mRNA Therapeutics: A Themed Issue in Honor of Professor Katalin Karikó

A special issue of Pharmaceutics (ISSN 1999-4923). This special issue belongs to the section "Gene and Cell Therapy".

Deadline for manuscript submissions: closed (10 December 2021) | Viewed by 69495

Special Issue Editors


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Guest Editor
School of Pharmacy, University College Cork, Cork T12 YN60, Ireland
Interests: nanotechnology; drug delivery; biomaterials engineering; non-viral gene therapy; nucleic acid delivery; polymeric nanocarriers; cardiovascular disease; diabetes

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Guest Editor
BioNTech RNA Pharmaceuticals, BioNTech SE, D-55131 Mainz, Germany
Interests: mRNA manufacturing; optimization of synthetic mRNA; mRNA quality control; mRNA-based therapeutics; mRNA vaccines; DNA repair

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Guest Editor
1. RNA Protein Replacement Therapies, BioNTech SE, D-55131 Mainz, Germany
2. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Interests: nucleoside-modified RNA; RNA immune recognition; mRNA-based therapeutics; protein replacement therapy; mRNA vaccines

Special Issue Information

Dear Colleagues,

This Special Issue of Pharmaceutics is dedicated to Professor Katalin Karikó in recognition of her immense contribution to the development of mRNA Pharmaceutics. Professor Karikó graduated from the University of Szeged in Hungary, and first began her work on RNA at the Biological Research Center in 1978. She received her PhD in 1982 by studying the antiviral effects of 2’-5’-linked oligoadenylates. In 1985, she continued her career at Temple University, then moved to the University of Pennsylvania in 1989. For four decades, her research has been focusing on RNA-mediated mechanisms with the goal of developing in vitro-transcribed mRNA for protein therapy. She investigated RNA-mediated immune activation and co-discovered that nucleoside modifications suppress the immunogenicity of RNA, which broadened the therapeutic potential of mRNA. In 2013, she moved to Germany and as the Senior Vice President she leads BioNTech RNA Pharmaceuticals to develop mRNA-based therapeutics. Patents co-invented by Professor Karikó on nucleoside-modified mRNA were used to create the anti-SARS-CoV-2 mRNA vaccines.

Professor Karikó co-authored over 80 peer-reviewed articles and is the co-inventor on many mRNA-related patents, including ten granted in the USA for application of non-immunogenic, nucleoside-modified RNA. Professor Karikó also co-founded and served as CEO of RNARx from 2006-2013.

In recognition of Professor Karikó's outstanding career contributions, this Special Issue of Pharmaceutics welcomes the submissions of unpublished original research articles or reviews in the field of RNA therapeutics. Submissions will be accepted until 10 December 2021.

Dr. Piotr Kowalski
Dr. Gábor Boros
Prof. Dr. Katalin Karikó
Guest Editors

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Keywords

  • nucleoside-modified RNA
  • RNA immune recognition
  • mRNA-based therapeutics
  • RNA delivery
  • gene therapy
  • vaccines
  • gene editing
  • protein replacement therapy

Published Papers (10 papers)

