Recombinant therapeutic FMR1 constructs and methods for treating fragile X syndrome and related disorders

Abstract

The present disclosure provides recombinant human FMR1 constructs and related methods for treating Fragile X syndrome and related disorders in a subject.

Description

[Background technology]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to UK Patent Application Publication No. 2206336.6, filed April 29, 2022, which is incorporated by reference in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing having 13 sequences, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on April 19, 2023 is named 55225WO_CRF_sequencelisting.xml and is 61,563 bytes.

[0003] (Background technology) Fragile X syndrome (FXS) is an X-linked disorder that affects approximately 1 in 4,000 males and 1 in 8,000 females worldwide. It is the leading genetic cause of intellectual disability and autism. Many patients with FXS also experience attention-deficit hyperactivity disorder (ADHD), increased seizure susceptibility, anxiety, and language disorders. FXS is caused by an expansion of a CGG trinucleotide repeat within the 5' untranslated region (UTR) of the fragile X gene FMR1.

[0004] The usual length of this locus is 5-44 CGG repeats. However, longer stretches of CGG repeats tend to be unstable when passed from parent to child. Premutation alleles (55-200 CGG repeats) are the causative mutations for Fragile X-associated Tremor / Ataxia Syndrome (FXTAS), a late-onset neurodegenerative disorder, and Fragile X-associated Primary Ovarian Insufficiency (FXPOI), a disorder resulting in reproductive problems in women. Full mutations (>200 CGG repeats) are the primary cause of Fragile X Syndrome (FXS). FXS is the most common monogenic cause of intellectual disability and autism spectrum disorders. Individuals with FMR1 premutations are at risk of developing FXTAS or FXPOI (1:130-1:256 in women, 1:250-1:810 in men), and individuals with full mutations are diagnosed with FXS (1:2500-1:8000 in women, 1:5000 in men).

[0005] When a premutation allele is present in the FMR1 gene, it is transcribed into messenger RNA (mRNA); however, because it is upstream of the start codon, it is not translated as part of the canonical protein isoform. The mechanisms that cause FXTAS and FXPOI are not fully understood, but it is thought that the longer CGG repeat stretch alters how the gene is expressed (elevated mRNA levels, alternative translation initiation) and how other proteins interact with the CGG repeat or alternative FMRP isoforms. On the other hand, when a full mutant allele is present in the FMR1 gene, epigenetic changes occur, including methylation of cytosine nucleotides within the CGG repeats and along the promoter region, and histones are modified to have a heterochromatin signature. These changes result in FMR1 silencing, where the gene is not significantly transcribed into mRNA and is not significantly translated into protein. Although the exact mechanisms and sequence of events that lead to transcriptional silencing are not yet fully understood, the end result of these epigenetic changes is the absence of the synaptic function regulator FMR1, also known as fragile X mental retardation protein (FMRP).

[0006] There is strong evidence that loss of FMRP causes FXS symptoms, and rare patients with mutations or deletions in other parts of FMR1 also present with FXS. Furthermore, Fmr1 knockout (KO) mouse models recapitulate many key features of the human disease, including learning disabilities, abnormal socialization and anxiety behaviors, increased seizure susceptibility, and abnormal dendritic spine morphology. FMRP is an RNA-binding protein that binds to approximately 4% of brain mRNAs, including enriched portions of synaptic transcripts from genes associated with autism.

[0007] Currently, there are no effective treatments for FXS and related disorders and therefore there is a need for effective therapies to treat these disorders and restore FMRP production. Summary of the Invention

[0008] FXS is a single gene disease with severe symptoms, and since there is no effective treatment, it is considered to be a good candidate for gene therapy.The purpose of the present disclosure is to develop an effective gene therapy cassette for the treatment of FXS.In one aspect, the present disclosure provides a therapeutic polynucleotide construct, comprising a human endogenous FMR1 promoter fragment, a human FMR1 isoform, a suitable human FMR1 isoform 7 sequence, an endogenous human 3'regulatory element, and a polyadenylation signal.

[0009] In some embodiments, the human FMR1 coding sequence comprises the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO:4. In some embodiments, the human FMR1 coding sequence comprises the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence lacking exon 12, particularly a nucleotide sequence lacking exon 12 and having at least 80%, at least 90%, at least 95% identity to SEQ ID NO:4. In some embodiments, the human FMR1 coding sequence comprises a nucleotide sequence that encodes a functionally equivalent nucleotide sequence of SEQ ID NO:4. In some embodiments, the human FMR1 coding sequence encodes a polypeptide or human FMRP comprising an amino acid sequence having at least 80%, at least 90%, at least 95% identity to SEQ ID NO:6.

[0010] In some embodiments, the promoter comprises SEQ ID NO:3 or SEQ ID NO:12, or a nucleotide sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO:3 or SEQ ID NO:12. In particular embodiments, the 3' regulatory element comprises SEQ ID NO:5 or SEQ ID NO:13, or a nucleotide sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO:5 or SEQ ID NO:13. Suitably, promoters and / or 3' regulatory elements functionally equivalent to SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:5, or SEQ ID NO:13 may be provided.

[0011] In some embodiments, the therapeutic construct comprises a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:9, or SEQ ID NO:11, or a nucleotide sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO:2, SEQ ID NO:9, or SEQ ID NO:11.

[0012] In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0013] In some embodiments, the polynucleotide comprises two AAV ITRs.

[0014] In additional embodiments, the disclosure provides a vector comprising a polynucleotide of any of the embodiments described herein.

[0015] In some embodiments, the vector is a viral vector.

[0016] In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0017] In some embodiments, the AAV vector is an AAV9 vector.

[0018] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising any of the polynucleotides or vectors described herein. In certain embodiments, the rAAV is AAV9. Adeno-associated Virus (AAV)

[0019] In another aspect, the disclosure provides a virion comprising a rAAV described herein.

[0020] In another aspect, the disclosure provides a transformed cell comprising any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, or the virions described herein.

[0021] In another aspect, the disclosure provides a pharmaceutical composition comprising any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, or the virions described herein, and optionally a pharma- ceutically acceptable carrier.

[0022] In another aspect, the present disclosure provides a method of treating FXS or FMR1-associated disease in a subject, the method comprising administering to the subject an effective amount of any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, the virions described herein, or the pharmaceutical compositions described herein. Thus, any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, the virions described herein, or the pharmaceutical compositions described herein are provided for use as pharmaceuticals. In particular, any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, the virions described herein, or the pharmaceutical compositions described herein are provided for use in a method of treating FXS or FMR1-associated disease in a subject. Furthermore, any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, the virions described herein, or the pharmaceutical compositions described herein are provided for use in the preparation of a medicament for treating FXS or FMR1-associated disease in a subject. [Brief description of the drawings]

[0023] [Figure 1] Figure 1A shows the FMR1 construct driven by expression from the constitutive CBh promoter (SEQ ID NO:7). Figure 1B shows low levels of transgenic FMRP expression observed by immunohistochemistry 70 days after injection into P0-2 wild type mice by ICV delivery. AAV9-CBh-hFMR1-Myc-bGHpA is delivered at two different doses and FMRP levels compared to vehicle-treated wild type mice. [Diagram 2]1 shows the FMR1 construct design of SEQ ID NO:2, which incorporates a 1050 bp fragment of the endogenous FMR1 promoter (hFMP1050), human wild-type FMR1 sequence encoding isoform 7 (hFMR1), a Myc tag, and a fragment of the endogenous 3'UTR of human FMR1 (FMpA1). [Diagram 3] Plasmid FMR1 construct containing the hMP1050-FMR1-Myc-FMpA1 construct flanked by wild-type AAV2 inverted terminal repeats (ITRs). The sequence of the plasmid is SEQ ID NO:1. [Figure 4] Experimental design and schematic diagram of the different phenotypic analyses used to test Fmr1- / y mice after administration of AAV9-hFMP1050-Myc-hFMR1-FMpA1. [Diagram 5] Figure 5A shows the marble-burying behavior data for WT vs. Fmr1- / y mice, and Figure 5B shows the observable therapeutic effect on marble-burying behavior of Fmr1- / y mice administered different doses of AAV9-hFMP1050-Myc-hFMR1-FMpA1. [Figure 6A] Illustrates the strong genotype effect observed in the Fmr1− / y mouse model for audiogenic seizures. [Figure 6B] Showing the therapeutic effect of AAV9-hFMP1050-Myc-hFMR1-FMpA1 (virus), increasing the dose of AAV9-hFMP1050-hFMR1-Myc-FMpA1 reduces the incidence of seizures in treated Fmr1- / y mice. [Figure 6C] Showing the therapeutic effect of AAV9-hFMP1050-Myc-hFMR1-FMpA1 (virus), increasing the dose of AAV9-hFMP1050-hFMR1-Myc-FMpA1 reduces the incidence of seizures in treated Fmr1- / y mice. [Figure 7]Immunoblot data from the cortex, hippocampus, and thalamus of wild-type (WT) and Fmr1- / y (KO) mice administered vehicle or 5E10vg / mouse of AAV9-hFMP1050-hFMR1-Myc-FMpA1 are shown. [Figure 8A] Quantification of FMRP expression levels in the cortex of wild-type mice treated with increasing doses of AAV9-hFMP1050-hFMR1-Myc-FMpA1 is shown. [Figure 8B] Quantification of FMRP expression levels in the cortex of Fmr1− / y mice treated with increasing doses of AAV9-hFMP1050-hFMR1-Myc-FMpA1. [Figure 9]FIG. 9A shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection with AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) mice and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. FIG. 9B shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection with AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) mice and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. FIG. 9C shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection with AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. Figure 9D shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection of AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. Figure 9E shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection of AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. Figure 9F shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection with AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. Figure 9G shows immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection with AAV9-hFMP1050-hFMR1-Myc-FMpA1.Wild-type (WT) and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. Figure 9H shows immunohistochemistry analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection of AAV9-hFMP1050-hFMR1-Myc-FMpA1. Wild-type (WT) and Fmr1- / y mice were treated by ICV delivery of 1E11vg / mouse at P0-2. [Figure 10] Figure 10A: FMRP expression increased after delivery of AAV9-hFMP1050-hFMR1-Myc-FMpA1 to Fmr1- / y mice. Representative tiled whole-brain sagittal micrographs immunolabeled for FMRP (green fluorescence) from vehicle-treated WT mice. Scale bar represents 1 mm. Figure 10B: FMRP expression increased after delivery of AAV9-hFMP1050-hFMR1-Myc-FMpA1 to Fmr1- / y mice. Representative tiled whole-brain sagittal micrographs immunolabeled for FMRP (green fluorescence) from vehicle-treated Fmr1- / y mice. Scale bar represents 1 mm. Figure 10C: FMRP expression increased after delivery. Representative tiled whole-brain sagittal micrographs immunolabeled for FMRP (green fluorescence) from Fmr1- / y mice treated with AAV9-hFMP1050-hFMR1-Myc-FMpA1 at a dose of 1E11vg / mouse. Scale bar represents 1 mm. FIG. 10D shows increased FMRP expression after delivery of AAV9-hFMP1050-hFMR1-Myc-FMpA1 to Fmr1- / y mice. Representative tiled whole-brain sagittal micrographs immunolabeled for FMRP (green fluorescence) from Fmr1- / y mice treated with AAV9-hFMP1050-hFMR1-Myc-FMpA1 at a dose of 3E11vg / mouse. Scale bar represents 1 mm. [Figure 11]The FMR1 construct design (SEQ ID NO:9) incorporates a 1050 bp fragment of the endogenous FMR1 promoter (hFMP1050), the human wild-type FMR1 sequence encoding isoform 7 (hFMR1), and the endogenous 3'UTR fragment of human FMR1 (FMpA1). [Figure 12] The plasmid FMR1 construct (SEQ ID NO:8) contains the hMP1050-FMR1-FMpA1 construct flanked by wild-type AAV2 inverted terminal repeats (ITRs). [Figure 13] FMR1 promoter region from UCSC genome browser. The 1050 bp fragment contains most of the major genetic control elements in this genomic region 5 required for endogenous expression and regulation of FMR1. [Figure 14] A dot plot generated by EMBOSS Dotmatcher is shown comparing human (hum) and mouse (mus) sequences in the genomic region upstream of the FMR1 coding region incorporating a 1050 bp fragment of the promoter. [Figure 15] The FMR1 construct design incorporates a shorter 734 bp fragment of the endogenous FMR1 promoter (hFMP734), the human wild-type FMR1 sequence encoding isoform 7 (hFMR1), and the endogenous 3'UTR fragment of human FMR1 (FMpA2) (hFMP734-hFMR1-FMpA2 construct, SEQ ID NO:11). [Figure 16] The plasmid FMR1 construct (SEQ ID NO:10) contains the hMP734-FMR1-FMpA2 construct flanked by wild-type AAV2 inverted terminal repeats (ITRs). [Figure 17]Immunoblot data of FMRP expression from plasmids containing hFMP1050-hFMR1-FMpA1 or hFMP734-hFMR1-FMpA2 are shown. HEK293A cells were transfected with various amounts of plasmid DNA and FMRP levels were detected using an anti-FMRP antibody. FMRP levels were calculated relative to FMRP levels in untransfected HEK293A cells and plotted against copy number per cell calculated from input DNA levels and number of cells per well. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] (definition) Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person of ordinary skill in the art to which this invention belongs. The terms used herein are merely for the purpose of describing particular embodiments and are not intended to be limiting of the invention. The following terms have the given meanings.

