2010

Breakthroughs of the Year

As it does every year, Science published its “Breakthrough of the Year” for 2010 in the 17 December issue of the journal.

For its “Breakthrough of the Year”, Science chose a non-life science innovation, the first quantum machine. Interestingly, the same issue of Science included a Perspective on biophysicist Britton Chance, who died last November at the age of 97. Among his many accomplishments, Dr. Chance discovered that biological electron transfer operates via quantum tunneling, a mechanism central to photosynthesis, respiration, and many oxidoreductase enzymes. Mitochondria, chloroplasts, and oxidoreductase enzymes thus constitute biological quantum machines of a sort.

Interestingly, Dr. Chance continued to work on metabolism all of his long working life, including in the era of molecular biology when interest in that field waned. By doing so, he made many important contributions, including the mechanism for the generation of the reactive oxygen species (ROS) superoxide and peroxide during normal mitochondrial respiration, and the  use of near-infrared (NIR) light for noninvasive diagnostics.

Although Science chose a non-life science advancement as its “Breakthrough of the Year”, the journal’s runners-up for “Breakthrough of the Year” were replete with life science items. The first runner-up was the synthetic Mycoplasma mycoides genome constructed by the J. Craig Venter Institute, which they used to create “the first synthetic cell”. As we discussed in a series of two articles on this blog (see here and here) although the creation of the synthetic genome and the “synthetic cell” represented a technical tour de force, it did not represent a true breakthrough. Many leading scientists, including leaders in the field of synthetic biology, agreed with us. However, at least several bioethicists and philosophers hailed this work as a milestone, calling it “the end of vitalism”. (As we noted in another blog post, however, not all bioethicists agree.)

Moreover, policy-makers were sufficiently alarmed by the “synthetic cell” that (as noted in the Science “Breakthrough of the Year” runners-up article) the Presidential Commission for the Study of Bioethical Issues held hearings on policy implications of this research. Nevertheless, the report of this commission (issued in December 2010) concluded that the Venter research “does not amount to creating life as either a scientific or a moral matter” and that synthetic biology remains “in the early stages,” with any dangers well into the future. The commission recommended continuing White House oversight, but a relatively mild set of regulatory measures.

As we said in our second article on the “synthetic cell”, we are much more impressed by the metabolic engineering studies of Jay Keasling, and by George Church’s automated method for optimizing metabolic engineering pathways, which we had discussed in an earlier blog post. The Science “Breakthrough of the Year” runners-up article mentioned Dr. Church’s automated system, among other synthetic biology advances made in 2009 and 2010.

Meanwhile, in a review of metabolic engineering published in the 3 December 2010 issue of Science, Dr. Keasling says that although minimal bacterial hosts such as Dr. Venter’s “synthetic” mycoplasma may be of scientific interest, they are not suitable to use in metabolic engineering studies whose goal is scale-up for industrial production of medicines, chemicals, or biofuels. This agrees with our statement that such applications require  “workhorse” organisms that can take the extensive genetic manipulation needed to engineer new metabolic pathways, and which are capable of scale-up.

We therefore believe that the “synthetic cell” is not the life science breakthrough of the year, despite its placement at the top of Science‘s “Breakthrough of the Year” runners-up article.

Our nominee for the life science breakthrough of the year is listed right under the “synthetic cell” in the Science “Breakthrough of the Year” runners-up article. It is the determination of the sequence of approximately two-thirds of the Neandertal genome by Svante Pääbo (Max-Planck Institute for Evolutionary Anthropology, Leipzig, Germany.) and his colleagues. This achievement is something that only a few years ago seemed completely impossible. Moreover, this work is of great cultural significance, since it indicates that Neandertals contributed some 1-4 percent of the genome sequences of non-African present-day humans. More recently, Dr. Pääbo and his colleagues followed up the Neandertal studies by using their DNA synthesis methods to identify a third species of humans, known as Denisovans. Denisovans, who were more closely related to Neandertals than to modern humans, were alive at the same time as modern humans emerged from Africa and also encountered the Neandertals. Dr. Pääbo’s new studies indicate that the Denisovans contributed some 4–6% of the genome sequences of present-day Melanesians.

Despite the importance of the Pääbo Neandertal studies, we have not blogged on this work simply because it has nothing to do with drug discovery and development. However, perhaps someday, for example, some of the products of genes that are found in present-day humans but not in Neandertals could emerge as potential drug targets. As discussed in the Science “Breakthrough of the Year” runners-up article, researchers have begun studying some of these gene products in cell culture systems.

