Drug Discovery and Biotechnology Trends – Genomics 1: Functional Genomics, Young but Growing

Although functional genomics remains young enough that people argue over its definition, few squabble over the value of this field. Advances in areas from gene expression to proteomics promise to push ahead basic research, biotechnology, and medicine. In fact, some experts predict an annual compound growth rate of 28 percent for the next six years in commercial sectors of functional genomics.

by Mike May and Gary Heebner

ADVERTISERS Carl Zeiss [USA] instruments and systems for imaging analysis, digital cameras 914-747-1800 www.carlzeiss.com/micro Carl Zeiss [Germany] +49 (0)551-5060 660 Genome Canada the primary funding and information resource relating to genomics and proteomics in Canada 613-751-4460 www.genomecanada.ca Leica [USA] instruments and systems for imaging analysis, digital cameras 847-405-0123 www.simply-microscopy.com Leica [Germany] +49 6441 29-0 Takara Bio, Inc. kits and reagents for molecular biology research +81 77 543 9254 www.takara-bio.co.jp/english

IN THIS ISSUE: • Open reading frames • Real-time quantitative PCR • Bioarrays for gene expression • Proteomics • Whole genome microarrays • Two types of two-hybrids • RNA interference • Bioinformatics software

The companies in this article were selected at random. Their inclusion in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Although having the human genome sequenced still seems new, genomics research rushes ahead to the next step, so-called functional genomics. What this field covers depends largely on who is being asked, but many scientists agree that functional genomics ranges from expression and sequencing of genes to proteomics and protein-protein interactions. (For a useful resource, see “News Flash: Functional Genomics,” later in this feature.) The ultimate range of this field will surely depend on how it evolves. As Manoj Kenkare, a senior analyst at Frost & Sullivan, said, “Functional genomics is a very young market.”

Nevertheless, functional genomics will not outgrow all previous technologies, especially gene expression. Kenkare sees growing demand for studying gene expression with microarrays, which increases the efficiency of toxicity testing in drug discovery. He added, “Controlling genetics expression and regulation may lead to treatments for diseases ranging from cancer to infection, and even to aging.” Genomics will also create continued benefits in agriculture, especially in terms of plant productivity and resistance to pests and harsh conditions. Overall, Kenkare said that the market for functional genomics is expected to increase at an annual compound growth rate of 28 percent for the next six years.

Executives in the commercial sector also expect advances emerging from functional genomics. At Amersham Biosciences, Mike Evans, vice president of marketing and strategy, anticipates that functional genomics will drive big benefits in drug discovery. “People doing drug discovery are ever so keen to get to biological significance very quickly,” said Evans.

Kenkare sees crucial advances lying just ahead for functional genomics. For one thing, the anticipated growth in this field makes this a market worthy of investment. “Application areas—such as clinical diagnostics, agrobiotech, and pharmacodynamics—are going to be of growing relevance to genomics in the future,” said Kenkare. He also sees microarrays becoming a research standard in genetics, which he said, “allows for very profitable partnerships between varied research areas.” For an applications oriented conference see “A Drug Discovery and Development Extravaganza,” later in this feature.

ORFs All Around
Understanding how a genome affects function at the gene and protein level makes up the foundation of functional genomics. “To accomplish that,” said Scott Provost, director of genomics at Invitrogen, “you need materials to work on and study. The raw material is going to be a copy of the gene of interest.” To get that gene, investigators either clone it or purchase it. If a scientist wants to develop a library of DNA clones, companies including Clontech, Epicentre, Invitrogen, and Promega provide kits and systems. In addition, Elchrom Scientific makes a DNA Recovery Kit that captures DNA after a polymerase chain reaction (PCR). That DNA can then be used in sequencing or cloning.

