DRUG-seq Provides Unbiased Biological Activity Readouts for Neuroscience Drug Discovery.

Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity.

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 ß-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.

Transcription Factor Elf3 Modulates Vasopressin-Induced Aquaporin-2 Gene Expression in Kidney Collecting Duct Cells.

Aquaporin-2 (AQP2) is a molecular water channel protein responsible for water reabsorption by the kidney collecting ducts. Many water balance disorders are associated with defects in AQP2 gene expression regulated by the peptide hormone vasopressin. Here, we studied roles of Elf3 (E26 transformation-specific (Ets)-related transcription factor 3) in AQP2 gene expression in the collecting duct cells (mpkCCD). Vasopressin increased AQP2 mRNA and protein levels without affecting AQP2 mRNA degradation, indicative of transcriptional regulation. Elf3 knockdown and overexpression, respectively, reduced and increased AQP2 gene expression under basal and vasopressin-stimulated conditions. However, the vasopressin-to-basal ratios of AQP2 gene expression levels remained constant, indicating that Elf3 does not directly mediate vasopressin response but modulates the level of AQP2 gene expression inducible by vasopressin. The Elf3-modulated AQP2 gene expression was associated with AQP2 promoter activity, in line with Elf3's ability to bind an Ets element in the AQP2 promoter. Mutation in the Ets element reduced both basal and vasopressin-stimulated AQP2 promoter activity, again without affecting vasopressin-to-basal ratios of the AQP2 promoter activity. Lithium chloride reduced both Elf3 and AQP2 mRNA in the mpkCCD cells as well as in mouse kidney inner medulla. We conclude that Elf3 modulates AQP2 promoter activity thereby gauging vasopressin-inducible AQP2 gene expression levels. Our data provide a potential explanation to lithium-induced nephrogenic diabetes insipidus where lithium reduces Elf3 and hence AQP2 abundance.

Generation of a Purified iPSC-Derived Smooth Muscle-like Population for Cell Sheet Engineering.

Induced pluripotent stem cells (iPSCs) provide a potential source for the derivation of smooth muscle cells (SMCs); however, current approaches are limited by the production of heterogeneous cell types and a paucity of tools or markers for tracking and purifying candidate SMCs. Here, we develop murine and human iPSC lines carrying fluorochrome reporters (Acta2hrGFP and ACTA2eGFP, respectively) that identify Acta2+/ACTA2+ cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2hrGFP+ and ACTA2eGFP+ cells can be sorted to purity and are enriched in markers characteristic of an immature or synthetic SMC. We characterize the resulting GFP+ populations through global transcriptomic profiling and functional studies, including the capacity to form engineered cell sheets. We conclude that these reporter lines allow for generation of sortable, live iPSC-derived Acta2+/ACTA2+ cells highly enriched in smooth muscle lineages for basic developmental studies, tissue engineering, or future clinical regenerative applications.

DRUG-seq for miniaturized high-throughput transcriptome profiling in drug discovery.

Here we report Digital RNA with pertUrbation of Genes (DRUG-seq), a high-throughput platform for drug discovery. Pharmaceutical discovery relies on high-throughput screening, yet current platforms have limited readouts. RNA-seq is a powerful tool to investigate drug effects using transcriptome changes as a proxy, yet standard library construction is costly. DRUG-seq captures transcriptional changes detected in standard RNA-seq at 1/100th the cost. In proof-of-concept experiments profiling 433 compounds across 8 doses, transcription profiles generated from DRUG-seq successfully grouped compounds into functional clusters by mechanism of actions (MoAs) based on their intended targets. Perturbation differences reflected in transcriptome changes were detected for compounds engaging the same target, demonstrating the value of using DRUG-seq for understanding on and off-target activities. We demonstrate DRUG-seq captures common mechanisms, as well as differences between compound treatment and CRISPR on the same target. DRUG-seq provides a powerful tool for comprehensive transcriptome readout in a high-throughput screening environment.

Stem cells and kidney regeneration.

Kidney disease is an escalating burden all over the world. In addition to preventing kidney injury, regenerating damaged renal tissue is as important as to retard the progression of chronic kidney disease to end stage renal disease. Although the kidney is a delicate organ and has only limited regenerative capacity compared to the other organs, an increasing understanding of renal development and renal reprogramming has kindled the prospects of regenerative options for kidney disease. Here, we will review the advances in the kidney regeneration including the manipulation of renal tubular cells, fibroblasts, endothelial cells, and macrophages in renal disease. Several types of stem cells, such as bone marrow-derived cells, adipocyte-derived mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells are also applied for renal regeneration. Endogenous or lineage reprogrammed renal progenitor cells represent an attractive possibility for differentiation into multiple renal cell types. Angiogenesis can ameliorate hypoxia and renal fibrosis. Based on these studies and knowledge, we hope to innovate more reliable pharmacological or biotechnical methods for kidney regeneration medicine.