain 3 million cells per 1 ml of medium. • Add 1 ml of this cell suspension to the previously prepared AggreWell plate, which contains 1 ml of E8 medium. Each well of AggreWellTM800 plate contains 300 microwells, and one microwell will have 10,000 cells. • Centrifuge the AggreWellTM800 plate at 100 x g for 3 minutes to distribute the cells i^{n the microwells and incubate for 24 hours at 37°C, in a 5% CO} ₂ ^incubator. [00114] At Day 0, iPSC-derived spheroids were harvested from the microwells by firmly pipetting the medium in the well up and down with a cut 1 ml plastic tip.1 ml of DMEM/F- 12 medium was pipetted across the entire surface of the well to dislodge any remaining spheroids. The suspension of spheroids was passed through a 40 um strainer. The process was repeated 3 times. Spheroids were collected by washing the strainer with Essential 6™ (E6) medium for neural induction. [00115] Harvested spheroids were placed in ultra-low attachment 100 mm plates in E6 medium supplemented with 2.5 μM Dorsomorphin (DM) and 10 μM SB-431542 (SB). Media changes are performed daily, except for day 1. On day 6, E6 medium containing DM and SB was replaced with neural medium (NM) composed of Neurobasal™-A Medium (500 mL), B-27T™ Supplement (50X) minus vitamin A, and GlutaMAX™ Supplement (1:100). This NM was also supplemented with EGF2 (20 ng/ml) and FGF2 (20 ng/ml) for 19 days with daily medium change in the first 10 days, and every other day medium changes for the subsequent 9 days. To promote differentiation of the progenitors, FGF2 and EGF were replaced with 20 ng/ml BDNF and 20 ng/ml NT-3 starting at day 25 (with media changes every other day). From day 43 onwards only NM without growth factors was used for medium changes every four days or as needed. Example 4. AAV gene therapy in NPCs [00116] NPCs were transduced at a confluence of 70% in 6-well plates with AAV-7m8 vectors at a MOI of 5e+3 vg/cell (low concentration) and/or 1e+4 vg/cell (high concentration) in a volume of 1 mL/well. 1 mL was added to each well 1 hour post- transduction and media was replaced 24 hours post-transduction. NPCs were harvested 72 hours post-transduction and messenger RNA was extracted using Direct-zol™ RNA MicroPrep (ZYMO Search) following manufacturer’s instructions. 100 ng of extracted mRNA was subject to DNAse treatment in a final volume of 20 µL containing 2 U of TURBO DNAse (Thermo Fisher Scientific), TURBO DNAse Buffer 10X, and DEPC- treated water. The mixture was incubated at 37 °C for 1 hour and treated with DNAse Inactivation Reagent for 5 minutes at RT. Samples were centrifuged for 2 minutes at 10,000 g and supernatant was transferred to a new tube in ice. [00117] Reverse transcription was performed in two steps using the SuperScript™ IV First-Strand Synthesis System (Invitrogen). Firstly, 10 μL of a buffer solution containing 150 ng of random hexamers, 0.4 mM of dNTP mix and DEPC-treated water was added to the sample and the mixture was incubated for 1 minute at 80 °C, 65 °C for 5 minutes and in ice for 1 minute.10 μL of SuperScript IV RT mix containing 5X RT buffer, 100 mM DTT, 40 U of Ribonuclease Inhibitor, and 200 U of SuperScript IV RT were added to 10 μL of the previous mixture. Samples were incubated 10 minutes at 35 °C, 50 minutes at 53 °C, and 10 minutes at 80 °C. Finally, samples were stored at 4 °C. The qPCR reaction was performed _{in a thermal cycler CFX384} ^™ _{Real-Time System (Bio- Rad) using the commercial mixture iTaq Universal SYBR} ^® _{Green Supermix (Bio-Rad). Each PCR reaction was carried out in a final volume of 10 μL which included: 5 μL of the mixture iTaq Universal SYBR} ^® _Green Supermix, 1 μL of each sense and antisense primers at 10 µM (below), 1 μL of sterile water, and 2 μL of each synthesised cDNA: CTNNB1 Forward primer- 5' CCTTCAACTATTTCTTCCATGCG 3' (SEQ ID NO:22) CTNNB1 Reverse primer- 5' CTAGTTCAGTTGCTTGTTCGTG 3' (SEQ ID NO:23) Exogenous CTNNB1 Forward primer– 5' CTAGAACGCGTCAAACACC 3' (SEQ ID NO:24) Exogenous CTNNB1 Reverse primer- 5' CTAGAAGGCACAGCTCGA 3' (SEQ ID NO:25). [00118] PCR conditions for amplification were 95 ^oC (3 mins) followed by 35 cycles of 95 ^oC (0:05 min), 60 ^oC (0:30 min), 72 ^oC (0:15 min), 77 ^oC (0:10 min). This was finally followed by a denaturing protocol, which consisted of an 0.5 ^oC increasing temperature from 65 ^oC to 95 ^oC. [00119] As shown in Figure 2, morphological differences exist between neuro-progenitor cells derived from iPSCs generated from cells from a healthy donor (Figure 2A) and neuro- progenitor cells derived from iPSCs generated from a patient carrying a mutant CTNNB1 (CTNNB1^mut) allele (Figure 2B). Patient-derived neuro-progenitor cells are more rounded and do not develop the prolongations that start to be present in healthy neuro-progenitor cells, demonstrating the presence of disease phenotype in the in vitro model. Figure 2C shows the increased basal levels of CTNNB1 mRNA expression in healthy individual (WT)-derived neuro-progenitor cells in comparison to patient (CTNNB1^mut)-derived cells. This demonstrates that the patient-derived neuro-progenitor cells are able to recapitulate the loss of CTNNB1 expression resulting from the CTNNB1 mutation.. Figure 2D shows vector- derived (exogenous) CTNNB1 mRNA expression in patient-derived neuro-progenitor cells transduced with constructs #1 to #6 (see Example 1 and Figure 1). Substantially higher expression was observed using constructs #2, #4, and #6. [00120] Figure 3 shows increased basal levels of CTNNB1 mRNA expression in healthy individual (WT)-derived neuro-progenitor cells in comparison to

