Self-regulating aav vectors for safe expression of mecp2 in rett syndrome

Abstract

In some aspects, the disclosure relates to compositions and methods of engineering a transgene. In some embodiments, the disclosure provides self-regulating recombinant nucleic acids, viral vectors and pharmaceutical compositions comprising a MeCP2 transgene. In some embodiments, compositions and methods described by the disclosure are useful for treating diseases and disorders associated with a loss of function mutation, for example Rett syndrome.

What is claimed is: 
     
         1 . A method of engineering a transgene, the method comprising:
 (a) selecting a first gene encoding a first product in a cell;   (b) selecting an miRNA, the expression of which is positively regulated by the first product in the cell; and,   (c) engineering a transgene that expresses a transcript having a coding region encoding the first product and a 3′-non-coding region comprising one or more binding sites for the miRNA.   
     
     
         2 . A method of engineering a transgene, the method comprising:
 (a) selecting a first gene encoding a first product in a cell;   (b) selecting a second gene encoding a second product in the cell;   (c) determining that expression of the second product is positively regulated by the first product in the cell;   (d) selecting an miRNA;   (e) determining that expression of the miRNA is positively regulated by the second product in the cell; and,   (f) engineering a transgene to express in the cell a transcript having a coding region encoding the first product and a 3′-non-coding region comprising one or more binding sites for the miRNA.   
     
     
         3 . The method of  claim 1  or  2 , wherein the first product is a protein. 
     
     
         4 . The method of  claim 3 , wherein the protein is MeCP2, optionally MeCP2 isoform e1 or MeCP isoform e2. 
     
     
         5 . The method of any one of  claims 2  to  4 , wherein the second product is a protein, or nucleic acid, optionally wherein the nucleic acid is an miRNA. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the miRNA is miR-132. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein the method further comprises engineering the 3′-non-coding region of the transcript to comprise one or more binding sites for one or more de-targeting miRNAs. 
     
     
         8 . The method of  claim 7 , wherein the one or more de-targeting miRNAs inhibit expression of the transgene from liver, heart, lung, muscle, pancreas, or antigen presenting cells. 
     
     
         9 . The method of  claim 7  or  8 , wherein the one or more de-targeting miRNA is miR-122, miR-1, or miR-122 and miR-1. 
     
     
         10 . The method of any one of  claims 1  to  9 , wherein the step of engineering the transgene comprises inserting the transgene into a vector. 
     
     
         11 . The method of  claim 10 , wherein the vector is a cloning vector, expression vector, plasmid, or viral vector. 
     
     
         12 . A method of engineering a transgene, the method comprising:
 (a) selecting a first gene encoding a first product in a cell;   (b) selecting an miRNA, the expression of which is positively regulated by the first product in the cell; and,   (c) engineering a transgene that expresses a transcript having a coding region encoding the first product and having one or more binding sites for the miRNA.   
     
     
         13 . A method of engineering a transgene, the method comprising;
 (a) selecting a first gene encoding a first product in a cell;   (b) selecting a second gene encoding a second product in the cell;   (c) determining that expression of the second product is positively regulated by the first product in the cell;   (d) selecting an miRNA;   (e) determining that expression of the miRNA is positively regulated by the second product in the cell; and,   (f) engineering a transgene to express in the cell a transcript having a coding region encoding the first product and having one or more binding sites for the miRNA.   
     
     
         14 . A recombinant AAV (rAAV) vector for self-regulated expression of a protein, the rAAV vector comprising a nucleic acid engineered to express in a cell of a target tissue a transcript encoding the protein, wherein the transcript comprises at least one first miRNA binding site specific for a first miRNA, wherein expression of the first miRNA is positively regulated by expression of the protein in the cell. 
     
     
         15 . A recombinant AAV comprising a capsid harboring the rAAV vector of  claim 14 , wherein the capsid comprises a capsid protein that facilitates selective transduction of the cell of the target tissue. 
     
     
         16 . A recombinant nucleic acid encoding a transcript having i) a coding region encoding a protein and ii) two or more miRNA binding sites, wherein the two or more miRNA binding sites comprise:
 (a) at least one first miRNA binding site specific for a first miRNA that is positively regulated by expression of the protein in a cell of a target tissue; and   (b) at least one second miRNA binding site specific for a second miRNA that is expressed, independent of expression of the protein, in cells of a non-target tissue.   
     
     
         17 . A recombinant nucleic acid encoding a transcript having a coding region encoding human MeCP2 protein or a functional fragment thereof and a 3′-non-coding region comprising two or more miRNA binding sites, wherein the two or more miRNA binding sites comprise:
 (a) at least one miRNA binding site specific for an miRNA that negatively regulates expression of the transcript; and 
 (b) at least one miRNA binding site specific for an miRNA that inhibits expression of the transcript in a cell of a non-target tissue. 
 
     
     
         18 . The recombinant nucleic acid of  claim 17 , wherein the coding region encodes MeCP2 isoform e1. 
     
     
         19 . The recombinant nucleic acid of  claim 17  or  18 , wherein the human MeCP2 comprises the sequence set forth in SEQ ID NO: 1. 
     
     
         20 . The recombinant nucleic acid of any one of  claims 17  to  19 , wherein the at least one miRNA binding site specific for an miRNA that negatively regulates expression of the transcript comprises a miR-132 binding site, optionally wherein the at least one miRNA binding site is two or three miR-132 binding sites. 
     