[0025] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0026] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus, or its modifications, derivatives, or pseudotypes. The term covers all subtypes and both native and recombinant forms, unless otherwise specified. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, and modifications, derivatives, or pseudotypes thereof. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects mammals other than primates, and "bovine AAV" refers to AAV that infects bovine mammals. In some embodiments, the AAV particles are selected from the group consisting of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV. 7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AA V.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.In some embodiments, the AAV particles include AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0027] The various serotypes of AAV are attractive for several reasons, most notably because AAV is believed to be nonpathogenic and because wild-type virus can integrate its genome site-specifically into human chromosome 19 (Linden et al., 1996, Proc Natl Acad Sci USA 93:11288-11294). The site at which AAV inserts into the human genome is called AAVS1. Site-specific integration, as opposed to random integration, is more likely to result in a predictable long-term expression profile.

[0028] The genome sequences of various serotypes of AAV, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art.Such sequences can be found in the literature or in public databases such as GenBank.See, for example, GenBank accession numbers NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC-006261 (AAV-8).The disclosures of these are incorporated herein by reference. See, e.g., Srivistava et al., 1983, J. Virology 45:555; Chiorini et al., 1998, J. Virology 71:6823; Chiorini et al., 1999, J. Virology 73:1309; Bantel-Schaal et al., 1999, J. Virology 73:939; Xiao et al., 1999, J. Virology 73:3994; Muramatsu et al., 1996, Virology 221:208; Shade et al., 1986, J. Virol. 58:921; Gao et al., 2002, Proc. Nat. Acad. Sci. USA 99:11854; Morris et al., 2004, Virology 33:375-383; see also WO 00 / 28061, WO 99 / 61601, WO 98 / 11244, WO 2013 / 063379, WO 2014 / 194132, WO 2015 / 121501, and U.S. Pat. Nos. 6,156,303 and 7,906,111.

[0029] As used herein, "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide that is heterologous to AAV), and is usually a sequence that is targeted for genetic conversion of a cell. In some embodiments, the heterologous polynucleotide may be flanked by at least one, and possibly two, AAV inverted terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors are either single-stranded (ssAAV) or self-complementary (scAAV). "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a viral particle that is composed of at least one AAV capsid protein (usually all capsid proteins of wild-type AAV) and an encapsidated polynucleotide rAAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to a mammalian cell), it is usually referred to as a "rAAV vector particle" or simply a "rAAV vector." Thus, production of rAAV particles necessarily involves production of rAAV vectors, as such vectors are contained within the rAAV particles.

[0030] By "vector" is meant a recombinant plasmid or virus containing a polynucleotide that is delivered to a host cell either in vitro or in vivo.

[0031] As used herein, "recombinant" means that a vector, polynucleotide, polypeptide, or cell is the product of various combinations of cloning, restriction, or ligation steps (e.g., relating to the polynucleotide or polypeptide contained therein) and / or other procedures that result in a construct that differs from the product found in nature. A recombinant virus or vector is a viral particle that contains a recombinant polynucleotide. These terms include copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0032] By "recombinant viral vector" is meant a recombinant polynucleotide vector that contains one or more heterologous sequences (ie, polynucleotide sequences that are not of viral origin).

[0033] "Recombinant" as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that differs from AAV particles found in nature.

[0034] By "AAV Rep" is meant AAV replication proteins and analogs thereof.

[0035] "AAV Cap" refers to the AAV capsid proteins VP1, VP2, VP3 and analogs thereof. In wild-type AAV virus, the three capsid genes vp1, vp2, vp3 overlap with each other. See Grieger and Samulski, 2005, J. Virol. 79(15):9933-9944. A single P40 promoter expresses all three capsid proteins in a ratio of about 1:1:10, vp1, vp2, vp3, respectively, to complement the production of rAAV. For the production of recombinant AAV vectors, the desired ratio of VP1:VP2:VP3 is in the range of about 1:1:1 to about 1:1:100, preferably in the range of about 1:1:2 to about 1:1:50, more preferably in the range of about 1:1:5 to about 1:1:20. The preferred ratio of VP1:VP2 is 1:1, but the range of VP1:VP2 ratios can vary from 1:50 to 50:1.

[0036] A comprehensive list and alignment of the amino acid sequences of the capsids of known AAV serotypes is provided in Marsic et al., 2014, Molecular Therapy 22(11):1900-1909, particularly Supplementary Figure 1.

[0037] For illustrative purposes only, wild-type AAV2 comprises the small (20-25 nm) icosahedral viral capsid of AAV, which is composed of three proteins with overlapping sequences (VP1, VP2, and VP3, a total of 60 capsid proteins that make up the AAV capsid). Proteins VP1 (735 aa; Genbank accession number AAC03780), VP2 (598 aa; Genbank accession number AAC03778), and VP3 (533 aa; Genbank accession number AAC03779) are present in a 1:1:10 ratio within the capsid. That is, in the case of AAV, VP1 is a full-length protein, while VP2 and VP3 are progressively shorter versions of VP1, with more N-terminal truncations compared to VP1.

[0038] "AAV TR" refers to inverted terminal repeat sequences at or near the termini of the AAV genome, including sequences that are largely complementary and symmetrically arranged, and include analogs of the native AAV TR and their analogs. In the case of recombinant parvovirus vectors, the recombinant polynucleotide is flanked by at least one, and preferably two, inverted terminal repeats (ITRs).

[0039] "Cis motifs" include conserved sequences found at or near the ends of a genomic sequence that are recognized for the initiation of replication, cryptic promoters or sequences at internal locations that may be used for transcription initiation, splicing, or termination.

[0040] "Therapeutically effective amount" refers to the minimum amount of an active agent required to provide a therapeutic effect to a subject. For example, a "therapeutically effective amount" for a patient is an amount that induces, improves, stabilizes, retards, or otherwise ameliorates pathological symptoms, disease progression, or resistance associated with or to succumbing to a disease.

[0041] By "gene" is meant a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0042] "Coding sequence" means a sequence that encodes a specific protein or "encoding nucleic acid," and refers to a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control (operably linked) of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.

[0043] "Chimeric" means, with respect to a viral capsid or particle, that the capsid or particle contains sequences of different parvoviruses, preferably different AAV serotypes, as described in U.S. Patent No. 6,491,907 to Rabinowitz et al., the disclosure of which is incorporated herein by reference in its entirety. See also Rabinowitz et al., 2004, J. Virol. 78(9):4421-4432. A particularly preferred chimeric viral capsid is the AAV2.5 capsid, which has the following mutations in the sequence of the AAV2 capsid: 263Q to A, 265 insertion T, 705N to A, 708V to A, and 716T to N. Here, the nucleotide sequence encoding such a capsid is defined as SEQ ID NO:15, as described in WO 2006 / 066066. Other preferred chimeric AAVs include, but are not limited to, AAV2i8, described in WO 2010 / 093784, AAV2G9 and AAV8G9, described in WO 2014 / 144229, and AAV9.45 (Pulicherla et al., 2011, Molecular Therapy 19(6):1070-1078).

[0044] "Flanking" refers to the presence of one or more adjacent elements upstream and / or downstream, i.e., 5' and / or 3', to a sequence flanked by other elements. The term "flanking" does not indicate that the sequences are necessarily contiguous. For example, there may be an intervening sequence between the nucleic acid encoding the transgene and the adjacent element. A sequence (such as a transgene) that is "adjacent" to two other elements (such as a TR) indicates that one element is located 5' of the sequence and the other element is located 3' of the sequence, although there may be an intervening sequence therebetween.

[0045] "Polynucleotide" refers to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are referred to herein in a 5' to 3' orientation. Polynucleotides of the invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. When the polynucleotide is a DNA molecule, the molecule can be a gene or a cDNA molecule. Nucleotide bases are referred to herein by the single letter code of adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). Polynucleotides of the invention can be prepared using standard techniques known to those skilled in the art.

[0046] Human FMRP or synaptic function regulator FMR1, described in UniProtKB-Q06787 (FMR1_Human), is a multifunctional polyribosome-associated RNA-binding protein that plays a central role in neuronal development and synaptic plasticity through regulation of alternative mRNA splicing, mRNA stability, mRNA dendritic transport, and postsynaptic local protein synthesis of a subset of mRNAs.

[0047] "Transduction" of a cell by a virus refers to the transfer of nucleic acid from a viral particle to the cell.

[0048] "Transfection" of a cell means the introduction of genetic material into a cell for the purpose of genetically modifying the cell. Transfection can be carried out by a variety of means known in the art, such as calcium phosphate, polyethyleneimine, electroporation, etc.

[0049] "Polypeptide" includes both peptides and proteins, unless otherwise specified.

[0050] "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting foreign DNA into a host cell. Such methods may result in transient expression of non-integrated introduced DNA, extrachromosomal replication and expression of introduced replicons (e.g., episomes), or integration of the introduced genetic material into the genomic DNA of the host cell.

[0051] "Transgene" is used to mean a heterologous nucleotide sequence, including a viral vector, that is delivered to a target cell (hereinafter also referred to as a "host cell") and incorporated into a vector for expression in the target cell, and associated expression control sequences, such as a promoter. Those skilled in the art will understand that the expression control sequence is selected based on its ability to promote the expression of the transgene in the target cell. Examples of transgenes include nucleic acids that code for therapeutic polypeptides.

[0052] The term "cell culture" refers to adherent cultured cells or cells cultured in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., as well as components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, lipids, flocculants, etc. Large-scale approaches such as suspension cultures and bioreactors involving adherent cells grown attached to microcarriers or macrocarriers in stirred bioreactors are also included in the term "cell culture." Cell culture procedures for both large-scale and small-scale production of proteins are included in this disclosure.

