What is Gene Therapy?
The ideal medicine is not a perfect treatment - it is a cure. For the first time, humankind has the tools to correct the genetic causes of disease.
Gene therapy gives us the opportunity to add, remove, and change our genes, influencing how protiens are made and better provide care for those who suffer from debilitating diseases.
Typically, gene therapy is performed using a virus to introduce the treatment into the body. Viruses are greatly successful at penetrating cells, and thus work as an excellent vector for introducing genetic material. Provided that the treatment is successful, functioning proteins will be secreted from the repaired cell, as diagrammed below.
How We're Moving Towards a Safer Gene Therapy
While viral vectors have successfully demonstrated the potential of gene therapy, safety concerns, such as the random integration of therapeutic DNA, which can lead to cancer and severe immune responses, constrain their use to severe diseases. In order for gene therapies to benefit a greater number of people, for a wider range of indications, the risks of viral delivery must be overcome.
Targeted integration of DNA in mammalian genomes has been made simple with CRISPR/Cas9. Unfortunately, the insertion of large (> 1 kb) DNA inserts has proven too inefficient for therapeutic applications, restricting the use of CRISPR-mediated knock-in to relatively small inserts. In light of these constraints, the University of Calgary iGEM team has designed a targeted gene integration platform to build a safer gene therapy.
There are two pillars at the core of our platform. The first consists of a two-phase integration system, based on CRISPR/Cas9 and Flp recombinase. By inserting small Flp recombinase target (FRT) sites using CRISPR/Cas9, Flp can be used to integrate large inserts. To minimize the risk of off-target effects, we will target a "genomic safe harbor" where DNA can safely be introduced.
Furthermore, we are minimizing off-target integration by employing Cas9 nickase with an ssDNA template. This second pillar leverages regulatory elements (REs) found naturally in the human genome to keep therapeutic DNA active and isolated from its genomic context. In particular, we are using ubiquitously-acting chromatin-opening elements (UCOEs) to maintain internal control of transgene expression, while preventing disruption of the host genome with insulators.
How Snip, Equip, Flip Works
1. Snip: Paired nicks near the genomic target to initiate DNA repair.
2. Equip: Single-stranded DNA as a repair template to introduce distinct target sites.
3. Flip: FLP recombinase-mediated cassette exchange (RMCE) between pairs of target sites.
What is iGEM?
iGEM, the International Genetically Engineered Machine, is a non-profit organization dedicated to the advancement of synthetic biology. Every year, iGEM holds a competition in Boston where over 300 teams from all over the world come to compete.
This year, team Calgary is competing at the iGEM competition in Boston with our project, Snip, Equip, Flip: Towards a Safer Gene Therapy. In preparation for the competition, the team is completing a number of requirements, which include building software solutions and generating a microfluidic system.
The requirements to succeed in the competition include demonstrating that our engineered system works, collaborating with other teams, and considering the social implications of our system (human practices).
The dry lab team is developing two tools for making software easily accessible for iGEM teams, as well as exploring microfluidic solutions for delivering gene therapy.
A tool for aggregating software created by past iGEM teams into a readable, editable, and searchable database. SARA (Software Aggregating Research Assistant) is being made available for online and local use. SARA is intended to make software developed by the iGEM community more valuable by improving access and awareness.
A lab management tool that is compatible with google assistant. The features of LARA (LAb Research Assistant) include step-by-step reading of protocols, a speech to text lab notebook, and access to SARA. LARA is intended to allow for the hands-free exchange of information in a lab, improving the efficiency and ease of lab work.
The applied design group is developing a GMP-in-a-box prototype to deliver the modification method in a clinical setting. This microfluidic system encapsulates target cells in droplets and transfects them via electroporation.
The team has been reaching out to thought leaders in the community with the goal of mitigating concerns regarding gene therapy. As a relatively new treatment, gene therapy has been met with mixed reactions. Concerns range from the ethics of enhancement to the accessibility of the treatment. The team would like to address two specific concerns: the act of playing god with respect to western religions, and the relationship between gene therapy treatments and traditional First Nations medicine practices.
While gene therapy and its applications are the focus of the human practices portion, the team is also working towards improving education in synthetic biology at the high school level. As an emerging science, the team is working to develop lesson plans and curriculum proposals for Alberta education and proposing a heavier focus on synthetic biology for the international baccalaureate (IB) curriculum, which operates all over the world.
Ethics and Safety
As with all new technology there are increased points of risk that need to be addressed. Particularly, the team has taken strides to increase awareness of the safety precautions that should be taken, while also implementing safeguards where possible. Careful consideration of the ethical implications of gene therapy through introspection is being done and extended via discussions with individuals in the realm of bioethics. These accounts inform our work along with meetings with the public, stakeholders and thought leaders.
The team is addressing the project in its clinical context. Meetings at Innovate Calgary are useful for consideration of the project's value from a product level view. The team is also speaking with clinicians and genetic counselors, who are the primary liason between the laboratory, our product, and patients. Collectively these meetings ground our work in reality, and allow us to enage meaningfully with potential stakeholders in our project.