Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as Antiviral Therapeutic Agents

Abstract

Provided herein are chemically ligated guide RNA oligonucleotides (lgRNA) which comprise two functional RNA modules (crgRNA and tracrgRNA) joined by non-nucleotide chemical linkers (nNt-linker), their complexes with CRISPR-Cas9, and cells comprising lgRNAs, preparation methods of Cas9-1gRNA complexes, and their uses for prevention and treatments of viral infections in humans. Also disclosed are processes and methods for preparation of these compounds.

What is claimed is: 
     
         1 . A cell treated with a composition comprising at least one lgRNA, and said lgRNA comprises:
 a. a synthetic crRNA, comprising a spacer and a second oligonucleotide, wherein
 i. said spacer is an oligonucleotide of greater than 12 bases that targets a DNA sequence, and said second oligonucleotide is an RNA segment of 8-25 nucleotides, 
 ii. said spacer and said second oligonucleotide are joined between the 3′-end of said spacer and the 5′-end of said second oligonucleotide via an internucleotide phosphate diester, a thiophosphate diester 
   
       
         
           
           
               
               
           
         
         
            a boranophosphate diester 
         
       
       
         
           
           
               
               
           
         
         
            a phosphonoacetate 
         
       
       
         
           
           
               
               
           
         
         
            a thiophosphonoacetate 
         
       
       
         
           
           
               
               
           
         
         
            a phosphoramidate 
         
       
       
         
           
           
               
               
           
         
         
            or a thiophosphoramidate 
         
       
       
         
           
           
               
               
           
         
         
            and 
           iii. said spacer and said second oligonucleotide comprise a single or a plurality of nucleosides or nucleotides modified at sugar moieties selected from the group consisting of: 
         
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein Q is a natural or modified nucleobase,
 b. a synthetic tracrRNA, comprising:
 i. a single or a plurality of oligonucleotides, 
 ii. said oligonucleotides of b. i. are sequentially joined between the 3′-end and the 5′-end via an internucleotide phosphate diester, a thiophosphate diester 
 
 
       
         
           
           
               
               
           
         
         
            a boranophosphate diester 
         
       
       
         
           
           
               
               
           
         
         
            a phosphonoacetate 
         
       
       
         
           
           
               
               
           
         
         
            a thiophosphonoacetate 
         
       
       
         
           
           
               
               
           
         
         
            a phosphoramidate 
         
       
       
         
           
           
               
               
           
         
         
            or a thiophosphoramidate 
         
       
       
         
           
           
               
               
           
         
         
            or an nNt-Linker set forth as in c., and 
           iii. said oligonucleotides of b. i. comprise a single or a plurality of nucleosides or nucleotides modified at sugar moieties, wherein said nucleosides and nucleotides are selected from the group consisting of II-1 to 11-37, 
         
         c. one or more nNt-Linkers, comprising:
 i. an optionally substituted M core structure of Formula M-1 to M-18: 
 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
            wherein X=O, S, NH, or CH 2 , X 1 ═N or CH, X 2 ═N or CH, R M ═H, CH 3 , alkyl, aryl, or heteroaryl, m=0 to 3 and n=0 to 3, 
           ii. two L linkers, and each said L linker comprises absent or more structures selected from the group consisting of L-1 to L-24: 
         
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
            where m=0 to 16 and n=0 to 16, and wherein 
           iii. said L linkers and said M core structure are joined as L-M-L, wherein the two L linkers are the same or different, and attached to two terminal nucleotides of Formula Nuc-1 to Nuc-19: 
         
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
            wherein the attached positions are 
         
       
       
         
           
           
               
               
           
         
         
            to L-M-L and 
         
       
       
         
           
           
               
               
           
         
         
            or 
         
       
       
         
           
           
               
               
           
         
         
            to upstream and downstream oligonucleotides, respectively, and wherein R is H, OH, 
         
       
       
         
           
           
               
               
           
         
         
            CH 2 OH, 
         
       
       
         
           
           
               
               
           
         
         
            F, NH 2 , OMe, CH 2 OMe, OCH 2 CH 2 OMe, an alkyl, a cycloalkyl, an aryl, or a heteroaryl, R′ is H, OH, 
         
       
       
         
           
           
               
               
           
         
         
            CH 2 OH, 
         
       
       
         
           
           
               
               
           
         
         
            F, NH 2 , OMe, CH 2 OMe, OCH 2 CH 2 OMe, an alkyl, a cycloalkyl, an aryl, or a heteroaryl, and Q is a natural or a non-natural nucleic acid base, wherein an nNt-Linker of c. joins the 3′-terminal nucleotide of said synthetic crRNA of a. and the 5′-terminal nucleotide of said synthetic tracrRNA of b. and said at least one nNt-Linkers of c. are positioned outside the bound regions of said lgRNA by a CRSPR-associated-protein. 
         
       
     
     
         2 . Said cell of  claim 1  comprising at least one said lgRNA. 
     
     
         3 . Said cell of  claim 1 , wherein said composition further comprises a polypeptide selected from the group consisting of a Cas9, a Cas9 with reduced nuclease activity, a Cas9 with nickase activity, a Cas9 with no nuclease activity, and a fusion protein comprising a Cas9 domain, wherein the Cas9 domain is capable of binding with the activating duplex region and wherein the fusion protein further comprises a domain from a polypeptide other than Cas9. 
     
