The Interview with Dr. Reynolds is the first of two parts. The second part will be devoted exclusively to his role in the CIMMYT and the yeoman service this organization has carried out in taking the world closer to food security.

Biotechwiz presents excerpts from an exclusive interview with Dr. Matthew Reynolds, who has been intimately connected with improvement of Wheat strains, on the release of the draft sequence of the reference strain of the Chinese Spring Wheat:

Dr Matthew Reynolds: CIMMYT

Biotechwiz: You are described as a wheat physiologist. Can you elaborate on the nature of your work?

Dr. Matthew Reynolds:  As wheat physiologist at CIMMYT the main task is to uncover ways to improve the ability of wheat to be more productive in a range of the environments from those with high yield -such as the Punjab- to those with heat and drought stress, with a special focus on developing countries. Activities encompass the following broad objectives: Through wide consultation define factors that limit current productivity; Develop breeding technologies through collaborative research encompassing novel and conventional approaches; Coordinate multidisciplinary elements of projects over different target countries thereby facilitating relevance and delivery of products; Lead a team of scientists and technicians in Mexico to address specific research objectives as well as human capacity development.

BW: The draft sequence of the genome of the Chinese Spring wheat has just been released. What, according to you, will be the most immediate benefit of this work?

Dr. Reynolds: There will be no immediate benefit as the job of sequencing must be completed thoroughly, however, the long term benefits will be that we can use genetic information to more precisely move useful physiological traits into good agronomic cultivars.

BW: You have spoken in earlier articles of the need to develop crops that will be more resistant to climate change. Do you think that the draft sequence will reveal sufficient data in order to be able to develop such plants or will we need to wait for the finished sequence?

Dr. Reynolds: It will most likely need to wait for the finished sequence.

BW: Breeders have reacted to the news saying they will be able to select for specific traits such as drought or salinity resistance using ‘Macro- assisted selection’. Can you tell us how they would carry out such selection and what does macro-assisted selection actually mean?

Dr. Reynolds: I have not heard of macro-assisted selection before. I assume it refers to whole genome selection, which is a way of taking into account the diversity of the whole genome as opposed to focusing on a few loci. Certainly complex traits like drought adaptation will benefit from considering the whole genome as their genetic basis is complex.

BW: Could you tell us a bit about the CIMMYT and the role you play in the organisation? Also will your organisation be working on developing newer strains of wheat using the sequence data that has become available?

Dr. Reynolds: CIMMYT -a member of the Consultative Group on International Agricultural Research (CGIAR)- partners with hundreds of breeders worldwide and delivers new crop genotypes to developing countries on a large scale as freely available global public goods. The impact of this work on the livelihoods of resource-poor farmers in less developed countries is well documented. The value of the international wheat breeding effort coordinated by CIMMYT is estimated at several billion dollars of extra revenue annually, spread among millions of farmers. While the impact of the so called Green Revolution cultivars were initially in relatively favourable environments, subsequent breeding and dissemination effort has resulted in economic benefits in more marginal environments, including those affected by drought and heat stress. This breeding-evaluation-delivery pipeline encompasses the following elements: (i) free exchange of germplasm with national agricultural research services worldwide, (ii) a centralized breeding effort that focuses on generic needs –i.e. yield potential, yield stability, genetic resistance to range of biotic and abiotic stresses, consumer-oriented quality traits-, (iii) distribution of international nurseries targeted to a number of major wheat agro-ecosystems via national wheat programs in over 120 countries, (iv) analysis of international yield trials and global disease monitoring to ensure relevance of current local, regional and global breeding activities, (v) capacity building and training of research partners, (Reynolds and Borlaug, 2006; attached).

BW: Finally, would you like to speculate on the time-frame it is likely to take for the benefits of the sequence data to become obvious?

Dr. Reynolds: I was told it will take around 5 years to complete the project, then their will be a research phase, followed by application and breeding. Could be around 20 years before benefits are felt by farmers.

Image Credit:

http://blog.cimmyt.org/?s=mathew++reynold

]]> http://biotechwiz.com/?feed=rss2&p=677 0 http://biotechwiz.com/?p=665 http://biotechwiz.com/?p=665#comments Tue, 31 Aug 2010 11:14:17 +0000 Dr Shyamala Pillai Shah http://biotechwiz.com/?p=665 Scientists at Liverpool, University of Bristol and the John Innes centre have released the draft sequence of the entire wheat genome. They were working in collaboration with the International Wheat Genome Consortium. This research has been funded by the Biotechnology and Biological Sciences Research Council. The work was carried out at the University’s Centre for Genomic Research, which is home to 5 next generation analyzers that can read sequences 100 times faster than those used to sequence the human genome!

This work has been received with great excitement and is expected to help wheat breeders to be able to select for strains of Wheat having desired characteristics. The reference variety used for the sequencing is the Chinese Spring Wheat (Triticum aestivum L. cv Chinese Spring) Strain. The availability of this sequence is expected to highlight natural Genetic variants between wheat types to help breeding programs. Wheat breeders have had precious little genetic information in the past to be able to make a choice as to the variety of wheat to be selected.

Wheat: One of the most important Food Crops in the World

The sheer size of the wheat genome has been daunting in terms of whole genome sequencing. The Wheat genome is about five times the size of the human genome and hence was considered close to impossible to sequence. In Comparison to other important crop plants such as Soyabean and Rice, the difficulty of working with such a large genome has left wheat lagging behind in the race of genome sequencing. However, using advanced sequencing techniques employed by Roche’s 454 sequencers, the effort has managed to cover about 95% of the known wheat genes. The results of the study are now available for public use via Genbank, EMBL and CerealsDB. Nevertheless, there are those who warn that the gene map is far from complete and that the first high quality complete map data will be available only within five years. The full sequenced genome requires further read-throughs, assembly of the data into chromosomes and significant work to fully annotate the sequence data.

According to Dr. Neil Hall of the University of Bristol, within the next 40 years the food production should be increased by at 50 % of the current value. This can only be achieved if we are able to produce wheat strains resistant to drought conditions, pesticides and salinity. Traditional methods require time consuming crosses and painstaking selection of desired characteristics sometimes after several generations. The use of genetic techniques would hopefully reduce the time frame and enable the breeder to efficiently select desired traits. These traits may include disease resistance, the ability to grow under extremes of whether and soil characteristics, & producing increased yields with minimum inputs in terms of fertilizers and other growth factors.

