Bile-duct proliferation as an unexpected side-effect after AAV2-LDLR gene transfer to rabbit liver.

Familial hypercholesterolemia (FH) is an inherited disease of lipoprotein metabolism caused by a defect in the LDL receptor (LDLR) leading to severe hypercholesterolemia, and associated with an increased risk of coronary heart disease and myocardial infarction. We have developed a gene therapy protocol for FH using AAV2, AAV9 and lentiviral vectors and tested safety and efficacy in LDL receptor deficient Watanabe Heritable Hyperlipidemic rabbits. We show that LV-LDLR produced a significant long-lasting decrease in total serum cholesterol whereas AAV9-LDLR resulted only in a transient decrease and AAV2-LDLR failed to reduce serum cholesterol levels. A significant pathological side effect, bile-duct proliferation, was seen in the liver of AAV2-LDLR rabbits associated with an increased expression of Cyr61 matricellular protein. Special attention should be given to liver changes in gene therapy applications when genes affecting cholesterol and lipoprotein metabolism are used for therapy.

A single-surgeon randomized trial comparing three meshes in lichtenstein hernia repair: 2- and 5-year outcome of recurrences and chronic pain.

BACKGROUND: Chronic pain may be a major long-term problem related to mesh material and operative trauma in inguinal hernioplasty. STUDY DESIGN: Lichtenstein hernioplasty was performed under local anaesthesia in 312 patients by the same surgeon and technique between 2003 and 2005. The patients were randomized to receive a partly absorbable polypropylene-polyglactin mesh (Vypro II(®) 50 g/m(2), 104 hernias), a lightweight polypropylene mesh (Premilene Mesh LP(®) 55 g/m(2), 107 hernias) or a conventional densely woven polypropylene mesh (Premilene(®) 82 g/m(2), 101 hernias). The 2- and 5-year recurrences and pain scores were analysed. RESULTS: Patient's characteristics and the mean duration of operation (30-32 min) were similar between the three groups. After two years, there were 6 recurrences (2 in each group) of which 3 patients were re-operated. A feeling of a foreign body and sensation of pain were comparable with all meshes. After five years, overall recurrence rate was 10/312 (3.2%) with only 4 re-operations. A feeling of a foreign body (6.5-8.1%), chronic pain (13-23%) as well as use of analgesics (0-2.9%) were similar in all groups. CONCLUSION: There were no statistical differences between the three meshes in pain, a feeling of a foreign body or use of analgesics after 5 years of Lichtenstein hernioplasty, when the same surgeon operated all patients with exactly the same surgical technique. CLINICAL TRIAL REGISTER: NCT01295437.

Antiangiogenic gene therapy with soluble VEGF-receptors -1, -2 and -3 together with paclitaxel prolongs survival of mice with human ovarian carcinoma.

We compared effects of antiangiogenic gene therapy with a combination of soluble sVEGFR-1, sVEGFR-2 and sVEGFR-3 to chemotherapy with carboplatin and paclitaxel and to antiangiogenic monoclonal anti-VEGF-antibody bevacizumab in an intraperitoneal ovarian cancer xenograft model in mice (n = 80). Gene therapy was also combined with chemotherapy. Therapy was initiated when sizable tumors were confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously (2 × 109 pfu), while chemotherapy and monoclonal anti-VEGF-antibody were dosed intraperitoneally. The study groups were as follows: AdLacZ control (n = 21); combination of AdsVEGFR-1, -2 and -3 (n = 21); combination of AdsVEGFR-1, -2, -3 and paclitaxel (n = 9); bevacizumab (n = 14); paclitaxel (n = 9) and carboplatin (n = 5). Effectiveness was assessed by survival time and surrogate measures such as sequential MRI, immunohistochemistry, microvessel density and tumor growth. Antiangiogenic gene therapy combined with paclitaxel significantly prolonged the mean survival of mice (25 days) compared to the controls (15 days) and all other treatment groups (p = 0.001). Bevacizumab treatment did not have any significant effect on the survival. Tumors of the mice treated by gene therapy were significantly smaller than in the control group (p = 0.021). The mean vascular density and total vascular area were also significantly smaller in the tumors of the gene therapy group (p = 0.01). These results show potential of the antiangiogenic gene therapy to improve efficacy of chemotherapy with paclitaxel and support testing of this approach in a phase I clinical trial for the treatment of ovarian cancer.

