Project description:Retroviral gene delivery is widely used in CAR-T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semi-random patterns, and their transduction efficiency is highly variable. In contrast, non-viral delivery methods could offer a potentially less expensive and safer alternative for CAR-T cell gene delivery. In this study, several different cationic vehicles, promoters, and additional culture conditions were compared to optimize non-viral transgene delivery and expression in both Jurkat and primary T cells. In addition, confocal microscopy and next-generation sequencing experiments were also conducted to detect putative methods of transfection resistance in Jurkat and primary T cells. Transfecting Jurkat cells in X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection efficiency in Jurkat cells (63.0±10.9% EGFP+). However, this protocol yielded a much lower transfection efficiency (8.07±0.76% EGFP+) in primary T cells. Confocal microscopy and mRNA-sequencing experiments revealed that a majority of lipoplexes did not enter the primary T cells, perhaps due to relatively low expression levels of heparan sulfate proteoglycans (HSPGs). Primary T cells also expressed high levels of PYHIN DNA sensors (e.g., AIM2, IFI16), which can induce apoptosis when bound to cytoplasmic DNA. Primary T cells are more resistant to transfection with Lipofectamine than Jurkat T cells. Transfection of primary T cells appears to be hindered by decreased expression of HSPGs and high expression of PYHIN DNA sensors. Both of these factors should be considered in the development of future viral and non-viral T cell gene delivery methods.
Project description:CRISPR-based epigenome editing was recently used to activate gene expression through direct transcriptional activation or site-specific DNA demethylation. Viral delivery of guide RNAs for these purposes remains to be developed. Furthermore, currently available viral delivery tools for genome editing show meager rates of heritability. Here, we have developed a tobacco rattle virus (TRV)-based guide RNA delivery system for both transcriptional activation and targeted DNA demethylation. To promote heritable epigenome editing specifically within plant meristems and the germline, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome, thus facilitating cell-to-cell movement of the RNA in plants. We achieved up to ~8% heritability of the induced phenotype in the progeny of virus-inoculated plants and 25% in the following generation, indicating high rates of heritability for targeted DNA demethylation. Thus, TRV delivery, in combination with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.
Project description:Cell cycle progression into mitosis induce cellular rearrangements such as mitotic spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Like certain retroviruses, nuclear delivery of HPV16 genomes is facilitated by these processes during entry into host cells by tethering of the viral DNA to mitotic chromosomes through the minor capsid protein L2. However, the mechanism of delivery onto and tethering to the condensed chromosomes is barely understood on a mechanistic level. To date it is unclear, which cellular proteins facilitate this process in interaction with L2 or how this process is regulated. Here, we discovered that HPV16 minor capsid protein L2 is phosphorylated during entry upon mitosis onset on conserved residues within the chromosome-binding region (CBR) that is responsible for nuclear import. The crucial L2 phosphorylations occurred sequentially by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, not only regulated timely delivery of HPV16 vDNA to mitotic chromatin at mitosis onset, but also likely resulted in a conformational switch in L2 that allowed engagement of cellular proteins for this purpose. In summary, our work demonstrates for the first time a crucial role of mitotic kinases for nuclear entry of a DNA virus and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.
Project description:In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with a polycationic vector based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetic model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.
Project description:Analysis of gene expression changes due to nonviral gene delivery of DNA lipoplexes versus control in human HEK293T cells. Human HEK293T RNA was isolated from control, non-transfected cells (CTR) and transfected (GFP) samples for analysis on microarrays with three biological replicates.
Project description:A pressing clinical challenge is identifying the etiologic basis of acute respiratory illness. Without reliable diagnostics, the uncertainty associated with this clinical entity leads to a significant, inappropriate use of antibacterials. Use of host peripheral blood gene expression data to classify individuals with bacterial infection, viral infection, or non-infection represents a complementary diagnostic approach. Patients with respiratory tract infection along with ill, non-infected controls were enrolled through the emergency department or undergraduate student health services. Whole blood was obtained to generate gene expression profiles. These profiles were then used to generate signatures of bacterial acute respiratory infection, viral acute respiratory infection, and non-infectious illness. 273 subjects were ascertained for this analysis. This included 88 patients with non-infectious illness, 115 with viral acute respiratory infection, and 70 with bacterial acute respiratory infection. Samples were obtained at the time of enrollment, which was at initial clinical presentation. Total RNA was extracted from human blood using the PAXgene Blood RNA Kit. Microarray data were generated using the GeneChip Human Genome U133A 2.0 Array. Microarrays were generated in two microarray batches with seven overlapping samples giving rise to 280 total microarray experiments.