Myocardial infarction creates a critical time window for AAV-based cardiac gene transfer

Abstract

Approaches to enhance adeno-associated virus (AAV)-based cardiac gene transfer are the key to successful cardiac gene therapy, but factors influencing AAV transduction remain poorly investigated. This study showed that myocardial infarction (MI) enhanced cardiac AAV transduction, peaking at the third day post-MI in mice. The excessive AAV enrichment at the border zone is due to local vascular permeabilization and cardiomyocyte metabolic remodeling, which is independent of AAV dosage, serotypes and promoters. This effect was harnessed to boost cardiac base editing and improve the outcome of gene therapy for MI in mice. Thus, heart disease itself is a non-negligible factor that alters AAV-based cardiac gene transfer, which provides a new inroad to develop approaches to enhance cardiac gene therapy.

Graphical abstract

1. Introduction

Gene therapy delivers genetic materials into patient cells to treat genetic or acquired diseases [1]. The capacity and application of the gene delivery vehicles, also named vectors, are essential for successful gene therapy [2]. Recombinant adeno-associated virus (AAV) is a popular gene delivery vector in cardiovascular research due to its relatively high tropism toward cardiovascular tissues and good safety profiles [3]. However, further improvement of AAV transduction efficiency is necessary to fully realize the potential of this therapeutic modality [4]. Common strategies to enhance AAV gene transfer include capsid engineering, administration route improvement and neutralizing antibody/immune response management [5], but whether other factors could be harnessed to enhance AAV applications in the heart remains poorly investigated.

Myocardial infarction (MI) is a critical manifestation of ischemic heart disease, the leading cause of death and disability worldwide. Many studies have utilized AAV to study heart protection or regeneration strategies in MI via direct injection into the myocardium [6,7]. However, this administration route is highly invasive and barely performed in clinics. Instead, catheter-based intracardiac infusion was routinely conducted to deliver AAV in patients [8,9]. A major difference between intramyocardial injection versus catheter-based infusion is that the former approach could physically penetrate the coronary vascular permeability barrier and achieve higher local transduction efficiency [10]. Thus, fully understanding the impact of vascular permeability on cardiac AAV transduction would be necessary to further enhance AAV transduction and eventually reach a consensus on the AAV administration strategies [[11], [12], [13]].

MI is associated with complex and dynamic events such as vascular damage, hypoxia, cell death and immune responses [[14], [15], [16]]. Whether these pathological changes influence cardiac gene transfer remains rarely investigated. Recently, accumulating evidence has implied a non-negligible impact of MI on cardiac gene delivery. For example, AAV was reported to exhibit more gene expression in MI hearts in mice [17]. Lipid nanoparticle was shown to exhibit increased cardiac expression upon cardiac ischemia-reperfusion (I/R) or MI injury [18,19]. However, the key components in MI that contribute to these observations remain unclear. Whether the impact of MI on cardiac gene transfer influences therapeutic outcomes in MI gene therapy remains unexplored.

In this study, we extensively studied the enhancing effect of MI on AAV transduction in the heart. Based on the recent development of base editing therapy for ischemic heart diseases [20,21], we also elucidated the effect of MI on the outcome of cardiac AAV gene therapy for itself.

2. Materials and methods

2.1. Animals

Animal experiments conformed to the mouse protocol authorized by the IACUC of Peking University with the approval number DLASBD0022. Mice were obtained from the Department of Laboratory Animal Science of Peking University Health Science Center, and were kept in ventilated cages with appropriate temperature (23 ± 1 °C), suitable humidity (50 ± 5%), controlled illumination (12 h dark/light cycle), and unrestricted access to water and food. All procedures involving mice were performed according to the animal protocol (No. DLASBD0022) approved by the Institutional Animal Care and Use Committee of Peking University.

