Stanford ChEM-H solicited seed grant proposals from the Stanford community of postdoctoral fellows for exploratory projects that align with the Institute’s mission. Successful proposals combined the complementary expertise of two or more postdoctoral researchers (including Ph.D. holders, M.D. residents and M.D. clinical fellows) seeking to collaboratively explore a potentially transformative new idea with the support of their mentors. The five proposals selected were each funded with $50,000 for one year.
"CRISPRouting Neural Development in a Zebrafish Model"
P. C. Dave P. Dingal, Qi Lab
Karen Mruk, Chen Lab
James Chen, Chemical and Systems Biology & Developmental Biology
Stanley Qi, Bioengineering & Chemical and Systems Biology
The Notch-Delta receptor signaling – triggered via direct cell-to-cell contact – plays a major role in embryonic development. In the central nervous system (CNS), Notch-Delta signaling controls the cell numbers and fate choice of specific neurons. However, both loss-of-function and gain-of-function mutants in this pathway lead to developmental defects. Conditional expression systems are perhaps our best tools, for understanding the role of Notch-Delta signaling in regulating neuronal cell fate.
This proposal aims to apply molecular engineering and synthetic biology approaches to engineer the Notch receptor and the CRISPR-Cas9 system for conditional knockout and overexpression studies in zebrafish. The zebrafish is a powerful animal model for these studies as they are completely transparent permitting real-time visualization of development. We have recently developed a class of human Notch-Cas9 chimeric receptors (CRISPRouters) that binds to extracellular Delta and releases receptor-tethered Cas9, a DNA-binding enzyme that we can program to target specific genes. Applying to zebrafish models, we will introduce CRISPRouters to perturb and examine the dynamic role of the Notch-Delta pathway during nervous system development. The modular CRISPRouter toolkit will allow rewiring of cell-cell signaling and cell fate, providing a novel approach to developmental studies.
"Genome-Wide Screen for Regulators of Adipogenesis"
Kyuho Han, Bassik Lab
Keren Hilgendorf, Jackson Lab
Michael Bassik, Genetics
Peter Jackson, Microbiology and Immunology & Baxter Laboratories
Obesity and co-morbidities like diabetes are a major unmet medical need, and there are few effective therapies targeting obesity or adipocyte pathology. To better understand the pathogenesis of unhealthy fat, we are interested in understanding how fat differentiation is initiated and regulated. Specifically, we will perform a genome-wide knockout screen using a fat progenitor cell line that robustly differentiates in vitro into lipid-laden fat cells upon exposure to differentiation factors. This screen will identify both known and novel genes required for fat differentiation. We are particularly interested in identifying genes related to primary cilia function. The cilium is a hair-like cellular protrusion that senses biochemical signals. In fat progenitor cells, it senses insulin and new factors we have identified. How primary cilium signaling is integrated to initiate fat differentiation remains unclear. In parallel, we will investigate the epigenetic changes underlying differentiation. In response to differentiation cues, fat progenitor cells undergo two rounds of cell division, which cue expression of differentiation-specific proteins. The regulation of these divisions and the specific transition that changes cell fate is also not known. Understanding the molecular factors regulating fat differentiation may uncover novel targets that lessen the burden of obesity.
"LRET Based Single Molecule ISH for Genomic Imaging in Tissues"
Nicholas Juul, Desai Lab
Monica Nagendran, Harbury Lab
Tushar Desai, Medicine - Pulmonary & Critical Care Medicine
Pehr Harbury, Biochemistry
We are developing a rapid, scalable, automated, and easy-to-use technology for measuring global gene expression at the single-cell level in intact tissues. This technology, which we call ‘Genomic Imaging,’ will rapidly replace single-cell RNA-sequencing and conventional in situ hybridization techniques, because it merges the deep gene coverage of the former with the spatial resolution of the latter. The technology is based on directly marking transcripts in fixed tissues, which avoids the reverse transcription and amplification steps that introduce bias in sequencing workflows. Genomic Imaging is compatible with concurrent antibody staining, and works robustly on formalin-fixed, paraffin-embedded (FFPE) tissue. We predict that the generation of “molecular histology” atlas's will become a widely adopted research assay, and the standard for molecular profiling across all medical disciplines.
"Immunostimulatory Synthetic Glycopeptides Targeting a Novel Pathway for Cancer Immunotherapy"
Justin Kenkel, Engleman Lab
Jessica Kramer, Bertozzi Lab
Carolyn Bertozzi, Chemistry
Edgar Engleman, Pathology & Medicine - Immunology & Rheumatology
Cancer immunotherapies show great promise in the clinic, but only a fraction of patients with certain cancers responds to existing immunotherapies. We recently discovered a new way to stimulate tumor immunity by targeting a pattern recognition receptor expressed by tumor-associated macrophages (TAMs). We found that triggering this receptor with microbial extracts reprograms TAMs into immunostimulatory cells that induce antitumor responses. The natural ligands for this receptor inhibit tumor progression as single agents and synergize with conventional therapies (e.g. chemotherapy) and other immunotherapies (e.g. checkpoint blockers) to induce tumor regression. Encouraged by these results, we decided to use a novel synthetic approach to prepare chemically defined, glycopeptide-based receptor ligands. With this platform, we can easily modify glycopeptide structure and function to generate strong TAM stimuli that can be targeted to tumor tissues and combined with other immunostimulatory molecules. We have already synthesized a series of glycopeptides, and our preliminary studies fully support this strategy. We are now developing glycopeptide agonists optimized for in vivo activity and functionalized with tumor-targeting properties. We expect this exciting project combining chemistry and tumor immunology to lead to the development of adaptable TAM activators that will form the foundation of a new class of cancer immunotherapeutics.
"A Novel Proteolytic Profiling Assay Based on Spectrally Encoded Beads to Guide the Design of Selective Chemical Probes for Bacterial Pathogens"
Christian Lentz, Bogyo Lab
Huy Nguyen, Fordyce Lab
Matthew Bogyo, Pathology & Microbiology and Immunology
Polly Fordyce, Genetics & Bioengineering
Chemical probes that visualize the activity of members of the protease enzyme family are useful tools for biomedical research and in vivo imaging. One of our long-term goals is to develop probes that would specifically recognize single proteases of bacterial pathogens and could be used for non-invasive in vivo imaging of infection. Protease-directed probes are usually based on amino acid sequences that mimic the natural substrates of a protease. However, probes based on the limited combinations of the 20 naturally occurring amino acids may lack specifity. Introducing chemical diversity by incorporating some of the >100 available non-natural amino acids may enhance these probes tremendously, but there are currently no flexible, cost-effective, high-throughput screening systems available to identify which non-natural amino acids individual proteases prefer.
In the proposed work we are going to establish a microfluidics platform based on peptide libraries on spectrally-encoded microbeads to enable proteolytic profiling of individual bacterial proteases and complex bacterial lysates in high throughput and at minimal costs and sample volumes. The gained structural information will then be used to guide the design of highly selective chemical probes for bacterial pathogens Mycobacterium tuberculosis and Staphylococcus aureus to address unmet clinical needs for imaging bacterial infections.