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Posts Tagged ‘MIT’


Vassar Street and Main Street, in the new world’s Cambridge, Massachusetts, would be a leading candidate.

According to the article published in Wired Magazine in November 2015 “when the Whitehead got too small for genomicist Eric Lander’s ambitions, he launched a flashier and brasher newcomer next door. The Broad Institute’s gargantuan gleaming glass lobby is filled with early gene-sequencing instruments. Its multimedia screens boast that this is one of the world’s largest gene-sequencing and research factories. The Broad’s strategy is different from that of the Whitehead; instead of concentrating a few in an ultra-exclusive bioclub, Broad bridges MIT, Harvard and most of the hospitals in Boston. Its 2,000 members extend outwards, partnering with tens of thousands of others globally. Those working at the Broad are not averse to commerce; its director alone helped to build Foundation Medicine, Verastem, Millennium, Fidelity Biosciences, Courtagen and Aclara among many other leading companies.

The sixth building on this extraordinary corner, Novartis, focuses on private research, and represents a huge migration from Basel in Switzerland towards the MIT campus, becoming Cambridge’s largest employer. Pfizer, Sanofi, Amgen, Biogen-Idec and hundreds of others cluster nearby. ”

Attracting the best and the brightest, one can change not just a city but the world.

 

Source

http://www.wired.co.uk/magazine/archive/2015/11/ideas-bank/vassar-main-cambridge-massachusetts-innovaton

 

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CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

 

Curator: Stephen J. Williams, Ph.D.

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987), their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007). The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

Jennifer Doudna, PhD Professor of Molecular and Cell Biology and Chemistry, University of California, Berkeley Investigator, Howard Hughes Medical Institute has recently received numerous awards and accolades for the discovery of CRISPR/Cas9 as a tool for mammalian genetic manipulation as well as her primary intended research target to understand bacterial resistance to viral infection.

A good post on the matter and Dr. Doudna can be seen below:

https://pharmaceuticalintelligence.com/2014/06/13/215-245-6132014-jennifer-doudna-the-biology-of-crisprs-from-genome-defense-to-genetic-engineering/

In Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting inheritable metabolic disorders in which may benefit from a CRISPR-Cas9 mediated therapy is discussed. However this curation is meant to focus on CRISPR/CAS9 AS A TOOL IN PRECLINICAL DRUG DEVELOPMENT.

Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:

 

  1. Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
  2. CRISPR/CAS9 Use in Developing Models of Disease
  • Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

 

 

I.     Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE

 

The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other’omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.

However, some of the experimental issues with this methodology involved

 

  • Most transfections experiments result in NON ISOGENIC cell lines – by definition the insertion of a transgene alters the genetic makeup of a cell line. Simple transfection experiments with one transgene compared to a “null” transfectant compares non-isogenic lines, possibly confusing the interpretation of gene-function studies. Therefore a common technique is to develop cell lines with inducible gene expression, thereby allowing the investigator to compare a gene’s effect in ISOGENIC cell lines.
  1. Use of CRSPR in Highthrough-put Screening of Genetic Function

A very nice presentation and summary of CRSPR’s use in determining gene function in a high-throughput manner can be found below

www.rna.uzh.ch/events/journalclub/20140429JCCaihong.pdf

  1. Determining Off-target Effects of Gene Therapy Simplified with CRSPR

In GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases (from This Journal’s series on Live Meeting Coverage) at a 2014 Koch lecture

Shengdar Q Tsai and J Keith Joung describe

an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint ‘hotspots’.

SOURCE http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3117.html

II. CRISPR/Cas9 Use in Developing Models of Disease

 

  1. Developing Animal Tumor Models

In a post this year I discussed a talk at the recent 2015 AACR National Meeting on a laboratories ability to use CRISPR gene editing in-vivo to produce a hepatocarcinoma using viral delivery. The post can be seen here: Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

 

1) In this talk Dr. Tyler Jacks discussed his use of CRSPR to generate a mouse model of liver tumor in an immunocompetent mouse. Some notes from this talk are given below

  1. B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research
  • Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
  • Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
  • then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
  • figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
  • he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
  • also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin

 

See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”

The original research article can be found in the August 6, 2014 issue of Nature[1]

And see also on the Jacks Lab site under Research

2)     In the Upcoming Meeting New Frontiers in Gene Editing multiple uses of CRISPR technology is discussed in relation to gene knockout/function studies, tumor model development and

 

 

New Frontiers in Gene Editing

Session Spotlight:
BUILDING IN VIVO MODELS FOR DRUG DISCOVERY

Genome Editing Animal Models in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Biology-Discovery, Genetics and Pharmacogenomics, Merck Research Laboratories

Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice. In this talk, we will discuss the application of ZFN and CRISPR to generate various animal models for drug discovery programs.

In vivo Cancer Modeling and Genetic Screening Using CRISPR/Cas9
Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratories of Dr. Phillip A. Sharp and Dr. Feng Zhang, Koch Institute for Integrative Cancer Research at MIT and Broad Institute of Harvard and MIT

Here we describe a genome-wide CRISPR-Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library. The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late stage primary tumors were found to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis.

FEATURED PRESENTATION: In vivo Chromosome Engineering Using CRISPR-Cas9
Andrea Ventura, M.D., Ph.D., Assistant Member, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center

We will discuss our experience using somatic genome editing to engineer oncogenic chromosomal rearrangements in vivo. More specifically, we will present the results of our ongoing efforts aimed at modeling cancers driven by chromosomal rearrangements using viral mediated delivery of Crispr-Cas9 to adult animals.

RNAi and CRISPR/Cas9-Based in vivo Models for Drug Discovery
Christof Fellmann, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Jennifer Doudna, Department of Molecular and Cell Biology, The University of California, Berkeley

Genetically engineered mouse models (GEMMs) are a powerful tool to study disease initiation, treatment response and relapse. By combining CRISPR/Cas9 and “Sensor” validated, tetracycline-regulated “miR-E” shRNA technology, we have developed a fast and scalable platform to generate RNAi GEMMs with reversible gene silencing capability. The synergy of CRISPR/Cas9 and RNAi enabled us to not only model disease pathogenesis, but also mimic drug therapy in mice, providing us capability to perform preclinical studies in vivo.

In vivo Genome Editing Using Staphylococcus aureus Cas9
Fei Ann Ran, Ph.D., Post-doctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and Junior Fellow, Harvard Society of Fellows

The RNA-guided Cas9 nuclease from the bacterial CRISPR/Cas system has been adapted as a powerful tool for facilitating targeted genome editing in eukaryotes. Recently, we have identified an additional small Cas9 nuclease from Staphylococcus aureus that can be packaged with its guide RNA into a single adeno-associated virus (AAV) vector for in vivo applications. We demonstrate the use of this system for effective gene modification in adult animals and further expand the Cas9 toolbox for in vivo genome editing.

OriGene, Making the Right Tools for CRISPR Research
Xuan Liu, Ph.D., Senior Director, Marketing, OriGene

CRISPR technology has quickly revolutionized the scientific community. Its simplicity has democratized the genome editing technology and enabled every lab to consider its utility in gene function research. As the largest tool box for gene functional research, OriGene created a large collection of CRISPR-related tools, including various all-in-one vectors for gRNA cloning, donor vector backbones, genome-wide knockout kits, AAVS1 insertion vectors, etc. OriGene’s high quality products will accelerate CRISPR research.

 

  1. Transgenic Animals : Custom Mouse and Rat Model Generation Service Using CRISPR/Cas9 by AppliedStem Cell Inc. (http://www.appliedstemcell.com/)

A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION  Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

  • In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

 

 

 

The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.

Applied StemCell, Inc. offers various services related to animal models including conventional transgenic rats, and phenotype analysis using knock-in, knock-out strategies.