Moreover, the types of advanced, next-generation DNA sequencing methods used by Dr. Pääbo and his colleagues are being applied to studies that are relevant to drug discovery. These include the 1000 Genomes Project, which seeks to find all single-nucleotide polymorphisms (SNPs) present in at least 1% of humans. This and other next-generation genomics projects were listed in the Science “Breakthrough of the Year” runners-up article, as the third runner-up. The 1000 Genomes Project, as well as genome-wide association studies (GWAS) that use high-throughput DNA sequencing methods, may enable researchers to identify rare mutations that are involved in complex human diseases. This might in turn lead to the discovery of novel drugs and diagnostics.

Among other life science items in the Science “Breakthrough of the Year” runners-up article was the production of knockout rats. We discussed knockout rats in an October 1, 2010 blog post.

Newsmaker of the Year

Nature also had an end-of-2010 special article, “The Newsmaker of the Year”, in its 23/30 December issue. Unfortunately, Nature chose a U.S. government official as its Newsmaker of the Year.

We would prefer that Nature stick to what it does so very well, and stay out of U.S. politics, whether in its “opinionated editorials” [sic] or elsewhere. Perhaps the low point in Nature‘s political forays was its November 2010 editorial calling for what amounts to a new version of Prohibition. This is despite the ample evidence that moderate consumption of red wine (for example) is healthy for most adults. Readers would be well advised not to believe everything they read in Nature editorials.

Our nominee for Newsmaker of the Year in the life sciences is Dr. Svante Pääbo, for the reasons we discussed earlier.

Deals of the Year

Also as an end-of-year feature, the IN VIVO blog has been running a Deal of the Year competition. The nominees are grouped in three categories: M&A Deal of the Year, Alliance Deal of the Year, and Exit/Financing Deal of the Year.

Only one of the nominees had been featured in an article on our blog: the Celgene/Agios alliance (April 23, 2010).

The IN VIVO Blog invited readers to vote on the Deal of the Year in each of the three categories, by going to their website. The voting closed at 12:00pm on 6 January 2011 (Eastern Standard Time).

The winners of the vote were:

• M&A Deal of the Year: Celgene/Abraxis (50.31% of 1,799 votes)
• Alliance Deal of the Year: Celgene/Agios (55.32% of 3,176 votes)
• Exit/Financing Deal of the Year: Ablexis (46.54% of 1,631 votes)

Congratulations to all the winners, especially Agios and Celgene, which were featured in our blog post.

Happy New Year!

This is our own version of an end-of-year special article, and will be our last blog post of 2010. Best wishes to all of you for a happy, productive, and innovative New Year in 2011.

As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

In Chapter 7 of our March 2010 book-length report, Animal Models for Therapeutic Strategies (published by Cambridge Healthtech Institute), we discussed recently-developed methods for producing knockout rats. These methods included zinc-finger nuclease (ZFN) genome editing and transposon mutagenesis in cultured spermatogonial stem cells. Our most extensive discussion was of the ZFN editing technology, which was developed by Sangamo BioSciences (Richmond, CA), and is the basis of the knockout rat models marketed by Sigma-Aldrich Advanced Genetic Engineering (SAGE). We also mentioned the SAGE knockout rat platform in an earlier blog post.

In Chapter 7 of our report, we also mentioned that it would now also be possible to construct knockout rats “the good old way”–using the same homologous recombination technology that researchers use to create knockout mice. Drs. Mario R. Capecchi, Martin J. Evans and Oliver Smithies were awarded the Nobel Prize in Physiology or Medicine for 2007 for having developed this technology in the late 1980s. To construct knockout mice, researchers isolate and culture mouse embryonic stem (ES) cells. These are derived from the inner cell masses of preimplantation mouse blastocyst embryos, and grown under particular culture conditions. These cells are subjected to homologous recombination with a vector containing a truncated version of the gene to be targeted, to eventually yield knockout mouse strains.

It has not been possible to develop knockout rats because the conditions for culturing ES cells worked only for a few inbred mouse strains, and not at all for either most mouse strains or for the rat. Conditions for culturing mouse ES cells are complex. They involve the use of feeder fibroblasts and/or the cytokine leukemia inhibitory factor (LIF), together with selected batches of fetal calf serum or bone morphogenetic protein (BMP). These culture conditions had been determined empirically.

