Modifying VEGF-A mRNA by Combinatorial Optimization to Enhance Therapeutic Efficacy for Myocardial Infarction

Modifying VEGF-A mRNA by Combinatorial Optimization to Enhance Therapeutic Efficacy for Myocardial Infarction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Modifying VEGF-A mRNA by Combinatorial Optimization to Enhance Therapeutic Efficacy for Myocardial Infarction Wei Wang, Zhenping Zhan, Lan Chen, Zhonghua Wang, Lei Wang, Shizheng Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8268281/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Vascular Endothelial Growth Factor A (VEGF-A) is a mitogen with high endothelial cell specificity, playing a key regulatory role in angiogenesis and vasculature formation. Administration of VEGF-A mRNA can facilitate dose-dependent protein expression, promoting therapeutic angiogenesis without genome modification. However, unmodified VEGF-A mRNA is susceptible to degradation and induces immunogenicity, limiting its efficacy. In this study, we designed and synthesized a carefully modified VEGF-A mRNA construct, designated Km10566, which exhibits enhanced VEGF-A protein expression during in vitro transcription (IVT). Intracardiac injection of Km10566, formulated in a biocompatible citrate saline solution, into a rat model of myocardial infarction resulted in significant improvement in left ventricular ejection fraction (LVEF) and reduced myocardial fibrosis after 21 days. A pharmacodynamic analysis further supports Km10566 as a promising therapeutic candidate, highlighting its potential for further application in preclinical and clinical research. Biological sciences/Biotechnology Health sciences/Cardiology Health sciences/Diseases Health sciences/Medical research In Vitro Transcription synthetic mRNA mRNA-based therapeutics VEGF-A mRNA myocardial infarction Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Messenger RNA (mRNA) technology has emerged as key focus within the scientific community, largely owing to its pivotal role in the development of vaccines for the Coronavirus Disease 2019 (COVID-19) [ 1 ]. Its favorable properties-such as safety, rapid manufacturing, and cost-effectiveness-have driven increased attention to mRNA compared to DNA or protein-based therapeutics [ 2 ]. Preliminary studies have demonstrated the broad potential of mRNA-based therapies across various applications, including vaccination, gene therapy, and cancer immunotherapy [ 3 ]. However, conventional mRNA pharmaceuticals still face significant challenges, notably immunogenicity, mRNA instability, and a short half-life [ 4 , 5 ]. It is evident that key structural elements of mRNA - including the 5'-cap, 5'-untranslated region (UTR), coding region, 3'-UTR, and poly(A) tail [ 6 ] play crucial roles in the translation process. For instance, UTRs have been shown to influence mRNA translation efficiency and resistance to degradation by various domains [ 7 ]. Chen et al . engineered branched oligonucleotides containing dual cap structures through chemical capping and click chemistry resulted in multi-cap mRNA, which significantly enhanced protein expression levels [ 8 ]. Additionally, circular RNA, owing to its unique ring structure, exhibited higher stability and longer half-life than linear RNA, enabling sustained expression in vivo and eliciting more enduring immune responses [ 9 – 11 ]. VEGF-A plays a critical role in angiogenesis and a variety of biological processes [ 12 ]. It has been demonstrated that VEGF-A165 is the most abundant and potent isoform [ 13 – 18 ]. In 1983, VEGF-A was first identified as a tumor pro-angiogenic cytokine, initially known as the vascular permeability factor. After its successful isolation in 1989, it was uniformly named vascular endothelial growth factor [ 20 ]. It is widely recognized that angiogenesis is a highly complex process in which VEGF-A functions as a central regulator, directly stimulating the proliferation of vascular endothelial cells, inhibiting their apoptosis, enhancing blood vessel permeability providing substrates for both fibroblasts and endothelial cells, and promoting tumor angiogenesis. Similar to other members of the VEGF family, VEGF-A mediates intracellular signaling through its interaction with VEGF receptors (VEGFRs), exhibiting different degrees of binding affinity [ 21 ]. Research into methods of mRNA delivery for inducing neovascularization in ischemic tissues has explored the encoding of angiogenic factors, including VEGF-A mRNA (AZD8601), which was developed through a collaboration between Moderna and AstraZeneca. AZD8601 yields VEGF-A165 by replacing uridine with N1-methylpseudouridine (N1-Ψ), a modification. This modification has been shown to reduce binding affinity for the Prkra dimer markedly, thereby diminishing recognition of double-stranded RNA (dsRNA) and attenuating innate immune responses [ 34 ]. Extensive studies have demonstrated that modified mRNA can facilitate efficient, dose-dependent, and transient protein expression while exhibiting low innate immunogenicity [ 33 , 35 ]. The commencement of clinical trials in 2016 signified a pivotal moment for mRNA therapeutics. AZD8601 completed Phase II clinical trials and has been proposed as a treatment for heart failure [ 25 ]. Furthermore, subsequent studies have shown that, in addition to mRNA modification such as N1-Ψ, changes to other components-including poly(A) tails, untranslated regions (UTRs), and coding sequences (CDSs)-can significantly enhance translation efficiency and molecular stability [ 36 , 37 ]. The development of new drugs for cardiovascular disease — such as myocardial infarction, heart failure, stroke, and skin injuries — remains a significant healthcare challenge, and the potential of neovascularization may inform these efforts [ 22 ]. However, as a secreted protein with a short in vivo half-life, VEGF-A poses risks of excessive angiogenesis and increased bleeding when administered chronically [ 23 ]. As an emerging class of therapeutic agents, mRNA offers broad application prospects due to its excellent programmability, rapid production, transient expression, and superior safety profile compared to DNA or protein-based drugs [ 24 ]. In this study, we evaluated the expression efficiency of various VEGF-A mRNA constructs to identify a highly expressed and stable VEGF-A mRNA therapeutic with improved clinical potential. We ultimately identified a linear VEGF-A mRNA transcript that surpasses control sequences in expression levels. In animal models, both linear and circular mRNA formulations significantly improved cardiac functions, including ejection fraction, in rat models of myocardial infarction. The modified linear mRNA demonstrated superior collagen volume fraction recovery and gene expression of Wt1, Tnnt2, and KDR in comparison to both the original mRNA and circular mRNA. These findings suggest that this identified mRNA has promising future for further drug development, with potential to reduce medication costs while maintaining therapeutic efficacy. Results Superior translational efficiency capabilities obtained from the optimized mRNA To confirm the structure of VEGF-A mRNA with a enhanced translation efficiency and prolonged half-life, we systematically designed and screened extensive component combinations (Fig. 1 A). The mRNA was synthesized via in vitro transcription (IVT) and subsequently transfected into HEK293T cells (see Materials and Methods for details). We employed a stepwise approach by fixing all components except one, then sequentially testing 30 optimized codon sequences (Fig. 1 B), 20 combinations of untranslated regions (UTRs) (Fig. 1 C), and four types of Poly (A) tails (Fig. 1 D). Detailed sequence information is provided in Supplementary Table 1, Fig. 1 B shows in CDS sheet, Fig. 1 C in UTR sheet, and Fig. 1 D in the Poly(A) & Modification sheet. AZD8601 served as a control in these experiments, and its protein expression levels were used as a benchmark for comparison. The elements selected were CDS105, Cyn5U6, Cyn3U5, and A120 (Fig. 1 B, C, D). Furthermore, chemical modifications of RNA bases have been shown to alter biophysical properties, influencing RNA secondary structure, folding, or interactions with specific RNA-binding proteins [ 26 ]. Prior studies demonstrated that synthetic mRNA incorporating modified nucleotides exhibits improved stability and reduced immunogenicity [ 27 , 28 ]. In this study, we synthesized mRNA using five previously reported efficacious modified bases: N6-Methyl-ATP (m6A), 5-Methoxy-UTP (5moU), Pseudouridine (Ψ), 5-Methyl-CTP (m5C), and N1-Methylpseudouridine (m1Ψ). The translational efficiency of each modification was assessed using the same method described above. Results indicated that m1Ψ modification achieved the highest protein expression level (Fig. 1 D). This optimal combination, designated Km10566, exhibited approximately two-fold increase in protein expression compared to AZD8601 in HEK293T cells (Fig. 1 E). Km10566 rapidly and sustainably improves cardiac function in myocardial infarction We evaluated the effects of optimized mRNA on cardiac function in vivo in a rat model of MI, induced by ligation of the left anterior descending (LAD) artery. Following the intracardiac injection of a single dose luciferase mRNA in citrate saline buffer into the peri-infant region, we observed successful transfection and protein expression. In vivo imaging showed that the mRNA was normally expressed in the rats’ hearts by Day 3 (Fig. 2 B). The LVEF, left ventricular fractional shortening (LVFS), heart weight, and heart-to-body weight ratio of the rat model were compared between the treatment and control groups at 1,2-, and 3-weeks post-injection (Fig. 2 A). To further elucidate the efficacy and safety of Km10566, its effects were compared with AZD8601. Km10566 significantly improved both LVEF and LVFS at 14 days p < 0.0001), with beneficial effects persisting up to 3 weeks. No significant differences were observed in heart weight (Fig. 2 E) or heart-to-body weight (Fig. 2 F) ratio in either group. Notably, Km10566 showed the most pronunced improvement in cardiac function, outperforming AZD8601 significantly (p < 0.01) (Fig. 2 C, 2 D). These results suggest that the design and assembly of synthetic mRNA — without altering the protein sequence — can achieve comparable therapeutic effects to those of mRNA drugs. Although no significant changes in heart weight or ratio were observed, the marked improvements in LVEF and LVFS indicate a beneficial effect on cardiac function following myocardial infartion (MI). This efficacy may be mediated through non-structural mechanisms, such as modulating cellular metabolism or reducing inflammation, which are not reflected by macroscopic indicators. Based on prior studies on circular mRNA, we circularized the optimized sequence to generate C6V, which contains the CDS105 element, using type I intron splicing. We plan to compare its effect with those of the