Further explanation of their use of CRSPR can be found at the site below:

https://pharmaceuticalintelligence.com/2014/10/29/gene-editing-at-crispr-speed-services-and-tools/

In addition, ReproCELL Inc., a Tokyo based stem cell company, uses CRSPR to develop

· Tailored disease model cells (hiPSC-Disease Model Cells)

  • 2 types of services
  • ReproUNUS™-g:human iPS cell derived functional cells involving gene editing by CRISPR/Cas9 system
  • eproUNUS™-p:patient derived iPS cell derived functional cells

III. Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

 

As of now it is unclear as to the strategy of pharma in how to use this technology for toxicology testing however a few companies have licensed the technology to use across their R&D platforms including

A recent paper used a sister technique TALEN to generate knock-in pigs which suggest that it would be possible to generate pigs with human transgenes, especially in human liver isozymes in orer to study hepatotoxicity of drugs.

 

Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

Jing Yao, Jiaojiao Huang, Tang Hai, Xianlong Wang, Guosong Qin, Hongyong Zhang, Rong Wu, Chunwei Cao, Jianzhong Jeff Xi, Zengqiang Yuan, Jianguo Zhao

Sci Rep. 2014; 4: 6926. Published online 2014 November 5. doi: 10.1038/srep06926

 

Other related articles on CRISPR/Cas9 were published in this Open Access Online Scientific Journal, include the following:

Search Results for ‘CRISPR’

Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

CRISPR/Cas9 genome editing tool for Staphylococcus aureus Cas9 complex (SaCas9) @ MIT’s Broad Institute

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Using CRISPR to investigate pancreatic cancer

Simple technology makes CRISPR gene editing cheaper

RNAi, CRISPR, and Gene Editing: Discussions on How To’s and Best Practices @14th Annual World Preclinical Congress June 10-12, 2015 | Westin Boston Waterfront | Boston, MA

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality

GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases

The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century

CRISPR: Applications for Autoimmune Diseases @UCSF

 

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Chapter 2 in 

R&D Alliances between Big Pharma and Academic Research Centers: Pharma’s Realization that Internal R&D Groups alone aren’t enough 

Israel’s Innovation System: A Triple Helix with Four Sub-helices

Prof. Henry Etzkowitz

It is fitting that the Triple Helix, with universities as a key innovation actor, along with industry and government, has been taken up in Israel, a knowledge-based society, rooted in Talmudic scholarship and scientific research. Biblical literature provided legitimation for the creation of the Jewish state while science helped create the economic base that made state formation feasible. In this case, the establishment of a government followed the creation of  (agricultural) industry and academia as the third element in a triple helix.  Nevertheless, a triple helix dynamic can be identified in the earliest phases of the formation of Israeli society, well before a formal state apparatus was constructed. Founding a state was a key objective of industry and academia but these intertwined helical strands did not accomplish the objective without assistance from other sources nor is innovation in contemporary Israel, along with many other societies, solely a triple helix phenomenon.

Several analysts have identified additional helices as relevant to innovation (Drori, Ch. 1). However, if everything is relevant than nothing is especially significant and a model that originally posited the transformation of the university from a secondary supporting institution of industrial society to a primary institution of a knowledge based society is vitiated. A second academic revolution expanded academic tasks from education and research to include entrepreneurship as a third mission. An entrepreneurial university, interacting closely with industry and government, is the core of a Triple Helix. By engaging in such relations an academic sector may, depending upon its previous experience, maintain or gain, relative independence. Triple Helix actors must also continually renew their commitment to entrepreneurship and innovation, lest they fall back into traditional roles and relationships.

What is the source of the Israeli Triple Helix? The contributors to this volume have identified seven helical strands as constitutive of the Israeli innovation system. I suggest that these strands may be grouped into primary and secondary categories: the primary strands are the classic triple helix (university-industry-government) while the secondary strands are supporting linkages, like the two diasporas (Israeli and foreign), or hybrid organizations like the military and non-governmental organizations (NGO’s). Thus, the resulting Israeli innovation system takes the form of a Trivium and a Quadrivium consisting of three primary and four secondary strands, in a variety of relationships with each other in different historical periods. The Innovation Trivium and Quadrivium are the constellation of core and supporting actors that constitute a knowledge-based innovation system. [1]

2.1 Triple Helix Origins

The triple helix innovation model originated in the analysis of MIT’s role in the renewal of New England, a region suffering industrial decline from the early 20th century (Etzkowitz, 2002).  MIT was founded in the mid 19th century, with industry and government support to raise the technological level of the regions’ industries but by the time it had developed research capabilities many of those industries had already left the region, to move closer to sources of raw materials, lines of distribution and less expensive labor. It was in this context, during the 1920’s, that the governors of New England called together the leadership of the region in a Council to address the region’s economic decline. Given a unique feature of the region, its extensive network of academic institutions, it is not surprising that the governors included the academic leadership of the region in their call.

However, their inclusion of academia had an unexpected consequence, transforming the usual public-private partnership model into a unique configuration- a proto-triple helix with a proclivity to originality. Triads are more flexible than dyads that typically take a strong common direction or devolve into opposition and stasis (Simmel, 1950).  Industry-government groups typically repeat timeworn strategies to attract industries from other regions in a zero sum game or attempt to revive local declining industries that may be beyond resuscitation. The inclusion of academia along with industry and government introduced an element of novelty into the government-industry dyad.  A moment of collective creativity occurred, during the discussions of the New England Council, inspired by the leadership of MIT’s President Karl Compton.  A triple helix dynamic, with the university as a key actor in an innovation strategy, was instituted that was highly unusual at the time.

The Council made an analysis of the strengths and weakness of the New England region and invented the venture capital firm to fill a gap in its innovation system, expanding a previously sporadic and uneven process of firm-formation from academic research into a powerful stream of start-ups and growth firms. A coalition of industry, government and university leaders invented a new model of knowledge-based economic and social development, building upon the superior academic resources of the region. This was not an isolated development but built upon previous financial and organizational innovations in the whaling industry and in academia.  In New England, industry and government, inspired by an academic entrepreneur and visionary, William Barton Rogers, earlier came together in the mid 19th century to found MIT, the first entrepreneurial university, thereby establishing the preconditions for a triple helix dynamic in that region.

2.2 From a Double to a Triple Helix

In a remote province of the Ottoman Empire in the early 20th century, Jewish agricultural settlements and an agricultural research institute created a triple helix dynamic that assisted the formation of the State of Israel. An industry-academia double helix provided the knowledge-based foundation for the Israeli triple helix. It preceded the founding of the state of Israel and indeed supplied many of the building blocks from which it was constructed. In a possibly unique configuration, state formation built upon scientific research and an agricultural industrial base. Before the Technion, the Weizmann Institute and the Hebrew University, there was the Jewish Agricultural Experiment Station in Atlit, founded in 1909 by agronomist Aaron Aaronsohn, with the support of Julius Rosenwald, an American-Jewish philanthropist (Florence, 2007).

Hints in the Bible of agricultural surplus, a land flowing with “milk and honey,” were investigated in an early 20th century context of desertification in Palestine.  The station’s researchers hypothesized that a seeming desert had a greater carrying capacity than was expected and thus could support a much larger population. Aronsohn and his colleagues’ advances in  “arid zone agriculture” opened the way to the transformation of a network of isolated agricultural settlements into a modern urban society.  The Atlit research program, conducted in collaboration with the US Department of Agriculture, was then introduced to California.

However, in California, arid zone methods were soon made superfluous by hydraulic transfer projects, from north to south, of enormous water resources. Arid agricultural methods remained relevant in the Israeli context of scarce water resources. Israel’s first high tech industry was based upon the development of drip irrigation techniques in the late 1950’s that preceded the IT wave by decades. Labor saving methods of agricultural production were also driven by ideological concerns of not wanting to be dependent upon hired Arab labor.  Science-based technology was thus at the heart of a developing Israeli society as well as a key link to a Diaspora that supplied infusions of support from abroad.