In 2008, Dr. Austin Smith (Director of the Wellcome Trust Centre for Stem Cell Research, University of Cambridge [Cambridge, UK]) and his colleagues developed culture conditions that allowed them to culture rat ES cells that were capable of transmitting their genomes to offspring. These ES cells could also be used to produce knockout rats.

Dr. Smith and his colleagues realized that the standard conditions for culturing mouse ES cells expose the cells to inductive stimuli (e.g., fibroblast growth factor 4 [FGF4]), which can activate ES cell commitment and differentiation. The aim of ES cell culture is to expand the cell population while maintaining pluripotency.  The researchers therefore cultured rat ES cells with leukemia inhibitory factor (LIF)-expressing mouse fibroblast feeder cells, in a medium containing two or three small-molecule inhibitors of pathways involved in ES cell commitment and differentiation, plus human LIF. (LIF supports proliferation of ES cells in an undifferentiated state.) This medium is known as 2i (for 2-inhibitors) or 3i medium.

Rat ES cells cultured in this manner expressed key molecular markers found in mouse ES cells. They also, when injected into blastocysts, can give rise to chimeric rats; i.e., they transmute their genomes into offspring. Such cultured rat ES cells thus are capable of being used to construct knockout rats.

In the 9 September 2010 issue of Nature, Dr. Qi-Long Ying (University of Southern California, Los Angeles CA) and his colleagues published the first study describing construction of a knockout rat strain via homologous recombination. (Dr. Ying, then at the University of Edinburgh, had been on the team led by Austin Smith that developed culture methods for rat ES cells.) This rat strain is a p53 gene knockout. The researchers designed a targeting vector to disrupt the p53 tumor suppressor gene via homologous recombination; the vector allowed for antibiotic selection for cells that had been successfully targeted. They transfected this vector into rat ES cells cultured in 2i medium, performed the antibiotic selection, and cultured the resistant cells. These cells were shown to have one of their two (since they were diploid) p53 genes disrupted. The researchers were able to routinely generate p53-targeted rat ES cells by this method. They also injected p53-targeted rat ES cells into rat blastocysts, transferred the blastocysts into pseudo-pregnant female rats, and obtained chimeric offspring. However, in the first studies, the p53-targeted rat ES cells exhibited low germline transmission efficiency.

In the mouse system, the failure of cultured ES cells to contribute to the germline is often caused by chromosomal abnormalities in the ES cells. This was also the case with the rat ES cells. In the case of mouse ES cell culture, cells with chromosomal abnormalities have a selective growth advantage over those with normal karyotypes. The smaller, slower-growing mouse ES cell clones tend to have normal karyotypes, and to give improved germline transmission. The researchers therefore subcloned their p53 gene-targeted rat ES cells, and selected for small, slower-growing subclones. These rat ES cell subclones were euploid. When injected into blastocysts, these rat ES cell clones gave rise to chimeric rats that the researchers further bred to generate homozygous p53 gene-targeted (i.e., p53 knockout, or p53 homozygous null) rats.

Using these methods, it should be possible to generate knockout rats for other genes routinely, including sophisticated knockouts such as tissue-specific gene knockouts.

Meanwhile, SAGE has generated p53 knockout rats, using its ZFN technology. As with the original p53 knockout mice, these rats develop normally, but are prone to development of spontaneous tumors. p53 knockout rats generated via homologous recombination should also be susceptible to spontaneous generation of tumors. However, as yet no data has been published. It remains to be seen which of these systems–p53 knockout mice or p53-knockout rats generated via either homologous recombination or ZFN editing, will be most useful in basic cancer research, or in such applications as carcinogenicity screening of compounds.

Why is the ability of researchers to generate knockout rats, as opposed to knockout mice, so important? The anatomy and physiology of the rat is closer to humans than is the mouse. There are also many rat models of complex human diseases (especially cardiovascular and metabolic diseases) that are better disease models than those based on inbred mouse strains. In addition, the larger size of the rat facilitates experimental procedures that involve surgery, getting blood samples for analysis, or isolation of specific cell populations. Researchers usually prefer rats over mice for physiological and nutritional studies, studies of psychiatric diseases, and in cases when a particular rat disease model is more applicable to a project than mouse strains. The rat is also widely used in preclinical efficacy and safety studies.

