Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System

Erythropoiesis-stimulating agents are widely used to treat anemia for chronic kidney disease (CKD) and cancer, however, several clinical limitations impede their effectiveness. Nonviral gene therapy systems are a novel solution to these problems as they provide stable and low immunogenic protein expression levels. Here, we show the application of an arginine-grafted bioreducible poly(disulfide amine) (ABP) polymer gene delivery system as a platform for in vivo transfer of human erythropoietin plasmid DNA (phEPO) to produce long-term, therapeutic erythropoiesis. A single systemic injection of phEPO/ABP polyplex led to higher hematocrit levels over a 60-day period accompanied with reticulocytosis and high hEPO protein expression. In addition, we found that the distinct temporal and spatial distribution of phEPO/ABP polyplexes contributed to increased erythropoietic effects compared to those of traditional EPO therapies. Overall, our study suggests that ABP polymer-based gene therapy provides a promising clinical strategy to reach effective therapeutic levels of hEPO gene.     

Preparation of a PEGylated liposome that co-encapsulates l-arginine and doxorubicin to achieve a synergistic anticancer effect

Strategies that combine chemotherapies with unconventional agents such as nitric oxide (NO) have been shown to enhance cancer therapies. Compared with small molecule chemotherapy drugs, nanosized particles have improved therapeutic efficacies and reduced systemic side effects because of the enhanced permeability and retention effect. In this report, we prepared PEGylated liposomes (LP) that incorporated l-arginine (Arg) and the anticancer drug doxorubicin (Dox) to yield a co-delivery system (Dox–Arg-LP). On the basis of our previous research, we hypothesized that Dox–Arg-LP should achieve a synergistic anticancer effect because Arg conversion to NO by activated M1 macrophages augments the chemotherapeutic activity of Dox. Dox–Arg-LP showed comparable physical properties to those of conventional Dox-only liposomes (Dox-LP). In vitro assessment revealed that the cytotoxicity of Dox–Arg-LP toward cancer cells was significantly higher than that of Dox-LP. In vivo application of Dox–Arg-LP in mice enhanced the chemotherapeutic effect with a 2 mg kg⁻¹ dose of Dox–Arg-LP achieving the same therapeutic efficacy as a two-fold higher dose of Dox-LP (i.e., 4 mg kg⁻¹). Therefore, co-encapsulation of dual agents into a liposome formulation is an efficient strategy to enhance chemotherapy while reducing systemic toxicity.

Strategies that combine chemotherapies with unconventional agents such as nitric oxide (NO) have been shown to enhance cancer therapies.     

Targeted Expression of miR-34a Using the T-VISA System Suppresses Breast Cancer Cell Growth and Invasion

Recurrence and metastasis result in a poor prognosis for breast cancer patients. Recent studies have demonstrated that microRNAs (miRNAs) play vital roles in the development and metastasis of breast cancer. In this study, we investigated the therapeutic potential of miR-34a in breast cancer. We found that miR-34a is downregulated in breast cancer cell lines and tissues, compared with normal cell lines and the adjacent nontumor tissues, respectively. To explore the therapeutic potential of miR-34a, we designed a targeted miR-34a expression plasmid (T-VISA-miR-34a) using the T-VISA system, and evaluated its antitumor effects, efficacy, mechanism of action, and systemic toxicity. T-VISA-miR-34a induced robust, persistent expression of miR-34a, and dramatically suppressed breast cancer cell growth, migration, and invasion in vitro by downregulating the protein expression levels of the miR-34a target genes E2F3, CD44, and SIRT1. In an orthotopic mouse model of breast cancer, intravenous injection of T-VISA-miR-34a:liposomal complex nanoparticles significantly inhibited tumor growth, prolonged survival, and did not induce systemic toxicity. In conclusion, T-VISA-miR-34a lead to robust, specific overexpression of miR-34a in breast cancer cells and induced potent antitumor effects in vitro and in vivo. T-VISA-miR-34a may provide a potentially useful, specific, and safe-targeted therapeutic approach for breast cancer.     

Microbial transglutaminase nanoflowers as an alternative nanomedicine for breast cancer theranostics

