RESUMEN
Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a rare progressive neurodegenerative disorder caused by mutations in CLN3. Patients present with early-onset retinal degeneration, followed by epilepsy, progressive motor deficits, cognitive decline, and premature death. Approximately 85% of individuals with Batten disease harbor at least one allele containing a 1.02 kb genomic deletion spanning exons 7 and 8. This study demonstrates CRISPR-Cas9-based homology-dependent repair of this mutation in induced pluripotent stem cells generated from two independent patients: one homozygous and one compound heterozygous for the 1.02 kb deletion. Our strategy included delivery of a construct that carried >3 kb of DNA: wild-type CLN3 sequence and a LoxP-flanked, puromycin resistance cassette for positive selection. This strategy resulted in correction at the genomic DNA and mRNA levels in the two independent patient lines. These CRISPR-corrected isogenic cell lines will be a valuable tool for disease modeling and autologous retinal cell replacement.
RESUMEN
Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.