The Atlit agricultural research institute transformed itself into an intelligence network on behalf of the British during the First World War, betting that assisting the exit of Palestine from the Ottoman Empire could provide a pathway for the creation of a Jewish state (Florence, 2007). The Atlit network was uncovered, and some of its members perished, but it had already provided significant information on invasion routes that assisted the British takeover of Palestine. Its leader, Aaron Aaronsohn, died in a plane crash over the English channel in 1919 while bringing maps to the post-war Paris peace conference. The Institute itself did not survive its repurposing but its mission was taken up by other agricultural research units.

A linkage between helices and the translation of social capital from one sphere to another was another element of the state building project. The Balfour Declaration, issued by the British government in 1917, favored a “national home” for the Jewish people in Palestine, without prejudicing the rights of other peoples, and was the first such statement by a major power. Although the Declaration was part of a geopolitical balancing act to gain support for the British war effort, and may have occurred for that reason alone, British-Jewish scientist Chaim Weizmann’s accomplishments gave it a boost (Weizmann, 1949).

Weizmann’s invention of a bacterial method of producing the feedstock for explosives assisted the British war effort. Weizmann, a professor at Manchester University was able to transmute this discovery into support for a projected Jewish state through his relationship with Arthur Balfour, the Foreign Secretary, and an MP from Manchester. Weizmann dual roles as an eminent scientist and as a political leader in the Zionist movement coincided and he used an achievement in one arena to advance his goals in another. The Diaspora, of which he was a member in that era, aggregated international support for the state-building project.

Science also served to legitimate the new state of Israel. Albert Einstein was offered the presidency of the newly founded state of Israel. While the aura of his renown was one reason for the offer, that fame was primarily based on his scientific achievements. When Einstein turned down the position, the presidency was offered to another scientist, Chaim Weizmann, who accepted. The fact that the position was offered to two scientists in a row suggests that science was implicitly seen as legitimating the state, while also recognizing its role in the founding of Israel.

2.3 Innovation Trivium and Quadrivium

Identification of additional secondary contributors to innovation is a useful task but their relationship to the primary helices, and the roles that they play, should be specified. For example, the Israeli military may be viewed as a hybrid entity. In addition to the usual functions of a military, the Israel Defense Forces also serves as an educational institution for virtually the entire society, intermediating between secondary and university education and as an industrial development platform, spinning off aircraft and software industries. It has some of the characteristics of an independent helix but remains a part of the state, embodying hybrid elements that give it some of the characteristics of an independent institutional sphere.

It is a significant actor in Israeli society, having a significantly higher profile than the militaries in most societies. Therefore we locate it in the “Quadrivium” of support helices that comprise hybrid organizations or links with other societies. The military derived from the “Shomrim”, watches mounted by isolated settlements while nascent governmental institutions were a confluence between the networks of settlements and more general support structures such as the Jewish Agency, a mix of local and Diaspora efforts. A proto-state was constructed from these elements prior to independence.

The Israeli Diaspora played a key role, along with government, in founding Israel’s venture capital industry. After several unsuccessful attempts at developing a venture industry, government hit on the idea of combining public and private elements, providing government funds to encourage private partners to participate by reducing their risk. Key to the efforts success was the recruitment of members of the Israeli Diaspora, working in financial and venture capital firms in the US, to return to Israel and participate in the Yozma project and the funds that emanated from it. [2]

2.4 Israel: A Triple Helix Society

This volume, analyzing Israel’s innovation actors, makes a significant contribution to triple helix theory and practice by providing evidence of their relative salience. Identifying multiple contributors to the innovation project is a useful exercise but not all helices are equal. A key contribution of the triple helix model is that it identified the increased significance of the university in a knowledge based society and the fundamental importance of creative triple helix interactions and relationships to societies that wish to increase their innovation potential (Durrani et al., 2012).

We can also identify the qualities of an emergent social structure that encourages innovation. Multiple sources of initiative, organizational venues that combine different perspectives and experiences and persons with dual roles across the helices are more likely to produce innovation and hybridization than isolated rigid structures, even with great resources behind them. The Israeli experience takes the triple helix model a step beyond organizational innovation by demonstrating the significance of triple helix roles and relationships to the creation of an innovative society.

 References

Durrani, Tariq and Jann Hidajat Tjakraatmadja and Wawan Dhewanto Eds. 2012. 10th Triple Helix Conference 2012 Procedia – Social and Behavioral Sciences, Volume 52.

Etzkowitz, Henry. 2002. MIT and the Rise of Entrepreneurial Science. London: Routledge.

Etzkowitz, Henry, Marina Ranga and James Dzisah, 2012. “Wither the University? The Novum Trivium and the transition from industrial to knowledge society.” Social Science Information June 2012 51: 143-164.

Florence, Ronald. 2007. Lawrence and Aaronsohn: 
T. E. Lawrence, Aaron Aaronsohn, and the Seeds of the Arab-Israeli Conflict 
 
New York: Viking.

Simmel, Georg. 1950. Conflict and the Web of Group Affiliations. Glencoe: Free Press.

Weizmann, Chaim. 1949. Trial and Error: the autobiography of Chaim Weizmann. New York: Harper & Bros.

[1] The classic Trivium and Quadrivium were the core and supporting academic disciplines that constituted the knowledge-base of medieval Europe. See Etzkowitz, Ranga and Dzisah, 2012.

[2] Author discussion with Yozma founders at the 3rd Triple Helix Conference in Rio de Janeiro, 1999. FINEPE, the Brazil Development Agency invited Yozma representatives to the conference and held side meetings to arrange transfer of the Yozma model to Brazil. FINEPE added an additional element, “FINEPE University,” a series of workshops held around the country to train entrepreneurs in “pitching” to venture firms.

 

Other articles by same author were published in this Open Access Online Scientific Journal, include the following:

BEYOND THE “MALE MODEL”: AN ALTERNATIVE FEMALE MODEL OF SCIENCE, TECHNOLOGY AND INNOVATION

 Professor Henry Etzkowitz 8/1/2012

https://pharmaceuticalintelligence.com/2012/08/01/beyond-the-male-model-an-alternative-female-model-of-science-technology-and-innovation/

BEYOND THE “MALE MODEL”: AN ALTERNATIVE FEMALE MODEL OF SCIENCE, TECHNOLOGY AND INNOVATION

THE TRIPLE HELIX ASSOCIATION NEWSLETTER, VOLUME 1 ISSUE 3 JULY 2012

Hélice www.triplehelixassociation.org  Triple Helix X, 2012, Bandung,Indonesia . . . www.th2012.org

by Professor Henry Etzkowitz, President of the Triple Helix Association,  Senior Researcher, H-STAR Institute, Stanford University, Visiting Professor, Birkbeck, London University and Edinburgh University Business School

henry.etzkowitz@stanford.edu

Professor Henry Etzkowitz paper is based on his Keynote Address to the FemTalent Conference, Barcelona, Spain 2011

 

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Precision Medicine: The Future of Medicine?

Reporter: Aviva Lev-Ari, PhD, RN

Dr. Laurie Glimcher, dean of Weill Cornell Medical College, and Dr. Robert Langer, the Koch Institute Professor at MIT, talk to the “CBS This Morning” co-hosts about what’s next in the fight against diseases like Alzheimer’s, cancer, and diabetes.

VIEW VIDEO

http://www.cbsnews.com/video/watch/?id=50149783n

Free Webinar:

The Economics of Precision Medicine: 

How Personalizing Treatment can Bend the Cost Curve by 

Improving the Value Delivered by Healthcare Innovations

In a world where it is clear that healthcare costs must be contained, how can we afford to pay for innovation? This webinar will explore how personalizing treatment can offer an escape from the innovation-cost conundrum. By simultaneously increasing clinical development efficiency and the treatment effectiveness, targeting clinical innovations to the patients most likely to benefit can improve healthcare value per dollar spent while maintaining the ROI levels needed to support investment in innovation. We believe precision medicine should play a more prominent role in the cost containment discussion of healthcare reform.

