InSilixa developing drug-resistance TB test showcasing potential for hundreds of targets

The TB test is intended to showcase the firm's core technology, which can perform highly multiplexed nucleic acid amplification and analysis of hundreds of targets simultaneously.

InSilixa received a two-year, $1.5 million Phase II Small Business Innovation and Research grant from the National Human Genome Research Institute in 2013. The company also received $224,764 in funding from the National Institutes of Health last month to develop a point-of-care test for cancer-causing strains of human papillomavirus. In addition to a total of about $3 million in NIH funding overall, InSilixa has won $1.4 million in early seed funding and completed a $13 million Series A financing round in 2014. The firm is now seeking $35 million in Series B funding.

InSilixa CEO Arjang Hassibi told GenomeWeb in an interview that the general area of focus for the firm's technology is infectious diseases.

Specifically, the firm is exploring "applications in which you require an actionable test in a quick amount of time ... a complex test looking at drug resistivity with different strains, where the level of multiplexing is beyond simple PCR platforms that are out there."

The InSilixa technology uses semi-conductor-based chips for solid-phase array-based detection as the amplification is happening. It's not a microarray, but it overcomes a bottleneck other technologies encounter with the detection step of NAAT testing, Hassibi said.

"The problem of multiplex PCR has been you can always increase the level of multiplexing — the record for sequencing is up to a few thousand — so, you can create multiple amplifications, but the challenge is detecting them in parallel," he said.

By using optical methods, with different colors and channels for detection, other technologies can multiplex to a certain level. But, Hassibi said, commercial multiplex panels often divide samples and do multiple reactions to get the required number of targets.

InSilixa's method instead uses multiplex PCR to amplify all the regions in which mutations exist in one reaction chamber, and then it uses a CMOS, or complementary metal-oxide semiconductor, chip to capture and detect during the reaction, and perform a melt curve analysis in parallel.

The melt curve component is able to distinguish single-base pair changes and provides "the sensitivity of sequencing," Hassibi said.

The firm's first chip, the Hydra 1K, can examine 1,024 mutations in parallel, and the company envisions this technology will be unique in the white space between sequencing and individual PCR tests or small multiplex PCR panels.

The TB test, which is being developed with undisclosed partners, is intended to showcase this technology, Hassibi said. The firm is aware that the TB testing market is commercially challenging, but believes this test could be impactful and potentially benefit from funding set aside for that infection.

"We decided to pick up TB about two and a half years ago," Hassibi said, adding that he believes surmounting the challenge of massively multiplexed testing for TB will prove to the industry the value of the technology. "If we can show it, everybody will say, 'Wow, OK, they can probably do other stuff too.'"

The TB genome does indeed provide a challenge. It is more than 60 percent GC rich, with mutations in many genes conferring resistance to any of about five different drugs used to treat infection.

"We showed our preliminary data to NIH, and the grant that we have been working on for a while is looking at more than 120 mutations across 10 genes to be able to detect them all in one test. So, it's a pretty comprehensive system," he said.

A dominant player in the molecular TB testing field is Cepheid. That firm has a test for TB strains resistant to the first-line drug rifampin which detects 20 mutations in a hemi-nested RT-PCR using five different nucleic acid hybridization probes. It is also developing two new tests that are anticipated to have improved specificity and measure dozens of mutations.

However, when a mutant strain was recently found in Swaziland to have gone undetected by the Cepheid platform, the firm noted it would need to decide whether that target could be added to the tests already in development. The implication was that adding a new target to a multiplex PCR assay takes time and requires additional clinical validation.

InSilixa, on the other hand, can theoretically start with up to 500 or 1,000 mutations in its test and may be able to add or subtract them more easily.

"We have the bandwidth to look at all known mutations, but after a while it becomes a diminishing return," Hassibi said. "Our strategy in the case of TB and other panels that we have been developing is to go for the set of mutations which are confirmed and actionable, put it on the panel to get it through a regulatory process ... but because we do have the bandwidth to increase it, it will be incremental efforts, adding a few to a few hundred in the future."

The addition of mutation targets within known resistance-conferring regions, such as rpoB, for example, is "extremely easy if the region is within what we already have, because we're not restricted by the multiplexing level," Hassibi said.

The chip, meanwhile, is run on a platform, but the essence of the workflow is in the chip itself, Hassibi said. The system is also compact, about the size of a shoebox. "We don't have any sophisticated optics or any instrumentation except what is needed to drive the cartridge — the detector, the electronics, the heater, and everything is integrated in the disposable chip."

He added that the cost is "very competitive, and not all that expensive," but he did not provide a specific price.

For the TB assay, initial results would come in about one hour, while 30 minutes to one hour more is needed for subsequent melt curve analysis to validate the results.

InSilixa is also developing other tests for infectious diseases and is partnering with outside firms to allow them to develop their panels on the InSilixa chip.

The in-house infectious disease tests "generally require looking at a panel of organisms for a large number of mutations," Hassibi said. Some of these already exist, and "they have a high reimbursement rate ... but there are things that the Cepheids of the world cannot address, and those are done with targeted sequencing or multiple tests in parallel."

Hassibi declined to provide too much detail on the assays in development, but noted that "one of the applications includes about 21 organisms and a handful of mutations, and is going to be used in the clinical setting, for near-patient applications, and be a one-hour test."

In terms of partners developing assays on the InSilixa chip, while declining to name them, Hassibi noted they are both large and small companies that require solutions to develop the next-generation of their assays.

"About two years ago, this [business model] sounded really crazy. People said why are you doing this, you should keep all the applications for yourself, these are very valuable, but in the last six months to a year it is completely different," said Hassibi.

There are so many potential panels out there for HIV, HPV, and different GI panels, he said. "These are not onesie, twosie tests, but these are all orphans currently; which platform is going to take that, who is going to be able to detect 30 or 40?

"I think we are one of the companies that does have a solution for that, and that's our model," he said. "When companies do have panels and they want to go after them, we are opening our chip platform for them to adopt it."

The ultimate product of this collaboration will be the InSilixa chip and technology, with some of these companies then packaging that into their own private-label instrument.

Hassibi noted that beyond networking the firm developed partnerships and "really saw some traction" after informally presenting to multiple diagnostics companies at the JP Morgan Healthcare Conference in January.

Now, InSilixa is rising to the challenge of addressing all of the partnership possibilities without getting distracted. "Startups generally die out of indigestion, not out of starvation," Hassibi said.

Source: GenomeWeb