Friday, March 22, 2024

Branched chemically modified poly(A) tails enhance the translation capacity of mRNA - Nature.com - Translation

Abstract

Although messenger RNA (mRNA) has proved effective as a vaccine, its potential as a general therapeutic modality is limited by its instability and low translation capacity. To increase the duration and level of protein expression from mRNA, we designed and synthesized topologically and chemically modified mRNAs with multiple synthetic poly(A) tails. Here we demonstrate that the optimized multitailed mRNA yielded ~4.7–19.5-fold higher luminescence signals than the control mRNA from 24 to 72 h post transfection in cellulo and 14 days detectable signal versus <7 days signal from the control in vivo. We further achieve efficient multiplexed genome editing of the clinically relevant genes Pcsk9 and Angptl3 in mouse liver at a minimal mRNA dosage. Taken together, these results provide a generalizable approach to synthesize capped branched mRNA with markedly enhanced translation capacity.

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Fig. 1: Conceptualization of chimeric mRNA bearing multiple poly(A) tails.
Fig. 2: Mechanistic characterization of mRNA stabilization by branched poly(A) tails.
Fig. 3: Multitail mRNA exhibited prolonged protein expression in vivo.
Fig. 4: Multiplexed genome editing in vivo using stabilized Cas9 mRNA.

Data availability

NGS data were deposited to the NCBI Sequence Read Archive database under the accession code PRJNA107297178. All data supporting the findings of the presented study are listed in the article and Supplementary Information is available upon reasonable request.

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Acknowledgements

We thank the MIT Department of Chemistry Instrument Facility for providing instrument access and N. Ye (MIT) for assistance on protein purification. We thank H. Zeng (Broad Institute) for his help in designing the STARmap/RIBOmap probes. We also thank other members of X.W.’s laboratory for helpful discussion throughout the project. X.W. acknowledges the support from the Searle Scholars Program, Thomas D. and Virginia W. Cabot Professorship, E. Scolnick Professorship, Ono Pharma Breakthrough Science Initiative Award, Merkin Institute Fellowship, and NIH DP2 New Innovator Award (1DP2GM146245-01). A.H. is a National Science Foundation Graduate Research Fellow. A.H. and D.R.L. were supported by NIH U01AI142756, R35GM118062, RM1HG009490 and the Howard Hughes Medical Institute (HHMI). This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Hongyu Chen, Dangliang Liu, Jianting Guo, Yiming Zhou, Jiakun Tian, Shuchen Luo, Jingyi Ren, Jiahao Huang, Franklin Kostas, Mingrui Wu & Xiao Wang

  2. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Hongyu Chen, Dangliang Liu, Jianting Guo, Abhishek Aditham, Yiming Zhou, Jiakun Tian, Shuchen Luo, Jingyi Ren, Jiahao Huang, Franklin Kostas, Mingrui Wu & Xiao Wang

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Abhishek Aditham

  4. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Yiming Zhou & Xiao Wang

  5. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Alvin Hsu & David R. Liu

  6. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Alvin Hsu & David R. Liu

  7. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Alvin Hsu & David R. Liu

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  1. Hongyu Chen
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Contributions

X.W. conceived the project. H.C. and X.W. designed experiments. H.C., D.L., J.G., A.A., Y.Z., J.T., J.R., A.H., F.K. and M.W. performed the experiments. H.C., J.G., S.L., A.H. and J.H. performed data analysis. X.W. supervised the work. H.C. and X.W. wrote the paper with input from all authors.

Corresponding author

Correspondence to Xiao Wang.

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Competing interests

X.W., H.C., A.A. and J.G. are inventors on patent applications related to branched RNA. X.W. is a scientific cofounder, consultant and equity holder of Stellaromics and Convergence Therapeutics. D.R.L. is a cofounder, consultant and equity holder of Beam Therapeutics, Prime Medicine, Pairwise Plants, Chroma Medicine, Exo Therapeutics and Nvelop Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Screening of oligonucleotide crosslinking chemistry.

(a) Summary of oligonucleotide chemical conjugation methods. Screening was performed using 15-nt poly-deoxyadenosine model substrates at micromolar concentrations. Modification handles were incorporated through oligonucleotide solid phase synthesis, followed by amine-NHS labeling and HPLC purification. (b) Gel electrophoresis of crude thiol-ene/yne oligonucleotide conjugation of 15-nt model substrates containing only one conjugation handle. (c) Gel electrophoresis of crude CuAAC and IEDDA 30-nt oligonucleotides bearing three alkyne/trans-cyclooctene handles reacting with 30-nt azide/tetrazine modified oligo. (d) Preliminary dual luciferase assay using branched mRNA prepared from CuAAC product mixture without HPLC purification using different equivalents of branched oligos versus stem oligo. Branching oligos contained modifications on the last six bases with either natural (5′ to 3′) or reversed (3′ to 5′) directionality. Time-course assay was performed as outlined in Fig. 1. n = 3 independent transfections for each construct. Mean ± s.e.m. P values were calculated by ordinary two-sided one-way ANOVA (comparison of means across time points). Gels are representative of at least two experiments.