linear mRNA. As shown in Fig. 2 , C6V did not differ significantly from AZD8601 in terms of heart weight and heart weight-to-body weight ratio. While C6V exhibited a significant impact on key cardiac function indicators—LVEF and LVFS (p < 0.05) — it was less effective than Km10566. Km10566 significantly reduces the expression of genes related to myocardial cell repair Myocardial infarction (MI) resulted from acute and sustained ischemia and hypoxia of coronary artery, leading to myocardial necrosis. The formation of new coronary vessels is to revascularize the ischemic myocardium by inducing various angiogenic factors, such as Wilms' Tumor 1 (Wt1), Troponin T type 2 (Tnnt2), and Kinase Insert Domain Receptor (KDR) [ 27 ]. We focused on comparing the Km10566 group and the AZD8601 group. For Wt1 (Fig. 3 A), Km10566 showed a sustained and significant down regulation from day 7 to day 21. This prolonged reduction likely favors Wt1-mediated endothelial proliferation and vascular network formation, potentially exerting a long-lasting effect on coronary revascularization. In contrast, AZD8601 only induced transient significant down regulation on day 14, suggesting its impact on Wt1-driven angiogenic processes is short-lived. Regarding KDR (Fig. 3 C), Km10566 presented a sharp, durable decrease ( initial p < 0.0001, then p < 0.01), which may more profoundly and persistently impact KDR-VEGF-A signaling and subsequent neovascularization. Conversely, AZD8601 showed a significant reduction that disappeared before day 21, indicating a transient influence on KDR-dependent vascular repair. For Tnnt2 (Fig. 3 B), no significant differences were observed during most of the study period, except at day 21, when both AZE8601 and Km10566 showed substantial changes (p < 0.05). This may reflect late-stage modifications in in the regulation of myocardial contractile proteins. In the T3-C6V group, the expressions levels of Wt1 (Fig. 3 A) and Tnnt2 (Fig. 3 B) were comparable to those of the control group throughout the experiment. The differential expression of KDR (Fig. 3 C), however, persisted until day 14 and normalized by day 21. Compared to AZD8601 and C6V, Km10566 exhibited the most potent and sustained regulatory effects on these three genes.These results indicate that the sequence modifications in Km10566 markedly enhance its efficacy, prolonging VEGF-A mRNA activity in vivo and potentially exerting more persistent effects on coronary vascular regeneration. Km10566 can significantly improve cellular morphology in the heart and effectively reduce collagen volume fraction Both cellular morphology and collagen volume fraction are important indicators for evaluating the therapeutic efficacy myocardial infarction. They are closely associated with the extent of myocardial fibrosis. They can reflect changes in cardiac structure and function, which are crucial for assessing treatment outcomes and prognostication. Hematoxylin and Eosin (HE) staining was performed on rat heart tissues at 7, 14, and 21 days post-treatment to observe histological changes in cell morphology following drug administration in myocardial infarction models.(Fig. 4 A). All three treatment groups showed some degree of improvement in tissue necrosis, indicating drug efficacy. To assess collagen volume fraction, Masson's trichrome staining was applied, quantifying the precentage of collagen content within the total tissue area. At 7 days, Km10566 demonstrated an initial improvement in collagen volume fraction (p < 0.05), with a significant and sustained decrease observed up to 3 weeks post-treatment (p < 0.0001). In contrast, AZD8601’s effects became significant only after 21 days. The efficacy of C6V was intermediate between the two (Fig. 4 B, C). These results reinforce the rapid and enduring therapeutic effects of Km10566 in the treatment of myocardial infarction. Discussion In this study, we developed an optimized VEGF-A mRNA construct, designated Km10566, which demonstrates enhanced translation efficiency and stability while minimizing unwanted immunogenicity. Through systematic engineering of the 5′- and 3′-UTRs, codon optimization of the coding sequence (CDS), and incorporation of a stabilizing poly(A) tail, we obtained a candidate capable of sustaining high levels of VEGF-A expression in vitro . Subsequent modification with N1-methylpseudouridine (m1Ψ) significantly reduced innate immune activation, consistent with prior reports highlighting nucleoside modifications as a strategy to evade Toll-like receptor (TLR)-mediated recognition. The functional superiority of Km10566 was validated in a murine model of myocardial ischemia, where it significantly improved left ventricular ejection fraction and reduced collagen deposition compared to both AZD8601 and the reference C6V construct. These findings suggest that our integrated optimization approach not only enhances protein expression but also translates into meaningful physiological improvements in a therapeutically relevant setting. Notably the m1Ψ-modified Km10566 did not elicit significant interferon-alpha or TNF-α responses in treated animals, supporting its favorable safety profile. Our results align with recent advances in nucleoside-modified mRNA therapeutics. For instance, Pardi et al . demonstrated that m1Ψ-modified mRNAs exhibit reduced immunogenicity and enhanced translation efficiency in vivo , supporting their use in repetitive dosing regimens. Furthermore, the selection of specific UTR elements derived from highly expressed endogenous genes—such as human α-globin—has been shown to prolong mRNA half-life and increase protein yield, consistent with our observation of sustained VEGF-A expression from Km10566. Compared to existing VEGF-A therapies, Km10566 offers several advantages. Although AZD8601 has shown promise in early clinical trials for wound healing and cardiovascular repair, its translational efficiency appears suboptimal in direct comparison. Similarly, viral vector-based VEGF delivery systems, though effective in preclinical models, raise concerns regarding immunogenicity and insertional mutagenesis. In contrast, Km10566 leverages non-viral, chemically defined mRNA technology, allowing for controlled, transient expression without genomic integration — a crucial consideration for regenerative applications. Several limitations should be noted. First, the translatability of m1Ψ-modified mRNA in human cells requires further validation, as recent reports suggest that it has context-dependent effects on ribosomal frameshifting. Second, while murine models provide valuable insights into safety and efficacy, species differences in mRNA sensing and VEGF signaling necessitate caution when extrapolating results to humans. Finally, long-term stability and storage conditions of formulated Km10566 require further characterization, which is essential for clinical translatin. Future work will focus on advancing Km10566 into GMP-compliant manufacturing and IND-enabling studies. Concurrently, we are exploring combination strategies with biomaterial-based delivery systems to enhance tissue retention and achieve controlled release. Additionally, the potential application of Km10566 in other VEGF-responsive conditions, such as chronic limb ischemia and diabetic ulcers, represents an exciting avenue for ongoing research. In conclusion, we have developed a novel VEGF-A mRNA construct with optimized efficacy and safety profiles. Km10566 is a promising candidate for further development as a regenerative therapeutic. Methods Sequence design and gene synthesis The VEGF-A candidate mRNA sequence was designed based on the corresponding amino acid sequence and includes following elements: a 5’-cap structure (7-Methyl GTP), 5’ untranslated region (5’-UTR), the coding region encoding (CDS), a peptide tag (Flag-tag), a 3’-untranslated region (3'-UTR), and a Poly(A) tail. Subsequently, the DNA template, containing the T7 promoter necessary for in vitro transcription was synthesized de novo. DNA template preparation for IVT A plasmid DNA working solution was prepared using nuclease-free water. A 50 μL PCR reaction mixture was assembled as follows: 6.6 μL of 2x Reaction Buffer, 1 μL of dNTP Mix (10 mM), 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 1 μL of DNA Polymerase, and 3 μL of plasmid DNA template, and 16 μL of nuclease-free water. After thorough mixing, PCR amplification was performed according to the optimized protocol. PCR program: At the end of the procedure, an appropriate amount of purified magnetic beads was added to the PCR reaction mixture and thoroughly mixed. The beads were then incubated for 10 minutes at room temperature to allow DNA binding. Following incubation, the supernatant was discarded. The beads were washed twice with 80% ethanol, and the supernatant was completely removed after each wash. Once the residual ethanol had evaporated completely, the beads were resuspended in nuclease-free water and placed on a magnetic stand until fully adsorbed. The supernatant was then carefully transferred as the linearization product of the DNA. The concentration and quality of the DNA were assessed by agarose gel electrophoresis. IVT reaction for mRNA synthesis Prepared 20 μL of IVT reaction system: 1 μL of 10x Reaction Buffer, 1 μL ATP, 1 μL CTP, 1 μL GTP, 1 μL of N1-methyl pseudouridine, 0.8 μL of Cap1 AG, 1 μg of linear DNA template, and 2 μL of T7 RNA Polymerase. Adjust the volume to 20 μL with nuclease-free water. Mix thoroughly and incubate d at 37°C for 2 hours. Next, 1 µL of DNase I was added to the reaction mixture and incubated/incubate at 37°C for 15 minutes to digest/ degrade the DNA template. Add 30 µL of nuclease-free water, 2-fold volume of Binding Buffer, and 3-fold volume of anhydrous ethanol, then mix well. Transfer the mixture to an RNA purification column and centrifuge at 13,000g for 1 min at room temperature, and discard the flow-through. Add 500 µL of Wash Buffer and centrifuge at 13,000 g for 1 min at room temperature; discard the flow-through. Repeat this step with an additional 500 µL of Wash Buffer and centrifugation at 13,000g for 1 minute, discarding the flow-through. Transfer the column to a new centrifuge tube, then elute the mRNA by adding 50-100 μL of nuclease-free water. Incubate for 5 minutes at room temperature, and then centrifuge at 13,000g for 1 minute. Determine mRNA concentration and quality via agarose gel electrophoresis. Transfection of HEK293T cells HEK293T cells were purchased from American Type Culture Collection (ATCC). Resuscitated fresh HEK293T cells were cultured in 37°C in a 5% CO 2 incubator and passaged to the second generation for use. Cells were seeded in 24-well plates at a density of 7×10⁴ cells per well, with 500 μL of complete medium (10% FBS, 1% double antibody) added to each well and cultured continuously for 24 hours. Preparation of the Lipo-mRNA MIX involved the following steps: first, 5µL of RNAiMAX was mixed with 50 µL of Opti-MEM (OMEM) and allowed