Researchers report on what they claim is an accurate and efficient method for selectively correcting disease-causing mutations in human induced pluripotent stem cells (hIPSCs) or adult stem cells generated from patients with genetic diseases. Using hIPSCs derived from a patient with Hutchinson-Gilford progeria syndrome (HGPS) as an example, the team showed that a single helper-dependent adenoviral vector (HDAdV) was capable of selectively correcting the lamin A gene (LMNA) mutation responsible for HGPS.
Targeted correction of LMNA led to the restored expression of wild-type lamin A and to the abolishment of progerin expression. This in turn corrected the disease-associated cellular phenotypes, decelerated cell senescence, and restored normal nuclear morphology. In fact, the team claims, using the HDAdV technique it was possible to correct different mutations spanning a substantially large region of the LMNA gene with very high efficiency and without causing any obvious genetic or epigenetic abnormalities.
The multidisciplinary team, at the Salk Institute for Biological Sciences, the University of California at San Diego (UCSD), the Scripps Research Institute, and the Center for Regenerative Medicine in Barcelona, report their findings in Cell Stem Cell. The paper, which will be published in June but is now available online, is titled “Targeted Gene Correction of Laminopathy-Associated LMNA Mutations in Patient-Specific iPSCs.”
Gene-editing methodologies reported to date for human cells suffer from technical problems. Classical nonviral vectors and delivery approaches are associated with low efficiency of gene targeting in human pluripotent stem cells, especially for transcriptionally silenced genomic loci.
Additionally the need to generate double-stranded DNA breaks carries the potential for unexpected and unwanted mutations and chromosomal aberrations. Moreover, the researchers note, “for genes bearing mutational ‘hotspots,’ efficient tools targeting multiple sites spanning a large DNA sequence still need to be developed.”
The HDAdV approach developed by Dr. Belmonte’s team is designed to address these issues to allow targeted gene editing in stem cells. Fortunately, the researchers had at hand a recently established LMNA-based iPSC model of disease in which to test the technology. LMNA is a known transcriptionally inactive locus in pluripotent cells. This coupled with the fact that over 300 mutations in the gene have already been reported means LMNA represented an ideal candidate for the study of hot-spot gene corrections using single vectors targeting large genomic regions, the authors explain.
LMNA mutations are associated with a range of human laminopathies, including the premature aging disease HGPS, which is caused by a specific LMNA mutation that results in production of an abnormal progerin protein. Scientists have now generated iPSCs from HGPS patient fibroblasts.
The HDAd-based gene correction vector tested on the HGPS iPSCs had previously been shown to mediate efficient and precise gene editing in human embryonic stem cells, without the requirement for artificial double-stranded DNA breaks, the team notes. The vector retains no viral genes, so shows low cytotoxicity, but retains a large cloning capacity and allows for insertion of long homologous DNA regions designed to facilitate targeted integration of the delivered gene via homologous recombination.
Dr. Belmonte’s team first confirmed that their LMNA-c-HDAdV vector could correct the specific HGPS-associated LMNA mutation in the hIPSCs. The overall system used positive and negative drug resistance selection steps to choose the cells in which the correct gene had been targeted by the vector.
In this first round of tests the researchers found that the vector targeted the correct gene in 78-100% of cells, and in 46% of the drug-resistant colonies the HGPS mutation had been successfully corrected. Genome-wide SNP genotyping demonstrated that the genetic background of the corrected HGPS-iPSCs was the same as the parental HGPS fibroblasts, while copy number variant analysis suggested there were no apparent duplications or deletions in the HGPS-iPSC cells, compared with the original fibroblasts.
Additional genome-wide DNA methylation analysis indicated the cHGPS-iPSCs showed a highly similar methylation profile to their parental iPSCs. “Thus, our gene-correction approach effectively maintained genetic and epigenetic cell integrity,” the authors claim.
To see whether correcting the LMNA mutation in HGPS-iPSCs could prevent the development of disease-associated phenotypes, they differentiated the HDAdV-corrected cells into vascular smooth muscle cells and fibroblasts, which are known to be affected by HGPS. These differentiated cells demonstrated no progerin mRNA or progerin protein, which correlated with a significant rescue of the senescence phenotype. The corrected HGPS-iPSC-derived fibroblasts also showed a more than 60% reduction in the number of abnormal nuclei compared with their non-corrected counterparts.
Dr. Belmonte’s achieved similarly encouraging results when they tested the technique on iPSCs derived from a patient with atypical Werner syndrome AWS, which is caused by a different LMNA mutation. Moreover, additional analyses of the corrected AWS iPSCs showed the vector had inserted a long enough stretch of the wild-type LMNA gene into the AWS iPSCs to replace two additional SNPs located in the LMNA gene 3.3 kb and 4.4 kb downstream of the original target insertion site.
“Thus, our method could potentially be used to correct more than 200 different mutations described to accumulate in the LMNA locus,” they claim.
As a final evaluation, the researchers tested whether the HDAd-based vector could be used to edit genes in human adult mesenchymal stem cells. Focusing on olfactory ectomesenchymal stem cells (OE-MSCs), which represent a potentially attractive population for clinical applications, they confirmed that the LMNA-c-HDAdV demonstrated a gene-editing efficiency of 54% in wild-type OE-MSCs without disrupting normal lamin A/C expression.
“Our results demonstrate that the long homology arms used in HDAdVs have the capability to edit the targeted genomic locus without off-target effects and/or introduction of additional mutations, thus presenting an advantageous alternative to the use of other gene-editing technologies,” the authors conclude.
They point out that while bacterial artificial chromosome-mediated vectors also use long homology arms, the ability of these constructs to target the genome appears restrictred to transcriptionally active loci. “Thus, the HDAdV represents a robust and versatile tool that could be applied toward the correction of multiple monogenic diseases. Finally, this approach could serve to generate appropriate genotype-matched iPSC lines in disease modeling and drug discovery studies.”