Lipid nanoparticles for mRNA delivery

In the paper

“Chromatographic purification can remove double-stranded RNA, an analogue of viral genome, in IVT mRNA preparations, diminishing immune activation and increasing translational efficiency2,4,7,183.”

What the sources say

• Supported

  The Box 1 passage says high-performance liquid chromatography eliminated dsRNA contaminants and the remaining immune stimulation in modified mRNA. The Protein-replacement therapies passage says improved purification protocols, used with nucleoside-modified IVT mRNA, increased translation. (LLM, conf=0.91)

  cites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Supported

  The “Modulation of immunogenicity” section says chromatographic methods remove dsRNA contaminants from IVT mRNA, which otherwise trigger type I interferon production and inhibit translation. The “Optimization of mRNA translation and stability” section states that FPLC purification decreases immune activation and increases translation. (LLM, conf=0.94)

  cites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Not checked yetcites [7] Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017). source
• Supported

  The Discussion states that HPLC purification removes dsRNA and other contaminants from in vitro-transcribed mRNA containing Ψ or m5C/Ψ, increases translation, and prevents the release of type I interferons and TNF-α. The claim is supported as a statement of what chromatographic purification can do; the reported elimination of immune activation applies to nucleoside-modified mRNA. (LLM, conf=0.94)

  cites [183] Kariko, K., Muramatsu, H., Ludwig, J. & Weissman, D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 39, e142 (2011). source

In the paper

“First, mRNA is a non-infective and non-integrating agent with the ability to encode a broad range of antigens4,15,17.”

What the sources say

• Supported

  The Abstract calls mRNA a “non-infectious, non-integrating platform.” The Basic mRNA vaccine pharmacology section says mRNA vaccines encode an antigen of interest, and the Self-amplifying mRNA vaccines section describes vaccines encoding antigens from diverse viruses and other pathogens. The provided passages do not specify page numbers. (LLM, conf=0.93)

  cites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Supported

  The mRNA VACCINES section states that mRNA is non-infectious and non-integrating. It also describes encoding protein antigens from different pathogens, supporting the claim that mRNA can encode a broad range of antigens. (LLM, conf=0.94)

  cites [15] Gebre, M. S. et al. Novel approaches for vaccine development. Cell 184, 1589–1603 (2021). source
• Supported

  The “Advantages of mRNA vaccines” section says mRNA vaccines avoid infection concerns and do not pose a risk of genomic integration. The Introduction says mRNA can encode virtually any protein, supporting the claim that it can encode a broad range of antigens. No page numbers are available in our copy. (LLM, conf=0.94)

  cites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source

In the paper

“Second, mRNA vaccines can combine different antigen mRNAs; for example, six separate mRNAs have been incorporated in a cytomegalovirus vaccine, five of which encode a single pentameric antigen and one encodes a glycoprotein antigen185.”

What the sources say

• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [185] John, S. et al. Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity. Vaccine 36, 1689–1699 (2018). source

In the paper

“Circular RNA (circRNA) is a single-stranded RNA with a closed-loop structure.”

What the sources say

• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Supported

  The Introduction describes circRNAs as lacking free ends, and Results (‘Long RNA circularization is assisted by homology’) describes the RNA transcript being excised as a covalently 5′-to-3′ linked circle. Together, these passages support the description of a single RNA strand with a closed-loop structure. (LLM, conf=0.89)

  cites [245] Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). source
• Supported

  The Results section (page not provided) describes the RNA product as a covalently linked circle and reports tests confirming its circularity. The paper’s description of circRNAs as single-stranded RNAs with a contiguous structure supports the closed-loop description. (LLM, conf=0.91)

  cites [246] Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019). source

In the paper

“The excessive accumulation of metabolites then results in clinical symptoms and may lead to death12.”

What the sources say

• Supported

  The “Liver delivery of mRNA” section links excess methylmalonic acid in MMA to clinical symptoms and reports lifelong mortality. It also states that hyperammonemia in ornithine transcarbamylase deficiency can lead to neurological injury, coma, and death. The copy provides no page numbers for these passages. (LLM, conf=0.90)

  cites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source

In the paper

“The sequences of these elements regulate translation initiation, translation termination and post-transcriptional modification of mRNA molecules2,4.”

