Mouse Retina Has Oligonucleotide-Induced Gene Repair Activity

Ciavatta, Vincent T.; Padove, S.A.; Boatright, Jeffrey H; Nickerson, John M.

doi:10.1167/iovs.04-1220

Cited by 9 publications

(5 citation statements)

References 34 publications

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“…While they found the chimera to be reliable in directing conversion, an evaluation of the stability of the molecule revealed that the “active molecule” was structurally intact, whereas a substantial amount of degradation produced an inactive, nonfunctional molecule. These results prompted (quite correctly) the notion that “… any researcher attempting [gene repair] should show the quality of their oligonucleotide….”10…”

Section: Background and Historical Developmentmentioning

confidence: 80%

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Regulation of targeted gene repair by intrinsic cellular processes

2009

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Targeted gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous genes. The process of how to accurately repair a genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the gene repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of repair.

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Section: Background and Historical Developmentmentioning

confidence: 80%

“…Unfortunately, this structure lacked robustness and reproducibility in its targeting activity 9. Recently, Ciavatta et al 10. carried out experiments that resolved some of the original problems associated with chimera‐directed gene repair.…”

Section: Background and Historical Developmentmentioning

confidence: 99%

Regulation of targeted gene repair by intrinsic cellular processes

2009

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“…This methodology can be used for dominant or recessive alleles. Evidence from earlier approaches in mouse and cell models of retinal degeneration 30–32 suggest that strategies invoking HDR/HR may eventually be clinically relevant if repair rates can be significantly enhanced.…”

Section: A New Gene Editing Toolmentioning

confidence: 99%

“…42–46 In vivo in retina, our target cells are quiescent, therefore HDR/HR enzymes are expressed at a much lower level than in dividing cells where HDR/HR enzymes are normally upregulated during mitosis. 27,32,47 Further research needs to be done to suppress NHEJ 46,48–51 or increase expression of endogenous HDR/HR if we hope to use gene editing to restore functional alleles in vivo for treatment. 49 It is notable that one approach to circumventing the challenges inherent in treating quiescent cells is the use of CRISPR/Cas9 to edit retinitis pigmentosa genes in induced pluripotent stem cells (iPSCs) generated from patient fibroblasts, with the eventual goal being to transplant the “repaired” cells back into patients’ retinas.…”

Section: Technical Challenges To Gene Editingmentioning

confidence: 99%

Clustered Regularly Interspaced Short Palindromic Repeats

Chrenek

Nickerson

Boatright³

2016

Asia-Pacific Journal of Ophthalmology

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Ophthalmic researchers and clinicians arguably have led the way for safe, effective gene therapy, most notably with adeno-associated viral gene supplementation in the treatment of Leber congenital amaurosis type 2 (LCA 2) patients with mutations in the RPE65 gene. These successes notwithstanding, most other genetic retinal disease will be refractory to supplementation. The ideal gene therapy approach would correct gene mutations to restore normal function in the affected cells. Gene editing in which a mutant allele is inactivated or converted to sequence that restores normal function is hypothetically one such approach. Such editing involves site-specific digestion of mutant genomic DNA followed by repair. Previous experimental approaches were hampered by inaccurate and high rates of off-site lesioning and by overall low digestion rates. A new tool, clustered regularly interspaced short palindromic repeats coupled with the nuclease Cas9 (CRISPR/Cas9), may address both shortcomings. Some of the many challenges that must be addressed in moving CRISPR/Cas9 therapies to the ophthalmic clinic are discussed here.

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“…Correction has been successfully demonstrated in hepatocytes (13–16), retinal cells (17,18), bone marrow-derived cells (19) and muscle cells (5,9,10,20–22). The level of correction varies depending on the cell type being targeted suggesting that the repair process involved is differentially regulated in different cell types.…”

Section: Introductionmentioning

confidence: 99%

Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

Bertoni

Rando

2009

Nucleic Acids Research

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Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.

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Mouse Retina Has Oligonucleotide-Induced Gene Repair Activity

Abstract: These in vitro assay data suggest that murine retina nuclei contain all the DNA repair factors necessary for OMGR, a finding that is prerequisite to attempting endogenous gene repair in mouse retina.

Cited by 9 publications

References 34 publications

Regulation of targeted gene repair by intrinsic cellular processes

Regulation of targeted gene repair by intrinsic cellular processes

Clustered Regularly Interspaced Short Palindromic Repeats

Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

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