CHAPTER 1
INTRODUCTION
1.1 WHAT IS GENETIC ENGINEERING?
Genetic engineering or genetic modification is the alteration or modification of an organism’s genome using modern DNA technology (Biotechnology). It usually involves the introduction of foreign DNA or synthetic genes into the organism; the new resulting organism is often referred to as transgenic and or genetically modified (GM). New DNA is obtained either by isolating or copying the genetic material of interest or by artificially synthesizing the DNA (Muntaha et al., 2016).
The headways that have been made in Genetic Engineering have its foundation in the discovery of DNA molecule in 1953 (Watson-Cricks-Wilkins-Franklin model WCWF Model) (Muntaha et al., 2016).
The first genetically modified organisms (GMOs) were bacteria in 1973 and the first genetically modified (GM) animals were mice in 1974.
The term ‘Genetic Engineering’ was first coined by Jack Williamson in his science fiction novel ‘Dragon’s Island’, published in 1951, but genetic engineering as the direct manipulation of DNA by humans outside breeding and mutations did not start until the 1970s.
1.2 DEFINITION OF SOME KEYWORDS
- BIOTECHNOLOGY: American Chemical Society defines biotechnology as the application of biological organisms, systems, or processes by various industries to learning about the sciences of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops and livestock. Basically, biotechnology is the use of or the manipulation of living cells for research to understand science of life and to produce new and improved products.
- Medical biotechnology specifically involves the use of living cells to research and produce diagnostic and treatment products that help to treat and prevent human diseases.
- RECOMBINANT DNA: This is a DNA molecule that has been produced from the combination of multiple genetic materials to create sequences that would ordinarily not be found in the genome.
- GENOME: An organism’s genome is it’s compete DNA set, including all its genes. It contains all of the organism’s information.
- DNA: Deoxyribonucleic acid is the hereditary material in humans and almost every other living organism because it carries the genetic instructions of growth, reproduction, development and general functioning of all living organisms and many viruses. The DNA has a Double Helix structure.
- MEDICINE: This is simply the science of healing. It involves diagnosis, treatment and prevention of diseases.
1.3 APPLICATION OF GENETIC ENGINEERING
Genetic engineering has numerous applications in numerous fields including research, agriculture, industrial biotechnology and medicine.
With the development of recombinant deoxyribonucleic acid (DNA) technology, the metabolic potentials of living organisms are being discovered and put to use in a variety of new ways. Today, genetically modified microorganisms (GMMs) are applied extensively in human health, agriculture, and bioremediation and in industries such as food, paper, and textiles. Genetic engineering offers the advantages over traditional methods of increasing molecular diversity and improving chemical selectivity. In addition, genetic engineering offers an abundant supply of desired products, cheaper product production prices, and safe handling of otherwise dangerous agents (Lei et al., 2004).
Genetic engineering in agriculture for example has lead to the production of transgenic plants, plants that had foreign traits incorporated into them that made them have better yield and enhanced nutritive value of crop has gathered much interest to combat malnutrition in developing countries. A very interesting trait that has been genetically engineered into crops is the resistance to pesticides or insecticides (Singh et al., 2014).
1.4 APPLICATION OF GE IN MEDICINE
In medicine, the advent of genetic engineering and biotechnology has greatly helped in diagnosis, cure or treatment, and prevention of several diseases. Medical Biotechnology has four specific applications, which are:
1. Gene therapy
2. Pharmacology
3. Stem cells
4. Tissue engineering
Genetic Engineering has made some remarkable achievements in medical science dealing with diseases and reducing the chances of it being passed to the next generation (Sandel, 2014).