[0053] As used herein, the terms "purifying," "purifying," "separating," "separating," "isolation," "isolating," "isolating" or "isolation" refer to increasing the purity of rAAV particles from a sample that contains a target product and one or more impurities. Typically, the purity of the target product is increased by removing (fully or partially) at least one impurity from the sample. In some embodiments, the purity of rAAV in a sample is increased by removing (fully or partially) one or more impurities from the sample using the methods described herein.

[0054] "Homologous" as used in reference to peptides refers to the similarity of amino acid sequences between two peptides. When an amino acid position in both peptides is occupied by the same amino acid, they are homologous at that position. Thus, "substantially homologous" means a largely, but not completely, homologous amino acid sequence that retains most or all of the activity of the homologous sequence.

[0055] As used herein, "substantially homologous" means that the sequence is at least 50% identical to the reference peptide, preferably at least 75%, more preferably 95% identical. Further peptide sequence modifications include minor mutations, deletions, substitutions or derivatizations of the amino acid sequence of the sequences disclosed herein, so long as the peptide has substantially the same activity or function as the unmodified peptide. Derivatives of amino acids include trifluoroleucine, hexafluoroleucine, 5,5,5-trifluoroisoleucine, 4,4,4-trifluorovaline, p-fluorophenylalanine, o-fluorotyrosine, m-fluorotyrosine, 2,3-difluorotyrosine, 4-fluorohistidine, 2-fluorohistidine, 2,4-difluorohistidine, fluoroproline, difluoroproline, 4-hydroxyproline, selenomethionine, telluromethionine, selenocysteine, selenatryptophan, 4-aminotryptophan, 5-aminotryptophan, 5-hydroxy ... Modified tryptophans may include, but are not limited to, tryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, homoallylglycine, homopropargylglycine, 2-butynylglycine, cis-crotylglycine, allylglycine, dehydroleucine, dehydroproline, 2-amino-3-methyl-4-pentenoic acid, azidohomoalanine, acidoalanine, azidonorleucine, p-ethynylphenylalanine, p-azidophenylalanine, p-bromophenylalanine, p-acetylphenylalanine, and benzofuranylalanine. In particular, modified peptides retain an activity or function associated with the unmodified peptide, although the modified peptides generally have an amino acid sequence that is "substantially homologous" to that of the unmodified sequence.

[0056] In some embodiments, recombinant human FMR1 constructs are provided herein. Additional embodiments provided herein include nucleic acid constructs, such as vectors, that include recombinant human FMR1 as part of their sequence. For example, the invention includes plasmids and / or other vectors that include recombinant human FMR1 sequences along with other elements, such as regulatory elements. Additionally, the invention provides packaged gene delivery vehicles, such as viral capsids, that include recombinant human FMR1 sequences. The invention also includes methods of delivering and preferably expressing recombinant human FMR1 genes by delivering modified sequences into cells along with the necessary elements to facilitate expression within the cells. The invention also provides gene therapy methods in which recombinant human FMR1 gene sequences are packaged and administered to a subject, for example, as a component of a vector and / or as a component of a viral gene delivery vehicle. Certain embodiments include those in which the recombinant human FMR1 sequence has 75% identity to SEQ ID NO:4 (human FMR1 isoform 7). In certain embodiments, the recombinant human FMR1 construct exhibits greater than 75%, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:4 (human FMR1 isoform 7).

[0057] (Recombinant FMR1 Design) Over 20 human FMR1 transcript isoforms have been detected in vivo. Of these, isoform 7 is the most abundant in adult brain tissue. Isoform 7 also lacks exon 12 (due to alternative splicing of human FMR1), and isoforms lacking exon 12 have a higher affinity for kissing complex RNA. Therefore, human FMR1 isoform 7 was selected as the transgene sequence. In the first iteration of recombinant FMR1 construct design, transgene expression was driven by a strong constitutive promoter CBh (cytomegalovirus enhancer, chicken beta actin promoter) and the transcript was stabilized by a universal polyadenylation signal bGHpA (bovine growth hormone polyadenylation signal). This sequence is set forth in SEQ ID NO:7. Expression of the human FMR1 transgene from this cassette was poor and barely detectable above endogenous levels of FMRP treated in wild-type mice. For this purpose, a new recombinant FMR1 construct design was engineered in which transgene expression is driven by a fragment of the endogenous human FMR1 promoter, a 1050 bp fragment immediately upstream of the transcription start site (TSS) and 5'UTR of FMR1. This 1050 bp fragment contains a high degree of sequence conservation with other species (including mouse and cynomolgus monkey) and incorporates the core promoter elements of the human FMR1 gene, driving FMR1 transgene expression with endogenous control elements (elements not present in the constitutive promoter). The transgene is further regulated by endogenous regulatory elements present in the 3'UTR. This 1400 bp fragment of human FMR1 3'UTR contains putative regulatory elements, including miRNA recognition sites and a polyadenylation signal that is most commonly used by FMR1 transcript isoforms.

[0058] For example, treatment can be affected to increase FMRP levels in a subject.

[0059] Modified Nucleic Acids for Expression of FMRP "Optimized" or "codon-optimized," as used interchangeably herein, refers to a coding sequence that has been optimized to enhance expression of the coding sequence compared to a wild-type coding sequence (such as the coding sequence for FMRP) by minimizing the use of rare codons, reducing the number of CpG dinucleotides, removing cryptic splice donor or acceptor sites, removing Kozak sequences, removing ribosome entry sites, etc.

[0060] As used herein, "codon adaptation index" refers to the adaptation of codons from tRNA codons that occur rarely in a particular organism or cell to tRNAs that occur more frequently in a particular organism or cell. A non-limiting example of calculating the codon adaptation index is using the "GenScript Rare Codon Analysis Tool" (genscript.com / tools / rare-codon-analysis).

[0061] As used herein, "percentage of identity" refers to the numerical score of two particular polynucleotides and / or polypeptides that have identical nucleic acids and / or amino acids in the same positions as provided by typical sequence alignment programs (i.e., the BLAST method).

[0062] (Codon optimization) There are 64 codons. 61 of them code for the 20 common amino acids, and the other three act as stop codons. The large number of codons compared to the number of amino acids they code for means that an amino acid can be coded for by more than one codon. In fact, common amino acids such as arginine and leucine are coded for by as many as six codons.

[0063] Organisms tend to preferentially use certain codons over others for the same amino acid. Some species are known to almost completely avoid certain codons. Such biases can affect protein expression. Therefore, codon optimization is important to consider when designing gene therapy constructs.

[0064] Although successful protein expression depends on many factors, codon optimization plays a key role, especially when proteins are expressed in heterologous systems. For example, when expressing human genes in E. coli, selecting codons that are preferentially used by the bacteria can increase the success rate of protein expression. This is especially true when rare codons are eliminated.

[0065] (Codon Adaptation Index) One method to analyze codon usage bias is the Codon Adaptation Index (CAI), which calculates an index that indicates how well a "foreign" sequence adapts to the host's protein expression machinery. The CAI compares the codon usage of the GOI to the most frequent codon usage in a set of highly expressed genes of a model expression organism. The relative adaptability of codons ranges from 0 to 1, with 1 being the closest value, depending on how well the GOI matches a reference set of highly expressed gene sequences.

[0066] Reference codon usage examples are contained in various databases, including the codon usage database kazusa.or.jp / codon / .

[0067] (Array Modification) Exemplary modifications include the removal of one or more cis-acting motifs and the introduction of one or more Kozak sequences. In one embodiment, one or more cis-acting motifs are removed and one Kozak sequence is introduced.

[0068] Examples of cis-acting motifs that may be removed include internal TATA boxes, Chi sites, ribosome entry sites, ARE, INS, and / or CRS sequence elements, repeat sequences and / or RNA secondary structures, (potential) splice donor and / or acceptor sites, branch points, and restriction sites (e.g., Sall).

[0069] Furthermore, in certain embodiments, the codon adaptation index of the modified nucleic acid encoding FMRP (i.e., the modified FMR1 gene) is preferably at least 0.74, preferably at least 0.76, even more preferably at least 0.77, even more preferably at least 0.80, preferably at least 0.82, even more preferably at least 0.84, even more preferably at least 0.85, even more preferably at least 0.89, even more preferably at least 0.90, and most preferably at least 0.91.

[0070] In another embodiment, the modified FMR1 sequence has about a 10%, 20%, 30%, 50% or more reduced level of CpG dinucleotides compared to the wild-type nucleic acid sequence encoding FMR1 (e.g., SEQ ID NO:4).

[0071] Methylation of CpG dinucleotides is known to play an important role in the regulation of gene expression in eukaryotes.Specifically, methylation of CpG dinucleotides in eukaryotes plays a role in suppressing gene expression by interfering with transcription machinery.Thus, due to gene silencing caused by methylation of CpG motifs, the nucleic acid and vector of certain embodiments with reduced number of CpG dinucleotides provide high and long-term transgene expression levels, potentially reducing the risk of immunological response and / or toxicity in host / subject.

[0072] In one embodiment, the modified recombinant human FMR1 gene contains fewer potential CpG dinucleotides than the wild-type FMR1 gene.

[0073] In certain embodiments, the recombinant human FMR1 gene sequence may also include flanking restriction sites to facilitate subcloning into an expression vector, many of which are known in the art and include, but are not limited to, those shown in Figure 2 (plasmid map of hFMP1050-hFMR1-Myc-FMpA1).

[0074] The present disclosure includes nucleic acid vectors comprising recombinant human FMR1 isoform 7 gene sequences and various regulatory or control elements. The exact nature of the regulatory elements useful for gene expression varies from organism to organism and from cell type to cell type.

[0075] In certain embodiments, the promoter comprises a 1050 bp fragment of the human core promoter of human FMR1 (SEQ ID NO:3) (e.g., a fragment of the endogenous or "native" core promoter of human FMR1). Suitably, a promoter functionally equivalent to the 1050 bp fragment of the human core promoter of human FMR1 may be provided.

[0076] In certain embodiments, the promoter comprises a 734 bp fragment of the human core promoter of human FMR1 (SEQ ID NO:12) (e.g., a fragment of the endogenous or "native" core promoter of human FMR1). Suitably, promoters functionally equivalent to the 734 bp fragment of the human core promoter of human FMR1 may be provided, as well as promoters exhibiting 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity thereto.

[0077] Generally, a promoter is used that directs the initiation of RNA transcription in the cells of interest. The promoter can be constitutive or regulated. A constitutive promoter is a promoter that causes the operably linked gene to be expressed essentially all the time. A regulated promoter is a promoter that can be activated or deactivated. Regulatory promoters include inducible promoters, which are normally "off" but can be induced to be "on", and "repressible" promoters, which are normally "on" but can be turned "off". A variety of regulators are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute, and constitutive promoters can often be controlled to some degree. In some cases, endogenous pathways can be utilized to control transgene expression, such as using a promoter that is naturally downregulated upon improvement of the disease state.

[0078] Examples of suitable promoters include adenovirus promoters such as the adenovirus major late promoter, heterologous promoters such as the cytomegalovirus (CMV) promoter, inducible promoters such as the respiratory syncytial virus promoter, the Rous sarcoma virus (RSV) promoter, the albumin promoter, the mouse mammary tumor virus (MMTV) promoter, metallothionein promoters, heat shock promoters, alpha-1-antitrypsin promoter, the hepatitis B surface antigen promoter, the transferrin promoter, the apolipoprotein A-1 promoter, the chicken beta-actin (CBA) promoter, the CBh promoter, the CAG promoter (cytomegalovirus early enhancer element and promoter, the first exon and first intron of the chicken beta-actin gene, and the splice acceptor of the rabbit beta-globin gene) (Alexopoulou et al., 2008, BioMed. Central Cell Biol. 9:2), and the human FMR1 promoter. The promoter can be a tissue-specific promoter, such as the mouse albumin promoter active in hepatocytes, as well as the transthyretin promoter (TTR). In certain embodiments, a liver-non-targeted promoter can be used. It will be clear to those skilled in the art how to utilize and adapt any of these features described herein.

