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UK's GeneFirst Developing Array of MDx Products Using Novel PCR Technologies

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NEW YORK (GenomeWeb) — GeneFirst, a three-year-old UK-based molecular diagnostics firm, is looking to parlay its portfolio of proprietary PCR technologies into products for highly multiplexed infectious disease diagnosis and blood-based cancer mutation detection.

Leveraging three active grants totaling £670,000 (about $1.1 million) from the UK government, Abingdon-based GeneFirst will seek CE marking within the next 18 months for an assay that can detect, quantify, and genotype all 14 high-risk human papillomavirus strains in a single, closed-tube reaction.

In addition, the company is attempting to apply one of its multiplex PCR technologies to detect lung cancer driver mutations in archived patient samples, and integrating another of its proprietary primer technologies with next-generation sequencing to detect ultra-rare cancer gene mutations in circulating cell-free DNA from patient plasma samples.

GeneFirst CSO Guoliang Fu discussed these initiatives and more in an email interview with PCR Insider this week.

Fu is the lead inventor on all four of GeneFirst's core technologies: polymerase chain displacement reaction (PCDR); multiplex probe amplification (MPA); multiplex mutation detection (MMD)-PCR; and targeted bi-directional (TBD)-seq. Fu developed the intellectual property surrounding PCDR and MPA while he was employed at insect-control company Oxitec, from which GeneFirst subsequently secured exclusive, global licensing rights to the technologies.

Meantime, Fu developed the IP surrounding MMD-PCR and TBD-seq while at GeneFirst. Patents for all four technologies have been granted in one or more territories, including the EU and US, Fu said.

The most mature of GeneFirst's technologies is PCDR. First described in a Biotechniques paper in February 2013, PCDR is "a simple amplification technology … [that] gives an incredibly high level of amplification compared to standard PCR," Fu said. As opposed to conventional PCR, PCDR uses more than one pair of primers to generate up to four-fold greater amplification per cycle, although it still can be run on conventional PCR platforms.

Specifically, GeneFirst and its collaborators have demonstrated that PCDR produces twice as much material in each round of thermal cycling, resulting in at least a 10-fold increase in sensitivity. In addition, as reported last month in PCR Insider, PCDR's sensitivity may be improved even further with the use of a novel DNA polymerase called SD Polymerase, sold by German molecular biology firm Bioron.

"Our view is that PCDR is next-generation PCR, and so we'd like to have the broad application and licensing success that [Applied Biosystems] and Roche had with PCR … [and] hope to license [it] out on a non-exclusive basis to many, many licensees," Fu said.

Meantime, GeneFirst's MPA technology, which the company described in a PLOS One paper in January 2012, is a "platform PCR-based technology with many potential applications," Fu said. MPA enables the detection and quantification of multiple nucleic acid targets, independently of one another, within one fluorescence channel in a standard real-time PCR platform, Fu noted.

Due to its high multiplexing ability, GeneFirst is attempting to use this technology to develop its HPV assay. The company last year garnered a £150,000 feasibility grant from the UK Technology Strategy Board's Biomedical Catalyst program to support this effort, and was recently awarded an additional £500,000 follow-on grant from the agency to continue the project.

HPV, Fu noted, is one of the most common sexually transmitted infections, with some but not all types known to cause cervical cancer. "Since early-stage cervical carcinomas are nearly 100 percent curable, early detection is very important," Fu said. "Accurate molecular diagnosis is needed to inform patient management and follow-up treatment. However, current methods suffer from sub-optimal sensitivity, discrimination, and/or complex hybridization-based procedures."

GeneFirst hopes to address these issues with its MPA HPV test, which is designed to detect 14 of these high-risk HPV types in a single reaction using existing real-time PCR platforms. "In this project, we set out to analyze the clinical performance of this novel HPV molecular diagnostic test to identify patients who have [high-risk] HPV," Fu said. "Such an assay would offer physicians better means to identify women at risk and optimize treatment strategies."

Fu added that the company aims to obtain CE marking for this assay and begin sales in the EU "during or soon after the completion" of the 18-month grant.

GeneFirst's third core technology, MMD-PCR, is an in vitro real-time PCR-based diagnostic technology designed to detect cancer-associated mutations in DNA extracted from blood or formalin-fixed paraffin-embedded tissue.

Current commercial PCR-based products for cancer mutation detection, such as Qiagen's Therascreen kits, claim a sensitivity of detecting 1 to 10 percent of mutated DNA in wild-type DNA background, Fu said. However, this usually requires a large quantity of clinical material.

"This sensitivity and requirement of large [amounts of] clinical material are unacceptable when aiming to identify rare mutants in genetically heterogeneous mixtures, such as tumors and plasma," Fu said. "Based on proprietary primer/probe design, MMD-PCR is ultra-sensitive, inexpensive, and consumes minimal clinical material."

GeneFirst has been awarded a small feasibility grant from the TSB Biomedical Catalyst program to investigate the specific use of this technology to detect lung cancer driver mutations, Fu noted.

Although the MMD-PCR technology is currently optimized for FFPE samples, Fu said that it would also be ideal for cancer gene mutation detection in plasma, and as such "we are looking beyond lung cancer to breast, colorectal, and prostate cancers."

Finally, under a third small feasibility grant from TSB Biomedical Catalyst that kicks off next month, GeneFirst is developing its TBD-seq technology as a way to improve NGS as a tool for cancer diagnosis and monitoring.

Fu noted the growing interest in circulating tumor DNA as a non-invasive biomarker to detect the presence of malignancy, follow treatment response, gauge prognosis, or monitor for recurrence. He also said that NGS has emerged as one of the most promising methods for detecting and analyzing ctDNA.

"However, the sensitivity of [NGS] is limited by the inherent error rate of the sequencer, as incorrectly read bases might be mistaken for true mutant copies," he said.

To overcome this limitation, TBD-seq uses primers that specially amplify targeted DNA in each separate strand such that sequencing variations can be read as genuine if they appear in both strands, thus greatly reducing NGS error rates.

Fu said that this technology "needs some further proving before we get too excited about it."

GeneFirst currently has several products and services based on its core technologies, however none of them has regulatory approval for clinical use in the EU, US, or elsewhere, and thus are "strictly for research use only," Fu said. "Thus sales are primarily to academic or industrial bench-based researchers, with PCDR and MMD leading the way," he added. "TBD-seq isn't ready and MPA tends to be exploited on a contract-research basis at present. Our aim is certainly to get our labs ISO13485 certified by developing appropriate quality systems and [to] get all our technologies out there [as] CE marke[d] kits. We'd hope that we can also get US approval via [the] 510(k) route as [pre-market approval] is too expensive for a [small company] like us."

To that end, GeneFirst is currently seeking "an additional £2.5M from private, corporate, and/or institutional investors" to complement its TSB grants. And while the company has closed on some private seed funding, "we do need substantial investment to reach long-term commercial viability," Fu said. "While this non-dilutive funding is great, it does need to be matched by either investments or sales."

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