Breast cancer is the most common malignancy among women. With the aim of decreasing the toxicity of conventional breast cancer treatments, an alternative that could provide appropriate and effective drug utilization was envisioned. Thus, we contemplated and compared the in vitro effects of microbial transglutaminase nanoflowers (MTGase NFs) on breast cancer cells (MCF-7). Transglutaminase is an important regulatory enzyme acting as a site-specific cross-linker for proteins. With the versatility of MTGase facilitating the nanoflower formation by acting as molecular glue, it was demonstrated to have anti-cancer properties. The rational drug design based on a transglutaminase enzyme-assisted approach led to the uniform shape of petals in these nanoflowers, which had the capacity to act directly as an anti-cancer drug. Herein, we report the anti-cancer characteristics portrayed by enzymatic MTGase NFs, which are biocompatible in nature. This study demonstrated the prognostic and therapeutic significance of MTGase NFs as a nano-drug in breast cancer treatment. The results on MCF-7 cells showed a significantly improved in vitro therapeutic efficacy. MTGase NFs were able to exhibit inhibitory effects on cell viability (IC₅₀-8.23 μg ml⁻¹) within 24 h of dosage. To further substantiate its superior anti-proliferative role, the clonogenic potential was measured to be 62.8%, along with migratory inhibition of cells (3.76-fold change). Drastic perturbations were induced (4.61-fold increase in G0/G1 phase arrest), pointed towards apoptotic induction with a 58.9% effect. These results validated the role of MTGase NFs possessing a cytotoxic nature in mitigating breast cancer. Thus, MTGase bestows distinct functionality towards therapeutic nano-modality, i.e., nanoflowers, which shows promise in cancer treatment.

Development of a novel therapeutic nano-modality in the form of enzymatic transglutaminase nanoflowers; endowed with anti-cancerous action against breast cancers.     

Synergistic antitumoral effect of combination E gene therapy and Doxorubicin in MCF-7 breast cancer cells

The low effectiveness of conventional therapies to achieve the long-term survival of metastatic breast cancer patients calls for the development of novel options. Genes encoding cytotoxic proteins have been proposed as a new strategy to enhance the antiproliferative activity of drugs. Combined therapy using these genes and classical antitumoral drugs are under intensive study. The E gene from ?X174 encodes a membrane protein with a toxic domain that leads to a decrease in the tumour cell growth rate. With the aim of improving the anti-tumour effect on breast cancer cells of the currently used chemotherapeutic drugs (Paclitaxel, Docetaxel and Doxorubicin), we investigated the association of E suicide gene with these drugs. The effect of the combined therapy (gene therapy and cytotoxic) was determined by treating transfected MCF-7 cells and multicellular tumour spheroids (MTS) with drugs gradient concentrations. Our results showed that E gene has a direct oncolytic effect inducing a significant decrease in the proliferation rate of the MCF-7 cells. The E gene antitumoral activity was mediated by the induction of apoptosis (mitochondrial pathway). In addition, a significant enhancement of proliferation inhibition was observed when E gene transfection was associated with cytotoxic drugs in comparison to single treatments. The use of the combined therapy E gene-Doxorubicin obtained the greatest effect on the MCF-7 growth arrest. This therapeutic association also induced a significant enhancement of the MTS volume growth inhibition. Anti-tumour activity of the chemotherapeutic drugs classically used in the treatment of breast cancer was enhanced by E gene. Our in vitro results indicate that experimental therapeutic strategy based in the combined therapy E gene and cytotoxic drugs may be of potential therapeutic value as a new strategy for patients with advanced breast cancer.     

Novel tocotrienol-entrapping vesicles can eradicate solid tumors after intravenous administration

The therapeutic potential of tocotrienol, a vitamin E extract with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration. In this work, we demonstrated that novel transferrin-bearing, tocopheryl-based multilamellar vesicles entrapping tocotrienol significantly improved tocotrienol uptake by cancer cells overexpressing transferrin receptors. This led to a dramatically improved therapeutic efficacy in vitro, ranging from 17-fold to 72-fold improvement depending on the cell lines, compared to the free drug.In vivo, the intravenous administration of this novel tocotrienol formulation led to complete tumor eradication for 40% of B16-F10 murine melanoma tumors and 20% of A431 human epidermoid carcinoma tumors. Animal survival was improved by more than 20 days compared to controls, for the two tumor models tested. These therapeutic effects, together with the lack of toxicity, potentially make transferrin-bearing vesicles entrapping tocotrienol a highly promising therapeutic system as part as an anti-cancer therapeutic strategy.     

Performance comparison of two nucleic acid amplification systems for SARS‐CoV‐2 detection: A multi‐center study

BackgroundMany rapid nucleic acid testing systems have emerged to halt the development and spread of COVID‐19. However, so far relatively few studies have compared the diagnostic performance between these testing systems and conventional detection systems. Here, we performed a retrospective analysis to evaluate the clinical detection performance between SARS‐CoV‐2 rapid and conventional nucleic acid detection system.MethodsClinical detection results of 63,352 oropharyngeal swabs by both systems were finally enrolled in this analysis. Sensitivity (SE), specificity (SP), and positive and negative predictive value (PPV, NPV) of both systems were calculated to evaluate their diagnostic accuracy. Concordance between these two systems were assessed by overall, positive, negative percent agreement (OPA, PPA, NPA) and κ value. Sensitivity of SARS‐CoV‐2 rapid nucleic acid detection system (Daan Gene) was further analyzed with respect to the viral load of clinical specimens.ResultsSensitivity of Daan Gene was slightly lower than that of conventional detection system (0.86 vs. 0.979), but their specificity was equivalent. Daan Gene had ≥98.0% PPV and NPV for SARS‐CoV‐2. Moreover, Daan Gene demonstrated an excellent test agreement with conventional detection system (κ = 0.893, p = 0.000). Daan Gene was 99.31% sensitivity for specimens with high viral load (C _t < 35) and 50% for low viral load (C _t ≥ 35).ConclusionsWhile showing an analytical sensitivity slightly below than that of conventional detection system, rapid nucleic acid detection system may be a diagnostic alternative to rapidly identify SARS‐CoV‐2‐infected individuals with high viral loads and a powerful complement to current detection methods.     