What the sources say

• Partly supported

  The 5′- and 3′-UTRs section says regulatory sequence elements modulate mRNA translation and stability, and the 5′ cap section links the cap to translation initiation. Figure 2 describes translation ending through mRNA degradation, but does not establish that the sequences of these elements regulate translation termination. The passages do not support the claim that those sequences regulate post-transcriptional modification of mRNA; Figure 2 instead describes post-translational modification of the protein product. Page numbers were not available in our copy. (LLM, conf=0.86)

  cites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). sourceExperimental — accuracy not yet measured
• Partly supported

  The “Optimization of mRNA translation and stability” section says that UTR sequences affect translation and that the 5′ cap interacts with a translation initiation factor. It does not say that the sequences of these elements regulate translation termination or post-transcriptional modification of mRNA. Page numbers are not available in our copy. (LLM, conf=0.86)

  cites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). sourceExperimental — accuracy not yet measured

In the paper

“To function in vivo, mRNA requires safe, effective and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release.”

In the paper

“This structural feature protects mRNA molecules from nuclease degradation and increases nanoparticle stability in physiological fluids17,46.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source
• Not checked yetcites [46] Blakney, A. K., McKay, P. F., Yus, B. I., Aldon, Y. & Shattock, R. J. Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA. Gene Ther. 26, 363–372 (2019). source

In the paper

“Ionizable lipids are protonated at low pH, which makes them positively charged, but they remain neutral at physiological pH (refs7,11,14).”

What the sources say

• Not checked yetcites refs7,11,14

In the paper

“However, targeted delivery and endosomal escape remain challenging for mRNA delivery systems, highlighting the need for safe and effective mRNA delivery materials.”

In the paper

“Lipid nanoparticle–mRNA formulations manufactured by rapid mixing exhibit a stable nanostructure17,130,131, in which mRNA molecules can be encapsulated in the interior core through electrostatic interactions with the lipids17,131.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source
• Not checked yetcites [130] Karnik, R. et al. Microfluidic platform for controlled synthesis of polymeric nanoparticles. Nano Lett. 8, 2906–2912 (2008). source
• Not checked yetcites [131] Leung, A. K., Tam, Y. Y. C., Chen, S., Hafez, I. M. & Cullis, P. R. Microfluidic mixing: a general method for encapsulating macromolecules in lipid nanoparticle systems. J. Phys. Chem. B 119, 8698–8706 (2015). source

In the paper

“Trapped in endosomes, in which the pH is lower than in the extracellular environment, ionizable lipids are protonated and, therefore, become positively charged, which may promote membrane destabilization and facilitate endosomal escape of the nanoparticles7,11,14”

What the sources say

• Not checked yetcites [7] Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017). source
• Not checked yetcites [11] Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019). source
• Not checked yetcites [14] Meng, C., Chen, Z., Li, G., Welte, T. & Shen, H. Nanoplatforms for mRNA therapeutics. Adv. Ther. 4, 2000099 (2021). source

In the paper

“To translate lipid nanoparticle–mRNA systems from bench to bedside, good manufacturing practice (GMP) is crucial to ensure drug quality and therapeutic effects, in addition to considerations such as storage conditions and safety profiles.”

In the paper

“Indeed, both i.m. and i.d. administration of lipid nanoparticle–mRNA vaccines produce robust immune responses at a well-tolerated dose in human trials156,157.”

What the sources say

• Not checked yetcites [156] Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses. Mol. Ther. 25, 1316–1327 (2017). source
• Not checked yetcites [157] Feldman, R. A. et al. mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials. Vaccine 37, 3326–3334 (2019). This paper reports a clinical trial on lipid nanoparticle–mRNA formulations as influenza vaccines. source

In the paper

“mRNA normally contains five structural elements, that is, a 5′ cap, a 3′ poly(A) tail, a protein-coding sequence and 5′ and 3′ untranslated regions (UTRs)2,4,7,13.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Not checked yetcites [7] Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source

