CRISPR Technique Offers Precise Blindness Therapy by Repairing Stem Cells

Health In Focus   - G J E 4
A regenerative gene-corrected iPSC transplantation could be the next targeted therapy for treating inherited retinal degenerations like retinitis pigmentosa (RP). Retinitis Pigmentosa GTPase Regulator gene (RPGR) mutations cause an aggressive, X-linked variant of RP (XLRP). It leads to loss of night vision or blindness.
CRISPR Technique Offers Precise Blindness Therapy by Repairing Stem Cells
CRISPR Technique Offers Precise Blindness Therapy by Repairing Stem Cells

The RPGR gene associated with X-linked retinitis pigmentosa is located on the X chromosome, which is one of the two sex chromosomes. In men, one altered copy of the gene is sufficient to cause retinitis pigmentosa while in women, mutations usually have to occur in both copies of the gene to cause the disorder.

Retinitis pigmentosa is a one of the common inherited diseases of the retina and affects 1 in 3,500 to 1 in 4,000 people in the United States and Europe.

The study published in the Journal Scientific Reports examined whether altering the stem cells of patients with XLRP using CRISPR/Cas9 technique and transplanting it back to the patient will help treat this disorder. The study was led by Dr. Alexander Bassuk from the University of Iowa along with the Columbia University Medical Center ophthalmologists.

Obtaining Induced Pluripotent Stem Cells (iPSC's)

Since embryonic stem cells possess the risk of immune-mediated rejection, researchers derived the induced pluripotent stem cells (iPSCs) directly from patients with XLRP. They cultured fibroblasts taken using skin biopsy of the patients and were transduced to produce induced pluripotent stem cells. These iPSCs still carried the gene mutation causing XLRP.

Selection of Genes for Editing

Researchers screened 21 gRNAs with high specificity to the Open Reading Frame -15 (ORF15) region containing the patient's RPGR mutation. The ORF15 region accounts for 60% of all XLRP mutations. They selected g58 and g59 as they had high activity for the mutation site.The DNA of the test cells was amplified by Polymerase Chain Reaction (PCR) using primers for the RPGR region and were analyzed by the SURVEYOR assay.

Use of CRISPR/ Cas9 Technique

Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 system is considered to be the breakthrough of 2015. This gene editing technique helps in correcting gene mutations easily, precisely, and relatively cheaply with the help of a guide RNA (gRNA). This technology has not yet been used in humans and there are concerns about the risks of unanticipated genetic changes.

Jennifer Doudna and Emmanuelle Charpentier were the first ones to discover that with the help of a guiding RNA, the Cas9 enzyme could be directed to a particular part of the DNA and cut it with precision, thus modifying the genome.

The researchers used this technique to correct the pathogenic mutations exhibited by the RPGR gene and produce corrected iPSCs for further use in autologous transplantation.

The selected gene58 and g59 were inserted into the gRNA/Cas9 expression vector pBT-U6-Cas9-2A-GFP and were transfected into the patient-derived iPSC alongside a single-stranded donor oligonucleotide (ssODN) template.

After ten days, the transfected cells were amplified using PCR. Since the RPGR region was highly repetitive, they found some non-specific sequences, which were categorized under "non-specific/error" and the nontransfected cells were used as controls.

After the gene validation, they found that the g58 was highly active than g59. Therefore, g58 was selected for gene editing. Researchers then transfected patient iPSCs with the g58 gRNA/Cas9 expression plasmid and an anti-sense ssODN.

After deep sequencing of transfected cells, they found that 13% of reads contained precise correction of the mutation, wherein the patient's original "TAG" stop codon point mutation was replaced with the correct "GAG" codon.


The study was the first report of successful gene correction in human iPSCs associated with inherited retinal degeneration. Researchers have generated patient-specific iPSCs from an XLRP patient and found that the transfection of CRISPR/Cas9 along with a donor homology template corrects a point mutation within the RPGR gene ORF15 region.

After the successful correction of the pathogenic mutation of RPGR gene in the patient-iPSCs using CRISPR/Cas9 technique, researchers are now working on converting this corrected iPSCs into photoreceptors capable of integrating within the retina.

"With CRISPR gene editing of human stem cells, we can theoretically transplant healthy new cells that come from the patient after having fixed their specific gene mutation. And retinal diseases are a perfect model for stem cell therapy, because we have the advanced surgical techniques to implant cells exactly where they are needed," said Mahajan, assistant professor of ophthalmology at the University of Iowa.

Researchers conclude that their findings provide a step towards combining CRISPR/Cas9 technique with autologous iPSCs to develop a personalized transplantation treatment for various retinal diseases.


1. Bassuk, A. G. et al. Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells. Sci. Rep. 6, 19969; doi: 10.1038/srep19969 (2016).

Source: Medindia

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