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PentaBase Demonstrates qPCR Method for Direct DNA Methylation Analysis


NEW YORK – Danish life sciences technology firm PentaBase has developed a qPCR-based method, dubbed EpiDirect, for the direct analysis of DNA methylation without the need for pre-treatment of the DNA.

The company has commercialized the technology as an assay, EpiDirect MGMT, to detect and quantify 5-methylcytosine (5mC) in the MGMT promoter region of brain tumor samples. In a paper published in Nature Communications last month, PentaBase researchers and their collaborators demonstrated its sensitivity and specificity and compared it with two methyl-specific PCR assays that require bisulfite treatment.

"We try to push the limits of what is possible with real-time PCR," said Ulf Bech Christensen, PentaBase's founder and CEO. "Our main focus is on these technologies that can give a very sensitive, very specific, and fast answer."

EpiDirect was built upon proprietary technology, called Intercalating Nucleic Acids (INAs), that Christensen originally developed at the University of Southern Denmark.

INAs are synthetically manufactured oligonucleotides that incorporate at least one hydrophobic base analog, called intercalating pseudo-nucleotide (IPN). The integrated IPN enhances the base stacking effect in a DNA double helix, which increases its thermal stability and melting temperature. In addition, since INAs stack differently with 5'-methylated cytosine (5mC) than with unmethylated cytosine, the thermal stability of methylated DNA is greater than that of unmethylated DNA.

Harnessing this property, the company developed the EpiDirect technology, which selectively amplifies methylated DNA using a special primer design.

Named EpiPrimer, the primer consists of an anchor, a loop, and a starter sequence. The anchor sequence contains IPNs that bind to a complementary methylated target with significantly higher thermal stability than to its unmethylated version. Meanwhile, the starter sequence, which is located at the 3’ end of the primer, is designed to have low thermal stability and not bind to the template DNA without the anchor sequence’s support.

As the anchor sequence will bind to methylated DNA with higher thermal stability, it is possible to selectively PCR-amplify it by using an annealing temperature higher than that of anchor sequence bound to unmethylated DNA.

Compared with traditional bisulfite-based methods, the approach comes with several advantages, Christensen said.

For one, the method offers a direct readout of the DNA methylation signature, whereas methods that rely on bisulfite treatment risk over- or under-conversion of DNA molecules.

In addition, EpiDirect requires significantly less sample input compared to bisulfite-based approaches, which can degrade the sample given the harsh chemical treatment. "This makes a lot of difference," Christensen said. "If you take a sample from a brain tumor, you want to make the biopsy as small as possible."

Currently, PentaBase offers the EpiDirect technology as a clinical qPCR kit for 5mC analysis of the MGMT promoter region. Christensen said the company chose this assay for its clinical relevance, especially for treatment selection and prognosis of glioblastoma patients.

When tested on formalin-fixed paraffin-embedded (FFPE) tumor samples from glioblastoma patients, the limit of detection for the EpiDirect MGMT assay was 3 percent methylation, according to the firm, and the DNA input ranged from 0.5 ng to 50 ng.

Christensen said the list price for the EpiDirect MGMT kit is around €55 ($58) per sample, and each kit contains 48 reactions. The kit was CE-IVD marked under Europe’s old In Vitro Diagnostic Medical Device Directive (IVDD), and the company is working to obtain clinical approval for the product under the new In Vitro Diagnostic Medical Device Regulation (IVDR).

In addition to the EpiDirect MGMT kit, PentaBase has developed a research-use-only EpiDirect test for MLH1 and has tests for additional targets in development, in collaboration with other firms.

A number of other companies, such as Boulder, Colorado-based Watchmaker Genomics and Cambridge, UK-based Biomodal, have been developing next-generation sequencing-based assays to more broadly profile epigenetic signatures.

Compared to these NGS-based technologies, Christensen said, PentaBase's approach is more sensitive toward a specific target, as well as less expensive and faster. The EpiDirect assay only requires a real-time PCR instrument to run as opposed to an NGS sequencer, he noted, and the turnaround time for the EpiDirect test is only about two hours, versus up to several days for a sequencing run. Additionally, the EpiDirect assay does not require bioinformatics expertise to analyze the data, he said.

"We believe NGS is a very good tool for making the first analysis of a tumor," Christensen said. "But then you do monitoring using real-time PCR assays like ours."

Even so, the company is not discounting sequencing and is exploring ways it may incorporate the EpiDirect technology into NGS assays as the field continues to evolve.

"We focus on the technologies that we think can bring patients the best outcome here and now, but it can change over time," he said. "As the NGS technologies continue to evolve and the speed goes up, prices go down, and bioinformatics problems are solved, then we will also move into that space."

Moving forward, Christensen said PentaBase will continue to develop EpiDirect to accommodate more targets while "pushing the boundaries" for the method’s multiplexing capability. As the newest platform technology for the company, EpiDirect is expected to be the "main growth driver" for the company from 2025, he noted.

Besides EpiDirect, PentaBase also offers several other products based on the INA technology that focus on detecting somatic mutations, especially in cancer and mastocytosis patient samples. For example, it has developed clinical assays to detect single copies of mutated circulating tumor DNA (ctDNA), Christensen said.

Another key product is MicroSight MSI, a CE-IVD kit designed to detect microsatellite instability from solid biopsies using real-time PCR. During COVID, the company also developed a qPCR test for detecting SARS-CoV-2 variants.

Beyond the IVD products, the company also produces INA-modified oligonucleotides for researchers, Christensen added.

A privately funded company, PentaBase has been cash flow positive since 2017, he said. However, he noted that some of the cash is being reinvested in growth of the company, and management is expecting a deficit in the next two to three years.

PentaBase, which has around 40 employees, also plans to expand its commercial presence and is interested in teaming up with other firms that have a larger commercial footprint in order to supply them with attractive assays, he said.

"We have some fantastic technologies," he said. "We now want to focus on actually getting them out into the market."