In Vivo Gene Editing of B cells with NICE-AAV Vectors

使用 NICE-AAV 载体对 B 细胞进行体内基因编辑

• 批准号: 10593435
• 负责人: Christopher W Peterson
• 金额: $ 46.21万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-16 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593435
• 关键词: 2019-nCoV; AIDS/HIV problem; Address; Anatomy; Animals; Antibodies; Antibody Therapy; Antigens; Autoimmune Diseases; B-Lymphocytes; Biological Assay; CRISPR/Cas technology; Capsid; Cells; Clinical; Clinical Research; Communicable Diseases; Data; Dependovirus; Disease remission; Dose; Engineering; Failure; Gene Transduction Agent; Genes; Genetic; Goals; HIV; HIV Antibodies; HIV-1; Human; IGH@ gene cluster; Immune response; Immune system; Immunocompetent; Immunodeficient Mouse; Immunoglobulin G; Individual; Infection; Injections; Knock-in; Libraries; Link; Lymphocyte; Macaca mulatta; Malignant Neoplasms; Measures; Methodology; Methods; Modality; Modeling; Monoclonal Antibodies; Monoclonal Antibody Therapy; Mus; Nature; Pathology; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phenotype; Proteins; Publications; Publishing; Regimen; Resource-limited setting; Secondary to; Serum; Site; Specificity; Technology; Testing; Therapeutic; Therapeutic Effect; Therapeutic antibodies; Time; Tissues; Transgenes; Tropism; Variant; Viral Antigens; Viremia; Virus; Virus Replication; Withdrawal; adeno-associated viral vector; antiretroviral therapy; base; chimeric antigen receptor T cells; clinical application; delivery vehicle; design; experimental study; fighting; gene therapy; genetic information; global health; humanized mouse; immunogenic; immunogenicity; in vivo; innovation; neutralizing antibody; nonhuman primate; novel; preclinical study; screening; simian human immunodeficiency virus; success; tool; trafficking; vector; vector biodistribution; viral rebound

ABSTRACT HIV-specific gene therapies are a powerful and promising means to achieve HIV cure/stable remission in the absence of antiretroviral therapy (ART). Broadly neutralizing antibodies (bNAbs) and analogous molecules such as eCD4-Ig offer one of the clearest paths to a cure, but are hindered by three key obstacles. First, passive administration of bNAb/eCD4-Ig proteins is by definition a transient therapy; when circulating levels of these potent anti-HIV factors decline, virus replication is able to resume. Second, gene therapy vector-based approaches including adeno-associated virus (AAV) support prolonged expression of bNAbs and other antiviral transgenes, but are frequently limited by host immune responses. Third, potent ART regimens suppress viral replication to extremely low levels, rendering engineered HIV-specific lymphocytes unable to recognize and clear persistently infected cells. We have generated an exciting set of tools and preliminary data that directly addresses each of these barriers. To overcome the transient nature of bNAbs and associated immunogenicity of vectored delivery approaches, we have performed an in vivo screen in nonhuman primates (NHP) and identified engineered AAV variants that persist long term (consistent with a lack of recognition by the host immune system), and specifically target B cells. B cell tropic vectors will be packaged with CRISPR-Cas9 gene editing machinery, applying highly innovative covalent linkage methodology to double our vectors’ packaging capacity. We refer to our novel in vivo delivery approach as Non-Immunogenic, Cargo-Enhanced (NICE) AAV: in a single dose, NICE- AAV vectors will specifically reprogram B cells with bNAb or eCD4-Ig sequences targeted to the native IgG locus. Finally, we will overcome the significant problem of insufficient viral antigen by supplying cell-associated HIV-1 Env in trans. Our recent publication in the NHP model demonstrates the immense success of this strategy to stimulate HIV-1-specific chimeric antigen receptor (CAR) T cells and should similarly boost and trigger expansion of our gene-edited B cells. The central goals of our proposal are to validate the efficiency and specificity of B cell-targeted NICE-AAV (AIM 1), to demonstrate that this in vivo delivery approach enables persistent bNAb/eCD4-Ig expression in HIV anatomical compartments and reservoir sites (AIM 2), and most importantly, to achieve a therapeutic impact in humanized mouse and NHP models of HIV persistence (AIM 3). We will merge one of the most promising therapeutic modalities for HIV cure (bNAbs/eCD4-Ig) with our extremely unique in vivo delivery platform (NICE-AAV). Importantly, this approach will be applicable not only for HIV-1, but for the broad range of pathologies where monoclonal antibody therapies offer clinical benefit.

抽象的 HIV特异性基因疗法是实现HIV治愈/稳定缓解的强大且有前途的手段 缺乏抗逆转录病毒疗法（ART）。广泛中和抗体（bNAb）和类似分子，例如 因为 eCD4-Ig 提供了最清晰的治愈途径之一，但受到三个主要障碍的阻碍：首先，被动。 根据定义，当 bNAb/eCD4-Ig 蛋白处于循环水平时，施用是一种短暂的治疗； 强效抗HIV因子下降，病毒复制得以恢复 二、以载体为基础的基因治疗。 包括腺相关病毒 (AAV) 在内的方法支持 bNAb 和其他抗病毒药物的延长表达 第三，有效的 ART 方案可抑制病毒。 复制到极低水平，导致工程化的 HIV 特异性淋巴细胞无法识别和清除 我们已经生成了一组令人兴奋的工具和初步数据，可以直接解决这些问题。 克服 bNAb 的瞬时性和载体化的相关免疫原性。 对于递送方法，我们在非人类灵长类动物 (NHP) 中进行了体内筛选，并确定了 工程化的 AAV 变体可以长期存在（与宿主免疫系统缺乏识别一致）， 专门针对 B 细胞的 B 细胞趋向性载体将与 CRISPR-Cas9 基因编辑机器一起包装， 我们指的是应用创新的高度共价连接方法使载体的包装能力加倍。 我们新颖的体内递送方法，即非免疫原性、货物增强型 (NICE) AAV：单剂量，NICE- AAV 载体将使用靶向天然 IgG 基因座的 bNAb 或 eCD4-Ig 序列特异性重编程 B 细胞。 最后，我们将通过提供细胞相关的HIV-1来克服病毒抗原不足的重大问题 我们最近在 NHP 模型中发表的 Env 展示了这一策略的巨大成功。 刺激 HIV-1 特异性嵌合抗原受体 (CAR) T 细胞，同样会促进和触发扩增 我们提案的中心目标是验证 B 细胞的效率和特异性。 细胞靶向 NICE-AAV (AIM 1)，以证明这种体内递送方法能够持久 HIV 解剖区室和储存部位中的 bNAb/eCD4-Ig 表达 (AIM 2)，最重要的是， 为了在 HIV 持续存在的人源化小鼠和 NHP 模型中实现治疗效果（AIM 3）。 治疗艾滋病毒 (bNAbs/eCD4-Ig) 的最有前途的治疗方式之一，我们在 重要的是，这种方法不仅适用于 HIV-1，也适用于 HIV-1。 单克隆抗体疗法可提供临床益处的广泛病理学。