The ABO blood groups – comprising the A, B, AB and O blood types – are vitally important in blood transfusion and organ transplantation. The four types are differentiated by the presence or absence of two sugar antigens on the surface of red blood cells: a terminal alpha-1,3-linked N-acetylgalactosamine (A-antigen) or an alpha-1,3-linked galactose (B-antigen), both of which are absent in the O-blood type. As all individuals have antibodies to the antigen(s) they lack, transfusion with an incorrect blood type results in destruction of the incompatible blood cells, which can result in death. The enzyme EABase is capable of releasing both the A and B trisaccharides from the surface of red blood cells, giving it the potential to be used to convert blood cell types by the addition or removal of their antigens. Fathima Shaikh’s studies seek to determine the mechanisms underlying EABase activity, and identify the residues that are created as a result. Knowledge of these enzyme properties is crucial for the next stage of the project: engineering EABase into a glycosynthase, which is a mutant form of the enzyme that can synthesize (form) antigens, rather than removing them. She will conduct further work to optimize the efficiency of this glycosynthase, as well as increasing its synthetic utility by broadening its ability to transfer different sugars. If Shaikh’s experiments are successful, this process would allow for the conversion between blood groups. These enzymes could be of great benefit to human health, helping to overcome shortages in donated blood, and helping in the modification of related antigens on other cell types.
Research Pillar: Biomedical
Microfluidic technologies for high-throughput selection and production of monoclonal antibodies from single cells
Antibodies are proteins produced by the immune system. They work by selectively and tightly attaching themselves to infectious bacteria, viruses, and other pathogens, neutralizing their disease-causing abilities. The natural role of antibodies in clearing infections has prompted the pharmaceutical industry to invest billions of dollars in attempts to produce new antibody therapies to treat rheumatoid arthritis, cancer, cardiovascular disease, HIV/AIDS, and other diseases. As a result, antibodies have become the most rapidly growing class of therapeutic drugs over the last decade. One successful example of an antibody-based therapy is the drug Herceptin, which treats a highly-aggressive form of breast cancer. In order to produce vast quantities of antibodies required for research and therapeutic use, antibody-producing cells are currently fused to immortal cancer cells to allow them to be grown in laboratory culture. Successfully creating antibody-producing fused cells can involve hundreds to thousands of attempts, requiring many years and millions of dollars in research money. MSFHR previously funded Anupam Singhal as he used micron-sized fluid-handling devices and nanotechnology to allow the study of stem cells at the single cell level. He is now using these approaches to rapidly and inexpensively produce antibodies. He is working on a novel technology that is sensitive enough to detect antibodies produced by single cells, and determine which ones are producing the optimal antibody. Then, the genes responsible for antibody production in these cells can be isolated, cloned and inserted into cell lines for production. As a demonstration of this technology, human antibodies against the influenza virus will be developed. This technology could have far-reaching impacts on the rapid and inexpensive development of breakthrough therapeutic drugs and diagnostic agents.
Effects of matrix metalloproteinase processing of chemokines on inflammation
Inflammation is the response of the body to infection or injury, triggered by the release of proteins that initiate inflammation. Amongst these proteins are members of the chemokine family. Chemokines act as biological beacons, guiding the migration of white blood cells (WBC) from blood vessels to the affected tissue. The structure of chemokines is important to their function. One end of the chemokine binds to tissue and vessels to create a path for white blood cells to follow; the other end interacts with a receptor on the surface of the WBC, prompting the release of further inflammatory mediators that spread the inflammatory response. Recruited and activated WBC attempt to isolate and destroy infectious agents and to prevent further damage. WBC – and in particular cells called macrophages – then help resolve the inflammatory response. In chronic inflammation, continual recruitment and activation of macrophages results in excessive production of reactive molecules that cause host tissue damage, as observed in diseases such as rheumatoid arthritis, multiple sclerosis and chronic obstructive pulmonary disease. The apparent deregulation of macrophage movement may be a result of a change in the structure and function of chemokines. Matrix metalloproteinases (MMPs) are a family of enzymes that modulate chemokine activity by cutting off a portion of either end of the chemokine, which keeps it from functioning properly. Amanda Starr is looking at the functional effects of MMP cleavage on chemokines that recruit and activate macrophages, determining whether cut chemokines are more able to attract and activate macrophage-like cells. She is also developing a technique for detection and quantification of both full-length and cut forms of chemokines from tissue samples. Ultimately, this knowledge could lead to more appropriate and directed therapeutics for the treatment of chronic inflammatory diseases.
