Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands

T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein‐ and aptamer‐based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting ligands can deliver RNA to T cells in vivo. To test this hypothesis, the delivery of siRNA to 9 cell types in vivo by 168 nanoparticles using a novel siGFP‐based barcoding system and bioinformatics is quantified. It is found that nanomaterials containing conformationally constrained lipids form stable LNPs, herein named constrained lipid nanoparticles (cLNPs). cLNPs deliver siRNA and sgRNA to T cells at doses as low as 0.5 mg kg^?1 and, unlike previously reported LNPs, do not preferentially target hepatocytes. Delivery occurs via a chemical composition‐dependent, size‐independent mechanism. These data suggest that the degree to which lipids are constrained alters nanoparticle targeting, and also suggest that natural lipid trafficking pathways can promote T cell delivery, offering an alternative to active targeting approaches.     

Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses

Using mRNA to produce therapeutic proteins is a promising approach to treat genetic diseases. However, systemically delivering mRNA to cell types besides hepatocytes remains challenging. Fast identification of nanoparticle delivery (FIND) is a DNA barcode‐based system designed to measure how over 100 lipid nanoparticles (LNPs) deliver mRNA that functions in the cytoplasm of target cells in a single mouse. By using FIND to quantify how 75 chemically distinct LNPs delivered mRNA to 28 cell types in vivo, it is found that an LNP formulated with oxidized cholesterol and no targeting ligand delivers Cre mRNA, which edits DNA in hepatic endothelial cells and Kupffer cells at 0.05 mg kg^?1. Notably, the LNP targets liver microenvironmental cells fivefold more potently than hepatocytes. The structure of the oxidized cholesterols added to the LNP is systematically varied to show that the position of the oxidative modification may be important; cholesterols modified on the hydrocarbon tail associated with sterol ring D tend to outperform cholesterols modified on sterol ring B. These data suggest that LNPs formulated with modified cholesterols can deliver gene‐editing mRNA to the liver microenvironment at clinically relevant doses.     

Implementing communication protocols in Java

As the number and variety of Web- and network-based applications continues to increase, so does the need for flexible communication protocols and services to support them. Traditionally, a major impediment to deployment of new protocols and services is the need to upgrade millions of end systems with compatible implementations. At the same time, Java-a language explicitly designed to support development and distribution of new applications via the Web-is emerging as a (potentially) ubiquitous system platform. It is therefore natural to consider whether Java might speed the introduction of protocols to better support new applications. We discuss the suitability of Java as an environment for implementing and deploying communication protocols. Using insights from a Java-based protocol suite and supporting protocol subsystem we have implemented, we describe the benefits of using Java for protocol development and deployment, and how protocol programmers can implement protocols taking advantage of those benefits     

Increasing the portability and re-usability of protocol code

Deploying protocols is an expensive and time-consuming process today. One reason is the high cost of developing, testing, and installing protocol implementations. To reduce this difficulty, protocols are developed and executed within environments called protocol subsystems, and protocol software is often ported instead of being coded from scratch. Unfortunately, today a variety of protocol subsystems offer a plethora of features, functionality, and drawbacks; the differences among them often reduce the portability and reusability of protocol code, and therefore present barriers to the deployment of new protocols. In this paper, we consider differences in subsystems and their effect on the portability and reusability of protocols and protocol implementations. We then propose two different approaches, each optimized for a different situation, that allow protocol code implemented in one subsystem to be used without modification within other subsystems, and thus reduce the barriers to protocol deployment. We relate our experiences designing, implementing, and measuring the performance of each approach using, as a baseline, an AppleTalk protocol stack we have developed