Genetics Lecture 22 Applications Applications Since the dawn of recombinant DNA technology in the 1970s, scientists have harnessed genetic engineering not only for biological research, but also for applications in medicine, agriculture, and biotechnology. Genetic engineering refers to the alteration of an organism s genome and typically involves the use of recombinant DNA technologies to add a gene or genes to a genome, but it can also involve gene removal. The ability to manipulate DNA in vitro and to introduce genes into living cells has allowed scientists to generate new varieties of plants, animals, and other organisms with specific gene traits, and to manufacture cheaper and more effective therapeutic products. These new varieties of organisms are called genetically modified organisms, or GMOs. Industry analysts estimate that genetic engineering is worth over $55 billion. 2 Biotechnology is the use of living organisms to create a product or a process that helps improve the quality of life for humans or other organisms. Biotechnology as a modern industry began in earnest shortly after recombinant DNA technology developed. But biotechnology is actually a science dating back to ancient civilization and the use of microbes to make many important products, including beverages such as wine and beer, vinegar, breads, and cheeses. Modern biotechnology relies heavily on recombinant DNA technology, genetic engineering, and genomics applications Existing products and new developments that occur seemingly every day make the biotechnology industry one of the most rapidly developing branches of the workforce world wide, encompassing nearly 5000 companies in 54 countries. 3 1
Genetically Engineered Organisms Synthesize a wide range of biological and pharmaceutical products The most successful and widespread application of recombinant DNA technology has been production by the biotechnology industry of recombinant proteins as biopharmaceutical products particularly, therapeutic proteins to treat diseases. Prior to the recombinant DNA era, biopharmaceutical proteins such as insulin, clotting factors, or growth hormones were purified from tissues such as the pancreas, blood, or pituitary glands. Clearly, these sources were in limited supply, and the purification processes were expensive. In addition, products derived from these natural sources could be contaminated by disease agents such as viruses Now that human genes encoding important therapeutic proteins can be cloned and expressed in a number of nonhuman host cell types, we have more abundant, safer, and less expensive sources of biopharmaceuticals. 4 5 Insulin Production in Bacteria Many therapeutic proteins have been produced by introducing human genes into bacteria. In most cases, the human gene is cloned into a plasmid, and therecombinant vector is introduced into the bacterial host. Large quantities of the transformed bacteria are grown, and the recombinant human protein is recovered and purified from bacterial extracts. 6 2
The first human gene product manufactured by recombinant DNA technology was human insulin, called Humulin, which was licensed for therapeutic use in 1982 by the U.S. Food and Drug Administration (FDA In 1977, scientists at Genentech, the San Francisco biotechnology company cofounded in 1976 by Herbert Boyer (one of the pioneers of using plasmids for recombinant DNA technology) and Robert Swanson isolated and cloned the gene for insulin and expressed it in bacterial cells. Genentech, short for genetic engineering technology, is also generally regarded as the world s first biotechnology company. Previously insulin was chemically extracted from the pancreas of cows and pigs obtained from slaughterhouses. Insulin is a protein hormone that regulates glucose metabolism. 7 8 Transgenic Animal Hosts and Pharmaceutical Products Although bacteria have been widely used to produce therapeutic proteins, there are some disadvantages in using prokaryotic hosts to synthesize eukaryotic proteins. One problem is that bacterial cells often cannot process and modify eukaryotic proteins. As a result, they frequently cannot add the carbohydrates and phosphate groups to proteins that are needed for full biological activity. In addition, eukaryotic proteins produced in prokaryotic cells often do not fold into the proper three dimensional configuration and are therefore inactive. To overcome these difficulties and increase yields, many biopharmaceuticals are now produced in eukaryotic hosts. A herd of goats or cows serve as very effective bioreactors or biofactories living factories that will continuously make milk containing the desired therapeutic protein that can then be isolated in a noninvasive way 9 3
Yeast are also valuable hosts for expressing recombinant proteins. Even insect cells are valuable for this purpose, through the use of a gene delivery system (virus) called baculovirus. Recombinant baculovirus containing a gene of interest is used to infect insect cell lines, which then express the protein at high levels. Baculovirus insect cell expression is particularly useful for producing human recombinant proteins that are heavily glycosylated. Regardless of the host, therapeutic proteins may then be purified from the host cells or when transgenic farm animals are used, isolated from animal products such as milk. 10 An example of a biopharmaceutical product synthesized in transgenic animals is the human protein 041 antitrypsin. A deficiency of the enzyme 041 antitrypsin is associated with the heritable form of emphysema, a progressive and fatal respiratory disorder common among people of European ancestry. To produce antitrypsin for use in treating this disease, the human gene was cloned into a vector at a site adjacent to a sheep promoter sequence that specifically activates transcription in milkproducing cells of the sheep mammary glands. Genes placed next to this promoter are expressed only in mammary tissue. This fusion gene was microinjected into sheep zygotes fertilized in vitro. The fertilized zygotes were transferred to surrogate mothers. The resulting transgenic sheep developed normally and produced milk containing high concentrations of functional human antitrypsin. 