Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study.
BMC Blood Disord
; 12: 5, 2012 May 14.
Article
em En
| MEDLINE
| ID: mdl-22583379
ABSTRACT
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 (-ßS) gene. METHODS ANDRESULTS:
First, using the complete 1606 genomic DNA base pair (bp) sequences of the normal hemoglobin-beta (ßA) 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 ßA 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 6th codon) of the ßA 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 ßA 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.
Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
BMC Blood Disord
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Uganda