With respect to models for central nervous system (CNS) diseases, gene-targeted and transgenic rat models may be expected to be better than mouse models. The rat is more intelligent than the mouse, and has a bigger brain. Unlike mice, rats are sociable and easily trained. Moreover, there are some new rat models of cognition, which enable researchers to perform studies that they previously thought could only be done in nonhuman primates. And optogenetics technology, which allows researchers to engineer specific neurons so that their activity can be switched on or off with laser light, in order to dissect the role of these neurons in behavior, is being implemented in rats. These new developments, together with knockout and transgenic technologies, should allow researchers to develop new rat models of psychiatric diseases, as well as of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. The lack of good animal models is a major factor in the high clinical attrition rate of CNS drugs, so new models are needed. There are of course no guarantees that novel rat models will help lower CNS drug attrition rates, but it is well worth trying these new approaches.

As we also discussed in Chapter 7 of Animal Models for Therapeutic Strategies, researchers are also interested in developing animal models based on mammalian species other than the mouse and the rat. We discussed methods for gene targeting by recombinant adeno-associated virus (rAAV) in pigs and ferrets in that chapter. In principle, ZFN editing technology could be also used to generate gene knockouts in mammalian species other than rodents. Moreover, the type of research done in the rat by Austin Smith, Qi-Long Ying, and their colleagues might be applied to developing culture conditions for ES cells of other mammalian species, which could set the stage for developing gene knockouts in these species via homologous recombination.

What causes obesity? To many people, the answer is obvious. Obesity is caused by eating too much food and/or not getting enough exercise. Obese and overweight people lack “personal responsibility” or have become addicted to food the same way that one becomes addicted to smoking. The alarming worldwide rise in obesity over the past several decades is due to an increasing lack of personal responsibility, perhaps as the result of the lure of bad eating habits and lack of exercise caused by increasing affluence.

This “common-sense view” of obesity is gaining increased currency, as the result of rising health care costs and health insurance premiums, and the drive to rein in these costs. Even some leaders of health insurance and health care providers blame the lack of personal responsibility of the obese for rising health care costs, and advocate using education, exhortation, and “economic incentives” (i.e., penalizing the obese, perhaps by raising their insurance rates) to combat obesity.

However, genetic and physiological research shows that obesity is a disease, not just the result of bad habits. This research has shown that weight is as heritable as height, and has uncovered a set of complex pathways that control energy balance. According to this “enlightened, science-based view”, the worldwide epidemic of obesity is mainly the result of the interaction between a set of social and economic factors (e.g., increased consumption of meals away from home, decreased prices for unhealthy versus healthy food, and decreased requirements for physical activity at work and for transportation) and genetic factors that make some people more susceptible to obesity than others. In the industrialized world, between 60%–70% of the variation in obesity-related phenotypes such as body mass index (BMI) and hip circumference appears to be heritable. People who undertake even the best systematic weight-loss programs are fighting a set of complex physiological pathways that have evolved to combat starvation. These pathways are only partially understood. Most people who manage to lose a significant amount of weight usually regain it over the medium to long term.

The “enlightened, science-based view” is discussed, for example, in our 2008 book-length report on obesity, and in our October 25, 2009 article on this blog. It is also the view of pharmaceutical and biotechnology companies that are developing antiobesity drugs, and the view of basic researchers who are endeavoring to understand pathways that control energy balance and may render individuals subject to obesity and its comorbidities.

Recent genetic studies provide an increasing amount of evidence that favors the  “enlightened, science-based view”. For example, researchers have recently identified associations between common variants in the fat mass and obesity-associated (FTO) geneand increases in BMI and waist circumference in several human populations. The FTO gene codes for a 2-oxoglutarate-dependent nucleic acid demethylase. It is expressed in the hypothalamus, a region of the brain that is involved in regulation of feeding and energy metabolism. Hypothalamic FTO appears to be involved in the regulation of energy intake, but not feeding reward. However, the mechanism of action of FTO in control of energy balance is not understood.

The findings on FTO adds to the large amount of evidence for the genetic determination of obesity, and thus for the “enlightened, science-based view” of this condition. Many academic and corporate researchers, including most of the recognized leaders in obesity research, believe that continued basic and translational research on the genetic and molecular basis of obesity will lead to new therapeutic strategies to control this disease.