to stand for 5 minutes. Second, 1 µg of mRNA (at 0.5 µg/µL) was mixed with 50 µL of OMEM. Finally, the RNAiMAX solution was added to the mRNA mixture, thoroughly mixed by pipetting, and incubated at room temperature for 15 minutes. Before transfection, 400 μL of fresh complete medium was replaced in each well. Then, 105 μL of the prepared Lipo-mRNA MIX was added to each well for transfection. After 48 hours, samples of cytosol and medium supernatant were collected separately. Medium supernatant: Centrifuge at 4,000 g for 10 minutes at 4°C, then transfer the supernatant and store it appropriately. Cytosol: Rinse the cells once with cold PBS, add 25 μL of cell lysate (containing protease inhibitors), and lyse on ice for 5 minutes. Then, pipette to collect the sample. Centrifuge the lysate at 10,000 g for 10 minutes at 4 °C, then transfer the supernatant and store it at -80°C. Detection of protein expression by Western Blot Thawed the supernatant samples to be tested, added 5×SDS Buffer, heated the samples at 99°C for 5 minutes. The samples were then centrifuged at 12,000 rpm for 5 minutes. A 10 μL aliquot was then loaded onto SDS-PAGE gels for electrophoresis. After electrophoresis, the proteins were transferred onto PVDF membranes using semi-dry blotting with iBlot 2 for 7 minutes. Following transfer, the membranes were blocked in 5% skimmed milk for 1 hour at room temperature with gentle shaking. After blocking, membranes were washed once with PBET. The membranes were then incubated with primary antibodies diluted appropriately at 4℃ overnight. The next day, the membranes were washed five times with PBST at 100 rpm for 10 minutes each on a horizontal shaker. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature in the dark followed by five washes with PBST at 100 rpm for 10 minutes each. Finally, chemiluminescent signals were detected and imaged using an ECL detection system on a protein imaging system. Myocardial infarction (MI) modeling and experimental design Healthy SPF-grade Sprague–Dawley rats (male, 6–8 weeks old, 180–220 g) were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. Upon arrival, animals were housed in the barrier facility of Shanhai RAT&MOUSE Biotech.,Ltd. under a 12 h light/12 h dark cycle at (22 ± 2) °C and 50–60 % relative humidity, with ad libitum access to food and water. After 5 days of acclimation, rats were used for experiments. All procedures were approved by the Laboratory Animal Ethics Committee of Shanhai RAT&MOUSE Biotech.,Ltd. and were conducted in accordance with the Regulations for the Administration of Laboratory Animals and the ARRIVE guidelines. Rapid induction with 5% isoflurane, followed by maintenance with 3% isoflurane after achieving deep anesthesia. After the absence of pedal withdrawal reflex was confirmed to ensure adequate surgical anesthesia, the thoracic and axillary fur was shaved. The surgical site was then aseptically prepared using alternating scrubs of povidone-iodine and 75% ethanol. Myocardial infarction surgery was subsequently performed. The external light source, surgical microscope, and ventilator were prepared, with the ventilator set to a respiratory ratio of 2:1, a tidal volume of 6–8 mL, and a frequency of 70 breaths per minute. A tracheal tube was carefully inserted into the trachea along the vocal folds, after which the rat was connected to the ventilator, and the respiratory status was monitored. Successful intubation was indicated by chest movements synchronized with the ventilator rhythm. The rat was placed in the proper lateral position. The left axillary region was incised using ophthalmic scissors to open the thoracic cavity. A microscopic scalpel was employed to separate the third and fourth ribs, exposing the heart entirely. The pericardium was gently dissected using microscopic forceps and torn open under the guidance of a microscope to expose the anterior descending branch of the left coronary artery (LAD) or its anatomical vicinity. The course or potential location of the LAD was identified under microscopic visualization. A 5-0 suture needle was then used to pass a non-absorbed suture through the LAD just below the pulmonary artery root near the apex of the left auricle, effectively occluding blood flow in the LAD. After confirming ligation, the thoracic incision was closed meticulously with sutures to ensure no gaps or misalignment. The muscular layer and skin were sutured sequentially from the inside outward. Postoperative monitoring included close observation of respiratory function and general health status. Once the rats regained consciousness naturally, tracheal intubation was removed, and the animals were returned to their normal housing conditions. For luciferase-mRNA delivery, three 8-week-old male rats received injections of 150 µg per rat (20 µL per injection) via intracardiac administration, with a total of three injections. In vivo bioluminescence imaging was conducted on day three post-injection. Successful modeling was confirmed if target gene expression was detectable in more than two of the three rats, indicating the procedure met the predefined experimental criteria. The sham-operated group underwent thoracotomy without LAD ligation. In the drug treatment group, 150 µg of the drug dissolved in citrate buffer was administered via intracardiac injection as a single dose. Cardiac function was assessed, and tissue samples were collected from the ischemic region of the heart at 1-, 2-, and 3-weeks post-treatment. Portions of the tissue were processed for histological analysis, including sectioning and staining to evaluate cardiac cell morphology and collagen deposition. The remaining tissue was used for RNA extraction to examine gene expression changes associated with MI. The sham group received no treatment, while the vehicle group was injected with an equivalent volume of citrate buffer. Following the experimental endpoint, animals were humanely euthanized. Euthanasia was performed under inhaled isoflurane anesthesia. Animals were initially placed in an induction chamber supplied with 5% isoflurane and 1–2 L/min oxygen until the loss of righting reflex was confirmed. Subsequently, the concentration of isoflurane was gradually increased (or maintained at an elevated level) and the exposure was continued until complete respiratory and cardiac arrest occurred, ensuring death prior to any tissue collection. Gene expression assay Myocardial tissue samples were collected from different experimental groups for RNA extraction and subsequent quantitative real-time PCR (qRT-PCR) analysis. Gene expression was detected using SYBR qPCR Master Mix from Novozymes. Gene expression levels were measured using SYBR Green Master Mix (Novozymes). The RT-qPCR reaction mixture was prepared as follows: 6.6 μL of ddH 2 O, 10 μL of 2×SYBR qPCR Master Mix, 0.2 μL of upstream primer (10 μM), 0.2 μL of downstream primer (10 μM), and 3 μL of cDNA template. The reaction was performed under the following cycling conditions: (1) pre-denaturation at 95℃for 3 minutes; (2) 40 amplification cycles consisting of denaturation at 95°C for 10 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds. At the end of the reaction, the relative expression level of the target gene was calculated using the of 2-ΔΔCt method, with GAPDH serving as the internal reference gene. Three technical replicates were performed for both the target and reference gene, and the Ct values among the replicates were required to vary by no more than 0.5 to ensure reproducibility. Hematoxylin-eosin (HE) staining Tissue samples were subjected to routine paraffin embedding. Added liquid paraffin to the mold, and placed the tissue samples to be embedded into the paraffin, making sure that the tissue position was regular. After replenishing a little liquid paraffin, the paraffin was cooled down and frozen, so that the paraffin becomes solid to achieve the effect of tissue fixation, and then sliced into a thickness of about 4-8 μm using a paraffin slicer. The sliced tissue sections are placed on slides and soaked in warm water at 40°C to fully stretch the tissue. Tissue samples were processed for routine paraffin embedding. Liquid paraffin was added to a mold, and the tissue samples were carefully placed to ensure proper positioning. Additional liquid paraffin was added to fully cover the tissue, which was then cooled and solidified to achieve fixation. The embedded tissues were sliced into sections approximately 4–8 μm thick using a rotary microtome. The sections were mounted onto glass slides and soaked in warm water at 40°C to stretch and flatten them gently. The sections of tissue samples to be tested were placed in xylene and fully soaked for 10 minutes, after soaking, the xylene was replaced and continued to soak for another 10 minutes. The xylene-soaked tissue was first soaked in anhydrous ethanol for 5 minutes so that the xylene used in dewaxing could be eluted out and water can enter the tissue. The samples were then soaked in 95%, 85%, and 70% ethanol for 5 minutes each to achieve full hydration. Sections of the hydrated tissue samples were washed by soaking in PBS solution for 5 minutes each time for a total of 3 times. For dewaxing, the tissue sections were first immersed in xylene for 10 minutes, with the solution replaced, and the soaking process repeated for an additional 10 minutes. Following xylene treatment, the sections were immersed in anhydrous ethanol for 5 minutes to remove residual xylene and facilitate rehydration. Next, they were sequentially soaked in 95%, 85%, and 70% ethanol solutions for 5 minutes each to ensure complete hydration. The hydrated sections were then washed three times in phosphate-buffered saline (PBS) for 5 minutes each. Afterward, 100 μL pre-prepared hematoxylin staining solution was added dropwise to each tissue section, and the staining is fully carried out for 10 minutes. Excess hematoxylin staining solution was washed away using distilled water after staining is completed. Differentiation was then carried out using 1% ethanol hydrochloride so that excess staining solution bound in the nucleus and excess staining solution in the cytoplasm were removed. After differentiation was completed, the tissue sections were then rinsed with double-distilled water. To stain hematoxylin blue, a weakly alkaline pro-blue solution was used and added to the tissue sections to allow the nuclei to stain blue. At the end of the antibullying process, the tissue sections were first washed with water and then rinsed clean with double-distilled water. Eosin staining solution was added to the tissue sample sections to be fully stained for 3 minutes. Hematoxylin staining was performed by dropping 100 μL of pre-prepared hematoxylin solution onto each section, which was left to stain for 10 minutes. Excess hematoxylin was washed away with distilled water. Differentiation was carried out using 1% hydrochloric acid ethanol to remove excess stain from the cytoplasm and nuclei. The sections were then rinsed with double-distilled water and stained with a weakly alkaline pro-blue solution to color the nuclei blue. After rinsing with water, eosin solution was applied to stain the cytoplasm for 3 minutes. After the staining was completed, the tissue sections were then subjected to gradient dehydration using concentrations of 80%, 95%, and anhydrous ethanol, respectively. 