[0079] In another embodiment, the recombinant human FMR1 construct further comprises an enhancer to enhance the expression of protein. Many enhancers are known in the art, including but not limited to the cytomegalovirus major immediate early enhancer. More specifically, the CMV MIE promoter is composed of three regions: a modulator, a unique region, and an enhancer (Isomura and Stinski, 2003, J. Virol. 77(6):3602-3614). The CMV enhancer region can be combined with other promoters or parts thereof to form hybrid promoters to further increase the expression of the nucleic acid operably linked thereto. For example, the chicken beta-actin (CBA) promoter or a portion thereof can be combined with a CMV promoter / enhancer or a portion thereof, and a hybrid intron of chicken beta-actin (CBA) and minute virus of mice (MMV) intron to create a version of the CBA called the "CBh" promoter, which stands for chicken beta actin hybrid promoter, as described in Gray et al. (2011, Human Gene Therapy 22:1143-1153).

[0080] Introns can also be used to improve the efficiency of mammalian expression vectors.Examples of introns include mouse cytomegalovirus (MCMV) immediate early (IE) promoter, human cytomegalovirus (HCMV) immediate early (IE) promoter, and human elongation factor 1 alpha (EF-1 alpha) promoter.Introns can be modified according to the gene of interest.

[0081] Additionally, regulatory elements can include collagen stabilizing sequences (CSS), stop codons, terminator sequences, and polyadenylation signal sequences, such as, but not limited to, the bovine growth hormone polyA signal sequence (bGHpA), which efficiently adds a polyadenosine "tail" to the 3' end of eukaryotic mRNA (see Goodwin and Rottman, 1992, J. Biol. Chem. 267(23):16330-16334).

[0082] In certain embodiments, the recombinant construct comprises an endogenous fragment of the human 3'UTR and polyadenylation sequence (SEQ ID NO:5, or SEQ ID NO:13).

[0083] A polyA tail is a long chain of adenine nucleotides that is added to messenger RNA (mRNA) molecules during RNA processing to increase the stability of the molecule, similar to what happens in vivo. The polyA tail makes the RNA molecule more stable and prevents it from being degraded. In addition, the polyA tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into protein by ribosomes in the cytoplasm.

[0084] The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) increases transgene expression from various viral vectors. The WPRE is most effective when placed downstream of the transgene, near the polyadenylation signal. The WPRE may reduce read-through transcription of viral mRNA by improving transcription termination, resulting in increased viral titer and expression (Gene Therapy volume 14, pages 1298-1304 (2007)).

[0085] (Non-viral vector) In certain embodiments, the vector used according to the present invention is a non-viral vector. Typically, the non-viral vector can be a plasmid comprising a nucleic acid sequence containing recombinant human FMR1 or a mutant thereof.

[0086] (Packaged recombinant FMR1 sequence) The recombinant human FMR1 gene sequence may be provided as a component of a packaged viral vector. Generally, a packaged viral vector includes a viral vector packaged in a capsid. Viral vectors and viral capsids are described in the next section. The nucleic acid packaged in the rAAV vector may be single-stranded (ss), self-complementary (sc), or double-stranded (ds). It is expected that any construct containing SEQ ID NO:2, SEQ ID NO:9, or SEQ ID NO:11 will be capable of desired packaging and expression.

[0087] (Viral Vector) Usually, the viral vector carrying transgene is assembled from the polynucleotide encoding the transgene, the appropriate regulatory elements, and the elements required for the production of viral proteins that mediate cell transduction.Examples of viral vectors include, but are not limited to, adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV) vectors.

[0088] The viral vector component of the packaged viral vector produced according to the methods of the present invention includes at least one transgene, e.g., a recombinant human FMR1 gene sequence, and an associated expression control sequence for controlling expression of the recombinant human FMR1 gene sequence.

[0089] In a preferred embodiment, the viral vector includes a portion of a parvovirus genome, such as an AAV genome, in which rep and cap have been deleted and / or replaced with recombinant human FMR1 and its associated expression control sequences. The recombinant human FMR1 gene sequence is usually inserted adjacent to (i.e., flanked by) one or two AAV TR or TR elements suitable for viral replication, in place of the nucleic acid encoding the viral rep and cap proteins (Xiao et al., 1997, J. Virol. 71(2):941-948). Other regulatory sequences suitable for promoting tissue-specific expression of the recombinant human FMR1 gene sequence in target cells may also be included.

[0090] Those skilled in the art will understand that AAV vectors that contain transgenes and lack viral proteins (e.g., cap and rep) necessary for viral replication cannot replicate because such proteins are necessary for viral replication and packaging. Furthermore, AAV is a dependant virus in that it cannot replicate in a cell without co-infection of the cell with a helper virus. The helper virus typically includes adenovirus or herpes simplex virus. Alternatively, as described below, helper functions (E1a, E1b, E2a, E4, and VA RNA) can be provided to the packaging cell by transfecting the cell with one or more nucleic acids encoding various helper elements and / or the cell can include nucleic acids encoding helper proteins. For example, HEK293 was generated by transforming human cells with adenovirus 5 DNA and now expresses a number of adenovirus genes, including but not limited to E1 and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72). Thus, these helper functions can be provided by the HEK 293 packaging cells without the need to supply them to the cells, such as by plasmids encoding them.

[0091] The viral vector may be any suitable nucleic acid construct, such as a DNA or RNA construct, and may be single-stranded, double-stranded, or duplex (i.e., self-complementary, as described in WO 2001 / 92551).

[0092] Those skilled in the art will appreciate that rAAV vectors may further include "stuffer" or "filler" sequences (filler / stuffer) in which the nucleic acid constituting the transgene is less than about 4.1-4.9 kb in size for optimal packaging of the nucleic acid into the AAV capsid. See Grieger and Samulski, 2005, J. Virol. 79(15):9933-9944. That is, AAV vectors accept insertions of DNA in a defined size range, typically about 4 kb to about 5.2 kb, or slightly larger. Thus, for shorter sequences, the length is adjusted to be at or near the normal size of the viral genome sequence that is acceptable for packaging the AAV vector into a viral particle by including the filler / stuffer in the insertion fragment. In various embodiments, the filler / stuffer nucleic acid sequence is a non-translated (non-protein-coding) segment of nucleic acid. In certain embodiments of the rAAV vector, the length of the heterologous polynucleotide sequence is less than 4.7 Kb and the length of the filler / stuffer polynucleotide sequence, when combined with the heterologous polynucleotide sequence (e.g., inserted into the vector), results in a total length of about 3.0-5.5 Kb, or about 4.0-5.0 Kb, or about 4.3-4.8 Kb.

[0093] Introns can also function as filler / stuffer polynucleotide sequences to achieve the length required for packaging AAV vectors into viral particles. Introns and intron fragments that function as filler / stuffer polynucleotide sequences can also enhance expression. For example, inclusion of intron elements can enhance expression compared to expression without intron elements (Kurachi et al., 1995, J. Biol. Chem. 270(10):5276-5281). Furthermore, filler / stuffer polynucleotide sequences are known in the art, including, but not limited to, those described in WO 2014 / 144486.

[0094] (Viral Capsid) The viral capsid component of the packaged viral vector can be a parvovirus capsid. AAV Cap and chimeric capsids are preferred. Examples of suitable parvovirus viral capsid components include capsid components of Parvoviridae, such as autonomous parvoviruses or dependoviruses. For example, the viral capsid can be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAVrh10, AAVrh74, RHM4-1 (SEQ ID NO: 1 in WO 2015 / 013313), or a combination thereof. NO:5), AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, AAVrh10, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV2.GL, AAV2.NN, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, and other AAVs now known or hereafter discovered. For example, see Fields et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers. The capsids may be derived from a number of AAV serotypes as disclosed in U.S. Pat. No. 7,906,111; Gao et al., 2004, J. Virol. 78:6381; Morris et al., 2004, Virol. 33:375; WO 2013 / 063379; WO 2014 / 194132, including the true AAV (AAV-TT) variants disclosed in WO 2015 / 121501, and RHM4-1, RHM15-1 through RHM15-6 and variants thereof as disclosed in WO 2015 / 013313, although one of skill in the art will recognize that there are likely other variants yet to be identified that perform the same or similar functions or may include components of more than one AAV capsid. The full complement of AAV Cap proteins includes VP1, VP2, and VP3.An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein can consist of less than a full complement of AAV Cap proteins, or a full complement of AAV Cap proteins can be provided.

[0095] One or more AAV Cap proteins can be chimeric proteins, which include the amino acid sequence of AAV Cap from two or more viruses, preferably two or more AAVs. This is described in U.S. Patent No. 6,491,907 by Rabinowitz et al., the entire disclosure of which is incorporated herein by reference. For example, a chimeric virus capsid can include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. A chimeric capsid can include, for example, an AAV capsid that includes one or more B19 Cap subunits. For example, an AAV Cap protein or subunit can be replaced with a B19 Cap protein or subunit. For example, in a preferred embodiment, the Vp3 subunit of an AAV capsid can be replaced with the Vp2 subunit of B19.

[0096] Another embodiment includes a synthetic chimeric virus strain that includes an AAV backbone, such as AAV2, AAV3, AAV6, AAV8, in combination with the galactose (Gal)-binding footprint of AAV9. Adeno-associated viruses (AAV) are helper-dependent parvoviruses that utilize heparan sulfate (HS), galactose (Gal), or sialic acid (Sia) as the primary receptor for cell surface binding. For example, AAV serotypes 2 and 3b utilize HS. AAV1, 4, and 5 bind Sia with different binding specificities. AAV serotype 6 recognizes both Sia and HS, whereas AAV9 utilizes Gal to attach to host cells. Specifically, the galactose (Gal)-binding footprint of AAV9 is grafted onto the heparin sulfate-binding AAV serotype 2, and transduction efficiency is improved by only grafting the orthogonal glycan-binding footprint. Using structural alignment and site-directed mutagenesis, we generated a new dual glycan binding strain (AAV2G9) and a chimeric muscle-tropic strain (AAV2i8G9) by incorporating the Gal-binding footprint of AAV9 into the AAV2 VP3 backbone or chimeric AAV2i8 capsid template. In vitro binding and transduction assays confirmed that AAV2G9 utilizes both HS and Gal receptors for cell entry. Subsequent characterization of in vivo gene expression kinetics and vector genome biodistribution profiles revealed rapid, sustained, and enhanced gene expression by this rationally designed chimeric AAV strain. Similar improved transduction profiles were observed with the liver-nontargeted, muscle-specific AAV2i8G9 chimera (Shen et al., 2013, J. Biol. Chem. 288(4):28814-28823). Such new transplantation combinations are described in detail in International Publication No. WO 2014 / 144229, the contents of which are incorporated herein by reference. Additional liver-nontargeted AAVs, such as AAV9.45, are described in Pulicherla et al., 2011, Molecular Therapy 19(6):1070-1078, the contents of which are incorporated herein by reference as if set forth in their entirety.

