Structural exploration of locked nucleic acids (LNAs) for incorporation into anticancer oligonucleotides

Antisense oligonucleotides (AON) are short lengths of RNA or DNA molecules which are used to change gene expression to treat diseases like cancer and Parkinson’s disease. Like DNA, AONs are made up of chains of nucleotide units, but to make them useful as drugs, these nucleotides have to be structurally changed. Locked nucleic acids (LNAs) are a very useful type of altered nucleotide unit, since they are not broken down as quickly in the body, and attach strongly to the gene they are targeting. The problem with LNAs is that they are very difficult to make, so it is hard for chemists to make a lot of different changes to the structure of LNAs in order to find the best one to use in AONs.
The Britton research team recently discovered a new way to make LNAs very quickly and easily, in large amounts, from simple compounds. Using this new technology, we want to make a large number of structurally unique LNAs and, test them to find the best ones to use in AONs for the treatment of cancer.

Comprehensive dissection of tumor evolution in pediatric acute myeloid leukemia using single cell methylation sequencing

Pediatric acute myeloid leukemia (pAML) is a common type of cancer in children and is diagnosed in roughly 40 Canadian children each year. Although 90% of all children respond well to the initial treatment the cancer comes back for 20% of the children while being resistant to treatment, leading to a poor outcome. Current studies of treatment resistant cancers are not able to detect rare but important cells that form the cancer, which may be especially important in how treatment resistance occurs. Fortunately, new technologies allow for measurements from each of the thousands of individual cancer cells that form the tumor allowing us to detect rare cancer cells, including those that may result in treatment resistant disease. For the first time, we aim to use these technologies to focus on chemical properties of the DNA that influence how the DNA is interpreted, or read, by the cell. By studying patterns of these chemical properties in rare cancer cells and also normal cells we aim to learn if, and how, these patterns contribute to the phenomenon of treatment resistance in pediatric AML. With this knowledge, our ultimate goal is to prevent the formation of treatment resistant disease in this vulnerable population of patients.

Defining the landscape of genetic variation underlying rare human disease using nanopore long-read sequencing

Collectively, rare diseases affect millions of people worldwide. Understanding the molecular cause of rare disease has important implications for clinical management. However, although most rare diseases are suspected to be genetic in origin, the causal genes are not known in a majority of affected families. This study will use emerging technologies to better understand the molecular basis of rare genetic diseases. Long-read genome sequencing, a recent genetic testing technology, will help us to identify rare and complex genetic changes in individuals suspected to have harmful genetic variation. These findings will allow us to study how specific genes lead to congenital disorders and adult-onset cancer predisposition syndromes, genetic syndromes that increase the risk of developing specific types of cancers. This research will improve our understanding of normal and disease-causing genetic variation and help establish a foundation for the broader application of new technologies in the clinic.

Evaluation of SMPD3 as a quarterback for extracellular vesicle-mediated metastasis in oral carcinoma

Mouth cancer remains an under-studied and significant global cancer killer; dismal survival rates (~50% over 5 years) have not changed in decades. Potential spread to neck lymph nodes (metastasis) is the single most important prognostic factor but clinical assessment has not been very accurate. This results in insufficient surgery or over-treatment for many patients. A better understanding of mouth cancer and its way to spread is needed to improve treatment for the patients.
The SMPD3 gene is frequently dysregulated in mouth cancer it has been linked to metastasis. SMPD3 expression can impact microRNA (miRNA: small non-coding RNA molecules that regulates gene expression) cargo within extracellular vesicles (EVs). Many of these miRNAs have been linked to tumor invasion and metastasis. I hypothesize that mouth cancer cells that exhibit decreased SMPD3 expression plays a role in lymph node metastasis via specific miRNA EV content and that SMPD3 expression can be used as a biological marker for lymph node spread in mouth cancer.
We hope this project will lead to novel tools to identify the patients at highest risk for lymph node involvement, ultimately increasing survival rate and quality of life for mouth cancer patients.

The influence of podocalyxin expression on immune response to ovarian cancer and the efficacy of an antibody-drug conjugate in immunotherapy

Despite significant advances in the treatment of many cancers, ovarian cancer still claims hundreds of lives in Canada every year. A molecule called podocalyxin is “switched on” by a high percentage of tumors from various cancer types including ovarian cancer and its expression is associated with poor prognosis. Since the immune system has a key influence in the control of tumor growth, one of my objectives will be to study how podocalyxin influences the immune response against tumors.
In addition, Dr. McNagny’s team recently developed an antibody, called PODO447, which recognizes an exquisitely tumor-specific form of podocalyxin. Accordingly, my second objective will be to explore the use of this antibody as a method to either attract immune cells to cancer cells and kill them or as a tool to deliver toxins and chemotherapeutic agents specifically to tumor cells while sparing normal tissue. Preliminary experiments in animal models already are suggesting the efficacy of the latter approach. In conclusion, the results obtained in this project will allow us to take one more step toward the objective of ultimately treating ovarian cancer patients with the podocalyxin targeting therapies.

