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Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model for Duchenne muscular dystrophy. Two different AAV viruses were delivered to the mdx muscle. Similar to previous reports in normal muscle, coinfection efficiency reached approximately 90% in the diseased muscle. This result suggests that coinfection is not a hurdle in dystrophic muscle. Another critical step in the trans-splicing method is the transcription and splicing across the inverted terminal repeat (ITR) junction in the reconstituted genome. To test whether this represented a significant obstacle, we systematically evaluated the transcription, pre-mRNA stability and splicing, and translation in a synthetic lacZ construct that mimicked the reconstituted genome. Although inserting an intron in the lacZ gene had no effect on its expression, inclusion of the ITR junction in the intron reduced expression by 50%. In construct containing the ITR junction, the mRNA transcript level was significantly reduced. This mRNA level reduction was associated with decreased pre-mRNA stability. These data suggest that the accumulation of mRNA is a rate-limiting factor in trans-splicing vector-mediated gene therapy.

A PEGylated multistimuli-responsive dendritic copolymer–doxorubicin (DOX) prodrug-based nanoscale system was developed as a delivery model for hydrophobic drugs. In this system, PEGylation did not only prolong circulation of the nanoscale system in the body (average half-life of 14.6 h, four times longer than that of the free drug), but also allowed the system to aggregate into nanoparticles (NPs) because of interactions between hydrophilic (polyethylene glycol) and hydrophobic (dendritic prodrug) moieties for better uptake through endocytosis (around 150 nm of particle size with a neutrally charged surface for the PEGylated dendritic prodrug with 12.1 wt % of DOX). The dendritic structure was built by bridging poly­[N-(2-hydroxypropyl)­methacrylamide] segments with enzyme-responsive GFLG (Gly-Phe-Leu-Gly tetrapeptide) linkers. DOX was released by hydrolyzing the hydrazone bond between DOX and the copolymer framework in the acidic endosomes/lysosomes. In vitro studies on DOX released from the NPs induced mitochondrial dysfunction during apoptosis. By imaging the main organs and tumor tissues from mice treated with the NPs, boosted accumulation of this nanoscale medicine was found in tumor tissues, leading to a decrease in toxicity and side effects to normal tissues and enhancement in drug tolerance. In the 4T1 breast cancer model, these NPs exhibited a superior antitumor efficacy confirmed by inhibiting angiogenesis, proliferation of tumor tissues, and inducing procedural apoptosis of tumor cells. The highest tumor growth inhibition value mediated by the NPs was up to 86.5%. Therefore, this PEGylated multistimuli-responsive dendritic copolymer–DOX prodrug-based nanoscale system may be further explored as an alternative to traditional chemotherapy for breast cancer treatment.

The aim of this study was to analyse the clinical features, microbiology results, management and outcomes of patients with endogenous endophthalmitis caused by Klebsiella pneumoniae in western China. Methods: A retrospective review of medical records of 10 eyes in 10 subjects diagnosed with endogenous K. pneumoniae endophthalmitis from January 2008 to December 2018 was undertaken. Results: The top 3 predisposing medical conditions included diabetes mellitus (50%), malignancy (20%) and cardiac stent implantation (10%). Extraocular infective foci were mainly found in the liver (40%), lungs (20%) and kidneys (10%). The positive culture rate was 85.71% (6/7) in vitreous samples, 83.33% (5/6) in blood samples and 100% (4/4) in body fluid samples. Only 20% of the patients, who had good initial visual acuity (VA) better than hand movement (HM), achieved a final VA better than 1.0 (log MAR). The mortality rate was 10%. Conclusions: Though the prognosis of endogenous K. pneumoniae endophthalmitis is often poor, patients with an initial VA better than HM may have a good prognosis under comprehensive treatments, including vitrectomy, systemic sensitive antibiotic injection and drainage of the primary infection loci.

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