Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription.Materials and methods:In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed.Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs.Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary

Charakterisierung der Apolipoprotein J-Genregulation in Fibroblasten und glatten Gefäßmuskelzellen durch nekrotische Zellen und Toll-like Rezeptoren

Apolipoprotein J (ApoJ) ist ein sezerniertes heterodimeres 80kDa Glykoprotein mit zytoprotektiven und antiinflammatorischen Eigenschaften, das ein nahezu ubiquitäres Expressionsmuster aufweist. Eine stark erhöhte ApoJ-Expression ist mit neurodegenerativen Erkrankungen, Atherosklerose, myokardialem Infarkt sowie einer Vielzahl anderer pathophysiologischer Bedingungen assoziiert. Die potentielle Bedeutung von ApoJ umfasst eine Funktion als extrazelluläres Chaperon, Komplementinhibitor, NF-kB-Inhibitor sowie eine Beteiligung an der Endozytose von nekrotischen Zellfragmenten. Unter Bedingungen, die zu einer massiven Akkumulation von absterbenden Zellen führen, ist eine vermehrte Expression von ApoJ auf die überlebenden Nachbarzellen in den betroffenen Geweben beschränkt. Die molekularen Mechanismen, die dieser gesteigerten ApoJ-Genexpression zugrunde liegen, sind jedoch unbekannt. Untersuchungen unserer Arbeitsgruppe konnten zeigen, dass eine Inkubation mit nekrotischem Zellmaterial in vitro eine Akkumulation von ApoJ-mRNA in Fibroblasten der Zelllinie Rat1 induziert, was darauf hindeutet, dass unter pathophysiologischen Bedingungen von nekrotischen Zellen exponierte bzw. freigesetzte Faktoren zu einer gesteigerten ApoJ-Genexpression in umliegenden vitalen Zellen beitragen können. Die im Rahmen der vorliegenden Arbeit durchgeführten Untersuchungen zeigen eine Korrelation zwischen der Expression von Toll-like Rezeptoren (TLRs) in Fibroblasten (Rat1), glatten Gefäßmuskelzellen (CRL2018) sowie embryonalen Dottersackzellen (10A) und einer durch nekrotische Zellen induzierten ApoJ-mRNA-Expression in diesen Zelllinien. Es wird angenommen, dass TLRs neben pathogenassoziierten Strukturen (PAMPs) auch durch körpereigene Agonisten wie Hitzeschockproteine und Nukleinsäuren aktiviert werden. In weiterführenden Experimenten stellte sich unter anderem heraus, dass neben nekrotischen Zellen auch der TLR3-spezifische Agonist Poly(I:C), eine synthetische doppelsträngige RNA, ausschließlich in den beiden TLR3-exprimierenden Zelllinien CRL2018 und Rat1, nicht jedoch in TLR3-defizienten 10A-Zellen, die ApoJ-mRNA-Expression induziert. Darüber hinaus führt auch die Inkubation mit eukaryotischer RNA (Gesamt-RNA, t-RNA) zu einer Akkumulation von ApoJ-mRNA in CRL2018-Zellen. Die Ergebnisse dieser Arbeit zeigen erstmals, dass die Expression von ApoJ-mRNA durch extrazelluläre Ribonukleinsäuren in TLR3-abhängiger Weise induziert wird, was darauf hindeutet, dass in verletzten Geweben aus post-apoptotischen oder nekrotischen Zellen freigesetzte Ribonukleinsäuren zu einer vermehrten ApoJ-Genexpression in vitalen Nachbarzellen beitragen.Apolipoprotein J (apoJ) is an 80 kDa secreted glycoprotein with nearly ubiquitous tissue distribution that has cytoprotective and anti-inflammatory properties. Strong expression of apoJ is associated with neurodegenerative conditions, atherosclerosis, myocardial infarction and other patho-physiological situations. ApoJ is suggested to inhibit complement-mediated cell lysis and NF-kB dependent signalling, to act as an extracellular chaperone and to facilitate the endocytic uptake of necrotic cell fragments. In tissues with massive cell death apoJ expression is increased and restricted to the surviving bystander cells. The molecular mechanisms leading to this response, however, are not known. Previous work by our group could show that exposure to necrotic cell debris induces apoJ-mRNA expression in vital Rat1-fibroblasts in vitro suggesting that in pathophysiological situations enhanced expression of apoJ in surviving cells is, at least in part, mediated by factors released by or associated with necrotic cells. By using fibroblasts (Rat1), vascular smooth muscle cells (CRL2018) and an embryonic yolk sac cell line (10A) the present study shows a correlation between the expression of Toll-like receptors (TLRs) and the induction of apoJ-mRNA by necrotic cells. Beside pathogen-associated molecular patterns (PAMPs) it is suggested, that TLRs are also activated by host derived agonists like heat shock proteins and nucleic acids. Further work demonstrated that induction of apoJ-mRNA by necrotic cells and by the TLR3-specific agonist Poly(I:C), a synthetic dsRNA, is restricted to TLR3-expressing CRL2018 and Rat1 cells. Exposure to both stimuli does not lead to enhanced apoJ-mRNA expression in TLR3-deficient 10A cells. In addition expression of apoJ in CRL2018 cells is induced upon incubation with eukaryotic ribonucleic acids (total RNA, t-RNA). The results of this work show for the first time that apoJ expression is regulated in a TLR3-dependent manner and is induced by extracellular ribonucleic acids indicating that in injured tissues RNA released from post-apoptotic or necrotic cells contributes to increased apoJ synthesis by vital bystander cells

