AAV9-Galns Gene Therapy Reverses MPS IVA Pathologies

AAV9-mediated Galns expression prevents skeletal alterations

Administration of AAV9-Galns to MPSIVA rats increased Galns expression in bones, such as the femur, tibia, and humerus (Fig. 4a), and decreased keratan sulfate (KS) accumulation in the growth plate (GP) and diaphysis (Fig. 4b–d).

Sections of GP cartilage from 2-month-old MPSIVA rats showed significant reductions in both GP area and calcification area, suggesting changes in ossification as the rats aged (Fig. 4e). Analysis of MPSIVA rat tibial GP revealed chondrocyte hypertrophy in both quiescent and proliferative zones, and chondrocyte hypertrophy was significantly reduced in AAV9-Galns-treated MPSIVA rats (Fig. 4f).

This was further confirmed by transmission electron microscopy (TEM), which showed a substantial reduction in storage vacuoles in proliferating chondrocytes from treated animals (Fig. 4g). These results were consistent with the normalization of body size observed in AAV9-Galns gene-treated MPSIVA rats (Fig. 3a,b).

Rats treated with MPSIVA showed normal bone density and content, as well as trabecular thickness, number, and spacing. In contrast, all bone parameters were altered in untreated MPSIVA rats (Fig. 4h–j). These results provide strong evidence for this gene therapy to treat bone pathology in MPSIVA.

Treatment with AAV9-Galns vectors prevents dental alterations

Abnormal changes in teeth are a common feature of MPSIVA patients. In 2-month-old MPSIVA rat models (MPS), tooth malocclusion, fragility, and enamel hypoplasia were already evident (Fig. 5a). In contrast, AAV9-Galns-treated MPS rats had intact incisors with normal occlusion and enamel (Fig. 5a).

Lysosomal integral membrane protein type-2 (LIMP2) immunostaining of incisors in mandibular sections revealed marked lysosomal expansion in enamel-producing ameloblasts and papillary layer cells, resulting in enamel dysplasia (Fig. 5a,b). AAV9-Galns treatment corrected the storage lesions of these cells (Figure 5b). Ultrastructural analysis of MPS rat incisors revealed abundant storage vacuoles in ameloblasts. Pathological lysosomal accumulation was reduced after gene therapy with AAV9-Galns (Fig. 5c).

Treatment of articular cartilage pathology with AAV9-Galns gene therapy

KS accumulation alters articular cartilage in MPSIVA patients leading to severe osteoarthritis that, in time, will require orthopedic surgery. In MPSIVA rats at 4 weeks of age, analysis of tibial cartilage sections with both Safranin O staining and LIMP2 immunostaining already demonstrated increased chondrocyte hypertrophy (about 2-fold) compared with WT rats, indicating early storage pathology (Fig. 6a–c). At 8 weeks of age, exacerbated LIMP2 signal was observed in articular cartilage sections from MPSIVA rats, consistent with progressive KS accumulation and lysosomal distension (Fig. 6d–e). AAV9-Galns treatment of MPSIVA rats resulted in marked correction of the storage pathology in articular cartilage (Fig. 6d–e). In agreement, analysis of distal femoral epiphysis in MPSIVA rats showed increased KS levels (~4-fold) that were significantly reduced after AAV9-Galns treatment (Fig. 6f).

Histological analysis of humerus cartilage sections from 6-month-old MPSIVA rats, stained with Safranin O, evidenced an osteoarthritic phenotype with a high number of hypertrophic chondrocytes in the most superficial layer of the articular cartilage (Fig. 6g). AAV9-Galns treatment was able to reduce the number of hypertrophic chondrocytes in this layer (Fig. 6g). Ultrastructural analysis of chondrocytes from the superficial layer of tibial articular cartilage revealed a lack of electrolucent storage vesicles in AAV9-Galns-treated MPSIVA rats compared with untreated animals (Fig. 6h).

Similarly, AAV9-Galns treatment reduced chondrocyte hypertrophy and ultimately prevented articular cartilage loss in knee joints 11 months after vector administration to MPSIVA rats (Fig. 6j–k). This agreed with the normalization of matrix metalloproteinase 13 expression in articular cartilage of AAV9-Galns-treated rats, contributing to osteoarthritis prevention (Supplementary Fig. 6l). Moreover, the lysosomal distension observed in the synovial membrane was also normalized by AAV9-Galns administration (Fig. 6m). Consistent with joint alterations, MPSIVA rats already showed reduced grip strength at 2 months of age (Fig. 6i). AAV9-Galns-treated MPSIVA rats behaved like WT rats (Fig. 6i), suggesting that this gene therapy would ameliorate osteoarthritic alterations of MPSIVA disease.

AAV9-Galns treatment of trachea and heart pathology

The most severe non-skeletal manifestations observed in MPSIVA patients were respiratory and cardiovascular complications, which were also the leading causes of death. Changes in tracheal hyaline cartilage, respiratory epithelium, and lamina propria were observed in MPSIVA rats. AAV9-Galns gene therapy increased Galns expression and activity in the trachea and lung, corrected storage abnormalities in chondrocytes, ciliary cells, and fibroblasts (Fig. 7a–f), and reversed airway lesions in MPSIVA rats.

Likewise, high levels of Galns expression and activity were observed in MPSIVA rat hearts following injection of AAV9-Galns (Fig. 7g). However, the main cardiac abnormality in Morquio patients is the valve. TEM analysis revealed abnormal storage of the mitral valve in MPSIVA rats (Fig. 7h), whereas AAV9-Galns treatment could correct the storage abnormality of mitral valve cells (Fig. 7h).

In addition, lysosomal expansion of smooth muscle fibers in aortic wall sections was also observed in LIMP2 immunostaining of MPSIVA rats, which was normalized in MPSIVA rats treated with AAV9-Galns gene therapy. Ultrastructural analysis of aortic fibers in AAV9-Galns-treated MPSIVA rats revealed no vacuoles, suggesting that storage abnormalities were corrected (Fig. 7i).

Valve dysfunction and alterations in the aortic wall in MPSIVA rats may lead to increased heart rate (Fig.7j), similar to the heart rate alterations seen in patients. In contrast, treatment of MPSIVA rats with AAV9-Galns gene therapy also normalized heart rate (Fig. 7j).