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|Abeona Therapeutics, Inc. to Present on CRISPR/CAS9 Technology Platform Progress at CRISPR Precision Gene Editing Congress|
Introduction: Fanconi anemia (FA) is a rare inherited disease manifested by bone marrow failure and increased risk of malignancy. The c.456 + 4A>T (IVS4 + 4A>T) point mutation in FA complementation group C (FA-C) gene results in a cryptic splice site and causes aberrant splicing and in-frame deletion of FANCC exon 4. Gene editing is highly desirable alternative to allogeneic hematopoietic cell transplantation (HCT) for FA. In the present study, we have generated a CRISPR/Cas9 system for FANCC locus and demonstrated its usefulness in repairing the FANCC c.456+4A>T mutation.
Methods and Results: To test the ability of our custom-designed CRISPR/Cas9 reagents to mediate FANCC gene HDR, a transformed skin fibroblast culture was derived from an FA-C patient homozygous for the c.456+4 A>T mutation. The cells were treated with a donor plasmid and either the CRISPR/Cas9 nuclease or nickase. To determine whether genome editing by CRISPR/Cas9 resulted in restoration of exon 4 expression, HDR-specific PCR was performed using an allele specific RT-PCR. Interestingly, CRISPR/Cas9 nuclease and nickase clones each identified correction of c.456+4A>T compared to untreated and WT controls. Furthermore, to evaluate functional capability of our gene editing method, H2AX staining clearly demonstrated inability of untreated FA-C cells to phosphorylate γ-H2AX, however, the clones that were corrected by the nickase or the nuclease showed clear evidence to phosphorylate γ-H2AX. These findings confirm correction of the c.456+4A>T mutation at DNA, RNA and protein level.
An important safety concern of gene editing based correction strategy is potential for off target (OT) effects. To assess this important safety issue, a surveyor assay and an integration deficient lenti virus (IDLV) reporter gene trapping assay was performed and no OT activity for the nuclease or nickase was observed. Moreover, to identify the sites of integration of the IDLV, the samples were tested using LAM PCR and nonrestrictive (nr) LAM PCR, these results documented only on target events. In total, the data suggests highly specific CRISPR/Cas9 reagents.
Conclusions: To summarize, this data show that CRISPR/Cas9 mediated direct c.456 + 4A>T mutation repair resulted in normalization of the FANCC gene. This study also demonstrates that nickase was more efficient and reliable compared to nuclease. Furthermore, the gene editing model system established here provides support for a favorable safety profile using these synthetic molecules for correction of FA and other genetic disease in human cells.
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