Wheat is one of the most important food crops around the world (though most of the wheat produces is what is known as red wheat and not the one that has been used for the study) with an estimated annual production close to 550 million tonnes. Mike Bevan of the John Innes institute has placed emphasis on the importance of the study in the light of a sharp spike in the international prices of wheat following a ban on wheat exports by Russia (due to droughts and wildfires) and the overall decrease in wheat production by countries such as Pakistan and China due to heavy rains and floods.

The wheat genome holds secrets aplenty waiting to be unlocked. We are racing against time as far as food security is concerned and any step forward is all for the best. We are waiting eagerly for the promise to be fulfilled and for the time when wheat breeders can easily and quickly select varieties that will pave the way for the next revolution. Countries like India that are struggling to meet the demands of burgeoning populations and where cultivable land is at a premium are sure to benefit from this research.

]]> http://biotechwiz.com/?feed=rss2&p=665 1 http://biotechwiz.com/?p=637 http://biotechwiz.com/?p=637#comments Sat, 28 Aug 2010 13:22:11 +0000 Dr Shyamala Pillai Shah http://biotechwiz.com/?p=637 An Interview with Dr. Krishanu Saha from the Whitehead Institute of Biomedical Research: On the invention of a new Synthetic Surface for the Cultivation of Human Stem cells for up to three months.

Scientists at MIT have developed a novel synthetic surface for the cultivation of human stem cells. The research team, led by Professors Robert Langer, Rudolf Jaenisch and Daniel G. Anderson, describes the new material in the Aug. 22 issue of Nature Materials. First authors of the paper are postdoctoral associates Krishanu Saha and Ying Mei. The new material was singled out of almost 500 polymers designed during the course of the study, and was found to be optimal after analysing  several chemical and physical properties of surfaces, including roughness, stiffness, and affinity for water that might play a role in stem cell growth. The new surface not only enabled Stem Cells to be grown for up to three months but also enabled harvesting of cells in the millions. Both of these attributes are very important to researchers as the in vitro culture of human Stem cells is fraught with difficulty. The surface also enables clonal growth of a stem cell allowing for easy selection of a particular cell with attributes of interest. As Researchers laud this important invention, Biotechwiz is proud to present an exclusive interview with Dr. Krishanu Saha, one of the authors of this seminal work. An excerpt of the interview is presented below:

Dr. Krishanu Saha

Biotechwiz: Why did you feel the need to develop a new material for the growth of stem cells?

Dr. Krishanu Saha: When we started this work, there were only a handful of culturing materials that were used to grow human embryonic stem cells. Most of these materials included components from animal sources. These animal-derived components are problematic for any cell therapy applications envisioned with these cells, because such components utilized during cell culture can increase the risk of immune rejection when such cells are injected into a patient.  We therefore sought to explore whether a library of synthetic polymers coated with human-derived proteins could replace and improve on the conventional methods of growing human embryonic stem cells.

We also wanted to gain more molecular insight into how human embryonic stem cells grow outside of the body. Mouse embryonic stem cells have particular properties of cell growth and genetic manipulation that make them easier to work with in the lab.  We wondered whether we could devise better culture conditions for human embryonic stem cells by systematically exploring stem cell growth on a diverse set of polymeric materials.

BW: Can you elaborate a bit on the nature of this new surface that you have developed and what is the most unique feature of your invention according to you?

Dr. Saha: The new surfaces can be synthesized entirely from standard chemicals.  They utilize a particular chemistry that was not defined before this work to interact with a human protein, Vitronectin.  The most unique feature is that it can support the long-term culture of fully dissociated human embryonic stem cells as well as the recently ‘reprogrammed’ human induced pluripotent stem cells.

BW:  How soon do you think the research you have done will be available as a commercially viable product?

Dr. Saha: This question of technology transfer is a difficult one to predict. There are already a few commercial products based on other work with novel stem cell culture materials that was just published in May. So if we extrapolate from those cases, our work could be translated into products in less than a year.  I believe the MIT technology transfer office is dedicated to ensuring that the materials get widely used.

BW:  What is the trend your future research is likely to take?

Dr. Saha: I am generally interested in combining this work with recent advances in cellular reprogramming. Cellular reprogramming can produce embryonic stem cell-like cells called induced pluripotent stem (iPS) cells from virtually any human cell source, such as a blood sample or biopsy.  I believe there is a key role of materials and engineering to play in developing these iPS cells for disease modelling and regenerative medicine applications.

BW: Can you tell us about any one hurdle that bugged you the most during your work?

Dr. Saha: Finding common patterns in the material characteristics that controlled the growth of the human embryonic stem cells was challenging.  We had hundreds of polymers with lots of data about surface chemistry, stiffness, and roughness that needed to be sorted and globally analyzed. At times, this seemed tedious, but it is part of the research process.

Yes! You read that right. An unusual proof-of-concept experiment was carried out by researchers at the U.S. Department of Agriculture led by scientist Bruce A. Kimball, Ph.D. the results of these experiments were presented today at the 240th National Meeting of the American Chemical Society (ACS). This study was a part of about 8000 scientific reports to be presented at the meet, making it easily one of the largest such conventions in 2010. The ACS is a non-profit organization chartered by the US Congress.

Dr. Kimbal, USDA

“Based on our results, we believe dogs, as well as mice, could be trained to identify a variety of diseases and health conditions,” said U.S. Department of Agriculture scientist Bruce A. Kimball, Ph.D., who presented the study results. “In fact, we envision two broad, real-world applications of our findings,” Kimball added. “First, we anticipate use of trained disease-detector dogs to screen feces, soil, or other environmental samples to provide us with an early warning about the emergence and spread of flu viruses. Second, we can identify the specific odor molecules that mice are sensing and develop laboratory instruments and in-the-field detectors to detect them.”

Dr. Kimball speculated that a “suite of chemicals, rather than a single compound” might responsible for the difference in fecal odor between healthy and infected ducks. The team is now investigating instrumentation methods to detect these volatile chemicals. Once this has been accomplished, statistical and pattern analysis could reveal the presence of an infection.