In the paper

“This technique allows reproducible and scalable production of lipid nanoparticle–mRNA formulations that show high encapsulation efficiency and homogeneous size distribution17,131.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source
• Not checked yetcites [131] Leung, A. K., Tam, Y. Y. C., Chen, S., Hafez, I. M. & Cullis, P. R. Microfluidic mixing: a general method for encapsulating macromolecules in lipid nanoparticle systems. J. Phys. Chem. B 119, 8698–8706 (2015). source

In the paper

“mRNA-1273 is stored at −15 °C to −20 °C and is directly injected after thawing17, whereas BNT162b2 is stored at −60 °C to −80 °C and requires thawing and dilution by saline before injection17.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source

In the paper

“To ameliorate safety concerns, numerous natural and synthetic polymers have been investigated as alternatives to PEG, of which several are under evaluation in clinical trials127,169.”

What the sources say

• Not checked yetcites [127] Knop, K., Hoogenboom, R., Fischer, D. & Schubert, U. S. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Ed. 49, 6288–6308 (2010). source
• Not checked yetcites [169] Lila, A. S. A., Kiwada, H. & Ishida, T. The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. J. Control. Release 172, 38–47 (2013). source

In the paper

“The accelerated blood clearance phenomenon may change the bioavailability and biodistribution of the drug encapsulated in PEGylated nanoparticles and, thus, cause side effects127,169.”

What the sources say

• Not checked yetcites [127] Knop, K., Hoogenboom, R., Fischer, D. & Schubert, U. S. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Ed. 49, 6288–6308 (2010). source
• Not checked yetcites [169] Lila, A. S. A., Kiwada, H. & Ishida, T. The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. J. Control. Release 172, 38–47 (2013). source

In the paper

“mRNA is susceptible to degradation and, thus, formulation buffers should be free of any ribonuclease contaminations2,4.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source

In the paper

“mRNA-1273 is stored at −15 °C to −20 °C and is directly injected after thawing17, whereas BNT162b2 is stored at −60 °C to −80 °C and requires thawing and dilution by saline before injection17.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source

In the paper

“Of note, the authorized COVID-19 mRNA vaccines are both stored in freezing conditions in the presence of sucrose17.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source

In the paper

“Finally, mRNA-1273 obtained Emergency Use Authorization from the FDA and conditional marketing authorization from the EMA within a year15,17.”

What the sources say

• Not checked yetcites [15] Gebre, M. S. et al. Novel approaches for vaccine development. Cell 184, 1589–1603 (2021). source
• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source

In the paper

“About 2, 5 and 6 months from sequence availability, clinical trial phases I, II and III were initiated, respectively15,17.”

What the sources say

• Not checked yetcites [15] Gebre, M. S. et al. Novel approaches for vaccine development. Cell 184, 1589–1603 (2021). source
• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source

In the paper

“Nevertheless, immune responses to IVT mRNA may also suppress antigen expression and negatively affect vaccine efficacy175,180,181.”

What the sources say

• Not checked yetcites [175] Lokugamage, M. P. et al. Mild innate immune activation overrides efficient nanoparticle-mediated RNA delivery. Adv. Mater. 32, 1904905 (2020). source
• Not checked yetcites [180] Karikó, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008). source
• Not checked yetcites [181] Pollard, C. et al. Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines. Mol. Ther. 21, 251–259 (2013). source

In the paper

“The IVT mRNA molecules used in the mRNA-1273 and BNT162b2 COVID-19 vaccines were prepared by replacing uridine with m1ψ17,19,21, and their sequences were optimized to encode a stabilized pre-fusion spike protein with two pivotal proline substitutions17,19,21.”

