Another process used by bacteria to obtain DNA fragments from other bacteria is conjugation (Figure 25.2). This genetic recombination process occurs between two living bacteria: a donor and a recipient. The donor and recipient form a cytoplasmic bridge by which single-stranded DNA of the donor crosses to the recipient. On rare occasions the DNA may be integrated into the recipient’s chromosome, but most often the DNA remains in the cytoplasm as a free-floating loop, called a plasmid (see Chapter 6, Bacteria and Archaea).

FIGURE 25.2 Conjugation in bacteria.
*(1)* A cytoplasmic bridge forms between the donor cell and the recipient; *(2)* the cells contact each other; *(3)* one strand of donor plasmid DA transfers to the recipient; and *(4)* a strand complementary to the plasmid is formed in both the recipient and donor cells.

A third form of recombination is transduction, which involves bacterial viruses called bacteriophages (see Chapter 7, Viruses). The virus enters a bacterial cell and integrates its DNA into the bacterial chromosome.

Viruses other than bacteriophages are also useful in genetic engineering experiments. Viruses (see Chapter 7) consist of nucleic acid fragments, either DNA or RNA, surrounded by a protein coat. They can replicate only in living cells, taking over the host cell machinery. Some viruses integrate their own DNA into the host cell’s DNA and become part of the cell’s genome. An example is herpesvirus, which integrates into a nerve cell genome and remains within that cell for years, causing recurrent herpes infections. DNA technologists realized that this was a way to bring genes into a cell of choice. The tools of genetic engineering include the following:

• Restrictions enzymes

• Gene libraries

• Plasmids

• Complementary DNA (cDNA)

• Polymerase chain reaction

Restriction Enzymes

In the 1950s Salvador Luria and colleagues discovered that E. coli could resist destruction by bacteriophages; the organism could “restrict” the replication of the virus. Later, in 1962 Werner Arber and his research group identified the mechanism by which E. coli could achieve this restriction. They showed that an enzyme system was able to cleave the phage DNA before it could get to the bacterial cytoplasm. Subsequently they isolated the DNA-cleaving enzyme, which they called endonuclease, now known as a restriction enzyme. The enzyme is capable of cutting viral DNA but does not cut the host DNA because the organism modifies the bacterial DNA by adding a methyl group to it. Since the work of these two research groups other restriction enzymes have been discovered. More than 800 restriction enzymes have been identified so far, recognizing more than 100 different nucleotide sequences. Restriction enzymes are named after the bacterium from which they have been isolated. The name includes three letters from the bacterium, one from the strain, and roman numerals indicating the order in which enzymes from the same type of bacterium were identified (Box 25.1).

A restriction enzyme scans a DNA molecule, recognizing and cutting DNA only at a particular sequence of nucleotides. Some enzymes cut the DNA straight across both of the strands of the double helix and therefore produce “blunt” ends (Table 25.1). On the other hand, many restriction enzymes cut in an offset fashion and the ends of the cut have an overhanging piece of single-stranded DNA. These ends are called “sticky” ends (Table 25.2) because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Every cleavage performed with the same enzyme will occur at the same restriction site, regardless of the source of the DNA. When such molecules from different DNA sources are mixed together they will join with each other by base pairing between their sticky ends (Figure 25.3, A). This combination can be made permanent by DNA ligase, an enzyme that forms covalent bonds along the backbone of each strand. The result of this reaction is a molecule of recombinant DNA (rDNA) (Figure 25.3, B). The ability to produce rDNA molecules has revolutionized the study of genetics and formed the foundation for the biotechnology industry.

