Nuclease Resistant Polynucleotides and Uses Thereof

Abstract

The invention provides, among other things, methods of stabilizing mRNA and nuclease resistant mRNA prepared in accordance with such methods. hi certain embodiments, the nuclease resistant mRNA encodes a functional protein, such as enzyme, and is characterized by its resistance to nuclease digestion, increased half-life and/or its ability to produce increased amounts of the functional protein (e.g., enzyme) encoded thereby.

1 .- 39 . (canceled) 
     
     
         40 . A nuclease resistant mRNA comprising mRNA having a coding region and a non-coding region and a complementary stabilizing oligonucleotide hybridized to at least a portion of the non-coding region of the mRNA, wherein the stabilizing oligonucleotide comprises at least one modified nucleobase, and wherein the nuclease resistant mRNA is more resistant to nuclease degradation relative to the un-hybridized mRNA. 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . The nuclease resistant mRNA of  claim 40 , wherein the nuclease resistant mRNA encodes a protein selected from the group consisting of erythropoietin, human growth hormone, cystic fibrosis transmembrane conductance regulator (CFTR), alpha-galactosidase A, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase, heparan sulfamidase, hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). 
     
     
         44 . The nuclease resistant mRNA of  claim 40 , wherein the stabilizing oligonucleotide is perfectly complementary to the at least a portion of the non-coding region of the mRNA. 
     
     
         45 . The nuclease resistant mRNA of  claim 40 , wherein the stabilizing oligonucleotide is perfectly complementary to a portion of the coding region of the mRNA. 
     
     
         46 . The nuclease resistant mRNA of  claim 44 , wherein the non-coding region is selected from the group of regions consisting of a 3′ untranslated region (UTR), a 5′ untranslated region (UTR), a poly-A tail and a terminal cap. 
     
     
         47 . The nuclease resistant mRNA of  claim 40 , wherein the stabilizing oligonucleotide is at least about 1 to about 50 nucleotides in length. 
     
     
         48 . The nuclease resistant mRNA of  claim 45 , wherein the mRNA is an endogenous mRNA of a subject that is insufficiently expressed by the subject. 
     
     
         49 . (canceled) 
     
     
         50 . The nuclease resistant mRNA of  claim 48 , wherein the stabilizing oligonucleotide is administered to the subject. 
     
     
         51 . (canceled) 
     
     
         52 . The nuclease resistant mRNA of  claim 47 , wherein the stabilizing oligonucleotide is about 15 nucleotides in length. 
     
     
         53 . The nuclease resistant mRNA of  claim 40 , wherein the stabilizing oligonucleotide comprises two or more modified nucleobases. 
     
     
         54 . The nuclease resistant mRNA of  claim 53 , wherein the modified nucleobase comprises a sugar modification. 
     
     
         55 . The nuclease resistant mRNA of  claim 54 , wherein the sugar modification is a 2′-O-alkyl modification. 
     
     
         56 . The nuclease resistant mRNA of  claim 54 , wherein the sugar modification comprises at least one modification selected from the group consisting of a locked polynucleotide (LNA), a peptide polynucleotide (PNA), and combinations thereof. 
     
     
         57 . The nuclease resistant mRNA of  claim 54 , wherein the sugar modification is a 2′-sugar modification selected from the group consisting of a 2′-deoxy-2′-fluoro modification, a 2′-O-methyl modification, a 2′-O-methoxyethyl modification, a 2′-deoxy modification, and combinations thereof. 
     
     
         58 . The nuclease resistant mRNA of any one of  claim 40 , wherein the stabilizing oligonucleotide comprises at least one modified internucleoside linkage. 
     
     
         59 . The nuclease resistant mRNA of  claim 58 , wherein the modified internucleoside linkage is a phosphorothioate bond. 
     
     
         60 . The nuclease resistant mRNA of  claim 40 , wherein the modification comprises at least one nucleobase modification. 
     
     
         61 . The nuclease resistant mRNA of  claim 60 , wherein the nucleobase modification is selected from the group consisting of 5-methyl cytidine, pseudouridine, 2-thio uridine, and combinations thereof. 
     
     
         62 . The nuclease resistant mRNA of  claim 40 , wherein the non-coding region comprises a poly(A) tail. 
     
     
         63 . The nuclease resistant mRNA of  claim 62 , wherein the stabilizing oligonucleotide comprises a poly-U sequence. 
     
     
         64 .- 119 . (canceled)