Drug Discovery and Biotechnology Trends - Genomics 4: Cellular Invasions

A plethora of new means of introducing genes into cells promises benefits for researchers aiming to understand cellular function and clinicians working on gene therapy.

by Peter Gwynne and Gary Heebner

ADVERTISERS 21st Century Biochemicals custom peptides, phosphospecific antibodies, and bioactive peptides, 508-303-8222 http://www.21stcenturybio.com Genome Canada the primary funding and information resource relating to genomics and proteomics in Canada, 613-751-4460 http://www.genomecanada.ca SANYO Sales & Marketing Corporation / SANYO Electric Biomedical Co., Ltd. constant temperature equipment (incubators, ovens, refrigerators, and freezers), http://www.sanyo-biomedical.co.jp Takara Bio, Inc. cell and gene therapy, +81 77 543 7247 http://www.takara-bio.co.jp/english

CONTENTS • Chemical transfection reagents • Virus-based transfection systems • Electroporation devices • Microinjection systems • Protein delivery systems • clinical applications of transfection

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.

This is this year’s final supplement on genomics. The first three appeared in the 6 February, 9 April, and 17 September issues of Science. You can find these and other supplements at http://www.science-benchtop.org.

The idea of transferring genes into mammalian cells, otherwise known as transfection, has intrigued the life science community for more than a quarter of a century. For basic researchers in academe and industry, a reliable method of transfection offers insights into the workings of cells. “To look at what’s going on in cells, you have to put genes into the cell,” observes Andrew Farmer of BD Biosciences Clontech.

Clinicians, meanwhile, regard a safe, effective method of delivering genes into the cells of human patients as an essential step in gene therapy for patients afflicted with a range of illnesses, from hemophilia to Parkinson’s disease. “Transfection is important for gene therapy and vaccination,” adds John Archdeacon, head of R&D at Active Motif.

Within the past five years, the idea has taken on several fresh forms of reality. A range of new transfection tools, including lipid based reagents, electroporation, and specialized viral delivery systems, now complements the established chemical methods that worked with only limited success after much trial and error. As a result, the process has found broader applications than in the past. “Initially people used transfection for looking at expression, promoter activities, and promoter bashing,” says Balwant Patel, marketing manager at Roche Applied Science. “Recently, transfecting cells with genes to study proteins has become really important. In the proteomics age, people are using the same techniques to rapidly overexpress, isolate, and study proteins.”

“There are three major fields into which gene transfer into mammalian cells is getting more important,” says Ignacio Anegon, a scientist in the French government’s research organization Inserm who is editor in chief of Current Gene Therapy, a peer reviewed journal published by Bentham Science that is indexed in PubMed. “Medically it is used in gene therapy, treatment of cancer, and vaccinations. As a research tool it can overexpress or inhibit gene expression. And as a biotechnology tool it can produce proteins and generate transgenic animals.”

Increase in Publications
The expansion of research has led to a steady increase in publications. Data gathered by Takara Bio reveal jumps from 733 papers on transfection or gene transfer in 1985 to 5,921 in 1995 and 10,393 last year. Current Gene Therapy will soon increase its frequency from quarterly to six times a year. Most recently, papers have started to appear on clinical trials of gene therapies that involve the use of transfection.

A revolutionary new life science technology has also benefited from and has stimulated new methods of transfection. “Especially in the past couple of years, the development of RNA interference [RNAi] has helped to make transfection into mammalian cells a very big subject,” says Constanze Kindler, senior global product manager for transfection at Qiagen. “Transfection has been the key technology for functional genomics research for a long time,” agrees Rob Bennett, director of research and development for proteomics at Invitrogen Corporation. “It’s now going forward in terms of an increased number of experiments on proteins and the use of RNAi.”

The general need for precision has also contributed to the popularity of new methods of inserting material into cells. “Transfection into mammalian cells has been critical for a long time,” says Rainer Christine of Amaxa. “What has become very important in the past few years is the ability to transfect primary mammalian cells. More and more, peer review boards are asking that experiments use primary cells, which represent a model system much closer to the in vivo situation than long established and often highly transformed cell lines.”

