Receptors that respond to hormones, neurotransmitters, chemokines and growth factors traffic to the cell surface where they are activated by ligands and signal via coupling to molecules such as G proteins or Grb2. For most G protein-coupled receptors (GPCRs), arrestins then bind and inhibit plasma membrane G protein coupling, facilitate internalization via clathrin-mediated endocystosis, and potentially generate a secondary signalling complex. Signalling potentially continues in endosomes, and receptors are then either recycled to the cell surface or are targeted for degradation. Furthermore, heteromerization can lead to transactivation, transinhibition, or modulation of receptor signalling pathways. Because of the importance of kinetics, being able to measure receptor function in live cells and in real time is highly advantageous, and this can be achieved through the use of technologies such as bioluminescence resonance energy transfer (BRET). BRET uses a donor luciferase enzyme fused to a protein of interest that transfers resonance energy to an acceptor, such as a small fluorescent moiety like BODIPY, or a derivative of green fluorescent protein like Venus. This only occurs if donor and acceptor are within 10 nm of each other. We have utilised BRET to monitor ligand binding, G protein and Grb2 coupling, arrestin recruitment and receptor trafficking. We have done this both with transient transfection and through tagging endogenous receptors with luciferase using CRISPR/Cas9 gene editing. We have also assessed numerous heteromer complexes, and our molecular pharmacology investigations have provided the rationale for both pre-clinical and clinical studies.

Learning Objectives:

1. Explain how the bioluminescence resonance energy transfer (BRET) technology can be used to monitor receptor complexes

2. Explain the implications of receptor heteromerization and how it can be investigated.

3. Explain the benefits of utilizing CRISPR/Cas9 to enable assessment of proteins under endogenous promotion.

Flow cytometry is an essential tool for in-process control and release testing of cell therapy products. Standardization of these analytical assays is critical for the success of the manufacturing process and final product quality. However, achieving standardization in flow cytometry can be full of hurdles. In this talk, we will review the common challenges in developing a cell therapy from pre-clinical research to the clinic. Highlights will include details on flow cytometry instrumentation, software reporting and analysis, and top quality recombinant antibody technology - all integrated into seamless, automatic workflows.

Learning Objectives:

1. Explain the challenges of flow analytics in pre-clinical and clinical research.

2. Discuss quality control methods to combat sources of variation during cell analyses.

3. Explain how mitigation strategies improved data quality and time savings.

Integrating external and internal ‘omics data is a key element of hypothesis generation, target identification, and biomarker prioritization when it comes to drug discovery. It is never as straightforward as it seems and usually costs more and takes more time than anticipated, often with poor results. In this talk, Joseph Pearson, PhD, from QIAGEN Digital Insights will discuss approaches and resources to overcome these obstacles and improve outcomes. 

Learning Objectives: 

1. Identify where automated curation approaches can leave you short.

2. Discuss why data scientists spend most of their time not doing data science.

3. Identify where bioinformaticians and data scientist spend most of their time.

4. Explain what is required of high-quality ‘omics data.

5. Analyze the example of high-quality TCGA data.

6. Explain how to extend these practices to internal data structures.

Genetically engineered cellular models are an integral part of the drug discovery process from early discovery through clinical biomanufacturing. The various applications of these cellular models are seemingly endless, including evaluating efficacy of CAR-T cells, screening drug candidates, and generating potency assays for therapeutic testing. Often research organizations must overcome resource and technical hurdles to generate complex cellular models quickly. Gene editing experts within the Cell Design Studio® team provide premier custom cell line engineering services to create and deliver unique cell-based assays tailored for drug discovery and preclinical manufacturing. We utilize state of the art editing technologies including CRISPR, zinc finger nucleases, lentiviruses, and custom plasmids to generate cell lines suitable for a range of applications. We partner closely with our customers at all stages of the process with regular updates and communication. In this presentation, we will discuss the technology used to generate custom cell lines, for both research use only and pre-clinical workflows, and the advantages to working with the Cell Design Studio® team.

Learning Objectives:

1. Discuss current gene editing technologies and how to apply them.

2. Identify the advantages to working with a research partner for model generation.

3. Discuss applications of cellular models for drug discovery research.

Current drug discovery workflows are inefficient. 90% of therapeutic agents reaching clinical trials fail, predominantly because traditional laboratory experiments fail to adequately predict a drug’s safety or efficacy in human. 

Building more predictive, human-relevant models is key to reducing drug attrition rates and the financial burden to pharmaceutical companies, however, this is not an insignificant task. Microphysiological systems (MPS), also known as organ-on-a-chip (OOC), have been designed to meet this need, filling the gap between preclinical testing and the clinic. MPS enable us to grow 3D organs and tissues from human cells with flow circulating through them to simulate blood and deliver nutrients. These lab-cultured mimics function and respond to drugs in the same way as in humans.

