RNA Stabilization

Design overview

We inserted a 4x RNA stabilizing sequence derived from Venezuelan equine encephalitis virus (VEEV RTSS in short) at the 3’ UTR region of the mRNA construct. With the assistance of the former three designs (in Exosome biogenesis, RNA packaging & RNA delivery), the therapeutic RNA transcript with 4x VEEV RTSS is integrated into the exosomes and delivered into neurons in the central nervous system, thereafter expressing its therapeutic gene for a prolonged time period.

Aim of the design

Intracellular delivery and stability are two major obstacles for mRNA-based therapy (Kaczmarek, Kowalski & Anderson, 2017). Quite a few RNA molecules have been substantiated to be therapeutically effective in vitro but fail to function appreciably in vivo, which is largely ascribed to their limited half-life and delivery method. Therefore, we manage to solve the RNA intracellular stability issue by attaching the RNA stabilizing element from VEEV to the 3’ UTR of various therapeutic RNA transcripts.

VEEV RTSS

This sequence is derived from the 3’ UTR of the single-stranded RNA virus VEEV upstream of its poly A tail (Garneau, et al., 2008). The specific function of this sequence in the virus is still not fully uncovered. However, according to the Hayashi’s research group (2010), adding this element to the 3’ end of any protein-encoding sequence could prolong the expression duration. Moreover, they also observed an enhanced RNA stabilizing capacity by arranging it in multiple repeats. The stabilizing mechanism demonstrated by the Hayashi’s group (2010) is that the sequence can impede the poly A shortening process mediated by deadenylase enzymes, which is thought to be the major RNA decay pathway (Houseley & Tollervey, 2009). A 38-kiloDalton protein in mammalian cells was reported to be involved in this block of deadenylation, however the identity of this protein still remains unspecified (Garneau, et al., 2008).

Hayashi et al (2010) tested the stabilizing abilities of different configurations of this sequence. From the results of Hayashi’s study, 4x VEEV RTSS appeared to be most capable of prolonging mRNA expression (see the figure below). Hence, in the iGEM project, we adopt using 4 copies of the VEEV sequence as our mRNA stabilizer.

Hayashi et al (2010) [Online] Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/

Design of testing

We constructed three plasmids for the RNA stabilization test. The L7Ae protein and C/D box (belonging to the RNA packaging part) are also involved in this test to ensure that after purifying exosomes by ultracentrifugation, there is detectable amount of RNA packaged in the exosomes for its expression in target neurons.

1. Co-transfection of HEK293T cells.

	pcDNA3.1-CD63-nanoluc	pcDNA3.1-STEAP3-IRES-SDC4-IRES-NadB	pcDNA3.1-nSMase
Group 1	+	-	-
Group 2	+	+	-
Group 3	+	+	+
Group 4	+	-	+

After co-transfection, exosomes are collected through ultracentrifugation.

2. Application of the collected exosomes to the neuronal cells.

3. The mRNA stabilizing ability (protein expression prolonging ability) is estimated by using luciferase assay over a time course.

References

Garneau, N. L., Sokoloski, K. J., Opyrchal, M., Neff, C. P., Wilusz, C. J., and Wilusz, J. (2008) The 3' untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and Mammalian cells. Journal of virology 82, 880-892

Hayashi, T., Lamba, D. A., Slowik, A., Reh, T. A., and Bermingham-McDonogh, O. (2010) A method for stabilizing RNA for transfection that allows control of expression duration. Developmental dynamics : an official publication of the American Association of Anatomists 239, 2034-2040

Houseley, J., and Tollervey, D. (2009) The many pathways of RNA degradation. Cell 136, 763-776

Kaczmarek, J. C., Kowalski, P. S., and Anderson, D. G. (2017) Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome medicine 9, 60