Examination of Long QT Syndrome causing variants in induced pluripotent stem cell-derived cardiomyocytes to evaluate novel therapeutic treatments

The rhythmic beating of the heart requires coordinated electrical activity that causes the heart to contract and relax. The electrical activity is controlled by proteins in the membranes of heart cells that form ion channels. Failure of channels to work properly is associated with abnormal heart rhythm, heart attack and sudden death. Long QT Syndrome (LQTS) is a condition that affects 1:2000 people and often results from inherited mutations in one of the heart channels. However, determining whether a mutation will cause the individual serious heart problems is still a major challenge. By using cutting edge technology, like induced pluripotent stem cells and CRISPR, we can recreate patient mutations in cells in the lab and turn them into beating heart cells. Specific techniques can be used to look at individual heart cells, as well as heart cells in a layer that beat together. The properties of the cells can be measured so that the effects of the mutations can be understood, and so that newer specific drugs can be tested to see if they are effective against different mutation types. The results from this research will help inform clinicians on how to better help patients with LQTS and potentially identify new, better treatments.

Effect of age-related spinal degeneration on older adult spinal cord injuries

Spinal cord injuries (SCIs) are becoming more prevalent in older adults, and the number of older adults is rapidly increasing. This is a challenge for healthcare professionals because the existing health issues and poor health of older adults may limit invasive surgical treatments. The most common form of SCI seen in older adults is caused by the neck extending beyond its typical range, damaging the spinal cord in a pattern that is different pattern than what is seen in younger adults. It is known that the risk of spinal cord injury and observed tissue damage is worsened by age-related degeneration in the spine; however, there is limited understanding of how these degenerative changes alter tissue damage caused by an SCI. The proposed study will consist of three objectives: (1) to measure the type and amount of degeneration typically found in older adults, (2) to simulate the spinal cord injury and use it to predict how tissue will be damaged (3) to predict how the tissue damage changes when the model includes spinal degeneration.

Developing the next generation of therapeutic regulatory T cells using CRISPR/Cas9

The immune system is critical for fighting infections but left unchecked, can attack healthy tissues resulting in autoimmunity or transplant rejection. Regulatory T cells (Tregs) are the immune cells responsible for controlling immune responses, so Treg transfusions are being investigated as treatments for these conditions. Unlike immunosuppressive drugs, Tregs are customisable and can have long-lasting effects.

Tailoring Tregs to treat specific diseases typically involves genetically modifying the cells. One approach involves incorporating synthetic proteins called chimeric antigen receptors (CARs) to help the Tregs migrate to where they are required in the body and specifically suppress harmful targets. I will build on this approach and explore the potential of using novel precise gene editing techniques (CRISPR) to maximise the survival and function of CAR Tregs following infusion.

This work will inform ongoing clinical studies that are investigating CAR Treg therapy in kidney transplantation, as well as future studies with other diseases. Fine-tuning personalised Treg therapy is key for its wide-scale implementation and potential to transform the life quality of autoimmune disease patients and transplant recipients.

Neural and muscular mechanisms of age- and sex-related impairments in muscle function and fatigue in old and very old adults

Even in the absence of disease, ageing leads to impairments in muscle function, limiting the abilities of many older adults to perform daily activities, such as walking. These functional declines are due to ageing-related impairments in the brain, spinal cord, and muscles. However, these declines in function are poorly understood in adults over 80 years of age, which is especially true for older females, as these groups are typically omitted from human physiology research.

To improve our understanding of ageing-related changes in muscle function, we will evaluate brain, spinal cord, and muscle function during force or power production and compare differences among young (18-30 years), old (60-69 years), and very old (over 80 years) females and males. The inclusion of very old adults is critical, as these individuals are most susceptible to impairments in muscle function. Furthermore, we are focusing our efforts on the thigh muscles, as they are vital for daily activities and mobility, and are greatly impacted by advancing age. This project will provide foundational knowledge to guide the development of interventions, such as age- and sex-specific exercise prescriptions, to restore muscle function and quality of life for older adults.

Role of SASH1 in generation of hematopoietic stem cells

For many patients with a serious blood disorder or malignancy the primary treatment option is a stem cell transplant (SCT), which involves destroying the unhealthy blood cells of the patient and replacing them with healthy donor stem cells. Unfortunately, a large number of patients are unable to find a suitable donor, and die as a result. Thus, there is an urgent need to identify new sources of healthy blood stem cells for these patients.
One promising solution is to harvest other types of cells from the patient and reprogram them to become blood stem cells, which can then be reintroduced later. Key to the success of this approach is placing the cells in an environment which mimics how the first blood cells are generated during embryonic development (called endothelial to hematopoietic transition [EHT]). To date little research has focused on the external cues needed for EHT, and this presents a bottleneck to producing stem cells for SCT. Therefore, our project will use models of EHT to identify external drivers of EHT, and the mechanisms by which they program cells to transition into blood cells. The knowledge from this project will help to create protocols to reproducibly reprogram patient-derived cells into blood cells for SCT.