TABLE 25.1

Examples of Restriction Enzymes That Produce “blunt” Ends

Enzyme	Source	Recognition Site
AluI	Arthrobacter luteus	5′-AG↓CT-3′ 3′-TC↑GA-5′

EcoRII Escherichia coli R245

5′-CC↓GG-3′

3′-GG↑CC-5′

HaeIII Haemophilus aegyptius

5′-GG↓CC-3′

3′-CC↑GG-5′

HpaI Haemophilus parainfluenzae

5′-GTT↓AAC-3′

3′-CAA↑TTG-5′

SmaI Serratia marcescens

5′-CCC↓GGG-3′

3′-GGG↑CCC-5′

TABLE 25.2

Examples of Restriction Enzymes That Produce “Sticky” Ends

Enzyme	Source	Recognition Site
BamHI	Bacillus amyloliquefaciens H	5′-G↓GATCC-3′ 3′-CCTAG↑G-5′
EcoRI	Escherichia coli RY13	5′-G↓AATTC-3′ 3′-CTTAA↑G-5′
HindIII	Haemophilus influenzae Rd	5′-A↓AGCTT-3′ 3′-TTCGA↑A-5′
PstI	Providencia stuartii	5′-CTGCA↓G-3′ 3′-G↑ACGTC-5′
SalI	Streptomyces albus	5′-G↓TCGAC-3′ 3′-CAGCT↑G-5′

FIGURE 25.3 Action of restriction enzymes.
A, Production of “sticky” ends. B, Formation of recombinant DNA—the DNA fragments combine and are sealed by DNA ligase.

Gene Libraries

A gene library is composed of a series of cells or unicellular organisms that are used to store genes that have been obtained from foreign cells. To form a gene library a mass of cells that contain the desired gene must be acquired. The desired gene must then be isolated from the total DNA of the cells. For example, human cells have a total of 100,000 genes and only 1 gene may be the target of interest, such as the gene for insulin (pancreatic cells), or the gene for human growth hormone (anterior pituitary).

Creating a Gene Library

To establish a cell line containing the gene of interest, the particular DNA containing the gene must be isolated. A restriction enzyme is used to cut the cell’s DNA into fragments. After this process a host organism for the fragments needs to be chosen. An example of such an organism is Escherichia coli. The plasmids of E. coli are then isolated and the DNA fragments that were previously obtained are inserted into the plasmids (Figure 25.4). The recombined plasmids contain all the DNA fragments and are then inserted into E. coli cells, and together these E. coli cells now are a warehouse of all the DNA of the original cell of interest. The cell collection is the library, the individual cells represent the books of the library, and the individual DNA fragments contain the information in each of the books. This type of library is referred to as a genomic library. The next step is to find the particular gene of interest and therefore the library must be screened.

FIGURE 25.4 Establishing a gene library.
*(1),* The DNA of interest is isolated; *(2),* the DNA is cut into fragments by restriction enzymes; *(3),* the DNA fragments of interest are inserted into plasmids; *(4),* the recombined plasmids are inserted into *Escherichia coli*.

Screening the Gene Library

The screening process begins with growing the transformed E. coli cells on nutrient agar. Colonies derived from each individual cell will be visible within 24 to 48 hours, and each colony contains cells with the DNA insert. Now each colony needs to be examined for the presence of the insert that contains the gene of interest. To identify the specific colony a copy of the plate must be made for further work. This is done by a process called replica plating (Figure 25.5). This technique is performed by pressing a piece of sterile velvet onto the gel containing the colonies, followed by pressing the velvet to a new petri dish of nutrient agar to establish another plate, which then can be used for analysis of the target gene.

The analysis can be done a number of ways, one of which is by using a radioactive mRNA probe to identify the gene of interest. Bacteria from a particular colony are taken; their DNA is fragmented and then separated in a process called electrophoresis. The radioactive mRNA probe is then applied to find out whether any of the fragments on the gel contain the DNA (gene) for the complementary mRNA. If no radioactivity occurs, the gene of interest is not present in that particular colony. If radioactivity accumulates at one site, the DNA fragment has been identified and the colony with the DNA of interest has been identified on the working (replica) plate. By working backward the colony in the original plate, the one containing the gene of interest, can be identified and then extracted far more easily than from the original cells (e.g., cells of the pancreas producing the insulin).

cDNA Libraries

Cloning genes from a eukaryotic cell is more problematic than cloning genes from prokaryotes. As described in Chapter 3 (Cell Structure and Function), eukaryotic genes contain exons, the stretches of DNA that encode a protein, and introns, portions of DNA that do not encode a protein and are removed from the mRNA before leaving the nucleus. To clone eukaryotic genes it is best to use a version of the gene that lacks the introns, because once a gene is placed into a bacterial cell, the bacterium will be unable to remove these introns, and therefore will be unable to produce the intended protein. With the advances of genetic engineering it is now possible to create artificial genes that contain only exons. This is achieved with the aid of an enzyme called reverse transcriptase to synthesize complementary DNA (cDNA) from an mRNA template (Figure 25.6). This process is the reverse of the normal interaction between DNA and RNA, in that the isolated mRNA is used to produce a complementary DNA molecule. After the synthesis of the first strand of DNA, the mRNA is digested by reverse transcriptase, and the addition of DNA polymerase will produce the second, complementary strand of DNA, resulting in a double-stranded DNA molecule. These cDNA molecules can then be cloned to form a cDNA library.

