Periodic Reporting for period 1 - MAGIC (Next-generation models and genetic therapies for rare neuromuscular diseases)
Periodo di rendicontazione: 2023-06-01 al 2024-11-30
The MAGIC Project aims to generate models of human skeletal pathophysiology to be used in the development of new vectors for gene therapy and genome editing. MAGIC partners aim to create novel and vigorous skeletal muscle-on-chip models that echo multiple muscle pathologies, with quantitative and reproducible phenotypic readouts at the cellular scale. These cellular models will allow phenotypic assessment of cellular pathways and mechanism that are central to the pathophysiology of multiple neuromuscular disorders using high temporal and super resolution microscopy.
WP2:
T2.2 : We have analyzed human single cell RNA-seq data from Tabula sapiens to assess relative specificity of cell surface markers known to be specific for mouse satellite cells. This analysis revealed good specificity for Calcitonin receptor and Mcadherin. However, other markers, including CD34 and Integrin alpha 7 were also expressed by non-satellite cells in human muscle. Based on these analyses we recommend Calcitonin receptor and Mcadherin as targets for satellite cell-centric approaches.
T2.4 : We have recently cloned a fragment of the rat Pax7 promoter into an AAV-Cherry plasmid designed in our lab, along with two putative Pax7 enhancers from the mouse genome and one synthetic enhancer that contains multiple binding sites for motifs found in Pax7 putative enhancers. We plan to use the same approach to pair enhancers with their respective gene promoters, specifically for Myf5, Calcitonin receptor and Mcadherin. We have begun transfecting rat myoblasts with these constructs to test their activity. In the near future we plan to test the same constructs in rat bulk muscle cultures and primary human myoblasts.
WP3:
T3.1 : To conserve the functions of DMD as much as possible, while being widely applicable, strategies to skip exons 47-53, 49-53, and 53 have been selected. Several alternative variants of guide RNAs have been designed and tested as pairs, together with a strong universal promoter, in the L6 rat myoblast cell line, to evaluate the efficiency of creating these large deletions. This was done, keeping in mind the size constraints of the AAV vectors. Editing has been evaluated for all designed guide RNA combinations, using sequencing and digital PCR, and the best pair has been selected for each of the three strategies, exhibiting efficiencies comparable to published data.
We are currently designing alternative constructs, utilizing approaches that work best specifically for large deletions, to improve the efficiency and precision of the desired editing.
We plan to test the most efficient constructs for the three initial editing strategies in the DMD rat model, evaluating the editing efficiency and precision by sequencing and digital PCR, as well as the changes in DMD protein level (by WB and immunohistochemistry) and associated phenotypic improvements. We will also test the new constructs in the L6 cell line to assess if they show improved efficiency.
T2.2 : We have analyzed human single cell RNA-seq data from Tabula sapiens to assess relative specificity of cell surface markers known to be specific for mouse satellite cells. This analysis revealed good specificity for Calcitonin receptor and Mcadherin. However, other markers, including CD34 and Integrin alpha 7 were also expressed by non-satellite cells in human muscle. Based on these analyses we recommend Calcitonin receptor and Mcadherin as targets for satellite cell-centric approaches.
T2.4 : We have recently cloned a fragment of the rat Pax7 promoter into an AAV-Cherry plasmid designed in our lab, along with two putative Pax7 enhancers from the mouse genome and one synthetic enhancer that contains multiple binding sites for motifs found in Pax7 putative enhancers. We plan to use the same approach to pair enhancers with their respective gene promoters, specifically for Myf5, Calcitonin receptor and Mcadherin. We have begun transfecting rat myoblasts with these constructs to test their activity. In the near future we plan to test the same constructs in rat bulk muscle cultures and primary human myoblasts.
WP3:
T3.1 : To conserve the functions of DMD as much as possible, while being widely applicable, strategies to skip exons 47-53, 49-53, and 53 have been selected. Several alternative variants of guide RNAs have been designed and tested as pairs, together with a strong universal promoter, in the L6 rat myoblast cell line, to evaluate the efficiency of creating these large deletions. This was done, keeping in mind the size constraints of the AAV vectors. Editing has been evaluated for all designed guide RNA combinations, using sequencing and digital PCR, and the best pair has been selected for each of the three strategies, exhibiting efficiencies comparable to published data.
We are currently designing alternative constructs, utilizing approaches that work best specifically for large deletions, to improve the efficiency and precision of the desired editing.
We plan to test the most efficient constructs for the three initial editing strategies in the DMD rat model, evaluating the editing efficiency and precision by sequencing and digital PCR, as well as the changes in DMD protein level (by WB and immunohistochemistry) and associated phenotypic improvements. We will also test the new constructs in the L6 cell line to assess if they show improved efficiency.
T2.2 ==> Selection of Calcitonin receptor and Mcadherin as targets for satellite cell-centric approaches
T2.4 ==> Obtention of AAV to test enhancers
T3.1 ==> Selection of the best pair for each of the three strategies, exhibiting efficiencies comparable to published data
T2.4 ==> Obtention of AAV to test enhancers
T3.1 ==> Selection of the best pair for each of the three strategies, exhibiting efficiencies comparable to published data