A meta-analysis: laparoscopic versus open liver resection for large hepatocellular carcinoma.

BACKGROUND: The indication of laparoscopic liver resection (LLR) for treating large hepatocellular carcinoma (HCC) is controversial. In this study, we compared the short-term and long-term outcomes of LLR and open liver resection (OLR) for large HCC. MATERIAL AND METHODS: We searched eligible articles about LLR versus OLR for large HCC in PubMed, Cochrane Library, and EMBASE and performed a meta-analysis. RESULTS: Eight publications involving 1,338 patients were included. Among them, 495 underwent LLR and 843 underwent OLR. The operation time was longer in the LLR group (MD: 22.23, 95% CI: 4.14-40.33, p = 0.02). but the postoperative hospital stay time was significantly shorter (MD : -4.88, CI: -5.55 to -4.23, p < 0.00001), and the incidence of total postoperative complications and major complications were significantly fewer (OR: 0.49, 95% CI:0.37-0.66, p < 0.00001; OR: 0.54, 95% CI:0.36 - 0.82, p = 0.003, respectively). Patients in the laparoscopic group had no significant difference in intraoperative blood loss, intraoperative transfusion rate, resection margin size, R0 resection rate, three-year overall survival (OS) and three-year disease-free survival (DFS). CONCLUSION: LLR for large HCC is safe and feasible. This surgical strategy will not affect the long-term outcomes of patients.

Anticancer effects of a single intramuscular dose of a minicircle DNA vector expressing anti-CD3/CD20 in a xenograft mouse model.

Bispecific antibodies (BsAbs) are a class of promising anticancer immunotherapies. Among them, the US Food and Drug Administration (FDA)-approved blinatumomab (BLI) is very effective in eliminating the minimum residual disease (MRD) of acute lymphoblastic leukemia (ALL), resulting in long-term remission in many individuals. However, the need for months-long intravenous delivery and high cost limit its clinical acceptance. Here we demonstrate that these problems can be solved by a BsAb expressed by one intramuscular (i.m.) dose of a minicircle DNA vector (MC). In a human B lymphoma xenograft mouse model, when microcancers became detectable in bone marrow, the mice received an i.m. dose of the MC encoding the BsAb anti-CD3/CD20 (BsAb.CD20), followed by 8 subsequent intravenous (i.v.) doses, one every other day (q2d), of human T cells to serve as effectors. The treatment resulted in persistent expression of a therapeutic level of serum BsAb.CD20 and complete regression or growth retardation of the cancers in the mice. These results suggest that the i.m. MC technology can eliminate the physical and financial burdens of i.v. delivered BLI without compromising anticancer efficacy and that cancer can be treated as easily as injecting a vaccine. This, together with other superior MC features, such as safety and affordability, suggests that the i.m. MC BsAb technology has great clinical application potential.

Sexual variation in two species of Helorus Latreille (Hymenoptera, Heloridae) from NW China, with description of female of Helorus caii He amp; Xu.

The intra-specific variation of two species of Helorus Latreille (Hymenoptera, Heloridae) from NW China is studied and for the first time the sexual variation in Chinese Helorus species is described. Both sexes of Helorus antefurcalis He Xu, 2015 (new for Shaanxi) and H. caii He Xu, 2015 (up to now only known from one male), are described and illustrated. Helorus xinjiangensis He Xu, [June] 2015, is a new junior synonym of H. alborzicus Izadizadeh, van Achterberg Talebi, [April] 2015 and H. elgoni Risbec, 1950, is re-instated as valid species. A revised key to the Old World species of Helorus Latreille (except Australian region) is included.

Calcium phosphate nanoneedle based gene delivery system for cancer genetic immunotherapy.

Ovarian cancer has become one of the most common gynecological cancers with a high mortality. However, conventional surgery together with combination chemotherapy is difficult to achieve ideal therapeutic effect. Although genetic immunotherapy is applied to active immune responses against cancer, the absence of efficient in vivo gene delivery technique is still an obstacle in clinical application. To overcome these problems, a minicircle DNA vector encoding humanized anti-EpCAM/CD3 bispecific antibody (BsAbEPH) has been constructed. Moreover, different shapes of calcium phosphate (CaPO) biomaterials were prepared. Specifically, the CaPO-nanoneedle-mediated "cell perforation" transfection technology achieves high levels of gene expression in peritoneal cavity. In an intraperitoneal xenograft model with human ovarian cancer cell line SKOV3, the CaPO-nanoneedle/minicircle DNA system expressed BsAbEPH resulted in significant retardation of cancer growth and extension of mouse life-span with limited toxicity. And this system can be made as off-the-shelf and easy-to-use products. Therefore, CaPO-nanoneedle based non-viral gene delivery technology will have great potential in clinical application.

The synthesis of amphiphilic polyethyleneimine/calcium phosphate composites for bispecific T-cell engager based immunogene therapy.