The experiment used the classical reward when correct method that has been time-honored in animal studies. In-bred mice were allowed to navigate a maze and when they identified feces of an infected duck, they were rewarded with a Drink of water! Needless to say there were no rewards for picking out the excrement of a healthy duck. Eventually, the mice were trained to spot the infected feces. This method could be extrapolated to train dogs in a similar fashion. SOURCE

Bird flu viruses carried by migratory birds can be spread far and wide, even across continents. Given the close proximity of human and bird populations (wild or domesticated) in many countries including India, an inter-species jump by the virus could result in serious infections in humans, some even being fatal. The prospect of simply using trained sniffer dogs to sniff out the soil near large bird farms might serve to detect the emergence of bird flu and to put in place systems to prevent an epidemic of the human or avian kind.

Well, this does not really seem so outrageous in the light of earlier research involving dogs and cancers. Dogs have been shown to be able to sniff out cancers like melanoma and bladder cancers. Here again certain volatile benzene derivatives present in the breath of cancer patients seem to be the indicator molecules. So researchers at the Pine Street Foundation in San Anselmo, California, US, selected three Labrador retrievers and two Portuguese water dogs with no previous training, and over several weeks trained them using breath samples that had been exhaled into tubes by cancer patients.

To test how well the dogs had learned, they used a new batch of samples and had the dogs attempt to distinguish among 55 lung cancer patients, 31 breast cancer patients and 83 healthy controls. The patients had all had their cancers confirmed by biopsy. The tests were double-blind, so neither the dog handlers nor the experimenters knew which tubes were which. (Integrative Cancer Therapies : vol 5, p 1).

James C Walker, director of the Florida State University Sensory Research Institute in Tallahassee, US, In 2004 showed that dogs could sniff out melanomas. He says that the next step is to see if dogs are really detecting cancer, or if they might be sensing a more general disease symptom, such as one that comes from inflammation. Walker says he would like, eventually, to see a long, large-scale trial designed to test whether dogs can detect cancer even earlier than standard screening tests.

The development of dogs as “biosensors” of the olfactory type will be a tremendously useful technology on the ground for early detection of diseases such as cancers or virus infections. One way or the other, these animals will be able to prevent mortality and will be instrumental in saving lives of people and other animals as the case may be.

The researchers across the world in different study centers need to be applauded for their brilliant and simple deductions in using animals, specifically dogs for detecting illness. We hope that this research can soon be brought to the fields and will be instrumental in saving lives and preventing epidemics or even pandemics like the Avian Flu or the Swine flu pandemics that are so fresh in our minds. SOURCE

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Image Credit: http://www.ars.usda.gov/pandp/people/people.htm?personid=3005

]]> http://biotechwiz.com/?feed=rss2&p=630 0 http://biotechwiz.com/?p=616 http://biotechwiz.com/?p=616#comments Thu, 19 Aug 2010 13:12:32 +0000 Dr Shyamala Pillai Shah http://biotechwiz.com/?p=616 For all those who were waiting with bated breath for the first ever Human Embryonic stem cell (HESC) phase I clinical trials to begin, well there’s good news. On the 30^th of July 2010, the US based Geron Corporation announced the FDA’s approval to its HESC based clinical trials in humans. The announcement comes after a year-long set-back to the proposed trials, after the company discovered during some of its tests that the rats treated with the stem cell line developed cysts. This set off a spate of further tests to ensure efficacy and safety of the therapy. After a year, the company seems to have effectively allayed fears of tumorigenicity and has obtained a green signal from the US FDA and will be beginning the first human trials of Human Embryonic stem cell therapy in the world.

We are pleased with the FDA’s decision to allow our planned clinical trial of GRNOPC1 in spinal cord injury to proceed,” said Thomas B. Okarma, Ph.D., M.D., Geron’s president and CEO. “Our goals for the application of GRNOPC1 in subacute spinal cord injury are unchanged – to achieve restoration of spinal cord function by the injection of hESC-derived oligodendrocyte progenitor cells directly into the lesion site of the patient’s injured spinal cord. Additionally, we are now formally exploring the utility of GRNOPC1 in other degenerative CNS disorders including Alzheimer’s, multiple sclerosis and Canavan disease.”¹

The clinical hold was placed following results from a single preclinical animal study in which Geron observed a higher frequency of small cysts within the injury site in the spinal cord of animals injected with GRNOPC1 than had previously been noted in numerous foregoing studies. In response to those results, Geron developed new markers and assays as additional release specifications for GRNOPC1. The company completed an additional confirmatory preclinical animal study to test the new markers and assays, and subsequently submitted a request to the FDA for the clinical hold to be lifted.¹

The year-long delay has been a serious set-back and I can only speculate the frustration of the many people who are anxiously awaiting the trials to begin. If the therapy manages to even partially restore function in paralyzed patients, we are talking some serious progress here. The trials are only in their first phase now, but its a giant step for mankind. We could be looking at potential therapy for debilitating diseases in the near future.

The therapy developed by Geron draws upon the work of Dr. Hans Keirstead and his team at the University of California. In a revolutionary paper published in 2005 in the Journal of Neurosciences, the authors “undertook a proof of concept experiment to determine the feasibility and efficacy of using human stem cell derivatives to promote remyelination and functional recovery in the injured adult rat spinal cord.” The authors go on to elucidate, “human embryonic stem cells (hESCs) were induced to differentiate into high-purity oligodendrocyte progenitor cells (OPCs) before transplantation. This strategy obviates the potential problems posed by the capability of embryonic stem cells to form teratomas after implantation and to differentiate in ways that are determined by environmental signals at the site of implantation, which are difficult or impossible to control. High-purity OPCs were then transplanted into spinal cord injury (SCI) sites in rats 7 d or 10 months after injury. We show that rats receiving OPCs at 7 d after injury exhibited enhanced remyelination and recovery of motor function, whereas rats that received OPCs at 10 months did not. These studies demonstrate for the first time that derivatives of hESCs have therapeutic potential for spinal cord injury and indicate that there may be a limited therapeutic window for this treatment.²

About Human Embryonic Stem Cells:

Human embryonic stem cells (hESCs) are a self‑renewing source for the scalable manufacturing of functional replacement cells for every tissue and organ in the body. The hESCs with which Geron works were derived from surplus in vitro fertilized embryos originally created as part of an in vitro fertilization (IVF) procedure. The embryos, which would otherwise have been destroyed, were donated for research by the parental donors under informed consent. The hESC line that is used to produce GRNOPC1 is the H1 line. The hESCs are immortal cells, having Telomerase enzyme allowing them to grow indefinitely in culture and further, they retain the capacity to differentiate into any one of the almost 200 different types of cells present in our bodies. The official website of Geron Corp claims that their scientists can differentiate their stem cells into almost 7 distinct types of specialized cells, each of which has a potential as a treatment for some disorder. These are listed below:

• Neural cells to treat chronic degenerative diseases of the nervous system;
• Cardiomyocytes for the treatment of congestive heart failure and myocardial infarction;
• Islets for the treatment of diabetes;
• Chondrocytes for the treatment of osteoarthritis;
• Hepatocytes for ADME drug testing;
• Dendritic cells cells for immunotherapy for cancer and infectious diseases; and
• Osteoblasts for the treatment of osteoporosis and bone fractures.³

The GRNOPC1 cell line is a Oligodendrocyte progenitor cell line derived from the hESC and is supposed to heal damage caused by extensive demyelination in the case of spinal cord injuries. Oligodendrocytes are naturally occurring cells in the nervous system that have several functions. Oligodendrocytes produce myelin (insulating layers of cell membrane) that wraps around the axons of neurons to enable them to conduct electrical impulses. Myelin enables efficient conduction of nerve impulses in the same manner as insulation prevents short circuits in an electrical wire. Without myelin, many of the nerves in the brain and spinal cord cannot function properly. Oligodendrocytes also produce neurotrophic factors (biologicals that enhance neuronal survival and function) to support the maintenance of nerve cells. Oligodendrocytes are lost in spinal cord injury, resulting in myelin and neuronal loss that cause paralysis in many patients with spinal cord injuries.³

It has been observed in the animal trials that if the GRNOPC1 cell line was injected into the damaged spinal cord of a rat about seven days after the injury, the treatment significantly increased the locomotary functions of the rat and improved its weight carrying capacity. With these findings, the FDA had agreed to allow Human trials to begin. However, Geron found in a single animal study that the rats developed cysts. These were microscopic and did not seem out of the ordinary as far as spinal injuries were concerned, the FDA did not want to take any chances. The trials were stalled till Geron came up with further tests and assays to prove that the cysts did not recur. For details refer this site.

The trials will now begin this year. The website provides the following information regarding the Clinical trials to be held at 7 different medical centres:

The GRNOPC1 Clinical Program

Patients eligible for the Phase I trial must have documented evidence of functionally complete spinal cord injury with a neurological level of T3 to T10 spinal segments and agree to have GRNOPC1 injected into the lesion sites between seven and 14 days after injury.

Although the primary endpoint of the trial is safety, the protocol includes secondary endpoints to assess efficacy, such as improved neuromuscular control or sensation in the trunk or lower extremities. Once safety in this patient population has been established, Geron plans to seek FDA approval to extend the study to increase the dose of GRNOPC1, enroll subjects with complete cervical injuries and expand the trial to include patients with severe incomplete (ASIA Impairment Scale grade B or C) injuries to enable access to the therapy for as broad a population of severe spinal cord-injured patients as is medically appropriate.

Geron has selected up to seven U.S. medical centers as candidates to participate in this study and in planned protocol extensions. The sites will be identified as they come online and are ready to enroll subjects into the study.⁴

The beauty of this treatment is that it shows promise not only for the treatment of paralyzed patients, but also for those suffering from multiple sclerosis.  The Geron study stands out even among other stem cell trials because of the capacity for healing nerve damage. In the trials, human embryonic stem cell derived glial progenitor cells will be injected directly into lesions along a patient’s spinal cord. Animal models indicate that the glial progenitor cells should work to promote nerve growth and repair the myelin sheaths on the nerves. This ‘remyelination’ of the nerves is a critical component of healing spinal cord injury as it allows signals to be passed along the nerve.

Though the current trial is being carried out primarily for safety evaluation and only those patients who have been injured after the trials commence can be a part of the trial (as the stem cells need to be injected 7 days after the injury for best results), the trial is still a landmark step and I want to wish all the very best to the scientists, the health care professionals and of course the patients who are going to be a part of this trial.

We are all waiting to see what the results will hold for the future of medicine!

References:

1. http://www.geron.com/media/pressview.aspx?id=1229
2. http://www.jneurosci.org/cgi/content/full/25/19/4694
3. http://www.geron.com/grnopc1trial/grnopc1-sec1.html
4. www.geron.com

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Judge Robert Sweet

Molecular Pathology v. U.S. Patent and Trademark Office, that the patents For BRCA1 and BRCA2 held by the company Myriad Genetics, were invalid. The decision was a highly anticipated one since this particular lawsuit has been hailed by many as being a direct attack on the company and the USPTO (The United States Patent and Trademarks Office). The issue of gene patenting has always been a controversial one and there have been heated debates for and against it. However, in recent times, we have seen with increasing unease, the extent to which essential health care testing, diagnostics and even treatments have slowly but steadily passed the truly needy patients by because of prohibitively high costs and monopolistic trade practices by many such companies in the name of millions of dollars sunk into research for the same.

The current judgement is a 156 page decision and invalidates about seven of the patents held by the company Myriad Genetics related to the BRCA1 and 2 genes that have been implicated in breast and ovarian cancers. If this decision is upheld by the Higher courts, it has the power to completely challenge many of the existing gene patents and change the face of Intellectual property law as we know it forever.

So what really is a gene patent. Wikipedia defines it thus: A gene patent is a patent on a specific isolated gene sequence, its chemical composition, processes for obtaining or using it, or a combination of such claims. Thus in essence, a gene existing within an organism is not patentable and neither is it of any use to us clinically if it remains as such. The moment it has been isolated, purified, it can be put to commercial use, as in for diagnosing clinical conditions such as cancers or treating them. This was presumably the premise underlying the grants of such patents in the first place. However, the premise seems to be rather ridiculous to me. Most of the genes under patent protection today (about 20 % of the genome is patent-protected) are not inventions but discoveries. If the mere act of isolating them from the body turns them into something ”novel” , then by that token a large number of chemical entities can be considered novel by simply isolating them from their parent source. This will lead to patents on a large number of ridiculous ‘inventions’. Writer Michael Crichton makes an emphatic point in an article in the New York Times. He says and I quote,” Gene patents are now used to halt research, prevent medical testing and keep vital information from you and your doctor. Gene patents slow the pace of medical advance on deadly diseases. And they raise costs exorbitantly: a test for breast cancer that could be done for $1,000 now costs $3,000. Why? Because the holder of the gene patent can charge whatever he wants, and does. Couldn’t somebody make a cheaper test? Sure, but the patent holder blocks any competitor’s test. He owns the gene. Nobody else can test for it. In fact, you can’t even donate your own breast cancer gene to another scientist without permission. The gene may exist in your body, but it’s now private property.”