By attending this Webinar, you will learn how to:

Help clients develop product development and commercialization strategies that get leverage from the benefits of precision medicine 

Support positioning of innovations as part of the healthcare solution, not the problem 

Understand and communicate the value proposition of precision medicine for payers, government decision makers, and legislators

The Economics of Precision Medicine: How Personalizing Treatment can Bend the Cost Curve by Improving the Value Delivered by Healthcare Innovations

Thursday, July 25, 2013

11:30 am PDT / 2:30 pm EDT

1 hour

Who should attend:

Franchise and Marketing Leaders

Therapeutic Area Leads

Medical Affairs

Government Affairs/Public Policy

Health Economics and Market Access

Webinar agenda:

Is the high cost of healthcare innovation incompatible with control of healthcare costs?

Cost-effectiveness criteria and how they can be met

Taking cost out of clinical development

Case Example: How everyone can win

Practical impact on development and commercialization strategies

Q&A

Speaker information: 

David Parker, Ph.D., Vice President, Market Access Strategy, Precision for Medicine

Vicki L. Seyfert-Margolis, Chief Scientific and Strategy Officer, Precision for Medicine

Harry Glorikian, Managing Director, Strategy, Precision for Medicine

Cambridge Healthtech Institute, 250 First Avenue, Suite 300, Needham, MA 02494

Tel: 781-972-5400 | Fax: 781-972-5425

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Curator: Aviva Lev-Ari, PhD, RN

On 3/13/2013 Forbes Science Writer, Metthew Herper, presented a curated article about the protein Cas9. With a compelling title like 

This Protein Could Change Biotech Forever, we drew over 40 comments. 

A tiny molecular machine used by bacteria to kill attacking viruses could change the way that scientists edit the DNA of plants, animals and fungi, revolutionizing genetic engineering. The protein, called Cas9, is quite simply a way to more accurately cut a piece of DNA.

“This could significantly accelerate the rate of discovery in all areas of biology, including gene therapy in medicine, the generation of improved agricultural goods, and the engineering of energy-producing microbes,” says Luciano Marraffini of Rockefeller University.

The ability to make modular changes in the DNA of bacteria and primitive algae has resulted in drug and biofuel companies such as Amyris and LS9. But figuring out how to make changes in the genomes of more complicated organisms has been tough.

http://www.forbes.com/sites/matthewherper/2013/03/19/the-protein-that-could-change-biotech-forever/?goback=.gde_48920_member_227143277

In this article we bring all the pieces to one place, telling the evolution of a series of discoveries, which together may have the Protein, Cas9,  changing the Biotech Industry forever with its contributions to Diagnosing Diseases and Gene Therapy by Precision Genome Editing and Cost-effective microRNA Profiling. 

MicroRNA detection on the cheap

MIT alumni’s startup provides rapid, cost-effective microRNA profiling, which is beneficial for diagnosing diseases.
Rob Matheson, MIT News Office
March 28, 2013
Current methods of detecting microRNA (miRNA) — gene-regulating molecules implicated in the onset of various diseases — can be time-consuming and costly: The custom equipment used in such tests costs more than $100,000, and the limited throughput of these systems further hinders progress.
Two MIT alumni are helping to rectify these issues through their fast-growing, Cambridge-headquartered startup, Firefly BioWorks Inc., which provides technology that allows for rapid miRNA detection in a large number of samples using standard lab equipment. This technology has helped the company thrive — and also has the potential to increase the body of research on miRNA, which could help lead to better disease diagnosis and screening.The company’s core technology, called Optical Liquid Stamping (OLS) — which was invented at MIT by Firefly co-founder and Chief Technical Officer Daniel C. Pregibon PhD ’08 — works by imprinting (or stamping) microparticle structures onto photosensitive fluids. The resulting three-dimensional hydrogel particles, encoded with unique “barcodes,” can be used for the detection of miRNAs across large numbers of samples. These particles are custom-designed for readout in virtually any flow cytometer, a cost-effective device that’s accessible to most scientists.“Our manufacturing process allows us to make very sophisticated particles that can be read on the most basic instruments,” says co-founder and CEO Davide Marini PhD ’03.The company’s first commercial product, FirePlex miRSelect, an miRNA-detection kit that uses an assay based on OLS-manufactured particles and custom software, began selling about a year ago. Since then, the company has drawn a steady influx of customers (primarily academic and clinical scientists) while seeing rapid revenue growth.

To date, most of the company’s revenue has come from backers who see value in Firefly’s novel technology. In addition to a cumulative $2.5 million awarded through Small Business Innovation Research grants — primarily from the National Cancer Institute — the company has attracted $3 million from roughly 20 independent investors. Its most recent funding came from a $500,000 grant from the Massachusetts Life Sciences Center.

Pregibon developed the technology in the lab of MIT chemical engineering professorPatrick Doyle, a Firefly co-founder who serves on the company’s scientific advisory board. Firefly’s intellectual property is partially licensed through the Technology Licensing Office at MIT, along with several other Firefly patents. Firefly’s technology, from OLS to miRNA detection, has been described in papers published in several leading journals, including ScienceNature MaterialsNature Protocols and Analytical Chemistry.

Shifting complexity from equipment to particle

The success of the technology, Marini says, derives from an early business decision to focus attention on the development of the hydrogel particle instead of the equipment needed. Essentially, this allowed the co-founders to focus on developing a high-quality miRNA assay and hit the market quickly with particles that are universally readable on basic lab instrumentation.

“Imagine sticking a microscopic barcode on a microscopic product,” Marini says. “How do you scan it? At the beginning we thought we would have to build our own scanner. This would have been an expensive proposition. Instead, by using a few clever tricks, we redesigned the barcode to make it readable by existing instruments. You can write these ‘barcodes,’ and all you need is one scanner to read different codes. To quote an investor: ‘It shifts the complexity from the equipment to the particle.’”

Firefly’s particles appear to a standard flow cytometer as a series of closely spaced cells; these data are recorded and the company’s FireCode software then regroups them into particle information, including miRNA target identification and quantity.

But why, specifically, did the company choose a flow cytometer as its primary “scanner”? Pregibon answers: “To start, there are nearly 100,000 cytometers worldwide. In addition, we are now seeing a trend where flow cytometers are getting smaller and closer to the bench — closer to the actual researcher. We’re finding that people are tight for money because of the economy and are trying to conserve capital as much as possible. In order to use our products, they can either buy a very inexpensive bench-top flow cytometer or use one that already exists in their core facility.”

In turn, opting out of equipment development and manufacturing costs has helped the company stay financially sound, says Marini, who worked in London’s financial sector before coming to MIT. As an additional perk, the manufacturers of flow cytometers have begun “courting” Firefly, Marini says, because “our products help expand the capability of their systems, which are now exclusively used to analyze cells.”

The company’s FirePlex kit allows researchers to assay (or analyze) roughly 70 miRNA targets simultaneously across 96 samples of a wide variety — including serum, plasma and crude cell digests — in approximately three hours.

This is actually a “middle-ground” assaying technique, Pregibon says, and saves researchers time and money: Until now, scientists were forced to use separate techniques to look at a few miRNA targets over thousands of samples, or vice versa.

Marini adds that if a scientist suspects a number of miRNAs, perhaps 50 or so, could be involved in a pancreatic-cancer pathway, the only way to know for sure is to test those 50 targets over hundreds of samples. “There’s nowhere to do this today in a cost-effective, timely manner. Our tech now allows that,” he says.

‘Over the bridge of validation’

Because miRNAs are so important in the regulation of genes, and ultimately proteins, they have implications in a broad range of diseases, from cancer to Alzheimer’s disease. Several studies have suggested these relationships, but the field currently lacks the validation required to definitively demonstrate clinical utility.

With that in mind, Pregibon hopes that Firefly’s technology will help push miRNA-based diagnoses “over the bridge of validation,” giving scientists the means to validate miRNA signatures they discover in diagnosing diseases such as cancer. “That’s where we want to fit in,” he says. “With the help of a technology like ours, you’ll start to see more tests hitting the market and ultimately, more people benefitting from early cancer detection.”

