The Technologies and Their Applications

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Here are a few of the new biotechnologies that use cells and biological molecules and examples of their applications in medicine, agriculture, food processing, industrial manufacturing and environmental management.

Bioprocessing Technology

The oldest of the biotechnologies, bioprocessing technology, uses living cells or the molecular components of their manufacturing machinery to produce desired products. The living cells most commonly used are one-celled microorganisms, such as yeast and bacteria; the biomolecular components we use most often are enzymes, which are proteins that catalyze biochemical reactions.

A form of bioprocessing, microbial fermentation, has been used for thousands of years-unwittingly-to brew beer, make wine, leaven bread and pickle foods. In the mid-1800s, when we discovered microorganisms and realized their biochemical machinery was responsible for these useful products, we greatly extended our use of microbial fermentation. We now rely on the remarkably diverse manufacturing capability of naturally occurring microorganisms to provide us with products such as antibiotics, birth control pills, amino acids, vitamins, industrial solvents, pigments, pesticides and food-processing aids.

Today, we are using recombinant DNA technology, coupled with microbial fermentation, to manufacture a wide range of biobased products including human insulin, the hepatitis B vaccine, the calf enzyme used in cheese-making, biodegradable plastics, and laundry detergent enzymes. Bioprocessing technology also encompasses tissue engineering and manufacturing as well as biopharmaceutical formulation and delivery.

Monoclonal Antibodies

Monoclonal antibody technology uses immune-system cells that make proteins called antibodies. We have all experienced the extraordinary specificity of antibodies: Those that attack a flu virus one winter do nothing to protect us from a slightly different flu virus the next year. (Specificity refers to the fact that biological molecules are designed so that they bind to only one molecule.)

The specificity of antibodies also makes them powerful diagnostic tools. They can locate substances that occur in minuscule amounts and measure them with great accuracy. For example, we use monoclonal antibodies to

• locate environmental pollutants.
• detect harmful miroorganisms in food.
• distinguish cancer cells from normal cells.
• diagnose infectious diseases in humans, animals and plants more quickly and more accurately than ever before.

In addition to their value as detection devices, monoclonal antibodies (MAbs) can provide us with highly specific therapeutic compounds. Monoclonal antibodies joined to a toxin can selectively deliver chemotherapy to a cancer cell while avoiding healthy cells. We are developing monoclonal antibodies to treat organ-transplant rejection and autoimmune diseases by targeting them specifically to the type of immune system cell responsible for these attacks, leaving intact the other branches of the immune system.

MAbs FOR IMMUNE-RELATED CONDITIONS

• Muromomab-CD3 (OKT3) is used to prevent acute rejection of organ transplants. A modified version of OKT3 shows promise in inhibiting the autoimmune destruction of beta cells in Type 1 diabetes mellitus.
• Infliximab (Remicade®) binds to tumor necrosis factor-alpha and has shown promise against some inflammatory diseases such as rheumatoid arthritis.
• Omalizumab (Xolair®) binds to IgE and prevents it from binging to mast cells. The drug is used against allergic asthma.
• Daclizumab (Zenapax®) binds to part of the IL-2 receptor and is used to prevent acute rejection of transplanted kidneys. The drug also shows promise against T-cell lymphoma.

MAbs USED TO KILL OR INHIBIT CANCER CELLS

• Rituximab (Rituxan®) binds to the CD20 molecule that is found on most B-cells and is used to treat B-cell lymphomas
• Ibritumomab tiuxetan (Zevalin®) is used against the CD20 molecule on B-cells (and lymphomas) conjugated to either of two radioactive isotopes in conjunction with Rituxan.
• Tositumomab (Bexxar®) is a conjugate of a monoclonal antibody against CD20 and the radioactive isotope iodine-131. It has been approved to treat lymphoma.
• Trastuzumab (Herceptin®) binds to HER2, a receptor for epidermal growth factor found on some breast cancers and lymphomas.
• Cetuximab (Erbitux®) blocks HER1, another epidermal growth factor receptor, and has been approved to treat colorectal cancer.
• Gemtuzumab ozogamicin (Mylotarg®) is a conjugate of a monoclonal antibody that binds to CD33, a cell-surface molecule expressed by the cancerous cells in acute myelogenous leukemia, and calicheamicin, a complex oligosaccharide that makes double-stranded breaks in DNA. The drug is the first immunotoxin that shows promise in the fight against cancer.
• Alemtuzumab (Campath®) binds to CD52, a molecule found on white blood cells, and has produced complete remission of chronic lymphocytic leukemia (for 18 months and counting).