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Research

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17 pages, 3509 KiB  
Article
Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA
by Irena Vlatkovic, János Ludwig, Gábor Boros, Gábor Tamás Szabó, Julia Reichert, Maximilian Buff, Markus Baiersdörfer, Jonas Reinholz, Azita Josefine Mahiny, Uğur Şahin and Katalin Karikó
Pharmaceutics 2022, 14(2), 328; https://doi.org/10.3390/pharmaceutics14020328 - 29 Jan 2022
Cited by 20 | Viewed by 11922
Abstract
The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT [...] Read more.
The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics. Full article
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19 pages, 3206 KiB  
Article
Novel N7-Arylmethyl Substituted Dinucleotide mRNA 5′ cap Analogs: Synthesis and Evaluation as Modulators of Translation
by Radoslaw Wojcik, Marek R. Baranowski, Lukasz Markiewicz, Dorota Kubacka, Marcelina Bednarczyk, Natalia Baran, Anna Wojtczak, Pawel J. Sikorski, Joanna Zuberek, Joanna Kowalska and Jacek Jemielity
Pharmaceutics 2021, 13(11), 1941; https://doi.org/10.3390/pharmaceutics13111941 - 16 Nov 2021
Cited by 10 | Viewed by 3885
Abstract
Dinucleotide analogs of the messenger RNA cap (m7GpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position [...] Read more.
Dinucleotide analogs of the messenger RNA cap (m7GpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position with benzyl (Bn) produces a dinucleotide cap with superior properties. Here, we followed up on this finding by synthesizing 17 novel Bn7GpppG analogs and determining their structure–activity relationship regarding translation and translational inhibition. The compounds were prepared in two steps, including selective N7-alkylation of guanosine 5′-monophosphate by arylmethyl bromide followed by coupling with imidazole-activated GDP, with total yields varying from 22% to 62%. The compounds were then evaluated by determining their affinity for eukaryotic translation initiation factor 4E (eIF4E), testing their susceptibility to decapping pyrophosphatase, DcpS—which is most likely the major cellular enzyme targeting this type of compound—and determining their translation inhibitory properties in vitro. We also synthesized mRNAs capped with the evaluated compounds and tested their translational properties in A549 cells. Our studies identified N7-(4-halogenbenzyl) substituents as promising modifications in the contexts of either mRNA translation or translational inhibition. Finally, to gain more insight into the consequences at the molecular level of N7-benzylation of the mRNA cap, we determined the crystal structures of three compounds with eIF4E. Full article
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13 pages, 2497 KiB  
Article
Hydrophobic Optimization of Functional Poly(TPAE-co-suberoyl chloride) for Extrahepatic mRNA Delivery following Intravenous Administration
by Xueliang Yu, Shuai Liu, Qiang Cheng, Sang M. Lee, Tuo Wei, Di Zhang, Lukas Farbiak, Lindsay T. Johnson, Xu Wang and Daniel John Siegwart
Pharmaceutics 2021, 13(11), 1914; https://doi.org/10.3390/pharmaceutics13111914 - 12 Nov 2021
Cited by 7 | Viewed by 3672
Abstract
Messenger RNA (mRNA) has generated great attention due to its broad potential therapeutic applications, including vaccines, protein replacement therapy, and immunotherapy. Compared to other nucleic acids (e.g., siRNA and pDNA), there are more opportunities to improve the delivery efficacy of mRNA through systematic [...] Read more.
Messenger RNA (mRNA) has generated great attention due to its broad potential therapeutic applications, including vaccines, protein replacement therapy, and immunotherapy. Compared to other nucleic acids (e.g., siRNA and pDNA), there are more opportunities to improve the delivery efficacy of mRNA through systematic optimization. In this report, we studied a high-throughput library of 1200 functional polyesters for systemic mRNA delivery. We focused on the chemical investigation of hydrophobic optimization as a method to adjust mRNA polyplex stability, diameter, pKa, and efficacy. Focusing on a region of the library heatmap (PE4K-A17), we further explored the delivery of luciferase mRNA to IGROV1 ovarian cancer cells in vitro and to C57BL/6 mice in vivo following intravenous administration. PE4K-A17-0.2C8 was identified as an efficacious carrier for delivering