Worksheet - NEW TECHNOLOGIES Part 1 1
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1 Worksheet - NEW TECHNOLOGIES Part 1 1 New medicines and new approaches: GENE THERAPY Genetic disorders such as cystic fibrosis, albinism, Huntington s chorea, phenylketonuria, haemophilia and muscular dystrophy result from mutations in the genetic code. The mutated gene gives out faulty instructions leading to the production of an abnormal protein. Phenylketonuria is usually tested for soon after birth and if detected this early it can be successfully treated by careful control of diet. Unfortunately treatment is not usually this straightforward. One solution under investigation for genetic disorders involving single gene defects is gene therapy, where the defective gene is compensated for by introducing a normal one. This is a very attractive proposition because it tackles the cause of the disorder rather than the symptoms. Every cell in the affected individual carries the faulty copy of the gene, but the gene only needs to be replaced in cells which produce the protein encoded for by this gene and cause disease symptoms. In somatic cell gene therapy, a normal copy of the gene is delivered to those cells so they can produce the normal protein. Germ line gene therapy is much more controversial as it involves modifying a gamete, fertilised egg or embryo before the germline has split off from the cells that will make the rest of the body, so any change made will be passed on to subsequent generations. Germ line gene therapy is currently prohibited because of the ethical concerns it raises. Before gene therapy can even be considered, the gene or genes responsible for the disorder need to be identified from the estimated 70,000 to 140,000 genes present in the human genome. In 1989 the human gene responsible for cystic fibrosis was identified on chromosome 7 and named CFTR. The aim of gene therapy is to get normal copies of this gene into the epithelial cells lining the airways of the lungs. CF is regarded as a good candidate for gene therapy because the cells affected line the lung passages so they can be treated directly without being removed from the body. Scientists are working on a way of administering the normal gene using the type of inhaler used commonly by asthmatics. This type of delivery is ideal since it causes the patient no discomfort. Since the DNA is not integrated into the nuclear DNA, it is not replicated when the cell divides. This means that the treatment has to be repeated every 2 to 3 months. KEY TERMS phenylketonuria haemophilia muscular dystrophy Huntington s disease gene therapy somatic cell germline bacterial plasmid vectors adenovirus liposomes lipid bilayer
2 Worksheet - NEW TECHNOLOGIES Part 1 2 The normal form of the human CFTR gene has been isolated and inserted into a bacterial plasmid (circular piece of DNA) and cloned to produce vast numbers of identical copies of the gene for experimental use. The next step is to find a safe way to deliver this normal gene into the epithelial cells of CF sufferers. Two carrier systems, or vectors, have been developed: The normal CFTR gene has been attached to the DNA in an adenovirus which has been treated to make it completely safe to use. This is an example of a viral vector. Human epithelial cell Adenovirus Cell surface receptor Nucleus Viral and normal CFTR DNA Normal CFTR protein expressed and correcting ion defect in plasma membrane Fig.1 Adenovirus vector. The normal CFTR gene is inserted into the virus which infects the cell by binding to a cell surface receptopr. DNA enters the cell and is transported to the nucleus where the normal CFTR gene is expressed and the normal protein produced. liposomes can also be used as vectors for the CFTR gene. Liposomes are tiny hollow spheres made up of a positively-charged lipid bilayer. They can be complexed with DNA and pass easily through cell membranes. Liposome sphere made up of a positively charged lipid bilayer Normal CFTR gene Liposome/ DNA complex Human epithelial cell Normal CFTR protein expressed and correcting ion defect in plasma membrane nucleus Fig. 2 Positively charged liposomes combine with the negatively charged DNA to form a complex which fuses with the plasma membrane and enters the cell. Inside the cell the DNA is released and transported to the nucleus where it is inserted into the cell s DNA, so the normal protein can be produced.
3 Worksheet - NEW TECHNOLOGIES Part 1 3 PROBLEM SOLVING ACTIVITIES (Option 1) 1. How might the Human Genome Project be used to help sufferers of genetic disorders? 2. What is meant by the term clone as used in this passage? 3a) Explain why viruses are being used in the development of gene therapy. b) Why is it important that the adenovirus be made safe before use? c) Are there any disadvantages of using a viral delivery method? 4a) Explain why the composition of liposomes make them ideal vectors for gene therapy. b) Give one reason why liposomes might be more popular than a virus for use as a vector. 5. Why should the type of gene therapy described here for CF be referred to as a treatment rather than a cure? (Option 2) You are the parents of a three year old son with CF. The disorder is being managed quite well with conventional treatment - drugs and physiotherapy. There is still the constant worry of lung infections and hospitalisation. You are however very hopeful that your son will grow up and lead as near a normal life as possible, his happiness and quality of life are very important to you. You have discussed your concerns with your doctor and she has suggested the possibility of your son joining a gene therapy trial. You are offered the chance of putting your son into a trial using either the liposome or the adenoviral vectors. Both vectors carry the same corrected form of the CFTR gene. Would you wish your son to be part of a trial and if so which one would you choose? Make an argument to persuade the doctors to enter your son in one of the trials - there are limited spaces available. You may wish to consider the following points in your argument: your son s age the current state of your son s health whether or not you live close to the centre where the trials are being run how the therapy works the relative safety of the different vectors the results of other gene therapy trials the form treatment will take how long the trial will last
4 Worksheet - NEW TECHNOLOGIES Part 2 1 New medicines and new solutions: BIOPHARMACEUTICALS Human pharmaceutical proteins currently in use include; human insulin for diabetes; human growth hormone for dwarfism; Factor VIII for haemophilia and Factor IX for haemophilia. Several hundred other human proteins are currently at various stages of clinical evaluation, including; blood proteins; monoclonal antibodies; growth factor; soluble receptors and nutraceuticals. Human pharmaceutical proteins are usually expensive to produce and demand outstrips supply. In the past the main source of many of these proteins was cadavers and donated blood and there have been several problems associated with each of these (for example transmission of hepatitis and HIV viruses and the agents responsible for Creutzfeldt-Jakob disease). Genetic engineering has made it possible to clone protein-encoding genes. These recombinant genes can be expressed in a variety of systems ranging from simple microbial systems such as bacteria, yeast and cultured mammalian cells to complex eukaryotic systems such as transgenic plants and animals. Most of the proteins pharmaceutical industries are interested in are secretory proteins which undergo complex post-translational modifications as they pass through the secretory pathway in the cell. Often the protein will not function properly unless it has gone through these modifications. Unfortunately for the genetic engineer, prokaryotic systems like bacteria cannot perform most of these modifications. Yeast and higher plants can make some of them, but when the protein is intended for therapeutic use, a nearly perfect match is required, so often only mammalian cells will do per litre plasma cell culture yeast transgenics Figure 1 Shows the cost of producing raw material from which the pharmaceutical protein is extracted, assuming 40,000 litres are produced per annum.
