Poster 2: Cell and Tissue Studies
Idlv-Based Crispr-Cas9 System Mediates Highly Efficient Gene Editing In The In Vitro Model Of Morquio Syndrome A
FNU Nidhi 1,2, Shaukat A Khan 1, Shunji Tomatsu 1,3,4,5*
1 Department of Biomedical Research, Nemours/Alfred I. DuPont Hospital for Children; 2 Department of Biological Sciences, University of Delaware; 3 Department of Pediatrics, Shimane University; 4 Department of Pediatrics, Graduate School of Medicine, Gifu University; 5 Department of Pediatrics, Thomas Jefferson University
Background: Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive, lysosomal storage disorder (LSD) caused by the deficiency of the N-acetylgalactosamine-6-
sulfatase (GALNS). Deficiency of GALNS causes glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), to which accumulate in cartilage and its extracellular matrix as well as cornea and heart valves, leading to short stature and neck, pectus carinatum, laxity of joints, kyphoscoliosis, knock-knee, tracheal obstruction, corneal clouding, and heart valvular disease. No effective treatment for this skeletal disease is available; therefore, an unmet challenge to develop an effective therapy. The most revolutionary genome editing platform, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated System (Cas), provided consistent gene expression and successive positive findings in various LSDs. Even though lentiviral vectors is are the first choice to accommodate a large CRISPR-Cas9 system, problems still exist with undesirable off-target effects. The Integrase-deficient lentiviral vector (IDLV) presents an attractive alternate means to deliver CRISPR-Cas9 system due to its superior packaging and low integration capacity, transient expression, and capability to transduce different cells and tissues. Here, we aimed to evaluate IDLV-based CRISPR/Cas9 systems. Materials; In vitro expression levels of GALNS and GAG levels in transduced mouse MPS IVA fibroblasts were assessed for IDLV-based CRISPR/Cas9 systems using two synthetic guide RNAs (sgRNA) and human GALNS cDNA. The target site was before ATG of exon1 in mouse galns gene.
Results and Discussion: . The results demonstrated successful homologous recombination and stable, long-term GALNS expression up to a 6.4-fold change as compared to WT. In addition, we observed marked amelioration in MPS IVA fibroblasts, evident in the normalization of lysosomal mass and total GAGs. These findings suggested IDLV-based CRISPR-Cas9 system as a highly efficient and specific platform for a novel therapy for MPS IVA.
Research Area: Bone