Dataset Information

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

ABSTRACT: Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human cystic fibrosis transmembrane conductance regulator (CFTR) minigene (cDNA) into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we were able to detect CFTR mRNA expression using qPCR and function correction using fluorometric image plate reader (FLIPR) and iodide efflux assays. Taken together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

SUBMITTER: Xia E

PROVIDER: S-EPMC6356284 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

Xia Emily E Zhang Yiqian Y Cao Huibi H Li Jun J Duan Rongqi R Hu Jim J

Genes 20190111 1

Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription act ...[more]

PMID: 30641980

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Project description:Gene therapy holds promise for the treatment of a range of inherited diseases, such as cystic fibrosis. However, efficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of cystic fibrosis pulmonary disease has proved elusive. There are many reasons for this lack of progress, both macroscopically in terms of airway defence mechanisms and at the molecular level with regard to effective cDNA delivery. This review of approaches to cystic fibrosis gene therapy covers these areas in detail and highlights recent progress in the field. For gene therapy to be effective in patients with cystic fibrosis, the cDNA encoding the cystic fibrosis transmembrane conductance regulator protein must be delivered effectively to the nucleus of the epithelial cells lining the bronchial tree within the lungs. Expression of the transgene must be maintained at adequate levels for the lifetime of the patient, either by repeat dosage of the vector or by targeting airway stem cells. Clinical trials of gene therapy for cystic fibrosis have demonstrated proof of principle, but gene expression has been limited to 30 days at best. Results suggest that viral vectors such as adenovirus and adeno-associated virus are unsuited to repeat dosing, as the immune response reduces the effectiveness of each subsequent dose. Nonviral approaches, such as cationic liposomes, appear more suited to repeat dosing, but have been less effective. Current work regarding non-viral gene delivery is now focused on understanding the mechanisms involved in cell entry, endosomal escape and nuclear import of the transgene. There is now increasing evidence to suggest that additional ligands that facilitate endosomal escape or contain a nuclear localization signal may enhance liposome-mediated gene delivery. Much progress in this area has been informed by advances in our understanding of the mechanisms by which viruses deliver their genomes to the nuclei of host cells.

Dataset Information

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

Publications

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

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