Now comes a new research report that might result in a “game-changing view” of obesity, published in the 9 April issue of Science. Andrew Gewirtz (Emory University, Atlanta, GA) and his colleagues studied T5KO mice, which are genetically deficient in TLR5, a Toll-like receptor (TLR) that recognizes bacterial flagella as a ligand and is expressed on the surface of both intestinal epithelial cells and cells that mediate innate immunity. TLRs are receptors that recognize conserved molecules derived from bacteria or viruses, and activate immune responses, thus serving as a first line of defense against infection. Researchers hypothesize that TLR5 in gut mucosa may have a role in maintaining a harmonious relationship between the host and the complex population of intestinal microbes.

The researchers found that as compared to wild-type mice, T5KO mice showed increased fat mass and body weights 20% higher than wild-type mice, and features of the metabolic syndrome (insulin resistance, elevated serum cholesterol and triglycerides, and elevated blood pressure).  The adipose tissue of TK5KO mice exhibited higher production of the proinflammatory cytokines interferon-γ and interleukin-1β.  T5KO mice were also hyperphagic, eating 10% more than wild type mice. When the researchers restricted the food fed to T5KO mice to the amount eaten by wild type mice, they no longer exhibited increased fat mass or body weight, or abnormalities in blood glucose and lipids. However, they still were insulin resistant.

When both wild type and T5KO mice were fed a high-fat diet, both populations showed increases in fat mass and body weight, as well as elevated levels of blood lipids. However, unlike wild type mice, T5KO mice fed a high-fat diet has blood glucose levels of greater than 120 milligrams per deciliter, and thus were diabetic. The T5KO mice also showed inflammatory infiltrates in their pancreatic islets, and hepatic steatosis. Thus a high-fat diet exacerbated the metabolic syndrome shown by T5KO mice.

The researchers asked whether other mediators of the immune system were involved in the induction of metabolic syndrome shown by T5KO mice. Deletion of the Toll-like receptors TLR2 and/or TLR4 in T5KO mice had no effect on their metabolic syndrome.  Deletion of RAG1 (which is necessary for development of the T and B cells of the adaptive immune system) also had no effect. However, deletion of the intracellular protein MyD88 in T5KO mice resulted in normalization of the metabolic syndrome. Since MyD88 is necessary for signaling by all TLRs except for TLR3, and for signaling by receptors for interleukin-1β and interleukin-18, this suggests that another TLR and/or signaling by one or both of these two cytokines might be necessary, together with TLR5 knockout, for induction of the metabolic syndrome.

Since TLR5 is expressed in the gut mucosa and recognizes bacterial flagellin, the researchers tested the hypothesis that the metabolic syndrome seen in T5KO mice might be due to alterations in the population of gut microbes as the result of the loss of TLR5 function. When the researchers treated newly weaned T5KO mice with broad-spectrum antibiotics, the number of gut bacteria was reduced by 90%. This treatment eliminated the metabolic syndrome, hyperphagia, and obesity of the T5KO mice.  Conversely, when the researchers transplanted the gut microbiota of T5KO mice to the guts of wild type germ-free mice, the recipient mice exhibited hyperphagia, obesity, metabolic syndrome, and elevated levels of proinflammatory cytokines in their adipose tissue. Analysis of the gut microbiota of T5KO and wild type mice showed that the species composition of the gut bacteria of T5KO mice was significantly different from that found in wild type mice.

These results suggest that obesity and metabolic syndrome may be caused at least in part by genetically determined differences in interactions between the innate immune system of the gut mucosa and the intestinal flora. Obesity-prone individuals may develop a gut microbe population that interacts with the immune system in such a way as to promote obesity. Interactions between gut microbes and innate immunity that promote obesity might result in changes in proinflammatory cytokines and in adipokines in adipose tissue (and perhaps also in muscle and liver) that not only cause increased inflammation and metabolic syndrome, but also disrupt signals within the brain that promote appetite control and energy balance. They further suggest that treatments that target intestinal microbes may be effective therapies for obesity and its sequelae.

These conclusions are based on a mouse model, which may or may not have much to do with the pathogenesis of human obesity. However, there is evidence that the the composition of gut bacteria differs between obese and nonobese humans in similar ways to differences in gut flora between obese and nonobese mice. Colonization of germ-free mice with the gut microbiota of obese mice results in significantly greater increase in body fat than colonization with the gut microbiota of lean mice. Researchers obtained evidence that gut microbes from obese mice have an increased ability to harvest energy from food than do the gut bacteria of lean mice. They therefore hypothesize that this extra energy harvest may help promote obesity, in both mice and humans. But it is also possible that obesity-associated gut microbe populations might promote systemic low-grade inflammation that contributes to the pathogenesis of metabolic syndrome and obesity.