[0097] In yet another embodiment, the present invention provides the use of ancestral AAV vectors for use in therapeutic in vivo gene therapy. Specifically, computer-generated sequences are newly synthesized and characterized for biological activity. This effort has generated nine putative functional ancestral AAVs and identified Anc80, the predicted ancestor of AAV serotypes 1, 2, 8, and 9 (Zinn et al., 2015, Cell Reports 12:1056-1068). Such prediction and synthesis of ancestral sequences and assembly into viral particles can be achieved by using the methods described in WO2015 / 054653, the contents of which are incorporated herein by reference. In particular, the use of viral particles assembled from ancestral viral sequences shows less susceptibility to pre-existing immunity in modern human populations than modern viruses or parts thereof.

[0098] (Production of packaged viral vectors) The present invention includes packaging cells encompassed by "host cells" that can be cultured to produce packaged viral vectors of the invention. Packaging cells of the invention generally include cells that have heterologous (1) viral vector functions, (2) packaging functions, and (3) helper functions. The function of each of these components is described in the following sections.

[0099] First, the vector can be made by several methods known to the skilled artisan (see, e.g., WO 2013 / 063379). A preferred method is described in Grieger et al., 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. Briefly, using efficient transfection of HEK293 cells as a starting point, adherent HEK293 cell lines from a certified clinical master cell bank are grown in animal component-free suspension conditions in shaker flasks and WAVE bioreactors to allow rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), suspension HEK293 cell lines can produce up to 1 × 10 rAAV when harvested 48 hours post-transfection. 5 More than 1 x 10 vector genome-containing particles (vg) / cell 14 vg / L of cell culture. More specifically, triple transfection means that three plasmids are introduced into the packaging cells: one plasmid encodes the AAV rep and cap genes, another encodes various helper functions (e.g., adenovirus or HSV proteins (E1a, E1b, E2a, E4, VA RNA, etc.)), and another encodes the transgene and its various control elements (e.g., recombinant FMR1 gene and human endogenous promoter, e.g., SEQ ID NO:3).

[0100] To achieve the desired yield, several variables are optimized, including the selection of a compatible serum-free suspension medium that supports both growth and transfection, the choice of transfection reagent, transfection conditions, and cell density. A universal purification strategy based on ion-exchange chromatography methods was also developed to obtain highly pure vector preparations of AAV serotypes 1-6, 8, 9 and various chimeric capsids. This user-friendly process can be completed within a week and results in high packed particle-to-empty particle ratios (>90% packed particles) and high post-purification yields (1 × 1013 It yields yields of rAAV at concentrations greater than 10000 vg / L and purity suitable for clinical use, and is universal for all serotypes and chimeric particles. This scalable manufacturing technology has been utilized to manufacture GMP Phase I clinical AAV vectors for retinal neovascularization (AAV2), hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered to patients. Furthermore, by implementing a perfusion method to collect rAAV from the culture medium at multiple time points post-transfection, overall vector production is increased by at least 5-fold.

[0101] (Function of viral vectors) The packaging cells of the present invention include a viral vector function in addition to packaging and vector functions. The viral vector function typically includes a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced with a recombinant human FMR1 sequence and associated expression control sequences. The viral vector function includes sufficient expression control sequences to replicate the viral vector for packaging. Typically, the viral vector includes a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced with a transgene and associated expression control sequences. The transgene is typically flanked by two AAV TRs, in place of the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as tissue-specific promoters and other regulatory sequences suitable for promoting tissue-specific expression of the transgene in target cells. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic or marker polypeptide.

[0102] As used herein, a "duplex vector" may be interchangeably referred to as a "dimeric" or "self-complementary" vector. A double-stranded parvovirus particle may, for example, comprise a parvovirus capsid containing virion DNA (vDNA). Because vDNA is self-complementary, it may form a hairpin structure when released from the viral capsid. The double-stranded vDNA appears to provide the host cell with double-stranded DNA that can be expressed (i.e., transcribed and, if necessary, translated) by the host cell without the need for second strand synthesis as required by conventional parvovirus vectors. Double-stranded / self-complementary rAAV vectors are well known in the art and are described, for example, in WO 2001 / 92551, WO 2015 / 006743, and many others.

[0103] The viral vector functions may be suitably provided as a double-stranded vector template, as described in U.S. Patent No. 7,465,583 to Samulski et al., the entire disclosure of which is incorporated herein by reference for teachings regarding double-stranded vectors. A double-stranded vector is a dimeric self-complementary (sc) polynucleotide, usually DNA. The double-stranded vector genome preferably contains sufficient packaging sequences for encapsidation within a selected parvovirus capsid (e.g., AAV capsid). Those skilled in the art will appreciate that the double-stranded vDNA does not exist in double-stranded form under all conditions, but is capable of existing in double-stranded form under conditions that favor annealing of complementary nucleotide bases. "Double-stranded parvovirus particles" include hybrid, chimeric, and targeted viral particles. Preferably, the double-stranded parvovirus particle has an AAV capsid, which may be a chimeric or targeted capsid, as described above.

[0104] Viral vector functions can be suitably provided as a double-stranded vector template, as described in U.S. Patent No. 7,465,583 to Samulski et al., the entire disclosure of which is incorporated herein by reference for teachings regarding double-stranded vectors. A double-stranded vector is a dimeric self-complementary (sc) polynucleotide (usually DNA). For example, the DNA of the double-stranded vector can be selected to form a double-stranded hairpin structure by intrastrand base pairing. Both strands of the double-stranded DNA vector can be packaged within the viral capsid. A double-stranded vector can provide functions comparable to double-stranded DNA viral vectors, and can alleviate the need for target cells to synthesize DNA complementary to the single-stranded genome that is usually encapsulated by the virus.

[0105] The TRs (degradable and non-degradable) selected for use in the viral vector are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. A resolvable AAV TR need not have a wild-type TR sequence (e.g., the wild-type sequence can be altered by insertion, deletion, truncation, or missense mutation), so long as the TR mediates the desired function, such as viral packaging, integration, and / or proviral rescue. The TR can be a synthetic sequence that functions as an AAV inverted terminal repeat, such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al., the disclosure of which is incorporated herein by reference in its entirety. Usually, but not necessarily, the TRs are from the same parvovirus. For example, both TR sequences are from AAV2.

[0106] The packaging functions include capsid components. The capsid components are preferably derived from parvovirus capsids, such as AAV capsids or chimeric AAV capsid functions. Examples of suitable parvovirus viral capsid components include capsid components of Parvoviridae, such as autonomous parvoviruses and dependoviruses. For example, the capsid components may be AAV capsids, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV The capsid component may be selected from Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03 (see U.S. Pat. No. 10,548,947, and other novel capsids yet to be identified or derived from non-human primates). The capsid component may include components of two or more AAV capsids.

[0107] In another embodiment, one or more VP capsid proteins are chimeric proteins comprising amino acid sequences from two or more viruses, preferably two or more AAVs, as described in U.S. Patent No. 6,491,907 to Rabinowitz et al. A chimeric capsid is described herein as having at least one amino acid residue from one serotype in combination with another serotype sufficient to alter (a) viral yield, (b) immune response, (c) targeting, (d) detargeting, etc.

[0108] Further chimeric proteins can be made following the instructions provided in Li et al., 2008, Mol. Ther. 16(7):1252-1260, the contents of which are incorporated herein by reference. Specifically, a DNA shuffling-based approach was used to develop cell type-specific vectors by directed evolution. The capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimera 1829) containing AAV1, 2, 8, and 9 genomic fragments was isolated from an integrin-deficient hamster melanoma cell line previously shown to be poorly permissive to AAV. Molecular modeling studies have shown that AAV2 contributes surface loops to the icosahedral three-fold symmetry axis, while AAV1 and 9 contribute interactions with two-fold and five-fold symmetry, respectively. The C-terminal domain (AAV9) was identified by rational mutagenesis as a key structural determinant of melanoma tropism. Chimeric-1829 utilizes heparan sulfate as its primary receptor and transduces melanoma cells more efficiently than all serotypes. Applying this technology to additional cell / tissue types using AAV or other viral capsid sequences may result in a new class of biological nanoparticles as vectors for human gene transfer.

[0109] The packaged viral vector typically contains a recombinant human FMR1 sequence and expression control sequences flanked by TR elements, termed a "transgene" or "transgene expression cassette," and is sufficient for packaging of the vector DNA and subsequent expression of the modified FMR1 sequence in transfected cells. Viral vector functions can be supplied to cells, for example, as components of a plasmid or amplicon.

[0110] The viral vector functions can exist extrachromosomally within the cell line and can also be integrated into the chromosomal DNA of the cells.

[0111] Any method can be used to introduce the nucleotide sequences carrying the viral vector functions into the cellular host for replication and packaging, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, liposomes combined with nuclear localization signals, etc. In embodiments where the viral vector functions are provided by transfection with a viral vector, standard methods for generating viral infection can be used.

[0112] (Packaging function) Packaging functions include genes for replication and packaging of viral vectors. Thus, for example, packaging functions include functions required for expression of viral genes, replication of viral vectors, rescue of viral vectors from integration state, expression of viral genes, and packaging of viral vectors into viral particles, as required. Packaging functions can be provided together or separately to packaging cells using gene constructs such as plasmids or amplicons, baculoviruses, or HSV helper constructs. Packaging functions can also be present extrachromosomally in packaging cells, but are preferably integrated into the chromosomal DNA of cells. Examples include genes encoding AAV Rep and Cap proteins.

[0113] (rAAV production system) Numerous cell culture-based systems for the production of rAAV particles are known in the art, any of which may be used to practice the methods disclosed herein, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. All rAAV production cultures for producing rAAV viral particles require (1) a suitable host cell, including, for example, a human-derived cell line such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), a mammalian cell line such as Vero, CHO cells or CHO-derived cells, or an insect-derived cell line such as SF-9 in the case of baculovirus production systems; (2) appropriate helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and (5) appropriate media and media components to support rAAV production.

[0114] The skilled artisan will recognize numerous ways in which rAAV may be generated or packaged by introducing AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela, Elb, E4, E2a, VA genes), and the rAAV genome (including one or more genes of interest flanked by inverted terminal repeats (ITRs)) into a cell. The phrase "adenovirus helper functions" refers to numerous viral helper genes that are expressed (as RNA or protein) in a cell and allow AAV to propagate efficiently within the cell. The skilled artisan will recognize that helper viruses, such as adenovirus and herpes simplex virus (HSV), facilitate AAV replication, and specific genes that provide essential functions have been identified. For example, the helpers may induce changes to the cellular environment that facilitate the expression and replication of such AAV genes. In some embodiments, the AAV rep and cap genes, helper genes, and rAAV genome are introduced into a cell by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments, the AAV rep and cap genes, helper genes, and rAAV genome can be introduced into a cell by transduction with a viral vector, such as an rHSV vector encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments, one or more of the AAV rep and cap genes, helper genes, and rAAV genome are introduced into a cell by transduction with an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome.In some embodiments, the rHSV vector encodes helper genes as well as the AAV rep and cap genes.

[0115] Any suitable medium known in the art may be used for the production of rAAV particles. These medium include, but are not limited to, modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), and media manufactured by Hyclone Laboratories and JRH, including Sf-900 II SFM medium, described in U.S. Patent No. 6,723,551, which is incorporated herein by reference in its entirety. In some embodiments, the medium comprises Invitrogen / ThermoFisher's Dynamis™ medium, FreeStyle™ 293 Expression medium, or Expi293™ Expression medium. In some embodiments, the medium comprises Dynamis™ medium. In some embodiments, the methods disclosed herein use cell cultures that include serum-free media, animal component-free media, or chemically defined media. In some embodiments, the medium is an animal component-free medium. In some embodiments, the medium comprises serum. In some embodiments, the medium comprises fetal bovine serum. In some embodiments, the medium is a glutamine-free medium. In some embodiments, the medium comprises glutamine. In some embodiments, the medium is supplemented with one or more of nutrients, salts, buffers, and additives (e.g., antifoam agents). In some embodiments, the medium is supplemented with glutamine. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is supplemented with fetal bovine serum. In some embodiments, the medium is supplemented with poloxamer, e.g., Kolliphor® P 188 Bio. In some embodiments, the medium is a basal medium. In some embodiments, the medium is a feed medium.

