Pharmaceutical Application of Plant Genetic Engineering Lectures 9 & 10 PDF

Biotechnological Production of Herbal Drugs [PPC505] Dr. Marwa Saeed Galaa Goda | PhD Lecturer of Pharmacognosy, Faculty of Pharmacy, GU gu.edu.eg Sylla...

Biotechnological Production of Herbal Drugs [PPC505] Dr. Marwa Saeed Galaa Goda | PhD Lecturer of Pharmacognosy, Faculty of Pharmacy, GU gu.edu.eg Syllabus Week Lecture 1st week: 28/9/2024 Advising & registration 2nd week: 5/10/2024 Biotechnology: Four colors of biotechnology, and Pros & cons of green biotechnology 3rd week: 12/10/2024 I. Plant tissue culture: Advantages & disadvantages, and stages 4th week: 19/10/2024 I. Plant tissue culture: Nutritional requirements, and common problems 5th week: 26/10/2024 I. Plant tissue culture: Culture types, plant regeneration, and genetic assessment I. Plant tissue culture: Extraction & quantification of metabolites, and strategies to increase 6th week: 2/11/2024 secondary metabolites production Day 7th week: 9/11/2024 I. Plant tissue culture: Biotechnological application of plant tissue culture 8th week: 16/11/2024 Midterm Exam 9th week: 23/11/2024 II. Plant genetic manipulation: Steps of plant genetic engineering/ transgenic biotechnology 10th week: 30/11/2024 II. Plant genetic manipulation: Advantages & disadvantages of genetically modified (GM) crops 11th week: 7/12/2024 II. Plant genetic manipulation: Different pharmaceutical applications of biotechnology 1 12th week: 14/12/2024 II. Plant genetic manipulation: Different pharmaceutical applications of biotechnology 2 13th week: 21/12/2024 Student presentations I 14th week: 28/12/2024 Student presentations II 15th week: 4/1/2025 Final Exam Total marks Items Marks Midterm exam 30 marks Semester activity Quizzes (26/10 & 14/12) 10 marks (Class work) Activity/ presentation 10 marks Student’s attendance 5 marks Student’s portfolio 5 marks Final exam 40 marks Total marks 100 marks  Learning Outcomes (LOs) K1. Describe different types of biotechnology, their scope and importance. K2. Recognize the basic principles, advantages and disadvantages of plant tissue culture. K3. Illustrate the fundamental concepts to increase the production of pharmaceuticals. Domain 1: K4. Demonstrate the relation between various growth media composition, culture Fundamental environment, or growth regulators and the yield of phytochemicals knowledge K5. Describe pros and cons of genetically modified crops as well as the basic steps. K6. Record the recent advances in the fields of plant biotechnology. K7. Explain different analytical methods that are used in the quantitative determination of the bioactive metabolites in different in vitro cultures. S1. Assess experimental scheme to suggest solutions for micropropagation of endangered Domain 2: plants. Professional and S2. Analyze different methods and elicitation protocols for enhancement of natural products ethical practice productions. S3. Interpret different protocols of transgenic plants. C1. Develop team-working ability through group projects. Domain 4: C2. Demonstrate time-management and presentation skills. Personal practice C3. Use computer technology to get relevant information.  Plant biotechnology - Pharmaceutical application of biotechnology.  Medical/pharmaceutical biotechnology & its application Medical or pharmaceutical biotechnology is a branch of medicine that uses living cells or cell materials to produce pharmaceutical and diagnosing products, helping in treatment or prevention of diseases. General applications of modern biotechnology in pharmaceutical field involve I. Creation of new pharmaceuticals, II. Creation safer and/or more effective versions of conventionally produced pharmaceuticals, or III. Production of substances identical to conventionally made pharmaceuticals more cost-effectively than the latter pharmaceuticals are produced. 7  Medical/pharmaceutical biotechnology & its application For example, before the development of recombinant human insulin - which became the first manufactured, or commercial, recombinant pharmaceutical in 1982 - animals (notably pigs and cattle) were the only nonhuman sources of insulin. Animal insulin, however, differs slightly but significantly from human insulin and can elicit troublesome immune responses. Recombinant human insulin (using Escherichia coli bacteria to produce human insulin) is at least as effective as insulin of animal origin, is safer than animal-source insulin, and can satisfy medical needs more readily and more affordably. 