FIGURE 25.6 Complementary DNA.
Complementary DNA (cDNA) can be synthesized from an mRNA template with the help of the enzyme reverse transcriptase.

Synthetic DNA

DNA technology has advanced to the point that DNA can be produced in the laboratory with instruments called DNA synthesis machines. The nucleotide sequence of the desired DNA is entered via keyboard, and a microprocessor controls the synthesis of the DNA from stored nucleotides and other vital reagents. Because only a relatively small number of nucleotides can be created in this way, several chains may have to be produced and then linked together to form a desired gene. To use this technique the nucleotide sequence of the gene must be known. If the gene has not been isolated previously the technique can still be used by predicting the DNA sequence if the amino acid sequence of the protein product of the gene is known. However, because the degeneracy of the genetic code prevents a definite outcome, the makeup of the gene cannot be 100% predicted. For example, how can it be determined which of the six codons for arginine is part of the gene (Figure 25.7)?

FIGURE 25.7 The genetic code.
This illustration shows the various transfer RNA (tRNA) molecules that encode the amino acid arginine—only one will base pair with the codon on the complementary mRNA.

Plasmids

In the early 1970s, Stanley Cohen and his team at Stanford University studied small loops of DNA found in the cytoplasm of bacteria, which are now know as plasmids (Figure 25.8) (see Chapter 6, Bacteria and Archaea). At about the same time Herbert Boyer at the University of California San Francisco studied restriction enzymes found in E. coli. In 1973, Herbert Boyer and Stanley Cohen teamed up to produce the world’s first recombinant DNA organism, using gene splicing. They removed plasmids from E. coli and, using restriction enzymes, they cut the plasmid at precise positions. The segments could then be inserted into a different spliced plasmid and recombined using DNA ligase. The altered DNA (recombinant) could then be inserted into E. coli. With their studies with restriction enzymes and plasmids Boyer and Stanley set the foundation of modern biotechnology/genetic engineering (see Why You Need to Know at the beginning of this chapter).

Once a recombinant plasmid is in the bacterial cell, the single plasmid multiplies itself, independent from the chromosomal DNA, forming dozens of replicas among the normal plasmids. At the time of cell division, the plasmid with the foreign DNA is also copied. In certain bacteria cell division occurs as often as every 20 minutes, and each new bacterium will contain several new plasmids. A single bacterium can breed millions of others—this population is referred to as a clone. Because all cells of a clone have the identical plasmids, millions of copies of the foreign gene have been generated. The gene has been cloned.

Polymerase Chain Reaction

The polymerase chain reaction (PCR) is a technique, developed in 1986, that can produce DNA copies without a host cell. It is a technique by which small DNA fragments can quickly be amplified to quantities large enough to allow analysis. PCR is used for many applications such as in molecular biology, human genetics, evolution, development, conservation, and forensics. Before starting the PCR process with just one piece of DNA, generally the size of a gene, information concerning the nucleotide sequence of the target DNA is required. This information is necessary to be able to produce two nucleotide primers: one complementary to the 5′ end and one complementary to the 3′ end. PCR can be used to make billions of DNA copies in only a few hours and the amount of amplified DNA produced is limited only to the number of times the steps in the PCR process are repeated. These steps are as follows (Figure 25.9):

• Denaturation: The double-stranded target DNA to be cloned is denatured into two single strands. This process takes place when heating the double-stranded DNA from 90° C to 95° C for about 1 minute.

• Priming: Subsequently the temperature of the reactions is lowered to about 50° C to 70° C, which causes the primers to bind to the single-stranded DNA molecules. These primers, synthetic oligonucleotides, are complementary to the sequences flanking the DNA. They serve as the starting points for the synthesis of new DNA strands, complementary to the original strands.