Responding to scientists’ demands, suppliers have started to offer use-tested transfection reagents and user-friendly kits. “We want to apply all sorts of transfection to different uses,” says David Roth, vice president of research and development at Gene Therapy Systems. “You need to develop a product that will be less toxic to cells and yet give you the highest transfer efficiency.” Scientists plainly believe that vendors have achieved those goals. “Practically every laboratory of molecular biology is using some sort of transfection method,” Archdeacon says. “It’s one of the staple technologies like PCR.”

No Single Approach
Unlike PCR, scientists have not yet agreed on a single optimal approach to transfecting mammalian cells. “Nobody has really cracked what makes a good transfection reagent,” Roche’s Patel comments. Categories of transfection techniques include those that involve carrier molecules, methods that introduce DNA directly into cells, and approaches that use viruses to deliver genes of interest. “There’s not one method that will work in all cell lines,” Qiagen’s Kindler explains. “The method varies very much with the cell type, plasma conditions, and other factors,” Archdeacon summarizes the situation. “There’s no one, golden method of delivering DNA into cells,” he says.

Several transfection methods use carrier molecules to enhance uptake into cells and protect the DNA from degradation by nucleases. When researchers first began transfecting cells, they relied on chemicals such as DEAE-Dextran and calcium phosphate that they prepared themselves. Some laboratories continue to use those reagents, which date back to the mid 1960s and the early 1970s respectively. But they have some serious disadvantages. “For home-made methods they are well established, relatively simple, but toxic,” Kindler says. “Handling is fairly easy for calcium phosphate and, unlike Dextran, it allows you to do stable transfections. But it has low reproducibility.” The method also depends heavily on the correct pH, a factor that can make it difficult to control. Nevertheless, demand for calcium phosphate remains high enough for such suppliers as GE Healthcare, Promega, and Sigma-Aldrich to continue to offer the reagent.

Where Science Meets Society Under the title “The Nexus: Where Science Meets Society,” next year’s annual meeting of AAAS (American Association for the Advancement of Science, which publishes this journal) will focus on topics that range from emerging diseases in developing nations to genomics, nutrition, public safety, and disappearing cultures. The meeting will represent “a means of connection, a link where science will meet society and sponsors and exhibitors will meet their markets,” says Jill Perla, senior manager of marketing and meeting operations at AAAS. The event, which will take place in Washington, D.C., from 17 to 21 February, will cover a variety of seminar topics, including nanotechnology and “the omics problem” of huge amounts of data created in life science experiments. On 20 February, the meeting will focus on the physical sciences, by kicking off the World Year of Physics with symposia on such issues as string theory, dark energy, Bose-Einstein condensates, and gravitational waves. “It will culminate in a gala reception with prominent physicists as characters from physics history,” Perla adds. The annual meeting will include familiar, student-oriented features from previous years, among them forums on school science, a career fair, and career workshops. Students will also have the chance to win cash prizes for entries to a poster competition. And Family Science Days, on Saturday and Sunday, the 19th and 20th of February, will echo the meeting’s theme by providing firsthand examples of the link between science and society. http://www.aaas.org/meetings/Annual_Meeting/

In the late 1980s, lipid based protocols began to take over from Dextran and calcium phosphate as transfection agents of choice. Scientists create them by combining DNA with cationic lipids to create lipid-DNA complexes that fuse with the cell membrane and are then transported into the cell. “Cationic lipids are easy to use,” Invitrogen’s Bennett says. “You simply mix the DNA with your reagent and deliver it.”

Lipid Based Methods
Lipid based methods have several other advantages over the viral methods that emerged later. They do not include proteins that might cause an immune response when injected into an organism; they can be synthesized chemically to provide better definition of the components; and they can be chemically modified to alter the characteristics of the transfection reagent. But lipid based transfection reagents have some drawbacks. For example, much of the DNA that enters a cell remains in endosomes, resulting in decreased rates of transfection. “Cationic lipids are ideal for the standard cells you want to transfect,” says Bennett. “But there’s a cell dependency; certain cell types will be resistant.” Adds Kindler: “In the presence of serum, you usually get lower transfection efficiencies when using liposomes.”

