Project description:Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86-216 nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies.
Project description:The astonishing findings that active and passive immunization against amyloid-β (Aβ) in mouse models of Alzheimer's disease (AD) dramatically decreased amyloid burden led to a rapid initiation of human clinical trials with much enthusiasm. However, methodological issues and adverse effects relating to these clinical trials arose, challenging the effectiveness and safety of these reagents. Efforts are now underway to develop safer immunotherapeutic approaches toward Aβ and the treatment of individuals at risk for AD before or in the earliest stages of cognitive decline with new hopes. Furthermore, several studies have shown tau as a potential immunotherapeutic target for the treatment of tauopathy-related diseases including frontotemporal lobar dementia (FTLD). Both active and passive immunization targeting tau in mouse models of tauopathy effectively decreased tau pathology while improving cognitive performance. These preclinical studies have highlighted tau as an alternative target with much anticipation of clinical trials to be undertaken.
Project description:Alzheimer's disease (AD) is characterized by extracellular amyloid β (Aβ) deposition and intracellular tau aggregation. Many studies have indicated some association between these processes, but it remains unknown how the two pathologies are linked. In this study, we investigated whether expression of amyloid precursor protein (APP) influences extracellular seed-dependent intracellular tau accumulation in cultured cells. Treatment of tau-expressing SH-SY5Y cells with Aβ fibrils did not induce intracellular tau aggregation. On the other hand, in cells expressing both tau and APP, treatment with tau fibrils or Sarkosyl-insoluble tau from AD brains induced intracellular tau aggregation. The seed-dependent intracellular tau aggregation was not induced by expression of APP lacking the extracellular domain. The amount of phosphorylated tau aggregates in cultured cells was dose dependently elevated in response to increased levels of APP on the cell membrane. Our results indicate that the extracellular region of APP is involved in uptake of tau fibrils into cells, raising the possibility that APP, but not Aβ, influences cell-to-cell spreading of tau pathologies in AD by serving as a receptor of abnormal tau aggregates.
Project description:Pathologically altered tau protein is a common denominator of neurodegenerative disorders including Alzheimer's disease (AD) and other tauopathies. Therefore, promising immunotherapeutic approaches target and eliminate extracellular pathogenic tau species, which are thought to be responsible for seeding and propagation of tau pathology. Tau isoforms in misfolded states can propagate disease pathology in a template-dependent manner, proposed to be mediated by the release and internalization of extracellular tau. Monoclonal antibody DC8E8, binding four highly homologous and independent epitopes in microtubule-binding domain (MTBD) of diseased tau, inhibits tau-tau interaction, discriminates between healthy and pathologically truncated tau and reduces tau pathology in animal model in vivo. Here, we show that DC8E8 antibody acts via extracellular mechanism and does not influence viability and physiological functions of neurons. Importantly, in vitro functional assays showed that DC8E8 recognises pathogenic tau proteins of different size and origin, and potently blocks their entry into neurons. Next, we examined the mechanisms by which mouse antibody DC8E8 and its humanized version AX004 effectively block the neuronal internalization of extracellular AD tau species. We determined a novel mode of action of a therapeutic candidate antibody, which potently inhibits neuronal internalization of AD tau species by masking of epitopes present in MTBD important for interaction with neuron surface Heparan Sulfate Proteoglycans (HSPGs). We show that interference of tau-heparane sulfate interaction with DC8E8 antibody via steric hindrance represents an efficient and important therapeutic approach halting tau propagation.
Project description:Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR). ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5' untranslated region. After excision of the intron, transfer RNA ligase joins the severed exons, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue. Hac1 in turn drives the UPR gene expression program comprising 7-8% of the yeast genome to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3' untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3' untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.
Project description:The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.
Project description:Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.
Project description:Silver has been widely used for disinfection. The cellular accumulation of silver ions (Ag+) is critical in these antibacterial effects. The direct cellular measurement of Ag+ is useful for the study of disinfection mechanisms. Herein, we reported a novel genetically encoded RNA-based sensor to image Ag+ in live bacterial cells. The sensor is designed by introducing a cytosine-Ag+-cytosine metallo base pair into a fluorogenic RNA aptamer, Broccoli. The binding of Ag+ induces the folding of Broccoli and activates a fluorescence signal. This sensor can be genetically encoded to measure the cellular flux and antibacterial effect of Ag+.
Project description:Splicing and nuclear export are vital components of eukaryotic gene expression. Defects in splicing due to cis mutations are known to cause a number of human diseases. Here we present a dual reporter system that can be used to look at splicing or export deficiencies resulting from an insufficiency in components of the cotranscriptional machinery. The constructs use a bidirectional promoter to coexpress a test reporter and a control reporter. In the splicing construct, maximal expression of the test reporter is dependent on efficient splicing and splicing-related nuclear export, whereas the control reporter is an intronless complementary DNA expression cassette. The dual reporters allow a robust ratiometric output that is independent of cell number or transfection efficiency. Therefore, our construct is internally controlled and amenable to high-throughput analysis. As a counterscreen, we have a nonsplicing control construct in which neither reporter bears an intron. We demonstrate the sensitivity of our construct to defects in nuclear export by depleting UAP56 and NXF1, essential components of the cotranscriptional machinery.
Project description:Extracellular vesicles (EVs) have been shown as key mediators of extracellular small RNA transport. However, carriers of cell-free messenger RNA (cf-mRNA) in human biofluids and their association with cancer remain poorly understood. Here, we performed a transcriptomic analysis of size-fractionated plasma from lung cancer, liver cancer, multiple myeloma, and healthy donors. Morphology and size distribution analysis showed the successful separation of large and medium particles from other soluble plasma protein fractions. We developed a strategy to purify and sequence ultra-low amounts of cf-mRNA from particle and protein enriched subpopulations with the implementation of RNA spike-ins to control for technical variability and to normalize for intrinsic drastic differences in cf-mRNA amount carried in each plasma fraction. We found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. We observed specific enrichment patterns of cancer-associated cf-mRNA in each particle and protein enriched subpopulation. The EV-enriched differentiating genes were associated with specific biological pathways, such as immune systems, liver function, and toxic substance regulation in lung cancer, liver cancer, and multiple myeloma, respectively. Our results suggest that dissecting the complexity of EV subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.