Source data

Extended Data Fig. 2 Synthesis and characterization of topologically augmented branched mRNA.

(a-c) Representative HPLC purification and gel electrophoresis characterizations of branched oligonucleotides (oligo) containing 1-3 branching poly(A) tails. Fractions containing the desired products (boxed) were pooled and isolated. (d) Schematics of preparation of firefly luciferase (FLuc) mRNA constructs lacking poly(A) tail before ligation. FLuc mRNA without template encoded poly(A) tail was ligated to a scramble (non-polyA) stem oligo with 3′ end modifications (six phosphorothioate/2MOE at 3′ and terminal dideoxycytidine modifications) and internal alkyne (5-octadiynyl deoxyuridine or OU), with or without conjugation to 5′ azide labeled scramble or poly(A) branches with 3′ end modifications (the same as the stem oligo). (e) RNase H characterization of branched mRNA-oligo conjugates with branched poly(A) or scramble tails. The branching topology was confirmed by further band shift on TBU gel. (f) Branched poly(A) sequence, rather than chemical modifications alone, conferred enhanced protein expression over time. Relative FLuc luminescence was normalized to the scramble OU only oligo ligated mRNA at indicated time points. n = 3 independent transfections for each construct. Mean ± s.e.m. P values were calculated by two-sided unpaired t-test (with Welch’s correction) against the scramble OU + scramble azide construct at corresponding time points. (g) RNase H characterization of branched mRNA-oligo conjugates with multiple ploy(A) tails. mRNA with full-length hemoglobin UTRs and template encoded 100A-tail was ligated to 0 (mock ligation), 30 A, 60 A, or oligos with one, two, or three branched poly(A) tails and characterized by RNase H assay. The branching topology was confirmed by further band shift on TBU gel. Gels are representative of at least two experiments.

Source data

Extended Data Fig. 3 Dissecting the effects of branched poly(A) tails on mRNA stability and translation efficiency.

(a) Representative STARmap/RIBOmap images were acquired under the same confocal imaging settings from three independent experiments for each condition. STARmap versus RIBOmap characterization of different modified Firefly luciferase (FLuc) mRNA constructs and STARmap characterization of internal control Renilla luciferase (RLuc) mRNA were performed as outlined in Fig. 2. DAPI (blue), nuclei; FLuc amplicons (magenta); RLuc amplicons (yellow). Colocalized FLuc/RLuc amplicons in STARmap (white dots) were lipid transfection vesicles and were excluded from downstream quantification. Scale bar = 100 µm. (b-c) Violin plots of single-cell quantification of FLuc STARmap/RIBOmap amplicons. P values were calculated by ordinary two-sided two-way ANOVA. (d) Decay kinetics of internal control RLuc luminescence outlined in Fig. 2c normalized to luminescence at 8 hrs post transfection. n = 3 independent transfections. P values were calculated by ordinary two-sided two-way ANOVA (with Geisser-Greenhouse correction, decay across time points).

Source data

Extended Data Fig. 4 Branched poly(A) tails bind PABPC1 protein.

(a) Electrophoretic mobility shift assay of three branched poly(A) oligo with varying concentrations of GST-tagged recominant human PABPC1 protein (PABPC1-GST). The stem oligo was labeled with Alexa Fluore 546 at 5′end. Both stem and branching poly(A)’s were 30 nt and modified with PS-2MOE at the last six bases and ddC at the 3′ end. (b) Gel shift assay of the same modified poly(A) oligo without branching. Gels are representative of at least two experiments.

Source data

Extended Data Fig. 5 Chemical and topological augmentation stabilized poly(A) tails against nuclease degradation in vitro.

(a-d) Evaluation of chemically and topologically modified poly(A) tails in CAF1-CCR4 deadenylation assay. Four different Alexa-546 labeled poly(A) oligos were subjected to deadenylation using recombinant CAF1/CCR4 protein complex over the course of 110 min: linear unmodified 60 A oligo (a), end modified (six PS-2MOE at 3′ and terminal dideoxycytidine, the same set of modifications for all other oligos) 30 A oligo (b), end modified 30 A stem oligo + unmodified 30 A branches (c), and end modified stabilized 30 A stem oligo + end modified 30 A branches (d). (e-h) Evaluation of chemically and topologically modified poly(A) tails in HeLa cell lysate. Alexa-546 labeled poly(A) oligos (the same four constructs used in a-d) were subjected to digestion in HeLa cytosolic lysate for 220 min: linear unmodified 60 A oligo (e), end modified 30 A oligo (f), end modified 30 A stem oligo + unmodified 30 A branches (g), and end modified 30 A stem oligo + end modified 30 A branches (h). Gels were representative of two experiments.

