Optimization of 5′ Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury

Abstract

Modified mRNA (modRNA) is a gene-delivery platform for transiently introducing a single gene or several genes of interest to different cell types and tissues. modRNA is considered to be a safe vector for gene transfer, as it negligibly activates the innate immune system and does not compromise the genome integrity. The use of modRNA in basic and translational science is rising, due to the clinical potential of modRNA. We are currently using modRNA to induce cardiac regeneration post-ischemic injury. Major obstacles in using modRNA for cardiac ischemic disease include the need for the direct and single administration of modRNA to the heart and the inefficient translation of modRNA due to its short half-life. Modulation of the 5 0 untranslated region (5 0 UTR) to enhance translation efficiency in ischemic cardiac disease has great value, as it can reduce the amount of modRNA needed per delivery and will achieve higher and longer protein production post-single delivery. Here, we identified that 5 0 UTR, from the fatty acid metabolism gene carboxylesterase 1D (Ces1d), enhanced the translation of firefly luciferase (Luc) modRNA by 2-fold in the heart post-myocardial infarction (MI). Moreover, we identified, in the Ces1d, a specific RNA element (element D) that is responsible for the improvement of modRNA translation and leads to a 2.5-fold translation increment over Luc modRNA carrying artificial 5 0 UTR, post-MI. Importantly, we were able to show that 5 0 UTR Ces1d also enhances modRNA translation in the liver, but not in the kidney, post-ischemic injury, indicating that Ces1d 5 0 UTR and element D may play a wider role in translation of protein under an ischemic condition.

Modified mRNA as a Therapeutic Tool for the Heart

“…Mechanistically, Ces1d is involved in lipid metabolism, and changes in this metabolic activity under MI conditions trigger Ces1d mRNA, leading to better translation. Further, this altered modRNA structure also enhanced translation in other organs, including the liver, under ischemic conditions [28]. These promising studies thus indicate modRNA technology can be used in future cardiac regenerative applications.…”

“…1), particularly changing uridine with naturally occurring pseudouridine and 5-methyl-cytosine for cytosine [25], combined with advances in in vitro transcription technologies led to less recognition by TLRs and nucleases. Additionally, the stability and in vivo translation efficiency of modRNA have [26,27] and replacing 5′UTR with one from the fatty acid metabolism gene carboxylesterase 1D (Ces1d) [28].…”

“…As was recently reported, modRNAdelivered acid ceramidase (AC) overexpression is associated with lower CM death rates and increased cell survival after hypoxia or MI. AC primarily frees fatty acids and sphingosine in ceramide hydrolysis, and elevated AC has been reported to lessen the negative effects of [28] elevated ceramides. In addition, elevated levels of this enzyme reduced detrimental neutrophil levels in the LV, thus decreasing the inflammation associated with MI and promoting cell survival.…”

“…1 ), particularly changing uridine with naturally occurring pseudouridine and 5-methyl-cytosine for cytosine [ 25 ], combined with advances in in vitro transcription technologies led to less recognition by TLRs and nucleases. Additionally, the stability and in vivo translation efficiency of modRNA have been increased by capping the molecule with the 3′-O-Mem7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) at its 5′end [ 26 , 27 ] and replacing 5′UTR with one from the fatty acid metabolism gene carboxylesterase 1D (Ces1d) [ 28 ]. ModRNA has the capacity to target the gene of interest via multiple mechanisms including: (i) gain of function by overexpressing a target molecule [ 13 , 29 ], (ii) loss of function by either using dominant negative molecules or introducing miRNA [ 30 ], (iii) correcting gene deletions at the mRNA level [ 31 ], and (iv) inhibiting intracellular signaling pathways by introducing decoy receptors to dilute the ligands [ 30 ].…”

m6A RNA methylation regulators contribute to progression and impact the prognosis of breast cancer

“…As early as the 1970s, people had discovered m 6 A modifications in the mRNA and lncRNA of eukaryotes (5) . It is known that in most eukaryotes, the methylation modification that occurs in the mRNA region of 5’UTR plays a significant role in mRNA splicing, editing, stability, degradation, and polyadenylation (6-8) ; while the modification of the 3’UTR region Methylation modification contributes to the nuclear transport of mRNA, the initiation of translation, and the structural stability of the mRNA together with the polyA binding protein (9) . m 6 A methylase is widely involved in mammalian development, immunity, tumor generation and metastasis, stem cell renewal, fat differentiation, and other life processes.…”