Firefly’s aim is to strengthen preventive medicine in the United States. “In the long term, we see these products helping in the shift from reactive to preventative medicine,” Marini says. “We believe we will see a proliferation of tools for detection of diseases. We want to move away from the system we have now, which is curing before it’s too late.”

Pregibon says Firefly’s technology can be used across several molecule classes that are important in development and disease research: proteins, messenger RNA and DNA, among many others. “Essentially, the possibilities are endless,” Pregibon says.

Editing the genome with high precision

New method allows scientists to insert multiple genes in specific locations, delete defective genes.
Anne Trafton, MIT News Office
 
Researchers at MIT, the Broad Institute and Rockefeller University have developed a new technique for precisely altering the genomes of living cells by adding or deleting genes. The researchers say the technology could offer an easy-to-use, less-expensive way to engineer organisms that produce biofuels; to design animal models to study human disease; and  to develop new therapies, among other potential applications.To create their new genome-editing technique, the researchers modified a set of bacterial proteins that normally defend against viral invaders. Using this system, scientists can alter several genome sites simultaneously and can achieve much greater control over where new genes are inserted, says Feng Zhang, an assistant professor of brain and cognitive sciences at MIT and leader of the research team.“Anything that requires engineering of an organism to put in new genes or to modify what’s in the genome will be able to benefit from this,” says Zhang, who is a core member of the Broad Institute and MIT’s McGovern Institute for Brain Research.Zhang and his colleagues describe the new technique in the Jan. 3 online edition ofScience. Lead authors of the paper are graduate students Le Cong and Ann Ran.Early effortsThe first genetically altered mice were created in the 1980s by adding small pieces of DNA to mouse embryonic cells. This method is now widely used to create transgenic mice for the study of human disease, but, because it inserts DNA randomly in the genome, researchers can’t target the newly delivered genes to replace existing ones.

In recent years, scientists have sought more precise ways to edit the genome. One such method, known as homologous recombination, involves delivering a piece of DNA that includes the gene of interest flanked by sequences that match the genome region where the gene is to be inserted. However, this technique’s success rate is very low because the natural recombination process is rare in normal cells.

More recently, biologists discovered that they could improve the efficiency of this process by adding enzymes called nucleases, which can cut DNA. Zinc fingers are commonly used to deliver the nuclease to a specific location, but zinc finger arrays can’t target every possible sequence of DNA, limiting their usefulness. Furthermore, assembling the proteins is a labor-intensive and expensive process.

Complexes known as transcription activator-like effector nucleases (TALENs) can also cut the genome in specific locations, but these complexes can also be expensive and difficult to assemble.

Precise targeting

The new system is much more user-friendly, Zhang says. Making use of naturally occurring bacterial protein-RNA systems that recognize and snip viral DNA, the researchers can create DNA-editing complexes that include a nuclease called Cas9 bound to short RNA sequences. These sequences are designed to target specific locations in the genome; when they encounter a match, Cas9 cuts the DNA.

This approach can be used either to disrupt the function of a gene or to replace it with a new one. To replace the gene, the researchers must also add a DNA template for the new gene, which would be copied into the genome after the DNA is cut.

Each of the RNA segments can target a different sequence. “That’s the beauty of this — you can easily program a nuclease to target one or more positions in the genome,” Zhang says.

The method is also very precise — if there is a single base-pair difference between the RNA targeting sequence and the genome sequence, Cas9 is not activated. This is not the case for zinc fingers or TALEN. The new system also appears to be more efficient than TALEN, and much less expensive.

The new system “is a significant advancement in the field of genome editing and, in its first iteration, already appears comparable in efficiency to what zinc finger nucleases and TALENs have to offer,” says Aron Geurts, an associate professor of physiology at the Medical College of Wisconsin. “Deciphering the ever-increasing data emerging on genetic variation as it relates to human health and disease will require this type of scalable and precise genome editing in model systems.”

The research team has deposited the necessary genetic components with a nonprofit called Addgene, making the components widely available to other researchers who want to use the system. The researchers have also created a website with tips and tools for using this new technique.

Engineering new therapies

Among other possible applications, this system could be used to design new therapies for diseases such as Huntington’s disease, which appears to be caused by a single abnormal gene. Clinical trials that use zinc finger nucleases to disable genes are now under way, and the new technology could offer a more efficient alternative.

The system might also be useful for treating HIV by removing patients’ lymphocytes and mutating the CCR5 receptor, through which the virus enters cells. After being put back in the patient, such cells would resist infection.

This approach could also make it easier to study human disease by inducing specific mutations in human stem cells. “Using this genome editing system, you can very systematically put in individual mutations and differentiate the stem cells into neurons or cardiomyocytes and see how the mutations alter the biology of the cells,” Zhang says.

In the Science study, the researchers tested the system in cells grown in the lab, but they plan to apply the new technology to study brain function and diseases.

The research was funded by the National Institute of Mental Health; the W.M. Keck Foundation; the McKnight Foundation; the Bill & Melinda Gates Foundation; the Damon Runyon Cancer Research Foundation; the Searle Scholars Program; and philanthropic support from MIT alumni Mike Boylan and Bob Metcalfe, as well as the newscaster Jane Pauley.

SOURCE:
Published online 2012 September 4. doi:  10.1073/pnas.1208507109
PMCID: PMC3465414
PNAS Plus

Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

ABSTRACT

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.

Keywords: nuclease, site-directed mutagenesis, RNA interference, DNA interference

Comparison with Other RNAi Complexes

The mechanism proposed here for the cleavage of dsDNA by the Cas9–crRNA complex differs significantly from that for the type I-E (former “Ecoli”) system (7). In the E. coli type I-E system crRNA and Cas proteins assemble into a large ribonucleoprotein complex, Cascade, that facilitates target recognition by enhancing sequence-specific hybridization between the crRNA and complementary target sequences (7). Target recognition is dependent on the PAM and governed by the seed crRNA sequence located at the 5′ end of the spacer region (24). However, although the Cascade–crRNA complex alone is able to bind dsDNA containing a PAM and a protospacer, it requires an accessory Cas3 protein for DNA cleavage. Cas3 is an ssDNA nuclease and helicase that is able to cleave ssDNA, producing multiple cuts (10). It has been demonstrated recently that Cas3 degrades E. coli plasmid DNA in vitro in the presence of the Cascade–crRNA complex (25). Thus, current data clearly show that the mechanistic details of the interference step for the type I-E system differ from those of type II systems, both in the catalytic machinery involved and the nature of the molecular mechanisms.

In type IIIB CRISPR/Cas systems, present in many archaea and some bacteria, Cmr proteins and cRNA assemble into an effector complex that targets RNA (612). In Pyrococcus furiosus the RNA-silencing complex, comprising six proteins (Cmr1–Cmr6) and crRNA, binds to the target RNA and cleaves it at fixed distance from the 3′ end. The cleavage activity depends on Mg2+ ions; however, individual Cmr proteins responsible for target RNA cleavage have yet to be identified. The effector complex of Sulfolobus solfataricus, comprising seven proteins (Cmr1–Cmr7) and crRNA, cuts invading RNA in an endonucleolytic reaction at UA dinucleotides (13). Importantly, these two archaeal Cmr–crRNA complexes perform RNA cleavage in a PAM-independent manner.