S-nitrosylation: integrator of cardiovascular performance and oxygen delivery

Delivery of oxygen to tissues is the primary function of the cardiovascular system. NO, a gasotransmitter that signals predominantly through protein S-nitrosylation to form S-nitrosothiols (SNOs) in target proteins, operates coordinately with oxygen in mammalian cellular systems. From this perspective, SNO-based signaling may have evolved as a major transducer of the cellular oxygen-sensing machinery that underlies global cardiovascular function. Here we review mechanisms that regulate S-nitrosylation in the context of its essential role in “systems-level” control of oxygen sensing, delivery, and utilization in the cardiovascular system, and we highlight examples of aberrant S-nitrosylation that may lead to altered oxygen homeostasis in cardiovascular diseases. Thus, through a bird’s-eye view of S-nitrosylation in the cardiovascular system, we provide a conceptual framework that may be broadly applicable to the functioning of other cellular systems and physiological processes and that illuminates new therapeutic promise in cardiovascular medicine.     

Application of Phi29 Motor pRNA for Targeted Therapeutic Delivery of siRNA Silencing Metallothionein-IIA and Survivin in Ovarian Cancers

Ovarian cancer is a highly metastatic and lethal disease, making it imperative to find treatments that target late-stage malignant tumors. The packaging RNA (pRNA) of bacteriophage phi29 DNA-packaging motor has been reported to function as a highly versatile vehicle to carry small interference RNA (siRNA) for silencing of survivin. In this article, we explore the potential of pRNA as a vehicle to carry siRNA specifically targeted to metallothionein-IIa (MT-IIA) messenger RNA (mRNA), and compare it to survivin targeting pRNA. These two anti-apoptotic cell survival factors promote tumor cell viability, and are overexpressed in recurrent tumors. We find that pRNA chimeras targeting MT-IIA are processed into double-stranded siRNA by dicer, are localized within the GW/P-bodies, and are more potent than siRNA alone in silencing MT-IIA expression. Moreover, knockdown of both survivin and MT-IIA expression simultaneously results in more potent effects on cell proliferation in the aggressive ovarian tumor cell lines than either alone, suggesting that therapeutic approaches that target multiple genes are essential for molecular therapy. The folate receptor–targeted delivery of siRNA by the folate-pRNA dimer emphasizes the cancer cell–specific aspect of this system. The pRNA system, which has the capability to assemble into multivalent nanoparticles, has immense promise as a highly potent therapeutic agent.     

ORFV: A Novel Oncolytic and Immune Stimulating Parapoxvirus Therapeutic

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. Parapoxvirus ovis or Orf virus (ORFV) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent T_h-1-dominated immune response. Here, we show that live replicating ORFV induces an antitumor immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumoricidal natural killer (NK) cells. We have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer.     

Polydopamine-doped virus-like structured nanoparticles for photoacoustic imaging guided synergistic chemo-/photothermal therapy

The therapeutic diagnosis effect of cancer commonly depends on the cellular uptake efficiency of nanomaterials. However, the morphology of nanomaterials significantly affects cellular uptake capability. Herein, we designed a polydopamine-doped virus-like structured nanoparticle (GNR@HPMO@PVMSN) composed of a gold nanorod (GNR) core, hollow periodic mesoporous organosilica (HPMO) shell and polydopamine-doped virus-like mesoporous silica nanoparticle (PVMSN) outer shell. Compared with conventional gold nanorod@hollow periodic mesoporous organosilica core–shell nanoparticles (GNR@HPMO), GNR@HPMO@PVMSN with its virus-like structure was proved to enhance the efficiency of cellular uptake. GNR@HPMO@PVMSN with the virtues of high photothermal conversion efficiency and good photoacoustic imaging (PAI) ability was expected to be a promising nanotheranostic agent for imaging guided cancer treatment. The experiments in vitro and in vivo proved that GNR@HPMO@PVMSN had good biocompatibility as well as photothermal conversion ability. In addition, DOX loading and pH-/NIR-response DOX release abilities of GNR@HPMO@PVMSN were also verified in vitro. Therefore, the GNR@HPMO@PVMSN offers a promising strategy for PAI directed synergistic chemo-/photothermal therapy, which improves the therapeutic effect of the nanomaterial on tumors. This work explores the effects of rough surfaces on cellular uptake and provides a versatile theranostic platform for biomedical applications.

