Tianhua Zhou

Genes encoding pathogenesis-related (PR-) proteins isolated from a cDNA library of Fusarium graminearum-infected wheat spikes of scab-resistant cultivar 'Sumai-3' were transformed into susceptible spring wheat, 'Bobwhite' using a biolistic transformation protocol, with the goal of enhancing levels of resistance against scab. Twenty-four putative transgenic lines expressing either a single PR-protein gene or combinations thereof were regenerated. Transgene expression in a majority of these lines (20) was completely silenced in the T(1) or T(2) generations. Four transgenic wheat lines showed stable inheritance and expression of either a single transgene or transgene combinations up to four generations. One line co-expressing a chitinase and beta-1,3-glucanase gene combination, when bioassayed against scab showed a delay in the spread of the infection (type II resistance) under greenhouse conditions. This line and a second transgenic line expressing a rice thaumatin-like protein gene (tlp) which had moderate resistance to scab in previous greenhouse trials, along with susceptible and resistance checks were evaluated for resistance to scab under field conditions. None of the transgenic lines had resistance to scab in the field under conditions of strong pathogen, suggesting these plants lacked effective resistance to initial infection (type I resistance) under these conditions. As far as is known, this is the first report of field evaluation of transgenic wheat expressing genes for PR-proteins against disease resistance.

CRISPR-Cas9 gene editing has emerged as a powerful therapeutic technology, but the lack of safe and efficient in vivo delivery systems, especially for tissue-specific vectors, limits its broad clinical applications. Delivery of Cas9 ribonucleoprotein (RNP) owns competitive advantages over other options; however, the large size of RNPs exceeds the loading capacity of currently available delivery vectors. Here, we report a previously unidentified genome editing delivery system, named exosome RNP , in which Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells through electroporation. Exosome RNP facilitated effective cytosolic delivery of RNP in vitro while specifically accumulated in the liver tissue in vivo. Exosome RNP showed vigorous therapeutic potential in acute liver injury, chronic liver fibrosis, and hepatocellular carcinoma mouse models via targeting p53 up-regulated modulator of apoptosis ( PUMA ), cyclin E1 ( CcnE1 ), and K (lysine) acetyltransferase 5 ( KAT5 ), respectively. The developed exosome RNP provides a feasible platform for precise and tissue-specific gene therapies of liver diseases.