Methods and compositions for rna-guided treatment of hiv infection

Abstract

A personalized method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, by determining a nucleic acid sequence of the HIV-1 proviral DNA harbored by a subject, designing two or more different multiplex guide RNAs (gRNAs) complementary to the HIV-1 proviral DNA sequences in the subject, administering to the subject a therapeutically effective amount of a composition comprising a CRISPR-associated endonuclease, and the two or more different multiplex gRNAs, 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, excising an entire HIV-1 proviral genome, and eradicating the HIV-1 proviral DNA from the host cell.

What is claimed is: 
     
         1 . A personalized method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, including the steps of:
 determining a nucleic acid sequence of HIV-1 proviral DNA harbored by a subject;   designing two or more different multiplex guide RNAs (gRNAs) complementary to the HIV-1 proviral DNA sequences in the subject;   administering to the subject a therapeutically effective amount of a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and the 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 a 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;   excising an entire HIV-1 proviral genome; and   eradicating the HIV-1 proviral DNA from the host cell.   
     
     
         2 . The method of  claim 1 , wherein said existing step includes excising the target sequences spanning from the 5′- to 3′-LTRs of the sequence in the virus. 
     
     
         3 . 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.   
     
     
         4 . The method of  claim 1 , 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. 
     
     
         5 . The method of  claim 1 , wherein the CRISPR-associated endonuclease is Cas9 or a human-optimized Cas9. 
     
     
         6 . 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. 
     
     
         7 . 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. 
     
     
         8 . 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. 
     
     
         9 . The method of  claim 1 , further including the step of immunizing the host cell against new viral infection. 
     
     
         10 . The method of  claim 1 , 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.