On the other hand, companies including ATCC, Invitrogen, and OriGene make large numbers of clones that can be ordered. More than just providing a clone, however, Provost said, “We believe it is important to be able to take a clone from one expression system to another, be able to move it around seamlessly and make sure it did not get damaged genetically.” To do that, Invitrogen developed its Gateway Technology for cloning and expression, which allows rapid cloning of one or more genes into a wide range of protein expression systems—from bacteria to yeast to C. elegans to mammalian systems. Rob Bennett, director of research and development at Invitrogen, said, “We see Gateway as the biological operating system.” He added that Gateway provides ORFs, or open reading frames, so that investigators can study them in multiple expression systems, look at protein-protein interactions, or identify targets for RNA interference (described below).

Recently, Invitrogen added another feature to Gateway—making the results less restrictive. “If you have Gateway clones,” Provost said, “we want you to share that. Researchers can take building blocks of Gateway and distribute those with no licensing fees or royalties.”

Jon Chesnut, group leader in charge of cloning technologies at Invitrogen, said that many scientists already use Gateway to move clones between expression systems. For example, he said, “Stephan Wiemann’s group at the EMBL [European Molecular Biology Laboratory] in Heidelberg took a pool of genes—about 100 cDNAs—and used Gateway to move the clones into mammalian expression constructs which fused fluorescent tags with the genes. After transfection, the expressed proteins were followed by microscopy to examine their compartmentalization and to classify them by where they go in the cell.”

Express Your Self
In functional genomics, today’s scientists often study two areas of gene expression: profiling and quantitation, said Julie Moore, marketing manager for the gene expression division of Bio-Rad Laboratories. She said, “Microarrays assess expression patterns of lots of genes at once for profiling, so that scientists can examine associations between different genes and biochemical pathways.”

A key improvement in expression analysis comes from real-time quantitative PCR, which lets an investigator watch gene expression occur and measure its levels. Bio-Rad offers that capability in its iCycler iQ real-time PCR detection system. Moore said, “This system can multiplex up to four targets in a single tube, so you can compare one to three genes to a housekeeping, or control, gene.” Bio-Rad also offers the MyiQ real-time PCR detection system for scientists who work with the most common detection chemistries—SYBR Green I and FAM. USB also makes kits for real-time PCR in one-tube and two-tube versions, plus tools for PCR amplification, purification, and analysis.

A Drug Discovery and Development Extravaganza IBC’s ScreenTech World Summit will take place in San Diego, March 21-24. This conference will include four in-depth programs: protein kinases and phosphatases; protease inhibitors; neurodegenerative diseases; and high throughput screening and assay technologies. This format of parallel conference programming will allow delegates to attend a program in their specific discipline, and increase their knowledge and contacts through joining in additional talks from the other sessions. Plus, attendees will receive documentation from all programs at no additional charge. Organizers also expect more than 40 industrial exhibitors. Apply online at the conference website. www.LifeSciencesInfo.com/ScreenTech

Amersham also provides tools for gene expression. For example, Evans said, “Our CodeLink is a bioarray approach to gene expression. It provides increased sensitivity and better reproducibility.” These arrays use a surface technology that Amersham acquired from Motorola about a year ago. In addition, scientists at the U.S. Environmental Protection Agency use CodeLink when searching for biomarkers related to aquatic toxicity.

Pulling Out the Proteins
“Some people include proteomics as a major subset of functional genomics,” said, Dave Hicks, senior director of the proteomics business group at Applied Biosystems. To understand the function of a specific gene, researchers want to know what protein that gene encodes, and when and where the protein gets expressed. To follow protein expression, researchers often turn first to two-dimensional gel electrophoresis and then to mass spectrometry (MS) techniques. A number of companies like Amersham Biosciences, Bio-Rad Laboratories, and Invitrogen offer complete systems to measure protein expression.

Next, a scientist recovers proteins from a spot on a gel and studies them further with MS, which can create peptide mass fingerprints. These systems are offered by companies like Agilent, Bruker Daltonics, and Waters. At Applied Biosystems, Hicks said, “The most significant move is the introduction of our 4700 Proteomics Discovery System.” He went on: “In proteomics, one key application is the need to measure and identify large numbers of proteins between and across samples and, if possible, measure the quantity or relative quantity between samples. The 4700 system provides that capability.” The 4700 analyzer also comes with a six-month subscription to the Celera Discovery System, which provides mouse and human genome data from public and proprietary databases, and a variety of other genomic tools.

