| Autor: |
Yakovlev IA; Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia.; PJSC Human Stem Cells Institute, 129110 Moscow, Russia., Emelin AM; Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia., Slesarenko YS; Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia., Limaev IS; Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia., Vetrova IA; Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia., Belikova LD; Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119992 Moscow, Russia., Grafskaia EN; Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia., Bobrovsky PA; Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia., Pokrovsky MV; Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia., Kuzubova EV; Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia., Pokrovsky VM; Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia., Lebedev PA; Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia., Bardakov SN; PJSC Human Stem Cells Institute, 129110 Moscow, Russia., Isaev AA; PJSC Human Stem Cells Institute, 129110 Moscow, Russia., Deev RV; Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia.; PJSC Human Stem Cells Institute, 129110 Moscow, Russia.; Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia. |
| Abstrakt: |
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. |
