BIG 2022

EPIGENOME EDITING TECHNOLOGIES FOR FUNCTIONAL GENOMICS AND CELL PROGRAMMING

The advent of genome engineering technologies, including the RNA-guided CRISPR/Cas9 system, has enabled the precise targeting of genomic locations with molecular machinery. While most widely used for editing DNA sequences, we believe these technologies can have even greater and broader impact by programming other functions at specific genomic locations. For example, we have adapted and applied these tools to robustly and precisely manipulate gene expression, program the epigenome, annotate the function of the non-coding genome, and control cell fate decisions. Specifically, we have engineered CRISPR/Cas9-based tools to regulate the expression of endogenous genes and applied these tools to control diverse genes relevant to disease, development, and differentiation. Genome-wide analysis of the DNA-binding, gene regulation, and chromatin remodeling by these targeted epigenome modifiers has demonstrated their exceptional specificity. We have applied these technologies to control the decisions of stem cells to become specific cell fates and reprogram cell types into other lineages for drug screening, disease modeling, and in vivo tissue regeneration. Genome-wide screens of epigenetic modulation of target gene expression have enabled the discovery of novel distal regulators of target gene expression and modulators of cell fate commitment. We have used in vivo epigenome editing to alter expression of genes associated with complex disease phenotypes. We also developed new transgenic mice carrying inducible dCas9-based transcriptional activators and repressors to enable study of function and regulation of genes and non-coding regulatory elements in diverse tissues and cell types. Collectively, these studies demonstrate the potential of modern genome engineering technologies to capitalize on the products of the Genomic Revolution and transform medicine, science, and biotechnology.

LIPID NANOPARTICLES AND THE PFIZER/BIONTECH COVID-19 VACCINE

Gene therapies employing genetic drugs such as small interfering RNA (siRNA) for gene silencing and mRNA for gene expression have the potential to cure most diseases. However, sophisticated delivery systems are required to enable clinical use of nucleic acid polymers as they are readily broken down in biological fluids, do not accumulate at sites of disease and cannot penetrate target cells even if they arrive at target tissues. Lipid nanoparticle (LNP) technology is increasingly enabling the clinical potential of genetic drugs by packaging the nucleic acid polymer in well-defined nanoparticles that protect the nucleic acid payload in vivo and facilitate intracellular delivery following uptake into target cells by endocytosis. This approach has received clinical validation with the approval of Onpattro by the FDA in 2018. Onpattro consists of an LNP containing siRNA to silence transthyretin in hepatocytes, thereby arresting and reversing the disease transthyretin induced amyloidosis (hATTR), a disease that was previously untreatable and was fatal within five years of diagnosis. In this talk I will describe the design features that were followed to develop Onpattro and how related technology is being employed to enable mRNA-based drugs. A notable example is the development of the Pfizer/BioNTech mRNA COVID-19 vaccine, which is playing a leading role in the global response to Covid-19.

CONCURRENT SESSIONS II

Lunch & Learn

Getting Gene Therapies to the Clinic: The CLIC Experience

Genetically engineered immune effector cell therapies such as chimeric antigen receptor (CAR)-T cells have led to deep and durable remissions for patients with B cell malignancies and myeloma that were refractory to conventional therapies. This has led to FDA approvals for CAR-T cell therapies; however, ~50% are refractory to CAR-T cells or relapse within a 1 year. Therefore, novel therapies and approaches are needed to advance the field. Canadian researchers, however, have little in terms of infrastructure to support translation of novel cell therapies to the clinic. Recognizing this need, the Canadian Led Immunotherapies in Cancer (CLIC) network of investigators have established clinical cell therapy infrastructure to support early phase clinical trial development. I will discuss the background, current projects, and future directions of this program.

