HIV-1 continues to be a world-wide health problem with an estimated incidence of ~50,000 new cases each year in the United States alone. While antiretroviral therapy has changed the face of HIV infection from terminal illness to a chronic manageable disease, it does not restore a normal immune function or lifespan in infected subjects. A cure to permanently eliminate the virus from the body is yet to be found. HIV can remain latent in several sites within the body including the CNS where it establishes viral reservoirs and continues to persist despite treatment. A targeted genome editing approach that allows for precise and efficient deletion of integrated HIV DNA from the infected cell will be a significant advancement toward HIV cure. We propose to develop engineered exosomes loaded with CRISPR/Cas9 genome editing machinery designed to target integrated HIV DNA in latently-infected cells. These engineered exosomes will also be programmed using surface ligand display technologies to address the vesicles to adhere and deliver the cargo to cell types of interest. The overall goal of this project is to create a system where personalized exosome shuttles can be utilized as therapeutic delivery vehicles to eradicate HIV in viral cellular reservoirs.
Exosomes are a class of extracellular vesicles that are formed in the multivesicular bodies in the cells and range between 30-100nm in diameter. They function as messengers for intercellular communication by transporting nucleic acids and proteins. Recent advances in targeted genome editing using engineered nucleases have generated considerable excitement for its potential in curing HIV infection. The RNA-guided Cas9 nuclease from the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used for precise and efficient genome editing in eukaryotic cells by guiding it to specific locations in the genome using a 20 nucleotide RNA sequence. An increasing line of evidence suggests that targeting the CRISPR/Cas9 machinery to the LTR region of the provirus can successfully disrupt the HIV genome and provide a long-term adaptive resistance against new infection. If harnessed to its full potential, a genome editing approach to excise out integrated HIV genome can lead to a paradigm shift in treatment strategies for HIV infection. Given that exosomes are efficient in delivering functionally active biological cargo and can be engineered and targeted, we propose to package the CRISPR/Cas9 machinery into the vesicles and investigate their ability to disrupt latently integrated HIV genome from infected cells.