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Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study
BACKGROUND: Sickle cell disease (or simply, SCD) is an inherited hemoglobinopathy which is mostly prevalent among persons of African descent. SCD results from a monogenic (Hemoglobin, beta) point-mutation (substitution of the base Adenine with Thymine at position six) that leads to replacement of th...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2012
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407482/ https://www.ncbi.nlm.nih.gov/pubmed/22583379 http://dx.doi.org/10.1186/1471-2326-12-5 |
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author | Wayengera, Misaki |
author_facet | Wayengera, Misaki |
author_sort | Wayengera, Misaki |
collection | PubMed |
description | BACKGROUND: Sickle cell disease (or simply, SCD) is an inherited hemoglobinopathy which is mostly prevalent among persons of African descent. SCD results from a monogenic (Hemoglobin, beta) point-mutation (substitution of the base Adenine with Thymine at position six) that leads to replacement of the amino acid glutamic acid (E) with valine (V). Management of SCD within resource-poor settings is largely syndromic, since the option of cure offered by bone-marrow transplantation (BMT) is risky and unaffordable by most affected individuals. Despite previous reports of repair and inhibition of the sickle beta-globin gene and messenger ribonucleic acids (mRNAs), respectively in erythrocyte precursor cells via gene-targeting using an oligomer-restriction enzyme construct and either ribozyme- or RNA-DNA chimeric oligonucleotides (or simply third strand binding), gene-therapy to treat SCD still remains largely preclinical. In the wake of the advances in target- gene- mutagenesis and repair wrought by zinc finger nuclease (ZFN) technology, it was hypothesized that SCD may be cured by the same. The goal of this study thus, was constructing a database of zinc finger arrays (ZFAs) and engineering ZFNs, that respectively bind and cleave within or around specific sequences in the sickle hemoglobin, beta (−β(S)) gene. METHODS AND RESULTS: First, using the complete 1606 genomic DNA base pair (bp) sequences of the normal hemoglobin-beta (β(A)) chain gene, and the ZiFiT-CoDA-ZFA software preset at default, 57 three-finger arrays (ZFAs) that specifically bind 9 base-pair sequences within the normal hemoglobin-beta chain, were computationally assembled. Second, by serial linkage of these ZFAs to the Flavobacterium okeanokoites endonuclease Fok I― four ZFNs with unique specificity to >24 bp target-sequences at the genomic contextual positions 82, 1333, 1334, and 1413 of the β(A) chain-gene were constructed in-silico. Third, localizing the point-mutation of SCD at genomic contextual position −69-70-71- bp (a position corresponding to the 6(th) codon) of the β(A) chain-gene, inspired the final design of five more ZFNs specific to >24 bp target-sequences within the 8,954 bp that are genomically adjacent to the 5′ end of the β(A) chain-gene. CONCLUSIONS: This set of 57 ZFAs and 9 ZFNs offers us gene-therapeutic precursors for the targeted mutagenesis and repair of the SCD mutation or genotype. |
format | Online Article Text |
id | pubmed-3407482 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-34074822012-07-29 Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study Wayengera, Misaki BMC Blood Disord Research Article BACKGROUND: Sickle cell disease (or simply, SCD) is an inherited hemoglobinopathy which is mostly prevalent among persons of African descent. SCD results from a monogenic (Hemoglobin, beta) point-mutation (substitution of the base Adenine with Thymine at position six) that leads to replacement of the amino acid glutamic acid (E) with valine (V). Management of SCD within resource-poor settings is largely syndromic, since the option of cure offered by bone-marrow transplantation (BMT) is risky and unaffordable by most affected individuals. Despite previous reports of repair and inhibition of the sickle beta-globin gene and messenger ribonucleic acids (mRNAs), respectively in erythrocyte precursor cells via gene-targeting using an oligomer-restriction enzyme construct and either ribozyme- or RNA-DNA chimeric oligonucleotides (or simply third strand binding), gene-therapy to treat SCD still remains largely preclinical. In the wake of the advances in target- gene- mutagenesis and repair wrought by zinc finger nuclease (ZFN) technology, it was hypothesized that SCD may be cured by the same. The goal of this study thus, was constructing a database of zinc finger arrays (ZFAs) and engineering ZFNs, that respectively bind and cleave within or around specific sequences in the sickle hemoglobin, beta (−β(S)) gene. METHODS AND RESULTS: First, using the complete 1606 genomic DNA base pair (bp) sequences of the normal hemoglobin-beta (β(A)) chain gene, and the ZiFiT-CoDA-ZFA software preset at default, 57 three-finger arrays (ZFAs) that specifically bind 9 base-pair sequences within the normal hemoglobin-beta chain, were computationally assembled. Second, by serial linkage of these ZFAs to the Flavobacterium okeanokoites endonuclease Fok I― four ZFNs with unique specificity to >24 bp target-sequences at the genomic contextual positions 82, 1333, 1334, and 1413 of the β(A) chain-gene were constructed in-silico. Third, localizing the point-mutation of SCD at genomic contextual position −69-70-71- bp (a position corresponding to the 6(th) codon) of the β(A) chain-gene, inspired the final design of five more ZFNs specific to >24 bp target-sequences within the 8,954 bp that are genomically adjacent to the 5′ end of the β(A) chain-gene. CONCLUSIONS: This set of 57 ZFAs and 9 ZFNs offers us gene-therapeutic precursors for the targeted mutagenesis and repair of the SCD mutation or genotype. BioMed Central 2012-05-14 /pmc/articles/PMC3407482/ /pubmed/22583379 http://dx.doi.org/10.1186/1471-2326-12-5 Text en Copyright ©2012 Wayengera; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Wayengera, Misaki Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study |
title | Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study |
title_full | Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study |
title_fullStr | Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study |
title_full_unstemmed | Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study |
title_short | Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study |
title_sort | zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: an in-silico study |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407482/ https://www.ncbi.nlm.nih.gov/pubmed/22583379 http://dx.doi.org/10.1186/1471-2326-12-5 |
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