Bispecific T-cell engagers (BiTEs) are single chain variable fragments with specific structures, which could connect the surface antigen on cancer cells and CD3 ligands on T cells, and then engage the T cells for cancer immunotherapy. In this report, a novel organic-inorganic hybrid gene delivery system composed of stearic acid modified polyethyleneimine (stPEI) and calcium phosphate (CaP) was used to deliver MC.DNA into cells to express BiTE antibodies. This gene delivery system exhibits high transfection efficiency, long-term effects and low cytotoxicity in vitro. Furthermore, the gene production, anti-IGF1R/CD3 bispecific T-cell engager, exhibited a rapid redirection activity in T cells to induce cancer cell apoptosis. In summary, the results confirmed that stPEI-CaP could be an efficient gene delivery system for BiTE encoding MC.DNA based gene immunotherapy.

Whole-exome sequencing predicted cancer epitope trees of 23 early cervical cancers in Chinese women.

Emerging evidence suggest that the heterogeneity of cancer limits the efficacy of immunotherapy. To search for optimal therapeutic targets for enhancing the efficacy, we used whole-exome sequencing data of 23 early cervical tumors from Chinese women to investigate the hierarchical structure of the somatic mutations and the neo-epitopes. The putative neo-epitopes were predicted based on the mutant peptides' strong binding with major histocompatibility complex class I molecules. We found that each tumor carried an average of 117 mutations and 61 putative neo-epitopes. Each patient displayed a unique phylogenetic tree in which almost all subclones harbored neo-epitopes, highlighting the importance of individual neo-epitope tree in determination of immunotherapeutic targets. The alterations in FBXW7 and PIK3CA, or other members of the significantly altered ubiquitin-mediated proteolysis and extracellular matrix receptor interaction related pathways, were proposed as the earliest changes triggering the malignant progression. The neo-epitopes involved in these pathways, and located at the top of the hierarchy tree, might become the optimal candidates for therapeutic targets, possessing the potential to mediate T-cell killing of the descendant cells. These findings expanded our understanding in early stage of cervical carcinogenesis and offered an important approach to assist optimizing the immunotherapeutic target selection.

Erythrocyte membrane based cationic polymer-mcDNA complexes as an efficient gene delivery system.

Gene therapy has great promise for the treatment of obtained and inherited serious diseases. However, the lack of safe and efficient gene delivery systems remains a barrier for their clinical application. Here, we reported a potential gene delivery vehicle composed of the erythrocyte membrane and cationic polymers, for example the XtremeGENE from Roche and the ε-caprolactone modified polyethylenimine. In addition to high efficiency, this system showed negligible cytotoxicity compared to the two cationic polymers alone in various cell lines, including human embryonic kidney cells (293T), human liver cancer cells (Huh7 and HepG2), murine dendritic cells (DC2.4) and human umbilical cord mesenchymal stem cells (Hu-MSCs). Moreover, the results of confocal laser scanning microscopy and flow cytometry suggested that the cell uptake of this gene vector was improved and might be introduced by the fusion interaction between the erythrocyte membrane and targeted cells.Thus, all the results revealed that the erythrocyte membrane based gene delivery system might be able to serve as an excellent gene delivery system.

Synthesis of Amphiphilic Poly(ß-amino ester) for Efficiently Minicircle DNA Delivery in Vivo.

Minicircle DNA (mcDNA) is a kind of enhanced nonviral DNA vector with excellent profiles in biosafety and transgene expression. Herein, we reported a novel amphiphilic polymer comprising polyethylenimine(PEI) modified Poly(ß-amino ester) PEI-PBAE(C16) for efficient mcDNA delivery in vivo. The synthesized polymer could condense mcDNA into nanoscaled structure and exhibited efficient gene transfection ability without detectable cytotoxicity. Importantly, when injected into mouse intraperitoneally, these PEI-PBAE(C16) nanocomplexes were able to result in high level of trangene expression which lasted at least 72 h. Overall, these results demonstrated the PEI-PBAE(C16) can mediate effective and safe gene delivery in vivo with clinical application potential.

The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.

Synthesis and characterization of low molecular weight polyethyleneimine-terminated Poly(ß-amino ester) for highly efficient gene delivery of minicircle DNA.

Gene therapy has held great promise for treating specific acquired and inherited diseases. However, the lack of safe and efficient gene delivery systems remains as the major challenge. Poly(ß-amino ester)s (PBAEs) have attracted much attention due to their outstanding properties in biosafety, DNA delivery efficiency and convenience in synthesis. In this paper, we reported the further enhancement of the PBAE functions by increasing its positive charge through conjugating with low molecular weight polyethylenimine (LPEI). The resulted LPEI-PBAE polymer was able to condense minicircle DNA (mcDNA) forming nanoparticles with a diameter of 50-200nm. Furthermore, as compared to parental PBAE and a commercial transfection reagent very common in laboratory application, the LPEI-PBAE demonstrated significantly higher transfection efficiency with little cytotoxicity. These results suggested LPEI-PBAEs are worthy of further optimization for gene therapy applications.

