Methods and compositions for rnra-guided treatment of hiv infection

Abstract

A pharmaceutical composition for use in inactivating an HIV-1 proviral DNA integrated into the genome of a host cell latently infected with a retrovirus including a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the HIV-1 proviral DNA, whereby treating the host cell with the composition cleaves a double strand of the HIV-1 proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease and cleaves a double strand of the HIV-1 proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease and thereby excises an entire HIV-1 proviral genome and eradicates the HIV-1 proviral DNA from the host cell, and a pharmaceutically acceptable carrier.

What is claimed is: 
     
         1 . A pharmaceutical composition for use in inactivating an HIV-1 proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, comprising:
 a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the HIV-1 proviral DNA, whereby treating the host cell with the composition cleaves a double strand of the HIV-1 proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease and cleaves a double strand of the HIV-1 proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease and thereby excises an entire HIV-1 proviral genome and eradicates the HIV-1 proviral DNA from the host cell; and   a pharmaceutically acceptable carrier.   
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the pharmaceutically acceptable carrier comprises a lipid-based or polymer-based colloid. 
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein said colloid is chosen from the group consisting of a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. 
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein said composition is formulated for topical application. 
     
     
         5 . The pharmaceutical composition of  claim 4 , wherein said composition is contained within a condom. 
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein said CRISPR-associated endonuclease is Cas9. 
     
     
         7 . The pharmaceutical composition of  claim 1 , wherein said CRISPR-associated endonuclease sequence is optimized for expression in a human cell. 
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein at least one of said first target protospacer sequence and said second target protospacer sequence is situated within the U3 region of said LTR. 
     
     
         9 . The pharmaceutical composition of  claim 1 , wherein said first spacer sequence and said second spacer sequence each include a sequence complementary to a target protospacer sequence selected from the group consisting of SEQ ID NO: 96, SEQ ID NO: 121, SEQ ID NO: 87, and SEQ ID NO: 110. 
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein said first spacer sequence and said second spacer sequence include, respectively, a sequence complementary to target protospacer sequences SEQ ID NO: 96 and SEQ ID NO: 121. 
     
     
         11 . The pharmaceutical composition of  claim 1 , wherein said first spacer sequence and said second spacer sequence each include, respectively, a sequence complementary to target protospacer sequences SEQ ID NO: 87 and SEQ ID NO: 110. 
     
     
         12 . The pharmaceutical composition of  claim 1 , wherein said composition is encoded in at least one expression vector. 
     
     
         13 . The pharmaceutical composition of  claim 12 , wherein said at least one expression vector is selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector. 
     
     
         14 . The pharmaceutical composition of  claim 1 , wherein at least one of said gRNAs comprises a CRISPR RNA (crRNA) and a trans-activated small RNA (tracrRNA), which are expressed as separate nucleic acids. 
     
     
         15 . The pharmaceutical composition of  claim 1 , wherein at least one of said gRNAs is engineered as an artificial fusion small guide RNA (sgRNA) comprised of a crRNA and a tracrRNA.