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

针对 HBV/HIV 双重感染的纳米颗粒递送的双特异性基因编辑药物

• 批准号: 10403587
• 负责人: Zhi Q. Yao
• 金额: $ 18.56万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-10 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10403587
• 关键词: Address; Area; Caliber; Cations; Cell model; Cells; Chronic Hepatitis B; Cirrhosis; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Data; Disease Progression; Drug Targeting; Drug usage; Encapsulated; Endothelium; Ensure; Environment; Face; Formulation; Foundations; Frequencies; Gene Delivery; Genes; Genetic Transcription; Genome; Goals; Guide RNA; HIV; HIV Infections; Hepatitis B Virus; High Prevalence; Human; Human Genome; Immunology; In Vitro; Individual; Injections; Insertional Mutagenesis; Intervention; Liposomes; Mediating; Modality; Modeling; Morbidity - disease rate; Nucleic Acids; Particle Size; Patients; Persons; Pharmaceutical Preparations; Pharmacologic Substance; Polyethylene Glycols; Polymers; Primary carcinoma of the liver cells; Prodrugs; Production; Proteins; Reaction; Readiness; Research Personnel; Ribonucleoproteins; Risk; Safety; System; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Viral; Viral Vector; Virus; Virus Diseases; Virus Replication; Work; antiretroviral therapy; arm; base; clinical application; co-infection; cytotoxic; cytotoxicity; design; drug development; drug release kinetics; efficacy testing; experience; immunogenicity; in vivo; innovation; mortality; multimodality; nanoparticle; nanoparticle delivery; new technology; novel; novel strategies; particle; research and development; success; translational study; viral DNA; virology

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.

慢性乙型肝炎病毒 (HBV) 的患病率较高，全球为 7.4%，在高流行地区为 15% 至 28%。 在艾滋病毒感染者（PLWH）中观察到。虽然目前的联合抗逆转录病毒疗法（cART）可以限制 HBV/HIV 复制时，cART 无法消除整合到宿主基因组中的 HIV/HBV DNA。像这样， HBV 和 HIV 在 cART 控制的个体中持续存在，并且 cART 停止很容易导致病毒重新激活和 疾病进展。因此，任何治疗策略都应包括消除整合病毒 DNA 的方法。 携带 HIV 和/或 HBV (HBV/HIV) DNA 且没有附带细胞毒性反应的储存细胞。基因编辑技术 （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 编辑是解决这个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，同时避免对人类产生脱靶影响 基因组并确保基因编辑药物有效递送至靶细胞。当前的 CRISPR/Cas9 交付 技术通常需要病毒载体，这给人类治疗应用带来了安全问题。 合成 Cas9-核糖核蛋白 (RNP) 对于 CRISPR/Cas9 系统来说是一种有吸引力的非病毒制剂，因为 DNA 切割活性快、脱靶效应频率低、插入突变风险低、易于 生产和临床应用准备。然而，现有的 Cas9-RNP 递送非病毒策略 面临细胞毒性高、体内稳定性差、粒径大、缺乏特异性等诸多挑战 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 在体内系统应用中的应用。因此，发现的进展 迫切需要针对整合病毒 DNA 的新型干预措施来治愈 HBV/HIV 双重感染。 为了满足这些需求，我们：1）选择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) 合成向导RNA (gRNA) 和Cas9-RNP 作为治疗药物； 3）开发了具有更长可裂解聚乙二醇（PEG）臂的新型纳米颗粒（NP） 修饰 HBV/HIV gRNA-Cas9 RNP 并减缓前药在细胞内的释放； 4) 建立 HBV/HIV 细胞模型用于测试我们生成的 HBV/HIV gRNA-RNP 的功效和细胞毒性。在这项研究中， 我们将使用 HBV/HIV 测试我们新设计的基因编辑药物，这些药物针对病毒 DNA，但不针对人类基因组 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。我们提出两个具体目标来检验我们的假设： 1 将使用核转染方法在我们的细胞 HBV/HIV 中筛选和测试 CRISPR/Cas9 基因编辑药物 模型；目标 2 将生成并测试 HBV/HIV gRNA-Cas9 NP，并比较它们在我们的研究中的功效和细胞毒性。 细胞 HBV/HIV 模型。该项目的目标是收集关键信息、建立新技术、 并为实现我们发现治疗 HBV/HIV 合并感染的长期目标奠定基础。

项目成果

Oxidative Stress Induces Mitochondrial Compromise in CD4 T Cells From Chronically HCV-Infected Individuals.

