A technology developed by researchers at Iowa State University that marries DNA hybridization and fluorescence-based spectroscopy can detect single molecules of DNA from human papillomavirus, the virus associated with cervical cancer, according to a recently published research paper.
The technology could be developed into a test that has 50-fold greater sensitivity than current PCR-based HPV tests, and may have a built-in market as a complement to Pap tests, according to the scientist primarily responsible for developing the technology.
However, commercialization of the test also faces a huge roadblock in that the basic invention surrounding the technology is not patentable in its current form due to the existence of prior patents.
Edward Yeung, professor and chair of the chemistry department at Iowa State, led the research team that developed the technique, in which fluorescently labeled DNA probes are hybridized to specific HPV DNA sequences and then detected using single-molecule spectroscopy techniques. The method can work in single-probe mode as described above, or in dual-probe mode in which a second probe is introduced that recognizes and binds the first DNA probe.
In a paper published in the Nov. 1 issue of Analytical Chemistry, Yeung and doctoral students Jiangwie Li and Ji-Young Lee wrote that they were able to use the method to detect specific HPV DNA sequences at a level of about one copy per cell.
The current most widely used test for detecting and quantifying viral nucleic acids is based on PCR, which requires somewhere in the neighborhood of 10 to 50 virus molecules, according to the researchers. Furthermore, they said that PCR can lead to false positives due to the amplification of contaminants, takes more time to perform, and can in some cases make quantification of the viral DNA difficult.
Single-molecule spectroscopy and DNA hybridization are both proven laboratory techniques and are described in numerous scientific publications. However, Yeung believes that his lab is one of the first to show how the techniques can be made specific and sensitive enough to detect a given nucleic acid target in a way that would be clinically useful.
“Several things must be involved,” Yeung said. “One is that the instrumentation needs to be in a form that can detect the small amount of light coming from these molecules, yet with confidence that you are seeing something well above the noise level of the detector and the chemical system.
“The other part of the technology is DNA hybridization – how to introduce a probe to target these specific DNA sequences in the HPV so you know that you’re detecting these and not anything else,” he added. “The paper that we published basically deals with making sure you can do this sensitively enough that you can count molecules anywhere from one copy per cell up to several million molecules, and to know that you are detecting the specific genetic area of HPV.”
Such an advance, in Yeung’s opinion, should be patentable and could form the basis of a diagnostic test that would complement Pap smears.
Yeung said that he has not done detailed market research, but that “it is clearly something that can be combined with all of the Pap smear tests that are being done today. Right now all the Pap smears are histological – they look at the shape of the cells, and so forth. But you might submit a sample to this kind of single-molecule test, which would allow you to have a much earlier diagnosis of the potential of cervical cancer.”
In addition, he said the test should be applicable to other viruses, including HIV, avian flu, and common influenza, although HPV testing is the most achievable and potentially lucrative application.
According to an April 2007 brochure issued by the US Centers for Disease Control entitled “Human Papillomavirus: HPV Information for Clinicians,” The combination of molecular testing for high-risk types of HPV together with a Pap test is considered by the [American Cancer Society] and the [American College of Obstetricians and Gynecologists] to be an acceptable approach to cervical cancer screening of women age 30 and older,” but the US Preventive Services Task Force found insufficient evidence to recommend for or against the routine use of HPV DNA testing as a primary screening test for cervical cancer.
In addition, the brochure states that the American Society for Colposcopy and Cervical Pathology also recommends use of the HPV DNA test for follow up of cervical abnormalities, but that there is no FDA-approved indication for this use.
Digene currently sells the only molecular diagnostic test for HPV cleared for marketing in the US and Europe. Other notable companies looking to enter the HPV testing market include Qiagen, Third Wave, and Cepheid. According to a recent article in BTW sister publication BioCommerce Week, Cepheid has estimated the market for HPV diagnostic preventative testing to be between $600 million and $700 million, while some competitors have put it as high as $1 billion.
Yeung’s opinion regarding the commercial potential of his HPV test appears to differ from that of his school’s tech-transfer office and, more importantly, from that of the US Patent and Trademark Office.
Yeung, through the Office of Intellectual Property and Technology Transfer at the Iowa State University Research Foundation, applied for a patent surrounding the technology in 2002.
“If the basic invention was rejected, I don’t know how we can actually patent anything here.”
The patent application, No. 10/031,353, entitled “High-throughput methods of distinguishing at least one molecule individually in a sample comprising multiple molecules and systems for use therein,” covers both the single-molecule detection method and related instrumentation systems. However, the application was rejected by the USPTO early last year in a final office action following a series of amendments by Yeung and colleagues and subsequent USPTO rebuffs.
According to the final USPTO action document, the patent was rejected as being unpatentable over claims in US Patent Nos. 6,485,625, entitled “Apparatus and method for the generation, separation, detection, and recognition of biopolymer fragments;” and 5,188,963, entitled “Device for processing biological specimens for analysis of nucleic acids.”
The ‘625 patent was filed in June 1999 and awarded in November 2002 to Conn.-based biopharma company CuraGen. Meanwhile, the ‘963 patent was filed in November 1989 and awarded in February 1993 to North Carolina biotech Gene Tec Corporation.
“We failed to get a patent on the actual detection methodology itself … so it doesn’t look like we’ll be able to commercialize this one,” said Ken Kirkland, director of the OIP at the ISURF. “I don’t know where we are on this. If the basic invention was rejected, I don’t know how we can actually patent anything here.”
Kirkland said that other routes for commercialization might be possible, but they likely won’t provide a financial return to Yeung or Iowa State.
“We’re getting it out there,” Kirkland said. “Somebody may look at [the publication] and try and improve upon it. If they can improve upon it sufficiently, then that new invention might be separately patentable.”
It is unlikely that a company would license the technology, Kirkland added, because “if we don’t have a proprietary position on it, then they’re afraid to use it without a license” from either CuraGen or GeneTec. “We have nothing to license if we don’t have a proprietary position on it,” Kirkland said.
Despite this, Yeung said that a few undisclosed small biotech companies that play in the protein- and DNA-analysis arenas have expressed interest in the technology. And although the initial patent application was rejected, Yeung remains optimistic that aspects of the technology remain patentable.
“There are other [patents] that we are thinking of filing for,” he said. “There are many different angles to this technology, and of course we must find an angle that is broad enough to be useful, but does not intrude on any other existing technologies.
“The rejected patent application covers one aspect of the technology,” he added. “There are other inventive components and trade secrets that can form the basis of future patent applications.”
Yeung has a commercialization track record: In 1999 he co-founded life science tool vendor CombiSep, which merged in December with another biotech called Advanced Analytical. CombiSep’s core technology enables researchers to analyze drug compounds, oligonucleotides, and proteins in parallel by combining capillary electrophoresis with UV absorbance detection.
However, he told BTW that CombiSep is not a likely partner for the HPV-detection technology because of differing market strategies. In addition, he said that he would prefer to find an existing partner to commercialize the current technology as opposed to starting another spinout company himself – at least for the time being.
“I would be interested as long as someone is going to do the administrative work, and raise money and so forth,” he said. “As a scientist I would [consult], but I just don’t have the energy right now to get personally involved in founding a new company.”