CHAPTER 2
GENE THERAPY
Gene therapy a GE process is used to treat genetic diseases like Alzheimer’s, Cystic Fibrosis or cardiovascular diseases that can be passed from one generation to the next. The advancements being made in biotechnology and GE have brought gene therapy in the limelight. Gene therapy which is developed with the establishment of recombinant DNA and gene cloning methods is considered an innovative therapeutic technology. It is basically associated with alteration of human hereditary material used to deal with biomedical abnormalities (Hongxin and Yuguan, 2015). The working principle of gene therapy is the correction of the faulty gene that is causing the genetic disease. For instance, there has been success achieved by researchers in altering the lymphocytes of patients having cancer, eye, immune system or blood diseases genetically (Aubourg, 2016). Gene therapy’s importance in cancer treatment is increasing as new technologies are developed. Gene therapy can be carried out by either of the following methods:
• Inserting a normal gene into a nonspecific location
• Swapping abnormal gene for normal gene
• Repairing abnormal gene
• Turning a gene on or off
CHAPTER 3
PHARMACOLOGY:
Drugs are also produced by genetic engineering; these drugs are called Biopharmaceuticals. Generally speaking, any drug produced by using microorganisms or process of genetic engineering falls under the category of Biopharmaceuticals (Muntaha et al., 2016). The production of genetically engineered human insulin was one of the pioneer breakthroughs of biotechnology in the pharmaceutical industries. The first time insulin produced through recombinant DNA technology entered clinical trials was in 1980. Genetic engineering has been used in medicine to produce or synthesize the following biopharmaceuticals:
• Antibiotics,
• Hormones (e.g. Insulin, Human growth hormones),
• Enzymes,
• Vaccines,
• Follistim (for treating infertility),
• Human albumin,
• Monoclonal antibodies,
• Antihemophilic factors,
• Drugs from plants.
3.1 GENETIC ENGINEERING IN INSULIN PRODUCTION
3.1.1 WHAT IS INSULIN?
Insulin is an important hormone that is secreted by the pancreas. Insulin regulates the metabolism of carbohydrates, proteins, fats; but specifically it controls the blood sugar level, it regulates the blood sugar preventing it from getting too high (hyperglycaemia) or too low (hypoglycaemia).
After eating, blood sugar level rise, beta cells in the pancreas receive a signal and they release insulin into the blood stream, the insulin then bind to signal cells to absorb sugar from the bloodstream. If there’s more sugar in the body than it needs, the storage of the excess sugar is controlled by insulin.
The secretion of insulin by the beta cells of the pancreas is in response to various ‘stimuli’ like glucose, arginine, and sulphonylureas though physiologically glucose is the major determinant. Various neural, endocrine and pharmacological agents can also exert stimulatory effect. Glucose is taken up by beta cells through GLUT-2 receptors. Glucose in the beta cell is oxidized by glucokinase, which acts as a glucose sensor. Glucose concentration below 90 mg/dl does not cause any insulin release (Shashank et al., 2007).
3.1.2 Insulin as a treatment for diabetes:
People with type 1 or type 2 diabetes need insulin injections to allow their body to process glucose and avoid complications from hyperglycaemia (high sugar level).
In type 1 diabetes patients, the pancreas cannot make insulin because the beta cells are either damaged or completely destroyed while type 2 diabetes patients are resistant to insulin. Therefore insulin injections have to be administered so that their sugar levels will be controlled and complications from hyperglycaemia will not occur.
Subsequently, there are various analogues of insulin classified based on their mode and duration of action (Gualandi-Signorini et al., 2001), they include:
Rapid-acting insulin
Short-acting insulin
Intermediate-acting insulin
Long-acting insulin
3.1.3 SYNTHESIS OF HUMAN INSULIN
Insulin extracted from the pancreas tissue of animals was used for therapy from 1922 until 1974, when semi-synthetic human insulin became available in limited quantity by modification of animal insulin (Ruttenberg, 1972). This method is based on the identification of the structure of human insulin gotten from autopsy and the subsequent engineering of animal insulin’s structure into human insulin.
3.1.3.1 Humulin:
Humulin is synthetic human insulin produced by using genetic engineering using the recombinant DNA technology in the laboratory. Genetically engineered insulin can carry out all the functions the natural human insulin carries out. In 1978, scientists synthesized human insulin from Escherichia coli bacteria using recombinant DNA technology, by preparing two DNA sequences for A and B chains of human insulin and introduced them into the plasmid of Escherichia coli. This led to production of human insulin chain.
Insulin can also be produced from yeast like (Saccharomyces cerevisiae, Pichia pastoris) by recombinant DNA technology.
The simple principle is the introduction of human insulin or proinsulin into the organism i.e. Escherichia coli, Saccharomyces cerevisiae after which the organism mass produce insulin when it undergoes cell division and growth. The process for recombinant human insulin was initiated by Genentech in 1978 (Miller, and Baxter, 1980).
Production of recombinant human insulin starts with the insertion of a gene encoding the precursor protein pre-pro-insulin into a DNA vector that is transferred into a host i.e. Escherichia coli or yeast (Frank et al., 1983). During the product synthesis, the culture and fermentation conditions are controlled optimally to optimise yields (Frank et al., 1981). A fusion protein obtained by fermentation is converted to bioactive human insulin by post-translational processing.
