Orthogonal multicolour high-affinity tags for RNA imaging and manipulation

RNA plays a very important role in the regulation of gene expression. Yet, the spatial and temporal dynamics of RNA are still poorly understood, mainly due to the scarcity of effective and simple RNA imaging and purification techniques.

The development of technologies that simultaneously allow imaging, purification and manipulation of multiple RNAs in live cells promises to enable the study of RNA in development, metabolism and disease, which is essential for understanding the control of gene expression in diseases such as autism, cancers and type II diabetes.

Dr. Dolgosheina will develop a multicolour RNA-based imaging method that will allow researchers to simultaneously visualize two RNAs in living cells, while concurrently purifying and/or manipulating RNA interactions with other biomolecules. This new technology will build on, and dramatically increase the capabilities of the bright, high affinity RNA Mango system that she developed during her PhD.

The proposed project is working on an outstanding international problem, and since these tools are urgently needed, the research has attracted significant national and international attention.

This research project will 1) result in international level talks and publications, 2) bring together some of the best international researchers in RNA biophysics and 3) result in intellectual property development, industrial research and training and commercialization via a rapidly growing Canadian biotechnology company, Applied Biological Materials (Richmond, BC).

Novel 18F-fluorinated amino acids as oncological PET radiotracers

Positron emission tomography (PET) is a non-invasive imaging technique used to detect tumours and provide information about a patient’s response to treatment. PET generates a 3D image of the inside of a patient’s body and highlights the location of tumors through detection of a radiotracer administered before generating the image. One of the most common forms of radiotracers are small, drug-like molecules containing a radioisotope that bind to or accumulate in cancer cells, precisely locating tumours. 

While many radioisotopes can be used for PET imaging, [18F] is arguably the most desirable due to its high positron output, small atomic size, metabolic stability and worldwide network of production facilities. Despite these advantages, the synthesis of [18F] radiotracers presents many challenges that have limited the scope of radiotracers available for oncological PET imaging. Thus, the majority of oncological PET imaging relies on a single radiotracer: [18F]-FDG, a sugar analogue that preferentially accumulates in cells that have increased metabolism (i.e., cancer cells). 

Unfortunately, [18F]-FDG is not cancer-specific and also tends to bind to other tissues such as brain and bladder, and at sites of inflammation, limiting its utility for detecting tumors in those areas. In recent years there has been considerable interest in identifying complementary radiotracers to FDG, and much attention has focused on the synthesis of 18F-labelled amino acids, which also accumulate in rapidly dividing cancer cells. Dr. Britton’s lab has recently discovered a method for incorporating the [18F] radioisotope into complex drug precursors without the need for elaborate precursor synthesis. 

Dr. Britton aims to:

  • Rapidly expand the number of available amino acid radiotracers using new unique capabilities.
  • Evaluate promising lead radiotracers for oncological PET imaging.
  • Advance selected radiotracers into preclinical animal studies.

In addition to these research aims, Dr. Britton has filed a provisional patent application and will work with the SFU Innovation Office to identify an industrial partner for this new technology. These new amino acid radiotracers could have a profound impact on the early detection of cancer and positively impact the lives of many British Columbians.

Elucidating the effect of O-GlcNAc modification on protein stability

The glycosylation of proteins with O-GlcNAc is a ubiquitous post-translational modification found throughout the metazoans. Deregulation of O-GlcNAcylation is implicated in several human diseases including type II diabetes, Alzheimer’s disease, and cancer.

However, the basic biochemical roles of O-GlcNAcylation remain largely unanswered. Several recent studies have demonstrated a clear link between O-GlcNAc and cellular thermotolerance.

It is likely that a basic function of the O-GlcNAc modification prevents the unfolding or aggregation of target proteins. Dr. King will investigate its role in protein stability through series of biochemical and biophysical experiments to probe the effect of O-GlcNAc on protein unfolding, folding, and aggregation. The results of this research will provide important insights into the basic molecular mechanisms governing O-GlcNAc deregulation in human disease. 

Development of improved substrates for live cell imaging to aid in discovering new glucocerebrosidase therapeutic agents

Parkinson’s disease (PD) is a neurodegenerative disorder that affects millions of people worldwide, with no standard treatment currently available. Therefore, there is a major need for new therapeutic agents to treat or prevent the progression of PD. One promising solution involves targeting the protein glucocerebrosidase (GCase) encoded by the gene GBA1. Studies have shown small molecules that increase GCase activity could help prevent the progression of PD.

Dr. Ashmus will use a combination of organic chemistry, chemical biology, and cell biology to discover new therapeutic agents that increase GCase activity. Fluorescently-quenched substrates will be chemically synthesized and used in enzymatic assays to monitor GCase activity in vitro and in neuroblastoma cells. The assay will then be adapted and optimized for use in a high-throughput screen of compounds from the Canadian Glycomics Network and from a natural products collaborator, Roger Linington, at SFU.

The results of this research could produce new lead compounds that increase GCase activity. In addition, the compound screen could aid in identifying new therapeutic targets for PD, which would drive preclinical translation research in this area.


