A scalable kit-based assay for multi-omic analyses of transcriptional protein binding and chromatin interactions from ultra-low input frozen and FFPE samples at single-cell resolution

A scalable kit-based assay for multi-omic analyses of transcriptional protein binding and chromatin interactions from ultra-low input frozen and FFPE samples at single-cell resolution Arima Genomics Project Summary/Abstract Precise regulation of gene expression is paramount to establishing cellular identities, and mis-regulation of genes causes human disease. Cells regulate gene expression using regulatory elements (REs), short DNA sequences embedded throughout the genome, who are bound by transcriptional proteins (TBPs) to facilitate their regulatory function. Molecular mapping tools, such as Chromatin immunoprecipitation with sequencing (ChIP-seq), produce “maps” of REs along the genome and have been a ubiquitous approach towards understanding gene regulation. However, REs mapped using ChIP-seq are only understood in context of a linear genome. In reality, REs execute gene control within a three dimensional (3D) genome. Therefore to truly understand gene regulation – gene regulation must be mapped in 3D. Indeed, high throughput chromatin interaction capture (HiC) was developed to produce 3D interaction maps of all 3 billion bases in the human genome, however, HiC does measure transcriptional protein binding, nor whether an interaction is regulatory, thus having limited utility in advancing our understanding of 3D gene regulation. To truly obtain 3D gene regulation maps, a multi-omic assay that concurrently captures the binding of transcriptional proteins and their mediated interactions is necessary. Recently, novel approaches attempt to combine the molecular steps of ChIP-seq and chromatin interaction capture to measure transcriptional protein binding and mediated chromatin interactions in a single, multi-omic assay. However these approaches, termed ChIA-PET and HiChIP, do not efficiently capture chromatin interactions or transcriptional protein binding, respectively. Consequently, there is need for improved assays that produce true multi-omic maps of 3D gene regulation. To satisfy this unmet need, we have developed and commercialized our optimized minimal viable product (MVP) Arima-HiChIP (A-HiChIP) solution. This phase-1 product incorporates innovations designed to meet the needs of early adopter customers, achieving efficient multi-omic mapping of TBP and chromatin interactions in higher input frozen cells and tissues, and a defined subset of transcriptional proteins. We have also developed our phase-1 product for workflow integration, leveraging industry and academic partnerships to reduce barriers in ChIP and bioinformatics components of the workflow, respectively. Our team has deep expertise in the science of chromatin interaction capture, gene regulation, and its commercialization. In 2018, we commercialized Arima- HiC kits for studying general principles of chromatin interactions and within 2 years have accumulated 500+ customers, providing tools to enable published discoveries across a host of basic science and disease research. Based on VOC analytics, we shifted our focus to develop the A-HiChIP kit - a more relatable product to the gene regulation market that customers wanted and that represented a larger market opportunity. Indeed, after our self-funded phase-1 RandD and product developments, we launched our MVP A-HiChIP solution into the market and have seen remarkable success – measured by an increase in our revenue contributions, increased quality of revenue, and traction with key opinion leaders (KOLs), large consortia, and COVID research. However, our phase-1 A-HiChIP has known limitations. In particular, the product falls short of meeting the needs of researchers utilizing common clinical samples types or quantities in their research, or seeking single-cell resolution in their analyses of heterogeneous tissues. As part of this direct-2-phase II program, we propose to further develop our technology to overcome these limitations, meet customer need, and enable broader adoption and application of this powerful multi-omic assay in the form of our second-generation A-HiChIP solution. Specifically, we propose assay developments to enable compatibility with pervasive clinical sample characteristics - ultra-low cell inputs (lt100K cells) and FFPE tissues. Further, we propose development of a first-of-its-kind single-cell HiChIP assay and companion bioinformatics tools. We also propose essential product developments, to ensure commercialization of a robust, premium- performance kit-based product that is optimally integrated into the bulk and single-cell sequencing ecosystems. Upon successful completion of these technical and product-oriented aims, we propose to benchmark and validate our phase-2 A-HiChIP solution through collaboration and prototype (beta) kit and bioinformatics evaluations with KOLs across customer segments.