Jijun Wang

Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery

Chitosan is a polysaccharide that demonstrates much potential as a gene delivery system. The ability of a commercially available chitosan and depolymerized chitosan oligomers to condense plasmid was determined using TEM and microtitration calorimetry, while the diameter and stability of the resultant complexes were measured using laser light scattering. Selected complexes were physically stable to challenge with both serum and salt solutions. Parameters such as chitosan molecular weight, plasmid concentration and charge ratio influenced such stability. The effect of including a pH-sensitive endosomolytic peptide on the physicochemical properties of the complex was determined. The presence of a pH-sensitive endosomolytic peptide enhanced the levels of reporter gene expression in Cos-1 cells 4-fold. A selected complex containing a lytic peptide was administered in the upper small intestine and colon of rabbits, and reporter gene expression was measured in defined intestinal tissues. Reporter gene expression was enhanced in defined intestinal tissues, although levels of expression remained low. The combination of strong complex stability and low in vivo expression levels suggest that uptake and/or decomplexation, but not endosomal release, may be the critical rate-limiting steps in the uptake process.

Systemic inhibition of tumor growth and tumor metastases by intramuscularadministration of the endostatin gene

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of angiostatic factors should be a viable approach for cancer gene therapy. Endostatin, a potent angiostatic factor, was expressed in mouse muscle and secreted into the bloodstream for up to 2 weeks after a single intramuscular administration of the endostatin gene. The biological activity of the expressed endostatin was demonstrated by its ability to inhibit systemic angiogenesis. Moreover, the sustained production of endostatin by intramuscular gene therapy inhibited both the growth of primary tumors and the development of metastatic lesions. These results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers.

Induction of apoptosis in frog virus 3-infected cells.

The ability of frog virus 3 (FV3), the type species of the family Iridoviridae, to induce apoptosis was examined by monitoring DNA cleavage, chromatin condensation, and cell-surface expression of phosphotidylserine (PS) in fathead minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h postinfection. As with some other viruses, FV3-induced apoptosis did not require de novo viral gene expression as both heat-inactivated and UV-inactivated virus readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK, suggesting that virus infection triggers programmed cell death through activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK cells as monitored by TUNEL and annexin V binding assays. To determine whether FV3, similar to other large DNA viruses, encoded proteins that block or delay apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether or not shocked cells were previously infected with FV3, indicating that infection with FV3 did not block apoptosis induced by osmotic shock in FHM cells. The above results demonstrate that iridoviruses triggered apoptosis and that the induction of programmed cell death did not require viral gene expression. However, it remains to be determined if virion attachment to target cells is sufficient to induce cell death, or if apoptosis is triggered directly or indirectly by one or more virion-associated proteins.

Synergistic effect of formulated plasmid and needle-free injection for genetic vaccines.

AbstractPurpose. A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC™) polymer system and administered with Medi-Jector™, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. Methods. Human growth hormone (hGH) or bacterial β-galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. β-galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. Results. Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15−20-fold higher than needle injection when pDNA was complexed with PVP, and only 3−4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by β-galaclosidase expression, following i.m. injection of pCMV-β-galaclosidase/PVP, was not significantly different between needle and NFID-injected groups. Conclusions. These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.