Scalable single-cell workflow for multiomic analyses of chromatin interactions, accessibility, gene expression and cell surface proteins to unravel mechanisms of cellular diversity

可扩展的单细胞工作流程，用于染色质相互作用、可及性、基因表达和细胞表面蛋白的多组学分析，以揭示细胞多样性的机制

基本信息

批准号：
10604121
负责人：
Anthony Schmitt
金额：
$ 101.34万
依托单位：
ARIMA GENOMICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-15 至 2025-01-31
项目状态：
未结题

项目摘要

Scalable single-cell workflow for multiomic analyses of chromatin interactions, accessibility, gene expression and cell surface proteins to unravel mechanisms of cellular diversity Arima Genomics Project Summary/Abstract All cells in the human body carry the same DNA sequence and yet individual cells are highly diverse in identity, morphology, proliferation, and function, leading to enormous heterogeneity in the context of tissues, organs, and organisms. Individual cells achieve this diversity via unique gene regulatory programs – where in, unique sets of regulatory elements (REs) precisely instruct each cell which genes to express and when. Mapping such gene regulatory programs are central to molecular biology and genomics, as mis-regulation is a major cause of disease – mapping not only helps in diagnosis but also enables therapies that can intervene and correct mis-regulation. Single cell ATAC sequencing (scATAC) has emerged as the popular mapping assay to delineate REs unique to each cell. When scATAC is performed alongside single cell RNA sequencing (scRNA), the researcher has access to both REs and gene expression, allowing them to obtain unprecedented insight into the gene regulatory programs of living cells. There is only one problem – there are often multiple REs in the neighborhood of a gene and without the ability to link specific REs to its target genes, a mechanistic view of gene regulation is lacking, limiting our ability to enable precise diagnosis and drug discovery programs. High throughput chromatin interaction capture assay and sequencing (HiC) presents a three-dimensional view of the genome, often informing the missing link between RE and their target genes. Indeed, several KOLs – e.g., Dr. Tomi Pastinen calls scATAC, scRNA and scHiC as the “trifecta of modalities” that can truly delineate gene regulatory programs of individual cells (see Dr. Pastinen’s letter and 30+ additional letters of support). Recognizing the value, several academic labs have developed scHiC protocols, which has already unraveled incredibly detailed mechanistic insights of gene regulation of complex microenvironments including breast cancer, prostate cancer, hippocampus – several of these studies are discussed in this application. Despite the enthusiasm around scHiC data, adoption has been restricted to a few labs because of (1) severe experimental inefficiencies that result in exorbitant costs (upwards of $20,000 per sample), and (2) because current scHiC protocols involve complex plate- or combinatorial indexing workflows that are challenging to setup and execute. Via a self-funded phase-1 program, we tackled problem (1) to drastically improve efficiency of scHiC and consequently, drive costs down from earlier $10 per cell, to <$2 per cell, details of which are discussed both in research and commercial plans. We then used our rigorous product development expertise to translate the resultant scHiC chemistry into kits that were extensively validated by multiple KOLs (see letters from Joe Ecker, Longzhi Tan and others). Upon validation, these KOLs have become customers using Arima’s scHiC, referred to as A-scHiC kits, in their single cell workflows instead of the inefficient former academic protocols. Webinars and conference presentations from these early adopters created a ripple in the community and in a span of few months, we have sold >1,000 reactions of A-scHiC kits to tens of academic labs (despite no marketing activity from Arima), who have embedded our kits within both the plate- and combinatorial indexing single cell workflows. The scope of phase-2 program is to tackle problem (2) to enable widespread adoption. In particular, we propose to build off the A-scHiC chemistry toward what we refer to as the sc3DGR chemistry that is performed upstream of 10X genomics (10XG) Chromium – i.e., the output of sc3DGR kit will be an input into the 10X ATAC (flavor1) or 10X Multiome (flavor2) kits, to concurrently capture scHiC and scATAC (flavor1), or, scHiC, scATAC and scRNA (flavor2), respectively. Such a chemistry will not only solve the ease to use problem (2) given its combability with the market leader 10XG, but importantly, it will enable multiomic analyses of the “trifecta” from the same individual cell concurrently, thus enabling cell perturbation, characterization and screening use-cases for precision mapping, diagnosis, and therapy. Once the sc3DGR chemistry is finalized, we translate it into robust kits, to be validated by 14 KOLs (see letters of support) across academia and pharma (AbbVie, AstraZeneca & Genentech). For Arima, the chemistry, the informatics, the easy end-to-end workflows, the overall workflow cost, the KOL-based go-to-market strategy – all play major factors in a seamless commercialization process of this leapfrog technology for delineating gene regulation programs of individual cells.

