Targeted Gene Therapy of Xeroderma Pigmentosum Cells Using Meganuclease and TALEN™

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.

CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

“…The XPC protein is an initiator of global nucleotide excision repair 21 . Thus, XPC-deficient cells accumulate DNA damage when they are exposed to chemical or physical stimuli including ultraviolet irradiation 22 . GM14867 cells have a homozygous C>T nonsense mutation at nucleotide 1840 in the XPC gene, which creates a 5'-TGA-3' stop codon that replaces a codon for arginine (Arg579) (1840C>T, Arg-579-UGA stop codon) ( Fig.…”

The Development of TALE Nucleases for Biotechnology

“…For example, we developed TALENs to generate small insertions and deletions to restore the reading frame of the dystrophin gene as a novel method to correct the molecular basis of Duchenne muscular dystrophy (50). TALENs have also been used to correct mutations associated with epidermolysis bullosa (108), sickle cell disease (109,110), beta-thalassemia (111), xeroderma pigmentosum (112), and alpha-1 antitrypsin deficiency (113) by homologous recombination and to correct mitochondrial DNA disorders (114) by deletion of aberrant sequences.…”

Advances in New Technology for Targeted Modification of Plant Genomes

“…Taken together, a large number of engineered meganucleases have been successfully developed for targeted genome modifi cation in numerous organisms, including human, mouse, maize, cotton, tobacco, and rice (Arnould et al 2007 ;Grizot et al 2009 ;Daboussi et al 2012 ;Valton et al 2012 ;D'Halluin et al 2008 ;D'Halluin et al 2013 ;Gao et al 2010 ;Menoret et al 2013 ;Rousseau et al 2011 ;Munoz et al 2011 ;Popplewell et al 2013 ;Dupuy et al 2013 ;Grosse et al 2011 ). This list is expected to expand as nuclease effi cacy and engineering capacity are constantly improved (Jarjour et al 2009 ;Thyme et al 2009 ).…”