As Hicks said, “It is not good enough to simply give lists of proteins that have been identified in samples. With the Celera Discovery System, scientists get the list of identified proteins plus functional classification and information on the genes and the genetic analysis associated with the proteins.” The 4700 Proteomics Analyzer can also be used with software from other companies, such as Spotfire, which is described below.

Nonetheless, really understanding the proteome demands more than knowledge of protein expression. Hicks said, “Posttranslational modifications [PTM] will play an important role.” Applied Biosystems’ 4000 Q TRAP LC/MS/MS System combines ion trapping, which is a form of liquid chromatography, and triple quadrupole technology. “The ion trap provides protein identification,” said Hicks, “and the triple quadrupole identifies and measures any modifications after translation, and potentially quantifies it. This enables whole new approaches to PTM analysis and other key applications in proteomics such as accurate protein quantitation.”

Advancing Arrays
Growth seems always under way in microarrays. Several companies have introduced or announced plans to market DNA microarrays that contain the entire human genome. Applied Biosystems, for instance, announced that it would offer a whole genome chip by the end of 2003. Shortly after that, NimbleGen Systems reported that it was running tests for customers with its own version of a full human genome chip. Then in October of last year, Affymetrix reported that it was accepting orders for its own whole genome chip. Microarrays can also be made in a laboratory, with products from Genetix, Hitachi Genetic Systems, and Thermo CRS.

Toward the end of 2003, Clark Mason, product line manager at Applied Biosystems, said, “We have a number of worldwide sites where our whole genome chip is up and running.” At that time, he expected unrestricted release of that microarray by the start of 2004.

Mason said, “The Applied Biosystems Expression Array System can detect gene expression at extremely low levels and with small amounts of sample, which is important to those researchers working with limited amounts of samples, as often required for cancer research. Also, the system unambiguously interprets those data into gene expression events.” Mason pointed out that a customer can use this system for target screening, target identification, and target validation. This whole genome microarray will include 30,000 genes and more than 60,000 transcripts. “It’s the best view of the genome that we have right now,” Mason said.

In addition, some companies use microarrays to follow proteins. Ciphergen Biosystems, for example, developed a number of systems based on microarrays and mass spectrometry for both research and clinical applications.

A Choice of Hybrids
Proteins usually function in pathways and interact with other related proteins. Researchers often begin studying proteins by attempting to understand how a protein with unknown function relates to other known proteins. That can be accomplished with the yeast two-hybrid method.

This yeast technique is based on a transcription factor that binds to a gene and causes it to produce RNA and then protein. This transcription factor consists of two parts: the binding domain, which attaches to a gene, and the activation domain, which turns on RNA production. The two domains can be separated and each fused to a different protein of interest. The two hybrid proteins can then be tested to see if they interact with each other. If they are related, the two domains will bind to each other and cause RNA production. This system has been well developed in the yeast model and is now offered by several companies, including Clontech, Invitrogen, and Stratagene.

News Flash: Functional Genomics To keep up with the latest news surrounding functional genomics, go to Science’s Functional Genomics online. This site provides daily updates under headline news, as well as an archive of previous stories. To delve even deeper, visitors can explore the research section, which includes links to articles arranged by topic. Links to Science’s genome special issues provide a broad background on many topics. This site includes a section on the business side of biotech plus links to genomic information on education and ethics. www.sciencegenomics.org

Mary Buchanan, director of product management at Stratagene, called the Cyto Trap Two-Hybrid System “a unique twist on yeast two-hybrid products. It lets a researcher introduce different ways to look at a protein.” In conventional yeast two-hybrid systems, activation takes places in the nucleus, but Cyto Trap works in the cytoplasm, where proteins can exhibit posttranslational modifications.

Stratagene also offers BacterioMatch, another two-hybrid system. With this technique, however, all screening is done in bacteria. Since bacteria grow so fast, a researcher can have colonies in one day. Buchanan said, “This system looks for protein-protein interactions through two screens: an initial one for histidine prototrophy, basically needing this as a nutrient, and a second for resistance to streptomycin, which reduces the likelihood of false positives.”

