Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes

Terunuma, Atsushi; Ye, J; Emmert, Steffen; Khan, Sikandar G.; Kraemer, Kenneth H.; Vogel, JC

doi:10.1038/sj.gt.3302344

Cited by 3 publications

(4 citation statements)

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“…An unrestricted autopsy was performed. Compound heterozygous mutations were detected in the XPA DNA repair gene (Table 1) with G>T intron 3 splice acceptor, and G>C splice donor of exon 4 [37,38]. The post-UV DNA repair rate (unscheduled DNA synthesis (UDS)) of the cultured skin fibroblasts was about 1% of normal [4,35,39,40].…”

Section: Resultsmentioning

confidence: 99%

The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D)

Lai

Liu

Alimchandani

et al. 2013

acta neuropathol commun

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BackgroundTo investigate the association of DNA nucleotide excision repair (NER) defects with neurological degeneration, cachexia and cancer, we performed autopsies on 4 adult xeroderma pigmentosum (XP) patients with different clinical features and defects in NER complementation groups XP-A, XP-C or XP-D.ResultsThe XP-A (XP12BE) and XP-D (XP18BE) patients exhibited progressive neurological deterioration with sensorineural hearing loss. The clinical spectrum encompassed severe cachexia in the XP-A (XP12BE) patient, numerous skin cancers in the XP-A and two XP-C (XP24BE and XP1BE) patients and only few skin cancers in the XP-D patient. Two XP-C patients developed internal neoplasms including glioblastoma in XP24BE and uterine adenocarcinoma in XP1BE. At autopsy, the brains of the 44 yr XP-A and the 45 yr XP-D patients were profoundly atrophic and characterized microscopically by diffuse neuronal loss, myelin pallor and gliosis. Unlike the XP-A patient, the XP-D patient had a thickened calvarium, and the brain showed vacuolization of the neuropil in the cerebrum, cerebellum and brainstem, and patchy Purkinje cell loss. Axonal neuropathy and chronic denervation atrophy of the skeletal muscles were observed in the XP-A patient, but not in the XP-D patient.ConclusionsThese clinical manifestations and autopsy findings indicate advanced involvement of the central and peripheral nervous system. Despite similar defects in DNA repair, different clinicopathological phenotypes are seen in the four cases, and therefore distinct patterns of neurodegeneration characterize XP-D, XP-A and XP-C patients.

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Section: Resultsmentioning

confidence: 99%

The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D)

Lai

Liu

Alimchandani

et al. 2013

acta neuropathol commun

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“…The situation becomes even more complicated when the exogenous modifying/therapeutic DNA (referred to as ''therapeutic DNA'' from this point forward) is either single-stranded DNA (ssDNA) or double-stranded DNA, is of varying lengths, and/or contains a variable number of mismatches, modifications of the bases, sugars, 5 0 or 3 0 ends, or the backbone; placed in different regions and sequence contexts within the therapeutic DNA ( Jain et al, 2008). A number of studies have begun to investigate potential pathways for specific gene targeting approaches and have shed some light on the involvement of general pathway and enzymatic features of a given approach Kucherlapati, 1987;Gruenert, 1998Gruenert, , 2003Yanez and Porter, 1999;Vasquez et al, 2001b;Igoucheva et al, 2004Igoucheva et al, , 2006aTerunuma et al, 2004;Ferrara and Kmiec, 2006;Goncz et al, 2006;Knauert et al, 2006;Radecke et al, 2006b) (Table 2). Understanding the role that these pathways play in modulating oligo/polynucleotide-based sequence modification will be critical for development and optimization of therapeutic efficacy and assessing potential adverse outcomes.…”

Section: Oligo/polynucleotide Gene Modification Strategiesmentioning

confidence: 97%

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

2011

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Oligonucleotide- and polynucleotide-based gene modification strategies were developed as an alternative to transgene-based and classical gene targeting-based gene therapy approaches for treatment of genetic disorders. Unlike the transgene-based strategies, oligo/polynucleotide gene targeting approaches maintain gene integrity and the relationship between the protein coding and gene-specific regulatory sequences. Oligo/polynucleotide-based gene modification also has several advantages over classical vector-based homologous recombination approaches. These include essentially complete homology to the target sequence and the potential to rapidly engineer patient-specific oligo/polynucleotide gene modification reagents. Several oligo/polynucleotide-based approaches have been shown to successfully mediate sequence-specific modification of genomic DNA in mammalian cells. The strategies involve the use of polynucleotide small DNA fragments, triplex-forming oligonucleotides, and single-stranded oligodeoxynucleotides to mediate homologous exchange. The primary focus of this review will be on the mechanistic aspects of the small fragment homologous replacement, triplex-forming oligonucleotide-mediated, and single-stranded oligodeoxynucleotide-mediated gene modification strategies as it relates to their therapeutic potential.

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“…For example, Alexeev and Yoon quantified oligonucleotide-based gene repair in the tyrosinase gene in melanocytes derived from albino mice by assessing the extent of black-pigmented clones among the treated cells [Alexeev and Yoon, 1998]. On the other hand, Terunuma et al have reported recently a sophisticated ultraviolet light selection assay to optimize oligonucleotide-based correction of mutations in endogenous xeroderma pigmentosum genes [Terunuma et al, 2004]. However, for most therapeutically important endogenous genes there is no possibility for direct and reliable quantification of gene repair through a functional assay.…”

Section: Reporter/selectable Systems For the Validation And Quantificmentioning

confidence: 99%

“…Terunuma et al [Terunuma et al, 2004] used the XP-UVC selection assay to assess the ability of chimeraplasts to correct point mutations on the XPA gene. The authors only detected gene repair in one experiment evidencing again the irreproducibility of chimeraplasty.…”

Section: Lack Of Reproducibility Consistency and Persistence In Genementioning

confidence: 99%

Targeted Gene Repair: The Ups and Downs of a Promising Gene Therapy Approach

Semir¹,

Aran

2006

CGT

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As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

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Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes

Cited by 3 publications

References 31 publications

The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D)

The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D)

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

Targeted Gene Repair: The Ups and Downs of a Promising Gene Therapy Approach

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