Methods and compositions for circular rna molecules

Abstract

This invention is directed to AAV compositions for circular RNA expression and methods of expressing covalently closed, circular RNA.

1 . A nucleic acid molecule encoding a circular RNA (circRNA) that is covalently closed, wherein the nucleic acid molecule comprises:
 a) a gene of interest which can be transcribed into noncoding RNA or a translatable mRNA;   b) intronic elements that flank the gene of interest, wherein the intronic elements are backspliced by the cellular splicing machinery to yield a circular RNA that is covalently closed;   c) an internal ribosome entry site (IRES) driving translation of the translatable mRNA transcribed from the gene of interest;   d) a promoter region in the 5′untranslated region (UTR) and outside of the intronic elements that flank the gene of interest; and   e) a translation regulating region in the 3′ UTR and outside of the intronic elements that flank the gene of interest.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the intronic elements of (b) comprise the nucleotide sequence of any of SEQ ID NOs:13-24 and 29-32, in any combination thereof, and in any multiples and/or ratios. 
     
     
         3 . The nucleic acid molecule of  claim 1 , wherein the IRES of (c) is a viral IRES listed in Table 5, a cellular IRES listed in Table 6, in any combination thereof, and in any multiples and/or ratios. 
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein the translation regulating region of (e) is a polyadenylation (polyA) sequence and/or a structural element that stabilizes the circRNA. 
     
     
         5 . An adeno-associated virus (AAV) genome comprising the nucleic acid molecule of  claim 1 , flanked by AAV inverted terminal repeats (ITRs). 
     
     
         6 . An AAV capsid or particle comprising the AAV genome of  claim 5 . 
     
     
         7 . An AAV capsid or particle comprising the nucleic acid molecule of  claim 1 . 
     
     
         8 . A composition comprising the nucleic acid molecule of  claim 1 , in a pharmaceutically acceptable carrier. 
     
     
         9 . A method of expressing a covalently closed circular RNA molecule in a cell, comprising introducing into the cell the nucleic acid molecule of  claim 1 , under conditions wherein the covalently closed circular RNA molecule is transcribed. 
     
     
         10 . A method of expressing a covalently closed circular RNA molecule in a cell, comprising introducing into the cell the AAV genome of  claim 5 , under conditions wherein the covalently closed circular RNA molecule is transcribed. 
     
     
         11 . A method of expressing a covalently closed circular RNA molecule in a cell, comprising introducing into the cell the AAV capsid or particle of  claim 6 , under conditions wherein the covalently closed circular RNA molecule is transcribed. 
     
     
         12 . A method of expressing a covalently closed circular RNA molecule in a cell, comprising introducing into the cell the composition of  claim 8 , under conditions wherein the covalently closed circular RNA molecule is transcribed. 
     
     
         13 . A method of expressing a covalently closed circular RNA molecule in a tissue specific and/or cell specific manner, comprising contacting the tissue and/or the cell with the nucleic acid molecule of  claim 1 , under conditions wherein the covalently closed, circular RNA molecule is expressed. 
     
     
         14 . A method of expressing a covalently closed circular RNA molecule in a tissue specific and/or cell specific manner, comprising contacting the tissue and/or the cell with the AAV genome of  claim 5 , under conditions wherein the covalently closed, circular RNA molecule is expressed. 
     
     
         15 . A method of expressing a covalently closed circular RNA molecule in a tissue specific and/or cell specific manner, comprising contacting the tissue and/or the cell with the AAV capsid or particle of  claim 6 , under conditions wherein the covalently closed, circular RNA molecule is expressed. 
     
     
         16 . A method of expressing a covalently closed circular RNA molecule in a tissue specific and/or cell specific manner, comprising contacting the tissue and/or the cell with the composition of  claim 8 , under conditions wherein the covalently closed, circular RNA molecule is expressed. 
     
     
         17 . The method of  claim 9 , wherein the covalently closed circular RNA molecule is a therapeutic mRNA molecule encoding a protein, an RNA silencing molecule, a guide RNA molecule that can target a genomic element, a guide RNA molecule that can target an RNA transcript, a tRNA molecule, a long noncoding RNA molecule, an antisense RNA molecule, or any combination thereof. 
     
     
         18 . The method of  claim 9 , wherein the cell and/or tissue is from a mammal.