氧化应激会导致慢性 HCV 感染者的 CD4 T 细胞线粒体受损。

• 发表时间: 2021
• 作者: Schank, Madison;Zhao, Juan;Wang, Ling;Nguyen, Lam Ngoc Thao;Cao, Dechao;Dang, Xindi;Khanal, Sushant;Zhang, Jinyu;Zhang, Yi;Wu, Xiao Y;Ning, Shunbin;Gazzar, Mohamed El;Moorman, Jonathan P;Yao, Zhi Q
• 通讯作者: Yao, Zhi Q

TRF2 inhibition rather than telomerase disruption drives CD4T cell dysfunction during chronic viral infection.

在慢性病毒感染期间，TRF2 抑制而不是端粒酶破坏导致 CD4T 细胞功能障碍。

• 发表时间: 2022-07-01
• 影响因子: 4
• 作者: Nguyen, Lam Ngoc Thao;Nguyen, Lam Nhat;Zhao, Juan;Schank, Madison;Dang, Xindi;Cao, Dechao;Khanal, Sushant;Wu, Xiao Y;Zhang, Yi;Zhang, Jinyu;Ning, Shunbin;Wang, Ling;El Gazzar, Mohamed;Moorman, Jonathan P;Yao, Zhi Q
• 通讯作者: Yao, Zhi Q

Mitochondrial topoisomerase 1 inhibition induces topological DNA damage and T cell dysfunction in patients with chronic viral infection.

线粒体拓扑异构酶 1 抑制会导致慢性病毒感染患者的拓扑 DNA 损伤和 T 细胞功能障碍。

• 发表时间: 2022
• 作者: Dang, Xindi;Cao, Dechao;Zhao, Juan;Schank, Madison;Khanal, Sushant;Nguyen, Lam Ngoc Thao;Wu, Xiao Y;Zhang, Yi;Zhang, Jinyu;Jiang, Yong;Ning, Shunbin;Wang, Ling;El Gazzar, Mohamed;Moorman, Jonathan P;Yao, Zhi Q
• 通讯作者: Yao, Zhi Q

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.

选择性氧化应激会引起人类 T 细胞端粒和线粒体的双重损伤。

• 发表时间: 2021
• 影响因子: 7.8
• 作者: Wang, Ling;Lu, Zeyuan;Zhao, Juan;Schank, Madison;Cao, Dechao;Dang, Xindi;Nguyen, Lam Nhat;Nguyen, Lam Ngoc Thao;Khanal, Sushant;Zhang, Jinyu;Wu, Xiao Y;El Gazzar, Mohamed;Ning, Shunbin;Moorman, Jonathan P;Yao, Zhi Q
• 通讯作者: Yao, Zhi Q

Plasma biomarkers for systemic inflammation in COVID-19 survivors.

COVID-19 幸存者全身炎症的血浆生物标志物。

• 发表时间: 2022-09
• 作者: Zhao, Juan;Schank, Madison;Wang, Ling;Dang, Xindi;Cao, Dechao;Khanal, Sushant;Nguyen, Lam N T;Zhang, Yi;Wu, Xiao Y;Adkins, James L;Pelton, Benjamin J;Zhang, Jinyu;Ning, Shunbin;Gazzar, Mohamed El;Moorman, Jonathan P;Yao, Zhi Q
• 通讯作者: Yao, Zhi Q