Suppliers such as Gene Therapy Systems, Mirus Bio, Qiagen, and Roche Applied Science have developed different approaches to solving that problem. “Transfection reagents from the major players enable you to transfect in the presence of serum without reducing the efficiency,” Kindler continues. “So you can keep the cells happy and healthy.”

Fugent, a Wisconsin-based biotech, has developed a proprietary transfection reagent called FuGENE 6, which Roche Applied Science is marketing worldwide. “The major advantage of FuGENE 6 is that it has probably the lowest toxicity of any reagent,” Patel says. “We have recently proved that using gene expression profiling studies. Customers have told us that FuGENE 6 works in more than 700 different cell types, shown on our website, although the level of performance varies. We still have customers who buy DOTAP because in some cells it outperforms FuGENE.” One blind spot of FuGENE 6 is its failure to work with synthetic small interfering RNA (siRNA) molecules. For that, Roche has recently introduced what it calls X-treme GENE siRNA transfection reagent.

Two Distinct Technologies
Qiagen offers transfection reagents based on two distinct transfection technologies – activated-dendrimer and nonliposomal lipid technologies – for different cell types and different applications. “Activated dendrimers are highly branched spheroid molecules that tend to condense DNA in very small complexes, which makes it easier for cells to take them up,” Kindler says. “They don’t interfere with your serum. Also, they are very robust and can be stored at or above room temperature.”

The company has optimized its PolyFect reagent specifically for transfection of COS-7, NIH/3T3, HeLa, 293, and CHO cells. “It is a one-component system that is easy to use,” Kindler says. Other Qiagen dendrimers, such as its SuperFect reagent, allow high transfection efficiencies in a broad range of cell lines. TransMessenger, Effectene, and RNAiFect are Qiagen’s nonliposomal lipid transfection reagents; RNAiFect is specifically designed for the delivery of siRNA molecules and TransMessenger for the transfection of RNA molecules. Effectene is also particularly effective for primary cells and sensitive cell lines. All three reagents offer a fast and easy procedure, transfection in the presence of serum, and low cytotoxicity. And all are available in bulk quantities and are suitable for high throughput transfection.

Invitrogen’s LipoFectamine 2000 and AlgoFectamine reagents, both cationic lipid mixtures, achieve high transfection efficiencies and protein expression levels. They offer good results for both transient transfection and the generation of stable cell lines. And their proven efficacy in the presence of serum eliminates the need to change media following transfection. “You choose the reagent according to the cell type you want to transfect,” Bennett says. “Recently we have chemically defined animal-free versions of LipoFectamine 2000 and a version called OptiFect for RNAi research, which works with cells of lower density.”

Gene Therapy Systems, self-described as “the transfection company,” has developed a method of targeting transfection for more efficient gene delivery via peptide nucleic acid (PNA) complexes that recognize specific DNA sequences. PNAs are DNA mimics that are not degraded by nucleases and are more stable in solution than DNA. The PNA strands hybridize with host DNA, causing it to uncoil and permitting the foreign DNA to integrate into that of the host cell. “It’s a way of modifying DNA to make it more bioavailable,” Roth explains. “You can attach any kind of probe you want to track plasmid distribution. It’s more of a trouble-shooting method than a pure transfection method.”

Physics to the Fore
Life scientists have more than chemical techniques at their disposal for introducing matter into cells. Physics comes to the fore in methods such as electroporation and microinjection, while biology takes responsibility for viral transfection.

Electroporation devices use bursts of direct current to destabilize the membranes of mammalian cells temporarily, thereby opening the way for the DNA fragments to insinuate themselves into the cells. “Conventional electroporation works mainly for cell lines and not primary cells,” Amaxa’s Christine says. “Its advantage is that it doesn’t differentiate between adherent and suspension cells.” The technology can work with a wide variety of cell types. However, the fact that it destroys a significant number of cells in the process limits its usefulness to samples that contain large numbers of cells. Firms that offer electroporation systems include Bio-Rad and BTX.

