Riassunto analitico
In the last 20 years gene editing tools were proposed to treat rare genetic diseases. CRISPR/Cas9, the newest gene editing technology, allows the correction of pathogenic genetic mutations and the engineering of any DNA or RNA molecules. CRISPR/Cas9 tool consists of the bacterial Cas9 endonuclease complexed with a guide RNA (gRNA) molecule able to direct Cas9 protein on the target region of the genome. Cas9 introduces a double-strand break (DSB) which is repaired through non-homologous end joining (NHEJ) mechanism, leading to gene disruption, or homology directed repair (HDR) which allows the introduction of a desired sequence in the target region. The aim of this thesis is the correction of a EMD gene mutation in tenocytes from a type-I Emery-Dreifuss muscular dystrophy (EDMD1) patient using CRISPR/Cas9 system. EMD gene maps on chromosome X and encodes for emerin protein, which is located into the inner nuclear membrane where interacts with lamin-A protein. Emerin regulates myogenic differentiation and proliferation interacting with transcription factor such as Lmo7 and β-catenin. Moreover, emerin has a crucial role in keeping the nuclear structural integrity and nuclear envelope elasticity. However, despite its relevance, emerin depletion in mice causes only a mild phenotype which does not recapitulate the disease. EDMD is a rare disease caused by genetic mutations occurring in genes involved in the maintenance of nuclear envelope structure. EDMD is characterized by a progressive muscle wasting, joint contractures and cardiac symptoms such as arrhythmias, palpitations and congestive heart failure which eventually cause patients death. To date, only few symptomatic therapies are available for the treatment of the disease. This thesis describes a CRISPR/Cas9 strategy to correct a 5 nucleotides duplication causing the loss of the proper reading frame in EMD gene. The strategy proposed to correct the genetic defect consists of a couple of gRNAs designed to address Cas9 cleavages upstream and downstream the target mutation inducing the excision of a DNA segment containing the mutation and restoring the correct reading frame of the gene. The 3 designed gRNAs were separately cloned into an effector plasmid expressing a human codon-optimize S. pyogenes Cas9 (hSpCas9) and tested separately and in combination. A target plasmid containing the mutated exon of the gene was generated in order to test the designed strategy in a surrogate co-transfection assay in HEK293T cells. Target and effector plasmids were co-transfected in HEK293T cells and the cleavage efficiency of each gRNA was determined by using Tracking of Indels by Decomposition (TIDE) analysis webtool. Moreover, two combinations of gRNAs were co-transfected in HEK293T cells to assess the efficiency of double editing. Once selected the gRNAs combination leading to higher editing efficiency and restoring the correct gene frame, primary tendon’s fibroblasts (tenocytes) from a patient suffering EDMD1 were electroporated with ribonucleoprotein (RNP) carrying two gRNAs and the SpCas9. The editing efficiency was determined by Sanger sequencing. COSMID webtool was used to computationally determine the potential off-target sites for each gRNA of the selected combination. Finally, the restoration of emerin protein production in tenocytes and the proper localization of the protein in the nuclear membrane after the CRISPR treatment was evaluated by Western blotting and immunofluorescence. In conclusion, this thesis shows a CRISPR/Cas9 approach to correct a mutation in EMD gene causing type I Emery-Dreifuss muscular dystrophy. The gene corrected tenocytes represent isogenic cells which will be used, in the near future, to identify biomarkers for the evaluation of disease progression. Moreover, the correction of primary tenocytes with CRISPR tool offers a proof of principle for a future application of gene editing to treat EDMD patients.
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Abstract
In the last 20 years gene editing tools were proposed to treat rare genetic diseases. CRISPR/Cas9, the newest gene editing technology, allows the correction of pathogenic genetic mutations and the engineering of any DNA or RNA molecules. CRISPR/Cas9 tool consists of the bacterial Cas9 endonuclease complexed with a guide RNA (gRNA) molecule able to direct Cas9 protein on the target region of the genome. Cas9 introduces a double-strand break (DSB) which is repaired through non-homologous end joining (NHEJ) mechanism, leading to gene disruption, or homology directed repair (HDR) which allows the introduction of a desired sequence in the target region. The aim of this thesis is the correction of a EMD gene mutation in tenocytes from a type-I Emery-Dreifuss muscular dystrophy (EDMD1) patient using CRISPR/Cas9 system. EMD gene maps on chromosome X and encodes for emerin protein, which is located into the inner nuclear membrane where interacts with lamin-A protein. Emerin regulates myogenic differentiation and proliferation interacting with transcription factor such as Lmo7 and β-catenin. Moreover, emerin has a crucial role in keeping the nuclear structural integrity and nuclear envelope elasticity. However, despite its relevance, emerin depletion in mice causes only a mild phenotype which does not recapitulate the disease. EDMD is a rare disease caused by genetic mutations occurring in genes involved in the maintenance of nuclear envelope structure. EDMD is characterized by a progressive muscle wasting, joint contractures and cardiac symptoms such as arrhythmias, palpitations and congestive heart failure which eventually cause patients death. To date, only few symptomatic therapies are available for the treatment of the disease. This thesis describes a CRISPR/Cas9 strategy to correct a 5 nucleotides duplication causing the loss of the proper reading frame in EMD gene. The strategy proposed to correct the genetic defect consists of a couple of gRNAs designed to address Cas9 cleavages upstream and downstream the target mutation inducing the excision of a DNA segment containing the mutation and restoring the correct reading frame of the gene. The 3 designed gRNAs were separately cloned into an effector plasmid expressing a human codon-optimize S. pyogenes Cas9 (hSpCas9) and tested separately and in combination. A target plasmid containing the mutated exon of the gene was generated in order to test the designed strategy in a surrogate co-transfection assay in HEK293T cells. Target and effector plasmids were co-transfected in HEK293T cells and the cleavage efficiency of each gRNA was determined by using Tracking of Indels by Decomposition (TIDE) analysis webtool. Moreover, two combinations of gRNAs were co-transfected in HEK293T cells to assess the efficiency of double editing. Once selected the gRNAs combination leading to higher editing efficiency and restoring the correct gene frame, primary tendon’s fibroblasts (tenocytes) from a patient suffering EDMD1 were electroporated with ribonucleoprotein (RNP) carrying two gRNAs and the SpCas9. The editing efficiency was determined by Sanger sequencing. COSMID webtool was used to computationally determine the potential off-target sites for each gRNA of the selected combination. Finally, the restoration of emerin protein production in tenocytes and the proper localization of the protein in the nuclear membrane after the CRISPR treatment was evaluated by Western blotting and immunofluorescence. In conclusion, this thesis shows a CRISPR/Cas9 approach to correct a mutation in EMD gene causing type I Emery-Dreifuss muscular dystrophy. The gene corrected tenocytes represent isogenic cells which will be used, in the near future, to identify biomarkers for the evaluation of disease progression. Moreover, the correction of primary tenocytes with CRISPR tool offers a proof of principle for a future application of gene editing to treat EDMD patients.
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