Biology subjects

Kirchner, R.

Publications and source records attributed to Kirchner, R..

6 recordsLinked to original sources

bioRxiv2024

ML-driven design of 3' UTRs for mRNA stability

Using mRNA as a therapeutic has received enormous attention in the last few years, but instability of the molecule remains a hurdle to achieving long-lasting therapeutic levels of protein expression. In this study, we describe our approach for designing stable mRNA molecules by combining machine learning-driven sequence design with high-throughput experimental assays. We developed a high-throughput massively parallel reporter assay (MPRA) that, in a single experiment, measures the half-life of tens of thousands of unique mRNA sequences containing designed 3 UTRs. Over multiple design-build-test iterations, we have accumulated mRNA stability measurements for 180,000 unique genomic and synthetic 3 UTRs, representing the largest such dataset of sequences. We trained highly-accurate machine learning models to map from 3 UTR sequence to mRNA stability, and used them to guide the design of synthetic 3 UTRs that increase mRNA stability in cell lines. Finally, we validated the function of several ML-designed 3 UTRs in mouse models, resulting in up to 2-fold more protein production over time and 30-100-fold higher protein output at later time points compared to a commonly used benchmark. These results highlight the potential of ML-driven sequence design for mRNA therapeutics.

synthetic biology↗

bioRxiv2023

Screening the human druggable genome identifies ABHD17B as an anti-fibrotic target in hepatic stellate cells

Hepatic stellate cells (HSCs) are activated with chronic liver injury and transdifferentiate into myofibroblasts, which produce excessive extracellular matrices that form the fibrotic scar. While the progression of fibrosis is understood to be the cause of end-stage liver disease, there are no approved therapies directed at interfering with the activity of HSC myofibroblasts. We performed a high-throughput small interfering RNA (siRNA) screen in primary human HSC myofibroblasts to identify gene products necessary for the fibrotic phenotype of HSCs. We found that depletion of ABHD17B promotes the inactivation of HSCs, characterized by reduced COL1A1 and ACTA2 expression and accumulation of lipid droplets. Mice deficient for Abhd17b are also protected from fibrosis in the setting of in vivo liver injury. While ABHD17B is a depalmitoylase, our data suggest that ABHD17B promotes fibrosis through pathways independent of depalmitoylation that include interaction with MYO1B to modulate gene expression and HSC migration. Together, our results provide an analysis of the phenotypic consequences for siRNAs targeting RNAs from >9,500 genes in primary human HSCs and identify ABHD17B as a potential therapeutic target to inhibit liver fibrosis.

cell biology↗

bioRxiv2022

Quantifying oligomer populations in real time during protein aggregation using single-molecule mass photometry

Protein aggregation is a hallmark of many neurodegenerative diseases. The early stages of the aggregation cascade are crucial because small oligomers are thought to be key neurotoxic species, but they are difficult to study because they feature heterogeneous mixtures of transient states. We show how the populations of different oligomers can be tracked as they evolve over time during aggregation by using single-molecule mass photometry to measure individually the masses of the oligomers present in solution. Applying the approach to tau protein, whose aggregates are linked to diseases including Alzheimers and frontotemporal dementia, we found that tau existed in an equilibrium between monomers, dimers, and trimers before aggregation was triggered. Once aggregation commenced, the monomer population dropped continuously, paired first with a rise in the population of the smallest oligomers and then a steep drop as the protein was incorporated into larger oligomers and fibrils. Fitting these populations to kinetic models allowed different models of aggregation to be tested, identifying the most likely mechanism and quantifying the microscopic rates for each step in the mechanism. This work demonstrates a powerful new approach for characterizing previously inaccessible regimes in protein aggregation and building quantitative mechanistic models.

biophysics↗

bioRxiv2021

Nanchangmycin regulates FYN, FAK and ERK to control the fibrotic activity of hepatic stellate cells

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic drugs, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, FAK, ERK1/2, HSP27 and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. The identification of the antifibrotic effects of NCMC and the pathways by which NCMC inhibits fibrosis provides new tools and therapeutic targets to combat the development and progression of liver fibrosis.

cell biology↗

bioRxiv2021

An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

plant biology↗

bioRxiv2018

A cell atlas of the adult Drosophila midgut

Studies of the adult Drosophila midgut have provided a number of insights on cell type diversity, stem cell regeneration, tissue homeostasis and cell fate decision. Advances in single-cell RNA sequencing (scRNA-seq) provide opportunities to identify new cell types and molecular features. We used inDrop to characterize the transcriptome of midgut epithelial cells and identified 12 distinct clusters representing intestinal stem cells (ISCs), enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs) from different regions, and cardia. This unbiased approach recovered 90% of the known ISCs/EBs markers, highlighting the high quality of the dataset. Gene set enrichment analysis in conjunction with electron micrographs revealed that ISCs are enriched in free ribosomes and possess mitochondria with fewer cristae. We demonstrate that a subset of EEs in the middle region of the midgut expresses the progenitor marker esg and that individual EEs are capable of expressing up to 4 different gut hormone peptides. We also show that the transcription factor klumpfuss (klu) is expressed in EBs and functions to suppress EE formation. Lastly, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing novel functions of genes in the midgut epithelium.

genetics↗