|Applying the results of genome sequencing for human benefit demands a series of new tools and technologies. They range from the now common approach of high throughput screening to the exciting new development of RNA interference.|
|By Peter Gwynne and Gary Heebner
DNA microarrays, based on the principles of semiconductor technology
Takara Bio, Inc.
IN THIS ISSUE:
|•||High throughput screening|
|•||Biochemical and cellular assays|
|•||Gene expression kits|
|•||In vitro transfection|
As the initial phases of sequencing the human genome come to a close, researchers have started to shift their focus to determining the compounds for which each of the many newly discovered human genes code. This pursuit, generally called functional genomics, consists of all the research undertaken to bridge the knowledge gap from DNA (or gene) to protein (or function).
"Functional genomics starts by having a physical clone," explains Gilbert Jay, chief executive officer of OriGene Technologies. "Complementary DNA clones are really the drivers." The application of cDNA leads to both basic understanding of genetics and applications of the field. "Increasingly we realize that genes work in combination with other genes. Scientists are creating matrices of genes to discover their functions," Jay continues. "Genes will also be used to generate downstream products such as proteins and antibodies. When you have genes in bulk you'll be able to get proteins in bulk."
The discipline has extraordinary breadth. "It involves understanding both the biology and the medical implications," says Robert Waterston, head of the Department of Genome Sciences at the University of Washington. "I would also include items such as understanding human variations as a clinical part of genomics."
Functional genomics brings together researchers from many different disciplines. Chemists and bioinformatics specialists join with molecular biologists and cellular physiologists to study the dynamics of the living cell. Their work relies heavily on tools and technologies adapted from the physical sciences as well as life science. "Functional genomics took off with the creation of large gene expression databases by Human Genome Sciences, Incyte Corporation, Millennium Pharmaceuticals, and other companies," says John Burczak, head of R&D at Amersham Biosciences. "Researchers had all those genes; to rationalize which ones they wanted to look at for drug targets and other uses they had to understand function."
As part of that effort, scientists generate huge volumes of sequence, conformation, and functional data. In response, manufacturers are scrambling to upgrade their products into state-of-the-art form, to serve the needs of leading edge researchers more effectively.
The successful sequencing of the human genome has greatly increased the number of potential targets - the specific points for drug intervention in biochemical pathways. Pharmaceutical companies screen for new targets as the first phase of drug development. They hope to develop some of the compounds that affect the targets into drugs that block or enhance a desired activity or function. As new proteins are identified and their activities determined through functional genomics, each will represent a potential new target for drug therapy.
In the past, finding appropriate targets represented a serious bottleneck in drug discovery. No more. Pharmas and other companies in the business of finding potent drugs to inhibit or regulate targets' activity find themselves flooded with new potential targets. To increase their productivity and decrease their costs, therefore, they must rely on advances in discovering, screening, and validating the leads that act on the targets. The methods used to analyze and manage data must continue to progress at impressive rates to enable researchers to stay in control of databanks that have grown rapidly in both overall number and the amount of data that individual collections contain. Scientists also need databases to be interconnected to permit them to perform comparative analyses among different databases most efficiently.
One of the most exciting recent developments in functional genomics is the application of RNA interference - the use of small stretches of double-stranded RNA to inhibit the expression of genes that carry their complementary sequences. First discovered as a natural phenomenon, it has since become a significant tool. "it's a hot topic with enormous potential for accelerating functional genomics research," says David Roth, vice president of R&D for Gene Therapy Systems. "It really has direct bearing on what will be important for genomics, proteomics, and drug discovery."