The final step in the manufacturing process is the multi-step purification of human insulin until the necessary high extent of purification is obtained. This is followed by crystallisation of the final product and pharmaceutical manufacture of the different insulin products (Sandow et al., 2015).
FIG. 1: Structure of Human Insulin. (Photo credit: PharmaFactz.com)
FIG. 2: Summary of insulin production. (Photo credit: www.ied.edu.hk)
3.1.4 ADVANTAGES OF INSULIN PRODUCED BY GENETIC ENGINEERING
The availability of insulin injection for diabetes patients has reduced the mortality rate of diabetes.
There are different analogues which have better working characteristics than the natural insulin, therefore it is more efficient.
The insulin produced by recombinant DNA technology is much better than the one gotten from animal pancreas.
3.2. VACCINE PRODUCTION
3.2.1 What are Vaccines?
The World Health Organization defined Vaccine as a biological preparation that improves immunity to a particular disease (WHO). Vaccine produces adaptive immunity in the individual to a specific disease. A vaccine is usually made from the live attenuated pathogen or a killed microorganism or the surface protein of the pathogen. The source of vaccine or the method of production also dictates the type of vaccines. There are five types of vaccines currently in use, they are:
1. Live attenuated vaccines – these are vaccines produced by reducing the virulence of a pathogen, but still keeping it viable (or live). (Badgett et al., 2002) examples include measles vaccine, typhoid vaccine e.t.c.
2. Killed/inactivated vaccines – As the name implies these are vaccines created using pathogens like viruses, bacteria and other pathogens that have been killed. The method used to kill is usually either heat or formaldehyde. The pathogens are grown under controlled condition and then killed as a means to reduce virulence (infectivity). (Petrovsky et al., 2004)
3. Sub unit vaccines: defined in the med lexicon dictionary as a vaccine which, through chemical extraction, is free from viral nucleic acid and contains only specific protein subunits of a given virus. These vaccines are free of the adverse reactions associated with vaccines containing the whole virion.
4. Toxoids: these are inactivated bacterial toxins which stimulate antibody production (Ingolotti et al., 2012).
5. Gene based vaccines: DNA vaccines are the gene based vaccines which involves recombinant DNA technology, in which a gene of interest (transgene), coding for target protein from a pathogen is carried by a plasmid vector under the control of a strong eukaryotic promoter (Sandoval et al., 2001).
Vaccines work by stimulating or inducing immune response to recognize the vaccine component as a threat and destroy it. Vaccine prepares the body from future attacks by the same pathogen because the body already knows the pathogen and is fully equipped to destroy it.
Vaccination is simply the administration of vaccine while Immunization is the process whereby a person is made immune or resistant to an infectious disease (WHO).
The properties of a good vaccine include;
Ability to provide long term protection
Ability to effect the appropriate immune response for the particular pathogen
Stability and safety
Inexpensiveness
Table 1 A comparative feature of the vaccines in current use
Type of Vaccines / Advantages / Disadvantages / Examples
Live attenuated Induce both arms of immunity
Produces memory Less stable
Reversion to virulence BCG vaccine
Killed Can be used in immune compromised patients
Sufficient No cellular immunity
Need boosters Typhoid vaccine
Subunit
Can be prepared against capsulated organism
Can be produced in large scale Serological variability
Need boosters Haemophilus influenza B (Hib) vaccine
Toxoids Induce humoral response
Can be produced in large scale Don’t induce long lasting immunity
Possible adverse reactions Tetanus toxoid
DNA vaccines
Induce both arms of immunity
Designing is simple
Highly stable even at room temperature Tolerance induction
Less cost of making
Storage and transport are easy
Plasmid integration
Mutagenesis
Canine Melanoma Vaccine
Source: (Kumaragurubaran and Kaliaperumal, 2013).
3.2.2 Biotechnology of Vaccine Development
Live-attenuated micro-organisms, inactivated bacteria, purified microbial components, polysaccharide- carrier protein conjugates, recombinant proteins or DNA are used as vaccines (Freddy et al., 2004).
Some of the most profound and far-reaching discoveries in the past decade in biological sciences in general and in immunology in particular are the recombinant DNA techniques (Gilbert and Villa-Kornaroff, 1980).