Overall, we have shown that the Cas9–crRNA complex in type II CRISPR/Cas systems is a functional homolog of Cascade in type I systems and represents a minimal DNAi complex. The simple modular organization of the Cas9–crRNA complex, in which specificity for DNA targets is encoded by crRNAs and the cleavage enzymatic machinery is brought by a single, multidomain Cas protein, provides a versatile platform for engineering universal RNA-guided DNA endonucleases. Indeed, by altering the RNA sequence within the Cas9–crRNA complex, programmable endonucleases can be designed both for in vitro and in vivo applications. To provide proof of principle of such a strategy, we engineered de novo into a CRISPR locus a spacer targeted to a specific sequence on a plasmid and demonstrated that such a plasmid is cleaved by the Cas9–crRNA complex at a sequence specified by the designed crRNA. Experimental demonstration that RuvC and HNH active-site mutants of Cas9 are functional as strand-specific nicking enzymes opens the possibility of generating programmed DNA single-strand breaks de novo. Taken together, these findings pave the way for the development of unique molecular tools for RNA-directed DNA surgery.

SOURCE:

Cheap and easy technique to snip DNA could revolutionize gene therapy

By Robert Sanders, Media Relations | January 7, 2013

BERKELEY —A simple, precise and inexpensive method for cutting DNA to insert genes into human cells could transform genetic medicine, making routine what now are expensive, complicated and rare procedures for replacing defective genes in order to fix genetic disease or even cure AIDS.

Cas9 protein on DNA
The bacterial enzyme Cas9 is the engine of RNA-programmed genome engineering in human cells. Graphic by Jennifer Doudna/UC Berkeley.
IMAGE SOURCE:

Discovered last year by Jennifer Doudna and Martin Jinek of the Howard Hughes Medical Institute and University of California, Berkeley, and Emmanuelle Charpentier of the Laboratory for Molecular Infection Medicine-Sweden, the technique was labeled a “tour de force” in a 2012 review in the journal Nature Biotechnology.

That review was based solely on the team’s June 28, 2012, Science paper, in which the researchers described a new method of precisely targeting and cutting DNA in bacteria.

Two new papers published last week in the journal Science Express demonstrate that the technique also works in human cells. A paper by Doudna and her team reporting similarly successful results in human cells has been accepted for publication by the new open-access journal eLife.

“The ability to modify specific elements of an organism’s genes has been essential to advance our understanding of biology, including human health,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute Investigator at UC Berkeley. “However, the techniques for making these modifications in animals and humans have been a huge bottleneck in both research and the development of human therapeutics.

“This is going to remove a major bottleneck in the field, because it means that essentially anybody can use this kind of genome editing or reprogramming to introduce genetic changes into mammalian or, quite likely, other eukaryotic systems.”

“I think this is going to be a real hit,” said George Church, professor of genetics at Harvard Medical School and principal author of one of the Science Express papers. “There are going to be a lot of people practicing this method because it is easier and about 100 times more compact than other techniques.”

“Based on the feedback we’ve received, it’s possible that this technique will completely revolutionize genome engineering in animals and plants,” said Doudna, who also holds an appointment at Lawrence Berkeley National Laboratory. “It’s easy to program and could potentially be as powerful as the Polymerase Chain Reaction (PCR).”

The latter technique made it easy to generate millions of copies of small pieces of DNA and permanently altered biological research and medical genetics.

Cruise missiles

Two developments – zinc-finger nucleases and TALEN (Transcription Activator-Like Effector Nucleases) proteins – have gotten a lot of attention recently, including being together named one of the top 10 scientific breakthroughs of 2012 by Science magazine. The magazine labeled them “cruise missiles” because both techniques allow researchers to home in on a particular part of a genome and snip the double-stranded DNA there and there only.

Researchers can use these methods to make two precise cuts to remove a piece of DNA and, if an alternative piece of DNA is supplied, the cell will plug it into the cut instead. In this way, doctors can excise a defective or mutated gene and replace it with a normal copy. Sangamo Biosciences, a clinical stage biospharmaceutical company, has already shown that replacing one specific gene in a person infected with HIV can make him or her resistant to AIDS.

Both the zinc finger and TALEN techniques require synthesizing a large new gene encoding a specific protein for each new site in the DNA that is to be changed. By contrast, the new technique uses a single protein that requires only a short RNA molecule to program it for site-specific DNA recognition, Doudna said.

In the new Science Express paper, Church compared the new technique, which involves an enzyme called Cas9, with the TALEN method for inserting a gene into a mammalian cell and found it five times more efficient.

“It (the Cas9-RNA complex) is easier to make than TALEN proteins, and it’s smaller,” making it easier to slip into cells and even to program hundreds of snips simultaneously, he said. The complex also has lower toxicity in mammalian cells than other techniques, he added.

“It’s too early to declare total victory” over TALENs and zinc-fingers, Church said, “but it looks promising.”

Based on the immune systems of bacteria

Doudna discovered the Cas9 enzyme while working on the immune system of bacteria that have evolved enzymes that cut DNA to defend themselves against viruses. These bacteria cut up viral DNA and stick pieces of it into their own DNA, from which they make RNA that binds and inactivates the viruses.

UC Berkeley professor of earth and planetary science Jill Banfield brought this unusual viral immune system to Doudna’s attention a few years ago, and Doudna became intrigued. Her research focuses on how cells use RNA (ribonucleic acids), which are essentially the working copies that cells make of the DNA in their genes.

Doudna and her team worked out the details of how the enzyme-RNA complex cuts DNA: the Cas9 protein assembles with two short lengths of RNA, and together the complex binds a very specific area of DNA determined by the RNA sequence. The scientists then simplified the system to work with only one piece of RNA and showed in the earlier Science paper that they could target and snip specific areas of bacterial DNA.

“The beauty of this compared to any of the other systems that have come along over the past few decades for doing genome engineering is that it uses a single enzyme,” Doudna said. “The enzyme doesn’t have to change for every site that you want to target – you simply have to reprogram it with a different RNA transcript, which is easy to design and implement.”

The three new papers show this bacterial system works beautifully in human cells as well as in bacteria.

“Out of this somewhat obscure bacterial immune system comes a technology that has the potential to really transform the way that we work on and manipulate mammalian cells and other types of animal and plant cells,” Doudna said. “This is a poster child for the role of basic science in making fundamental discoveries that affect human health.”

Doudna’s coauthors include Jinek and Alexandra East, Aaron Cheng and Enbo Ma of UC Berkeley’s Department of Molecular and Cell Biology.

Doudna’s work was sponsored by the Howard Hughes Medical Institute.

RELATED INFORMATION

SOURCE:
http://newscenter.berkeley.edu/2013/01/07/cheap-and-easy-technique-to-snip-dna-could-revolutionize-gene-therapy/

Matthew Herper, Forbes Staff on 3/24/2013

 A Cancer Patient’s Quest Hits DNA Pay Dirt

 

Kathy Giusti

Kathy Giusti has faced her cancer with the verve of an entrepreneur. Now her fight with multiple myeloma has moved to a new front: DNA.

Giusti was a 37-year-old marketing executive at Searle (now part of Pfizer) when she was diagnosed in 1996 with myeloma, a deadly blood and bone marrow cancer. She had a 1-year-old daughter. Sixty percent of myeloma patients die within five years, but Giusti beat the odds, living for a decade and a half through multiple rounds of drug therapy and a bone marrow transplant from her twin sister.

She has also changed the way her disease is treated. Giusti founded an advocacy group, the Multiple Myeloma Research Foundation, that works with companies like NovartisCelgene, and Merck to develop new treatments. It played a key role in the development of Velcade and Revlimid, two of the biggest advances in treating the disease, which is diagnosed in 20,000 patients a year.

Now a new research effort, funded with $14 million of MMRF money, has revealed new hints at what causes the disease and potential avenues for treating it. “This is going to be the next wave of how health care gets changed over time,” Giusti says. The results are published in the current issue of Nature.

Working with patient samples collected by the MMRF and using DNA sequencers made by Illumina of San Diego, researchers at the Broad Institute of MIT and Harvard sequenced the genes of 38 myeloma tumors and the DNA of the patients in whom they were growing. Tumors are twisted versions of the people in which they are growing; their DNA is mutated and disfigured, turning them deadly. By comparing DNA from healthy cells with malignant ones, researchers can find genetic differences that might be what led the tumors to go bad in the first place.

