RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of Transcribed mRNA in Human Whole Blood.

The mRNA therapeutics is a new class of medicine to treat many various diseases. However, transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required.

Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34 HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.

β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production.

Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases.

Protein supplementation therapy using in vitro-transcribed (IVT) mRNA for genetic diseases contains huge potential as a new class of therapy. From the early ages of synthetic mRNA discovery, a great number of studies showed the versatile use of IVT mRNA as a novel approach to supplement faulty or absent protein and also as a vaccine. Many modifications have been made to produce high expressions of mRNA causing less immunogenicity and more stability.

A bioactive collagen membrane that enhances bone regeneration.

Membranes are an integral component of guided bone regeneration protocols. This pre-clinical study was aimed at enhancing the bioactivity of collagen membranes by incorporating plasmid DNA (pDNA) or chemically modified RNA (cmRNA) encoding bone morphogenetic protein-9 (BMP-9). In addition, we also endeavored to harness the regenerative potential of the periosteum by creating perforations in the membrane.

Gene correction of HBB mutations in CD34 hematopoietic stem cells using Cas9 mRNA and ssODN donors.

Background: β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control.

Chemically modified hCFTR mRNAs recuperate lung function in a mouse model of cystic fibrosis.

Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug.

Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification.

The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses.

Correction: mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo.

[This corrects the article DOI: 10.1371/journal.pone.

Transcriptomic profile of cystic fibrosis patients identifies type I interferon response and ribosomal stalk proteins as potential modifiers of disease severity.

Cystic Fibrosis (CF) is the most common monogenic disease among people of Western European descent and caused by mutations in the CFTR gene. However, the disease severity is immensely variable even among patients with similar CFTR mutations due to the possible effect of 'modifier genes'. To identify genetic modifiers, we applied RNA-seq based transcriptomic analyses in CF patients with a mild and severe lung phenotype.

A Comparative Study of the Bone Regenerative Effect of Chemically Modified RNA Encoding BMP-2 or BMP-9.

Employing cost-effective biomaterials to deliver chemically modified ribonucleic acid (cmRNA) in a controlled manner addresses the high cost, safety concerns, and lower transfection efficiency that exist with protein and gene therapeutic approaches. By eliminating the need for nuclear entry, cmRNA therapeutics can potentially overcome the lower transfection efficiencies associated with non-viral gene delivery systems. Here, we investigated the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2.

Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling.

Objective: The generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment.

Design: We designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny.

Results: Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish.

mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo.

Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation.

The oral and craniofacial relevance of chemically modified RNA therapeutics.

Several tissue engineering strategies in the form of protein therapy, gene therapy, cell therapy, and their combinations are currently being explored for oral and craniofacial regeneration and repair. Though each of these approaches has advantages, they all have common inherent drawbacks of being expensive and raising safety concerns. Using RNA (encoding therapeutic protein) has several advantages that have the potential to overcome these limitations.

Modified mRNA as a new therapeutic option for pediatric respiratory diseases and hemoglobinopathies.

Background: The immunogenicity and limited stability of conventional messenger RNA (mRNA) has traditionally restricted its potential therapeutic use. In 1992, the first clinical application of mRNA was reported as a potential protein-replacement therapy; however, subsequent investigations have not been made for almost two decades. Recent developments, including increased stability, controlling immunogenicity, as well as utilization of mRNA encoding zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9, have implicated modified mRNA as a very promising option for cancer immunotherapy, vaccines, protein expression replacement, and genome editing.

Chemically modified RNA activated matrices enhance bone regeneration.

There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2).

Airway mucus obstruction triggers macrophage activation and matrix metalloproteinase 12-dependent emphysema.

Whereas cigarette smoking remains the main risk factor for emphysema, recent studies in β-epithelial Na(+) channel-transgenic (βENaC-Tg) mice demonstrated that airway surface dehydration, a key pathophysiological mechanism in cystic fibrosis (CF), caused emphysema in the absence of cigarette smoke exposure. However, the underlying mechanisms remain unknown. The aim of this study was to elucidate mechanisms of emphysema formation triggered by airway surface dehydration.

Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism.

Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models.

Flagellin induces myeloid-derived suppressor cells: implications for Pseudomonas aeruginosa infection in cystic fibrosis lung disease.

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin.

Expression and regulation of interferon-related development regulator-1 in cystic fibrosis neutrophils.

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects.

Neutrophils express distinct RNA receptors in a non-canonical way.

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells.

CXCR1 and CXCR2 haplotypes synergistically modulate cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity.

The chitinase-like protein YKL-40 modulates cystic fibrosis lung disease.

The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity.

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.