2.2. Induction of MI and I/R models

Mouse myocardial infarction models were established by ligation of the left anterior descending (LAD) coronary artery. Briefly, under sterile conditions, adult male mice (6–8 weeks) were anesthetized by intraperitoneal injection of tribromoethanol (30 µl/g, MA0478, MeilunBio, China). After the left chest was opened, the LAD was ligated using 7–0 silk sutures. Sham-operated mice underwent an analogous surgical operation without LAD ligation.

In the I/R mouse model, adult male mice (6–8 weeks) were anesthetized with 1% pentobarbital sodium (10 µl/g) and ventilated before left lateral thoracotomy to expose the heart. Next, the LAD was ligated with a 7–0 silk suture tied around a 30-gauge needle. After 45 min of ischemia, the slipknot was released for heart tissue reperfusion. The same procedures without LAD ligation were performed in mice from the sham group.

2.3. rAAV vector construction and production

The pAAV-HA-luciferase plasmids with the Tnnt2 or CMV promoter were cloned by seamless cloning (B632219–0040, Sangon Biotech, China) into the pAAV-CMV-MCS-noATG-GFP or pAAV-Tnnt2-St-GFP plasmids [22]. The pAAV-Tnnt2-Atp2a2-HA-miR122TS vector was constructed by synthesizing the Atp2a2 coding sequence and subcloning it into the previously published pAAV-Tnnt2-Cre-miR122TS [21]. The vectors to deliver adenine base editors (ABEs) to knockout Camk2d [23] or ablate CaMKIIδ oxidation [21] were reported. The proper expression of newly constructed plasmids was verified in HEK293T cells before AAV packaging. AAV1, AAV2, AAV6 or AAV9 were produced by PackGene Biotech company (Guangzhou, China).

2.4. Vascular permeability assay and nitroglycerin treatment

Cardiac vascular permeability was measured by leakage of Evans blue-labeled plasma proteins [24]. 200 µl Evans blue solution (E808783, Macklin, China) was slowly injected into the tail vein. At 4 h after Evans blue injection, hearts were harvested and weighed. Evans blue dye was extracted with formamide (105096, Beijing Tong Guang Fine Chemicals Co., Ltd., China) from tissue by incubating hearts in a 70 °C-heating block for 18 h Next, the concentration of the Formamide/Evans blue mixture was measured by the absorbance at 610 nm with a spectrophotometer (Multiskan SkyHigh, Thermo Scientific, USA).

Nitroglycerin was acquired from Beijing Yimin Pharmaceutical, China (H11020289). 10 mg/kg nitroglycerin was administered intraperitoneally 10 min before the following AAV or Evans blue treatments.

2.5. Oxygen-glucose deprivation (OGD) experiment

NMVCs cultured for 48 h were divided into a control group and an OGD group. The control group was cultured with complete DMEM and maintained in a normal cell incubator (95% air and 5% CO₂) at 37 °C. To mimic the low oxygen-low glucose condition, the OGD group cells were incubated with DMEM with low glucose (1 g/L) and exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) at 37 °C. AAV9 was administered to two groups with a multiplicity of infection of 1E4. DNA and RNA were extracted from the cardiomyocytes at 60 h after AAV transduction for qPCR analysis.

2.6. Quantitative real-time PCR

Total RNA was extracted from mouse hearts or cardiomyocytes using the TransZol Up Plus RNA Kit (ER501–692 01, TransGen, China). Reverse transcription was performed to convert RNA to cDNA using Hiscript III Reverse Transcriptase kit (R302–01, Vazyme Biotech, China). Quantitative real-time PCR was performed with SYBR Green Qpcr mix (Q712–02, Vazyme Biotech, China) using the AriaMx Real-Time PCR System (Agilent Technologies, USA) with Agilent AriaMx software (version 1.8). The relative gene expression of mRNA was normalized to Gapdh as an internal control. See Table S1.1 for primer information.

