Multiplex nucleosome-based profiling for the development of next-generation chromatin labeling reagents

基于多重核小体的分析，用于开发下一代染色质标记试剂

• 批准号: 10094217
• 负责人: Zu-Wen Sun
• 金额: $ 92.02万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094217
• 关键词: Acylation; Address; Antibodies; Antibody Specificity; Arginine; Bar Codes; Binding; Biological Assay; Biomedical Research; Chromatin; Chromatin Structure; Collaborations; Complement; Couples; Data; Detection; Development; Diagnostic; Epigenetic Process; Family; Generations; Genomics; Goals; Histones; K562 Cells; Label; Letters; Libraries; Licensing; Lysine; Methods; Methylation; Nucleosomes; Peptides; Performance; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Production; Reaction; Reagent; Recombinant Antibody; Recombinants; S-nitro-N-acetylpenicillamine; Sampling; Science; Signal Transduction; Site-Directed Mutagenesis; Specificity; Technology; Testing; Time; Validation; Variant; Voice; base; candidate selection; chromatin immunoprecipitation; combinatorial; cost; cross reactivity; drug development; human disease; innovation; interest; next generation; phase 2 study; screening; success; technology development

PROJECT SUMMARY Alterations in chromatin structure are associated with many human diseases and often characterized by changes in histone post-translational modifications (PTMs). Histone PTMs are commonly analyzed by chromatin immunoprecipitation (ChIP), which relies on antibodies to enrich chromatin subsets. However, the accuracy of such antibodies is an increasing concern in the biomedical field, impeding advancements in chromatin science and related drugs / diagnostics. EpiCypher® is pioneering the development of technologies that use recombinant designer nucleosomes (dNucs), with an early focus on PTM antibody specificity testing. Using EpiCypher’s disruptive quantitative ChIP platform (SNAP-ChIP®; Sample Normalization and Antibody Profiling), we found that >80% of commercially-available PTM antibodies display a striking amount of off-target binding and low binding efficiency. These results underscore substantial problems in the selection and validation of antibodies to histone PTMs, which currently utilize histone peptides for candidate selection. Ideally, we would integrate SNAP-ChIP assays early in antibody development; however, SNAP-ChIP is low-throughput, labor intensive, and thus not suitable for antibody development. In this Direct-to-Phase II proposal we will address these deficiencies with NucleoPlex™, which couples modified dNucs to Luminex® xMAP® beads for multiplexed, high-throughput antibody screening. The innovation of this proposal is the creation of dNuc-xMAP bead panels (i.e. NucleoPlex panels) that enable both on- and off- target antibody profiling in a single reaction. In Phase I equivalent proof-of-concept studies, we conjugated barcoded xMAP beads to dNucs containing histone methyl-lysine PTMs, and used this panel to interrogate the binding of >50 commercially available ChIP-grade PTM antibodies. NucleoPlex data demonstrated strong concordance with SNAP-ChIP, at fraction of the time and cost, indicating that NucleoPlex provides accurate and rapid specificity screening of PTM antibodies in a nucleosomal context. EpiCypher is poised to make a significant breakthrough with the NucleoPlex platform, redefining histone PTM antibody development, screening, and validation. A major goal of this Phase II study is to integrate NucleoPlex into a recombinant antibody development pipeline, and demonstrate how this approach increases production of highly specific antibodies. To this end, we will first validate and scale manufacturing of NucleoPlex panels for distinct histone PTM families (Aim 1), and then apply NucleoPlex toward the development of recombinant antibodies (Aim 2). Finally, we will develop combinatorially-modified nucleosomes to interrogate antibody binding specificity in the context of adjacent PTMs, which have been shown to impact antibody binding (Aim 3). The development of NucleoPlex as a low-cost multiplexed antibody screening platform will accelerate the generation of highly specific histone PTM-targeting reagents, which will have a lasting impact on the epigenetics field and save millions of dollars annually that are wasted on low-quality detection reagents.

项目摘要 染色质结构的改变与许多人类疾病有关，并且通常以 组蛋白翻译后修饰（PTMS）的变化。组蛋白PTM通常通过染色质分析 免疫沉淀（芯片），依赖于富集染色质亚群的抗体。但是，准确性 这种抗体在生物医学领域越来越关注，阻碍了染色质科学的进步 和相关药物 /诊断。 Epicypher®正在开发使用重组的技术的发展 设计师核小组（DNUCS），早期侧重于PTM抗体特异性测试。使用Epicypher 破坏性定量芯片平台（Snap-Chip®；样品归一化和抗体分析），我们发现 > 80％的市售PTM抗体显示出攻击量的脱靶结合和低约束力 效率。这些结果突显了组蛋白抗体的选择和验证中的重大问题 PTM，当前利用组蛋白肽进行候选。理想情况下，我们将整合Snap-Chip 抗体开发早期的测定；但是，Snap-chip是低通量，劳动密集的，因此不是 适用于抗体开发。 在此直接到相关的II提案中，我们将使用nucleoplex™解决这些缺陷，这是夫妻的 修改为Luminex®XMAP®珠的DNUCS用于多重高通量抗体筛选。创新 该建议的是创建DNUC-XMAP珠面板（即核次面板） 单一反应中的靶抗体分析。在第一阶段等效概念验证研究中，我们结合了 条形码的Xmap珠至包含组蛋白甲基赖氨酸PTM的DNUC，并使用此面板询问 结合> 50个市售的芯片级PTM抗体。核因数据显示出很强的数据 在时间和成本的一部分中，与快照芯片一致，表明NucleOplex提供了准确的和 在核小体环境下PTM抗体的快速特异性筛选。 Epicypher被中毒，可以与NucleOplex平台取得重大突破，重新定义 组蛋白PTM抗体的开发，筛选和验证。这一第二阶段研究的主要目标是 将核孔插入重组抗体发育管道中，并证明这种方法如何 增加高度特异性抗体的产生。为此，我们将首先验证和规模制造 用于不同组蛋白PTM家族的Nucleoplex面板（AIM 1），然后将核孔应用于发育 重组抗体（AIM 2）。最后，我们将开发组合修饰的核小体以询问 相邻PTM的背景下的抗体结合特异性，这些特异性已被证明会影响抗体结合 （目标3）。 NucleOplex作为低成本多路复用抗体筛选平台的发展将加速 高度特异的组蛋白靶向试剂的产生，这将对持久影响 表观遗传学领域并节省数百万美元，这些资金浪费在低质量检测试剂中。

项目成果

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

• DOI: 10.1016/j.molcel.2021.02.012
• 发表时间: 2021-04-15
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Radzisheuskaya A;Shliaha PV;Grinev VV;Shlyueva D;Damhofer H;Koche R;Gorshkov V;Kovalchuk S;Zhan Y;Rodriguez KL;Johnstone AL;Keogh MC;Hendrickson RC;Jensen ON;Helin K
• 通讯作者: Helin K

Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

• DOI: 10.1111/jth.15003
• 发表时间: 2020-10
• 期刊: Journal of thrombosis and haemostasis : JTH
• 作者: Thålin C;Aguilera K;Hall NW;Marunde MR;Burg JM;Rosell A;Daleskog M;Månsson M;Hisada Y;Meiners MJ;Sun ZW;Whelihan MF;Cheek MA;Howard SA;Saxena-Beem S;Noubouossie DF;Key NS;Sheikh SZ;Keogh MC;Cowles MW;Lundström S;Mackman N;Wallén H;Johnstone AL
• 通讯作者: Johnstone AL