• Extension: A heat-stable DNA polymerase is added and DNA synthesis takes place at temperatures between 70° C and 75° C.

After each cycle the DNA is heated up again to denature all newly formed DNA into single strands and the multiplication can continue. Thirty cycles can be completed in just a few hours and will increase the amount of the original DNA by more than 1 billion times. PCR is applied in many situations, including the identification of infectious agents that would otherwise stay undetected because of low numbers in a sample.

Vectors in Biotechnology

One of the goals of biotechnology is to insert foreign or modified genes into a cell to produce a new phenotype. Various types of DNA molecules can serve as vectors, which are carriers intended to deliver the newly inserted gene into a given cell. All vectors have certain criteria in common:

• Vectors need to be small so that they can be manipulated in the laboratory. A large DNA molecule, such as an entire chromosome, is usually too frail to serve as a vector.

• Vectors need to survive in the target cell.

• Vectors need to contain a recognizable genetic marker so that the biotechnologist can identify the cells that have received the vector and the associated gene of interest.

• Vectors need to provide the required promoters to ensure genetic expression of the target gene in the cell.

Plasmid Vectors

The first vectors developed and still being used were genetically modified plasmids, derived from naturally occurring plasmids. Many plasmids have been genetically modified to serve as vectors and are now commercially available. As previously stated, once a plasmid enters a bacterial cell it will multiply and therefore enhance the number of DNA clones within that cell. In addition, these vectors have been genetically engineered and contain a number of different restriction enzyme recognition sequences, as well as marker genes that can express their presence in the host cell. In general, plasmids that are used in DNA technology should be as small as possible because they are less easily damaged than large plasmids. Furthermore, small plasmids are more easily taken up by the host cell. The ease of uptake of a plasmid into the host cell also requires that the inserted cDNA cannot be unreasonably large. A simplified version of the insertion of DNA into a plasmid and subsequent cloning is illustrated in Figure 25.10.

FIGURE 25.10 Plasmid vector.
*(1)* A plasmid vector is cut by a specific restriction enzyme and the DNA fragment of interest is inserted to form a recombinant plasmid; *(2)* the recombinant plasmid is inserted into a bacterial cell (e.g., *E. coli*) and the bacterium is transformed; *(3)* within the bacteria the recombinant plasmids multiply independent of cell division; and *(4)* as the bacterial cells divide the plasmids continue to multiply and produce the desired gene product.

Lambda (λ) Phage Vectors

Plasmid vectors are generally used for cloning small DNA segments consisting of up to about 25 kilobases (kb) of inserted DNA. To prepare a genomic library for a eukaryote, the clone fragments need to be large enough to contain a whole gene. The requirements to clone larger segments of DNA can be fulfilled by both the lambda (λ) phage and cosmid vectors; the former allows cloning of segments up to 25 kb and the latter can accommodate segments up to 45 kb.

The lambda phage is a bacteriophage (see Chapter 7, Viruses) that infects E. coli. The genome of the phage has been completely mapped and sequenced, and experiments with this phage have shown that the central part of its chromosome can be replaced with foreign DNA without affecting the phage and its ability to infect E. coli. This makes this phage an ideal vector for larger DNA segments. To clone the phage and use it as a vector, the phage DNA needs to be isolated and cut with a restriction enzyme, to produce three chromosomal fragments, with the middle one being replaced by the DNA of another source. Once the foreign DNA has been inserted, DNA ligase is used to produce the recombinant λ vector that is then enclosed into the phage protein head (Figure 25.11). The recombinant phage will then be introduced to a culture of E. coli, and once in the bacteria the vectors replicate, forming many phages with the inserted DNA. As the phages reproduce, they lyse their bacterial host cells, and the cloned DNA can be recovered in multiple copies.

FIGURE 25.11 Lambda phage.
The lambda phage can act as a vector once a desired gene has been inserted into its DNA. The recombinant phage will then infect *E. coli* and the bacterium will produce the desired gene product.

