DSCI112 Lecture 18 Biotechnology PDF

DSCI112: Molecules, Cells and Organisms Lecture 18: Biotechnology 1  Lecture 18: Biotechnology Biotechnology is a broad term generally used to describe the use of biology in industrial processes such as agriculture, brewing and drug...

DSCI112: Molecules, Cells and Organisms Lecture 18: Biotechnology 1  Lecture 18: Biotechnology Biotechnology is a broad term generally used to describe the use of biology in industrial processes such as agriculture, brewing and drug development. Traditional applications include animal breeding, brewing beer with yeast, and cheese making with bacteria. The term also refers to the production of genetically modified organisms (GMOs) or the manufacture of products from GMOs. Learning outcomes: Define biotechnology and gene technology. Describe the covid-19 vaccines – generated through mRNA technology and adenovirus vector technology. Describe gene editing using the CRISPR / Cas9. This lecture is based upon Describe examples of CRISPR / Cas9 use in content within Chapter 20 of human health. Campbell Biology, 12th Edition 2 Lecture outline What is biotechnology? Gene technology Generation of Covid-19 vaccines  adenovirus vector vaccines  mRNA vector vaccines Gene editing CRISPR  CRISPR defence mechanism  CRISPR Cas9 gene editing  CRISPR in human health Gene Therapy. (2021, January 12). https://k12.libretexts.org/@go/page/13355 3 What is biotechnology Biotechnology is a broad term generally used to describe the use of biology in industrial processes such as: Agriculture Brewing with yeast Drug development The term also refers to the production of genetically modified organisms (GMOs) or the manufacture of Cheese making products from GMOs. with bacteria 4 Gene technology Modern biotechnology includes the use of gene technology  Which can refer to the movement of genetic material from one species to another:  Or the removal (or ‘silencing’) of a gene within an organism: e.g. silencing a viral gene within prawns to inhibit infection  Or the modification of a gene within an organism: e.g. increasing the nicotine potency and Nicotine production in tobacco 5 Gene technology Can also be used to describe the manipulation of natural variation in a population Over the last few thousand years, farmers have bred Brassica Oleracea into six ‘cultivars’ that eventually became many of the vegetables we eat  Selective breeding has been done for thousands of years 6  Gene technology regulation In Australian the use of GMOs and products from GMOs are regulated by the Gene Technology Regulator. http://www.ogtr.gov.au/ Commonwealth Gene Technology Act 2000. 7 Genetically Modified (GM) crops In Australia, ingredients from GMOs are typically only found in highly processed foods and cannot be purchased as fresh food. Australian laws require food containing 1 per cent or more GM ingredients to be labelled In Australia, the following GM products have been approved for use in food: corn safflower canola soybean sugarbeet cotton rice potato  They have been modified to be insect resistant, herbicide tolerant or both  All GM food products available in Australia have been assessed for safety by Food Standards Australia New Zealand (FSANZ). 8 Current crop trials Field trials of wheat, barley, sugarcane, safflower, sorghum, pineapple, cotton, canola and banana are currently underway in Australia.  These crops have been modified for insect resistance, herbicide tolerance, colour, greater oil production, sugar composition, improved flowering and fruit development. 9 Genetically Modified Organisms: Uses in the environment GM crops that have been modified for insect resistance or herbicide tolerance allow farmers to use more herbicide and less pesticide on their farms. Bollgard II (Bt) cotton:  Insect resistant (Helicoverpa, the main pest of cotton)  Reduces need for insecticides (by up to 85%) ▪ Minimises the use of indiscriminate, hazardous insecticides that cause harm to welcome insects and contaminate the environment This: Instead of this: 10 Livestock research Gene technology used to improve the efficiency of animal production in Australia  Current research into using traditional and mRNA vaccines to treat livestock diseases 11 Gene technology: Uses in human health Development of more effective therapies for diseases like cancer, diabetes, hepatitis C and influenza.  