Adeno-associated virus vectors for the treatment of rett syndrome

Abstract

To provide nucleic acids (including AAV expression cassettes), AAV vectors, and compositions for use in methods of treating and / or delaying the onset of diseases associated with mutations in mecp2 genes, such as Rett Syndrome.SOLUTION: Also provided herein are methods for treating a brain-derived neurotrophic factor (BDNF) - associated disease and / or delaying the onset of a brain-derived neurotrophic factor (BDNF) - associated disease. The present disclosure provides a nucleic acid comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette comprises, in a 5' to 3' direction, a 5' inverted terminal repeat (ITR), a synthetic activity-dependent promoter, a Rett syndrome-associated gene, and a 3' ITR.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 067,668, filed August 19, 2020. No. 60 / 699,999, filed on Oct. 1, 2003, the contents of which are incorporated herein by reference in their entirety. can be.

[0002] The present disclosure relates to the fields of molecular biology and gene therapy. More specifically, the present disclosure relates to: The present invention relates to compositions and methods for producing recombinant viral vectors.

[0003] Incorporating a sequence listing The contents of the text files submitted electronically herein are incorporated herein by reference in their entirety. The sequence listing (STRD_020_01WO_SeqList_ST25.t xt, Recorded on: August 19, 2021, File size: Approximately 89,555 bytes) Pewter readable copy. [Background technology]

[0004] Rett syndrome is a genetic neurological disorder that primarily affects girls and women between the ages of approximately 6 and 18 months. Affecting approximately 1 in 8,500 women, Rett syndrome is a rare disease. Patients with Stroke syndrome suffer from speech impairment, poor coordination, microcephaly, repetitive movements, seizures, scoliosis, and Women with Rett syndrome often have a wide range of symptoms, including cognitive impairment and cognitive impairment. While most women with Rett syndrome die in their mid-teens, males with Rett syndrome often die in infancy.

[0005] Rett syndrome is caused by mutations in the mecp2 gene and results in defects in neuronal function. Many cases of Rett syndrome result from de novo mutations in the mecp2 gene. However, in a minority of cases, mecp2 mutations show an X-linked dominant inheritance pattern. Mutations in mecp2 are also associated with other disorders, such as MECP2 duplication syndrome and PPM-X syndrome. It can also cause dementia and may be associated with autism spectrum disorders.

[0006] There is no known cure for Rett syndrome, or any other MECP2-related disorder. Symptoms of Stroke syndrome are typically managed with anticonvulsant medications, special education, physical therapy, and braces. Gene therapy may be a useful method for treating Rett syndrome. However, gene therapy targeting the nervous system may provide the correct treatment to alleviate symptoms upon onset. Identification of neural target genes, promotion of high levels of expression of these genes in a regulated manner, and Several techniques, including delivery of gene therapy constructs in a neuron-targeted manner, have been developed. Therefore, the development of Rett syndrome and other MECP2-related There is an unmet need for compositions and methods for treating disease. Summary of the Invention

[0007] The present disclosure provides nucleic acids comprising adeno-associated virus (AAV) expression cassettes, The current cassette contains, in the 5' to 3' direction, a 5' inverted repeat (ITR), a synthetic activity-dependent promoter sequence, and a nucleotide sequence. In some embodiments, the promoter, the Rett syndrome-associated gene, and the 3' ITR. The promoter drives expression of a Rett syndrome-associated gene. The promoter is an MECP2-independent promoter. comprises a nucleic acid sequence derived from a promoter of a neuronal immediate early gene. In the mitotic state, neuronal immediate early genes are Arc, c-fos, and egr. In some embodiments, the promoter is selected from the group consisting of the A -1 gene. Contains the rc gene promoter (ArcMin).

[0008] In some embodiments, ArcMin is human ArcMin (hArcMin). In some embodiments, hArcMin is the nucleic acid sequence of SEQ ID NO: 12, or In some embodiments, the promoter comprises a sequence that is at least 90% identical to the sequence of AMP-like response element (CRE), serum response element (SRE), synaptic activity response element (SARE), MEF2 response element, or a combination thereof. In this embodiment, the promoter comprises a synaptic activity response element (SARE). In some embodiments, the synaptic activity response element (SARE) is a human synaptic activity response element. In some embodiments, the hSARE is a nucleotide sequence of SEQ ID NO: 11. The nucleic acid sequence may be at least 90% identical to the sequence of SEQ ID NO: 11. In one embodiment, the promoter is human ArcMin (hArcMin) and at least one In some embodiments, the promoter comprises hArcMin and one hSARE. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 6, or or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 6. The promoter comprises hArcMin and five hSAREs. The promoter may comprise the nucleic acid sequence of SEQ ID NO: 16 or a nucleic acid sequence of SEQ ID NO: 16 and at least Contains 90% identical sequences.

[0009] In some embodiments, the promoter binds to a neuronal activity-dependent transcription factor. In some embodiments, the neuronal activity-dependent transcription factor is a cAMP response element binding Protein (CREB), myocyte enhancer factor 2 (MEF2), serum response factor (SR F), or Elk-1. In some embodiments, the Rett syndrome associated gene is a brain-derived Brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF1), methyl-CpG binding protein 2 (MECP2), huntingtin protein, huntingtin-associated protein 1 , orthodenticle homeobox 2 (OTX-2), FXYD domain-containing ion transporter transporter regulator 1 (FXYD1), neurexin-2-alpha (NRXN2), or In some embodiments, the gene encoding the protein kinase C gamma (PRKCG) is a gene encoding ... The syndrome-associated gene encodes brain-derived neurotrophic factor (BDNF). In some embodiments, the BDNF is human BDNF. In some embodiments, the BDNF is the nuclear BDNF of SEQ ID NO: 7. or encoded by a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO:7 .

[0010] In some embodiments, at least one of the 5' ITR and the 3' ITR has a length of about 110 to about In some embodiments, the 5' ITR is the same length as the 3' ITR. In some embodiments, the 5' ITR and the 3' ITR have different lengths. In some embodiments, at least one of the 5' ITR and 3' ITR is selected from the group consisting of AAV1, AAV 2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33 isolated from the genome of AAVrh74, avian AAV or bovine AAV, or In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO: 1. In one embodiment, the 3' ITR comprises the sequence of SEQ ID NO:2.

[0011] In some embodiments, the AAV cassette comprises a brain-derived neurotrophic factor (BDNF) short 3 In some embodiments, the BDNF short 3'UTR is a BDNF short 3'UTR. The BDNF long 3'UTR is a 3'UTR homolog of the Rett syndrome-associated gene. In some embodiments, the AAV cassette is located between the BDNF short 3'U and the BDNF short 3'U. In some embodiments, the BDNF short 3'UTR comprises the nucleic acid sequence of SEQ ID NO: 8. or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the AAV cassette comprises the BDNF long 3'UTR. The NF long 3'UTR has the nucleic acid sequence of SEQ ID NO: 10 or a sequence at least equal to the nucleic acid sequence of SEQ ID NO: 10. contain sequences that are at least 90% identical.

[0012] In some embodiments, the AAV cassette comprises a polyadenylation signal. In the form, polyadenylation signals are found in the following genes: simian virus 40 (SV40), rBG, α-globin, β-globin, human collagen, human growth hormone (hGH), Polyomavirus, human growth hormone (hGH) or bovine growth hormone (bGH) One or more polyadenylation signals may be isolated from or provided by the gene. In some embodiments, the AAV cassette comprises a bGH polyadenylation signal. In embodiments, the bGH polyadenylation signal is the nucleic acid sequence of SEQ ID NO: 9, or No. 9.

[0013] In some embodiments, the AAV cassette comprises at least one stuffer sequence. In some embodiments, at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 13, or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 13. The AAV expression cassette contains a Kozak sequence, which is a sequence encoding a Rett syndrome-associated gene. In some embodiments, the Kozak sequence overlaps the start codon of the gene. a nucleic acid sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 14, or a sequence 15, or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 3, or the sequence A sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3, the sequence of SEQ ID NO: 4 or SEQ ID NO: 4 a sequence at least 90% identical to the sequence of SEQ ID NO: 5 or the sequence of SEQ ID NO: 5 It contains a sequence that is at least 90% identical to the sequence.

[0014] The present disclosure provides a plasmid comprising any one of the nucleic acids disclosed herein. The disclosure also provides a method for the preparation of any one of the nucleic acids disclosed herein or any one of the primers disclosed herein. The present disclosure also provides a method for producing a recombinant AAV vector, comprising the steps of: Further provided is a method for transfecting AAV producing cells with any of the nucleic acids disclosed herein. or any one of the plasmids disclosed herein. The present disclosure also provides any of the methods for producing a recombinant AAV vector disclosed herein. In some embodiments, the vector -AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AA V8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10 , AAVrh32.33, AAVrh74, avian AAV, and bovine AAV. In some embodiments, the recombinant AAV vector is a single-stranded AAV ( In some embodiments, the recombinant AAV vector is a self-complementary AAV (ssAAV). In some embodiments, the AAV vector is AAV1, AAV2, or V(scAAV). , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV1 0, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, Contains capsid proteins of AAVrh74, avian AAV, or bovine AAV. In terms of morphology, the AAV vector may have one or more capsid proteins, as compared to wild-type AAV capsid proteins. It includes a capsid protein with multiple substitutions or mutations.

[0015] The present disclosure provides a method for the preparation of a nucleic acid molecule comprising: (a) any one of the nucleic acids disclosed herein; any one of the plasmids disclosed herein, any one of the cells disclosed herein, or any one of the disclosed recombinant AAV vectors; and (b) a pharmaceutically acceptable carrier. The present disclosure provides a composition comprising: A method for expressing a gene is provided, comprising administering to a subject a therapeutically effective amount of a nucleic acid disclosed herein. Any one of the plasmids disclosed herein, any one of the plasmids disclosed herein Any one of the cells or any one of the recombinant AAV vectors disclosed herein or any one of the compositions disclosed herein. In embodiments, the subject has Rett Syndrome.

[0016] The present disclosure provides a method for administering to a subject a therapeutically effective amount of any one of the nucleic acids disclosed herein, any one of the plasmids disclosed herein; any one of the cells disclosed herein; or any one of the recombinant AAV vectors disclosed herein, or A method for treating Rett syndrome in a subject, comprising administering any one of the disclosed compositions. The present disclosure provides methods for treating or delaying the onset of Rett Syndrome, comprising administering to a subject: A therapeutically effective amount of any one of the nucleic acids disclosed herein, any one of the mides, any one of the cells disclosed herein, or any one of the cells disclosed herein any one of the recombinant AAV vectors described herein, or any one of the compositions disclosed herein. 2. The method of claim 1, wherein the patient is a subject in need thereof, and the patient is a patient in need thereof. The present invention provides a method for expressing BDNF.

[0017] In some embodiments, the subject has a cognitive disorder or a stress-related disorder. In embodiments, the subject is diagnosed with depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, and Dementia, anorexia nervosa and bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder have diabetes, obesity, Rett syndrome, or post-chemotherapy cognitive impairment.

[0018] The present disclosure provides a method for administering to a subject a therapeutically effective amount of any one of the nucleic acids disclosed herein, any one of the plasmids disclosed herein; any one of the cells disclosed herein; or any one of the recombinant AAV vectors disclosed herein, or BDNF-related disorders in a subject, comprising administering any one of the disclosed compositions. The present invention provides a method for treating or delaying the onset of BDNF-related disorders. In one embodiment, the BDNF-related disorder is a cognitive disorder and / or a stress-related disorder. In some embodiments, the BDNF-related disorder is depression, obsessive-compulsive disorder, Alzheimer's disease, Menton's disease, and dementia, anorexia nervosa, bulimia nervosa, schizophrenia, epilepsy, trauma Post-chemotherapy stress disorder, bipolar disorder, Rett syndrome, major depressive disorder, or post-chemotherapy cognitive impairment It is harmful.

[0019] The present disclosure provides a method for administering to a subject a therapeutically effective amount of any one of the nucleic acids disclosed herein, any one of the plasmids disclosed herein; any one of the cells disclosed herein; or any one of the recombinant AAV vectors disclosed herein, or A method for treating an MECP2-associated disease in a subject, comprising administering any one of the disclosed compositions. The present invention provides methods for treating or delaying the onset of MECP2-associated diseases. In embodiments, the MECP2-associated disease is MECP2 duplication syndrome, MECP2-associated severe neoplasia, In some embodiments, the subject has: In some embodiments, the nucleic acid, plasmid, cell, recombinant AAV vector, or Alternatively, the composition is administered by injection into the central nervous system. In some embodiments, the rhesus malabsorption syndrome-associated gene is expressed in neurons of the subject. A neuron is an active neuron.

[0020] These and other embodiments are addressed in more detail in the detailed description set forth below. [Brief explanation of the drawings]

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with any drawings are available upon request and payment of the necessary fee. The Patent Office will provide such information as necessary.

[0022] [Figure 1] Figure 1 shows an AAV expression cassette containing the hSARE-hArcMin promoter (SEQ ID NO: 6), the BDNF gene (SEQ ID NO: 7), the BDNF short 3'UTR (SEQ ID NO: 8) and a stuffer sequence (SEQ ID NO: 13), all of which are flanked by the 5'ITR (SEQ ID NO: 1) and 3'ITR (SEQ ID NO: 2).

[0023] [Figure 2] Figure 2 shows an AAV expression cassette containing the hSARE-hArcMin promoter (SEQ ID NO: 6), BDNF gene (SEQ ID NO: 7), bGH polyA signal (SEQ ID NO: 9) and stuffer sequence (SEQ ID NO: 13), all of which are flanked by the 5' ITR (SEQ ID NO: 1) and 3' ITR (SEQ ID NO: 2).

[0024] [Figure 3] Figure 3 shows an AAV expression cassette containing the hSARE-hArcMin promoter (SEQ ID NO: 6), the BDNF gene (SEQ ID NO: 7), and the BDNF long 3'UTR (SEQ ID NO: 10), all of which are flanked by the 5'ITR (SEQ ID NO: 1) and 3'ITR (SEQ ID NO: 2).

[0025] [Figure 4]Figure 4 shows a schematic diagram of an AAV expression cassette for reporter gene expression containing a constitutive ("C") promoter, hSyn, or an activity-dependent ("AD") promoter, hSARE-hArcMin. Elements of the expression cassette are also listed in Table 3.

[0026] [Figure 5A] FIG. 5A is a bar graph showing reporter protein fluorescence in cells not treated with tetrodotoxin (TTX) compared to fluorescence in cells treated with 2 μM TTX. [Figure 5B] FIG. 5B is a bar graph showing reporter protein fluorescence in cells treated with 150 mM KCl compared to fluorescence in cells treated with 2 μM TTX. [Figure 5C] Figure 5C is a bar graph showing reporter protein fluorescence in cells treated with 30 μM bicuculline (BIC) compared to that of cells treated with 2 μM TTX. Each bar shows the relative fluorescence of cells transduced with an AAV vector containing the indicated AAV expression cassette at the multiplicity of infection (MOI) indicated on the x-axis.

[0027] [Figure 6A] Figure 6A is a graph showing relative reporter gene expression in cells transduced with AAV vectors containing the indicated AAV expression cassettes and either treated with 30 μM BIC and further treated with 2 μM TTX or left untreated with TTX, as indicated. [Figure 6B] Figure 6B is a graph showing relative reporter gene expression in cells transduced with an AAV vector containing an activity-dependent (AD) AAV expression cassette and either treated with 30 μM BIC and further treated with 2 μM TTX, or left untreated with TTX, as indicated.