- derived cells, confirming that patient-derived neuro-progenitor cells recapitulate the loss of CTNNB1 expression. Levels of CTNNB1 mRNA expression were determined in patient- derived neuro-progenitor cells transduced with constructs #2, #4, and #6 at two different MOIs, the high dose (HD) MOI as used above (see Figure 2) and a low dose (LD) MOI. As shown in Figure 3, the lower concentration of each of the three constructs is effective in restoring CTNNB1 mRNA expression in patient-derived neuro-progenitor cells. A dose- dependent response was observed, as the higher MOI led to higher CTNNB1 expression levels. Example 5. AAV gene therapy in brain organoids [00121] Organoids were transferred to 6-well plates (6 organoids per well) and transduced with AAV vectors at days 23 and 127 of differentiation. Organoids were transduced on Thursday morning in a total volume of 1 mL/well. Plates were left tilted until afternoon, when 2 mL of medium were added to each well and plates were kept on the shaker. On Friday afternoon, 2 mL of medium were added to each well (5 mL per well in total). Media was aspirated and replaced on Monday (2 mL) and from that day onwards, organoids were fed every Monday, Wednesday, and Friday. Organoids were harvested two weeks post-transduction to analyse mRNA levels. Messenger RNA was extracted using AllPrep® DNA/RNA Mini Kit (QIAGEN) following manufacturer’s instructions and DNAse treatment, cDNA Synthesis and qPCR were performed as previously described. [00122] Basal levels of CTNNB1 mRNA expression were increased in healthy individual (WT)-derived cortical brain organoids in comparison to patient (CTNNB1^mut)-derived organoids (Figure 4), confirming that 37-day-old cortical brain organoids recapitulate the loss of CTNNB1 expression present in patients with the mutant CTNNB1 allele. Construct #4 was shown to significantly increase CTNNB1 mRNA expression in patient-derived organoids to levels similar to those observed in WT organoids (Figure 4). [00123] Figure 5 shows that similar basal levels of CTNNB1 mRNA expression were observed in healthy individual (WT)- and patient (CTNNB1^mut)-derived organoids, demonstrating that 141-day-old cortical brain organoids do not constitute an appropiate model for recapitulating the loss of CTNNB1 expression. Constructs #2 and #4 were able to significantly increase CTNNB1 mRNA expression in patient-derived organoids. Table 3 - Sequences SEQ ID Description Sequence NO: MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSL SGKGNPEEEDVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQ RVRAAMFPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVN LINYQDDAELATRAIPELTKLLNDEDQVVVNKAAVMVHQLSKKEAS RHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLA IFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLA GGLQKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIILASGGPQ Wild-type ALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGL human 1 HLTDPSQRLVQNCLWTLRNLSDAATKQEGMEGLLGTLVQLLGSDDI CTNNB1 NVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDREDI polypeptide TEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPL IKATVGLIRNLALCPANHAPLREQGAIPRLVQLLVRAHQDTQRRTS MGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPL FVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGATAPLTELL HSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPMAW NETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMME HEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDL Wild-type ATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAAC human CAGACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCT CTNNB1 GGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTCTG coding AGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAG sequence TCCTGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGA ACAAGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAG AGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGC 2 AGATCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCA GCGTTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAAC TTGATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCC CTGAACTGACAAAACTGCTAAATGACGAGGACCAGGTGGTGGTTAA TAAGGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCC AGACACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTAC GTACCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGC TGGGACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCC ATCTTTAAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTT CACCAGTGGATTCTGTGTTGTTTTATGCCATTACAACTCTCCACAA CCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCT GGTGGGCTGCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTA AATTCTTGGCTATTACGACAGACTGCCTTCAAATTTTAGCTTATGG CAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAA GCTTTAGTAAATATAATGAGGACCTATACTTACGAAAAACTACTGT GGACCACAAGCAGAGTGCTGAAGGTGCTATCTGTCTGCTCTAGTAA TAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTT CACCTGACAGATCCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGA CTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGGAAGGGATGGA AGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATA AATGTGGTCACCTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCA ATAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGTGGTATAGA GGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATC ACTGAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGACACC AAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTATGGACT ACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTG ATAAAGGCTACTGTTGGATTGATTCGAAATCTTGCCCTTTGTCCCG CAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGACTAGT TCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCC ATGGGTGGGACACAGCAGCAATTTGTGGAGGGGGTCCGCATGGAAG AAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGGGA TGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTG TTTGTGCAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAG CTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGA AGCTATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTT CACTCTAGGAATGAAGGTGTGGCGACATATGCAGCTGCTGTTTTGT TCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTC AGTTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGG AATGAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAAC CCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTC TGGTGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAA CATGAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATG GGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCC TCCAGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAA Codon- ATGGCTACGCAGGCTGACCTGATGGAACTGGATATGGCGATGGAAC optimized CGGATCGCAAGGCCGCCGTCTCTCATTGGCAACAGCAATCATATCT CTNNB1 CGATTCAGGTATTCACAGCGGGGCAACAACAACTGCACCGTCCCTC coding AGCGGCAAGGGTAACCCAGAAGAGGAAGACGTCGACACAAGCCAGG sequence TGCTCTACGAATGGGAGCAAGGGTTCAGCCAATCTTTTACCCAGGA GCAGGTGGCCGACATAGACGGGCAATACGCCATGACAAGGGCCCAA CGGGTCAGGGCCGCCATGTTTCCAGAAACGCTCGACGAAGGCATGC AAATTCCCTCAACTCAATTCGACGCCGCACACCCAACCAACGTGCA AAGACTGGCCGAGCCCAGTCAAATGCTCAAGCACGCGGTAGTGAAT CTGATCAATTACCAGGACGACGCTGAGCTGGCTACTAGAGCCATAC CCGAGCTTACTAAGCTTCTGAACGATGAAGATCAAGTAGTCGTGAA CAAAGCAGCGGTAATGGTTCACCAACTCAGCAAGAAAGAGGCAAGC CGTCATGCCATTATGAGGTCACCGCAAATGGTTTCAGCAATCGTGA GAACTATGCAAAACACCAACGACGTGGAAACCGCAAGATGCACAGC GGGCACTCTGCACAATCTCTCTCACCACCGCGAAGGGCTCCTTGCA ATTTTCAAATCCGGTGGAATCCCGGCTCTTGTCAAGATGTTAGGGT CCCCTGTCGACAGCGTTCTTTTCTACGCGATCACTACACTGCATAA TCTCCTCTTGCACCAAGAAGGGGCCAAGATGGCTGTACGGCTGGCC GGCGGTTTACAAAAGATGGTGGCGCTCCTGAATAAGACCAACGTGA AGTTTCTGGCCATCACAACGGATTGTCTGCAGATCCTGGCCTACGG AAATCAGGAGTCCAAACTGATTATTTTGGCCAGCGGCGGCCCGCAG GCACTTGTCAACATCATGCGCACGTACACCTATGAGAAGCTCCTCT GGACAACTTCTCGCGTCCTCAAAGTCTTGAGCGTGTGTTCCAGCAA CAAACCAGCCATCGTTGAGGCCGGCGGCATGCAGGCCCTGGGCCTT CATCTCACTGACCCTTCCCAGAGGCTGGTGCAAAATTGCCTCTGGA CCCTGCGTAACCTGTCCGACGCAGCCACAAAGCAAGAGGGCATGGA AGGGCTGCTGGGAACACTGGTCCAATTACTCGGGAGTGACGACATT AACGTCGTGACTTGCGCCGCAGGCATCCTGTCCAAcCTGACcTGcA ACAACTACAAGAAcAAgATGATGGTgTGTCAGGTTGGCGGCATCGA AGCATTAGTCCGGACGGTTCTCAGAGCAGGGGATCGAGAGGATATT ACAGAACCAGCTATTTGCGCGCTCCGACACCTTACAAGTAGACATC AGGAAGCCGAAATGGCGCAAAACGCTGTCCGACTGCATTACGGGCT GCCCGTGGTCGTCAAATTACTTCATCCGCCCTCACATTGGCCCCTT ATCAAAGCCACCGTAGGCCTTATCAGAAACTTGGCACTCTGCCCTG CTAACCACGCTCCCCTGAGAGAACAAGGAGCAATACCGAGATTAGT ACAACTGCTGGTGCGCGCGCACCAAGACACACAACGGCGCACTAGT ATGGGCGGCACCCAACAACAGTTCGTCGAAGGAGTTCGGATGGAAG AGATTGTAGAGGGCTGCACTGGCGCACTCCATATTTTGGCCAGAGA CGTCCATAATCGGATAGTAATACGCGGGTTGAACACGATCCCTCTG TTCGTCCAATTGCTGTACAGTCCTATAGAGAATATTCAGAGGGTGG CCGCTGGCGTGCTTTGCGAGTTGGCCCAAGATAAAGAGGCCGCTGA GGCCATCGAGGCAGAAGGCGCTACCGCGCCCCTCACTGAACTGTTG CATAGTCGGAACGAGGGAGTAGCTACCTACGCTGCAGCCGTGCTGT TTCGCATGTCAGAAGATAAACCGCAGGACTATAAGAAGCGACTGTC TGTGGAACTTACTTCCAGTCTGTTTCGGACTGAACCTATGGCGTGG AACGAAACAGCAGACTTGGGCCTGGACATCGGAGCTCAAGGTGAGC CATTAGGCTACAGGCAAGACGACCCAAGTTACCGGTCCTTCCATTC CGGCGGCTACGGTCAAGACGCTCTGGGCATGGACCCAATGATGGAG CACGAAATGGGTGGACATCATCCAGGAGCAGATTACCCTGTGGACG GACTCCCCGACCTCGGTCACGCACAAGATTTGATGGACGGACTCCC GCCTGGCGATTCAAACCAATTGGCTTGGTTCGACACAGATTTGTAA Sequence CAGTTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTG encoding GAATGAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAA CTNNB1 CTD CCCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACT domain CTGGTGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGA ACATGAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGAT GGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGC CTCCAGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTA A 5' end of GTTTGTGTCATTAAATCTTTAGTTACTGAATTGGGGCTCTGCTTCG CTNNB1 intron TTGCCATTAAGCCAGTCTGGCTGAGATCCCCCTGCTTTCCTCTCTC 2 CCTGCTTA 3' end of TTTCTAAAAATATTTCAATGGGTCATATCACAGATTCTTTTTTTTT CTNNB1 intron AAATTAAAGTAACATTTCCAATCTACTAATGCTAATACTGTTTCGT 2 ATTTATAG 5' and 3' GTTTGTGTCATTAAATCTTTAGTTACTGAATTGGGGCTCTGCTTCG CTNNB1 intron TTGCCATTAAGCCAGTCTGGCTGAGATCCCCCTGCTTTCCTCTCTC 2 sequences CCTGCTTATTTCTAAAAATATTTCAATGGGTCATATCACAGATTCT TTTTTTTTAAATTAAAGTAACATTTCCAATCTACTAATGCTAATAC TGTTTCGTATTTATAG _{CBh promoter} ^{TCCTGCAGGCGACATTGATTATTGACTAGTTATTAATAGTAATCAA} TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTC TCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTAT TTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCG CGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCG GAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAA GCGCGCGGCGGGCGGGAGCAAGCTTGAACTGAAAAACCAGAAAGTT AACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATC CGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCT TTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTG CGGAATTGTAC _{5' UTR} ^{AAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCG} GAGAGCGAGGGGAGGCGGAGACGGAGGAAGGTCTGAGGAGCAGCTT CAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTCGGACTCC CGCGGCGGGAGGAGCCTGTTCCCCTGAGGGTATTTGAAGTATACCA TACAACTGTTTTGAAAATCCAGCGTGGACA Truncated 3' CATCATCCTTTAGGTAAGAAGTTTTAAAAAGCCAGTTTGGGTAAAA UTR TACTTTTACTCTGCCTACAGAACTTCAGAAAGACTTGGTTGGTAGG GTGGGAGTGGTTTAGGCTATTTGTAAATCTGCCACAAAAACAGGTA TATACTTTGAAAGGAGATGTCTTGGAACATTGGAATGTTCTCAGAT TTCTGGTTGTTATGTGATCATGTGTGGAAGTTATTAACTTTAATGT