Recombinant optimized MECP2 cassettes and methods for treating Rett syndrome and related disorders

Abstract

The present disclosure provides optimized therapeutic MECP2 polynucleotide constructs used to replace or compensate for loss of MeCP2 function in patients with Rett Syndrome. Preferably, the present disclosure provides gene therapy cassettes that allow for better regulatory control of MeCP2 protein, including tunable systems that allow MECP2 gene therapy to be expressed at desirable moderate levels and that provide vector-derived transgene expression within a window that alleviates the disease-causing genetic defect without causing undesirable side effects such as overexpression toxicity.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of, and priority to, UK patent application No. GB2201242.1, filed on January 31, 2022, the entire contents of which are incorporated by reference into this application.

[0002] [Array List] This application contains a Sequence Listing having 25 sequences, which has been submitted electronically in XML format and is incorporated by reference in its entirety into this application. A copy of said XML, created on January 24, 2023, is named P265708.WO.01_sequencelisting.xml and is 90k bytes in size. [Background technology]

[0003] [background]

[0004] DNA methylation is a major modification of eukaryotic genomes and plays a key role in mammalian development. The human proteins MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of nuclear proteins related by the presence of a methyl-CpG binding domain (MBD) in each. Each of these proteins, except MBD3, can specifically bind to methylated DNA. MeCP2, MBD1, and MBD2 can also repress transcription from methylated gene promoters. In contrast to other MBD family members, MeCP2 (methyl-CpG binding protein 2) is X-linked and undergoes X-inactivation. MeCP2 is dispensable in stem cells but is essential for embryonic development.

[0005] Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. Currently, there is no approved treatment for RTT. Rett syndrome, a progressive neurodevelopmental disorder, is one of the most common causes of cognitive impairment in women. Alternative splicing results in multiple transcript variants that encode different isoforms.

[0006] Gene therapy provides delivery of therapeutic transgenes that affect correction in genetic diseases, but many genes, such as MECP2, are highly dosage sensitive, and therefore too little or too much expression of the gene product can have deleterious effects. Viral-mediated gene transfer is a powerful method for delivering therapeutic transgenes to target tissues and cells, such as cells of the nervous system. However, high viral titers are typically required to allow effective system-wide transduction for maximum therapeutic effect. As a result, at these same high titers, transgene expression may exceed physiological levels in some cells, causing overexpression toxicity. A transgene system that provides dosage control of therapeutic transgenes is described in WO / 2022 / 003348. There is a need for additional therapeutic constructs that provide optimal expression levels and additional control of MeCP2 suitable for treating Rett syndrome. Summary of the Invention [Problem to be solved by the invention]

[0007] [overview] The present disclosure provides optimized therapeutic MECP2 polynucleotide constructs used to replace or compensate for loss of MeCP2 function in patients with Rett Syndrome. Preferably, the present disclosure provides gene therapy cassettes that allow for better regulatory control of MeCP2 protein, including a tunable system that allows MECP2 gene therapy to be expressed at desirable moderate levels, as shown in Figures 5A-C. Preferably, the present MECP2 gene therapy constructs can demonstrate efficacy and clear improvement in the motor and respiratory phenotypic domains in mouse models of RTT (Figures 6A-B, and Figures 7A-F). Preferably, polynucleotide constructs such as RTT252, RTT253, RTT254 (also known as NGN-401), RTT269, RTT270, RTT271, and RTT272 can exhibit vector-derived transgene expression within a window that alleviates disease-causing genetic defects without producing undesirable side effects such as overexpression toxicity (Figures 3A-C and Figures 4A-C).

[0008] Preferably, provided in the present application is a polynucleotide which comprises, from 5' to 3': ·promoter; at least one non-mammalian miRNA or synthetic miRNA expressed within the intron; · Protein translation start site (Kozak sequence); the human MECP2 coding sequence of SEQ ID NO:7, a nucleotide sequence containing at least 90% identity to SEQ ID NO:7, a codon-optimized or wild-type human MECP2 coding sequence; at least one 3' stabilizing element; at least three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA; optionally, the miRNA binding sites contain at least one mismatch, optionally a single mismatch; · and a polyadenylation signal.

[0009] In certain embodiments provided herein, provided herein is a polynucleotide that includes, from 5' to 3': ·promoter; at least one non-mammalian miRNA or synthetic miRNA expressed within the intron; · Protein translation start site (Kozak sequence); · codon-optimized or wild-type human MECP2 coding sequence; at least one 3' stabilizing element; at least three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA; optionally, where the miRNA binding sites comprise at least one mismatch, optionally a single mismatch, optionally three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA; optionally, where the miRNA binding sites comprise a single mismatch; · and a polyadenylation signal.

[0010] MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNAs ~22 nucleotides in length. Most miRNAs are transcribed by RNA polymerase II either as independent transcripts or as RNA embedded within introns of mRNAs. The primary miRNA transcript is processed into ~70 nt hairpin precursor miRNAs, which are then finally processed into ~22 nt mature miRNAs by two RNase III enzymes (Drosha and Dicer). miRNAs function by targeting messenger RNAs (mRNAs) to repress translation and / or to degrade the messenger RNAs (mRNAs) and regulating protein levels. We have developed non-mammalian, or synthetic, miRNAs that can knock-down the expression of transcripts containing their respective binding regions. In some instances, these are insect-derived miRNA sequences originally designed to target the firefly luciferase protein. In other examples, they are synthetic miRNA sequences that have no orthology to naturally occurring miRNAs. In some examples, synthetic miRNA sequences are designed to target codon-optimized coding sequences, where the coding sequences are modified at the DNA level while retaining the same amino acid sequence. In the context of gene therapy, this allows the synthetic miRNA to exclusively target an exogenously delivered transgene, while the endogenous gene is unaffected. Preferably, the miRNAs may be embedded within different introns. Preferably, the polynucleotide comprises one non-mammalian miRNA, or synthetic miRNA, expressed within an intron. Preferably, the non-mammalian miRNA, or synthetic miRNA, comprises SEQ ID NO:4. Preferably, the human MECP2 coding sequence may comprise a nucleotide sequence having at least 90% identity, at least 95%, at least 97%, at least 99%, at least 100% identity to SEQ ID NO:7.Preferably, the MECP2 sequence may be a codon-optimized human MECP2 sequence. Preferably, the protein translation initiation site may be a Kozak sequence comprising SEQ ID NO: 13. Preferably, the polynucleotide may comprise a human MECP2 coding sequence or any active fragment thereof, preferably a functionally active fragment thereof similar to the complete sequence, such as a minigene encoding such a functional fragment, wherein the coding sequence comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 7, or to SEQ ID NO: 23 encoding the Methyl-CpG Binding Domain (MBD) of MeCP2, or to SEQ ID NO: 24 encoding the NCoR / SMRT Interaction Domain (NID) of MeCP2. [ka] [ka]

[0011] Preferably, the promoter may comprise a CBM or a CBE (SEQ ID NO: 21 or 22).

[0012] Preferably, the CBM promoter may comprise a nucleotide sequence having at least 90% identity, at least 95%, at least 97%, at least 99%, at least 100% identity to SEQ ID NO:21. Preferably, the CBE promoter may comprise a nucleotide sequence having at least 90% identity, at least 95%, at least 97%, at least 99%, at least 100% identity to SEQ ID NO:22. Preferably, the at least one 3' stabilizing element may be a WPRE. Preferably, the polynucleotide comprises three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA. Preferably, the miRNA binding site may comprise SEQ ID NO:8. Preferably, the miRNA binding site may comprise one mismatch. Preferably, the polyadenylation signal may be a simian vacuolating virus 40 polyadenylation signal (SV40pA). Preferably, the SV40pA signal comprises the nucleotide sequence of SEQ ID NO:12.

[0013] Preferably, the polynucleotide may comprise: a CBM promoter, one non-mammalian miRNA or a synthetic miRNA expressed within an intron, a wild-type human MECP2 coding sequence with an optimized Kozak sequence, a WPRE stabilization element, three miRNA binding sites for the non-mammalian miRNA or for the synthetic miRNA, and an SV40pA signal.

[0014] Preferably, the polynucleotide may comprise: a CBM promoter, at least one non-mammalian miRNA or synthetic miRNA expressed within an intron, a codon-optimized human MECP2 coding sequence with an optimized Kozak sequence, a WPRE stabilization element, three miRNA binding sites for the non-mammalian miRNA or for the synthetic miRNA, and an SV40pA signal.

[0015] Preferably, the polynucleotide construct may comprise SEQ ID NO: 25 (RTT254). Preferably, the polynucleotide construct may comprise a nucleotide sequence having at least 90% identity, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 25.

[0016] Preferably, the polynucleotide may further comprise at least one adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0017] Preferably, the polynucleotide may comprise two AAV ITRs.

[0018] Preferably, the present disclosure provides a vector comprising a polynucleotide of any of the embodiments described in this application.

[0019] Preferably, the vector may be a viral vector.

[0020] Preferably, the vector may be an adeno-associated virus (AAV) vector.

[0021] Preferably, the AAV vector may be an AAV9 vector.

[0022] In another aspect, a recombinant adeno-associated virus (rAAV) is provided that includes any of the polynucleotides or vectors described herein. In certain embodiments, the rAAV is AAV9.

[0023] In another aspect, the disclosure provides a virion comprising a rAAV described in this application.

[0024] In another aspect, the present 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.

[0025] In another aspect, the present 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.

[0026] In another aspect, the disclosure provides a method of treating an MECP2-associated disorder in a subject, the method comprising administering to the subject an effective amount of any of a polynucleotide described herein, a vector described herein, a rAAV described herein, or a virion described herein, or a pharmaceutical composition described herein.

[0027] An effective amount of any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, or the virions described herein, or the pharmaceutical compositions described herein, is also provided for use as a medicament, particularly for use in treating an MECP2-associated disorder in a subject.

[0028] Further provided is the use of any of the polynucleotides described herein, the vectors described herein, the rAAVs described herein, or the virions described herein, or the pharmaceutical compositions described herein in the preparation of a medicament for treating an MECP2-associated disorder in a subject.

[0029] In another embodiment, the treated subject exhibits improvement in one or more symptoms associated with the MECP2-associated disorder.

[0030] In another embodiment, the subject receives a 1.0 x 10 15vg, 1.0 x 10 14 Preferably, the subject is a human. Preferably, the subject is administered 1.0×10 14 to 1.0×10 16 Preferably, the subject receives a 1.0×10 14 to 1.0×10 16 Preferably, the dose is delivered via ICV injection, particularly via a 10 mL ICV injection. Preferably, the dose includes NGN-401 and is delivered via ICV injection, particularly via a 10 mL ICV injection. Preferably, the dose includes NGN-401 and is delivered via ICV injection, particularly via a 10 mL ICV injection. Preferably, the dose includes NGN-401 and is delivered via ICV injection, particularly via a 10 mL ICV injection. 13 to 1.0×10 15 vg / mL and delivered via 10 mL ICV injection.

[0031] In another embodiment, the effective dose is 8.3×10 11 vg / g brain. Preferably, the effective dose is 8.2×10 11 vg / g brain = 8.4 × 10 11 vg / g brain. Preferably, the effective dose is 8.3×10 10 vg / g brain = 8.3 × 10 12 vg / g brain. Preferably, the subject is a human. Preferably, the polynucleotide construct may comprise SEQ ID NO: 25 (NGN-401 / RTT254).

[0032] In another embodiment, the subject is substantially free of MECP2 overexpression toxicity. [Brief description of the drawings]

