MicroRNA-511-3p Mediated Modulation of the Peroxisome Proliferator-Activated Receptor Gamma (PPAR?) Controls LPS-Induced Inflammatory Responses in Human Monocyte Derived DCs

Abstract: The peroxisome proliferator-activated receptor gamma (PPAR) is a ligand-activated transcription factor expressed in dendritic cells (DCs), where it exerts anti-inflammatory responses against TLR4-induced inflammation. Recently, microRNA-511 (miR 511) has also emerged as a key player in controlling TLR4-mediated signalling and in regulating the function of DCs. Interestingly, PPAR has been previously highlighted as a putative target of miR-511 activity; however, the link between miR-511 and PPAR and its influence on human DC function within the context of LPS-inducedinflammatory responses is unknown. Using a selection of miR-511-3p-specific inhibitors and mimics, we demonstrate for the first time that knockdown or overexpression of miR-511-3p inversely correlates with PPAR mRNA levels and affects its transcriptional activity following treatment with rosiglitazone (RSG; PPAR agonist), in the presence or absence of LPS. Additionally, we show that PPAR-mediated suppression of DC activation and pro-inflammatory cytokine production in miR-511-3p knockdown DCs is abrogated following overexpression of miR-511-3p. Lastly, PPARactivation suppressed LPS-mediated induction of indoleamine 2,3-dioxygenase (IDO) activity in DCs, most likely due to changes in miR-511-3p expression. Our data thus suggests that PPAR-induced modulation of DC phenotype and function is influenced by miR-511 3p expression, which may serve as a potential therapeutic target against inflammatory diseases

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Adeno-associated viruses (AAVs) are useful vehicles for gene therapy because of their stability, low immunogenicity. and non-pathogenicity. However, disparity in AAV sample preparations (e.g., in capsid composition, DNA packaging, and impurities) gives rise to product heterogeneity, with possibly undesired effects on gene delivery. Ideally, AAV production should be with full control of AAV structure and genetic payload. Therefore, robust, efficient, and low material consuming methods are essential to characterize AAVs. Here, we use two emerging single-molecule techniques, mass photometry and Orbitrap-based charge-detection mass spectrometry, and show how they may efficiently and accurately characterize AAVs. We were able to resolve heterogeneous pools of particles, evaluating AAVs from two different serotypes (AAV8 and AAV2), produced by three independent production platforms, either lacking a genome or packed with a transgene. Together our data confirm that the different AAV production methods result in rather different and diverse AAV particle distributions. Especially for the packed AAVs, frequently additional subspecies were observed, next to the expected packed genome, mostly resulting from under- or overpackaging of genome material and/or residual empty particles. This work further establishes that both these single-particle techniques may become valuable tools in characterizing AAVs before they are used in gene therapy

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Adeno-associated viruses (AAVs) are useful vehicles for gene therapy because of their stability, low immunogenicity. and non-pathogenicity. However, disparity in AAV sample preparations (e.g., in capsid composition, DNA packaging, and impurities) gives rise to product heterogeneity, with possibly undesired effects on gene delivery. Ideally, AAV production should be with full control of AAV structure and genetic payload. Therefore, robust, efficient, and low material consuming methods are essential to characterize AAVs. Here, we use two emerging single-molecule techniques, mass photometry and Orbitrap-based charge-detection mass spectrometry, and show how they may efficiently and accurately characterize AAVs. We were able to resolve heterogeneous pools of particles, evaluating AAVs from two different serotypes (AAV8 and AAV2), produced by three independent production platforms, either lacking a genome or packed with a transgene. Together our data confirm that the different AAV production methods result in rather different and diverse AAV particle distributions. Especially for the packed AAVs, frequently additional subspecies were observed, next to the expected packed genome, mostly resulting from under- or overpackaging of genome material and/or residual empty particles. This work further establishes that both these single-particle techniques may become valuable tools in characterizing AAVs before they are used in gene therapy