Compositions and methods for in vivo excision of HIV-1 proviral DNA
Inventors
Howell, Alexandra L. • Eszterhas, Susan K.
Assignees
Dartmouth College • US Department of Veterans Affairs
Publication Number
US-11274305-B2
Publication Date
2022-03-15
Expiration Date
2034-03-25
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Abstract
Methods and kits for excising HIV-1 DNA in vivo are provided, which employ Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated (cas) proteins. Vectors harboring nucleic acids encoding one or more guide RNA, wherein said guide RNA hybridizes with a target HIV-1 DNA are also provided.
Core Innovation
This invention provides methods and kits employing the CRISPR/CRISPR-associated (Cas) system for in vivo excision or inhibition of the function or presence of integrated HIV-1 DNA sequences in eukaryotic cells, particularly human cells. The technology uses one or more guide RNA molecules that hybridize uniquely to target sequences in the HIV-1 DNA, coupling with Cas proteins such as Cas9 to specifically cleave and disrupt HIV-1 proviral DNA within the host genome, thereby preventing production of new viral particles.
The problem being addressed arises from the persistence of integrated HIV-1 DNA sequences (proviral DNA) in infected human cells, which serve as the source of new infectious viral particles. Current approaches to treatment do not target these integrated sequences for excision or functional inhibition at the DNA level, limiting efficacy in eradicating the virus. The invention overcomes this by providing targeted CRISPR-based methods to cleave and excise the HIV-1 proviral DNA, reducing viral burden and spread within the host.
Claims Coverage
The patent includes three main independent claims covering a method of inhibiting HIV-1 DNA in eukaryotic cells using guide RNA and Cas9 protein, delivered via nucleic acids or directly, targeting specific HIV-1 DNA sequences.
Method for inhibiting HIV-1 DNA using guide RNA and Cas9 protein
A method comprising contacting a eukaryotic cell harboring integrated HIV-1 DNA with one or more guide RNA or nucleic acids encoding them, plus a Cas9 protein or its encoding nucleic acids, wherein the guide RNA uniquely hybridizes with the target HIV-1 DNA sequence selected from SEQ ID NO:3, 6, and 4, thereby inhibiting function or presence of the HIV-1 DNA.
Guide RNA and Cas9 complex formation and cleavage
The method wherein the guide RNA and Cas9 protein form a complex inside the cell that cuts the HIV-1 DNA sequence, thus inhibiting its function or presence.
Targeting HIV-1 LTR sequences with guide RNA
The guide RNA targets an HIV-1 LTR sequence complementary to SEQ ID NO:3 or 6, with specific guide RNA or nucleic acid sequences comprising SEQ ID NO:3 or 6.
Use of codon-optimized Cas9 protein
The Cas9 protein used in the method is codon-optimized for expression in human cells to enhance effectiveness.
Cas9 protein including nuclear localization sequence
The Cas9 protein further comprises a nuclear localization sequence to facilitate transport into the nucleus where the target HIV-1 DNA resides.
Delivery via viral vectors
The nucleic acids encoding the guide RNA and Cas9 protein are contained in viral vectors for delivery to the eukaryotic cell.
In vitro application
The method can be carried out in vitro by contacting the eukaryotic cell with guide RNA and Cas9 protein.
Expanded targeting of HIV-1 DNA sequences
Additional guide RNAs or nucleic acids encoding guide RNAs targeting HIV-1 DNA sequences selected from SEQ ID NO:7, 8, and 9 are included in the method.
The claims cover a comprehensive method employing specific guide RNA sequences hybridizing to defined HIV-1 DNA targets and Cas9 proteins optimized for human expression and nuclear localization, forming complexes that cleave proviral HIV-1 DNA, delivered via nucleic acids or vectors, applicable in vitro or potentially in vivo.
Stated Advantages
Provides a method to excise or inhibit the function of integrated HIV-1 DNA sequences within human cells, reducing viral burden.
Targets the HIV-1 DNA specifically with minimal off-target effects due to guide RNA hybridization to unique viral sequences, minimizing cytotoxicity to uninfected cells.
Uses codon-optimized Cas9 and nuclear localization sequences to enhance efficacy in human cells.
Delivery via various viral and non-viral vectors allows flexibility and potential for in vivo therapeutic application.
Documented Applications
Therapeutic treatment or prevention of HIV-1 infection by targeting and cleaving integrated HIV-1 proviral DNA in infected human cells to reduce viral load and prevent spread.
In vitro screening or research applications to assess guide RNA efficacy in cleaving HIV-1 LTR regions using reporter systems based on HIV-1 LTR-driven GFP expression.
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