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.

: Agarose gel electrophoresis of the different recombinant mRNA products. 1 µg recombinant mRNA of each construct was loaded on 1% agarose gel. RiboRuler High Range RNA ladder (L) was used to determine the correct length of the transcripts (right corner). As shown here, all transcripts showed only one single band as expected.
Table S1: Size of transcripts coding for MetLuc. The size, length in bases (b), of each recombinant mRNA construct furnished with cellular UTRs is listed above. Additionally, all produced transcripts showed a single band on 1% agarose gel and were free of protein and organic contamination verified by agarose gel electrophoresis and spectrophotometric measurement, respectively. This recombinant mRNA starting material was used for the entire screening studies in vitro.

Screening of different transfection reagents for mRNA delivery
In an initial experiment, different transfection reagents (TfR) were compared for mRNA delivery in NIH3T3 and A549 cell lines. Four different commercially available TfRs namely, Dreamfect Gold (DFG, OzBiosciences), Lipofectamine TM 2000 (Invitrogen), MetafectenePro (Biontex) and Dogtor (OzBiosciences) were investigated with respect to the resulting protein translation and cell viability post-transfection. Both transfection efficiency and cell viability were evaluated to compare the different TfR. Cell viability was quantified by MTT assay. In both tested cell lines, MetafectenePro resulted in significant higher protein translation over a broad range of transfected mRNA doses ( Figure S2 a, b). Nonetheless mRNA delivery with MetafectenePro resulted in extensive cell death with cell viability falling to below 50% at higher doses in both cell lines. DFG instead showed an overall survival rate of more than 75% in NIH3T3 (up to 500 ng/well) and A549 cells (up to 250 ng/well) compared to MetafectenePro ( Figure S2 c, d).

Determination of mRNA half-life of MetLuc RNA and hBMP2 RNA
To determine the mRNA half-life of MetLuc in both cell lines including NIH3T3 and A549, mRNA decay kinetic data were obtained by qRT-PCR (Fig. 4a, b) and were fitted in one-phase decay equation. The same was performed for hBMP2 transcript in C2C12 cells. As a result we could not observe any significant changes in mRNA T1/2 of CYBA UTR bearing transcripts in contrast to the control without UTRs (Fig. S3 a, b) with the exception of hBMP2-CYBA 2X3 transcript showing a decrease in mRNA T1/2 in C2C12 cells (Fig. S3 c). Mean values of physical mRNA half-life are listed in Table S2.

Screening of hBMP2-CYBA constructs in C2C12 cells
The integrity of the modified mRNA constructs coding for hBMP2 was checked on 1% agarose gel ( Fig. S4a). mRNA magnetofection studies were conducted by using SoMag 5 nanoparticles at a dose of 20 pg mRNA/cell. At 24 and 48 hours post-transfection, hBMP2 was quantified using ELISA. The hBMP2-CYBA 2X3 transcript showed the highest and significant increase in hBMP2 protein amounts at both time points compared to the control (Fig. S4b). Messenger RNA constructs with 3' and 5'+2x3' CYBA UTR also resulted in significantly higher levels of hBMP2 at 48h post-transfection compared to the control. Recombinant mRNA constructs with 5'-UTR alone or hBMP2-CYBA 5+3 resulted in the lowest protein amount compared to the other tested mRNA constructs with CYBA UTR combinations. Values present mean ± standard error of three replicates. Statistical significance was assessed by 2way ANOVA test with p values: ** p<0.01, *** p<0.001, ****p<0.0001.