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.

慢性丙型肝炎病毒（HBV）的患病率更高，全球7.4％，高度流行地区15％至28％是 在艾滋病毒（PLWH）的人中观察到。虽然当前的抗逆转录病毒疗法（CART）可以限制 HBV/HIV复制，CART无法消除整合到宿主基因组中的HIV/HBV DNA。像这样， HBV和HIV一直存在于手推车控制的个体中，并且卡车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一种消除从中消除综合病毒DNA的方法 含有HIV和/或HBV（HBV/HIV）DNA的储层细胞，无副细胞毒性反应。 CRISPR （群集定期间隔短的短质体重复序列）Cas9（CRISPR相关蛋白9）介导的基因 编辑是解决此问题的一种吸引人的方法。 CRISPR/CAS9方法成功的关键是 选择对病毒复制至关重要但避免对人的脱靶影响至关重要的病毒特异性靶基因 基因组并确保有效递送基因编辑药物向靶细胞。当前的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-核核蛋白（RNP）是CRISPR/CAS9系统的一种有吸引力的非病毒配方 它的快速DNA裂解活性，脱靶效应的低频率，插入诱变的低风险，易于 生产和临床应用的准备。但是，现有的CAS9-RNP交付的非病毒策略 面临许多挑战，例如高细胞毒性，体内稳定性差，大粒径，缺乏特定 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，发现的进步 靶向拟合病毒DNA的新型干预措施迫切需要治愈HBV/HIV共同感染。 为了满足这些需求，我们有：1）选择特定的HBV/HIV靶基因，这些基因对于病毒复制至关重要 但是与人类基因组（不靶向）没有重叠； 2）合成的指南RNA（GRNA）和CAS9-RNP 作为治疗药物； 3）开发了具有较长可裂解聚乙烯乙二醇（PEG）臂的新型纳米颗粒（NP） 要装饰HBV/HIV GRNA-CAS9 RNP并减慢前药细胞内的释放；和4）建立 HBV/HIV细胞模型测试我们生成的HBV/HIV GRNA-RNP的功效和细胞毒性。在这项研究中， 我们将测试我们新设计的基因编辑药物，该药物靶向病毒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基因编辑药物将废除HBV/HIV复制 并在这些细胞模型中引起最小细胞毒性。我们提出了两个特定的目的来检验我们的假设：目标 1将使用我们的细胞HBV/HIV筛选和测试CRISPR/CAS9基因编辑药物 模型； AIM 2将产生和测试HBV/HIV GRNA-CAS9 NP，并比较其在我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， 并奠定了实现我们发现HBV/HIV共同感染的长期目标的基础。

项目成果

Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.

• DOI: 10.3390/v14091902
• 发表时间: 2022-08-28
• 期刊: Viruses
• 作者: Khanal S;Cao D;Zhang J;Zhang Y;Schank M;Dang X;Nguyen LNT;Wu XY;Jiang Y;Ning S;Zhao J;Wang L;Gazzar ME;Moorman JP;Yao ZQ
• 通讯作者: Yao ZQ

ROS-Induced Mitochondrial Dysfunction in CD4 T Cells from ART-Controlled People Living with HIV.

• DOI: 10.3390/v15051061
• 发表时间: 2023-04-26
• 期刊: Viruses
• 作者: Schank M;Zhao J;Wang L;Nguyen LNT;Zhang Y;Wu XY;Zhang J;Jiang Y;Ning S;El Gazzar M;Moorman JP;Yao ZQ
• 通讯作者: Yao ZQ

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

• DOI: 10.3389/fcimb.2022.1026293
• 发表时间: 2022
• 期刊: Frontiers in cellular and infection microbiology
• 影响因子: 5.7

Identification of virus-specific B-cell epitopes by convalescent plasma from COVID-19 patients.

• DOI: 10.1016/j.molimm.2022.10.016
• 发表时间: 2022-12
• 期刊: MOLECULAR IMMUNOLOGY
• 影响因子: 3.6
• 作者: Wang, Ling;Zhao, Juan;Schank, Madison;Khanal, Sushant;Dang, Xindi;Cao, Dechao;Nguyen, Lam N. T.;Zhang, Yi;Wu, Xiao Y.;Adkins, James L.;Brueggeman, Justin;Zhang, Jinyu;Ning, Shunbin;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.

• DOI: 10.1111/acel.13513
• 发表时间: 2021-12
• 期刊: Aging cell
• 影响因子: 7.8
• 作者: Wang L;Lu Z;Zhao J;Schank M;Cao D;Dang X;Nguyen LN;Nguyen LNT;Khanal S;Zhang J;Wu XY;El Gazzar M;Ning S;Moorman JP;Yao ZQ
• 通讯作者: Yao ZQ