G Model
ARTICLE IN PRESS
MUT-11382; No. of Pages 7
Mutation Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Review
DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease Aurélie Dupuy a , Alain Sarasin a,b,∗ a b
Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif, France Service de Génétique, Institut Gustave Roussy, France
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 11 August 2014 Accepted 22 August 2014 Available online xxx Keywords: Xeroderma pigmentosum Gene therapy Engineered nucleases TALEN UV light Nucleotide excision repair
a b s t r a c t Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient’s cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
DNA damage and repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xeroderma pigmentosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic correction of xeroderma pigmentosum cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. XPC gene correction using meganucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. XPC gene correction using TALE-nucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
00 00 00 00 00 00 00 00 00
1. DNA damage and repair
∗ Corresponding author at: UMR8200 CNRS, Laboratory of Genetic Instability and Oncogenesis, PR2, Institut Gustave Roussy, 114, rue Edouard Vaillant, 94805 Villejuif, France. Tel.: +33 1 42 11 63 28; fax: +33 1 42 11 50 08. E-mail addresses: [email protected], [email protected] (A. Sarasin).
Mammalian genome is constantly damaged by a wide variety of environmental agents (ultraviolet, ionizing radiations, genotoxic chemicals. . .), its intrinsic instability or products of normal cellular metabolism (reactive oxygen species). UV light induces two major types of photolesions: cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PP). They are the predominant forms of premutagenic damage following sun
http://dx.doi.org/10.1016/j.mrfmmm.2014.08.007 0027-5107/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: A. Dupuy, A. Sarasin, DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrfmmm.2014.08.007
G Model MUT-11382; No. of Pages 7
ARTICLE IN PRESS A. Dupuy, A. Sarasin / Mutation Research xxx (2014) xxx–xxx
2
exposure [1]. In the absence of repair, these DNA lesions remain in the genome, lead to mutagenesis, which is implicated in skin cancer development. Several mechanisms exist to repair the various DNA lesions and prevent carcinogenesis. For example, UV-induced damage are removed by the nucleotide excision repair pathway (NER) [2–4] and double-strand breaks (DSB) are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). The consequences of deficiencies in DNA repair mechanisms for human health are exemplified by the existence of rare, autosomal human disorders such as xeroderma pigmentosum (XP) or high cancer predisposition [5] (Table 1). NER is a versatile DNA repair system that removes a broad spectrum of structurally-unrelated lesions, including UV-induced photoproducts as well as other bulky DNA adducts [6,7] that can be induced by numerous chemical compounds. Although these lesions do not share common chemical structures, they induce a sufficient distortion of the DNA helix to be recognized by the first protein complex of NER. NER operates through two subpathways, depending on whether the damage is located anywhere in the genome (global genome repair, GG-NER) or in an activelytranscribed gene (transcription-coupled repair, TC-NER) [7–9]. The NER mechanism involves several successive steps: (i) DNA damage recognition by the XPC-HHR23B-Centrin2 complex in GG-NER or following stalling of RNA polymerase II at the DNA lesion in TC-NER. (ii) Formation and stabilization of the multiprotein TFIIH complex comprising XPB and XPD helicases. (iii) Incision on both sides of the DNA lesion by XPG and XPF-ERCC1 3 - and 5 -endonucleases, respectively. (iv) Removing of the damage-containing oligonucleotide and (v) filling in the gap by DNA synthesis with DNA polymerase ␦/ and ligation [10] (Fig. 1). The TC-NER pathway allows a fast repair of lesions in transcribed regions in order to rapidly recover RNA transcription and protein translation after genotoxic attack and to protect cells against acute deleterious effects such as apoptosis. GG-NER pathway contributes to reduce mutation load in the overall genome and to maintain chromosomal stability. Mutations in genes encoding NER proteins lead to several dramatic human disorders (Table 1), among them we will focus on xeroderma pigmentosum.