This experiment would have been unthinkable just a few years ago, when sequencing a human being was so expensive that all the people whose DNA had been read out could fit in a small room. In 2005, the idea of producing 38 DNA sequences was laughable. Now it’s par for the course, and researchers expect thousands of genomes will be sequenced by the end of the year – and experiments like this are expected to become commonplace.

What’s so exciting is that sometimes the DNA changes scientists find are completely unexpected. “There were genes we found to be recurrently mutated and yet no one had any clue that they had anything to do with multiple myeloma or any other cancer,” says Todd Golub, the Broad researcher who led the study. He splits his time with the Dana-Farber Cancer Institute.

One gene, called FAM46C, was mutated in 13% of the cancers, but has never been studied in humans. “It appears no one had been working on it,” says Golub, but from studies in yeast and bacteria it appears that it has to do with how the recipes in genes are used to make proteins, the building blocks of just about everything in the body.

Another surprise gene, called BRAF, is generating excitement because it is the target of a skin cancer drug developed by Plexxikon, a small biotech firm that is partnered with Roch and is being purchased by Daiichi Sankyo. For the 4% of myeloma patients who have this mutation, this drug might be an option. The challenge will be testing it: it will be difficult to find enough of these patients to conduct a clinical trial. The MMRF says early discussions on such a study are moving forward. Giusti imagines that in the future, the MMRF may fund studies not of myeloma, but of a mix of different cancers caused by similar genetic mutations.

Several of the genes seem involved in the proteins that help guide epigenetics, a kind of molecular code written on DNA that may represent another kind of genetic code. The MMRF is already supporting some small drug companies that hope to create cancer drugs that target this second code.

Golub, the Broad scientist, says that right now it doesn’t make sense for most multiple myeloma patients to get their full DNA sequences outside of clinical trials, although he can imagine that for patients who have failed every available treatment it might make sense as a way to come up with another drug to try.

Giusti says, however, that the kinds of genetic tests that are done are changing the way that patients understand their disease. “Patients like me are starting to know, ‘I have this DNA translocation, maybe a proteasome inhibitor [a type of drug] is better for me.’ We become forerunners in the role patient can plan and the importance it has in drug development.”

Moving past old ways of thinking about inventing new medicines to a new path that is based on genetics and a flood of biological data is going to be difficult. But Giusti has never been afraid of hard — and she is sure there will be ways to drive the science forward.

SOURCE:

http://www.forbes.com/sites/matthewherper/2011/03/24/a-cancer-patients-quest-hits-dna-pay-dirt/

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465414/

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Curator: Aviva Lev-Ari, PhD, RN

Chaperon Protein Mechanism inspired MIT Team to Model the Role of Genetic Mutations on Cancer Progression, proposing the next generation of Oncology drugs to aim at Suppression of Passenger Mutations. Current drug, in clinical trials, use the Chaperon Protein Mechanism to suppress Driver Mutations.

Deleterious Mutations in Cancer Progression

Kirill S. Korolev1, Christopher McFarland2, and Leonid A. Mirny3

1Department of Physics, MIT, Cambridge, MA.

E-mail: papers.korolev@gmail.com

2Graduate Program in Biophysics, Harvard University, Cambridge, MA.

3Health Sciences and Technology, MIT, Cambridge, MA

The research was funded by the National Institutes of Health/National Cancer Institute Physical Sciences Oncology Center at MIT.

SOURCE:

http://cnls.lanl.gov/q-bio/wiki/images/4/40/Abstract.pdf

Deleterious passenger mutations significantly affect evolutionary dynamics of cancer. Including passenger mutations in evolutionary models is necessary to understand the role of genetic diversity in cancer progression and to create new treatments based on the accumulation of deleterious passenger mutations.

Evolutionary models of cancer almost exclusively focus on the acquisition of driver mutations, which are beneficial to cancer cells. The driver mutations, however, are only a small fraction of the mutations found in tumors. The other mutations, called passenger mutations, are typically neglected because their effect on fitness is assumed to be very small. Recently, it has been suggested that some passenger mutations are slightly deleterious. We find that deleterious passengers significantly affect cancer progression. In particular, they lead to a critical tumor size, below which tumors shrink on average, and to an optimal mutation rate for cancer evolution.

ANCER is an outcome of somatic evolution [1-3]. To outcompete their benign sisters, cancer cells need to acquire many heritable changes (driver mutations) that enable proliferation. In addition to the rare beneficial drivers, cancer cells must also acquire neutral or slightly deleterious passenger mutations [4]. Indeed, the number of possible passengers exceeds the number of possible drivers by orders of magnitude. Surprisingly, the effect of passenger mutations on cancer progression has not been explored. To address this problem, we developed an evolutionary model of cancer progression, which includes both drivers and passengers. This model was analyzed both numerically and analytically to understand how mutation rate, population size, and fitness effects of mutations affect cancer progression.

RESULTS

Upon including passengers in our model, we found that cancer is no longer a straightforward progression to malignancy. In particular, there is a critical population size such that smaller populations accumulate passengers and decline, while larger populations accumulate drivers and grow. The transition to cancer for small initial populations is, therefore, stochastic in nature and is similar to diffusion over an energy barrier in chemical kinetics. We also found that there is an optimal mutation rate for cancer development, and passengers with intermediate fitness costs are most detrimental to cancer. The existence of an optimal mutation rate could explain recent clinical data [5] and is in stark contrast to the predictions of the models neglecting passengers. We also show that our theory is consistent with recent sequencing data.

SOURCE:

http://cnls.lanl.gov/q-bio/wiki/images/4/40/Abstract.pdf

Just as some mutations in the genome of cancer cells actively spur tumor growth, it would appear there are also some that do the reverse, and act to slow it down or even stop it, according to a new US study led by MIT.

Senior author, Leonid Mirny, an associate professor of physics and health sciences and technology at MIT, and colleagues, write about this surprise finding in a paper to be published online this week in the Proceedings of the National Academy of Sciences.

In a statement released on Monday, Mirny tells the press:

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers.”

“Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations,” he suggests.

Cancer Cell‘s Genome Has “Drivers” and “Passengers”

Your average cancer cell has a genome littered with thousands of mutations and hundreds of mutated genes. But only a handful of these mutated genes are drivers that are responsible for the uncontrolled growth that leads to tumors.

Up until this study, cancer researchers have mostly not paid much attention to the “passenger” mutations, believing that because they were not “drivers”, they had little effect on cancer progression. 

Now Mirny and colleagues have discovered, to their surprise, that the “passengers” aren’t there just for the ride. In sufficient numbers, they can slow down, and even stop, the cancer cells from growing and replicating as tumors. 

New Drugs Could Target the Passenger Mutations in Protein Chaperoning

Although there are already several drugs in development that target the effect of chaperone proteins in cancer, they are aiming to suppress driver mutations.

Recently, biochemists at the University of Massachusetts Amherst“trapped” a chaperone in action, providing a dynamic snapshot of its mechanism as a way to help development of new drugs that target drivers.

But Mirny and colleagues say there is now another option: developing drugs that target the same chaperoning process, but their aim would be to encourage the suppressive effect of the passenger mutations.

They are now comparing cells with identical driver mutations but different passenger mutations, to see which have the strongest effect on growth.

They are also inserting the cells into mice to see which are the most likely to lead to secondary tumors (metastasize).

Written by Catharine Paddock PhD
Copyright: Medical News Today

SOURCE:

http://www.medicalnewstoday.com/articles/255920.php

After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.
Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.

Drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

REFERENCE

Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

Biochemists Trap A Chaperone Machine In Action

Main Category: Biology / Biochemistry
Article Date: 11 Dec 2012 – 0:00 PST

Molecular chaperones have emerged as exciting new potential drug targets, because scientists want to learn how to stop cancer cells, for example, from using chaperones to enable their uncontrolled growth. Now a team of biochemists at the University of Massachusetts Amherst led by Lila Gierasch have deciphered key steps in the mechanism of the Hsp70 molecular machine by “trapping” this chaperone in action, providing a dynamic snapshot of its mechanism.

