Gene Technology & Synthetic Biology /Biosafety & Biosecurity
Yee‐Joo TAN, PhDMember of GMAC Subcommittee for
Research on GMOsDepartment of Microbiology and Immunology, NUS& Institute of Molecular and Cell Biology, A*STAR
Genetically Modified Organisms (GMOs)
• Any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology (Cartagena Protocol, 2000)
Singapore’s GMAC research guideline :“… all biological entities (cells, prions, viroids, viruses ororganisms) which have been made by genetic manipulation and are of a novel genotype and which are unlikely to occur naturally, or which could cause public health or environmental hazards ...”
Importance of GMOs in different areas
• Genetically modified salmon grows faster, cow producing human‐like milk, low lactose milk
• Genetically modified mosquitoes: Release into the environment to reduce the transmission of infectious diseases such as dengue fever and malaria
• Genetically modified mice for research use as well as new drug testing (toxicity, preclinical)
FOOD
ENVIRONMENT
MEDICINE
Examples: GMO plants
GM food = food produced from or using GMO
• Flavr‐Savr Tomato is the first GM food ‐>reduced the cost to produce canned tomato products about 20% ‐> economic benefit for farmers planting GM crops.
• Livestock can be raised less expensively, using feed made from GM crops
• Cheaper fish production: AquaBounty salmon is the first GM animal approved to be sold as food.
GM food ‐ a solution to food shortage?
http://www.nature.com/news/specials/gmcrops/index.html
http://www.isaaa.org/resources/publications/pocketk/16/
Example: http://www.oxitec.com
• Aedes aegypti, the main vector for several of viruses including Zika, dengue, and chikungunya
• OX513A male mosquitoes are engineered with a self‐limiting gene to cause their offspring to die
• Males, which do not bite or transmit disease, are released to mate with wild females
Science & public survey on genetically modified mosquito
Offspring inherited the
artificial gene and will die before adulthood
Antidote given to larva in lab & the mosquito grows to adult
Field trials being conducted in different countries
Preclinical research in medical science
• Understand disease pathways & identify drug targets
• Test drugs for toxicity/pharmacokinetics
• Improve animal models to mimic human diseases e.g. cancer
Cell 2015 163, 39-53DOI: (10.1016/j.cell.2015.08.068) Copyright © 2015 Elsevier Inc. Terms and Conditions
Preclinical Mouse Cancer Models: A Maze of Opportunities and Challenges
Chi-Ping Day, Glenn Merlino, Terry Van Dyke
Figure 3
Cell 2015 163, 39-53DOI: (10.1016/j.cell.2015.08.068) Copyright © 2015 Elsevier Inc. Terms and Conditions
Common Genetic Engineering Techniques
• Recombinant DNA technology (gene transfer)
• Genome editing
• Synthetic biology
Recombinant‐DNA (rDNA) technology• The way in which genetic material from one organism is artificially introduced into the genome of another organism and then replicated and expressed by that other organism
https://www.chemheritage.org/historical‐profile/herbert‐w‐boyer‐and‐stanley‐n‐cohen
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2383885/
Transfection of plasmids into human cells
Gene Therapeutic antibody
https://www.eurostemcell.org/ips‐cells‐and‐reprogramming‐turn‐any‐cell‐body‐stem‐cell
Therapeuticcells
Transgenic mice (DNA pronuclear microinjection technology)
Permanentintegration of gene into
chromosome
Genome Editing• Technique to precisely and efficiently modify (delete/insert/replace) DNA within a cell
• Make cuts at specific DNA sequences with “engineered nucleases”
• Versatile: more than just add “missing” or “additional copy” in gene transfer
https://www.yourgenome.org/facts/what‐is‐genome‐editing
Common techniques
http://cshperspectives.cshlp.org/content/8/12/a023754.full
An emerging field related to GMOs
Synthetic Biology
Synthetic Biology
– Interdisciplinary area that involves the application of engineering principles to biology.
–Modify and/or construct new biological parts, devices and systems for improvement
Ref: http://www.synbioproject.org/topics/synbio101/definition/https://www.bio.org/articles/synthetic‐biology‐explained
Biosafety and Biosecurity
Biosafety and Biosecurity
• Biosafety is the discipline addressing the safe handling and containment of infectious microorganisms and hazardous biological materials
• Biosecurity is the discipline addressing the security of microbiological agents and toxins and the threats posed to human and animal health, the environment, and the economy by deliberate misuse or release
https://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
Genetic Modification Advisory Committee (Singapore)(https://www.gmac.sg/)
• Established in 1999 under the purview of the Ministry of Trade and Industry
• Members are from multiple agencies• Objective: To ensure public safety while maintaining an
environment that is conducive for commercial exploitations of GMOs and GMO‐derived products
Subcommittee for Release of Agriculture‐related GMOsSubcommittee for Research on GMOsSubcommittee for Labelling of GMOsSubcommittee for Public Awareness
Guidelines– Singapore Guidelines for Release of Agriculture‐Related Genetically Modified Organisms: Safe import, release and use in Singapore of agriculture‐related organisms that have been genetically modified.
