Interviews

Dr. Athanasios Zovoilis, University of Lethbridge

Test Arc-Gag in cell culture on primary mouse neural cell lines

We spoke with Dr. Athanasios Zovoilis, a faculty member at the University of Lethbridge’s department of Chemistry and Biochemistry. Dr. Zovoilis has vast experience in cell transfection. He explained to us some of the challenges associated with cell transfections in eukaryotic cell lines as well as how cell transfections vary significantly between cell lines. Some of the cell transfection techniques that he mentioned were lipofection: using lipid membranes with an epitope tag for delivery, electroporation: electrically inducing pores in the cell membranes of cells for ready uptake, the use of transfecting agents (which vary in composition), and even some encapsulating proteins such as Qiagen’s dendrimer based SuperFect Transfection Reagent. The typical challenges associated with these techniques are that they can cause stress for the cells, leading to cell death, or unintended gene expression. Primary neural cell lines from mice however, face difficulty in their transfection affinity as well as consistency in their transfection rates. Using a protein nanocompartment such as Arc-Gag could be a good step in making such improvements since it has a natural tropism for mouse neural cells. Testing a fluorescence transfection of a primary mouse neural cell line would help to show if Arc-Gag could be used as a transfecting agent in mouse cell culture.

Dr. Justin Pahara, Amino Bio Labs

Increase specificity and accessibility of nanocompartments

We spoke with Dr. Justin Pahara, the CSO for Amino Labs to discuss the utility of our project for researchers and industrial applications. He said that protein nanocompartments would be a useful delivery system that can be tuned to deliver a variety of cargos with increased specificity depending on their chemical structure and functionalization. One possible application could be in increasing transformation efficiency during transfections of various cell types. Further, compartmentalization at the molecular scale using phages is often difficult and intimidating from a biosafety perspective. A simple plasmid-based expression system of capsid proteins built for bacteria or yeast could make nano-encapsulation much more accessible to non-experts who are keen to learn molecular biology techniques or researchers who want to become familiar with biological encapsulation and delivery. The “tuning” of the capsid proteins to have different chemical characteristics is an advanced undertaking. Having a software application to help with the capsid protein design would enable those with less experience and the broader scientific community to begin working with encapsulation. Effectively, the software application would give the user a starting point to learn about how protein capsids work. Overall, this interview was helpful for informing our construct design and confirming this would be a useful tool for researchers. Justin and Julie Legault, the CEO of Amino Labs, are also releasing a book called From Zero to Genetic Engineering Hero this month (https://amino.bio/products/learn-genetic-engineering-the-genetic-engineering-hero-book). This book is the perfect journey for students to learn the basics of engineering bacteria - From covering the basics and learning about DNA, extracting proteins that you’ve engineered bacteria to create, to even control genetic circuits, these are the topics you need to know before you start innovating on your iGEM project.

Dr. Vanessa Meier-Stephenson, Postdoctoral Fellow

Need a more effective tool for cell transfection

We spoke with Dr. Vanessa Meier-Stephenson, an MD-PhD working as a Postdoctoral Fellow in collaboration effort at the University of Lethbridge and the University of Calgary. During her PhD, she worked on drug design and told us that packaging methods for small molecules are designed to ensure that the drug can survive the acidic conditions of the stomach. Additionally, some larger drugs such as vancomycin cannot diffuse naturally across cell membranes. She suggested that for our protein nanocompartment system, it would be effective for this application by helping with moving larger molecules across membranes and for ensuring a smaller concentration of molecules would be required for certain cell types. In her own work, she said that there are difficulties in doing transfection assays using cell lines for Hepatitis B. She suggested that it would be useful if tags could be added to capsids to make them specific for different cell lines to increase the modularity of our system. Vanessa also suggested that we would need to look into toxicity levels in the cells and the potential immune response for safety considerations. Overall, this interview helped identify that there are issues with trying to transfect some cell lines, helped inform our construct design, and safety issues we may encounter for the drug delivery application.

