Natures et applications des vecteurs viraux dans les thérapies géniques

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Targeted gene therapy of Xeroderma pigmentosum cells using meganuclease and TALEN™

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 (Delta TG) 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 DTG mutation in XP-C cells using engineered meganuclease and TALEN (TM). 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 (TM) insensitive to CpG methylation enable successful correction of the Delta TG 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

Homologous gene targeting (HGT) induced by the TALEN™ (XPCT1) in XP4PA cells.

<p>(<b>A</b>) Western blot performed on protein extracts from cells transfected with XPCT1 (+) or empty vector (−). Each monomer, XPCT1R and XPCT1L was tagged with S-tag and HA-tag, respectively. (<b>B</b>) HGT frequency was determined from XP4PA cells transfected with XPCT1 (+) or non-related TALEN™ (−) in the presence of the DNA correction matrix described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078678#pone-0078678-g002" target="_blank">Figure 2</a>. The transfected cells were seeded at a density of 20 cells/well or lower, enabling the formation of individual clones.</p

Phenotypic correction of XP4PA cells.

<p>(<b>A</b>) Western blot performed on protein extracts from clones derived from transfection with the meganuclease XPCm in the presence of demethylating treatment (left panel) or from transfection with the TALEN™ XPCT1 (right panel). XPC expression of corrected clones (Corr) was compared to negative controls, XP4PA (1), to uncorrected ΔTG clones (3), and to a positive control MRC5, proficient for XPC (2). In the left panel, an additional band is revealed by the XPC antibody. This band is most probably due to the non-specific binding of the antibody. Furthermore, this could be heightened by the 5-aza-dC treatment, as the band seems to appear only in treated samples. (<b>B</b>) UV-C survival assay on clones derived from gene correction experiment using XPCm (left panel) or using XPCT1 (right panel). The percentage of cell survival after exposure to UV-C of XPC corrected clones (closed triangle and lozenge)) was compared to two negative controls, XP4PA and uncorrected ΔTG clone (open triangle and lozenge, respectively) and one positive control MRC5 (closed square).</p

Efficacy of XPCm in XP4PA cells after 5-aza-dC treatment.

<p>(<b>A</b>) Chromatogram showing the impact of 5-aza-dC treatment on methylating status of the XPCt. Cells were grown with 0.2 µM (+) or without (−) 5-aza-dC and transfected with empty vector under the same conditions as in TM or HGT expriments. While the CpGs present in XPCt was fully methylated under non-treated conditions, the 5-aza-dC treatment induced partial demethylation as shown by the presence of a double peak. (<b>B</b>) TM frequency was determined from XP4PA cells grown with 0.2 µM (+) or without (−) 5-aza-dC and transfected with XPCm (+) or empty vector (−). (<b>C</b>) Design of the DNA correction matrix used for HGT experiments, which was composed of two arms of 1,579 bp and 1,830 bp, homologous to the <i>XPC</i> sequences and separated by the underlined meganuclease-recognizing site (part of the normal wild type sequence of <i>XPC</i>). The DNA sequence that was recognized by the meganuclease was modified by producing silent mutations (in red letters) to avoid any cleavage of the matrix by XPCm. (<b>D</b>) Sequencing of HGT-PCR products from one corrected population (CP). These sequences were compared to the sequences obtained in the MRC5 cell proficient for XPC (+) and in the parental cell line XP4PA carring the TG deletion (−).</p

Impact of demethylating treatment on targeted mutagenesis (TM) and homologous gene targeting (HGT) frequencies induced by engineered meganucleases in 293-H cells.

<p>(<b>A</b>) TM frequency was determined from cells grown with or without 0.2 µM or 1 µM 5-aza-dC and transfected with the engineered meganuclease (MN) XPCm, or RAG1 m and CAPNS1 m, two meganucleases targeting DNA sequences that lack methylated CpG. (<b>B</b>) Distribution of TM events in methylated (white) and unmethylated (black) sequences from cells transfected with XPCm with and without 5-aza-dC. (<b>C</b>) HGT frequency was determined from cells grown with 0.2 µM (+) or without (−) 5-aza-dC and co-transfected with the DNA repair matrix (RM) and the XPCm engineered meganuclease (+) or empty vector (−).</p