Aging-induced changes in microdomains of vascular smooth muscle lead to alterations in vasomotor function
Cardiovascular diseases are the primary cause of death and disability in Canada, accounting for one-third of all deaths in 2002. Age significantly increases the risk of developing cardiovascular diseases. As our elderly population increases, it is very important to improve our understanding of how and why cardiovascular diseases occur with age. Most cardiovascular diseases are caused by problems with the way our blood vessels work, especially changes in the muscle layer (made of cells called smooth muscle) that surrounds the blood vessels. The calcium level in smooth muscle is critical for proper cell function, allowing the muscles to contract and generate force. Within the cell, the way that internal compartments called organelles are arranged in relation to each other allows high concentrations of calcium to be localized to small pockets of the cell called microdomains. Microdomains are important for smooth muscle cells to function properly, because certain adjacent proteins are only activated by high concentrations of calcium. Disintegration of microdomains leads to the development of various health conditions. Harley Syyong was previously funded by MSFHR for his Master’s research training. He hypothesizes that smooth muscle cells undergo major changes in organelle arrangement as we age – making it difficult or impossible for cells to form calcium microdomains. Furthermore, proteins that generate microdomains may also be affected, including changes in where they are located or how much of the protein is made. Syyong is monitoring different blood vessels across time, identifying changes to organelles and proteins and determining how they affect blood vessel function. He hopes a better understanding of these changes could lead to ways to decrease the effects of aging on blood vessels, alleviating cardiovascular disease in the growing older population.
Novel algorithms for in vitro gene synthesis and gene optimization with applications to therapeutics and health research
In the development of a vaccine against four strains of the human papilloma virus (HPV), of particular note were studies involving innate immune response when genes of the virus were introduced into host cells. It was observed that increased levels of antibodies were produced by inoculation that used synthetic versions of two HPV genes. The application of gene vaccines, not only for cancer immunization, but also to aid the treatment of infectious diseases, is an ongoing and very active area of study. Developing an efficient and reliable method to produce synthetic DNA is a necessary tool for these studies to succeed. Due to the inherent difficulty of creating long strands of DNA, current technologies for gene synthesis use computational methods for design of shorter DNA fragments called oligos (oligonucleotides), which can be reliably synthesized and assembled. However, ensuring a set of oligos will self-assemble correctly into a longer DNA strand is difficult and complex, and previous software programs have failed to solve this issue. Chris Thachuk is a computer scientist who develops synthetic gene design algorithms. He and his colleagues are developing algorithms to successfully assemble long strands of DNA from oligos. These new algorithms outperform the current state-of-the-art and their effectiveness has been demonstrated through computational experiments on a large set of genes. Three average size genes have been produced with the aid of these algorithms. He now intends to build upon this success by extending the algorithms to produce reliable designs for synthesizing long genes and for synthesizing multiple genes in one step. By providing researchers with more advanced algorithms and accurate modeling software for gene synthesis, Thachuk hopes to contribute to new insights for treatment, detection and/or prevention of diseases.
Identification of genetic signatures predictive of progression risks in oral pre-malignant lesions
Squamous cell carcinoma (SCC), a cancer of the epithelium, is the most common human cancer throughout the body. When SCC occurs in the oral cavity, the five-year survival rate is less than 50 per cent. Before SCC develops, pre-malignant changes often become visible to patients or clinicians. While this offers the opportunity for early intervention to prevent the progression to cancer, only 15-20 per cent of oral pre-malignant lesions (OPLs) will progress to invasive carcinomas. Currently, it is not possible to determine which lesions will develop into cancer. It may be possible to predict cancer risk by studying the molecular features of OPLs. The progression to cancer is caused by genetic alterations to cells; some changes are the “driver” genetic alterations that lead to cancer, while others are random genetic changes. Determining the specific genetic events that are associated with progression to cancer would help identify those at greatest risk for cancer. Ivy Tsui is studying the initiating genetic events at the pre-malignant stage to identify genetic markers that can predict whether an OPL will become malignant. To do this, she is studying the molecular mechanisms of oral cancer progression. Using DNA from banked samples, she is assessing DNA alterations across the whole genome of late stage OPLs and tumours. Once she has identified recurring alterations, she will compare them with the genomes of early stage OPLs – both those that are known to have progressed to cancer, and those that did not. Once validated, these genetic markers will used to develop a clinical diagnostic tool. If DNA from a patient’s OPL can be assessed, patients at risk could be treated early and immediately to prevent progression to cancer. The identified genes critical for oral cancer development could also be used to develop therapeutic targets to treat oral cancer patients.
