U of T Technology & Startup Explorer

Serum-free cell culture media design is important because it offers the opportunity to construct chemically defined media that possess greater batch-to-batch reproducibility and clinically compatible reagents; benefits not necessarily associated with serum-containing media.  As such, it is an ideal method for producing media for the growing cell therapy market segment where high quality and reproducibility standards influence clinical translation.  However, the use of defined chemical factors often creates a complex, large-scale optimization problem where the number of possible combinations of factors across different dose concentrations creates a search space so large that only a millionth or less of it can be sampled.  Consequently, this renders traditional media optimization techniques ineffective and necessitates the development of new techniques to handle this problem.

Small molecules like metabolites and drugs are at the core of healthcare. The levels of vitamins, hormones, sugars and other critical metabolites gives a snapshot of our health and small molecules like antibiotics, cancer drugs, and antidepressants are vital for treating disease. However, detecting these diverse small molecules is a massive bottleneck in every area of healthcare and medical research. We have developed a new technology that allows us to detect small molecules using DNA sequencing instead of mass spectroscopy. We use small DNA sensors called aptamers to detect each small molecule — different aptamers detect different small molecule targets with high specificity and when any target is detected it gives a unique signal in the form of a DNA sequence readout.

Fibrosis, or scarring of tissue, is not limited to the skin after an injury. In many chronic conditions, such as hypertension, autoimmune lung disease, and alcohol abuse (affecting the heart, lungs, and liver, respectively), tissue becomes progressively fibrotic over time, a condition that often goes undetected but ultimately results in organ failure and even death. Unfortunately, there is no cure for late-stage fibrosis, and tissue biopsy remains the only reliable diagnostic option, despite the deficiencies of limited sampling and mis-sampling.  At the University of Toronto, our inventors have developed a non-invasive magnetic resonance imaging (MRI) compound that can be administered to the patient to “light up” fibrotic tissue, including newly developing scars, in 3D throughout the body, to find fibrosis at early stage when the opportunity to interrupt disease progression is the greatest.

Cell-free protein synthesis involves the production of proteins in the absence of living cells by using a mixture of extracted machinery and molecular components from chassis organisms (e.g. E. coli).  It is an emerging format for the rapid testing of protein properties and function (e.g. functional screening).  It is also advantageous in situations where protein synthesis is enhanced by conditions (e.g. pH, redox potentials, temperatures) outside the normal range of cells, and also enables on demand co-and-post-translational modifications of proteins, incorporation of non-natural amino acids, selective and site-specific labelling, and production of toxic or difficult proteins.  A key limitation though is the dependence of cell-free systems on plasmid DNA templates which are both timely (>3 days) and costly to generate, reducing the throughput of protein production and slowing the research and development process.  A linear DNA template on the other hand can be cheaply and quickly (3 hrs) generated but is susceptible to rapid degradation by exonucleases in cell-free reactions.  If this degradation rate could be reduced or eliminated to make cell-free systems compatible with linear DNA templates, the throughput of cell-free protein synthesis could be dramatically increased and production costs sharply reduced.

Boron-containing compounds play an indispensable role in biological and organic chemistry, functioning as intermediates, catalysts and biological probes. The success of several boron-based medicines further emphasizes the potential of incorporating boron into therapeutic agents. Such agents typically contain a considerable number of heteroatoms, thus the development of technologies that introduce boron into these environments are growing in demand.

Protein phosphorylation is major molecular mechanism through which protein function is regulated in response to extracellular stimuli, it often acts as a switch to proteins' activation and is frequently perturbed in diseases. Currently there are a number of commercialized techniques to detect phospho-proteins, however none of them offers information about the relative spatial arrangement of phosphorylated residues. In biological systems, the extent of phosphorylation of a target is often associated different physiological response and disease state. This invention is the first turn-on fluorescent sensor for the selective detection of proximally phosphorylated protein sites, suitable for application in both aqueous solutions and polyacrylamide gels.

Complex multi-protein clusters play vital roles in many aspects of biology. Deregulation of these clusters may lead to several human diseases including cancer, cystic fibrosis, cardiovascular and neurodegenerative disorders. This technology uses a bait (protein of interest) - prey (candidate molecules) system to detect and identify novel interacting partners in a mammalian genetic system. Dr Stagljar has built upon his initial patented Membrane Yeast Two-Hybrid (MYTH) invention to propose a unique platform to discover new target molecules for research and drug development purposes. This technology provides a new tool to examine membrane proteins in their natural environment of the human cell. It is sensitive enough to detect minor changes upon the introduction of drugs and thus should prove useful in the development of therapeutics, especially for cancer and neurological diseases.

