Methods and compositions for RNA-guided treatment of HIV infection
Compositions for specifically cleaving target sequences in retroviruses include nucleic acids encoding a Clustered Regularly Interspace Short Palindromic Repeat (CRISPR) associated endonuclease and a guide RNA sequence complementary to one or more target nucleic acid sequences in a retrovirus genome.
1. A composition comprising:
(a) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(b) a second nucleic acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence;
(c) a third nucleic acid sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence; and
(d) an excipient.
2. The composition of claim 1 , wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.
3. The composition of claim 1 , wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.
4. The composition of claim 1 , wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.
5. A pharmaceutical composition comprising:
(a) an expression vector comprising:
(i) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(ii) a second nucleic acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence; and
(iii) a third nucleic sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence; and
(b) a pharmaceutically acceptable excipient.
6. The pharmaceutical composition of claim 5 , wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.
7. The pharmaceutical composition of claim 5 , wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.
8. The pharmaceutical composition of claim 5 , wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.
9. An expression vector comprising:
(a) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(b) a second acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence;
(c) a nucleic acid sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence; and
(d) a regulatory region.
10. The expression vector of claim 9 , wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.
11. The expression vector of claim 9 , wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.
12. The expression vector of claim 9 , wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.
13. A polynucleotide encoding:
(a) a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas9 endonuclease;
(b) a second nucleic acid sequence encoding a first guide RNA (gRNA), the first gRNA being complementary to a first target sequence within a 5′ long terminal repeat (LTR) or a 3′ LTR of a retroviral sequence; and
(c) a third nucleic acid sequence encoding a second gRNA, the second gRNA being complementary to a second target sequence within a GagD region of the retroviral sequence.
14. The polynucleotide of claim 13 , wherein the retroviral sequence is a human immunodeficiency virus (HIV) sequence integrated into a mammalian genome.
15. The polynucleotide of claim 13 , wherein the first gRNA comprises a nucleic acid sequence having a sequence identity of 100% to any one of SEQ ID NOS: 1-8.
16. The polynucleotide of claim 13 , wherein the second gRNA comprises a nucleic acid sequence having a sequence identity of 100% to SEQ ID NO: 47 or SEQ ID NO: 48.