ANGIOGENESIS INHIBITOR

• Bevacizumab (Avastin®) blocks the vascular endothelial growth factor (VEGF) receptor and has been approved for the treatment of colorectal cancer.

OTHER

• Abciximab (ReoPro®) inhibits the clumping of platelets by binding the receptors on their surface that normally are linked by fibrinogen. This therapy is helpful in preventing the re-clogging of the coronary arteries in patients who have undergone angioplasty.

Monoclonal antibodies can be created in mice, but mouse antibodies are "seen" by the human immune system and often the human patient mounts an immune response, which not only eliminates the therapeutic MAb administered, but also causes damage to the kidneys. To reduce the problem of human anti-mouse antibodies (HAMA), scientists use chimeric, or humanized, antibodies. To form a chimeric antibody, one must combine the antigen-binding parts (variable regions) of the mouse antibody with the effector parts (constant regions) of a human antibody. Infliximab, rituximab and abciximab are examples. To create human antibodies, one combines only the amino acids responsible for making the antigen binding site (the hypervariable regions) of a mouse antibody and the rest of a human antibody molecule, thus replacing its own hypervariable regions. Zenapax®, Vitaxin, Mylotarg®, Herceptin®, and Xolair® are examples.

Cell Culture

Cell culture technology is the growing of cells outside of living organisms.

PLANT CELL CULTURE
An essential step in creating transgenic crops, plant cell culture also provides us with an environmentally sound and economically feasible option for obtaining naturally occurring products with therapeutic value, such as the chemotherapeutic agent paclitaxel, a compound found in yew trees and marketed under the name Taxol®. Plant cell culture is also an important source of compounds used as flavors, colors and aromas by the food-processing industry.

INSECT CELL CULTURE
Insect cell culture can broaden our use of biological control agents that kill insect pests without harming beneficial insects or having pesticides accumulate in the environment. Even though we have recognized the environmental advantages of biological control for many decades, manufacturing biological control products in marketable amounts has been impossible. Insect cell culture removes these manufacturing constraints. In addition, like plant cell culture, insect cell culture is being investigated as a production method of therapeutic proteins. Insect cell culture is also being investigated for the production of VLP (virus-like particle) vaccines against infectious diseases such as SARS and influenza, which could lower costs and eliminate the safety concerns associated with the traditional egg-based process. A patient specific cancer vaccine that utilizes insect cell culture has reached Phase III clinical trials.

MAMMALIAN CELL CULTURE
Livestock breeding has used mammalian cell culture as an essential tool for decades. Eggs and sperm, taken from genetically superior bulls and cows, are united in the lab, and the resulting embryos are grown in culture before being implanted in surrogate cows. A similar form of mammalian cell culture has also been an essential component of the human in vitro fertilization process.

Our use of mammalian cell culture now extends well beyond the brief maintenance of cells in culture for reproductive purposes. Mammalian cell culture can supplement-and may one day replace-animal testing to assess the safety and efficacy of medicines. Like plant cell culture and insect cell culture, we are relying on the manufacturing capacity of mammalian cells to synthesize therapeutic compounds, in particular, certain mammalian proteins too complex to be manufactured by genetically modified microorganisms. For example, monoclonal antibodies are produced through mammalian cell culture.

Scientists are also investigating the use of mammalian cell culture as a production technology for vaccines. In 2005, the Department of Health and Human Services awarded a $97 million contract to Sanofi Pasteur to develop mammalian cell culturing techniques to speed the production process for new influenza vaccines and thereby enhance pandemic preparedness.

Therapies based on cultured adult stem cells, which are found in certain tissues like the bone marrow and brain, are on the horizon as well. Researchers have found that adult stem cells can be used by the body to replenish tissues. Adult hematopoietic stem cells already are being transplanted into bone marrow to stimulate the generation of the various types of blood cells necessary to rejuvenate an immune system. These stem cells can be harvested in large quantities from umbilical cord blood, but they are difficult to isolate and purify.

Researchers also are working on ways to harvest stem cells from placentas and from fat. Some are looking at cellular reprogramming as a way to get specialized body cells, like skin cells, to revert to a primordial state so that they can be coaxed into various types of tissues.

Embryonic stem cells are also under study as potential therapies. As the name suggests, embryonic stem cells are derived from embryos-specifically those that develop from eggs that have been fertilized in vitro (in an in vitro fertilization clinic) and then donated by consent for research purposes. The embryos are typically four or five days old and are each a hollow microscopic ball of cells called the blastocyst.

