Description:
Background
Highly active antiretroviral therapy is now optimized to control HIV-1 replication long-term, but the virus remains integrated into the host genome in a latent form and poses a threat for re-emergence. In search for more potent therapeutic agents geared towards HIV cure, newly developed chimeric nucleases, which allow the precise modification of viral and human genomes, have recently been explored for HIV reservoir elimination. These designer enzymes have the ability to disrupt the integrated HIV genome by double-stranded DNA break, so integrated proviruses become permanently defective, and to modify host genes essential for HIV replication, so cells become resistant to HIV infection. The gene-editing enzymes currently available to the scientific community are zinc finger nucleases (ZFNs), transcription activator like-effector nucleases (TALENs), homing endonucleases, and clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9. Each of these restriction enzymes is associated with unique strengths, but also with off-target effects.
Project Goals
The primary goal is to design improved nucleases for disruption of integrated HIV provirus and/or modification of host genes, so HIV replication is no longer supported. There is also a need for alternative delivery strategies for these nucleases to substitute for lentiviral gene delivery or plasmid transfections. An additional goal is to evaluate off-target effects and immune responses induced by the delivered nucleases and their vectors.
Phase 1 activities may include
· Design and test chimeric nucleases that irreversibly disrupt or excise HIV provirus in infected cell lines and peripheral blood mononuclear cells (PBMC)
· Design modified host genes and test their ability to impede HIV infection
· Evaluate off-target effects in cell lines and primary PBMC
· Develop strategies for eliminating off-target effects, including software tools for designing nucleases with reduced off-target sites
Phase 2 activities may include
· Develop and test improved delivery strategies
· Evaluate efficacy and adverse reactions of delivered nucleases in humanized mouse and nonhuman primate models
· Evaluate immune responses against nucleases and vectors in vivo and develop strategies to reduce the immunogenicity of the delivered constructs