What the sources say

• Not checked yetcites [17] Kim, J., Eygeris, Y., Gupta, M. & Sahay, G. Self-assembled mRNA vaccines. Adv. Drug Deliv. Rev. 170, 83–112 (2021). source
• Not checked yetcites [19] Baden, L. R. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 384, 403–416 (2021). source
• Not checked yetcites [21] Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383, 2603–2615 (2020). References 19, 20, and 21 report safety and efficacy data about the SARS-CoV-2 lipid nanoparticle–mRNA vaccines BNT162b2 and mRNA-1273. source

In the paper

“To address these concerns, the engineering of mRNA molecules (Box 1) and the design of lipid nanoparticles have been optimized2,4,7–17, which has contributed to the rapid development and clinical assessment15,17 of the two COVID-19 mRNA vaccines, mRNA-1273 (NCT04470427)19,20 and BNT162b2 (NCT04368728)21.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Not checked yetcites [7] Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017). source
• Not checked yetcites [8] Xiong, Q., Lee, G. Y., Ding, J., Li, W. & Shi, J. Biomedical applications of mRNA nanomedicine. Nano Res. 11, 5281–5309 (2018). source
• Not checked yetcites [9] Guan, S. & Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Ther. 24, 133–143 (2017). source
• Not checked yetcites [10] Li, B., Zhang, X. & Dong, Y. Nanoscale platforms for messenger RNA delivery. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 11, e1530 (2019).
• Not checked yetcites [11] Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019). source
• Not checked yetcites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [14] Meng, C., Chen, Z., Li, G., Welte, T. & Shen, H. Nanoplatforms for mRNA therapeutics. Adv. Ther. 4, 2000099 (2021). source

In the paper

“Moreover, mRNA-based cellular reprogramming, tissue regeneration and genome editing have shown therapeutic potential in preclinical studies2,4,8,12–14.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Not checked yetcites [8] Xiong, Q., Lee, G. Y., Ding, J., Li, W. & Shi, J. Biomedical applications of mRNA nanomedicine. Nano Res. 11, 5281–5309 (2018). source
• Not checked yetcites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [14] Meng, C., Chen, Z., Li, G., Welte, T. & Shen, H. Nanoplatforms for mRNA therapeutics. Adv. Ther. 4, 2000099 (2021). source

In the paper

“Both vaccines use ionizable lipid nanoparticles to deliver nucleoside-modified mRNA encoding the full-length spike protein of SARS-CoV-2 (Fig. 4).”

What the sources say

• Not checked yetcites [19] Baden, L. R. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 384, 403–416 (2021). source
• Not checked yetcites [20] Anderson, E. J. et al. Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults. N. Engl. J. Med. 383, 2427–2438 (2020). source
• Not checked yetcites [21] Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383, 2603–2615 (2020). References 19, 20, and 21 report safety and efficacy data about the SARS-CoV-2 lipid nanoparticle–mRNA vaccines BNT162b2 and mRNA-1273. source

In the paper

“Lipid nanoparticle–mRNA vaccines are also being investigated as influenza vaccines29,41,156,157,159,184,189–191, with some formulations in clinical trials (Table 1).”

What the sources say

• Not checked yetcites [29] Brazzoli, M. et al. Induction of broad-based immunity and protective efficacy by self-amplifying mRNA vaccines encoding influenza virus hemagglutinin. J. Virol. 90, 332–344 (2016). source
• Not checked yetcites [41] Perche, F. et al. Neutral lipopolyplexes for in vivo delivery of conventional and replicative RNA vaccine. Mol. Ther. Nucleic Acids 17, 767–775 (2019). source
• Not checked yetcites [156] Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses. Mol. Ther. 25, 1316–1327 (2017). source
• Not checked yetcites [157] Feldman, R. A. et al. mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials. Vaccine 37, 3326–3334 (2019). This paper reports a clinical trial on lipid nanoparticle–mRNA formulations as influenza vaccines. source
• Not checked yetcites [159] Zhuang, X. et al. mRNA vaccines encoding the HA protein of influenza A H1N1 virus delivered by cationic lipid nanoparticles induce protective immune responses in mice. Vaccines 8, 123 (2020). source
• Not checked yetcites [184] Martinon, F. et al. Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA. Eur. J. Immunol. 23, 1719–1722 (1993). source
• Not checked yetcites [189] Pardi, N. et al. Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies. Nat. Commun. 9, 3361 (2018). source
• Not checked yetcites [190] Pardi, N. et al. Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses. J. Exp. Med. 215, 1571–1588 (2018). source
• Not checked yetcites [191] Goswami, R. et al. Mannosylation of LNP results in improved potency for self-amplifying RNA (SAM) vaccines. ACS Infect. Dis. 5, 1546–1558 (2019). source

In the paper

“Applying a prime–boost vaccination method, the vaccines induce high levels of antigen-specific antibodies and elicit robust T helper 1 cell responses19–21.”

