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Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid.

ABSTRACT: Gene therapy is a therapeutic strategy of delivering foreign genetic material (encoding for an important protein) into a patient's target cell to replace a defective gene. Nucleic acids are embedded within the adeno-associated virus (AAVs) vectors; however, preexisting immunity to AAVs remains a significant concern that impairs their clinical application. Extracellular vesicles (EVs) hold great potential for therapeutic applications as vectors of nucleic acids due to their endogenous intercellular communication functions through their cargo delivery, including lipids and proteins. So far, small RNAs (siRNA and micro (mi)RNA) have been mainly loaded into EVs to treat several diseases, but the potential use of EVs to load and deliver exogenous plasmid DNA has not been thoroughly described. This review provides a comprehensive overview of the principal methodologies currently employed to load foreign genetic material into EVs, highlighting the need to find the most effective strategies for their successful clinical translations.

SUBMITTER: Orefice NS

PROVIDER: S-EPMC7464422 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid.

Orefice Nicola Salvatore NS Orefice Nicola Salvatore NS

Pharmaceutics 20200726 8

Gene therapy is a therapeutic strategy of delivering foreign genetic material (encoding for an important protein) into a patient's target cell to replace a defective gene. Nucleic acids are embedded within the adeno-associated virus (AAVs) vectors; however, preexisting immunity to AAVs remains a significant concern that impairs their clinical application. Extracellular vesicles (EVs) hold great potential for therapeutic applications as vectors of nucleic acids due to their endogenous intercellul ...[more]

PMID: 32722622

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Project description:BackgroundSmall extracellular vesicles from adipose-derived stem cells (ASC-sEVs) have gained remarkable attention for their regenerative and protective properties against skin aging. However, the use of ASC-sEVs to further encapsulate certain natural anti-aging compounds for synergistic effects has not been actively explored. For large-scale production in skincare industry, it is also crucial to standardize cost-effective methods to produce highly pure ASC-sEVs.MethodsHuman ASCs were expanded in serum-free media with different compositions to first optimize the sEV production. ASC-sEVs from different batches were then purified using tangential flow filtration and sucrose cushion ultracentrifugation, followed by extensive characterization for identity and content profiling including proteomics, lipidomics and miRNA sequencing. ASC-sEVs were further loaded with nicotinamide riboside (NR) and resveratrol by sonication-incubation method. The therapeutic effect of ASC-sEVs and loaded ASC-sEVs was tested on human keratinocyte cell line HaCaT exposed to UVB by measuring reactive oxygen species (ROS). The loaded ASC-sEVs were later applied on the hand skin of three volunteers once a day for 8 weeks and skin analysis was performed every 2 weeks.ResultsOur standardized workflow produced ASC-sEVs with high yield, high purity and with stable characteristics and consistent biocargo among different batches. The most abundant subpopulations in ASC-sEVs were CD63+ (∼30%) and CD81+ -CD63+ (∼35%). Purified ASC-sEVs could be loaded with NR and resveratrol at the optimized loading efficiency of ∼20%. In UVB-exposed HaCaT cells, loaded ASC-sEVs could reduce ROS by 38.3%, higher than the sEVs (13.3%) or compounds (18.5%) individually. In human trial, application of loaded ASC-sEVs after 8 weeks substantially improved skin texture, increased skin hydration and elasticity by 104% and reduced mean pore volume by 51%.ConclusionsThis study demonstrated a robust protocol to produce ASC-sEVs and exogenously load them with natural compounds. The loaded ASC-sEVs exhibited synergistic effects of both sEVs and anti-aging compounds in photoaging protection and skin rejuvenation.

Project description:The aim of this study was to investigate cell source-dependent nucleic acids repertoire of diverse subpopulations of plasma extracellular vesicles (EVs). Blood plasma from nine healthy volunteers was used for the analysis. Samples of EVs were obtained by differential centrifugation of plasma. The application of high-sensitivity fluorescence-activated vesicles sorting (hs-FAVS) using fluorophore-conjugated anti-CD41-FITC (Fluorescein isothiocyanate) and anti-CD235a-PE antibodies allowed the isolation of three subpopulations of EVs, namely CD41+ CD235a-, CD41-CD235a+ and CD41-CD235a dim. The high purity (>97%) of the sorted subpopulations was verified by high-sensitivity flow cytometry. Presence of nanosized objects in sorted samples was confirmed by combination of low-voltage scanning electron microscopy and dynamic light scattering. The amount of material in sorted samples was enough to perform Quantitative polymerase chain reaction (qPCR)-based nucleic acid quantification. The most prominent differences in the nucleic acid repertoire were noted between CD41+ CD235- vs. CD41-CD235a+ vesicles: the former contained significantly (p = 0.004) higher amount of mitochondrial DNA, and platelet enriched miR-21-5p (4-fold), miR-223-3p (38-fold) and miR-199a-3p (187-fold), but lower amount of erythrocyte enriched miR-451a (90-fold). CD41-CD235a+ and CD41-CD235a dim vesicles differed in levels of miR-451a (p = 0.016) and miR-21-5p (p = 0.031). Nuclear DNA was below the limit of detection in all EV subpopulations. The hs-FCM-based determination of the number of sorted EVs allowed the calculation of per single-event miRNA concentrations. It was demonstrated that the most abundant marker in CD41+ CD235a- subpopulation was miR-223-3p, reaching 38.2 molecules per event. In the CD41-CD235+ subpopulation, the most abundant marker was miR-451a, reaching 24.7 molecules per event. Taken together, our findings indicate that erythrocyte- and platelet-derived EVs carry different repertoires of nucleic acids, which were similar to the composition of their cellular sources.

Dataset Information

Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid.

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Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid.

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