Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Development and Delivery Systems of mRNA Vaccines

doi: 10.3389/fbioe.2021.718753

Figure Lengend Snippet: mRNA COVID-19 vaccines undergoing clinical testing.

Article Snippet: In the mRNA influenza vaccine studied by , a poly(A) tail with a length of 101 nt was used, while BioNTech disclosed in a published patent that the mRNA stability and translation efficiency of the 120 nt 3' poly(A) tail was higher than that of the 16 nt, 42 nt, and 67 nt mRNAs (patent WO 2017/059902 Al, BioNTech RNA Pharmaceuticals GmbH).

Techniques: Binding Assay, Produced, Modification

Journal: Molecular Therapy. Nucleic Acids

Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

doi: 10.1016/j.omtn.2025.102453

Figure Lengend Snippet: mRNA boosters enhance haploinsufficiency-associated mRNAs (A) Schematic of the guide sequences position on the 3′ UTR of their target genes: MECP2 , CTNNB1 , PURA , and SYNGAP1 mRNAs, used throughout the study. Guide sequences spanning distinct regions were evaluated and assigned. (B) Levels of M E CP2 mRNA measured by qRT-PCR in SH-SY5Y cells transfected with 40 nM booster V.2.2 (MB2) against 3′ UTR of the M E CP2 or a control booster not targeting M E CP2 (control) and harvested 16 h after transfection. Welch’s t test, ∗ p = 0.05. (C and D) Levels of CTNNB1 (C) and PURA (D) mRNAs measured by qRT-PCR in HEK293 cells transfected with boosters V.2.2 targeting distinct regions of the 3′ UTR (B1 and B2) or a non-specific control (control), 24 h after transfection. Ordinary one-way ANOVA, ∗ p = 0.05, ∗∗ p = 0.005. (E) S YN GAP1 mRNA levels measured by qRT-PCR in SH-SY5Y cells transfected with two versions of booster V.2.0 (SB1 and SB2) targeting S YN GAP1 mRNA 3′ UTR or a non-specific control (control). SB2 showed a significant (∗) up to 4-fold increase in the mRNA level. Welch’s t test, ∗ p = 0.05.

Article Snippet: Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control.

Techniques: Quantitative RT-PCR, Transfection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

doi: 10.1016/j.omtn.2025.102453

Figure Lengend Snippet: Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( Figure S3 ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control. To screen and select the most effective booster for CTNNB1 , HEK293-STF cells were transfected with different doses (10, 20, 30, 40, 50, and 100 nM) of five distinct CTNNB1 boosters V.1.0 (CB3, CB4, CB5, CB6, and CB7). Booster CB7 increases the protein level up to 2-fold compared to control at 40 nM booster or higher. (C) Representative western blotting and quantifications for three biological replicates showing the significant dose-dependent increase of CTNNB1 protein levels upon exposure to booster CB7 compared to control (for more blots and loading control, see Figure S3 ). Two-way ANOVA, ∗ p = 0.05, ∗∗∗ p = 0.0005. (D) qRT-PCR analysis confirms booster CB7 efficiency to increase CTNNB1 mRNA levels in a dose-dependent manner. Two-way ANOVA, ∗ p = 0.01. (E and F) mRNA levels of Wnt signaling pathway markers measured by qRT-PCR, illustrating the functional enhancement of CTNNB1 following the increase in CTNNB1 expression, in two different cell lines: HEK293-STF (E) and SH-SY5Y (F) cells. Welch’s t test, ∗ p < 0.05. (G and H) Protein levels of B-catenin and its downstream effector EN2 in β-catenin heterozygote, human iPSC-derived neurons, 48 h after transfection with LNP-packed boosters against CTNNB1 (CB7) or a non-specific control. Three different LNP formulations were tested (LNP1, LNP2, and LNP10; see Figure S2 ). (G) A representative western blot and (H) a quantitation of CTNNB1 and EN2 from western blots from two biological replicates.

Article Snippet: Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control.

Techniques: Expressing, Western Blot, Control, Transfection, Quantitative RT-PCR, Functional Assay, Derivative Assay, Quantitation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

doi: 10.1016/j.omtn.2025.102453

Figure Lengend Snippet: Optimization of mRNA Booster to enhance stimulatory activity (A) Chemical structure of the modifications (phosphorothioate and 2′-O-methyl) that increase oligonucleotide specificity and resistance to nucleases. (B) Booster version 3.0 schematic including 100 adenosines in the 5′ end of the 30-nucleotide guide sequence, which targets the 3′ UTR of the mRNA of interest. The first three nucleotides in both 5′ and 3′ ends have been modified by phosphorothioate and 2′-O-methyl. Below are the sequences of the chemically modified SYNGAP1 and non-specific scrambled control boosters version 3.0. (C) Comparison of estimated dissociation constant (K D ) means and standard deviations of PABPC between modified (fully modified and 3NT mod) and unmodified poly(A) RNA. No statistical significance in binding affinity was found when comparing modified poly(A) RNAs to unmodified poly(A) RNA by one-way ANOVA. (D) S YN GAP1 mRNA levels 48 h after transfection with the chemically modified S YN GAP1 -specific or scrambled control boosters, measured by qRT-PCR. n = 3 biological repeats, including two technical repeats per experiment. ∗∗∗∗Benjamin, Krieger, and Yekutieli t test, p < 0.00001. (E) Detection of SYNGAP1 booster V.1.0 and V.3.0 by northern blot. SH-SY5Y cells were transfected with 40 nM boosters and the cells were washed after 24 h. The medium was changed every 3 days and the cells harvested a week after transfection. Whole-tRNA signal from ethidium bromide staining is used as a loading control. (F) S YN GAP1 mRNA levels in SH-SY5Y cells 48 h after transfection with SYNGAP1 booster V.3.0 (SB7) with different poly(A) stretch lengths: 3(A) tail, 25, 50, 75, and 100 nucleosides. qRT-PCR analysis shows a robust significant increase in the level of S YN GAP1 mRNA in the presence of poly(A) tail compared to 3(A) tail. Ordinary one-way ANOVA. (G) Protein levels of β-catenin in CTNNB1 heterozygote, human iPSC-derived neurons, 5 days after transfection with LNP-packed boosters against CTNNB1 or a non-specific control. Unpaired t test, ∗∗ p < 0.005.

Article Snippet: Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control.

Techniques: Activity Assay, Sequencing, Modification, Control, Comparison, Binding Assay, Transfection, Quantitative RT-PCR, Northern Blot, Staining, Derivative Assay