80% ethanol was dehydrated for 5 seconds, 95% ethanol was dehydrated for 2 minutes, and anhydrous ethanol was dehydrated for 2 minutes. Sections of dehydrated tissue samples were soaked using xylene twice for 4 minutes each time, and then the tissue samples were dried and sealed using neutral gum. Finally, the sections were observed and photographed under a microscope. Dehydration was achieved through graded ethanol immersion: 80% ethanol for 5 seconds, 95% ethanol for 2 minutes, and absolute ethanol for 2 minutes. The sections were then cleared with xylene (twice for 4 minutes each), dried, and sealed with neutral balsam. Finally, the stained sections were observed and imaged under a microscope. Masson staining Prepare paraffin sections containing tissue, making sure that the sections were deparaffinized and ready for staining. Place the deparaffinized sections into a preheated acidic fuchsin solution to stain them red. The staining time and temperature could be adjusted according to the needs of the experiment. The sections were rinsed using hydrochloric acid or acidic ethanol solution to remove excess dye. Paraffin-embedded tissue sections were prepared by deparaffinization to render them suitable for staining. The deparaffinized sections were then immersed in preheated acidic fuchsin solution to stain them red. The staining duration and temperature were adjusted as needed according to the specific requirements of the experiment. Following staining, the sections were washed with hydrochloric acid or acidic ethanol solution to remove excess dye. The sections were placed in a hematoxylin solution to give the tissue sections a yellow or light green color. Then the sections were gradually dehydrated by placing them in increasingly concentrated alcohol solutions (e.g., 70%, 95%, 100%). The sections were then placed in a clearing agent (e.g., Inouye's solvent) to make them transparent and fixed. Next, the sections were immersed in hematoxylin solution, which imparted a yellow or light green coloration to the tissue. The sections were then gradually dehydrated by sequential immersion in increasing concentrations of ethanol (e.g., 70%, 95%, and 100%). After dehydration, the sections were placed in a clearing agent, such as Inouye’s solvent, to render the tissue transparent and prepare it for mounting. Then placed the sections on the slides, added the transparent sealer, and covered the slides. Lastly, the stained tissue sections were observed and analyzed using a microscope. Finally, the sections were mounted onto slides, covered with a transparent sealing medium, and cover-slipped. The stained tissue sections were then examined and analyzed under a microscope. Data Statistics and Analyses All data is analyzed and plotted using GraphPad Prism software. Declarations Acknowledgments This study was financially supported by Knature Bio-pharm Co., Ltd. through internal research and development funds. And we thank Dr. Qi Fang from Shanghai Cyn Biotechnology Co., Ltd for providing AZD8601 and C6V Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wei Wang, Zhenping Zhan and Lan Chen. The first draft of the manuscript was written by Wei Wang, Zhenping Zhan and Zhonghua Wang. Lan Chen, Lei Wang and Shizheng Liu commented on previous versions of the manuscript. Methodology and project administration were preformed by Lei Wang, Shizheng Liu and Zirong Lin. Conceptualization and writing editing was performed by Kanglin Wang and Zhaoyi Yang. All authors read and approved the final manuscript. Data availability statement The data and metarial that support the finding of this study are available from the corresponding author upon reasonable request. Funding information This study was supported by the Anhui Provincial Key Research and Development Plan (grant number: 2023s07020012). Competing Interests Statement The authors have no relevant financial or non-financial interests to disclose. Code availability Not applicable. Ethics approval All animal use was approved by the Institutional Animal Care and Use ommittee(IACUC). Animal welfare conformed to the Guide for the Care and Use of Laboratory Animals(8th ed., 2011) and Chinese national standard Guideline for Ethoical Review of Laboratory Animal Welfare (GB/T 35892-2018). Consent to participate Not applicable. Consent for publication Not applicable. References Wang, A. Y. L. Modified mRNA-Based Vaccines Against Coronavirus Disease 2019. Cell. 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Additional Declarations No competing interests reported. Supplementary Files SupplementalTable1sequence.xlsx SupplementalTable2PCRprimer.xlsx Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Mar, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 11 Feb, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 24 Jan, 2026 Reviewers agreed at journal 16 Jan, 2026 Reviewers invited by journal 12 Jan, 2026 Editor assigned by journal 12 Jan, 2026 Editor invited by journal 15 Dec, 2025 Submission checks completed at journal 12 Dec, 2025 First submitted to journal 12 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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 component group. (E) The mRNA constructs incorporating these optimal elements were synthesized and their expression levels were compared with those of AZD8601. Protein expression and grayscale value calculations were performed using the same methods as described previously. Significant differences were determined using an unpaired Student’s t-test. Data are presented as means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/6adef2bffe75cf0bda23ce24.png"},{"id":100270749,"identity":"ba613c73-44c1-4b40-91cb-16f8677cefd8","added_by":"auto","created_at":"2026-01-14 20:03:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1202952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKm10566 improves cardiac function rapidly and sustainably in MI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic 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 cm\u003csup\u003e2\u003c/sup\u003e 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). \u003cem\u003eP\u003c/em\u003e values were calculated by an ordinary one-way ANOVA.\u003c/p\u003e","description":"","filename":"OnlineFigure220251104.png","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/21979081c52e0bdf8ff7e6af.png"},{"id":100373211,"identity":"62b54c72-2f1e-42a6-b478-68c045a1157e","added_by":"auto","created_at":"2026-01-16 08:13:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":635042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKm10566 significantly reduces the expression of genes related to myocardial cell repair\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess gene expression changes in ischemic cardiac tissue, the expression levels of \u003cem\u003eWt1\u003c/em\u003e(A), \u003cem\u003eTnnt2\u003c/em\u003e (B), and \u003cem\u003eKDR\u003c/em\u003e (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 \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.)\u003c/p\u003e","description":"","filename":"OnlineFigure320251105.png","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/62cc6b2911888550e7ccd863.png"},{"id":100373103,"identity":"7d7e17f5-b995-469c-966e-004796e0f3a5","added_by":"auto","created_at":"2026-01-16 08:13:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":383082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKm10566 can significantly improve cellular morphology in the heart and effectively reduce collagen volume fraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative micrographs of H\u0026amp;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 \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.)\u003c/p\u003e","description":"","filename":"Figure414.png","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/63ab89f27dea443ca76bbe00.png"},{"id":106343739,"identity":"d05a8abe-87f6-453a-b66d-26df1a0b5b18","added_by":"auto","created_at":"2026-04-07 16:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1809164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/a98ecb6c-06be-41cb-a046-c76ee07fd299.pdf"},{"id":100270754,"identity":"382e730d-6939-4947-8008-468e9925097f","added_by":"auto","created_at":"2026-01-14 20:03:34","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19429,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1sequence.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/0084b76b2d2edc566d987cf5.xlsx"},{"id":100371936,"identity":"7bb708fd-ff54-45ec-ab38-966d58b4ae79","added_by":"auto","created_at":"2026-01-16 08:11:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11270,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable2PCRprimer.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8268281/v1/780d688bd8fdcf0a684eeac7.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modifying VEGF-A mRNA by Combinatorial Optimization to Enhance Therapeutic Efficacy for Myocardial Infarction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMessenger RNA (mRNA) technology has emerged as key focus within the scientific community, largely owing to its pivotal role in the development of vaccines for the Coronavirus Disease 2019 (COVID-19) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its favorable properties-such as safety, rapid manufacturing, and cost-effectiveness-have driven increased attention to mRNA compared to DNA or protein-based therapeutics [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Preliminary studies have demonstrated the broad potential of mRNA-based therapies across various applications, including vaccination, gene therapy, and cancer immunotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, conventional mRNA pharmaceuticals still face significant challenges, notably immunogenicity, mRNA instability, and a short half-life [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is evident that key structural elements of mRNA - including the 5'-cap, 5'-untranslated region (UTR), coding region, 3'-UTR, and poly(A) tail [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] play crucial roles in the translation process. For instance, UTRs have been shown to influence mRNA translation efficiency and resistance to degradation by various domains [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Chen \u003cem\u003eet al\u003c/em\u003e. engineered branched oligonucleotides containing dual cap structures through chemical capping and click chemistry resulted in multi-cap mRNA, which significantly enhanced protein expression levels [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, circular