Determinants of intracellular RNA pharmacokinetics: Implications for RNA-based immunotherapeutics

Kuhn, Andreas N.; Diken, Mustafa; Kreiter, Sebastian; Vallazza, Britta; Türeci, Özlem; Şahin, Uğur

doi:10.4161/rna.8.1.13767

Cited by 34 publications

(26 citation statements)

References 101 publications

(121 reference statements)

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“…It has been suggested that the immunobioavailability of antigen mRNA is a critical success-limiting factor in view of cancer therapy (34). First, we showed high antigen expression when mRNA was delivered in Ca 2þ -containing HBSS or the clinically applied 0.8 RL, confirming the Ca 2þ dependency for efficient uptake of mRNA (29).…”

Section: Discussionsupporting

confidence: 55%

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

Goyvaerts²,

et al. 2012

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The use of tumor-associated antigen (TAA) mRNA for therapeutic purposes is under active investigation. To be effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In this study, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encoding CD40 ligand, constitutive active Toll-like receptor 4 and CD70, results in the in situ modification and maturation of DCs, hence, priming of TAA-specific T cells. We showed selective uptake and translation of mRNA in vivo by lymph node resident CD11c þ cells. This process was hampered by codelivery of classical maturation stimuli but not by TriMix mRNA. Importantly, TriMix mRNA induced a T-cell-attracting and stimulatory environment, including recruitment of antigen-specific CD4 þ and CD8 þ T cells and CTLs against various TAAs. In several mouse tumor models, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNAelectroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together with mRNA encoding immunomodulating molecules is a promising vaccination strategy. Cancer Res; 72(7); 1661-71. Ó2012 AACR.

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Section: Discussionsupporting

confidence: 55%

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

Goyvaerts²,

et al. 2012

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“…Furthermore, Holtkamp et al ., have shown that 2 copies of 3′ UTR from β-globin result in increased protein levels and mRNA stability only in immature dendritic cells, whereas in mature dendritic cells, no effect could be observed18. Cell specificity of UTRs when combined with mRNA has been also observed for other cells and tissues and has been discussed in the recent reviews4142.…”

Section: Discussionmentioning

confidence: 93%

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Ferizi

Aneja

Balmayor

et al. 2016

Sci Rep

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Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

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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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Determinants of intracellular RNA pharmacokinetics: Implications for RNA-based immunotherapeutics

Cited by 34 publications

References 101 publications

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

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

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