Protamine is a natural cationic peptide mixture mostly known as a drug for the neutralization of heparin and as a compound in formulations of slow-release insulin. Protamine is also used for cellular delivery of nucleic acids due to opposite charge-driven coupling. This year marks 60 years since the first use of Protamine as a transfection enhancement agent. Since then, Protamine has been broadly used as a stabilization agent for RNA delivery. It has also been involved in several compositions for RNA-based vaccinations in clinical development. Protamine stabilization of RNA shows double functionality: it not only protects RNA from degradation within biological systems, but also enhances penetration into cells. A Protamine-based RNA delivery system is a flexible and versatile platform that can be adjusted according to therapeutic goals: fused with targeting antibodies for precise delivery, digested into a cell penetrating peptide for better transfection efficiency or not-covalently mixed with functional polymers. This manuscript gives an overview of the strategies employed in protamine-based RNA delivery, including the optimization of the nucleic acid’s stability and translational efficiency, as well as the regulation of its immunostimulatory properties from early studies to recent developments.

Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding “untranslated” regions (UTRs) that are involved in the stabilization and translation of the mRNA. However, UTRs are often complex structures, which may complicate the entire production process. To eliminate this obstacle, we managed to reduce the total amount of nucleotides in the UTRs to only four bases. In this way, we generate minimal ivt mRNA (“minRNA”), which is less complex than the usual optimized ivt mRNAs that are contained, for example, in approved vaccines. We have compared the efficacy of minRNA to common augmented mRNAs (with UTRs of globin genes or those included in licensed vaccines) in vivo and in vitro and could demonstrate equivalent functionalities. Our minimal mRNA design will facilitate the further development and implementation of ivt mRNA-based vaccines and therapies.

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.

Recombinant in vitro-transcribed mRNA is broadly used for vaccination and is evaluated in numerous clinical studies for multiple indications. Typical features of mRNA are the 5′ cap, 5′ untranslated region, start and stop codons, 3′ untranslated region and 3′ poly(A) tail. Here, contrary to current dogma, we show that short, chemically synthesized RNA oligonucleotides lacking some or all of these features are efficiently translated when they encode epitopes recognized by CD8 ⁺ T cells. In particular, one design that we termed ChemRNA with the structure 5′-OH-AUG-coding sequence-3′-OH strongly stimulates antigen-specific CD8 ⁺ T cells both in vitro and in vivo. Our results challenge the current understanding of canonical mRNA structure and introduce the possibility that defective or supposedly non-coding RNA may encode human and murine major histocompatibility complex class I-associated peptides. Moreover, ChemRNA could help overcome challenges associated with the design and purification of individualized anti-cancer vaccines.

Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3′ poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)₁₂ oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)₁₂ oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials.

Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3’ poly-A tail required for optimal stabilization and translation of the mRNA. The average length of the poly-A tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly-A tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly-A tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. A poly-dT(12) oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly-A sequence available. Using a titration curve made with known amounts of poly-A RNA oligonucleotide, the thiazole orange oligo dT probe can instantly indicate the average length of the poly-A tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly-A tail in in vitro transcribed mRNA produced for research, pre-clinical studies and clinical trials.