NEW YORK (GenomeWeb) – By using the CRISPR/Cas9 genome editing approach, researchers from the University of California, San Francisco report that they were able to correct β-thalassemia gene mutations in patient-specific iPS cells.
As UCSF's Yuet Wai Kan and his colleagues reported in Genome Research today, they combined the CRISPR/Cas9 technology with the piggyBac transposon to target and correct mutations in the hemoglobin beta gene with, they noted, seemingly no off-target effects.
"Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step towards the future application of stem cell-based gene therapy of monogenic disease," the researchers said in their paper.
β-thalassemia is a common genetic disorder in people from areas stretching from the Mediterranean through the Middle East to Southeast Asia. Some 200 genetic mutations have been linked to disease, with the variants in the HBB gene commonly affecting the promoter or RNA splicing and, subsequently, hemoglobin function. People with homozygous mutations have severe anemia that can be treated with transfusions and iron chelation and possibly cured through hematopoietic stem cell transplantation, if a suitable donor is available.
Similarly, correcting the mutation at the root of the disease, the researchers noted, could potentially cure it.
While other genome editing approaches could be implemented, they argued that the CRISPR/Cas9 approach is less expensive than a zinc finger nuclease-based method and likely is more efficient than a TALEN-based one.
For this study, Kan and his colleagues used iPS cells derived from a β-thalassemia patient who was a double heterozygote for a promoter mutation and a deletion in exon two.
They designed three guide RNA sequences to target the HBB gene, and then focused their efforts on the one gRNA that had high transfection and double-strand break efficiency and targeted an intron 1 TTAA sequence that is used by the piggyBac transposon for integration.
Meanwhile, they constructed a donor plasmid with a corrected HBB gene that was inserted into the piggyBac intron. The intron also contained a bi-functional hybrid puroΔTK gene that could be used for positive and negative selections.
They then transfected the donor plasmid, gRNA, and Cas9 vectors into the iPS cells.
By screening for clones resistant to puromycin, the researchers plucked out the ones that underwent homologous recombination and took up the vectors, which they confirmed using PCR amplification and Southern blot analysis.
Further, they found that of the 12 clones that underwent homologous recombination, four corrected the promoter mutation and five corrected the deletion.
The piggyBac transposon was removed from the iPS cells by a combination of transposase and negative selection of the puroΔTK gene.
DNA sequencing of the cells, the researchers reported, indicated the "seamless removal" of the transposon and the "restoration of the original intron without any exogenous sequences."
Kan and his colleagues further said that they detected no off-site nicking or targeting in the six genomic regions that are most similar to the gRNA sequences they used. However, they noted that other genomic changes could not be ruled out.
The corrected iPS cells, the researchers reported, retained their pluripotency. To determine whether these cells could be used to correct HBB expression, they differentiated two of the corrected iPS cell lines into hematopoietic progenitors and erythroblasts in a monolayer culture.
The cells, the researchers found, differentiated into hematopoietic progenitors efficiently. After nearly a month, they noted through mRNA expression analysis that HBB expression in the corrected cells was about 16-fold higher than in the parental cells.
However, the iPS cells preferentially differentiated into fetal globin rather than into adult globin — an effect that bears further research, the researchers said.
More work, the researchers noted, will be also needed before such cells could be transplanted into patients as a therapy.
"Although we and others are able to differentiate iPSCs into blood cell progenitors as well as mature blood cells, the transplantation of the progenitors into mouse models to test them has so far proven very difficult," Kan said in a statement. "I believe it will take quite a few more years before we can apply it in a clinical setting."