8  Medical/pharmaceutical biotechnology & its application General applications of modern biotechnology involve production of hormones, genes, antibiotics, vaccines, interferons, vitamins, immunological proteins, and an antenatal diagnosis cure in preventing genetic disease. 9  Medical/pharmaceutical biotechnology & its application 1. Pharmaceutical biotechnology and hormone production 2. Pharmaceutical biotechnology and enzyme production 3. Pharmaceutical biotechnology and vaccine production 4. Pharmaceutical biotechnology and vitamins production 5. Pharmaceutical biotechnology and gene therapy 10  Medical/pharmaceutical biotechnology & its application 1. Medical biotechnology & hormone production E.g.1. Human recombinant insulin is the first human hormone produced by recombinant DNA technology. Scientists cut and paste the human insulin gene into a plasmid, which can be used to transfer the gene into bacteria (E. coli or Saccharomyces cerevisiae). The bacteria produce the insulin, which can then be isolated from the bacterial culture and given to patients. Insulin lispro is an analog insulin that is a genetically modified human insulin. The peptide structure of Lispro insulin is identical to that of human insulin except for transposition of the proline residue in position B28 and the lysine residue in position B29 (Fig. 69). Lispro is formerly called LYSPRO from the chemical nomenclature LYS(B28), PRO(B29). Regular insulin ordinarily must be injected 30 to 45 minutes before meals to control blood glucose levels. Lispro (Humalog®) -a recombinant insulin like substance- is faster-acting than regular insulin. Because injection of lispro is appropriate within 15 minutes before meals, using it instead of regular insulin may be more convenient for some patients. 11  Medical/pharmaceutical biotechnology & its application 1. Medical biotechnology & hormone production 12   Medical/pharmaceutical biotechnology & its application 1. Medical biotechnology & hormone production E.g.2. Erythropoietin (EPO), a hormone produced by the kidneys, stimulates the bone marrow to produce red blood cells. The FDA has approved recombinant EPO for the treatment of anemia due to chronic renal failure. E.g.3. Human growth hormone (hGH) is used to counter growth failure in children, that is due to a lack of hGH production by the body. Before the introduction of recombinant human growth hormones, the hormone was derived from human cadavers, dead human bodies. Cadaver- derived hGH was linked to progressive and fatal degenerative neurologic disorders like Alzheimer. Recombinant hGH has greatly improved the long- term treatment of children whose bodies do not produce enough hGH. 13  Medical/pharmaceutical biotechnology & its application 2. Medical biotechnology & enzyme production E.g.1. Amylase enzyme makes up approximately 25% of the world enzyme market. The α-amylases have been widely used in the baking industry and as a digestive aid in case of dyspepsia. Recombinant DNA technology for amylase production involve selection of an efficient amylase gene, insertion of the gene into an appropriate vector system (Bacillus subtilis or Bacillus stearothermophilus), transformation into an efficient bacterial system to produce a higher amount of recombinant mRNA, and overproduction of amylase from the bacterial system. In this technology, high-copy numbers of the gene promote higher yields of amylase. 14  Medical/pharmaceutical biotechnology & its application 2. Medical biotechnology & enzyme production E.g.2. Thrombolytic alteplase enzyme (Activase® or Altelase®) dissolves blood clots in the circulatory system via conversion of the protein plasminogen to the proteolytic enzyme plasmin. A recombinant version of one of the enzymes that accelerate this conversion can contribute to the treatment of heart attacks, strokes, and pulmonary emboli. This recombinant enzyme is recombinant tissue-type plasminogen activator (alteplase) that binds to fibrin with a greater affinity than streptokinase or urokinase. once bound, it starts to convert plasminogen to plasmin on the fibrin surface. Hence it is relatively clot selective (more localized). 