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

Probst, Jochen; Weide, Benjamin; Scheel, Birgit; Pichler, Bernd J.; Hoerr, Ingmar; Rammensee, Hans Georg; Pascolo, Steve

doi:10.1038/sj.gt.3302964

Cited by 174 publications

(177 citation statements)

References 17 publications

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“…The only study that investigated the uptake of RNA in vivo proposed a saturable Ca +2 ion-dependent process without defining the exact mechanism and cell type. 22 Similarly, for other naked nucleic acid based compounds, there is a lack of knowledge on mechanisms of internalization by APCs. For siRNA only uptake by metazoan cells has been studied, 23 whereas for plasmid DNA circumstantial evidence supports the hypothesis of uptake into macrophages via scavenger receptor A.…”

Section: Discussionmentioning

confidence: 99%

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

et al. 2011

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Even though it is known for more than one decade that antigen-encoding RNA can deliver antigenic information to induce antigen-specific immunity against cancer, the nature and mechanism of RNA uptake have remained enigmatic. In this study, we investigated the pharmacokinetics of naked RNA administered into the lymph node. We observed that RNA is rapidly and selectively uptaken by lymph node dendritic cells (DCs). Furthermore, in vitro and in vivo studies revealed that the efficient internalization of RNA by human and murine DCs is primarily driven by macropinocytosis. Selective inhibition of macropinocytosis by compounds or as a consequence of DC maturation abrogated RNA internalization and delivery of encoded antigens. Our findings imply that bioavailability of recombinant RNA vaccines in vivo highly depends on the density and the maturation stage of DCs at the administration site and are of importance for the design of RNA-based clinical immunotherapy protocols. Gene Therapy (2011) Keywords: vaccination; dendritic cells; RNA; macropinocytosis INTRODUCTION Antigen-encoding RNA has emerged as an attractive new vaccine format. Major advantages of RNA are efficient delivery of the complete antigenic information, transient expression and lack of genome integration, as well as cost-efficient and easy production in large amounts and high purity. 1 Moreover, through activation of the immune system via TLR3, TLR7 and TLR8, 2,3 RNA supports the induction of immune responses against the encoded protein.Of several in vitro and in vivo strategies utilizing antigen-encoding RNA as a vaccine format, 4 two are in clinical stage. One is based on adoptive transfer of autologous dendritic cells (DCs) transfected ex vivo with antigen-encoding RNA. [5][6][7] The other employs direct injection of naked RNA. 8,9 Feasibility and safety of vaccination protocols based on intradermal administration of naked RNA have been shown in preclinical and clinical settings and partial protection from tumor challenge has been achieved in mice. 10,11 However, at present, neither in animals nor in men, systemic immune responses let alone therapeutic effects were unequivocally demonstrated. It is assumed that rapid degradation by ubiquitous RNAses constraining pharmacologically efficient dosing of unprotected RNA contributes to the still suboptimal clinical effects. Knowledge about the nature and pharmacokinetics of RNA uptake in vitro and in vivo as well as the cell-type specificity is still scarce. Recently, we discovered that the administration route of naked RNA has a crucial impact on its efficacy as a vaccine. By injecting RNA directly into lymph nodes of mice, we achieved for the first time strong systemic antigen-specific Th1 type immunity and cancer cure in preclinical animal models. 12

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Section: Discussionmentioning

confidence: 99%

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

et al. 2011

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“…In contrast, it was reported that transfection on intradermal injection of messenger RNA is saturated at a concentration of 0.05 mg=ml. This suggests a different uptake mechanism between intradermal mRNA injection and the tattooing of double-stranded DNA (Probst et al, 2007).…”

Section: Van Den Berg Et Almentioning

confidence: 99%

Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin

et al. 2009

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The intradermal administration of DNA vaccines by tattooing is a promising delivery technique for genetic immunization, with proven high immunogenicity in mice and in nonhuman primates. However, the parameters that result in optimal expression of DNA vaccines that are applied by this strategy to human skin are currently unknown. To address this issue we set up an ex vivo human skin model in which DNA vaccine-induced expression of reporter proteins could be monitored longitudinally. Using this model we demonstrate the following: First, the vast majority of cells that express DNA vaccine-encoded antigen in human skin are formed by epidermal keratinocytes, with only a small fraction (about 1%) of antigen-positive epidermal Langerhans cells. Second, using full randomization of DNA tattoo variables we show that an increase in DNA concentration, needle depth, and tattoo time all significantly increase antigen expression ( p < 0.001), with DNA concentration forming the most critical variable influencing the level of antigen expression. Finally, in spite of the marked immunogenicity of this vaccination method in animal models, transfection efficiency of the technique is shown to be extremely low, estimated at approximately 2 to 2000 out of 1Â10 10 copies of plasmid applied. This finding, coupled with the observed dependency of antigen expression on DNA concentration, suggests that the development of strategies that can enhance in vivo transfection efficacy would be highly valuable. Collectively, this study shows that an ex vivo human skin model can be used to determine the factors that control vaccine-induced antigen expression and define the optimal parameters for the evaluation of DNA tattoo or other dermal delivery techniques in phase 1 clinical trials.

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“…4,5 Preclinical experiments and human clinical trials testing administration of autologous DCs loaded in vitro with RNA as well as direct injection of RNA via different routes to reach DCs in situ showed feasibility, lack of toxicity and induction of the expected antigen-specific immune response. [6][7][8][9][10][11] Improvement of immunobioavailability of RNA-based vaccines in DCs has been a recurrent subject of our research, because the dose of the antigen may be one of the critical factors for generating strong and sustained antigen-specific immune responses. 12 In previous work, we have developed plasmid templates for in vitro transcription of RNA-encoded antigen with modified 3 0 structures (3 0 UTR and poly(A)tail) stabilizing the RNA and optimizing its translational performance.…”

Section: Introductionmentioning

confidence: 99%

“…15 This cap has important roles in all aspects of mRNA metabolism such as specific recognition of mature mRNA by the translational initiation factor eIF4E for formation of the 48S complex, translation initiation and stability of the RNA in the process of protein synthesis. 16 Capping of recombinant antigen-encoding RNA manufactured for preclinical development and clinical use is achieved by transcribing the DNA template with a bacteriophage RNA polymerase in the presence of all four nucleotide triphosphates and the cap dinucleotide m 7 GpppG as structural homolog of the endogenous cap structure. 3 However, not all of the RNA is capped, because the cap dinucleotide has to compete with guanosine triphosphate (GTP) for the initiation of transcription.…”

Section: Introductionmentioning

confidence: 99%

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

et al. 2010

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Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5 0 mRNA cap analogs on the kinetics of the encoded protein, we found that m 2 7,2 0 ÀO Gpp S pG (b-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as b-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5 0 mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

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

Cited by 174 publications

References 17 publications

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

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