Cosmid Vectors

Another class of useful vectors is cosmids, hybrid vectors created by combining a fragment of DNA (cDNA) from a virus, usually a bacteriophage, with parts of a plasmid. Cosmids have sequences allowing them to pack phage DNA into the phage protein coat, and the plasmid sequence necessary for replication. A marker, such as an antibiotic resistance gene, is also inserted into the cosmid for identification of the host cell. The cosmid is then packed into the protein head of the bacteriophage and is ready to infect a bacterial cell. Once in the bacterial host cell the linear DNA becomes a plasmid-like loop and replicates as a plasmid.

Biotechnology in Human Medicine

Today’s biotechnology allows the production of a variety of pharmaceutical and therapeutic substances to assist in a variety of medical tasks. These include protein replacement such as for insulin, aiding in therapy such as with interferons, vaccines such as the hepatitis B vaccine, screening for genetic diseases, and gene therapy, which for the most part is still in the developmental stage. Traditional biotechnology is now supplemented by recombinant DNA technology to produce a variety of pharmaceutical and therapeutic products, performing many medically important tasks. A summary of products produced by recombinant DNA technology and used in human medicine is shown in Table 25.3.

TABLE 25.3

Products From Recombinant DNA Technology Used in Human Medicine

Product	Use
Insulin	Treatment for some type 1 and type 2 diabetes mellitus
Human growth hormone (hGH)	Dwarfism, growth hormone deficiency, adult growth hormone deficiency
Factor VIII	Replacement for blood-clotting factor in type A hemophilia
Tissue plasminogen activator (tPA)	Can dissolve blood clots in heart attack and stroke
Interferon	Treatment of viral infections such as hepatitis C and genital warts, some types of cancer, and multiple sclerosis
Interleukins	Cancer treatment
Orthoclone	Immune suppressant in transplant patients
Macrophage colony-stimulating factor	Stimulation of red bone marrow activity after bone marrow grafting
Tumor necrosis factor (TNF)	Treatment of cancer
Granulocyte colony-stimulating factor	Treatment of cancer patients experiencing low neutrophil counts
Antisense molecules	Possible way to treat genetic disorders
Erythropoietin	Treatment of anemia; hormone stimulates red blood cell production
rhDNase	Treatment that can break down thick lung secretions in cystic fibrosis
Antitrypsin	Replacement therapy to benefit emphysema patients
Vaccines	Hepatitis B, Haemophilus influenzae type b meningitis, experimental HPV and AIDS vaccine

Human Protein Replacements

Although the Human Genome Project was finished in 2003 and the sequence of the human chromosome is essentially known, the exact number of genes within the genome is still not known. In contrast to the previously believed number of 100,000 genes in a human cell, in 2003 at the time of the completion of the project this number was reduced to 35,000 protein-coding genes. Then, in a National Institutes of Health (NIH) news release on October 20, 2004, the International Human Genome Sequencing Consortium reduced the estimated number of human protein-coding genes to only 20,000 to 25,000. Sometimes a gene can be defective and produce an incorrect protein, or does not produce the protein at all. A protein deficiency can lead to disease and generally this is inherited. At present, certain protein deficiencies can be overcome via biotechnology/genetic engineering.

Insulin

Diabetes mellitus is a disease in which insulin is either not produced by the pancreas (diabetes I), or is produced in insufficient amounts (diabetes II). Insulin is an important protein hormone that facilitates the uptake of glucose by cells for use in cellular metabolism. In the absence of insulin, glucose remains in the bloodstream, resulting in the insufficient production of ATP within the cells. This then leads to weakness of the patient due to the lack of cellular ATP.

Millions of diabetics must take insulin to avoid the adverse affects of diabetes. The hormone insulin is a protein consisting of two polypeptide chains, an A chain and a B chain, linked together by disulfide bonds. The A chain consists of 21 amino acids and the B chain contains 30 amino acids, a total of 51 amino acids per insulin molecule. Before the advent of modern biotechnology insulin was purified from the pancreases of pigs and cows, obtained from slaughterhouses. Although the animal insulin is similar to human insulin, there is a difference in one or three amino acids, which can result in an allergic reaction in human patients. With current methods human insulin can now be mass-produced by the application of recombinant DNA technology and with the help of a plasmid from E. coli. The steps of insulin production involve the tools of genetic engineering previously discussed:

• Isolation of the human insulin gene

• Preparation of the target DNA, the plasmid

• Insertion of the human gene into the plasmid

• Incorporation of the recombinant plasmid back into the bacteria

• Multiplication of the plasmid

• Target cell reproduction, and production of human insulin

• Harvesting of human insulin

Millions of diabetics now use the human insulin produced by bacteria.