Some authors says that subunit vaccines are of three different types, these are; toxoids, recombinant subunit vaccines and non recombinant subunit vaccines.
Recombinant subunit vaccines are those vaccines in which genes for the desired or required antigens are inserted into a vector, usually a virus that isn’t very virulent i.e. virulence is low. The vector (low virulence virus) which is now expressing the desired antigen may then be used as the vaccine or the antigen may be inserted in a subunit vaccine after purification.
Paralleling the development of new, more efficacious, stable, and safe recombinant vaccines has been the study of vaccine delivery methods and immunostimulating adju¬vant compounds that enhance the im¬mune response (Mark et al., 2008).
The overriding reason for using the recombinant DNA method to produce vaccines is the lack of immunogenic materials. This is certainly the case of hepatitis B virus (HBV) vaccine (Liew, 1990). Hepatitis B virus vaccine is the first vaccine produced by the recombinant DNA technology. The shortcoming of the other methods of vaccine development is the antigenic variability of some pathogenic organisms or the inconsistent ability of the antigen to produce the desired effect, these problems are usually overcome with the use of recombinant DNA for example; in Asia the formalin killed Pasteurella multocida does not provide a lasting immunity against the pathogen due to its high antigenic variability (Qureshi & Saxena, 2014), but recombinant technology seems promising and may be used to develop an efficient vaccine towards protection against most strains of Pasteurella multocida (Abubakar, 2014).
Several genes from different etiologic agents have been cloned, expressed and purified to be tested as vaccines. There are a variety of expression systems for antigenic protein components, such as bacteria, yeast, mammalian cells and insect cells, in which the DNA encoding the antigenic determinant can be inserted and expressed. However, several factors must be taken into account before selecting the system for antigen expression. The level of expression obtained using each specific expression vector and promoter, the selection marker of choice, the presence or absence of post-translational modification by the recombinant vector, among others, are essential features that interfere in the efficacy of production of recombinant antigens as vaccines. Bacterial expression systems are the most used due to the ease of handling and to their capacity for high level expression. However, for antigens in which post-translational modifications (e.g., glycosylation) are necessary, the use of mammalian or insect cells should be considered (Hansson et al, 2000; Clark et al., 2005; Nascimento and Leite, 2012).
A classic recombinant vaccine is the Hepatitis B virus vaccine which is a recombinant protein vaccine (Michel and Tiollais, 2010). Hepatitis B virus (HBV) causes a variety of liver diseases.
The current vaccines are produced by expressing the hepatitis B surface antigen (HBsAg) in yeast cells. The HBsAg assembles into virus-like particles (VLPs), which are extremely immunogenic, making the HBV vaccine a very efficacious vaccine. The yeast expression system may secrete the antigen into the culture supernatant that can facilitate its purification (Dertzbaugh, 1998; Adkins and Wagstaff, 1998; Nascimento and Leite, 2012).
3.2.3 Advantages of Sub unit Vaccines
Recombinant subunit vaccines have the following advantages over other vaccines
• There is no risk of pathogenicity
• There are various delivery options or systems available
• The composition is defined
• Large scale production is simple and easy
CHAPTER 4
STEM CELL THERAPY
4.1 What is a stem cell?
A stem cell is a cell that has the ability to continuously divide and develop or differentiate into other different types of cells or tissues (Ashutosh Tiwari, 2014). Stem cells give rise to all other cells e.g. nerve cells, blood cells e.t.c.
They are usually found in multicellular organisms and divide through mitosis giving rise to more stem cells. There are two broad classifications of stem cells; the embryonic stem cells that are found in the inner cell mass of blastocyst; and the adult stem cells found in various tissues e.g. liver, muscles e.t.c. The embryonic stem cells can also be called Pluripotent and the adult stem cells can be called multipotent (Thomson et al., 1998; Reubinoff et al., 2000).
4.2 Potency Level of Stem Cells
Stem cells are defined by their capabilities to self renew and give rise to various types of differentiated cells depending on their potency. They are classified as:
• Totipotent,
• Pluripotent,
• Multipotent, and
• Unipotent.
Totipotent stem cells can differentiate into an entire organism totipotent cells are from the fusion of egg and sperm. Pluripotent stem cells may give rise to all types of cells in an organism. Multipotent and Unipotent stem cells remain restricted to the particular tissue or lineages. The potency of these stem cells can be defined by using a number of functional assays along with the evaluation of various molecular markers (Singh et al., 2016).