[0033] [Figure 1]Figure 1A illustrates the MeCP2 dosage-sensitive gene therapy cassette, designed to reduce dosage sensitivity, prevent overexpression, and achieve therapeutic set-point transgene levels. Figure 1B is a graph of flow cytometry data showing the effect of various modifications of the therapy cassette (feed-forward circuit) to modulate MeCP2 protein expression levels. A reporter construct, in which the reporter mNeonGreen is fused to hMeCP2, and a second expression cassette that allowed measurement of mRuby as a transfection control, were transfected into HEK cells, and 48 hours later cells were treated and analyzed by flow cytometry, and levels of mRuby (transfection efficiency) and mNeonGreen (MeCP2) were measured. [Diagram 2] FIG. 2 is a schematic diagram showing the polynucleotide cassette elements that are regulated to tailor the dosage insensitivity and set point of MeCP2 expression. [Diagram 3] Figures 3A-C show a table depicting the modular polynucleotide sequence elements and design strategy for MeCP2 constructs (Figure 3A), along with MeCP2 expression data. Twenty-one to 23 days after administration of AAV9-RTT252, AAV9-RTT253, AAV9-RTT254, AAV9-RTT269, AAV9-RTT270, AAV9-RTT271, or AAV9-RTT272 to wild-type mice, tissue samples were collected and analyzed by Western blot to measure levels of MeCP2 expression in the WT cortex (Figure 3B) and WT hippocampus (Figure 3C). [Figure 4]Figures 4A-C are graphs comparing the survival (Figure 4A), body weight (Figure 4B), and RTT clinical score (Figure 4C) of therapeutic MEPC2 constructs in Mecp2- / y (KO) mice after injection of 3x1011 vg / mouse of therapeutic AAV9-MECP2 constructs at P1. The RTT clinical score is an observational scoring system used to determine the severity of the Rett phenotype in mice. Scoring ranges from 0 (similar to wild type) to 5 (most severe) for each individual component of the phenotype. [Diagram 5] Figures 5A-C show the results of systematically adjusting the expression levels using various polynucleotide cassette components to identify and titrate the expression levels for optimal efficacy, which are intermediate or moderate levels of expression. Survival plots and RTT clinical scores are shown for Mecp2- / y animals expressing weak (Figure 5A), moderate (Figure 5B), or strong (Figure 5C) levels of transfected MeCP2 after administration of AAV9-MECP2 constructs at 3x1011 vg / mouse. [Figure 6] Figures 6A-B show improved survival (Figure 6A) and efficacy (RTT phenotypic score, Figure 6B) for AAV9-RTT254-treated KO animals compared to vehicle-treated KO animals. [Figure 7] Figures 7A-F show decomposition of the individual components of the RTT score and include graphs of improved motor and respiratory phenotypes in KO mice treated with AAV9-RTT254 compared to controls at two doses (1 x 1011 vg and 3 x 1011 vg). [Figure 8] FIG. 8 is a plasmid map showing the elements of construct SEQ ID NO:14 (RTT252_CBE-ffluc1-hsaMECP2-3x ligation-SV40pA). [Figure 9]FIG. 9 is a plasmid map showing elements of construct SEQ ID NO:15 (RTT253_CBE-ffluc1-hsaMECP2-3x ligation-WPRE3-SV40pA). [Figure 10] FIG. 10 is a plasmid map showing elements of construct SEQ ID NO:16 (RTT254_CBM-ffluc1-hsaMECP2-3x ligation-WPRE3-SV40pA). [Figure 11] FIG. 11 is a plasmid map showing the elements of construct SEQ ID NO:17 (RTT269_CBE-ffluc1-hsaMECP2-3xjunction_mut3-WPRE3-SV40pA). [Figure 12] FIG. 12 is a plasmid map showing the elements of construct SEQ ID NO:18 (RTT270_CBE-ffluc1-hsaMECP2-3xjunction_mut6-WPRE3-SV40pA). [Figure 13] FIG. 13 is a plasmid map showing the elements of construct SEQ ID NO:19 (RTT271_CBE-ran1g-hsaMECP2-3x-WPRE3-SV40pA). [Figure 14] FIG. 14 is a plasmid map showing elements of construct SEQ ID NO:20 (RTT272_CBE-ran2g-hsaMECP2-3x-WPRE3-SV40pA). [Figure 15] FIG. 15 is a graph showing the survival curves of Mecp2− / y mice treated with NGN-401 compared to vehicle-treated mice. [Figure 16] 16 is a graph showing weekly body weight assessments following ICV delivery of vehicle or NGN-401 at P0-2. Animal body weights were measured weekly starting at P28. Group size numbers are indicated in the figure legend. [Figure 17]Figure 17 is a graph showing the weekly assessment of RTT phenotypic scores after ICV delivery of vehicle or NGN-401 at P0-2. For each parameter, a score from 0 (normal) to 5 (most severe) was assigned every week starting at P28 (4 weeks of age). Scores were combined to obtain an aggregated RTT phenotypic score. Numbers of group sizes are indicated in the figure legend. [Figure 18] FIG. 18 is a survival curve showing safety over 26 weeks of survival for vehicle-treated WT mice and for Mecp2+ / − mice treated with NGN-401 or AAV9-RTT251 at either 1.0×1011 vg / mouse or 3.0×1011 vg / mouse. [Figure 19] FIG. 19 is a graph showing weekly assessment of body weight for Mecp2+ / − mice treated with NGN-401 and for vehicle-treated WT and Mecp2+ / − mice. [Figure 20] FIG. 20 is a graph showing weekly assessment of MeCP2 overexpression toxicity of regulated NGN-401 or unregulated AAV9-RTT251 vectors in Mecp2+ / − female mice at P1 / 2. [Figure 21] FIG. 21 is a graph showing vector biodistribution in Mecp2+ / − mice treated with NGN-401. [Figure 22] FIG. 22 is a graph showing vector biodistribution for Mecp2+ / − mice treated with AAV9-RTT251. [Figure 23] 23A-C are graphs of Western blot protein expression data in the cortex (FIG. 24A), cerebellum (FIG. 24B), and liver (FIG. 24C) for Mecp2+ / − mice treated with NGN-401. [Figure 24] Figures 24A-B are graphs of Western blot protein expression data in the cortex (Data 25A) and liver (Data 25C) for Mecp2+ / - mice treated with AAV9-RTT251. [Diagram 25]FIG. 25 shows in-life survival data following ICV administration of NGN-401 at a dose of 7.4×10 11 vg / mouse in Mecp2+ / − female mice on P1 / 2. [Figure 26] 26 is a graph showing in-life body weight data following ICV administration of NGN-401 at a dose of 7.4×10 vg / mouse in Mecp2+ / − female mice on P1 / 2. Mice were evaluated weekly after P28 (4 weeks of age). [Figure 27] Figure 27 is a graph showing MeCP2 overexpression toxicity score data in Mecp2+ / - female mice following ICV administration of NGN-401 at a dose of 7.4x1011 vg / mouse on P1 / 2. Mice were evaluated weekly after P28 (4 weeks of age). [Figure 28] Figure 28 is a graph showing in-life RTT phenotypic score data in Mecp2+ / - female mice following ICV administration of NGN-401 on P1 / 2. Mice were assessed weekly after P28 (4 weeks of age). [Figure 29] Figure 29 is a graph showing the biodistribution of vector DNA in the cortex, cerebellum, thoracic spinal cord, and liver at 8 weeks after ICV delivery of NGN-401 at a dose of 7.4x1011 vg / mouse. Results are shown as vector genome copy numbers per diploid genome, determined by a qPCR assay targeting the WPRE3 element of the NGN-401 vector and normalized per diploid genome using an assay targeting the mouse actin gene. Numbers for population size are indicated in the figure legend. vg = vector genomes. [Diagram 30] Figure 30 is a graph showing Western blot quantification of MeCP2 protein levels in the cortex, cerebellum, and liver at 8 weeks after ICV delivery of NGN-401 at a dose of 7.4x1011 vg / mouse. Results are shown as the ratio of MeCP2 levels compared to levels in vehicle-treated Mecp2+ / - mice. Group size numbers are indicated in the figure legend. [Diagram 31]Figure 31 is a graph showing data on transgene mRNA levels in NHPs treated with NGN-401 or AAV9-RTT251 at 5.0x1011 or 1.5x1012 vg / g brain weight assessed 29 / 30 days post-dosing. mRNA levels in treated NHPs were determined by qRT-PCR using an assay targeting the WPRE3 element of the MECP2 transcript produced by the NGN-401 vector. Transgene mRNA levels are plotted for each animal relative to the mean value of the NGN-401 low dose group (n=3 / group. vg=vector genome). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] [Detailed Description] Rett syndrome (RTT) is a neurological disorder caused by mutations in the X-linked MECP2 gene. Mecp2-deficient mice recapitulate the cardinal features of the disorder, and studies reactivating the gene with conditional alleles have led to robust phenotypic correction. This makes RTT an attractive gene therapy target, but MECP2 is a dosage-sensitive gene according to both animal studies and human overlap disorders, suggesting that MeCP2 levels must therefore be maintained within a narrow range to achieve efficacy while avoiding overexpression-associated toxicity. These challenges are magnified by the biodistribution patterns of commonly used AAV vectors, which result in hotspots of expression and in excessive transgene expression in sensitive cell types.

[0035] To overcome these challenges, applicants have developed an optimized polynucleotide cassette that utilizes a single gene circuit in which transgene expression is regulated by a miRNA-based feed-forward loop. This circuit provides cells with an autonomous mechanism that prevents overexpression in strongly transduced cells while still allowing expression of therapeutic protein levels in more mildly transduced targets. Importantly, the miRNA sequence is not based on any existing mammalian miRNA, thus preventing interference with endogenous miRNA-mRNA gene regulation in transduced cells. Optimized therapeutic polynucleotide MECP2 constructs and methods for treating Rett syndrome and related disorders in a subject are provided. The coding sequence, including splice variants of human MECP2, can be found in the NCBI database under gene ID 4204.

[0036] In certain embodiments, utilization of a wild-type MEPC2 gene in the therapeutic constructs described herein results in improved protein expression, e.g., the protein encoded thereby is expressed at a more desirable or preferred level in a cell compared to the protein expression levels provided by MECP2 in various codon-optimized, otherwise identical therapeutic polynucleotide cassettes.

[0037] [Definition] Unless otherwise defined, all technical and scientific terms used in this application have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The following terms have the meanings given:

[0038] AAV "rep" and "cap" refer to polynucleotide sequences encoding the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to in this application as AAV "packaging genes."

[0039] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or to variants, derivatives, or pseudotypes thereof. The term covers all subtypes and both native and recombinant forms, unless otherwise required. 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 (AAV 8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AV, as well as variants, derivatives, or pseudotypes thereof. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so forth. In some embodiments, the AAV particles include AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV 14, AAV 15 and AAV 16, 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, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.In some embodiments, the rAAV particles include AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV-PHP.B, AAV-PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, 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.

[0040] The various serotypes of AAV are attractive for several reasons, most notably, AAV is believed to be non-pathogenic, and wild-type virus can integrate its genome in a site-specific manner into human chromosome 19 (Linden et al., 1996, Proc Natl Acad Sci USA 93:11288-11294). The insertion site of AAV into the human genome is called AAVS1. It is believed that site-specific integration, as opposed to random integration, is more likely to result in a predictable long-term expression profile.

[0041] The genomic sequences of the various AAV serotypes, as well as the sequences of the naturally occurring terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art and can be found in the literature or in public databases such as GenBank. See, e.g., 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 which are incorporated by reference into this application. For example, 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; Moris et al., 2004, Virology 33:375-383; International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; WO 2013 / 063379; WO 2014 / 194132; WO 2015 / 121501, and U.S. Patent Nos. 6,156,303 and 7,906,111.

[0042] As used herein, "rAAV vector" refers to an AAV vector that includes a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide that is heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. In some embodiments, the heterologous polynucleotide may be flanked by at least one, and sometimes two, AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors may be 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 (typically with all of the capsid proteins of wild-type AAV) and an encapsidated polynucleotide, the rAAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle," or simply an "rAAV vector." Thus, production of an rAAV particle necessarily includes production of an rAAV vector, and such a vector is contained within the rAAV particle.

[0043] "Vector" means a recombinant plasmid or virus containing a polynucleotide that is delivered into a host cell, either in vitro or in vivo.

[0044] "Recombinant" as used herein means that a vector, polynucleotide, polypeptide, or cell is the product of various combinations of cloning, restriction or ligation steps (e.g., of 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 the progeny of the original viral construct, respectively.

[0045] "Recombinant viral vector" means a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., polynucleotide sequences not of viral origin).

[0046] "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 a naturally occurring AAV particle.

[0047] "AAV Rep" means AAV replication proteins and analogs thereof.

[0048] "AAV Cap" refers to the AAV capsid proteins, VP1, VP2, and VP3, and their analogs. In wild-type AAV virus, the three capsid genes, vp1, vp2, and vp3, overlap each other. See Grieger and Samulski, 2005, J. Virol. 79(15):9933-9944. A single P40 promoter allows expression of all three capsid proteins, vp1, vp2, and vp3, respectively, in a ratio of about 1:1:10, which is sufficient for rAAV production. To produce a recombinant AAV vector, the desired ratio of VP1:VP2:VP3 ranges from about 1:1:1 to about 1:1:100, preferably from about 1:1:2 to about 1:1:50, and more preferably from about 1:1:5 to about 1:1:20. The desired ratio of VP1:VP2 is 1:1, but the range of VP1:VP2 ratios can vary from 1:50 to 50:1.

[0049] A comprehensive list and alignment of the capsid amino acid sequences of known AAV serotypes is provided by Marsic et al., 2014, Molecular Therapy 22(11):1900-1909, in particular in Supplementary Figure 1.

[0050] For illustration purposes only, wild-type AAV2 contains 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 make up the AAV capsid). Proteins VP1 (735 aa; Genbank Accession No. AAC03780), VP2 (598 aa; Genbank Accession No. AAC03778), and VP3 (533 aa; Genbank Accession No. AAC03779) are present in the capsid in a ratio of 1:1:10. That is, for AAV, VP1 is the full-length protein, and VP2 and VP3 are progressively shorter versions of VP1, with increasing N-terminal truncations compared to VP1.

[0051] "AAV TR" refers to palindromic terminal repeat sequences at or near the termini of the AAV genome, including sequences that are nearly complementary, symmetrically arranged, and include analogs of the native AAV TR and its analogs. In the case of recombinant parvovirus vectors, the recombinant polynucleotide is flanked by at least one, and preferably two, inverted terminal repeat sequences (ITRs).

[0052] "Cis-motifs" include conserved sequences found at or near the ends of genomic sequences that are recognized to initiate replication; potential promoters, or sequences at internal locations likely to be used for transcription initiation, splicing, or transcription termination.

[0053] "Therapeutically effective amount" refers to the minimum amount of an active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" for a patient is an amount that induces, ameliorates, stabilizes, slows the progression of, or otherwise causes an improvement in a pathological symptom, progression of a disease, or a physiological condition associated with a disorder, or resistance to succumbing to a disorder.

[0054] "Gene" means a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0055] "Coding sequence" means a sequence that codes for 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, either 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 even synthetic DNA sequences.

[0056] "Chimeric" means, with respect to a viral capsid or particle, that the capsid or particle comprises sequences from different parvoviruses, preferably from 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 sequence of the AAV2 capsid with the following mutations: 263 Q to A; 265 inserted T; 705 N to A; 708 V to A; and 716 T to N), where 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).

[0057] "Flanking" refers to the presence of one or more elements that are adjacent to another element, either upstream and / or downstream, i.e., 5' and / or 3', to the sequence. The term "flanking" is not intended to indicate that the sequences must be contiguous. For example, there may be an intervening sequence between the nucleic acid encoding the transgene and the flanking element. A sequence (e.g., a transgene) that is "flanked" by two other elements (e.g., TRs) indicates that one element is located 5' and the other is located 3' of the sequence, although there may be an intervening sequence between them.

