Abstract
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.
Highlights
Another cis-acting element located in the mRNA with major impact on stability is the poly(A) tail
Results obtained from the magnetofection of C2C12 cells indicated that human bone morphogenetic protein 2 (hBMP2)-cytochrome b-245 alpha polypeptide (CYBA) 2X3
Both of these recombinant transcripts were selected for further stability analysis via Quantitative real time PCR (qRT-PCR). hBMP2 transcripts bearing CYBA untranslated region (UTR) did not result in any increased T1/2 by comparison to control hBMP2 transcript without UTRs (Fig. 5a and Supplementary Fig. S3c and Supplementary Table S2)
Summary
Another cis-acting element located in the mRNA with major impact on stability is the poly(A) tail. Other studies have reported that modified nucleotide(s) within the mRNA structure have a strong impact on protein translation and enable reprogramming of human cells to pluripotency, vascular regeneration, as well as bone regeneration in vitro and in vivo[20,21,22,23]. In the latter example, modified mRNA coding for human bone morphogenetic protein 2 (hBMP2) was used to induce bone regeneration[22,23]. Further ex vivo and in vivo experiments, in human adipose tissue and long bone defects in rats, respectively, have confirmed transcript therapy as a potential therapeutic strategy[22]
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