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Giovanni Pasquini, Anka Kempe, Mike O. Karl, Knut Stieger, Volker Busskamp; Transcriptomic assessing and guiding DSB repair pathway activity towards precise genomic engineering of post-mitotic neurons. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3123.
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© ARVO (1962-2015); The Authors (2016-present)
Over 200 retina specific genes have been associated with inherited retinal diseases (IRDs). Although genome editing represents a promising emerging field in the treatment of monogenic disorders, it relies on the capacity of the cell to repair double-strand breaks (DSB). When repairing exonic mutations or replacing exons, homology-dependent repair (HR) pathways (i.e. HDR and MMEJ) allow to preserve either the open reading frame or the splice site, whereas NHEJ will likely introduce indels. Nevertheless, HDR (G2) and MMEJ (G1) seem to be up-regulated during certain phases of the cell-cycle, whilst NHEJ is the predominant pathway at all stages in mitotic cells. Although robust data exist showing the complexity of DNA repair mechanisms and their regulation, almost nothing is known about post-mitotic neurons and photoreceptors (PRs).
We analyzed transcriptomes at several timepoints of in vitro developing retinal organoids and hiPS-derived neurons to monitor gene expression level of the three major DSB repair pathway members during cell differentiation and in post-mitotic state. Furthermore, we compared in vivo rodent and human PR transcriptomes to elucidate differential DSB repair gene level abundance related to inverted nuclear architecture in nocturnal species. Datasets were collected from published work as well as self-generated by LCM of PRs. Two bioinformatic RNA-seq pipelines were adopted for annotation-based transcript level quantification and precise quality assessment of raw reads.
Developing in vitro retinal organoids and neurons show that genes participating in HR are associated to the expression of cell-cycle active markers, while NHEJ genes appeared to be the most abundant up to post-mitotic state. In both in vitro and in vivo post-mitotic transcriptomes, HDR showed to be inactive, while NHEJ key genes were always highly expressed. Besides, MMEJ determinant component were consistently up regulated among post-mitotic PR and neurons.
Homologous dependent DSB repair pathways are prominently regulated within first stages of differentiation of retinal organoids and neurons. Nevertheless, MMEJ is active in post-mitotic PRs. Further experiments will be focused on pathway manipulation, i.e. inhibiting NHEJ and boosting MMEJ. This will ultimately enabling control of DSB repair, allowing precise genome editing treatments of monogenic IRDs.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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