Science and Security The AAAS Center for Science, Technology and Security Policy, created in May 2004, has the goal of helping to integrate science with public policy related to national and international security affairs. To head the new center, AAAS recruited Norman Neureiter, a prominent scientist, diplomat, and technology executive. Neureiter discusses the activities of the new organization in the current issue of the web newsletter AAAS Advances. You can find the interview at: http://www.aaas.org/news/releases/2004/0915center.shtml

Amaxa has adapted electroporation in its Nucleofector technology and has expanded it to efficient primary cell transfection. “It’s a combination of a device based on electroporation and solutions that provide a very specific environment in cells that makes them amenable to transfection,” Christine explains. “We developed the technology with the aim of making nuclear transport of DNA plasmids possible. That’s important for primary cells because they normally don’t divide. In order to transfect them, you need to deliver the DNA plasmids directly into the nucleus. With our Nucleofector technology you can do your DNA transfer or siRNA knockdown in primary cells, giving your experiments more in vivo relevance.”

Microinjection and Microscopy
An even more direct approach uses a microinjection device to insert a sample of DNA into a cell. When they have only limited numbers of cells, researchers can turn to microinjection coupled with light microscopy to visualize the process. This approach uses a very small hollow bore needle to pierce through the cell membrane and deliver the needle’s contents directly into the cell. Narishige and Bio-Rad offer microinjection systems.

Other physical approaches have begun to emerge. “We’ve looked into aeroporation, a kind of barometric chamber that uses air pressure to drive genes into cells,” Gene Therapy Systems’ Roth says. Bio-Rad uses an air gun to blow the genes into cells.

Because mammalian cell membranes resist the entry of most proteins and peptides, scientists have generally used microinjection or electroporation to study proteins’ effects on cellular function. Recently, however, companies such as Active Motif and Stratagene have introduced reagents that deliver proteins to the nucleus and the cytoplasm of a cell. Active Motif bases its Chariot reagent on a short synthetic signaling peptide called Pep-1. “It is noncovalent and much faster, as no cloning or chemical reaction is involved,” Archdeacon explains. “The peptide forms a ‘cage’ around the cargo, allowing the complex to be shuttled through pores in the plasma membrane. Reducing conditions in the cytoplasm help the complex to ‘decage’ and release the cargo.” The noncytotoxic reagent works for many cell types, including endothelial cells and primary human fibroblasts, that can be difficult to transfect.

Viruses’ Virtues and Vices
Viral transfection systems have an obvious benefit: They produce rates of transfection that approach 100 percent. On the other hand, the infectious or potentially infectious nature of viruses, coupled with undesirable immune responses that they might elicit, counterbalance the high transfection rates. “There was probably a fear factor originally in using viruses,” says Farmer of BD Biosciences Clontech. “But as we have progressed and people have understood better how to use the viral methods, they have realized that there aren’t any safety concerns.” The net result: “Lipid techniques are still more popular,” Invitrogen’s Bennett says. “But viral methods are gaining in popularity because of the need to get into primary cells.”

Scientists can use any of several viral systems for transfecting cells. So far, retroviruses and adenoviruses have proved the most popular. Retroviruses insert their genetic material into the genomes of their hosts; but host cells must be actively dividing to make them usable. In contrast, adenoviruses are episomal: They can infect both actively dividing and quiescent cells. “The advantage of retroviruses is that they stably integrate into the cell,” Farmer says. “If you want to make stable cell lines, they’ll do it. That might not be an advantage on the therapy side; you might want to avoid mutations. So you can choose adenoviruses, which don’t survive as cells divide.”

Qbiogene, Retrogen, and Stratagene offer adenovirus-based transfection systems. BD Biosciences Clontech carries both adenoviral and retroviral systems, along with purification systems and complementary expression systems. The company also offers adeno-associated viruses. “These co-infect parasitically with adenoviruses,” Farmer explains. “They represent a possibility for gene therapy for safety reasons. They are also attracting growing interest in the research arena.” The company also carries a pantropic virus that, as Farmer says, “will get into virtually anything.”

Other viral systems include lentiviruses, a class of retroviruses that, unlike most of that class, can infect nondividing cells. “We offer lentivirus and adenovirus products under our Virapower name,” Bennett says.