Microorganisms such as yeast and bacteria have been modified to produce vaccines for hepatitis B and insulin for diabetics. Hepatitis virus Yeast Study causes of disease:  Heart disease, Alzheimer’s disease, motor neurone disease, cancer, rheumatoid arthritis, Huntington’s, and lupus- among many, many others 12 Summary #1 Biotechnology  Use of biology in industrial processes: including brewing, cheesemaking Gene Technology is one kind of biotechnology  Removal of genes  Modification of genes  Insertion of genes  Selective breeding Gene technology outcomes:  Plants that are herbicide tolerant and/or insecticide resistant  Organisms that are resistant to viruses  Production of vaccines or therapeutic proteins (e.g. insulin)  Investigations of gene/protein function to better understand and combat disease 13 COVID-19 Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Most people infected with the virus will experience mild to moderate respiratory illness and recover without requiring special treatment. However, some will become seriously ill and require medical attention. The World Health Organisation declared the COVID-19 outbreak a pandemic on 11th March 2020  Effects are still being felt today 14 The basics of coronavirus infection (more next lecture) The SARS-CoV-2 is a single-stranded RNA virus It consist of: Infection pathway:  A fatty acid coat 1. Virus enters cell (stolen from the host) 2. Viral RNA enters cytoplasm  A protein shell 3. Viral RNA makes viral proteins  Spike proteins 4. Proteins assemble into viruses  RNA genome 5. Cell releases viruses 6. Viruses infect new cells 7. Infection eliminated by immune response 15 COVID-19 Vaccines Most vaccines function through the introduction of antigens to the immune system (more to come in lectures 21 & 22)  Usually in the form of either live-attenuated or inactivated/heat-killed viruses In the case of SARS-CoV-2, the antigen targeted was the spike glycoprotein  The protein that allows the virus to enter cells The basis for the COVID-19 vaccines:  Translated spike protein is expressed on the host cell surface or parts of the protein are ‘presented’ on the cell surface (antigen-presenting cells)  Spike proteins recognised by the immune system  Immune system then builds antibodies and memory T and B cells  If infected with real SARS-CoV-2 virus, the immune system produces antibodies which bind to the virus and stop it from replicating or binding/attaching to other cells 16 What vaccines have been made? 1. The Oxford/AstraZeneca vaccine  Adenovirus vector (ChAdOx1) engineered to be harmless and cannot replicate ▪ Does not have the enzymatic machinery to integrate into host cell DNA  SARS-CoV-2 spike protein gene added into viral vector’s modified genome 1. The adenovirus genome enters the nucleus (normal path of infection) 2. DNA is transcribed into mRNA, which is exported to the cytoplasm and translated on ribosomes  Includes gene for the SARS-CoV-2 spike protein 3. Some spike proteins migrate to the cell membrane 4. Others are broken down and presented on the cell’s surface by other protein complexes 5. These protruding spikes/spike protein fragments are then recognized by the immune system  Neither the original virus shell or modified viral genome persist long enough to cause disease 17 What vaccines have been made? 2. BioNTech/Pfizer and Moderna vaccines  mRNA for spike protein is modified (to mimic human mRNA) and coated in a lipid nanoparticle (LNP, liposome) that is positively charged (facilitates attachment & entry via the negatively charged cell surface) 1. The mRNA enters the cell  Does not enter nucleus 2. Is translated on ribosomes to produce the SARS-CoV-2 spike protein 3. Some spike proteins migrate to the cell membrane 4. Others are broken down and presented on the cell’s surface by other protein complexes 5. These protruding spikes/spike protein fragments are then recognized by the immune system  Problem: mRNA is less stable than DNA  mRNA vaccines need to be stored at -80°C 18 A different approach Novavax The Novavax vaccine uses a version of the spike protein made in the lab. The spike proteins are assembled into tiny nanoparticles with lipids that resemble the structure of the coronavirus –  They cannot replicate once injected and the vaccine cannot cause COVID-19. In order for these ‘subunit’ vaccines to generate strong protective responses, they need to include molecules that boost your immune system, called adjuvants.  