Researchers at Oregon Health & Science University have developed a mouse model that can be serially repopulated with human hepatocytes in order to produce a steady source of human liver cells for drug testing and toxicity screening.
The work, led by Markus Grompe, a professor of molecular and medical genetics and pediatrics at OHSU School of Medicine in Portland, was published in the August issue of Nature Biotechnology.
Anticipating high demand for the technology from drugmakers, and pegging the global market for human hepatocyte-based drug testing at around $2 billion a year, Grompe and OHSU have spun out a company, called Yecuris, that hopes to have the mouse models on the market next year.
The mice are Fah-/-/Rag2-/-/Il2-/- (FRG) triple mutants in which healthy hepatocytes have a growth advantage. “We can routinely make mice that have a high percentage of actual human hepatocytes in them, functioning as hepatocytes should, to support the metabolism of the animal,” Grompe told CBA News this week.
Currently, researchers studying human hepatocytes must get their cells from leftover human liver specimens such as cadavers and surgical specimens, but these cells are often of poor quality and their availability is unpredictable.
While there have been prior efforts to produce primary human hepatocytes in rodents, the resulting models have had a number of disadvantages including poor breeding efficiency, a narrow window for transplantation, and a tendency to develop renal disease.
Grompe said that the OHSU mice offer a number of benefits over previous attepmts, and can supply fresh cells upon request.
In addition, a mouse with human hepatocytes enables in vivo toxicology testing, said Grompe. Drug metabolites in the blood, urine, and bile can be evaluated over time, and researchers can determine if a drug candidate is toxic only to the mice that contain human hepatocytes compared to controls with mouse hepatocytes. Such a finding would indicate human-specific toxicity.
Furthermore, because the mice are Fah mutants, they develop liver disease when the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, or NTBC, is withdrawn. This means that researchers can essentially dial-in the degree of liver disease in the hepatocytes by increasing or decreasing the amount of NTBC.
Yecuris, the spin-out based on the technology, is currently seeking investors, Grompe said.
OHSU holds the rights to the intellectual property and has granted an option agreement to the company, Arundeep Pradhan, OHSU’s director of technology and research collaborations, recently told CBA News sister publication Biotech Transfer Week.
Yecuris was created through OHSU’s Springboard program, an entrepreneurial mentorship program for OHSU faculty interested in creating a spin-off company, Pradhan said. A portion of Springboard pays for the legal expertise that a company will require, such as articles of incorporation, bylaws, operation agreements, employment agreements, and appropriate corporate structure.
In addition, an intern in OHSU’s technology and research collaborations office, through a grant from a National Science Foundation program called Lab-to-Market, helped to develop Yecuris’ business plan.
While the company was incorporated in April, Pradhan said it has actually been in development “for the better part of nine months.” OHSU has been “putting together some of the issues related to the business plan: identifying all of the necessary components it will take to make the company a success; identifying the target markets; identifying the areas of opportunity; and identifying the different product mix that it will take to make this successful,” Pradhan said.
Grompe said that the financing of the company is in the early stages, but declined to elaborate further.
Yecuris expects to make the hepatocytes available for commercial purposes next year, Grompe said. The company will produce a limited supply of them at first, because the whole operation needs to be scaled up and expanded. However, it expects to be able to produce a steady supply of these “humanized” mice and hepatocytes for potential users sometime during the second half of 2008.
Although the company does not have any employees on a regular payroll at this point, it is planning to hire a business development manager and two scientists to actually produce the mice, said Grompe.
Grompe is the scientific founder of Yecuris, but the company will have its own management and expertise, said Pradhan. He added, “A lot of individuals are available to provide that expertise, and I think for something like this, personalities become very important in terms of finding a good fit. Also, a number of individuals are very good at taking the company to a certain stage, versus coming in at a later stage. It’s a question of defining what the company really wants and needs, and going out and actively looking for that.”
Healthy Cells, Healthy Market
Grompe said that the potential US market for human hepatocytes will be $5 million to $10 million annually in about five years. Grompe said this estimate is based on the current US market for human hepatocytes. He said that Yecuris plans to sell its mouse-derived hepatocytes for the same price as companies that procure hepatocytes from cadavers and surgical specimens.
The OHSU office of technology and research collaborations business team estimated the worldwide market for using human liver cells in drug testing assays for the pharmaceutical industry at $2 billion per year.
“We can routinely make mice that have a high percentage of actual human hepatocytes in them, functioning as hepatocytes should, to support the metabolism of the animal.”
Yecuris’ customer base would mostly comprise toxicology labs and pharmaceutical companies that would use them for drug metabolism testing and virology research, Grompe said. He pointed out that the academic market for human hepatocytes is fairly small, representing probably less than five percent of the total US human hepatocyte market.
Yecuris is currently housed in an incubator space at OHSU, Grompe said. It is not planning to move to its own facility until 2009. Pradhan pointed out that because the mice are immunodeficient, facilities have to be set up accordingly.
Pradhan said that initially, Yecuris will likely contract OHSU to breed the FRG mice, because the university already has the facilities and the capacity to do that. Over time, that commercial aspect of the operation will be transitioned completely back to the company, he said.
Grompe and his team generated a Fah-/- strain of mice that completely lacked T cells, B cells, and natural killer cells. The resulting mice are immunodeficient, so they cannot reject human cells, said Grompe.
In a pilot experiment, the researchers observed primary human hepatocyte engraftment only in FRG mice that had received a urokinase-expressing adenovirus prior to transplantation. They continued the uPA pre-treatment regimen in subsequent experiments.
The transplantation protocol began with adult FRG mice receiving an injection of the uPA virus when the liver cells arrived. One million human hepatocytes were injected intrasplenically 24 to 48 hours after the uPA injection, and NTBC was gradually withdrawn over the next five days.
Two weeks after NTBC was discontinued, the mice were put back on the drug for five days and then taken off of it permanently.
The researchers also found that serial transplantation of the FRG mice with human hepatocytes is feasible – a “major advantage” of the system, according to the paper, because it allows human cells of the same genotype to be expanded through several generations of mice. “It also means that a high-quality source of human hepatocytes for further transplantation is always at hand,” the authors wrote.
In the paper, the OHSU team demonstrated that four rounds of transplantation were feasible with a 150-fold in vivo expansion of hepatocytes in each round, meaning that the total expansion was at least 500 million-fold.