Public Health Agency of Canada

Look through regulations carefully and at the end applications

We spoke with Brigitte Cadieux, a senior policy analyst for new and emerging technologies, Jennifer Mihowich, who works in the biosafety risk assessment unit, and Melanie Sabourin, a biosafety education officer from the Public Health Agency of Canada (PHAC) to discuss the regulations surrounding protein nanocompartments. They suggested we look through the regulations across the different agencies carefully as terminology is not always the same. In Canada products and pathogens are regulated and not technologies so there are currently no regulations for the use of our system as a technology. However, under some legislation, virus-like particles are considered microorganisms and that legislation would still apply to them. They told us that under the acts that PHAC monitors, our PNCs would be classified as Risk Group 1 and thus exempt from their regulations. In terms of explaining our project to non-scientist audiences, we were advised to change our language to keep it simple. We were also told that even though our project is not using viruses, it would be good to have an example of viruses being used in a positive way when speaking to the public. Overall, this interview was helpful in examining if our system would be safe and what the regulatory landscape is surrounding it.

Nicole Kimmel, Alberta Environment and Parks

Compare your project to current methods in use

We spoke with Nicole Kimmel, the aquatic invasive species specialist with Alberta Environment and Parks. She helps to oversee the five components of the aquatic invasive species program: policy and legislation, education and outreach, monitor, inspection, and response. She said that one of the larger threats that they are setting up prevention methods are zebra mussels. Current detection methods that they employ are substrate monitoring in various lakes in the province, tow nets through lakes, and are starting to explore environmental DNA methods. For prevention, the province is using boat inspection stations and have done studies on using potash (potassium chloride) as a treatment though is still in the process of being registered as a treatment with the federal government of Canada. When asked if they had heard about Zequanox, she said that they have, but it is too cost prohibitive for Alberta to use. She suggested that we consider the advantages and disadvantages of our project with these methods. For our project, she said that we would need to ensure that our PNCs with the toxin would be stable within the water. With this type of project, you need to be conscious of the users of waterways and where the water is distributed. Overall, this interview was helpful in learning more about how the province is currently handling this problem and that our system would need to be safe to ensure no unintended effects would occur.

Shannon Frank, Oldman Watershed Council

More research needs to be conducted on preventative measures for invasive species

We spoke with Shannon Frank, the Executive Director of the Oldman Watershed Council, a local not-for-profit group that works on finding solutions to environmental issues in Southern Alberta. The group has 8 goals and their main focuses are currently enhancing the commitment of the community to taking care of the watershed, to restore headwaters, and to educate recreationists about their impact on the watershed. One of their goals they plan on working on in the future is to prevent and control invasive species. Shannon mentioned that one of the invasive species that is current threat to Alberta waterways is zebra mussels. Current prevention methods include educating the public and boat inspection stations. A potash treatment is also being investigated as a viable treatment option. We are the first biotechnology project that has approached the Oldman Watershed Council. Shannon said that for our project the main considerations would be that it is specific for zebra mussels and would not harm native species and also what the long-term effects would be on the ecosystem. In terms of the implementation of our system, she suggested that it may be more practical for government agencies to implement our system into pipelines. If zebra mussels were to invade Alberta, infestation in city and agriculture pipes would cost millions of dollars to repair this infrastructure. Overall, this interview was helpful in determining what we would need to do to have our system be useful for an environmental application.

Ryan Dyck, Agriculture and Agri-Food Canada

Southern Alberta would not recover from an aquatic invasive species infestation

We spoke with Ryan Dyck, a research technician in Cereal Agronomy at the Lethbridge Research and Development Centre, a branch of the Department of Agriculture Agri-Food Canada. Cereal Agronomy conducts research experiments in the field to determine the optimum agronomic methods for producing the highest quality of crop of existing crop varieties the public has access to. Due to the climate in Southern Alberta, the agriculture industry here utilizes 65% of S. Alberta’s total fresh water consumed making this a very important resource for the economy. With over 40 crop varieties supported by the irrigation systems involving 50 reservoirs, any infestation of invasive species or contamination of the water with biological control agents would have a debilitating effect on the industry. If an infestation of zebra mussels colonized any part of the system, the entire network of reservoirs and over 8,000 km of pipelines would be at risk. The value of the irrigation network is estimated to be worth $3.6 billion and the cost to replace the existing infrastructure could be in the hundreds of billions of dollars. Additionally, the current method of controlling zebra mussels involves dumping KCl into the water supply and the turbidity of the water would have a severe impact on certain sensitive life stages of crop development.