Adjuvants stimulate receptors on immune cells (the way the real virus would) initiating a rapid but non-specific immune response ▪ Enhances the response to other antigens present!  Novavax includes an adjuvant based on a natural product known as saponin, an extract from the bark of the Chilean soapbark tree. Source: https://www.sydney.edu.au/news-opinion/news/2021/04/14/what-is-novavax--australia-s-third-covid-vaccine-option--.html 19 Additional Video Resources Viral vector vaccine Explainer: How the AstraZeneca-Oxford COVID vaccine works (simple animation) https://www.youtube.com/watch?v=LSudYGwzOFI Covid conversations with Sarah Gilbert from Oxford University (3:46-8:27 most relevant, hear from an instrumental scientist) https://www.youtube.com/watch?v=MKNavonhXyk AstraZeneca DNA COVID 19 Vaccine Explained (vs. Pfizer / BioNTech, Moderna) (a good comparison from an MD) https://www.youtube.com/watch?v=GOq8-FR8s1E mRNA vaccine Why it actually took 50 years to make Covid mRNA vaccines (good overall picture) https://www.youtube.com/watch?v=XPeeCyJReZw Inside the lab that invented the Covis-19 vaccine (good to see the importance of understanding the protein structure of the spike protein) https://www.youtube.com/watch?v=-92HQA0GcI8 How the Covid-19 vaccines were created so quickly (simple animation) https://www.youtube.com/watch?v=v-NEr3KCug8 The Story Behind mRNA Vaccines: Katalin Karikó and Drew Weissman (hear from 2 instrumental scientists) https://www.youtube.com/watch?v=DyCmhKMd148&t=10s 20 Summary #2 Covid-19 vaccines: Adenovirus viral vector  Spike protein DNA added to a genetically engineered adenovirus that is non-replicable and non- harmful; DNA cannot be incorporated into host DNA as it lacks the machinery.  Viral vector enters cell and releases DNA into cytoplasm.  DNA enters nucleus where it is transcribed and then is translated in the cytoplasm (on ribosomes).  Spike protein then activates the immune system. mRNA  Modified spike protein mRNA encapsulated in a lipid nanoparticle (LNP), that is positively charged to attach to negatively charged cell surface,  Enters cell by endocytosis, and LNP is disassembled;  The mRNA is translated into the spike protein then activates the immune response.  mRNA does not enter the nucleus, cannot alter or integrate into host DNA. Other vaccines exist, including the Novavax protein subunit vaccine. 21 Development of techniques for editing specific genes What if you want to specifically edit a pre-existing gene in a genome  Restriction site/plasmid approach requires many steps (need to isolate target gene from the rest of the genome to edit it to avoid off-target effects…) Previous technologies for introducing cuts/editing DNA based on N u c l e a s e s Nucleic Enzymes acid All of these require custom engineering of specific proteins for making very specific cuts to specific DNA sequence  To disrupt the gene via insertions/deletions  To insert a new gene Development of CRISPR/Cas9 system  Allows for very specific cuts 22 CRISPR Clustered Regularly Interspaced Short Palindromic Repeats  First discovered in bacteria  Identified in many bacterial (40%) and archaeal (90%) genomes Escherichia coli Viruses can infect virtually every type or organism Image source: Digestive System Infections. (2023, January 9). https://bio.libretexts.org/@go/page/102722  Bacteria too!  Plasmids or bacterial viruses (bacteriophages) can invade bacteria  CRIPSR is a natural bacterial defence mechanism (bacterial “immunity”) against infection Image source: Mu- A Double-Stranded Transposable DNA Bacteriophage. (2020, April 22). Boundless. https://bio.libretexts.org/@go/page/28881 23 How does CRISPR work? 1. Bacteriophage (virus) infects bacterial cell and injects viral DNA into cytoplasm 2. A Cas nuclease will cleave a short sequence out of the viral DNA 3. Other Cas  enzymes will Short incorporate this palindromic ‘Cas’ genes: repeats sequence into the CRISPR array Cas9 as a spacer 6. When this complex encounters complementary DNA, it cuts it! 4. DNA is transcribed into RNA: Cas nucleases Spacer RNAs are translated are processed 5. Cas nuclease complexes with RNA 24 The components of CRISPR: 1. The ‘Cas’ genes: Adaptation proteins: process invading viral DNA for incorporation into the CRISPR array as spacers Interference proteins: use spacers from CRISPR array to target and