Human embryonic stem cells are isolated by transferring the inner cell mass into a nutrient rich culture medium. There the human stem cells proliferate. Over the course of several days, the cells of the inner cell mass divide and spread all over the dish. Researchers then must remove the growing cells and divide them into fresh culture dishes. This process of replating the cells, called subculturing, is repeated many times over many months. Each cycle of subculturing cells is called a passage. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating (i.e., remain pluripotent) and appear genetically normal are referred to as an embryonic stem cell line.

The inner surface of the culture dish may be coated with mouse embryonic skin cells that have been engineered not to divide. This is called the "feeder layer." It provides a sticky surface to which the human embryonic cells attach. Recently scientists have been figuring out ways to grow embryonic stem cells without using mouse feeder cells-a significant advance because of the risk of viruses and other macromolecules in the mouse cells being transmitted to the human cells.

The potential value of stem cell therapy and tissue engineering can best be realized if the therapeutic stem cells and the tissues derived from them are genetically identical to the patient receiving them. Therefore, unless the patient is the source of the stem cells, the stem cells need to be "customized" by replacing the stem cell's genetic material with the patient's before cueing the stem cells to differentiate into a specific cell type. To date, this genetic material replacement and reprogramming can be done effectively only with embryonic stem cells.

Recombinant DNA Technology

Recombinant DNA technology is viewed by many as the cornerstone of biotechnology. The term recombinant DNA literally means the joining or recombining of two pieces of DNA from two different species.

Humans began to preferentially combine the genetic material of domesticated plants and animals thousands of years ago by selecting which individuals would reproduce. By breeding individuals with valuable genetic traits while excluding others from reproduction, we changed the genetic makeup of the plants and animals we domesticated. Now, in addition to using selective breeding to combine valuable genetic material from different organisms, we combine genes at the molecular level using the more precise techniques of recombinant DNA technology.

Genetic modification through selective breeding and recombinant DNA techniques fundamentally resemble each other, but there are important differences:

• Genetic modification using recombinant DNA techniques allows us to move single genes whose functions we know from one organism to any other.
• In selective breeding, large sets of genes of unknown function are transferred between related organisms.

By making our manipulations more precise and our outcomes more certain, we decrease the risk of producing organisms with unexpected traits and avoid the time-consuming, trial-and-error approach of selective breeding. By increasing the breadth of species from which we can obtain useful genes, we can access all of nature's genetic diversity.

Techniques for making selective breeding more predictable and precise have been evolving over the years. In the early 1900s, Hugo DeVries, Karl Correns and Eric Tshermark rediscovered Mendel's laws of heredity. In 1953, James Watson and Francis Crick deduced DNA's structure from experimental clues and model building. In 1972, Paul Berg and colleagues created the first recombinant DNA molecules, using restriction enzymes. Ten years later, the first recombinant DNA-based drug (recombinant human insulin) was introduced to the market. By 2000 the human genome had been sequenced and today we use recombinant DNA techniques, in conjunction with molecular cloning to

• produce new medicines and safer vaccines.
• treat some genetic diseases.
• enhance biocontrol agents in agriculture.
• increase agricultural yields and decrease production costs.
• decrease allergy-producing characteristics of some foods.
• improve food's nutritional value.
• develop biodegradable plastics.
• decrease water and air pollution.
• slow food spoilage.
• control viral diseases.
• inhibit inflammation.

Cloning

Cloning technology allows us to generate a population of genetically identical molecules, cells, plants or animals. Because cloning technology can be used to produce molecules, cells, plants and some animals, its applications are extraordinarily broad. Any legislative or regulatory action directed at "cloning" must take great care in defining the term precisely so that the intended activities and products are covered while others are not inadvertently captured.

MOLECULAR OR GENE CLONING
Molecular or gene cloning, the process of creating genetically identical DNA molecules, provides the foundation of the molecular biology revolution and is a fundamental and essential tool of biotechnology research, development and commercialization. Virtually all applications in biotechnology, from drug discovery and development to the production of transgenic crops, depend on gene cloning.

The research findings made possible through molecular cloning include identifying, localizing and characterizing genes; creating genetic maps and sequencing entire genomes; associating genes with traits and determining the molecular basis of the trait. For a full discussion, see the next section.

