Design of in vitro Transcribed mRNA Vectors for Research and Therapy

Tusup, Marina; French, Lars E.; Matos, Mara De; Gatfield, David; Kündig, Thomas M.; Pascolo, Steve

doi:10.2533/chimia.2019.391

Cited by 31 publications

(29 citation statements)

References 24 publications

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“…The Cap 1 structure is achieved by the methylation of the mRNA first nucleotide at the ribose 2′-O position. Both caps can be added during in vitro mRNA transcription using a synthetic cap analogue [27] or the proprietary Cap dinucleotide CleanCap® [28] . Another capping approach uses a post-transcription enzymatic reaction based on the vaccinia capping system [29] .…”

Section: The Rise Of Mrna Technologymentioning

confidence: 99%

“…These regions strongly affect translation efficiency as the sequences used are involved in the translation machinery recognition, recruitment, and mRNA trafficking. Strategies to modulate the innate immune response, such as the introduction of unnatural nucleosides (NTPs), and to improve translation efficiency, by using codon optimisation, are also commonly used in mRNA production [27] , [28] .…”

Section: The Rise Of Mrna Technologymentioning

confidence: 99%

“…Nevertheless, a cost analysis should be performed to compare the costs of the one-step and two-step production options [93] . Alternatively, co-transcriptional capping can be performed using CleanCap® Reagent AG [28] . Although this method does not compete with GTP and delivers a Cap 1 construct, it requires the use of templates with a modified T7 promoter.…”

Section: Mrna Manufacturing: From Upstream To Downstreammentioning

confidence: 99%

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mRNA vaccines manufacturing: Challenges and bottlenecks

Rosa

Prazeres

Azevedo

et al. 2021

Vaccine

285

255

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Vaccines are one of the most important tools in public health and play an important role in infectious diseases control. Owing to its precision, safe profile and flexible manufacturing, mRNA vaccines are reaching the stoplight as a new alternative to conventional vaccines. In fact, mRNA vaccines were the technology of choice for many companies to combat the Covid-19 pandemic, and it was the first technology to be approved in both United States and in Europe Union as a prophylactic treatment. Additionally, mRNA vaccines are being studied in the clinic to treat a number of diseases including cancer, HIV, influenza and even genetic disorders. The increased demand for mRNA vaccines requires a technology platform and cost-effective manufacturing process with a well-defined product characterisation. Large scale production of mRNA vaccines consists in a 1 or 2-step in vitro reaction followed by a purification platform with multiple steps that can include Dnase digestion, precipitation, chromatography or tangential flow filtration. In this review we describe the current state-of-art of mRNA vaccines, focusing on the challenges and bottlenecks of manufacturing that need to be addressed to turn this new vaccination technology into an effective, fast and cost-effective response to emerging health crises.

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Section: The Rise Of Mrna Technologymentioning

confidence: 99%

Section: The Rise Of Mrna Technologymentioning

confidence: 99%

Section: Mrna Manufacturing: From Upstream To Downstreammentioning

confidence: 99%

See 1 more Smart Citation

mRNA vaccines manufacturing: Challenges and bottlenecks

Rosa

Prazeres

Azevedo

et al. 2021

Vaccine

285

255

View full text Add to dashboard Cite

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“…The 5′ end consisted of a CleanCap TM (Trilink) followed by an eIF4G aptamer as the 5′ untranslated region (Tusup and Pascolo, 2018 ) and by a codon-optimized firefly luciferase open reading frame. The 3′ end consisted of a tandem repeat of the mouse beta-globin 3′ UTR and a poly-A tail (Tusup et al., 2019 ). The transcription was made in the presence of the four canonical bases (alanine(A), cysteine (C), glycine (G) , and uracil (U)) to obtain immunostimulatory RNA and in the presence of pseudo-uridine instead of uridine to obtain immuno-silent mRNA used for luciferase expression assays.…”

Section: Methodsmentioning

confidence: 99%

Functional differences between protamine preparations for the transfection of mRNA

et al. 2020

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Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20-1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.

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“…These are of great interest for anticancer and antiviral therapies. On the other hand, larger particles activate monocytes and production of TNF-α [270].…”

Section: Protamine In Vaccine Developmentmentioning

confidence: 99%

Use of Protamine in Nanopharmaceuticals—A Review

et al. 2021

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Macromolecular biomolecules are currently dethroning classical small molecule therapeutics because of their improved targeting and delivery properties. Protamine-a small polycationic peptides represent a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interactions between the negatively charged DNA-phosphate backbone and the positively charged protamine. Researchers are mimicking this technique to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as a carrier for biologically active components such as DNA or RNA. The first part of this review highlights ongoing investigations in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are those that lead to the second key part, which is protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed, and we provide an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines.

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Design of in vitro Transcribed mRNA Vectors for Research and Therapy

Cited by 31 publications

References 24 publications

mRNA vaccines manufacturing: Challenges and bottlenecks

mRNA vaccines manufacturing: Challenges and bottlenecks

Functional differences between protamine preparations for the transfection of mRNA

Use of Protamine in Nanopharmaceuticals—A Review

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