RNA, owing to its unique ring structure, exhibited higher stability and longer half-life than linear RNA, enabling sustained expression \u003cem\u003ein vivo\u003c/em\u003e and eliciting more enduring immune responses [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVEGF-A plays a critical role in angiogenesis and a variety of biological processes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been demonstrated that VEGF-A165 is the most abundant and potent isoform [\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In 1983, VEGF-A was first identified as a tumor pro-angiogenic cytokine, initially known as the vascular permeability factor. After its successful isolation in 1989, it was uniformly named vascular endothelial growth factor [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It is widely recognized that angiogenesis is a highly complex process in which VEGF-A functions as a central regulator, directly stimulating the proliferation of vascular endothelial cells, inhibiting their apoptosis, enhancing blood vessel permeability providing substrates for both fibroblasts and endothelial cells, and promoting tumor angiogenesis. Similar to other members of the VEGF family, VEGF-A mediates intracellular signaling through its interaction with VEGF receptors (VEGFRs), exhibiting different degrees of binding affinity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearch into methods of mRNA delivery for inducing neovascularization in ischemic tissues has explored the encoding of angiogenic factors, including VEGF-A mRNA (AZD8601), which was developed through a collaboration between Moderna and AstraZeneca. AZD8601 yields VEGF-A165 by replacing uridine with N1-methylpseudouridine (N1-Ψ), a modification. This modification has been shown to reduce binding affinity for the Prkra dimer markedly, thereby diminishing recognition of double-stranded RNA (dsRNA) and attenuating innate immune responses [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Extensive studies have demonstrated that modified mRNA can facilitate efficient, dose-dependent, and transient protein expression while exhibiting low innate immunogenicity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The commencement of clinical trials in 2016 signified a pivotal moment for mRNA therapeutics. AZD8601 completed Phase II clinical trials and has been proposed as a treatment for heart failure [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, subsequent studies have shown that, in addition to mRNA modification such as N1-Ψ, changes to other components-including poly(A) tails, untranslated regions (UTRs), and coding sequences (CDSs)-can significantly enhance translation efficiency and molecular stability [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe development of new drugs for cardiovascular disease — such as myocardial infarction, heart failure, stroke, and skin injuries — remains a significant healthcare challenge, and the potential of neovascularization may inform these efforts [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, as a secreted protein with a short \u003cem\u003ein vivo\u003c/em\u003e half-life, VEGF-A poses risks of excessive angiogenesis and increased bleeding when administered chronically [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. As an emerging class of therapeutic agents, mRNA offers broad application prospects due to its excellent programmability, rapid production, transient expression, and superior safety profile compared to DNA or protein-based drugs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the expression efficiency of various VEGF-A mRNA constructs to identify a highly expressed and stable VEGF-A mRNA therapeutic with improved clinical potential. We ultimately identified a linear VEGF-A mRNA transcript that surpasses control sequences in expression levels. In animal models, both linear and circular mRNA formulations significantly improved cardiac functions, including ejection fraction, in rat models of myocardial infarction. The modified linear mRNA demonstrated superior collagen volume fraction recovery and gene expression of Wt1, Tnnt2, and KDR in comparison to both the original mRNA and circular mRNA. These findings suggest that this identified mRNA has promising future for further drug development, with potential to reduce medication costs while maintaining therapeutic efficacy.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSuperior translational efficiency capabilities obtained from the optimized mRNA\u003c/h2\u003e \u003cp\u003eTo confirm the structure of VEGF-A mRNA with a enhanced translation efficiency and prolonged half-life, we systematically designed and screened extensive component combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The mRNA was synthesized via \u003cem\u003ein vitro\u003c/em\u003e transcription (IVT) and subsequently transfected into HEK293T cells (see Materials and Methods for details). We employed a stepwise approach by fixing all components except one, then sequentially testing 30 optimized codon sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), 20 combinations of untranslated regions (UTRs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and four types of Poly (A) tails (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Detailed sequence information is provided in Supplementary Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows in CDS sheet, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC in UTR sheet, and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD in the Poly(A) \u0026amp; Modification sheet. AZD8601 served as a control in these experiments, and its protein expression levels were used as a benchmark for comparison. The elements selected were CDS105, Cyn5U6, Cyn3U5, and A120 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, chemical modifications of RNA bases have been shown to alter biophysical properties, influencing RNA secondary structure, folding, or interactions with specific RNA-binding proteins [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Prior studies demonstrated that synthetic mRNA incorporating modified nucleotides exhibits improved stability and reduced immunogenicity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, we synthesized mRNA using five previously reported efficacious modified bases: N6-Methyl-ATP (m6A), 5-Methoxy-UTP (5moU), Pseudouridine (Ψ), 5-Methyl-CTP (m5C), and N1-Methylpseudouridine (m1Ψ). The translational efficiency of each modification was assessed using the same method described above. Results indicated that m1Ψ modification achieved the highest protein expression level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This optimal combination, designated Km10566, exhibited approximately two-fold increase in protein expression compared to AZD8601 in HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKm10566 rapidly and sustainably improves cardiac function in myocardial infarction\u003c/h3\u003e\n\u003cp\u003eWe evaluated the effects of optimized mRNA on cardiac function in vivo in a rat model of MI, induced by ligation of the left anterior descending (LAD) artery. Following the intracardiac injection of a single dose luciferase mRNA in citrate saline buffer into the peri-infant region, we observed successful transfection and protein expression. \u003cem\u003eIn vivo\u003c/em\u003e imaging showed that the mRNA was normally expressed in the rats\u0026rsquo; hearts by Day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The LVEF, left ventricular fractional shortening (LVFS), heart weight, and heart-to-body weight ratio of the rat model were compared between the treatment and control groups at 1,2-, and 3-weeks post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the efficacy and safety of Km10566, its effects were compared with AZD8601. Km10566 significantly improved both LVEF and LVFS at 14 days p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with beneficial effects persisting up to 3 weeks. No significant differences were observed in heart weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) or heart-to-body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) ratio in either group. Notably, Km10566 showed the most pronunced improvement in cardiac function, outperforming AZD8601 significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese results suggest that the design and assembly of synthetic mRNA \u0026mdash; without altering the protein sequence \u0026mdash; can achieve comparable therapeutic effects to those of mRNA drugs. Although no significant changes in heart weight or ratio were observed, the marked improvements in LVEF and LVFS indicate a beneficial effect on cardiac function following myocardial infartion (MI). This efficacy may be mediated through non-structural mechanisms, such as modulating cellular metabolism or reducing inflammation, which are not reflected by macroscopic indicators.\u003c/p\u003e \u003cp\u003eBased on prior studies on circular mRNA, we circularized the optimized sequence to generate C6V, which contains the CDS105 element, using type I intron splicing. We plan to compare its effect with those of the linear mRNA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, C6V did not differ significantly from AZD8601 in terms of heart weight and heart weight-to-body weight ratio. While C6V exhibited a significant impact on key cardiac function indicators\u0026mdash;LVEF and LVFS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u0026mdash; it was less effective than Km10566.\u003c/p\u003e\n\u003ch3\u003eKm10566 significantly reduces the expression of genes related to myocardial cell repair\u003c/h3\u003e\n\u003cp\u003eMyocardial infarction (MI) resulted from acute and sustained ischemia and hypoxia of coronary artery, leading to myocardial necrosis. The formation of new coronary vessels is to revascularize the ischemic myocardium by inducing various angiogenic factors, such as Wilms' Tumor 1 (Wt1), Troponin T type 2 (Tnnt2), and Kinase Insert Domain Receptor (KDR) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe focused on comparing the Km10566 group and the AZD8601 group. For Wt1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), Km10566 showed a sustained and significant down regulation from day 7 to day 21. This prolonged reduction likely favors Wt1-mediated endothelial proliferation and vascular network formation, potentially exerting a long-lasting effect on coronary revascularization. In contrast, AZD8601 only induced transient significant down regulation on day 14, suggesting its impact on Wt1-driven angiogenic processes is short-lived.