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

慢性B型肝炎病毒（HBV）的患病率较高，全球为7.4%，在高度流行地区为15 - 28%， 艾滋病毒感染者（PLWH）。虽然目前的联合抗逆转录病毒疗法（cART）可以限制 在HBV/HIV复制中，cART不能消除整合到宿主基因组中的HIV/HBV DNA。因此，在本发明中， HBV和HIV在cART控制的个体中持续存在，cART停止容易导致病毒再激活， 疾病进展。因此，任何治疗策略都应该包括从细胞中消除整合的病毒DNA的手段。 携带HIV和/或HBV（HBV/HIV）DNA而无附带细胞毒性反应的储库细胞。CRISPR （成簇规则间隔短回文重复序列）Cas9（CRISPR相关蛋白9）介导的基因 编辑是解决这一问题的一种有吸引力的方法。CRISPR/Cas9方法成功的关键是 选择对病毒复制至关重要的病毒特异性靶基因，同时避免对人类的脱靶效应 基因组，并确保基因编辑药物有效地传递到靶细胞。目前的CRISPR/Cas9交付 技术通常需要病毒载体，这对人类的治疗应用提出了安全问题。 合成的Cas9-核糖核蛋白（RNP）是CRISPR/Cas9系统的一种有吸引力的非病毒制剂，这是由于 其快速DNA切割活性、低频率脱靶效应、低插入突变风险、容易 生产和准备临床应用。然而，现有的Cas9-RNP递送的非病毒策略 面临着许多挑战，如高细胞毒性、体内稳定性差、大颗粒尺寸、缺乏特异性 组织和/或细胞靶向能力、RNP货物的可变负载和潜在的免疫原性。这些 这些挑战限制了Cas9-RNP在体内系统性应用中的应用。因此，发现的进展 因此，迫切需要一种新的针对掺入的病毒DNA的干预措施来治愈HBV/HIV合并感染。 为了满足这些需求，我们：1）选择了对病毒复制至关重要的特定HBV/HIV靶基因 但与人类基因组没有重叠（脱靶）; 2）合成的向导RNA（gRNA）和Cas9-RNP 作为治疗药物; 3）开发了具有较长可裂解聚乙二醇（PEG）臂的新型纳米颗粒（NP） 修饰HBV/HIV gRNA-Cas9 RNP并减缓细胞内前药的释放;和4）建立 HBV/HIV细胞模型来测试我们产生的HBV/HIV gRNA-RNP的功效和细胞毒性。在本研究中， 我们将测试我们新设计的基因编辑药物，这些药物靶向病毒DNA，而不是使用HBV/HIV的人类基因组。 细胞模型我们假设特定的CRISPR/Cas9基因编辑药物将消除HBV/HIV复制 并在这些细胞模型中引起最小的细胞毒性。我们提出了两个具体的目标来检验我们的假设： 1将在我们的细胞HBV/HIV中使用核转染方法筛选和测试CRISPR/Cas9基因编辑药物 目标2将产生和测试HBV/HIV gRNA-Cas9 NPs，并在我们的研究中比较它们的功效和细胞毒性。 细胞HBV/HIV模型。该项目的目标是收集关键信息，建立新技术， 并为实现我们发现HBV/HIV合并感染的治愈方法的长期目标奠定基础。

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.