A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of ß-Cell Glucolipotoxicity.

Type 2 diabetes (T2D) is caused by loss of pancreatic ß-cell mass and failure of the remaining ß-cells to deliver sufficient insulin to meet demand. ß-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on ß-cell function and survival, contributes to T2D-associated ß-cell failure. Drugs and mechanisms that protect ß-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected ß-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured ß-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve ß-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

Thermal and Physico-Mechanical Characterizations of Thromboresistant Polyurethane Films.

Hemocompatibility remains a challenge for injectable and/or implantable medical devices, and thromboresistant coatings appear to be one of the most attractive methods to down-regulate the unwanted enzymatic reactions that promote the formation of blood clots. Among all polymeric materials, polyurethanes (PUs) are a class of biomaterials with excellent biocompatibility and bioinertness that are suitable for the use of thromboresistant coatings. In this work, we investigated the thermal and physico-mechanical behaviors of ester-based and ether-based PU films for potential uses in thromboresistant coatings. Our results show that poly(ester urethane) and poly(ether urethane) films exhibited characteristic peaks corresponding to their molecular configurations. Thermal characterizations suggest a two-step decomposition process for the poly(ether urethane) films. Physico-mechanical characterizations show that the surfaces of the PU films were hydrophobic with minimal weight changes in physiological conditions over 14 days. All PU films exhibited high tensile strength and large elongation to failure, attributed to their semi-crystalline structure. Finally, the in vitro clotting assays confirmed their thromboresistance with approximately 1000-fold increase in contact time with human blood plasma as compared to the glass control. Our work correlates the structure-property relationships of PU films with their excellent thromboresistant ability.

Automated detection and sorting of microencapsulation via machine learning.

Microfluidic-based microencapsulation requires significant oversight to prevent material and quality loss due to sporadic disruptions in fluid flow that routinely arise. State-of-the-art microcapsule production is laborious and relies on experts to monitor the process, e.g. through a microscope. Unnoticed defects diminish the quality of collected material and/or may cause irreversible clogging. To address these issues, we developed an automated monitoring and sorting system that operates on consumer-grade hardware in real-time. Using human-labeled microscope images acquired during typical operation, we train a convolutional neural network that assesses microencapsulation. Based on output from the machine learning algorithm, an integrated valving system collects desirable microcapsules or diverts waste material accordingly. Although the system notifies operators to make necessary adjustments to restore microencapsulation, we can extend the system to automate corrections. Since microfluidic-based production platforms customarily collect image and sensor data, machine learning can help to scale up and improve microfluidic techniques beyond microencapsulation.

Engineering Approaches to Prevent Blood Clotting from Medical Implants.

Injectable and/or Implantable medical devices are widely used in the treatment of diseases. Among them, vascular stents provide the medical solution to treat blood clotting. However, traditional metallic stents, even with current improvements in anticoagulation properties, have potential drawbacks in local inflammation when first implanted into the body and undesirable protein adsorption and cell adhesion after a prolonged period of time in the body. In this perspective, we discuss several engineering approaches, including drug-eluting materials, polymeric and non-polymeric coatings, and surface modifications to coating materials that can be applied to the surface of medical implants to significantly improve the hemocompatibility. These coatings are expected to have a slow degradation rate with the ability to either load drugs or attach biomacromolecules to form an architecture that mimics the surrounding cells. In general, our perspective provides a current view on the achievements of hemo-compatible coatings and future trends in coating materials that will extend the life of the medical implants.

A Scalable Pipeline for High-Throughput Flow Cytometry.

Flow cytometry (FC) provides high-content data for a variety of applications, including phenotypic analysis of cell surface and intracellular markers, characterization of cell supernatant or lysates, and gene expression analysis. Historically, sample preparation, acquisition, and analysis have presented as a bottleneck for running such types of assays at scale. This article will outline the solutions that have been implemented at Novartis which have allowed high-throughput FC to be successfully conducted and analyzed for a variety of cell-based assays. While these experiments were generally conducted to measure phenotypic responses from a well-characterized and information-rich small molecular probe library known as the Mechanism-of-Action (MoA) Box, they are broadly applicable to any type of test sample. The article focuses on application of automated methods for FC sample preparation in 384-well assay plates. It also highlights a pipeline for analyzing large volumes of FC data, covering a visualization approach that facilitates review of screen-level data by dynamically embedding FlowJo (FJ) workspace images for each sample into a Spotfire file, directly linking them to the metric being observed. Finally, an application of these methods to a screen for MHC-I expression upregulators is discussed.