ANIMAL CLONING
Animal cloning has helped us to rapidly incorporate improvements into livestock herds for more than two decades and has been an important tool for scientific researchers since the 1950s. Although the 1997 debut of Dolly, the cloned sheep, brought animal cloning into the public consciousness, the production of an animal clone was not a new development. Dolly was considered a scientific breakthrough not because she was a clone, but because the source of the genetic material that was used to produce Dolly was an adult cell, not an embryonic one.

Recombinant DNA technologies, in conjunction with animal cloning, are providing us with excellent animal models for studying genetic diseases, aging and cancer and, in the future, will help us discover drugs and evaluate other forms of therapy, such as gene and cell therapy. Animal cloning also provides zoo researchers with a tool for helping to save endangered species.

There are two different ways to make an exact genetic copy of an organism such as a sheep or a laboratory mouse.

Artificial embryo twinning (AET) is the old-fashioned way to clone. AET mimics the natural process of creating identical twins, only in a Petri dish rather than the mother's womb. Researchers manually separate a very early embryo into individual cells and then allow each cell to divide and develop on its own. The resulting embryos are placed into a surrogate mother, where they are carried to term and delivered. Since all the embryos come from the same zygote, they are genetically identical.

Somatic cell nuclear transfer (SCNT) involves the isolation of a somatic (body) cell, which is any cell other then those used for reproduction (sperm and egg, known as the germ cells). In mammals, every somatic cell has two complete sets of chromosomes, whereas the germ cells have only one complete set. To make Dolly, scientists transferred the nucleus of a somatic cell taken from an adult female sheep and transferred it to an egg cell from which the nucleus had been removed. After some chemical manipulation, the egg cell, with the new nucleus, behaved like a freshly fertilized zygote. It developed into an embryo, which was implanted into a surrogate mother and carried to term.

Protein Engineering

Protein engineering technology is used, often in conjunction with recombinant DNA techniques, to improve existing proteins, such as enzymes, antibodies and cell receptors, and to create proteins not found in nature. These proteins may be used in drug development, food processing and industrial manufacturing.

The most pervasive uses of protein engineering to date are applications that alter the catalytic properties of enzymes to develop ecologically sustainable industrial processes. Enzymes are environmentally superior to most other catalysts used in industrial manufacturing because, as biocatalysts, they dissolve in water and work best at neutral pH and comparatively low temperatures. In addition, because biocatalysts are more specific than chemical catalysts, they also produce fewer unwanted byproducts. The chemical, textile, pharmaceutical, pulp and paper, food and feed, and energy industries are all benefiting from cleaner, more energy-efficient production made possible by incorporating biocatalysts into their production processes.

The characteristics that make biocatalysts environmentally advantageous may, however, limit their usefulness in certain industrial processes. For example, most enzymes fall apart at high temperatures. Scientists are circumventing these limitations by using protein engineering to increase enzyme stability under harsh manufacturing conditions.

In addition to industrial applications, medical researchers have used protein engineering to design novel proteins that can bind to and deactivate viruses and tumor-causing genes; create especially effective vaccines; and study the membrane receptor proteins that are so often the targets of pharmaceutical compounds. Food scientists are using protein engineering to improve the functionality of plant storage proteins and develop new proteins as gelling agents.

In addition, new proteins are being developed to respond to chemical and biological attacks. For example, hydrolases detoxify a variety of nerve agents as well as commonly used pesticides. Enzymes are safe to produce, store and use, making them an effective and sustainable approach to toxic materials decontamination.

Biosensors

Biosensor technology couples our knowledge of biology with advances in microelectronics. A biosensor is composed of a biological component, such as a cell, enzyme or antibody, linked to a tiny transducer-a device powered by one system that then supplies power (usually in another form) to a second system. Biosensors are detecting devices that rely on the specificity of cells and molecules to identify and measure substances at extremely low concentrations.

When the substance of interest binds with the biological component, the transducer produces an electrical or optical signal proportional to the concentration of the substance. Biosensors can, for example,

• measure the nutritional value, freshness and safety of food.
• provide emergency room physicians with bedside measures of vital blood components.
• locate and measure environmental pollutants.
• detect and quantify explosives, toxins and biowarfare agents.

Nanobiotechnology

Nanotechnology, which came into its own in 2000 with the birth of the National Nanotechnology Initiative, is the next stop in the miniaturization path that gave us microelectronics, microchips and microcircuits. The word nanotechnology derives from nanometer, which is one-thousandth of a micrometer (micron), or the approximate size of a single molecule. Nanotechnology-the study, manipulation and manufacture of ultra-small structures and machines made of as few as one molecule-was made possible by the development of microscopic tools for imaging and manipulating single molecules and measuring the electromagnetic forces between them.

