PROJECT SUMMARY/ ABSTRACT The AIDS pandemic took a life every minute in 2021 and the UNAIDS estimated that over 1.5 million new infections occurred, marking the smallest annual decline in new HIV-1 infections since 2016. Today almost 29 million of the world's 38 million HIV-infected people have access to life-saving combination antiretroviral drug therapies (cART) drugs, according to UNAIDS. Despite the absence of a cure that would completely eliminate replication-competent HIV-1 proviruses in the body, cART suppresses plasma viremia in infected individuals to undetectable levels, however viral load rapidly rebounds upon cART interruption, requires lifelong administration, and can have undesirable side effects. This indicates an additional therapeutic agent is needed to eliminate HIV- infected cells and remove the viral reservoir. The major obstacle that impedes HIV-1 eradication in cART-treated individuals is the establishment of long lived and proliferating, latently infected cells. A lack of HIV-1 transactivator of transcription (tat) protein is important in maintaining latency in resting CD4+ T-cells and therefore the HIV-1 tat protein is not readily available during the initial reactivation of provirus. Moreover, the HIV-1 tat protein is a potent activator of HIV-1 gene expression and is essential for integrated viral genome expression, which represents an opportunity for the HIV-1 tat protein to be used as a molecular tool. The Gardner lab has expertise in using gene therapy strategies for HIV prevention and cure approaches. The lab has traditionally used adeno- associated virus vectors for delivery and expression of HIV-1 entry inhibitors and broadly neutralizing antibodies (bNAbs). We have recently started expanding our gene therapy repertoire to include virus-like particle (VLP) delivery of therapeutic protein cargos in HIV-1 cure strategies. VLPs are known as viral “empty shells” that maintain the same structural properties of virions without any genome, therefore these constructs are considered very efficient as therapeutic delivery systems. VLPs can also be modified to target specific cell types, while having good cell penetrating properties to mediate cargo passing the cell membrane allowing for release of proteins of interest into cells, making the VLP an ideal tool for intracellular delivery of proteins3. Therefore, VLPs can be applied to improve HIV-1 cure strategies. In aim 1, we will use VLPs to deliver the HIV-1 tat protein as a latency reversing agents (LRA). This aim will utilize a shock and kill strategy, first with VLPs to transport the HIV- 1 tat protein as a LRA (the “shock”) and secondly these reactivated cells will be targeted for elimination with AAV vectors that express bNAbs (the “kill”). Additionally, in aim 2, we will utilize VLPs to deliver Cas12k fused to HIV- 1 integrase (VLP-Cas12k) to target integration of a transgene cassette into an integrated HIV-1 provirus within CD4+ T-cells triggering apoptosis and immun...