MEDICAL HIGHLIGHTS

Noninjectible Insulin

Millions of people with type 1 or type 2 diabetes are dependent on insulin injection to control their blood sugar levels. Many pharmaceutical companies have been working on a noninjectible form of insulin—a dream for diabetes patients. Generex, a Canadian biotechnology company, developed an oral insulin spray called Oral-lyn. In 2005, the formulation was approved for commercial marketing and sale in Ecuador by the Ministry of Public Health. Oral-lyn has not been approved in the United States and Europe at this time.

Another noninjectible insulin preparation was developed by Nektar Therapeutics (San Carlos, CA) and Pfizer (New York, NY). The preparation Exubera was approved by the U.S Food and Drug Administration (FDA) in January 2006. Exubera is fast-acting and meant to be used at mealtime; however, some diabetics will still have to take slow-acting injectable insulin to work between meals. Lately, concerns have arisen that the inhaled product may cause lung problems and lung cancer. According to Pfizer, the label update states that all patients who develop lung cancer had a history of cigarette smoking. On October 18, 2007, Pfizer announced that it is returning its Exubera rights to Nektar Therapeutics and that it would discontinue the marketing of the product. According to the company the decision was not based on any safety problems; rather, too few patients are taking Exubera. The production of Exubera stopped January 16, 2008.

Human Growth Hormone

Human growth hormone (HGH) is a protein hormone of about 190 amino acids, synthesized and secreted by somatotroph cells of the anterior pituitary. The hormone plays a major role in several metabolic processes, including growth and metabolism. It stimulates overall body growth by increasing cellular uptake of amino acids and encouraging protein synthesis.

Dwarfism, a metabolic disorder, occurs when the anterior pituitary fails to secrete sufficient amounts of HGH. Growth hormone deficiency (GHD) in children is also a condition that occurs when not enough HGH is produced and released. Children with GHD may grow less than 2 in. per year. Once an adult reaches full height, growth hormone helps to maintain the proper amounts of fat, muscle, and bone. If an adult does not produce enough growth hormone, the individual may have a condition called adult growth hormone deficiency, or aGHD.

In the past, generally only dwarfism was treated with growth hormone, which had to be obtained from cadavers. Approximately 80 cadavers had to be used to obtain enough HGH for a year’s therapy. This method of treatment unfortunately also brought the risk of the development of Creutzfeldt-Jakob syndrome, a central nervous system disease caused by a prion (see Chapter 7, Viruses). Because of the possibility of transmission of this disease, the use of cadaver tissue in the United States and Britain was restricted in 1985. HGH can now be produced by genetic engineering, using cDNA encoding growth hormone. The cDNA is then expressed in a bacterial expression vector. A problem with producing a short polypeptide hormone such as HGH is its susceptibility to protease digestion. This problem is being resolved by using bacterial host strains that lack several proteases.

Interferon

Interferon is a small protein produced naturally by certain white blood cells and by cells that have been infected with a virus (see Chapter 20, The Immune System). Originally, the interferon system was believed to be a defense system against viruses only, but it is now known to be involved in the defense against other microbes and also in immune regulation. The interferon system consists of three main groups: interferon-α, interferon-β, and interferon-γ. Interferon-α is primarily a product of lymphocytes and macrophages, interferon-β is produced by fibroblasts and epithelial cells, and interferon-γ is produced by T cells, B-cells, and NK cells. Interferon was not readily available until 1980, when the interferon gene was inserted into bacteria by recombinant DNA technology; this allowed mass cultivation and purification from bacterial cultures or from yeast. Synthetic interferons are now used in the treatment of viral infections, multiple sclerosis, and cancer (see Health Care Application: Examples of Interferon Therapy in Humans).

