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

Abstract

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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