mRNA to mouse lungs. The delivery selectivity between organs (lungs versus spleen) was found to be tunable through chemical modification of polyesters (both alkyl chain length and molar ratio in the formulation). Cre recombinase mRNA was delivered to the Lox-stop-lox tdTomato mouse model to study potential application in gene editing. Overall, we identified a series of polymer-mRNA polyplexes stabilized with Pluronic F-127 for safe and effective delivery to mouse lungs and spleens. Structure–activity relationships between alkyl side chains and in vivo delivery were elucidated, which may be informative for the continued development of polymer-based mRNA delivery. Full article
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11 pages, 1328 KiB  
Article
Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice
by Natsuko Oyama, Maho Kawaguchi, Keiji Itaka and Shigeru Kawakami
Pharmaceutics 2021, 13(11), 1810; https://doi.org/10.3390/pharmaceutics13111810 - 29 Oct 2021
Cited by 14 | Viewed by 2515
Abstract
Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive [...] Read more.
Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney. Full article
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24 pages, 6680 KiB  
Article
Pharma 4.0 Continuous mRNA Drug Products Manufacturing
by Andreas Ouranidis, Christina Davidopoulou, Reald-Konstantinos Tashi and Kyriakos Kachrimanis
Pharmaceutics 2021, 13(9), 1371; https://doi.org/10.3390/pharmaceutics13091371 - 31 Aug 2021
Cited by 18 | Viewed by 6772
Abstract
Continuous mRNA drugs manufacturing is perceived to nurture flow processes featuring quality by design, controlled automation, real time validation, robustness, and reproducibility, pertaining to regulatory harmonization. However, the actual adaptation of the latter remains elusive, hence batch-to-continuous transition would a priori necessitate holistic [...] Read more.
Continuous mRNA drugs manufacturing is perceived to nurture flow processes featuring quality by design, controlled automation, real time validation, robustness, and reproducibility, pertaining to regulatory harmonization. However, the actual adaptation of the latter remains elusive, hence batch-to-continuous transition would a priori necessitate holistic process understanding. In addition, the cost related to experimental, pilot manufacturing lines development and operations thereof renders such venture prohibitive. Systems-based Pharmaceutics 4.0 digital design enabling tools, i.e., converging mass and energy balance simulations, Monte-Carlo machine learning iterations, and spatial arrangement analysis were recruited herein to overcome the aforementioned barriers. The primary objective of this work is to hierarchically design the related bioprocesses, embedded in scalable devices, compatible with continuous operation. Our secondary objective is to harvest the obtained technological data and conduct resource commitment analysis. We herein demonstrate for first time the feasibility of the continuous, end-to-end production of sterile mRNA formulated into lipid nanocarriers, defining the equipment specifications and the desired operational space. Moreover, we find that the cell lysis modules and the linearization enzymes ascend as the principal resource-intensive model factors, accounting for 40% and 42% of the equipment and raw material, respectively. We calculate MSPD 1.30–1.45 €, demonstrating low margin lifecycle fluctuation. Full article
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22 pages, 4494 KiB  
Article
In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary-Based Alpha-1-Antitrypsin Deficiency Treatment
by Shan Guan, Max Darmstädter, Chuanfei Xu and Joseph Rosenecker
Pharmaceutics 2021, 13(8), 1281; https://doi.org/10.3390/pharmaceutics13081281 - 17 Aug 2021
Cited by 7 | Viewed by 2411
Abstract
In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for [...] Read more.
In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for alpha-1-antitrypsin deficiency in the future. Here we show that lipoplexes containing A1AT-mRNA prepared 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 number 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. Our data indicate that aerosolization of A1AT-mRNA therapy constitutes 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. Full article
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Review