In addition to the research report itself, two commentaries on the report were published in April 2010–one by Darleen A Sandoval and Randy J Seeley (University of Cincinnati in Ohio) and the other by Ping Li and Gökhan Hotamisligil (Harvard School of Public Health, Boston MA). Drs. Sandoval and Seeley conclude that the new findings may allow researchers to develop means of preventing obesity by manipulating gut microbe-immune system interactions by such means as drugs, diet, or probiotics.  Drs.  Li and Hotamisligil take a more nuanced view. (Dr. Hotamisligil is a leader in the study of pathways involved in control of energy metabolism, and their relationship to inflammation and metabolic diseases.) They state that the results seen in T5KO mice were relatively mild, and thus probably cannot account for the full spectrum of metabolic dysfunction seen in obesity. They essentially see the gut microbiota/innate immunity interaction as one factor in the complex networks that determine obesity and metabolic syndrome. They call for more research into the gut microbe/immune system relationship, and believe that such research will lead to a better understanding of metabolic syndrome.

What if the gut microbiota/immune system interaction is a major factor in obesity, at least in a subpopulation of obese subjects? That would resemble the situation with peptic ulcers. Peptic ulcers were once considered a disease of “lifestyle”, due to the “type A personality”, “a stressful lifestyle”, and/or eating spicy foods. However, eventually Drs.  Barry J. Marshall and Robin Warren of Australia discovered that a high percentage of ulcers were caused by chronic inflammation due to infection with Helicobacter pylori. It is now accepted that this bacterium is responsible for 60% of gastric ulcers and up to 90% of duodenal ulcers. Treatment involves administration of combinations of antibiotics together with a proton pump inhibitor and sometimes a bismuth compound.

The majority of scientists and physicians resisted the idea that peptic ulcers were caused by a microbial infection for a long time. Dr. Marshall even had to do and publish a self-experiment, drinking a culture of bacteria from a patient, before the scientific community would accept his findings. Finally, In 2005, Drs. Marshall and Warren received the Nobel Prize in Physiology or Medicine for their [re]discovery of H. pylori and its role in gastritis and peptic ulcers.

The role of gut microbes in obesity and metabolic syndrome may not be simple as the role of H. pylori in gastric ulcers. Nevertheless, this hypothesis deserves intensive investigation, and it may lead to a game-changing view of metabolic disease and eventually important new treatments. In any event, it would be wise for the scientific, medical, and policy communities to take the advice of Dr. Jeffrey Friedman (Rockefeller University, New York, NY), who is arguably the founder of the “enlightened, science-based view” of obesity and metabolic syndrome, with his breakthrough discovery of the hormone leptin in 1994:  “A war on obesity, not the obese.”

The cover of the 30 April 2010 issue of Science bears a photo of a tadpole of the western clawed frog Xenopus tropicalis. In that issue is a report on the draft sequence of the genome of this organism, and a short companion news feature. The report on the genome emphasizes X. tropicalis’ role as an emerging animal model in developmental and evolutionary biology and in comparative genomics.

X. tropicalis is also an emerging animal model in biomedical research, potentially including development of disease models for drug discovery. We emphasize that potential role in Chapter 5 (“Xenopus tropicalis: an emerging model system”) of our book-length report, Animal Models for Therapeutic Strategies, published by Cambridge Healthtech Institute in March 2010.

The Nature news feature, authored by Elizabeth Pennisi, also cites the potential role of this frog in biomedical research. X. tropicalis has about 1700 genes that are related to human genes that have been linked to disease. Some of these diseases are type 2 diabetes, acute myeloid leukemia, congenital muscular dystrophy, alcoholism, and sudden infant death syndrome. In our book chapter, we discuss efforts to develop an X. tropicalis model of congenital spinal muscular atrophy (SMA). We also discuss studies aimed at using the frog as an animal model of human congenital heart disease, and for developing novel therapies for these conditions.

The related frog Xenopus laevis (known as the African clawed frog) is an old animal model that has long been used in developmental and cell biology research. However, X. laevis (pictured above) is genetically intractable, since its genome is allotetraploid, having been formed by fusion of diploid genomes from two different species. This makes genetic and genomic studies with this frog difficult. In contrast, X. tropicalis is diploid. X tropicalis also has a much shorter generation time than X. laevis, and is much smaller, thus requiring less space and making breeding and experimentation much more feasible than with X. laevis.

