Self-regulating aav vectors for safe expression of mecp2 in rett syndrome
AssigneeUniversity of Massachusetts
InventorsMiguel Sena Esteves, Guangping Gao, Michael R. Green, Dan Wang, Tessa Mercedes Simone
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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.
Claims49
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.
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.
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.
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.
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.
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.
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.
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).
Dependencies are inferred from the claim text.