Total genomic DNA (gDNA) extraction obtained from mouse organ tissues (heart, liver, skeletal muscle) or cardiomyocytes was prepared using the TIANGEN Genomic DNA Kit (DP304, TIANGEN, China). The copy number of AAV DNA encoding luciferase was measured by quantitative real-time PCR and was normalized to the Tnni3 gene as an internal control. See Table S1.1 for primer information.

2.7. Amplicon sequencing

Total genomic DNA extraction obtained from mouse hearts was prepared using TIANGEN Genomic DNA Kit (DP304, TIANGEN, China). The sgRNA-targeted loci of the Camk2d gene were amplified using Taq PCR MasterMix (KT211, TIANGEN, China) and purified by TIANgel Purification Kit (DP219, TIANGEN, China). Sequencing was performed on an Illumina NovaSeq 6000 platform at Novogene, China. The sequencing results were analyzed by CRISPResso2 [25]. See Table S1.2 for primer sequences.

2.8. Western blot analysis

Heart tissues were homogenized in RIPA lysis buffer (P0013, Beyotime, China), mixed with protease inhibitor cocktail (4693116001, Roche, USA) and PhosSTOP phosphatase inhibitor cocktail (4906845001, Roche, USA). After centrifugation (12,000 g, 4 °C, 30 min), protein extracts were quantified with BCA Protein Assay Kits (PC0020, Solarbio, China) and then denatured (70 °C, 10 min) in Protein Loading Buffer (P1016, Solarbio, China). Oxidized proteins were prepared in 4X LDS Sample Buffer (M00676, GenScript, China). Proteins were electrophoresed in 10% SDS-PAGE gels and transferred onto PVDF membranes (IPVH00010, Merck Millipore, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween (TBST) for 1h at room temperature, then incubated with primary antibodies at 4 °C overnight, followed by the appropriate HRP-conjugated secondary antibodies for 1h at room temperature. Blots were detected by ECL reagent (P1050–500, APPLYGEN, China) on the Invitrogen iBright™ CL1500 system. Target protein expression levels were quantified using ImageJ. See Table S2 for antibody and dye information.

2.9. Statistical analysis

Statistical analysis was performed with GraphPad Prism (version 10.1.2). Comparisons between the two groups were calculated by Welch’s t-test. For comparisons of multiple groups with changes over time, mixed-effect ANOVA with Tukey’s multiple comparison test was used. A P-value < 0.05 was considered statistically significant.

See supplementary information for additional Materials and Methods.

3. Results

3.1. Myocardial infarction enhances AAV transduction in the heart

To determine the effect of MI on AAV-based cardiac gene transfer, MI and sham mice were administered with 2 × 10¹¹vg (vector genome) AAV serotype 9 (AAV9) carrying a HA-luciferase transgene driven by the cardiac-specific Tnnt2 promoter at day 3 post infarction via tail vein injection (Fig. 1a). One week later, bioluminescence imaging demonstrated a robust increase of luciferase activity in MI hearts versus sham (Fig. 1b, Fig. S1a). Western blot analysis of HA-luciferase in the heart validated this significant increase of AAV expression in MI mice (Fig. 1c). DNA and RNA were purified from the heart for real-time quantitative PCR (RT-qPCR). While AAV vector DNA and its corresponding mRNA were both significantly higher in the MI group, the mRNA/DNA ratio, a metric for AAV promoter activity, exhibited no significant change (Fig. 1d). The Tnnt2 promoter restricted AAV expression to cardiomyocytes. No AAV expression was detected in isolated non-myocytes (Fig. S1b). Therefore, MI enhanced AAV gene expression by increasing the viral load in cardiomyocytes.

Fig. 1.