Some of the same researchers that have been participating in the X. tropicalis genome sequencing project have been developing genetic tools such as transgenics, genetic screening, and gene knockdown using antisense morpholinos. With the determination of the genome sequence, X. tropicalis may join the zebrafish as a lower vertebrate animal model in developing novel therapeutic strategies for human diseases.

Elsewhere on the animal model genome front, researchers recently published a draft sequence of the genome of Hydra magnipapillata. Hydra, a freshwater cnidarian or polyp, has long been a staple of high school and university biology lab courses, so is a favorite of many biologists. The University of California at Irvine, whose researchers participated in the Hydra genome project along with many others (e.g., leading genomics researcher J. Craig Venter), has long been a center of Hydra research, beginning in the late 1960s.

Hydra is used as an animal model in the study of regeneration, body patterning, and stem cell biology. The determination of the genome sequence of Hydra will facilitate these studies, as well as studies of comparative genomics and evolutionary biology.

Hydra may also be of interest for biomedical research. As discussed in the genome report, Hydra possesses four homologues of the Myc oncogene, which is involved in human cancers and also regulates pluripotency and self-renewal of mammalian stem cells. Myc is also central to the pluripotentency of Hydra stem cells. The researchers also found genes in the Hydra genome that are linked with Huntington’s disease and with the beta-amyloid pathway of Alzheimer’s disease.

The April 1, 2010 issue of The Scientist has an article, entitled “Building a better mouse”, on efforts of researchers to develop improved mouse models of cancer.

Current mouse models of cancer, mainly xenograft models in which human cancer cell lines are transplanted into immune deficient mice, are notoriously unpredictive of efficacy when oncology drug candidates are tested in them. This is a major factor in the high failure rate of oncology drugs in clinical trials. It is estimated that oncology drugs that enter human clinical trials have a 95 percent attrition rate, as compared to the 89 percent attrition rate for all clinical candidates. (Poorly predictive animal models are a major factor in the failure of clinical candidates in all therapeutic areas, but cancer models are particularly unpredictive.)

The Scientist article focuses on the ongoing “co-clinical mouse/human trials” now being led by Pier Paolo Pandolfi, MD, PhD (Director, Cancer and Genetics Program, Beth Israel-Deaconess Medical Center Cancer Center and the Dana-Farber/Harvard Cancer Center). Dr. Pandolfi and his colleagues have constructed genetically engineered transgenic mouse strains that have genetic changes that mimic those found in human cancers. These mouse models spontaneous develop cancers that resemble the corresponding human cancers. In the co-clinical mouse/human trials, researchers simultaneous treat a genetically engineered mouse model and patients with tumors that exhibit the same set of genetic changes with the same experimental targeted drugs. The goal is to determine to what extent the mouse models are predictive of patient response to therapeutic agents, and of tumor progression and survival. The studies may thus result in validated mouse models that are more predictive of drug efficacy than the currently standard xenograft models.

The human clinical trials being “shadowed” by simultaneous studies in mice include Phase III trials of several targeted therapies for lung and prostate cancer. Xenograft models in which tumor tissue from the patients have been transplanted into immunosuppressed mice are being tested in parallel with the genetically engineered mouse models. This two-year project represents the most rigorous test to date of how well genetically engineered mouse models of cancer can predict clinical outcomes.

Dr. Pandolfi started in the mouse cancer model field with his studies of acute promyelocytic leukemia (APL). Unlike humans, mice do not naturally develop APL. Chromosomal translocations, in which the gene for the retinoic acid receptor alpha (RARα) (located on chromosome 17) becomes fused to one of several partner genes (known as “X genes”) on different chromosomes, are involved in the causation of APL. In over 98% of cases of APL, RARα is fused to the promyelocytic leukemia (PML) gene, located on chromosome 15. In a relatively small percentage of cases, RARα is fused to other X genes. An example of one of these other genes is the promyelocytic leukemia zinc finger (PLZF) gene, located on chromosome 11.