Functional genomics promises advantages of several types, from improved agricultural crops and animal health to targeted therapies and better diagnosis of human illness. "The benefits are already arriving," says Waterston. "You see it for better or worse in genetically modified organisms. And in terms of health, researchers are already finding molecular signals that allow them to stratify diseases in a way that couldn't be done in terms of morphology. Medicine used to be descriptive. Now you can do molecular biology on humans as a real experimental approach."THE ASSAYS RACE
GENOMES TO LINKAGE TO LIFE
The 19th iteration of the International Congress of Genetics, an event that originated in 1899, will take place in Melbourne, Australia, on 6-11 July of this year. Titled Genomes - the Linkage to Life, the event will look at past, present, and future. It will commemorate the 50th anniversary of the discovery of DNA's structure. It will present reports on present-day progress in genetics and genomics. And it will permit life scientists to peer into the crystal ball. "we're in the position of having both sequencing and the associated technologies of gene silencing and arrays to begin to work out what genes are doing," says Congress Chair Philip Batterham, a senior lecturer in the University of Melbourne's Department of Genetics.
The event, which includes Science among its sponsors, will feature 54 symposia, 280 invited speakers from around the world, and up to 1,500 posters. It will cover clinical genetics, cancer genetics, mutation detection, gene silencing, gene networks, and much more. Forums will include a discussion of the issue ±Public Science vs. Private Science: Who Wins?" Eight Nobel Laureates, among them all three 2002 winners of the prize in medicine and physiology, will join up to 3,000 other scientists at the congress. "it's like the Olympics of genetics," Batterham says.
Attendees might consider a precongress stop between 30 June and 3 July in Brisbane for Intelligent Systems for Molecular Biology, a meeting that Batterham describes as ±the most significant bioinformatics conference held in the world on an annual basis."
The tools used in functional genomics include reagents and supplies, instruments, and computer software. Stimulated by the need to improve efficiency and throughput and to lower costs, researchers want to run larger numbers of simultaneous tests, to decrease variability between tests, and to use smaller and smaller sample volumes.
Responding to those needs, manufacturers now adapt their instruments and sampling systems, such as microwell plates, to handle ever decreasing sample sizes and ever increasing numbers of samples. "There's been a huge race to get new assays up and running," says John Watson, director of marketing and screening applications for PanVera Corporation.
High throughput screening (HTS) has become the functional genomics approach of choice for the giants of drug discovery. "The real volume users at this point are the pharmaceutical and biotechnology companies and large genomic centers," says Amersham's Burczak. However, other customers have emerged recently. "Our main customers are the very large pharmas," says Watson. "But we're trying to get the technology to smaller pharmas, biotechnology firms, and some academic departments. The technology is so powerful that it's essential for them." At the high end, meanwhile, HTS is now transforming to ultrahigh throughput screening, with sample volumes in the microliter range and the ability to handle 1,536 samples per plate.
Several companies specialize in instruments for high throughput screening. Applied Biosystems offers a wide range of core instruments and systems necessary for functional genomics, such as DNA sequencers and synthesizers. "Our newest products are off-the-shelf, predesigned gene-specific primer and probe sets," says director of genomic assays Chris Walworth. "We preformulate them and functionally test them; users know that they're going to work before they buy them."
Other companies, such as Invitrogen, PanVera, and Roche Applied Science, have developed complete biochemical and cellular assays that are compatible with high throughput systems. "Our strongest technology is beta lactamase," says Watson. "It produces a transcriptional readout for monitoring the expression of important genes, especially for high throughput drug discovery. The real advantage from a technical standpoint is that the beta lactamase substrate can be retained in a living cell. To find out which clones are expressing, you do flow cytometry."
PanVera has also developed a series of high throughput screening assays for kinases, nuclear receptors, and other families of molecules. "Our Z±LYTE technology can readily meet the growing demand for new assays to screen for inhibitors of a broad array of tyrosine and serine/threonine protein kinases and phosphatases," says Watson. "The technology is highly compatible with automated high throughput screening systems."
ITEMS FOR GENE EXPRESSION
Suppliers such as Amersham Biosciences, Gene Therapy Systems, and Stratagene provide a variety of kits and reagents for gene expression studies. "Functional genomics is central to our business strategy," says Amersham's Burczak. "We bring a lot of tools and technologies to bear, such as DNA sequencing capabilities, microarrays, gene expression, SNP detection and analysis, and our capabilities in high throughput sequencing for cells and proteomics."
