Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration

Abstract

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.

Transplantation of iPSC-TM stimulates division of trabecular meshwork cells in human eyes

“…Human ipSc-tM exhibit morphology resembling ptM. Dermal fibroblasts or keratinocytes were obtained from three donors and induced pluripotent stem cells (iPSC) were generated using methods described earlier 13,14 . These iPSC were then differentiated into a cell type resembling TM cells (designated iPSC-TM) through co-culture with human primary trabecular meshwork cells (pTM).…”

“…iPSC were derived from fibroblasts (lines A and C) or keratinocytes (line B) derived from 3 individuals without a history of glaucoma and reprogrammed using the CytoTune ® -iPS Sendai Reprogramming Kit (Invitrogen, MA, USA), as described earlier 13,14 . iPSCs were seeded in Corning Synthemax coated plates and cultured in TeSR-E8 media until they reached 5-10% confluency.…”

53BP1 nuclear body-marked replication stress in a human mammary cell model of BRCA2 deficiency

“…While alleles can be effectively distinguished via CRISPR approaches in cases where a unique PAM is present in one allele [19,20], discrimination between alleles with single nucleotide mismatches within the sgRNA site is conceptually challenging given the promiscuity of sgRNA recognition [16,17,18]. In this regard, previous studies have shown promise in utilizing SNPs in the sgRNA target regions to achieve allele-specific mutagenesis [21,22,23,24,25]. Our results now extend allele-specific applications to HR-mediated gene targeting.…”

CRISPR Interference–Potential Application in Retinal Disease

“…In cases of autologous transplantation, CRISPRi could be used to suppress the pathogenic gene in induced pluripotent stem cells (iPSCs) pre-implantation before reprogramming into the desired retinal cell type, and autologous transplantation. The potential of this treatment has been demonstrated with successful CRISPR/Cas9 gene correction of pathogenic retinal mutations in iPSCs for retinitis pigmentosa and Leber congenital amaurosis [59][60][61]. Following correction of three different RPGR frame shift mutations in patient iPSCs, Deng et al cultured the iPSCs into retinal organoids and found that the X-linked retinitis pigmentosa phenotype was completely alleviated [60].…”