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

Sahu, Itishri; Haque, Akm Ashiqul; Weidensee, Brian; Weinmann, Petra; Kormann, Michael

doi:10.1016/j.ymthe.2019.02.019

Cited by 67 publications

(66 citation statements)

References 327 publications

(351 reference statements)

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“…We observed that both chemically modified or unmodified mRNA were accumulated in high abundance in two separate donors compare to the control samples. The mRNA entering the cell in this procedure make the whole blood assay a viable technique to check TLR (3,7 and 8), RIG‐1, and MDA5 mediated immune responses . However, we did not clearly understand the mechanism of reduced DsRed protein expression despite of the presence of magnitude of mRNA transcripts in whole blood cells.…”

Section: Discussionmentioning

confidence: 59%

RNA ImmunoGenic Assay: Simple method for detecting immunogenicity of in vitro transcribed mRNA

et al. 2020

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In vitro transcribed (IVT) mRNA therapies is a promising approach for the effective and safe treatment of various diseases. However, IVT‐mRNA triggers immune responses due to recognition by human RNA sensors, but incorporation of chemically modified nucleosides have been shown to reduce such responses. Nonetheless, an assay reflecting the complexity of the human immune system is still needed. Here, we present a simple and fast ex vivo method called “RNA Immunogenic Assay” for measuring the immunogenicity of IVT‐mRNA in human whole blood. Chemically modified and unmodified mRNA were complexed with a transfection reagent (TransIT), and co‐incubated in human whole blood and specific cytokines were quantified (TNF‐α, INF‐α, IL‐6, and IL‐12p70) using ELISAs. The qPCR analysis was conducted to unveil the activation of specific immune pathway. Our findings demonstrated that the complete replacement of uridine with pseudouridine reduced TNF‐α, IL‐6, and IL‐12p70 levels and did not elevate the expression of genes involved in immune response against RNA including IRF7/3, EIF2A, & RNASEL. In addition, we observed that the transcript length was not found to be a confounding factor in RNA immunogenicity generation and our assay provide the feasibility to dissect donor specific immune response against mRNA therapeutics.

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Section: Discussionmentioning

confidence: 59%

RNA ImmunoGenic Assay: Simple method for detecting immunogenicity of in vitro transcribed mRNA

et al. 2020

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“…Establishing safe, e cient and reproducible nebulized IVT-mRNA formulations will become the basis of successful gene therapy for various lung based and respiratory diseases, such as cystic brosis and AATD. However, challenges remain in optimizing IVT-mRNA delivery to the lung [19,20], and there are yet very few studies focusing on the nebulized IVT-mRNA formulations designed for AATD treatment. As a result, we set out to investigate optimal conditions for the preparation of A1AT-mRNA/Lipofectamine2000 complexes that can produce su cient A1AT after the nebulization process.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary based Alpha-1-Antitrypsin Deficiency Treatment

Guan

Darmstaedter

et al. 2021

Preprint

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Background: In vitro transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The pulmonary delivery of IVT-mRNA encoding alpha-1-antitrypsin (A1AT) is a promising strategy for protein replacement therapy of alpha-1-antitrypsin deficiency (AATD). The nebulized A1AT-mRNA formulations would be a highly acceptable and tolerable remedy for the AATD patients in the future. Method: we first optimized parameters that influencing the transfection efficiency of formulations containing IVT-mRNA and Lipofectamine2000 based on human bronchial epithelial cells transfection. Cell viability was evaluated by performing MTT assay after transfection with different IVT-mRNA lipoplexes. Functional analysis was employed to assess the biological function of A1AT proteins produced from optimized formulations using anti-trypsin assay and anti-elastase assay. Results: Lipoplexes prepared by IVT-mRNA encoding A1AT (A1AT-mRNA) in optimum conditions could successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial amount of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and could successfully inhibit the enzyme activity of trypsin as well as elastase. Conclusion: Aerosolization of A1AT-mRNA therapeutic constitute a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT while bringing along the unique advantages of IVT-mRNA.

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“…mRNA expression of hACE2 removes these barriers allowing for hACE2 expression in multiple species using a single construct. Additionally, mRNAs can be targeted to specific organs including the liver and lungs [ 49 , 50 ], allowing for localized expression of hACE2 to study the impact of SARS-CoV-2 on specific organs. Finally, as we and others have shown, the mRNA transfection is poorly immunogenic, so repeated administration of the same or different constructs can be administered to the same animal over a prolonged period of time.…”

Section: Discussionmentioning

confidence: 99%

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2

et al. 2020

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The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Critical to the rapid evaluation of vaccines and antivirals against SARS-CoV-2 is the development of tractable animal models to understand the adaptive immune response to the virus. To this end, the use of common laboratory strains of mice is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Importantly, using this system, we functionally identified the CD4+ and CD8+ structural peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice and confirmed their existence in an established model of SARS-CoV-2 pathogenesis. We demonstrated that, identical to what has been seen in humans, the antigen-specific CD8+ T cells in mice primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. As the focus of the immune response in mice is highly similar to that of the humans, the identification of functional murine SARS-CoV-2-specific T cell epitopes provided in this study will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2 infection.

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

Cited by 67 publications

References 327 publications

RNA ImmunoGenic Assay: Simple method for detecting immunogenicity of in vitro transcribed mRNA

RNA ImmunoGenic Assay: Simple method for detecting immunogenicity of in vitro transcribed mRNA

In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary based Alpha-1-Antitrypsin Deficiency Treatment

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2

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