Last year Amersham expanded its TempliPhi product, used to prepare samples for DNA sequencing, to include two new DNA template preparations for core laboratories and individual researchers. Now there's more to come. "We will soon be launching GenomePhi for sample preparation up front of any analysis for genetic variation," says Burczak. "This technology amplifies entire genomes. You could amplify a drop of blood up to the entire genome and have enough DNA to do a lot of analysis."
To study gene function, researchers can insert DNA sequences into living cells; there, the DNA sequence can be read and expressed as protein. Building a library of DNA clones is no small task. However, researchers can turn to several companies that provide the tools to help customize their own libraries. Alternatively, they can source large numbers of clones from such companies as ATCC.
OriGene Technologies has produced a large collection of human full-length clones for research and drug discovery work. The company also provides custom services in this area. "We have basically 800,000 independent clones in our possession from proprietary, high-quality cDNA libraries," says Jay. "We also have a state-of-the-art 5± EST database. That's particularly important for identifying and validating or disproving new genes. We released 10,000 genes in 2002 and another 10,000 in January of this year. To facilitate selection of genes from such a large collection, researchers can search for genes with specific function domains of interest to them." OriGene makes its genes available to all sectors of the life science industry, from large pharmas to academic departments. "The majority will be getting genes in bulk - from hundreds up to 20,000 - on a totally nonexclusive basis for research use only," Jay explains.
The most significant introduction of a new technology to functional genomics within the past two years has involved RNA interference. Referred to as RNAi, this approach uses small segments of double-stranded RNA, 21 to 23 base pairs in length, to inhibit the expression of genes that carry the segments' complementary sequences. It has drawn huge interest from researchers. "We cannot make products fast enough for this market," says Stephen Scaringe, chief executive officer of Dharmacon, Inc.
"The earliest publications describing RNA interference in animal cells occurred in about 1998, but it didn't work well with mammalian cells," recalls Jim Hagstrom, vice president of scientific operations for Mirus Corporation. "But the functional discovery describing the use of small interfering RNAs (siRNAs) in mammalian cells was published in May 2001. It showed that small interfering RNA could knock out genes without causing any deleterious effects on the cells containing the genes." Since that publication, adds Scaringe, "researchers have rushed to validate the technology and to see how this new regulatory role of RNA is going to help identify gene and protein functions."
The technology has several major advantages over other posttranscriptional gene silencing techniques such as antisense and gene knockouts. RNAi can inactivate a gene at almost any stage of development. "Rather than having an on-off switch like a regular light bulb, it's like having a rheostat that can adjust the brightness of the bulb," explains Roth of Gene Therapy Systems. "When you knock out a gene and the animal dies, you don't know precisely what caused it. What RNAi does is give you the chance to regulate the process. I hope that we'll see a whole new brand of animal models that are alive but sick, so that we can explore disease."SMALL DOSES
FOCUS ON FUNCTIONAL GENOMICS
The ScienceFunctional Genomics website serves as an entry point for the worlds of genomics and postgenomics. The site's editors don't expect to provide a comprehensive view of the subject. Rather, as a starting point for scientists' own explorations they aim to provide a representative sample of happenings in the field, along with ±enough news and breaking information to keep things fresh and interesting."
In addition to current news and a news archive, the site includes a collection of relevant research papers organized in genomic categories, a page that contains Science's special issues on genomics, pointers to scientific and educational resources on the World Wide Web, and a special section of news and information about the biotechnology business. Access to the site is free, although several of the site's links require free registration or a paid subscription.
RNAi technology has another key advantage. Scientists need very small doses of siRNA to carry out their experiments; only a small number of the molecules need to enter a cell to exert their silencing effect. On the other hand, says Hagstrom, "the real bottleneck is that you have to get siRNA into cells." That's not particularly difficult for cells in vitro. But transfecting siRNA into cells in vivo presents a more serious challenge.