The therapeutic diagnosis effect of cancer commonly depends on the cellular uptake efficiency of nanomaterials.     

Development of a prostate-specific promoter for gene therapy against androgen-independent prostate cancer

Androgen ablation has been the standard treatment for metastasized prostate cancer. In most cases, however, prostate cancer cells eventually lose androgen dependency and become refractory to the conventional endocrine therapy. Androgen-independent prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells. Prostate-specific promoters such as prostate-specific antigen and rat probasin (rPB) promoters have been examined in the development of gene therapy targeted to prostate cancer. However, those promoters require binding of the androgen–AR complex to the androgen-response element and are active only in the androgen-dependent prostate cancer cell lines and not in the androgen-independent cell lines. To target transgene expression in androgen-independent prostate cancer, we designed a prostate-specific promoter that is activated by the retinoids–retinoid receptor complex instead of the androgen–AR complex. The modified rPB promoters expressed transgenes in response to retinoid in both androgen-dependent and androgen-independent prostate cancer cells and not in other cancer cell lines or in human normal cells, in vitro and in vivo. Furthermore, the combination of retinoid treatment and adenovirus-mediated gene transfer of the modified rPB-driven HSV-tk gene resulted in a significant growth suppression of the androgen-independent prostate cancer cells in the presence of the prodrug ganciclovir. This study suggests that tailoring of the hormone-responsive elements may offer a new therapeutic opportunity against the hormone-refractory stage of prostate cancer.     

Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets

The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer.     

Vaginal delivery of siRNA using a novel PEGylated lipoplex-entrapped alginate scaffold system

Sustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administered intravaginally are unable to reach the vaginal epithelium under normal physiological conditions. Here we have developed a novel alginate scaffold system containing muco-inert PEGylated lipoplexes to provide a sustained vaginal presence of lipoplexes in vivo and to facilitate the delivery of siRNA/oligonucleotides into the vaginal epithelium. These PEGylated lipoplex-entrapped alginate scaffolds (PLAS) were fabricated using a freeze-drying method and the entrapment efficiency, release rate, and efficacy were characterized. We demonstrated that the PLAS system had an entrapment efficiency of ~ 50%, which released PEGylated lipoplexes gradually both in vitro and in vivo. While the presence of alginate diminished the cell uptake efficiency of PEGylated lipoplexes in vitro, as expected, we showed a six-fold increase their uptake into the vaginal epithelium compared to existing transfection systems following intravaginal administration in mice. A significant knockdown of Lamin A/C level was also observed in vaginal tissues using siLamin A/C-containing PLAS system in vivo. Overall, our results indicated the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium.     

A doxorubicin–platinum conjugate system: impacts on PI3K/AKT actuation and apoptosis in breast cancer cells

In recent years, the development of a nano-conjugate system for drug delivery applications has gained attention among researchers. Keeping this in mind, in this study, we developed a doxorubicin–platinum conjugate system that targeted breast cancer cell lines. To achieve this, we developed platinum nanoparticles using polyvinylpyrrolidone (PVP). High resolution-transmission electron microscopy (HR-TEM) revealed the occurrence of octopod-shaped platinum nanoparticles. Subsequently, doxorubicin (DOX) was conjugated on the surface of the as-prepared platinum octopods via an in situ stirring method. The physicochemical characterization of the doxorubicin–platinum conjugate system revealed that the PVP of PtNPs interacts with the NH₂ group of doxorubicin via electrostatic interaction/hydrogen bonding. Besides, the doxorubicin–platinum conjugate system exhibited a sustained drug release profile within the cancer cells. Furthermore, the evaluation of the in vitro anticancer efficacy of the doxorubicin–platinum conjugate system in breast cancer cells (MCF-7 and MDA-MB-231) unveiled the induction of apoptosis via intracellular ROS and DNA damage, rather than free DOX and PtNPs. Remarkably, we also perceived that the doxorubicin–platinum conjugate system was strong enough to down-regulate the PI3K/AKT signalling pathway. As a result, the tumour suppressor gene PTEN was activated, which led to the stimulation of a mitochondrion-based intrinsic apoptotic pathway and its downstream caspases, triggering cell death. Hence, our findings suggested that a biologically stable doxorubicin–platinum conjugate system could be an imperative therapeutic agent for anticancer therapy in the near future.

In recent years, the development of a nano-conjugate system for drug delivery applications has gained attention among researchers.     

Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR.     

GOLGA2/GM130, cis-Golgi Matrix Protein,is a Novel Target of Anticancer Gene Therapy

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras^LA1 mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.