Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.
Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.
The N 6 -(isopentenyl)adenosine (i 6 A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codonspecific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the D 2 -isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1 + in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA Trp CCA from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA Trp CCA is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA Trp CCA to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA Cys GCA crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA Trp CCA consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.
Mesenchymal stem cell (MSC)‐derived extracellular vesicles (EVs) have emerged as potential therapeutic agents for numerous applications. EVs offer potential advantages over cell‐based therapies with regard to safety, stability and clearance profiles, however production and potency limitations must be addressed to enable eventual translation of EV‐based approaches. Thus, we sought to examine the role of specific cell culture parameters on MSC EV production and bioactivity toward informing rational design parameters for scalable EV biomanufacturing. We report significantly reduced MSC EV vascularization bioactivity, as measured by an endothelial cell gap closure assay, with increasing passage in culture by trypsinization, especially beyond passage 4. We further show that increased frequency of EV collection yielded higher numbers of EVs from the same initial number of MSCs over a 24 hr period. Finally, we demonstrate that decreased cell seeding density in culture flasks resulted in increased production of EVs per cell in MSCs and other cell types. Overall, these studies highlight the need for careful consideration of the parameters of cell passage number and cell seeding density in the production of therapeutic EVs at laboratory scale and for rational design of large‐scale EV biomanufacturing schemes.
In all kingdoms of life, RNAs undergo specific post-transcriptional modifications. More than 100 different analogues of the four standard RNA nucleosides have been identified. Modifications in ribosomal RNAs are highly prevalent and cluster in regions of the ribosome that have functional importance, a high level of nucleotide conservation, and that typically lack proteins. Modifications also play roles in determining antibiotic resistance or sensitivity. A wide spectrum of chemical diversity from the modifications provides the ribosome with a broader range of possible interactions between ribosomal RNA regions, transfer RNA, messenger RNA, proteins, or ligands by influencing local ribosomal RNA folds and fine-tuning the translation process. The collective importance of the modified nucleosides in ribosome function has been demonstrated for a number of organisms, and further studies may reveal how the individual players regulate these functions through synergistic or cooperative effects.In all kingdoms of life, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other RNAs undergo specific post-transcriptional modification by a wide variety of enzymes (1). To date, >100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (2). These modifications can be organized into four main types (Figure 1) (1). The first involves isomerization of uridine to pseudouridine (5-ribosyluracil, Ψ), which contains a C-rather than the typical N-glycosidic linkage, as well as an additional imino group that is available for unique hydrogen-bonding interactions. The second includes alterations to the bases, such as methylation (typically on carbon, primary nitrogen, or tertiary nitrogen), deamination (e.g., inosine), reduction (e.g., dihydrouridine), thiolation, or alkylation (e.g., isopentenylation or threonylation). The third involves methylation of the ribose 2′ hydroxyl (Nm). The fourth type includes more complex modifications, such as multiple modifications (e.g., 5-methylaminomethyl-2-thiouridine; 3-(3-amino-3-carboxypropyl)uridine, acp 3 U; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, m 1 acp 3 Ψ) or "hypermodifications" that can be incorporated by specific exchange mechanisms (e.g., queuosine). The possible electronic and steric effects of the nucleoside modifications on base pairing, base stacking, and sugar pucker in RNA have been discussed in detail by Davis (3) and Agris (4), among others (1). The effects of modifications such as Ψ on RNA hydration and dynamics have also been considered (4).Modified nucleotides in the ribosome are varied in their identity, but highly localized in their positions (5). If the sites of modification are mapped on the secondary structures of the small and large subunit (SSU and LSU) rRNAs, they might appear to be random; however, if the same modifications are located within the ribosome tertiary structures from high-resolution Xray crystal structures (6,7), they occur in the mo...
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