End of Award Update – March 2022

Most exciting outputs

An exciting and successful specific output as part of the project was that we were able to develop a newly designed probe that performs better than the original probe the Vocadlo Lab published and patented back in 2015. The new probe is also capable of being used in a high-throughput screening in live cells. Moreover, the new design led to the development of probes that could for the first-time target other disease-related enzymes of interest in live cells and led to a high-impact publication in Nature Chemical Biology.

Impacts so far

While the main purpose of the research project failed to discover any lead compounds that could be developed as a potential therapeutic agent for Gaucher/Parkinson’s disease, the steps (develop a better probe and optimize use for screening) required to reach the point of running the screen were successful. The data collected (unpublished) has helped secure funding for the Vocadlo Lab and led to collaborations with biotech companies interested in targeting the same enzyme.

Potential future influence

I think some of the work described briefly will start to gain more attention in the next few years. Over the past year or so, I have noticed an increased interest from research institutes and biotech companies in studying enzymes found within the lysosome. This is in part because more of these lysosomal enzymes are being linked to neurological diseases so having biochemical tools that can study them in live cells will be desired. I think some of the probes we have developed over the past couple of years will be of interest to a broader scientific community.

Next steps

The work searching for potential therapeutic agents for Gaucher/Parkinson’s disease is currently ongoing. The majority of my research efforts have shifted to developing and evaluating novel probes targeting other disease-related enzymes. One notable example is a new project collaborating with an expert clinician in Fabry’s Disease. Using one of our recently developed probes, we aim to advance current diagnostic methods and improve dosing and timing of current therapeutics for Fabry Disease patients. I am excited to see some of my work being used in a clinical setting and hope this can lead to something more fruitful in time. Dissemination of the work will be continued through publications, presentations at conferences and through social media platforms.

Useful links

In vivo multi-resolution functional optical imaging for investigation of age-related macular degeneration (AMD) process

Vision loss from age-related macular degeneration (AMD) and other retinal degeneration diseases is due to the loss of the light sensitive photoreceptor cells in the eye. This is often secondary to dysfunction of the retinal pigment epithelium (RPE). 

The photoreceptor and RPE cells are arranged in a characteristic mosaic. The mosaic is an accurate clue to how healthy these layers are. However, attempts to visualize these mosaics have been so far unsuccessful, despite technological advancements in conventional ophthalmic imaging. 

Dr. Ju will develop a novel clinical ophthalmic imaging system to visualize and quantify the changes of the structure and function of photoreceptor and RPE cells in humans. The results of this research will provide a clearer understanding of the pathological processes of AMD, which will help clinicians establish more reliable clinical treatment options.

 

Development of a flow cytometry assay for accurate and selective measurement of lysosomal GBA1 activity in PBMC

Recently, loss-of-function mutations of the GBA1 gene, which encodes glucocerebrosidase (GCase), have been characterized as a major genetic risk for Parkinson’s disease (PD). Patients carrying these mutations have a much higher incidence of PD, earlier onset, and more severe disease.

These data strongly suggest that GCase activity may be useful for early diagnosis as well as monitoring the progression of PD. Dr. Gros will build on her previous work describing a substrate that specifically measures GCase activity both in vitro and in neuronal cells in microscopy. This research will lead into a proof-of-concept clinical study, using a flow cytometry assay to establish correlations between the progression of PD, GBA1 mutant status and GCase activity.

The results of this study will lead to the development of a new assay for clinical studies that will benefit Parkinson’s patients and deepen our overall understanding of the disease.

 

Studying genetic mechanisms of treatment resistance in non-Hodgkin lymphomas

Dr. Morin's research program will develop and apply laboratory and computational genomic methodologies that use DNA sequencing and other sensitive platforms to study the drivers of tumour onset, progression and treatment resistance in solid cancers in order to understand the somatic drivers of non-Hodgkin lymphomas (NHLs). Using massively parallel (next-generation) DNA and RNA sequencing, Dr. Morin will be able to identify somatic alterations and gene expression signatures in tumour tissue and liquid biopsies (circulating tumour DNA). To properly study such large data sets, he will utilize cutting-edge bioinformatics techniques and develop novel analytical approaches and pipelines that will allow leverage of unique sample processing techniques and applications.

Moving forward, this research will investigate aggressive subtypes of NHL including patients who typically fail standard-of-care treatments. Dr. Morin will rely on features of this malignancy such as high somatic point mutation rate, a well established list of known lymphoma-related genes, and the presence of clonal immunoglobulin rearrangements to develop assays to study the genetics of specimens from NHL patients in various ways. These include deep sequencing using a novel molecular barcoding system and digital PCR-based methods. He will continue to push the limits of sequencing technology by applying deep sequencing and whole exome sequencing to circulating tumour DNA. Under this research program, he will also continue to use a variety of laboratory and computational approaches to understand the clonal structure of NHLs, especially in the context of serial samples collected over the course of disease progression and after treatment failure or relapse. 

Dr. Morin's lab, along with the BC Cancer Agency, plan to pursue options to commercialize these strategies so that a broader group of users can use these techniques for research and clinical applications. Some of the research under this program will involve evaluating the performance of novel ctDNA-based methods to study tumour genetics and evaluate treatment responsiveness. This will be conducted in the context of prospective and retrospective samples from multi-centre clinical trials in Canada. This engagement with clinicians and publications describing these trials will help accelerate the adoption of such emerging technologies to the clinic.

