February 15, 2007 (Vol. 27, No. 4)

Sue Pearson Ph.D. Freelance Writer GEN

New Applications Promise to Boost Drug Development and Manufacturing

Until recently, almost all the talk about stem cells revolved around the use of human embryonic stem (ES) cells as a therapeutic modality. Now, noted Majid Mehtali, Ph.D., CSO of Vivalis (www.vivalis.com), there is a growing realization that other mammalian and avian ES cells offer good utility in areas such as drug screening and manufacturing.

Dr. Mehtali was one of a number of speakers at “EuroBio 2006”, which was held in Paris late last year. A key topic of discussion centered upon applications of ES cells, which cut across both drug discovery and development.

Marc Peschanski, Ph.D., director of research at Inserm (www.inserm.fr), presented some interesting work on the use of human ES cells to study myotonic dystrophy (Steinert’s disease). “The human ES cell-line we have developed carries the DM1 mutation, which causes Steinert’s disease and is derived from IVF-generated embryos that are rejected following preimplantation genetic diagnosis,” Dr. Peschanski explained.

According to Dr. Peschanski, this cell line is being mass produced using a batch culture bioreactor from Synthecon (www.synthecon.com), and the ES cells are being kept in long-term culture and differentiated into specific cell types in 96-well plates, a process that Dr. Peschanski’s group hopes to automate using a BioCel system from Velocity 11 (www.velocity11.com).

The DNA from these cell lines is being compared using DNA arrays from normal cell lines to identify mutation-linked biomarkers. “We have screened 25,000 genes and found less than 40 abnormally expressed,” Dr. Peschanski commented. “Four of these genes are common to the 11 known to be associated with Steinert’s disease, and we hope to study these to identify disease mechanisms and also possible therapies.”

To do this, the group is developing functional genomics tools, such as a lentiviral vector with a luciferase marker, which it will use to determine if there are any compounds that cause up or down regulation of these genes. “We are hopeful that a handful of the compounds we are working on could eventually be assessed in Phase I trials as new treatments for Steinert’s disease,” Dr. Peschanski added.

Drug Screening

Another use for ES cells was discussed by Elena Cattaneo, Ph.D., founder of Dialectica (www.dialectica.com). According to Dr. Cattaneo, it is difficult to culture 100% neural stem cells capable of producing neurons, which is why Dialectica is using mouse ES cells to determine if these have better utility in this application.

“Our mouse ES-derived, neural cell line has been through 170 passages, yet it is still neurogenic and contains approximately 80 percent neurons,” Dr. Cattaneo stated. “Action potential is present in these cells that also express sodium channels and the major inhibitory neurotransmitter, GABA, which means they could be a useful model for identifying compounds to treat conditions including Huntington’s disease.”

Like Dialectica, Axiogenesis (www.axiogenesis.com) is using mouse ES cells but for toxicity testing rather than compound screening. Heribert Bohlen, Ph.D., founder and CEO of Axiogenesis, explained the rationale for developing in vitro testing. “Animal-based toxicity testing is mandatory prior to the registration of a new compound. Current EU regulations require testing on 100 animals at several doses, a process that can take up to six months to complete and be as much as 30 percent of the registration cost.

“Our in vitro method, RETOX, uses mouse ES cells transfected with a vector containing a green fluorescent protein (GFP) marker and an actin promoter. During the in vitro cell-differentiation process into cardiac cells, GFP expression begins, and the cells fluoresce,” Dr. Bohlen continued.

“Adding toxic or teratogenic compounds at this point alters the amount of fluorescent cardiac cells produced, which can be measured by fluorometry, microscopy, or cytofluorometry.”

Dr. Bohlen presented data to show that RETOX was able to correctly identify the category of 93% of 70 known toxic compounds tested, and even detected the teratogenic effect of thalidomide. He also discussed a case study where Grunenthal (www.gruenthal.com) used RETOX to screen 30,000 compounds for E15,000 and saved E55, 000 on the cost of animal testing.

“If we can get RETOX accepted in a regulatory setting, it could save the lives of millions of lab animals every year and reduce testing costs by billions of euros,” Dr. Bohlen concluded.

An Alternative to Eggs

In contrast, Dr. Mehtali discussed the rationale for using ES cells in drug manufacturing. “In the past 15 years, we have seen the number of biologicals on the market increase from five to over 40, and this rise will continue as there is a new upward trend for pharmas to produce more vaccines, as well as protein- and antibody-based drugs.

“Many vaccines, such as flu and recombinant pox viruses, are still grown on eggs or primary cells derived from eggs. Although such cells are genetically stable and nontumorigenic, they are not immortal, so vaccine manufacturing has to be carefully planned or a shortage of vaccine can occur due to an egg shortage or egg contamination,” Dr. Mehtali explained. “Some biological drugs can be made using cell lines, such as CHO or PerC6, but these also have the drawback of being genetically unstable and tumorigenic. ES cells are the only genetically stable, nontumorigenic, and immortal cells available, which makes them a sensible choice for manufacturing biologicals.”

According to Dr. Mehtali, to fill this niche, Vivalis has developed a nontumorigenic, chicken ES-based cell line known as EBx. EBx has been adapted for large-scale manufacture by maintaining the cells at around 10–20 x106/mL in serum-free media co-developed by Vivalis and SAFC Biosciences (sigmaaldrich.com) in 20-L, stirred-tank bioreactors.

“EBx has been tested with 17 different flu vaccines and routinely yields 20 to 50 micrograms per milliliter,” stated Dr. Mehtali.

“So in a 100-liter bioreactor, we can, in principle, rapidly produce a thousand 15-microgram doses of vaccine, which means that for a situation like an avian flu pandemic where there may be a need to manufacture many doses at short notice, the EBx cell line is ready to be utilized. Additionally, there are no ethical issues using EBx for production in Europe or the U.S. as this is a chicken ES-derived line.”

The EBx cells could be used for manufacturing antibody drugs too, and Dr. Mehtali presented mass spectrometry evidence to show that human IgG transfected into EBx is much less fucosylated and postulated; this could offer a method of making more active antibody drugs. “We have issued 16 commercial and research licenses for producing protein and vaccine products in EBx cells. We have also generated an EBx cell line from ducks and are planning to proceed similarly with primates, so EBx offers a viable alternative to using transgenic animals or plant bioreactors and could solve short-term bottlenecks in vaccine manufacture, as well as improve potency in antibody production,” stated Dr. Mehtali.

The Future for ES Cell Research

Many of the applications discussed at “EuroBio” using ES cells were based on research with mammalian rather than human ES cells. “Until two years ago in France, we could not use any human embryo to produce human ES cells. Now we can use those derived from IVF cell lines that have been given a preimplantation diagnosis of a serious disease,” Dr. Mehtali commented.

“In Italy, human ES cells can be imported rather than derived, and in Germany, the situation is even more difficult because researchers cannot derive human ES cells nor import any that were generated after 2001,” commented Dr. Mehtali. “This is why in many European countries, biotechs are developing applications for use now in drug discovery with mammalian ES cells only. For example, we have not developed our EBx line in humans, even though it is technically possible, because of the regulations in France.”

Many speakers at “EuroBio” were hopeful that by 2009 a common EU law on human ES cell research could be in place to even out the disparity of regulations and make human ES cell research more accessible across Europe. “Who knows, when we have one rule for all in Europe, scientific progress in human ES cell research may accelerate so much that we can find ways to not just treat diseases but actually cure them,” concluded Dr. Mehtali.

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