11 ln 2006, recombinant human antithrombin, an anticlotting protein, became the world s first drug extracted from the milk of farm animals to be approved for use in humans. Scientists at GTC Biotherapeutics of Framingham, Massachusetts, introduced the human antithrombin gene into goats. By placing the gene adjacent to a promoter for beta casein, a common protein in milk, GTC scientists were able to target antithrombin expression in the mammary gland. As a result, antithrombin protein is highly expressed in the milk. In one year, a single goat will produce the equivalent amount of antithrombin that in the past would have been isolated from ~90,000 blood collections. 12 4
Recombinant DNA Approaches for Vaccine Production and Transgenic Plants with Edible Vaccines One of the most promising applications of recombinant DNA technology for therapeutic purposes may be the production of vaccines. Vaccines stimulate the immune system to produce antibodies against disease causing organisms and thereby confer immunity against specific diseases. Traditionally, i two types of vaccines have been used: inactivated i vaccines, which are prepared from killed samples of the infectious virus or bacteria; and attenuated vaccines, which are live viruses or bacteria that can no longer reproduce but can cause a mild form of the disease. Inactivated vaccines include the vaccines for rabies and influenza; vaccines for tuberculosis, cholera, and chickenpox are examples of attenuated vaccines. 13 Genetic engineering is being used to produce a relatively new type of vaccine called a subunit vaccine, which consists of one or more surface proteins from the virus or bacterium but not the entire virus or bacterium. This surface protein acts as an antigen that stimulates the immune system to make antibodies that act against the organism from which it was derived. One of the first subunit vaccines was made against the hepatitis B virus, which causes liver damage and cancer. The gene that encodes the hepatitis B surface protein was cloned into a yeast expression vector, and the cloned gene was expressed in yeast host cells. 14 In 2005, the FDA approved Gardasil, a subunit vaccine produced by the pharmaceutical company Merck and the first cancer vaccine to receive FDA approval. Gardasil targets four strains of human papillomavirus (HPV) that cause ~70 percent of cervical cancers. Approximately 70 percent of sexually active women will be infected by an HPV strain during their lifetime. Gardasil is designed to provide immune protection againsthpv prior to infectionbut is not effective against existing infections. You may have heard of Gardasil through media coverage of the legislation pending in several states that would require all adolescent school girls to receive a Gardasil vaccination regardless of whether or not they are sexually active. 15 5
Developing countries face serious difficulties in manufacturing, transporting, and storing vaccines. Most vaccines need refrigeration and must be injected under sterile conditions. In many rural areas, refrigeration and sterilization facilities are not available. In addition, in many cultures people are fearful of being injected with needles. To overcome these problems, scientists are attempting to develop vaccines that can be synthesized in edible food plants. These vaccines would be inexpensive to produce, would not require refrigeration, and would not have to be given under sterile conditions by trained medical personnel. 16 17 Plants offer several other advantages for expressing recombinant proteins. For instance, once a transgenic plant is made, it can easily be grown and replicated in a greenhouse or field, and it will provide a constant source of recombinant protein. 18 6
No recombinant proteins expressed in transgenic plants have yet been approved for use by the FDA as therapeutic proteins for humans, although about a dozen products are close to making it through final clinical trials. Some edible vaccines are now in clinical trials. For example, a vaccine against a bacterium that causes cholera has been produced in genetically engineered potatoes and used to successfully vaccinate human volunteers. 19 Genetic Engineering of Plants Has Revolutionized Agriculture For millennia, farmers have manipulated the genetic makeup of plants and animals to enhance food production. Until the advent of genetic engineering 30 years ago, these genetic manipulations were primarily restricted to selective breeding the selection and breeding of naturally occurring or mutagen induced variants. In the last 50 to l00 years, genetic improvement of crop plants through the traditional methods of artificial selection and genetic crosses has resulted in dramatic increases in productivity and nutritional enhancement. For example, maize yields have increased fourfold over the last 60 years, and more than half of this increase is due to genetic improvement by artificial selection and selective breeding. Modern maize has substantially larger ears and kernels than the predecessor crops, including hybrids from which it was bred. 20 21 7