[0058] "Polynucleotide" refers to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented in the present application in a 5' to 3' orientation. The polynucleotides of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. When the polynucleotide is a DNA molecule, the molecule may be a gene or a cDNA molecule. Nucleotide bases are referred to in the present application by the single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). Polynucleotides of the present invention may also be prepared using techniques well known to one of ordinary skill in the art.

[0059] "Transduction" of a cell by a virus means that there is transfer of nucleic acid from the viral particle to the cell.

[0060] "Codon-optimized MECP2" means a modified nucleic acid encoding a MECP2 gene having at least one modification compared to the wild-type nucleic acid encoding MECP2 (SEQ ID NO:7), including, but not limited to, an MECP2 gene having a reduced GC content, or a reduced CpG content. Human MECP2 can be found in the NCBI database as gene ID 4204 (considered to be wild-type).

[0061] "Transfection" of a cell means the introduction of genetic material into said cell in order to genetically modify said cell. Transfection can be carried out by a variety of methods known in the art, such as calcium phosphate, polyethyleneimine, electroporation, etc.

[0062] "Polypeptide" includes both peptides and proteins, unless otherwise indicated.

[0063] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting foreign nucleic acid, e.g., DNA or RNA, into a host cell. Such methods may result in transient expression of the non-integrated transferred DNA, extrachromosomal replication and expression of the transferred replicon (e.g., episome), or integration of the transferred genetic material into the genomic DNA of the host cell.

[0064] "Transgene" is used to mean any heterologous nucleotide sequence incorporated into a vector, such as a viral vector, for delivery to and expression in a target cell (also referred to in this application as a "host cell"), and associated with an expression control sequence, such as a promoter. One of skill in the art will understand that the expression control sequence will be selected based on its ability to promote expression of the transgene in the target cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide.

[0065] The term "cell culture" refers to cells grown attached or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., as well as the 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, and lipids, and flocculants. Larger scale approaches, such as adherent cells grown attached to microcarriers or macrocarriers in bioreactors, such as suspension cultures, and stirred bioreactors, are also encompassed by the term "cell culture." The present disclosure encompasses cell culture procedures for both large and small scale production of proteins.

[0066] As used herein, the terms "purifying," "purification," "separate," "separating," "separation," "isolate," "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 methods described herein.

[0067] "Homologous" when used in reference to peptides refers to the similarity of amino acid sequences between two peptides. If an amino acid position in both peptides is occupied by the same amino acid, then they are homologous at that position. Thus, "substantially homologous" refers to an amino acid sequence that is largely, but not entirely, homologous and that retains most or all of the activity as the homologous sequence.

[0068] As used herein, "substantially homologous" means that the sequence is at least 50% identical to the reference peptide, and preferably at least 75% and more preferably 95% homologous. Further peptide sequence modifications can 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, but are not limited to, 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 ... Tryptophan, 5-hydroxytryptophan, 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, asidoalanine, 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, and the modified peptides generally have an amino acid sequence that is "substantially homologous" to the amino acid sequence of the unmodified sequence.

[0069] In certain embodiments, the therapeutic polynucleotide construct comprises a wild-type MECP2 gene. In another embodiment, a modified MECP2 gene is provided herein. Further embodiments provided herein include nucleic acid constructs (e.g., vectors) that include as part of their sequence a modified MECP2 gene (e.g., a GC content optimized MECP2 gene sequence that includes a greater or lesser amount of GC nucleotides compared to the wild-type MECP2 gene sequence, and / or a MECP2 gene sequence that has reduced or increased levels of CpG dinucleotides compared to the levels of CpG dinucleotides present in the wild-type MECP2 gene). For example, embodiments include plasmids and / or other vectors that include either the wild-type or modified MECP2 sequence, along with other elements such as regulatory elements. Further embodiments provide packaged gene delivery vehicles, such as viral capsids, that include either the wild-type or modified MECP2 sequence. Also provided herein are methods of delivering a wild-type or modified MECP2 gene, and preferably expressing the wild-type or modified MECP2 gene by delivering the modified sequence into a cell together with elements required to facilitate expression in said cell. The invention also provides gene therapy methods in which the wild-type or modified MECP2 gene sequence is administered to a subject, for example, as a component of a vector and / or packaged as a component of a viral gene delivery vehicle. Certain embodiments include those in which the modified nucleic acid sequence has 90% identity to SEQ ID NO: 7 (wild-type human MECP2). In certain embodiments, the MECP2 construct exhibits greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 7 (wild-type human MECP2).

[0070] Treatment may be effected, for example, to increase the level of MeCP2 in the subject, an amount that provides therapeutic levels of MeCP2 without undesirable toxic effects.

[0071] Modified Nucleic Acids for Expression of MECP2 "Optimized" or "codon optimized," as referred to interchangeably in this application, refers to a coding sequence that has been optimized relative to a wild-type coding sequence (e.g., the coding sequence of MECP2) such that expression of the coding sequence is increased (e.g., by minimizing rare codon usage, reducing the number of CpG dinucleotides, removing potential splice donor or acceptor sites, removing Kozak sequences, removing ribosome entry sites, etc.).

[0072] "Percent identity" as used herein refers to a numerical score for two given polynucleotides and / or polypeptides that have identical nucleic acids and / or amino acids in the same positions, as obtained by a typical sequence alignment program (i.e., the BLAST method). Preferably, the "percent identity" is determined over the full length of the polynucleotide and / or polypeptide, or over the length of a functional fragment of the polynucleotide and / or polypeptide. A functional fragment may be provided as a shorter portion of said polynucleotide and / or polypeptide that provides the same desired function as the full-length polynucleotide and / or polypeptide.

[0073] Codon Optimization There are 64 different codons. 61 of them code for the 20 common amino acids, while the other three function 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, some common amino acids, such as arginine and leucine, are coded for by as many as six codons.

[0074] Different organisms exhibit biases in using 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. Thus, in certain embodiments, it can be beneficial to consider codon optimization when designing gene therapy constructs.

[0075] While many factors contribute to successful protein expression, codon optimization plays a key role, especially when proteins are expressed in heterologous systems. For example, when expressing human genes in E. coli, the success rate of protein expression can be increased by selecting codons that are preferentially used by the bacteria. This is especially true when rare codons are removed.

[0076] In certain embodiments, as described below in this application, it has been determined that a wild-type MECP2 coding sequence provides an optimal, moderate level of expression in certain therapeutic cassettes, as shown and described herein.

[0077] Sequence Modifications and Polynucleotide Cassette Elements Exemplary modifications include the removal of one or more cis-acting motifs and the introduction of one or more Kozak sequences, hi some embodiments, one or more cis-acting motifs are removed and one Kozak sequence is introduced.

[0078] Examples of cis-acting motifs that can 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 enzyme sites (e.g., Sall).

[0079] In certain embodiments, the MeCP2 gene sequence may also contain flanking restriction enzyme sites to facilitate subcloning into an expression vector. Many such restriction enzyme sites are well known in the art.

[0080] The present disclosure includes nucleic acid vectors that include MECP2 gene sequences and various regulatory or control elements. The exact nature of the regulatory elements that aid in gene expression varies from organism to organism and from cell type to cell. Broadly, they include a promoter that directs the initiation of RNA transcription in the cells of interest. The promoter may be constitutive or regulatable. A constitutive promoter is one that causes an operably linked gene to be expressed essentially all the time. Regulatable 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"). Many different regulators are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. This distinction is not absolute; constitutive promoters are often regulated to some degree. In some cases, endogenous pathways may be utilized to regulate the expression of the transgene (e.g., using a promoter that is naturally down-regulated when a pathological condition improves).

[0081] Examples of preferred promoters include adenovirus promoters, such as the adenovirus major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus promoter; Rous sarcoma virus (RSV) promoter; albumin promoter; inducible promoters, such as the mouse mammary tumor virus (MMTV) promoter; metallothionein promoters; heat shock promoters; alpha-1-antitrypsin promoter; hepatitis B surface antigen promoter; transferrin promoter; apolipoprotein A-1 promoter; chicken beta-actin (CBA) promoter, CBh promoter, and CAG promoter (cytomegalovirus early enhancer element and promoter, first exon and first intron of chicken beta-actin gene, and splice acceptor of rabbit beta-globin gene) (Alexopoulou et al., 2008, BioMed. Central Cell Biol. 9:2), CBE promoter (cytomegalovirus early enhancer element and chicken beta-actin promoter, and the first exon, first intron, and second exon of human elongation factor 1 alpha), CBM promoter (cytomegalovirus early enhancer element and chicken beta-actin promoter, and the first exon, first intron, and second exon of MINIX), and human MECP2 promoter. The promoters are tissue-specific promoters, such as the mouse albumin promoter, which is active in liver cells, and the transthyretin promoter (TTR). In certain embodiments, liver-detargeted promoters may be used. It will be clear to one of skill in the art how to utilize and adapt any of these features described in this application.

[0082] In another embodiment, the modified nucleic acid encoding MECP2 further comprises an enhancer to increase expression of the 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 comprises 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 portions thereof to form hybrid promoters to further increase expression of the nucleic acid operably linked thereto. For example, as described by Gray et al. (2011, Human Gene Therapy 22:1143-1153), 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) introns to create a type of CBA called the "CBh" promoter, which stands for chicken beta-actin hybrid promoter.

[0083] In some embodiments, a synthetic RNA circuit may be used to regulate expression of a transgene. The circuit includes a single-gene microRNA (miRNA)-based feed-forward loop. It provides a non-mammalian miRNA, or a synthetic (not naturally occurring) miRNA, expressed within an intron, that targets its own transcript, where the miRNA is not expected to target a human mRNA. The miRNA is non-mammalian or synthetic. Expression of a miRNA from within a different intron (hEF1a vs. MINIX) may be used to fine-tune the circuit. Expression of a different miRNA (EXACT1 vs. EXACT2 vs. EXACT3) may be used to fine-tune the circuit. The binding site in the 3'UTR of the construct mRNA is specific for a non-mammalian miRNA, or a synthetic miRNA, expressed from within an intron of the circuit, allowing expression of the transgene to be controlled. It is not expected that the non-mammalian miRNA binding site or the synthetic miRNA binding site will allow binding of human endogenous miRNAs at all. Providing different numbers of miRNA binding sites (one or more) may be used to fine-tune the circuit.

[0084] Introns may be used to increase efficiency in mammalian expression vectors. Examples of introns are the murine cytomegalovirus (MCMV) immediate early (IE) promoter, the human cytomegalovirus (HCMV) immediate early (IE) promoter, and the human elongation factor 1 alpha (EF-1 alpha) promoter. The introns may vary depending on the gene of interest.

[0085] Further control elements include collagen stabilization sequences (CSS), stop codons, termination sequences, and poly-adenylation signal sequences, such as, but not limited to, the bovine growth hormone polyA signal sequence (bGHpA), which drives the efficient addition of poly-adenosine "tails" at the 3' ends of eukaryotic mRNAs (see, e.g., Goodwin and Rottman, 1992, J. Biol. Chem. 267(23):16330-16334).

[0086] A poly-A 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 poly-A tail makes the RNA molecule more stable and prevents its degradation. Furthermore, the poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into protein by ribosomes in the cytoplasm.

[0087] The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression from various viral vectors. WPRE is most effective when placed downstream of the transgene and proximal to the polyadenylation signal. By improving transcription termination, WPRE can reduce read-through translation of viral mRNA, which in turn increases viral titer and expression. (Gene Therapy volume 14, pages 1298-1304 (2007)).

[0088] Non-viral vectors In certain embodiments, the vector used according to the present invention is a non-viral vector. Typically, the non-viral vector may be a plasmid comprising a nucleic acid sequence encoding MECP2 or a variant thereof.

[0089] Packaged MECP2 sequence The MECP2 gene sequence may also 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 discussed in the next section. The nucleic acid packaged into the rAAV vector may be single-stranded (ss), self-complementary (sc), or double-stranded (ds). It is expected that constructs containing any of the polynucleotide constructs described in this application will be capable of desired packaging and expression. Furthermore, single-stranded vectors exhibit similarly desirable packaging capabilities.

[0090] Viral Vectors Typically, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, suitable regulatory elements, and elements required for the production of viral proteins that mediate cell transduction. Examples of viral vectors include, but are not limited to, adenoviruses, retroviruses, lentiviruses, herpes viruses, and adeno-associated viruses (AAV).

[0091] The viral vector components of the packaged viral vectors produced according to the methods of the invention include at least one transgene, e.g., an MECP2 gene sequence, and an associated expression control sequence for controlling expression of a modified MECP2 therapeutic cassette.

[0092] In a preferred embodiment, the viral vector comprises a portion of a parvovirus genome, e.g., an AAV genome in which rep and cap have been deleted and / or replaced by MECP2 gene sequences and associated expression control sequences. The MECP2 gene sequences are typically inserted adjacent to (i.e., adjacent to) one or two AAV TR or TR elements suitable for viral replication, in place of nucleic acid encoding the viral rep and cap proteins (Xiao et al., 1997, J. Virol. 71(2): 941-948). Other regulatory sequences suitable for use in promoting tissue-specific expression of the MECP2 cassette in target cells may also be included.

[0093] One of skill in the art will appreciate that an AAV vector containing a transgene and lacking viral proteins necessary for viral replication (e.g., cap and arep) cannot replicate because such proteins are essential for viral replication and packaging. Furthermore, AAV is a dependant virus in that it cannot replicate in a cell without a helper virus co-infecting the cell. The helper virus typically includes adenovirus or herpes simplex virus. Alternatively, as discussed below, helper functions (E1a, E1b, E2a, E4, and VA RNA) may be provided to the packaging cell, such as by transfecting the cell with one or more nucleic acids encoding various helper elements, and / or the cell may contain nucleic acids encoding the helper proteins. For example, HEK 293 was generated by transforming human cells with adenovirus 5 DNA and now expresses many of the 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 may be conferred by the HEK 293 packaging cells without the need to supply them to the cells, e.g., by plasmids encoding them.

