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

慢性乙肝病毒(乙肝)的高流行率，全球为7.4%，在高流行地区为15%至28%， 在艾滋病毒携带者(PLWH)中观察到。而目前的联合抗逆转录病毒疗法(CART)可以限制 在病毒/艾滋病毒复制过程中，CART不能消除整合到宿主基因组中的艾滋病毒/HBVDNA。因此， 乙肝病毒和艾滋病毒在CART控制的个人中持续存在，CART的停止很容易导致病毒重新激活和 疾病的发展。因此，任何治疗策略都应该包括一种方法，从 携带艾滋病毒和/或乙肝病毒(乙肝病毒/艾滋病)DNA而没有伴随的细胞毒性反应的存储细胞。CRISPR (规则排列的短回文重复序列)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但又避免对人类产生非靶点影响的病毒特异性靶基因 并确保有效地将基因编辑药物输送到靶细胞。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构成了安全问题。 合成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，原因是 其DNA切割活性快，脱靶频率低，插入突变风险低，易于实现 生产，并准备好临床应用。然而，现有的非病毒策略用于Cas9-RNP传递 面临着细胞毒性高、体内稳定性差、粒径大、缺乏特异性等挑战 组织和/或细胞靶向能力，RNP货物的可变负载，以及潜在的免疫原性。这些 挑战限制了Cas9-RNP在体内全身应用中的应用。因此，这一发现的进展 迫切需要针对整合的病毒DNA的新的干预措施来治愈乙肝病毒/艾滋病毒混合感染。 为了满足这些需求，我们有：1)选择了对病毒复制至关重要的特定的乙肝病毒/艾滋病病毒靶基因 但与人类基因组没有重叠之处；2)合成的引导RNA(GRNA)和Cas9-RNP 作为治疗药物；3)开发具有更长可切割聚乙二醇臂的新型纳米颗粒(NP) 修饰HBVgRNA-Cas9RNP并减缓前药物在细胞内的释放；以及4)建立 建立乙肝病毒/艾滋病细胞模型，测试我们制备的乙肝病毒/艾滋病病毒gRNA-RNP的有效性和细胞毒性。在这项研究中， 我们将测试我们新设计的基因编辑药物，这些药物针对的是病毒DNA，而不是使用乙肝病毒/艾滋病毒的人类基因组 细胞模型。我们假设特定的CRISPR/Cas9基因编辑药物将消除乙肝病毒/艾滋病毒的复制 并在这些细胞模型中产生最小的细胞毒性。我们提出了两个具体的目标来验证我们的假设： 1将在我们的细胞乙肝病毒/艾滋病病毒中使用核转染法筛选和测试CRISPR/Cas9基因编辑药物 模型；Aim 2将产生和测试HBVgRNA-Cas9 NPs，并比较它们在我们的 细胞乙肝病毒/艾滋病病毒模型。该项目的目标是收集关键信息，建立新技术， 并为实现我们发现治愈乙肝病毒/艾滋病毒混合感染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.