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. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.
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. Recent advancements in the in vivo lung delivery of mRNA complexed with various carriers encouraged the whole mRNA community to tackle various genetic lung diseases. This review gives a comprehensive overview of cells associated with various lung diseases and recent advancements in mRNA-based protein replacement therapy. This review also covers a brief summary of developments in mRNA modifications and nanocarriers toward clinical translation.
mRNA has gathered attention as a new class of therapeutics for different diseases. Progress has been made in the field of protein replacement therapy by developing mRNA into a safe and efficient therapy. This article focuses on the delivery of mRNA therapeutics in the lung, and it provides an overview of lung architecture and associated diseases, such as asthma, cystic fibrosis, COPD, and SP-B deficiency.
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. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.
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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. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for in vivo gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient's individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is-in contrast to conventional viral vectors-non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either Tlr1/2 mRNA or Tlr2/6 mRNA, but not Tlr2 mRNA alone, resulted in better lung function as well as reduced airway inflammation in vivo. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis.
Background Early exposure to microbes reduces the risk for asthma. Toll-like receptors (TLRs) represent a major group of receptors for the specific recognition of pathogen-associated molecular ...patterns of microbes capable of activating innate and adaptive immunity. Objective Because TLRs can influence key events in the induction and perpetuation of asthma and atopy, we sought to determine whether genetic alterations in TLR genes affect asthma risk. Methods We systematically evaluated putatively functional genetic variants in all 10 human TLR genes for their association with different asthma phenotypes in a case-control study (n = 1872) by using matrix-assisted laser desorption/ionization time-of-flight genotyping. For polymorphisms showing association with atopic asthma, effects on gene and protein expression were studied by means of RT-PCR and flow cytometry ex vivo . T-cell cytokine production was evaluated by means of ELISA after stimulation of the respective TLRs with specific ligands. Results Protective effects on atopic asthma were identified for single nucleotide polymorphisms in TLR1 (odds ratio OR, 0.54; 95% CI, 0.37-0.81; P = .002), TLR6 (OR, 0.54; 95% CI, 0.37-0.79; P = .003), and TLR10 (OR, 0.58; 95% CI, 0.39-0.86; P = .006), all capable of forming heterodimers with TLR2 . Effects remained significant after correction for multiple comparisons. PBMCs of minor allele carriers showed increased levels of the respective TLR mRNA and proteins, augmented inflammatory responses, increased TH 1 cytokine expression, and reduced TH 2-associated IL-4 production after specific stimulation. Conclusion These results suggest that functional relevant TLR1 and TLR6 variants are directly involved in asthma development.
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. Polyethylenimine (PEI) was used as a vector to increase
in vitro
transfection efficacy. Complexes of PEI-cmRNA (encoding BMP-2 or BMP-9) were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficacy
in vitro
using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these complexes was determined by evaluating the expression of bone-specific genes as well as through the detection of bone matrix deposition. It was found that alkaline phosphatase (ALP) expression 3 days post transfection in the group treated with
BMP-9
-cmRNA was significantly higher than that in the group that received
BMP-2-
cmRNA treatment. Alizarin red staining and atomic absorption spectroscopy demonstrated enhanced osteogenic differentiation as evidenced by increased bone matrix production by the BMSCs treated with
BMP-9
-cmRNA when compared to cells treated with
BMP-2-
cmRNA.
In vivo
studies showed increased bone formation in calvarial defects treated with the
BMP-9
-cmRNA and
BMP-2-
cmRNA collagen scaffolds when compared to empty defects. The connectivity density of the regenerated bone was higher (2-fold-higher) in the group that received
BMP-9
-cmRNA compared to
BMP-2-
cmRNA. Together, these findings suggest that cmRNA-activated matrix encoding osteogenic molecules can provide a powerful strategy for bone regeneration with significant clinical translational potential.
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. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNA
in vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNA
in vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNA
together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNA
was quantified by hCFTR ELISA, and cmRNA
values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV
, suggesting NP-cmRNA
as promising therapeutic option for CF patients independent of their CFTR genotype.
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. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.
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