FEEDBACK ENABLED SYNTHETIC GENES, TARGET SEED MATCH CASSETTES, AND THEIR USES
This invention relates to feedback-enabled synthetic genes, polynucleotide target cassettes, vectors, and pharmaceutical compositions for the purpose of providing transgene expression in target tissues that is capable of endogenous regulation for treating disorders such as dose-sensitive intellectual ability disorders, as well as methods of making and methods of using the same.
1 . A synthetic gene comprising:
a polynucleotide comprising a coding region encoding MECP2 and one or more regulatory regions, wherein the regulatory region comprises the nucleotide sequence of SEQ ID NO:1 (“Reg2”).
2 . A vector comprising the synthetic gene of claim 1 .
3 . The vector of claim 2 , wherein the vector is a viral vector.
4 . The vector of claim 3 , wherein the vector is an adeno-associated virus (AAV) vector.
5 . The vector of claim 4 , wherein the AAV vector is an AAV9 or AAV9-derived vector.
6 . A pharmaceutical composition comprising the synthetic gene of claim 1 and a pharmaceutically acceptable carrier.
7 . A polynucleotide target cassette for providing dose dependent inhibitory feedback to a synthetic gene, the cassette comprising a regulatory region comprising the sequence of SEQ ID NO:1.
8 . A method of preparing a synthetic gene comprising a polynucleotide comprising a coding region encoding MECP2 and one or more regulatory regions, comprising the step of inserting the polynucleotide target cassette of claim 7 into a regulatory region of the synthetic gene.
9 . A method of making the synthetic gene of claim 1 , comprising inserting one or more nucleic acid segments each comprising a seed match identified as a binding site for an endogenous miRNA and 5′ and 3′ flanking sequences neighboring said seed match into a regulatory region of the polynucleotide comprising a coding region encoding MECP2 and one or more regulatory regions such that the regulatory region comprises the nucleotide sequence of SEQ ID NO:1.
10 . A method of delivering a synthetic gene to a subject, the method comprising administering to the subject the synthetic gene of claim 1 , thereby delivering the synthetic gene to the subject.
11 . A method of treating Rett Syndrome in a subject in need thereof, the method comprising administering to the subject the synthetic gene of claim 1 .
12 . A method of treating Rett Syndrome in a subject, the method comprising genetically knocking down an endogenous MECP2 gene in a cell of the subject, and administering the synthetic gene of claim 1 .