Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study.

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 (-?S) gene. METHODS AND RESULTS:First, using the complete 1606 genomic DNA base pair (bp) sequences of the normal hemoglobin-beta (?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 ?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 6th codon) of the ?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 ?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.

SUBMITTER: Wayengera M 

PROVIDER: S-EPMC3407482 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature