Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.

Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

Twenty-Year Clinical Progression of Dysferlinopathy in Patients from Dagestan.

To date, over 30 genes with mutations causing limb-girdle muscle dystrophy have been described. Dysferlinopathies are a form of limb-girdle muscle dystrophy type 2B with an incidence ranging from 1:1,300 to 1:200,000 in different populations. In 1996, Dr. S. N. Illarioshkin described a family from the Botlikhsky district of Dagestan, where limb-girdle muscle dystrophy type 2B and Miyoshi myopathy were diagnosed in 12 members from three generations of a large Avar family. In 2000, a previously undescribed mutation in the DYSF gene (c.TG573/574AT; p. Val67Asp) was detected in the affected members of this family. Twenty years later, in this work, we re-examine five known and seven newly affected family members previously diagnosed with dysferlinopathy. We observed disease progression in family members who were previously diagnosed and noted obvious clinical polymorphism of the disease. A typical clinical case is provided.

Glu20Ter Variant in PLEC 1f Isoform Causes Limb-Girdle Muscle Dystrophy with Lung Injury.

Plectinopathies are orphan diseases caused by PLEC gene mutations. PLEC is encoding the protein plectin, playing a role in linking cytoskeleton components in various tissues. In this study, we describe the clinical case of a 26-year-old patient with an early onset plectinopathy variant "limb-girdle muscle dystrophy type 2Q," report histopathological and ultrastructural findings in m. vastus lateralis biopsy and a novel homozygous likely pathogenic variant (NM_201378.3:c.58G>T, NP_958780.1:p.Glu20Ter) in isoform 1f of the gene PLEC. The patient had an early childhood onset with retarded physical development, moderate weakness in pelvic girdle muscles, progressive weakening of limb-girdle muscles after the age of 21, pronounced atrophy of axial muscles, and hypertrophy of the gastrocnemius, deltoid, and triceps muscles, intermittent dyspnea, and no skin involvement. Findings included: non-infectious bronchiolitis and atelectasis signs, biopsy revealed myodystrophal pattern without macrophage infiltration, muscle fiber cytoskeleton disorganization resulted from the plectin loss, incomplete reparative rhabdomyogenesis, and moderate endomysial fibrosis. We have determined a novel likely pathogenic variant in PLEC 1f isoform that causes limb-girdle muscle dystrophy type 2Q and described the third case concerning an isolated myodystrophic phenotype of LGMD2Q with the likely pathogenic variant in PLEC 1f isoform. In addition, we have demonstrated the presence of severe lung injury in a patient and his siblings with the same myodystrophic phenotype and discussed the possible role of plectin deficiency in its pathogenesis.