CRISPR-Mediated Correction of ABCA4 Mutations in Stem Cells May Provide Treatment for Incurable Inherited Disease Causing Vision Loss

Stargardt disease is an inherited macular neurodegenerative disease that causes retinal dystrophy and vision loss. Stargardt disease is caused by mutations in the ABCA4 gene, which encodes a protein involved in the visual cycle and transportation of toxic photoproducts out of the retina.

Currently, there are no treatments for Stargardt disease, and although restoring ABCA4 expression could help prevent or treat Stargardt disease retinal dystrophy, gene replacement therapy has not yet been successful in preventing retinal dystrophy or improving vision.

In a recent study, a team of researchers led by Esther Pomares, PhD, at the Department of Genetics, IMO Foundation (Barcelona, Spain), used the CRISPR-Cas9 system to edit pathogenic ABCA4 mutations in human induced pluripotent stem cells (iPSCs) from two patients with Stargardt disease. This approach led to successful correction of ABCA4 mutations without causing detectable off-target effects.

»For the first time, we have successfully corrected two ABCA4 pathogenic variants in iPSCs derived from patients affected with Stargardt disease. In addition, we have achieved a very high percentage of gene editing, which is important for a potential future therapeutic application,« said Laura Siles Mena, PhD, the first author of the study.

Siles also noted that the use of single-stranded oligodeoxynucleotides (ssODNs) as donor templates for CRISPR-mediated ABCA4 editing ensured permanent restoration of the ABCA4 sequence. She explained that this approach results in higher knock-in efficiency and reduced off-target effects, as well as avoids the use of external sources of DNA. Their findings were published recently in Molecular Therapy Nucleic Acids.

Sanger sequencing also showed that there were no insertions, deletions, or single-nucleotide alterations at off-target sites in any of the 35 genetically-engineered clones screened.

»Our results strongly suggest that specifically designed sgRNAs can effectively discriminate between the two DNA alleles in case of heterozygosity. These analyses also suggest that sgRNAs with more than one mismatch in sequence homology decrease DNA recognition and Cas9-induced DSB,« Siles noted.

Commenting on how many patients are likely to benefit from such an approach considering the number of pathogenic mutations in ABCA4, Siles said: »Stargardt disease accounts for 12% of IRD-related vision loss cases, and more than the 60% of ABCA4 pathogenic variants correspond to missense and nonsense variants while splicing substitutions and small deletions, insertions, duplications, and indels account for approximately 35% of pathogenic mutations. All these pathogenic variants are suitable for ssODN-mediated correction by CRISPR-Cas9 approach performed in our study, providing a potential cure for almost 95% of patients with Stargardt disease.«

Patient-derived hiPSCs remain pluripotent after CRISPR-Cas9-mediated gene editing

Comparison of hiPSC clones and parental hiPSCs demonstrated that engineered hiPSCs retained their stem cell characteristics, such as colony-like morphology and proliferation (Figure 2). In addition, edited and parental hiPSCs exhibited similar expression levels of the pluripotency markers SOX2, SSEA4, TRA-160, and NANOG. This finding was confirmed at both the mRNA (quantitative PCR) and protein (immunoblotting, immunofluorescence) levels.

The team also analysed the differentiation potential of hiPSCs and found that genetically engineered hiPSCs retained their ability to differentiate into ectodermal, endodermal, and mesodermal lineages despite having been subjected to gene editing. Siles explained that the pluripotency of genetically-engineered hiPSCs is critical to the feasibility of differentiating hiPSCs with restored ABCA4 function into the right cell type for effective reversal of retinal degeneration.

Challenges and future steps

The results of this study demonstrate, for the first time, that IRD-related pathogenic variants can be efficiently and precisely corrected using CRISPR-Cas9; however, this approach is still in the very early stages.

»Further increase in the specificity and precision of gene editing is crucial. Another potentially challenging aspect of developing this approach further is finding a way to avoid re-cutting of the DNA by Cas9 after ABCA4 repair, as multiple rounds of DNA cutting could trigger undesired genomic abnormalities,« she said.

The team also plans to validate the feasibility of this method using in vitro 3D cultures of retinas. »We want to characterise CRISPR-Cas9 effectiveness in organoid models that mimick a potential future in vivo CRISPR delivery strategy for human treatment,« Siles concluded.

Link to the original article in Molecular Therapy Nucleic Acids:

Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients

Christos Evangelou, PhD, is a freelance medical writer and science communications consultant.