– Singapore Biosafety Guidelines for Research on GMOs: Provides safe containment, handling and transport of GMOs used in research and to provide a common framework for assessment and notification of research on GMOs
• Ministry of Health (MOH)– Laboratory Biosafety Manual (3rd Edition, World Health Organisation)
• National Advisory Committee for Laboratory Animal Research– Guidelines on the Care and Use of Animals for Scientific Purposes, NACLAR
Does genetic modification affect biosafety risk?
• Transient or permanent • Host (intended or not) receiving the transgene• Accurate prediction of phenotype and impact• Vector versus transgene
Subcommittee for Release of Agriculture‐related GMOsSubcommittee for Research on GMOsSubcommittee for Labelling of GMOsSubcommittee for Public Awareness
Regulatory Agencies Involved and their Relevant Acts and Laws (Life Sciences Research)
Agri‐Food & Veterinary Authority of Singapore (AVA)• The importation of plants, animals, plant‐related pathogens, animal‐related
pathogens falls under AVA’s purview.• Animals and Birds Act (Cap 7, Part II, Section 9), Control of Plants Act (Cap 57A)
etc.
Ministry of Health (MOH)• The importation and/or possession of BATA‐controlled biological agents and
toxins falls under the MOH’s purview.• Biological Agents and Toxins Act (BATA) (Cap 24A)
National Environment Agency (NEA)• Any person who breed, keep, collect, distribute, sell, import or export any vectors
(e.g. Mosquitoes, flies, rodents, rat fleas, cockroaches) is required to obtain permission in writing from the Director‐General of Public Health.
• Control of Vectors and Pesticides Act (Cap 59, Part IV, Section 16), Infectious Diseases Act (Cap 137, Part V, Section 40)
Ministry of Manpower (MOM)• In protecting the workplace safety and health of laboratory workers, the WSHA
requires laboratory operators to identify hazards and to take reasonable efforts to minimize the risks.
• The Workplace Safety and Health Act (Cap 354A) (WSHA)
Biosafety considerations for transgene
http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
Transgenic and “knock‐out” animals
http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
Virus as “gene transfer” vector
• Most viruses are small, ~100 nanometers • Each virus has a genome (DNA or RNA)
Virus as a GMO: Reverse genetics method
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx
‐ A gene sequence is known, but its function is unknown‐Make mutation and check the phenotype of the organism‐ Infer the gene function
Influenza A virus (RNA, respiratory): isolation vs reverse genetics
Transfection
https://courses.lumenlearning.com/microbiology/chapter/isolation‐culture‐and‐identification‐of‐viruses/https://www.publichealthontario.ca/en/ServicesAndTools/LaboratoryServices/Pages/Kit%20Instruction%20Sheets/Virus‐Respiratory.aspx
Extract RNA, convert to DNA and sequence
Usefulness of genetically modified viruses
• As viral vector for gene transfer/genome editing – both therapy and research
• Vaccine development• Studying the importance of virus mutations found in patients – e.g. drug resistance
• Relevance for surveillance – pandemic preparedness (vaccine and drug stockpiling)
Modified virus genome• Viral vectors are engineered to give a safe profile
‐ Infectious but replication‐defective{Viral genomes are separated & cannot be
packaged}
‐ Eliminate viral pathogenesis {Minimize insertional mutagenesis}{Delete non‐essential viral genes}
Viral vector for gene transfer
First approved gene therapy in Europe or the US – based on viral vector (AAV)
Glybera was first approved in October 2012 for hereditary lipoprotein lipase deficiency (LPLD), a genetic disorder that uniQure acknowledges is “ultra‐rare.” Indeed, only about 1 person in a million suffers the disease, which manifests as pancreatitis, recurrent abdominal pain and eruptive fat‐filled spots that result from very high triglyceride levels. Glybera provided a one‐off solution by introducing copies of the relevant gene to produce the deficient lipase indefinitely; the longest term study has proven its efficacy for at least six years.
http://www.uniqure.com/gene‐therapy/glybera.phphttps://labiotech.eu/uniqure‐glybera‐marketing‐withdrawn/
Reconstructed 1918 Spanish flu virus• “Spanish Flu” killed up to 50 million people worldwide in 1918‐1919
• Live virus was never isolated from patients• Fragment of virus genome was found in tissue archives in Pathology department
• More virus RNA was found in one flu victim unburied from an Alaskan permafrost cemetery
• After sequencing & whole genome was transfected into cells to create virus
• Virus found to be deadly in animal models• Can “Spanish Flu” be re‐created for bioterrorism?
Tumpey et al. (2005). Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310: 77–80.
Thank you for your attention!