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

Kuhn, Andreas N.; Diken, Mustafa; Kreiter, Sebastian; Selmi, Abderraouf; Kowalska, Joanna; Jemielity, Jacek; Darżynkiewicz, Edward; Huber, Christoph; Türeci, Özlem; Şahin, Uğur

doi:10.1038/gt.2010.52

Cited by 203 publications

(179 citation statements)

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“…34 pSTI-eGFP-120 (enhanced green fluorescence protein (eGFP)), pSTI-SIIN-FEKL-MITD (SIINFEKL), pSTI-Influenza-HA-MITD (HA) and pSTI-Luciferase-A120 (Luc) vectors were described previously. [34][35][36] SIINFEKL construct encodes for the dominant epitope (aa 257-264) of chicken Ovalbumin, and HA construct contains a codon optimized partial sequence of influenza haemagglutinin (aa 60-285 fused to aa 517-527; influenza strain A/PR/8/34) designed to combine all major immunodominant MHC epitopes.…”

Section: Rna Constructsmentioning

confidence: 99%

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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: Rna Constructsmentioning

confidence: 99%

“…34 MHC-peptide tetramer staining from blood samples was performed with H-2K b /SIINFEKL tetramer (Beckman Coulter, Brea, CA, USA) and CD8 antibody (Invitrogen, Carlsbad, CA, USA). 36 Flowcytometric data were acquired on a FACSCalibur flow cytometer and analyzed by FlowJo (Tree Star, Ashland, OR, USA) software.…”

Section: Flow Cytometric Analysismentioning

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 vitro-synthesized mRNAs encoding tumor antigens can be used to elicit an immune response in dendritic cells, and vaccination with such mRNAs has already entered clinical testing for the treatment of cancer (Weide et al 2008;Diken et al 2011). The use of a cleavage-resistant cap increases mRNA stability and translational efficiency of exogenous mRNA in mice and enhances priming and expansion of naive antigen-specific T-cells (Kuhn et al 2010(Kuhn et al , 2011. In Saccharomyces pombe, oligouridylation of poly(A)-containing mRNA by the noncanonical poly(A) polymerase Cid1 has been demonstrated Norbury 2008, 2009).…”

Section: Blocking the 39 End Stabilizes Luc-slmentioning

confidence: 99%

mRNAs containing the histone 3′ stem–loop are degraded primarily by decapping mediated by oligouridylation of the 3′ end

Slepenkov

Slevin

et al. 2012

RNA

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Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 59/39 and 39/59 processes, but the relative contributions are not known. The 39 end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 39 UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of~40 min with the uncleavable cap compared to~8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 59/39 decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 59 decay intermediates were oligouridylated. Preventing oligouridylation by 39-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 39/59 degradation machinery stalls during initial degradation, whereupon reuridylation occurs.

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“…46 Others have explored different strategies for improving the mRNA-construct and the antigen presentation on HLA class I and II. [47][48][49] The vaccine concept may be further developed by novel strategies for enhancing DC-migration and immune stimulation. [50][51][52][53][54] We currently test the clinical use of DCs generated with a 3D protocol, based on a collaboration with Prof. Schendel and collegues.…”

Section: Discussionmentioning

confidence: 99%

Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells

et al. 2016

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The most effective anticancer immune responses are probably directed against patient-specific neoantigens. We have developed a melanoma vaccine targeting this individual mutanome based on dendritic cells (DCs) loaded with autologous tumor-mRNA. Here, we report a phase I/II trial evaluating toxicity, immune response and clinical outcome in 31 metastatic melanoma patients. The first cohort (n D 22) received the vaccine without any adjuvant; the next cohort (n D 9) received adjuvant IL2. Each subject received four weekly intranodal or intradermal injections, followed by optional monthly vaccines. Immune response was evaluated by delayed-type hypersensitivity (DTH), T cell proliferation and cytokine assays. Data were collected for 10 y after inclusion of the last patient. No serious adverse events were detected. In the intention-to-treat-cohort, we demonstrated significantly superior survival compared to matched controls from a benchmark meta-analysis (1 y survival 43% vs. 24%, 2 y 23% vs. 6.6%). A tumorspecific immune response was demonstrated in 16/31 patients. The response rate was higher after intradermal than intranodal vaccination (80% vs. 38%). Immune responders had improved survival compared to non-responders (median 14 mo vs. 6 mo; p D 0.030), and all eight patients surviving >20 mo were immune responders. In addition to the tumor-specific response, most patients developed a response against autologous DC antigens. The cytokine profile was polyfunctional and did not follow a Th1/Th2 dichotomy. We conclude that the favorable safety profile and evidence of a possible survival benefit warrant further studies of the RNA/DC vaccine. The vaccine appears insufficient as monotherapy, but there is a strong rationale for combination with checkpoint modulators.

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

Cited by 203 publications

References 30 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

mRNAs containing the histone 3′ stem–loop are degraded primarily by decapping mediated by oligouridylation of the 3′ end

Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells

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