[0028] [Figure 7]Figure 7 shows microscopy images showing mScarlet fluorescence (upper images) and staining with the neuronal marker anti-beta III tubulin antibody (lower images) of neurons transduced with AAV vectors containing the indicated constitutive (C) AAV expression cassettes.

[0029] [Figure 8] Figure 8 shows microscopy and overlay images of neurons transduced with an AAV vector containing the "AD-dmScar-bGH" AAV expression cassette, showing Hoechst DNA staining, dmScarlet fluorescence, and anti-beta III tubulin antibody staining. The upper panel shows images of cells treated with 2 μM TTX and 30 μM BIC, while the lower panel shows images of cells not treated with TTX but treated with 30 μM BIC.

[0030] [Figure 9A] Figure 9A shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO:26 and containing the following elements: hSyn promoter (SEQ ID NO:38), mScarlet gene (SEQ ID NO:36), BDNF short 3'UTR (SEQ ID NO:8) and stuffer sequence (SEQ ID NO:13), all of which are flanked by 5'ITR (SEQ ID NO:1) and 3'ITR (SEQ ID NO:2). [Figure 9B] Figure 9B shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO:27 and containing the following elements: hSyn promoter (SEQ ID NO:38), mScarlet gene (SEQ ID NO:36), and BDNF long 3'UTR (SEQ ID NO:10), all of which are flanked by 5'ITR (SEQ ID NO:1) and 3'ITR (SEQ ID NO:2). [Figure 9C] Figure 9C shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO:28 and containing the following elements: hSyn promoter (SEQ ID NO:38), mScarlet gene (SEQ ID NO:36), bGH polyA signal (SEQ ID NO:9) and stuffer sequence (SEQ ID NO:13), all of which are flanked by 5' ITR (SEQ ID NO:1) and 3' ITR (SEQ ID NO:2).

[0031] [Figure 10A] Figure 10A shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO:29 and containing the following elements: hSyn promoter (SEQ ID NO:38), dmScarlet gene (SEQ ID NO:35), BDNF short 3'UTR (SEQ ID NO:8) and stuffer sequence (SEQ ID NO:13), all of which are flanked by 5'ITR (SEQ ID NO:1) and 3'ITR (SEQ ID NO:2). [Figure 10B] Figure 10B shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO: 30 and containing the following elements: hSyn promoter (SEQ ID NO: 38), dmScarlet gene (SEQ ID NO: 35), and BDNF long 3'UTR (SEQ ID NO: 10), all of which are flanked by 5'ITR (SEQ ID NO: 1) and 3'ITR (SEQ ID NO: 2). [Figure 10C] Figure 10C contains an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO: 31 and including the following elements: hSyn promoter (SEQ ID NO: 38), dmScarlet gene (SEQ ID NO: 35), bGH polyA signal (SEQ ID NO: 9) and stuffer sequence (SEQ ID NO: 13), all of which are flanked by 5' ITR (SEQ ID NO: 1) and 3' ITR (SEQ ID NO: 2).

[0032] [Figure 11A] Figure 11A shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO: 32 and containing the following elements: hSARE-hArcMin promoter (SEQ ID NO: 6), dmScarlet gene (SEQ ID NO: 35), BDNF short 3'UTR (SEQ ID NO: 8) and stuffer sequence (SEQ ID NO: 13), all of which are flanked by 5'ITR (SEQ ID NO: 1) and 3'ITR (SEQ ID NO: 2). [Figure 11B] Figure 11B shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO: 33 and containing the following elements: the hSARE-hArcMin promoter (SEQ ID NO: 6), the dmScarlet gene (SEQ ID NO: 35), and the BDNF long 3'UTR (SEQ ID NO: 10), all of which are flanked by the 5'ITR (SEQ ID NO: 1) and 3'ITR (SEQ ID NO: 2). [Figure 11C] Figure 11C shows an AAV expression cassette comprising the nucleic acid sequence of SEQ ID NO: 34 and containing the following elements: hSARE-hArcMin promoter (SEQ ID NO: 6), dmScarlet gene (SEQ ID NO: 35), bGH polyA signal (SEQ ID NO: 9) and stuffer sequence (SEQ ID NO: 13), all of which are flanked by 5' ITR (SEQ ID NO: 1) and 3' ITR (SEQ ID NO: 2).

[0033] [Figure 12] Figure 12 shows microscopy and overlay images of neurons transduced with an AAV vector containing the "AD-dmScar-longUTR" AAV expression cassette, showing Hoechst DNA staining, dmScarlet fluorescence, and anti-beta III tubulin antibody staining. The upper panel shows images of cells treated with 2 μM TTX and 30 μM BIC, and the lower panel shows images of cells not treated with TTX but treated with 30 μM BIC. DETAILED DESCRIPTION OF THE INVENTION

[0034] Rett syndrome is a rare genetic neurological disorder that primarily affects girls between 6 and 18 months of age. Symptoms of the syndrome, which become apparent later in life, include: speech problems, coordination problems, repetitive movements, Slow growth, walking problems, small head size, seizures, scoliosis, cognitive impairment, Patients with Rett syndrome may experience one or more of these symptoms: A person may present with any combination of symptoms, each of which may vary in severity. In an embodiment of the present invention, for example, the following items are provided: (Item 1) A nucleic acid comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, in a 5' to 3' direction: 5' inverted repeat (ITR); synthetic activity-dependent promoter; Rett syndrome-associated genes; and A nucleic acid comprising a 3' ITR. (Item 2) 2. The nucleic acid of item 1, wherein the promoter directs expression of the Rett syndrome-associated gene. (Item 3) 3. The nucleic acid of item 1 or 2, wherein the promoter is an MECP2-independent promoter. (Item 4) 4. The nucleic acid according to any one of items 1 to 3, wherein the promoter comprises a nucleic acid sequence derived from a promoter of a neuronal immediate early gene. (Item 5) 5. The nucleic acid according to item 4, wherein the neuronal immediate early gene is selected from the group consisting of the Arc gene, the c-fos gene, and the egr-1 gene. (Item 6) 6. The nucleic acid of any one of items 1 to 5, wherein the promoter comprises a minimal Arc gene promoter (ArcMin). (Item 7) 7. The nucleic acid of item 6, wherein the ArcMin is human ArcMin (hArcMin). (Item 8) 8. The nucleic acid of item 7, wherein the hArcMin comprises the nucleic acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 12. (Item 9) 9. The nucleic acid of any one of items 1 to 8, wherein the promoter comprises a cyclic AMP response element (CRE), a serum response element (SRE), a synaptic activity response element (SARE), a MEF2 response element, or a combination thereof. (Item 10) 10. The nucleic acid of item 9, wherein the promoter comprises a synaptic activity response element (SARE). (Item 11) 11. The nucleic acid of item 10, wherein the synaptic activity response element (SARE) is a human synaptic activity response element (hSARE). (Item 12) 12. The nucleic acid of item 11, wherein the hSARE comprises the nucleic acid sequence of SEQ ID NO: 11 or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 11. (Item 13) 13. The nucleic acid of any one of items 1 to 12, wherein the promoter comprises human ArcMin (hArcMin) and at least one hSARE. (Item 14) 14. The nucleic acid of item 13, wherein the promoter comprises hArcMin and one hSARE. (Item 15) 15. The nucleic acid of item 14, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 6 or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 6. (Item 16) 14. The nucleic acid of item 13, wherein the promoter comprises hArcMin and five hSAREs. (Item 17) 17. The nucleic acid of item 16, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 16 or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 16. (Item 18) 18. The nucleic acid of any one of items 1 to 17, wherein the promoter binds to a neuronal activity-dependent transcription factor. (Item 19) 19. The nucleic acid of item 18, wherein the neuronal activity-dependent transcription factor is cAMP response element binding protein (CREB), myocyte enhancer factor 2 (MEF2), serum response factor (SRF), or Elk-1. (Item 20) 20. The nucleic acid of any one of items 1 to 19, wherein the Rett syndrome-associated gene encodes brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF1), methyl-CpG-binding protein 2 (MECP2), huntingtin protein, huntingtin-related protein 1, orthodenticle homeobox 2 (OTX-2), FXYD domain-containing ion transport regulator 1 (FXYD1), neurexin-2-alpha (NRXN2), or protein kinase C gamma (PRKCG). (Item 21) 21. The nucleic acid of item 20, wherein the Rett syndrome-associated gene encodes brain-derived neurotrophic factor (BDNF). (Item 22) 22. The nucleic acid of item 21, wherein the BDNF is human BDNF. (Item 23) 23. The nucleic acid of item 22, wherein the BDNF is encoded by the nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 7. (Item 24) 24. The nucleic acid according to any one of items 1 to 23, wherein at least one of the 5' ITR and the 3' ITR is about 110 to about 160 nucleotides in length. (Item 25) 25. The nucleic acid of any one of items 1 to 24, wherein the 5' ITR is the same length as the 3' ITR. (Item 26) 25. The nucleic acid of any one of items 1 to 24, wherein the 5' ITR and the 3' ITR have different lengths. (Item 27) 27. The nucleic acid of any one of paragraphs 1 to 26, wherein at least one of the 5' ITR and the 3' ITR is isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. (Item 28) 24. The nucleic acid according to any one of items 1 to 23, wherein the 5'ITR comprises the sequence of SEQ ID NO: 1. acid. (Item 29) 24. The nucleic acid of any one of items 1 to 23, wherein the 3′ ITR comprises the sequence of SEQ ID NO: 2. (Item 30) 30. The nucleic acid of any one of items 1 to 29, wherein the AAV cassette comprises a brain-derived neurotrophic factor (BDNF) short 3'UTR or a BDNF long 3'UTR. (Item 31) 31. The nucleic acid of item 30, wherein the BDNF short 3'UTR or the BDNF long 3'UTR is located between the Rett syndrome-associated gene and the 3'ITR. (Item 32) 32. The nucleic acid of claim 30 or 31, wherein the AAV cassette comprises a BDNF short 3'UTR. (Item 33) 33. The nucleic acid of item 32, wherein the BDNF short 3'UTR comprises the nucleic acid sequence of SEQ ID NO: 8 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 8. (Item 34) 32. The nucleic acid of claim 30 or 31, wherein the AAV cassette comprises the BDNF long 3'UTR. (Item 35) 35. The nucleic acid of item 34, wherein the BDNF long 3'UTR comprises the nucleic acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 10. (Item 36) 36. The nucleic acid of any one of items 1 to 35, wherein the AAV cassette comprises a polyadenylation signal. (Item 37) 37. The nucleic acid of item 36, wherein the polyadenylation signal is a polyadenylation signal isolated from or derived from one or more of the following genes: simian virus 40 (SV40), rBG, α-globin, β-globin, human collagen, human growth hormone (hGH), polyoma virus, human growth hormone (hGH), or bovine growth hormone (bGH). (Item 38) 37. The nucleic acid of item 36, wherein the AAV cassette comprises a bGH polyadenylation signal. (Item 39) 39. The nucleic acid of item 38, wherein the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 9 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 9. (Item 40) 40. The nucleic acid of any one of items 1 to 39, wherein the AAV cassette comprises at least one stuffer sequence. (Item 41) 41. The nucleic acid of item 40, wherein the at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 13. (Item 42) 42. The nucleic acid of any one of items 1 to 41, wherein the AAV expression cassette comprises a Kozak sequence, and the Kozak sequence overlaps with the start codon of the Rett syndrome-associated gene. (Item 43) 43. The nucleic acid of item 42, wherein the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 14, or the nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 15. (Item 44) 44. The nucleic acid of any one of items 1 to 43, wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 3, or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3, the nucleic acid sequence of SEQ ID NO: 4, or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4, or the nucleic acid sequence of SEQ ID NO: 5, or a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 5. (Item 45) 45. A plasmid comprising the nucleic acid according to any one of Items 1 to 44. (Item 46) A cell comprising the nucleic acid according to any one of Items 1 to 44 or the plasmid according to Item 45. (Item 47) 46. ​​A method for producing a recombinant AAV vector, comprising contacting an AAV-producing cell with the nucleic acid of any one of items 1 to 44 or the plasmid of item 45. (Item 48) A recombinant AAV vector produced by the method described in item 47. (Item 49) 49. The recombinant AAV vector of item 48, wherein the vector is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV. (Item 50) 50. The recombinant AAV vector of claim 48 or 49, wherein the recombinant AAV vector is a single-stranded AAV (ssAAV). (Item 51) 51. The recombinant AAV vector according to any one of items 48 to 50, wherein the recombinant AAV vector is a self-complementary AAV (scAAV). (Item 52) 52. The recombinant AAV vector of any one of items 48 to 51, wherein the AAV vector comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. (Item 53) 53. The recombinant AAV vector of any one of items 48 to 52, wherein the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to a wild-type AAV capsid protein. (Item 54) 53. A composition comprising: (a) the nucleic acid of any one of items 1 to 44, the plasmid of item 45, the cell of item 46, or the recombinant AAV vector of any one of items 48 to 53, and (b) a pharmaceutically acceptable carrier. (Item 55) 53. A method for expressing a Rett syndrome-associated gene in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of items 1 to 44, the plasmid of item 45, the cell of item 46, the recombinant AAV vector of any one of items 48 to 53, or the composition of item 54. (Item 56) 56. The method of claim 55, wherein the subject has Rett Syndrome. (Item 57) Methods of Treating or Delaying the Onset of Rett Syndrome in a Subject 54. A method comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of Items 1 to 44, the plasmid of Item 45, the cell of Item 46, the recombinant AAV vector of any one of Items 48 to 53, or the composition of Item 54. (Item 58) 53. A method for expressing brain-derived neurotrophic factor (BDNF) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of items 1 to 44, the plasmid of item 45, the cell of item 46, the recombinant AAV vector of any one of items 48 to 53, or the composition of item 54. (Item 59) 59. The method of claim 58, wherein the subject has a cognitive disorder or a stress-related disorder. (Item 60) 59. The method of claim 58, wherein the subject has depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, dementia, anorexia nervosa and bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder, obesity, Rett syndrome, or post-chemotherapy cognitive impairment. (Item 61) 53. A method for treating a BDNF-related disorder or delaying the onset of a BDNF-related disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid according to any one of items 1 to 44, the plasmid according to item 45, the cell according to item 46, the recombinant AAV vector according to any one of items 48 to 53, or the composition according to item 54. (Item 62) Item 62. The method of item 61, wherein the BDNF-related disorder is a cognitive disorder and / or a stress-related disorder. (Item 63) 62. The method of item 61, wherein the BDNF-related disease is depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, dementia, anorexia nervosa, bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder, bipolar disorder, Rett syndrome, major depressive disorder, or post-chemotherapy cognitive impairment. (Item 64) 53. A method of treating an MECP2-associated disease or delaying the onset of an MECP2-associated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of Items 1 to 44, the plasmid of Item 45, the cell of Item 46, the recombinant AAV vector of any one of Items 48 to 53, or the composition of Item 54. (Item 65) 65. The method of item 64, wherein the MECP2-associated disease is MECP2 duplication syndrome, MECP2-associated severe neonatal encephalopathy, PPM-X syndrome, or Rett syndrome. (Item 66) 66. The method of any one of items 55 to 65, wherein the subject is a human subject. (Item 67) Item 68. The method of any one of Items 55 to 66, wherein the nucleic acid, the plasmid, the cell, the recombinant AAV vector, or the composition is administered by injection into the central nervous system. 68. The method of any one of items 55 to 67, wherein the Rett syndrome-associated gene is expressed in neurons of the subject. (Item 69) 69. The method of claim 68, wherein the neuron is an active neuron.

