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: 10374629
• 负责人: Christopher W Peterson
• 金额: $ 27.11万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374629
• 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.

摘要 HIV特异性基因疗法是在HIV感染者中实现HIV治愈/稳定缓解的有力且有前途的手段。 没有抗逆转录病毒治疗（ART）。广泛中和抗体（bNAb）和类似分子， 因为eCD 4-IG提供了一条最清晰的治愈途径，但受到三个关键障碍的阻碍。第一，被动 bNAb/eCD 4-IG蛋白的施用根据定义是瞬时疗法;当这些蛋白的循环水平升高时， 强效抗艾滋病毒因子下降，病毒复制就能恢复。二、基于载体的基因治疗 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。 转基因，但经常受到宿主免疫反应的限制。第三，有效的抗逆转录病毒疗法抑制了 复制到极低水平，使工程化的HIV特异性淋巴细胞无法识别和清除 持续感染的细胞我们已经生成了一套令人兴奋的工具和初步数据， 每一个障碍。为了克服bNAb的瞬时性质和载体的相关免疫原性， 我们已经在非人灵长类动物（NHP）中进行了体内筛选， 长期持续存在的工程化AAV变体（与缺乏宿主免疫系统的识别一致）， 并且特异性靶向B细胞。B细胞嗜性载体将用CRISPR-Cas9基因编辑机器包装， 采用高度创新的共价键方法，使我们的载体包装能力翻倍。我们称 我们的新型体内递送方法为非免疫原性、货物增强（NICE）AAV：在单剂量中，NICE- AAV载体将用靶向天然IgG基因座的bNA B或eCD 4-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 我们最近发表的NHP模型证明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，并应类似地加强和触发扩增 基因编辑的B细胞。我们建议的中心目标是验证B的效率和特异性 细胞靶向的NICE-AAV（AIM 1），以证明这种体内递送方法能够实现持久的细胞靶向的NICE-AAV（AIM 1）。 bNAb/eCD 4-IG在HIV解剖区室和储库部位的表达（AIM 2），最重要的是， 以在HIV持续存在的人源化小鼠和NHP模型中实现治疗效果（AIM 3）。我们将合并 一种最有前途的HIV治愈治疗方式（bNAbs/eCD 4-IG），我们在 体内递送平台（NICE-AAV）。重要的是，这种方法不仅适用于HIV-1， 单克隆抗体疗法提供临床益处的广泛病理学。