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding KDR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), Km10566 presented a sharp, durable decrease ( initial p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, then p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which may more profoundly and persistently impact KDR-VEGF-A signaling and subsequent neovascularization. Conversely, AZD8601 showed a significant reduction that disappeared before day 21, indicating a transient influence on KDR-dependent vascular repair. For Tnnt2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), no significant differences were observed during most of the study period, except at day 21, when both AZE8601 and Km10566 showed substantial changes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This may reflect late-stage modifications in in the regulation of myocardial contractile proteins.\u003c/p\u003e \u003cp\u003eIn the T3-C6V group, the expressions levels of Wt1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and Tnnt2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) were comparable to those of the control group throughout the experiment. The differential expression of KDR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), however, persisted until day 14 and normalized by day 21. Compared to AZD8601 and C6V, Km10566 exhibited the most potent and sustained regulatory effects on these three genes.These results indicate that the sequence modifications in Km10566 markedly enhance its efficacy, prolonging VEGF-A mRNA activity in vivo and potentially exerting more persistent effects on coronary vascular regeneration.\u003c/p\u003e\n\u003ch3\u003eKm10566 can significantly improve cellular morphology in the heart and effectively reduce collagen volume fraction\u003c/h3\u003e\n\u003cp\u003eBoth cellular morphology and collagen volume fraction are important indicators for evaluating the therapeutic efficacy myocardial infarction. They are closely associated with the extent of myocardial fibrosis. They can reflect changes in cardiac structure and function, which are crucial for assessing treatment outcomes and prognostication. Hematoxylin and Eosin (HE) staining was performed on rat heart tissues at 7, 14, and 21 days post-treatment to observe histological changes in cell morphology following drug administration in myocardial infarction models.(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). All three treatment groups showed some degree of improvement in tissue necrosis, indicating drug efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess collagen volume fraction, Masson's trichrome staining was applied, quantifying the precentage of collagen content within the total tissue area. At 7 days, Km10566 demonstrated an initial improvement in collagen volume fraction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a significant and sustained decrease observed up to 3 weeks post-treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, AZD8601\u0026rsquo;s effects became significant only after 21 days. The efficacy of C6V was intermediate between the two (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). These results reinforce the rapid and enduring therapeutic effects of Km10566 in the treatment of myocardial infarction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we developed an optimized VEGF-A mRNA construct, designated Km10566, which demonstrates enhanced translation efficiency and stability while minimizing unwanted immunogenicity. Through systematic engineering of the 5\u0026prime;- and 3\u0026prime;-UTRs, codon optimization of the coding sequence (CDS), and incorporation of a stabilizing poly(A) tail, we obtained a candidate capable of sustaining high levels of VEGF-A expression \u003cem\u003ein vitro\u003c/em\u003e. Subsequent modification with N1-methylpseudouridine (m1\u0026Psi;) significantly reduced innate immune activation, consistent with prior reports highlighting nucleoside modifications as a strategy to evade Toll-like receptor (TLR)-mediated recognition.\u003c/p\u003e\n\u003cp\u003eThe functional superiority of Km10566 was validated in a murine model of myocardial ischemia, where it significantly improved left ventricular ejection fraction and reduced collagen deposition compared to both AZD8601 and the reference C6V construct. These findings suggest that our integrated optimization approach not only enhances protein expression but also translates into meaningful physiological improvements in a therapeutically relevant setting. Notably the m1\u0026Psi;-modified Km10566 did not elicit significant interferon-alpha or TNF-\u0026alpha; responses in treated animals, supporting its favorable safety profile.\u003c/p\u003e\n\u003cp\u003eOur results align with recent advances in nucleoside-modified mRNA therapeutics. For instance, Pardi \u003cem\u003eet al\u003c/em\u003e. demonstrated that m1\u0026Psi;-modified mRNAs exhibit reduced immunogenicity and enhanced translation efficiency\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e, supporting their use in repetitive dosing regimens. Furthermore, the selection of specific UTR elements derived from highly expressed endogenous genes\u0026mdash;such as human \u0026alpha;-globin\u0026mdash;has been shown to prolong mRNA half-life and increase protein yield, consistent with our observation of sustained VEGF-A expression from Km10566.\u003c/p\u003e\n\u003cp\u003eCompared to existing VEGF-A therapies, Km10566 offers several advantages. Although AZD8601 has shown promise in early clinical trials for wound healing and cardiovascular repair, its translational efficiency appears suboptimal in direct comparison. Similarly, viral vector-based VEGF delivery systems, though effective in preclinical models, raise concerns regarding immunogenicity and insertional mutagenesis. In contrast, Km10566 leverages non-viral, chemically defined mRNA technology, allowing for controlled, transient expression without genomic integration \u0026mdash; a crucial consideration for regenerative applications.\u003c/p\u003e\n\u003cp\u003eSeveral limitations should be noted. First, the translatability of m1\u0026Psi;-modified mRNA in human cells requires further validation, as recent reports suggest that it has context-dependent effects on ribosomal frameshifting. Second, while murine models provide valuable insights into safety and efficacy, species differences in mRNA sensing and VEGF signaling necessitate caution when extrapolating results to humans. Finally, long-term stability and storage conditions of formulated Km10566 require further characterization, which is essential for clinical translatin.\u003c/p\u003e\n\u003cp\u003eFuture work will focus on advancing Km10566 into GMP-compliant manufacturing and IND-enabling studies. Concurrently, we are exploring combination strategies with biomaterial-based delivery systems to enhance tissue retention and achieve controlled release. Additionally, the potential application of Km10566 in other VEGF-responsive conditions, such as chronic limb ischemia and diabetic ulcers, represents an exciting avenue for ongoing research.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we have developed a novel VEGF-A mRNA construct with optimized efficacy and safety profiles. Km10566 is a promising candidate for further development as a regenerative therapeutic.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSequence design and gene synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe VEGF-A candidate mRNA sequence was designed based on the corresponding amino acid sequence and includes following elements: a 5\u0026rsquo;-cap structure (7-Methyl GTP), 5\u0026rsquo; untranslated region (5\u0026rsquo;-UTR), the coding region encoding (CDS), a peptide tag (Flag-tag), a 3\u0026rsquo;-untranslated region (3\u0026apos;-UTR), and a Poly(A) tail. Subsequently, the DNA template, containing the T7 promoter necessary for in vitro transcription was synthesized de novo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA template preparation for IVT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA plasmid DNA working solution was prepared using nuclease-free water. A 50 \u0026mu;L PCR reaction mixture was assembled as follows: 6.6 \u0026mu;L of 2x Reaction Buffer, 1 \u0026mu;L of dNTP Mix (10 mM), 2 \u0026mu;L of upstream primer (10 \u0026mu;M), 2 \u0026mu;L of downstream primer (10 \u0026mu;M), 1 \u0026mu;L of DNA Polymerase, and 3 \u0026mu;L of plasmid DNA template, and 16 \u0026mu;L of nuclease-free water. After thorough mixing, PCR amplification was performed according to the optimized protocol.\u003c/p\u003e\n\u003cp\u003ePCR program:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"536\" src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1768420432.png\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the procedure, an appropriate amount of purified magnetic beads was added to the PCR reaction mixture and thoroughly mixed. The beads were then incubated for 10 minutes at room temperature to allow DNA binding. Following incubation, the supernatant was discarded. The beads were washed twice with 80% ethanol, and the supernatant was completely removed after each wash. Once the residual ethanol had evaporated completely, the beads were resuspended in nuclease-free water and placed on a magnetic stand until fully adsorbed. The supernatant was then carefully transferred as the linearization product of the DNA. The concentration and quality of the DNA were assessed by agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIVT reaction for mRNA synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrepared 20 \u0026mu;L of IVT reaction system: 1 \u0026mu;L of 10x Reaction Buffer, 1 \u0026mu;L ATP, 1 \u0026mu;L CTP, 1 \u0026mu;L GTP, 1 \u0026mu;L of N1-methyl pseudouridine, 0.8 \u0026mu;L of Cap1 AG, 1 \u0026mu;g of linear DNA template, and 2 \u0026mu;L of T7 RNA Polymerase. Adjust the volume to 20 \u0026mu;L with nuclease-free water. Mix thoroughly and incubate\u003cs\u003ed\u003c/s\u003e at 37\u0026deg;C for 2 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, 1 \u0026micro;L of DNase I was added to the reaction mixture and incubated/incubate at 37\u0026deg;C for 15 minutes to digest/ degrade the DNA template.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdd 30 \u0026micro;L of nuclease-free water, 2-fold volume of Binding Buffer, and 3-fold volume of anhydrous ethanol, then mix well. Transfer the mixture to an RNA purification column and centrifuge at 13,000g for 1 min at room temperature, and discard the flow-through.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdd 500 \u0026micro;L of Wash Buffer and centrifuge at 13,000 g for 1 min at room temperature; discard the flow-through. Repeat this step with an additional 500 \u0026micro;L of Wash Buffer and centrifugation at 13,000g for 1 minute, discarding the flow-through. Transfer the column to a new centrifuge tube, then elute the mRNA by adding 50-100 \u0026mu;L of nuclease-free water. Incubate for 5 minutes at room temperature, and then centrifuge at 13,000g for 1 minute. Determine mRNA \u0026nbsp;concentration and quality via agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection of HEK293T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T cells were purchased from American Type Culture Collection (ATCC). Resuscitated fresh HEK293T cells were cultured in 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator and passaged to the second generation for use. Cells were seeded in 24-well plates at a density of 7\u0026times;10⁴ cells per well, with 500 \u0026mu;L of complete medium (10% FBS, 1% double antibody) added to each well and cultured continuously for 24 hours.