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.

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 in vitro 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, CLU1‑449, CLU21‑449 and CLU34‑449, 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.

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

This article is available online at http://www.jlr.org complications, atherosclerosis, and Alzheimer's disease (AD) ( 1 ). Proteins and peptides like advanced glycation end products (AGEs), amyloid-␤ peptides (A ␤ s), S100/ calgranulin family members, and HMGB1 (amphoterin) have been identifi ed as ligands for RAGE ( 2 ). Diabetes is characterized by a high blood glucose level. This enhanced concentration of glucose is responsible for the nonenzymatic generation of AGEs. AGEs represent a heterogeneous group of proteins, lipids, and nucleic acids resulting from chemical reactions between reducing sugars and amino groups. Studies with animal models have shown that RAGE is the best known target for AGEs in the vasculature and it is well established that the AGE/RAGE interaction contributes to the progression of atherosclerotic plaques ( 3-5 ). Ligand/RAGE interaction induces activation of various pro-infl ammatory and pro-atherogenic mediators, such as the nuclear factor-B (NF-B)-dependent mediators vascular cell adhesion molecule-1, tumor necrosis factor-␣ , interleukin-6, and RAGE ( 6 ). Because RAGE itself is regulated by NF-B, this further increases its expression and promotes cellular dysfunction ( 7 ).Blockade of RAGE by using the soluble form of the receptor ameliorates the vascular complications of diabetes in animal models ( 8-10 ) and suppresses the accumulation of A ␤ s in the brain of an AD mouse model ( 11 ). These benefi cial effects of soluble RAGE are thought to be mediated by trapping ligands, thus preventing ligand binding of membrane-bound RAGE. The circulating soluble form of RAGE containing only the extracellular part of fulllength RAGE, is either produced by alternative splicing The type I transmembrane protein receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, has been shown to play a crucial role in chronic infl ammatory diseases, late diabetic Press, August 21, 2013 DOI 10.1194 Statins stimulate the production of a soluble form of the receptor for advanced glycation end products Abbreviations: A ␤ , amyloid-␤ peptide; AD, Alzheimer's disease; AGE, advanced glycation end product; APP, amyloid precursor protein; esRAGE, endogenous secretory receptor for advanced glycation end products; FCS, fetal calf serum; LDS, lipid-defi cient serum; M ␤ CD, methyl-␤ -cyclodextrin; NF-B, nuclear factor-B; RAGE, receptor for advanced glycation end products; RNAi, RNA interference; siRNA, small interfering RNA; SQS, squalene synthase; sRAGE, shed receptor-the soluble form of the receptor for advanced glycation end products after proteolytic shedding of the full length receptor; ZA, zaragozic acid A . Published, JLR Papers in

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