Neonatal gene therapy with AAV2/8-LSPhGAA improves hypertrophic cardiomyopathy in the Gaac.1826dupA knock-in murine model

Fig. 1.. AAV2/8-LSPhGAA gene therapy ameliorates left ventricular hypertrophy in Gaac.1826dupA KI mice after 12 weeks of treatment. (A) Representative M-mode echocardiography images from an age-matched wild-type (WT; left), an untreated KI (middle), and an AAV2/8-LSPhGAA–treated KI mouse (Gene Tx; right). Untreated KI mice exhibit increased IVSd (blue) and LVPWd (red) alongside decreased LVIDd (white). These markers indicate significant left ventricular wall thickening, which was near-completely reversed in the treated mice. (B) Quantitative structural parameters (IVSd, LVPWd and LVIDd), are presented separately for male (top) and female (bottom) cohorts. Gene therapy–treated KI mice showed near-normalization of IVSd and LVPWd, and recovery of LVIDd toward WT levels, indicating substantial improvement in ventricular wall morphology and chamber dimensions in both sexes. (C) Functional parameters including LV mass index (LVMI; LVM/BW, mg/g) and fractional shortening (%FS) are shown by sex. Both parameters were markedly improved in treated KI mice, with values trending toward WT levels, consistent with amelioration of hypertrophic cardiomyopathy. Heart rate (HR) was maintained greater than…

Fig. 2.. Biochemical evaluation of tissues of Gaac.1826dupA KI mice after 12 weeks of AAV2/8-LSPhGAA gene therapy. (A) GAA enzyme activity (U/mg protein) was significantly elevated in heart and liver of treated KI animals, whereas no significant increase was detected in either diaphragm or quadriceps. (B) Quantification of glycogen content (μmole/mg protein) revealed a marked reduction in glycogen accumulation in heart tissue, reaching levels comparable to WT mice. A partial glycogen reduction was observed in the quadriceps, though levels remained higher than WT. Each dot represents data from one animal. WT (hashed bars; n = 3; 3 M); KI (white bars; n = 5; 3 M, 2 F); Gene Tx (gray bars; for heart and liver, n = 18; 9 M, 9 F; for diaphragm and quadriceps, n = 13; 5 M, 8 F). Statistical significance was determined using one-way ANOVA with Tukey’s HSD test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3.. Strength assessment and motor coordination of Gaac.1826dupA KI mice after 12 weeks of AAV2/8-LSPhGAA gene therapy. (A) Forelimb grip strength (N) showed no significant improvement in gene therapy–treated KI mice compared with untreated KI controls. (B) Gait analysis revealed a significant improvement in hind paw base of support, while no change was observed in the front paw base of support, suggesting partial restoration of motor coordination following gene therapy. In (A) each dot represents an average of replicates from one animal. WT (hashed bars; n = 18; 9 M, 9 F); KI (white bars; n = 21; 10 M, 11 F); Gene Tx (gray bars; n = 18; 9 M, 9 F). In (B) each dot represents one measured distance (cm) between paws from pooled steps across all animals; individual step-level measurements preclude sex-based stratification. WT (hashed bars; n = 71); KI (white bars; n = 93); Gene Tx (gray bars; n = 50). Statistical significance was determined using one-way ANOVA with Tukey’s HSD test (*p < 0.05, ***p < 0.001, ****p < 0.0001).

Fig. 4.. Cardiac tissue pathology demonstrates marked reduction of glycogen accumulation in Gaac.1826dupA KI mice following AAV2/8-LSPhGAA gene therapy. (A) Representative hematoxylin/PAS-stained sections of heart, gastrocnemius, and quadriceps from study cohort animals. Purple-magenta precipitates indicate tissue glycogen accumulation. Representative regions of abnormal glycogen deposition in cardiac and skeletal muscle are highlighted by yellow arrowheads. (B) Quantification of PAS staining intensity by optical density (OD) using color deconvolution analysis. Increased PAS signal was observed in untreated KI mice compared with WT. A significant reduction in PAS staining intensity was observed in heart tissue following gene therapy, while a partial but not significant decrease was detected in quadriceps sections. Statistical significance was determined using one-way ANOVA with Tukey’s HSD test (*p < 0.05, ****p < 0.0001). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)