5'-cap compounds and their uses in stabilizing rna, expressing proteins and in therapy

Abstract

The present invention relates to 5′-cap compounds, in particular the stabilization of RNA by such 5′-cap compounds, and provides compositions, such as pharmaceutical compositions, and cells comprising an RNA which is modified with such a 5′-cap compound, as well as methods for producing a peptide or protein of interest using the compositions or cells according to the present invention. Furthermore, the present invention provides the RNA, compositions, or cells for use in therapy, in particular for use in a method of treating a disease or disorder by protein replacement therapy, genome engineering, genetic reprogramming, and immunotherapy; a method for increasing the stability of RNA in cells; a method for increasing the expression of RNA in cells; and a method for providing an RNA with a 5′-cap structure.

1 . A 5′-cap compound having the 5′-cap structure according to formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; 
         R 2  and R 3  are independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2  and R 3  together form O—X—O, wherein X is selected from the group consisting of optionally substituted CH 2 , optionally substituted CH 2 CH 2 , optionally substituted CH 2 CH 2 CH 2 , optionally substituted CH 2 CH(CH 3 ), and optionally substituted C(CH 3 ) 2 , or R 2  is combined with the hydrogen atom at position 4′ of the ring to which R 2  is attached to form —O—CH 2 — or —CH 2 —O—; 
         R 4  and R 6  are independently selected from the group consisting of O, S, Se, and BH 3 ; 
         R 5  is selected from the group consisting of S, Se, and BH 3 ; 
         R 7  is a mononucleotide or an oligonucleotide having 2 to 9 bases; 
         R 8  is H, halo, or optionally substituted alkoxy; 
         n is 1, 2, or 3; and 
         B is a purine or pyrimidine base moiety. 
       
     
     
         2 . The 5′-cap compound of  claim 1 , wherein R 1  is selected from the group consisting of optionally substituted C 1-4  alkyl, optionally substituted C 2-4  alkenyl, and optionally substituted aryl. 
     
     
         3 . The 5′-cap compound of  claim 1 , wherein R 1  is optionally substituted C 1-4  alkyl. 
     
     
         4 . The 5′-cap compound of  claim 1 , wherein R 1  is C 1-4  alkyl. 
     
     
         5 . The 5′-cap compound of  claim 1 , wherein R 2  and R 3  are independently selected from the group consisting of H, F, OH, methoxy, ethoxy, propoxy, and 2-methoxyethoxy. 
     
     
         6 . The 5′-cap compound of  claim 1 , wherein R 2  is OH and R 3  is methoxy or R 2  is methoxy and R 3  is OH. 
     
     
         7 . The 5′-cap compound of  claim 1 , wherein R 4  and R 6  are independently selected from the group consisting of O and S. 
     
     
         8 . The 5′-cap compound of  claim 1 , wherein R 4  and R 6  are O. 
     
     
         9 . The 5′-cap compound of  claim 1 , wherein R 5  is S. 
     
     
         10 . The 5′-cap compound of  claim 1 , wherein R 5  is S, n is 1 or 2, and R 4  and R 6  are O. 
     
     
         11 . The 5′-cap compound of  claim 10 , wherein n is 1. 
     
     
         12 . The 5′-cap compound of  claim 1 , wherein B is selected from the group consisting of guanine, adenine, cytosine, thymine, uracil, and modified forms thereof. 
     
     
         13 . The 5′-cap compound of  claim 1 , wherein B is selected from the group consisting of guanine, adenine, and modified forms thereof. 
     
     
         14 . The 5′-cap compound of  claim 1 , wherein B is guanine or adenine. 
     
     
         15 . The 5′-cap compound of  claim 1 , wherein R 7  is *pGpN or *pG. 
     
     
         16 . The 5′-cap compound of  claim 1 , wherein R 8  is optionally substituted alkoxy. 
     
     
         17 . The 5′-cap compound of  claim 1 , wherein R 8  is methoxy. 
     
     
         18 . The 5′-cap compound of  claim 1 , wherein the 5′-cap compound has a structure according to formula (III): 
       
         
           
           
               
               
           
         
       
     
     
         19 . The 5′-cap compound of  claim 18 , wherein R 1  is methyl or ethyl. 
     
     
         20 . The 5′-cap compound of  claim 18 , wherein one of R 2  and R 3  is OCH 3  and the other is OH. 
     
     
         21 . The 5′-cap compound of  claim 18 , wherein n is 1. 
     
     
         22 . The 5′-cap compound of  claim 18 , wherein B is guanine or adenine. 
     
     
         23 . The 5′-cap compound of  claim 18 , wherein R 7  is a ribomononucleotide having a free OH group at position 2′. 
     
     
         24 . The 5′-cap compound of  claim 18 , wherein R 8  is methoxy. 
     
     
         25 . The 5′-cap compound of  claim 18 , wherein R 1  is methyl or ethyl; one of R 2  and R 3  is OCH 3  and the other is OH; n is 1; R 7  is a ribomononucleotide having a free OH group at position 2′; the internucleotide linkage between the ribomononucleotide and the ring to which R 7  is attached is phosphate; and B is guanine or adenine. 
     
     
         26 . An RNA which is modified with a 5′-cap compound of  claim 1 . 
     
     
         27 . The RNA of  claim 26 , wherein the RNA further comprises a nucleotide sequence encoding a peptide or protein of interest. 
     
     
         28 . A composition or cell comprising an RNA of  claim 26 . 
     
     
         29 . The composition or cell of  claim 28 , wherein the RNA further comprises a nucleotide sequence encoding a peptide or protein of interest. 
     
     
         30 . A method of treating a disease or disorder in a subject comprising the step of administering to said subject:
 (i) an RNA modified with a 5′-cap compound having the 5′-cap structure according to formula (I), wherein the RNA further comprises a nucleotide sequence encoding a peptide or protein of interest, or   (ii) a composition or cell comprising an RNA modified with a 5′-cap compound having the 5′-cap structure according to formula (I), wherein the RNA further comprises a nucleotide sequence encoding a peptide or protein of interest;   wherein formula (I) is:   
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; 
         R 2  and R 3  are independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2  and R 3  together form O—X—O, wherein X is selected from the group consisting of optionally substituted CH 2 , optionally substituted CH 2 CH 2 , optionally substituted CH 2 CH 2 CH 2 , optionally substituted CH 2 CH(CH 3 ), and optionally substituted C(CH 3 ) 2 , or R 2  is combined with the hydrogen atom at position 4′ of the ring to which R 2  is attached to form —O—CH 2 — or —CH 2 —O—; 
         R 4  and R 6  are independently selected from the group consisting of O, S, Se, and BH 3 ; 
         R 5  is selected from the group consisting of S, Se, and BH 3 ; 
         R 7  is a mononucleotide or an oligonucleotide having 2 to 9 bases; 
         R 8  is H, halo, or optionally substituted alkoxy; 
         n is 1, 2, or 3; and 
         B is a purine or pyrimidine base moiety.