I am sure most of my readers would agree with me that this situation is bizarre. It begs the question how could the USPTO be so irresponsible as to grant such patents in the first place? In the current ruling, Myriad Genetics, that holds the patents with the University of Utah Research Foundation, asked the court to dismiss the case, claiming that the work of isolating the DNA from the body transforms it and makes it patentable. Such patents, it said, have been granted for decades; the Supreme Court upheld patents on living organisms in 1980. In fact, many in the patent field had predicted the courts would throw out the suit. However, Judge Sweet made the point that such patents were “improperly granted” because they involved a “law of nature.” He said that many critics of gene patents considered the idea that isolating a gene made it patentable “a ‘lawyer’s trick’ that circumvents the prohibition on the direct patenting of the DNA in our bodies but which, in practice, reaches the same result.”

In an important observation, the court ruled as follows, “The identification of the BRCA1 and BRCA2 gene sequences is unquestionably a valuable scientific achievement for which Myriad deserves recognition, but that is not the same as concluding that it is something for which they are entitled to a patent.”

While browsing the net I came across an interesting perspective on the judgement, I quote from the author’s post, “This lawsuit against Myriad signifies a change in that it finally places the patient and the administration of genetic testing at the centre of the stage.  Although the Court’s holding focuses on patent subject matter the court dedicates a significant part of the opinion to access to BRCA1/2 genetic testing. Myriad charges about $3,000 for testing an exorbitant amount compared to other genetic tests. Furthermore, Myriad does not allow other laboratories to conduct the testing – all samples have to be sent to its headquarters in Salt Lake City. The opinion tells the stories of women who were unable to test to find out whether they carry the BRCA1/2 genes because Myriad would not accept their insurance. It recounts the ordeals of women who could not get definitive answers through Myriad’s testing and were precluded from seeking testing elsewhere. It underscores that women were unable to get a second opinion of the test results because tests are conducted only by Myriad. It also discusses the efforts of doctors and laboratories that were willing and able to offer BRCA1/2 testing but were precluded by Myriad from conducting the testing.”

Many are not comfortable with the idea of patenting genes. Michael Crichton in his article talks about the need to have open access to information. He cites the poignant case of a disease known as Canavan Disease. This is a debilitating disease that is inherited and in which infants are unable to crawl or walk, suffer seizures and rarely survive beyond their adolescence. He goes on to document the ironic and heart-breaking history of the research that led to the development of the diagnostic test for this disease. Because of the lack of such a test in the first place, families who had borne children with the Canavan disease came together and donated tissue samples for study. They engaged a researcher to identify the gene and donated their time and money for the same. Eventually the efforts met with success. The gene was identified in 1993. The families managed to get the commitment of a New York Hospital that it would offer a diagnostic test for the defective gene to anyone who needed it for free. In a twist of fate however, the employer of the hospital, Miami Children’s Hospital Research Institute patented the gene and refused to allow any health care worker to use the test without paying a royalty. The families who had contributed did not believe in patenting and so did not put their names on the patent and consequently had no claims whatsoever on the fate of the research.

If companies were denied access to essential material for research like for example tissue and blood samples or even clinical data or the questionnaires filled out by the patients and/or their families, would they be able to pursue their research at all? Let alone come to a point where their “inventions” become money spinning prospects for the companies? If recognising their efforts and granting them patent protection means that they are going to use this as a license to charge whatever they please for the tests, prevent anyone else from coming up with a cheaper alternative and effectively making efficient healthcare the privilege of the few, I think this goes against the spirit of the law.

This is exactly the position taken by Judge Sweet in the above landmark decision. He has placed emphasis on the fact that accessibility of genetic testing is a key concern and that granting patent protection for superfluous claims is a dangerous trend. Whilst not passing any ruling on the larger constitutional issue of why the USPTO granted such patents in the first place, he has underscored a larger more important issue; One of the plight of the patients. It is more important now that we learn to strike a balance between protecting an inventor and the extent to which this protection should be granted in the face of abuse of exclusive rights and grants.

In 2007, the European Patent Office (EPO) rejected an appeal by Myriad Genetics and the University of Utah, and upheld an earlier decision  to revoke some claims of patent EP705902 relating to the BRCA1 gene and its applications. There was widespread objection to the extent of this claim among European researchers, since it granted an effective monopoly to Myriad which they believed unjustified; six bodies filed objections, leading to a decision by the EPO in 2005 to substantially amend the patent, retaining only the claims relating to a specific nucleic acid probe and vectors containing gene sequences. The EPO has now rejected an appeal by Myriad and the University of Utah, and amended the patent, meaning that European laboratories retain the right to perform diagnostic tests for mutations in the BRCA1 gene sequence, which are associated with increased susceptibility to breast and ovarian cancer.

Thus, in countries where gene patenting is not recognised, testing facilities can be made cheaper and probably even more efficient than the original one. The point that we need to mull over here is this: can there be another method to make this system work for both the researchers and the end-users? For example, can we reduce the term of patent protection in such cases or put a ceiling on how much a company with patent protection can charge for a particular test/ treatment so that it covers its costs but does not profiteer and do so at the expense of patients for whom the research was meant to be in the first place. Also, if a company is found to be recklessly profiteering, can we not have punitive measures for the same? Or make exceptions in costs for patients who might not be able to spend so much for a single diagnostic test? If a patient desires to have another lab carry out the test in order to verify/ confirm/ have a second opinion, instead of the patent owning company blocking it out, can it not work out a cost sharing with other laboratories so that the system becomes more transparent?

This is one of those questions that begs deeper debate. We cannot afford to get defensive or offensive and merely argue. This problem needs urgent and fair solutions. One the one hand, the fate of Biotechnology companies hangs in the balance, as experts say newer companies will find it difficult to raise venture capitalist funding if they are not sure they will get strong patent protection. On the other, monopolistic patent protection granted on frivolous grounds is like playing with the lives of patients. I laud Judge Sweet for his gumption in giving the kind of decision that he has. It is indirectly a comment on the haphazard and mindless policy of the USPTO (contrast this with the behaviour of the EPO on the same issue).

I will write another article where I will attempt to analyse a few aspects of the judgment. But as for now, I will content myself with just saying this…… We are at the brink of a major change. How we use this opportunity will decide our fate in more ways than one!