     
         4 . Said cell of  claim 3 , wherein said polypeptide is replaced with its encoding nucleic acid. 
     
     
         5 . Said cell of  claim 1 , wherein said composition further comprising a fusion protein comprising a Cas9 domain, wherein the Cas9 domain is capable of binding with the activating duplex region and wherein the fusion protein further comprises a domain from a polypeptide other than Cas9 and which confers an additional activity on the site-directed polypeptide selected from the group consisting of nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity and demyristoylation activity. 
     
     
         6 . Said cell of  claim 5 , wherein said fusion protein is replaced with its encoding nucleic acid. 
     
     
         7 . Said cell of  claim 1 , wherein said composition further comprises one or more DNA repair templates selected from the group consisting of ssDNA and dsDNA. 
     
     
         8 . Said DNA repair template of  claim 7  comprising an oligonucleotide sequence to introduce insertion(s) of one or more stop codons selected from the group consisting of 5′-(tga)-3′, 5′-(taa)-3′, 5′-(tag)-3′, 5′-(tga-ntga-ntga)-3′, 5′-(tga-ntga-ntaa)-3′, 5′-(tga-ntga-ntag)-3′, 5′-(tga-ntaa-ntga)-3′, 5′-(tga-ntaa-ntaa)-3′, 5′-(tgantaa-ntag)-3′, 5′-(tga-ntga-ntga)-3′, 5′-(tga-ntga-ntaa)-3′, 5′-(tga-ntga-ntag)-3′, 5′-(taa-ntga-ntga)-3′, 5′-(taa-ntga-ntaa)-3′, 5′-(taa-ntga-ntag)-3′, 5′-(taa-ntaa-ntga)-3′, 5′-(taa-ntaa-ntaa)-3′, 5′-(taa-ntaa-ntag)-3′, 5′-(taa-ntga-ntga)-3′, 5′-(taa-ntga-ntaa)-3′, 5′-(taa-ntga-ntag)-3′, 5′-(tag-ntga-ntga)-3′, 5′-(tag-ntga-ntaa)-3′, 5′-(tag-ntga-ntag)-3′, 5′-(tag-ntaa-ntga)-3′, 5′-(tag-ntaa-ntaa)-3′, 5′-(tag-ntaa-ntag)-3′, 5′-(tag-ntga-ntga)-3′, 5′-(tag-ntga-ntaa)-3′, 5′-(tag-ntga-ntag)-3′, wherein n is any nucleotide, and said more stop codons comprises repetitive said sequence separated by absent or more nucleotides in between or different said sequences separated by absent or more nucleotides in between. 
     
     
         9 . Said DNA repair template of  claim 7  comprising an oligonucleotide sequence to introduce insertion(s) of one or more transcription cis-regulatory elements, and said more elements comprises repetitive sequence separated by absent or more nucleotides in between or different sequences separated by absent or more nucleotides in between. 
     
     
         10 . Said cell of  claim 7 , wherein said DNA repair template is covalently linked to said lgRNA. 
     
     
         11 . Said cell of  claim 1 , wherein said lgRNA is covalently linked with one or more molecules selected from the group consisting of fluorescent molecules, PEGs, non-PEG polymers, ligands of cellular receptors, lipids, oligonucleotides, polysaccharides, glycans, peptides, aptamers and antibodies to form an lgRNA conjugate, and the said more molecules can be the same or different. 
     
     
         12 . Said cell of  claim 1 , wherein said composition further comprises a carrier containing a DNA repair template, and said carrier is selected from the group consisting of an AAV vector, a plasmid and a retron. 
     
     
         13 . Said cell of  claim 1 , wherein the cell is selected from the group consisting of a bacterial cell, an archaeal cell, a plant cell, an algal cell, a fungal cell, an invertebrate cell, a vertebrate cell, a mammalian cell, and a human cell. 
     
     
         14 . Said cell of  claim 1 , wherein the cell comprises deactivated integrated viral DNAs or deactivated pathogenic genes. 
     
     
         15 . Said cell of  claim 1 , wherein the cell comprises deactivated integrated HBV DNAs. 
     
     
         16 . Said cell of  claim 1 , wherein the cell comprises deactivated integrated HIV DNAs. 
     
     
         17 . Said cell of  claim 1 , wherein the cell comprises a deactivated CCR5 gene. 
     
     
         18 . Said cell of  claim 1 , wherein the cell comprises deactivated integrated HSV DNAs. 
     
     
         19 . Said cell of  claim 1 , wherein the cell comprises an edited host gene. 
     
     
         20 . Said cell of  claim 1 , wherein the cell comprises one or more inserted genes. 
     
     
         21 . Said cell of  claim 1 , wherein the cell further comprises other therapeutic agents. 
     
     
         22 . Said cell of  claim 2 , wherein said at least one lgRNA is introduced into the cell by lipofection, electroporation, nucleofection, microinjection, biolistics, in liposomes, immunoliposomes, with polycations, as nucleic acid conjugates, or combinations thereof. 
     
     
         23 . A cell comprising at least one said ligated tracrRNA of  claim 1 . 
     
     
         24 . Said cell of  claim 23 , wherein said tracrRNA is covalently linked to one or more molecules selected from the group consisting of fluorescent molecules, PEGs, non-PEG polymers, ligands of cellular receptors, lipids, oligonucleotides, polysaccharides, glycans, peptides, aptamers and/or antibodies to form a tracrRNA conjugate, and the said more molecules can be the same or different.