rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency.

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.

Ethiadin Induces Apoptosis and Suppresses Growth of MCF-7 Breast Cancer Cells by Regulating the Phosphorylation of Glycogen Synthase Kinase 3 Beta (GSK3beta).

Glycogen synthase kinase 3 beta (GSK3ß) has emerged as a therapeutic target for breast cancer. As inhibitors of GSK-3ß, 1,2,4-thiadiazole-3,5-dione (TDZD) family members have been reported as potential candidates for cancer treatment. In this study, the anticancer effects of ethiadin (ETD-174), one of the chemical synthesis compounds of TDZD, were investigated in MCF-7 human breast cancer cells. MCF-7 cells incubated with different doses of ETD-174 for different time periods. CCK-8 assays were carried out to test the effect of ETD-174 on the proliferation of MCF-7 cells. The occurrence of apoptosis was detected by Hoechst 33258 staining and flow cytometry. ETD-174 on cell migration and colony formation were examined by wound healing experiments and soft agar assays. Relative protein expressions were conducted with immunoblot assay. ETD-174 demonstrated a higher degree of cytotoxicity in MCF-7 cells. Topical morphological changes of apoptotic body formation after ETD-174 treatment were observed. Meanwhile, apoptosis was elicited by ETD-174. Also, ETD-174 could inhibit the migration and clonality of MCF-7 cells. After the treatment with ETD-174, the level of phosphorylation of GSK3ßSer9 in MCF-7 cells increased significantly, and the enzymatic activity of GSK3ß decreased. ETD-174 is likely to have an effective suppressor role in breast cancer, suggesting that pharmacological inhibition of GSK3ß as a novel treatment modality for breast cancer should warrant further investigation.