Functional differences between protamine preparations for the transfection of mRNA

Abstract

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.

Hybridization Networks of mRNA and Branched RNA Hybrids

“…Genetic material that does not carry the risk of being inserted into the patient genome, and that can be designed to induce the expression of specific proteins, such as mRNA, is a most valuable tool in therapy. [27,33] To achieve the desired effect in vivo, the RNA has to be formulated. This is no easy task for a well soluble class of linear, polyanionic biopolymers.…”

“…Instead, biomaterial‐ or nanoparticle‐based strategies are being sought [27] . Here again, the cationic peptides, polymers or lipids commonly used to prepare the materials can be quite toxic or poorly defined on a molecular level [27,33] . This prompted us to ask whether less toxic agents that bind to the mRNA through more selective interactions could be employed to create biomaterials with the potential to be used in the clinic.…”

Use of Protamine in Nanopharmaceuticals—A Review

“…Thanks to the nuclear localization signals in its sequence, protamine is effectively taken to the cell nucleus, which is why it represents a great carrier for DNA molecules. However, the cargo can be released in the cytoplasm as well, which facilitates the use of protamine as a carrier for RNA molecules, that need to be released in the cytoplasm in order to be effective [269].…”

Multifunctional Immunoadjuvants for Use in Minimalist Nucleic Acid Vaccines

“…The vaccine showed good tolerability but was only efficacious when administered using a needle-free injection device. The mechanism of the adjuvanticity of protamine-RNA was reported to be related to TLR7/8 activation and was found to be strictly contingent upon complexation with mRNA [ 174 ]. Human PBMCs exposed to either mRNA or protamine alone did not produce any measurable cytokine production, while a combination of both resulted in a significant production of IFN-α, regardless of the protamine source or its salt form [ 174 ].…”