In studies in the late 1990s, Dr. Pandolfi and his colleagues constructed transgenic mice that expressed either PML-RARα or PLZF-RARα transgenes, in a promyelocytic-specific manner. (Expression of these transgenes in every cell of a mouse embryo results in embryonic lethality, and their expression in all early hematopoietic progenitors results in impaired myelopoiesis but no leukemia; these transgenic mice are thus not informative with respect to APL. The researchers were able to model PML only by expressing the transgenes specifically and exclusively in promyelocytes.)

The promyelocytic-specific PML-RARα-transgenic mice exhibit abnormal hematopoiesis over their first year of life, and between 12-14 months of age 10% of them develop APL.The promyelocytic-specific PLZF-RARα transgenic mice also exhibit a long latency period, and a subset of these mice eventually develops a leukemia that has features of human chronic myelogenous leukemia (CML).

Importantly, the above transgenic mouse models were useful in designing therapies for human patients. The leukemias in both the PML-RARα-transgenic mice and in patients with the PML-RARα translocation were responsive to treatment with all-trans retinoic acid (ATRA) (Genentech’s Vesanoid, generics). However, both the PLZF-RARα transgenic mice and patients with APL bearing the PLZF-RARα translocation were not responsive to ATRA. APL patients who initially responded to ATRA developed resistance to the drug, as did the PML-RARα transgenic mice. Using the PML-RARα transgenic mice, the researchers found that a combination of ATRA with arsenic trioxide (As₂O₃) (Cephalon’s Trisenox) cured the mice of leukemia. This later proved to also be true for human patients with APL bearing the PML-RARα translocation. Thus a cancer that once was uniformly fatal now has an approximately 90% survival rate.

Leukemic mice with the PLZF-RARα transgene were not responsive to As₂O₃. However, later studies have indicated that histone deacetylase inhibitors such as phenylbutyrate, in combination with ATRA, may be effective in treating these transgenic mice. These drug combinations may therefore be effective in APL patients with the PLZF-RARα translocation.

The success of Dr. Pandolfi’s genetically engineered mouse model in designing an effective therapy for the major type of APL illustrates the potential power of improved mouse models for cancer. Of course, this is a special case, since researchers were able to use the model to design an effective therapy using already-approved drugs. In most cases, researchers use the models to develop novel therapeutic strategies for a particular cancer, which involves discovery and development of new drugs or design of clinical trials using experimental drugs that have yet to be approved. The “co-clinical mouse/human trials” being run by Dr. Pandolfi and his colleagues may result in additional validation of the power of genetically engineered mouse models of cancer, and may thus encourage their adoption by companies developing new oncology drugs.

Our recently published book-length report, Animal Models for Therapeutic Strategies, includes a case study on a genetically engineered model of pancreatic cancer. Pancreatic cancer is one of the most lethal of cancers. Although models bearing transplanted human pancreatic tumors (i.e., xenograft models) are sensitive to numerous chemotherapeutic agents, human pancreatic cancers are insensitive to the same agents. Using a genetically engineered mouse model of pancreatic cancer, researchers hypothesized that the reason for the insensitivity of human pancreatic cancer (and of tumors in the mouse model) is impaired drug delivery. Researchers have been using the mouse model to develop novel therapeutic strategies to enhance drug delivery and thus to achieve improved treatment of this disease.

Our 2009 book-length report, Approaches to Reducing Phase II Attrition, includes a case study on adoption of genetically engineered cancer models by industry. Most animal models designed to enable researchers to develop novel therapeutic strategies for complex human diseases are developed by academic researchers. This includes genetically engineered cancer mouse models. However, most drugs are developed by industry, not academia. Industrial researchers are hampered in their ability to develop successful new oncology drugs by the poorly predictive xenograft models. Genetically engineered mouse models of cancer may help biotechnology and pharmaceutical company researchers to be more productive in oncology drug development, provided the corporate researchers can adopt these animal models for use in their discovery research and preclinical studies. However, for several reasons, industry has not widely adopted these models.

Our report discusses the barriers to adoption of these models, large pharmaceutical companies that are beginning to adopt the models, and the biotechnology company Aveo Pharmaceuticals, whose technology platform is based on in-licensing genetically engineered mouse cancer models from its principals’ academic laboratories and developing new models in-house. Aveo uses its models in its own internal drug discovery and development, and also collaborates with several large pharmaceutical companies. Aveo thus serves as a means of technology transfer from academia to industry, including both to its own internal programs and to its partners. The article in The Scientist also discusses Aveo’s research on genetically engineered mouse cancer models, and their use in the company’s internal drug development programs.