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

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.

Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.

Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays.

The measurement of mRNA turnover in living cells plays an important role in the search for stable mRNA constructs for RNA-based therapies. Here we show that automated time-lapse microscopy combined with micropatterned arrays allows for efficient high-throughput monitoring of fluorescent reporter protein expression at the single-cell level. The fluorescence time courses after mRNA transfection yield the distribution of individual mRNA expression and degradation rates within a population. We compare mRNA constructs with combinations of 5' and 3' UTR sequences and find a systematic broadening and shift towards longer functional half-lives for UTR stabilized mRNA. At the same time the life time distribution of the destabilized EGFP reporter protein was found to be constant and narrowly distributed. Using mathematical modeling, we show that mRNA functional life-time predicts the time-integrated protein level, i.e. the area under the curve (AUC) of mRNA translation. Our approach paves the way for quantitative assessment of hitherto unexplored mRNA functional life time heterogeneity, possibly predicated on multiple mRNA secondary structures and its dependence on UTR sequences.