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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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Autor principal: Wayengera, Misaki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
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.
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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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