Dual specific gene editing drugs delivered by nanoparticles targeting HBV/HIV coinfection

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A higher prevalence of chronic hepatitis B virus (HBV), 7.4% globally and 15 to 28% in highly endemic areas, is observed in people living with HIV (PLWH). While current combined antiretroviral therapy (cART) can restrict HBV/HIV replication, cART cannot eliminate the HIV/HBV DNAs that are integrated into the host genome. As such, HBV and HIV persist in cART-controlled individuals, and cART cessation readily leads to viral reactivation and disease progression. Thus, any curative strategy should include a means to eliminate integrated viral DNA from the reservoir cells that harbor HIV and/or HBV (HBV/HIV) DNA without collateral cytotoxic reactions. CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 (CRISPR-associated protein 9)-mediated gene editing is an appealing approach to tackle this problem. The keys to success in the CRISPR/Cas9 approach are to select virus-specific target genes that are critical for viral replication yet avoid off-target effects on the human genome and ensure efficient delivery of the gene-editing drugs to target cells. The current CRISPR/Cas9 delivery technologies often require viral vectors, which pose safety concerns for therapeutic applications in humans. Synthetic Cas9-ribonucleoprotein (RNP) is an attractive non-viral formulation for the CRISPR/Cas9 system due to its quick DNA cleavage activity, low frequency of off-target effects, low risk of insertional mutagenesis, easy production, and readiness for clinical application. However, existing non-viral strategies for Cas9-RNP delivery face a number of challenges, such as high cytotoxicity, poor in vivo stability, large particle sizes, lack of specific tissue- and/or cell-targeting abilities, variable loading of the RNP cargo, and potential immunogenicity. These challenges limit the application of Cas9-RNP for in vivo systemic application. Therefore, advances in the discovery of novel interventions targeting incorporated viral DNA are urgently needed for the cure of HBV/HIV co-infection. To address these needs, we have: 1) selected specific HBV/HIV target genes that are crucial for viral replication but share no overlap with (off-targeting) the human genome; 2) synthesized guide-RNAs (gRNA) and Cas9-RNP as therapeutic drugs; 3) developed novel nanoparticles (NP) with longer cleavable polyethylene glycol (PEG) arms to decorate the HBV/HIV gRNA-Cas9 RNP and slow the release of the prodrug intracellularly; and 4) established HBV/HIV cellular models to test the efficacy and cytotoxicity of our generated HBV/HIV gRNA-RNP. In this study, we will test our newly designed gene editing drugs that target viral DNA but not the human genome using HBV/HIV cellular models. We hypothesize that specific CRISPR/Cas9 gene editing drugs will abolish HBV/HIV replication and elicit minimum cytotoxicity in these cellular models. We propose two specific aims to test our hypothesis: Aim 1 will screen and test CRISPR/Cas9 gene editing drugs using a nucleofection approach in our cellular HBV/HIV models; Aim 2 will generate and test HBV/HIV gRNA-Cas9 NPs and compare their efficacy and cytotoxicity in our cellular HBV/HIV models. The objectives of this project are to collect critical information, establish new techniques, and lay the foundation for achieving our long-term goal of discovery a cure for HBV/HIV co-infection.