In vivo engineering of B cells for the secretion of HIV broadly neutralizing antibodies

用于分泌 HIV 广泛中和抗体的 B 细胞体内工程

• 批准号: 10533802
• 负责人: Adi Barzel
• 金额: $ 73.86万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-03 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10533802
• 关键词: Affinity; Animal Model; Animals; Antibodies; Antibody Response; Antibody Specificity; Antibody titer measurement; Antigen Receptors; Antigens; B-Cell Antigen Receptor; B-Lymphocytes; Biodistribution; CRISPR/Cas technology; Capsid; Cell secretion; Cells; Cellular biology; Choristoma; Clonal Expansion; DNA; DNA cassette; Data; Development; Engineering; Episome; Evaluation; Evolution; Gene Delivery; Gene Targeting; Genes; Genetic Transcription; Goals; HIV; HIV vaccine; Human; Humoral Immunities; Immune system; Immunity; Immunocompetent; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Switch Recombination; Immunoglobulins; Immunotherapy; In Vitro; Infection; Injections; Laboratories; Liver; MS4A1 gene; Macaca; Macaca mulatta; Membrane; Memory; Modeling; Monitor; Monkeys; Mus; Muscle; Plasma Cells; Protein Isoforms; Protocols documentation; Publishing; Reagent; Receptor Cell; Recombinant Antibody; Recombinants; Route; Safety; Series; Serum; Signal Transduction; Site; Specificity; Structure of germinal center of lymph node; System; Testing; Therapeutic Effect; Time; Transgenes; Vaccination; Vaccine Design; Viral; Viral Antigens; Viremia; Virus; Virus Replication; adeno-associated viral vector; antiviral immunity; cellular engineering; cost; delivery vehicle; design; donor-specific antibody; env Gene Products; gene therapy; genome editing; improved; in vivo; nanobodies; neutralizing antibody; nonhuman primate; novel; preservation; prevent; programs; promoter; response; simian human immunodeficiency virus; therapeutic gene; vector; viral rebound

PROJECT SUMMARY We have developed a novel gene therapeutic approach to achieve an HIV functional cure, wherein broadly neutralizing antibody (bnAb) genes are targeted to the endogenous heavy chain locus in B cells for expression as functional antigen-receptors (BCRs). This takes advantage of the precision of gene editing conferred by CRISPR-Cas9 reagents and homology-directed gene editing. We have shown that these broadly neutralizing BCRs respond to HIV Env immunogens by signaling clonal expansion and germinal center maturation of the engineered cells and thereby generate durable, isotype-switched memory bnAb responses that could be deployed for long-term control of HIV. Given these features of B cell biology, which are preserved by our gene editing approach, we hypothesize that sufficient neutralizing antibody titers in serum could be generated from very few engineered B cells, including the levels achieved by in vivo delivery of genome-editing reagents directly to the target cells. In support of this approach, we have now shown that high-titer bnAb responses can be elicited in mice by vaccination with HIV-Env immunogens when the animals are injected with a transcriptionally-targeted dual-AAV vector system carrying the bnAb editing reagents. Unlike injection of recombinant bnAbs, or AAV- vectored delivery of bnAb-IgG expression cassettes to liver or muscle for long-term secretion from viral episomes, bnAbs generated from in vivo gene-edited B cells in response to vaccination are expected to have the following advantages: 1) be boostable, as needed, in response to viral antigen, 2) express bnAbs as all effector isotypes to expand antiviral immunity, 3) affinity-mature or evolve the bnAb response to keep-pace with a rapidly evolving virus, and 4) be better tolerated by the immune system. To further develop this approach, we have assembled a team that brings together expertise in B cell engineering, vector development, HIV vaccine design, and animal models to further develop this in vivo delivery approach to specifically and efficiently engineer B cells in vivo and to evaluate this approach as an in vivo HIV cure.

项目总结 我们已经开发了一种新的基因治疗方法来实现艾滋病毒的功能性治愈，其中广泛地 中和抗体(BNab)基因针对B细胞的内源性重链基因进行表达 作为功能性抗原受体(BCR)。这利用了基因编辑的精确性 CRISPR-Cas9试剂和同源基因编辑。我们已经证明，这些广泛的中和 BCR对HIV Env免疫原的反应是通过信号传递克隆扩张和生发中心成熟 工程细胞，从而产生持久的、同种类型切换的存储器bNab响应 部署用于长期控制艾滋病毒。考虑到B细胞生物学的这些特征，我们的基因保存了这些特征 编辑方法，我们假设血清中可以产生足够的中和抗体效价 很少有经过工程改造的B细胞，包括直接体内注射基因组编辑试剂所达到的水平 目标单元格。为了支持这一方法，我们现在已经证明了可以引起高滴度的bNab反应 在小鼠接种HIV-Env免疫原时，给动物注射转录靶向的 携带bNab编辑试剂的双AAV载体系统。与注射重组bNAbs或AAV不同- 通过载体将bNab-Ig G表达盒输送到肝脏或肌肉以实现病毒的长期分泌 由体内基因编辑的B细胞对疫苗的反应产生的episome，bNAbs预计会有 以下优点：1)根据需要是可启动的，对病毒抗原作出反应，2)将bNAbs表达为ALL 效应器亚型以扩大抗病毒免疫，3)亲和力-成熟或进化bNab反应以跟上 快速进化的病毒，以及4)更好地被免疫系统耐受。为了进一步发展这一方法，我们 组建了一个团队，汇集了B细胞工程、媒介开发、艾滋病毒疫苗等方面的专业知识 设计和动物模型，以进一步开发这种体内递送方法，以具体和高效地设计 B细胞体内，并评估这种方法作为体内治愈艾滋病毒的方法。