Methods and compositions for rna-guided treatment of hiv infection

Abstract

A method of treating a subject having or at risk for having a virus infection, by administering a therapeutically effective amount of a composition comprising a vector encoding a CRISPR-associated endonuclease and at least two guide RNAs that are complementary to two target sequences spanning from the 5′- to 3′-LTRs of the sequence in the virus, and completely excising a fragment of greater than 9000-bp of integrated proviral DNA that spanned from its 5′- to 3′-LTRs. A method of treating a subject having or at risk for having a genetic caused disease, by administering a therapeutically effective amount of a composition comprising a vector encoding a CRISPR-associated endonuclease and at least two guide RNAs that are complementary to two target sequences spanning from the sequence of the subjects DNA greater than 9000-bp that is chromosomally integrated and causes the genetic caused disease, and excising the chromosomally integrated sequence.

What is claimed is: 
     
         1 . A method of treating a subject having or at risk for having an HIV-1 virus infection, including the steps of:
 administering to the subject a therapeutically effective amount of a composition 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 proviral DNA of the virus that is unique from the genome of the host cell;   cleaving a double strand of the proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease;   cleaving a double strand of the proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease;   completely excising a fragment of greater than 9000-bp of integrated HIV-1 proviral DNA that spanned from its 5′- to 3′-LTRs; and   eradicating the HIV-1 proviral DNA from the host cell.   
     
     
         2 . The method of  claim 1 , wherein said administering step further includes the steps of:
 exposing a host cell to a composition including an isolated nucleic acid encoding the CRISPR-associated endonuclease; an isolated nucleic acid sequence encoding a first gRNA having a first spacer sequence that is complementary to a first target protospacer sequence in a proviral DNA; and an isolated nucleic acid encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the proviral DNA;   expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA;   assembling, in the host cell, a first gene editing complex including the CRISPR-associated endonuclease and the first gRNA; and a second gene editing complex including the CRISPR-associated endonuclease and the second gRNA;   directing the first gene editing complex to the first target protospacer sequence by complementary base pairing between the first spacer sequence and the first target protospacer sequence; and   directing the second gene editing complex to the second target protospacer sequence by complementary base pairing between the second spacer sequence and the second target protospacer sequence.   
     
     
         3 . The method of  claim 2 , wherein at least one of the first target protospacer sequence and the second target protospacer sequence is situated within the U3 region of the LTR. 
     
     
         4 . The method of  claim 3 , wherein the first spacer sequence and the 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. 
     
     
         5 . The method of  claim 3 , wherein the first spacer sequence and the second spacer sequence include, respectively, a sequence complementary to the target protospacer sequences SEQ ID NO: 96 and SEQ ID NO: 121. 
     
     
         6 . The method of  claim 3 , wherein the first spacer sequence and the second spacer sequence each include, respectively, a sequence complementary to the target protospacer sequences SEQ ID NO: 87 and SEQ ID NO: 110. 
     
     
         7 . The method of  claim 1 , wherein the CRISPR-associated endonuclease is Cas9 or a human-optimized Cas9. 
     
     
         8 . The method of  claim 1 , wherein the composition is encoded in a vector selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector. 
     
     
         9 . The method of  claim 1 , wherein at least one of the gRNAs comprises a CRISPR RNA (crRNA) and a trans-activated small RNA (tracrRNA), which are expressed as separate nucleic acids. 
     
     
         10 . The method of  claim 1 , wherein at least one of the gRNAs is engineered as an artificial fusion small guide RNA (sgRNA) comprised of a crRNA and a tracrRNA. 
     
     
         11 . The method of  claim 2 , wherein said step of expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA, is further defined as stably expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA, and the method additionally includes the step of immunizing the host cell against new retroviral infection. 
     
     
         12 . The method of  claim 2 , wherein the host cell is chosen from the group consisting of a CD4+ T cell, a macrophage, a monocyte, a gut associated lymphoid cell, a microglial cell, and an astrocyte. 
     
     
         13 . A method of treating a subject having or at risk for having a genetic caused disease, including the steps of:
 administering to the subject a therapeutically effective amount of a composition 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 proviral DNA that is unique from the genome of the host cell, and wherein the gRNAs are complementary to two target sequences spanning from the sequence of the subjects DNA greater than 9000-bp that is chromosomally integrated and causes the genetic caused disease;   cleaving a double strand of the DNA at a first target protospacer sequence with the CRISPR-associated endonuclease;   cleaving a double strand of the DNA at a second target protospacer sequence with the CRISPR-associated endonuclease;   excising the entire chromosomally integrated sequence; and   eradicating the chromosomally integrated sequence from the host cell.   
     
     
         14 . The method of  claim 13 , wherein said administering step further includes the steps of:
 exposing a host cell to a composition including an isolated nucleic acid encoding the CRISPR-associated endonuclease; an isolated nucleic acid sequence encoding a first gRNA having a first spacer sequence that is complementary to a first target protospacer sequence in the DNA; and an isolated nucleic acid encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the DNA;   expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA;   assembling, in the host cell, a first gene editing complex including the CRISPR-associated endonuclease and the first gRNA; and a second gene editing complex including the CRISPR-associated endonuclease and the second gRNA;   directing the first gene editing complex to the first target protospacer sequence by complementary base pairing between the first spacer sequence and the first target protospacer sequence; and   directing the second gene editing complex to the second target protospacer sequence by complementary base pairing between the second spacer sequence and the second target protospacer sequence.   
     
     
         15 . The method of  claim 13 , wherein at least one of the first target protospacer sequence and the second target protospacer sequence is situated within the U3 region of the LTR. 
     
     
         16 . The method of  claim 13 , wherein the CRISPR-associated endonuclease is Cas9 or a human-optimized Cas9. 
     
     
         17 . The method of  claim 13 , wherein the composition is encoded in a vector selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector. 
     
     
         18 . The method of  claim 13 , wherein at least one of the gRNAs comprises a CRISPR RNA (crRNA) and a trans-activated small RNA (tracrRNA), which are expressed as separate nucleic acids. 
     
     
         19 . The method of  claim 13 , wherein at least one of the gRNAs is engineered as an artificial fusion small guide RNA (sgRNA) comprised of a crRNA and a tracrRNA. 
     
     
         20 . The method of  claim 13 , wherein the host cell is chosen from the group consisting of a CD4+ T cell, a macrophage, a monocyte, a gut associated lymphoid cell, a microglial cell, and an astrocyte. 
     
     
         21 . The method of  claim 13 , wherein said method is performed prenatally.