Advantageous Interference
In most cases, interference makes things harder to see, like a fuzzy image on a television. RNA interference (RNAi), though, helps scientists see more clearly what a specific gene does. In this process, short lengths—21 to 23 base pairs—of naturally occurring double-stranded RNA (dsRNA) inhibit the expression of a gene bearing its complementary sequence. So-called small interfering RNA (siRNA) can induce RNAi in mammalian cells. A number of companies have developed kits and reagents for RNAi studies, including Ambion, Dharmacon, and Mirus. According to David Dorris, business development manager at Ambion, RNAi is especially useful for the early research phase of drug discovery and for pure functional genomics.

Dorris pointed out that RNAi can answer a wide variety of questions. What happens if a given gene is not there? What happens if six genes get knocked out at once? RNAi tackles such questions. Still, Dorris said, “RNAi is not a perfect technique. You need to make sure that an effect comes from knocking down a gene, not from the effect of the RNAi itself.” So, Ambion products use as little RNA as possible.

With so many genes and so many model organisms, companies need to provide many forms of siRNA. Dorris said, “We have many predesigned siRNAs. If a researcher has a gene, then we more than likely already have the siRNA designed.” Ambion has more than eighteen thousand siRNAs for human, about eighteen thousand for mouse, and five thousand for rat. This company also carries siRNAs to knock down entire classes of genes, including kinases and other drugable target classes. If Ambion does not have the siRNA on the shelf, the company will make it. “Send us the sequence by 2 p.m.,” said Dorris, “and then we start synthesis this afternoon.”

Many companies plunged into this rapidly growing field. At Invitrogen, for instance, Bennett said, “We recently launched a line of RNAi expression vectors, which are Gateway-adapted so that you can place short hairpin RNA [shRNA] in an expression cassette and test it to knock down mRNA and a protein.” Then, when a researcher finds an shRNA that knocks down the appropriate gene, it can be moved to various vectors through the Gateway technology.

Much of today’s high throughput screening for therapeutics takes place in vitro. Evans of Amersham said, “Customers want to see how purified proteins interact with candidate drug compounds in vitro, and then take this primary screening and understand how the ‘hit’ compounds interact with target proteins in the cell.” To follow the action inside cells, Amersham developed its IN Cell Analyzer 3000. Evans said, “This instrument platform is an automated fluorescence analyzer. It lets you see events inside the cell and track proteins moving in the cell.”

Several companies already use the IN Cell Analyzer 3000 for a variety of purposes. Norak Biosciences, for example, uses the IN Cell system for drug discovery. Scientists at Norak combine their Transfluor technology with the IN Cell system. Transfluor uses G-protein-coupled receptors, often called GPCRs, in a drug discovery assay. Specifically, this system looks for drugs against GPCR targets.

Firming Up the Software Side
With so many high throughput instruments, scientists also need rapid forms of storing and analyzing the data. Bill Ladd, senior director of analytical applications at Spotfire, said, “Bioinformatics plays a role in two parts of functional genomics. First, in the sequence analysis world, scientists are trying to understand gene function based on sequence homology with genes of known functions. The next level is on the protein side—building databases to identify proteins based on mass spectroscopy. Here, researchers want to know what protein species are in a sample and to what extent.”

Those tasks create large volumes of data. Ladd said, “Now, we are looking at ten thousand or tweny thousand genes at a shot, and who knows how many proteins.” That all creates more information than anyone can juggle in a single brain. Consequently, a variety of companies—including Accelrys, DNAStar, and Spotfire—make bioinformatics packages.

Spotfire’s DecisionSite for Functional Genomics, just out in a new version, enables research groups to quickly find and categorize important genes and gene expression patterns. Ladd said, “We have added the ability to connect to gene ontologies. That is, this program makes it easier to go from a cluster of genes that behave similarly in an experiment to understanding where this group of genes is found—in a statistically significant way—in pathways or cellular locations.” In addition, this program incorporates visualization capabilities that let users examine the expression and annotation dimensions of their data. DecisionSite also allows researchers to take advantage of an open-source statistical environment called R. Ladd said, “We have an R integration that lets customers incorporate any R algorithm they might want.”

