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CRISPR-based diagnostics are poised to make an early debut amid COVID-19 outbreak

COVID-19 could give CRISPR diagnostics their first proof of principle

CRISPR could see its first direct application to human health much earlier than expected as the COVID-19 outbreak accelerates development timelines for CRISPR-based diagnostics.

Diagnostic testing for COVID-19 is currently conducted with real-time PCR tests that need to be run in centralized labs. Capacity and speed of readout are highly limited. While China has rushed ahead to produce and distribute PCR-based tests, doubts have been raised about the level of clinical validation (see “Plotting a Scientific Path to Counter COVID-19”).

Sherlock Biosciences Inc. and Mammoth Biosciences are shooting for improved diagnostics with CRISPR-based technologies that could increase and accelerate the detection of the novel coronavirus to better treat patients and control the spread of the virus. In addition to being quickly adaptable to new targets and offering a potentially rapid and specific readout, the tests are being designed for the point of care setting.

“This could be it for sure. The first real demonstration of value of the CRISPR technology.”

Rahul Dhanda, Sherlock

Prior to the outbreak, Sherlock had been targeting a 2023 launch for a CRISPR-based diagnostic test; it now hopes to submit its first COVID-19 test for FDA clearance this season, said CEO Rahul Dhanda.

Mammoth co-founder and CEO Trevor Martin described the company’s new timescale as “rapid,” but declined to give more specifics about the revised or original timelines.

Both companies are seeking partners earlier than planned to bring COVID-19 diagnostics to market.

The accelerated timelines for CRISPR-based diagnostics mean that the unplanned use could give the first hints at the transformative potential of the gene editing technology in human health applications. CRISPR-based therapeutics are likely years away from clinical proof of concept for in vivo applications, where CRISPR could produce broad-ranging benefits.

“This could be it for sure. The first real demonstration of value of the CRISPR technology,” said Rahul Danda, Sherlock president and CEO. “We’ll really see the actual impact when the product is out, but from what I’ve seen, this technology certainly has that potential.”

Two shots on goal at Sherlock

Sherlock is developing two diagnostic platforms and plans to deploy both for COVID-19. The company was founded in 2018 around CRISPR diagnostic technology developed by Feng Zhang and colleagues at the Broad Institute of MIT and Harvard.

The first platform, dubbed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), uses Cas13a to target specific RNA sequences. The platform is expected to yield the company’s lead COVID-19 product.

The test involves a paper strip in a test tube with a Cas enzyme, a guide RNA and a short reporter. When the enzyme encounters its target in a biological sample, it becomes activated to cut up all nearby RNA, including the reporter, resulting in a signal such as a measurable increase in fluorescence (see "Sherlock: Leveraging CRISPR, Synthetic Biology for Diagnostics").

Dhanda told BioCentury the tests should read out in less than 30 minutes. At a global online forum sponsored by WuXi AppTech Co Ltd. earlier this week, Victor Shi, CEO of Adicon Clinical Laboratories, said there is a two to three hour turnaround for existing COVID-19 screens.

While the SHERLOCK platform offers potentially unprecedented speed and specificity, Dhanda said it requires a “small instrument component to maintain temperature for a period of time,” meaning that initial tests will likely need to be sent to a central lab or used in a hospital setting with the right equipment and technicians.

“All CRISPR-based detection requires pre-amplification of DNA or RNA prior to paper-based detection,” said Dhanda. According to a white paper from Zhang and colleagues at the Broad Institute, Sherlock can be used after an isothermal recombinase polymerase amplification (RPA) procedure, which uses simpler equipment than PCR but requires temperature control.

On Friday, Sherlock announced a collaboration with Cepheid to run SHERLOCK tests for infectious diseases on the Cepheid’s GeneXpert cartridge-based system. COVID-19 will be the initial proof of concept for the research collaboration.

The tests developed with Cepheid can be used anywhere Cepheid’s hardware is available, which may be a centralized lab setting or hospitals. Over 23,000 GeneXpert systems have been installed.

Cepheid also announced earlier this month it would develop an automated point-of-care test for COVID-19 using GeneXpert, but that test is independent of the new collaboration.

Dhanda expects point-of-care use will be possible for future iterations with an engineering solution. The company does not have timeline for a point-of-care diagnostic, but is exploring the application with SHERLOCK and its other platform.

“A test done at home means that nobody who comes up positive needs to leave the house.”

Rahul Dhanda, Sherlock

Further behind is a synthetic biology platform called INSPECTR (INternal Splint-Pairing Expression Cassette Translation Reaction) that doesn’t involve CRISPR and doesn’t require any instrumentation.

INSPECTR uses a DNA hybridization-based sensor and a paper-based synthetic gene network that can integrate nucleic acids. When it detects a genetic match, the network translates the sequence into proteins that generate a fluorescent signal or an enzyme.

“INSPECTR is instrument-free from the beginning and can be done without a trained technician, so we see this as something that could be used at home. It wasn’t ready for this season though,” said Dhanda. The company has not disclosed a timeline for testing or submitting an INSPECTR-based test to FDA.

He added that current diagnosis of COVID-19 requires patients to leave the home to get the result. “A test done at home means that nobody who comes up positive needs to leave the house, which could dramatically reduce transmission and spread.”

Furthermore, point-of-care tests could potentially be cheap and accessible enough for widespread testing and even screening in asymptomatic individuals.

