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, preparation methods of Cas9-lgRNA complexes, and their uses for prevention and treatments of HIV infections in humans. Also disclosed are processes and methods for preparation of these compounds.

What is claimed is: 
     
         1 . A method of cleaving an HIV viral DNA and proviral DNA chromosomally integrated into the genome of a host cell latently infected with HIV, including the steps of:
 a. treating the host cells with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one lgRNA having a spacer sequence that is complementary to a target sequence in HIV genome; and   b. cleaving the HIV viral and proviral DNA.   
     
     
         2 . The method according to  claim 1 , wherein said at least one lgRNA comprises a nucleic acid sequence complementary to a target nucleic acid sequence having a sequence identity of at least 75% to one or more including SEQ ID NOs: 47-50. 
     
     
         3 . The method according to  claim 1 , wherein said lgRNA further comprises short peptide motifs of nuclear localization signals targeting nucleus, and said short peptide motifs are attached at its 5′- and/or 3′-end, and/or ligation sites of said lgRNA. 
     
     
         4 . The method according to  claim 1 , wherein said lgRNA further comprises small molecular ligands, and said small molecular ligands are attached at 5′- and/or 3′-end, and/or ligation sites of said lgRNA. 
     
     
         5 . The method according to  claim 1 , wherein said lgRNA comprises a spacer in crgRNA selected from sequences in the sense strand (5′→3′) of HIV genome, and namely its RNA transcript, or the antisense strand (5′→3′) of said HIV genome, and namely its antisense RNA transcript. 
     
     
         6 . The method according to  claim 1 , wherein said lgRNA comprises a spacer in crgRNA selected from the group of sequences immediately before a PAM in HIV genome targeting Gag conserved sequences. 
     
     
         7 . The method according to  claim 1 , wherein said lgRNA comprises a spacer in crgRNA selected from the group of sequences immediately before a PAM in HIV genome targeting overlapping Tat/Rev and Envelope conserved sequences. 
     
     
         8 . The method according to  claim 1 , wherein said lgRNA comprises a spacer in crgRNA selected from the group of sequences immediately before a PAM in HIV genome targeting 5′/3′-LTR sequences. 
     
     
         9 . The method according to  claim 1 , wherein said composition comprises at least one lgRNA, for contacting and cleaving the target polynucleotide(s) in HIV genome. 
     
     
         10 . The method according to  claim 1 , wherein said composition comprises mixtures of lgRNAs with various spacers targeting different loci of target genomes, and/or to variants of a single locus of target HIV genome. 
     
     
         11 . The method according to  claim 1 , wherein said composition comprises cationic lipids for therapeutic delivery to animal or human cells. 
     
     
         12 . The method according to  claim 1 , wherein said composition comprises cell-penetrating peptide (CPP) for therapeutic delivery to animal or human cells. 
     
     
         13 . The method according to  claim 1 , wherein said composition comprises transfecting agents for therapeutic delivery to host human cells. 
     
     
         14 . The method according to  claim 1 , wherein said composition comprises CRSPR-associated-proteins. 
     
     
         15 . The method according to  claim 1 , wherein said composition comprises recombinant engineered CAS9 protein. 
     
     
         16 . The method according to  claim 1 , wherein said composition is used in combination with other direct antiviral agents. 
     
     
         17 . The method according to  claim 1 , wherein said composition is used in combination with other direct antiviral agents selected from tenofovir disoproxil fumarate, tenofovir alafenamide, abacavir, lamivudine, emtricitabine, zidovudine, didanosine, stavudine, 4′-ethynyl-2-fluoro-2′-deoxyadenosine, efavirenz, etravirine, rilpivirine, nevirapine, delavirdine mesylate, atazanavir, cobicistat, darunavir, lopinavir, fosamprenavir, tipranavir, enfuvirtide, ritonavir, dolutegravir, bictegravir, raltegravir, elvitegravir, and cabotegravir. 
     
     
         18 . A method of eliminating a proviral DNA integrated into the genome of ex vivo cultured host cells latently infected with human immunodeficiency virus (HIV), including the steps of:
 a. obtaining a population of host cells latently infected with HIV, wherein a proviral HIV DNA is integrated into the host cell genome;   b. culturing the host cells ex vivo;   c. treating the host cells with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one lgRNA having a spacer sequence that is complementary to a target sequence in a long terminal repeat (LTR) of the proviral HIV DNA;   d. cleaving the proviral DNA from the host cell genome;   e. infusing the HIV-eliminated cell population into the patient; and   f. treating the patient.   
     
     
         19 . The method according to  claim 18 , wherein said step of obtaining a population of host cells is further defined as obtaining a population of human peripheral blood mononuclear cells, or obtaining a population of CD4+T cells. 
     
     
         20 . A kit for the treatment or prophylaxis of HIV infection, including a measured amount of a composition comprising at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease or CRISPR endonuclease, and at least one lgRNA, wherein each of said one or more lgRNA includes a spacer sequence complementary to a target sequence of an HIV viral and proviral DNA, and one or more items selected from the group consisting of packaging material, a package insert comprising instructions for use, a sterile fluid, a syringe and a sterile container.