Ferrand, M. | Search bioRxiv

bioRxiv2019

The R2TP chaperone assembles cellular machineries in intestinal CBC stem cells and progenitors

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium, and death within 10 days. Levels of R2TP substrates were decreased, with strong effects on mTOR, ATM and ATR. Rpap3-deficient CBC stem cells and progenitors also failed to import RNA polymerase II in the nucleus. This correlated with p53 activation, cell cycle arrest and apoptosis. Interestingly, post-mitotic, differentiated cells did not display any of those alterations, indicating that R2TP clients are built in actively proliferating cells. Analyses of tissues from colorectal cancer patients revealed that high RPAP3 levels correlate with bad cancer prognosis. Thus, in the intestine, the R2TP chaperone functions in physiologic and pathologic proliferation.

developmental biology↗

bioRxiv2019

Syncytins enable novel possibilities to transduce human or mouse primary B cells and to achieve well-tolerated in vivo gene transfer

Syncytins are cellular transmembrane glycoproteins with fusogenic and immunosuppressive properties that are encoded by endogenous retroviral envelope sequences in mammalian genomes. Based on their properties, syncytins may be useful to pseudotype lentiviral gene transfer vectors (LV) and to obtain well-tolerated in vivo gene delivery but their cellular targets are unknown in this context. We pseudotyped LV with human or murine syncytins. Such LV-Syn particles were infectious in vitro but required a transduction additive, as do other retroviral envelope LV pseudotypes. In these conditions, LV-Syn remarkably transduced quiescent human or murine primary B cells at high level in vitro including naive blood B cells or B cell precursors from murine bone marrow. Transduced human B cells could be expanded in culture and were functional. Human or murine T cells were transduced less efficiently than B cells, in agreement with lower levels of syncytin receptors on T cells compared to B cells. Well-tolerated in vivo gene transfer was possible without additive, as demonstrated with murine syncytin A-mediated gene delivery in C57BL/6 mice. A single intravenous injection of LV-SynA vector to mice led to stable gene transfer into spleen germinal center B cells. LV-SynA were also intrinsically less immunogenic than LV-VSVG, leading to low antibody responses against the vector capsid. This is the first evidence of interactions between syncytins and B cells, providing novel opportunities for B cell genetic engineering and for well-tolerated gene transfer in vivo. The findings also suggest that some immunosuppressive properties of syncytins could be mediated by B cells.\n\nOne Sentence SummarySyncytins are fusogenic cellular proteins that can pseudotype lentiviral gene transfer vector particles, achieving efficient gene transfer into primary quiescent B cells and reducing the in vivo immunogenicity of the particles following systemic administration.

immunology↗