MI-mediated AAV-enhancing effect in the heart. (a) A diagram of AAV-luciferase vector and experimental design. AAV, adeno-associated virus; ITR, inverted terminal repeats; HA, hemagglutinin tag; MI, myocardial infarction; DPI, day post MI. (b) Representative bioluminescence images (left) and quantification of signals (right) in isolated hearts. Surg., surgery; Luc, luciferase; RLU, relative luminometer unit (Sham group n = 8, MI group n = 7). (c) Western blot images (left) and quantification (right) showing HA-Luciferase proteins in the heart (Sham group n = 6, MI group n = 7). (d) RT-qPCR fold changes (FC) of AAV vector DNA (left), luciferase mRNA (middle) and mRNA/DNA ratio (right) in the heart. (Sham group n = 7, MI group n = 8). All plots: mean ± SEM; Welch’s t-test: *P < 0.05, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, non-significant P value in parentheses.

Next, we examined whether this MI-mediated AAV-enhancing effect was dependent on AAV dosage by applying 5 × 10¹⁰vg or 1 × 10¹¹vg AAV. Consistent with the prior experiments, MI hearts also exhibited significantly increased AAV vector DNA (Fig. 2a) and AAV-expressed proteins (Fig. 2b). MI was also observed to enhance the cardiac transduction of AAVs carrying the ubiquitously active CMV promoter, but no changes of AAV load were detected in liver or skeletal muscle by applying 2 × 10¹¹vg AAV (Fig. 2c). This enhancing effect could also be observed with AAV1, AAV2 or AAV6 capsids which were applied 2 × 10¹¹vg respectively (Fig. 2d–e), although the AAV2 < AAV1 < AAV6 < AAV9 rank of cardiac tropisms was retained [26]. Therefore, MI locally enhanced cardiac AAV transduction independent of promoters or serotypes.

Fig. 2.

Cardiac AAV-enhancing effects independent of dosage, promoters or serotypes. (a) RT-qPCR analysis of relative AAV DNA in the heart from mice treated with varying doses of AAV. (b) Western blot images (left) and quantification (right) analysis of HA-Luciferase expression in cardiac tissues. (n = 4 per group). (c) A diagram of vector design and AAV DNA FC in variant organs. Sk. Mus., skeletal muscle. (Sham group n = 4, MI group n = 5). (d-e) AAV vector DNA (d) and luciferase mRNA (e) fold changes in hearts treated with various AAV serotypes. FCs are relative to the AAV9-MI group. (n = 4–9 hearts per group). All plots: mean ± SEM; Welch’s t-test: *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, non-significant P value in parentheses.

3.2. The AAV-enhancing effect peaks at the border zone at day 3 post-MI

Myocardial remodeling post MI progresses through the early pro-inflammatory phase (0–3 days post MI), followed by the reparative phase (3–7 days post MI) and the maturation phase (> 7 days post MI) [16]. We hypothesized that AAV transduction efficiency exhibited dynamic changes in parallel with myocardial remodeling. Hence, AAV was next administered at serial time points after MI and then examined one week later (Fig. 3a). While the viral DNA in the heart increased as the AAV was injected in the first three days after MI, it gradually declined afterwards and returned to the basal level by about a week after MI (Fig. 3a). This observation was also validated by RT-qPCR and western blot analysis (Fig. 3a–b). A similar assay was performed in a cardiac I/R model. Despite the significantly enhanced AAV transduction in the heart by 5 days post I/R, this effect was much milder than that in the MI model (Fig. 3c). Thus, prolonged cardiac ischemia for up to 3 days was required to trigger a robust AAV-enhancing effect.

Fig. 3.

Spatiotemporal effects of MI-mediated AAV enhancement. (a) A diagram of experimental design (left) and quantitative analysis (right) of vector DNA and RNA in the hearts when AAV was injected at serial time points (n = 3 per group). (b) Representative immunoblots of cardiac HA-luciferase expression (left) and quantification (right) (n = 3 per group). (c) A diagram of experimental design (left) and RT-qPCR analysis (right) of AAV DNA load in the I/R model. DPR, day post reperfusion (n = 4 per group). (d) Immunofluorescence images (left) and quantification of fluorescence intensity (FI) in cardiac subdomains of MI hearts (right). Arrows point to zones with enhanced HA signals. Grey dashed line indicates mean background signal in non-AAV-treated control samples. Scale bar, 500 µm; a.u., arbitrary unit; MZ, marginal zone; DZ, distal zone. (Sham group n = 6, MI group n = 9). Plots in (a): mean ± SEM; Plots in (b–d): mean ± SD; Welch’s t-test: *P < 0.05, ^⁎⁎⁎P < 0.001, non-significant P value in parentheses.