This technology relates to a novel method to discover receptors for extracellular proteins in an unbiased fashion. The approach is based on a simple concept: bacterial exotoxin, when fused to a secreted protein, intoxicates cells in a receptor-dependent manner, which facilitates the identification of the cognate receptor through genome-wide CRISPR/Cas9-based positive selection screen.

Our researchers have developed an in vitro stem cell mediated skeletal muscle repair platform referred to as “MEndR” (Muscle Endogenous Repair), that accurately recapitulates the timing of key phases of the stem cell (SC) mediated skeletal muscle repair process in a dish (SC-expansion, SC cell cycle exit, SC nascent fiber formation, fiber maturation). Furthermore, this in vitro platform accurately predicts the performance of stem cells at mediating in vivo repair in standard in vivo mouse transplantation assays and has the potential for parallel stratification of the potency of many (100s) drug or cell therapy candidates at enhancing muscle regeneration.

Our researchers have developed a novel culture substrate combination that robustly supports human pluripotent stem cell (hPSC) proliferation and pluripotency in chemically defined conditions. hPSCs grown on this novel substrate culture display higher proliferation rates and pluripotency marker expression than current gold-standard culture substrates Geltrex- and vitronectin-coated plastic. Because this invention uses human recombinant proteins in combination with chemically defined media, it addresses the unmet need for defined culture platforms (culture substrate and media) required to generate clinical-grade cells for future cell therapy and broader medical applications. Furthermore, these substrates provide additional benefit to basic research through avoiding lot-to-lot variation and potential confounding effects associated with using undefined matrices (Geltrex and Matrigel).

The Recombinant Antibody Network (RAN) is a multi-institutional research consortium (member institutions: The University of Toronto, University of California San Francisco and The University of Chicago) created accelerate the identification and development of recombinant antibodies raised against valuable and challenging targets to support drug development and cutting edge biomedical research.  The RAN was founded by three ex-Genentech antibody engineers.  To date the team has screened and validated over 880 antibodies, some of which are now available for licensing as therapeutics.

Motivated by the current urgency due to the pandemic, University of Toronto researchers (with extensive expertise in protein engineering) are developing a set of novel immunoassays for detecting anti-CoV-2 antibodies (IgM and IgG) directly in patients sera based on protein complementation assay (PCA), specifically on tri-part split NanoLuc. For this, they are repurposing their recently developed and patented Split Intein-Mediated Protein Ligation (SIMPL) (1) detection assay to develop an innovative diagnostic immunoassay for detecting anti-SARS-CoV-2 Abs directly from COVID-19 patient sera by adapting the tri-part split NanoLuc assay.

Precisely controlled regulation of gene expression in cells, tissues, and organisms is one of the major challenges in tissue engineering, cell-based therapies, and gene therapy. Controlled gene expression in cells is achieved either by exogenously introducing cDNAs by transfection or viral transduction, or by introducing sequence-specific transcriptional regulators. These regulators include zinc-finger nucleases, transcription activator like effectors (TALEs), or enzymatically inactive Cas9 (dCas9) which are fused to transcriptional activation or repression domains to achieve the desired effect on gene expression. Of these techniques, CRISPR interference (CRISPRi) based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression.  However, it suffers from incomplete silencing of target genes.

Access to a patient’s GI tract, either for purposes of delivery of therapeutic agents or collecting samples for diagnostic purposes faces a number of challenges. For the former, oral administration, requiring transit through the stomach and upper GI, can impact the stability and solubility of therapeutic agents such as antibodies or probiotics. For the latter, current procedures for retrieving microbiome samples rely on the use of highly invasive endoscopic procedures. Consequently, many diagnoses of gut health (for example to monitor diseases such as inflammatory bowel disease (IBD) or cancer) rely on analysis of a patient’s stool, material that does not reflect the conditions that occur higher up in the GI tract. There is therefore an urgent need for both drug delivery and collection (GI sampling) systems for more effective and precise disease intervention.