     
         21 . The recombinant nucleic acid of any one of  claims 17  to  20 , wherein the at least one miRNA binding site specific for an miRNA that inhibits expression of the transcript in a non-target tissue comprises a miR-1 binding site, mir-122 binding site, or miR-1 and miR-122 binding site. 
     
     
         22 . The recombinant nucleic acid of any one of  claims 17  to  21 , wherein each of the one or more miRNA binding sites is located between the last codon of the coding region and the poly-A tail of the transcript. 
     
     
         23 . The recombinant nucleic acid of any one of  claims 17  to  22 , further comprising a promoter, optionally a mouse MeCP2 promoter. 
     
     
         24 . The recombinant nucleic acid of  claim 23 , wherein the mouse MeCP2 promoter comprises the sequence set forth in SEQ ID NO: 3. 
     
     
         25 . The recombinant nucleic acid of any one of  claims 17  to  24 , wherein the recombinant nucleic acid is located on a plasmid. 
     
     
         26 . A viral vector comprising the recombinant nucleic acid of any one of  claims 17  to  24 , optionally wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, a lentiviral vector, a herpesvirus vector, or a baculovirus vector. 
     
     
         27 . A recombinant nucleic acid encoding a transcript having
 (a) a coding region encoding human MeCP2 or a functional fragment thereof; and,   (b) a 3′-non-coding region comprising one or more miRNA binding sites, wherein transcript is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).   
     
     
         28 . The recombinant nucleic acid of  claim 27 , wherein the coding region encodes MeCP2 isoform e1. 
     
     
         29 . The recombinant nucleic acid of  claim 27  or  28 , wherein the human MeCP2 comprises the sequence set forth in SEQ ID NO: 1. 
     
     
         30 . The recombinant nucleic acid of any one of  claims 27  to  29 , wherein the one or more miRNA binding sites are miR-1, miR-122, or miR-132 binding sites, or any combination thereof. 
     
     
         31 . The recombinant nucleic acid of  claim 30 , wherein the transcript comprises a miR-1 binding site, a mir-122 binding site, and at least one miR-132 binding site. 
     
     
         32 . The recombinant nucleic acid of  claim 31 , wherein the at least one miR-132 binding site is two or three mir-132 binding sites. 
     
     
         33 . The recombinant nucleic acid of any one of  claims 27  to  32 , wherein the one or more miRNA binding sites are located between the last codon of the coding region and the poly-A tail of the transcript. 
     
     
         34 . The recombinant nucleic acid of any one of  claims 27  to  33 , further comprising a promoter, optionally a mouse MeCP2 promoter. 
     
     
         35 . The recombinant nucleic acid of  claim 34 , wherein the mouse MeCP2 promoter comprises the sequence set forth in SEQ ID NO: 3. 
     
     
         36 . The recombinant nucleic acid of any one of  claims 27  to  35 , wherein the ITRs are AAV2 ITRs. 
     
     
         37 . A recombinant adeno-associated virus (rAAV) comprising:
 a capsid harboring the recombinant nucleic acid of any one of  claims 17  to  24 , or  27  to  36 .   
     
     
         38 . The rAAV of  claim 37 , wherein the nucleic acid comprises at least one ITR selected from an AAV2, AAV3, AAV4, AAV5, or AAV6 ITR. 
     
     
         39 . The rAAV of  claim 37  or  38 , wherein the capsid comprises a capsid protein that facilitates passage of the rAAV across the blood-brain barrier. 
     
     
         40 . The rAAV of  claim 39 , wherein the capsid protein has a serotype selected from the group consisting of AAV-PHP.B, AAV1, AAV2, AAV2i8, AAV2.5, AAV5, AAV6, AAV8, AAVrh8, AAV9, AAVrh10, AAV-B1, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-CAM130, and AAV9HR. 
     
     
         41 . The rAAV of any one of  claims 37  to  40 , wherein the capsid protein comprises or consists of a sequence set forth in SEQ ID NO: 14 or 15 (AAV-PHP.B, AAV9). 
     
     
         42 . A composition comprising the recombinant nucleic acid of any one of  claims 17  to  24 , or  27  to  36 , or the rAAV of any one of  claims 37  to  41 , and a pharmaceutically acceptable excipient. 
     
     
         43 . The composition of  claim 42 , wherein the composition is formulated for injection, optionally wherein the injection is systemic injection (e.g., intravenous injection) or intrathecal injection. 
     
     
         44 . A method of treating Rett syndrome in a subject, the method comprising, administering to a subject having or suspected of having Rett syndrome an effective amount of:
 (a) the recombinant nucleic acid of any one of  claims 17  to  24 , or  27  to  36 ;   (b) the rAAV of any one of  claims 37  to  41 ; or,   (c) the composition of  claim 42  or  43 .   
     
     
         45 . The method of  claim 44 , wherein the subject is a human subject, optionally wherein the subject is less than one year old. 
     
     
         46 . The method of  claim 44  or  45 , wherein the subject is characterized by a mutation in at least one copy of the MeCP2 gene, optionally wherein the mutation is a loss of function mutation. 
     
     
         47 . The method of any one of  claims 44  to  46 , wherein the administration is injection, optionally systemic injection (e.g., intravenous injection) or intrathecal injection. 
     
     
         48 . The method of any one of  claims 44  to  47 , wherein the administration results in the effective amount of (a), (b), or (c) crossing the blood-brain barrier of the subject. 
     
     
         49 . The method of any one of  claims 44  to  48 , wherein the administration results in a non-toxic level of MeCP2 expression in the brain of the subject.