Combination Therapy Using CRISPR/Cas Gene Editing Plus Human Monoclonal Antibodies for a Functional HIV Cure

使用 CRISPR/Cas 基因编辑加人单克隆抗体的联合疗法实现功能性 HIV 治愈

• 批准号: 9032718
• 负责人: ALEXANDRA L HOWELL
• 金额: --
• 依托单位: WHITE RIVER JUNCTION VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-10-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9032718
• 关键词: Animal Model; Anti-HIV Therapy; Anti-Retroviral Agents; Antibodies; Antigens; Binding; CCR5 gene; CD34 gene; CD4 Lymphocyte Count; CD4 Positive T Lymphocytes; Cell Line; Cell Surface Proteins; Cells; Cellular Assay; Cleaved cell; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Combined Modality Therapy; Communicable Diseases; Complement; Coupled; Development; Disease; Effectiveness; Engineering; Epitopes; Fc Receptor; Fc domain; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Guide RNA; HIV; HIV Antibodies; HIV Envelope Protein gp120; HIV Infections; HIV resistance; HIV-1; Health; Hematopoietic stem cells; Human; Immune; Immune system; In Vitro; Infection; Interruption; Lead; Lentivirus Vector; Leukocytes; Life Cycle Stages; Measures; Mediating; Modification; Monitor; Mono-S; Monoclonal Antibodies; Mus; Myeloid Cells; Natural Killer Cells; Patients; Peripheral; Phagocytosis; Pharmaceutical Preparations; Plasma; Production; Proviruses; Reagent; Regimen; Resistance; Specificity; Staging; Testing; Therapeutic; Therapeutic Uses; Toxic effect; Transgenes; Validation; Viral; Viral Envelope Proteins; Viral Genome; Viral Load result; Viremia; Virion; Virus; Withdrawal; Work; antibody-dependent cell cytotoxicity; cellular transduction; cytotoxicity; design; human disease; human monoclonal antibodies; humanized mouse; improved; in vitro Assay; in vitro testing; in vivo; innovation; macrophage; mathematical model; mouse model; particle; peripheral blood; pre-clinical; progenitor; receptor; receptor function; stem cell population; targeted treatment; therapeutic development

 DESCRIPTION (provided by applicant) The persistence of the latent reservoir in HIV-infected patients is a major barrier to the eradication of this disease. Although combination anti-retroviral therapy (cART) is highly effective at suppressing viral production from the latent reservoir, cART withdrawal leads to a rapid viral rebound driven by the reactivation of viral transcription from the latent pool. In this project, we will develop a multi-targeted therapy that we expect will significantly reduce and potentially eliminate the ability of HIV-1 to infect target cells in the absence of cART. This combination therapeutic will target different key aspects of HIV infection, each of which contributes to sustained viremia in the absence of cART. These components include viral particles, virus producing cells, the CCR5 co-receptor expressed on target cells, and the integrated provirus. To clear HIV and HIV-producing cells that emerge during viral rebound, we have engineered a panel of anti-HIV monoclonal antibodies. These antibodies bind to various epitopes on HIV- 1 and have been modified to express functionally enhanced Fc domains. These altered Fc domains promote Fc receptor functions on myeloid cells such as antibody dependent cellular cytotoxicity (ADCC), phagocytosis, and complement recruitment. We have developed CRISPR/Cas constructs that target and cleave the CCR5 gene. CCR5 gene cleavage in the CD34+ hematopoietic stem cell (HSC) population will lead to the differentiation and proliferation of CCR5-negative immune progeny that are resistant to infection with R5-tropic HIV-1. Moreover, CCR5 gene cleavage in differentiated leukocytes will also result in HIV-1 resistance. We have developed CRISPR/Cas gene editing constructs to cleave the integrated proviral sequence, so that infected cells transduced with these genes are unable to transcribe intact viral genomes. To deliver these CRISPR/Cas transgenes, we have developed lentiviral (LV) vectors that we will pseudotype with different viral envelope proteins and anti-receptor antibodies to direct LV to specific target cells. CRISPR genes delivered by LV are stably integrated into the target cell genome so that the transduced cells and their progeny are permanently protected from HIV infection. Typically, each of these approaches would be proposed independently, and efficacy measured against the absence of treatment. However, a key innovation of this application is to combine these approaches into one therapeutic regimen. In the first 3 aims, we will develop and select the most efficacious reagents for each of these approaches using a combination of in vitro cellular assays and in vivo assessments in the HIV-infected humanized mouse (hu-mouse). In Aim 4, we will combine the therapeutics and test them in the HIV-infected hu-mouse model, and determine their ability to suppress and delay viral production following interruption of cART treatment. We expect that these studies will not only develop clinic-ready reagents that will have undergone rigorous testing and validation in a pre-clinical animal model, but we will also demonstrate that combination targeting, like combination anti-retroviral therapy, is more effective than a single therapeutic in reducing the viral burden in HIV patients and in developing an HIV-resistant immune system. This work brings together the combined expertise of Drs. Susan Eszterhas and George O'Toole in the design of CRISPR gene targeting constructs, Dr. Bryan Luikart in the development of LV vectors for primary cell targeting in vivo, Dr. Margaret Ackerman in the optimization of antibodies for therapeutic development, and Dr. Dorothy Wallace in mathematical modeling of human diseases.