Increasing the minicircle DNA purity using an enhanced triplex DNA technology to eliminate DNA contaminants.

DNA vectors for human gene therapy have to meet the efficacy and safety requirements. Minicircles (MCs), a class of optimized DNA vectors free of plasmid backbone (PB) DNAs, have emerged as promising candidates because of their superior transgene expression profiles. However, the existence of impure DNAs, including the unrecombined MC producing plasmid (PP) and PB circle, in the MC products made using the current technologies exceed the safety limit. Here, we report the development of an enhanced triplex DNA (TriD) technology to eliminate almost all the impure DNAs from the MC products. To do this, a pair of optimized TriD forming sequences was placed to flank the kanamycin resistance gene in the PP. The MC products were incubated with a biotinylated TriD forming DNA oligonucleotide (olig), and the resulted TriDs were removed by binding to streptovidin-coated magnetic beads. Consequently, the residual impure DNAs were 0.03% or less in the final MC products. The reproducibility of this technique was confirmed with MCs of various transgene expression cassettes, sizes, and quantities, suggesting its great potential in making high quality MC for human gene therapy.

Efficacy of MRI visible iron oxide nanoparticles in delivering minicircle DNA into liver via intrabiliary infusion.

Gene therapy is a very promising technology for treatment of liver diseases. Minicircle DNA (MCDNA) is a versatile gene vector, which possesses excellent features in bio-safety and duration of transgene expression. However, its application has been hampered by the lack of an efficient gene delivery system. In this study, we developed a magnetic resonance imaging (MRI) visible nanoparticle to monitor MCDNA gene delivery and explore the potential of gene therapy in vivo. The nanoparticle was constructed via self-assembly of stearic acid modified low molecular polyethyleneimine (stPEI) and superparamagnetic iron oxide (SPIO) nanocrystals. The multiple SPIO nanocrystals with a controlled clustering structure in the nanoparticles were designed to achieve high MRI sensitivity. Furthermore, the MCDNA was combined with the nanoparticles via electronic interaction. Through intrabiliary infusion, the stPEI-SPIO/MCDNA nanoparticles were efficiently delivered to liver that was visualized by MRI in vivo and confirmed by histology. Moreover, the MCDNA nanoparticles exhibited non-cytotoxicity with no obvious inflammation in the transfection sites. These results indicate that stPEI-SPIO nanoparticle is able to serve as both an efficient DNA vector delivery system and a sensitive agent for MRI visualization.

Self-assembled magnetic theranostic nanoparticles for highly sensitive MRI of minicircle DNA delivery.

As a versatile gene vector, minicircle DNA (mcDNA) has a great potential for gene therapy. However, some serious challenges remain, such as to effectively deliver mcDNA into targeted cells/tissues and to non-invasively monitor the delivery of the mcDNA. Superparamagnetic iron oxide (SPIO) nanoparticles have been extensively used for both drug/gene delivery and diagnosis. In this study, an MRI visible gene delivery system was developed with a core of SPIO nanocrystals and a shell of biodegradable stearic acid-modified low molecular weight polyethyleneimine (Stearic-LWPEI) via self-assembly. The Stearic-LWPEI-SPIO nanoparticles possess a controlled clustering structure, narrow size distribution and ultrasensitive imaging capacity. Furthermore, the nanoparticle can effectively bind with mcDNA and protect it from enzymatic degradation. In conclusion, the nanoparticle shows synergistic advantages in the effective transfection of mcDNA and non-invasive MRI of gene delivery.

A robust system for production of minicircle DNA vectors.

Minicircle DNA vectors allow sustained transgene expression in quiescent cells and tissues. To improve minicircle production, we genetically modified Escherichia coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, ΦC31 integrase and I-SceI homing endonuclease. This bacterial strain produces purified minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation, making it feasible to use minicircles in place of plasmids in mammalian transgene expression studies.

Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo.

We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The oC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.

Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.

The loss of transgene expression has been a major obstacle to the development of nonviral vectors for the treatment of human diseases. We previously demonstrated that bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo. To confirm these studies and develop a means to produce a robust DNA vector that is not silenced in vivo, we developed a phage phiC31 integrase-mediated intramolecular recombination technology to prepare minicircle vector DNA devoid of the bacterial backbone and then compared the transgene expression profile of the minicircle with different molecular forms of plasmid DNAs in mice. We demonstrate that minicircular DNAs devoid of bacterial sequences expressed 45- and 560-fold more serum human factor IX and alpha1-antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver. Our data suggest that minicircles are capable of expressing high and persistent levels of therapeutic products in vivo and have a great potential to serve as episomal vectors for the treatment of a wide variety of diseases.