Mirus, a company that specializes in gene transfer technologies, has developed TransIT-TKO to carry out the task in vitro. "Our formulation is a combination of a polymer and an amphipathic polyamine," says Hagstrom. "we're very far along with the technology for in vitro transfection. For in vivo transfection we have had one of the first publications showing highly effective nonviral gene delivery to a mouse liver. In this procedure the nucleic acid is delivered into the bloodstream in a manner that facilitates its delivery to cells outside the blood vessel wall. The liver is particularly effective here as the blood vessels in the liver have small holes. By using this delivery method in the mouse you can target the siRNA to the liver with a single tail vein injection. In humans you would probably have to deliver it via a catheter into a vessel that goes straight into the target areas, such as the liver or the heart."
Dharmacon, an 8-year-old company with proprietary 2'-ACE RNA synthesis chemistry, focuses on the siRNA reagents that transfection technologies carry into cells. "One of the key bottlenecks with RNAi is how to design a highly functional siRNA. We have introduced a custom siRNA design service that employs the proprietary SMARTselection algorithm maximizing silencing efficiencies," says Scaringe. "The algorithm takes into consideration over 30 variables that help predict the most functional siRNA target sequences in any messenger RNA. Short interfering RNAs designed with these rules have a 99.9 percent probability of being functional, with more than 50 percent of the mRNA degraded, or knocked down. An additional sophisticated algorithm gives a greater than 75 percent probability of being exceptionally functional, with more than 95 percent of the mRNA knocked down. Our goal is to provide a complete solution for RNAi researchers."
Gene Therapy Systems recently announced a new product, the Dicer siRNA Generation Kit, that generates a large number of siRNAs from full-length target genes. The kit mimics the natural RNA interference process by using recombinant human dicer enzyme, a dsRNA-specific endonuclease, to cut large molecules of dsRNA templates into small, 22±base pair siRNAs. It also includes the company's GeneSilencer transfection reagent. Other companies that have developed kits and reagents for RNAi studies include Ambion and Stratagene.
ANALYSIS AND COMPARISON
As they gather more information on living cells, scientists find it increasingly difficult to analyze and compare all their data, particularly as the data can reside in different locations and on different platforms. "it's critical for researchers to have at their fingertips all the information about the gene or genes of interest to them," says Anthony Kerlavage, senior director of bioinformatics applications at Celera, a unit of Applied Biosystems. "We pull together annotations around chemical regions, genes, and transcripts. If you're trying to functionally analyze your gene, you want to know as much about it as possible before you start your experimental work."
Applied Biosystems offers a program called Rapid Integration Solutions that combines state-of-the-art software products and services to provide complete informatics solutions for integrating and automating laboratories. "We work with our customers to determine their specific needs," says Ray Stonecipher, the firm's senior manager for software integration marketing. "The idea is to develop genotyping, gene expression, and other solutions."
Independent R&D organization SRI International is developing research programs that promise to provide significant data resources for functional genomics. "We are about to release HumanCyc, a new database of pathways. it's a research tool and analysis framework for analyzing gene expression and proteomics data," explains Peter Karp, head of SRI's bioinformatics group. "we're using an algorithm to predict an organism's metabolic pathways. Once we have the initial computational derivation we'll use manual curation to refine it against experimental findings and flesh out the database. we'll create a one-page overview diagram to show the entire metabolic map of humans that people can use to interpret functional genomics data." SRI has also put together a commercial consortium to provide advice on the use of the database.
Another SRI program, called Pathway Logic, generates and analyzes complex biological signaling networks. "We view it as a potential drug discovery tool," says Keith Laderoute, director of SRI's cancer biology program. "We hope to have a project launch this year with a commercial client." Pathway Logic software tools originate in SRI's computer science laboratory, directed by Patrick Lincoln.
A major reason scientists are interested in understanding the function of each gene is to gain a better understanding of the disease process and what role these genes might play in altering a person's metabolism. "Ultimately," says Waterston of the University of Washington, "we want to understand the genome sufficiently to be able to predict the response of an individual member of a species on the basis of knowledge of its genetic information."
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 18 April 2003 issue of Science