NEW YORK – Sherlock Biosciences announced last week that it has licensed nucleic acid amplification technology enabling ambient temperature reactions, which the firm plans to combine with its other proprietary technologies to create infectious disease diagnostic tests for home use and point-of-care testing.
The ambient amplification technology was developed in the lab of Sherlock Cofounder James Collins at Harvard's Wyss Institute and licensed from Harvard.
Reaction temperature varies among nucleic acid amplification technologies. Loop-mediated amplification (LAMP) can run at the temperature of scalding hot water, or approximately 65 degrees Celsius, while recombinase polymerase amplification (RPA) can run at body temperature, or 37 degrees Celsius, for example.
The new ambient amplification method, which does not yet have a name, runs at even cooler temperatures, according to Bryan Dechairo, president and CEO of Cambridge, Massachusetts-based Sherlock.
Specifically, it works between 18 and 39 degrees Celsius, Dechairo said in an interview, adding that room temperature is typically 22 degrees Celsius.
Amplification at room temperature may help overcome some critical hurdles to decentralizing highly accurate diagnostics and potentially improve global health equity.
"When you don't require heat, you don't require any form of electricity, or circuit boards, or batteries," Dechairo said. "You can do the whole test in paper wrapped in plastic with a lateral flow or colorimetric readout, which means you get very low cost of goods."
The Specific High-sensitivity Enzymatic Reporter unlocking, or SHERLOCK method has been utilized in malaria assays for point-of-care multispecies detection by the Collins lab at the Wyss Institute and Massachusetts Institute of Technology, but these tests used RT-RPA combined with CRISPR.
According to Nico Angenent-Mari, a scientist at Sherlock who helped invent the ambient technology in the Collins lab, the new method is quite different from those described in previous work, in that it uses "a combination of enzymes that are robust at low temperatures and new chemistries."
The method can also be run without extraction, and lends itself to techniques like single-tube reactions with colorimetric readout or lateral flow test formats, Dechairo said.
While isothermal amplification methods are sometimes dinged for having lower specificity and the higher potential for false-positive results than thermal cycling, room temperature reactions tend to increase the likelihood of incorrect amplification even further. But Dechairo noted that combining isothermal chemistries with CRISPR detection counterbalances these limitations.
"CRISPR brings back the specificity, because it is one of the most specific enzymes; that is why it can be used for gene editing," he said.
The Sherlock team has also increased the performance of the system since a Clinical Chemistry study on it was published in 2021, Dechairo noted.
Also developed in the Collins lab, INSPECTR is a synthetic biology-based molecular diagnostics system to distinguish targets based on a single nucleotide using a DNA hybridization-based sensor coupled with a paper-based synthetic gene network that translates the sensor's detection into a bioluminescent signal.
The CRISPR-based SHERLOCK system along with a high-throughput LAMP-based SARS-CoV-2 assay showed 100 percent concordance with the CDC's qPCR assay in the Clinical Chemistry study published last year. When used with direct saliva samples, the system also demonstrated a negative percent agreement of 100 percent and a positive percent agreement of 88 percent compared to lab testing using the Abbott Alinity or Biocartis Idylla platforms.
The path to commercialization
While Sherlock has previously been focused on research and development, the firm is now pivoting to a focus on commercialization. Favoring this pivot, the regulatory path for home-use isothermal molecular tests is more straightforward than in the past.
A handful of companies have received authorization for lab-based COVID tests using LAMP, and there is also precedent for over-the-counter isothermal molecular tests as well.
For example, Detect received Emergency Use Authorization from the US Food and Drug Administration for an over-the-counter COVID test using LAMP technology and is expanding into additional menu. Lucira has also obtained EUA for a COVID test and is developing tests for sexually transmitted infections (STIs). And, a disposable molecular COVID test from Aptitude Medical that can test from saliva samples was recently authorized.
But developing LAMP-based assays is notoriously tricky, and since each target requires six primers and probes, multiplex assays can require development of dozens of oligos. Thus, LAMP assay development "becomes complex really fast, and there is a lot of primer dimers and misamplification," Dechairo said.
The Sherlock team has previously developed an artificial intelligence-based workflow to help accelerate this design process. In a poster at the Next Generation Dx Summit in August, the firm said it's in silico design tools could accelerate assay development from weeks to hours.
There are other isothermal amplification methods besides RT-LAMP that are used for diagnostics and may have simpler assay design. For example, researchers in India have developed a CRISPR system called Feluda — in honor of a famous fictional Bengali detective — and paired it with RPA to detect SARS-CoV-2. That system has been licensed to a diagnostics subsidiary of Tata Group. Meanwhile, startup Sense Biodetection uses an undisclosed isothermal method, while Innova Medical has recently licensed an isothermal method from the University of Birmingham in the UK called Reverse Transcription-Free Exponential Amplification Reaction (RTF-EXPAR).
But, Sherlock's new ambient amplification method makes development simpler, Dechairo said, because it uses many fewer primers, and the simpler design enables even higher multiplexing than LAMP tests.
The team now plans to develop the ambient method into paper-based tests, which will align with its overall mission to "decentralize and democratize diagnostics globally," Dechairo said.
The low cost of goods and high accuracy support tests for influenza and respiratory syncytial virus, he said, as well as at-home chlamydia and gonorrhea infection testing.
With respect to sexually transmitted infections, which have been on the rise due to the pandemic, the Sherlock technology "allows a price point and ease of use to bring the testing into the home," he said. These infections are also a scourge of global health, and Sherlock has collaborators in low- and middle-income countries and sees its tests used in these spaces as well.
The sale of millions of rapid antigen tests for home use during the pandemic was dependent on the cost of goods in the $2 range, Dechairo said, and to his mind assays that require heaters, readers, or other complex technology quickly become unaffordable for many households.
"Low cost of goods is the most important thing, and to get that you have to move away from complex technologies," he said.
Overall, Sherlock plans to ramp up the commercialization phase in the new few years, Dechairo said.
He joined the company in July 2021 from Myriad Genetics and has since expanded the device development and commercial team, for example, adding Karen Davies from Quidel as chief development officer and hiring Julie Garlikov, a commercialization expert who previously worked at Grail and Procter & Gamble, to be Sherlock's first chief commercial officer.
The firm recently closed an $80 million Series B funding round, which included Illumina Ventures and others as new investors and added Paul Meister from lead investor Novalis LifeSciences to its board.
Dechairo's perspectives on molecular diagnostics are informed by his tenure at Roche Diagnostics in the early 1990s, just after that firm acquired the patent for PCR from Cetus. Although development in molecular diagnostics has been remarkable over the ensuing 30 years, the decentralization is the missing piece.
Now, the new technology may finally bridge this gap. "When I saw the ability of CRISPR paired with ambient temperature methods, I realized that this is the first chance to actually bring highly accurate molecular diagnostic tests out of the lab," he said.