We next wondered if there is a spatial effect by performing immunostaining of HA-luciferase on MI and sham heart sections. This experiment showed that MI elevated AAV expression preferentially at the marginal zone (MZ) adjacent to the infarcted area labelled by strong wheat germ agglutinin staining (Fig. 3d, arrows). By contrast, distal zones (DZ) further away from the injured region exhibited less enhancement of the HA-luciferase signal (Fig. 3d). Notably, this local enhancing effect was so robust that when low acquisition time was applied to avoid saturated signals at BZ, the normal AAV signals at DZ become barely visible (Fig. 3d).

3.3. Vascular permeabilization and metabolic remodeling facilitate AAV transduction

MI triggered a myriad of vascular [14], metabolic [27] and inflammatory [16] changes that were intense at day 3 post-MI. We wondered which of these factors influenced AAV transduction in the heart. We first performed an Evans Blue permeabilization assay and confirmed the temporarily enhanced cardiac vascular permeability at 3 days after MI (Fig. 4a). Sham and MI mice were next pretreated with nitroglycerin (NTG), a classic vasodilatory drug, followed by AAV administration via tail vein injection. The vascular permeabilization effect of NTG was validated in the heart by the Evans Blue assay (Fig. 4b). Interestingly, NTG increased cardiac AAV transduction in sham hearts as expected [28]. However, no such additional effect was observed in the MI mice (Fig. 4c), suggesting that MI already saturated the permeabilization effect so NTG did not exert extra benefit on AAV transduction.

Fig. 4.

The impact of vascular permeability and metabolic stress on AAV transduction. (a) Quantification of Evans blue in myocardial extracts. 1% Evans blue was tail-vein injected at 4 h before the dyes were extracted from the heart and quantified via 620 nm absorption (n = 4 per group). (b) Representative images of myocardial extracts (left) and quantification of Evans blue concentration (right). 10 mg/kg nitroglycerin (NTG) was administered 10 min before Evans blue injection (n = 3–5 hearts per group). (c) RT-qPCR analysis of AAV vector counts in sham vs. MI mice following 10 mg/kg NTG pre-treatment for 10 min (Sham group n = 8, MI group n = 6). (d) A sketch of the experiment workflow. NMVCs, neonatal mouse ventricular cardiomyocytes; Con, control; OGD, oxygen-glucose deprivation. (e) Analysis of vector DNA FC in NMVCs (Con group n = 8, OGD group n = 6). (f) AAV-delivered luciferase expression at the mRNA level (n = 7 per group). Plots in (a): mean ± SD; Plots in (b-f): mean ± SEM; Welch’s t-test: *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001, ^⁎⁎⁎⁎P < 0.0001, non-significant P value in parentheses.

Next, neonatal mouse ventricular cardiomyocytes (NMVCs) were isolated to test the effect of metabolic changes on AAV transduction. NMVCs were exposed to hypoxia (1% O₂) and low-glucose (1 g/L) media for 12 h before AAV9-CMV-HA-luciferase was applied to these cells for another 60 h (Fig. 4d). AAV-delivered DNA and RNA were extracted from NMVCs for RT-qPCR, which covered a significant increase of AAV transduction by this oxygen-glucose deprivation (OGD) condition (Fig. 4e-f).