HEALTHCARE APPLICATION
Examples of Interferon Therapy in Humans

Generic Interferon	Trade Name	Source	Used in Treatment of:
Interferon alfa-n3	Alferon N	Human leukocytes	Genital warts
Interferon alfa-2b	Intron A	Recombinant (E. coli)	Chronic hepatitis C Hairy cell leukemia Malignant melanoma Follicular lymphoma Condylomata acuminatum AIDS-related Kaposi’s sarcoma

Interferon alfa-2a Roferon A (discontinued for the U.S. market in 2008) Recombinant

Chronic hepatitis C

Hairy cell leukemia

AIDS-related Kaposi’s sarcoma

Interferon beta-1a

Avonex

Rebif

CinnoVex

Recombinant (E. coli) Multiple sclerosis Interferon beta-1b Betaseron Recombinant (E. coli) Relapsing forms of multiple sclerosis Interferon Alfacon-1 Infergen Recombinant (E. coli) Chronic hepatitis C Interferon gamma-1b Actimmune Recombinant (E. coli) Chronic granulomatous disease

Antisense Molecules

A sense strand is a 5′-to-3′ mRNA or DNA molecule. The complementary strand to the sense strand is called antisense. Antisense technology uses antisense strands to bind with the sense strand. When an antisense strand binds to an mRNA sense strand, the cell will identify the now double-stranded RNA as foreign to the cell and degrade the defective mRNA molecule, and therefore prevent the production of the undesired protein.

Antisense therapy is a possible way to treat genetic disorders or infections. If the genetic sequence of a gene is known to be the cause of a particular disease, and antisense molecules can be produced and inactivate the functioning of that gene—the gene is turned “off.” Antisense drugs are being investigated to treat cancer, diabetes, amyotrophic lateral sclerosis (ALS), and diseases with an inflammatory component, such as asthma and arthritis. Although many potential therapies are under investigation, most of them have not yet produced significant clinical results. At present, one antisense drug, fomivirsen (Vitravene) has been approved by the FDA as a treatment for cytomegalovirus retinitis in patients with AIDS. This approval is an important step in demonstrating that antisense drugs can be effective in the treatment of specific diseases.

Erythropoietin

Erythropoietin is a hormone produced by the kidneys to stimulate bone marrow stem cells to mature into red blood cells. If erythropoietin levels are low, anemia (low erythrocyte count) will occur. This is especially a problem in patients with kidney failure, in cancer patients undergoing chemotherapy, and in patients who use drugs for the treatment of AIDS.

In 1989 the FDA approved the genetically engineered version of the hormone Epogen produced by Amgen (Thousand Oaks, CA). Another genetically engineered version of erythropoietin, epoetin alfa, received FDA approval in 1997. The drug is marketed as Procrit by Ortho Biotech (Raritan, NJ) and is designed to be an alternative to blood transfusions in anemic patients who undergo noncardiovascular surgery. Because the drug stimulates the production of erythrocytes, the drug is administered before surgery to counteract the effects of blood loss, and therefore limiting the need for preoperative blood transfusions.

LIFE APPLICATION

Blood Doping

Blood doping is the practice of illegally boosting the number of erythrocytes (red blood cells [RBCs]) in the circulation in order to enhance athletic performance. Because the main function of RBCs is to carry oxygen from the lungs to the muscles, a higher RBC count dramatically improves an athlete’s performance by increasing their aerobic capacity and stamina. Blood doping was traditionally done by harvesting red blood cells from the athlete or a compatible donor, freezing and saving the cells until needed, and then transfusing the cells back into the body.

Genetically engineered erythropoietin (EPO) has changed this practice. There now is no need for transfusions, just an EPO shot. For many years accurate testing was not possible because the synthetic EPO is practically identical to the naturally occurring form. However, the testing technology now is improved with an accurate urine test that can detect the differences between normal and synthetic EPO. Blood doping has been especially problematic in professional cycling (for more reading see http://www.rice.edu/~jenky/sports/epo.html).

Vaccines

Vaccines are preparations that provide/improve immunity against certain infectious diseases (see Chapter 20, The Immune System). Before the development of genetically engineered vaccines, vaccines included attenuated microbes, killed or fragmented microbes, and toxoids. In addition to genetically engineered vaccines, traditional vaccines are still available and in use to this date.