Preprint Article Version 1 This version is not peer-reviewed

TALEN Mediated Gene Targeting for CF Gene Therapy

Version 1 : Received: 16 November 2018 / Approved: 19 November 2018 / Online: 19 November 2018 (10:14:02 CET)

A peer-reviewed article of this Preprint also exists.

Xia, E.; Zhang, Y.; Cao, H.; Li, J.; Duan, R.; Hu, J. TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy. Genes 2019, 10, 39. Xia, E.; Zhang, Y.; Cao, H.; Li, J.; Duan, R.; Hu, J. TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy. Genes 2019, 10, 39.

Journal reference: Genes 2019, 10, 39
DOI: 10.3390/genes10010039


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 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 CFTR minigene 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 have detected both CFTR mRNA and protein expression by qPCR and Wetern analysis, respectively. We have also confirmed CFTR function correction by flurometric Image Plate Reader (FLIPR) and iodide efflux assays. Taking together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

Subject Areas

cystic fibrosis; gene therapy; gene targeting; gene integration

Comments (1)

Comment 1
Received: 9 February 2019
Commenter: Martin L. Nelwan (Click to see Publons profile: )
The commenter has declared there is no conflict of interests.
Comment: The authors introduced cystic fibrosis (CF) disease and the use of TALEN for treating CF. They engineered HD-Ad vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. The authors explained the use of TALEN to edit the mutated gene. It is an interesting manuscript. However, this manuscript still needs a minor revision.

The authors introduced that their study demonstrated the success of the use of TALEN in correcting a human CFTR mutant cell line as well as the rescue of CFTR channel activity. They stated that their method was not mutation-specific and was applicable to correcting all CFTR mutations. However, the authors should discuss the prevalence of cystic fibrosis and should add more information regarding the CFTR gene, and mutations in the CFTR gene. In addition, the authors should explain how TALEN edits incorrect sequences in the mutated gene. For example, how the minigene edited the incorrect sequences in the target site or mutated gene. I suggest the Discussion section can be the place in explaining a brief correction for the mutated gene. Moreover, the writing style of this manuscript still needs to be improved. There are many passive voices. For example, the authors wrote, “the major CF gene therapy at present is directed to the lung.” This sentence is a passive voice. I suggest, “the major CF gene therapy is for the lung.” The authors should use active voice when possible. “CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.” It is a passive voice.

There are many incorrect spaces between paragraphs. For example, the last paragraph of the Results section and the Discussion section is too wide. The authors should rewrite it into fit line spacing between those paragraphs, and the manuscript should have line numbers. At this preprint server, it seems that line numbers will make easy for authors, reviewers, editors, and other readers to find a specific sentence in the manuscript. Six figures are in this manuscript. Each one has a caption and an informative legend. In the Discussion section, the authors can add a figure about how TALENs to edit the mutated gene that results in healthy patients. Authors can also add a table in the Discussion section. This table can include platforms (i.e., ZFNs, TALENs), safety, sources, and references.

Other points:

Remove the last sentence of the last paragraph in the sub-section of the Integration of the CFTR gene into the AAVS1 locus allowed persistent CFTR expression and presence of CFTR channel activity.

Remove the last two sentences of the paragraph in the Transient TALEN expression sub-section.

Interpretation and inference do not belong to in the Results section.
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