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
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
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
Obtaining Induced Pluripotent Stem Cells
Since embryonic stem cells
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
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
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
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
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).