Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5'-UTRs.

The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These findings were confirmed with a human erythropoietin (hEPO)-encoding mRNA. Significantly, higher levels of hEPO could be quantified in supernatants from A549 cells transfected with minimal 5'-UTR7 containing RNA when compared to commonly used benchmarks 5'-UTRs. Our results demonstrate the superior potential of synthetic minimalistic 5'-UTRs for use in transcript therapies.

Magnetized aerosols comprising superparamagnetic iron oxide nanoparticles improve targeted drug and gene delivery to the lung.

PURPOSE: Targeted delivery of aerosols could not only improve efficacy of inhaled drugs but also reduce side effects resulting from their accumulation in healthy tissue. Here we investigated the impact of magnetized aerosols on model drug accumulation and transgene expression in magnetically targeted lung regions of unanesthetized mice. METHODS: Solutions containing superparamagnetic iron oxide nanoparticles (SPIONs) and model drugs (fluorescein or complexed plasmid DNA) were nebulized to unanesthetized mice under the influence of an external magnetic gradient directed to the lungs. Drug accumulation and transgene expression was subsequently measured at different time points. RESULTS: We could demonstrate 2-3 fold higher accumulation of the model drug fluorescein and specific transgene expression in lung regions of mice which had been exposed to an external magnetic gradient during nebulization compared to the control mice without any exposure to magnetic gradient. CONCLUSIONS: Magnetized aerosols present themselves as an efficient approach for targeted pulmonary delivery of drugs and gene therapeutic agents in order to treat localized diseases of the deeper airways.

Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.

Aerosol gene delivery holds great therapeutical potential for many inherited and acquired pulmonary diseases. The physical instability of aqueous suspensions of non-viral vector complexes is a major limitation for their successful application. In this study, we investigated dry powder aerosols as novel gene vector formulations for gene transfer in vitro and murine lungs in vivo. Lyophilization was used to produce dry powder cakes followed by powderization to produce dry powder aerosols. Different sugars, namely lactose, sucrose and trehalose, were tested as lyoprotectants for gene delivery complexes consisting of branched polyethylenimine 25 kDa and plasmid DNA. Biophysical particle characterization demonstrated that lyophilization and powderization in the presence of lyoprotectants were well tolerated. In vitro transfection efficiency remained unaffected by the choice of lyoprotectant and subsequent lyophilization and/or powderization. In vivo screening of powderized samples, by applying the powder with an insufflator, resulted in highest gene expression with lactose as lyoprotectant. Delivering a plasmid coding for murine erythropoietin together with lactose as lyoprotectant resulted in increased blood hematocrit values post application thereby demonstrating the potential of dry powder aerosol as a promising method for pulmonary gene delivery.

Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.