Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta.

BACKGROUND: Gene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. METHODS AND RESULTS: Aortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0. 57+/-0.04. VEGF-C gene transfer reduced I/M to 0.38+/-0.02 (P:<0.05 versus lacZ group). I/M in VEGF-A-treated animals was 0.49+/-0.17 (P:=NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73+/-0.16, the VEGF-C group 0.44+/-0.14, and the VEGF-A group 0. 63+/-0.21 (P:=NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9% saline-treated animals. CONCLUSIONS: Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.

Thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses.

Herpes simplex virus thymidine kinase (HSV tk) gene therapy combined with ganciclovir (GCV) medication is a potential new method for the treatment of malignant glioma. We have used both retrovirus-packaging cells (PA317/tk) and adenoviruses (Adv/tk) for gene therapy for malignant glioma. Retrovirus-packaging cells were used for eight tumors in seven patients and adenoviruses were used for seven tumors in seven patients. As a control group, seven tumors in seven patients were transduced with lacZ marker gene 4-5 days before tumor resection. Safety and efficacy of the gene therapy were studied with clinical evaluation, blood and urine samples, MRI follow-up, and survival of the patients. Four patients with adenovirus injections had a significant increase in anti-adenovirus antibodies and two of them had a short-term fever reaction. Frequency of epileptic seizures increased in two patients. No other adverse events possibly related to gene therapy were detected. In the retrovirus group, all treated gliomas showed progression by MRI at the 3-month time point, whereas three of the seven patients treated with Adv/tk remained stable (p < 0.05). Mean survival times for retrovirus, adenovirus, and control groups were 7.4, 15.0, and 8. 3 months, respectively. The difference in the survival times between the adenovirus and retrovirus groups was significant (p < 0.012). It is concluded that HSV tk gene therapy is safe and well tolerated. On the basis of these results further trials are justified, especially with adenovirus vectors.

Gene encoding the catalytic subunit p110beta of human phosphatidylinositol 3-kinase: cloning, genomic structure, and screening for variants in patients with type 2 diabetes.

Phosphatidylinositol (PI) 3-kinase is a key signaling molecule in insulin-stimulated glucose transport. Therefore, we investigated the catalytic subunit p110beta, of human PI 3-kinase as a candidate gene for type 2 diabetes. Human p110beta gene was cloned from the placental genomic library. All 22 exons, intronic regions flanking the exons and 1.5 kb of the proximal/5' region of the p110beta gene, were screened for variants by single-strand conformation polymorphism analysis in 79 Finnish patients with type 2 diabetes . Allele frequencies of the variants were also determined in 77 nondiabetic control subjects. No variants were found in exons in diabetic patients. However, we identified two nucleotide polymorphisms in the proximal/5' region of the p110beta gene and a variation in the number of 2-bp repeat sequence (TA)n in intron 4. The allele frequencies did not differ between diabetic and control subjects. Our results may indicate that the catalytic subunit p110beta of PI 3-kinase plays such a fundamental role in the insulin-signaling pathway that structural variants are not likely to exist in that gene. The importance of the polymorphisms in the proximal/5' region of the p110beta gene for insulin signaling remains to be determined.

Beta-galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses.

Both retro- and adenovirus-mediated gene therapy have been suggested as a novel approach to the treatment of malignant brain tumors. However, little information is available about the gene transfer efficiency in human malignant glioma in vivo. We compared the feasibility and safety of retrovirus- and adenovirus-mediated beta-galactosidase gene transfer in human malignant glioma. Beta-galactosidase gene was transferred to 10 patients with malignant glioma via a catheter inserted into the tumor. The catheter was left in place until the tumor resection. To maximize gene transfer efficiency, gene transfer vectors (BAG retroviruses, titer, 6 x 10(5) CFU; and adenoviruses, titer from 3 x 10(8) to 3 x 10(10) PFU) were injected into the tumor via the catheter once a day for three consecutive days, followed by tumor resection 1-2 days later. Tumor was resected in such a way that the catheter was still in place inside the tumor, which permitted accurate histological analysis of the transduced tumors. X-Gal staining for beta-galactosidase activity was used to study gene transfer efficiency and distribution of the marker gene. Beta-galactosidase gene transfer was well tolerated with both vectors. Except for two patients with clear increases in serum adenovirus antibody titers, no adverse tissue responses or systemic complications were noticed in any of the patients. Gene transfer was successful in all patients. Gene transfer efficiency varied between <0.01 and 4% with retroviruses and between <0.01 and 11% with adenoviruses. However, the transgene activity was not evenly distributed in the tumors. Both glioma cells and endothelium in the tumor blood vessels were transduced with retro- and adenovirus vectors. In conclusion, the safety and feasibility of in vivo gene transfer to human malignant glioma was established with retro- and adenovirus vectors. Adenoviruses were more efficient than retroviruses in achieving in vivo gene transfer. Transduction of endothelial cells may have important consequences for the proposed treatment strategies and selection of treatment genes. The results justify clinical gene therapy trials for malignant glioma.

Adenovirus-mediated gene transfer to lower limb artery of patients with chronic critical leg ischemia.

Arterial gene transfer offers a promising new approach for the treatment of vascular disorders. However, no data are available about the gene transfer efficiency in human arteries in vivo. The aim of this study was to evaluate the safety and feasibility of catheter-mediated adenoviral gene transfer in human peripheral arteries. Ten patients (8 females, 2 males, mean age 80 +/- 8 years) suffering from chronic critical leg ischemia with a prior decision for amputation were recruited in the study. Gene transfer was performed in eight patients in conjunction with a conventional percutaneous transluminal angioplasty, using a perfusion coil balloon catheter. Two patients served as controls. Increasing concentrations of replication-deficient adenoviruses (titers from 1 x 10(8) to 4 x 10(10) PFU) containing a nuclear-targeted beta-galactosidase marker gene were administered into the arteries over 10 min via the catheter. Amputations were performed 20 to 51 hr after the procedures and gene transfer efficiency was evaluated in the transduced arteries using X-Gal staining for beta-galactosidase activity. Beta-galactosidase gene transfer was well tolerated and no adverse tissue responses or systemic complications were observed in any of the patients. Gene transfer was successful in six of the eight patients. Gene transfer efficiency varied between 0.04 and 5.0% of all arterial cells. Transgene expression was detected in smooth muscle cells, endothelial cells, and macrophages and in tunica adventitia. However, transgene activity was not evenly distributed in the arterial wall and no transgene activity was found beneath advanced atherosclerotic lesions. The safety and feasibility of in vivo gene transfer by adenoviral vectors to human peripheral arteries were established. Although improvements are still required in gene transfer efficiency, these findings suggest that adenoviruses can be used to deliver therapeutically active genes into human arteries.