REFERENCES:

1. http://www.genomicslawreport.com/index.php/2010/03/30/pigs-fly-federal-court-invalidates-myriads-patent-claims/#comment-6174

2. http://www.phgfoundation.org/news/3854/

3. http://www.nytimes.com/2010/03/30/business/30gene.html

4. http://www.pbs.org/wgbh/pages/frontline/shows/snitch/procon/sweet.html ( A general Interview with Judge Robert Sweet)

Scientists at the Arizona State University have developed a reader that can discriminate between DNA’s four Nitrogenous bases, namely Adenine, Guanine, Cytosine and Thiamine. The detailed paper is to be published in Nano Letters and is entitled “Electronic Signatures of all Four DNA Nucleosides in a Tunnelling Gap.” The research was headed by ASU Regents’ professor Stuart Lindsay, Ph.D.

The team had earlier (2008) demonstrated the capability of reading individual DNA sequences but in order to do so they had to use four separate readers, each designed to recognize and record the presence of one of the four bases. Subsequently they fed DNA sequences through a Carbon nanotube and used scanning tunnelling and atomic force microscopy to make measurements. The microscopes have electrodes at the tips and these are held close to the DNA sample. Fluctuations in current are correlated to the presence of specific bases.

The latest invention modifies the same principle. Only in this case there are two electrodes one at the end of a microscope probe and the other on the surface, acting like a pair of charged tweezers. The ends of each are chemically modified to catch hold of the DNA between them. The beauty of the method is the gap between the electrodes is adjusted in such a way that when a single base of the DNA passes through 2.5 nanometer gap between 2 gold electrodes, it sticks on there and the fluctuation in current (a small increase in this case) is recorded. The accuracy is so adjusted that if the gap was any bigger, then smaller bases would not be captured and conversely if it were any larger then molecules could bind in varied configurations and the resultant signal would be highly confused. The reader strikes a balance to give just the right gap required to measure and identify each individual base. Each of the chemical bases of the DNA genetic code gives a unique electrical signature as they pass between the gap in the electrodes.

“What we did was to narrow the number of types of bound configurations to just one per DNA base,” Dr. Lindsay explains. “The beauty of the approach is that all the four bases just fit the 2.5 nanometer gap, so it is one size fits all but only just so!”

“We’ve now made a generic DNA sequence reader and are the first group to report the detection of all four DNA bases in one tunnel gap,” according to Dr. Lindsay. “Also, the control experiments show that there is a certain (poor) level of discrimination with even bare electrodes (the control experiments) and this is in itself a first, too.

“We were quite surprised about binding to bare electrodes because, like many physicists, we had always assumed that the bases would just tumble through. But actually, any surface chemist will tell you that the bases have weak chemical interactions with metal surfaces.”

Next, Lindsay’s group is hard at work trying to adapt the reader to work in water-based solutions, a critically practical step for DNA sequencing applications.

On the third of March 2010, UK based leading stem cell Therapy Company ReNeuron announced positive pre-clinical data for the treatment of Peripheral Arterial Disease (PAD) in Diabetic Patients using its indigenously developed Stem cell line ReN009. PAD occurs when there is a build-up of plaque in the arteries. This plaque generally is made up of fats and cholesterol, calcium and fibrous components of the body. Periodic build-up of such fats in the arteries can cause them to harden and narrow the lumen (Hollow space) of the artery. This narrowing prevents proper blood flow within the body. Known as Atherosclerosis, this generally affects blood flow to the legs but can also affect flow to kidneys hands and other parts of the body. PAD is a chronic and debilitating disease that progressively restricts blood flow in the limbs, causing cramping, chronic pain and in extreme cases, amputation.  PAD is commonly associated with other conditions, including diabetes, obesity and stroke.  At least 1 in 20 people over the age of 55 have some degree of PAD and it becomes more common with increasing age. For more on this disease please click here

The current research was conducted in collaboration with Professor Paolo Madeddu, Dr Rajesh Katare and colleagues at the Bristol Heart Institute, University of Bristol, UK, and is based on earlier successful pre-clinical efficacy studies with ReN009 conducted by that group.  In this latest study, researchers tested the newly-developed freeze-thaw formulation of ReNeuron’s CTX stem cell line, via intramuscular injection, in a recognized diabetic mouse model of hind limb ischaemia.  The CTX cell line forms the basis of ReNeuron’s ReN009 therapy for PAD as well as its ReN001 therapy for stroke.  Initial clinical trials with ReN001 are due to commence in the UK shortly, following following final regulatory approval last month.¹

The results of the new ReN009 study initially showed that the diabetic mice had reduced blood flow capacity compared to the non-diabetic control mice. When treated with the ReN009 cells, the diabetic mice exhibited a significant and dose-dependent recovery of blood flow to the ischaemic limb, with significantly increased re-vascularisation of the damaged tissue as measured by increased capillary and arteriole density.  These results were presented by poster at the Diabetes UK Annual Professional Conference in Liverpool, UK, running from 3-5 March.¹

This therapy is being developed as an allogenic (non-patient specific) stem cell treatment for late-stage PAD, or critical limb ischaemia, in diabetic patients for whom PAD is a side-effect of their diabetes.  In diabetic patients, PAD progresses rapidly with vascular surgery showing a poor prognosis and many a times leaving doctors with only amputation as the option for treatment.  A stem cell- based therapy like the ReN009, offers hope to be able to enable the rebuilding of the vascular system in the limb and hence restoring blood flow to that limb and probably preventing amputation.

This is one technology to look out for! !

RETINITIS PIGMENTOSA

On February eighteenth, 2010, ReNeuron announced that its collaborative research in the US with the Schepens Eye Research Institute at Harvard Medical School would receive a boost in terms of funding from a private company in the US. The aim this time is to take research to the clinics in the US for the treatment of the disease known as Retinitis Pigmentosa. Retinitis Pigmentosa is a group of inherited disorders characterized by progressive peripheral vision loss and night vision difficulties (nyctalopia) that can lead to central vision loss. For more on this disease, click here.

This will be the first phase of a two year translational programme to take human retinal progenitor cells (hRPCs), a cell line that has been designated as ReN003 and extrapolate results to human patients in the US. This phase will aim to demonstrate an improvement in vision after grafting of hRPCs in ophthalmic disease models.