MI-mediated immune responses are characterized by increased local concentration of inflammatory cytokines such as IL-6, IL-1β and TNFα and monocyte-macrophage infiltration into the myocardium [29]. To determine their impacts on AAV transduction, the clodronate liposome was applied to reduce monocyte/macrophage in the heart [30,31] before the hearts were subjected to MI and AAV (Fig. S2a). While the reduced monocyte/macrophage number was confirmed by flow cytometry of cardiac immune cells (Fig. S2b-c), no increase in AAV transduction was detected (Fig. S2d). Meanwhile, treatment with a mixture of IL-6, IL-1β and TNFα led to decreased AAV transduction into NMVCs (Fig. S3a-b). These data ruled out immune responses in the MI-mediated AAV-enhancing effect and supported immune suppression as an important component in AAV administration [32,33].

3.4. The AAV-enhancing effect could be harnessed to boost gene therapy for MI

Next, the AAV-enhancing effect was examined using potential gene therapy vectors. We first constructed a vector that expressed the classic cardiac therapeutic protein SERCA2 (sarcoplasmic/endoplasmic reticulum calcium ATPase 2, coded by Atp2a2 gene) [34] (Fig. 5a). Immunofluorescence validated the proper subcellular localization of AAV-expressed SERCA2-HA as the striated and perinuclear patterns of sarcoplasmic reticulum in cardiomyocytes (Fig. 5b). The increased AAV DNA load and RNA expression were observed when 2 × 10¹¹vg AAV-SERCA2 was applied at day 3 post MI rather than at 3 days before MI (Fig. 5c).

Fig. 5.

MI-mediated AAV-enhancing effects on distinct cargo genes. (a) A diagram of the AAV-Atp2a2 vector and the experimental design. Atp2a2 is the coding gene for SERCA2. AAV-MI, AAV injected 3 days before MI; MI-AAV, AAV injected 3 days after MI. (b) An immunofluorescence image of HA-SERCA2 in isolated cardiomyocytes. Scale bar, 10 µm. (c) RT-qPCR analysis of AAV-Atp2a2 vector DNA (left) and RNA expression (right) in MI hearts (n = 4 per group). (d) A diagram of the AAV-SauriABE vector and the experimental design. Myl2 is a small cardiac-specific promoter. SauriABE is a miniature ABE gene that fits into a single AAV. (e) RT-qPCR analysis of AAV-SauriABE DNA (left) and RNA (right) levels in MI hearts (n = 5 per group). Two AAV doses were tested. (f) Amplicon sequencing quantification of ABE-based genome editing fold changes in the heart (n = 5 per group). Plots in (c): mean ± SEM; Plots in (e–f): mean ± SD; Welch’s t-test: *P < 0.05, ^⁎⁎P < 0.01, non-significant P value in parentheses.

We recently established an AAV vector that expressed an adenine base editor (ABE) to silence CaMKIIδ (Fig. 5d) [23], another well-established therapeutic target for heart diseases [20]. When this vector was administered at day 3 post-MI, enhanced AAV DNA load and ABE expression were validated (Fig. 5d-e). Importantly, amplicon sequencing showed increased genome editing rates by MI at two different AAV doses (Fig. 5f). Thus, MI-enhanced AAV transduction could potentiate cardiac genome editing.

We then examined the impact of AAV-enhancing effect on gene therapy using a pre-validated AAV-ABE system that ablates CaMKIIδ oxidation (Fig. 6a) [20,21]. When 5 × 10¹¹vg AAV was injected at day 3 post-MI, amelioration of MI-caused cardiac dysfunction was observed as expected (Fig. 6b–d). However, early AAV administration prior to MI led to a significantly less effect (Fig. 6b–d). Western blot confirmed more efficient ablation of CaMKII oxidation and phosphorylation in the group that harnessed the AAV-enhancing effect, even though AAV was injected at a later time point (Fig. 6e–f). Thus, the outcome of AAV-based gene therapy for MI could be potentially improved by harnessing the MI-mediated AAV-enhancing effect.

Fig. 6.