TTTTTGCCACAGCTTTTGCAACTTAATACTCAAATGAGTAACATTT GCTGTTTTAAACATTAATAGCAGCCTTTCTCTCTTTATACAGCTGT ATTGTCTGAACTTGCATTGTGATTGGCCTGTAGAGTTGCTGAGAGG GCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGACACACTAAC CAAGCTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCT GGTCCTTTTTGGTCGAGGAGTAACAATACAAATGGATTTTGGGAGT GACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGATGGAAT TTATCAAACCCTAGCCTTGCTTGTTAAATTTTTTTTTTTTTTTTTT TAAGAATATCTGTAATGGTACTGACTTTGCTTGCTTTGAAGTAGCT CTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTTTTTAAGT CTCTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTA ATTTTTAAGAATTGAGTAATGGTGTAGAACACTAATTCATAATCAC TCTAATTAATTGTAATCTGAATAAAGTGTAACAATTGTGTAGCCTT TTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCC ^miR-122 CAAACACCATTGTCACACTCCA _miRTS ^miR-183 AGTGAATTCTACCAGTGCCATA _miRTS ^miR-199 GAACAGGTAGTCTGAACACTGGG _miRTS (miR-122 CAAACACCATTGTCACACTCCAAGTGAATTCTACCAGTGCCATAGA miRTS / miR- ACAGGTAGTCTGAACACTGGG 183 miRTS / miR-199 miRTS)₁ (miR-122 CAAACACCATTGTCACACTCCAAGTGAATTCTACCAGTGCCATAGA miRTS / miR- ACAGGTAGTCTGAACACTGGGCAAACACCATTGTCACACTCCAAGT 183 miRTS / GAATTCTACCAGTGCCATAGAACAGGTAGTCTGAACACTGGGCAAA miR-199 CACCATTGTCACACTCCAAGTGAATTCTACCAGTGCCATAGAACAG miRTS)₃ GTAGTCTGAACACTGGG _{BGH poly(A)} ^{CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT} GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAA TAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG _{5' ITR} ^{TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC} GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG GGTTCCT _{3' ITR} ^{AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG} CTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAG TGGCCAA Expression TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC construct #2 GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG GGTTCCTGGAGGGGTGGAGTCGTGACGTAAAGATCTGATATCTCCT GCAGGCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCC CACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCC CATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCC AGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGA GGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC GCGGCGGGCGGGAGCAAGCTTGAACTGAAAAACCAGAAAGTTAACT GGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGT GGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTAC TTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGA ATTGTACCCTAGGAAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCC GGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGGAGGAAGGTC TGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGG ACGGTCGGACTCCCGCGGCGGGAGGAGCCTGTTCCCCTGAGGGTAT TTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAATG GCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAG ACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGA CTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTCTGAGT GGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCC TGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACA AGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAGAGG GTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGA TCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCAGCG TTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTTG ATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTG AACTGACAAAACTGCTAAATGACGAGGACCAGGTGGTGGTTAATAA GGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGA CACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTA CCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGCTGG GACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATC TTTAAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCAC CAGTGGATTCTGTGTTGTTTTATGCCATTACAACTCTCCACAACCT TTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGT GGGCTGCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAAT TCTTGGCTATTACGACAGACTGCCTTCAAATTTTAGCTTATGGCAA CCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCT TTAGTAAATATAATGAGGACCTATACTTACGAAAAACTACTGTGGA CCACAAGCAGAGTGCTGAAGGTGCTATCTGTCTGCTCTAGTAATAA GCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCAC CTGACAGATCCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTC TCAGGAATCTTTCAGATGCTGCAACTAAACAGGAAGGGATGGAAGG TCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAAT GTGGTCACCTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCAATA ATTATAAGAACAAGATGATGGTCTGCCAAGTGGGTGGTATAGAGGC TCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACT GAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGACACCAAG AAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTATGGACTACC AGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATA AAGGCTACTGTTGGATTGATTCGAAATCTTGCCCTTTGTCCCGCAA ATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGACTAGTTCA GTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATG GGTGGGACACAGCAGCAATTTGTGGAGGGGGTCCGCATGGAAGAAA TAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGGGATGT TCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTT GTGCAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAGCTG CAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGAAGC TATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTTCAC TCTAGGAATGAAGGTGTGGCGACATATGCAGCTGCTGTTTTGTTCC GAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAGT TGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAAT GAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAACCCC TTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGG TGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACAT GAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATGGGC TGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCC AGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAtct agaACGCGTCAAACACCATTGTCACACTCCAAGTGAATTCTACCAG TGCCATAGAACAGGTAGTCTGAACACTGGGCAAACACCATTGTCAC ACTCCAAGTGAATTCTACCAGTGCCATAGAACAGGTAGTCTGAACA CTGGGCAAACACCATTGTCACACTCCAAGTGAATTCTACCAGTGCC ATAGAACAGGTAGTCTGAACACTGGGCTCGAGCTGTGCCTTCTAGT TGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAAT TGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGG GTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGC ATGCTGGGGATGCGGTGGGCTCTATGGTTAATTAATGCATCGCGAT CGATGATATCAGATCTGTTACGTAGATAAGTAGCATGGCGGGTTAA TCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG CGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGC GCAGAGAGGGAGTGGCCAA Expression TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC construct #4 GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG GGTTCCTGGAGGGGTGGAGTCGTGACGTAAAGATCTGATATCTCCT GCAGGCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCC CACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCC CATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCC AGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGA GGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC GCGGCGGGCGGGAGCAAGCTTGAACTGAAAAACCAGAAAGTTAACT GGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGT GGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTAC TTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGA ATTGTACCCTAGGAAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCC GGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGGAGGAAGGTC TGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGG ACGGTCGGACTCCCGCGGCGGGAGGAGCCTGTTCCCCTGAGGGTAT TTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAATG GCTACTCAAGGTTTGTGTCATTAAATCTTTAGTTACTGAATTGGGG CTCTGCTTCGTTGCCATTAAGCCAGTCTGGCTGAGATCCCCCTGCT TTCCTCTCTCCCTGCTTATTTCTAAAAATATTTCAATGGGTCATAT CACAGATTCTTTTTTTTTAAATTAAAGTAACATTTCCAATCTACTA ATGCTAATACTGTTTCGTATTTATAGCTGATTTGATGGAGTTGGAC ATGGCCATGGAACCAGACAGAAAAGCGGCTGTTAGTCACTGGCAGC AACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCAC AGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTG GATACCTCCCAAGTCCTGTATGAGTGGGAACAGGGATTTTCTCAGT CCTTCACTCAAGAACAAGTAGCTGATATTGATGGACAGTATGCAAT GACTCGAGCTCAGAGGGTACGAGCTGCTATGTTCCCTGAGACATTA GATGAGGGCATGCAGATCCCATCTACACAGTTTGATGCTGCTCATC CCACTAATGTCCAGCGTTTGGCTGAACCATCACAGATGCTGAAACA TGCAGTTGTAAACTTGATTAACTATCAAGATGATGCAGAACTTGCC ACACGTGCAATCCCTGAACTGACAAAACTGCTAAATGACGAGGACC AGGTGGTGGTTAATAAGGCTGCAGTTATGGTCCATCAGCTTTCTAA AAAGGAAGCTTCCAGACACGCTATCATGCGTTCTCCTCAGATGGTG TCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGAAACAG CTCGTTGTACCGCTGGGACCTTGCATAACCTTTCCCATCATCGTGA GGGCTTACTGGCCATCTTTAAGTCTGGAGGCATTCCTGCCCTGGTG AAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCATTA CAACTCTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGC AGTGCGTTTAGCTGGTGGGCTGCAGAAAATGGTTGCCTTGCTCAAC AAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAAA TTTTAGCTTATGGCAACCAAGAAAGCAAGCTCATCATACTGGCTAG TGGTGGACCCCAAGCTTTAGTAAATATAATGAGGACCTATACTTAC GAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTG TCTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCA AGCTTTAGGACTTCACCTGACAGATCCAAGTCAACGTCTTGTTCAG AACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAAC AGGAAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGG TTCAGATGATATAAATGTGGTCACCTGTGCAGCTGGAATTCTTTCT AACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAAG TGGGTGGTATAGAGGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGA CAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTCGTCATCTG ACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCC TTCACTATGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATC CCACTGGCCTCTGATAAAGGCTACTGTTGGATTGATTCGAAATCTT GCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCA TTCCACGACTAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCA GCGCCGTACGTCCATGGGTGGGACACAGCAGCAATTTGTGGAGGGG GTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACA TCCTAGCTCGGGATGTTCACAACCGAATTGTTATCAGAGGACTAAA TACCATTCCATTGTTTGTGCAGCTGCTTTATTCTCCCATTGAAAAC ATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACA AGGAAGCTGCAGAAGCTATTGAAGCTGAGGGAGCCACAGCTCCTCT GACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGCGACATATGCA GCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACA AGAAACGGCTTTCAGTTGAGCTGACCAGCTCTCTCTTCAGAACAGA GCCAATGGCTTGGAATGAGACTGCTGATCTTGGACTTGATATTGGT GCCCAGGGAGAACCCCTTGGATATCGCCAGGATGATCCTAGCTATC GTTCTTTTCACTCTGGTGGATATGGCCAGGATGCCTTGGGTATGGA CCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTGCTGAC TATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCA TGGATGGGCTGCCTCCAGGTGACAGCAATCAGCTGGCCTGGTTTGA TACTGACCTGTAAtctagaACGCGTCAAACACCATTGTCACACTCC AAGTGAATTCTACCAGTGCCATAGAACAGGTAGTCTGAACACTGGG CAAACACCATTGTCACACTCCAAGTGAATTCTACCAGTGCCATAGA ACAGGTAGTCTGAACACTGGGCAAACACCATTGTCACACTCCAAGT GAATTCTACCAGTGCCATAGAACAGGTAGTCTGAACACTGGGCTCG AGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTC TATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGG GAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGTTA ATTAATGCATCGCGATCGATGATATCAGATCTGTTACGTAGATAAG TAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGG AGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA Expression TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC construct #6 GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG GGTTCCTGGAGGGGTGGAGTCGTGACGTAAAGATCTGATATCTCCT GCAGGCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCC CACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCC CATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTT TAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCC AGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGA GGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGC GCGGCGGGCGGGAGCAAGCTTGAACTGAAAAACCAGAAAGTTAACT GGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGT GGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTAC TTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGA ATTGTACCCTAGGAAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCC GGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGGAGGAAGGTC TGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGG ACGGTCGGACTCCCGCGGCGGGAGGAGCCTGTTCCCCTGAGGGTAT TTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAATG GCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAG ACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGA CTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTCTGAGT GGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCC TGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACA AGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAGAGG GTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGA TCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCAGCG TTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTTG ATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTG AACTGACAAAACTGCTAAATGACGAGGACCAGGTGGTGGTTAATAA GGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGA CACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTA CCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGCTGG GACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATC TTTAAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCAC CAGTGGATTCTGTGTTGTTTTATGCCATTACAACTCTCCACAACCT TTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGT GGGCTGCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAAT TCTTGGCTATTACGACAGACTGCCTTCAAATTTTAGCTTATGGCAA CCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCT TTAGTAAATATAATGAGGACCTATACTTACGAAAAACTACTGTGGA CCACAAGCAGAGTGCTGAAGGTGCTATCTGTCTGCTCTAGTAATAA GCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCAC CTGACAGATCCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTC TCAGGAATCTTTCAGATGCTGCAACTAAACAGGAAGGGATGGAAGG TCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAAT GTGGTCACCTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCAATA ATTATAAGAACAAGATGATGGTCTGCCAAGTGGGTGGTATAGAGGC TCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACT GAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGACACCAAG AAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTATGGACTACC AGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATA AAGGCTACTGTTGGATTGATTCGAAATCTTGCCCTTTGTCCCGCAA ATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGACTAGTTCA GTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATG GGTGGGACACAGCAGCAATTTGTGGAGGGGGTCCGCATGGAAGAAA TAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGGGATGT TCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTT GTGCAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAGCTG CAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGAAGC TATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTTCAC TCTAGGAATGAAGGTGTGGCGACATATGCAGCTGCTGTTTTGTTCC GAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAGT TGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAAT GAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAACCCC TTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGG TGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACAT GAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATGGGC TGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCC AGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAtct agaACGCGTCAAACACCATTGTCACACTCCAAGTGAATTCTACCAG TGCCATAGAACAGGTAGTCTGAACACTGGGCAAACACCATTGTCAC ACTCCAAGTGAATTCTACCAGTGCCATAGAACAGGTAGTCTGAACA CTGGGCAAACACCATTGTCACACTCCAAGTGAATTCTACCAGTGCC ATAGAACAGGTAGTCTGAACACTGGGCTCGAGCTGTGCCTTCTAGG CGGCCGCATCATCCTTTAGGTAAGAAGTTTTAAAAAGCCAGTTTGG GTAAAATACTTTTACTCTGCCTACAGAACTTCAGAAAGACTTGGTT GGTAGGGTGGGAGTGGTTTAGGCTATTTGTAAATCTGCCACAAAAA CAGGTATATACTTTGAAAGGAGATGTCTTGGAACATTGGAATGTTC TCAGATTTCTGGTTGTTATGTGATCATGTGTGGAAGTTATTAACTT TAATGTTTTTTGCCACAGCTTTTGCAACTTAATACTCAAATGAGTA ACATTTGCTGTTTTAAACATTAATAGCAGCCTTTCTCTCTTTATAC AGCTGTATTGTCTGAACTTGCATTGTGATTGGCCTGTAGAGTTGCT GAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGACAC ACTAACCAAGCTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTT TGTTCTGGTCCTTTTTGGTCGAGGAGTAACAATACAAATGGATTTT GGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGA TGGAATTTATCAAACCCTAGCCTTGCTTGTTAAATTTTTTTTTTTT TTTTTTTAAGAATATCTGTAATGGTACTGACTTTGCTTGCTTTGAA GTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTTT TTAAGTCTCTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAA TTTCTAATTTTTAAGAATTGAGTAATGGTGTAGAACACTAATTCAT AATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTGTGT AGCCTTTTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTT TCCGGTTAATTAATGCATCGCGATCGATGATATCAGATCTGTTACG TAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCT AGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT GAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCC CGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA ^CTNNB1 CCTTCAACTATTTCTTCCATGCG _{forward primer} ^CTNNB1 CTAGTTCAGTTGCTTGTTCGTG _{reverse primer} Exogenous CTAGAACGCGTCAAACACC CTNNB1 forward primer Exogenous CTAGAAGGCACAGCTCGA CTNNB1 reverse primer

