National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)

The National Institutes of Biomedical Innovation, Health and Nutrition adopted Retrieva’s GGGenome packaged version to assess off-target risk for antisense oligonucleotides. The institute can now work in a secure offline environment, and because multiple nucleotide sequences can be searched at once, the workload has fallen substantially. Analysis and design are markedly faster and more efficient, and running other analyses in parallel has improved accuracy beyond what was previously possible.

Why the GGGenome packaged version

A project aiming for oligonucleotide therapeutics from Japan

The National Institutes of Biomedical Innovation, Health and Nutrition was formed in 2015 through the merger of the National Institute of Biomedical Innovation and the National Institute of Health and Nutrition. Its biomedical innovation arm pursues research on foundational technologies and on biological resources, and supports drug discovery drawing on those results, with the aim of contributing to the development of innovative pharmaceuticals for world-class medical care.

Within it, the Artificial Nucleic Acid Screening Project at the Center for Drug Design Research builds on nucleic acid technology: it adds nucleic acid technology seeds to drug discovery seeds from universities and other academic institutions, licenses them out to companies, and works to bring them into practical use as pharmaceuticals as quickly as possible.

We do not simply screen oligonucleotide therapeutic candidates against drug discovery seeds; we also develop new screening methods and new design approaches.

Dr. Yuya Kasahara, Sub-Project Leader, Artificial Nucleic Acid Screening Project, Center for Drug Design Research

In January 2021, a joint research group including Kasahara developed a synthetic enzyme (a modified polymerase) capable of transcribing and reverse-transcribing locked nucleic acid (LNA) accurately and rapidly, and became the first in the world to create artificial nucleic acid aptamers containing LNA through screening with that modified polymerase. Aptamers containing LNA resist degradation by nucleases and combine high in vivo stability with high duplex stability, so they are expected to find use in therapeutic development and in nucleic acid engineering.

Adopting the GGGenome packaged version for off-target risk assessment

The project had long used the GGGenome web edition published by the Database Center for Life Science (DBCLS) to assess off-target risk for the antisense oligonucleotides it designed. The web edition does allow analysis via a static API in Google Sheets, but limits on analysis speed and volume made it one of the bottlenecks in antisense sequence design. There were also requests to analyze offline rather than on the web, given the risk of information leakage.

When there are thousands or tens of thousands of targets to analyze, doing them one at a time is extremely difficult. Until now we narrowed thousands or tens of thousands of candidates down to a few hundred using various factors, then analyzed those one by one. Even so, a few hundred is a great deal of work, and we were wondering what could be done about it.

Dr. Yuya Kasahara

It was at that point, at the 5th Annual Meeting of the Japan Society of Nucleic Acid Therapeutics in July 2019, that the project learned Retrieva offered a packaged version of GGGenome, and it began evaluating it immediately.

The evaluation led to a decision to adopt the packaged version for off-target risk assessment of antisense oligonucleotides. Kasahara cites the reasons: analysis can be done offline, batch processing is fast, multiple sequences can be analyzed at once, and the D3G sequence databases are included.

The team uses Macs in its day-to-day research and was not fully accustomed to Windows, but Kasahara says careful deployment support from Retrieva made the rollout relatively straightforward.

Batch search cuts the workload substantially

Since adopting it in 2019, the Center for Drug Design Research has installed the updated edition each fiscal year and continues to use the packaged version. "We use it to assess off-target risk for the several hundred antisense oligonucleotides we design, and then use those results to narrow down which sequences we actually synthesize and evaluate," Kasahara says. It is operated by a technical assistant well versed in sequence design, and is used around two to three days a week.

I would say it leaves nothing to be desired. No particular specialist knowledge is needed; anyone who can operate a computer can use it easily. As for databases, we often use the D3G spliced and pre-spliced RNA databases for human, mouse, rat and cynomolgus monkey.

Adopting the packaged version has resolved a number of problems. Beyond secure offline use, it provides search support features for searching multiple nucleotide sequences at once and searching across databases. Previously the team repeated a simple manual task — entering each of several hundred candidate sequences, analyzing it and copying the result — which invited human error and made the search stage slow and inefficient. That has been resolved.

As it feels to us, the effort has dropped to about a tenth.

With analysis and design markedly faster, the team can run other analyses in parallel and run analyses across multiple patterns, raising both efficiency and accuracy. "Previously the flow was to narrow down to a few hundred and then analyze," Kasahara says. "Thanks to the packaged version we can now also evaluate every option, pick out those with low off-target risk, and take them forward to the next stage of evaluation."

Alongside the search support features, the team also uses gene-of-interest highlighting. "We are optimizing our sequence design methods on the assumption that we have the packaged version," Kasahara explains. "Including our use of gene-of-interest highlighting, we are looking at whether we can build new evaluation metrics for design."

What the Artificial Nucleic Acid Screening Project is working toward is personalized medicine tailored to each individual patient’s condition.

Our wish is to use the properties unique to oligonucleotide therapeutics to deliver a treatment made for one patient alone, based on that patient’s genetic information. There are difficult aspects, but this field is advancing worldwide and we intend to keep pace.