LMP student seminars: 18 March
Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.
Anyone is welcome. No need to register.
Location: Medical Sciences Building, rooms 4171 or 4279, see below.
As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.
Group 2: Cancer, Development and Aging
Location: MSB 4171
Kangni Zou
- Title: Investigating YAP/TAZ-mediated regulation of the spindle assembly checkpoint
- Supervisor: Dr. Kelsie Thu & Dr. Andras Kapus
Group 1: Brain and Neuroscience
Location: MSB 4279
Medha Krishnan
- Title: Building on a 1st generation CRISPR-Cas9 all-in-one recombinant AAV-delivered PrP-lowering gene therapy for prion diseases
- Supervisor: Dr. Gerold Schmitt-Ulms
Abstracts
Kangni Zou: Investigating YAP/TAZ-mediated regulation of the spindle assembly checkpoint
The spindle assembly checkpoint (SAC) is a critical cell cycle checkpoint that maintains genomic integrity during cell division. The SAC delays separation of sister chromatids until all chromosomes are properly attached to the mitotic spindle and thus safeguards against deleterious or tumour-promoting chromosome missegregation. YAP and TAZ are related transcriptional co-factors that regulate organ growth and tissue regeneration and are known to be aberrantly activated in cancer. Recent studies suggest that YAP/TAZ may regulate the SAC, but their precise role and the mechanism of this regulation remain unclear. Our lab has shown that TAZ binds to BUB3 and RAE1, homologous proteins involved in SAC activation, through a C-terminal region (327-341) which we have termed the M-motif. Thus, we hypothesize that YAP/TAZ regulate the SAC through a transcription-independent mechanism via competing for SAC proteins through direct interactions. To assess the overall effect of YAP/TAZ on SAC activation, we depleted or overexpressed YAP/TAZ in cells and quantified mitosis duration using live-cell microscopy. TAZ knockdown in cancer cells with high YAP/TAZ induced a 14% increase while combined YAP/TAZ knockdown induced a 35% increase in mitosis duration. Under conditions of SAC activation, overexpression of either the M-motif or transcriptionally inactive TAZ in cancer cells with low YAP/TAZ decreased mitosis duration by 20%. We observed a similar 20% reduction in mitosis duration in non-malignant cells (HEK293T) transiently overexpressing the TAZ M-motif. These results suggest that TAZ may inhibit SAC activity at least partially in a transcription-independent manner. Future work will investigate the effect of YAP/TAZ on recruitment of SAC proteins to unattached kinetochores during checkpoint activation. We expect to uncover a novel mechanism by which YAP/TAZ regulate SAC signaling which would clarify their role in cell cycle regulation and maintaining genomic integrity.
Medha Krishnan: Building on a 1st generation CRISPR-Cas9 all-in-one recombinant AAV-delivered PrP-lowering gene therapy for prion diseases
Prion diseases are fatal neurodegenerative disorders affecting several mammalian species. First identified in sheep as scrapie, later in cows as bovine spongiform encephalopathy (BSE), and in humans as Creutzfeldt-Jakob disease (CJD), these diseases are driven by infectious prions (PrPSc). PrPSc acts as a template, inducing the normal cellular prion protein (PrPC) to undergo a conformational change to PrPSc. Any therapy that profoundly reduces the brain levels of PrPC should be effective for the treatment of prion diseases. This project builds on the specific hypothesis that recent advances in the virus-mediated delivery of gene therapies can be harnessed for this objective. The aims of the study are to establish a 2nd generation all-in-one recombinant adeno-associated virus (rAAV) vector whose payload can potently induce a functional knockout of the prion gene. This project builds on a 1st generation implementation of an all-in-one rAAV vector that codes for a prion gene-specific guide RNA and a high-fidelity SlugCas9 endonuclease which was used to generate proof-of-concept data. To improve efficacy benchmarks of this original vector, the ongoing research program is pursuing three avenues of optimization: 1) The payload composition is being revised and optimized. 2) An unbiased comparative screen of gRNA is being conducted. 3) The method of assembly and purification of AAVs is being refined. First generation proof-of-concept prion gene editing experiments have been successfully conducted. This first iteration of the therapeutic vector had limited PrP lowering efficacy. Second generation payloads have been assembled that drive the expression of SauCas9 from one of several promoters with potent expression in neurons. To further increase potency, regulatory elements of the payload have been replaced. An array of payloads has been assembled to identify the most potent gRNAs. The key components of an rAAV-delivered gene therapy that generates a functional knockout of the prion gene are in place. Improvements to the first-generation payload are currently being assessed. Future work will need to establish the relative efficacy and safety of this approach for the treatment of prion diseases.
Contact
No need to register.
Contact lmp.grad@utoronto.ca with any questions.