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Rapid quantification of nuclear citrullination in human neutrophils

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 2R44AI134162-02
Agency Tracking Number: R44AI134162
Amount: $2,945,857.00
Phase: Phase II
Program: SBIR
Solicitation Topic Code: NIAID
Solicitation Number: PA18-574
Solicitation Year: 2018
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-02-25
Award End Date (Contract End Date): 2023-01-31
Small Business Information
Chapel Hill, NC 27514-4617
United States
DUNS: 078882699
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (509) 660-0107
Business Contact
Phone: (281) 760-3246
Research Institution

PROJECT SUMMARYChromatin structure and gene expression are controlled by histone post-translational modifications
(PTMs) on nucleosomes, the basic repeating unit of chromatin. Chromatin decondensation is controlled in part
by the PAD4-dependent conversion of arginine to citrulline on histones (i.e. citrullination). Significantly, PAD4
activation in neutrophils initiates a cell death program distinct from apoptosis or necrosis, wherein chromatin is
hypercitrullinated, decondensed, and extruded from the cell in neutrophil extracellular traps (i.e. NETosis). The
release of citrullinated chromatin into the blood is thought to contribute directly to PAD4-related pathologies.
Thus, citrullinated nucleosomes (Cit-Nucs) are a promising blood-accessible biomarker for a range of
autoimmune diseases, including rheumatoid arthritis (RA), cancer thrombosis, and sepsis. However, there are
no assays capable of reliably quantifying nucleosome citrullination from serum or plasma.Current assays that aim to quantify Cit-Nucs use citrullinated histones as standards for assay
development and calibration. These type of standards are problematic for two major reasons; First,
citrullinated histones fail to provide accurate quantification of nucleosomes, especially at low concentrations;
second, free histones are highly charged and readily aggregate in plasma, which significantly impacts their linear
recovery from plasma samples. By contrast, we (and others) have found that nucleosomes are highly stable in
plasma, suggesting that these substrates may provide superior standards for assay quantification. EpiCypher®
is pioneering the commercialization of recombinant designer nucleosomes (dNucs) carrying physiological
histone PTMs for next-generation epigenetics assays. Here, we are developing CitNucâ„¢, the first ELISA to
accurately quantify Cit-Nucs for NETosis research and preclinical biomarker development. Our innovative
assay uses recombinant citrullinated designer nucleosomes (Cit-dNucs) as quantification standards for antibody
pair selection / validation and reliable assay quantification. Importantly, unlike histones, Cit-dNucs can be
faithfully recovered from plasma samples, enabling development of a highly sensitive ELISA.In Phase I, we successfully used Cit-dNucs to identify highly specific antibodies and establish reliable
standards for quantification of Cit-Nucs in plasma. We also examined key bioanalytical parameters and validated
the ability of CitNuc ELISA to detect differences in Cit-Nucs between healthy and RA patient samples,
demonstrating the utility of our assay in a clinical environment. In Phase II, we will complete bioanalytical testing
and define reliable lot-release strategies for CitNuc ELISA kits (Aim 1), paving the way for commercialization. In
Aims 2 and 3, we will develop the preclinical application of this assay for biomarker discovery, focusing on RA
and cancer thrombosis, two diseases associated with high levels of PAD4-dependent nucleosome citrullination.
Together, these Aims will result in the commercial launch of a first-generation CitNuc ELISA assay, which will
be marketed for NETosis research and biomarker development applications.

* Information listed above is at the time of submission. *

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