Modifying VEGF-A mRNA by combinatorial optimization to enhance therapeutic efficacy for myocardial infarction

Figures

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  Fig. 1. Superior translational efficiency capabilities obtained from the optimized mRNA. (A) schematic representation of mRNA structure, including the 5′-UTR, CDS, 3′-UTR, and poly(A) tail, with the various elements of each component for expression screening, is depicted. Additionally, the synthesis of mRNA was conducted using five reported nucleotide modifications found in mRNA vaccines or drugs: m6A, 5moU, m5C, m1ψ, and ψ. Thirty mRNA sequences (B) were designed based on a known amino acid sequence. Twenty distinct element combinations were subsequently generated by incorporating four 5′-UTR variants, five 3′-UTR variants (C), diverse poly(A) sequences, and modified ribonucleotides (D). All mRNA sequences—including the unoptimized original AZD8601 mRNA sequence as the control—were subjected to in vitro expression; proteins from these expression systems were then extracted and analyzed by Western blot to quantify expression levels. The upper panel shows representative immunoblots, and the lower panel presents relative protein abundance (normalized to AZD8601). Data represent three independent experiments. The red star indicates the sequence with the highest expression level within each…

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  Fig. 2. Km10566 improves cardiac function rapidly and sustainably in MI. (A) Schematic of the experimental timeline. Rats with myocardial infarction (MI) were treated with the optimized mRNA Km10566, followed by echocardiographic assessments at 7, 14, and 21 days post-treatment. (B) Representative in vivo luminescence images at 3 days after injection. Radiance (photons per s per cm2 per sr). Quantification of cardiac function parameters across five groups (Sham, Vehicle, AZD8601, Km10566, C6V): changes in left ventricular ejection fraction (LVEF) at 7, 14, and 21 days post-treatment (C); and changes in left ventricular fractional shortening (LVFS) at 7, 14, and 21 days post-treatment (D). Data are presented as means ± SD of five biological replicates. Statistical significance of the data was analyzed by one-way ANOVA with Tukey’s multiple comparison test. E, F, Heart weight (E) and heart-to-weight ratio (F) on day 21 quantified (n = 5 biological replicates; mean). P values were calculated by an ordinary one-way ANOVA.

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  Fig. 3. Km10566 significantly reduces the expression of genes related to myocardial cell repair. To assess gene expression changes in ischemic cardiac tissue, the expression levels of Wt1 (A), Tnnt2 (B), and KDR (C) were measured in five groups of rat at 7, 14, and 21 days post-treatment, with GAPDH as the internal reference gene. Expression was quantified using the 2^(ΔΔCt) method. Statistical analysis was performed one-way ANOVA followed by Tukey’s multiple comparisons test (n = 9, samples collected from three animals analysed in triplicate; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.)

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  Fig. 4. Km10566 can significantly improve cellular morphology in the heart and effectively reduce collagen volume fraction. (A) Representative micrographs of H&E-stained sections. Tissues were harvested from rats cardiac, fixed in 4% paraformaldehyde, dehydrated through a graded series of ethanol and xylene, and then embedded in paraffin. Sections of 5-micron thickness were cut, dewaxed, stained with hematoxylin and eosin, dehydrated, cleared, and mounted with a coverslip. Scale bar = 50 μm. (B) The microscopic image displayed represents the results of Masson’s trichrome staining on sections obtained from rat heart tissue. Collagen fibers appear blue, while muscle fibers are red. Scale bar = 500 μm. (C) The collagen volume fraction (CVF) The collagen volume fractions (CVF) at three time points were demonstrated by analyzing images of Masson’s trichrome-stained sections, obtained by dividing the collagen area by the total area. Statistical analysis was performed using One Way ANOVA followed by Tukey’s multiple comparisons test (n = 4; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)