What the sources say

• Not checked yetcites [19] Baden, L. R. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 384, 403–416 (2021). source
• Not checked yetcites [20] Anderson, E. J. et al. Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults. N. Engl. J. Med. 383, 2427–2438 (2020). source
• Not checked yetcites [21] Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383, 2603–2615 (2020). References 19, 20, and 21 report safety and efficacy data about the SARS-CoV-2 lipid nanoparticle–mRNA vaccines BNT162b2 and mRNA-1273. source

In the paper

“Other COVID-19 vaccines based on lipid nanoparticle–mRNA formulations are also under evaluation in different clinical phases (Table 1).”

What the sources say

• Not checked yetcites Table 1

In the paper

“Haemagglutinin is an essential surface antigen of influenza viruses156,157.”

What the sources say

• Not checked yetcites [156] Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses. Mol. Ther. 25, 1316–1327 (2017). source
• Not checked yetcites [157] Feldman, R. A. et al. mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials. Vaccine 37, 3326–3334 (2019). This paper reports a clinical trial on lipid nanoparticle–mRNA formulations as influenza vaccines. source

In the paper

“In addition, lipid nanoparticle–mRNA formulations have been applied to produce therapeutic proteins or antibodies39,75,145,146, to edit virus genomes105 and to engineer immune cells136.”

What the sources say

• Not checked yetcites [39] Erasmus, J. H. et al. Intramuscular delivery of replicon RNA encoding ZIKV-117 human monoclonal antibody protects against Zika virus infection. Mol. Ther. Methods Clin. Dev. 18, 402–414 (2020). source
• Not checked yetcites [75] Kim, J., Mukherjee, A., Nelson, D., Jozi c, A. & Sahay, G. Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2. Preprint at bioRxiv https://doi.org/10.1101/2020.07.24.205583 (2020). source
• Not checked yetcites [145] Pardi, N. et al. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge. Nat. Commun. 8, 14630 (2017). source
• Not checked yetcites [146] Thran, M. et al. mRNA mediates passive vaccination against infectious agents, toxins, and tumors. EMBO Mol. Med. 9, 1434–1447 (2017). source
• Not checked yetcites [105] Jiang, C. et al. A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell Res. 27, 440–443 (2017). source
• Not checked yetcites [136] Hou, X. et al. Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis. Nat. Nanotechnol. 15, 41–46 (2020). source

In the paper

“According to mRNA-1893 interim phase I data (NCT04064905) reported by Moderna, 10-µg and 30-µg dose levels (prime–boost regimen) induced 94% and 100% seroconversion, respectively, and both dose levels were generally well tolerated.”

What the sources say

• Not checked yetcites NCT04064905

In the paper

“Compared with regular mRNA, self-amplifying RNA (also termed replicon), which was originally derived from an alphavirus genome, has similar basic elements (5′ cap, 5′ and 3′ untranslated regions, 3′ poly(A) tail) and a long coding region.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source

In the paper

“The half-life of CHKV-IgG was about 69 days and the dose levels were reasonably well tolerated, including 0.3 mg kg−1 given twice a week apart.”

What the sources say

• Not checked yetcites NCT03829384

In the paper

“Adoptive transfer of macrophages engineered by delivering mRNA that encodes an antimicrobial peptide considerably reduced bacterial burden and increased survival in mice with multidrug-resistant bacterial sepsis136.”

What the sources say

• Not checked yetcites [136] Hou, X. et al. Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis. Nat. Nanotechnol. 15, 41–46 (2020). source

In the paper

“The initial replication leads to negative-strand RNA, which acts as template for the generation of the positive-strand viral genome.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source

In the paper

“Similar to linear mRNA, the chemical modification of specific nucleotides and chromatographic purification can minimize immunogenicity of the RNA and increase translation.”

