Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six-to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay -referred to as repeat-anchored integration capture (RAIC) PCR -that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Although recent advances in therapeutic approaches for treating HCC have improved the prognoses of patients with HCC, this cancer is still associated with a poor survival rate mainly due to late diagnosis. Therefore, a diagnosis must be made sufficiently early to perform curative and effective treatments. There is a need for a deeper understanding of the molecular mechanisms underlying the initiation and progression of HCC because these mechanisms are critical for making early diagnoses and developing novel therapeutic strategies. Over the past decade, much progress has been made in elucidating the molecular mechanisms underlying hepatocarcinogenesis. In particular, recent advances in next-generation sequencing technologies have revealed numerous genetic alterations, including recurrently mutated genes and dysregulated signaling pathways in HCC. A better understanding of the genetic alterations in HCC could contribute to identifying potential driver mutations and discovering novel therapeutic targets in the future. In this article, we summarize the current advances in research on the genetic alterations, including genomic instability, single-nucleotide polymorphisms, somatic mutations and deregulated signaling pathways, implicated in the initiation and progression of HCC. We also attempt to elucidate some of the genetic mechanisms that contribute to making early diagnoses of and developing molecularly targeted therapies for HCC.

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1–7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal – indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.

CGRP is a neuropeptide that has previously been described to possess immunosuppressive activities. CGRP is released from peripheral nerves that, in the skin, are in close physical association with dendritic APC. We sought to investigate the mechanisms by which CGRP can inhibit immune responses by studying its effects on human peripheral blood mononuclear cells (PBMC). Using allogeneic monocytes as stimulator cells, CGRP could inhibit the proliferation of PBMC by 47% when CGRP was present for the duration of culture. Interestingly, when the stimulator monocytes were incubated with CGRP for 2 h prior to irradiation then washed, the observed inhibition increased to 85%, suggesting that CGRP was exerting a direct effect on the monocyte stimulator population. Finally, the recall response to tetanus toxoid (TT) by PBMC from individuals vaccinated with TT 14 d prior was inhibited by 25-50% in the presence of CGRP. Also, CGRP decreased the levels of B7.2 but not B7.1 on treated monocytes, and this inhibition could be abrogated by the addition of anti-IL-10 antibody, suggesting that the inhibition was mediated by an increase in IL-10 production. Moreover, increased IL-10 production was confirmed by ELISA. Both IL-12 p40 and IFN-gamma levels in CGRP-treated cultures were found to be decreased by approximately 30%. The decrease in IL-12 p40 levels could be reversed by addition of anti-IL-10. These data suggest that CGRP inhibits PBMC proliferation, in part, through the release of IL-10, which in turn can downregulate important co-stimulatory molecules and the cytokines IL-12 and IFN-gamma.

Long non-coding RNAs (lncRNAs) are a subgroup of non-coding RNA transcripts greater than 200 nucleotides in length with little or no protein-coding potential. Emerging evidence indicates that lncRNAs may play important regulatory roles in the pathogenesis and progression of human cancers, including hepatocellular carcinoma (HCC). Certain lncRNAs may be used as diagnostic or prognostic markers for HCC, a serious malignancy with increasing morbidity and high mortality rates worldwide. Therefore, elucidating the functional roles of lncRNAs in tumors can contribute to a better understanding of the molecular mechanisms of HCC and may help in developing novel therapeutic targets. In this review, we summarize the recent progress regarding the functional roles of lncRNAs in HCC and explore their clinical implications as diagnostic or prognostic biomarkers and molecular therapeutic targets for HCC.