[0094] 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 double-stranded (i.e. self-complementary as described in WO 2001 / 92551).

[0095] Those skilled in the art will appreciate that if the nucleic acid containing the transgene is less than the optimal size of about 4.1-4.9 kb for packaging the nucleic acid into an AAV capsid, the rAAV vector may further comprise a "stuffer" or "filler" sequence (filler / stuffer). See Grieger and Samulski, 2005, J. Virol. 79(15):9933-9944. That is, AAV vectors typically accept DNA inserts having a defined size range, generally about 4 kb to about 5.2 kb, or slightly larger. Thus, for shorter sequences, the inclusion of a filler / stuffer in the insert fragment brings the length closer to or to the normal size of the viral genomic sequence that is permissive for packaging of the AAV vector into a viral particle. 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 heterologous polynucleotide sequence has a length of less than 4.7 Kb and the filler / stuffer polynucleotide sequence, when combined with the heterologous polynucleotide sequence (e.g., inserted into the vector), has a total length of between about 3.0-5.5 Kb, or between about 4.0-5.0 Kb, or between about 4.3-4.8 Kb.

[0096] Introns may also function as filler / stuffer polynucleotide sequences to achieve length for packaging of AAV vectors into viral particles. Introns and fragments of introns that function as filler / stuffer polynucleotide sequences may also enhance expression. For example, inclusion of intron elements can enhance expression compared to expression in the absence of intron elements (Kurachi et al., 1995, J. Biol. Chem. 270(10):5276-5281). Additionally, filler / stuffer polynucleotide sequences are well known in the art and include, but are not limited to, those described in WO 2014 / 144486.

[0097] Viral Capsid The viral capsid component of the packaged viral vector may be a parvovirus capsid. AAV Cap and chimeric capsids are preferred. Preferred parvovirus capsid components include capsid components from the Parvoviridae family, such as autonomous parvoviruses or dependoviruses. For example, the viral capsid may 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: 5 in WO 2015 / 013313), 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, The AAV vector may be an equine AAV, an ovine AAV, a caprine AAV, a shrimp AAV, or any other AAV now known or later discovered (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69 (4 thed., Lippincott-Raven Publishers). The capsid 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; Moris et al., 2004, Virol. 33:375; WO 2013 / 063379; WO 2014 / 194132; and the true AAV (AAV-TT) variants as disclosed in WO 2015 / 121501, and RHM4-1, RHM15-1 through RHM15-6, and variants thereof, as disclosed in WO 2015 / 013313, as one of skill in the art would know, and there are likely other variants yet to be identified that perform the same or similar functions, or may contain components from more than one AAV capsid. The full complement of AAV Cap proteins includes VP1, VP2, and VP3. An ORF containing a nucleotide sequence encoding an AAV VP capsid protein may contain less than the full complement of AAV Cap proteins, or the full complement of AAV Cap proteins may be provided.

[0098] The one or more AAV Cap proteins may be chimeric proteins, such as the amino acid sequences of AAV Cap from two or more viruses, preferably two or more AAVs, as described in Rabinowitz et al., US Pat. No. 6,491,907, which is incorporated by reference in its entirety. For example, a chimeric virus capsid may include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. The chimeric capsid may include, for example, an AAV capsid having one or more B19 Cap subunits, e.g., an AAV Cap protein or subunit may be replaced by a B19 Cap protein or subunit. For example, in a preferred embodiment, the Vp3 subunit of the AAV capsid may be replaced by the Vp2 subunit of B19.

[0099] In another embodiment, the synthesized chimeric virus strains include combinations of AAV backbones from AAV2, AAV3, AAV6, AAV8, etc., with the galactose (Gal)-binding footprint from AAV9. Adeno-associated viruses (AAV) are helper-dependent parvoviruses that utilize heparan sulfate (HS), galactose (Gal) or sialic acid (Sia) as primary receptors 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, while AAV9 utilizes Gal for host cell binding. Specifically, the galactose (Gal)-binding footprint from AAV9 was grafted onto the heparin sulfate-binding AAV serotype 2, and simple grafting of the orthogonal glycan-binding footprint improved transduction efficiency. A new dual glycan-binding strain (AAV2G9) and a chimeric muscle-tropic strain (AAV2i8G9) were generated by incorporating the Gal-binding footprint from AAV9 into the AAV2 VP3 backbone or chimeric AAV2i8 capsid template using structural alignment and site-directed mutagenesis. In vitro binding and transduction assays confirmed that AAV2G9 utilizes both HS and Gal receptors for cell entry. Subsequent in vivo characterization of transgene expression kinetics and vector genome biodistribution profiles indicate that this rationally engineered chimeric AAV strain expresses transgenes rapidly, persistently, and enhancedly. Similar improved transduction profiles were observed with liver-detargeted, muscle-specific AAV2i8G9 chimeras (Shen, et al., 2013, J. Biol. Chem. 288(4):28814-28823). Such novel grafting combinations are fully described in WO2014 / 144229, the contents of which are incorporated by reference into the present application.Additional liver-detargeted AAVs, such as AAV9.45, are described in Pulicherla et al., 2011, Molecular Therapy 19(6):1070-1078, the contents of which are incorporated by reference as if set forth in their entirety herein.

[0100] In yet another embodiment, the present invention provides the use of ancestral AAV vectors for use in therapeutic in vivo gene therapy. Specifically, in silico-derived sequences were synthesized de novo and characterized for biological activity. This effort generated nine functional putative 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). Predicting and synthesizing such ancestral sequences, in addition to assembling them into viral particles, can be accomplished by using the methods described in WO2015 / 054653, the contents of which are incorporated by reference into this application. In particular, using viral particles constructed from ancestral viral sequences is less susceptible to pre-existing immunity in modern human populations than contemporary viruses or portions thereof.

[0101] 1. Production of Packaged Viral Vectors The present invention includes packaging cells, which are 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 a heterologous (1) viral vector function, (2) packaging function, and (3) helper function. Each of these component functions is described in the following sections.

[0102] To begin with, the vector can be made by several methods known to those skilled in the art (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 by reference into this application for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, where adherent HEK293 cell lines derived from qualified clinical master cell banks are grown under animal-component-free suspension conditions in shake flasks and WAVE bioreactors, allowing for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), suspension HEK293 cell lines can produce 1 x 10 cells when harvested 48 hours post-transfection. 5 Vector genome-containing particles (vg) / cell, or 1 x 10 14 vg / L of cell culture. More specifically, triple transfection refers to packaging cells being transfected with three plasmids: one plasmid encoding the AAV rep and cap genes, another encoding various helper functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA), and another encoding the transgene and its various control elements (e.g., the MECP2 gene and the CBM or CBE promoter).

[0103] To achieve the desired yield, numerous variables are optimized, including the choice 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 method based on an ion-exchange chromatography method was also developed, resulting in 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 whole-to-empty particle ratios (>90% whole particles) and post-purification yields (>1 x 10) suitable for clinical use. 13 vg / L) and purity, and is universal for all serotypes and chimeric particles. This scalable production technique has been utilized to produce 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 that involves harvesting rAAV from the culture medium at multiple time points post-transfection, overall vector production was increased by at least 5-fold.

[0104] Viral Vector Function The packaging cells of the invention contain viral vector functions along with packaging and vector functions. The viral vector functions typically include a portion of a parvovirus genome, e.g., an AAV genome, with rep and cap deleted and replaced by a wild-type or optimized MECP2 gene sequence and associated expression control sequences. The viral vector functions include sufficient expression control sequences to provide replication of the viral vector for packaging. Typically, the viral vectors contain a portion of a parvovirus genome, e.g., an AAV genome, with rep and cap deleted and replaced by 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. Suitable expression control sequences include, for example, tissue-specific promoters and other regulatory sequences suitable for use in 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.

[0105] A "duplexed vector" may be referred to interchangeably in this application as a "dimeric" or "self-complementary" vector. A duplexed parvovirus particle may, for example, include a parvovirus capsid containing virion DNA (vDNA). The vDNA may be self-complementary and, as a result, may form a hairpin structure upon release from the viral capsid. The duplexed vDNA appears to provide the host cell with double-stranded DNA that may be expressed (i.e., transcribed and, optionally, translated) in the host cell without the need for second strand synthesis, as is required for conventional parvovirus vectors. Duplexed / 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.

[0106] The viral vector function may preferably be provided as a duplexed 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 its teachings regarding duplexed vectors. Duplexed vectors are dimeric self-complementary (sc) polynucleotides, typically DNA. The duplexed vector genome preferably includes sufficient packaging sequences to encapsidate into a selected parvovirus capsid (e.g., an AAV capsid). One of skill in the art will appreciate that the duplexed vDNA may not exist in double-stranded form under all conditions, but is capable of becoming so under conditions that favor annealing of complementary nucleotide bases. "Duplexed parvovirus particles" encompass hybrid, chimeric, and targeted viral particles. Preferably, the duplexed parvovirus particles include an AAV capsid, which may further be a chimeric capsid or a targeted capsid, as described above.

[0107] The viral vector functions 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 its teachings on duplexed vectors, and is preferably provided as a duplexed vector template. A duplexed vector is a dimeric self-complementary (sc) polynucleotide, typically DNA. For example, the DNA of the duplexed vector may be selected to form a double-stranded hairpin structure by intrastrand base pairing. Both strands of the duplexed DNA vector may be packaged within the viral capsid. A duplexed vector may provide functionality comparable to a double-stranded DNA viral vector, and may relax the need for the target cell to synthesize complementary DNA to the single-stranded genome normally packaged by the virus.

[0108] The TRs (lytic and non-lytic) selected for use in the viral vector are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5 and 6 being preferred. A lytic AAV TR need not have a wild-type TR sequence (e.g., the wild-type sequence may be modified 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 may be a synthetic sequence that functions as an AAV inverted terminal repeat, such as the "double-D sequence" described in U.S. Pat. No. 7,465,583 to Samulski et al., the entire disclosure of which is incorporated herein by reference in its entirety. Typically, but not necessarily, the TRs are from the same parvovirus, e.g., both TR sequences are from AAV2.

[0109] The packaging function comprises a capsid component, preferably from a parvovirus capsid (e.g., an AAV capsid or a chimeric AAV capsid function). A preferred example of a parvovirus capsid component is a capsid component from a Parvoviridae family, such as an autonomous parvovirus or a dependovirus. For example, the capsid component can be an AAV capsid, e.g., 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 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. Patent No. 10,548,947), as well as other novel capsids yet to be identified, or from non-human primate sources. The capsid components may include components from more than one AAV capsid.

[0110] In another embodiment, one or more VP capsid proteins are chimeric proteins and contain amino acid sequences from more than one virus, preferably more than one AAV, as described by Rabinowitz et al., U.S. Patent No. 6,491,9071. A chimeric capsid is described herein as having at least one amino acid residue from one serotype in combination with sufficient of another serotype to modify a) viral yield, b) immune response, c) targeting, d) de-targeting, etc.

[0111] Additional chimeric proteins may be generated by following the instructions 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 genome fragments from AAV1, 2, 8, and 9 was identified from an integrin-minus hamster melanoma cell line previously shown to have low permissivity for AAV. Molecular modeling studies suggest that AAV2 contributes surface loops to the icosahedral 3-fold symmetry axis, while AAV1 and 9 contribute 2-fold and 5-fold symmetric interactions, respectively. The C-terminal domain (AAV9) was identified by rational mutagenesis as a key structural determinant for melanoma tropism. Chimera-1829 utilizes heparan sulfate as the primary receptor and transduces melanoma cells more efficiently than all serotypes. Application of this technology to alternative cell / tissue types using AAV or other viral capsid sequences will result in a new class of biological nanoparticles as vectors for human gene transfer.

[0112] The packaged viral vector generally contains a wild-type or modified MECP2 sequence flanked by TR elements, and expression control sequences, referred to herein as a "transgene" or "transgene expression cassette," sufficient to effect packaging of the vector DNA and subsequent expression of the wild-type or modified MECP2 sequence in a transduced cell. The viral vector functions may be provided to the cell, for example, as a component of a plasmid or amplicon.

[0113] The viral vector functions may be present extrachromosomally in the cell line and / or may be integrated into the chromosomal DNA of the cells.

[0114] Any method of introducing the nucleotide sequence carrying the viral vector functions into a cellular host for replication and packaging may be used, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with a nuclear localization signal. In embodiments where the viral vector functions are provided by transfection with a viral vector, standard methods for generating a viral infection may be used.

[0115] Packaging Features The packaging functions include genes for replication and packaging of viral vectors. Thus, for example, the packaging functions may include functions necessary for viral gene expression, viral vector replication, rescue of viral vectors from an integrated state, viral gene expression, and packaging of viral vectors into viral particles, as needed. The packaging functions may be provided to the packaging cell together or separately using genetic constructs such as plasmids or amplicons, baculoviruses, or HSV helper constructs. The packaging functions may be present extrachromosomally in the packaging cell, but are preferably integrated into the chromosomal DNA of the cell. For example, the genes encoding AAV Rep and Cap proteins.

[0116] rAAV Production System Numerous cell culture-based systems are known in the art for the production of rAAV particles, 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, such as, for example, a human-derived cell line, e.g., HeLa, A549, or HEK293 cells, and its derivatives (HEK293T cells, HEK293F cells), a mammalian cell line, e.g., Vero, CHO cells or CHO-derived cells, or an insect derived cell line, e.g., SF-9 in the case of a baculovirus production system; (2) suitable helper virus functions, which can be provided by a wild-type or mutant adenovirus (e.g., a 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) suitable media and media components to support rAAV production.

