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Eclipse Bioinnovations Aims to Help Researchers Understand Gene Regulation, RNA Processing

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SAN FRANCISCO (GenomeWeb) – Eclipse Bioinnovations, a spinout of the University of California, San Diego, plans to commercialize technology to help researchers better understand gene expression and regulation by analyzing RNA-binding proteins.

Last year the company received a two-year, $1.3 million National Institutes of Health Small Business Innovation Research grant to further develop and commercialize the technology, called enhanced UV crosslinking and immunoprecipitation (eCLIP). The company is currently offering a service based on the technology, and plans to launch a kit for researchers by early next year.

Gene Yeo, a principal investigator at UCSD whose lab developed the eCLIP technique, said that his lab had been working on various methods to study RNA-binding proteins for the last 10 years, but they were all "very laborious, required a trained technician with molecular biology background, and all had nine- to 10-day-long protocols."

Yeo said that better tools were needed because in the last 10 years there has been an explosion of discovery in the space, with RNA-binding proteins now thought to play critical roles in gene regulation and RNA processing. Recent research has begun linking RNA-binding proteins to many different diseases, but the specific functions of such ribonucleoprotein complexes are not well understood.

A decade ago, there were only an estimated 300 to 500 proteins suspected of binding RNA, Yeo said. Now, there are an estimated 1,500 to 2,000 proteins. In addition, he said, there were previously not known antibodies that could immunoprecipitate the proteins. Much of Yeo's research over the last 10 years has focused on identifying both the RNA-binding proteins and corresponding antibodies as part of the NIH's Encode project. As part of that, his lab mapped more than 200 RNA-binding proteins to the transcriptome and validated 300 to 400 antibodies that could immunoprecipitate those proteins, creating a publicly available repository.

That helped spur the field, Yeo said, but the methods were still a bottleneck and "not feasible to do at scale."

The CLIP method had been around for years, but it wasn't until costs for next-generation sequencing came down that researchers were able to use it in a high-throughput manner to study RNA-binding proteins at single-nucleotide resolution.

CLIP first involves crosslinking RNA and protein complexes using UV light, which causes nucleic acids and proteins that are near each other to link. Then, the crosslinked cells are lysed and the proteins are isolated via immunoprecipitation. The RNA and its protein are then separated, and the RNA molecule can be analyzed via NGS.

CLIP coupled with NGS enabled single-nucleotide resolution of RNA binding sites, but was initially challenging due to many sequencing reads having to be discarded because they were PCR duplicates. In addition, a previous iteration of the method used circular ligation, which was often inefficient at producing sequencing libraries.

Yeo's lab began working on the method and described an improved version of the protocol, eCLIP-seq, in a study published in Nature Methods in 2016.

Yeo's team modified the protocol to add adapters in two separate steps — first ligating a 3' RNA adapter to the crosslinked RNA fragment while it is still attached to the immunoprecipitation beads and then ligating a 3' single-stranded DNA adapter following reverse transcription.

The second major modification the group made was to reduce the amount of amplification used in order to reduce the number of reads that were PCR duplicates.

In the study, they found that they were able to reduce PCR duplicate reads by around 60 percent while still maintaining single-nucleotide resolution. In addition, the researchers have been able to reduce the turnaround time from more than a week to three to four days.

Following that study, Yeo said Eclipse Bioinnovations was spun out of UCSD to provide eCLIP-seq services and to continue to improve on the protocol and ultimately develop it into a kit. Yeo is a cofounder and scientific advisor to the company.

Peter Chu, CEO of Eclipse Bio, said that a kit is under development and will be sent to beta customers either this quarter or next quarter with a commercial launch planned for either the fourth quarter of 2018 or early 2019.

Eric Van Nostrand, a postdoc in Yeo's lab, and cofounder and CSO of Eclipse Bio, said that a kit would essentially "provide a researcher with all the essential reagents to perform eCLIP-seq." With the kit, researchers would be able to take a sample of interest and an antibody and make a sequencing library that contains the binding sites for that protein. The kit would "include buffers to isolate the protein as well as the reagents to take the RNA fragments and turn them into a sequencing library," he said. In addition, it would include the bioinformatics on the back end to make sense of the data. The goal is to make the method, "which is simpler than previous versions, but still complex, doable by someone with expertise in molecular biology but not necessarily in RNA processing," he said.

Van Nostrand said that the company also aims to validate the method on different tissue types and cells, and as such plans to develop reference datasets in various tissue types so researchers can compare data.

Van Nostrand anticipates that academic researchers will be the primary target market for the kit version of eCLIP-seq, while both academic and pharmaceutical researchers will be interested in the service.

"We're starting to really see that RNA-binding proteins and processing alterations are linked to many different diseases," he said, including different types of cancers, neurodegenerative diseases such as ALS, and neurological disorders like autism and schizophrenia. Whole-genome and exome sequencing studies are increasingly identifying RNA-binding proteins as being linked to such diseases.

Researchers are "interested in studying those further," Van Nostrand said, and the goal of the company is to help them "understand what is happening downstream — what targets the RNA-binding proteins are binding to and what that can tell us about how they are linked to disease."

Chu said that aside from the eCLIP-seq kit, down the line the company would continue to focus on the RNA space, including moving into the RNA therapeutics and RNA-targeting drugs space. The goal would be to use the revenues from the services business to fund in-house discovery work to "start to identify targets and potentially get those started down the track of RNA therapeutics," he said.

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