Following transplantation in a rodent model of damaged retina, the hRPCs were seen to integrate with the host retinal tissue and differentiate to express the protein rhodopsin, a marker for the light- sensitive rod cells found in healthy retina. This research has tremendous implications not only for Retinitis Pigmentosa, though this is the initial target, but also for other degenerative and age-related disorders affecting sight, such as age-related macular degeneration and diabetic retinopathy. The lead investigator of these studies at the Harvard medical School is Dr. Michael Young.

I hope that both the experimental treatments outlined above will be reality soon and we will be able to bid goodbye to debilitating and even life-threatening disorders such as the ones outlined here very soon.

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References:

1. http://www.reneuron.com/news__events/news/document_239_237.php
2. http://www.reneuron.com/news__events/news/document_237_237.php

Typically stem cells are divided into adult stem cells and embryonic stem cells. Embryonic stem cells are derived from the Blastocyst stage of Embryos. Embryonic stem cells can generally give rise to almost all the different cell types in the human body. Adult stem cells on the other hand, generally give rise only to all the different cells of the particular tissue from which they are derived. What does this mean? In simple terms, Hematopoetic Stem cells derived from bone marrow can give rise to all the different types of blood cells but not to cells of a very different organ system such as neurons of the brain. So in a sense they are of limited capacity. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be “reprogrammed” genetically to assume a stem cell-like state. This new type of stem cell is called induced pluripotent stem cells (iPSCs).

The unique properties of these cells have given rise to a new field of research known as Regenerative or Reparative medicine. This field is defined as the field of medicine dedicated to the use of stem cells for treatment of diseases by inducing them to differentiate into the tissue type that has been destroyed. A large number of Genetic diseases are now currently being treated by the use of Regenerative therapy. Acute Lymphoblastic Leukemia, Common Variable Immune Deficiency, Hunter’s Syndrome, Thalassemia, and Tay Sach Disease to name just a few are being treated using these therapies.

So What Is Cord Blood?

Cord Blood

After the birth of a baby the umbilical cord that forms the connection between the mother and child when still in the womb is cut. Even so, some blood remains in the blood vessels of the placenta and the attached umbilical cord. The baby now no longer has need for this blood which is called placental blood or umbilical cord blood: “cord blood” for short.

Cord blood contains all the normal elements of blood – red blood cells, white blood cells, platelets and plasma. But it is also rich in hematopoietic (blood-forming) stem cells, similar to those found in bone marrow. Cord blood, therefore, is being used increasingly on an experimental basis as a source of stem cells, as an alternative to bone marrow. Most cord blood transplants have been done to treat diseases of the blood and immune system. It has also been used to restore the functional deficiencies of several genetic metabolic diseases. Scientists are investigating the possibility that stem cells in cord blood may be able to replace cells of other tissues such as nerve or heart cells. Whether cord blood can be used to treat other kinds of diseases will be learned from this research.

In this context, I would like to quote from a 2007 document released by the American Diabetes Association with respect to a clinical trial carried out using Cord Blood cells. “In a small pilot study, transfusion of stored, autologous (i.e. the person’s own), umbilical cord blood into a group of children newly diagnosed with type 1 diabetes appears to have reduced their disease severity, possibly re-setting the immune system and slowing the destruction of their insulin-producing cells, according to a report presented at the American Diabetes Association’s 67th Annual Scientific Sessions.” “After only six months, it is too early to tell how long the children will benefit from this therapy, but early signs indicate that it may have helped enhance blood glucose control and management,” said Michael J. Haller, MD, Assistant Professor of Pediatric Endocrinology at the University of Florida College of Medicine and lead author of the study, “but more important than the potential benefit in these children, this first use of cord blood in diabetes will help us focus on what it is in the cord blood that yielded the benefit,” he said. “We then hope to isolate and grow that cell type to develop therapies for a larger pool of people, not just those who have stored cord blood.” He discussed how such a cellular therapy might be one component of a future immune-modulating “cocktail.” This I think underlines the basis of all stem cell related therapy.

In a study in rats, it was shown that, Intravenous injections of cells from human umbilical cord blood improved the neurological and motor function of the rats that were recovering from severe traumatic brain injury. This was demonstrated by researchers at Henry Ford Health Sciences Center (HFHSC), Detroit, and the University of South Florida (USF), Tampa. If this is indeed true, then we will have hit upon a relatively easily obtained source of stem cells that can be safely collected and screened and stored for future use.

What is even better is that unlike bone marrow or organ transplants an exact match of cord blood antigens is not necessary.

What Is Cord Blood Matching?

HLA matching

Whenever any type of transplantation is carried out, organ transplants or blood transfusions, it is necessary to “match” markers that are known as antigens of the donor and the recipients. What are these antigens? They are proteinaceous ‘tags’ or molecules that are present on surfaces of cells and tissue of our body. These tags define our uniqueness. The moment a foreign ‘tag’ enters our system; our body senses it and we mount an immunological response to the same. This is known as rejection. So if a person undergoing a kidney transplant for example, receives a kidney whose tissue has antigenic tags that are very different from those of his own kidney, his body will attack the transplanted kidney and he will have what is known as a Graft Rejection. Similar is the case with blood transfusion and hence only after determining the relevant blood group can the transfusion be successfully accomplished. Rejection reactions are extremely strong reactions and can be fatal. Thus, the advantages of not requiring a complete antigenic match while using cord blood for cure become evident. In a cord blood transfer, the antigens that are matched are known as HLA (Human Lymphocyte Antigens); the same ones that are matched during organ transplants. Now there are a large number of different HLAs found in our tissues so not all of them are matched for reasons of practicality. Out of all of these, 6 important antigenic clusters that are found to be of prime importance in rejection processes have been identified. These major groups are, HLA A, B and DRB1. Complete matches between the donor and the recipient would mean 2 pairs each Of HLA A,B and DRB1; a total of six. This complete match is known as a 6/6 match or a 6/6 HLA match. Sometimes an additional set of antigens are used for better matching. These are, HLA C and DQ. Thus, complete matching of these additional markers yield a total of 10 points. So, one may have a 6/10 match or a 10/10 match or a 5/10 match. Generally, the first 3 antigens are used and a 6/6 match is considered perfect. While a 5/6 match works best, a large number of successful procedures have been carried out using just a 3/6 match that is only 3 out of the stipulated 6 markers match! That is incredible and it exponentially increases chances of success in finding a donor.  Today almost 70 different genetic disorders can be treated using Cord Blood.