15  Medical/pharmaceutical biotechnology & its application 3. Medical biotechnology & vaccine production Vaccination is the most effective way to control infectious diseases. The principle of vaccination is mimicking an infection in such a way that the natural specific defense mechanism of the host against the pathogen will be activated and immunological memory is established, but the host will remain free of the disease. Vaccine consists of killed microorganisms, nonvirulent microorganisms, microbial products (e.g., toxins), or microbial components that have been purified. All these active ingredients are antigens: substances that can stimulate the immune system to produce specific antibodies. Such stimulation leaves the immune system prepared to destroy bacteria and viruses whose antigens correspond to the antibodies it has learned to produce. Although conventionally produced vaccines are generally harmless, some of them may, rarely, contain infectious contaminants. Vaccines whose active ingredients are recombinant antigens do not carry this slight risk. 16  Medical/pharmaceutical biotechnology & its application 3. Medical biotechnology & vaccine production Recombinant vaccines are produced as follows: finding the gene, insertion of the gene into a plasmid or other suitable carrier, introduction of this complex into bacteria, yeast, flowering plants or mammalian host cells and, finally, the expression and purification of the material desired. 17  Medical/pharmaceutical biotechnology & its application 3. Medical biotechnology & vaccine production E.g.1. For example, more than 350 million persons worldwide are infected with the virus that causes hepatitis B, a major cause of chronic inflammation of the liver, cirrhosis of the liver, and liver cancer. Hepatitis B kills a million people each year worldwide. The first hepatitis B vaccine available in the U.S. was made with derivatives of plasma from persons with chronic HBV infections. A recombinant vaccine has replaced it. To make the hepatitis B vaccine, part of the DNA from the hepatitis B virus is inserted into the DNA of yeast cells. Use of this vaccine is very cost- effective especially in North America, since interferon treatment of hepatitis B is very expensive. 18  Medical/pharmaceutical biotechnology & its application 3. Medical biotechnology & vaccine production E.g.2. Another prospect is effective inoculation by ingestion. In February 1998 U.S. researchers announced that they had genetically engineered potatoes to produce a vaccine against cholera. Every year five million people contract cholera, and 200,000 die from it. The vaccine is a nontoxic, relatively heat-stable protein that can elicit an immune response even when it is ingested as a potato constituent In the same manner, tomato-based edible vaccine (TOMAVAC) against COVID-19 was produced via developing transgenic tomato plants expressing the RBD subunit of the S1 protein of SARS-CoV-2. The S protein mediates viral entry into host cells by first binding to a host receptor through the receptor-binding domain. 19 https://youtu.be/RTy-JwhVWzI?si=UxLW2h-cKXIHChQY 20  Medical/pharmaceutical biotechnology & its application 4. Medical biotechnology & vitamin production Vitamins are defined as essential micronutrients required in trace quantities for metabolism of all living organisms. They are synthesized by microorganisms or plants, not by mammals. Apart from their in vivo nutritional-physiological roles, vitamins are now introduced as medical-therapeutic agents. 21  Medical/pharmaceutical biotechnology & its application 4. Medical biotechnology & vitamin production Chemical synthesis methods are usually environment- unfriendly as they often require high temperatures or pressurized reactors and use non-renewable chemicals or toxic solvents that cause product safety concerns, pollution, and hazardous waste. Microbial cell factories for the production of vitamins are green (easy waste recycling) and sustainable (low energy consumption) from both environmental and economic standpoints. Water-soluble vitamins (vitamin B complex and vitamin C) as well as fat-soluble vitamins (vitamins A/D/E and K) can potentially be produced using microbial cell factories or are already being produced in commercial fermentation processes. 22  4. Medical biotechnology & vitamin production  Vitamin B-12 Vitamin B-12 plays an essential role in red blood cell formation, cell metabolism, nerve function and the production of DNA. Industrial production of vitamin B12 (having a corrin ring with cobalt as the central atom) is achieved through aerobic or anaerobic fermentation of selected microorganisms such as Pseudomonas denitrificans and Propionibacterium freudenreichii subsp. Shermanii, respectively. During vitamin B12 biomanufacturing, cobalt, which is central to the function of the vitamin, is inserted into the core of the vitamin B12 molecule by a metal inserting enzyme. 23  4. Medical biotechnology & vitamin production  Vitamin B-12 After fermentation, the recovery and purification of the nutrient is important for commercial process. Corrin is converted into cyanocobalamine by the addition of potassium cyanide in presence of sodium nitrite and heat. Then, the vitamin solution is purified via precipitation, adsorption, ion-exchange chromatography and crystallization. This process can take a further week or so before the nutrient is pure enough to be used in vitamin supplements or to fortify food products. 