[0117] Those of skill in the art will recognize numerous ways in which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela, Elb, E4, E2a, and VA genes), and rAAV genomes (including one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. The term "adenovirus helper functions" refers to numerous viral helper genes that are expressed (as RNA or protein) in a cell, such that the AAV propagates efficiently in the cell. Those of skill in the art will recognize that helper viruses, such as adenovirus and herpes simplex virus (HSV), facilitate AAV replication, and that certain genes have been identified that provide essential functions (e.g., the helpers induce changes in the cellular environment that facilitate such AAV gene expression and replication). 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, e.g., 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, hi some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes.

[0118] Any suitable medium known in the art may be used to produce rAAV particles. These media include, but are not limited to, media manufactured by Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM medium, and are described in U.S. Patent No. 6,723,551, which is incorporated by reference in its entirety. In some embodiments, the medium is Dynamis TM from Invitrogen / ThermoFisher. TM Media, FreeStyle TM 293 Expression medium, or Expi293 TM In some embodiments, the medium comprises Dynamis TM In some embodiments, the methods disclosed herein use cell cultures comprising serum-free media, animal-component free media, or chemically defined media. In some embodiments, the media is an animal-component free media. In some embodiments, the media comprises serum. In some embodiments, the media comprises fetal bovine serum. In some embodiments, the media is a glutamine-free media. In some embodiments, the media comprises glutamine. In some embodiments, the media is supplemented with one or more of nutrients, salts, buffering agents, and additives (e.g., antifoam agents). In some embodiments, the media is supplemented with glutamine. In some embodiments, the media is supplemented with serum. In some embodiments, the media is supplemented with fetal bovine serum. In some embodiments, the media is supplemented with a poloxamer, e.g., Kolliphor (登録商標)In some embodiments, the medium is a basal medium. In some embodiments, the medium is a feed medium.

[0119] rAAV production cultures may be routinely carried out under a variety of conditions (over a wide range of temperatures, varying 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 roller bottles, hollow fiber filters, multilayer or multi-tray tissue culture flasks (or stacks, e.g., hyperstacks), microcarriers, and packed-bed or fluidized-bed bioreactors. For rAAV vector production cultures, suspension-adapted host cells, such as HeLa cells, HEK293 cells, HEK293 derivatives (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO derivatives, 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, Also included are Per.C6 cells, chicken embryo cells, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, those cultures disclosed in U.S. Patents 6,995,006, 9,783,826, and U.S. Patent Publication No. 20120122155, each of which is incorporated herein by reference in its entirety.

[0120] Packaging Cells Any cell or cell line known in the art for producing rAAV particles may be used in any one of the methods disclosed in this application. In some embodiments, the methods disclosed herein for producing rAAV particles or increasing rAAV particle production include those for HeLa cells, HEK293 cells, HEK293 derivative cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO derivative 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, 3T3 cells, 3T4 cells, 3T5 cells, 3T6 cells, 3T7 cells, 3T8 cells, 3T9 cells, 3T8 cells, 3T9 cells, 3T1 cells, 3T2 cells, 3T3 cells, 3T4 cells, 3T5 cells, 3T6 cells, 3T7 ... 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 adapted to grow in suspension culture.

[0121] 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 for 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 shake flasks, spinner flasks, cell bags, or bioreactors.

[0122] 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.

[0123] In some embodiments, the cell cultures disclosed herein are adherent cultures. In some embodiments, the cell cultures disclosed herein are adherent cultures comprising HEK293. In some embodiments, the cell cultures disclosed herein comprise serum-free media, animal-component free media, or chemically defined media. In some embodiments, the cell cultures disclosed herein comprise serum-free media.

[0124] In some embodiments, the cell culture disclosed herein comprises a high-density cell culture. In some embodiments, the culture is at a total cell density between about 1x10E+06 cells / ml and about 30x10E+06 cells / ml. In some embodiments, greater than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293 derivative 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 growth in suspension culture.

[0125] The cell line for use as packaging cell includes insect cell line. In the context of the present invention, any insect cell that allows AAV replication and can be maintained in culture may be used. For example, Spodoptera frugiperda (Sf9 or Sf21 cell line), Drosophila spp. cell line, or mosquito cell line, such as Aedes albopictus derived cell line. A preferred cell line is Spodoptera frugiperda Sf9 cell line. The following references are incorporated by reference into this application for their teaching regarding the use of insect cells to express heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: 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., J. Virol. 219:37-44; al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Patent No. 6,204,059.

[0126] For example, viral capsids for use in the embodiments described herein may be produced using any method known in the art, such as by baculovirus expression (Brown et al., (1994) Virology 198:477-488). As a further alternative, viral vectors of the invention may be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and rAAV template, as described, for example, in Urabe et al., 2002, Human Gene Therapy 13:1935-1943.

[0127] In another aspect, a method for producing rAAV in insect cells is provided in the present application, in which a baculovirus packaging system or vector may be constructed to carry AAV Rep and Cap coding regions by engineering these genes into the polyhedrin coding region of a baculovirus vector, and then transfected into a host cell to produce a viral recombinant. In particular, when using baculovirus production for AAV, preferably the AAV DNA vector product is a self-complementary AAV-like molecule that does not use mutations to the AAV ITRs. This is likely 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 may be a baculovirus-infected cell, or may further have introduced therein nucleic acid encoding or contain baculovirus helper functions. These baculovirus viruses may express AAV components, which in turn promote the production of capsids.

[0128] During production, the packaging cells generally contain one or more viral vector functions, along with helper and packaging functions sufficient to effect replication and packaging of the viral vector. These various functions may be provided to the packaging cells, together or separately, using genetic constructs such as plasmids or amplicons, and they may be present extrachromosomally in the cell line or integrated into the chromosome of the cell.

[0129] The cells may be supplied with any one or more of the stated functions already integrated, for example, a cell line with one or more vector functions either extrachromosomally integrated or integrated into the cell's chromosomal DNA, a cell line with one or more packaging functions either extrachromosomally integrated or integrated into the cell's chromosomal DNA, or a cell line with helper functions either extrachromosomally integrated or integrated into the cell's chromosomal DNA.

[0130] rAAV purification The produced rAAV particles may be isolated using methods known in the art. In some embodiments, the method of isolating rAAV particles includes downstream processing, such as harvesting the cell culture, clarification of 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, hydroxylapatite chromatography, sterile filtration, or any combination thereof. In some embodiments, the downstream processing includes at least two, at least three, at least four, at least five, or at least six of the following: harvesting the cell culture, clarification of 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, hydroxylapatite chromatography, and sterile filtration. In some embodiments, downstream processing comprises harvesting the cell culture, clarification of 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 comprises clarification of the harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises 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 comprises sterile filtration. In some embodiments, downstream processing does not comprise centrifugation.

[0131] 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, a method of isolating rAAV particles comprises clarification of a 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, a method of isolating rAAV particles disclosed herein comprises clarification of a 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), tangential flow filtration, and a second sterile filtration. In some embodiments, a method for isolating rAAV particles includes 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, 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 methods do not include centrifugation. In some embodiments, the clarification of the harvested cell culture includes sterile filtration.

[0132] Recombinant AAV particles can be harvested from a rAAV production culture by harvesting the production culture containing the host cells, or by harvesting spent medium from the production culture, where the cells are cultured under conditions known in the art to cause release of rAAV particles from intact host cells into the medium. Recombinant AAV particles may also be harvested from a 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.

[0133] 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 entities, empty virus capsids, aggregated virus particles or capsids, misfolded virus capsids, and degraded virus particles.

[0134] 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 accomplished by a variety of other standard techniques known in the art, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 mm or greater known in the art. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the production culture harvest does not comprise centrifugation.

[0135] In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, clarification of the harvested cell culture comprises depth filtration. In some embodiments, 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 a sterile filtration medium. In some embodiments, the depth filter medium is a porous depth filter. In some embodiments, the filter train comprises a Clarisolve (登録商標) 20MS, Millistak +(登録商標) In some embodiments, the filter train comprises Clarisolve (登録商標) 20MS, Millistak +(登録商標) C0HC and Sartopore (登録商標)2 XLG 0.2 pm. In some embodiments, the harvested cell culture is pretreated before contacting it with a depth filter. In some embodiments, the pretreatment comprises adding salts to the harvested cell culture. In some embodiments, the pretreatment comprises adding a flocculent chemical to the harvested cell culture. In some embodiments, the harvested cell culture is not pretreated before contacting it with a depth filter.

[0136] In some embodiments, the clarified feed is concentrated by tangential flow filtration ("TFF") prior to application 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 technically manageable volumes of the clarified feed to be subjected to chromatography, allowing columns to be of a more reasonable size without the need for long recirculation times. In some embodiments, the clarified feed is concentrated between at least 2-fold and at least 10-fold. In some embodiments, the clarified feed is concentrated between at least 10-fold and at least 20-fold. In some embodiments, the clarified feed is concentrated between at least 20-fold and at least 50-fold. In some embodiments, the clarified feed is concentrated about 20-fold. One of skill in the art will also 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 the clarified feed via 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 between about 3 and about 10 diafiltration volumes of buffer. In some embodiments, the diafiltration involves the use of about 5 diafiltration volumes of buffer. One of skill in the art will also recognize that TFF can be used at any step in the purification process where it is desirable to exchange buffer before performing the next step in the purification process. In some embodiments, the methods for isolating rAAV from a clarified feed disclosed herein involve the use of TFF to exchange buffer.

[0137] Affinity chromatography may be used to isolate rAAV particles from the composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from the clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from the 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. TM , POROS TM CaptureSelect TM AAVX affinity resin, POROS TM CaptureSelect TM AAV9 affinity resin and POROS TM CaptureSelect TM In some embodiments, the affinity chromatography medium is a POROS affinity resin. TM CaptureSelect TM In some embodiments, the affinity chromatography medium is a POROS AAV9 affinity resin. TM CaptureSelect TM In some embodiments, the affinity chromatography medium is a POROS AAV8 affinity resin. TM CaptureSelect TM AAVX affinity resin.

[0138] Anion exchange chromatography may be used to isolate 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, Calif.), 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 medium 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 a monolithic anion exchange chromatography medium, such as that available from CIMmultus TM QA-l Advanced Composite Column (Quaternary Amine), CIMmultus TM DEAE-l Advanced Composite Column (diethylamino), CIM (登録商標) QA Disk (quaternary amine), CIM (登録商標) DEAE and CIM (登録商標) In some embodiments, the monolith anion exchange chromatography medium is selected from the group consisting of CIMmultus TM QA-1 Advanced Composite Column (Quaternary Amine). In some embodiments, the monolithic anion exchange chromatography medium is a CIM(登録商標) QADisk (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 (登録商標) QA-80 (column volume is 80 mL). One of skill in the art can appreciate that a wash buffer of suitable ionic strength can be identified such that the rAAV remains bound to the resin while impurities are removed, such as, but not limited to, impurities that may be introduced by upstream purification steps.

[0139] In further embodiments, the disclosure provides a composition comprising an isolated rAAV particle produced according to the methods disclosed herein, hi some embodiments, the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0140] As used herein, the term "pharmaceutical acceptable" refers to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, that is suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutical 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, when administering rAAV isolated according to the methods of the present disclosure 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, dispersions and suspending media, coatings, isotonicity agents, and absorption enhancing or retarding agents, that 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 pharma- ceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterials, antivirals, and antifungals) may also be incorporated into the composition. Pharmaceutical compositions can also be formulated to be compatible with a particular route of administration or delivery, as described in this application or known to one of ordinary skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0141] rAAV particles, as well as pharmaceutical compositions and delivery systems suitable for the methods and uses of the invention, are known in the art (see, 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) l2th 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) l lth ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al, Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, NY, (see pp. 253-315).

[0142] As used in this application, the described rAAV may be used as a gene therapy to treat MECP2 deficiency. The method of treatment includes administering the rAAV by injection to a subject in need thereof. A person skilled in the art will understand the amount required to treat the subject, as it depends on several factors such as the size, age, and sex of the subject.

[0143] Treatment methods 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.

[0144] In some embodiments, the effective amount is at least 1×10 per dose. 8 In some embodiments, the effective amount is at least 5×10 8 Viral genomes / dose, 7.5 x 10 8 Virus genomes / dose, at least 1 x 10 9 Virus genomes / dose, at least 2.5 x 10 9 Virus genomes / dose, at least 5 x 10 9 viral genome / dose.

[0145] In some embodiments, the effective amount is at least 1×10 11 At least 5 x 10 viral genomes / kg patient body weight 11 At least 1 x 10 viral genomes / kg 12 At least 5 x 10 viral genomes / kg 12 At least 1 x 10 viral genomes / kg 13 At least 1 x 10 viral genomes / kg 14 viral genomes / kg, or at least 5 x 10 14 In some embodiments, the rAAV is administered based on brain weight rather than body weight. In some embodiments, the rAAV dose is considered a low dose, which is particularly beneficial for CNS applications.

[0146] In some embodiments, the rAAV is administered intravenously. In some embodiments, the rAAV is administered intrathecally. In some embodiments, the rAAV is administered by intracerebral ventricular injection administration. In some embodiments, the rAAV is administered by intracisternal magna administration. In some embodiments, the rAAV is administered by intravitreal injection administration.

[0147] In various embodiments, a method of treating an MECP2-associated disorder (Rett Syndrome) in a subject is disclosed, wherein the method comprises administering to the subject an effective amount of any of the polynucleotide constructs, or vectors, or rAAVs comprising said vectors, or virions, described herein, or any pharmaceutical composition comprising any of these elements described herein.

