Isolation of Fully Human Antagonistic RON Antibodies Showing Efficient Block of Downstream Signaling and Cell Migration.

RON belongs to the c-MET family of receptor tyrosine kinases. As its well-known family member MET, RON and its ligand macrophage-stimulating protein have been implicated in the progression and metastasis of tumors and have been shown to be overexpressed in cancer. We generated and tested a large number of human monoclonal antibodies (mAbs) against human RON.

ErbB2 genetic cancer vaccine in nonhuman primates: relevance of single nucleotide polymorphisms.

Aberrant Her2/neu expression is associated with the development of epithelial-derived human carcinomas and for this reason it is considered a good target for immunologic intervention. To define methods to circumvent immunologic tolerance and to elicit immunity against the Her2/neu tumor-associated antigen in a suitable animal model, we have isolated the cDNA encoding the rhesus monkey homolog of human Her2/neu (RhErbB2) to construct DNA plasmids and adenoviral vectors for the development of a cancer vaccine against this protein. To further increase the immunogenic potency of these vectors, a synthetic codon-optimized RhErbB2 cDNA (RhErbB2OPT) was constructed and characterized.

Synergistic effect of gene-electro transfer and adjuvant cytokines in increasing the potency of hepatitis C virus genetic vaccination.

Background: Gene electro-transfer (GET) increases DNA uptake and expression by muscle cells following intramuscular plasmid injection. This technology has been used to increase the production of therapeutic proteins, such as cytokines and growth factors, and to improve immunization efficiency following the injection of antigen-encoding plasmids.

Methods: Hepatitis C virus (HCV) E2 and cytokine encoding plasmids were co-injected in the mouse quadriceps with or without GET and vaccination outcome was monitored by analysis of antigen-specific cellular-mediated or antibody-mediated immunity.

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag.

In vivo DNA gene electro-transfer: a systematic analysis of different electrical parameters.

Background: Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction levels with several reporter genes.

Methods: In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production.

Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates.

Background: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates.

Gene electro-transfer improves transduction by modifying the fate of intramuscular DNA.

Background: Intramuscular gene delivery through injection of plasmid DNA has long been considered a promising approach for safe and simple in vivo gene expression for vaccination and gene therapy purposes. Recently, intramuscular gene delivery has been improved by applying low-voltage electric pulses after plasmid injection, a procedure that has been variably called gene electro-transfer, in vivo electroporation or electrical stimulation. Different types of electrical treatments have been used with excellent results both in terms of transgene expression levels and immunization outcome.

Construction of an rtTA2(s)-m2/tts(kid)-based transcription regulatory switch that displays no basal activity, good inducibility, and high responsiveness to doxycycline in mice and non-human primates.

The tetracycline (Tc)-dependent system in its "on" version (rtTA system) displays a baseline activity in the uninduced state, severely limiting its potential applicability in human gene therapy. So far, two different strategies to circumvent this limitation have been described. On one side, co-expression of the tetracycline regulated repressor tTS(kid) has proved capable of substantially reducing the baseline activity of rtTA.

Hyaluronidase increases electrogene transfer efficiency in skeletal muscle.

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment.