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Branched Chemically Modified Poly(A) Tails Enhance mRNA Translation

Branched Chemically Modified Poly(A) Tails Enhance mRNA Translation
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Branched Chemically Modified Poly(A) Tails Enhance mRNA Translation


Vaccines and therapeutics based on mRNA technology continue to rapidly advance, driven especially by discovery and development of new chemical modifications of mRNA to improve translation of encoded proteins. As reviewed elsewhere, this includes chemical modifications of the 5′-cap, coding sequence, and flanking 5′- and 3′-untranslated regions (UTRs). Only recently have chemically synthesized modifications to the 3′-poly(A) tail been explored (Aditham et al. 2022). 

The present blog discusses new discoveries for enhanced protein translation by use of branched chemically modified poly(A) tails, termed “multitails,” reported by Chen et al. at the Broad Institute and Massachusetts Institute of Technology. This Nature Biotechnology publication in May, 2024, which has already been accessed more than 26,000-times, used IVT mRNA prepared with CleanCap® AG analog and N1-methylpseudouridine(m1ψ)-5′-triphosphate from TriLink.  

Selected findings are presented in the following sections: 

  1. Design, synthesis, and screening of multitail mRNA 
  2. Mechanistic insights into how multitails increase mRNA translation 
  3. Prolonged protein production by multitail mRNA in vitro and in vivo  
  4. Multitail mRNA for enhanced CRISPR-based therapeutics  


(1) Design, synthesis, and screening of multitail mRNA 

Previous work at the Broad Institute (Aditham et al. 2022) demonstrated that ligation of exonuclease-resistant chemically modified synthetic oligonucleotides to the 3′ end of poly(A) tails increases mRNA stability and protein production in vitro. This suggested to Chen et al. that other types of 3′-end poly(A) tail modifications might further prevent deadenylation and initiate ribosomal translation by interacting with poly(A)-binding protein (PABP) to engage elongation factors (ElFs), forming a closed-loop complex (PABPC) (Figure 1).  


Figure 1. Closed-loop mRNA complex (PABPC) with PABP and ElFs for ribosomal translation (Taken from Du et al. 2018 and free to use under CC-BY license). 

Chen et al. hypothesized that multimerization of the poly(A) tail via a branched topology, with each individual poly(A) tail bearing extensive nuclease-resistant modifications, would further protect poly(A) tail integrity against RNA decay and preserve PABPC for prolonged translation. As depicted in Figure 2, firefly luciferase (FLuc)-encoding IVT mRNA incorporating TriLink’s CleanCap® AG analog and m1ψ was therefore enzymatically ligated (Aditham et al.) to a 5′-phosphorylated multitail precursor. This precursor was synthesized by click chemistry using a functionalized poly(A) “stem” and 3 modified poly(A) “branches”.  


Figure 2. Exemplary multitail FLuc mRNA synthesis by ligation of 5′-CleanCap IVT FLuc mRNA (m1ψ) with a 3-branch multitail; gold = rA, blue = 2′MOE/PS-modified rA; pink dot = 5′-phosphate; green dot = click chemistry linkage (Adapted from Chen et al.; Credit: Jerry Zon). 


Similar methodology was used to prepare precursor constructs having either 1 or 2 branches. In all three cases, the last six nucleotides of the 3′-ends of the branches were comprised of nuclease-resistant phosphorothioate (PS) and 2′-O-methoxyethyl (2′MOE) moieties. Details for this elegant chemistry and additional modified constructs can be found in Chen et al. 

To evaluate the effect of multitail structures on translation, the modified mRNAs were screened by co-transfected into HeLa cells with an unmodified linear Renilla luciferase (RLuc) mRNA as an internal transfection control to quantify protein production by the ratio of FLuc to RLuc bioluminescence.  

Among all constructs tested, the construct with 3 branches and synergistic incorporation of PS and 2′MOE (Figure 2) had the highest enhancements, yielding 4.7-, 10.7- and 19.5-fold higher luminescence signals than the control unmodified linear FLuc mRNA at 24, 48 and 72 h post transfection, respectively. This 3-branched multitail construct was therefore selected for use in the following studies. 

(2) Mechanistic insights into how multitails increase mRNA translation 

To gain mechanistic insights into how the branched poly(A) tails increased mRNA translation, Chen et al. dissected the overall effects of protein translation enhancement into two factors, namely, translation efficiency (TE) and mRNA stability (half-life, t1/2). For TE, they performed in situ profiling to quantify RNA copy numbers at subcellular resolution, with STARmap detecting all transcripts of the target sequence and RIBOmap detecting only the ribosome-bound fractions of mRNA copies. This allowed filtration of large clusters of endosomal non-translating transcripts in lipid transfection vesicles, thus providing more accurate quantification of cytosolic mRNAs than would traditional bulk northern blotting or RT-qPCR methods. 

The TE of FLuc mRNAs were approximated using the ratio of FLuc RIBOmap and STARmap signals of cells reseeded from the same biological conditions, with each normalized to the internal transfection-control RLuc STARmap signals. Interestingly, the difference in TE was insignificant regardless of chemical modification and topology at 24 h, indicating that multiple poly(A) branches did not hamper or enhance TE. However, at 48 h, multitail mRNA exhibited 1.5-fold higher TE than a control, which increased to 1.8-fold higher TE at 72 h, indicating that multitail mRNA preserved a more functional poly(A) tail at later time points to better sustain the cap/tail-dependent translation initiation. 

To further characterize the kinetics of RNA translation at different time points, Chen et al. synthesized mRNA transcripts encoding a degron-tagged FLuc, which effectively reduced luciferase protein t1/2 in HeLa cells from 20.4 h to 0.9 h for unmodified linear mRNA control, as expected for this tagging method to accelerate degradation. Luminescence decay kinetics showed that the t1/2 for 3-branch multitail degron-tagged FLuc mRNA increased to 16.8 h, which is approximately a 20-fold prolongation compared to the control. 

(3) Prolonged protein production by multitail mRNA in vitro and in vivo 

To compare multitail mRNA with a circular RNA (circRNA), which is inherently resistant to exonucleases, Chen et al. synthesized multitail and circular mRNA vectors encoding a very bright, secreted nanoluciferase (NLuc), which allowed repeatedly sampling of media and measurement of protein expression over a longer time course. 

In cell culture, the multitail mRNA outperformed the circRNA construct by more than 1,000-fold, suggesting that multitail-mediated cap-dependent translation initiation with optimized UTRs is far more efficient than circRNA with an internal ribosome entry site (IRES). Because of its longer t1/2, the superior performance of the multitail mRNA led to observable protein signal at day 14. 

Encouraged by these results, the NLuc multitail mRNA construct was next studied in mice by encapsulation in lipid nanoparticles (LNPs) and administration through retro-orbital (RO) injection. A single dose of 40 μg/kg of multitail mRNA yielded a luminescence signal approximately 4-fold higher than that obtained with control mock-ligated mRNA after 6 h and a remarkable 47-fold higher after 48 h.  

Immunogenicity was evaluated by serum level of tumor necrosis factor (TNF) and liver toxicity was measured by aspartate transaminase (AST) and alanine transaminase (ALT). Levels of all biomarkers were indistinguishable among mice treated by multitail mRNA, mock-ligated mRNA, and poly(C) negative control. Taken together, these results indicate that the branched poly(A) topology and unnatural linkage from click chemistry do not result in long-term immunotoxicity in vivo while enabling high protein expression at low doses, thus supporting their suitability for use as therapeutic RNAs. 

(4) Multitail mRNA for enhanced CRISPR-based therapeutics 

Chen et al. postulated that the enhanced translatability and stability of multitail mRNA would enable effective expression of Cas9 protein at lowered mRNA doses. To test this, chemically modified single-guide RNAs were designed to target Pcsk9 and Angptl3, genes implicated in lipoprotein homeostasis and whose knock-down has shown promise as a therapeutic strategy for treatment of familial hypercholesterolemia (Qui et al. 2021). 


Consistent with preliminary cell-based comparisons, 4.1-fold higher levels of Cas9 protein expression were found for the multitail construct compared to a control mRNA 24 h post-injection. A single dose of 0.7 mg/kg multitail Cas9 mRNA was sufficient to induce detectable editing levels at day 4, yielding significantly higher editing efficiency, as quantified by sequencing, at both Pcsk9 (46%) and Angptl3 (54%) compared to that obtained with mock-ligated mRNA (14% and 6%, respectively).  


Phenotypic analyses revealed significantly lower serum levels of both targeted proteins for mice treated with multitail mRNA, with a 71% decrease for Pcsk9 protein and 66% for Angptl3 

protein at week 4. By contrast, mice treated with mock-ligated mRNA had only a 45% reduction in Pcsk9 and a 27% reduction in Angptl3. Collectively, these protein level reductions translated to downstream decrease of serum lipids, with the multitail group showing a 63% decrease in serum-free cholesterol, a 51% decrease in serum total cholesterol, and a 49% decrease in 

triglycerides, more efficient than the 26% reduction in serum-free cholesterol, 18% reduction in total cholesterol and 9% reduction in triglycerides in the mock-ligated group at week 4. 


Concluding comments 

From a clinical perspective, Chen et al. envision that multitail mRNA holds promise for improving mRNA-based therapeutic platforms, in general, including gene therapy, multivalent vaccine formulation and personalized T-cell therapy. 

They note, however, that the yields and scalability of current multitail mRNA synthesis is limited by several factors: (a) the inefficiency of multisite click reactions at a submicromolar concentration, (b) requirement for HPLC purification, and (c) the super-stoichiometric nature of the ligation reaction, which requires use of a 10-fold excess of IVT mRNA.  

In addition to improving yield and scalability, they noted that further work is needed for optimization of multitail topology, including (a) the effects of chemical linkage for branching, (b) the spacing of branched tails, and (c) patterns of chemical modifications. 

Nevertheless, Chen et al. emphasize that their “results shed light on previously unexplored chemical space to design and develop next-generation mRNA therapeutics not only with enhanced stability but also with improved translatability, specificity, delivery, and pharmacokinetics.” 


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