Award amount - Direct
Although HIV eradication efforts have mainly focused on latently-infected CD4+ T cells, myeloid cells harbor virus during suppressive antiretroviral therapy and participate in HIV dissemination and persistence. Infection of monocytes and macrophages contributes to inflammation and HIV-associated neurocognitive disorder (HAND). CRISPR/Cas9-mediated anti-HIV gene editing has been applied to excise HIV provirus and knock out the CXCR4 and CCR5 HIV co-receptors in CD4+ T cells as a cure approach. However, anti-HIV gene editing in the myeloid compartment has not been reported as yet. In addition, most studies have used lentiviral or Adeno Associated Virus (AAV)-mediated delivery, typically necessitating integration of vector DNA into the host genome to drive expression of Cas9 and guide RNAs that steer Cas9 to its intended genomic targets. Therefore, these methods raise safety concerns due to the lack of control of integration site and Cas9 and guide expression, which could lead to undesired insertional and off-target mutations.
In this study, we propose to safely and effectively target HIV persistence in the myeloid compartment by delivering CRISPR-Cas9 ribonucleoprotein complexes (Cas9/RNPs) into infected monocyte-derived macrophages (MDMs) using synthetic lipid nanoparticles. This approach enables a convenient, efficient and nontoxic delivery method with transient activity of the Cas9 protein. In Aim 1, we will build off of our promising preliminary data to investigate the ability of engineered Cas9/RNPs targeting the HIV long terminal repeat (LTR) region to excise provirus in primary MDMs. We will further investigate the impact of antiviral gene editing on cellular immunological functions, including phagocytosis and cytokine production. In Aim 2, we will administer nanoparticles loaded with Cas9/RNPs in a mouse model to evaluate in vivo delivery and antiviral editing. Ultimately, in combination with other approaches, this gene therapy method may lead to HIV eradication.