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.

项目摘要 我们的实验室已经开发了用于编辑人类原代B细胞的B细胞受体的新技术。 使用新鉴定的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的内源性可变重链（VH）和可变轻链（VL）区段。 将BCR与广泛中和HIV抗体（bNabs）的可变基因重组。重要的是这些 可变基因被置于它们各自的天然基因座中，并且-除了新的VH和VL区段- 这些编辑的B细胞与未修饰的成熟、幼稚B细胞是不可区分的。我们描述这些编辑过的B 细胞称为“嵌合抗原受体B细胞”或CAR B细胞，引起更熟悉的CAR T细胞。 这些CAR B细胞在体内复制、分化、亲和力成熟和分泌抗体，提供有效的免疫调节。 bNabs的运载工具。CAR B细胞代表了被动输注或基因治疗的关键进展 因为它们不产生针对其新型BCR的抗药抗体，并且因为 可以在对抗原的反应中亲和力成熟，包括从重新激活的储存库中出现的HIV-1。他们 因此可以真实的适应于储存库中的特定病毒变体。 然而，迄今为止，我们仅通过从特定的细胞分离原代B细胞来离体转化CAR B细胞。 宿主，通过CRISPR/Cas RNP和DNA修复模板的电穿孔转化它们，并重新输注 CAR B细胞进入宿主。尽管离体CAR B转化可能在临床上可行，但它可能 对大多数艾滋病毒抗体阳性者来说，这是一个昂贵得令人望而却步的程序。在这里，我们建议执行CAR 通过开发基于B细胞的腺相关病毒（AAV）基因的体内B转化方法 治疗载体和CRISPS/Cas编辑盒，可以静脉内施用; 快速和廉价的程序。 该提案分为三个目标。在目标1中，我们利用我们丰富的经验修改AAV 衣壳靶向特定的细胞类型以产生B细胞嗜性衣壳。在目标2中，我们开发和评估一系列 AAV-递送的CRISPR/Cas编辑盒设计的能力，以有效转化CAR B细胞。 最后，在目标3中，我们优化免疫原和免疫策略以驱动增殖和亲和力 新转化的CAR B细胞的成熟。这些研究将使临床上可行的一种有前途的方法 用于抑制已确定的HIV-1感染或预防高危人群中的新感染。