Using Oncolytic Viruses to Potentiate CAR T Therapy

We have used oncolytic viruses (OVs) in combination with chimeric antigen receptor (CAR)-modified T cells with the goal of improving therapy in the immune suppressive solid tumour setting. We initially tested whether the inflammatory nature of OVs and their ability to remodel the tumor microenvironment may help to recruit and potentiate the functionality of CAR T cells. Contrary to our hypothesis, VSVmIFNβ infection was associated with attrition of murine EGFRvIII CAR T cells in a B16EGFRvIII model, despite inducing a robust proinflammatory shift in the chemokine profile. Mechanistically, type I interferon (IFN) expressed following infection promoted apoptosis, activation, and inhibitory receptor expression, and interferon-insensitive CAR T cells enable combinatorial therapy with VSVmIFNβ. Our study uncovers an unexpected mechanism of therapeutic interference, and prompts further investigation into the interaction between CAR T cells and oncolytic viruses to optimize combination therapy.

Pioneer the New Frontier in Spatial Biology

Discover what's new in spatial biology. Get the latest updates in NanoString’s spatial technologies and take innovation to a new level. Explore the powerful GeoMx Digital Spatial Profiler (DSP) the brand new CosMx Spatial Molecular Imager (SMI). GeoMx DSP ignited the spatial biology revolution and is the leading spatial profiling technology offering highly multiplexed, highly sensitive measurement of protein and RNA. CosMx SMI takes spatial biology one level deeper and enables you to profile RNA and protein at the single-cell and subcellular level. What novel discoveries can you learn from spatial biology.

HD Video Recorder of the Cell

The dynamic behaviors of cells during development, tumorigenesis, and other disorders remain largely unclear. Our lab is developing "DNA event recording" systems by which high-resolution information of cells is progressively stored in cell-embedded "DNA tapes." Using high-throughput single-cell sequencing, such a system enables access to molecular and cellular history information of cells at the time of observation and provides a way of observing the dynamics of complex biological systems in high resolution. We envision mapping the whole-body cell lineage and differentiation trajectories of mouse development and have been actively progressing towards this goal. I will share our grant vision and recent progresses in developing new genome editing tools and high-performance computing technologies.

Defining Developmental Pathways using Single Cell Analysis

Single cell analysis of transcriptomes has revolutionized the way we ask developmental questions. It has allowed us to examine cell diversity in small embryonic structures with a resolution not previously possible. In the laboratory, we are using single cell RNA-seq to examine cell specification in the mouse embryonic liver and heart valves. Our data has defined the types of cells present at different stages of embryogenesis and allowed us to build trajectories describing how gene expression changes as cells differentiate. In addition, we are using this data to predict cell-cell interactions in the developing tissues.

Fanning the flames in cancer cells under oxidative stress: identification and targeting of adaptive response mechanisms

Metastasizing cancer cells must overcome anoikis prior to colonization in distant organs. To pinpoint critical regulators of anoikis and metastasis, we performed an integrated analysis of the global proteome and acute translatome in transformed and non-transformed cells cultured under 3D conditions. We found that distinct oncoproteins such as mutant KRas, ETV6-NTRK3, and EWS-FLI1 each upregulate IL-1 receptor accessory protein (IL1RAP) to suppress anoikis. The chimeric transcription factor EWS-FLI1, the predominant oncogenic driver in Ewing sarcoma (EwS), a highly metastatic childhood sarcoma, directly promotes IL1RAP transcription via potent enhancer activation. IL1RAP inactivation impedes redox homeostasis, and triggers anoikis and ferroptosis in EwS cells. Mechanistically, IL1RAP binds the cell surface system Xc- transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and glutathione antioxidant pools. Moreover, under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Thus, IL1RAP maintains cyst(e)ine and glutathione pools which are vital for redox homeostasis and ferroptosis resistance. Notably, IL1RAP or CTH genetic inactivation each dramatically impedes metastatic dissemination of EwS cells in vivo. Finally, we found that IL1RAP is minimally expressed in normal tissues, and we have developed immunotherapeutic strategies to target IL1RAP, including highly specific chimeric antigen receptor (CAR) T cells and antibody-drug conjugates (ADCs). Therefore, we define IL1RAP as a new cell-surface target in EwS that is exploitable for immunotherapy.

Keeping Pace with a Pandemic: An Integrated Technology Stack to Discover and Develop Antibody Treatments