Currently, single-organ models that recapitulate either individual organ functions (such as the liver), or routes of entry into the body (such as gut and lung), are most common. While traditional in vitro approaches fail to recapitulate a human organs’ phenotype, architecture and function for more than seven days, MPSs extend culture longevity up to a month, enabling studies that were not previously possible, such as the ability to profile phase I and II drug metabolism, characterise slowly metabolised compounds, identify potential metabolite-driven toxicity, or detect chronic toxicity. In this presentation, we will discuss the use of liver and lung MPSs for drug safety, disease modelling and efficacy and provide insights into what is coming next to further improve preclinical drug development workflows.

Learning Objectives:

1. Identify preclinical drug development challenges.

2. Introduce MPSs and how/where they fit into the preclinical workflow.

3. Demonstrate how MPSs provide translatable insights into drug safety and efficacy.
 

Adhesion G protein-coupled receptors (aGPCRs) are an ancient class of receptors that represent some of the largest transmembrane-integrated proteins in humans. Besides classic features of GPCRs, aGPCRs display a distinct N-terminal structure that contributes essentially to the size of these proteins. Most of the aGPCRs are still considered to be orphan, as detailed information on their signal transduction and physiological function are scarce. The finding of the deletion mutation that renders aGPCRs constitutively active prompted a rapid increase in number of aGPCR signaling studies. This laid ground for the discovery of the tethered agonist sequence, whose role in the complex signaling mechanisms of aGPCRs as well as the characterization of its derived peptide agonists, has been studied extensively. We found that aGPCRs can be activated through various mechanical stimuli, with or without the interaction with an endogenous binding partner. The mechanical force-induced activation depends on the existence of an intact core agonistic region within the tethered agonist sequence and can be modified through the adjacent positioning region. Transferring these observations to synthetic peptides allows for the establishment of agonists with increased specificity and different downstream signaling preferences. As the type of binding partner at the N terminus defines the required mechanical stimuli for receptor activation ligands could be considered allosteric modulators. We propose that aGPCRs ‘sense’ their environment through binding to surrounding proteins to translate this context into a specific signal. This localized signal can be the basis for future therapeutic strategies when targeting this important receptor class. 

Learning Objectives:

1. Discuss how adhesion-type G protein-coupled receptors (aGPCRs) are metabotropic mechanosensors that govern essential physiological functions and that are implicated in a wide range of diseases.

2. Explain how modulation of aGPCRs can be achieved through synthetic peptides, derived from the tethered agonist sequence, extracellular binding partners and a diverse set of mechanical forces. The combination of these different stimuli has the potential to yield efficient and specific therapeutic strategies targeting these receptors.

Over the last few years, the space of advanced in vitro models has grown dramatically as researchers look to better mimic the in vivo environment within inexpensive and high throughput in vitro models. Of all tissue systems, one of the most modeled is the liver as drug induced liver injury accounts for over 20% of late-stage clinical failures and diseases like NASH and HBV affect millions within the US. The focus of this talk is on describing the current state of advanced liver cell culture models and providing guidance on how best to choose which liver in vitro model system is best for a particular research question. Three separate in vitro model systems will be discussed: 1) HUREL 2D Micro Livers, 2) Spheroid 3D cell culture and 3) Ex vivo tissue slices. The advantages and disadvantages of each model system will be thoroughly described, and researchers will be provided with guidance on how best to choose their models based on cost, in vivo relevancy and throughput.

Learning Objectives:

1. Explain the advantages and disadvantages of various liver in vitro model systems and the types of applications that they should be applied to.

2. Identify a framework for choosing the right liver in vitro model for a specific application based on balancing cost, throughput and in vivo relevancy.

Princess Margaret Living Biobank (PMLB) is a repository for patient-derived organoid models. PMLB has generated over200 organoid models from lung, pancreas, colon, ovary, esophageal and breast tumor tissues obtained from patients at University Health Network, Toronto, Canada. All deposited models have passed quality checks including Short Tandem Repeat (STR) genotyping, mycoplasma testing, growth analysis, and histological identification of organoids using a panel of tissue specific biomarkers. Our long-term organoid establishment rate varied between 10-80% depending on the tumor type. PMLB organoids closely recapitulated the histology, the mutation and copy number landscape of their parental tumors and were tumorigenic in mice. The majority of organoid drug responses were similar to that of the matched patient/PDX. Major challenges in next generation biobanking of patient-derived organoid models include normal organoid outgrowth, mouse-derived organoid contamination and the state of tissue used for organoid generation. The PMLB aims to provide researchers at the academic institutions and industrial collaborators with tools to answer many basic oncology research questions with the hope to positively impact cancer treatment.

Learning Objectives:

1. Discuss processes involved in biobanking of patient-derived organoid models.

2. Explain the advantages and challenges of using patient-derived organoid models for research.