Innovative addiction research program: Addressing polysubstance use

British Columbia (BC) faces a mental health and addiction crisis with an estimated cost of $100 million annually. In April 2016, a public health emergency was declared due to an alarming increase in opioid-related overdose deaths in recent years.

People who use illicit drugs (PWUD) bear a great burden of preventable morbidity and mortality from drug overdoses as well as other comorbidities including mental disorders and infectious diseases. While opioid agonist therapies (OAT) have proven effective in reducing heroin use, concomitant use of opioids and stimulant drugs (e.g., heroin and cocaine) is common among PWUD. Furthermore, recent research has suggested that many PWUD also suffer from untreated chronic pain, which may be driving prescription opioid (PO) misuse among this population. However, little is known about patterns of concomitant use of illicit opioids, POs and stimulants, and how OAT and other health services may serve to mitigate potential harms associated with such polydrug use. Currently, no approved pharmacotherapies exist for stimulant use disorder, necessitating urgent research effort in this area.

Dr. Hayashi's research will inform policies, programs and clinical practice to reduce harms associated with polydrug use. The primary research objectives are:

  • To investigate and address the impact of PO misuse, untreated chronic pain and concomitant opioid and stimulant use on patterns of drug-related harm.
  • Evaluate "naturally occurring" interventions and policy changes (i.e., new opioid addiction-related services and Vancouver Coastal Health's Downtown Eastside Second Generation Strategy) that are relevant to polydrug users.
  • Evaluate the efficacy of a novel pharmacotherapy to treat polydrug users. 

The research will employ vast longitudinal behavioural and biological data collected since 1996 via three ongoing prospective cohort studies of over 3000 PWUD in Vancouver. The findings are expected to inform care development and overdose prevention efforts for a high needs population in BC. One objective will involve implementing a clinical trial to evaluate whether an amphetamine-based medication reduces powder/crack cocaine use among 130 patients on OAT, who have both opioid and cocaine use disorders. If the study medication proves effective, Dr. Hayashi's research will potentially contribute to the identification of the first proven medication for cocaine addiction.

Transforming Urban Form for Mobility: Interventions to improve population health

Governments are making major investments in transit, cycling, and walking infrastructure to alleviate the pressures of traffic congestion and emissions. These changes may have lasting impacts on population health.

The aim of this five-year program is to generate new evidence on the impact of population health interventions on health and health equity along two lines: 
 

  1. "Population Health Intervention Research" will generate new knowledge on the impact of population-level interventions on mobility.
  2. "Methods and Tools for Intersectoral Action" will develop and apply novel methods and tools to study urban form, and to facilitate uptake by intersectoral stakeholders.

This work aims to generate new, locally-relevant evidence in order to understand how to enhance health and mobility in mid-size cities and suburbs. While these settings are very common in Canada, they are surprisingly absent from the literature.

The program will assess how changes to urban form, such as new cycling networks or education programs, influence how people choose to travel, and how safety-conscious and active they are. This will be studied in the population overall, and also specifically with groups facing greater mobility challenges (e.g. women, new immigrants, older adults). The work will focus on how an investment in a city-wide cycling network for people of all ages and abilities impacts uptake, safety and equity.

A telehealth intervention to promote healthy lifestyles after stroke: The Stroke COACH

Stroke is often associated with low levels of physical activity and poor nutrition habits and with related conditions such as obesity, hypertension and diabetes. Within five years of the initial stroke, 30 percent of stroke survivors will suffer a recurrent stroke.

We developed the telehealth Stroke COACH programme, a lifestyle modification intervention comprised of a self-management manual for stroke survivors, a self-monitoring kit (including a blood pressure monitor, pedometer, and health report card), and telephone-coaching sessions by trained ‘lifestyle coaches’. In this six month program, seven sessions of 30-60 minutes are delivered by the coaches who use motivational interviewing techniques to facilitate active patient engagement and enhance chronic disease self-management skills of problem solving, decision making, action planning, and resource utilization.

One hundred twenty-five community-dwelling individuals who have had a stroke of mild to moderate severity within the last twelve months will be enrolled in this single-blind randomized controlled trial. They will be randomly assigned to either: 1) Stroke COACH with a lifestyle coach, or 2) control group (memory training program with a memory coach).

We predict that the Stroke COACH will improve a global measure of lifestyle behaviour in community-dwelling stroke survivors compared to the control group. This will be measured at zero, six, and 12 months using the Lifestyle Profile II, a global lifestyle behaviour measure that considers physical activity, stress management, nutrition, health advocacy, interpersonal support, and spirituality.

We also predict improvement in physical activity and cardiovascular health outcomes, which we will measure using the StepWatch Activity Monitor and bloodwork results.

If testing is successful, the low-cost and remote delivery of the Stroke COACH would enable a large number of Canadians in both urban and rural regions to improve health behaviours of people living with stroke, potentially reducing the risk of subsequent stroke.