Diaphragmatic myoblasts (DM) are stem cells of the diaphragm, a muscle displaying high resistance to stress and exhaustion. We hypothesized that DM modified to overexpress connexin-43 (cx43), seeded on aligned poly (l-lactic acid) (PLLA) sheets would decrease infarct size and improve ventricular function in sheep with acute myocardial infarction (AMI). Sheep with AMI received PLLA sheets without DM (PLLA group), sheets with DM (PLLA-DM group), sheets with DM overexpressing cx43 (PLLA-DMcx43) or no treatment (control group, n = 6 per group). Infarct size (cardiac magnetic resonance) decreased ∼25% in PLLA-DMcx43 [from 8.2 ± 0.6 ml (day 2) to 6.5 ± 0.7 ml (day 45), p < .01, ANOVA-Bonferroni] but not in the other groups. Ejection fraction (EF%) (echocardiography) at 3 days post-AMI fell significantly in all groups. At 45 days, PLLA-DM y PLLA-DMcx43 recovered their EF% to pre-AMI values (PLLA-DM: 61.1 ± 0.5% vs. 58.9 ± 3.3%, p = NS; PLLA-DMcx43: 64.6 ± 2.9% vs. 56.9 ± 2.4%, p = NS), but not in control (56.8 ± 2.0% vs. 43.8 ± 1.1%, p < .01) and PLLA (65.7 ± 2.1% vs. 56.6 ± 4.8%, p < .01). Capillary density was higher (p < .05) in PLLA-DMcx43 group than in the remaining groups. In conclusion, PLLA-DMcx43 reduces infarct size in sheep with AMI. PLLA-DMcx43 and PLLA-DM improve ventricular function similarly. Given its safety and feasibility, this novel approach may prove beneficial in the clinic.

Background: Gene therapy has been proposed as a strategy to induce cardiac regeneration following acute myocardial infarction (AMI). Given that Tbx20, a transcription factor of the T‐box subfamily, stimulates cell proliferation and angiogenesis, we designed a baculovirus overexpressing Tbx20 (Bv‐Tbx20) and evaluated its effects in cultured cardiomyocytes and in an ovine model of AMI. Methods and Results: Cell proliferation and angiogenesis were measured in cardiomyocytes transduced with Bv‐Tbx20 or Bv‐Null (control). Subsequently, in sheep with AMI, Bv‐Tbx20 or Bv‐Null was injected in the infarct border. Cardiomyocyte cell cycle activity, angioarteriogenesis, left ventricular function, and infarct size were assessed. Cardiomyocytes transduced with BvTbx20 increased cell proliferation, cell cycle regulatory and angiogenic gene expression, and tubulogenesis. At 7 days posttreatment, sheep treated with Bv‐Tbx20 showed increased Tbx20 , promitotic and angiogenic gene expression, decreased levels of P21 , increased Ki67‐ (17.09±5.73 versus 7.77±7.24 cardiomyocytes/mm ² , P <0.05) and PHH3 (phospho‐histone H3)‐labeled cardiomyocytes (10.10±3.51 versus 5.23±2.87 cardiomyocytes/mm ² , P <0.05), and increased capillary (2302.68±353.58 versus 1694.52±211.36 capillaries/mm ² , P <0.001) and arteriolar (146.95±53.14 versus 84.06±16.84 arterioles/mm ² , P <0.05) densities. At 30 days, Bv‐Tbx20 decreased infarct size (9.89±1.92% versus 12.62±1.33%, P <0.05) and slightly improved left ventricular function. Baculoviral gene transfer‐mediated Tbx20 overexpression exerted angiogenic and cardiomyogenic effects in vitro. Conclusions: In sheep with AMI, Bv‐Tbx20 induced angioarteriogenesis, cardiomyocyte cell cycle activity, infarct size limitation, and a slight recovery of left ventricular function, suggesting that Bv‐Tbx20 gene therapy may contribute to cardiac regeneration following AMI.

Background/Objectives: Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10⁴ virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development.