5 Worksheet - NEW TECHNOLOGIES Part 2 2 Another option now available is the use of transgenic livestock. Eukaryotic genes have regulatory sequences attached to them to determine which tissues they are expressed in. Genetic engineers have identified those sequences which direct the expression of genes to the mammary gland. Any gene linked to these regulatory sequences is expressed only in the mammary gland of a lactating animal. The pharmaceutical protein is produced in the milk and can be purified from the milk. Transgenic animals can be made by inserting recombinant DNA directly into one of the pronuclei of a fertilised egg. The embryo is transferred to a foster mother, where it continues its development to birth. The efficiency of producing transgenic livestock by this method depends on the species, but usually only about 3-5% of the animals born carry the transgene. Only those animals which produce large amounts of the protein in their milk and can pass this ability onto future generations will become founder animals for the large production flocks (see figure 2). Production Diagram G0 G1 Male Transgenic founders Females G2 Production unit Figure 2: production of a herd of transgenic animals.
6 Worksheet - NEW TECHNOLOGIES Part 2 3 Choice of transgenic species Figure 3 Livestock generation times (in months) Species Go Birth Go Adult G1 birth (first milk) G1 adult G2 birth G3 birth Sheep Cow Pig Rabbit Figure 4 Relative merits of species Species Volume of milk Time to milk Ease of generating production founders Health Sheep Cow Pig Rabbit Recombinant protein Antithrombin III Protein C Antibiotics Fibrinogen AAT Expression level in g/litre AAA- alpha-1-antitrypsin Figure 5 Protein produced in the milk of transgenic livestock (Data for figures 1, 3, 4 and 5 kindly supplied by PPL Pharmaceuticals)
7 Worksheet - NEW TECHNOLOGIES Part 2 4 KEY TERMS Hepatitis HIV Creutzfeldt-Jakob disease Eukaryotic transgene Alpha-1-antitrypsin Activity Using the information above answer the following questions: Calcitonin is a peptide hormone secreted by the thyroid gland in mammals. It lowers the level of calcium in the blood by reducing release of calcium from bone. A pharmaceutical company is investigating the possibility of producing large quantities of calcitonin for the treatment of osteoporosis. 1. Use the information in figure 1 to answer the following questions: i) Which is the most expensive option and what other reason is there for not choosing this option? ii) The human calcitonin gene has been cloned. Why does the company not even consider the possibility of using bacteria to produce the calcitonin. It would be much cheaper to use bacteria iii) Compare the annual cost of producing 40,000 litres of raw material for calcitonin extraction from mammalian cell culture with that from transgenic livestock. iv) What hidden costs are there? v) What do the genetic engineers have to do to the human calcitonin gene before microinjection if they want the transgenic animal to produce human calcitonin in its milk? 2. Use the information in figure 2 to answer the following questions: a) Why choose a male animal to found G 2, the production flock? b) What is the purpose of producing the females in G 1? 3. Use figures 2, 3 and 4 to answer the following questions: a) How long does it take to go from microinjection of the transgene to production of raw material in (1) cattle; (2) sheep and (3) pigs? b) What are the main (1) advantages, (2) disadvantages of choosing cattle to produce recombinant protein in their milk? c) A biotechnology company decides to produce flocks of transgenic sheep to produce human therapeutic proteins, explain why they choose sheep rather than pigs or cattle.
8 Worksheet - NEW TECHNOLOGIES Part 2 5 Activity (continued) 4. Use figure 5 to answer the following questions: a) Complete the following table for annual production capacity: Rabbits Sheep Cows Milk volume per year 8 litres 300 litres 9,000 litres Yield at t 5g/l expression ession b) Complete the following table for protein yield: Recombinant protein Yield per year for flock of 50 ewes es AAT Antibiotics Protein C Fibrinogen Antithrombin III c) The annual market for AAT is estimated to be in the order of 1,000kg. The best mammalian cell culture system working at maximum production levels can only produce approximately 10-20kg protein per year. How many (1) mammalian cell culture systems (2) transgenic ewes, are required to meet the demand for AAT? d) Explain how the technique used to produce Dolly the sheep could be applied to this technology.
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