[0116] rAAV production cultures can be routinely cultured under a variety of conditions (wide ranges of temperatures, for various lengths of time, etc.) appropriate for the particular host cells utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, multi-layer or multi-tray tissue culture flasks (or stacks, e.g., hyperstacks), microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures also include suspension adapted host cells such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, SF-9 cells, which may be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wavebag system. Numerous suspension cultures for the production of rAAV particles are known in the art, including, for example, the cultures disclosed in U.S. Pat. Nos. 6,995,006, 9,783,826, and U.S. Patent Publication No. 20120122155, each of which is incorporated by reference in its entirety.

[0117] (Packaging Cell) Any cell or cell line known in the art to produce rAAV particles can be used in any of the methods disclosed herein. In some embodiments, the methods of producing rAAV particles or increasing production of rAAV particles disclosed herein use HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLPCK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the methods disclosed herein use mammalian cells. In some embodiments, the methods disclosed herein use insect cells, such as SF-9 cells. In some embodiments, the methods disclosed herein use HEK293 cells. In some embodiments, the methods disclosed herein use HEK293 cells that have been adapted to grow in suspension culture.

[0118] In some embodiments, the cell culture disclosed herein is a suspension culture. In some embodiments, the cell culture disclosed herein is a suspension culture comprising HEK293. In some embodiments, the cell culture disclosed herein is a suspension culture comprising HEK293 cells adapted to growth in suspension culture. In some embodiments, the cell culture disclosed herein comprises a serum-free medium, an animal component-free medium, or a chemically defined medium. In some embodiments, the cell culture disclosed herein comprises a serum-free medium. In some embodiments, the suspension-adapted cells are cultured in shaker flasks, spinner flasks, cell bags, or bioreactors.

[0119] In some embodiments, the cell cultures disclosed herein comprise cells attached to a substrate (e.g., a microcarrier) that is itself suspended in a medium. In some embodiments, the cells are HEK293 cells.

[0120] In some embodiments, the cell culture disclosed herein is an adherent culture. In some embodiments, the cell culture disclosed herein is an adherent culture comprising HEK293. In some embodiments, the cell culture disclosed herein comprises a serum-free medium, an animal component-free medium, or a chemically defined medium. In some embodiments, the cell culture disclosed herein comprises a serum-free medium.

[0121] In some embodiments, the cell culture disclosed herein comprises a high density cell culture. In some embodiments, the total cell density of the culture is about 1×10E+06 cells / ml to about 30×10E+06 cells / ml. In some embodiments, about 50% or more of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted to grow in suspension culture.

[0122] Cell lines used as packaging cells include insect cell lines. Any insect cell capable of replicating AAV and being maintained in culture may be used in the present invention. Examples include Spodoptera frugiperda (such as Sf9 or Sf21 cell lines), Drosophila spp. cell lines, or mosquito cell lines (e.g., cell lines derived from Aedes albopictus). A preferred cell line is the Spodoptera frugiperda Sf9 cell line. See the following references: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol. 63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and U.S. Pat. Nos. 5,611,110 and 6,204,059 to Samulski et al. are incorporated herein by reference for their teachings regarding the use of insect cells for expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture.

[0123] For example, the viral capsids utilized in the embodiments described herein can be produced by any method known in the art, such as expression from baculovirus (Brown et al., (1994) Virology 198:477-488). As yet another alternative, the viral vectors of the invention can be produced in insect cells using a baculovirus vector to deliver the rep / cap genes and rAAV template, for example as described in Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0124] In another aspect, provided herein is a method for producing rAAV in insect cells by incorporating these genes into the polyhedrin coding region of a baculovirus vector to construct a baculovirus packaging system or vector carrying the AAV Rep and Cap coding regions, and generating a viral recombinant by transfection into a host cell. In particular, when using baculovirus production for AAV, it is desirable for the AAV DNA vector product to be a self-complementary AAV-like molecule without using mutations to the AAV ITRs. This is believed to be a by-product of inefficient AAV rep nicking in insect cells, resulting in a self-complementary DNA molecule due to the lack of functional Rep enzyme activity. The host cell is a cell infected with a baculovirus, or has additional nucleic acid encoding baculovirus helper functions introduced into it, or contains these baculovirus helper functions. These baculovirus viruses can express AAV components and then promote the production of capsids.

[0125] During production, packaging cells typically contain one or more viral vector functions along with sufficient helper and packaging functions to replicate and package the viral vector. These various functions are delivered together or separately to the packaging cells using genetic constructs such as plasmids or amplicons, which are present extrachromosomally in the cell line or integrated into the cell chromosome.

[0126] The cells can be provided with one or more of the above functions already integrated, such as a cell line in which one or more vector functions have been integrated extrachromosomally or into the chromosomal DNA of the cell, a cell line in which one or more packaging functions have been integrated extrachromosomally or into the chromosomal DNA of the cell, or a cell line in which helper functions have been integrated extrachromosomally or into the chromosomal DNA of the cell.

[0127] (rAAV purification) The generated rAAV particles can be isolated using methods known in the art. In some embodiments, the method of isolating rAAV particles includes downstream processing such as, for example, harvesting cell culture, clarifying the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, sterile filtration, or any combination thereof. In some embodiments, downstream processing includes at least two, at least three, at least four, at least five, or at least six of harvesting cell culture, clarifying the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and sterile filtration. In some embodiments, downstream processing includes harvesting cell culture, clarifying the harvested cell culture (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, downstream processing does not include centrifugation.

[0128] In some embodiments, a method of isolating rAAV particles comprises harvesting a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles disclosed herein comprises harvesting a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method of isolating rAAV particles comprises clarification of the harvested cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, the method of isolating rAAV particles disclosed herein comprises clarification of the harvested cell culture, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a tangential flow filtration, and a second sterile filtration. In some embodiments, the method of isolating rAAV particles comprises clarification of the harvested cell culture by depth filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration.In some embodiments, the methods of isolating rAAV particles disclosed herein include clarification of the harvested cell culture by depth filtration, first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, the clarification of the harvested cell culture includes sterile filtration.

[0129] Recombinant AAV particles can be collected from rAAV production culture by harvesting the production culture containing host cells or by harvesting spent medium from the production culture. However, the cells are cultured under conditions known in the art that release rAAV particles from intact host cells into the medium. Recombinant AAV particles can also be collected from rAAV production culture by lysing the host cells of the production culture. Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0130] At harvest, rAAV production cultures may contain one or more of the following: (1) host cell proteins, (2) host cell DNA, (3) plasmid DNA, (4) helper virus, (5) helper virus proteins, (6) helper virus DNA, and (7) media components such as serum proteins, amino acids, transferrin, and other low molecular weight proteins. rAAV production cultures may further contain product-associated impurities such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

[0131] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtration through cellulose acetate filters with a pore size of 0.2 mm or greater. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the production culture harvest does not include centrifugation.

[0132] In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, the clarification of the harvested cell culture comprises depth filtration. In some embodiments, the clarification of the harvested cell culture further comprises depth filtration and sterile filtration. In some embodiments, the harvested cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises a depth filtration medium. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train comprises two depth filtration media. In some embodiments, the filter train comprises a sterile filtration medium. In some embodiments, the filter train comprises two depth filtration media and one sterile filtration medium. In some embodiments, the depth filtration medium is a porous depth filter. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and a sterilizing grade filter media. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and Sartopore® 2 XLG 0.2pm. In some embodiments, the harvested cell culture is pretreated prior to contacting with the depth filter. In some embodiments, the pretreatment comprises adding salt to the harvested cell culture. In some embodiments, the pretreatment comprises adding a chemical flocculant to the harvested cell culture. In some embodiments, the harvested cell culture is not pretreated prior to contacting with the depth filter.

[0133] In some embodiments, the purified feed is concentrated by tangential flow filtration ("TFF") before being applied to a chromatography medium, such as an affinity chromatography medium. Large-scale concentration of viruses using TFF ultrafiltration is described in Paul et al. (Human Gene Therapy 4:609-615 (1993)). TFF concentration of the clarified feed allows for a technically manageable amount of clarified feed to be chromatographed, allowing for more rational column sizing without the need for long recirculation times. In some embodiments, the clarified feed is concentrated at least 2-fold to at least 10-fold. In some embodiments, the clarified feed is concentrated at least 10-fold to at least 20-fold. In some embodiments, the clarified feed is concentrated at least 20-fold to at least 50-fold. In some embodiments, the clarified feed is concentrated about 20-fold. One of skill in the art will recognize that TFF can also be used to remove small molecule impurities (e.g., cell culture contaminants including media components, serum albumin, or other serum proteins) from a feed that has been clarified by diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration involves the use of about 3 to about 10 times the diafiltration volume of buffer. In some embodiments, the diafiltration involves the use of about 5 times the diafiltration volume of buffer. One of skill in the art will recognize that TFF can also be used at any step of the purification process where it is desirable to exchange buffer before performing the next step of the purification process. In some embodiments, the methods of separating rAAV from a clarified feed disclosed herein involve using TFF to exchange buffer.

[0134] Affinity chromatography can be used to separate rAAV particles from a composition. In some embodiments, affinity chromatography is used to separate rAAV particles from a clarified feed. In some embodiments, affinity chromatography is used to separate rAAV particles from a clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art and include, but are not limited to, AVB Sepharose™, POROS™ CaptureSelect™ AAVX affinity resin, POROS™ CaptureSelect™ AAV9 affinity resin, and POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAVX affinity resin.

[0135] Anion exchange chromatography can be used to separate rAAV particles from the composition. In some embodiments, anion exchange chromatography is used as a final concentration and polishing step after affinity chromatography. Suitable anion exchange chromatography media are known in the art and include, but are not limited to, Unosphere Q (Biorad, Hercules, CA), and N-charged amino or imino resins such as POROS 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Pat. No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media comprises a quaternary amine. In some embodiments, the anion exchange media is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography medium comprises glycidyl methacrylate-ethylene dimethacrylate or styrene-divinylbenzene polymer. In some embodiments, the monolithic anion exchange chromatography medium is selected from the group consisting of CIMmultus™ QA-1 Advanced Composite Column (quaternary amine), CIMmultus™ DEAE-1 Advanced Composite Column (diethylamino), CIM® QA Disk (quaternary amine), CIM® DEAE, and CIM® EDA Disk (ethylene diamino). In some embodiments, the monolithic anion exchange chromatography medium is CIMmultus™ QA-1 Advanced Composite Column (quaternary amine). In some embodiments, the monolithic anion exchange chromatography medium is CIM® QA Disk (quaternary amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia).In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (column volume is 80 mL). One of skill in the art will know how to identify a wash buffer of appropriate ionic strength to remove impurities that may be introduced by upstream purification steps, including but not limited to, while still retaining the rAAV bound to the resin.