 描述（由申请人提供） 艾滋病毒感染者体内潜伏宿主的持续存在是根除这一疾病的主要障碍。尽管联合抗逆转录病毒疗法（cART）在抑制潜伏库中的病毒产生方面非常有效，但cART停药会导致病毒快速反弹，这是由潜伏库中病毒转录的重新激活驱动的。在这 在该项目中，我们将开发一种多靶向治疗，我们预计这种治疗将显著降低并可能消除HIV-1在没有cART的情况下感染靶细胞的能力。这种联合治疗将针对HIV感染的不同关键方面，在没有cART的情况下，每一个方面都会导致持续的病毒血症。这些组分包括病毒颗粒、病毒产生细胞、在靶细胞上表达的CCR 5共受体和整合的前病毒。为了清除病毒反弹期间出现的HIV和HIV产生细胞，我们设计了一组抗HIV单克隆抗体。这些抗体结合HIV- 1上的各种表位，并已被修饰以表达功能增强的Fc结构域。这些改变的Fc结构域促进骨髓细胞上的Fc受体功能，例如抗体依赖性细胞毒性（ADCC）、吞噬作用和补体募集。我们已经开发了靶向和切割CCR 5基因的CRISPR/Cas构建体。在CD 34+造血干细胞（HSC）群体中的CCR 5基因切割将导致CCR 5阴性免疫子代的分化和增殖，所述免疫子代抵抗R5嗜性HIV-1的感染。此外，分化的白细胞中的CCR 5基因切割也会导致HIV-1抗性。我们已经开发了CRISPR/Cas基因编辑构建体来切割整合的前病毒序列，因此用这些基因转导的感染细胞无法转录完整的病毒基因组。为了递送这些CRISPR/Cas转基因，我们开发了慢病毒（LV）载体，我们将用不同的病毒包膜蛋白和抗受体抗体假型化，以将LV引导至特定的靶细胞。由LV递送的CRISPR基因稳定地整合到靶细胞基因组中，使得转导的细胞及其后代永久地免受HIV感染。通常情况下，这些方法中的每一种都是独立提出的，并根据缺乏治疗来衡量疗效。然而，本申请的关键创新是将这些方法联合收割机组合成一种治疗方案。在前3个目标中，我们将使用体外细胞测定和HIV感染的人源化小鼠（hu-小鼠）体内评估的组合来开发和选择用于这些方法中的每一种的最有效的试剂。在目标4中，我们将联合收割机组合治疗剂并在HIV感染的hu小鼠模型中测试它们，并确定它们在中断cART治疗后抑制和延迟病毒产生的能力。我们希望这些研究不仅能开发出在临床前动物模型中经过严格测试和验证的临床可用试剂，而且我们还将证明联合靶向治疗（如联合抗逆转录病毒治疗）在减少HIV患者的病毒负担和开发抗HIV免疫系统方面比单一治疗更有效。这项工作汇集了Susan Eszterhas博士和乔治O 'Toole博士在CRISPR基因靶向构建体设计方面的综合专业知识，Bryan Luikart博士在开发用于体内原代细胞靶向的LV载体方面的专业知识，Margaret阿克曼博士在优化用于治疗开发的抗体方面的专业知识，以及Dorothy Wallace博士在人类疾病数学建模方面的专业知识。