Genome wide knockout screens can be used to elucidate genetic pathways and identify novel drug targets.  They work by analyzing the impact of gene inactivation on a measured physiological readout, such as cell survival.  CRISPR-Cas9 is a simple and effective tool used to disrupt single genes on a large scale.  However, generating the necessary pooled libraries to simultaneously inactivate two or more genes (i.e. double knockout) is challenging.  Furthermore, other limitations that impact its efficiency exist, such as recombination between duplicated promoters and expression cassettes.  Cas12a has been proposed as an attractive alternative to Cas9 as its intrinsic RNAase activity can be used to generate multiple distinct gRNAs from a single concatemer.  However, it too has its deficiencies, such as a low double knockout efficiency (15%).  Thus, there is a need for improved techniques to probe genetic interactions (GIs) between two or more gene participants.

Detection of negatively charged phospholipids can serve as indicators of apoptosis, bacterial presence, or diseases such as phospholipidosis.  For example, phosphoserine, a negatively charged lipid, is largely present in the outer membrane of mammalian cells only in the event of apoptosis, while the negative net charge of bacterial membranes can serve to differentiate these cells from mammalian ones.  Consequently, the phosphoserine binding protein, AnnexinV, has been used for detection of apoptosis, while the small molecule Zn2+-containing fluorophore, PSVueTM380, has been used for generic detection of negatively charged phospholipids and their indicative states.  However, AnnexinV has several limitations (high Ca2+ levels required for binding, biochemical instability, slow rate of binding, need for washes) and the turn-on sensor PSVueTM380 has a short dynamic range.

Induced pluripotent stem cells (iPSCs), and iPSC-derived cardiomyocytes (iPSC-CMs) offer tremendous promise for basic cardiovascular science, disease modeling, drug efficacy and toxicity testing, personalized medicine, and cell therapy to restore myocardial function during heart failure or post-infarction. However, current iPSC-CMs are physiologically immature in all categories of relevant function, including morphology, contractility, calcium handling, action potential electrophysiology, and metabolism. This is a major limitation towards the application and commercialization of iPSC-CMs.

Targeted protein degradation (TPD) is an alternative approach to conventional paradigms (e.g. small molecule inhibitors) that target disease.  In this technique, a PROteolysis TArgeting Chimera (PROTAC) or a molecular glue is used to initiate the degradation process of the targeted protein.  While these two molecular classes are mechanistically distinct, they both enable degradation by recruiting an E3 ligase, an enzyme which enables the transfer of the ubiquitin tag needed for proteasome-mediated degradation.  One obstacle to the development of these treatment modalities is that the two E3 ligases largely used with this approach only work with some proteins.  Thus, identification of new E3 ligases can increase the disease targeting space of targeted protein degradation therapeutics.

Precisely controlled regulation of gene expression in cells, tissues, and organisms is one of the major challenges in tissue engineering, cell-based therapies, and gene therapy. Controlled gene expression in cells is achieved either by exogenously introducing cDNAs by transfection or viral transduction, or by introducing sequence-specific transcriptional regulators. These regulators include zinc-finger nucleases, transcription activator like effectors (TALEs), or enzymatically inactive Cas9 (dCas9) which are fused to transcriptional activation or repression domains to achieve the desired effect on gene expression. Of these techniques, CRISPR activation (CRISPRa), based on the fusion of inactive Cas9 (dCas9) to various trans-activation domains, is a powerful platform for activating gene expression. However, non-human activators (e.g. VP16) can elicit an immune response necessitating the development of human activators of comparable and tunable efficacy.

Gene therapy using space-constrained delivery vehicles like adeno-associated viral (AAV) vectors can benefit from the shortening of large tissue-specific promoters.  This motivated efforts to make promoters highly compact while maintaining activity and specificity.  However, the promoter design process is largely iterative and experimental.  It typically involves the synthesis of a promoter fragment roughly up to 3000 nucleotides upstream and ∼50 nucleotides downstream of the transcription start site.  Upstream fragments are then truncated until loss of function occurs, indicating the optimal short promoter.  However, it may still contain “subsequences” that do not contribute to function.  If an alternative solution can be designed to computationally predict the transcription factor binding sites, this would enable removal of non-functional “subsequences” yielding even shorter promoters and in a cost-effective manner.

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High-performance software solutions and advanced algorithms for cryo-EM data processing.

A method to extract, assess, and visualize information from electroencephalography (EEG) readings.