Statins stimulate the production of a soluble form of the receptor for advanced glycation end products

The beneficial effects of statin therapy in the reduction of cardiovascular pathogenesis, atherosclerosis, and diabetic complications are well known. The receptor for advanced glycation end products (RAGE) plays an important role in the progression of these diseases. In contrast, soluble forms of RAGE act as decoys for RAGE ligands and may prevent the development of RAGE-mediated disorders. Soluble forms of RAGE are either produced by alternative splicing [endogenous secretory RAGE (esRAGE)] or by proteolytic shedding mediated by metalloproteinases [shed RAGE (sRAGE)]. Therefore we analyzed whether statins influence the production of soluble RAGE. Lovastatin treatment of either mouse alveolar epithelial cells endogenously expressing RAGE or HEK cells overexpressing RAGE caused induction of RAGE shedding, but did not influence secretion of esRAGE from HEK cells overexpressing esRAGE. Lovastatin-induced secretion of sRAGE was also evident after restoration of the isoprenylation pathway, demonstrating a correlation of sterol biosynthesis and activation of RAGE shedding. Lovastatin-stimulated induction of RAGE shedding was completely abolished by a metalloproteinase ADAM10 inhibitor. We also demonstrate that statins stimulate RAGE shedding at low physiologically relevant concentrations. Our results show that statins, due to their cholesterol-lowering effects, increase the soluble RAGE level by inducing RAGE shedding, and by doing this, might prevent the development of RAGE-mediated pathogenesis

A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA

The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications. Keywords: double-stranded RNA, in vitro transcription, messenger RNA, nucleoside-modified RNA, RNA purification, cellulose-based purification, RNA immunogenicit

Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

<div><p>Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with <i>in vitro</i> mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU_1‑449, CLU_21‑449 and CLU_34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.</p> </div

Expression of CLU-V5 proteins from recombinant cDNAs.

<p>Lysates (upper panels) and culture media (lower panels) of HEK-293 cells transiently expressing the indicated CLU cDNA variants were analyzed by Western blotting. Lanes are labeled with circled numbers. Untransfected cells (HEK‑293) or cells transfected with empty pcDNA6 (mock) served as controls. Data shown are representative of at least three independent experiments. (A) Transfection of cDNA variants 1, 2 and 3 leads to expression and secretion of sCLU (lanes 3-5). Variant 1 [Δex2] produces a non-secreted 45 kDa CLU‑V5 protein corresponding to CLU_34‑449 (lane 6). This form is also present in low amounts in the lysates of cells transfected with the cDNA variants 1, 2 and 3. Furthermore, cells transfected with these variants express an additional non-secreted 50 kDa CLU‑V5 protein. (B) A schematic outline of the 5’-end of cDNA variant 3 is shown. Neither point-mutations (crossed out codons) of the sCLU start codon (framed, lane 5) nor the in-frame ATG on exon 1c (lane 4) completely block sCLU expression. Concurrent mutation of both codons eliminates sCLU synthesis (lane 6).</p

Proteasomal inhibition and heat stress modulate sCLU and intracellular CLU protein expression in cancer and non-cancer cells.

<p>HEK‑293, PC‑3, MCF‑7 and Caco-2 cells were treated with DMSO as control (C), 10 µM MG-132 (MG) or subjected to heat shock (45°C). Whole cell lysates (upper panel) and cell culture media (lower panel) of cells were analyzed for CLU expression by Western blot. 45-50 kDa CLU protein bands were detected primarily in stressed cells (*). Data shown are representative of three independent experiments.</p

Expression of CLU mRNA variants in cancer and non-cancer cells upon proteotoxic stress.

<p>(A) Semi-quantitative RT‑PCR analyses of the expression of different CLU mRNA variants in control and MG‑132-treated HEK‑293, MCF‑7, Caco-2 and PC‑3 cells. The different numbers of amplification cycles performed reflect the varying amounts of individual CLU mRNA variants expressed. Analysis of Hsp27 mRNA indicates the induction of a heat-shock response upon MG‑132 treatment. GAPDH RT‑PCR served as control to ensure the use of equal amounts of reverse transcribed mRNA. Data shown are representative of at least 3 independent experiments. (B, C, D) Quantification of CLU mRNA expression in control and MG‑132-treated HEK‑293 (B), MCF‑7 (C) and PC‑3 cells (D) by qPCR. The amounts of individual transcripts are expressed as copy number per ng of total RNA (mean ± SD, <i>n</i> = 3). Variant 1 is the pre-dominant CLU mRNA in all cell lines conforming to the amounts of total CLU mRNA. The other variants represent low abundant CLU mRNAs.</p