[0035] Rett syndrome is caused by a gene encoding the methyl-CpG-binding protein 2 (MECP2) protein. The disease is caused by a genetic mutation in the mecp2 gene, which is incorporated herein by reference in its entirety. See UniProt accession number P51608, which is incorporated herein by reference. The offspring had over 620 mutations, many of which were single base pair insertions or deletions. It was identified in a woman with Rett syndrome. MECP2 is involved in the normal function of nerve cells. It contributes to the regulation of methyl-Cp in the MECP2 domain and is present at high levels in mature neurons. G-binding (MBD) can recognize DNA regions with 5-methylcytosine modifications. Mutations in the mecp2 gene are associated with MECP2 duplication syndrome and MECP2-associated severe neonatal encephalopathy. Mutations in the mecp2 gene can also cause other disorders, such as phenotype-specific phenotype-specific phenotype-specific syndrome (PPM-X). There are no approved therapies to treat any of the associated diseases.

[0036] The present disclosure provides a method for treating diseases associated with mutations in the mecp2 gene, such as Rett syndrome, and and / or delaying the onset of a disease. The present invention provides a method for the production of brain-derived neurotrophic factor (BND) vectors, including BND-1, BND-2, BND-3, BND-4, BND-5, BND-6, BND-7, BND-8, BND-9, BND-11, BND-12, BND-13, BND-14, BND-15, BND-16, BND-17, BND-18, BND-19, BND- F) Treating related disorders and / or preventing the onset of brain-derived neurotrophic factor (BDNF)-related disorders Also provided herein are methods for delaying the disease.

[0037] definition The following terminology is used herein and in the appended claims.

[0038] The singular forms "a," "an," and "the" refer to the plural unless the context clearly indicates otherwise. It is intended to include shapes as well.

[0039] Furthermore, the length of the polynucleotide or polypeptide sequence, the amount of dosage, time, temperature, etc. When referring to a measurable value of 0.1, the term "about" as used herein refers to a range of values ​​within ±20 of the specified amount. %, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variation. This means that

[0040] Also as used herein, "and / or" means any of the associated listed items. Any and all possible combinations of one or more of these, as well as alternatives ( Refers to and encompasses the absence of a combination when interpreted as either or.

[0041] The term "wild type" is a term of the art that is understood by those skilled in the art and does not include mutant or is a naturally occurring organism, strain, gene, protein, or characteristic that is distinct from variant forms. For example, a wild-type protein refers to the typical form of a protein as it occurs in nature. This is a typical form of protein.

[0042] The term "mutant protein" is a term of the art that is understood by those skilled in the art, e.g. For example, amino acid substitutions, insertions, and / or deletions can be used to identify proteins. The term "mutant gene" refers to a protein that is distinct from the wild-type form of a protein. is a term of the art that is understood to include, for example, nucleic acid substitutions, insertions and / or deletions. It refers to a gene that is distinguished from the wild-type form of the gene based on the presence of nucleic acid modifications such as In embodiments, the mutant gene encodes a mutant protein.

[0043] A "nucleic acid" or "polynucleotide" refers to a sequence of nucleotide bases, e.g., RNA, DNA or DNA-RNA hybrid sequences (naturally occurring nucleotides and naturally occurring In some embodiments, the nucleic acids of the present disclosure are It is either a single-stranded or double-stranded DNA sequence. Nucleic acids can range from 1 to 1,000, 1,000, ~10,000, 10,000-100,000, 100,000-1 million, or 1 Nucleic acids are generally made up of phosphodiester bonds. In some cases, e.g., phosphoramides, phosphorothioates, phosphorodiamines, Thioates, O-methyl phosphoramidite linkages, and peptide nucleic acid backbones and linkages Other analog nucleic acids include nucleic acid analogs that may have alternative backbones, including: These include those with a ribose backbone, a non-ionic backbone, and a non-ribose backbone. Nucleic acids containing carbocyclic sugars are also included within the definition of nucleic acids. Modifications of the nucleotides may facilitate the addition of labels or may increase the stability of such molecules in physiological environments. The nucleic acids of the present disclosure can be linear or or circular (eg, a plasmid).

[0044] As used herein, the term "promoter" refers to a promoter that directs transcription of an operably linked nucleic acid. A promoter refers to one or more nucleic acid regulatory sequences that direct the transcription of a gene. A promoter may also contain nucleic acid sequences near the transcription start site, such as a promoter that encodes a transcription factor. It may also include cis-acting polynucleotide sequences that can be bound by a gene.

[0045] A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a nucleic acid expression control sequence (e.g., a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, and The sequence directs the transcription of a nucleic acid corresponding to the second sequence.

[0046] An "AAV expression cassette" is a nucleic acid that is packaged into a recombinant AAV vector. The AAV vector binds to the target cell and contains a sequence encoding one or more transgenes. When contacted, the transgene is expressed by the target cell.

[0047] As used herein, the terms "viral vector," "viral vector," "genetic vector," " "Gene delivery vector" refers to a viral particle that functions as a nucleic acid delivery vehicle, and includes a virion. Exemplary embodiments of the present disclosure include nucleic acids (e.g., AAV expression cassettes) packaged within the vector. Examples of useful viral vectors include adenovirus vectors and adeno-associated virus vectors ( AAV), lentiviral vectors, and retroviral vectors.

[0048] As used herein, the term "adeno-associated virus" (AAV) refers to AAV Type 1, AAV type 2, AAV type 3 (types 3A and 3B), AAV type 4, AAV type 5, AAV 6 type, AAV7 type, AAV8 type, AAV9 type, AAV10 type, AAV11 type, AAV12 Type, AAV13 type, AAVrh32.33 type, AAVrh8 type, AAVrh10 type, AA Vrh74 type, AAVhu.68 type, avian AAV, bovine AAV, canine AAV, equine AAV , sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, A AV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and currently including, but not limited to, any other AAV known or later discovered See, for example, Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0049] The terms "virus-producing cells," "virus-producing cell line," and "virus-producing cells" refer to Refers to the cells used to produce the vector. HEK293 and 239T cells are common virus-producing cell lines. Table 2 below shows examples of various viral vectors. List of representative virus-producing cell lines. [Table 2]

[0050] "HEK293" is a cell line originally derived from human embryonic kidney cells grown in tissue culture The HEK293 cell line is easily grown in culture and is commonly used for virus production. As used herein, "HEK293" also refers to one or more variants of HEK293 cell line, i.e., the original HEK293 cell line, further containing one or more genetic alterations. It may also refer to cell lines derived from the HEK293 cell line. Many variant HEK293 lines are Developed and optimized for one or more specific uses, e.g., 293T cells The strain undergoes episomal replication of transfected plasmids containing the SV40 replication origin. It contains the SV40 large T antigen, which allows for increased expression of the desired gene product. This brings about

[0051] "Sf9" is the parent of Spodoptera frugiperda Insect cell line IPLB-Sf-21-AE, a clonal isolate derived from the erda cell line IPLB-Sf-21-AE, Sf9 cells can grow in the absence of serum and can be grown either adherently or in suspension. It can be cultivated in

[0052] "Transfection reagent" means a composition that enhances the transfer of nucleic acids into cells. Several transfection reagents commonly used in the art are capable of transfecting nucleic acids and One or more antibodies that bind to the cell surface (e.g., Lipofectamine™) Contains lipids.

[0053] Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be measured using, but not limited to, the methods of Smith & Waterman, and the local sequence identity algorithm of Adv. Appl. Math. 2, 482 (1981). Zum, Needleman & Wunsch, J Mol. Biol. 48, 443 ( 1970) sequence identity alignment, Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988) Search methods, computerized implementations of these algorithms (Wisc onsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madiso n, WI's GAP, BESTFIT, FASTA, and TFASTA), Devere ux et al., Nucl. Acid Res. 12, 387-395 (1984) or by scanning the Best Fit sequence program described by This may be determined using standard techniques known in the art.

[0054] Another suitable algorithm is that of Altschul et al., J Mol. Biol .215,403-410,(1990) and Karlin et al.,Proc .Natl.Acad.Sci.USA 90,5873-5787(1993) The BLAST algorithm is a particularly useful BLAST program. chul et al.,Methods in Enzymology,266,46 0-480(1996);http: / / blast.wustl / edu / blast The WU-BLAST-2 program is available from / README.html. BLAST-2 uses several search parameters that are optionally set to default values. The parameters are dynamic values ​​that depend on the configuration and the specific sequence in which the target sequence is searched. The value is established by the program itself depending on the database and the configuration of the particular database. It may be adjusted to increase the intensity.

[0055] Further useful algorithms are described by Altschul et al. (1997) )Reported by Nucleic Acids Res.25,3389-3402 It's Gap Blast.

[0056] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms include, but are not limited to, treatment. Approaches to obtaining beneficial or desired results, including benefit and / or preventative benefits Therapeutic benefit refers to any improvement in one or more of the diseases, conditions, or symptoms being treated. Refers to a therapeutically relevant improvement or effect on a disease, condition, or symptom that is not yet apparent. Although the composition may not be localized, for prophylactic benefit, the composition may be used to treat a particular disease, condition, or subjects who are at risk of developing a condition or who report one or more physiological symptoms of the disease It may be administered to a subject undergoing

[0057] The terms "subject," "individual," and "patient" are used herein to refer to vertebrate animals, such as mammals. The terms "mammal" and "animal" are used interchangeably herein. Mammals include, for example, mice, rats, rabbits, and cats. , dogs, pigs, sheep, horses, non-human primates (e.g., cynomolgus monkeys, chimpanzees), or human. They may be obtained in vivo or cultivated in vitro. Also encompassed are tissues, cells, or derivatives thereof of a subject that are cultivated. , teen, child (2-14 years), toddler (1-24 months), or newborn (up to 1 month) In some embodiments, the adult may be about 65 years of age or older, or about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman intending to become pregnant. is.

[0058] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a result, e.g., beneficial or A therapeutically effective amount refers to an amount of an agent sufficient to produce a desired result. The disease state, weight and age of the subject, severity of the disease state, mode of administration, etc. may vary depending on several factors and can be readily determined by one of skill in the art. , the particular drug selected, the dosing regimen to be followed, and whether it will be administered in combination with other compounds. Whether and timing of administration, the tissue to be imaged, and the physical delivery to which it is transported This may vary depending on one or more of the systems.

[0059] As used herein, the term "gene therapy" refers to the use of a gene to correct an abnormal gene. It also refers to the process of introducing genetic material into cells to make therapeutic proteins.

[0060] As used herein, a neuron is a cell that has at least a portion of its cell membrane detached. When polarized, a neuron is said to be "active" or have "neuronal activity." In some embodiments, the depolarization is a voltage-gated Na + Opening the channel In some embodiments, depolarization of the neuronal cell membrane induces Ca 2+ Aeon This results in intracellular influx and induces downstream transcription of immediate early genes (IEGs). In vivo depolarization of at least part of the neuronal membrane induces synaptic responses in presynaptic neurons. Depolarization in vitro may be induced by signals from neurons. Depolarization can also be induced by treating the neurons with KCl. , bicuculline) to, for example, Cl - or K + Detachment by opening channels signaling receptors, such as gamma-aminobutyric acid type A (GABAA) receptors, which normally slow down the polarity. It may be enhanced in vitro by inactivation.

[0061] As used herein, a neuron is a cell that transports a net ion across the cell membrane of a neuron. When there is no flow, the neuron is said to be "resting." In some embodiments, the resting neuron is a neuron in which no portion of the neuron cell membrane is depolarized. It has a resting membrane potential of approximately -70 mV.

[0062] AAV expression cassette The present disclosure provides nucleic acid sequences comprising one or more adeno-associated virus (AAV) expression cassettes. In some embodiments, the AAV expression cassette comprises a 5' inverted repeat (ITR) sequence. ), a promoter, a transgene, and a 3' ITR. In some embodiments, the gene is a Rett syndrome associated gene. Contains a Kozak sequence, a polyadenylation sequence, and / or a stuffer sequence.

[0063] In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 3, or SEQ ID NO: 3 nucleic acid sequence at least 70% identical (e.g., all sequences and portions therebetween) to the nucleic acid sequence of Ranges include at least 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least Some of the sequences contain sequences that are at least 99%, at least 99.5%, or 100% identical. In embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO:4, or the nucleic acid sequence of SEQ ID NO:4. At least 70% identical to the column (e.g., includes all values ​​and subranges that fall between them) , at least 75%, at least 80%, at least 85%, at least 90%, less At least 95%, at least 96%, at least 97%, at least 98%, at least 99% %, at least 99.5%, or 100% identical). The AAV expression cassette may comprise the nucleic acid sequence of SEQ ID NO: 5, or at least the nucleic acid sequence of SEQ ID NO: 5. at least 70% identical (e.g., including all values ​​and subranges therebetween) At least 75%, at least 80%, at least 85%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99%, at least The sequences are 99.5% or 100% identical to each other.

[0064] (i) Inverted terminal sequences Inverted terminal repeat (ITR) sequences are essential for AAV proviral integration and the translation of AAV DNA. ITRs mediate packaging into virions. For example, the ITR sequences that can form hairpin structures are involved in various activities in transcription. removal from the plasmid after transfection, replication of the vector genome, and host cell genome replication It plays a role in the integration and rescue of cancer.

[0065] The AAV expression cassette of the present disclosure may comprise a 5' ITR and a 3' ITR. The sequence may be about 110 to about 160 nucleotides in length, e.g., 110, 111, 112, 113 , 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 , 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 , 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 , 154, 155, 156, 157, 158, 159 or 160 nucleotides in length In some embodiments, the ITR sequence may be about 141 nucleotides in length. In some embodiments, the 5' ITR is the same length as the 3' ITR. In some embodiments, the 5' ITR and 3' ITR have different lengths. R is longer than the 3' ITR, and in other embodiments, the 3' ITR is longer than the 5' ITR. .

[0066] ITRs may be isolated from the genome of any AAV, e.g., the AAVs listed in Table 1. In some embodiments, at least one of the 5' ITR and 3' ITR may be introduced. At least one is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAV rh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV genomes In some embodiments, the 5' ITR and 3' ITR are isolated or derived from a At least one of the ITRs comes from a member of another parvovirus species in addition to AAV. For example, in some embodiments, the ITR may be an isolated wild-type or mutant ITR. ITRs have been isolated from bocaviruses or parvoviruses B19 or The ITR may be a wild-type or mutant ITR.

[0067] In some embodiments, the ITRs comprise modifications to facilitate production of scAAV. In this embodiment, modifications to facilitate production of scAAV include altering the degree of terminal separation from the ITRs. In some embodiments, the 5' ITR is a wild-type ITR, The 3' ITR is a mutant ITR that lacks a terminal separation sequence. 5'ITR is a wild-type ITR, and 5'ITR is a mutant ITR lacking the terminal separation sequence. In some embodiments, the terminal separation sequence is absent from both the 5' ITR and the 3' ITR. In other embodiments, modifications to facilitate scAAV production include sequences such as shRNA-forming sequences. which is the replacement of the ITR with a different hairpin-forming sequence.

[0068] In some embodiments, the 5' ITR has the nucleic acid sequence of SEQ ID NO: 1 or the nucleic acid of SEQ ID NO: 1. Sequences at least 70% identical to (e.g., including all values ​​and subranges therebetween) At least 75%, at least 80%, at least 85%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 9 In some embodiments, the sequence of the sequence is at least 99.9%, at least 99.5%, or 100% identical. 3'ITR is a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence of SEQ ID NO: 2 and at least 7 0% identical (e.g., at least 75%, including all values ​​and subranges therebetween) %, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99%, at least 9 In some embodiments, the 5' ITR comprises a sequence that is 9.5%, or 100% identical to the 5' ITR. The 3'ITR comprises the sequence of SEQ ID NO:2.