Mammoth’s path

The competition again sees the Broad Institute’s Feng Zhang face off against the University of California Berkeley’s Jennifer Doudna. The two are at the center of the patent dispute over the inventorship of CRISPR-Cas9. (see “No Crisp Upper Hand in CRISPR”; “Plenty of CRISPR Pie”).

Mammoth was founded in 2017 around CRISPR-based diagnostic technology from Doudna’s lab, and has created a DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) platform to develop tests that can differentiate COVID-19 from other coronaviruses, and detect multiple coronavirus strains.

“With the right equipment, I could imagine using it in the field setting.”

Trevor Martin, Mammoth

Like Sherlock, DETECTR is a CRISPR-based test that uses a visual lateral flow strip format that reads out within 30 minutes. The test relies on a Cas12a, rather than the Cas 13a in Sherlock’s system, to detect viral RNA sequences in patient samples (see "Mammoth Makes Dx CRISPR").

In a February white paper, Mammoth described the steps to perform the test; RNA is extracted from patient samples according to the CDC protocol; samples undergo a 20-minute isothermal amplification (RT-LAMP) step; they are then combined with a Cas12a ribonucleoprotein complex and guide RNA; and a test strip is inserted for the readout.

Martin told BioCentury that the earliest version of the test will require those reagent-based steps and several tools that “will be most efficient in a centralized lab setting,” but that doesn’t mean it's limited to that. “With the right equipment, I could imagine using it in the field setting,” he added.

The first iteration of the test could enable real-time readouts in a hospital setting. Later generations will likely use a cartridge format to enable true point of need use, such as in the airport or home to allow real-time diagnostic access.

“I don’t think we need to wait until we have a cartridge format to make a test available. There is a huge potential for something with a fast turnaround time that is accurate and not complicated relative to trying to run a real-time PCR test,” Martin added.

Accelerated development

While development of assays using the SHERLOCK and DETECTR platforms are under way, the companies will need to access samples of the virus and find the right partners to get FDA clearance this season.

Both companies are designing COVID-19 assays based on the published virus sequences, but neither has access to viral samples yet.

For Sherlock, Dhanda said, “We can use negative samples to demonstrate that the system works, and we are continuing to develop in silico modeling that is highly predictive of actual performance against other targets." The company has started outreach to access samples, which will be required for validation.

Martin believes patient samples are required earlier in the development process, and Mammoth is collaborating with SAB member Charles Chiu at the University of California San Francisco to try to access samples.

“You can get to the first stage to see if a hypothesis works without the samples, but validation will require them, and there will always be differences between real patient samples and everything else so they’ll be required in the design too,” said Martin.

Both companies will rely on partners to translate their chemical expertise to commercialization.

Dhanda noted that Sherlock is capable of rapidly developing and validating assays, but “we need a partner with the scalability and infrastructure to manufacture and commercialize a product now.”

Prior to announcing the partnership with Cepheid, Dhanda said Sherlock was in active discussions with a short list of partners and planned to narrow that down to one or two in the next few weeks. The company did not disclose whether it is seeking additional partnerships, or whether the test it is pushing forward for potential clearance this season will use Cepheid’s GeneXpert technology.

It wasn’t always the plan to hand off the technology to a partner this early in development, but it’s necessary given the accelerated timeline, said Dhanda. “We are less than a year out from our company launch and haven’t fully developed our capabilities.”

“Before this happened, we were talking about developing and maturing two platforms and building the infrastructure ourselves. Everything from business development to operations to technology has gone through some degree of acceleration, and it’s helped us solidify certain parts of the organization we were planning to develop much later,” he added.

Sherlock plans to use the Emergency Use Authorization regulatory pathway to expedite clearance, which has fewer requirements than a standard 510(k) approval.

Mammoth is seeking partners who “are on the ground, in the thick of it and can bring our technology where it’s needed the most,” and “someone with a platform of manufacturing capacity interested in bringing the amazing chemistry to the points where its needed,” said Martin.

The company will use “the most rigorous and appropriate channels for clearance depending on where in the world it’s being used,” he added.

Jumping ahead in CRISPR

While CRISPR has proven its worth in the research setting, making genetic screens and animal model generation much more efficient, the industry is still waiting for the technology’s first proof of concept in the healthcare arena.

Several clinical trials evaluating CRISPR-based therapeutics are already under way, and two have read out initial data on a few patients, but those initial applications involve ex vivo edited cell therapies and an ophthalmic injection. The full proof of concept for CRISPR therapeutics may not come until an in vivo therapy reads out in the clinic, which could be years off.

CRISPR Therapeutics AG (NASDAQ:CRSP) and Vertex Pharmaceuticals Inc. (NASDAQ:VRTX) have the gene edited stem cell therapy CTX001 in Phase I for sickle cell disease and β thalassemia. CRISPR Therapeutics and a group from Tmunity Therapeutics Inc. and the University of Pennsylvania are each testing CRISPR-edited T cell therapies for cancer. However, benefits in the design and manufacture of the cells may be greater than the added benefit to patients.

Editas Medicine Inc. (NASDAQ:EDIT) and Allergan plc (NASDAQ:AGN) have AGN-151587 (EDIT-101) in Phase I/II for the ophthalmic indication Leber congenital amaurosis 10 (LCA10).

Further analysis of the coronavirus crisis can be found at https://www.biocentury.com/coronavirus. The COVID-19 content is free to all who visit the site.

Targets

Cas12a (Cpf1) - CRISPR from Prevotella and Francisella 1

Cas13a - CRISPR-associated protein 13a

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