Project description:The continuing reports of plastic pollution in various ecosystems highlight the threat posed by the ever-increasing consumption of synthetic polymers. Therefore, Pseudomonas capeferrum TDA1, a strain recently isolated from a plastic dump site, was examined further regarding its ability to degrade polyurethane (PU) compounds. The previously reported degradation pathway for 2,4-toluene diamine (2,4-TDA), a precursor and degradation intermediate of PU, could be confirmed by RNA-seq in this organism. In addition, different cell fractions of cells grown on a PU oligomer were tested for extracellular hydrolytic activity using a standard assay. Strikingly, purified outer membrane vesicles (OMV) of P. capeferrum TDA1 grown on a PU oligomer showed higher esterase activity than cell pellets. Hydrolases in the OMV fraction possibly involved in extracellular PU degradation were identified by mass spectrometry. On this basis, we propose a model for extracellular degradation of polyester-based PUs by P. capeferrum TDA1 involving the role of OMVs in synthetic polymer degradation.
Project description:Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for hemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralize F8 activity. Taking advantage of split inteins-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV split-inteins vectors whose co-administration to HemA mice results in F8 protein reconstitution and expression of therapeutic levels of F8 over time without anti-F8 antibodies development.
