Transcriptional Dynamics of NRF2 Overexpression and KEAP1-NRF2 Inhibitors in Human Cell Line and Primary Lung Cells.

Oxidative stress in the human lung is caused by both internal (e.g., inflammation) and external stressors (smoking, pollution, and infection) to drive pathology in a number of lung diseases.

Living Donor Liver Transplantation in a Cohort of Recipients With Left Ventricular Systolic Dysfunction.

Background: Data on feasibility, management, and outcomes of liver transplantation (LT) in patients with pre-existing left ventricular systolic dysfunction (LVSD), severe coronary artery disease (CAD) or cirrhotic cardiomyopathy (CCM) is scarce.

Methods: We reviewed outcomes of living donor liver transplantation (LDLT) in recipients with LVSD (ejection fraction [EF] < 50%) from our series of 1946 LDLT's performed between July 2010 and July 2018.

Results: LVSD was detected in 12 male patients with a mean age, BMI and MELD of 52 ± 9 years, 25 ± 5 kg/m, and 19 ± 4 respectively.

Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active.

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice.

Delivery of mRNA Therapeutics for the Treatment of Hepatic Diseases.

Promising improvements in the field of transcript therapeutics have clearly enhanced the potential of mRNA as a new pillar for protein replacement therapies. Synthetic mRNAs are engineered to replace mutated mRNAs and to be immunologically inconspicuous and highly stable while maximizing protein expression. Approaches to deliver mRNA into the cellular cytoplasm safely and efficiently have been further developed so that two mRNA-based approaches replacing vascular endothelial growth factor (VEGF) and cystic fibrosis transmembrane conductance regulator (CFTR) have now made it into clinical trials.

Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life.

Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in , resulting in extensive shortening of the poly(A) tail.

An Improved, Chemically Modified RNA Encoding BMP-2 Enhances Osteogenesis In Vitro and In Vivo.

The use of chemically modified RNA (cmRNA) with increased stability using translation initiator of short untranslated regions (TISU) offers the prospect of finally allowing us to unlock the potent osteogenic properties of BMP-2 in a clinically expedient manner. As noted, delivery of recombinant BMP-2 protein has had modest clinical efficacy, whereas gene delivery is effective but very difficult to translate into human clinical use. This study shows the great potential of cmRNA encoding BMP-2 with TISU in a long-bone critical-sized rat model.

Exploring Cytotoxic mRNAs as a Novel Class of Anti-cancer Biotherapeutics.

New treatments to overcome the obstacles of conventional anti-cancer therapy are a permanent subject of investigation. One promising approach is the application of toxins linked to cell-specific ligands, so-called immunotoxins. Another attractive option is the employment of toxin-encoding plasmids.

Chemically Modified Messenger RNA: Modified RNA Application for Treatment of Achilles Tendon Defects.

Different regenerative medicine approaches for tendon healing exist. Recently, especially gene therapy gained popularity. However, potential mutagenic and immunologic effects might prevent its translation to clinical research.

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.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34 cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size.

Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA.

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form.

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

Modified nucleotide chemistries that increase the half-life (T) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail.

Modified mRNA for BMP-2 in Combination with Biomaterials Serves as a Transcript-Activated Matrix for Effectively Inducing Osteogenic Pathways in Stem Cells.

Bone regeneration using stem cells and growth factors has disadvantages while needing to use supraphysiological growth factors concentrations. Gene therapy has been proposed as alternative, but also has limitation. Messenger RNA (mRNA)-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations.

Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA.

Chemically modified RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats.

Limitations associated to the use of growth factors represent a major hurdle to musculoskeletal regeneration. On the one hand, they are needed to induce neo-tissue formation for the substitution of a necrotic or missing tissue. On the other hand, these factors are used in supraphysiological concentrations, are short lived and expensive and result in many side effects.

Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays.

The measurement of mRNA turnover in living cells plays an important role in the search for stable mRNA constructs for RNA-based therapies. Here we show that automated time-lapse microscopy combined with micropatterned arrays allows for efficient high-throughput monitoring of fluorescent reporter protein expression at the single-cell level. The fluorescence time courses after mRNA transfection yield the distribution of individual mRNA expression and degradation rates within a population.

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.

Aerosolized BC-819 inhibits primary but not secondary lung cancer growth.

Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma.

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.

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice.

Efficient, specific and targeted delivery of genes to the lung.

Many inherited and acquired pulmonary disorders without satisfactory therapies may be amenable to gene therapy. Despite numerous advances, efficient delivery and expression of the therapeutic transgene at physiological levels for phenotypic correction of disease has proved elusive. This article focuses on various strategies aimed at achieving targeted delivery to the lungs.

Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues.

Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot.

Vectors for pulmonary gene therapy.

The success of gene transfer in preclinical animal models and proof of principle clinical studies has made gene therapy an attractive concept for disease treatment. A variety of diseases affecting the lung are candidates for gene therapy. Delivery of genes to the lungs seems to be straightforward, because of the easy accessibility of epithelial cells via the airways.