Sweta Gupta, Pharmacist

Be clear on what components are in your system

We interviewed Sweta Gupta, a pharmacist and manager of Wal-Mart pharmacies. She said that within Wal-Mart’s repositories, they do dispense biologics and that what is available from their distribution centre is how they choose what is available on their shelves. She suggested that our system would need to have clear stability information and the components of the reaction would need to be indicated to determine if there are any allergens present. Overall, this interview informed us that we would need to ensure our system would have to come in a stable form and identified a potential safety issue.

Regulatory Landscape

This year we explored the regulatory landscape within Canada regarding protein nanocompartments. Overall, the legislation shows that protein nanocompartments themselves are not high risk. However, when thinking of the applications of PNCs and the cargo choice, more regulations will apply for the user as Canada regulates based on the end-product and not the technology used to make it. This exploration was guided by our discussion with the Public Health Agency of Canada.

Table 1. Overview of select acts within Canadian legislation [1-10].

Regulation Area	Canada
Environment	Canadian Environmental Protection Act Report potential immediate or long-term effects on the ecosystem for release of toxic substances
Food/Pharmaceuticals	Food and Drugs Act Ensure prescribed information is made public within a set time frame For vaccines, identity and purity must be known and tested with a standardized method
Health	Assisted Human Reproduction Act Alterations to the genome of a cell in vivo or in vitro cannot be passed on
Laboratory Biosafety	Canadian Biosafety Standard Synthetic biology products can be classified as a microorganism Follow proper protocols depending on risk group classification
Agriculture	Canada Agricultural Products Act Require proper accreditation for work on biological products
Pests	Pest Control Products Act Must evaluate possible harm to the environment and human health
Microorganisms	Canadian Environmental Protection Act New Substances Regulations Notification Regulations (Organisms) Follow guidelines based on risk group classification Virus-like particles are classified as microorganisms under some legislation
Animals	Health of Animals Act Consider potential effects on animal well-being Risk Group 1 organisms are not regulated
Pathogens and Toxins	Human Pathogens and Toxins Act Risk Group 1 organisms are not regulated

Risk Assessment Rubric

Based on our interviews, we found that many people would likely have concerns if our system were to be used for one of the applications that we explored, such as using them with zebra mussels or for gene therapies. We decided to design a risk assessment rubric for future iGEM teams and researchers to use if they are considering the use of protein nanocompartments for their project. This rubric was reviewed by Brigitte Cadieux, Jennifer Mihowich, and Melanie Sabourin of the Public Health Agency of Canada and their input was used to revise our initial draft. Check out our rubric here!

Environmental Counter-Measures

Invasive species are very difficult to combat and a debate exists on whether or not it is ethical to wipe out a species once it has established itself in the ecosystem regardless if it is of invasive nature or not. However, preventing them from establishing themselves in the ecosystem is a tactic that is being explored by many government entities. An example of this is boat inspection stations in Alberta for zebra mussels as a primary method of prevention. However, in time, this may not be enough and the development of secondary countermeasures is necessary for future prevention of the spread of invasive species. The introduction of invasive species to Canadian industries alone costs an estimated $30 billion annually, and the introduction of zebra and quagga mussels to Alberta ecosystems is estimated to cost $75 million to the agricultural sector annually [11,12].
Since the introduction of invasive Dresseinia spp. mussels into Canada, the Alberta government has been exploring many secondary preventative measures including the use of potash (potassium chloride) at high and lethal concentrations. Consequently, it is expected to have negative effects on other freshwater mussel species in the environment, making it a less than ideal option in open water environments and on the crops that would be exposed via irrigation pipelines in Alberta.
Beyond Alberta, there are many other invasive species that affect the environment and economies of Canada. Well known examples include the lampreys of the great lakes, Varroa destructor mites in bees, and even goldfish that had been inappropriately released into Alberta storm drains. Safely preventing the spread of these destructive organisms is a priority that needs to be explored beyond prevention of exposure to the environment.
We would like to propose the use of protein nanocompartments produced cell free in an optimized, mechanistic pipetting strategy, partnered with extensive research in specific toxins for individual pests and invasive species in order to initiate rapid and efficable response [13]. Protein nanocompartments are useful for introducing specific toxins to organisms and would prevent off target effects as well as environmental exposure of the cargo. This would work only in strategy of consistent production and research to prevent imminent threats from becoming realistic hazards. The strategy of which, could be employed by our previous project Next Vivo, which exercised the simplification and improvement of cell free protein synthesis [14]. Using cell free protein synthesis simplifies purification, and if produced efficiently, would allow for more cost effective production.
If the implementation of secondary preventative measures is utilized, an infestation could be stopped before it gets out of control. This is essential to the ideal of preventing negative impact induced by human activity.