[0148] In certain embodiments, as an additional modification to the therapeutic cassette, the gene of interest (GOI) was tested as a codon-optimized gene and as a wild-type gene. Several different codon-optimized MECP2 sequences were compared to the wild-type human MECP2 sequence (SEQ ID NO:7) and it was determined that expression of the codon-optimized MECP2 was not improved over the wild-type MECP2 sequence (SEQ ID NO:7). Therefore, the lead therapeutic cassette uses wild-type human MECP2 codons. EXAMPLES

[0149] Working Example Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and variation should, of course, be allowed for.

[0150] 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, all of which are explained in the literature.

[0151] Example 1 Optimization of MECP2 Expression Cassette Figure 1A shows a MeCP2 dosage-sensitive gene therapy cassette designed to reduce dosage sensitivity, prevent overexpression, and achieve therapeutic set-point transgene levels. Figure 1B shows a graph of flow cytometry data showing the effect of various modifications of the therapy cassette (feed-forward circuit) to modulate MeCP2 protein expression levels. A reporter construct in which the reporter mNeonGreen was fused to hMeCP2, and a second expression cassette in which mRuby was measured as a transfection control, were transfected into HEK cells, and 48 hours later, cells were treated and analyzed by flow cytometry to measure mRuby (transfection efficiency) and mNeonGreen (MeCP2) levels.

[0152] FIG. 2 is a schematic diagram showing examples of polynucleotide cassette elements that can be regulated to tailor dosage insensitivity and set point expression of MeCP2.

[0153] Example 2 Mouse model demonstrating efficacy of therapeutic MECP2 cassette

[0154] EffectivenessTo demonstrate in vivo efficacy, either regulated or unregulated MECP2 constructs were delivered to male Mecp2 knockout mice using AAV. EXACT-regulated MECP2 constructs packaged in AAV9 were highly efficient at packaging viral genomes. This severe mouse model shows shortened survival (median survival ~11 weeks) and marked respiratory and motor impairment. Mice administered 1e11 vg / mouse of the lead regulated MECP2 construct by neonatal injection (ICV) showed significantly improved survival (median survival extension = 14 weeks) and concomitant improvement in RTT-like clinical scores. In contrast, mice administered the unregulated construct did not show any improvement in survival, likely due to overexpression toxicity. A higher dose of 3x10 11 In vg / mouse, mice treated with the regulated lead construct had a marked improvement in survival (75% survived beyond 35 weeks) and a significantly improved RTT-like phenotype. At this higher dose, mice treated with the unregulated construct showed severe signs of MeCP2 overexpression and were euthanized at -3 weeks. These data demonstrate that the EXACT circuit can enable robust efficacy and significantly improved safety profiles for MECP2 gene therapy vectors. A separate toxicity study conducted by Labcorp demonstrated safety in non-human primates treated at therapeutically relevant doses. Finally, the safety of non-mammalian miRNA elements was also evaluated in human cell lines by RNAseq, which showed that expression of most predicted human gene targets was not altered when transfected with miRNA.

[0155] These results are shown in Figure 3A-C, which shows the modular polynucleotide sequence elements and design strategy for MeCP2 constructs (Figure 3A), along with MeCP2 expression data. Twenty-one to 23 days after administration of AAV9-RTT252, AAV9-RTT253, AAV9-RTT254 (also called NGN-401), AAV9-RTT269, AAV9-RTT270, AAV9-RTT271, or AAV9-RTT272 to wild-type mice, tissue samples were collected and analyzed by Western blot to determine the levels of MeCP2 expression in WT cortex (Figure 3B) and WT hippocampus (Figure 3C).

[0156] Figure 4A-C shows Mecp2 - / y In (KO) mice, 3 × 10 11 4A-4C are graphs comparing therapeutic AAV9-MECP2 constructs in survival (FIG. 4A), body weight (FIG. 4B), and RTT clinical score (FIG. 4C) after injection of therapeutic AAV9-MECP2 constructs in vg / mouse. The RTT clinical score is an observational scoring system used to determine the severity of the Rett phenotype in mice. Scoring ranges from 0 (similar to wild type) to 5 (most severe) for each individual component of the phenotype.

[0157] Figures 5A-C show the systematic adjustment of the various polynucleotide cassette components to identify and titrate expression levels for optimal efficacy (which are low or moderate expression levels). For AAV9-MECP2 constructs expressing the transgene MeCP2 at weak (Figure 5A), moderate (Figure 5B), or strong (Figure 5C) levels, 3 × 10 11 vg / mouse administered Mecp2 - / y For, survival plots, and RTT clinical scores, are shown.

[0158] Figures 6A-B show the improvement in survival rate (Figure 6A) and efficacy (RTT phenotypic score (Figure 6B)) for AAV9-RTT254-treated KO animals compared to vehicle-treated KO animals.

[0159] Figure 7A-F shows the results of two doses (1 × 10 11 vg and 3×10 11 vg) Graphical results showing improved motor and respiratory phenotypic domains in AAV9-RTT254-treated KO mice compared to controls.

[0160] Example 3: Dose selection in a hemizygous male mouse model: Survival and scoring data for RTT254 / NGN-401 up to 52 weeks after injection A hemizygous male mouse model of Rett syndrome (RTT) is characterized by complete knockout of Mecp2 (Mecp2 - / y ), and rapidly develop a robust and reproducible RTT-like phenotype, including respiratory failure, debilitating apneic events, spasticity, incoordination, and lameness. The mice typically only survive to 5-20 weeks of age, with a median survival of approximately 10 weeks.

[0161] male Mecp2 - / y An in vivo efficacy study was performed to examine NGN-401 in a mouse model. - / y Mice were administered NGN-401 or vehicle via intracerebroventricular (ICV) injection on postnatal day P0-2. NGN-401 was administered at a dose of 1.0 × 10 11 Or 3.0 x 10 11 Either dose level of total vg / mouse, which was selected based on earlier, proof-of-concept, in vivo studies, was administered, and the NGN-401-treated mice were then followed for survival and disease phenotype.

[0162] A total of 10-29 animals per group were included in the study to assess the improvement in survival and RTT phenotype. RTT phenotype was assessed using a scoring system developed at the University of Edinburgh. Animals treated with NGN-401 had a significant increase in survival (median survival was 100% compared to vehicle control, Mecp2 - / y From 9 weeks in mice, 1.0 x 10 11 and 3.0 x 10 11 vg / mouse doses extended to 23 and 37 weeks, respectively). Improvement in the RTT phenotype was also observed, with the RTT-like phenotype being reduced compared to vehicle-treated mice. Greater efficacy was observed with higher doses of NGN-401.

[0163] [Table 1]

[0164] All clinical evaluations were performed by a person blinded to both genotype and treatment. Animal caretakers performed daily cage side observations of each animal and recorded any abnormal findings. Body weights were recorded once a week from P28 onwards. Individual body weights were measured using a countertop scale. RTT phenotype was assessed weekly from P28 onwards using the RTT score, a non-invasive observational scoring system; a modified version of a scoring system developed by Dr. Jacky Guy at the University of Edinburgh (Guy et al., 2007 (DOI: 10.1126 / science.1138389)). For each of six parameters, namely mobility, gait, hindlimb grasp, tremor, respiration, and general condition, animals were scored 0-5 by a blinded investigator. A score of 0 indicates the phenotype of wild-type animals, and a score of 5 indicates the most severe phenotype. These scores are then combined to obtain a pooled RTT phenotype score. Detailed records were collected for each animal. Animals were sacrificed when they reached human endpoint criteria, for euthanasia, or at scheduled terminal sacrifice at 30 weeks. - / y Due to the extended survival in mice, these cohorts were extended to 52 weeks to fully evaluate survival and phenotypic improvements.

[0165] In-life assessment: Survival rate Mice treated with NGN-401 showed a significant increase in survival compared to vehicle-treated mice (Figure 15). Median survival was 1.1-fold higher than that of vehicle control Mecp2 mice. - / y From 9 weeks in mice, 1.0 × 10 11 vg / mouse for 23 weeks at a dose of 3.0 × 10 11vg / mouse dose, to 37 weeks (p < 0.0001, Mantel-Cox test). All mice in the vehicle-treated cohort were found to have died or reached human endpoints by week 20 of the study. In contrast, the longest surviving mice reached 52 weeks of age with both doses of NGN-401 treatment, at which point the study was terminated. Animal survival curves show that mice died or reached human endpoints for RTT-like-phenotype after ICV delivery of vehicle or NGN-401 at P0-2. Pups lost before weaning or mice sacrificed for reasons unrelated to the RTT phenotype were not included. Numbers for group sizes are indicated in figure legends. ****p < 0.0001 Mantel-Cox test.

[0166] In-life assessment: weight Body weights were recorded for each animal weekly from P28 onwards. - / y There was a marked difference in body weight between mice, with significant differences between groups from 8 to 13 weeks of age (mixed-effects model with Sidak's multiple comparison test (REML)) (Figure 16). - / y Compared to mice, 1.0 x 10 11 Or 3.0 x 10 11 Mecp2 treated with either dose of NGN-401 vg / mouse - / y Mice showed no significant difference in body weight (mixed-effects model with Dunnett's multiple comparison test (REML)). - / y Mice continued to gain weight throughout the study.

[0167] In-life body weights following ICV delivery of vehicle or NGN-401 at P0-2. Animal body weights were measured weekly starting at P28. Numbers for group size are indicated in the figure legend.

[0168] In-life assessment: RTT phenotype score

[0169] Male Mecp2 - / y The RTT-like phenotype progresses rapidly from 4 weeks of age when mice develop pronounced motor, autonomic, and respiratory disorders. Mice were assessed weekly from P28 onwards for the RTT-like phenotype using an observational scoring system. The aggregate RTT score reflects the sum of individual scores derived from all six parameters assessed (motility, gait, hindlimb grasp, tremor, respiration, and general condition).

[0170] Vehicle-treated WT and vehicle-treated Mecp2 - / y There were significant differences in the pooled RTT scores between mice throughout the study, with significant differences between groups from 5 weeks to 13 weeks of age (mixed-effects model with Sidak's multiple comparison test (REML)) (Figure 17). Similarly, vehicle-treated Mecp2 - / y Compared to mice, 1.0 x 10 11 Or 3.0 x 10 11 Mecp2 treated with either dose of NGN-401 vg / mouse - / y Mice showed robust improvements in phenotypic scores throughout the study, with significant differences observed between NGN-401- and vehicle-treated mice from 5 to 13 weeks of age (mixed-effects model with Dunnett's multiple comparison test (REML)). - / y There were insufficient mice remaining in the cohort beyond 13 weeks of age to allow statistical comparisons to be made.

[0171] Combined RTT phenotypic scores after ICV delivery of vehicle or NGN-401 on P0-2. Starting on P28, animals were scored weekly for each parameter from 0 (normal) to 5 (most severe). Scores were combined to obtain an aggregated RTT phenotypic score. Numbers for group size are indicated in the figure legend (Figure 17).

[0172] In-life assessment: individual RTT phenotypes

[0173] Animals treated with NGN-401 showed a marked improvement in phenotypic scores. As shown in Figure 7A-F, analysis of each of the six RTT parameters evaluated (motility, ambulation, hindlimb grip, tremor, respiration, and general condition) showed that ICV delivery of NGN-401 reduced all parameters, especially mobility, ambulation, and respiration. In vehicle-treated animals, clinical scores rapidly increased to the highest level (score = 5) for mobility, ambulation, and hindlimb grip parameters, and presented a very severe phenotype with respect to akinesia and grip in both hindlimb. This rapid increase in clinical scores was prevented by NGN-401 treatment. Similarly, in respiratory parameters, NGN-401 treatment reduced the frequency of visible apnea.

[0174] Example 4

[0175] Tolerability in heterozygous female mouse model: Survival rate and scores up to 25 weeks after injection

[0176] Female heterozygous Mecp2 + / - The mouse model exhibits a mild and variable phenotype, making it less than ideal as a robust validation model, however, the mosaicism of sex, genotype, and MeCP2 expression in these animals is more representative of female Rett syndrome (RTT) patients.

[0177] An in vivo study was conducted in female heterozygous mice to evaluate the tolerability of NGN-401, a MECP2 gene therapy construct whose expression levels are regulated by the EXACT miRNA circuit. NGN-401 was compared to AAV9-RTT251, an unregulated MECP2 vector that does not contain the EXACT miRNA regulatory circuit. The vector was produced using a baculovirus production system and delivered at 1.0×10 11 vg / mouse or 3.0 × 10 11 vg / mouse at P1 / P2, administered via intracerebroventricular (ICV) injection.

[0178] A total of 9-20 animals per group were injected and NGN-401 was assessed for tolerability using the MeCP2 overexpression toxicity scoring system developed at the University of Edinburgh. As shown in Figure 20, animals treated with NGN-401 showed no evidence of toxicity over the 26-week survival period of the study. In contrast, mice treated with the unregulated MECP2 vector (AAV9-RTT251) showed severe toxicity at approximately 3 weeks of age, resulting in death or humane euthanasia.

[0179] As shown in Figures 21-22, biodistribution and Western blot analysis of vector DNA demonstrated that mice treated with either NGN-401 or AAV9-RTT251 at comparable doses exhibited similar levels of vector genome copies. However, as shown in Figures 23A-C-24A-B, MeCP2 expression levels were dramatically altered. Mice treated with NGN-401 expressed vector-derived MeCP2 protein in a 3D mouse model, whereas mice treated with AAV9-RTT251 expressed MeCP2 protein in a 3D mouse model. + / - Although expressed at up to 120% of the endogenous protein levels in mice, vector-derived MeCP2 protein levels in mice treated with AAV9-RTT251 were significantly lower than Mecp2 + / - In conclusion, NGN-401 inhibits heterozygous Mecp2 expression in mice, closely mimicking the relevant patient population. + / - In mouse models, it is well tolerated and overcomes the severe toxicity observed with a comparable, unregulated vector.

