Naked DNA Gene Transfer Research Articles

Introduction--translational research trends: urologic gene therapy.

GENE THERAPY, a rapidly growing field of interest in the basic sciences, is a relatively new area of research and clinical application within urology. With increasing technological advances, the number of gene therapy-based clinical trials has increased dramatically. As such, translational research involving gene therapy for a variety of urologic diseases has also increased. In this issue, the contributors present a review of the proceedings from the First Meeting of Japanese Urological Association for Gene Therapy, held on November 20, 1999 in Okayama, Japan. An overview of the meeting was presented by the society’s first president, Dr. Kumon. A guest speaker, Dr. Asano, defined gene therapy translational research objectives and emphasized the importance of applying appropriate basic science concepts in selected preclinical models. He also stressed the careful design of good manufacturing principles and clinical trial indications, patient selection, and logistics. Specific urologic applications of gene therapy were also presented and discussed at the meeting. Gene therapy strategies can essentially be classified into three basic categories: (1) immune augmentation; (2) as a mode of delivery of cytotoxic agents; and (3) specific gene replacement. One of the earliest gene therapy strategies to augment the immune system attempted to deliver high concentrations of cytokines to localize the host immune response. Two basic cytokine delivery strategies are currently utilized: one is to vaccinate patients with tumor cells expressing a particular cytokine, and the other is to directly express cytokines within the tumor. Vaccination with cytokine-expressing tumor cells enhances the efficacy of tumor cell vaccination strategies in rodent models. The most widely utilized cytokine in current clinical trials is granulocyte-macrophage colony-stimulating factor (GM-CSF). Kawai et al., review the data of trials using GM-CSF expressing autologous tumor cells in renal cell carcinoma, malignant melanoma, and prostate cancer. Thus far, despite demonstrating histologic confirmation of host immune response at the sites of immunization, clinically evident tumoricidal responses of the in situ tumor are not yet evident. However, multiple variables need to be addressed before disregarding this approach as a viable gene therapy strategy. These variables include vaccination dosage and frequency as well as the in situ tumor burden at the time of vaccination. An alternative method to augment the host immune response with local cytokines is direct expression of the cytokine with the tumor itself. Nishitani et al. review cytokine gene therapy using naked DNA gene transfer techniques. They presented promising preliminary preclinical data using interleukin-12 (IL12) transfected in situ into subcutaneous renal cell carcinoma tumors in a murine model. One potential limitation of this strategy centers on efforts to ensure delivery of the cytokine gene into the tumor. In particular, in situ targeting of renal cell carcinoma cells versus normal kidney cells may present a clinical problem. This could be overcome if the cytokine gene could be expressed in a tumor-specific manner. Nonetheless, gene therapy immune augmentation is one of the most promising immunotherapy strategies. An alternative gene therapy strategy to target tumors is to use cytotoxic genes or viruses. Cytotoxic gene therapy primarily locally targets tumors but may also have bystander and/or immune-stimulating effects in surrounding cells. The most widely utilized cytotoxic gene is the herpes simplex virus thymidine kinase (HSV-TK) gene. The HSV-TK gene converts a nontoxic metabolite into a toxic metabolite, killing cells expressing the HSV-TK gene, and thus this strategy is aptly named “suicide” gene therapy. Nasu et al., review the usage of the HSV-TK gene in gene therapy trials. Tumor-specific expression of the HSV-TK gene may limit toxicity to normal tissues lacking HSV-TK expression. Shirakawa et al. presented data using tissue-specific expression of the HSV-TK gene using the prostate-specific antigen (PSA) and osteocalcin promoters. The PSA promoter restricts expression primarily to prostate tissues, whereas the osteocalcin promoter targets prostate cancer and bone metastasis. Preliminary data of their Phase I clinical trial, using adenovirus encoding the osteocalcin promoter directed expression of the HSV-TK gene, demonstrated this strategy could be used with minimal toxicity. Cytotoxic gene therapy also includes the usage of cytotoxic viruses that target rapidly dividing tumor cells. Oyama et al. reviewed the usage of a replication competent herpes simplex virus, G207. The G207 virus replicates in (and kills) cells that are rapidly dividing, such as tumor cells, but does not appear to replicate in normal cells. Although cytotoxic agent gene therapy approaches have demonstrated promising results for local tumor control, further studies are warranted to investigate systemic bystander and/or immune effects.

Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy

Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy.

Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector

To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 μl over 10 min. of either HSVlac (1×10 5 b.f.u), AdLac (1×10 5 p.f.u), Tfx-10 or Tfx-20 (2.6 μg DNA in 2.0 μl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 μg/ μl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of β-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect β-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of β-gal in HSVlac infected animals ( p=0.0029); a 29% increase in Adlac infected animals ( p=0.01); a 56% increase in Tfx-10 transduced animals ( p=0.04); a 24% increase in Tfx-20 transduced animals ( p=0.01); and a 69% increase in naked DNA gene transfer ( p=0.05). Total β-galactosidase activity was greatest in HSVlac infected mice followed by Adlac>Tfx-20™>Tfx-10™=naked DNA.

Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA

Previous studies have demonstrated that arterial gene transfer of naked DNA encoding for a secreted protein may permit modulation of the host phenotype despite a low transfection efficiency. Acidic fibroblast growth factor (aFGF) is an angiogenic growth factor, but is not secreted by intact cells. In the current study, we investigated the hypothesis that addition of a hydrophobic leader sequence to achieve active secretion of the gene product would permit therapeutic angiogenesis following arterial gene transfer of naked DNA encoding for aFGF. Ten days following surgical induction of unilateral hindlimb ischemia, New Zealand white rabbits were randomized to intra-arterial gene transfer with one of three plasmids: p267 (encoding non-secreted aFGF, n = 10), pMJ35 (encoding secreted aFGF) (n = 10), or 500 micrograms of pGSVLacZ (control, n = 10) (500 micrograms each). All animals were studied at 30 days post-gene transfer for evidence of therapeutic angiogenesis. pMJ35 transfectants had more angiographically visible collaterals (angiographic score = 0.76 +/- 0.02) than either p267 (0.55 +/- 0.02, p < 0.01) or LacZ (0.47 +/- 0.02, p < 0.001). Limb blood pressure ratio for pMJ35 was 0.88 +/- 0.02 vs. 0.68 +/- 0.04 for p267 (p < 0.01) and 0.57 +/- 0.04 for LacZ (p < 0.001). Vascular resistance was significantly lower in the pMJ35 group, compared with that in pGSVLacZ group, both in resting state (3.2 +/- 0.4 vs. 7.4 +/- 1.4 respectively, p < 0.05) and after the administration of nitroprusside. Capillary density (per mm2) was also superior in pMJ35 group (274 +/- 10) vs. p267 (204 +/- 9, p < 0.01) and LacZ (177 +/- 6, p < 0.001). The paracrine effects of a secreted gene product may obviate the need for adjunctive vectors in strategies of arterial gene therapy.

Apoptosis in Xenopus tadpole tail muscles involves Bax-dependent pathways.

Apoptosis is a fundamental mechanism implicated in normal development. One of the most spectacular developmental events involving apoptosis is tail regression during amphibian metamorphosis. We analyzed how thyroid hormone (3, 5, 3'-triiodothyronine, T3), the orchestrator of metamorphosis, affects expression and function of the proapoptotic gene Bax in the tail muscle of free-living Xenopus tadpoles. During natural metamorphosis Bax mRNA was expressed in tail muscles and was spatially correlated with apoptosis. Precocious treatment of tadpoles with T3 induced Bax expression and apoptosis. To verify that Bax expression was causally related to apoptosis we used a naked DNA gene transfer method to express Bax in the dorsal tail muscle. This induced apoptosis, and the process was exacerbated by T3 treatment. To determine whether T3 effects on Bax expression involved transcriptional regulation, we injected a Bax promoter sequence into dorsal and caudal tail muscles. In the dorsal muscle, T3 treatment did not affect transcription from the Bax promoter. However, in the caudal muscle, T3 treatment significantly increased Bax transcription. We conclude that T3-induced apoptosis in Xenopus tadpole tail muscles involves Bax-activating and Bax-synergis tic mechanisms. These programs are induced in spatially and temporally distinct manners.