Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34 + cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34 + cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP + cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

Docosahexaenoic acid alleviates cell injury and improves barrier function by suppressing necroptosis signalling in TNF-α-challenged porcine intestinal epithelial cells.

Long-chain n-3 polyunsaturated fatty acids are known to have beneficial effects on intestinal health. However, the underling mechanisms are largely unknown. The present study was conducted to investigate whether docosahexaenoic acid (DHA) attenuates TNF-α-induced intestinal cell injury and barrier dysfunction by modulating necroptosis signalling. Intestinal porcine epithelial cell line 1 was cultured with or without 12.5 µg/ml DHA, followed by exposure to 50 ng/ml TNF-α for indicated time periods. DHA restored cell viability and cell number triggered by TNF-α. DHA also improved barrier function, which was indicated by increased trans-epithelial electrical resistance, decreased FD4 flux and increased membrane localisation of zonula occludins (ZO-1) and claudin-1. Moreover, DHA suppressed cell necrosis in TNF-α-challenged cells, as shown in the IncuCyte ZOOM™ live cell imaging system and transmission electron microscopy. In addition, DHA decreased protein expression of TNF receptor, receptor interacting protein kinase 1, RIP3 and phosphorylation of mixed lineage kinase-like protein, phosphoglycerate mutase family 5, dynamin-related protein 1 and high mobility group box-1 protein. Furthermore, DHA suppressed protein expression of caspase-3 and caspase-8. Collectively, these results indicate that DHA is capable of alleviating TNF-α-induced cell injury and barrier dysfunction by suppressing the necroptosis signalling pathway.