IL-10 mRNA Engineered MSCs Demonstrate Enhanced Anti-Inflammation in an Acute GvHD Model

Mesenchymal stem cells (MSCs) are used in various studies to induce immunomodulatory effects in clinical conditions associated with immune dysregulation such as graft versus host disease (GvHD). However, most of these clinical trials failed to go beyond early phase 2 studies because of limited efficacy. Various methods have been assessed to increase the potency of MSCs. IL-10 is an anti-inflammatory cytokine that is known to modulate immune responses in GvHD. In this study, we evaluated the feasibility of transfecting IL-10 mRNA to enhance MSC therapeutic potential. IL-10 mRNA engineered MSCs (eMSCs-IL10) maintained high levels of IL-10 expression even after freezing and thawing. IL-10 mRNA transfection did not appear to alter MSC intrinsic characteristics. eMSCs-IL10 significantly suppressed T cell proliferation relative to naïve MSCs in vitro. In a mouse model for GvHD, eMSCs-IL10 induced a decrease in plasma level of potent pro-inflammatory cytokines and inhibited CD4+ and CD8+ T cell proliferation in the spleen. In summary, our studies demonstrate the feasibility of potentiating MSCs to enhance their immunomodulatory effects by IL-10 mRNA transfection. The use of non-viral transfection may generate a safe and potent MSC product for treatment of clinical conditions associated with immune dysregulation such as GvHD.

Concentration of Na+-taurocholate-cotransporting polypeptide expressed after in vitro-transcribed mRNA transfection determines susceptibility of hepatoma cells for hepatitis B virus

Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system.

Identifiability analysis for models of the translation kinetics after mRNA transfection

Mechanistic models are a powerful tool to gain insights into biological processes. The parameters of such models, e.g. kinetic rate constants, usually cannot be measured directly but need to be inferred from experimental data. In this article, we study dynamical models of the translation kinetics after mRNA transfection and analyze their parameter identifiability. Previous studies have considered ordinary differential equation (ODE) models of the process, and here we formulate a stochastic differential equation (SDE) model. For both model types, we consider structural identifiability based on the model equations and practical identifiability based on simulated as well as experimental data and find that the SDE model provides better parameter identifiability than the ODE model. Moreover, our analysis shows that even for those parameters of the ODE model that are considered to be identifiable, the obtained estimates are sometimes unreliable. Overall, our study clearly demonstrates the relevance of considering different modeling approaches and that stochastic models can provide more reliable and informative results.

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities, but they possess limited genetic tools due to challenges in gene delivery, expression, and detection, despite decades of work. We developed a toolbox for heterologous protein expression in planarian cells and in live animals. Specifically, we identified and optimized nanotechnological and chemical transfection methods to efficiently deliver mRNA encoding nanoluciferase into somatic cells, including planarian adult stem cells (neoblasts). The use of a luminescent reporter allowed us to quantitatively measure protein expression through spectroscopy and microscopy, thus overcoming the strong autofluorescent background of planarian tissues. Using this platform, we investigated the use of endogenous untranslated region (UTR) sequences and codon usage bias to post-transcriptionally alter gene expression. Our work provides a strong foundation for advancing exogenous gene expression and for the rapid prototyping of genetic constructs to accelerate the development of transgenic techniques in planarians.

YAF2-mediated YY1-Sirtuin6 interactions responsible formitochondrial down-regulation in aging tunicates

In budding tunicates, aging accompanies a decrease in the gene expression of mitochondrial transcription factor A (Tfam), and the in vivo transfection of Tfam mRNA stimulates the mitochondrial respiratory activity of aged animals. The gene expression of both the transcriptional repressor Yin-Yang-1 (YY1) and corepressor Sirtuin6 (Sirt6) increased during aging, and the co-transfection of synthetic mRNA of YY1 and Sirt6 synergistically down-regulated Tfam gene expression. Pull-down assays of proteins indicated that YY1-associated factor 2 (YAF2) was associated with both YY1 and SIRT6. Protein cross-linking confirmed that YAF2 bound YY1 and SIRT6 with a molar ratio of 1:1. YY1 was bound to CCAT- or ACAT-containing oligonucleotides in the 5′ flanking region of the Tfam gene. ChIP-qPCR showed that SIRT6 specifically induced the histone H3K9 deacetylation of the Tfam upstream region. YY1 and YAF2 accelerated SIRT6-induced H3K9 deacetylation. YY1 and Sirt6 mRNA transfection attenuated mitochondrial respiratory gene expression and blocked MitoTracker fluorescence. In contrast, the SIRT6 inhibitor and Tfam mRNA antagonized the inhibitory effects of YY1 and Sirt6, indicating that Tfam acts on mitochondria downstream of YY1 and Sirt6. We concluded that in the budding tunicate, Polyandrocarpa misakiensis, YY1 recruits SIRT6 via YAF2 to the TFAM gene, resulting in aging-related mitochondrial down-regulation.

CD40 signaling augments IL-10 expression and the tolerogenicity of IL-10-induced regulatory dendritic cells

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells’ abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.