In vivo transformation of chimeric antigen receptor B cells for a functional cure of HIV

嵌合抗原受体 B 细胞的体内转化用于 HIV 功能性治愈

• 批准号: 10594208
• 负责人: Hyeryun Choe
• 金额: $ 79.07万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-07 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594208
• 关键词: Affinity; Animals; Antibodies; Antigens; Automobile Driving; B-Cell Antigen Receptor; B-Lymphocytes; BCAR1 gene; Back; Binding; CAR T cell therapy; CD19 gene; Capsid; Capsid Proteins; Cell Proliferation; Clinical; Clonal Expansion; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA Repair; DNA cassette; Dependovirus; Dose; Electroporation; Elements; Engraftment; Event; Gene Transduction Agent; Genes; Genetic Engineering; Guide RNA; HIV; HIV Antibodies; HIV Infections; HIV Seropositivity; HIV-1; Human; Immunity; Immunization; Immunoglobulin D; Immunoglobulin Idiotypes; Infection; Infusion procedures; Insulin Receptor; Intervention; Intravenous; Laboratories; Life; Light; Lymphoid Tissue; MS4A1 gene; Macaca; Macaca mulatta; Medical; Membrane; Messenger RNA; Methods; Modification; Mutation; Patients; Persons; Pharmaceutical Preparations; Procedures; Proliferating; Proteins; RNA Sequences; Reagent; Recombinant adeno-associated virus (rAAV); Research; Safety; Serotyping; Site; Specificity; System; Techniques; Technology; Testing; Time; Translating; Tropism; Vaccinated; Vaccination; Vaccines; Variant; Viral; Viremia; Writing; adeno-associated viral vector; antiretroviral therapy; cell transformation; cell type; chimeric antigen receptor; chimeric antigen receptor T cells; combat; comparative; compliance behavior; cost; delivery vehicle; demographics; design; disorder control; experience; gene therapy; high risk population; in vivo; individual patient; innovation; intravenous administration; manufacture; nanobodies; neutralizing antibody; novel; personalized medicine; prevent; repaired; response; side effect; social stigma; tool; transduction efficiency; vaccination strategy; vector

PROJECT SUMMARY Our laboratories have developed novel techniques for the editing B-cell receptors of human primary B cells. Using newly identified CRISPR/Cas proteins and innovative homology-directed repair templates, we can efficiently overwrite the endogenous variable heavy (VH) and variable light (VL) segments of a mature VDJ- recombined BCR with the variable genes of broadly neutralizing HIV antibodies (bNabs). Importantly, these variable genes are placed in their respective natural loci, and – excepting the new VH and VL segments – these edited B cells are indistinguishable from unmodified mature, naïve B cells. We describe these edited B cells as “chimeric antigen receptor B cells”, or CAR B cells, evoking the more familiar CAR T cells. These CAR B cells replicate, differentiate, affinity mature, and secrete antibodies in vivo providing an efficient delivery vehicle for bNabs. CAR B cells represent a key advance over passive infusion or gene therapy delivery of bNabs, because they do not raise anti-drug antibodies against their novel BCR, and because the can affinity mature in response to an antigen, including HIV-1 emerging from a reactivated reservoir. They can thus adapt in real time to the specific viral variants in the reservoir. To date, however, we have only transformed CAR B cells ex vivo by isolating primary B cells from a particular host, transforming them by electroporation of CRISPR/Cas RNPs and DNA repair templates, and re-infusing the CAR B cells into the host. Although ex vivo CAR B transformation could be clinically viable, it would likely be a prohibitively expensive procedure for most HIV-positive persons. Here we propose to perform the CAR B transformation procedure in vivo by developing a B cell-tropic adeno-associated virus (AAV)-based gene therapy vector and CRISPS/Cas editing cassette that could be administered intravenously; a comparatively fast and inexpensive procedure. This proposal is divided into three aims. In Aim 1, we draw upon our extensive experience modifying AAV capsids to target specific cell types to create a B cell-tropic capsid. In Aim 2, we develop and evaluate a range of AAV-delivered CRISPR/Cas editing cassette designs for their ability to efficiently transform CAR B cells. Lastly, in Aim 3, we optimize an immunogen and immunization strategy to drive proliferation and affinity maturation of newly transformed CAR B cells. These studies will make clinically viable a promising approach for suppressing an established HIV-1 infection or preventing a new one in high-risk persons.

项目总结 我们的实验室已经开发出编辑人类原代B细胞的B细胞受体的新技术。 使用新发现的CRISPR/Cas蛋白和创新的同源定向修复模板，我们可以 有效地覆盖成熟VDJ的内源可变重(VH)和可变轻(VL)段- 重组bcr与广谱中和HIV抗体(BNAb)的可变区基因。重要的是，这些 可变基因被放置在各自的自然基因座上，并且--除了新的VH和VL片段-- 这些经过编辑的B细胞与未经修饰的成熟、幼稚的B细胞没有什么区别。我们描述这些编辑过的B 细胞被称为“嵌合抗原受体B细胞”，或CAR B细胞，唤起更熟悉的CAR T细胞。 这些CAR B细胞在体内复制、分化、亲和成熟并分泌抗体，提供了有效的 Bnabs的运送工具。CAR B细胞代表着比被动输血或基因治疗更重要的进步 递送bNAb，因为它们不会针对其新的bcr产生抗药抗体，而且因为 亲和力是否会随着抗原的反应而成熟，包括从重新激活的储存库中冒出的HIV-1。他们 因此可以实时适应储存库中的特定病毒变体。 然而，到目前为止，我们只通过从特定的细胞中分离原代B细胞来体外转化CAR B细胞。 宿主，通过CRISPR/Cas RNPs和DNA修复模板的电穿孔转化，并重新注入 将Car B细胞送入宿主体内。虽然体外转化Car B在临床上是可行的，但很可能 对于大多数艾滋病毒阳性者来说，这是一个昂贵得令人望而却步的程序。在这里，我们提议表演赛车 基于B细胞嗜性腺相关病毒基因的B细胞体内转化过程 可静脉给药的治疗载体和CRIPS/CAS编辑磁带；比较 快速和廉价的程序。 这项提议分为三个目标。在目标1中，我们借鉴了我们修改AAV的丰富经验 衣壳以特定的细胞类型为靶标，产生嗜B细胞的衣壳。在目标2中，我们开发和评估了一系列 AAV提供的CRISPR/CA编辑盒式磁带设计的能力，以有效地转化CAR B细胞。 最后，在目标3中，我们优化了一种免疫原和免疫策略，以促进增殖和亲和力 新转化的CAR B细胞的成熟。这些研究将使临床可行成为一种有希望的方法。 用于在高危人群中抑制已确定的艾滋病毒-1感染或预防新的感染。