\u003c/p\u003e\n\u003cp\u003ePreparation of the Lipo-mRNA MIX involved the following steps: \u0026nbsp;first, 5\u0026micro;L of RNAiMAX was mixed with 50 \u0026micro;L of Opti-MEM (OMEM) and allowed to stand for 5 minutes. Second, 1 \u0026micro;g of mRNA (at 0.5 \u0026micro;g/\u0026micro;L) was mixed with 50 \u0026micro;L of OMEM. Finally, the RNAiMAX solution was added to the mRNA mixture, thoroughly mixed by pipetting, and incubated at room temperature for 15 minutes.\u003c/p\u003e\n\u003cp\u003eBefore transfection, 400 \u0026mu;L of fresh complete medium was replaced in each well. Then, 105 \u0026mu;L of the prepared Lipo-mRNA MIX was added to each well for transfection. After 48 hours, samples of cytosol and medium supernatant were collected separately.\u003c/p\u003e\n\u003cp\u003eMedium supernatant: Centrifuge at 4,000 g for 10 minutes at 4\u0026deg;C, then transfer the supernatant and store it appropriately.\u003c/p\u003e\n\u003cp\u003eCytosol: Rinse the cells once with cold PBS, add 25 \u0026mu;L of cell lysate (containing protease inhibitors), and lyse on ice for 5 minutes. Then, pipette to collect the sample. Centrifuge the lysate at 10,000 g for 10 minutes at 4 \u0026deg;C, then transfer the supernatant and store it at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of protein expression by Western Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThawed the supernatant samples to be tested, added 5\u0026times;SDS Buffer, heated the samples at 99\u0026deg;C for 5 minutes. The samples were then centrifuged at 12,000 rpm for 5 minutes. A 10 \u0026mu;L aliquot was then loaded onto SDS-PAGE gels for electrophoresis. After electrophoresis, the proteins were transferred onto PVDF membranes using semi-dry blotting with iBlot 2 for 7 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing transfer, the membranes were blocked in 5% skimmed milk for 1 hour at room temperature with gentle shaking. After blocking, membranes were washed once with PBET. The membranes were then incubated with primary antibodies diluted appropriately at 4℃ overnight. The next day, the membranes were washed five times with PBST at 100 rpm for 10 minutes each on a horizontal shaker. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature in the dark followed by five washes with PBST at 100 rpm for 10 minutes each.\u003c/p\u003e\n\u003cp\u003eFinally, chemiluminescent signals were detected and imaged using an ECL detection system on a protein imaging system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMyocardial infarction (MI) modeling and experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy SPF-grade Sprague\u0026ndash;Dawley rats (male, 6\u0026ndash;8 weeks old, 180\u0026ndash;220 g) were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. Upon arrival, animals were housed in the barrier facility of Shanhai RAT\u0026amp;MOUSE Biotech.,Ltd. under a 12 h light/12 h dark cycle at (22 \u0026plusmn; 2) \u0026deg;C and 50\u0026ndash;60 % relative humidity, with ad libitum access to food and water. After 5 days of acclimation, rats were used for experiments. All procedures were approved by the Laboratory Animal Ethics Committee of Shanhai RAT\u0026amp;MOUSE Biotech.,Ltd. and were conducted in accordance with the Regulations for the Administration of Laboratory Animals and the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003eRapid induction with 5% isoflurane, followed by maintenance with 3% isoflurane after achieving deep anesthesia. After the absence of pedal withdrawal reflex was confirmed to ensure adequate surgical anesthesia, the thoracic and axillary fur was shaved. The surgical site was then aseptically prepared using alternating scrubs of povidone-iodine and 75% ethanol. Myocardial infarction surgery was subsequently performed.\u003c/p\u003e\n\u003cp\u003eThe external light source, surgical microscope, and ventilator were prepared, with the ventilator set to a respiratory ratio of 2:1, a tidal volume of 6\u0026ndash;8 mL, and a frequency of 70 breaths per minute. A tracheal tube was carefully inserted into the trachea along the vocal folds, after which the rat was connected to the ventilator, and the respiratory status was monitored. Successful intubation was indicated by chest movements synchronized with the ventilator rhythm.\u003c/p\u003e\n\u003cp\u003eThe rat was placed in the proper lateral position. The left axillary region was incised using ophthalmic scissors to open the thoracic cavity. A microscopic scalpel was employed to separate the third and fourth ribs, exposing the heart entirely. The pericardium was gently dissected using microscopic forceps and torn open under the guidance of a microscope to expose the anterior descending branch of the left coronary artery (LAD) or its anatomical vicinity. The course or potential location of the LAD was identified under microscopic visualization.\u003c/p\u003e\n\u003cp\u003eA 5-0 suture needle was then used to pass a non-absorbed suture through the LAD just below the pulmonary artery root near the apex of the left auricle, effectively occluding blood flow in the LAD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter confirming ligation, the thoracic incision was closed meticulously with sutures to ensure no gaps or misalignment. The muscular layer and skin were sutured sequentially from the inside outward.\u003c/p\u003e\n\u003cp\u003ePostoperative monitoring included close observation of respiratory function and general health status. Once the rats regained consciousness naturally, tracheal intubation was removed, and the animals were returned to their normal housing conditions.\u003c/p\u003e\n\u003cp\u003eFor luciferase-mRNA delivery, three 8-week-old male rats received injections of 150 \u0026micro;g per rat (20 \u0026micro;L per injection) via intracardiac administration, with a total of three injections. \u003cem\u003eIn vivo\u003c/em\u003e bioluminescence imaging was conducted on day three post-injection. Successful modeling was confirmed if target gene expression was detectable in more than two of the three rats, indicating the procedure met the predefined experimental criteria.\u003c/p\u003e\n\u003cp\u003eThe sham-operated group underwent thoracotomy without LAD ligation. In the drug treatment group, 150 \u0026micro;g of the drug dissolved in citrate buffer was administered via intracardiac injection as a single dose. Cardiac function was assessed, and tissue samples were collected from the ischemic region of the heart at 1-, 2-, and 3-weeks post-treatment. Portions of the tissue were processed for histological analysis, including sectioning and staining to evaluate cardiac cell morphology and collagen deposition. The remaining tissue was used for RNA extraction to examine gene expression changes associated with MI. The sham group received no treatment, while the vehicle group was injected with an equivalent volume of citrate buffer.\u003c/p\u003e\n\u003cp\u003eFollowing the experimental endpoint, animals were humanely euthanized. Euthanasia was performed under inhaled isoflurane anesthesia. Animals were initially placed in an induction chamber supplied with 5% isoflurane and 1\u0026ndash;2 L/min oxygen until the loss of righting reflex was confirmed. Subsequently, the concentration of isoflurane was gradually increased (or maintained at an elevated level) and the exposure was continued until complete respiratory and cardiac arrest occurred, ensuring death prior to any tissue collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMyocardial tissue samples were collected from different experimental groups for RNA extraction and subsequent quantitative real-time PCR (qRT-PCR) analysis.\u003c/p\u003e\n\u003cp\u003eGene expression was detected using SYBR qPCR Master Mix from Novozymes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGene expression levels were measured using SYBR Green Master Mix (Novozymes).\u003c/p\u003e\n\u003cp\u003eThe RT-qPCR reaction mixture was prepared as follows: 6.6 \u0026mu;L of ddH\u003csub\u003e2\u003c/sub\u003eO, 10 \u0026mu;L of 2\u0026times;SYBR qPCR Master Mix, 0.2 \u0026mu;L of upstream primer (10 \u0026mu;M), 0.2 \u0026mu;L of downstream primer (10 \u0026mu;M), and 3 \u0026mu;L of cDNA template.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe reaction was performed under the following cycling conditions: (1) pre-denaturation at 95℃for 3 minutes; (2) 40 amplification cycles consisting of denaturation at 95\u0026deg;C for 10 seconds, annealing at 56\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 30 seconds. At the end of the reaction, the relative expression level of the target gene was calculated using the of 2-\u0026Delta;\u0026Delta;Ct method, with GAPDH serving as the internal reference gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree technical replicates were performed for both the target and reference gene, and the Ct values among the replicates were required to vary by no more than 0.5 to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin-eosin (HE) staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples were subjected to routine paraffin embedding. Added liquid paraffin to the mold, and placed the tissue samples to be embedded into the paraffin, making sure that the tissue position was regular. After replenishing a little liquid paraffin, the paraffin was cooled down and frozen, so that the paraffin becomes solid to achieve the effect of tissue fixation, and then sliced into a thickness of about 4-8 \u0026mu;m using a paraffin slicer. The sliced tissue sections are placed on slides and soaked in warm water at 40\u0026deg;C to fully stretch the tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTissue samples were processed for routine paraffin embedding. Liquid paraffin was added to a mold, and the tissue samples were carefully placed to ensure proper positioning. Additional liquid paraffin was added to fully cover the tissue, which was then cooled and solidified to achieve fixation. The embedded tissues were sliced into sections approximately 4\u0026ndash;8 \u0026mu;m thick using a rotary microtome. The sections were mounted onto glass slides and soaked in warm water at 40\u0026deg;C to stretch and flatten them gently.\u003c/p\u003e\n\u003cp\u003eThe sections of tissue samples to be tested were placed in xylene and fully soaked for 10 minutes, after soaking, the xylene was replaced and continued to soak for another 10 minutes. The xylene-soaked tissue was first soaked in anhydrous ethanol for 5 minutes so that the xylene used in dewaxing could be eluted out and water can enter the tissue. The samples were then soaked in 95%, 85%, and 70% ethanol for 5 minutes each to achieve full hydration. Sections of the hydrated tissue samples were washed by soaking in PBS solution for 5 minutes each time for a total of 3 times.