[0069] In some embodiments, the AAV expression cassette may contain one or more "surrogate" ITRs, i.e., That is, it contains non-ITR sequences that perform the same function as ITRs. For example, Xie, J. et al. ., Mol. Ther., 25(6):1363-1374(2017). In some embodiments, the ITRs in the AAV expression cassette are replaced by surrogate ITRs. In some embodiments, the surrogate ITR comprises a hairpin-forming sequence. The surrogate ITRs are short hairpin (sh)RNA-forming sequences.

[0070] (ii) activity-dependent promoter In some embodiments, the AAV expression cassettes described herein include a promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a synthetic promoter. The gene contains nucleic acid sequences derived from endogenous promoters and / or endogenous enhancers. In some embodiments, the promoter may be derived from a region upstream of the start codon of a gene. The nucleic acid sequence may include a nucleic acid sequence occurring within 1 base pair (bp) upstream of the start codon. It may be within about 8000 bp, including, for example, all subranges and values ​​therebetween. Approximately 10bp, 50bp, 100bp, 500bp, 1000bp upstream of the start codon 1500bp, 2000bp, 2500bp, 3000bp, 3500bp, 4000b p, 4500bp, 5000bp, 5500bp, 6000bp, 6500bp, 700 It may be within 0 bp, 7500 bp, or 8000 bp.

[0071] Without being bound by any theory, MECP2 function may be involved in the regulation of neuronal activity. These target genes are thought to contribute to the expression of target genes involved in the function of the thymus. These target genes are affected when MECP2 is mutated. associated with MECP2 mutations or MECP2 mutations by independently restoring Therefore, in some embodiments, As used herein, the term "MECP2 promoter" refers to a promoter that is MECP2-independent. "ECP2-independent promoters" are those whose function is due to reduced MECP2 function or the inability to function as MECP2. A promoter that is not impaired by loss of transcriptional activity.

[0072] Without being bound by any theory, it is believed that targets involved in neuronal function Constitutive expression of target genes is believed to be deleterious. In some embodiments, the promoter function is inducible. In some embodiments, the promoter is As used herein, a "neuronal activity dependent promoter" refers to a promoter that is a neuronal activity dependent promoter. The function of an "activity-dependent promoter," which is used interchangeably with "promoter," depends on neuronal activity. In some embodiments, the activity-dependent promoter is induced by calcium ions. In some embodiments, the activity-dependent promoter is induced by the influx of are toxic when constitutively expressed in cells (e.g., neurons) using , resulting in expression of any target gene.

[0073] In some embodiments, the promoter is a promoter of a neuronal immediate early gene and The identity of neuronal immediate early genes includes nucleic acid sequences derived from the promoter and / or enhancer. The present invention is not limited to any neurotransmitter known in the art or to be identified in the future. Non-limiting examples of neuronal immediate early genes include Ar c gene, c-fos gene, and egr-1 gene. In this embodiment, the promoter is selected from the group consisting of the Arc gene, the c-fos gene, and the egr-1 gene. It contains nucleic acid sequences derived from the promoter and / or enhancer of a gene. In embodiments, the promoter is a promoter of a neurotrophin gene and / or Non-limiting examples of neurotrophin genes include nucleic acid sequences derived from enhancers. The genes involved are the BDNF (brain-derived neurotrophic factor) gene, the NGF (nerve growth factor) gene, and the neurotransmitter These include the neurotrophin-3 gene and the neurotrophin-4 gene.

[0074] In some embodiments, the promoter is a promoter that encodes one or more neuronal activity-dependent transcription factors. As used herein, "neuronal activity-dependent transcription" refers to a transcription factor that can bind to a protein. A "transcription factor" is a transcription factor that is activated in response to neuronal activity. Neuronal activity-dependent transcription factors promote gene expression in response to neuronal activity. In some embodiments, the neuronal activity-dependent transcription factor is a transcription factor that is responsive to neuronal activity. In some embodiments, the neuronal activity-dependent transcription factor is It is activated by a calcium-dependent kinase cascade.

[0075] In some embodiments, the promoter is a promoter that encodes one or more neuronal activity-dependent transcription factors. The transcription factor comprises one or more nucleic acid sequences capable of binding to the target gene. Non-limiting examples of factors include cAMP response element binding protein (CREB), muscle Cellular enhancer factor 2 (MEF2), serum response factor (SRF), or Elk-1 are In some embodiments, the promoter may be more than one neuronal activity-dependent promoter. The promoter has the ability to bind transcription factors, e.g., two, three, four, or five In some embodiments, the promoter is capable of binding to a neuron activity-dependent transcription factor. is capable of binding to two neuronal activity-dependent transcription factors. The promoter can bind to three neuronal activity-dependent transcription factors. In this embodiment, the promoter is selected from the group consisting of the following neuronal activity-dependent transcription factors: CREB, M It has the ability to bind to EF2 and SRF.

[0076] In some embodiments, the promoter comprises one or more response elements. As used herein, a "response element" refers to a gene that results in gene expression in the presence of a stimulus. In some embodiments, the response element is a region of the promoter that contributes to the transcription of the promoter. The motor is important in driving gene expression in the presence of stimuli. Non-limiting examples of stimuli include: Examples include hormones, environmental cues such as heat or light, and chemical ions such as calcium. In some embodiments, the promoter inhibits gene expression in the presence of calcium. In some embodiments, the promoter comprises a response element that confers neuronal expression. In some embodiments, the promoter comprises a response element present in the promoter of an immediate early gene of The promoters are those of the Arc gene, the c-fos gene, and / or the egr-1 gene. Contains response elements present in the promoter.

[0077] In some embodiments, the response element is one of the following: cyclic AMP response element (CRE ), serum response element (SRE), synaptic activity response element (SARE), and In some embodiments, the MEF2-responsive element (MRE) comprises one or more of: The promoter may comprise one or more CREs, one or more SREs, or one or more may include multiple SAREs, one or more MREs, or any combination thereof. In some embodiments, the response element is one or more neuronal activity-dependent transcription factors In some embodiments, CRE binds to CREB. In some embodiments, the SRE binds to SRF. In some embodiments, the MRE binds to MEF2. In some embodiments, the SARE binds to CREB, MEF2 and SRF.

[0078] In some embodiments, the promoter comprises 1 to 20 SAREs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 In some embodiments, the promoter comprises 9 or 20 SAREs. In some embodiments, the promoter comprises five SAREs. In some embodiments, the SARE is a mouse SARE. In some embodiments, the SARE is a human SARE. In some embodiments, the hSARE is a synaptic activity response element (hSARE). , a nucleic acid sequence having at least 70% identity (e.g., all sequences present therebetween) with the nucleic acid sequence of SEQ ID NO: 11. At least 75%, at least 80%, at least 85%, inclusive of all values ​​and subranges %, at least 90%, at least 95%, at least 96%, at least 97%, at least at least 98%, at least 99%, at least 99.5%, or 100% identity In some embodiments, the hSARE comprises a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 11: or comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO:11.

[0079] In some embodiments, the promoter is a nucleic acid sequence derived from the promoter of the Arc gene. In some embodiments, the Arc gene is a mouse Arc gene. In some embodiments, the Arc gene is a human Arc gene. The target is a region spanning nucleic acid -300 to +300 of the Arc gene, or any subregion thereof. In some embodiments, the promoter comprises a minimal Arc gene promoter (A In some embodiments, human ArcMin (hArcMin) comprises A It contains a short upstream sequence of the rc gene and the 5'UTR (-276 to +208). In morphology, mouse ArcMin (mArcMin) contains a short upstream sequence of the Arc gene and and 5'UTR (-222 to +198). A nucleic acid sequence having at least 70% identity (e.g., all sequences therebetween) with the nucleic acid sequence of SEQ ID NO: 12. At least 75%, at least 80%, at least 85%, inclusive of all values ​​and subranges , at least 90%, at least 95%, at least 96%, at least 97%, at least (98%, at least 99%, at least 99.5%, or 100% identity) In some embodiments, hArcMin comprises the nucleic acid sequence of SEQ ID NO: 12. or a sequence that is at least 90% identical to the sequence of SEQ ID NO:12.

[0080] In some embodiments, the promoter comprises hArcMin and at least one hSA The number of hSAREs in a promoter is not limited, and can range from 1 hSARE to 20 hSAREs. hSARE, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, The hSARE may be in the range of 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the promoter comprises hArcMin and one hSARE (SAR In some embodiments, SARE-ArcMin comprises , a nucleic acid sequence having at least 70% identity (e.g., all sequences therebetween) with the nucleic acid sequence of SEQ ID NO:6. At least 75%, at least 80%, at least 85%, inclusive of all values ​​and subranges , at least 90%, at least 95%, at least 96%, at least 97%, at least (98%, at least 99%, at least 99.5%, or 100% identity) In some embodiments, SARE-ArcMin comprises a nucleic acid sequence encoding the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the sequence of SEQ ID NO: 6 is at least 90% identical to the sequence of SEQ ID NO: 6. In this study, the promoter contains hArcMin and five hSAREs (called E-SAREs). In some embodiments, the E-SARE comprises the nucleic acid sequence of SEQ ID NO: 16 and at least At least 70% identity (e.g., including all values ​​and subranges therebetween) at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99%, Some embodiments include nucleic acid sequences that have at least 99.5%, or 100% identity to the nucleic acid sequence. In some embodiments, the E-SARE has the nucleic acid sequence of SEQ ID NO: 16, or a sequence at least as long as SEQ ID NO: 16. Both contain 90% sequence identity. For more details on hArcMin and hSARE, see , Kawashima et al., Nat Methods 10,889-895 (2013), Kawashima et al., Front Neural Cir. cuits 2014 Apr 23;8:37, and Kawashima et a l., PNAS January 6,2009 106(1)316-32 No. 6,239,999, each of which is incorporated herein by reference in its entirety.

[0081] In some embodiments, the promoter is a promoter of the Arc gene, the c-fos gene, or e.g. Higher gene expression than the endogenous promoter of neuronal immediate early genes such as the r-1 gene In some embodiments, the promoter is a promoter for the Arc gene, c-fos genes, or the endogenous promoter of neuronal immediate-early genes such as egr-1 In some embodiments, the promoter induces gene expression levels that are at least 1.5-fold higher than those of the promoter. Motors are expressed by neurons, such as the Arc gene, c-fos gene, or egr-1 gene. Approximately 1.5 to 100 times (e.g., approximately 2 times) the endogenous promoter of the immediate early gene of the , about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, about 20 times, about 30 times, about 40 times, about 5 0-fold, approximately 60-fold, approximately 70-fold, approximately 80-fold, approximately 90-fold, or approximately 100-fold higher gene expression levels Guide the bell.

[0082] In some embodiments, the promoter is a promoter commonly used in the art for gene expression. The nucleic acid sequence may be derived from one or more promoters used in the In terms of form, the promoter may be a CMV promoter, an SV40 early promoter, an SV40 Late promoter, metallothionein promoter, mouse mammary tumor virus (MMTV) ) promoter, Rous sarcoma virus (RSV) promoter, polyhedrin promoter -, chicken β-actin (CBA) promoter, dihydrofolate reductase (DHFR) promoter, and the nuclear promoter derived from the phosphoglycerol kinase (PGK) promoter In some embodiments, the promoter further comprises a sequence encoding chicken β-actin (C BA) promoter, EF-1 alpha promoter, or EF-1 alpha short In some embodiments, the promoter comprises a nucleic acid sequence derived from a promoter. SEQ ID NOs: 17-20, or at least 70% identical to SEQ ID NOs: 17-20 (e.g., sequences existing therebetween) SEQ ID NOs: 17-20 and at least 75%, including all values ​​and subranges present at least 80%, at least 85%, at least 90%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, at least 99.5% , or 100% identical).

[0083] In some embodiments, the AAV expression cassettes described herein further comprise an enhancer. The enhancer may be, for example, a CMV enhancer. In this embodiment, the enhancer comprises the sequence of SEQ ID NO: 21 or the sequence of SEQ ID NO: 21 and at least 70% identical (e.g., at least 70%, including all values ​​and subranges therebetween) 5%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical).

[0084] In some embodiments, the promoter is the promoter of: HMG-COA reductase Promoter; sterol regulatory element 1 (SRE-1); phosphoenolpyruvate carboxykinase (PEPCK) promoter; human C-reactive protein (CRP) promoter Human glucokinase promoter; Cholesterol 7-alpha hydrolase promoter (CYP-7) promoter; beta-galactosidase alpha-2,6-sialyltransferase insulin-like growth factor binding protein 1 (IGFBP-1) promoter promoter; aldolase B promoter; human transferrin promoter; Prostatic acid phosphatase (PAP) promoter; prostatic acid phosphatase type I promoter; Secretory protein 94 (PSP94) promoter; prostate-specific antigen complex promoter Human glandular kallikrein gene promoter (hgt-1); muscle cell-specific enhancer binding MEF-2 promoter; muscle creatine kinase promoter; pancreatitis-associated protein promoter -(PAP); elastase 1 transcriptional enhancer; pancreatic specific amylase and elastase Pancreatic cholesterol esterase gene promoter; tereglobin promoter; cholesterol side chain cleavage (SCC) promoter; gamma- Gamma enolase (neuron-specific enolase, NSE) promoter; Neurophy NF-H promoter; human CGL-1 / granzyme B promoter terminal deoxytransferase (TdT), lambda 5, VpreB, and lck ( Lymphocyte-specific tyrosine protein kinase p561ck promoter; human CD2 promoter promoter and its 3' transcriptional enhancer; human NK and T cell-specific activation (NK G5) promoter; pp60c-src tyrosine kinase promoter; organ-specific O-antigen (OSN), mw 40 kDa (p40) promoter; colon-specific antigen P Promoter; Human alpha-lactalbumin promoter; Phosphoenolpyruvate Peptide carboxykinase (PEPCK) promoter, HER2 / neu promoter, Zein promoter, IgG promoter, chorionic embryo antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter , growth hormone factor promoter, tyrosine hydroxylase promoter, albumin Promoter, alpha-fetoprotein promoter, acetyl-choline receptor promoter promoter, alcohol dehydrogenase promoter, alpha or beta globin promoter -, T cell receptor promoter, osteocalcin promoter, IL-2 promoter , IL-2 receptor promoter, whey (wap) promoter, and MHC class The nucleic acid sequences may further comprise nucleic acid sequences derived from any one or more of the serotype II promoters.

[0085] (iii) Rett syndrome-related genes As used herein, a "Rett syndrome associated gene" refers to at least one gene that is associated with Rett syndrome. Rett syndrome, which may be targeted by gene therapy to alleviate another sign or symptom. In some embodiments, the gene is a gene encoding a gene associated with Rett syndrome. Levels of proteins encoded by the genes are reduced in subjects with Rett syndrome In some embodiments, the Rett syndrome-associated gene Levels of the encoded protein are decreased at the onset of at least one symptom of Rett syndrome. In some embodiments, the protein encoded by the Rett syndrome associated gene is Levels increase during normal postnatal development and / or neuronal development. In embodiments, the Rett syndrome associated genes encode proteins that contribute to normal neuronal function. In some embodiments, the Rett syndrome associated gene encodes a gene encoding a gene for promoting synaptic plasticity and In some embodiments, the gene encoding the gene encoding a protein is involved in the induction and / or maintenance of Rett syndrome. In some embodiments, the Rett syndrome-associated gene is a neurotrophin. Gene mutations or loss of function of Rett syndrome-associated genes are used to identify subjects with Rett syndrome. In some embodiments, a mutation in a Rett syndrome-associated gene or Rett syndrome is present. Loss of function of the associated gene causes Rett syndrome. Mutations in syndrome-related genes, loss of function of Rett syndrome-related genes, and especially forebrain stimulation neurotransmission Mutations in Rett syndrome-related genes and loss of function of Rett syndrome-related genes in m It causes symptoms similar to those caused by loss of the ecp2 gene. The type of mutation is not limited, and it can be insertion or deletion. , may be overlaps and / or substitutions.

[0086] The present disclosure provides AAV expression cassettes comprising Rett Syndrome-associated genes. In some embodiments, the AAV expression cassettes may be used for therapeutic (e.g., pharmaceutical or veterinary) or immunological purposes. Rett syndrome, encoding proteins containing epidemiological (e.g., vaccine) polypeptides In some embodiments, the AAV expression cassette comprises a mammalian Rett syndrome-associated gene. In some embodiments, the AAV expression cassette comprises a human Rett syndrome-associated gene. In some embodiments, the AAV expression cassette comprises a gene encoding brain-derived neurotrophic factor (BDNF). F), insulin-like growth factor 1 (IGF1), methyl-CpG binding protein 2 (MECP 2), Huntingtin protein, Huntington-related protein 1, Orthodenticleho Meobox 2 (OTX-2), FXYD domain-containing ion transport regulator 1 (FXYD 1), neurexin-2-alpha (NRXN2), or protein kinase C cancer In some embodiments, the Rett syndrome-associated gene encoding ribosomal kinase (PRKCG) is The gene associated with BB syndrome is KCNA1 (potassium voltage-gated channel subfamily A member) member 1), GABRA1 (gamma-aminobutyric acid type A receptor subunit alpha 1), MAPK1 (mitogen-activated protein kinase 1), NRXN2 (neurexin 2), RBFOX1 (RNA-binding Fox-1 homolog 1), GNAO1 (G protein subunit Unit alpha O1), NCAN (neurocan), PRKCG (protein kinase C C gamma), KCNJ4 (potassium inward rectifying channel J member 4), CAMK2B (Calcium / calmodulin-dependent protein kinase II beta), EFNB3 FurinB3), GABBR1 (gamma-aminobutyric acid type B receptor subunit 1), LY6 H (lymphocyte antigen 6 family member H), KCNA2 (potassium voltage-gated channel Subfamily A member 2), and NEFL (neurofilament light chain) The gene is selected from the group:

[0087] In some embodiments, the Rett syndrome associated gene is brain-derived neurotrophic factor (BDNF). BDNF encodes the neurotransmitter BDNF, which regulates neuronal survival and growth, synaptic development, and BDNF is a neurotrophin that functions in supporting plasticity. Loss of function of BDNF is altered in the BRCA1 / BRCA2 syndrome. BDNF function is affected upon loss of mecp2 function, resulting in the phenotype. For more information on this topic, see Eduardo E. Benarroch, Neurology A pr 2015,84(16)1693-1704;Chang et al.Neur on.2006;49(3):341-348; and Zhou et al., Neu ron 52, 255-269, October 19, 2006, and each of which is incorporated herein by reference in its entirety. In some embodiments, the BDNF is human BDNF. In some embodiments, the BDNF is the nuclear BDNF of SEQ ID NO: 7. and a sequence that has at least 70% identity (e.g., all sequences and portions therebetween) Ranges include at least 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least by a nucleic acid sequence having at least 99%, at least 99.5%, or 100% identity In some embodiments, BDNF is encoded by the nucleic acid sequence of SEQ ID NO: 7, or It is encoded by a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:7.

[0088] BDNF has either a short 3'UTR (approximately 0.35 kb long) or a long 3'UTR (approximately 2 Generate two distinct populations of bdnf mRNA, one with either .85 kb length , two alternative polyadenylation transcription termination sites exist. bdnf with a short 3'UTR The mRNA variant is primarily localized to the neuronal cell body but has a long 3'UTR. The bdnf mRNA variant is also localized to dendrites. The V expression cassette contains either the BDNF short 3'UTR or the BDNF long 3'UTR. In some embodiments, the BDNF short 3'UTR or the BDNF long 3'UTR R is located between the stop codon and the 3' ITR.

[0089] In some embodiments, the BDNF short 3'UTR comprises the nucleic acid sequence of SEQ ID NO: 8 and at least At least 70% identity (e.g., including all values ​​and subranges therebetween) at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, In some embodiments, the nucleic acid sequence includes a nucleic acid sequence having at least 99.5%, or 100% identity to the target nucleic acid. In one embodiment, the BDNF short 3'UTR comprises the nucleic acid sequence of SEQ ID NO: 8, or the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid sequence comprises a sequence that is at least 90% identical to the BDNF short sequence. The 3'UTR has at least 70% identity (e.g., 70% identity) with the nucleic acid sequence of SEQ ID NO: 10. Includes all values ​​and subranges present in at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% In some embodiments, the BDNF long 3' UTR comprises a nucleic acid sequence having a sequence identity of , the nucleic acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 10. Contains columns.

[0090] In some embodiments, the AAV expression cassette comprises a Kozak sequence. A nucleic acid sequence that serves as the protein translation initiation site in many eukaryotic mRNA transcripts In some embodiments, the Kozak sequence overlaps the start codon. In the Kozak sequence, the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 is a sequence of at least 70 % identity (e.g., at least 7, including all values ​​and subranges therebetween) 5%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence having a sequence identity to the target nucleic acid (e.g., 99.5%, or 100% identity). The Kozak sequence is the nucleic acid sequence of SEQ ID NO: 14, or at least the nucleic acid sequence of SEQ ID NO: 14. or a sequence 90% identical to the nucleic acid sequence of SEQ ID NO: 15, or the nucleic acid sequence of SEQ ID NO: 15 and a sequence that is at least 90% identical to the sequence of the sequence of the

[0091] (iv) polyadenylation (polyA) signal Polyadenylation signals are nucleotide sequences found in almost all mammalian genes. and a string of approximately 200 adenosine residues (poly(A) tail) of the gene transcript. The addition of poly(A) tails to the 3' end contributes to mRNA stability and mRNA lacking a poly(A) tail is rapidly degraded. Evidence that it positively contributes to mRNA translatability by affecting initiation There are also.

[0092] In some embodiments, the AAV expression cassettes of the present disclosure include a polyadenylation signal. Polyadenylation signals include those from simian virus 40 (SV40), rBG, and α-globin. , β-globin, human collagen, human growth hormone (hGH), polyomavirus, Polyadenylation signals of human growth hormone (hGH) and bovine growth hormone (bGH) The signal may be selected from the following:

[0093] In some embodiments, the AAV expression cassette comprises a bGH polyadenylation signal. In some embodiments, the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 9 and at least one At least 70% identity (e.g., including all values ​​and subranges therebetween) at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, In some embodiments, the nucleic acid sequence includes a nucleic acid sequence having at least 99.5%, or 100% identity to the target nucleic acid. In one embodiment, the bGH polyadenylation signal is the nucleic acid sequence of SEQ ID NO: 9, or The nucleic acid sequence of the present invention is at least 90% identical to the nucleic acid sequence of the present invention.

[0094] In some embodiments, the polyadenylation signal is an SV40 polyadenylation signal. In some embodiments, the polyadenylation signal is the rBG polyadenylation signal. In some embodiments, the polyadenylation signal is SEQ ID NO: 22 or SEQ ID NO: In some embodiments, the polyadenylation signal comprises the sequence of SEQ ID NO: 22 or The sequence of SEQ ID NO: 23 is at least 90%, at least 95%, at least 96%, or at least The sequences may be at least 97%, at least 98%, or at least 99% identical.

[0095] (v) Stuffer array AAV vectors are generally about 4 kb to about 5.2 kb, or slightly larger. They typically accept DNA inserts with a defined size range. For shorter sequences, to achieve the required length acceptable for AAV vectors, It may be necessary to include additional nucleic acid within the insert. Thus, in some embodiments Alternatively, the AAV expression cassette of the present disclosure may comprise a stuffer sequence. Columns can be, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-6 0, 60-75, 75-100, 100-150, 150-200, 200-250, 2 50-300, 300-400, 400-500, 500-750, 750-1,000 , 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,5 00-3,000, 3,000-3,500, 3,500-4,000, 4,000-4 ,500, or sequences between 4,500 and 5,000 nucleotides or longer The stuffer sequence may be any sequence that does not interfere with the function or activity of the vector. The stuffer may be located within the cassette at any predetermined position. For example, in some embodiments, a stuffer sequence is located upstream of the 3'UTR. The columns indicate the region between the BDNF short 3'UTR and 3'ITR, or between the bGH polyA signal and the 3'ITR. 'It exists between ITR.

[0096] In some embodiments, the AAV cassette comprises at least one stuffer sequence. In some embodiments, the stuffer sequence comprises a nucleic acid sequence of SEQ ID NO: 13 and at least 7 0% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99%, at least In some embodiments, the nucleic acid sequence includes a nucleic acid sequence having at least 99.5%, or 100% identity to the nucleic acid sequence. The stuffer sequence may be the nucleic acid sequence of SEQ ID NO: 13 or at least the nucleic acid sequence of SEQ ID NO: 13. The sequences contain sequences that are at least 90% identical.

[0097] (vi) intron sequences In some embodiments, the AAV expression cassettes of the present disclosure may include intron sequences. The inclusion of an intron sequence enhances expression compared to expression in the absence of the intron sequence. possible.

[0098] In some embodiments, the intron sequence is a hybrid or chimeric sequence. In some embodiments, the intron sequence is selected from the group consisting of SV40, β-globin, chicken beta- Actin, minute virus of mice (MVM), factor IX, and / or human IgG (heavy isolated from or derived from one or more intron sequences of the human ovarian or human bronchial In some embodiments, the intron sequence is chimeric. The intron sequence comprises the sequence of SEQ ID NO: 24 or SEQ ID NO: 25. The intron sequence has at least 70% identity with the nucleic acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. Identity (e.g., at least 75%, including all values ​​and subranges therebetween) , at least 80%, at least 85%, at least 90%, at least 95%, less At least 96%, at least 97%, at least 98%, at least 99%, at least 99 The present invention includes nucleic acid sequences having at least 0.5%, or 100% identity to the sequence.

[0099] (vii) Exemplary AAV Expression Cassettes In some embodiments, the AAV expression cassette comprises a 5' inverted repeat repeat (ITR), a transgene, In some embodiments, the AAV expression cassette comprises a 3' ITR, a 3' ITR, and a 3' ITR. (5'ITR), SEQ ID NO:7 (BDNF) and SEQ ID NO:2 (3'ITR).

[0100] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a transgene, , and a 3' ITR. In some embodiments, the AAV expression cassette comprises SEQ ID NO: 1 ( 5'ITR), SEQ ID NO: 6 (hSARE-ArcMin), SEQ ID NO: 7 (BDNF) and and SEQ ID NO:2 (3'ITR). In some embodiments, the AAV expression cassette comprises the sequence SEQ ID NO: 1 (5'ITR), SEQ ID NO: 16 (E-SARE), SEQ ID NO: 7 (BDNF), and Contains SEQ ID NO:2 (3'ITR).

[0101] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a transgene, In some embodiments, the AAV expression cassette comprises a nucleotide sequence, a polyadenylation sequence, and a 3' ITR. The sequences are SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE-ArcMin), SEQ ID NO: 7 (BDNF), SEQ ID NO: 9 (bGH polyadenylation sequence) and SEQ ID NO: 2 (3'IT In some embodiments, the AAV expression cassette comprises SEQ ID NO: 1 (5'ITR), SEQ ID NO: 16 (E-SARE), SEQ ID NO: 7 (BDNF), SEQ ID NO: 9 (bGH polyadenylation) ionization sequence) and SEQ ID NO: 2 (3' ITR).

[0102] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a Kozak sequence, In some embodiments, the AAV expression cassette comprises a nucleotide sequence, a transgene, and a 3' ITR. , SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE-ArcMin), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BDNF), and SEQ ID NO: 2 (3'ITR In some embodiments, the AAV expression cassette comprises SEQ ID NO: 1 (5'ITR), SEQ ID NO: 2 (5'ITR), SEQ ID NO: 3 (5'ITR), SEQ ID NO: 4 (5'ITR), SEQ ID NO: 5 (5'ITR), SEQ ID NO: 6 (5'ITR), SEQ ID NO: 7 (5'ITR), SEQ ID NO: 8 (5'ITR), SEQ ID NO: 9 (5'ITR), Sequence number 16 (E-SARE), sequence number 14 or 15 (Kozak sequence), sequence number 7 ( BDNF), and SEQ ID NO: 2 (3'ITR).

[0103] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a Kozak sequence, In some embodiments, the A The AV expression cassette is SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE-ArcMi n), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BDNF), SEQ ID NO: 9 (bGH polyadenylation sequence) and SEQ ID NO:2 (3'ITR). In the AAV expression cassette, SEQ ID NO: 1 (5'ITR), SEQ ID NO: 16 (E-SARE), ), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BDNF), SEQ ID NO: 9 ( bGH polyadenylation sequence) and SEQ ID NO:2 (3'ITR).

[0104] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a transgene, In some embodiments, the AAV expression cassette comprises a sequence Sequence number 1 (5'ITR), sequence number 6 (hSARE-ArcMin), sequence number 7 (BD NF), SEQ ID NO:8 (BDNF short 3'UTR), and SEQ ID NO:2 (3'ITR) In some embodiments, the AAV expression cassette comprises SEQ ID NO: 1 (5'ITR), the sequence SEQ ID NO: 16 (E-SARE), SEQ ID NO: 7 (BDNF), SEQ ID NO: 8 (BDNF short 3 In some embodiments, the AAV expression vector comprises SEQ ID NO: 1 (3'ITR), SEQ ID NO: 2 (4'ITR), and SEQ ID NO: 3 (5'ITR). The cassettes consisted of SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE-ArcMin), SEQ ID NO: Sequence number 7 (BDNF), sequence number 10 (BDNF long 3'UTR), and sequence number 2 (3' ITR). In some embodiments, the AAV expression cassette comprises SEQ ID NO:1 (5' ITR), SEQ ID NO: 16 (E-SARE), SEQ ID NO: 7 (BDNF), SEQ ID NO: 10 (B DNF long 3'UTR), and SEQ ID NO: 2 (3'ITR).

[0105] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a transgene, , a 3' UTR, a polyadenylation sequence and a 3' ITR. The V expression cassette is SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE-ArcMin ), SEQ ID NO: 7 (BDNF), SEQ ID NO: 8 or 10 (BDNF short or long 3'UTR), SEQ ID NO: 9 (bGH polyadenylation sequence) and SEQ ID NO: 2 (3'ITR In some embodiments, the AAV expression cassette comprises SEQ ID NO: 1 (5'ITR), SEQ ID NO: 2 (5'ITR), SEQ ID NO: 3 (5'ITR), SEQ ID NO: 4 (5'ITR), SEQ ID NO: 5 (5'ITR), SEQ ID NO: 6 (5'ITR), SEQ ID NO: 7 (5'ITR), SEQ ID NO: 8 (5'ITR), SEQ ID NO: 9 (5'ITR), Sequence number 16 (E-SARE), sequence number 7 (BDNF), sequence number 8 or 10 (BDNF) F short or long 3'UTR), SEQ ID NO: 9 (bGH polyadenylation sequence) and and SEQ ID NO: 2 (3'ITR).

[0106] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a Kozak sequence, In some embodiments, the AAV expression vector comprises a transgene, a 3' UTR, and a 3' ITR. The cassettes consisted of SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE-ArcMin), SEQ ID NO: Sequence number 14 or 15 (Kozak sequence), sequence number 7 (BDNF), sequence number 8 or 1 0 (BDNF short or long 3'UTR), and SEQ ID NO: 2 (3'ITR) In some embodiments, the AAV expression cassette comprises SEQ ID NO: 1 (5'ITR), SEQ ID NO: No. 16 (E-SARE), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BD NF), SEQ ID NO: 8 or 10 (BDNF short or long 3'UTR), and Contains SEQ ID NO:2 (3'ITR).

[0107] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a Kozak sequence, The sequence includes the transgene, 3'UTR, polyadenylation sequence, and 3'ITR. In this embodiment, the AAV expression cassette comprises SEQ ID NO: 1 (5'ITR), SEQ ID NO: 6 (hSARE -ArcMin), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BDNF) , SEQ ID NO: 8 or 10 (BDNF short or long 3'UTR), SEQ ID NO: 9 ( bGH polyadenylation sequence) and SEQ ID NO:2 (3' ITR). The AAV expression cassette is SEQ ID NO: 1 (5'ITR), SEQ ID NO: 16 (E-SARE) , SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BDNF), SEQ ID NO: 8 or SEQ ID NO: 10 (BDNF short or long 3'UTR), SEQ ID NO: 9 (bGH polyadenylation It contains SEQ ID NO: 2 (3'ITR) and SEQ ID NO: 3 (4'ITR).

[0108] In some embodiments, the AAV expression cassette comprises a 5' ITR, a stuffer sequence, a promoter sequence, and a Transformer, Kozak sequence, transgene, 3' untranslated region (3'UTR), polyadenylation sequence In some embodiments, the AAV expression cassette comprises the 3' ITR and the 3' ITR. 5'ITR), SEQ ID NO: 13 (stuffer sequence), SEQ ID NO: 6 (hSARE-ArcM in), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (BDNF), SEQ ID NO: 8 or 10 (BDNF short or long 3'UTR), SEQ ID NO: 9 (bGH poly adenylation sequence) and SEQ ID NO: 2 (3' ITR). The expression cassette contains SEQ ID NO: 1 (5'ITR), SEQ ID NO: 13 (stuffer sequence), SEQ ID NO: SEQ ID NO: 16 (E-SARE), SEQ ID NO: 14 or 15 (Kozak sequence), SEQ ID NO: 7 (B DNF), SEQ ID NO: 8 or 10 (BDNF short or long 3'UTR), SEQ ID NO: Contains sequence number 9 (bGH polyadenylation sequence) and sequence number 2 (3'ITR).

[0109] In some embodiments, the AAV expression cassette comprises a 5' ITR, a promoter, a Kozak sequence, sequence, transgene, 3' untranslated region (3'UTR), polyadenylation sequence, stuffer sequence In some embodiments, the AAV expression cassette comprises the 3' ITR and the 3' ITR. 5'ITR), SEQ ID NO: 6 (hSARE-ArcMin), SEQ ID NO: 14 or 15 ( SEQ ID NO: 7 (BDNF), SEQ ID NO: 8 or 10 (BDNF short or or long 3'UTR), SEQ ID NO: 9 (bGH polyadenylation sequence), SEQ ID NO: 13 (sequence Taffer sequence), and SEQ ID NO: 2 (3'ITR). The V expression cassette is composed of SEQ ID NO: 1 (5'ITR), SEQ ID NO: 13 (stuffer sequence), SEQ ID NO: Sequence number 16 (E-SARE), sequence number 14 or 15 (Kozak sequence), sequence number 7 ( BDNF), SEQ ID NO: 8 or 10 (BDNF short or long 3'UTR), SEQ ID NO: 9 (bGH polyadenylation sequence), SEQ ID NO: 13 (stuffer sequence), and SEQ ID NO: Contains sequence number 2 (3'ITR).

[0110] AAV production methods The AAV expression cassettes described herein can be expressed in vectors using standard molecular biology techniques. The vector may be incorporated into a vector (e.g., a plasmid or bacmid). The present invention provides a vector comprising any one of the AAV expression cassettes described herein. The vector (e.g., a plasmid or bacmid) contains, for example, AAV rep and cap genes, as well as helper virus protein sequences used during AAV production It may further comprise one or more genetic elements.

[0111] AAV expression cassettes and vectors comprising AAV expression cassettes as described herein - (e.g., plasmids) may be used to produce recombinant AAV vectors.

[0112] The present disclosure provides a method for transfecting AAV-producing cells (e.g., HEK293 cells) with an AAV expression cassette of the present disclosure. a recombinant AAV vector, including contacting the vector with a recombinant AAV vector (e.g., a plasmid) The present disclosure provides a method for producing an AAV expression cassette as disclosed herein. In some embodiments, the method further provides a cell comprising any one of the methods, The method involves transfecting AAV-producing cells with, for example, AAV rep and cap genes, as well as helper - contacting one or more additional plasmids encoding viral protein sequences In some embodiments, the method for producing a recombinant AAV vector further comprises: AAV-producing cells (e.g., insect cells such as Sf9 cells) are transfected with the AAV expression cassette of the present disclosure. "Insect cell compatible" includes contacting the insect with at least one insect cell compatible vector containing the vector. "Insect vector" refers to a vector that facilitates transformation or transfection of insect cells with nucleic acid. In some embodiments, the compound or agent is any compound or agent (biological or chemical) that The insect cell-compatible vector is a baculovirus vector. The method further comprises maintaining the insect cells under conditions such that the AAV is produced.

[0113] The present disclosure also provides recombinant A produced using any one of the methods disclosed herein. The recombinant AAV vectors produced can be of any serotype, e.g., , AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, A It may be of AVrh32.33, AAVrh74, avian AAV or bovine AAV. In some embodiments, the recombinant AAV vector produced contains a naturally occurring AAV capsid. containing one or more amino acid modifications (e.g., substitutions and / or deletions) compared to For example, the recombinant AAV vector may be AAV1, AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, A AV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, Modified AAV vectors derived from human AAV and bovine AAV may also be used. In embodiments, the recombinant AAV vector is a single-stranded AAV (ssAAV). In embodiments, the recombinant AAV vector is a self-complementary AAV (scAAV).

[0114] In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, and TriA In some embodiments, the AAV vector comprises a capsid protein of a bovine AAV or a bovine AAV. - contains one or more substitutions or mutations compared to the wild-type AAV capsid protein The recombinant AAV vectors disclosed herein contain capsid proteins having the following structure: For example, the transgene can be delivered by contacting a recombinant AAV vector with a target cell. The daughter sequences may be used to transduce target cells.

[0115] Expression and Treatment Methods The present disclosure relates to the nucleic acids, AAV expression cassettes, plasmids, cells, or provides a composition comprising any one of the recombinant AAV vectors. The compositions disclosed herein may comprise at least one pharmaceutically acceptable carrier, excipient, and / or vehicles, such as solvents, buffers, solutions, dispersion media, coatings, Antibacterial and antifungal agents, isotonic and absorption delaying agents. Acceptable carriers, excipients, and / or vehicles include saline, buffered saline, dextromethorphan, and the like. Contains cellulose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. In some embodiments, a pharmaceutically acceptable carrier, excipient, and / or vehicle may be used. Kuru contains phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate , dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grade mannitol , lactose, starch, magnesium stearate, sodium saccharin, cellulose magnesium carbonate, polyols (e.g., glycerol, propylene glycol, and and liquid polyethylene glycol, etc.) or suitable mixtures thereof. Therefore, the compositions disclosed herein can contain minor amounts of emulsifying or wetting agents, or pH buffering agents. Further includes:

[0116] In some embodiments, the compositions disclosed herein do not contain a preservative, or chlorobutanol. ethanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, paraben, ethyl vanillin, glycerin, phenol, parachlorophenol or albumin In some embodiments, the compositions disclosed herein further include other conventional pharmaceutical ingredients, such as chemical stabilizers. The compositions shown are free of parabens, chlorobutanol, phenol, sorbic acid or thimerosin. antibacterial and antifungal agents such as Rosal; isotonic agents such as sugars or sodium chloride; and / or may further contain absorption delaying agents such as aluminum monostearate and gelatin. Good too.

[0117] The present disclosure provides for the preparation of cells containing the nucleic acids, AAV expression cassettes, plasmids, and / or compositions disclosed herein. or a composition containing the recombinant AAV vector. In some embodiments, the cell is a cell line. In some embodiments, the cells are non-dividing cells, such as cultured cells in cell culture. In some embodiments, the Rett syndrome associated gene is delivered to cells in vitro. For example, in vitro or ex vivo gene therapy for Rett syndrome Produces a disease-associated polypeptide.

[0118] In some embodiments, the Rett syndrome associated gene is delivered to a subject in need thereof, For example, to express an immunogenic or therapeutic polypeptide. The peptide or functional RNA can be produced in vivo in a subject. Thus, the present disclosure provides a method for administering to a subject a therapeutically effective amount of a nucleic acid, an AAV gene, or a combination thereof, as disclosed herein. any one of the present cassette, plasmid, cell, recombinant AAV vector, or composition and administering to a subject in need thereof a method for expressing a Rett syndrome-associated gene, the method comprising administering to a subject in need thereof a method for expressing a Rett syndrome-associated gene. The present disclosure also provides a method for treating at least one symptom of Rett Syndrome in a subject. to treat and / or delay the onset of at least one symptom of Rett Syndrome In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a compound according to the present invention. Nucleic acids, AAV expression cassettes, plasmids, cells, and recombinant AAV vectors disclosed herein In some embodiments, the subject is administered one of: In some embodiments, the subject has Rett Syndrome. For example, the subject may be a newborn who has been identified as having a mutation in the mecp2 gene. In some embodiments, the Rett syndrome associated gene is targeted by gene therapy to In some embodiments, the expression and / or function of a Rett syndrome-associated gene is increased. The gene can be targeted by gene therapy to reduce its expression and / or function.

[0119] The present disclosure provides a method for administering to a subject a therapeutically effective amount of a nucleic acid, an AAV expression cassette, or a combination thereof, as disclosed herein. , a plasmid, a cell, a recombinant AAV vector, or a composition. and / or administering to a subject a MECP2-associated disease, comprising administering to a subject a MECP2-associated As used herein, "MECP2-related" refers to a method for delaying the onset of a MECP2-related disease. "Mecp2-associated diseases" are characterized by genetic alterations to the mecp2 gene compared to the wild-type mecp2 gene. (e.g., one or more deletions, insertions, duplications and / or substitutions), and / or The expression and / or expression of MECP2 protein is decreased compared to wild-type MECP2 protein. is a disease that correlates with or is caused by an alteration to activity.

[0120] In some embodiments, the MECP2-associated disease is MECP2 duplication syndrome, MECP2-associated Severe neonatal encephalopathy, PPM-X syndrome, or Rett syndrome. MECP2 duplication syndrome. is caused by duplication of the MECP2 gene and leads to intellectual disability, developmental delay, and seizures. Duplication of the MECP2 gene leads to excessive production of MECP2 protein, and increased protein function, resulting in abnormal neuronal function. MECP2-related severe neonatal encephalopathy is caused by mutations in the mecp2 gene. Most are single base pair insertions, deletions, or substitutions. The condition affects almost exclusively males. and is characterized by small head size (microcephaly), movement disorders, breathing problems, and seizures. Mutations in the mecp2 gene alter the structure of the MECP2 protein or inhibit its production. PPM-X syndrome can range from mild to severe. It is a disorder characterized by intellectual disability, bipolar disorder, and a pattern of movement disorders. Approximately half of all cases of PM-X syndrome are caused by one of eight mutations in the mecp2 gene. These mutations are caused by insertions and deletions of amino acids in the MECP2 protein. or causes a substitution or generates a premature termination signal in the mecp2 mRNA. do.

[0121] The present disclosure provides a method for administering to a subject a therapeutically effective amount of a nucleic acid, an AAV expression cassette, or a combination thereof, as disclosed herein. , a plasmid, a cell, a recombinant AAV vector, or a composition. and / or administering to a subject a BDNF-related disorder, comprising administering to said subject a BDNF-related disorder. Further provided are methods for delaying the onset of BDNF-related diseases. "BDNF-associated disorders" are characterized by genetic alterations to the bdnf gene and / or the BDNF protein. Diseases that are correlated with or caused by alterations in the expression and / or activity of In some embodiments, the BDNF-related disorder is a cognitive impairment and / or stress-related disorder. In some embodiments, the BDNF-related disorder is depression, obsessive-compulsive disorder, Alzheimer's disease, or a combination thereof. Marger's disease, Huntington's disease, dementia, anorexia nervosa, bulimia nervosa, schizophrenia, seizures post-traumatic stress disorder, bipolar disorder, Rett syndrome, major depressive disorder, or chemotherapy Post-cognitive impairment.

[0122] In some embodiments, the Rett syndrome associated gene is expressed in neurons of the subject. In some embodiments, the expression level of the Rett syndrome associated genes is higher in the brain than in non-neuronal cells of the body. In some embodiments, expression of Rett syndrome-associated genes is higher in neurons than in non-neurons. In some embodiments, expression of Rett syndrome associated genes is undetectable in human cells. The present level is at least about 1.2 times higher than that of non-neuronal cells of the body (e.g., Approximately 1.5x, approximately 2x, approximately 2.5x, approximately 3x, including all values ​​and subranges present in , about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times , about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, about 15 times, about 20 times times, approximately 30 times, approximately 40 times, approximately 50 times, approximately 60 times, approximately 70 times, approximately 80 times, approximately 90 times, or approximately 100-fold), and is higher in neurons.

[0123] In some embodiments, the neuron is an active neuron. Expression levels of TT-related genes are higher in active neurons than in resting neurons of the body. In some embodiments, expression of Rett syndrome associated genes is elevated in quiescent neurons. In some embodiments, the expression level of a Rett syndrome associated gene is undetectable. At least about 1.2 times (e.g., all values ​​in between) and partial ranges, including approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 times, approximately 5 times, approximately 5.5 times, approximately 6 times, approximately 6.5 times, approximately 7 times, approximately 7.5 times, approximately 8 times, approximately 8.5x, approx. 9x, approx. 9.5x, approx. 10x, approx. 15x, approx. 20x, approx. 30x, approx. 40x, Approximately 50 times, approximately 60 times, approximately 70 times, approximately 80 times, approximately 90 times, or approximately 100 times), active neurotransmitter High in Ron.

[0124] In some embodiments, the Rett syndrome associated gene is a central nervous system (CNS) neurotransmitter of the subject. In some embodiments, the expression level of a Rett syndrome associated gene is , at least about 1.2 times greater than non-neuronal cells of the body (e.g., Approximately 1.5x, approximately 2x, approximately 2.5x, approximately 3x, approximately 3.5x, including all values ​​and subranges times, approximately 4 times, approximately 4.5 times, approximately 5 times, approximately 5.5 times, approximately 6 times, approximately 6.5 times, approximately 7 times, approximately 7.5 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 30x 1x, approximately 40x, approximately 50x, approximately 60x, approximately 70x, approximately 80x, approximately 90x, or approximately 100x ), high in CNS neurons.

[0125] In some embodiments, the expression levels of Rett syndrome associated genes are measured in the peripheral nervous system (P It is higher in CNS neurons than in other non-CNS neurons, such as CNS (Central Neural Network) neurons. In some embodiments, expression of Rett syndrome associated genes is detected in PNS neurons. In some embodiments, the expression levels of Rett syndrome associated genes are determined by measuring the expression levels of the genes in the body. at least about 1% of CNS neurons, such as peripheral nervous system (PNS) neurons. .2 times (e.g., about 1.5 times, about 2 times, including all values ​​and subranges in between) 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x times, approximately 6.5 times, approximately 7 times, approximately 7.5 times, approximately 8 times, approximately 8.5 times, approximately 9 times, approximately 9.5 times, approximately 1 0x, approx. 15x, approx. 20x, approx. 30x, approx. 40x, approx. 50x, approx. 60x, approx. 70x, approx. 8 0-fold, approximately 90-fold, or approximately 100-fold), and higher in CNS neurons.

[0126] The dosage of the recombinant AAV vector to be administered to a subject depends on the method of administration, the treatment and / or the disease or condition to be prevented, the condition of the individual subject, the specific viral vector or The amount of the capsid depends on the nucleic acid to be delivered, and can be determined in a conventional manner. An exemplary dosage to achieve a therapeutic effect is at least about 10 5 , about 10 6 ,about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 1 0 14 , about 10 15 Transducing units, optionally about 10 8 ~about 10 13 Transducing unit power It is a price.

[0127] In certain embodiments, multiple administrations (e.g., two, three, four or more administrations) are utilized to A desired level of genetic activity can be achieved over a variety of intervals, e.g., daily, weekly, monthly, yearly, etc. Expression may be achieved.

[0128] Exemplary methods of administration include oral, rectal, transmucosal, intranasal, and inhalation (e.g., by aerosol). via the oral cavity (e.g., sublingually), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraembryonic) ), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [skeletal muscle, diaphragm, and / or cardiac muscle] administration to the surface of the respiratory tract, including intradermal, intrapleural, intracerebral, and intra-articular administration, both to the skin and mucosal surfaces, including the skin, and transdermally), intralymphatic, etc., as well as direct Tissue or organ injections (e.g., liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain) In some embodiments, administration is by injection into the central nervous system. In some instances, the most suitable route is the administration of It depends on the nature and severity, as well as the nature of the particular vector being used.

[0129] Delivery to target tissues can also involve the use of depots containing viral vectors and / or capsids. In an exemplary embodiment, this can also be achieved by delivery of a viral vector. and / or capsid-containing depots in skeletal muscle, cardiac muscle, and / or diaphragm muscle tissue. Implant or tissue containing a film containing viral vectors and / or capsids or other substrate.

[0130] In some embodiments, the methods disclosed herein comprise administering to a subject a therapeutically effective amount of a compound or compound(s) described herein. Nucleic acids, AAV expression cassettes, plasmids, cells, and recombinant AAV vectors disclosed therein or any one of the compositions, in combination with one or more second-line therapies targeting Rett Syndrome. In some embodiments, the present invention may include administering the compound disclosed herein in combination with treating at least one symptom of Rett Syndrome in a subject, and / or The method for delaying the onset of Rett Syndrome comprises administering one or more secondary therapies to treat Rett Syndrome. In some embodiments, the secondary therapy may further comprise administering glatiramer acetate. Further details regarding the use of glatiramer acetate can be found in Djukic A et al. al., Pediatr Neurol.2016 Aug;61:51-7 No. 6,239,999, the contents of which are incorporated herein by reference in their entirety.

[0131] In some embodiments, the secondary therapy includes administration of a drug to treat the seizures, In some embodiments, the secondary therapy comprises administering a drug to treat the onset of Non-limiting examples of medications for treating seizures include: These include levetiracetam, valproic acid, oxcarbazepine, and lamotrigine. In some embodiments, the secondary therapy comprises administering a drug to treat reflux. Non-limiting examples of drugs for treating include cimetidine, ranitidine, nizatidine, and and H2 blockers such as famotidine, omeprazole, esomeprazole, and lansoprazole. Proprietors such as rabeprazole, pantoprazole, and dextran. ATP pump inhibitors, as well as low-dose erythromycin, benzamide, domperidone, and and motility agents such as linaclotide.

[0132] As used herein, the term "administered in combination with" refers to a Two (or more) different treatments may be administered to treat a disorder (e.g., "Rett Syndrome" is understood to mean that the drug is delivered to a subject during the course of the subject's suffering from the drug. In certain embodiments, delivery of one therapy is initiated when delivery of a second therapy begins. This often occurs, resulting in overlap in administration. "simultaneous" or "concurrent" delivery In other embodiments, delivery of one treatment begins before delivery of the other treatment begins. In the end, this can be called "continuous" delivery.

[0133] In some embodiments, the treatments are more effective due to the combined administration. For example, the second treatment is , more effective, e.g., equivalent effects are seen with fewer second treatments, or The second treatment may be administered in the absence of the first treatment or under similar circumstances. The condition reduces symptoms to a greater extent than is seen with the first treatment. The effects of two treatments may be partially additive, totally additive, or greater than additive (synergistic). That's fine.

[0134] All articles, publications and patents cited herein are the property of their respective authors. , whether the publication or patent is specifically and individually indicated to be incorporated by reference As such, methods and are incorporated herein by reference to disclose and describe the methods and / or materials used in the present invention. While any references, articles, publications, patents, patent publications, and References in patent applications are not to be construed as constituting valid prior art in any country in the world or as being or form part of the common general knowledge. It cannot and should not be.

[0135] Unless the context indicates otherwise, the various features described herein may be used in any combination. It is specifically contemplated that the present invention may be used in conjunction with other methods.

[0136] Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present disclosure. The terms have the same meaning as commonly understood by a person skilled in the art to which they pertain.

[0137] The above description and the following examples are intended to illustrate, but not limit, the scope of the invention. It should be understood that this is not intended to limit the scope of the present invention. Modifications and variations will be apparent to those skilled in the art to which the present invention pertains. [Example]

[0138] The following examples are included herein for illustrative purposes only and are not intended to be limiting. It is not something that can be done.

[0139] Example 1: Preparation of AAV expression cassettes Activity-dependent h flanked by the 5'ITR (SEQ ID NO: 1) and 3'ITR (SEQ ID NO: 2) SARE-hArcMin promoter (SEQ ID NO: 6) and BDNF gene (SEQ ID NO: Three AAV expression cassettes containing the nucleotide sequences (nucleotides 7 and 8) were generated using standard cloning techniques. See Figures 1-3 for a schematic representation of the three cassettes. The first cassette contains the BDNF shot. The second sequence contains a 3' UTR (SEQ ID NO: 8) and a stuffer sequence (SEQ ID NO: 13). The cassette contains the bGH polyA signal (SEQ ID NO: 9) and stuffer sequence (SEQ ID NO: 1 3). The third cassette contains the BDNF long 3'UTR (SEQ ID NO: 10). The two AAV expression cassettes are SEQ ID NO: 3 (FIG. 1), SEQ ID NO: 4 (FIG. 2), and and the nucleic acid sequence of SEQ ID NO: 5 (Figure 3).

[0140] Example 2: Preparation of recombinant AAV vectors in mammalian cells Each of the three AAV expression cassettes was incorporated into a plasmid to produce three AAV expression cassettes. Three plasmids containing each of the sets are obtained. The Rep / Cap plasmid encoding the p and Cap genes, and the AAV production (E4 along with a helper plasmid containing various helper sequences required for the expression of the vector (E2a, E2b, and VA). Use an appropriate transfection reagent (e.g., Lipofectamine™). The virus-producing cells (e.g., HEK293) are transfected with the vector. After incubation at 37°C for 1 h, AAV particles were collected from the medium and the cells were lysed. The AAV particles are then purified, titered, and used later. Therefore, it may be stored at -80°C.

[0141] Example 3: Preparation of recombinant AAV vectors in insect cells Each of the three AAV expression cassettes was incorporated into a baculovirus vector. Obtain three baculovirus vectors containing one AAV expression cassette each. Cells (e.g., Sf9) were transfected with each of the three baculovirus vectors, as well as AA V at least one further sequence comprising sequences encoding the Rep and Cap proteins The recombinant baculovirus vectors are co-infected in suspension culture. After incubation at 28°C, AAV particles were collected from the medium, the cells were lysed, and AAV was added to the medium. The AAV particles are then purified, titered, and purified for further use. It may be stored at -80°C.

[0142] Example 4: The hSARE-hArcMin promoter mediates activity-dependent reporter gene expression Bringing reality The hSARE-hArcMin promoter is inherited in a neuronal activity-dependent manner. To verify whether expression of the gene can be induced, the following experiment was carried out.

[0143] Neuron (hSyn; human synapsin 1 gene promoter, comprising the nucleic acid sequence of SEQ ID NO: 38) Constitutive or activity-dependent promoters that provide long-term expression in the An AAV expression cassette containing hSARE-hArcMin (SEQ ID NO: 6) was transfected into the AAV using standard These were generated using cloning techniques (see Table 3). Figures 11A-11C show schematic diagrams of the three cassettes. TR sequence: BDNF short 3'UTR (SEQ ID NO: 8), bGH polyA signal ("bG HpA" or "bGH" (SEQ ID NO: 9), or BDNF long 3' The cassette also contains one of the constitutive or activity-dependent promoters. The reporter gene comprises a reporter gene operably linked to a monomeric red Fluorescent protein (mScarlet, encoded by the nucleic acid sequence of SEQ ID NO: 36), or m A destabilized version of Scarlet (dmScarlet, the nucleic acid sequence of which is SEQ ID NO: 35) The destabilized version of mScarlet encodes either mS Promotes faster turnover due to proteasomal degradation compared to carlet and a C-terminal PEST degron signal (encoded by the nucleic acid sequence of SEQ ID NO: 37) Therefore, dmScarlet fluorescence is expressed to a lesser extent in cells than mScarlet fluorescence. accumulates within. [Table 3]

[0144] Recombinant AAVs containing AAV expression cassettes 1, 2, 3, 7, 8, and 9 listed in Table 3 The V vector was prepared as described herein. Wild-type mouse primary neurons were transfected with this The transduced cells were cultured in 2 μM NaCl solution. The cells were treated overnight with tetrodotoxin (TTX), a thorium channel inhibitor. The treatment used inhibits neuronal activity. After that, the medium was completely aspirated and the following was performed: ) medium only, (b) medium + TTX, (c) medium + 150 mM KCl, or (d) medium + 30 μM bicuculline (BIC). While depolarizing, BIC acts as a competitive antagonist of γ-aminobutyric acid type A (GABAA) receptors. Therefore, either KCl or BIC is a neurotransmitter that induces neuronal disinhibition. Treating neurons with either of these promotes neuronal activity. Control neurons in the control group (according to theory (a)) are neither inhibited nor activated, remaining quiescent, while Inhibit neuronal activity in TTX-containing medium (as per treatment (b) above) The cells were further incubated for 2 hours at 37°C. The cells were then fixed and stained with Hoechst. 33342 and imaged using a fluorescent microscope.

[0145] Figure 5A shows the fluorescence of TTX compared to that of cells treated with 2 μM TTX, normalized to 1 μM. Cells not treated with TX (i.e., cells in which neuronal activity was neither stimulated nor inhibited) As shown in Figure 5A, the reporter protein fluorescence in the nuclei of the neurons was , when neither promoted nor inhibited, dm using the hSARE-hArcMin promoter. The relative fluorescence of cells transduced with AAV vectors leading to Scarlet expression was measured at constitutive The target gene was expressed in an AAV vector using the hSyn promoter to drive mScarlet expression. Equivalent to the relative fluorescence of transduced cells.

[0146] Figure 5B shows the fluorescence of TTX compared to that of cells treated with 2 μM TTX, normalized to 1 μM. Cells not treated with TX but instead treated with 150 mM KCl (i.e., nuclei) Figure 5C shows reporter protein fluorescence in cells in which the activity of IL-1 is promoted. is the fluorescence of cells treated with TTX compared to that of cells treated with 2 μM TTX, normalized to 1. Cells that were not treated with 30 μM bicuculline (BIC) instead (i.e., Figure 1 shows reporter protein fluorescence in cells in which neuron activity is promoted.

[0147] As shown in Figures 5B and 5C, neuronal activity was significantly increased using KCl or BIC treatment. When driven by the activity-dependent promoter hSARE-hArcMin, The relative fluorescence of cells transduced with AAV vectors leading to dmScarlet expression was The AAV vector used to drive mScarlet expression uses a constitutive promoter (hSyn). The relative fluorescence of cells transduced with the vector was higher.

[0148] These results suggest that the hSARE-hArcMin promoter regulates the expression of GABA-dependent genes in neurons. Furthermore, Figures 5B and 5C show that the α-glucanase gene can induce gene expression in a manner that is consistent with the α-glucanase gene. As shown in Figure 1, dmScarlet constructs containing bGH or long 3'UTRs were expressed in hSA It was similarly expressed by the RE-hArcMin promoter.

[0149] The activity-dependent expression of the hSARE-hArcMin promoter is further illustrated in Figures 6A-B. As shown in Figure 6A, the constitutive promoter hSyn is upregulated in the presence of TTX. Regardless, this results in high levels of mScarlet expression. The cells were not treated with TTX, but instead with 30 μM BIC, resulting in neuronal When the hSARE-hArcMin promoter stimulated the activity of the ribosomal ATPase, it was found that the hSARE-hArcMin promoter induced high levels of reporter gene expression. In particular, Figure 6B shows that the long 3'UTR in combination with the 3'UTR results in the expression of the target gene. The AAV expression construct containing the hSARE-hArcMin promoter was used to express bGHpA and Compared to an AAV expression construct containing the hSARE-hArcMin promoter in combination, showed that the presence of TTX resulted in approximately 5-fold higher levels of dmScarlet expression. These results demonstrate that the hSARE-hArcMin promoter is neuronal activity-dependent. The derivation is further shown.

[0150] Constitutively express hSyn to determine the subcellular localization and levels of the expressed fluorescent protein Reporter under promoter or activity-dependent hSARE-hArcMin promoter Neurons expressing the gene were observed using a microscope.

[0151] As shown in Figure 7, mScarlet was expressed using the constitutive promoter hSyn. The expressing cells were fluorescent, but the level of fluorescence was higher than that of the 3'UTR region of the expression cassette. or polyadenylation signal. For example, expression of genes containing long 3'UTRs The cassette results in higher levels of mScarlet gene expression and is The expression cassette containing the short 3'UTR was followed by the expression cassette containing the short 3'UTR. In contrast, activity-dependent hSARE expression was observed. Expression of reporter proteins under the -hArcMin promoter was achieved using the 3'UT In particular, Figures 5 and 6 show that the 3′UT An AAV expression construct containing the hSARE-hArcMin promoter in combination with R The presence of neuronal activity resulted in similar levels of dmScarlet expression. Shows.

[0152] Furthermore, Figure 7 shows that the intracellular localization of mScarlet expressed from a constitutive promoter is It was shown that the expression cassettes varied based on the 3'UTR region or polyadenylation signal. The expression cassette containing the long 3'UTR and bGHpA was expressed in the cell body and neurites. While expression cassettes containing short 3'UTRs resulted in mScarlet expression in resulted in mScarlet expression only in the cell body.

[0153] As shown in Figures 8 and 12, bGHpA (Figure 8) or long 3'UTR (Figure 12) AAV expression constructs containing the combined hSARE-hArcMin promoters were used to express the nuclease. The presence of agonist activity resulted in similar levels of dmScarlet expression. Furthermore, we used the activity-dependent hSARE-hArcMin promoter to express dmSca rlet-expressing cells were observed in the absence of TTX and the presence of BIC. The fluorescent signals in the cell body and neurites during the endothelial cell division are shown.

[0154] In summary, the above results indicate that the synthetic hSARE-hArcMin promoter is capable of inhibiting or Higher levels in active neurons compared to quiescent neurons Therefore, the synthetic hSARE-hArcMin promoter The motor can be used to transduce any target gene, such as a Rett syndrome-associated gene, as described herein. This can result in neuronal activity-dependent expression of the gene.

[0155] Example 5: Characterization of AAV expression cassettes containing BDNF The AAV particles prepared in Example 2 or 3 were used to treat m Administered by injection into ecp2 mutant mice, the activity-dependent promoter hSARE - Expression of BDNF using hArcMin reduces the severity of one or more symptoms Development of Rett syndrome symptoms in newborn mecp2 mutant mice The ability of activity-dependent BDNF expression to delay aging is also examined.

[0156] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof.

[0157] Numbered Embodiments The following list of embodiments is included herein for illustrative purposes only and is not intended to be exhaustive or limiting. Claimed subject matter is expressly limited to the following embodiments: It will not be done. Embodiment 1. A nucleic acid comprising an adeno-associated virus (AAV) expression cassette, But in the 5' to 3' direction, 5' inverted repeat (ITR); synthetic activity-dependent promoter; Rett syndrome-associated genes; and A nucleic acid comprising a 3' ITR. Embodiment 2. 2. The nucleic acid of embodiment 1, wherein the promoter drives expression of a Rett Syndrome-associated gene. Embodiment 3. 3. The nucleic acid of embodiment 1 or 2, wherein the promoter is an MECP2-independent promoter. Embodiment 4. The promoter comprises a nucleic acid sequence derived from a promoter of a neuronal immediate early gene. , the nucleic acid of any one of embodiments 1 to 3. Embodiment 5. Neuronal immediate early genes include Arc, c-fos, and egr-1. 5. The nucleic acid of embodiment 4, selected from the group consisting of: Embodiment 6.

[0023] Embodiments in which the promoter comprises the minimal Arc gene promoter (ArcMin). Any one of the nucleic acids 1 to 5. Embodiment 7. The nucleic acid of embodiment 6, wherein ArcMin is human ArcMin (hArcMin). Embodiment 8. hArcMin is a nucleic acid sequence of SEQ ID NO: 12 or at least the nucleic acid sequence of SEQ ID NO: 12. 8. The nucleic acid of embodiment 7, comprising a sequence 90% identical to both of Embodiment 9. The promoter contains a cyclic AMP response element (CRE), a serum response element (SRE), ), synaptic activity response element (SARE), MEF2 response element, or a combination thereof The nucleic acid of any one of embodiments 1 to 8, comprising a combination thereof. Embodiment 10. 10. The nucleic acid of embodiment 9, wherein the promoter comprises a synaptic activity response element (SARE). . Embodiment 11. Synaptic activity response element (SARE) is a human synaptic activity response element (hS 11. The nucleic acid of embodiment 10, wherein said nucleic acid is a nucleotide sequence selected from the group consisting of nucleotides Embodiment 12. hSARE is a nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence having at least one amino acid sequence identical to the nucleic acid sequence of SEQ ID NO: 11 12. The nucleic acid of embodiment 11, comprising a sequence 90% identical. Embodiment 13. The promoter comprises human ArcMin (hArcMin) and at least one hSA 13. The nucleic acid of any one of embodiments 1 to 12, comprising an RE. Embodiment 14. 14. The nucleic acid of embodiment 13, wherein the promoter comprises hArcMin and one hSARE. . Embodiment 15. The promoter is a nucleic acid sequence of SEQ ID NO: 6 or a nucleic acid sequence of SEQ ID NO: 6 and at least 9 15. The nucleic acid of embodiment 14, comprising a sequence identical to any one of the preceding claims. Embodiment 16. 14. The nucleic acid of embodiment 13, wherein the promoter comprises hArcMin and five hSAREs. . Embodiment 17. The promoter comprises the nucleic acid sequence of SEQ ID NO: 16 or a sequence at least identical to the nucleic acid sequence of SEQ ID NO: 16. 17. The nucleic acid of embodiment 16, comprising a sequence 90% identical to Embodiment 18. 18. Any of embodiments 1 to 17, wherein the promoter binds to a neuronal activity-dependent transcription factor. Any one of the nucleic acids. Embodiment 19. Neuronal activity-dependent transcription factors bind to cAMP response element binding protein (CRE) B), myocyte enhancer factor 2 (MEF2), serum response factor (SRF), or Elk 19. The nucleic acid of embodiment 18, wherein said nucleic acid is -1. Embodiment 20. Rett syndrome-related genes include brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF1), methyl-CpG binding protein 2 (MECP2), huntingtin protein Quality, Huntington-related protein 1, orthodenticle homeobox 2 (OTX-2 ), FXYD domain-containing ion transport regulator 1 (FXYD1), neurexin-2- alpha (NRXN2), or protein kinase C gamma (PRKCG) The nucleic acid of any one of embodiments 1 to 19. Embodiment 21.

[0023] Embodiments in which the Rett syndrome-associated gene encodes brain-derived neurotrophic factor (BDNF). 20 nucleic acids. Embodiment 22. 22. The nucleic acid of embodiment 21, wherein the BDNF is human BDNF. Embodiment 23. BDNF has a nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical to the nucleic acid sequence of SEQ ID NO: 7 23. The nucleic acid of embodiment 22, encoded by the same sequence. Embodiment 24. At least one of the 5'ITR and the 3'ITR is about 110 to about 160 nucleotides in length. 24. The nucleic acid of any one of embodiments 1 to 23, wherein Embodiment 25. 25. The nucleic acid of any one of embodiments 1 to 24, wherein the 5' ITR is the same length as the 3' ITR. . Embodiment 26. 25. Any one of embodiments 1 to 24, wherein the 5' ITR and the 3' ITR have different lengths. Two nucleic acids. Embodiment 27. At least one of the 5'ITR and 3'ITR is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV AV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74 , isolated or derived from the genome of avian AAV or bovine AAV, embodiments A nucleic acid of any one of forms 1 to 26. Embodiment 28. 24. The nucleic acid of any one of embodiments 1 to 23, wherein the 5' ITR comprises the sequence of SEQ ID NO:1. Embodiment 29. 24. The nucleic acid of any one of embodiments 1 to 23, wherein the 3′ ITR comprises the sequence of SEQ ID NO:2. Embodiment 30. The AAV cassette encodes the brain-derived neurotrophic factor (BDNF) short 3'UTR, or BD 30. The nucleic acid of any one of embodiments 1 to 29, comprising a NF long 3'UTR. Embodiment 31. BDNF short 3'UTR or BDNF long 3'UTR is associated with Rett syndrome 31. The nucleic acid of embodiment 30, which is located between the gene and the 3' ITR. Embodiment 32.

[0039] Embodiment 30 or embodiment, wherein the AAV cassette comprises a BDNF short 3'UTR. 31 nucleic acids. Embodiment 33. the BDNF short 3'UTR is the nucleic acid sequence of SEQ ID NO: 8, or the nucleic acid sequence of SEQ ID NO: 8 33. The nucleic acid of embodiment 32, comprising a sequence at least 90% identical to Embodiment 34. Embodiment 30 or embodiment 3, wherein the AAV cassette comprises a BDNF long 3'UTR. 1 nucleic acid. Embodiment 35. BDNF long 3'UTR is the nucleic acid sequence of SEQ ID NO: 10, or the nucleic acid sequence of SEQ ID NO: 10 35. The nucleic acid of embodiment 34, comprising a sequence at least 90% identical to the sequence. Embodiment 36. 36. Any one of embodiments 1 to 35, wherein the AAV cassette comprises a polyadenylation signal. of nucleic acids. Embodiment 37. Polyadenylation signals are found in the following genes: simian virus 40 (SV40), rBG , α-globin, β-globin, human collagen, human growth hormone (hGH), polio rhoma virus, human growth hormone (hGH) or bovine growth hormone (bGH) or a polyadenylation signal isolated from or derived from a plurality of Nucleic acid of state 36. Embodiment 38. 37. The nucleic acid of embodiment 36, wherein the AAV cassette comprises a bGH polyadenylation signal. Embodiment 39. The bGH polyadenylation signal is selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 9, 39. The nucleic acid of embodiment 38, comprising a sequence at least 90% identical to the sequence. Embodiment 40. 40. Any of embodiments 1 to 39, wherein the AAV cassette comprises at least one stuffer sequence. Any one of the nucleic acids. Embodiment 41. At least one stuffer sequence is the nucleic acid sequence of SEQ ID NO: 13, or 41. The nucleic acid of embodiment 40, comprising a sequence that is at least 90% identical to the nucleic acid sequence of Embodiment 42. The AAV expression cassette comprises a Kozak sequence, and the Kozak sequence is a sequence encoding a Rett syndrome-associated gene. 42. The nucleic acid of any one of embodiments 1 to 41, wherein the nucleic acid overlaps with the start codon of the target gene. Embodiment 43. The Kozak sequence is the nucleic acid sequence of SEQ ID NO: 14 or at least the nucleic acid sequence of SEQ ID NO: 14. a sequence 90% identical to the nucleic acid sequence of SEQ ID NO: 15, or the nucleic acid sequence of SEQ ID NO: 15 43. The nucleic acid of embodiment 42, comprising a sequence at least 90% identical to the sequence. Embodiment 44. The AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 3 or at least the nucleic acid sequence of SEQ ID NO: 3. a sequence at least 90% identical to the sequence of SEQ ID NO: 4 or a sequence at least 90% identical to the sequence of SEQ ID NO: 4 % identical to the sequence of SEQ ID NO:5 or at least 90% identical to the sequence of SEQ ID NO:5 44. The nucleic acid of any one of embodiments 1 to 43, comprising a single sequence. Embodiment 45. A plasmid comprising the nucleic acid of any one of embodiments 1 to 44. Embodiment 46. A cell comprising the nucleic acid of any one of embodiments 1 to 44 or the plasmid of embodiment 45. . Embodiment 47. A method for producing a recombinant AAV vector, comprising: 46. ​​A method comprising contacting a nucleic acid of any one of embodiment 44, or a plasmid of embodiment 45. Law. Embodiment 48. A recombinant AAV vector produced by the method of embodiment 47. Embodiment 49. Vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and AAV 7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAV Select from rh10, AAVrh32.33, AAVrh74, avian AAV and bovine AAV 49. The recombinant AAV vector of embodiment 48, wherein the vector is of a selected serotype. Embodiment 50. 49. The recombinant AAV vector of embodiment 48, wherein the recombinant AAV vector is a single-stranded AAV (ssAAV), or The recombinant AAV vector of embodiment 49. Embodiment 51. Embodiment 48 - The recombinant AAV vector is a self-complementary AAV (scAAV). Any one of 50 recombinant AAV vectors. Embodiment 52. AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV 52. The recombinant AAV vector of any one of embodiments 48 to 51, comprising a capsid protein of Tar. Embodiment 53. The AAV vector contains one or more substitutions compared to the wild-type AAV capsid protein. 53. The method of any one of embodiments 48 to 52, comprising a capsid protein having a substitution or mutation. Recombinant AAV vectors. Embodiment 54. (a) the nucleic acid of any one of embodiments 1 to 44, the plasmid of embodiment 45, or the embodiment 46, or the recombinant AAV vector of any one of embodiments 48 to 53, and (b) a pharmaceutically acceptable carrier. Embodiment 55. A subject is administered a therapeutically effective amount of a nucleic acid according to any one of embodiments 1 to 44, a nucleic acid according to embodiment 45, The plasmid of embodiment 46, the cell of embodiment 48 to 53, or the recombinant AAV vector of any one of embodiments 48 to 53. 54. In a subject in need thereof, comprising administering to a subject a therapeutic agent, or a composition of embodiment 54. A method for expressing Rett syndrome-associated genes. Embodiment 56. 56. The method of embodiment 55, wherein the subject has Rett Syndrome. Embodiment 57. A subject is administered a therapeutically effective amount of a nucleic acid according to any one of embodiments 1 to 44, a nucleic acid according to embodiment 45, The plasmid of embodiment 46, the cell of embodiment 48 to 53, or the recombinant AAV vector of any one of embodiments 48 to 53. 55. A method for treating Rett syndrome in a subject, comprising administering to a subject a composition comprising administering to a subject a composition comprising a compound selected from the group consisting of acetaminophen, ... A method of treating or delaying the onset of Rett Syndrome. Embodiment 58. A subject is administered a therapeutically effective amount of a nucleic acid according to any one of embodiments 1 to 44, a nucleic acid according to embodiment 45, The plasmid of embodiment 46, the cell of embodiment 48 to 53, or the recombinant AAV vector of any one of embodiments 48 to 53. 54. In a subject in need thereof, comprising administering to a subject a therapeutic agent, or a composition of embodiment 54. A method for expressing brain-derived neurotrophic factor (BDNF) in the mouse. Embodiment 59. The method of embodiment 58, wherein the subject has a cognitive disorder or a stress-related disorder. Embodiment 60. The subjects are those with depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, dementia, and Anorexia nervosa and bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder, obesity, 59. The method of embodiment 58, wherein the patient has Rett syndrome, or post-chemotherapy cognitive impairment. Embodiment 61. A subject is administered a therapeutically effective amount of a nucleic acid according to any one of embodiments 1 to 44, a nucleic acid according to embodiment 45, The plasmid of embodiment 46, the cell of embodiment 48 to 53, or the recombinant AAV vector of any one of embodiments 48 to 53. 55. A method for treating a BDNF-related disorder in a subject, comprising administering to a subject a method for treating a BDNF-related disorder in a subject, the method ... A method of treating or delaying the onset of a BDNF-related disorder. Embodiment 62. Embodiment 6, wherein the BDNF-related disorder is a cognitive disorder and / or a stress-related disorder. Method 1. Embodiment 63. BDNF-related disorders include depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, and Dementia, anorexia nervosa, bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder, Bipolar disorder, Rett syndrome, major depressive disorder, or post-chemotherapy cognitive impairment. Method of posture 61. Embodiment 64. A subject is administered a therapeutically effective amount of a nucleic acid according to any one of embodiments 1 to 44, a nucleic acid according to embodiment 45, or a nucleic acid according to embodiment 46. the mide of embodiment 46, the cell of embodiment 47, or the recombinant AAV vector of any one of embodiments 48 to 53. 55. A method for detecting an MECP2-associated tumor in a subject, comprising administering to a subject a tumor suppressor or a composition of embodiment 54. Methods of treating or delaying the onset of MECP2-associated diseases. Embodiment 65. MECP2-related diseases include MECP2 duplication syndrome, MECP2-related severe neonatal encephalopathy, and PP. 65. The method of embodiment 64, wherein the syndrome is MX syndrome or Rett syndrome. Embodiment 66. The method of any one of embodiments 55-65, wherein the subject is a human subject. Embodiment 67. The nucleic acid, plasmid, cell, recombinant AAV vector, or composition is capable of delivering the desired therapeutic agent to the central nervous system. The method of any one of embodiments 55-66, wherein the administration is by injection. Embodiment 68. Embodiments 55-6, wherein the Rett syndrome-associated gene is expressed in neurons of the subject. One of the seven methods. Embodiment 69. 69. The method of embodiment 68, wherein the neuron is an active neuron.

Claims

[Claim 1] The invention described in this specification.

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