Nanobiotechnology joins the breakthroughs in nanotechnology to those in molecular biology. Molecular biologists help nanotechnologists understand and access the nanostructures and nanomachines designed by 4 billion years of evolutionary engineering-cell machinery and biological molecules. Exploiting the extraordinary properties of biological molecules and cell processes, nanotechnologists can accomplish many goals that are difficult or impossible to achieve by other means.

For example, rather than build silicon scaffolding for nanostructures, DNA's ladder structure provides nanotechnologists with a natural framework for assembling nanostructures and its highly specific bonding properties bring atoms together in a predictable pattern to create a nanostructure.

Nanotechnologists also rely on the self-assembling properties of biological molecules to create nanostructures, such as lipids that spontaneously form liquid crystals.

DNA has been used not only to build nanostructures but also as an essential component of nanomachines. Most appropriately, DNA, the information storage molecule, may serve as the basis of the next generation of computers. As microprocessors and microcircuits shrink to nanoprocessors and nanocircuits, DNA molecules mounted onto silicon chips may replace microchips with electron flow-channels etched in silicon. Such biochips are DNA-based processors that use DNA's extraordinary information storage capacity. Conceptually, they are very different from the DNA chips discussed below. Biochips exploit the properties of DNA to solve computational problems; in essence, they use DNA to do math. Scientists have shown that 1,000 DNA molecules can solve in four months computational problems that would require a century for a computer to solve.

Other biological molecules are assisting in our continual quest to store and transmit more information in smaller places. For example, some researchers are using light-absorbing molecules, such as those found in our retinas, to increase the storage capacity of CDs a thousand-fold.

Some applications of nanobiotechnology include

• increasing the speed and power of disease diagnostics.
• creating bio-nanostructures for getting functional molecules into cells.
• improving the specificity and timing of drug delivery.
• miniaturizing biosensors by integrating the biological and electronic components into a single, minute component.
• encouraging the development of green manufacturing practices.

Microarrays

Microarray technology is transforming laboratory research because it allows us to analyze tens of thousands of samples simultaneously.

Researchers currently use microarray technology to study gene structure and function. Thousands of DNA or protein molecules are arrayed on glass slides to create DNA chips and protein chips, respectively. Recent developments in microarray technology use customized beads in place of glass slides.

DNA MICROARRAYS
DNA microarrays are used to

• detect mutations in disease-related genes.
• monitor gene activity.
• diagnose infectious diseases and identify the best antibiotic treatment.
• identify genes important to crop productivity.
• improve screening for microbes used in bioremediation.

DNA-based arrays will be essential for converting the raw genetic data provided by the Human Genome Project and other genome projects into useful products. Gene sequence and mapping data mean little until we determine what those genes do-which is where protein arrays come in.

PROTEIN MICROARRAYS
While going from DNA arrays to protein arrays is a logical step, it is by no means simple to accomplish. The structures and functions of proteins are much more complicated than that of DNA, and proteins are less stable than DNA. Each cell type contains thousands of different proteins, some of which are unique to that cell's job. In addition, a cell's protein profile varies with its health, age, and current and past environmental conditions.

Protein microarrays will be used to

• discover protein biomarkers that indicate disease stages.
• assess potential efficacy and toxicity of drugs before clinical trials.
• measure differential protein production across cell types and developmental stages, and in both healthy and diseased states.
• study the relationship between protein structure and function.
• assess differential protein expression in order to identify new drug leads.
• evaluate binding interactions between proteins and other molecules.

The fundamental principle underlying microarray technology has inspired researchers to create many types of microarrays to answer scientific questions and discover new products.

TISSUE MICROARRAYS
Tissue microarrays, which allow the analysis of thousands of tissue samples on a single glass slide, are being used to detect protein profiles in healthy and diseased tissues and validate potential drug targets. Brain tissue samples arrayed on slides with electrodes allow researchers to measure the electrical activity of nerve cells exposed to certain drugs.

WHOLE-CELL MICROARRAYS
Whole-cell microarrays circumvent the problem of protein stability in protein microarrays and permit a more accurate analysis of protein interactions within a cell.

SMALL-MOLECULE MICROARRAYS
Small-molecule microarrays allow pharmaceutical companies to screen ten of thousands of potential drug candidates simultaneously.

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