What the sources say

• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [245] Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). source
• Not checked yetcites [246] Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019). source

In the paper

“circRNA does not have a free 5′ cap or 3′ poly(A) tails and is, thus, unsusceptible to degradation by nucleases and more stable than linear RNA.”

What the sources say

• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [245] Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). source
• Not checked yetcites [246] Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019). source

In the paper

“The attempt of mRNA-based cancer immunotherapies dates back to 1995, when i.m. injection of mRNA encoding carcinoembryonic antigens was shown to induce antigen-specific immune responses in mice199.”

What the sources say

• Not checked yetcites [199] Conry, R. M. et al. Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res. 55, 1397–1400 (1995).

In the paper

“Moreover, circRNA without a stop codon reduces the frequency of ribosome detachment from the RNA, thereby enabling continuous translation and high protein expression.”

What the sources say

• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [245] Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). source
• Not checked yetcites [246] Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019). source

In the paper

“The transient replication generates double-stranded RNA (dsRNA) and, thus, self-amplifying RNA tends to activate innate immune pathways.”

What the sources say

• Not checked yetcites [2] Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source

In the paper

“Synthetic circRNA can be made by covalently linking the 3′ and 5′ ends of a linear precursor using enzymatic or chemical methods.”

What the sources say

• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [245] Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). source
• Not checked yetcites [246] Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019). source

In the paper

“After the eighth immunization, more than 75% of patients generated immune responses against at least one tumour-associated antigen, and CD8+ T cells played a major role in high-magnitude T cell responses200.”

What the sources say

• Not checked yetcites [200] Sahin, U. et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 585, 107–112 (2020). This is a clinical trial on lipid nanoparticle–mRNA formulations for cancer immunotherapy. source

In the paper

“Neoantigens, generated by somatic mutations in cancer cells, are usually tumour-specific and have high immunogenicity4,11.”

What the sources say

• Not checked yetcites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Not checked yetcites [11] Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019). source

In the paper

“Neoantigens are often different between individual patients, allowing the development of personalized vaccines4,11.”

What the sources say

• Not checked yetcites [4] Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018). source
• Not checked yetcites [11] Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019). source

In the paper

“In the combination group, the overall response rate in the cohort (human papillomavirus-negative, immune checkpoint inhibitor-naive, head and neck squamous cell carcinomas) was 50% and the median progression-free survival was 9.8 months210,211.”

What the sources say

• Not checked yetcites [210] Bauman, J. et al. 798 Safety, tolerability, and immunogenicity of mRNA-4157 in combination with pembrolizumab in subjects with unresectable solid tumors (KEYNOTE-603): an update. J. Immunother. Cancer https://doi.org/10.1136/jitc-2020-SITC2020.0798 (2020). source
• Not checked yetcites [211] Burris, H. A. et al. A phase I multicenter study to assess the safety, tolerability, and immunogenicity of mRNA-4157 alone in patients with resected solid tumors and in combination with pembrolizumab in patients with unreshectable solid tumors. J. Clin. Oncol. 37, 2523 (2019). source

In the paper

“i.v. injection of this vaccine resulted in CLDN6 expression on splenic dendritic cells and macrophages213, promoting the activation of adoptively transferred CLDN6-CAR T cells and leading to suppression of large tumours in mice at a sub-therapeutic CAR T cell dose213.”

What the sources say

• Not checked yetcites [213] Reinhard, K. et al. An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors. Science 367, 446–453 (2020). source

In the paper

“Genetic disorders are caused by inherited or acquired gene mutations, which can cause abnormal protein expression12.”

What the sources say

• Not checked yetcites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source

In the paper

“In the 22 patients (monotherapy: n = 15; combination: n = 7), six had stable disease, one had partial responses with 52% tumour reduction and five showed tumour shrinkage in treated and/or untreated sites215,216.”

