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Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells.

ABSTRACT: Over the past decade, numerous gene-editing platforms which alter host DNA in a highly specific and targeted fashion have been described. Two notable examples are zinc finger nucleases (ZFNs), the first gene-editing platform to be tested in clinical trials, and more recently, CRISPR/Cas9. Although CRISPR/Cas9 approaches have become arguably the most popular platform in the field, the therapeutic advantages and disadvantages of each strategy are only beginning to emerge. We have established a nonhuman primate (NHP) model that serves as a strong predictor of successful gene therapy and gene-editing approaches in humans; our recent work shows that ZFN-edited hematopoietic stem and progenitor cells (HSPCs) engraft at lower levels than CRISPR/Cas9-edited cells. Here, we investigate the mechanisms underlying this difference. We show that optimized culture conditions, including defined serum-free media, augment engraftment of gene-edited NHP HSPCs in a mouse xenograft model. Furthermore, we identify intracellular RNases as major barriers for mRNA-encoded nucleases relative to preformed enzymatically active CRISPR/Cas9 ribonucleoprotein (RNP) complexes. We conclude that CRISPR/Cas9 RNP gene editing is more stable and efficient than ZFN mRNA-based delivery and identify co-delivered RNase inhibitors as a strategy to enhance the expression of gene-editing proteins from mRNA intermediates.

SUBMITTER: Peterson CW

PROVIDER: S-EPMC8671732 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p=0.002) and 4.3±0.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis. Use Affymetrix microarrays to examine the effecs of knockout of the gene ADPGK using Zinc Finger nucleases in two cultured cell lines: (i) HCT116 cells (three wild type cultures compared to three ADPGK-knockout cultures), (ii) H460 cells (two wild type cultures compared to two ADPGK-knockout cultures). Ten microarrays in total.

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Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells.

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