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15 pages, 611 KiB  
Review
Stability Modelling of mRNA Vaccine Quality Based on Temperature Monitoring throughout the Distribution Chain
by Zoltán Kis
Pharmaceutics 2022, 14(2), 430; https://doi.org/10.3390/pharmaceutics14020430 - 17 Feb 2022
Cited by 19 | Viewed by 6222
Abstract
The vaccine distribution chains in several low- and middle-income countries are not adequate to facilitate the rapid delivery of high volumes of thermosensitive COVID-19 mRNA vaccines at the required low and ultra-low temperatures. COVID-19 mRNA vaccines are currently distributed along with temperature monitoring [...] Read more.
The vaccine distribution chains in several low- and middle-income countries are not adequate to facilitate the rapid delivery of high volumes of thermosensitive COVID-19 mRNA vaccines at the required low and ultra-low temperatures. COVID-19 mRNA vaccines are currently distributed along with temperature monitoring devices to track and identify deviations from predefined conditions throughout the distribution chain. These temperature readings can feed into computational models to quantify mRNA vaccine critical quality attributes (CQAs) and the remaining vaccine shelf life more accurately. Here, a kinetic modelling approach is proposed to quantify the stability-related CQAs and the remaining shelf life of mRNA vaccines. The CQA and shelf-life values can be computed based on the conditions under which the vaccines have been distributed from the manufacturing facilities via the distribution network to the vaccination centres. This approach helps to quantify the degree to which temperature excursions impact vaccine quality and can also reduce vaccine wastage. In addition, vaccine stock management can be improved due to the information obtained on the remaining shelf life of mRNA vaccines. This model-based quantification of mRNA vaccine quality and remaining shelf life can improve the deployment of COVID-19 mRNA vaccines to low- and middle-income countries. Full article
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23 pages, 8555 KiB  
Review
Lipid Nanoparticle Delivery Systems to Enable mRNA-Based Therapeutics
by Sean C. Semple, Robert Leone, Christopher J. Barbosa, Ying K. Tam and Paulo J. C. Lin
Pharmaceutics 2022, 14(2), 398; https://doi.org/10.3390/pharmaceutics14020398 - 11 Feb 2022
Cited by 33 | Viewed by 8423
Abstract
The world raced to develop vaccines to protect against the rapid spread of SARS-CoV-2 infection upon the recognition of COVID-19 as a global pandemic. A broad spectrum of candidates was evaluated, with mRNA-based vaccines emerging as leaders due to how quickly they were [...] Read more.
The world raced to develop vaccines to protect against the rapid spread of SARS-CoV-2 infection upon the recognition of COVID-19 as a global pandemic. A broad spectrum of candidates was evaluated, with mRNA-based vaccines emerging as leaders due to how quickly they were available for emergency use while providing a high level of efficacy. As a modular technology, the mRNA-based vaccines benefitted from decades of advancements in both mRNA and delivery technology prior to the current global pandemic. The fundamental lessons of the utility of mRNA as a therapeutic were pioneered by Dr. Katalin Kariko and her colleagues, perhaps most notably in collaboration with Drew Weissman at University of Pennsylvania, and this foundational work paved the way for the development of the first ever mRNA-based therapeutic authorized for human use, COMIRNATY®. In this Special Issue of Pharmaceutics, we will be honoring Dr. Kariko for her great contributions to the mRNA technology to treat diseases with unmet needs. In this review article, we will focus on the delivery platform, the lipid nanoparticle (LNP) carrier, which allowed the potential of mRNA therapeutics to be realized. Similar to the mRNA technology, the development of LNP systems has been ongoing for decades before culminating in the success of the first clinically approved siRNA-LNP product, ONPATTRO®, a treatment for an otherwise fatal genetic disease called transthyretin amyloidosis. Lessons learned from the siRNA-LNP experience enabled the translation into the mRNA platform with the eventual authorization and approval of the mRNA-LNP vaccines against COVID-19. This marks the beginning of mRNA-LNP as a pharmaceutical option to treat genetic diseases. Full article
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30 pages, 5247 KiB  
Review
RNA-Based Therapeutics: Current Developments in Targeted Molecular Therapy of Triple-Negative Breast Cancer
by Sakib Haque, Kiri Cook, Gaurav Sahay and Conroy Sun
Pharmaceutics 2021, 13(10), 1694; https://doi.org/10.3390/pharmaceutics13101694 - 15 Oct 2021
Cited by 19 | Viewed by 4156
Abstract
Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive cancer that has the highest mortality rate out of all breast cancer subtypes. Conventional clinical treatments targeting ER, PR, and HER2 receptors have been unsuccessful in the treatment of TNBC, which has led [...] Read more.
Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive cancer that has the highest mortality rate out of all breast cancer subtypes. Conventional clinical treatments targeting ER, PR, and HER2 receptors have been unsuccessful in the treatment of TNBC, which has led to various research efforts in developing new strategies to treat TNBC. Targeted molecular therapy of TNBC utilizes knowledge of key molecular signatures of TNBC that can be effectively modulated to produce a positive therapeutic response. Correspondingly, RNA-based therapeutics represent a novel tool in oncology with their ability to alter intrinsic cancer pathways that contribute to poor patient prognosis. Current RNA-based therapeutics exist as two major areas of investigation—RNA interference (RNAi) and RNA nanotherapy, where RNAi utilizes principles of gene silencing, and RNA nanotherapy utilizes RNA-derived nanoparticles to deliver chemotherapeutics to target cells. RNAi can be further classified as therapeutics utilizing either small interfering RNA (siRNA) or microRNA (miRNA). As the broader field of gene therapy has advanced significantly in recent years, so too have efforts in the development of effective RNA-based therapeutic strategies for treating aggressive cancers, including TNBC. This review will summarize key advances in targeted molecular therapy of TNBC, describing current trends in treatment using RNAi, combination therapies, and recent efforts in RNA immunotherapy, utilizing messenger RNA (mRNA) in the development of cancer vaccines. Full article
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13 pages, 2627 KiB  
Review
Lipid Nanoparticles for Organ-Specific mRNA Therapeutic Delivery
by Magdalena M. Żak and Lior Zangi
Pharmaceutics 2021, 13(10), 1675; https://doi.org/10.3390/pharmaceutics13101675 - 13 Oct 2021
Cited by 30 | Viewed by 14175
Abstract
Advances in the using in vitro transcribed (IVT) modRNA in the past two decades, especially the tremendous recent success of mRNA vaccines against SARS-CoV-2, have brought increased attention to IVT mRNA technology. Despite its well-known use in infectious disease vaccines, IVT modRNA technology [...] Read more.
Advances in the using in vitro transcribed (IVT) modRNA in the past two decades, especially the tremendous recent success of mRNA vaccines against SARS-CoV-2, have brought increased attention to IVT mRNA technology. Despite its well-known use in infectious disease vaccines, IVT modRNA technology is being investigated mainly in cancer immunotherapy and protein replacement therapy, with ongoing clinical trials in both areas. One of the main barriers to progressing mRNA therapeutics to the clinic is determining how to deliver mRNA to target cells and protect it from degradation. Over the years, many different vehicles have been developed to tackle this issue. Desirable vehicles must be safe, stable and preferably organ specific for successful mRNA delivery to clinically relevant cells and tissues. In this review we discuss various mRNA delivery platforms, with particular focus on attempts to create organ-specific vehicles for therapeutic mRNA delivery. Full article
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