Browsing by Subject "Genome editing"

Now showing 1 - 3 of 3
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    Gene Correction of Limb Girdle Muscular Dystrophy Type 2A Patient-Specific Induced Pluripotent Stem Cells
    (2019-09) Selvaraj, Sridhar
    Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes invariably display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease typically does not manifest in the embryonic muscle, but at more mature stages. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by increased expression of fetal, neonatal and adult myosin heavy-chain isoforms. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle bundles, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells. We applied this maturation model for in vitro validation of Calpain 3 (CAPN3) protein expression. CAPN3 mutations are associated with Limb Girdle Muscular Dystrophy type 2A (LGMD2A), which is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. Using a gene knock-in approach, here we applied CRISPR-Cas9 mediated genome editing to induced pluripotent stem (iPS) cells from three LGMD2A patients carrying three different CAPN3 mutations, to enable correction of mutations in the CAPN3 gene. CAPN3 protein rescue upon gene correction was validated in myotube-derivatives in vitro following the small molecule treatment. Transplantation of gene corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof concept for the integration of genome editing and iPS cell technologies to develop a novel autologous cell therapy for LGMD2A.
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    Glycopolymers for Targeted Gene Delivery and Genome Editing
    (2017-08) Dhande, Yogesh
    Targeted delivery of therapeutics is of great interest to reduce toxicity and immunogenicity of the treatment. In particular, the liver is an ideal target for nucleic acid therapeutics due to its large size, regenerative capacity, and the role in producing serum proteins. In this work, N-acetyl-D-galactosamine (GalNAc) ligands clustered into a polymeric architecture were studied for enhanced binding to the asialoglycoprotein receptors (ASGPRs) on hepatocytes. A series of cationic glycopolymers based on this architecture was used to encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes) and deliver them to receptor-specific cells. The GalNAc-derived polyplexes were colloidally stable and showed cell type-specific gene expression in cultured cells. This work demonstrated the versatility of glycopolymers in selective delivery of therapeutics to cells of interest. We sought to further understand the role of such gene-delivery vehicles in genome editing applications using the CRISPR/Cas9 system. Our results show that the gene delivery vehicle can play a role in promoting homology-directed repair over nonhomologous end joining based on its gene delivery properties. The frequency of editing correlates with the fraction of cells expressing Cas9 above a certain threshold and higher expression does not contribute to any gains in editing efficiency. Taken together, these observations suggest that future gene-delivery vehicles aimed for genome editing applications should be designed to deliver only a sufficient amount of DNA but to a large fraction of cells.
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    Production of induced regulatory T-cells through CRISPR/Cas9-based gene editing
    (2018-12) Tschann, Madison
    Regulatory T-cells (Tregs) are a subset of T-cells essential for maintaining immune tolerance and their dysregulation has been found to have a central role in the progression of various autoimmune diseases. The transplantations of Treg as a form of immune therapy has and continues to be an attractive method for the treatment of such disease based on their immuno-modulatory properties. Despite its potential, Treg adoptive cell transfer therapy is hampered by limited isolation efficiency due to low frequencies in human peripheral blood and poor in vitro expansion of a pure population. Herein, a novel CRISPR/Cas9 based technique is described utilizing AAV incorporation of strong transcriptional elements into the promoter region of the Treg master transcription factor, FOXP3, to upregulate expression in isolated primary T-cells and drive them toward a Treg phenotype.