Recombinant DNA technology provides powerful new tools for altering the genetic constitution of agriculturally important organisms. Scientists can now identify isolate, and clone genes that confer desired traits, then specifically and efficiently introduce these into organisms. As a result, it is possible to quickly introduce insect resistance, herbicide resistance, or nutritional characteristics into farm plants and animals, a primary purpose of agricultural biotechnology. 22 Worldwide, over 150 million hectares have been planted with genetically engineered crops as of 2010, (the most recent date for which such data are available), particularly herbicide and pest resistant soybeans, corn, cotton, and canola; over 50 different transgenic crop varieties are available, including alfalfa, corn, rice, potatoes, tomatoes, tobacco, wheat, and cranberries These crops are planted by over 8 million farmers in 17 countries. Both industrialized and developing countries are taking advantage of transgenic crops. Since 1996, there has been a 4000 percent increase in GM crop acreage worldwide. 23 24 8
Transgenic Animals Transgenic animals with genetically enhanced characteristics have the potential to serve important roles in biotechnology Although genetically engineered plants are major players in modern agriculture, commercial applications of transgenic animals are less widespread. 25 Makin a Transgenic Animal The method of creating transgenic animals is conceptually relatively simple, although there are species specific challenges associated with creating transgenics. Many of the prevailing techniques used to make transgenics were developed in mice. One method is to isolate newly fertilized eggs from a female mouse (or female of the desired danimal species) and to inject purified cloned DNA containing a vector and the transgene of interest into the nucleus of the egg. In a relatively small percentage of transgenic eggs, the transgenic DNA becomes inserted into the egg cell genome by recombination due to the action of naturally occurring DNA recombination enzymes. Newer approaches involving stem cells are also popular for creat ing transgenic animals 26 27 9
Examples 0f Transgenic Animals Oversize mice containing a human growth hormone trans gene were some of the first transgenic animals created. Attempts to create farm animals containing transgenic growth hormone genes have not been particularly successful, probably bbl because growth is a complex, multigene trait. One no table exception is the transgenic Atlantic salmon, bearing copies of a Chinook salmon growth hormone gene adjacent to a constitutive promoter. 28 29 Gene Therapy Although drug treatments are often effective in controlling symptoms of genetic disorders, the ideal outcome of medical treatment is to cure these diseases. In an effort to cure genetic diseases, scientists are actively investigating gene therapy a therapeutic technique that aims to transfer normal genes into a patient s cells. 30 10
One key to gene therapy is having a delivery system to transfer genes into a patient. In many gene therapy trials, scientists often used genetically modified retroviruses as vectors. An example is a vector based on a mouse virus called Moloney murine leukemia virus (MLV). Disabled forms of adeno associated virus (AAV), which in its native form infects ~80 9O percent of the population during childhood, and nonviral methods are being used to transfer genes into cells include chemically assisted transfer of genes across cell membranes, and fusion of cells with artificial vesicles containing cloned DNA sequences. Retroviral vectors are created by removing a cluster of three genes from the virus and inserting a cloned human gene. 31 After being packaged in a viral protein coat, the recombinant vector is used to infect cells. Once inside a cell, the virus cannot replicate itself because of the missing viral genes. In the cell, the recombinant virus with the inserted human gene moves to the nucleus, integrates into a site on a chromo some, and becomes part of the genome. If the inserted gene is expressed, it produces a normal gene product that may be able to correct the mutation carried by the affected individual. In initial attempts at gene therapy, several heritable disorders, including severe combined immunodehciency (SCID), familial hypercholesterolemia, and cystic fibrosis were treated. 32 Human gene therapy began in 1990 with the treatment of a young girl named Ashanti, who has a heritable disorder called severe combined immunodeficiency (SCID). Individuals with SCID have no functional immune system and usually die from what htwould normally be minor infections. Ashanti has an autosomal form of SCID caused by a mutation in the gene encoding the enzyme adenosine deaminase (ADA). Her gene therapy began when clinicians isolated some of her white blood cells, called T cells. 33 11
34 To date, gene therapy has successfully restored the health of about 20 children affected by SCID. Although gene therapy was originally developed as a treatment for single gene (monogenic) inherited diseases, the technique was quickly adapted for the treatment of acquired diseases such as cancer, neurodegenerative diseases, cardiovascular disease, and infectious diseases, such as HIV In the case of HIM scientists are exploring ways to deliver immune system stimulating genes that could make individuals resistant to HIV infection or cripple the virus in HIV positive persons. There are nearly 1000 gene therapy trials activelyunderway inthe United States alone. Over a 10 year period, from 1990 to 1999, more than 4000 people underwent gene therapy for a variety of genetic disorders. These trials often failed and thus led to a loss of confidence in gene therapy. 35 12