FIG.3 Classification of various types of pluripotent stem cells on the basis of their origin (Photo credit: Singh et al., 2016).
Embryonic stem cells (ES cells) can be expanded in culture indefinitely without losing their pluripotency; this makes them particularly attractive candidates for use in cell replacement therapy (Suter et al., 2006).
4.3 Stem Therapy and its Applications
Stem therapy is simply the use of stem cells (usually embryonic stems cells) to treat and in some cases to prevent disease.
In stem therapy, stem cells are introduced into injured or damaged area(s) of the body where, under healthy and right conditions, the stem cells will replace the damaged cells in the area.
The major areas where stem therapy has been used over the years include: replacing damaged nerve tissues usually in the case of spinal cord injuries, in replacing damaged heart tissues that have been damaged by heart attack and most especially in bone marrow transplants. Bone marrow transplant being the most widely used form of stem cell therapy used to treat cancer patients with conditions like leukaemia and lymphoma (Murnaghan, 2013).
Several tissues can now be engineered using stem cells; they include tissue in the epithelial surfaces (skin, cornea and mucosal membranes) to skeletal tissues (Bianco and Robey, 2001).
4.4 Genetic Engineering of Stem Cells
The stems cells used in stem cell therapy most of the time are genetically engineered stem cells. The engineering of stem cells is a new field that has the aim to find out new ways to apply stem cell therapies, treatment options and diagnostics by manipulating elements of stem cells that have been previously difficult to control.
Generation of transgenic stem cells has broad applications, ranging from development and cell differentiation studies to redirection of stem cells towards a specific phenotype. Transgenesis of stem cells could also be used to decrease the risk of rejection through down-regulation of immune recognition molecules and decrease the risk of tumour formation through expression of molecules which allow selective killing of transplanted cells (e.g. herpes simplex thymidine kinase would allow killing of transplanted cells through gancyclovir).
More recently, gene delivery by viral vectors in ES cells has proven to be increasingly attractive (Pfeifer et al., 2002; Ma et al., 2003; Suter et al., 2006). Lentiviral vectors are particularly promising, owing to their capacity to integrate transgenes into the host cell genome. Lentiviral vectors are based on the human immunodeficiency virus (HIV); instead of delivering genetic material of HIV, these vectors are used to introduce transgenes of interest into the host cells. Current generations of lentiviral vectors carry several modifications which render them completely incapable of replicating into the host cell, thus making them compatible with a high bio safety level (Zufferey et al., 1997).
However, current lentiviral vectors are limited in their cloning flexibility and the possibility of selecting cells expressing the transgene of interest. We have therefore developed a novel generation of lentiviral vectors which allows easy vector construction and rapid generation of mouse and human ES cell lines that homogeneously express a transgene of interest. The novelty of these vectors lies in several features:
i. they are based on recombinational cloning technology, rendering possible rapid insertion of a promoter and a transgene to express it ubiquitously or in a tissue-specific manner;
ii. They carry several elements to optimise transgene expression levels;
iii. They carry an antibiotic selection cassette whereby cells carrying the transgene can be selected, thus obtaining pure populations of transduced cells (Hartley et al., 2000; Suter et al., 2006).
The ease of combination of any promoter and gene of interest allows rapid construction of vectors to express proteins at different levels or in a specific cell type.
The expression levels of a transgene of interest can thus be fine-tuned by choosing the appropriate promoter (Suter et al., 2006).
Ethical concerns about the use of these cells have been voiced. In Switzerland the generation and use of human ES cells are authorised only if spare embryos from in vitro fertilisation are used to derive them (Suter et al., 2006).
4.5 Advantages of Stem Therapy
It offers a lot of medical benefits in the therapeutic sectors of regenerative medicine.
The stem cell therapy puts into use the cells of the patient’s own body and hence the risk of rejection is reduced because the cell belongs to the same human body.
Stem cell therapy can be used in treatment of a number of birth defects, infertility problems, and tissue damage.
CHAPTER 5
CONCLUSION
The application of Genetic Engineering to medicine has proved to be of great help, it has produced extremely good results. Life expectancy has increased since the introduction of genetically engineered products and processes in health care provision.
Even though there is still a lot of unfinished/ongoing research the few finished processes are still of great influence. Hopefully there will be an increase in the GE processes used in medical practice as time goes on and for this to be made possible scientists should be given the necessary support for efficient work.
2017-10-11-1507750977