Claims

CLAIMS: 1. A nucleic acid construct comprising: a promoter operably linked to a nucleotide sequence encoding a functional CTNNB1 polypeptide; a 5' untranslated region (UTR) of a human CTNNB1 gene or a fragment of said 5' UTR; a microRNA (miRNA) targeting region; and a polyadenylation signal.

2. The nucleic acid construct of claim 1, wherein the CTNNB1 nucleotide sequence encodes a human CTNNB1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence having at least or about 80% identity thereto.

3. The nucleic acid construct of claim 1 or 2, wherein the CTNNB1 nucleotide sequence comprises a CTNNB1 cDNA sequence comprising the sequence set forth in SEQ ID NO:2 or SEQ ID NO:3 or a sequence having at least or about 80% identity thereto.

4. The nucleic acid construct of any one of claims 1 to 3, wherein the CTNNB1 nucleotide sequence comprises one or more intronic sequences.

5. The nucleic acid construct of claim 4, wherein the intronic sequences comprise: at least about 50 nucleotides derived from the 5' end of the human CTNNB1 intron 2, comprising the sequence set forth in SEQ ID NO:5 or a sequence at least or about 80% identical thereto; and at least about 50 nucleotides derived from the 3' end of the human CTNNB1 intron 2, comprising the sequence set forth in SEQ ID NO:6 or a sequence at least or about 80% identical thereto.

6. The nucleic acid construct of claim 5, wherein the intronic sequences comprise the sequence set forth in SEQ ID NO:7 or a sequence at least or about 80% identical thereto, located at a position corresponding to the position between nucleotides 13 and 14 of the nucleotide sequence set forth in SEQ ID NO:2 or 3.

7. The nucleic acid construct of any one of claims 1 to 6, wherein the promoter is a hybrid form of the chicken β-actin promoter, optionally the CBh promoter comprising the nucleotide sequence set forth in SEQ ID NO:8, or a sequence having at least or about 80% sequence identity thereto.

8. The nucleic acid construct of any one of claims 1 to 7, wherein the 5' UTR sequence comprises a nucleotide sequence set forth in SEQ ID NO:2, or a sequence having at least or about 80% sequence identity thereto.

9. The nucleic acid construct of any one of claims 1 to 8, wherein the miRNA targeting region comprises one or more targeting sequences for one or more miRNAs, optionally selected from miR122, miR183 and/or miR199.

10. The nucleic acid construct of claim 9, wherein the miR122 targeting sequence comprises the sequence set forth in SEQ ID NO:11 or a sequence having at least or about 80% sequence identity thereto, the miR183 targeting sequence comprises the sequence set forth in SEQ ID NO:12 or a sequence having at least or about 80% sequence identity thereto, and the miR199 targeting sequence comprises the sequence set forth in SEQ ID NO:13 or a sequence having at least or about 80% sequence identity thereto.