[0136] In additional embodiments, the disclosure provides compositions comprising isolated rAAV particles produced according to the methods disclosed herein, hi some embodiments, the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0137] The term "pharmaceutically acceptable" as used herein means a biologically acceptable formulation, gas, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" composition is a substance that is not biologically or otherwise undesirable, e.g., the substance may be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions may be used, for example, in administering rAAV isolated according to the disclosed methods to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonicity agents and absorption enhancers or retarders, and are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, antifungal agents) can also be incorporated into the composition. Pharmaceutical compositions can be formulated to suit specific administration or delivery routes as described herein or known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0138] Pharmaceutical compositions and delivery systems suitable for rAAV particles and the methods and uses of the invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, pp. 253-315.

[0139] As described herein, recombinant polynucleotide constructs and rAAV can be used as gene therapy to treat fragile X syndrome or related diseases related to FMRP deficiency.The treatment method includes injecting any of the rAAV described herein into the subject that needs treatment.The amount required to treat a subject depends on multiple factors such as the size, age, and sex of the subject, and will be understood by those skilled in the art.

[0140] (Treatment method) In another aspect, a method of treatment is provided, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising any of the desired constructs or rAAV virions described above.

[0141] In some embodiments, the effective amount is at least 1×10 8 In some embodiments, the effective amount is at least 5×10 8 Viral genomes / dose, 7.5 × 10 8 Viral genomes / dose, at least 1 x 10 9 Viral genomes / dose, at least 2.5 x 10 9 Viral genomes / dose, at least 5 x 10 9 Viral genome / administration.

[0142] In some embodiments, the effective amount is at least 1×10 / kg of patient body weight. 11 Viral genomes, at least 5 × 10 11 Viral genomes / kg, at least 1 × 10 12 Viral genomes / kg, at least 5 × 10 12 Viral genomes / kg, at least 1 × 10 13 Viral genomes / kg, at least 1 × 10 14 viral genomes / kg, or at least 5 × 10 14 In some embodiments, rAAV is administered based on brain weight instead of body weight. In some embodiments, the dosage of rAAV is considered to be low, which is particularly beneficial for CNS indications.

[0143] In some embodiments, the rAAV is administered intravenously. In some embodiments, the rAAV is administered intrathecally. In some embodiments, the rAAV is administered by intracerebroventricular injection. In some embodiments, the rAAV is administered by intracisternal administration. In some embodiments, the rAAV is administered by intravitreal injection.

[0144] In various embodiments, methods of treating FXS and related disorders in a subject are disclosed, the methods comprising administering to the subject an effective amount of any of the polynucleotide constructs, or vectors, or rAAVs comprising the vectors, or virions, or any pharmaceutical composition comprising any of these elements described herein. EXAMPLES

[0145] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but of course, some experimental error and deviation should be allowed for.

[0146] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art, such techniques being fully explained in the literature.

[0147] Example 1 Optimization of recombinant FMR1 constructs

[0148] Figure 1 shows (Figure 1A) an FMR1 construct driven by expression from the constitutive CBh promoter (SEQ ID NO:7) and (Figure 1B) low levels of transgenic FMRP expression observed by immunohistochemistry 70 days after injection into P0-2 wild-type mice by ICV delivery. AAV9-CBh-hFMR1-Myc-bGHpA was delivered at two different doses and levels of FMRP were compared to vehicle-treated wild-type mice.

[0149] 2-3 show examples of FMR1 construct designs incorporating endogenous promoters and 3'UTR fragments, and plasmids. The sequence of the FMR1 construct containing hFMP1050-hFMR1-Myc-FMpA1 is SEQ ID NO: 2. The sequence of the plasmid containing the hFMP1050-hFMR1-Myc-FMpA1 construct is SEQ ID NO: 1.

[0150] A 1050 bp fragment of the human FMR1 promoter and 5'UTR immediately upstream of the ATG codon in exon 1 was predicted to contain most of the major gene regulatory elements required for near-endogenous control of FMR1 transgene expression. This 1050 bp fragment (SEQ ID NO:3) contained CpG islands in the core promoter region, transcription factor binding sites, cis-regulatory elements identified by ENCODE, H3K27Ac, ​​and regulatory elements from the Open Regulatory Annotation database (ORegAnno) (Figure 13). The H3K27Ac mark is defined as an active enhancer mark, as it indicates epigenetic modification of the DNA packaging protein histone H3. The ORegAnno regulatory elements represent the main DNase cluster in this genomic region, as well as important conserved transcription factor binding sites. No such elements are found in the genomic region upstream of the 1050 bp promoter fragment. Furthermore, the 1050 bp promoter fragment (SEQ ID NO:3) is well conserved between humans and mice, suggesting that this region contains important conserved sequences (Figure 14). Therefore, this 1050 bp fragment in the FMR1 promoter region (SEQ ID NO:3) may recapitulate the endogenous FMR1 promoter that drives the expression of FMR1 in the brain. Figure 9A-H shows FMR1 expression detected in different regions of the brain (cortex, hippocampus, thalamus, inferior colliculus) under the control of the hFMP1050 promoter fragment (hFMP1050-hFMR1-Myc-FMpA1) (SEQ ID NO:2).

[0151] Example 2 Evaluation of behavioral phenotype and immunoblot data for FMRP localization in brain tissue samples

[0152] Figure 4 shows the Fmr1 expression after administration of AAV9-hFMP1050-hFMR1-Myc-FMpA1. - / y Experimental design and schematic diagram of the different phenotypic analyses used to test Fmr1 mice. - / y FMR1 expression in mice was driven under the control of the hFMP1050 promoter fragment (SEQ ID NO:2). Individual mice were administered AAV9-hFMP1050-hFMR1-Myc-FMpA1 by intracerebroventricular injection (ICV) at week 0. Mice were grown for 8 weeks. From week 3, general weight changes of mice were monitored. At week 8, mice were evaluated in various behavioral tests. On day 1, the open field test, on day 2, the marble burying test, on day 3, the elevated plus maze (EPM) test, and on day 5, the audiogenic seizure test were performed. Tissues were harvested from individual mice and histological evaluation was performed. The results are shown in Figures 5-6.

[0153] FIG. 5A-B shows Fmr1 cells administered different doses of AAV9-hFMP1050-hFMR1-Myc-FMpA1. - / y The observable therapeutic effect on marble burying behavior in mice is shown in Figure 5A. - / y Mutant mice had a significantly higher tendency to marble-burying behavior compared to wild-type (WT) mice (p<0.0001, Mann-Whitney test). Expression of hFMR1 in mutant mice significantly reduced marble-burying behavior in a dose-dependent manner (Figure 5B). Data show that a dose (p value) of 1e11vg / mouse of AAV9-hFMP1050-hFMR1-Myc-FMpA1 was sufficient to significantly reduce marble behavior (p=0.0004, Kruskal-Wallis test and Dunn's post hoc analysis), and that a higher dose (3e11vg / mouse) further improved the therapeutic effect (p=0.0024, Kruskal-Wallis test and Dunn's post hoc analysis).

[0154] Figure 6A-C shows (Figure 6A) Fmr1 of audiogenic seizures. - / y The strong genotype effect observed in the mouse model (Figure 6B-C) shows the therapeutic effect of AAV9-hFMP1050-hFMR1-Myc-FMpA1 (virus). - / y Administration of AAV9-hFMP1050-hFMR1-Myc-FMpA1 to mutant mice significantly reduced seizure incidence at a dose of 5e10vg / mouse (p=0.0554, Fisher's exact test). The therapeutic effect was more pronounced at higher doses of 1e11 or 3e11vg / mouse (p<0.0001, Fisher's exact test). Figure 6C shows that at the highest dose of AAV9-hFMP1050-hFMR1-Myc-FMpA1 (3e11vg / mouse), clonic and tonic seizures disappeared compared to 81% of control vehicle-treated animals.

[0155] FIG. 7 shows wild-type (WT) and Fmr1 mice administered vehicle or 5E10vg / mouse of AAV9-AAV9-hFMP1050-hFMR1-Myc-FMpA1. - / y Immunoblot data from the cortex, hippocampus, and thalamus of (KO) mice are shown. Transgenic FMRP was detected in the cortex and hippocampus after administration of AAV9-hFMP1050-hFMR1-Myc-FMpA1 at 5E10vg / mouse. Myc expression was used as a positive control.

[0156] FIG. 8A-B shows wild-type (WT) and Fmr1 mice treated with increasing doses of AAV9-hFMP1050-hFMR1-Myc-FMpA1. - / y Quantification of FMRP expression levels in the cortex of WT (KO) mice. At a dose of 1e11vg / mouse, FMRP expression was significantly higher in the cortex of WT mice (Figure 8A) and Fmr1 (KO) mice. - / y (KO)-treated mice (Fig. 8B).

[0157] Figure 9A-H show immunohistochemical analysis of transgenic FMRP expression in various brain regions of mice 70 days after injection of AAV9-hFMP1050-hFMR1-myc-FMpA1. - / y Mice were treated by ICV delivery of 1E11vg / mouse at P0-2. FMRP expression was detected in the cortex (Figure 9B) and hippocampus (Figure 9D). The imaging data is consistent with the immunoblot data detection of FMRP expression in Figure 7.

[0158] FIG. 10A-D shows AAV9-hFMP1050-hFMR1-Myc-FMpA1 containing SEQ ID NO:2 expressed as Fmr1. - / y The increase in FMRP expression after delivery to mice is shown. (FIG. 10A) WT mice treated with vehicle, (FIG. 10B) Fmr1 mice treated with vehicle. - / y Fmr1 mice treated with AAV9-hFMP1050-hFMR1-Myc-FMpA1 at a dose of 1E11vg / mouse (FIG. 10C) or 3E11vg / mouse (FIG. 10D) - / y Representative tiled whole-brain sagittal micrographs immunolabeled for FMRP (green) from a mouse (FIG. 10C-D). Scale bar represents 1 mm.

[0159] Example 3 Further modified FMR1 constructs and elements

[0160] Figure 11 shows the design of the FMR1 construct incorporating a 1050 bp fragment of the endogenous FMR1 promoter (hFMP1050), the human wild-type FMR1 sequence encoding isoform 7 (hFMR1), and the endogenous 3'UTR fragment of human FMR1 (FMpA1). This construct lacks the Myc tag and is an example of a therapeutic FMR1 construct. Figure 12 shows the plasmid FMR1 construct containing the hMP1050-FMR1-FMpA1 construct flanked by wild-type AAV2 inverted terminal repeats (ITRs).

[0161] A modified FMR1 gene therapy construct was designed to contain additional regulatory elements identified in the 3'UTR of human FMR1. The new 3'UTR fragment, designated FMpA2 (SEQ ID NO:13), is an extended version of FMpA1 and retains an additional 181 bp of human 3'UTR sequence containing a putative CNS enhancer element.

[0162] To generate a construct that could be packaged into a single-stranded AAV, a 1050 bp fragment of the FMR1 promoter was truncated. Most of the major transcription factor binding sites and CNS-associated DNAse sites that showed enhancer-like activity were present in a 734 bp fragment of the FMR1 promoter (SEQ ID NO: 12, which also incorporates the 5'UTR immediately upstream of the FMR1 coding sequence). Figure 13 shows the FMR1 promoter region in the UCSC genome browser. The 1050 bp fragment contains most of the major gene control elements of this genomic region required for endogenous expression and regulation of FMR1. Figure 14 shows a dot plot generated by EMBOSS Dotmatcher, comparing human (hum) and mouse (mus) sequences in the genomic region upstream of the FMR1 coding region incorporating the 1050 bp fragment of the promoter.

