Spontaneous cellular uptake of exogenous messenger RNA in vivo is nucleic acid-specific, saturable and ion dependent.

The development of new treatments in the post-genomic era requires methods for safe delivery of foreign genetic information in vivo. As a transient, natural and controllable alternative to recombinant viruses or plasmid DNA (pDNA), purified or in vitro transcribed messenger RNA (mRNA) can be used for the expression of any therapeutic protein in vitro and in vivo. As it has been shown previously, the simple injection of naked mRNA results in local uptake and expression. We show here that this process, in the skin, can greatly be modulated according to the injection solution composition and blocked by an excess of competing nucleic acids or a drug affecting cytosolic mobility. Different cell types at the site of injection can take up the foreign nucleic acid molecules and the protein translated from this is detected for no more than a few days. To test this gene transfer method in humans, we produced in vitro transcribed mRNA under good manufacturing practice (GMP) conditions in a dedicated facility. After injection into the human dermis, we could document the translation of the exogenous mRNA. Our results pave the way toward the use of mRNA as a vehicle for transient gene delivery in humans.

Human peripheral blood mononuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro.

The efficiency of test vaccines needs to be evaluated by quantification of the triggered cellular immune response. Usually, for these assays, autologous target cells expressing the vaccine antigen are required. In the context of messenger RNA (mRNA)-based vaccinations, the target cells used for the read-out are mRNA-transfected monocyte-derived dendritic cells (Mo-DCs). Their production typically requires samples of 100 ml blood from the patients, and limits the number of assays that can be performed. We show here that fresh peripheral blood mononuclear cells (PBMCs) can be transfected with mRNA by electroporation. Such cells are as efficient as mRNA-transfected Mo-DCs for their ability to activate memory T cells in vitro. Thus, mRNA-transfected PBMCs are a convenient replacement of mRNA-transfected Mo-DCs for the in vitro monitoring of natural or vaccine-induced immune responses.

Polarization of immunity induced by direct injection of naked sequence-stabilized mRNA vaccines.

In the context of developing a safe genetic vaccination strategy we tested and studied globin-stabilized mRNA-based vaccination in mice. This vaccination strategy has the advantages of genetic vaccination (easy production, adaptability to any disease and inexpensive storage when lyophilized), but not the drawbacks of DNA vaccination (long-term uncontrolled expression of a transgene, possibility of integration into the host genome and possible induction of anti-DNA antibodies). We report here that injection of naked beta-globin untranslated region (UTR)-stabilized mRNA coding for beta-galactosidase is followed by detectable translation in vivo. In addition, we show that such a vaccination strategy primes a T helper 2 (Th2) type of response which can be enhanced and shifted to a Th1-type immune response by application of recombinant granulocyte/macrophage colony-stimulating factor 1 day after mRNA injection. Our data demonstrate that the administration of globin UTR-stabilized mRNA is a versatile vaccination strategy that can be manipulated to fit the requirement of antiviral, antibacterial or antitumor immunity.

In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies.

To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.