In some ways, the future of functional genomics unfolds day by day. Scientists now do things that researchers could not imagine a decade ago. As new information emerges, the advances could be exponential—more and more questions being resolved as more is known.

Mike May (mikemay@mindspring.com) is a freelance writer and editor based in Madison, Indiana, U.S.A. Gary Heebner (gheebner@cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

WEBLINKS ADVERTISERS Carl Zeiss [USA] instruments and systems for imaging analysis, digital cameras 914-747-1800 www.carlzeiss.com/micro Carl Zeiss [Germany] +49 (0)551-5060 660 Genome Canada the primary funding and information resource relating to genomics and proteomics in Canada 613-751-4460 www.genomecanada.ca Leica [USA] instruments and systems for imaging analysis, digital cameras 847-405-0123 www.simply-microscopy.com Leica [Germany] +49 6441 29-0 Takara Bio, Inc. kits and reagents for molecular biology research +81 77 543 9254 www.takara-bio.co.jp/english FEATURED COMPANIES Accelrys – a subsidiary of Pharmacopeia bioinformatics software www.accelrys.com Affymetrix oligo DNA microarrays www.affymetrix.com Agilent Technologies, Inc. mass spectrometry systems www.agilent.com Ambion, Inc. siRNA kits and reagents www.ambion.com American Type Culture Collection (ATCC) DNA clones www.atcc.org Amersham Biosciences instruments and supplies for genomics and proteomics research www.amershambiosciences.com Applied Biosystems instruments and supplies for genomics and proteomics research www.appliedbiosystems.com BD Biosciences Clontech DNA clones – kits and systems www.clontech.com Bio-Rad Laboratories instruments and reagents for genomics and proteomics research www.discover.bio-rad.com Bruker Daltonics, Inc. mass spectrometry systems www.bdal.com Celera Genomics drug discovery www.celera.com Ciphergen Biosystems, Inc. instruments and arrays for proteomics research www.ciphergen.com Dharmacon, Inc. siRNA kits and reagents www.dharmacon.com DNAStar bioinformatics software www.dnastar.com Elchrom Scientific AG kits and reagents for genomics and proteomics research www.elchrom.com Epicentre Technologies DNA clones – kits and systems www.epicentre.com Frost & Sullivan consulting/market research www.frost.com IBC Life Sciences scientific conference organizer - ScreenTech World Summit www.lifesciencesinfo.com/screentech Genetix instruments and supplies for array fabrication www.genetix.com Hitachi Genetic Systems instruments and supplies for array fabrication www.hitachi-soft.com/gs Invitrogen Corporation kits and reagents for genomics and proteomics research www.invitrogen.com Mirus Corporation siRNA kits and reagents www.genetransfer.com Motorola electronic communication and semiconductor products www.motorola.com NimbleGen Systems, Inc. oligo DNA microarrays www.nimblegen.com Norak Biosciences, Inc. biotechnology/drug discovery and development www.norakbioscience.com OriGene Technologies, Inc. DNA clones www.origene.com Promega Corporation DNA clones – kits and systems www.promega.com Science’s Functional Genomics online functional genomics news www.sciencegenomics.org Spotfire, Inc. bioinformatics software www.spotfire.com Thermo CRS (formerly CRS Robotics) instruments and supplies for array fabrication www.thermo.com USB Corporation biochemicals and molecular biology products www.usbweb.com U.S. Environmental Protection Agency (EPA) government agency www.epa.gov Waters Corporation mass spectrometry systems www.waters.com

Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. The companies and organizations in this article were selected at random. Their inclusion in this article does not indicate endorsement by either AAAS or Science nor is it meant to imply that their products or services are superior to those of other companies.

This article was published as a special advertising section in the 6 February 2004 issue of Science