[0163] The truncated FMR1 promoter region selected is the hMP734 promoter fragment, which is combined with the FMpA2 3'UTR sequence to generate the hFMP734-hFMR1-FMpA2 construct (SEQ ID NO:11) to drive and control the expression of FMRP. As shown in FIG. 15, the hFMP734-hFMR1-FMpA2 construct (SEQ ID NO:11) incorporates a 734 bp fragment of the endogenous FMR1 promoter (hFMP734), the human wild-type FMR1 sequence encoding isoform 7 (hFMR1), and a fragment of the endogenous 3'UTR of human FMR1 (FMpA2). FIG. 16 shows the hMP734-FMR1-FMpA2 plasmid (SEQ ID NO:10) containing the hMP734-FMR1-FMpA2 construct flanked by wild-type AAV2 inverted terminal repeats (ITRs).

[0164] Example 4 Comparison of the hFMP1050-hFMR1-FMpA1 and hFMP734-hFMR1-FMpA2 constructs

[0165] To compare FMRP expression from these two constructs, a plasmid (SEQ ID NO:8) expressing FMRP from the hFMP1050-hFMR1-FMpA1 construct (Figure 11, SEQ ID NO:9) and a plasmid (SEQ ID NO:10) expressing FMRP from the hFMP734-hFMR1-FMpA2 construct (Figure 15, SEQ ID NO:11) were transfected into HEK293A cells and FMRP expression levels were analyzed by immunoblotting.

[0166] For transfection, HEK293A cells maintained in supplemented Dulbecco's modified Eagle's medium (DMEM) were cultured to 80-90% confluency the day before transfection, trypsinized (TrypLE™ Express, Life Technologies, Carlsbad, CA), and seeded into 24-well plates coated with poly-L-ornithine and fibronectin. Cells were transfected using Lipofectamine 2000 (Life Technologies) with 2000ng, 1000ng, 500ng, 250ng, or 125ng of plasmid DNA mixed with Opti-MEM (ThermoFisher Scientific) per well according to the manufacturer's protocol. At 48 h post-transfection, media was removed and cells were transferred to NE1 buffer (20 mM HEPES, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, 20% glycerol, 0.5 mM DTT, Pierce protease inhibitor tablets, EDTA-free [A32965, ThermoFisher Scientific]) and prepared for immunoblotting. Genomic DNA was removed with benzonase digest (E1014, Merck, Rockville, MD, USA) for 15 min at room temperature. Protein concentrations were quantified in microtiter plates using a DC BioRad II (5000112, BioRad, Hercules, CA, USA) and a FLUOstar Omega plate reader (BMG Labtech, Cary, NC, USA). Samples were diluted with one-third the volume of 4x Laemmli buffer (0.5 M Tris-HCl, 8% SDS, 0.01% bromophenol blue, 40% glycerol, 10% beta-mercaptoethanol) and denatured at 95°C for 10 min before being stored at -80°C. Protein samples (20 μg / lane) were resolved and transferred to nitrocellulose membranes (BioRad).Total protein was measured using Revert 700 Total Protein Stain (926-11010, LI-COR, Lincoln, NB, USA) and membranes were blocked in Intercept (TBS) blocking buffer (927-60001, LI-COR) for 1 h at room temperature. Rabbit anti-FMRP primary antibody (ab17722, Abcam, 1:1,000) was incubated overnight at 4 °C. Secondary antibody (IRDye 800CW donkey anti-rabbit IgG secondary antibody (926-32213; LI-COR); 1:10,000) was incubated for 1 h at room temperature, followed by data acquisition on a LI-COR Odyssey Classic. Data were analyzed using Image Studio Lite (LI-COR) and Microsoft Excel.

[0167] FIG. 17 shows immunoblot data for FMRP expression from plasmids containing hFMP1050-hFMR1-FMpA1 (SEQ ID NO:8) or hFMP734-hFMR1-FMpA2 (SEQ ID NO:10). FMRP expression from hFMP1050-hFMR1-FMpA1 (SEQ ID NO:9) was not detected at significant levels above background FMRP expression (endogenous levels of FMRP in HEK293A cells) at any DNA input level tested. FMRP expression from hFMP734-hFMR1-FMpA2 (SEQ ID NO:11) was detected at levels significantly higher than background FMRP expression when DNA input levels were 250 ng or higher per 24 wells. A key expression test for therapeutic applications is indicated by the robust expression of the hFMP1050-hFMRP-FMpA1 construct observed in vivo (see FIGS. 4-11). However, in vitro expression in cell lines such as HEK293A also predicts expression from therapeutic transgene cassettes. FMRP expression from the more minimal hFMP734 promoter (from the hMFP734-hFMR1-FMpA2 construct) results in stronger expression in vitro compared to the larger hFMP1050 promoter (from the hFMP1050-hFMRP-FMpA1 construct) (Figure 17). This strong expression obtained with the more minimal promoter fragment (Figure 17) was unexpected and provides an alternative, stronger and more potent expression cassette sequence.

[0168] [Table 1]

[0169] Sequence of plasmid hFMP1050-hFMR1-Myc-FMpA1 · The bold text indicates the hFMP1050 promoter sequence · Bold and underlined are hFMR1 GOI · Bold, diagonal and underlined are Myc tag sequences The underlined sequence is FMpA1.

[0170] >hFMP1050-hFMR1-Myc-FMpA1(SEQ ID NO:1) JPEG2025514599000003.jpg182160JPEG2025514599000004.jpg245170JPEG2025514599000005.jpg80160

[0171] >hFMP1050-hFMR1-Myc-FMpA1(SEQ ID NO:2) JPEG2025514599000006.jpg146157JPEG2025514599000007.jpg145160

[0172] >hFMP1050 (human core promoter, SEQ ID NO:3) JPEG2025514599000008.jpg74163

[0173] >hFMR1 (human isoform 7, SEQ ID NO:4) JPEG2025514599000009.jpg124156

[0174] >FMpA1 (endogenous human 3'UTR and polyA, SEQ ID NO:5) JPEG2025514599000010.jpg82156JPEG2025514599000011.jpg28157

[0175] SEQ ID NO:6 Human FMRP amino acid sequence JPEG2025514599000012.jpg48158

[0176] >CBh-hFMR1-bGHpA (SEQ ID NO:7) JPEG2025514599000013.jpg140157JPEG2025514599000014.jpg135157

[0177] >hFMP1050-hFMR1-FMpA1 Plasmid (SEQ ID NO: 8) · The bold text indicates the hFMP1050 promoter sequence · The bold and underlined text is the hFMR1 GOI sequence The underlined sequence is FMpA1. JPEG2025514599000015.jpg75160JPEG2025514599000016.jpg231161JPEG2025514599000017.jpg233158JPEG2025514599000018.jpg184162

[0178] >hFMP1050-hFMR1-FMpA1(SEQ ID NO:9 JPEG2025514599000019.jpg220160JPEG2025514599000020.jpg130159

[0179] JPEG2025514599000021.jpg233157JPEG2025514599000022.jpg230162JPEG2025514599000023.jpg245170JPEG2025514599000024.jpg43161

[0180] >hFMP734-hFMR1-FMpA2(SEQ ID NO:11) JPEG2025514599000025.jpg175157JPEG2025514599000026.jpg170161

[0181] >hFMP734, human core promoter (SEQ ID NO:12) JPEG2025514599000027.jpg63155

[0182] > Endogenous human FMR1 3'UTR and polyA (FMpA2) (SEQ ID NO:13) JPEG2025514599000028.jpg135159

[0183] (Equivalents and Incorporation by Reference) All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (such as a Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. This incorporation by reference statement is intended to link, in accordance with 37 CFR §1.57(b)(1), to all individual publications, database entries (such as a Genbank sequence or GeneID entry), patent application, or patent that are clearly identified by the applicant in accordance with 37 CFR §1.57(b)(2), even if the citation is not immediately adjacent to the dedicated incorporation by reference statement. The inclusion of a dedicated incorporation by reference statement in the specification does not weaken this general statement of incorporation by reference. The citation of a reference herein is not an admission that the reference is relevant prior art, nor is it an admission of the contents or dates of these publications or documents.

[0184] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

1. A therapeutic polynucleotide construct comprising a human endogenous FMR1 promoter fragment, a human FMR1 coding sequence, an endogenous human 3' regulatory element, and a polyadenylation signal.

2. The polynucleotide of claim 1, wherein the human FMR1 coding sequence comprises any of the following sequences: a sequence having at least 80% identity to SEQ ID NO:4, a sequence having at least 90% identity to SEQ ID NO:4, a sequence having at least 95% identity to SEQ ID NO:4, SEQ ID NO:4, or an FMR1 isoform 7 sequence.

3. The polynucleotide of claim 1 or 2, wherein the human endogenous FMR1 promoter fragment comprises any of the following nucleotide sequences: a nucleotide sequence having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:12, a nucleotide sequence having at least 90% identity with SEQ ID NO:3 or SEQ ID NO:12, a nucleotide sequence having at least 95% identity with SEQ ID NO:3 or SEQ ID NO:12, or SEQ ID NO:3 or SEQ ID NO:

12.

4. The polynucleotide of any one of claims 1 to 3, wherein the 3' regulatory element comprises a nucleotide sequence having at least 80% identity to SEQ ID NO:5 or SEQ ID NO:13, a nucleotide sequence having at least 90% identity to SEQ ID NO:5 or SEQ ID NO:13, a nucleotide sequence having at least 95% identity to SEQ ID NO:5 or SEQ ID NO:13, or a nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:

13.

5. The polynucleotide construct according to any one of claims 1 to 4, comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO:2, a polynucleotide sequence having at least 90% identity to SEQ ID NO:9, a polynucleotide sequence having at least 90% identity to SEQ ID NO:11, any of the sequences of SEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:

11.

6. 6. The polynucleotide of claim 1, further comprising at least one adeno-associated virus (AAV) inverted terminal repeat (ITR).

7. The polynucleotide of claim 6, wherein the polynucleotide comprises two AAV ITRs.

8. A vector comprising a polynucleotide according to any one of claims 1 to 7.

9. The vector of claim 8 , wherein the vector is a viral vector.

10. The vector of claim 8 or 9, wherein the vector is an adeno-associated virus (AAV) vector.

11. The vector of claim 10, wherein the AAV vector is an AAV9 vector.

12. A recombinant adeno-associated virus (rAAV) comprising a polynucleotide according to any one of claims 1 to 7 or a vector according to any one of claims 8 to 11.

13. The rAAV of claim 12, wherein the rAAV is AAV9.

14. A virion comprising the rAAV of claim 12 or 13.

15. A transformed cell comprising a polynucleotide according to any one of claims 1 to 7, a vector according to any one of claims 8 to 11, an rAAV according to claim 12 or 13, or a virion according to claim 14.

16. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 7, a vector according to any one of claims 8 to 11, an rAAV according to claim 12 or 13, or a virion according to claim 14, and a pharma- ceutically acceptable carrier.

17. 17. A method of treating fragile X syndrome and related disorders in a subject comprising administering to the subject an effective amount of a polynucleotide according to any one of claims 1 to 7, a vector according to any one of claims 8 to 11, a rAAV according to claim 12 or 13, a virion according to claim 14, or a pharmaceutical composition according to claim 16.

18. 18. The method of claim 17, wherein the administration is by intraventricular injection or intracisternal cerebral administration to the subject.

19. 17. A polynucleotide according to any one of claims 1 to 7, a vector according to any one of claims 8 to 11, an rAAV according to claim 12 or 13, a virion according to claim 14, or a pharmaceutical composition according to claim 16, for use in a method of treating fragile X syndrome and related diseases in a subject.

20. 20. The polynucleotide, vector, rAAV, virion or pharmaceutical composition for use according to claim 19, wherein the polynucleotide, vector, rAAV, virion or pharmaceutical composition is administered to a subject by intraventricular injection or intracisternal administration.

Images