Abstract
Xeroderma pigmentosum group C (XP-C) is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide excision repair (NER) pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG) located in the middle of exon 9. This deletion leads to the expression of an inactive truncated XPC protein, normally involved in the first step of NER. New approaches used for gene correction are based on the ability of engineered nucleases such as Meganucleases, Zinc-Finger nucleases or TALE nucleases to accurately generate a double strand break at a specific locus and promote correction by homologous recombination through the insertion of an exogenous DNA repair matrix. Here, we describe the targeted correction of the ΔTG mutation in XP-C cells using engineered meganuclease and TALEN™. The methylated status of the XPC locus, known to inhibit both of these nuclease activities, led us to adapt our experimental design to optimize their in vivo efficacies. We show that demethylating treatment as well as the use of TALEN™ insensitive to CpG methylation enable successful correction of the ΔTG mutation. Such genetic correction leads to re-expression of the full-length XPC protein and to the recovery of NER capacity, attested by UV-C resistance of the corrected cells. Overall, we demonstrate that nuclease-based targeted approaches offer reliable and efficient strategies for gene correction.
Highlights
Xeroderma pigmentosum (XP) is a rare, autosomal, recessive syndrome characterized by hypersensitivity to UV light [1]
XP cells are deficient in the nucleotide excision repair (NER) pathway, a complex process involved in the recognition and removal of DNA lesions induced by UV light [3]
Clones negative for homologous gene targeting (HGT) events and TG correction displayed high sensitivity to UV-C. These results provided the first demonstration of a stable correction of XPC mutation using sequence-specific engineered nucleases. We showed that both meganuclease- and TALENTM-assisted targeted approaches allowed efficient correction of the XPC founder mutation in an XP4PA cell line derived from the fibroblasts of Xeroderma pigmentosum group C (XP-C) patients
Summary
Xeroderma pigmentosum (XP) is a rare, autosomal, recessive syndrome characterized by hypersensitivity to UV light [1] It is associated with a dramatic predisposition to skin neoplasms. Among the known genetic alterations, a founder mutation within exon 9 has been described in almost 90% of Maghrebian XP-C patients [4] and corresponds to the deletion of a TG dinucleotide leading to the expression of an inactive and undetectable XPC truncated protein. This lack of NER activity allows UV-dependent DNA damage to accumulate and is responsible for the development of high numbers of skin cancers
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have