cleave invading DNA with matching sequence (i.e. the Cas9 nuclease) Cas9 Spacers: short Repeats: Short (24- sequences (~35 base 2. the CRISPR array: 48 bp) palindromic pairs) acquired from sequence repeats previous viral infections The order of the spacers can be used to infer infection history! (acquired in the order of infection) 25 CRISPR/Cas9 Interference CRISPR uses a ‘guide RNA’ to target specific DNA sequences Nuclease function In the CRISPR/Cas9 system, the of Cas9 Cas9 nuclease cuts both Guide RNA strands of the sequence targeted by the guide RNA  Generates a double Target sequence stranded break that the cell attempts to repair 26  Engineering CRISPR/Cas9  By engineering guide RNAs Target sequence complementary to a target gene, it is possible to target Engineer sequence the nuclease to cleave into CRISPR array within that gene Cas9 = Can now digest target sequence Emmanuelle Charpentier Jennifer Doudna Nobel prize Chemistry 2020 27 Use of CRISPR/Cas9 in biotechnology Deleting a gene  Once a double-stranded break is made, error correction by the cell is poor…  Usually, DNA cannot be repaired without introducing mutations/deletions  = Gene loses function Targeted mutation of a gene  Can use the CRISPR/Cas9 system to make a cut, and then provide a modified sequence to replace it Insertion of a new gene  Can similarly introduce complete new transcriptional units to the cut site! Other variations  Example: a CRISPR/Cas9 complex with a deactivated Cas9 can be used to bind to a target sequence and simply stay there, blocking transcription! 28 CRISPR/Cas9 use: In humans Mother: HIV negative Father: HIV positive He Jiankui: Presented this in November 2018 Embryo: normally expresses CCR5 The audience, and then the broader A protein which aids HIV scientific community, was outraged entry onto host cells Wensheng Wei: Cas/CRISPR was used to “Why did you choose to cross this disable the CCR5 gene line? And … why did you choose to do HIV entry into host cells is all these clinical studies in secret?” impeded This work has prompted calls for Child: Protected tighter regulation of this technology against HIV (?) He Jiankui is being investigated 29  CRISPR/Cas9 use: In humans Human clinical trials currently planned/in progress utilising CRISPR-Cas9 First trial started October 2016 in China:  Patients with metastatic lung cancer for whom chemo- and radio- therapies have failed T-cells have a receptor called Programmed cell Death protein 1 (PD-1) 1: Extract cancer  Use to downregulate the immune system to patients T-cells No prevent cytotoxicity/autoimmune reactions more lies! Some cancers produce Programmed death ligand 1 (PD-L1) 2: Use CRISPR/Cas9 to disable the PD-1 gene PD-1Δ cells should show When PD-1 binds to PD-L1, the Hey! I’m PD-1 says you greater T-cell (cytotoxic) just like are, so OK – activation and proliferation of you! you can live! activity against cancer cells T-cells is inhibited – a means by which tumour cells can 4. Transplant 3. Grow many evade the immune system them back into PD-1Δ T-cells  Can sometimes use PD-1/PD-L1 antibodies, but some the patient patients are insensitive or suffer inflammatory side affects 30 Many applications for biotechnology Medical applications  Diagnosis and treatment of diseases  Development of personalised medicine  Gene therapy for hereditary diseases Pharmaceutical products  Synthesis of small molecule for use as drugs  Production of recombinant proteins Environmental cleanup  Plastic eating microbes  Extraction of heavy metals from sewerage Better agricultural applications  What could you do with gene editing? 31 Summary #3 CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats  Originally a bacterial defence mechanism CRISPR components:  Cas genes: ▪ Adaptation proteins will cleave invading DNA to create short “spacer” sequences ▪ Interference proteins will join with spacer RNA to cut invading DNA  CRISPR array: ▪ Short palindromic repeats ▪ Spacers (short pieces of DNA from previous infections) Cas9 (interference) proteins can complex with spacer RNA, and cut DNA at specific places By modifying the “guide” RNA in a Cas protein/RNA complex, CRIPSR can be used to edit genomes:  Delete, modify or insert genes Trials are underway using CRIPSR in humans  Previous use of CRISPR on embryos has been shunned  Clinical trials currently underway, modifying cell lines to fight cancer 32