Liability in iGEM

Like with all scientific technologies, protein nanocompartments have the potential for misuse such as the delivery of neurotoxins to humans, or the improper release of biological controls. If we are enabling these risks with our toolkit, we also want to address them. This has led us to explore liabilities in biotechnology, concerning the burden of responsibility; Is the liability dependent of the designer or the user? As well, what are the strategies that can be employed by scientists to limit the potential for liabilities.
It is typically assumed that people in science use technologies with the intention of good, but often is the case that the user makes an error and a technology is misused with unintended consequences. The 2002 gene therapy treatment of X-linked severe combined immunodeficiency (XSCID) in eleven children is an example of accidental misuse [15]. Fortunately, nine out of eleven patients were treated, showing significant improvement, and as such, led relatively normal lives. Contrarily, the remaining two had off target effects of the gene treatment, involving the integration of the vector into the LMO-2 gene, which is linked to T-cell hyperproliferation, causing childhood leukaemia[16].
As such, a question should be asked: are the developers of the technology liable for not exploring potential misuse before release, or is it the users who did not implement the technology without first considering the consequences involved?

We propose that the liability towards the technology designer should be considered on a case by case basis depending on three factors:

Did the designer provide proper literature for their findings, and were the findings tested appropriately before implementation?
Did the designer suggest potential for misuse and knowing such, did they attempt to remove the risks?
Did the designer allow correspondence to discuss the risks of their technology?

Similarly, we propose that the liability towards the user should be considered on a case by case basis depending on three factors:

Did the user consult appropriate literature for their intended application, and did they test the finding appropriately?
Did the user know about potential risks? If so, how did they attempt to remove the risks?
Did the user attempt correspondence with the designer to discuss the risks inherent in the technology?

In addition to these factors, it must be considered whether both the designer and the user followed the biosecurity requirements of both their own nation and organization.

References

[1] Canadian Environmental Protection Act 1999 (Fed) (CAN).
[2] Food and Drugs Act 1985 (Fed) (CAN).
[3] Assisted Human Reproduction Act 2004 (Fed) (CAN).
[4] Food and Drugs Act 1985 (Fed) (CAN).
[5] Canadian Biosafety Standard 2nd Edition 2015 (Fed) (CAN).
[6] Canada Agricultural Products Act 1985 (Fed) (CAN).
[7] Pest Control Products Act 2002 (Fed) (CAN).
[8] New Substances Notification Regulations (Organisms) 2005 (Fed) (CAN).
[9] Health of Animals Act 1990 (Fed) (CAN).
[10] Human Pathogens and Toxins Act 2009 (Fed) (CAN).
[11] https://ec.gc.ca/nature/default.asp?lang=En&n=b008265c-1 2018/10/13
[12] http://aep.alberta.ca/fish-wildlife/invasive-species/aquatic-invasive-species/default.aspx 2018/10/13
[13] Bundy BC, Franciszkowicz MJ, Swartz JR. Escherichia coli-Based Cell-Free Synthesis of Virus-Like Particles. Biotechnology and Biochemistry. 2007. Department of Chemical Engineering, Stanford University, Stanford, California.
[14] https://2017.igem.org/Team:Lethbridge 2018/10/14
[15] Aiuti A, et al. Correction of ADA-SCID by Stem Cell Gene Therapy Combined with Nonmyeloablative Conditioning. 2002. Science. Vol. 296, Issue 5577, pp. 2410-2413
[16] Alexander DR. Uses and abuses of genetic engineering. 2003. BMJ. Volume 79, Issue 931.

Team:Lethbridge/Human Practices