Compositions and methods for specific reactivation of hiv latent reservoir
AssigneeTemple University of the Commonwealth System of Higher Education
InventorsWenhui Hu, Kamel Khalili, Yonggang Zhang
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Abstract
The present invention relates to compositions and methods for reactivation of HIV in vivo or in vitro. latently infected cell. In one embodiment, the present invention uses the CRISPR/Cas9 system in combination with the MS2 bacteriophage coat protein-mediated synergistic activation mediator (SAM) system to provide enhanced transcriptional activation of the HIV genome.
Claims36
A composition for reactivation of a retrovirus in vitro or in vivo comprising:
- an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to one or more target sequences in the retroviral genome;
- an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease/Cas (CRISPR/Cas) fusion protein, comprising CRISPR/Cas and one or more transcriptional activators;
- and, an isolated nucleic acid encoding a fusion protein comprising an RNA binding protein, fragments, mutants, derivatives or variants thereof and one or more transcriptional activators.
An isolated nucleic acid encoding:
- a guide nucleic acid, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to one or more target sequences in the retroviral genome;
- a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease/Cas (CRISPR/Cas) fusion protein, comprising CRISPR/Cas and one or more transcriptional activators;
- and, an isolated nucleic acid encoding a fusion protein comprising an RNA binding protein, fragments, mutants, derivatives or variants thereof and one or more transcriptional activators.
An isolated nucleic acid molecule encoding at least one guide nucleic acid molecule (gRNA), an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease/Cas (CRISPR/Cas) fusion protein or functional fragment or derivative thereof, and an isolated nucleic acid encoding an RNA binding protein, functional fragments, mutants, derivatives or variants thereof.
A composition for reactivation of HIV in a latently infected cell comprising:
- a) one selected from the group consisting of an isolated guide nucleic acid and an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to a target sequence in the HIV genome;
- b) one selected from the group consisting of a catalytically deficient Cas9 (dCas9) fusion protein, and an isolated nucleic acid encoding a dCas9 fusion protein, wherein the dCas9 fusion protein comprises dCas9 and one or more transcriptional activators;
- and
- c) one selected from the group consisting of a MS2 fusion protein, and an isolated nucleic acid encoding a MS2 fusion protein, wherein the MS2 fusion protein comprises MS2 bacteriophage coat protein and one or more transcription activators.
A pharmaceutical composition comprising
- a) one selected from the group consisting of an isolated guide nucleic acid and an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to a target sequence in the HIV genome;
- b) one selected from the group consisting of a catalytically deficient Cas9 (dCas9) fusion protein, and an isolated nucleic acid encoding a dCas9 fusion protein, wherein the dCas9 fusion protein comprises dCas9 and one or more transcription activators;
- and
- c) one selected from the group consisting of a MS2 fusion protein, and an isolated nucleic acid encoding a MS2 fusion protein, wherein the MS2 fusion protein comprises MS2 bacteriophage coat protein and one or more transcription activators.
An expression vector encoding at least one of:
- a guide nucleic acid, a dCas9 fusion protein, and a MS2 fusion protein, wherein
- the guide nucleic acid comprises a targeting nucleotide sequence directed to a target sequence in the HIV genome;
- the dCas9 fusion protein comprises dCas9 and one or more transcription activators;
- and
- the MS2 fusion protein comprises MS2 bacteriophage coat protein and one or more transcription activators.
A host cell comprising the expression vector comprising:
- (i) an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to one or more target sequences in the retroviral genome;
- a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease/Cas (CRISPR/Cas) fusion protein, comprising CRISPR/Cas and one or more transcriptional activators;
- and, an isolated nucleic acid encoding a fusion protein comprising an RNA binding protein, fragments, mutants, derivatives or variants thereof and one or more transcriptional activators;
- or,
- (ii) an isolated nucleic acid molecule encoding at least one guide nucleic acid molecule (gRNA), an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease/Cas (CRISPR/Cas) fusion protein or functional fragment or derivative thereof, and an isolated nucleic acid encoding an RNA binding protein, functional fragments, mutants, derivatives or variants thereof;
- or,
- (iii) (a) an isolated guide nucleic acid and an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to a target sequence in the HIV genome;
- (b) a catalytically deficient Cas9 (dCas9) fusion protein, and an isolated nucleic acid encoding a dCas9 fusion protein, wherein the dCas9 fusion protein comprises dCas9 and one or more transcriptional activators;
- and
- (c) a MS2 fusion protein, and an isolated nucleic acid encoding a MS2 fusion protein, wherein the MS2 fusion protein comprises MS2 bacteriophage coat protein and one or more transcription activators;
- or,
- (iv) at least one of:
- a guide nucleic acid, a dCas9 fusion protein, and a MS2 fusion protein, wherein the guide nucleic acid comprises a targeting nucleotide sequence directed to a target sequence in the HIV genome;
- the dCas9 fusion protein comprises dCas9 and one or more transcription activators;
- and
- the MS2 fusion protein comprises MS2 bacteriophage coat protein and one or more transcription activators.
A method for reactivation of HIV in a latently infected cell in a subject, comprising contacting a cell of the subject with a therapeutically effective amount of a composition comprising
- a) one selected from the group consisting of an isolated guide nucleic acid and an isolated nucleic acid encoding a guide nucleic acid, where the guide nucleic acid comprises a targeting nucleotide sequence directed to a target sequence in the HIV genome;
- b) one selected from the group consisting of a catalytically deficient Cas9 (dCas9) fusion protein, and an isolated nucleic acid encoding a dCas9 fusion protein, wherein the dCas9 fusion protein comprises dCas9 and one or more transcription activators;
- and
- c) one selected from the group consisting of a MS2 fusion protein, and an isolated nucleic acid encoding a MS2 fusion protein, wherein the MS2 fusion protein comprises MS2 bacteriophage coat protein and one or more transcription activators.
The method of claim 38, wherein the subject is diagnosed with having HIV.
The method of claim 38, wherein the subject has a latent HIV infection.
Dependencies are inferred from the claim text.