Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing S gene from porcine epidemic diarrhea virus and VP7 gene from porcine rotavirus.

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Developments in Intralesional Therapy for Metastatic Melanoma.

BACKGROUND: Locoregional advanced melanoma poses a complex clinical challenge that requires a multidisciplinary, patient-centered approach. Numerous agents have been studied for their suitability as intralesional therapy in the past decades, but few have successfully completed phase 3 clinical trial testing. METHODS: The relevant medical literature was searched for articles regarding use of intralesional therapies in metastatic melanoma. Therapies with data from phase 2 or higher studies were selected for review. This review also summarizes the mechanisms of action, adverse-event profiles, and clinical data for these agents. RESULTS: Intralesional therapies demonstrate promising effects in select patients with advanced melanoma. The optimal approach should be individually tailored and consist of a combination of intralesional therapies, regional perfusions, systemic immunotherapies, targeted therapies, and surgery, if necessary. CONCLUSIONS: Due to its relatively good local response rates and tolerable adverse-event profile, intralesional therapy may be a treatment option for select patients with unresectable, locally advanced or metastatic melanoma.

In vivo delivery of bionanocapsules displaying Phaseolus vulgaris agglutinin-L4 isolectin to malignant tumors overexpressing N-acetylglucosaminyltransferase V.

Metastasis is a key aspect of tumor malignancy, and several malignant tumors show expression of various mature N-type glycans. In particular, beta1-6 branching N-acetylglucosamine (GlcNAc) is abundantly expressed as a part of high-mannose glycans in various highly metastatic cancers. Phaseolus vulgaris agglutinin-L(4) isolectin (L(4)-PHA), which adheres to beta1-6 GlcNAc specifically, has been used for in situ cancer diagnosis. Bionanocapsules (BNCs), hollow particles with a diameter of approximately 80 nm and composed of hepatitis B surface antigen (HBsAg) and a lipid bilayer, have been developed as human liver-specific nanocapsules for in vivo drug delivery system. In this study, we have generated L(4)-PHA-displaying BNCs (PHA-BNCs) and examined whether L(4)-PHA could retarget the BNCs to malignant tumors as a "biosensor" distinguishing tumor metastaticity. Fluorescence-labeled PHA-BNCs injected systemically into a mouse xenograft model were found to accumulate in beta1-6 GlcNAc-expressing malignant tumors. The PHA-BNCs were able to deliver DNA to the malignant cancer cells. These results open up the possibility of using L(4)-PHA lectin as a targeting molecule in a drug delivery system, and of using PHA-BNCs as a novel nanodevice for malignant tumor-specific bioimaging and drug delivery.

Optimization of 25 kDa linear polyethylenimine for efficient gene delivery.

A 25-kDa linear polyethylenimine (25 kDa L-PEI) has proven to be efficient and versatile agent for gene delivery. Therefore, we determined the optimal transfection conditions of 25 kDa L-PEI and examined whether it has comparable transfection efficiency with other commercially available reagents, ExGen 500, LipofectAMINE 2000, and Effectene by using EGFP expression vector in different cell lines. Transfection efficiency and cytotoxicity were measured by flow cytometry. First of all, we determined the optimal ratio of nitrogen to phosphorous (N/P) and DNA concentration. With the increase of N/P ratio and DNA amounts, transfection efficiency increased with a slight variation in cell types. The optimal amounts of 25 kDa L-PEI were determined at N/P ratio 40 and DNA concentration varied among the cell types. In addition, 25 kDa L-PEI worked efficiently and was less toxic than other reagents. However, the efficiency and toxicity of all these reagents varied according to cell types as well as the ratio of DNA to reagents and the amounts of DNA. Our finding illustrates the importance of optimal transfection conditions of 25 kDa L-PEI to obtain maximal transgene expression with less cytotoxicity. Importantly, the optimization of those conditions may make possible to perform transfection cost-effectively and efficiently.

Enhanced gene delivery to PC12 cells by a cationic polypeptide.

Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.

Gene gun bombardment with gold particles displays a particular Th2-promoting signal that over-rules the Th1-inducing effect of immunostimulatory CpG motifs in DNA vaccines.

The mode of administering a DNA vaccine can influence the type of immune response induced by the vaccine. For instance, application of a DNA vaccine by gene gun typically induces a Th2-type reaction, whereas needle inoculation triggers a Th1 response. It has been proposed that the approximately 100-fold difference in the amount of DNA administered by these two methods is the critical factor determining whether a Th1 or a Th2 response is made. To test this hypothesis, BALB/c mice were immunized with two plasmid DNA constructs encoding different proteins (OspC/ZS7 of Borrelia burgdorferi and Bet v 1a, the major birch pollen allergen). Both vaccines were applied by needle and/or by gene gun immunization at the same and at different sites of injection. An analysis of the IgG subclass distribution and measurement of IFN-gamma after antigen-specific lymphoproliferation does not support the widely accepted view that Th2-type immunity induced by gene gun application is solely due to the low amount of injected plasmid DNA thus falling below the critical concentration of CpG motifs necessary for Th1-induction. Furthermore, the data also indicate a strong and even systemic adjuvant effect of the gene gun shot itself.

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain.

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X(3) and P2X(2/3) receptors and between two P2X receptors in rat sensory neurones.

1. We have compared the antagonist activity of trinitrophenyl-ATP (TNP-ATP) and diinosine pentaphosphate (Ip(5)I) on recombinant P2X receptors expressed in Xenopus oocytes with their actions at native P2X receptors in sensory neurones from dorsal root and nodose ganglia. 2. Slowly-desensitizing responses to alpha,beta-methylene ATP (alpha,beta-meATP) recorded from oocytes expressing P2X(2/3) receptors were inhibited by TNP-ATP at sub-micromolar concentrations. However, Ip(5)I at concentrations up to 30 microM was without effect. 3. Nodose ganglion neurones responded to alpha,beta-meATP with slowly-desensitizing inward currents. These were inhibited by TNP-ATP (IC(50), 20 nM), but not by Ip(5)I at concentrations up to 30 microM. 4. In DRG neurones that responded to ATP with a rapidly-desensitizing inward current, the response was inhibited by TNP-ATP with an IC(50) of 0.8 nM. These responses were also inhibited by Ip(5)I with an IC(50) of 0.1 microM. Both antagonists are known to inhibit homomeric P2X(3) receptors. 5. Some DRG neurones responded to alpha,beta-meATP with a biphasic inward current, consisting of transient and sustained components. While the transient current was abolished by 1 microM Ip(5)I, the sustained component remained unaffected. 6. In conclusion, Ip(5)I is a potent antagonist at homomeric P2X(3) receptors but not at heteromeric P2X(2/3) receptors, and therefore should be a useful tool for elucidating the subunit composition of native P2X receptors.

Process and product development in the manufacturing of molecular therapeutics.

In the development of molecular therapies, a great deal of attention has focused on tissue targets, gene delivery vectors, and expression cassettes. In order to become an approved therapy, however, a molecular therapeutic has to pass down the same product registration pathway as any other biological product. Moving from research into industrial production requires careful attention to regulatory, manufacturing and quality concerns. Early work on developing and characterizing robust and scaleable manufacturing processes will ultimately be rewarded by ease of implementation as the product is successful in clinical trials. Regulatory agencies require solid process and product characterization studies to demonstrate control and understanding of the molecular therapeutic. As the gene therapy industry matures, standards will continue to rise, creating an industry that is capable of producing safe, high-quality and effective therapies for many of the world's most difficult disease targets.