24  4. Medical biotechnology & vitamin production  Vitamin B-12 However, the strains that are used for commercial vitamin B12 production have several shortcomings: long fermentation cycles as well as complex and expensive media requirements. Recently, scientists have shifted their attention to E. coli as a platform for vitamin B12 production. A recombinant E. coli bacterial strain that is capable of producing high levels of vitamin B12 accompanied by eliminating the creation of hazardous waste / trade effluent and making the manufacturing process more affordable and environmentally friendly. It now represents a stand-by option for the commercial collaborators in the case that costs of vitamin B12 become prohibitive. 25  4. Medical biotechnology & vitamin production  Vitamin C Reichstein–Grüssner process was first designed for vitamin C production on industrial scale in 1933. Glucose is catalytically hydrogenated to D-sorbitol, the D-sorbitol is oxidized and converted to its ketose sugar L- sorbose using a fermentation step by microorganism Acetobacter suboxydans or Gluconobacter oxydans and finally several chemical steps were carried out for conversion of L-sorbose to L-ascorbic acid. Figure 69. Industrial production of L- ascorbic acid (vitamin C). 26  4. Medical biotechnology & vitamin production  Vitamin D Vitamin D is synthesized in the skin through the action of ultra- violet light or is obtained from dietary sources. In the skin, there is 7- dehydrocholesterol which reacts, in the presence of UVB radiation to form previtamin D3. The first hydroxylation, which occurs in the liver, converts vitamin D to 25-hydroxyvitamin D [25(OH)D], also known as calcidiol. The second hydroxylation occurs primarily in the kidney and forms the physiologically active 1,25-dihydroxy vitamin D [1,25(OH)2D], also known as calcitriol (Fig. 70). 27  4. Medical biotechnology & vitamin production  Vitamin D Figure 70. Metabolism/activation of vitamin D The Actinomyces hyovaginalis bacterium is capable of converting vitamin D3 into calcitriol. This biotransformation is a possible tool to produce active form vitamin D3 [1,25(OH)2D] from the readily available vitamin D3 for patients with compromised kidney function. 28  Medical/pharmaceutical biotechnology & its application 5. Medical biotechnology & gene therapy Gene therapy is the insertion of genetic material into cells to prevent, control, or cure disease. It encompasses repairing or replacing defective genes and making tumors more susceptible to other kinds of treatment. Gene therapy can be introduced in two different ways. It can be directly infused into them (which is called in vivo gene therapy), or it can be used to modify cells in a lab that will then be transplanted into the child (called ex vivo gene therapy). 29 5. Medical biotechnology & gene therapy Concepts of in vivo and ex vivo gene transfer 30  5. Medical biotechnology & gene therapy  Multidrug resistance (MDR) gene therapy Introduction of the Multiple Drug Resistance (MDR) gene into the bone-marrow cells of patients with advanced cancer seems safe and may protect their bone marrow from the toxic side effects of chemotherapy. This makes stem cells, which are responsible for the production of blood cells, more immune to the toxic side effects of anticancer drugs. It may thus make high-dose chemotherapy safer and improve recovery.  Anti-angiogenesis gene therapy Another anticancer strategy undergoing investigation, anti-angiogenesis gene therapy, involves introducing genetic material to a limited area to decrease the formation of blood vessels there. Decreasing angiogenesis at the site of a tumor decreases the tumor’s ability to grow and spread. 31  5. Medical biotechnology & gene therapy  Angiogenesis gene therapy A form of gene therapy with the opposite effect on blood-vessel formation has also been developed. Preliminary research suggests that "therapeutic angiogenesis," or VEGF gene therapy, may be effective against sensory neuropathy (specifically, a loss of feeling in the feet) and critical limb ischemia (an arterial disease marked by a decrease in the supply of oxygen-rich blood to the legs). Such a decrease can result in gangrene and the need for amputation. VEGF stands for vascular endothelial growth factor, a protein that can induce angiogenesis. 32  Plant biotechnology Student presentations. Thank You gu.edu.eg