Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo.

The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.

Size matters: Functional differences of small extracellular vesicle subpopulations in cardiac repair responses.

Cardiac progenitor cell (CPC)-derived small extracellular vesicles (sEVs) exhibit great potential to stimulate cardiac repair. However, the multifaceted nature of sEV heterogeneity presents a challenge in understanding the distinct mechanisms underlying their regenerative abilities. Here, a dual-step multimodal flowthrough and size-exclusion chromatography method was applied to isolate and separate CPC-derived sEV subpopulations to study the functional differences related to cardiac repair responses. Three distinct sEV subpopulations were identified with unique protein profiles. Functional cell assays for cardiac repair-related processes demonstrated that the middle-sized and smallest-sized sEV subpopulations exhibited the highest pro-angiogenic and anti-fibrotic activities. Proteasome activity was uniquely seen in the smallest-sized subpopulation. The largest-sized subpopulation showed no effect in any of the functional assays. This research uncovers the existence of sEV subpopulations, each characterized by a distinct composition and biological function. Enhancing our understanding of sEV heterogeneity will provide valuable insights into sEV mechanisms of action, ultimately accelerating the translation of sEV therapeutics.

Fleet's Geode: A Breakthrough Sensor for Real-Time Ambient Seismic Noise Tomography over DtS-IoT.

As most of the outcropping and shallow mineral deposits have been found, new technology is imperative to finding the hidden critical mineral deposits required to transition to renewable energy. One such new technique, called ambient seismic noise tomography, has shown promise in recent years as a low-cost, low environmental impact method that can image under cover and at depth. Wireless and compact nodal seismic technology has been instrumental to enable industry applications of ambient noise tomography, but these devices are designed for the active seismic reflection method and do not have the required sensitivity at low frequencies for ambient noise tomography, and real-time data transmission in remote locations requires significant infrastructure to be installed. In this paper, we show the development and testing of the Geode-a real-time seismic node purpose-built by Fleet Space Technologies for ambient seismic noise tomography on exploration scales. We discuss the key differences between current nodal technology and the Geode and show results of a field trial where the performance of the Geode is compared with a commercially popular nodal geophone. The use of a 2 Hz high sensitivity geophone and low noise digitiser results in an instrument noise floor that is more than 30 dB lower below 5 Hz than nodes that are commonly used in the industry. The increased sensitivity results in signal-to-noise ratios in the cross-correlation functions in the field trial that are more than double that of commercially available nodal geophone at low frequencies. When considering the full bandwidth of retrievable correlations in our study, using the Geode would reduce the required recording time from 75 h to 32 h to achieve an average signal-to-noise ratio in the cross-correlation functions of 10. We also discuss the integration of a real-time direct-to-satellite Internet of Things (DtS-IoT) modem in the Geode, which, together with edge processing of seismic data directly on the Geode, enables us to image the subsurface in real-time. During the field trial, the Geodes successfully transmitted more than 90% of the available preprocessed data packets. The Geode is compact enough so that several devices can be carried and installed by one field technician, whilst the array of stations do not require a base station to transmit data to the cloud for further processing. We believe this is the future of passive seismic surveys and will result in faster and more dynamic seismic imaging capabilities analogous to the medical imaging community, increasing the pace at which new mineral deposits are discovered.

Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.