2. Xeroderma pigmentosum XP is a rare, autosomal, recessive, genetic disease characterized by hypersensitivity to UV exposure, associated with numerous skin abnormalities (like skin aging, hypopigmentation/hyperpigmentation, skin freckles), ocular abnormalities (irritation, eyelid atrophy, cataracts) and a very high predisposition for developing skin cancers. The median age for skin cancer appearance is about 8 years old that is 50–60 years earlier than in the general population. More than 90% of carcinomas are located on the face, neck and head in other words on sun-exposed sites. XP patients develop carcinoma (squamous- and basal cell carcinoma) and malignant melanoma in childhood (
ARTICLE IN PRESS
MUT-11382; No. of Pages 7
Mutation Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Review
DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease Aurélie Dupuy a , Alain Sarasin a,b,∗ a b
Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif, France Service de Génétique, Institut Gustave Roussy, France
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 11 August 2014 Accepted 22 August 2014 Available online xxx Keywords: Xeroderma pigmentosum Gene therapy Engineered nucleases TALEN UV light Nucleotide excision repair
a b s t r a c t Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient’s cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
DNA damage and repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xeroderma pigmentosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic correction of xeroderma pigmentosum cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. XPC gene correction using meganucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. XPC gene correction using TALE-nucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
00 00 00 00 00 00 00 00 00
1. DNA damage and repair
∗ Corresponding author at: UMR8200 CNRS, Laboratory of Genetic Instability and Oncogenesis, PR2, Institut Gustave Roussy, 114, rue Edouard Vaillant, 94805 Villejuif, France. Tel.: +33 1 42 11 63 28; fax: +33 1 42 11 50 08. E-mail addresses: [email protected], [email protected] (A. Sarasin).
Mammalian genome is constantly damaged by a wide variety of environmental agents (ultraviolet, ionizing radiations, genotoxic chemicals. . .), its intrinsic instability or products of normal cellular metabolism (reactive oxygen species). UV light induces two major types of photolesions: cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PP). They are the predominant forms of premutagenic damage following sun
http://dx.doi.org/10.1016/j.mrfmmm.2014.08.007 0027-5107/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: A. Dupuy, A. Sarasin, DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrfmmm.2014.08.007
G Model MUT-11382; No. of Pages 7
ARTICLE IN PRESS A. Dupuy, A. Sarasin / Mutation Research xxx (2014) xxx–xxx
2
exposure [1]. In the absence of repair, these DNA lesions remain in the genome, lead to mutagenesis, which is implicated in skin cancer development. Several mechanisms exist to repair the various DNA lesions and prevent carcinogenesis. For example, UV-induced damage are removed by the nucleotide excision repair pathway (NER) [2–4] and double-strand breaks (DSB) are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). The consequences of deficiencies in DNA repair mechanisms for human health are exemplified by the existence of rare, autosomal human disorders such as xeroderma pigmentosum (XP) or high cancer predisposition [5] (Table 1). NER is a versatile DNA repair system that removes a broad spectrum of structurally-unrelated lesions, including UV-induced photoproducts as well as other bulky DNA adducts [6,7] that can be induced by numerous chemical compounds. Although these lesions do not share common chemical structures, they induce a sufficient distortion of the DNA helix to be recognized by the first protein complex of NER. NER operates through two subpathways, depending on whether the damage is located anywhere in the genome (global genome repair, GG-NER) or in an activelytranscribed gene (transcription-coupled repair, TC-NER) [7–9]. The NER mechanism involves several successive steps: (i) DNA damage recognition by the XPC-HHR23B-Centrin2 complex in GG-NER or following stalling of RNA polymerase II at the DNA lesion in TC-NER. (ii) Formation and stabilization of the multiprotein TFIIH complex comprising XPB and XPD helicases. (iii) Incision on both sides of the DNA lesion by XPG and XPF-ERCC1 3 - and 5 -endonucleases, respectively. (iv) Removing of the damage-containing oligonucleotide and (v) filling in the gap by DNA synthesis with DNA polymerase ␦/ and ligation [10] (Fig. 1). The TC-NER pathway allows a fast repair of lesions in transcribed regions in order to rapidly recover RNA transcription and protein translation after genotoxic attack and to protect cells against acute deleterious effects such as apoptosis. GG-NER pathway contributes to reduce mutation load in the overall genome and to maintain chromosomal stability. Mutations in genes encoding NER proteins lead to several dramatic human disorders (Table 1), among them we will focus on xeroderma pigmentosum.
2. Xeroderma pigmentosum XP is a rare, autosomal, recessive, genetic disease characterized by hypersensitivity to UV exposure, associated with numerous skin abnormalities (like skin aging, hypopigmentation/hyperpigmentation, skin freckles), ocular abnormalities (irritation, eyelid atrophy, cataracts) and a very high predisposition for developing skin cancers. The median age for skin cancer appearance is about 8 years old that is 50–60 years earlier than in the general population. More than 90% of carcinomas are located on the face, neck and head in other words on sun-exposed sites. XP patients develop carcinoma (squamous- and basal cell carcinoma) and malignant melanoma in childhood (