She and colleagues describe this work in the current issue of Cell. Gierasch’s research on Hsp70 chaperones is supported by a long-running grant to her lab from NIH’s National Institute for General Medical Sciences.

Molecular chaperones like the Hsp70s facilitate the origami-like folding of proteins, made in the cell’s nanofactories or ribosomes, from where they emerge unstructured like noodles. Proteins only function when folded into their proper structures, but the process is so difficult under cellular conditions that molecular chaperone helpers are needed. 

The newly discovered information about chaperone action is important because all rapidly dividing cells use a lot of Hsp70, Gierasch points out. “The saying is that cancer cells are addicted to Hsp70 because they rely on this chaperone for explosive new cell growth. Cancer shifts our body’s production of Hsp70 into high gear. If we can figure out a way to take that away from cancer cells, maybe we can stop the out-of-control tumor growth. To find a molecular way to inhibit Hsp70, you’ve got to know how it works and what it needs to function, so you can identify its vulnerabilities.”

Chaperone proteins in cells, from bacteria to humans, act like midwives or bodyguards, protecting newborn proteins from misfolding and existing proteins against loss of structure caused by stress such as heat or a fever. In fact, the heat shock protein (Hsp) group includes a variety of chaperones active in both these situations.

As Gierasch explains, “New proteins emerge into a challenging environment. It’s very crowded in the cell and it would be easy for them to get their sticky amino acid chains tangled and clumped together. Chaperones bind to them and help to avoid this aggregation, which is implicated in many pathologies such as neurodegenerative diseases. This role of chaperones has also heightened interest in using them therapeutically.”

However, chaperones must not bind too tightly or a protein can’t move on to do its job. To avoid this, chaperones rapidly cycle between tight and loose binding states, determined by whether ATP or ADP is bound. In the loose state, a protein client is free to fold or to be picked up by another chaperone that will help it fold to do its cellular work. In effect, Gierasch says, Hsp70s create a “holding pattern” to keep the protein substrate viable and ready for use, but also protected.

She and colleagues knew the Hsp70’s structure in both tight and loose binding affinity states, but not what happened between, which is essential to understanding the mechanism of chaperone action. Using the analogy of a high jump, they had a snapshot of the takeoff and landing, but not the top of the jump. “Knowing the end points doesn’t tell us how it works. There is a shape change in there that we wanted to see,” Gierasch says.

To address this, she and her colleagues postdoctoral fellows Anastasia Zhuravleva and Eugenia Clerico obtained “fingerprints” of the structure of Hsp70 in different states by using state-of-the-art nuclear magnetic resonance (NMR) methods that allowed them to map how chemical environments of individual amino acids of the protein change in different sample conditions. Working with an Hsp70 known as DnaK from E. coli bacteria, Zhuravleva and Clerico assigned its NMR spectra. In other words, they determined which peaks came from which amino acids in this large molecule.

The UMass Amherst team then mutated the Hsp70 so that cycling between tight and loose binding states stopped. As Gierasch explains, “Anastasia and Eugenia were able to stop the cycle part-way through the high jump, so to speak, and obtain the molecular fingerprint of a transient intermediate.” She calls this accomplishment “brilliant.”

Now that the researchers have a picture of this critical allosteric state, that is, one in which events at one site control events in another, Gierasch says many insights emerge. For example, it appears nature uses this energetically tense state to “tune” alternate versions of Hsp70 to perform different cellular functions. “Tuning means there may be evolutionary changes that let the chaperone work with its partners optimally,” she notes.

“And if you want to make a drug that controls the amount of Hsp70 available to a cell, our work points the way toward figuring out how to tickle the molecule so you can control its shape and its ability to bind to its client. We’re not done, but we made a big leap,” Gierasch adds. “We now have a idea of what the Hsp70 structure is when it is doing its job, which is extraordinarily important.” 

Article adapted by Medical News Today from original press release. Click ‘references’ tab above for source.
Visit our biology / biochemistry section for the latest news on this subject.
SOURCE:

REFERENCES

[1] Michor F, Iwasa Y, and Nowak MA (2004) Dynamics of cancer

progression. Nature Reviews Cancer 4, 197-205.

[2] Crespi B and Summers K (2005) Evolutionary biology of cancer.

Trends in Ecology and Evolution 20, 545-552.

[3] Merlo LMF, et al. (2006) Cancer as an evolutionary and ecological

process. Nature Reviews Cancer 6, 924-935.

[4] McFarland C, et al. “Accumulation of deleterious passenger mutations

in cancer,” in preparation.

[5] Birkbak NJ, et al. (2011) Paradoxical relationship between

chromosomal instability and survival outcome in cancer. Cancer

Research 71,3447-3452.

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Hold on. Mutations in Cancer do good.

https://pharmaceuticalintelligence.com/2013/02/04/hold-on-mutations-in-cancer-do-good/

Rational Design of Allosteric Inhibitors and Activators Using the Population-Shift Model: In Vitro Validation and Application to an Artificial Biosensor

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LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2

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Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes

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SNAP: Predict Effect of Non-synonymous Polymorphisms: How well Genome Interpretation Tools could Translate to the Clinic

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Drugging the Epigenome

https://pharmaceuticalintelligence.com/2013/02/01/drugging-the-epigenome/

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Reporter: Prabodh Kandala, PhD

A typical cancer cell has thousands of mutations scattered throughout its genome and hundreds of mutated genes. However, only a handful of those genes, known as drivers, are responsible for cancerous traits such as uncontrolled growth. Cancer biologists have largely ignored the other mutations, believing they had little or no impact on cancer progression.

But a new study from MIT, Harvard University, the Broad Institute and Brigham and Women’s Hospital reveals, for the first time, that these so-called passenger mutations are not just along for the ride. When enough of them accumulate, they can slow or even halt tumor growth.

The findings, reported in this week’sProceedings of the National Academy of Sciences, suggest that cancer should be viewed as an evolutionary process whose course is determined by a delicate balance between driver-propelled growth and the gradual buildup of passenger mutations that are damaging to cancer, says Leonid Mirny, an associate professor of physics and health sciences and technology at MIT and senior author of the paper.

Furthermore, drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

Lead author of the paper is Christopher McFarland, a graduate student at Harvard. Other authors are Kirill Korolev, a Pappalardo postdoctoral fellow at MIT, Gregory Kryukov, a senior computational biologist at the Broad Institute, and Shamil Sunyaev, an associate professor at Brigham and Women’s.

Power struggle

Cancer can take years or even decades to develop, as cells gradually accumulate the necessary driver mutations. Those mutations usually stimulate oncogenes such as Ras, which promotes cell growth, or turn off tumor-suppressing genes such as p53, which normally restrains growth.

Passenger mutations that arise randomly alongside drivers were believed to be fairly benign: In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

To test this theory, the researchers created a computer model that simulates cancer growth as an evolutionary process during which a cell acquires random mutations. These simulations followed millions of cells: every cell division, mutation and cell death.

They found that during the long periods between acquisition of driver mutations, many passenger mutations arose. When one of the cancerous cells gains a new driver mutation, that cell and its progeny take over the entire population, bringing along all of the original cell’s baggage of passenger mutations. “Those mutations otherwise would never spread in the population,” Mirny says. “They essentially hitchhike on the driver.”

This process repeats five to 10 times during cancer development; each time, a new wave of damaging passengers is accumulated. If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers,” Mirny says. “Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

Tipping the balance

In computer simulations, the researchers tested the possibility of treating tumors by boosting the impact of deleterious mutations. In their original simulation, each deleterious passenger mutation reduced the cell’s fitness by about 0.1 percent. When that was increased to 0.3 percent, tumors shrank under the load of their own mutations.

The same effect could be achieved in real tumors with drugs that interfere with proteins known as chaperones, Mirny suggests. After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.

Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.

Ref:

Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

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