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Extended Data Fig. 6 Multi-tailed mRNA depends on the canonical eIF4-eIF3 translation initiation mechanism.

(a) Timeline of the knockdown (KD) experiments. HeLa cells were treated with siRNA cocktails targeting the corresponding eIFs 48 hrs post siRNA transfection, cells were reseeded and transfected with mRNA followed by protein quantification after 6 hrs. Successful knockdowns were confirmed by western blots and RT-qPCR at 48 hrs. (b,c,e) KD experiments for eIF4E/eIF4G/eIF3D. (d) Comparison of multi-tailed mRNA to regular mRNA in 4EGI-1 treated in vitro translation assay using rabbit reticulocyte lysate (RRL). Luciferase expressions were normalized to the wild-type condition for each construct. Mean± s.e.m. n = 3. P values were calculated by two-sided unpaired t-test for intra-construct comparison (KD vs WT) and by two-sided one-way ANOVA for cross-construct comparison. Western blots were representative of two experiments.

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Extended Data Fig. 7 Comparison of UTR optimized circRNA and multi-tailed mRNA using secreted NanoLuc reporter.

(a) Gel electrophoresis of circRNA encoding secreted Nanoluc (IL6-Nluc) generated through IVT, backsplicing, and enriched by RNase R treatment. Successful circularization was confirmed by RNase R resistance and slower mobility on TBU gel compared to corresponding intron-free linear RNA. (b) Comparison of UTR optimized circRNA and multi-tailed mRNA using IL6-NLuc 1 day after transfection with indicated amounts of RNA. Mock lig./mod-only/multi-tail mRNAs contained optimized UTRs, full m1ψ replacement and 100 A through IVT with mock ligation (mock lig.), ligated to modification-only oligo (mod-only), or modified multi-tailed oligo (multi-tail). CircRNAs were designed to contain optimized HRV IRES (with proximal loop Apt-eIF4G insertion) and 3′-PABP binding motif without addition of modified nucleotides. Cells were cultured in phenol red-free media. On each day, media was completely harvested and renewed with 150 μL fresh media and 5 μL of old media was diluted and used for luciferase assay. n = 3 independent transfections in each biological condition. Mean ± s.e.m. (c) IL6-Nluc signal over 14 days. n = 3 independent transfections in each biological condition. Mean ± s.e.m.

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Extended Data Fig. 8 Paired STARmap/RIBOmap effectively characterize the quantity and translatability of mRNA constructs delivered by LNP to murine liver.

(a) Schematics for evaluating two-gene STARmap/RIBOmap experiments in different lobes of murine liver. 2:1 NLuc/FLuc mRNA were co-encapsulated in LNP and administered by retro-orbital (R.O.) injection. 6 hrs post mRNA injection, the mouse liver was harvested in five lobes (caudate, left/right medial and lateral lobes). Each lobe was sections into two adjacent 10-μm slices, with one slice profiled by NLuc STARmap and FLuc STARmap and the other slice profiled by NLuc RIBOmap and Fluc STARmap. Whole sections were profiled in 291×291 μm2 tiles. (b, c) Quantification of numbers of NLuc and FLuc amplicons in each tile. n = 3379/3199 tiles. Slopes (NLuc/FLuc) were calculated by linear regression (least-square, Q = 5% for outlier rejection).

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Extended Data Fig. 9 Delivery of CRISPR editing using chemically and topologically modified mRNA.

(a) Comparison of Cas9 expression by western blot 48 hrs post transfection in HEK293T-uGFP cells. NLS, nuclear localization signal. Western blots were representative of two experiments. (b) FACS gating strategy for uGFP positive HEK293T cells. (c) Exemplary single-cell uGFP quantification for cells treated with unligated Cas9 mRNA only (Cas9-only) without sgRNAs, or with mock lig./mod-only/multi-tail Cas9 mRNA co-transfected with two sgRNAs. FACS were performed 72 hrs post mRNA transfection. n = 11845/10983/12334/12131 single cells. P value was calculated by two-sided one-way ANOVA. (d-h) Serum levels of Pcsk9 protein (d), Angptl3 protein (e), free cholesterol (f), total cholesterol (g), and triglyceride (h) over 4 weeks normalized to the Cas9-only group. Mean ± s.e.m. n = 4, biological replicates.

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Supplementary information

Reporting Summary

Supplementary Table 1

Compiled sequences of genes/oligos/probes used in the study.

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Source Data Figs. 1–4 and Extended Data Figs. 1–9

Compiled statistical source data for all figures presented.

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Chen, H., Liu, D., Guo, J. et al. Branched chemically modified poly(A) tails enhance the translation capacity of mRNA. Nat Biotechnol (2024). https://ift.tt/yUZ2p9t

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