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In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases.

ABSTRACT: The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.

SUBMITTER: Sebastiano V

PROVIDER: S-EPMC3285772 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases.

Sebastiano Vittorio V Maeder Morgan L ML Angstman James F JF Haddad Bahareh B Khayter Cyd C Yeo Dana T DT Goodwin Mathew J MJ Hawkins John S JS Ramirez Cherie L CL Batista Luis F Z LF Artandi Steven E SE Wernig Marius M Joung J Keith JK

Stem cells (Dayton, Ohio) 20111101 11

The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintainin ...[more]

PMID: 21898685

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Project description:BackgroundSickle 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 (-βS) gene.Methods and resultsFirst, using the complete 1606 genomic DNA base pair (bp) sequences of the normal hemoglobin-beta (βA) 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 βA 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 βA 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 βA chain-gene.ConclusionsThis set of 57 ZFAs and 9 ZFNs offers us gene-therapeutic precursors for the targeted mutagenesis and repair of the SCD mutation or genotype.

Dataset Information

In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases.

Publications

In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases.

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