Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system.
Site-specific antibody conjugates with a well-defined structure and superb therapeutic index are of great interest for basic research, disease diagnostics, and therapy. Here, we develop a novel proximity-induced antibody conjugation strategy enabling site-specific covalent bond formation between functional moieties and native antibodies without antibody engineering or additional UV/chemical treatment. A high conjugation efficiency and specificity was achieved with IgGs from different species and subclasses. The utility of this approach was demonstrated by site-specific conjugation of the small-molecule fluorophore to a native antibody and in vitro characterization of its activities.
With the increasing global popularity of selfdriving cars, there is an immediate need for challenging realworld datasets for benchmarking and training various computer vision tasks such as 3D object detection. Existing datasets either represent simple scenarios or provide only day-time data. In this paper, we introduce a new challenging A*3D dataset which consists of RGB images and LiDAR data with significant diversity of scene, time, and weather. The dataset consists of high-density images (≈ 10 times more than the pioneering KITTI dataset), heavy occlusions, a large number of nighttime frames (≈ 3 times the nuScenes dataset), addressing the gaps in the existing datasets to push the boundaries of tasks in autonomous driving research to more challenging highly diverse environments. The dataset contains 39K frames, 7 classes, and 230K 3D object annotations. An extensive 3D object detection benchmark evaluation on the A*3D dataset for various attributes such as high density, day-time/night-time, gives interesting insights into the advantages and limitations of training and testing 3D object detection in real-world setting.
Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging.
Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.
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