RESUMO
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.
Assuntos
Dependovirus , Distrofia Muscular do Cíngulo dos Membros , Animais , Camundongos , Dependovirus/genética , Disferlina/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/terapia , Códon , Fibras Musculares Esqueléticas , RNA MensageiroRESUMO
The recently discovered CRISPR-Cas9 modification, base editors (BEs), is considered as one of the most promising tools for correcting disease-causing mutations in humans, since it allows point substitutions to be edited without generating double-stranded DNA breaks, and, therefore, with a significant decrease in non-specific activity. Until recently, this method was considered the safest, but at the same time, it is quite effective. However, recent studies of non-specific activity of BEs revealed that some of them lead to the formation of a huge number of off-targets in both DNA and RNA, occurring due to the nature of the Cas9-fused proteins used. In this review article, we have considered and combined data from numerous studies about the most commonly used and more described in detail APOBEC-based BEs and Target-AID version of CBE, as well as ABE7 and ABE8 with their basic modifications into TadA to improve BEs' specificity. In our opinion, modern advances in molecular genetics make it possible to dramatically reduce the off-target activity of base editors due to introducing mutations into the domains of deaminases or inhibition of Cas9 by anti-CRISPR proteins, which returns BEs to the leading position in genome editing technologies.
Assuntos
Sistemas CRISPR-Cas , Citosina , Citosina/metabolismo , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Humanos , MutaçãoRESUMO
Cystic fibrosis is one of the most common inherited diseases caused by mutations in CFTR gene, of which F508del is the most frequent. Currently, the possibility of cell therapy including genome editing is widely discussed. We generated induced pluripotent stem cells from fibroblasts obtained from a 22-year-old woman with clinically manifested and genetically proven disease by using non-viral, non-integrating RNA reprogramming vector that contains five reprogramming factors: OCT4, KLF4, SOX2, GLIS1, and c-MYC. The established cell line can express endogenous pluripotency markers, possesses a normal karyotype, and has the ability to differentiate into three germ layers in spontaneous differentiation assay.
Assuntos
Fibrose Cística , Células-Tronco Pluripotentes Induzidas , Adulto , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/genética , Fibrose Cística/genética , Feminino , Fibroblastos , Humanos , Fator 4 Semelhante a Kruppel , Mutação , Adulto JovemRESUMO
Skin fibroblasts obtained from a 28-year-old man with clinically manifested and genetically proven (F508del/W1282X) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using non-viral, non-integrating, self-replicating RNA reprogramming vectorthat contains five reprogramming factors: OCT4, KLF4, SOX2, GLIS1, and c-MYC as well as a puromycin-resistance gene. Two iPSC lines showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. These iPSC lines may be subsequently used for development of a personalized etiotropic treatment,disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.
Assuntos
Fibrose Cística , Células-Tronco Pluripotentes Induzidas , Adulto , Diferenciação Celular , Reprogramação Celular , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibroblastos , Humanos , Fator 4 Semelhante a Kruppel , Masculino , MutaçãoRESUMO
Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out.