Overcoming the Fear Factor
Gene Therapy Systems, meanwhile, plans to develop fresh means of reducing concerns about viruses. “You can use components of viruses as your transfection agents,” Roth says. “People are generally scared of working with viruses; they want something in a can that they can squirt into the cell without hoods, pipetting, and other precautions. We’re also looking at hybrids that contain viral components and polymers.”

The fear factor that surrounds viral vectors emerges most strongly in the context of gene therapy. Viral transfection into patients’ cells causes concern because of its potential for unexpected activity. Despite that worry, and the negative results of some early human experiments, transfection technology is finding increased application to gene therapy.

Takara Bio has a leading role in the field with its development, in collaboration with Indiana University, of RetroNectin. This recombinant protein of human fibronectin, consisting of 574 amino acids, increases transduction efficiency in retrovirus-mediated gene therapy. “Since 1997 we have supplied RetroNectin to more than 30 institutes and hospitals in six countries for use in clinical trials of gene therapy,” says a company representative.

Takara has also licensed its RetroNectin-mediated gene transduction technology to VIRxSYS for the treatment of HIV/AIDS. VIRxSYS has initiated a phase 1 clinical trial of VRX496 in HIV patients in the United States at the University of Pennsylvania.

Today, basic and clinical researchers can purchase user-friendly, effective reagents and systems to deliver genes of interest into mammalian cells. As manufacturers continue to refine their products, they will reduce cell toxicity and increase the efficiency of transfection.

Peter Gwynne (//pgwynne767{at}aol.com) is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner (//gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

WEBLINKS ADVERTISERS 21st Century Biochemicals custom peptides, phosphospecific antibodies, and bioactive peptides, 508-303-8222 http://www.21stcenturybio.com Genome Canada the primary funding and information resource relating to genomics and proteomics in Canada, 613-751-4460 http://www.genomecanada.ca SANYO Sales & Marketing Corporation / SANYO Electric Biomedical Co., Ltd. constant temperature equipment (incubators, ovens, refrigerators, and freezers), http://www.sanyo-biomedical.co.jp Takara Bio, Inc. cell and gene therapy, +81 77 543 7247 http://www.takara-bio.co.jp/english FEATURED COMPANIES and ORGANIZATIONS AAAS (American Association for the Advancement of Science) nonprofit scientific society http://www.aaas.org Active Motif, Inc. protein expression products http://www.activemotif.com Amaxa Biosystems electroporation systems http://www.amaxa.com BD Biosciences Clontech transfection kits and reagents http://www.clontech.com Bentham Science Publishers scientific publisher http://www.bentham.org Bio-Rad Laboratories transfection kits and reagents http://www.expressionproteomics.com BTX – A Harvard Bioscience Company electroporation systems http://www.btxonline.com Fugent, LLC biotechnology company 608-241-7775 GE Healthcare (formerly Amersham Biosciences) transfection kits and reagents http://www.amershambiosciences.com Gene Therapy Systems, Inc. transfection kits and reagents http://www.genetherapysystems.com Indiana University university http://www.iu.edu Inserm research institute http://www.inserm.fr Invitrogen Corporation transfection kits and reagents http://www.invitrogen.com Mirus Bio Corporation transfection kits and reagents http://www.mirusbio.com Narishige International micromanipulators http://www.narishige.co.jp Promega Corporation transfection kits and reagents http://www.promega.com Qbiogene, Inc. transfection kits and reagents http://www.qbiogene.com Qiagen GmbH transfection kits and reagents http://www.qiagen.com Retrogen, Inc. transfection kits and reagents http://www.retrogen.com Roche Applied Science transfection kits and reagents http://www.biochem.roche.com Sigma-Aldrich Corporation transfection kits and reagents http://www.sigma-aldrich.com Stratagene two-hybrid systems http://www.stratagene.com Takara Bio, Inc. transfection kits and reagents http://www.takara-bio.co.jp/english University of Pennsylvania university http://www.upenn.edu VIRxSYS Corporation viral therapeutic systems http://www.virxsys.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 22 October 2004 issue of Science

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