[0180] [Table 2]

[0181] All clinical evaluations were performed by a person blinded to both genotype and treatment. An animal caretaker performed daily cage side observations for each animal and flagged any abnormal findings. Body weights were recorded weekly from P28 onwards for mice treated with NGN-401. Body weights were not obtained for mice treated with AAV9-RTT251 because they died or reached the human end point before P28. MeCP2 overexpression toxicity was assessed weekly from P28 onwards for mice treated with NGN-401 using a 6-point scoring system developed by Kamal Gadalla, University of Edinburgh. Mice treated with AAV9-RTT251 died or reached the human end point before scheduled toxicity scoring could begin. Mice that reached the human end point were scored for MeCP2 overexpression toxicity before sacrifice.

[0182] In-life assessment: Survival rate

[0183] In-life safety was monitored for 26 weeks. 11 vg / mouse or 3.0 × 10 11 WT mice and Mecp2 mice treated with either NGN-401 or vg / mouse + / - In mice, no natural mortality was observed and no animals had to be sacrificed due to reaching human endpoints. In contrast, 11 vg / mouse treated with AAV9-RTT251. + / - Mice were found to die or reach human endpoints by P19. 11 vg / mouse, more than half of the mice treated with AAV9-RTT251 were found to have died or reached human endpoints by P23 (at which point this arm of the study was discontinued for ethical reasons and the remaining mice were sacrificed). + / -One mouse was sacrificed due to reaching the human endpoint at approximately 10 weeks of age. + / - Mice survived to the end of the 26-week study (Figure 18).

[0184] In-life assessment: weight Body weights were recorded weekly for NGN-401 and vehicle treated mice from P28 onwards. A robust phenotypic effect was observed. Compared to vehicle treated WT animals, vehicle-treated Mecp2 + / - Body weight was increased (21.2 g in vehicle-treated Mecp2 mice compared to 21.2 g in vehicle-treated WT animals at week 26) + / - The average body weight of the mice was 29.1 g. In contrast, Mecp2 + / - Mice showed body weight correction to WT levels. Body weight was not measured for AAV9-RTT251 treated mice because they were found to die or reach human endpoints before phenotyping began (FIG. 19).

[0185] In-life assessment: MeCP2 overexpression toxicity score Toxicity phenotypes were monitored using a scoring system developed by Dr. Kamal Gadalla at the University of Edinburgh to classify adverse effects associated with MeCP2 overexpression. Severe toxicity was observed in both unmodified and AAV9-RTT251-treated groups. 11 In animals treated with the highest dose of 1.0×10 vg / mouse, all mice were found to have died or reached a maximum toxicity score of 6 by P19 and were sacrificed for humane reasons. 11For animals treated with 1000 mg / mouse, more than half of the mice were found to have died or reached a maximum toxicity score of 6 by P23. At this point, the AAV9-RTT251 arm of the study was discontinued for ethical reasons. In contrast, animals treated with a comparable dose of NGN-401 showed no observable in-life toxicity and maintained their mean toxicity scores around 0-26 weeks of age (Figure 20).

[0186] Vector biodistribution Using TaqMan qPCR assays targeting the WPRE3 component of the NGN-401 and AAV9-RTT251 vectors, vector DNA levels were determined across various regions. Biodistribution was measured in NGN-401-treated mice (Figure 21) at 26 weeks of age (the end of life period of the study). The presence of vector genomes was detected in a dose-dependent manner in the cortex, cerebellum, and liver in all NGN-401-treated animals, with levels highest in the cortex and lowest in the cerebellum. For vehicle-treated mice, there was an amplified background signal in individual samples, suggesting small levels of vector DNA that may have been introduced during tissue collection or nucleic acid extraction, but which were typically below the quantification limit of the assay.

[0187] Biodistribution was measured in AAV9-RTT251-treated mice at approximately 3 weeks of age (at which point mice had to be sacrificed as they had reached the human endpoint due to overexpression toxicity). The presence of vector genomes was detected in the cortex and liver in a dose-dependent manner in all AAV9-RTT251-treated animals (Figure 22). Vector genome levels in the cortex were broadly similar for equivalent doses of NGN-401 and AAV9-RTT251, indicating that the prevention of toxicity in NGN-401 is related to exact regulation of expression levels and not due to differences in vector biodistribution.

[0188] MeCP2 Expression: Western Blot Data Protein expression was measured across various regions using Western blot analysis using an anti-MeCP2 antibody. Because the anti-MeCP2 antibody recognizes both mouse and human forms of the protein, the protein levels measured were a combination of mouse endogenous MeCP2 protein and vector-derived human protein. Results showed that mice treated with either NGN-401 or AAV9-RTT251 exhibited similar levels of vector genome copies at comparable doses, but MeCP2 expression levels varied dramatically. In the cortex, which had the highest levels of vector biodistribution, mice treated with NGN-401 (Figure 23A) had over 1.0 × 10 11 and 3.0 x 10 11 vg / mouse, respectively, compared with vehicle-treated Mecp2 + / In the cerebellum, levels were lower, with no detectable vector-derived protein at the lower dose and Mecp2 at the higher doses than in vehicle-treated mice. + / 35% of the levels in WT mice (FIG. 23B). Importantly, even at the highest dose in fully transduced cortex, treatment with NGN-401 resulted in total MeCP2 protein levels that were only 70% above the levels in vehicle-treated WT mice, demonstrating that NGN-401 can maintain protein expression within physiological limits.

[0189] In contrast, mice treated with AAV9-RTT251 (Figure 24A) had a 1.0 x 10 11 and 3.0 x 10 11 vg / mouse, respectively, compared with vector-derived MeCP2 protein and vehicle-treated Mecp2 + / - The difference in the liver was less pronounced (Figure 24B), with mice treated with NGN-401 expressing Mecp2 at the highest dose, compared to 190% in mice treated with AAV9-RTT251.+ / - Mice expressed vector-derived MeCP2 protein at approximately 70% of endogenous MeCP2 levels, demonstrating that in the absence of the regulatory circuitry present in NGN-401, vector-derived MeCP2 protein levels were dramatically higher than normal physiological levels.

[0190] Example 5

[0191] Highest feasible dose in a heterozygous female mouse model: survival and scoring, as well as expression (Western) and histopathology, up to 8 weeks after injection.

[0192] Female heterozygous Mecp2 + / - Further in vivo studies were performed in mice to evaluate the tolerability of NGN-401, a MECP2 gene therapy construct whose expression levels are regulated by the EXACT miRNA circuit. 11 An extremely high dose of 1.0×10 vg / mouse was used ( FIG. 25 ), which is the maximum achievable dose based on vector titers and injection dose volume limitations. 11 and 3.0×10 11 Previous studies using lower doses of vg / mouse found no evidence of toxic MeCP2 overexpression effects.

[0193] The vector was produced using a baculovirus production system and administered via intracerebroventricular (ICV) injection on postnatal day 1 or 2 (P1 / P2). A total of 8-12 animals per group were used in the survival arm of the study for 8 weeks. The tolerability of NGN-401 was assessed using the MeCP2 toxicity scoring system (MeCP2 overexpression score) developed at the University of Edinburgh. In this study, animals treated with high doses of NGN-401 showed a very mild phenotype, exhibiting abnormal grasping of the hind limbs when the base of the tail was lifted. This represented a score of 1, the lowest achievable score on the overexpression toxicity score. The phenotype stabilized at 6 weeks and then reached a plateau.

[0194] Evaluation of vector DNA at 8 weeks of age showed widespread biodistribution across various CNS regions, with highest MeCP2 levels in the cortex and lowest in the cerebellum. Western blot analysis showed a similar pattern, with the highest MeCP2 levels observed in the cortex. Importantly, even in the highly transduced cortex, NGN-401-treated Mecp2 + / - Transgene-derived MeCP2 levels in mice were maintained within physiological levels and were similar to MeCP2 levels in untreated WT mice.

[0195] To identify any histopathological correlates of the mild hindlimb phenotype, eight mice per group were sacrificed at 8 weeks of age and tissues were evaluated by an expert veterinary neuropathologist. Results showed that NGN-401 did not induce adverse findings in the tissues evaluated.

[0196] In conclusion, even at the highest feasible vector doses that can be administered, Mecp2 + / - NGN-401 treatment in mice showed only a very mild, viable, hindlimb phenotype that was not associated with any histopathological changes. 11 This is in contrast to unregulated vectors, which have previously been shown to be highly toxic even at a dose of 1 vg / mouse (7.4-fold lower than that used for NGN-401 in this study), highlighting the significantly improved safety margin afforded by the EXACT regulated NGN-401 construct.

[0197] [Table 3]

[0198] All clinical assessments were performed by a person blinded to both genotype and treatment. Animal caretakers performed daily cage side observations for each animal and flagged any abnormal findings. Body weights were recorded once a week from P28 onwards. MeCP2 overexpression toxicity was assessed weekly from P28 onwards using a 6-point scoring system developed at the University of Edinburgh. RTT phenotype was assessed weekly from P28 onwards using the RTT score, a non-invasive observational scoring system modified from a previous scoring system developed at the University of Edinburgh (Guy et al., 2007). For each of six parameters, namely mobility, gait, hindlimb grasp, tremor, respiration, and general condition, animals were scored 0-5 by a blinded investigator. A score of 0 indicates the phenotype of wild-type animals, and a score of 5 indicates the most severe phenotype. These scores are then combined to obtain an aggregate RTT phenotype score.

[0199] In-life assessment: Survival rate In-life safety was monitored up to 8 weeks after injection (Figure 25). 11 vg / Mecp2 in mice treated with NGN-401 + / - Mice did not experience any natural deaths and no animals had to be sacrificed due to reaching human endpoints. + / - One mouse was sacrificed due to reaching the human endpoint at approximately 7 weeks of age and was found to have developed hydrocephalus at necropsy. For vehicle-treated WT mice, one mouse died at approximately 5 weeks of age due to severe hydrocephalus and one mouse reached the human endpoint at approximately 6 weeks of age.

[0200] In-life assessment: weight Body weights were recorded weekly for each animal from P28 onwards (Figure 26). + / -Body weight was slightly reduced in Mecp2 mice treated with NGN-401 compared to WT animals (16.8 g at week 8 compared to 16.8 g for vehicle-treated WT animals). + / - The average body weight of the mice was 15.2 g. In contrast, vehicle-treated Mecp2 + / - The mice had a slightly higher body weight of 17.4 g. These results suggest that treatment with NGN-401 results in a very slight loss of body weight.

[0201] In-life assessment: MeCP2 overexpression score To classify adverse effects associated with MeCP2 overexpression, the toxicity phenotype was monitored using a toxicity scoring system developed at the University of Edinburgh. From week 5 onwards, the toxicity phenotype was 7.4 × 10 11 Mecp2 treated with NGN-401 at a dose of vg / mouse + / - A very mild phenotype was detected in the majority of mice (Figure 27). This manifested as an abnormal posture of the hind limbs when the mice were suspended by the base of the tail, and met the criteria for a minimum score of 1 in the MeCP2 overexpression score. Apart from this specific phenotype, the mice otherwise appeared healthy and normal. This phenotype was not observed in vehicle-treated, WT or Mecp2 + / - To identify any histopathology correlating with this mild hindlimb phenotype, a cohort of mice was sacrificed at 8 weeks and an extensive set of tissues was collected for evaluation by a veterinary pathologist.

[0202] Live Evaluation: RTT Score Compared to the more severe male mouse model, Mecp2 + / Female mice display subtle and highly variable phenotypes. + / - In mice, the RTT phenotype was negligible, with a score of approximately 2.5 out of 30 at 8 weeks of age. At the same age, Mecp2 mice treated with NGN-401 + / -Mice showed a mild phenotype and received a score of 5 (Figure 28). The increase in RTT score in NGN-401-treated mice is due to the presence of a mild hindlimb phenotype. The hindlimb grasping phenotype was captured by both the RTT score and the MeCP2 overexpression score, which are usually observed in Mecp2-treated mice at 5-6 months of age. + / - Observed in mice, but may also occur due to overexpression toxicity. The phenotype is detectable but mild. Vector biodistribution Using a qPCR assay targeting the WPRE3 component of the NGN-401 vector, levels of vector DNA were determined across various regions. Biodistribution was measured in the necropsy arm of the study at 8 weeks of age in NGN-401-treated mice (Figure 29). The presence of vector genomes was detected at various levels in the cortex, cerebellum, thoracic spinal cord, and liver in all NGN-401-treated animals, with levels highest in the cortex (5.4 copies of NGN-401 per diploid genome) and lowest in the cerebellum (0.14 copies of NGN-401 per diploid genome). In Figure 29, NGN-401 is 7.4 x 10 11 Biodistribution of vector DNA in the cortex, cerebellum, thoracic spinal cord, and liver at 8 weeks after ICV delivery at a dose of 10 ... MeCP2 Expression: Western Blot Protein expression was measured across various regions using Western blot analysis with an anti-MeCP2 antibody. Because the anti-MeCP2 antibody recognizes both mouse and human forms of the protein, the protein levels measured were a combination of mouse endogenous MeCP2 protein and vector-derived human protein. In the cortex, where vector biodistribution is highest (Figure 30), mice treated with NGN-401 expressed more vector-derived MeCP2 protein than vehicle-treated Mecp2. + / - In the cerebellum, levels were lower and vector-derived MeCP2 protein was expressed at 200% of the levels in vehicle-treated Mecp2 cells. + / - The levels of MeCP2 in the WT mice were only 16% of those in the control mice. Importantly, even at this very high dose in fully transduced cortex, treatment with NGN-401 resulted in total MeCP2 protein levels that were only about 10% above those in vehicle-treated WT mice. This demonstrates that even after treatment with very high doses of NGN-401, NGN-401 can maintain protein expression within physiological limits. histopathology Histopathological evaluation of tissues taken at the 2-month interim sacrifice was performed by an expert neuropathologist. NGN-401 did not produce any adverse findings in the brain (site of injection administration) or any of the other organs evaluated: autonomic ganglia (variously located lateral and / or ventral to the vertebrae), bones with bone marrow (vertebrae), brain (one hemisphere), forebrain (major regions: cerebral cortex [frontal, parietal, temporal, occipital], striatum, hippocampus, hypothalamus, thalamus), midbrain, hindbrain (major regions: cerebellum, pons, or rarely medulla oblongata), dorsal root ganglia (DRG, cervical and lumbar spinal cord segments - with spinal nerve roots [mainly caudal lumbar segments]), heart (one longitudinal section through the ventricles), large intestine - colon section (located within the lumbar spinal cord section), small intestine - jejunum (or rarely duodenum) cross sections, kidney (one cross section through the hilus), liver, lungs, nerves (multiple cross sections of the caudal [tail], sciatic, and tibial shafts, and one cross section of the sciatic nerve), ovaries, fallopian tubes, spinal cord (cross sections of the cervical, thoracic, lumbar [variable], and sacral regions), skeletal muscles (gastrocnemius, quadriceps, various caudal muscles), spleen (one longitudinal section), uterus (one longitudinal section).