\u003c/p\u003e\n\u003cp\u003eFor dewaxing, the tissue sections were first immersed in xylene for 10 minutes, with the solution replaced, and the soaking process repeated for an additional 10 minutes. Following xylene treatment, the sections were immersed in anhydrous ethanol for 5 minutes to remove residual xylene and facilitate rehydration. Next, they were sequentially soaked in 95%, 85%, and 70% ethanol solutions for 5 minutes each to ensure complete hydration. The hydrated sections were then washed three times in phosphate-buffered saline (PBS) for 5 minutes each.\u003c/p\u003e\n\u003cp\u003eAfterward, 100 \u0026mu;L pre-prepared hematoxylin staining solution was added dropwise to each tissue section, and the staining is fully carried out for 10 minutes. Excess hematoxylin staining solution was washed away using distilled water after staining is completed. Differentiation was then carried out using 1% ethanol hydrochloride so that excess staining solution bound in the nucleus and excess staining solution in the cytoplasm were removed. After differentiation was completed, the tissue sections were then rinsed with double-distilled water. To stain hematoxylin blue, a weakly alkaline pro-blue solution was used and added to the tissue sections to allow the nuclei to stain blue. At the end of the antibullying process, the tissue sections were first washed with water and then rinsed clean with double-distilled water. Eosin staining solution was added to the tissue sample sections to be fully stained for 3 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHematoxylin staining was performed by dropping 100 \u0026mu;L of pre-prepared hematoxylin solution onto each section, which was left to stain for 10 minutes. Excess hematoxylin was washed away with distilled water. Differentiation was carried out using 1% hydrochloric acid ethanol to remove excess stain from the cytoplasm and nuclei. The sections were then rinsed with double-distilled water and stained with a weakly alkaline pro-blue solution to color the nuclei blue. After rinsing with water, eosin solution was applied to stain the cytoplasm for 3 minutes.\u003c/p\u003e\n\u003cp\u003eAfter the staining was completed, the tissue sections were then subjected to gradient dehydration using concentrations of 80%, 95%, and anhydrous ethanol, respectively. 80% ethanol was dehydrated for 5 seconds, 95% ethanol was dehydrated for 2 minutes, and anhydrous ethanol was dehydrated for 2 minutes. Sections of dehydrated tissue samples were soaked using xylene twice for 4 minutes each time, and then the tissue samples were dried and sealed using neutral gum. Finally, the sections were observed and photographed under a microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDehydration was achieved through graded ethanol immersion: 80% ethanol for 5 seconds, 95% ethanol for 2 minutes, and absolute ethanol for 2 minutes. The sections were then cleared with xylene (twice for 4 minutes each), dried, and sealed with neutral balsam. Finally, the stained sections were observed and imaged under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMasson staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrepare paraffin sections containing tissue, making sure that the sections were deparaffinized and ready for staining. Place the deparaffinized sections into a preheated acidic fuchsin solution to stain them red. The staining time and temperature could be adjusted according to the needs of the experiment. The sections were rinsed using hydrochloric acid or acidic ethanol solution to remove excess dye.\u003c/p\u003e\n\u003cp\u003eParaffin-embedded tissue sections were prepared by deparaffinization to render them suitable for staining. The deparaffinized sections were then immersed in preheated acidic fuchsin solution to stain them red. The staining duration and temperature were adjusted as needed according to the specific requirements of the experiment. Following staining, the sections were washed with hydrochloric acid or acidic ethanol solution to remove excess dye.\u003c/p\u003e\n\u003cp\u003eThe sections were placed in a hematoxylin solution to give the tissue sections a yellow or light green color. Then the sections were gradually dehydrated by placing them in increasingly concentrated alcohol solutions (e.g., 70%, 95%, 100%). The sections were then placed in a clearing agent (e.g., Inouye\u0026apos;s solvent) to make them transparent and fixed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, the sections were immersed in hematoxylin solution, which imparted a yellow or light green coloration to the tissue. The sections were then gradually dehydrated by sequential immersion in increasing concentrations of ethanol (e.g., 70%, 95%, and 100%). After dehydration, the sections were placed in a clearing agent, such as Inouye\u0026rsquo;s solvent, to render the tissue transparent and prepare it for mounting.\u003c/p\u003e\n\u003cp\u003eThen placed the sections on the slides, added the transparent sealer, and covered the slides. Lastly, the stained tissue sections were observed and analyzed using a microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the sections were mounted onto slides, covered with a transparent sealing medium, and cover-slipped. The stained tissue sections were then examined and analyzed under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Statistics and Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is analyzed and plotted using GraphPad Prism software.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Knature Bio-pharm Co., Ltd. through internal research and development funds. And we thank Dr. Qi Fang from Shanghai Cyn Biotechnology Co., Ltd for providing AZD8601 and C6V\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wei Wang, Zhenping Zhan and Lan Chen. The first draft of the manuscript was written by Wei Wang, Zhenping Zhan and Zhonghua Wang. Lan Chen, Lei Wang and Shizheng Liu commented on previous versions of the manuscript. Methodology and project administration were preformed by Lei Wang, Shizheng Liu and Zirong Lin. Conceptualization and writing editing was performed by Kanglin Wang and Zhaoyi Yang. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and metarial that support the finding of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Anhui Provincial Key Research and Development Plan (grant number: 2023s07020012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal use was approved by the Institutional Animal Care and Use ommittee(IACUC). Animal welfare conformed to the Guide for the Care and Use of Laboratory Animals(8th ed., 2011) and Chinese national standard Guideline for Ethoical Review of Laboratory Animal Welfare (GB/T 35892-2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang, A. Y. L. Modified mRNA-Based Vaccines Against Coronavirus Disease 2019. \u003cem\u003eCell. Transpl.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 9636897221090259 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVallazza, B. et al. 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Algorithm for optimized mRNA design improves stability and immunogenicity. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e621\u003c/b\u003e, 396\u0026ndash;403 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"In Vitro Transcription, synthetic mRNA, mRNA-based therapeutics, VEGF-A, mRNA, myocardial infarction","lastPublishedDoi":"10.21203/rs.3.rs-8268281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8268281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVascular Endothelial Growth Factor A (VEGF-A) is a mitogen with high endothelial cell specificity, playing a key regulatory role in angiogenesis and vasculature formation. Administration of VEGF-A mRNA can facilitate dose-dependent protein expression, promoting therapeutic angiogenesis without genome modification. However, unmodified VEGF-A mRNA is susceptible to degradation and induces immunogenicity, limiting its efficacy.\u003c/p\u003e \u003cp\u003eIn this study, we designed and synthesized a carefully modified VEGF-A mRNA construct, designated Km10566, which exhibits enhanced VEGF-A protein expression during \u003cem\u003ein vitro\u003c/em\u003e transcription (IVT). Intracardiac injection of Km10566, formulated in a biocompatible citrate saline solution, into a rat model of myocardial infarction resulted in significant improvement in left ventricular ejection fraction (LVEF) and reduced myocardial fibrosis after 21 days. A pharmacodynamic analysis further supports Km10566 as a promising therapeutic candidate, highlighting its potential for further application in preclinical and clinical research.\u003c/p\u003e","manuscriptTitle":"Modifying VEGF-A mRNA by Combinatorial Optimization to Enhance Therapeutic Efficacy for Myocardial Infarction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 20:03:29","doi":"10.21203/rs.3.rs-8268281/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-04T04:22:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T07:10:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T10:38:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301664125201332253014861908071058412294","date":"2026-01-26T06:43:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55400684433184945101006830336557404868","date":"2026-01-24T17:39:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301664125201332253014861908071058412294","date":"2026-01-16T05:10:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-12T18:25:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-12T09:19:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-15T14:48:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-12T09:02:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-12T08:48:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30218232-ee35-4c63-b5bd-a4e8e663dbc2","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":61092088,"name":"Biological sciences/Biotechnology"},{"id":61092089,"name":"Health sciences/Cardiology"},{"id":61092090,"name":"Health sciences/Diseases"},{"id":61092091,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-07T16:05:03+00:00","versionOfRecord":{"articleIdentity":"rs-8268281","link":"https://doi.org/10.1038/s41598-026-46680-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-31 15:59:50","publishedOnDateReadable":"March 31st, 2026"},"versionCreatedAt":"2026-01-14 20:03:29","video":"","vorDoi":"10.1038/s41598-026-46680-x","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46680-x","workflowStages":[]},"version":"v1","identity":"rs-8268281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8268281","identity":"rs-8268281","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}