What the sources say

• Not checked yetcites [215] Bauer, T. et al. Abstract CT210: A Phase I, open-label, multicenter, dose escalation study of mRNA-2752, a lipid nanoparticle encapsulating mRNAs encoding human OX40L, IL-23, and IL-36γ, for intratumoral injection alone and in combination with immune checkpoint blockade. Cancer Res. https://doi.org/10.1158/1538-7445.AM2019-CT210 (2019). source
• Not checked yetcites [216] Patel, M. R. et al. A phase I study of mRNA-2752, a lipid nanoparticle encapsulating mRNAs encoding human OX40L, IL-23, and IL-36γ, for intratumoral (iTu) injection alone and in combination with durvalumab. J. Clin. Oncol. 38, 3092–3092 (2020). source

In the paper

“For example, restoration of the tumour suppressor gene TP53 can induce tumour cell apoptosis and sensitize tumour cells to chemotherapeutics in vivo118,218.”

What the sources say

• Not checked yetcites [118] Zhang, C. et al. Chemotherapy drugs derived nanoparticles encapsulating mRNA encoding tumor suppressor proteins to treat triple-negative breast cancer. Nano Res. 12, 855–861 (2019). source
• Not checked yetcites [218] Kong, N. et al. Synthetic mRNA nanoparticle-mediated restoration of p53 tumor suppressor sensitizes p53-deficient cancers to mTOR inhibition. Sci. Transl Med. 11, eaaw1565 (2019). source

In the paper

“Moreover, the injected lesions showed an increase in OX40L expression and enhanced T cell activation214.”

What the sources say

• Not checked yetcites [214] Jimeno, A. et al. Abstract CT032: a phase 1/2, open-label, multicenter, dose escalation and efficacy study of mRNA-2416, a lipid nanoparticle encapsulated mRNA encoding human OX40L, for intratumoral injection alone or in combination with durvalumab for patients with advanced malignancies. Cancer Res. https://doi.org/10.1158/1538-7445.AM2020-CT032 (2020). source

In the paper

“Lipid nanoparticle–mRNA formulations have also been investigated for ex vivo engineering of CAR T cells217 and for the production of antibodies, such as anti-CD20 (ref.146), anti-human epidermal growth factor receptor 2 (HER2)95 and anti-CD3/claudin 6 (ref.147).”

What the sources say

• Not checked yetcites [217] Billingsley, M. M. et al. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Lett. 20, 1578–1589 (2020). source
• Not checked yetcites [146] Thran, M. et al. mRNA mediates passive vaccination against infectious agents, toxins, and tumors. EMBO Mol. Med. 9, 1434–1447 (2017). source
• Not checked yetcites [95] Rybakova, Y. et al. mRNA delivery for therapeutic anti-HER2 antibody expression in vivo. Mol. Ther. 27, 1415–1423 (2019). source
• Not checked yetcites [147] Stadler, C. R. et al. Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nat. Med. 23, 815–817 (2017). source

In the paper

“mRNA-2752 was designed to induce a pro-inflammatory tumour microenvironment and to simultaneously strengthen T cell expansion, as well as memory responses215,216.”

What the sources say

• Not checked yetcites [215] Bauer, T. et al. Abstract CT210: A Phase I, open-label, multicenter, dose escalation study of mRNA-2752, a lipid nanoparticle encapsulating mRNAs encoding human OX40L, IL-23, and IL-36γ, for intratumoral injection alone and in combination with immune checkpoint blockade. Cancer Res. https://doi.org/10.1158/1538-7445.AM2019-CT210 (2019). source
• Not checked yetcites [216] Patel, M. R. et al. A phase I study of mRNA-2752, a lipid nanoparticle encapsulating mRNAs encoding human OX40L, IL-23, and IL-36γ, for intratumoral (iTu) injection alone and in combination with durvalumab. J. Clin. Oncol. 38, 3092–3092 (2020). source

In the paper

“mRNA-based protein replacement therapies are also being explored for heart241, liver242, lung243 and other organ diseases12.”

