Dataset Information

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.

SUBMITTER: Burnight ER

PROVIDER: S-EPMC5589061 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Burnight Erin R ER Gupta Manav M Wiley Luke A LA Anfinson Kristin R KR Tran Audrey A Triboulet Robinson R Hoffmann Jeremy M JM Klaahsen Darcey L DL Andorf Jeaneen L JL Jiao Chunhua C Sohn Elliott H EH Adur Malavika K MK Ross Jason W JW Mullins Robert F RF Daley George Q GQ Schlaeger Thorsten M TM Stone Edwin M EM Tucker Budd A BA

Molecular therapy : the journal of the American Society of Gene Therapy 20170612 9

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 ( ...[more]

PMID: 28619647

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Project description:ImportanceMany common inherited retinal diseases are not easily treated with gene therapy. Gene editing with base editors may allow the targeted repair of single-nucleotide transition variants in DNA and RNA. It is unknown how many patients have pathogenic variants that are correctable with a base editing strategy.ObjectiveTo assess the prevalence and spectrum of pathogenic single-nucleotide variants amenable to base editing in common large recessively inherited genes that are associated with inherited retinal degeneration.Design, setting, and participantsIn this retrospective cross-sectional study, nonidentifiable records of patients with biallelic pathogenic variants of genes associated with inherited retinal degeneration between July 2013 and December 2019 were analyzed using data from the Oxford University Hospitals Medical Genetics Laboratories, the Leiden Open Variation Database, and previously published studies. Six candidate genes (ABCA4, CDH23, CEP290, EYS, MYO7A, and USH2A), which were determined to be the most common recessive genes with coding sequences not deliverable in a single adeno-associated viral vector, were examined. Data were analyzed from April 16 to May 11, 2020.Main outcomes and measuresProportion of alleles with a pathogenic transition variant that is potentially correctable with a base editing strategy and proportion of patients with a base-editable allele.ResultsA total of 12 369 alleles from the Leiden Open Variation Database and 179 patients who received diagnoses through the genetic service of the Oxford University Hospitals Medical Genetics Laboratories were analyzed. Editable variants accounted for 53% of all pathogenic variants in the candidate genes contained in the Leiden Open Variation Database. The proportion of pathogenic alleles that were editable varied by gene; 63.1% of alleles in ABCA4, 62.7% of alleles in CDH23, 53.8% of alleles in MYO7A, 41.6% of alleles in CEP290, 37.3% of alleles in USH2A, and 22.2% of alleles in EYS were editable. The 5 most common editable pathogenic variants of each gene accounted for a mean (SD) of 19.1% (9.5%) of all pathogenic alleles within each gene. In the Oxford cohort, 136 of 179 patients (76.0%) had at least 1 editable allele. A total of 53 of 107 patients (49.5%) with biallelic pathogenic variants in the gene ABCA4 and 16 of 56 patients (28.6%) with biallelic pathogenic variants in the gene USH2A had 1 of the 5 most common editable alleles.Conclusions and relevanceThis study found that pathogenic variants amenable to base editing commonly occur in inherited retinal degeneration. These findings, if generalized to other cohorts, provide an approach for developing base editing therapies to treat retinal degeneration not amenable to gene therapy.

Dataset Information

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

Publications

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

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