可扩展的单细胞工作流程，用于染色质相互作用，可及性，基因的多磁分析表达和细胞表面蛋白，以阐明细胞多样性的机制 Arima基因组学项目摘要/摘要人体中的所有细胞都具有相同的DNA序列，但单个细胞的身份高度潜水，形态，增殖和功能，在组织，器官和有机体。单个细胞通过独特的基因调节程序实现这种多样性 - 在其中，独特的集合调节元素（RES）精确指示每个基因表达和何时表达的细胞。映射此类基因监管程序是分子生物学和基因组学的核心，因为错误调节是疾病的主要原因 - 映射不仅有助于诊断，还可以实现可以干预和纠正错过调节的疗法。单细胞ATAC测序（SCATAC）已成为流行的映射测定法，以划定RES独特每个单元格。当与单细胞RNA测序（SCRNA）一起进行SCATAC时，研究人员已访问RES和基因表达，使它们能够获得对基因调节的前所未有的见解活细胞的程序。只有一个问题 - 基因附近通常有多个RES 并且没有将特定RES与其靶基因联系起来的能力，缺乏对基因调节的机械观点限制了我们实现精确诊断和药物发现计划的能力。高通量染色质相互作用捕获测定和测序（HIC）呈现三维视图基因组，经常告知RE与其目标基因之间缺失的联系。确实，几个科尔人 - 例如 Tomi Pastinen博士将Scatac，Scrna和Schic称为“模态的三连胜”，可以真正描述基因单个细胞的监管程序（请参阅Pastinen博士的信和30多封额外的支持信）。认识到该价值，几个学术实验室已经开发了SCHIC协议，该协议已经解开了复杂微环境（包括乳房）的基因调节基因调节的令人难以置信的详细的机械见解癌症，前列腺癌，海马 - 在本应用中讨论了其中一些研究。尽管有对SCHIC数据的热情，由于（1）严重的实验效率低下，导致成本高昂（每个样本超过20,000美元），并且（2）因为目前的SCIC 协议涉及要挑战和执行的复杂板或组合索引工作流。通过自资助的第1阶段计划，我们解决了问题（1），以极大地提高SCIC和SCHIC的效率因此，开车成本从每个单元的$ 10降至每个单元<$ 2，详细信息都在研究和商业计划。然后，我们使用严格的产品开发专业知识来翻译由此产生的SCHIC化学成分被多个KOL广泛验证的套件（请参阅Joe Ecker的来信， Longzhi Tan等）。验证后，这些KOL已成为使用Arima的Schic的客户，作为A-Schic套件，在其单细胞工作流程中，而不是效率低下的以前的学术方案。网络研讨会这些早期采用者的会议演讲在社区中产生了涟漪，很少有几个月，我们已经将A-Schic套件的1,000个反应卖给了数十实验室（尽管没有营销活动）来自Arima），他们将我们的套件嵌入了板和组合索引单细胞工作流程中。第2阶段计划的范围是解决问题（2）以实现宽度采用。特别是，我们建议为了建立A-Schic化学反应，我们称之为上游的SC3DGR化学反应 10倍基因组学（10xG）铬 - 即，SC3DGR试剂盒的输出将是10倍ATAC的输入（Floation1）或10倍多组件（味道2）套件，同时捕获Schic和scatac（floation1）或Schic，scatac和scatac和 scrna（flain2）。这样的化学反应不仅可以解决易于使用问题（2）与市场领导者10xG的组合性，但重要的是，它将能够对“ Trifecta”的多组分分析同时同时使用相同的单个细胞，从而实现细胞扰动，表征和筛选用例用于精确映射，诊断和治疗。 SC3DGR化学最终确定后，我们将其转化为强大套件，由学术界和制药公司的14个KOLS（请参阅支持信）进行验证（Abbvie，Astrazeneca＆ Genentech）。对于Arima，化学，信息信息，易于端到端的工作流程，整体工作流程成本，基于KOL的上市策略 - 所有在无缝商业化过程中都发挥了主要因素用于描述单个细胞基因调节程序的leapfrog技术。