Methods and compositions for rna-guided treatment of hiv infection

Abstract

A composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus including an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, an isolated nucleic acid sequence encoding a first guide RNA (gRNA) having a first spacer sequence that is complementary to a first target protospacer sequence in a proviral DNA, and an isolated nucleic acid sequence encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the proviral DNA, wherein said first target protospacer sequence and said second target protospacer sequence are situated in a long terminal repeat (LTR) of the proviral DNA. A pharmaceutical composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus.

What is claimed is: 
     
         1 . A composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, comprising:
 an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease;   an isolated nucleic acid sequence encoding a first guide RNA (gRNA) having a first spacer sequence that is complementary to a first target protospacer sequence in a proviral DNA; and   an isolated nucleic acid sequence encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the proviral DNA, wherein said first target protospacer sequence and said second target protospacer sequence are situated in a long terminal repeat (LTR) of the proviral DNA.   
     
     
         2 . The composition of  claim 1 , wherein said CRISPR-associated endonuclease is Cas9. 
     
     
         3 . The composition of  claim 1 , wherein said CRISPR-associated endonuclease sequence is optimized for expression in a human cell. 
     
     
         4 . The 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. 
     
     
         5 . The composition of  claim 1 , wherein said retrovirus is selected from the group consisting of human immunodeficiency virus-1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), and caprine arthritis/encephalitis virus (CAEV). 
     
     
         6 . The 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. 
     
     
         7 . The 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. 
     
     
         8 . The 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. 
     
     
         9 . The composition of  claim 1 , wherein the isolated nucleic acids encoding said CRISPR-associated endonuclease, said first gRNA, and said second gRNA, are encoded in at least one expression vector. 
     
     
         10 . The composition of  claim 9 , 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. 
     
     
         11 . The 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. 
     
     
         12 . The 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. 
     
     
         13 . A pharmaceutical composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, comprising:
 an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease;   an isolated nucleic acid sequence encoding a first guide RNA (gRNA) having a first spacer sequence that is complementary to a first target protospacer sequence in a proviral DNA;   an isolated nucleic acid sequence encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the proviral DNA, wherein said first target protospacer sequence and said second target protospacer sequence are situated in a long terminal repeat (LTR) of the proviral DNA; and   a pharmaceutically acceptable carrier.   
     
     
         14 . The pharmaceutical composition of  claim 13 , wherein the pharmaceutically acceptable carrier comprises a lipid-based or polymer-based colloid. 
     
     
         15 . The pharmaceutical composition of  claim 14 , wherein said colloid is chosen from the group consisting of a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. 
     
     
         16 . The pharmaceutical composition of  claim 13 , wherein said composition is formulated for topical application. 
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein said composition is contained within a condom. 
     
     
         18 . The pharmaceutical composition of  claim 13 , wherein said CRISPR-associated endonuclease is Cas9. 
     
     
         19 . The pharmaceutical composition of  claim 13 , wherein said CRISPR-associated endonuclease sequence is optimized for expression in a human cell. 
     
     
         20 . The pharmaceutical composition of  claim 13 , 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. 
     
     
         21 . The pharmaceutical composition of  claim 13 , wherein said retrovirus is selected from the group consisting of human immunodeficiency virus-1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), and caprine arthritis/encephalitis virus (CAEV). 
     
     
         22 . The pharmaceutical composition of  claim 13 , 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. 
     
     
         23 . The pharmaceutical composition of  claim 13 , 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. 
     
     
         24 . The pharmaceutical composition of  claim 13 , 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. 
     
     
         25 . The pharmaceutical composition of  claim 13 , wherein the isolated nucleic acids encoding said CRISPR-associated endonuclease, said first gRNA, and said second gRNA, are encoded in at least one expression vector. 
     
     
         26 . The pharmaceutical composition of  claim 25 , 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. 
     
     
         27 . The pharmaceutical composition of  claim 13 , 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. 
     
     
         28 . The pharmaceutical composition of  claim 13 , wherein at least one of said gRNAs is engineered as an artificial fusion small guide RNA (sgRNA) comprised of a crRNA and a tracrRNA.