What the sources say

• Not checked yetcites [241] Magadum, A., Kaur, K. & Zangi, L. mRNA-based protein replacement therapy for the heart. Mol. Ther. 27, 785–793 (2019). source
• Not checked yetcites [242] Trepotec, Z., Lichtenegger, E., Plank, C., Aneja, M. K. & Rudolph, C. Delivery of mRNA therapeutics for the treatment of hepatic diseases. Mol. Ther. 27, 794–802 (2019). source
• Not checked yetcites [243] Sahu, I., Haque, A. A., Weidensee, B., Weinmann, P. & Kormann, M. S. Recent developments in mRNA-based protein supplementation therapy to target lung diseases. Mol. Ther. 27, 803–823 (2019). source
• Not checked yetcites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source

In the paper

“Progress in mRNA technologies and lipid nanoparticle-based delivery systems has allowed the development of mRNA COVID-19 vaccines at unprecedented speed, demonstrating the clinical potential of lipid nanoparticle–mRNA formulations and providing a powerful tool against the SARS-CoV-2 pandemic.”

In the paper

“A variety of lipid nanoparticles have been explored and optimized for mRNA delivery, providing valuable information for the future design of mRNA therapeutics.”

In the paper

“MRT5005 was well tolerated at the 8-mg and 16-mg dose levels, and no serious side events were observed at any dose level.”

What the sources say

• Not checked yetcites NCT03375047

In the paper

“Patients with cystic fibrosis usually suffer from repeated airway infections and chronic respiratory problems because of the defective cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel on epithelial cells12.”

What the sources say

• Not checked yetcites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source

In the paper

“circRNA lacks the free ends necessary for nuclease-mediated degradation and, therefore, has a longer half-life than its linear mRNA counterpart245,246.”

What the sources say

• Not checked yetcites [245] Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018). source
• Not checked yetcites [246] Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019). source

In the paper

“Polymers, such as polyethyleneimine, protamine and polyaspartamide derivatives, are already widely used for mRNA delivery7–17.”

What the sources say

• Not checked yetcites [7] Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017). source
• Not checked yetcites [8] Xiong, Q., Lee, G. Y., Ding, J., Li, W. & Shi, J. Biomedical applications of mRNA nanomedicine. Nano Res. 11, 5281–5309 (2018). source
• Not checked yetcites [9] Guan, S. & Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Ther. 24, 133–143 (2017). source
• Not checked yetcites [10] Li, B., Zhang, X. & Dong, Y. Nanoscale platforms for messenger RNA delivery. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 11, e1530 (2019).
• Not checked yetcites [11] Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019). source
• Not checked yetcites [12] Zhao, W., Hou, X., Vick, O. G. & Dong, Y. RNA delivery biomaterials for the treatment of genetic and rare diseases. Biomaterials 217, 119291 (2019). source
• Not checked yetcites [13] Uchida, S., Perche, F., Pichon, C. & Cabral, H. Nanomedicine-based approaches for mRNA delivery. Mol. Pharm. 17, 3654–3684 (2020). source
• Not checked yetcites [14] Meng, C., Chen, Z., Li, G., Welte, T. & Shen, H. Nanoplatforms for mRNA therapeutics. Adv. Ther. 4, 2000099 (2021). source
• Not checked yetcites [15] Gebre, M. S. et al. Novel approaches for vaccine development. Cell 184, 1589–1603 (2021). source
• Not checked yetcites [16] Weng, Y. et al. The challenge and prospect of mRNA therapeutics landscape. Biotechnol. Adv. 40, 107534 (2020). source

In the paper

“In addition, charge-altering releasable transporters162,247 and modified dendrimers248 can effectively deliver mRNA molecules in vitro and in vivo.”

What the sources say

• Not checked yetcites [162] Haabeth, O. A. W. et al. Local delivery of Ox40l, Cd80, and Cd86 mRNA kindles global anticancer immunity. Cancer Res. 79, 1624–1634 (2019). source
• Not checked yetcites [247] McKinlay, C. J. et al. Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals. Proc. Natl Acad. Sci. USA 114, E448–E456 (2017). source
• Not checked yetcites [248] Chahal, J. S. et al. Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose. Proc. Natl Acad. Sci. USA 113, E4133–E4142 (2016). source

In the paper

“Biodegradable lipids enable fast elimination of lipid nanoparticles from plasma and tissues, improving their safety and tolerability.”

In the paper

“Notably, biodegradable lipids are part of the mRNA-1273 and BNT162b2 COVID-19 mRNA vaccines.”

In the paper

“In summary, mRNA has shown great therapeutic potential in a number of clinical trials and in clinical applications.”