Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - 'gene stacking', and cointegration of multiple engineered large vectors - 'combineering', to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

The replication timing of CFTR and adjacent genes.

Correlations between transcriptional activity and replication timing have been observed for the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, as well as for other tissue-specific genes. In addition, the patterns of histone modifications and the nuclear positioning of chromosomal loci appear to be related to their replication timing. It is not understood why and how these different features are functionally linked. To address this problem, we investigated the replication timing of the human CFTR gene and of adjacent genes. Recently, we could show that CFTR and adjacent genes associate independently from each other with different nuclear regions and chromatin fractions, in accordance with their individual transcriptional regulation. Together, the results show that not the transcriptional activity, but rather the nuclear position of CFTR and adjacent genes appears to be a major determinant of their replication timing. Furthermore, the results imply a specific functional order of nuclear changes related to switches in replication timing.