11. The nucleic acid construct of claim 9 or 10, wherein the miRNA targeting region comprises one or more copies of the sequence set forth in SEQ ID NO:14 or a sequence having at least or about 80% sequence identity thereto.

12. The nucleic acid construct of claim 11, wherein the miRNA targeting region comprises the sequence set forth in SEQ ID NO:15 or a sequence having at least or about 80% sequence identity thereto.

13. The nucleic acid construct of any one of claims 1 to 12, wherein the polyadenylation signal is a bovine growth hormone (BGH) poly(A) signal, optionally comprising the sequence set forth in SEQ ID NO:16 or a sequence having at least or about 80% sequence identity thereto.

14. The nucleic acid construct of any one of claims 1 to 12, wherein the polyadenylation signal is the endogenous polyadenylation signal residing within the 3' UTR of the human CTNNB1 gene, optionally comprising the sequence set forth in SEQ ID NO:10 or a sequence having at least or about 80% sequence identity thereto.

15. The nucleic acid construct of claim 1, comprising, from 5' to 3': a CBh promoter; a CTNNB1 5' UTR; a CTNNB1 coding sequence operably linked to the CBh promoter; a miRNA targeting region comprising one or more copies of targeting sequences for, sequentially, miR122, miR183 and miR199; and a BGH poly(A) signal.

16. The nucleic acid construct of claim 15, comprising the CBh promoter of SEQ ID NO:8, the CTNNB15' UTR sequence of SEQ ID NO:9, the CTNNB1 coding sequence of SEQ ID NO:2 or 3, the miRNA targeting region of SEQ ID NO:15, and the BGH poly(A) signal of SEQ ID NO:16.

17. The nucleic acid construct of claim 1, comprising, from 5' to 3': a CBh promoter; a CTNNB1 5' UTR; a CTNNB1 coding sequence operably linked to the CBh promoter, wherein the coding sequence comprises one or more intronic sequences; a miRNA targeting region comprising one or more copies of targeting sequences for, sequentially, miR122, miR183 and miR199, and a BGH poly(A) signal.

18. The nucleic acid construct of claim 17, comprising the CBh promoter of SEQ ID NO:8, the CTNNB15' UTR sequence of SEQ ID NO:9, the CTNNB1 coding sequence of SEQ ID NO:2 or 3 comprising the intronic sequences of SEQ ID NO:7 located between positions 13-14 of SEQ ID NO:2 or 3, the miRNA targeting region of SEQ ID NO:15, and the BGH poly(A) signal of SEQ ID NO:16.

19. The nucleic acid construct of claim 1, comprising, from 5' to 3': a CBh promoter; a CTNNB1 5' UTR; a CTNNB1 coding sequence operably linked to the CBh promoter; a miRNA targeting region comprising one or more copies of targeting sequences for, sequentially, miR122, miR183 and miR199, and a CTNNB13' UTR sequence comprising a poly(A) signal.

20. The nucleic acid construct of claim 19, comprising the CBh promoter of SEQ ID NO:8, the CTNNB15' UTR sequence of SEQ ID NO:9, the CTNNB1 coding sequence of SEQ ID NO:2 or 3, the miRNA targeting region of SEQ ID NO:15, and the 3' UTR sequence of SEQ ID NO:10.

21. The nucleic acid construct of any one of claims 1 to 20, further comprising an adeno- associated virus (AAV) inverted terminal repeat (ITR) 5' of the promoter and an AAV ITR nal.

22. The nucleic acid construct of claim 21, wherein the the AAV ITR 5' of the promoter comprises a sequence set forth in SEQ ID NO:17 and the AAV ITR 3' of the poly(A) signal comprises a sequence set forth in SEQ ID NO:18.

23. The nucleic acid construct of claim 1, comprising the sequence of SEQ ID NO:19 or a sequence having at least or about 80% sequence identity thereto, the sequence of SEQ ID NO:20 or a sequence having at least or about 80% sequence identity thereto, or the sequence of SEQ ID NO:21, or a sequence having at least or about 80% sequence identity thereto.

24. A vector comprising a nucleic acid construct of any one of claims 1 to 23.

25. The vector of claim 24, wherein the vector is an AAV vector.

26. A host cell, comprising or transduced with a nucleic acid construct of any one of claims 1 to 23 or a vector of claim 24 or 25.

27. A method for the expression of CTNNB1, comprising introducing a nucleic acid construct of any one of claims 1 to 23 or a vector of claim 24 or 25 into a host cell to facilitate expression of the CTNNB1 coding sequence present in the nucleic acid construct or vector in the host cell.

28. A method for the treatment of a CTNNB1-related disorder or inhibiting or ameliorating at least one symptom thereof, wherein the disorder is characterised by, or associated with, a deficiency or mutation in CTNNB1 in a subject, comprising administering to the subject a nucleic acid construct of any one of claims 1 to 23, a vector of claim 24 or 25 or a cell of claim 26.

29. The method of claim 28, wherein the CTNNB1-related disorder is CTNNB1 neurodevelopmental disorder (CTNNB1 syndrome), familial exudative vitreoretinopathy (FEVR) or neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV).