ADENO-ASSOCIATED VIRUS VECTORS FOR TREATMENT OF RETT SYNDROME
The disclosure provides nucleic acids (comprising AAV expression cassettes), AAV vectors, and compositions for use in methods for treating and/or delaying the onset of diseases associated with mutations in the mecp2 gene, such as Rett Syndrome. Also, provided herein are methods for treating and/or delaying the onset of brain-derived neurotrophic factor (BDNF)-associated diseases.
1 . A nucleic acid comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′:
a 5′ inverted terminal repeat (ITR);
a synthetic, activity-dependent promoter;
a Rett Syndrome-associated gene; and
a 3′ ITR.
2 . The nucleic acid of claim 1 , wherein the promoter drives expression of the Rett-syndrome-associated gene.
3 . The nucleic acid of claim 1 or 2 , wherein the promoter is a MECP2-independent promoter.
4 . The nucleic acid of any one of claims 1 - 3 , wherein the promoter comprises a nucleic acid sequence derived from a promoter of a neuronal immediate early gene.
5 . The nucleic acid of claim 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.
6 . The nucleic acid of any one of claims 1 - 5 , wherein the promoter comprises a minimal Arc gene promoter (ArcMin).
7 . The nucleic acid of claim 6 , wherein the ArcMin is a human ArcMin (hArcMin).
8 . The nucleic acid of claim 7 , wherein the hArcMin comprises a nucleic acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto.
9 . The nucleic acid of any one of claims 1 - 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.
10 . The nucleic acid of claim 9 , wherein the promoter comprises a synaptic activity response element (SARE).
11 . The nucleic acid of claim 10 , wherein the synaptic activity response element (SARE) is a human synaptic activity response element (hSARE).
12 . The nucleic acid of claim 11 , wherein the hSARE comprises a nucleic acid sequence of SEQ ID NO: 11, or a sequence at least 90% identical thereto.
13 . The nucleic acid of any one of claims 1 - 12 , wherein the promoter comprises a human ArcMin (hArcMin) and at least one hSARE.
14 . The nucleic acid of claim 13 , wherein the promoter comprises hArcMin and one hSARE.
15 . The nucleic acid of claim 14 , wherein the promoter comprises a nucleic acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto.
16 . The nucleic acid of claim 13 , wherein the promoter comprises hArcMin and five hSAREs.
17 . The nucleic acid of claim 16 , wherein the promoter comprises a nucleic acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto.
18 . The nucleic acid of any one of claims 1 - 17 , wherein the promoter binds to a neuronal activity dependent transcription factor.
19 . The nucleic acid of claim 18 , wherein the neuronal activity dependent transcription factor is cAMP-responsive element binding protein (CREB), myocyte enhancer factor 2 (MEF2), serum response factor (SRF), or Elk-1.
20 . The nucleic acid of any one of claims 1 - 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, Huntington-associated 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).
21 . The nucleic acid of claim 20 , wherein the Rett-syndrome-associated gene encodes brain-derived neurotrophic factor (BDNF).
22 . The nucleic acid of claim 21 , wherein the BDNF is a human BDNF.
23 . The nucleic acid of claim 22 , wherein the BDNF is encoded by a nucleic acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto.
24 . The nucleic acid of any one of claims 1 - 23 , wherein at least one of the 5′ ITR and the 3′ ITR is about 110 to about 160 nucleotides in length.
25 . The nucleic acid of any one of claims 1 - 24 , wherein the 5′ ITR is the same length as the 3′ ITR.
26 . The nucleic acid of any one of claims 1 - 24 , wherein the 5′ ITR and the 3′ ITR have different lengths.
27 . The nucleic acid of any one of claims 1 - 26 , wherein at least one of the 5′ ITR and the 3′ ITR is isolated 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.
28 . The nucleic acid of any one of claims 1 - 23 , wherein the 5′ ITR comprises the sequence of SEQ ID NO: 1.
29 . The nucleic acid of any one of claims 1 - 23 , wherein the 3′ ITR comprises the sequence of SEQ ID NO: 2.
30 . The nucleic acid of any one of claims 1 - 29 , wherein the AAV cassette comprises a brain-derived neurotrophic factor (BDNF) short 3′ UTR, or a BDNF long 3′ UTR.
31 . The nucleic acid of claim 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.
32 . The nucleic acid of claim 30 or claim 31 , wherein the AAV cassette comprises a BDNF short 3′ UTR.
33 . The nucleic acid of claim 32 , wherein the BDNF short 3′ UTR comprises a nucleic acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto.
34 . The nucleic acid of claim 30 or claim 31 , wherein the AAV cassette comprises a BDNF long 3′ UTR.
35 . The nucleic acid of claim 34 , wherein the BDNF long 3′ UTR comprises a nucleic acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto.
36 . The nucleic acid of any one of claims 1 - 35 , wherein the AAV cassette comprises a polyadenylation signal.
37 . The nucleic acid of claim 36 , wherein the polyadenylation signal is a polyadenylation signal isolated 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).
38 . The nucleic acid of claim 36 , wherein the AAV cassette comprises a bGH polyadenylation signal.
39 . The nucleic acid of claim 38 , wherein the bGH polyadenylation signal comprises a nucleic acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto.
40 . The nucleic acid of any one of claims 1 - 39 , wherein the AAV cassette comprises at least one stuffer sequence.
41 . The nucleic acid of claim 40 , wherein the at least one stuffer sequence comprises a nucleic acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto.
42 . The nucleic acid of any one of claims 1 - 41 , wherein the AAV expression cassette comprises a Kozak sequence, wherein the Kozak sequence overlaps with the start codon of the Rett-syndrome-associated gene.
43 . The nucleic acid of claim 42 , wherein the Kozak sequence comprises a nucleic acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto; or a nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto.
44 . The nucleic acid of any one of claims 1 - 43 , wherein the AAV expression cassette comprises a nucleic acid sequence SEQ ID NO: 3, or a sequence at least 90% identical thereto; SEQ ID NO: 4 or a sequence at least 90% identical thereto; or SEQ ID NO: 5 or a sequence at least 90% identical thereto.
45 . A plasmid comprising the nucleic acid of any one of claims 1 - 44 .
46 . A cell comprising the nucleic acid of any one of claims 1 - 44 or the plasmid of claim 45 .
47 . A method of producing a recombinant AAV vector, the method comprising contacting an AAV producer cell with the nucleic acid of any one of claims 1 - 44 , or the plasmid of claim 45 .
48 . A recombinant AAV vector produced by the method of claim 47 .
49 . The recombinant AAV vector of claim 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.
50 . The recombinant AAV vector of claim 48 or claim 49 , wherein the recombinant AAV vector is a single-stranded AAV (ssAAV).
51 . The recombinant AAV vector of any one of claims 48 - 50 , wherein the recombinant AAV vector is a self-complementary AAV (scAAV).
52 . The recombinant AAV vector of any one of claims 48 - 51 , wherein the AAV vector comprises a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, Avian AAV or Bovine AAV.
53 . The recombinant AAV vector of any one of claims 48 - 52 , wherein the AAV vector comprises a capsid protein with one or more substitutions or mutations, as compared to a wildtype AAV capsid protein.
54 . A composition, comprising: (a) the nucleic acid of any one of claims 1 - 44 , the plasmid of claim 45 , the cell of claim 46 , or the recombinant AAV vector of any one of claims 48 - 53 ; and (b) a pharmaceutically acceptable carrier.
55 . 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 claims 1 - 44 , the plasmid of claim 45 , the cell of claim 46 , the recombinant AAV vector of any one of claims 48 - 53 , or the composition of claim 54 .
56 . The method of claim 55 , wherein the subject has Rett syndrome.
57 . A method for treating or delaying the onset of Rett syndrome in a subject, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of claims 1 - 44 , the plasmid of claim 45 , the cell of claim 46 , the recombinant AAV vector of any one of claims 48 - 53 , or the composition of claim 54 .
58 . 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 claims 1 - 44 , the plasmid of claim 45 , the cell of claim 46 , the recombinant AAV vector of any one of claims 48 - 53 , or the composition of claim 54 .
59 . The method of claim 58 , wherein the subject has a cognitive disorder, or a stress-related disorder.
60 . The method of claim 58 , wherein the subject has depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, and dementia, anorexia nervosa and bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder, obesity, Rett syndrome, or post-chemotherapy cognitive impairment.
61 . A method of treating or delaying the onset of a BDNF-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of claims 1 - 44 , the plasmid of claim 45 , the cell of claim 46 , the recombinant AAV vector of any one of claims 48 - 53 , or the composition of claim 54 .
62 . The method of claim 61 , wherein the BDNF-associated disease is a cognitive disorder, and/or a stress-related disorder.
63 . The method of claim 61 , wherein the BDNF-associated disease is depression, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, and dementia, anorexia nervosa, bulimia nervosa, schizophrenia, epilepsy, post-traumatic stress disorder, bipolar disease, Rett syndrome, major depressive disorder, or post-chemotherapy cognitive impairment.
64 . A method of treating or delaying the onset of a MECP2-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of claims 1 - 44 , the plasmid of claim 45 , the cell of claim 46 , the recombinant AAV vector of any one of claims 48 - 53 , or the composition of claim 54 .
65 . The method of claim 64 , wherein the MECP2-associated disease is MECP2 duplication syndrome, MECP2-related severe neonatal encephalopathy, PPM-X syndrome, or Rett syndrome.
66 . The method of any one of claim 55 - 65 , wherein the subject is a human subject.
67 . The method of any one of claims 55 - 66 , wherein the nucleic acid, the plasmid, the cell, the recombinant AAV vector, or composition is administered by injection into the central nervous system.
68 . The method of any one of claims 55 - 67 , wherein the Rett Syndrome-associated gene is expressed in the neurons of the subject.
69 . The method of claim 68 , wherein the neurons are active neurons.