[0203] Example 6: Safety in NHPs (Labcorp); Safety and Regulated vs. Unregulated Expression Data Safety and biodistribution studies were initiated in cynomolgus monkeys to compare the safety, vector biodistribution, and MECP2 expression levels following delivery of a regulated NGN-401 construct (equivalent to RTT254) to an otherwise equivalent unregulated MECP2 construct (AAV9-RTT251) in a large animal model.

[0204] NGN-401 or AAV9-RTT251 were administered by ICV injection in two doses separated by a half-logarithm (Table 4) as determined using a qualified ddPCR titer assay targeting the human MECP2 transgene. The NGN-401 and AAV9-RTT251 vectors used in this study were produced using Virovek's baculovirus system. Doses were determined by the Mecp2 - / yThe doses were chosen to be within the range of NGN-401 dosages shown to be effective in efficacy studies. The study was designed to clarify any potential NGN-401-related toxicities (such as potential toxicity due to overexpression of MeCP2) by evaluating higher doses than previously tested in NHPs, including comparison with the unregulated vector AAV9-RTT251. The AAV9-RTT251-treated group was sacrificed one month after vector administration to avoid potential severe toxicity, given the known toxicity observed in previous mouse studies. Animals received daily oral prednisolone (1 mg / kg) starting 2 weeks prior to vector administration and continued throughout the duration of the study. [Table 4]

[0205] The study included evaluation of clinical and neurobehavioral observations, as well as anatomic pathology. All animals survived until scheduled sacrifice, and there were no test article-related clinical or neurobehavioral observations, changes in body weight or food consumption, or gross findings with NGN-401 or AAV9-RTT251.

[0206] Clinical pathology effects for both NGN-401 and AAV9-RTT251 consisted of minimal or mild increases in platelets, white blood cells, absolute neutrophil and monocyte counts, fibrinogen concentration, and alanine aminotransferase (ALT) activity. Increases in platelet and white blood cell counts, as well as increases in fibrinogen concentration, suggested an inflammatory response. For animals harvested on day 92, values ​​generally declined toward baseline by this time point. In the AAV9 study, a transient increase in alanine aminotransferase activity was reported, without correlating changes in liver weight or microscopic findings in the liver. An asymptomatic increase in ALT is an expected effect of AAV gene therapy. Transient increases in ALT activity have been reported in AAV studies involving intravenous (IV) and intrathecal (IT) routes of administration, with greater exposure to the liver that was well managed with steroid treatment.

[0207] Test article-related microscopic findings for both vectors were noted in the cervical, thoracic, and / or lumbar dorsal root ganglia (mononuclear cell infiltration, neuronal loss, and / or axonal degeneration) and / or spinal cord (axonal degeneration in the nerve roots and / or white matter). These findings were not dose-related in incidence or severity, were similar at interim and terminal sacrifice, and were not considered adverse. This is consistent with the current understanding of the safety profile of AAV gene therapy. Histopathological findings in the dorsal root ganglia (DRG) are an expected effect of AAV gene therapy, since clinically asymptomatic findings have been reported in AAV studies in NHPs. Currently available data suggest that in non-clinical studies with NHPs, DRG pathology was nearly universal after AAV gene therapy and did not result in clinical signs after treatment with therapeutic transgene doses.

[0208] Nerve conduction measurements were performed in the superior (radial and median) and inferior (sural, peroneal, and saphenous) sensory nerves at weeks 3 / 4 and 13 of the study. A decrease in nerve conduction velocity (NCV) of ≥3 m / s from baseline was considered an NCV slowing. A summary of the incidence of sensory NCV slowing in each treatment group is provided in Table 5.

[0209] [Table 5]

[0210] No loss of sensory function in the radial or median nerve was observed at any time point. At week 3 / 4, two animals receiving low dose AAV9-RTT251 exhibited no response in the sural nerve, and one animal exhibited slowing of NCV, which was considered severe and had an increased incidence compared to the other groups. All other findings indicate that the effects on each nerve function were mild or moderate, with low incidence, and each nerve is expected to remain within physiological functional parameters. Of note, one animal in the low dose AAV9-RTT251 group exhibited a slight prolongation of FM wave latency difference during week 3 / 4, potentially indicating slowing of motor conduction in the proximal portion of the tibial nerve.

[0211] Overall, the sural NCV data highlight the difference in the safety profile of NGN-401 when compared to the otherwise comparable MECP2 vector, which does not include the EXACT auto-regulatory technology, as 2 of 9 animals treated with NGN-401 exhibited sural NCV slowing, whereas 5 of 6 animals treated with AAV9-RTT251 exhibited sural NCV slowing.

[0212] One month after ICV administration of NGN-401 and AAV9-RTT251, selected tissues (brain, liver, spinal cord) were harvested to assess mRNA expression. Transgene expression was assessed using qRT-PCR analysis targeting the WPRE3 element in the 3' UTR of MECP2 mRNA. The impact of EXACT technology on NGN-401 is evident when mRNA levels produced by AAV9-RTT251 are directly compared to those produced by NGN-401. Transgene mRNA levels (copies per μg of RNA) for each dose group were normalized to the NGN-401 low dose group in each region evaluated (Figure 31). In most regions, AAV9-RTT251 produced mRNA levels several-fold higher than the levels in the corresponding groups receiving equivalent doses of NGN-401, and also exhibited greater variability between animals. This data provides evidence that the EXACT technology is capable of modulating expression levels in CNS tissues in a large animal model.

[0213] These data indicate that some optimized therapeutic polynucleotide cassettes are good candidates for effective gene therapy of Rett syndrome and circumvent safety / toxicity concerns.

[0214] Clinical Design A clinical dose was designed and proposed for clinical trials, where the subjects received 1.0×10 15 vg dose, which is 1.0 × 10 14 Delivered via ICV injection of 10 mL at 100 mg / mL.

[0215] Based on the average female brain weight, 1E15vg is 8.3 x 10 11 vg / g brain, but in women with Rett syndrome, the actual dose per gram of brain weight may be higher due to the smaller brain size. In some patients, it is approximately 8.3 × 10 11vg / g brain is reached. Consistent with GLP and the safety and toxicity studies in NHPs provided above in this application, dosed subjects are expected to be substantially free of MECP2 overexpression toxicity. Furthermore, the endpoints map to preclinical data of improvements in various domains in animal models, as shown below.

[0216] [Table 6] [Table 7-1] [Table 7-2] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

change

change

change

change

change

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[0217] Equivalents and Incorporation by Reference All references cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated. This statement of incorporation by reference is intended by the applicant to refer to each and every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is clearly identified as complying with 37 CFR § 1.57(b)(2), even if such citation is not immediately adjacent to the dedicated incorporation by reference statement, in accordance with 37 CFR § 1.57(b)(1). This dedicated incorporation by reference statement in this specification in no way weakens this general statement of incorporation by reference. The citation of any reference in this application is not intended as an admission that the reference is pertinent prior art, nor as an admission as to the contents or date of such publications or documents.

[0218] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, those skilled in the art will recognize that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

Claims

1. a polynucleotide comprising, from 5' to 3': - a promoter, comprising a CBM promoter comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 21, or a CBE promoter comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 22; - at least one non-mammalian or synthetic miRNA expressed within the intron; - Protein translation start site (Kozak sequence); a human MECP2 coding sequence comprising at least 90% identity to, optionally comprising, SEQ ID NO: 7, or a codon-optimized human MECP2 coding sequence; at least one 3' stabilizing element; at least three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA; optionally, the miRNA binding sites contain one to six mismatches, optionally a single mismatch; and - polyadenylation signal, or - a promoter, comprising a CBM promoter comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 21, or a CBE promoter comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 22; - at least one non-mammalian or synthetic miRNA expressed within the intron; - Protein translation start site (Kozak sequence); a human MECP2 coding sequence comprising at least 90% identity to, optionally comprising, SEQ ID NO: 7, or a codon-optimized human MECP2 coding sequence; at least three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA; optionally, the miRNA binding sites contain one to six mismatches, optionally a single mismatch; at least one 3' stabilizing element; and - Polyadenylation signal.

2. 10. The polynucleotide of claim 1, wherein the polynucleotide comprises a non-mammalian miRNA or a synthetic miRNA that is expressed within an intron.

3. The polynucleotide of claim 1, wherein the non-mammalian miRNA or synthetic miRNA comprises SEQ ID NO:

4.

4. The polynucleotide of claim 1, wherein the human MECP2 coding sequence comprises SEQ ID NO:

7.

5. 2. The polynucleotide of claim 1, wherein the human MECP2 coding sequence is a codon-optimized human MECP2 sequence.

6. 2. The polynucleotide of claim 1, wherein the protein translation initiation site is a Kozak sequence comprising SEQ ID NO:

13.

7. 2. The polynucleotide of claim 1, wherein the promoter comprises a CBM or CBE comprising SEQ ID NO: 21 or 22, respectively.

8. 8. The polynucleotide of claim 7, wherein the promoter comprises a CBM comprising SEQ ID NO:

21.

9. 2. The polynucleotide of claim 1, wherein the at least one 3' stabilizing element is a WPRE.

10. 2. The polynucleotide of claim 1, wherein the polynucleotide comprises three miRNA binding sites for a non-mammalian miRNA or for a synthetic miRNA.

11. 2. The polynucleotide of claim 1, wherein the miRNA binding site comprises SEQ ID NO:

8.

12. The polynucleotide of claim 1, wherein the miRNA binding site contains one mismatch.

13. 2. The polynucleotide of claim 1, wherein the polyadenylation signal is the simian vacuolating virus 40 polyadenylation signal (SV40pA).

14. The polynucleotide of claim 13, wherein the SV40pA signal comprises the nucleotide sequence of SEQ ID NO:

12.

15. 2. The polynucleotide of claim 1, wherein the polynucleotide comprises: a CBM promoter, one non-mammalian miRNA or synthetic miRNA expressed within an intron, a wild-type human MECP2 coding sequence with an optimized Kozak sequence, three miRNA binding sites for the non-mammalian miRNA or synthetic miRNA, a WPRE stabilization element, and an SV40pA signal.

16. 2. The polynucleotide of claim 1, wherein the polynucleotide comprises: a CBM promoter, one non-mammalian miRNA or synthetic miRNA expressed within an intron, a codon-optimized human MECP2 coding sequence with an optimized Kozak sequence, three miRNA binding sites for the non-mammalian miRNA or synthetic miRNA, a WPRE stabilization element, and an SV40pA signal.

17. 16. The polynucleotide of claim 15, wherein the polynucleotide comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

25.

18. 18. The polynucleotide of claim 17, wherein the polynucleotide comprises SEQ ID NO:

25.

19. 2. The polynucleotide of claim 1, wherein the polynucleotide further comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences.

20. 20. A vector comprising the polynucleotide of any one of claims 1 to 19, optionally wherein the vector is a viral vector, further optionally wherein the vector is an adeno-associated virus (AAV) vector, even more optionally wherein the AAV vector is an AAV9 vector.

21. A recombinant adeno-associated virus (rAAV) comprising a polynucleotide described in any one of claims 1 to 19, optionally wherein the rAAV is AAV9; or a virion comprising the rAAV; or a transformed cell comprising the virion or the rAAV, or comprising the polynucleotide described in any one of claims 1 to 19.

22. 20. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 19, and optionally a pharmaceutically acceptable carrier.

23. 20. A pharmaceutical composition comprising an effective amount of a polynucleotide according to any one of claims 1 to 19 for use as a medicament.

24. 20. A pharmaceutical composition comprising an effective amount of the polynucleotide of any one of claims 1 to 19 for use in treating Rett syndrome in a subject, optionally wherein the subject exhibits improvement in one or more symptoms associated with Rett syndrome and / or wherein the subject is substantially free of MECP2 overexpression toxicity.

25. 25. The polynucleotide for use according to claim 24, wherein the target is 1.0 x 10 15 SEQ ID NO: 25 containing vg was added at 1.0 x 10 14 administered by ICV injection of 10 mL at 8.3 x 10 vg / mL, optionally wherein the effective dose is 8.3 x 10 11 vg / g brain.

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