MI-mediated AAV-enhancing effects on gene therapy outcomes. (a) A scheme of AAV-ABE vector design. (b) Experimental design. AAV-MI, AAV was injected 3 days before MI. MI-AAV, AAV was injected 3 days post-MI. (c) Representative M-mode echocardiogram images at day 30 post MI. (d) Echocardiographic statistical analyses of the ejection fraction (EF), fractional shortening (FS) and left ventricular end-systolic volume (LVESV). Arrows point to pair-wise statistical analyses at each time point by mixed-effect ANOVA with Tukey’s multiple comparison correction. (e) Western blot analysis of CaMKII oxidation in the heart. oxCaMKII, oxidized CaMKII; tCaMKII, total CaMKII. (f) Western blot analysis of CaMKII phosphorylation. pCaMKII, phosphorylated CaMKII. All plots: mean ± SEM; Welch’s t-test in (e–f): *P < 0.05.

4. Discussion

AAV is widely used in basic and translational research in disease contexts, but the knowledge and guidance of AAV-based gene transfer were mainly built on healthy animals. In this study, we showed how heart disease itself could skew AAV-based cardiac gene transfer, adding a previously underestimated variable in AAV-applied cardiovascular research. Since vascular and metabolic remodeling are likely the key underlying mechanisms of MI-mediated AAV enhancement, future studies are warranted to test if other vascular or metabolic diseases could also influence AAV transduction.

In gene therapy, preventive AAV administration before the onset of the disease is usually expected to result in better outcomes than later treatments after the disease has already initiated [35]. However, this study showed an opposite case that the enhancing effect of MI on AAV transduction was sufficiently robust to result in better outcomes than preventative AAV injection prior to MI. This counter-intuitive result was due to the dynamic changes of AAV transduction rates along the course of disease progression. Thus, a more extensive understanding of the interactions between AAV and specific indications would likely facilitate the design of better gene therapy strategies in the future.

Since percutaneous coronary interventions are expected to be performed as early as possible, usually within a couple of hours after the onset of acute MI, it is impractical to wait for prolonged MI stages and harness the day-3 time point for AAV-based cardiac protection in the clinics. Instead, the discovery of MI-mediated AAV-enhancing effect offers a new direction for mechanistic investigation to identify potential approaches to enhance AAV transduction.

5. Conclusion

In conclusion, our study found that MI could enhance AAV-based cardiac gene transfer, especially achieving peak after AAV administration at three days post-MI, which can be employed for efficacious gene therapy for MI. The potential mechanisms, including vascular and metabolic remodeling, need further in-depth exploration.

Data availability

Data are available upon reasonable requests. Plasmids are available at Addgene. See Fig. S5 for original Western blots.

CRediT authorship contribution statement

Gonglie Chen: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. Yueyang Zhang: Investigation. Zhanzhao Liu: Visualization, Investigation. Jingdong Wu: Resources, Methodology, Investigation. Zhan Chen: Validation. Luzi Yang: Validation. Junxia Zhang: Methodology. Yufei Wu: Methodology. Jiting Li: Resources. Baochen Bai: Resources. Zhengyuan Lv: Investigation. Fei Gao: Supervision, Resources. Erdan Dong: Supervision. Yuxuan Guo: Writing – review & editing, Funding acquisition, Conceptualization.

Biographies

Gonglie Chen received her master's degree in medicine from Peking University in 2022. She is currently a doctoral candidate in the School of Basic Medicine, Peking University Health Science Center. Her main research interests are cardiac pathophysiology and the discovery of enhanced AAV cardiac delivery methods.

Yuxuan Guo (BRID: 07890.00.75931) is a researcher and an assistant professor at State Key Laboratory of Vascular Homeostasis and Remodeling, School of Basic Medical Sciences and Institute of Cardiovascular Sciences, Peking University Health Science Center. His research interests mainly include development of new approaches in cardiac gene delivery and gene therapy with the aim to develop novel therapy for heart diseases and cardiac regeneration.