A new patient‐derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α‐dystroglycan

Abstract

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of a-dystroglycan-laminin interaction due to defective glycosylation of a-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human-and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of a-dystroglycan in iPSCderived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of a-dystroglycan. Using human FKRP-iPSCderived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1 H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of a-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

CRISPR-mediated correction of skeletal muscle Ca2+handling in a novel DMD patient-derived pluripotent stem cell model

“…The genome editing strategy used in this study was as described [23]. A donor targeting vector was constructed using Gibson Assembly (NEB, E2611S) to incorporate the piggyBac ( PGK-puro Δ tk ) selection cassette flanked by two 1-kb homology arms specific to the DMD locus.…”

Dystroglycanopathy: From Elucidation of Molecular and Pathological Mechanisms to Development of Treatment Methods

“…In addition, low-molecular-weight compounds, rather than gene therapy, may be a viable option in certain cases. In fact, compounds that enhance matriglycan modification have been identified [ 77 , 78 ], and these, including compounds that enhance LARGE1 gene expression, are interesting therapeutic candidates. In addition, instead of targeting matriglycan, antibody therapy that connects cell membrane and basement membrane using a bivalent antibody against laminin and DG has been proposed [ 79 ].…”

The use of induced pluripotent stem cells in domestic animals: a narrative review

“…In vitro differentiation of iPSCs has allowed for study of the developmental processes and pathologies of tissues and may allow for preclinical testing of therapeutic drugs for veterinary and human medicine. With regard to drug screening, there has been success in human and mouse iPSC research in using differentiated iPSC lines to model disease and conduct high-throughput screening of small molecules for their effects on disease progression [ 120 ]. This technique allows for testing of potential therapeutics against disease-genotype cells specific to an individual or species without the need for interspecies comparisons or excessive lab animal use.…”