ABSTRACT Substantial evidence now indicates that HIV-infected individuals are at a significantly elevated risk for severe COVID-19 disease when infected with SARS-CoV-2. Moreover, persons who use and inject drugs (PWUD and PWID) have a high-risk of HIV infection and exposure to SARS-CoV-2 and face major barriers to accessing antiviral therapies (i.e., are often immunocompromised). The problem is further exacerbated by the emergence of SARS-CoV-2 variants that escape vaccine-mediated immunity—it is now evident that immunocompromised individuals promote the evolution of SARS-CoV-2 escape variants and HIV-infected PWUD/PWID with barriers to treatment represent such a population. Consequently, there is a critical unmet medical need for new therapeutics that could treat HIV as well as SARS-CoV-2—particularly the emerging variants of concern’—and could be effectively deployed in difficult-to-reach, high-risk populations (e.g., PWUD/PWID). The long-term goal of this work is to develop single-administration therapies for HIV-1 and SARS-CoV-2 variants to effectively reach PWID/PWUD populations. The specific objective of this supplement proposal is to test efficacy of our recently developed Gene Drive Therapies (GDT) against HIV and SARS-CoV-2 variants in patient cells from HIV+ PWID. This effort will build heavily off our recent success in engineering GDTs for HIV-1 (see Parent Award) and Zika Virus (ZIKV), as well as our extensive preliminary in vitro data showing efficacy of GDTs against SARS-CoV-2 variants. The central hypothesis—based on our extensive preliminary in vitro studies—is that our engineered GDT candidates will have the capacity to reduce both SARS-CoV-2 viral load and pathogenesis, including of SARS-CoV-2 variants of concern, and HIV viral load, thereby serving as a single-administration, combination therapeutic for HIV-1 and SARS-CoV-2. The rationale for a GDT for SARS-CoV-2 is based on our preliminary data showing that GDTs significantly reduce SARS-CoV-2 replication in cell culture, are equally effective against CoV-2 variants and from extensive studies on HIV-1 in humanized mice and positive FDA meetings. We will achieve our objectives via two specific aims: (i) Quantify in vivo efficacy of the recently developed GDT in reducing SARS-CoV-2 viral replication and pathogenesis in hamsters; and (ii) Develop a lung-organoid co- culture to test efficacy of GDTs against HIV-1 and SARS-CoV-2 in patient-derived cells from HIV+ PWIDs. While the GDT approach carries inherent risks, single-administration therapeutics active against both SARS-CoV-2 variants and HIV would be highly beneficial, particularly for treating difficult-to-reach, high-risk PWID populations. The studies proposed here will also have broad fundamental significance by establishing a novel culture model and tool to assay how SARS-CoV-2 and HIV infections interact in the PWUD/PWID setting (i.e., in the context of Substance Use Disorders (SUDs) in at-risk populations) and will pro...