Investigation into the factors mediating embryonic muscle migration in Caenorhaditis elegans
During embryonic development, precursor muscle cells (myoblasts) are generated in one region of the embryo and then dispersed throughout the body. These migrations are controlled by external signals that guide the migrating cells. Previous muscle research has identified genes for muscle differentiation and development, but the important genes regulating muscle cell migrations have are unknown. Muscle cell migration is of particular importance for a new treatment called myoblast transfer therapy, which is being developed to treat muscular dystrophy and hearts damaged by cardiac arrest. The treatment injects healthy myoblasts into the damaged area in order to repair the affected tissue. A key problem in this treatment is that, for both cardiac and muscle tissue, the myoblasts fail to migrate properly and effectively colonize the damaged area. Ryan Viveiros is studying the embryonic development of a small nematode worm called Caenorhabditis elegans. While substantially simpler than humans, these worms also have muscle and share a number of the same genes required for muscle development as mammals. Because the worm embryos develop inside clear eggs, Viveiros can record the developing embryo and watch the cell migrations in real time. Computer software then allows him to follow the cell migrations and determine which cell types are defective. Viveiros will search for the genes that cause improper muscle migration and determine where these genes are turned on in the embryo. In addition to determining the basics of how muscle migration occurs, Viveiros hopes his findings could lead to new insights for improving myoblast transfer therapy. In addition, because this research is also relevant to cell migration in general, his findings may also inform our understanding of how cancer cells migrate (metastasize).
Epigenetically silenced tumour suppressor genes in lung cancer
Lung cancer continues to kill more British Columbians each year than any other cancer. Diagnosis typically occurs late, leaving only toxic chemotherapy and radiotherapy as the treatment options. Medicine needs better ways to screen for lung cancer in high risk individuals, and better ways to treat them if lung cancer is found. Cancer cells have their cellular “brakes” cut, bypassing the checkpoints in normal cells that stop them from dividing too fast. Human DNA has two copies of each of these checkpoint genes, to ensure a backup in case one copy is damaged. In cancer cells, however, both copies are often damaged by two different mechanisms. One copy may be totally removed, and the other may be silenced by a mechanism called DNA methylation. Because cancer cells go to such great lengths to disrupt both copies of the gene, these genes are likely very important to the continued survival of the cancer. Identifying these genes in early cancers could lead to new screening and therapeutic targets. Ian Wilson is jointly funded by MSFHR and the BC Cancer Foundation. He is working to identify these gene targets, using approaches that enable the entire genome of the cancer cell, as well as every gene product of the cancer cell, to be analyzed simultaneously. He is searching for checkpoint genes in the lung cancer genome that are aggressively shut down in the cancer cell, determining the role of these genes in transforming normal cells into malignant ones. The identification of key checkpoint genes will be very useful as screening markers. This could lead to earlier diagnosis of lung cancer and new targets for therapeutic intervention.
Bioinformatic approaches for the interpretation of cancer genomes
The rapidly developing field of genomics is providing increasingly powerful tools to investigate our genetic make-up and provide a fundamental understanding into how cells and organisms function. Previously funded by an MSFHR Scholar award, Dr. Steven Jones’ ongoing research focus is to apply genomic and bioinformatic technologies to cancer research. Next-generation DNA sequencing machines at Canada’s Michael Smith Genome Sciences Centre provide the underlying technology platform for Jones to conduct a number of studies that will expand our knowledge about the fundamental mechanisms underlying health and disease. Jones will develop a number of studies around three key themes: • Understanding the genetic changes present in human cancer cells, as compared to the normal human genome, to improve drug screening and testing methods. • Investigating the changes that occur in cells in response to drug treatments to identify ways to improve the efficacy of these drugs. • Using the mouse liver as a model, identifying active regions of the genome in order to further understand the functional elements within our genetic material and how, in concert, they are able to coordinate and maintain the activity of a tissue or organ.
The role of the CD34 family of Sialomucins in Development and Disease
Dr. Kelly McNagny studies the CD34 family of molecules: CD34, Podocalyxin, and Endoglycan. First identified solely as markers of blood stem cells and blood vessels, McNagny’s research has shown that they are also present on a variety of other cell types in the body. In particular, they are found on cells that play an important role in inflammatory diseases like asthma, allergies, arthritis, multiple sclerosis, intestinal infections and cancer.
Previously supported by MSFHR as a Scholar, McNagny’s current focus is to determine whether these molecules are important in the development or progression of inflammatory disease. Developing mice that lack each of these molecules, then testing their susceptibility to disease, has shown that mice that that lack CD34 are strikingly resistant to asthma, allergies and other lung inflammatory diseases. McNagny has also shown that these mice are more resistant to colon cancer and to bacterial infections.
Inhibiting CD34 expression may be beneficial in preventing or treating these diseases. In studies of Podocalyxin, the second member of this family, it appears that this molecule is essential for normal kidney development and for regulating normal blood pressure. McNagny has also found that this protein is ‘turned on’ in a number of high-risk cancers (those with very poor outcomes). This molecule may be a particularly good diagnostic tool for identifying those high-risk cancers. He will further clarify how these molecules work under normal and disease conditions. The research could lead to new treatments for a variety of conditions and diseases.