Cord Blood Banking In India

The awareness about cord blood banking has seen a spurt in India, with a large number of companies entering the fray in order to set up such blood banks. Cord stem cell banks process leftover umbilical cord blood from the placenta and umbilical cord after the baby is delivered, and preserves by freezing them in liquid nitrogen at a temperature of -195 degrees celsius. Umbilical cord blood banks can be divided into two categories: private and public. In private banks, one can keep the umbilical cord for two decades, which can be claimed by the family or the patient. For example, At Lifecell, one such private bank, each client has to incur Rs 27,000 ($621 USD) as enrollment and processing fees and will be charged Rs 2,900 ($66 USD) every year as storage cost. For one-time payment, the cost comes to Rs 59,900 ($1,377 USD). The donor cannot stake claim from public banks. Similarly, The Apollo Group of Hospitals has also recently signed a memorandum of understanding with the American blood bank Histostem Inc to offer stem cell therapy to patients from South Asia. As part of the deal, Histostem India will train doctors and nurses at Apollo for stem cell treatment protocols and develop new treatment protocols with relevant regulatory approval. (Source). For a list of cord blood banks in India, please refer to this link.

The costs are still prohibitively high and out of reach of most middle class families in India. There are some incentives available through the banking sector in the form of loans, for example, StemOne Biologicals Private Limited formerly Cord Life Biotech had a strategic alliance with the bank ICICI.  Cord blood clients could obtain personal loans from ICICI to cover the cost of cord blood banking.  According to Stem Cells Research Forum of India (SCRFI), India’s stem cell market is growing at a rate of 15% per annum and is estimated to hit US $ 40 Million by the year 2010. As of 2008, StemOne Biologicals Pvt Ltd. is the only company in India offering the entire spectrum of stem cell related services: In addition to cord blood banking, they offer other forms of autologous stem cell cryopreservation.  StemOne is expanding to offer cord blood banking through more marketing associates and to support stem cell based therapies at medical centres. (Source).

The cost remains a major concern in India as also the putative benefits of the technology. However, If one has a history of genetic disorders in the family, onemight want to consider this technology that has changed millions of lives across the globe. Maybe, in time, we might be able to come up with schemes to subsidise the cost for poor and needy families and then Cord Blood Therapy will really have achieved a miracle ! !

REFERENCES:

Image Credit: 1.  Cord Blood: www.afroromance.com/blog/cord-blood-banking.htm

2. HLA match Diagram: www.marrow.org.

THe Humanised Mouse !

The problem with viruses is their host specificity. What this simply means is, viruses attacking a human being will generally have no effect on a mouse and vice versa. While this is good as the non- human viruses will not spread rapidly and infect humans, it does present substantial problems to scientists trying to study the pathology of human viruses and developing vaccines and drugs for the same. Since one cannot test therapies directly on human beings, a viable alternative needs to be provided. In the past, we have experimented with using in vitro cell cultures; liver cells grown in a Petri dish. However, this model has severe limitations due to the lack of proper organ structure and complete absence of the kind of interactions between cells and organs seen in a complete organism. Alternatives to this were to use animals such as mice that completely lacked a viable immune system (nude mice) for studying tumors of human origin. The tumors would be transplanted into these mice and their effects and probable curative measure would be studied.

However, this model has its side effects too. As Dr. Inder says, clinically speaking, a tumor does not start by acquiring millions of tumor cells from outside the system. It starts with one or two or ten cells that have lost control of their cell division cycle and hence keep on multiplying uncontrollably to give rise to a tumor. So, to that extent, transplanting a tumor into a mouse is really a fundamentally different process and may not yield real-time data as needed. However, with this new model where the mouse liver cells are literally taken over by human liver cells (Hepatocytes) so that they overgrow to give a liver that is almost entirely human, we will be able to study the actual process of tumorigenesis or viral infections along with the possible cures and vaccination methods.

This physiological change was confirmed when these ‘humanised’ mice were subjected to challenge with the Hepatitis B and C viruses. The normally resistant mice developed the infection clinically. As we have already discussed, the initial resistance to these viruses was due to the host specificity of the viruses. The receptors required by the HBV or HCV were not to be found in the mouse liver and hence the immunity. Now, however, with a human liver, these mice became susceptible to the infection just like their human counterparts.

Dr. Inder Verma: Salk Institute

What was even better, the current first line of treatment for Hepatitis C worked just as well as it does with the human liver. This treatment consists of using Pegylated Interferon. What is Pegylated interferon? Well, interferon is a natural protein produced by our bodies in response to a viral infection. It works by inducing resistance to the infecting virus in the cells surrounding a virally infected cell in the body. It is therefore like a chemical signal sent out by a cell that has already been infected to the other healthy cells to raise their defences. Now, this natural interferon molecule was coupled with the chemical Polyethylene Glycol via a process called Pegylation (Technical definition of the process¹: PEGylation is the technique that involves the modification of a protein, peptide or nonpeptide molecule (drug) by covalent attachment of one or more PEG molecules for the purpose of enhancing therapeutic value. Advanced PEGylation10 involves attaching specific, modified polyethylene glycol (PEG) polymers to biomolecules) to yield what is known as Pegylated Interferon. The commercially available molecule is Pegasys (Pegylated Interferon Alpha 2a). The mode of action of this drug is as yet unclear. It is thought to bind to specific cell-surface receptors, suppressing cell proliferation and viral replication. It has also been observed to increase the effector protein levels and to reduce white blood cell (WBC) and platelet counts.

PEGylation is necessary for the reasons such as increasing the solubility, stability, circulation half-life, for minimizing the rapid kidney clearance, for achieving the controlled release, etc. Advanced PEGylation overcomes the drawbacks of the earlier technology. Four types of bioconjugates of PEG comprise conjugation with peptides and proteins, with lipids, with low molecular weight drugs and with biological macromolecules. PEGylation is widely applied in various fields viz. medical, diagnostic, synthesis of peptides and carrying out some organic synthetic reactions.¹

The applications of this chimeric mouse are tremendous and it will boost research and investigations into viral disease, vaccination and cure. This study has been published on February 22 in the Journal of Clinical Investigation. Dr Inder M. Verma is a Professor, Irwin and Joan Jacobs Chair in Exemplary Life Science,  Laboratory of Genetics, at the Salk Intstitute. For a detailed profile please click here.

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REFERNCES:

1. www.kppub.com