The administration of an adenoviral thymidine kinase suicide gene to the uterine artery of rabbits does not affect fertility: a safety study of pregnant and nonpregnant rabbits and their offspring.

BACKGROUND: Adenoviral gene therapy, based on herpes simplex virus thymidine kinase (AdvTK) is being developed for clinical use but no safety data are available with respect to the effects on female germ cells should the virus accidentally be released into systemic circulation. We studied the effects of AdvTK gene therapy on ovaries and germ cells in pregnant and nonpregnant rabbits and also the potential transmission of a transgene to offspring. METHODS: To mimic the severest of conditions, gene transfer was made by direct catheter-mediated injection into the uterine artery of pregnant and nonpregnant rabbits. AdvTK or AdvLacZ at 1 x 10(10) pfu were used for gene transfer. Ganciclovir was administered to AdvTK-treated rabbits to induce gene therapy. The rabbits were mated 6 and 12 weeks following gene transfer and the surviving young (89 from a total of 114) were analysed. RESULTS: No change in fertility was observed in the two matings after the gene transfer. In addition, no change was observed in ovarian histology between the AdvTK group, the AdvLacZ group and the nontreated controls. Southern blotting analysis showed no genomic integration of the transgene. However, in PCR analysis, transgene DNA was found in 9.3% of the litter samples. This was not the case for results from the reverse transcription-PCR assay. CONCLUSIONS: Although AdvTK gene therapy may initially affect ovarian cells, the influence appears to be transient. However, after direct exposure of the ovarian cells in high concentration of adenoviruses, transmission of a transgene in the offspring cannot be excluded.

Transduction patterns and efficiencies in rabbit uterine tissues after intraluminal uterine adenovirus administration vary with the reproductive cycle.

BACKGROUND: The options for destroying the full thickness of endometrium are very limited, relying mainly on surgical endometrial ablation. A nonsurgical, safe method would benefit many women in clinical settings. This study was undertaken to investigate the potential of gene therapy to transfect and destroy endometriun at different stages of the reproductive cycle in rabbits. METHODS: We used adenoviruses carrying the LacZ (AdvLacZ) marker gene, indicating the transfection efficiency, and adenoviruses carrying thymidine kinase (AdvTK) followed by intravenous ganciclovir therapy as a potential treatment for excess endometrial growth. Ganciclovir was given intravenously after AdvTK treatment. Adenoviruses were injected intraluminally into the uterus of nonpregnant, pseudopregnant, and pregnant New Zealand White rabbits (n=25). RESULTS: After AdvLacZ gene transfer into the uterus of intact rabbits, transduced cells were observed in uterine muscle and endometrial stroma. In pseudopregnant and pregnant rabbits the endometrium was proliferative and transduction was restricted to endometrial epithelium. However, no treatment effect was observed after AdvTK gene therapy although the transduction with AdvLacZ was clearly detectable. CONCLUSIONS: It is concluded that the transduction pattern of uterine tissues varies significantly with the reproductive cycle. Secondly, although the transduction efficiency was relatively high in the endometrial epithelium, the effect of the AdvTK and ganciclovir treatment was poor and not sufficient for clinical applications.

Long-term lowering of plasma cholesterol levels in LDL-receptor-deficient WHHL rabbits by gene therapy.

Lentiviral vectors encoding rabbit low-density lipoprotein receptor (LDLR) or green fluorescent protein (GFP) under the control of a liver-specific promoter (LSP) were used for intraportal gene transfer into the liver of hypercholesterolemic LDLR-deficient Watanabe Heritable Hyperlipidemic rabbits. In vitro cell culture analysis demonstrated functionality of the LSP-LDLR vector in mediating increased degradation of LDL in transduced liver cells. Twenty-five rabbits were each injected with 1 x 10(9) infectious virus particles into the portal vein. Liver biopsy samples were collected 4 weeks after the gene transfer and the rabbits were followed up for 2 years. Histological and RT-PCR analyses showed the expression of GFP and LDLR transgenes in the biopsy samples. Clinical chemistry and histological analyses revealed normal liver function and morphology during the 2-year follow-up with no safety issues. LSP-LDLR-treated rabbits demonstrated an average of 14 +/- 7% decrease in serum cholesterol levels during the first 4 weeks, 44 +/- 8% decrease at 1 year, and 34 +/- 10% decrease at the 2-year time point compared to the control rabbits. This study demonstrates the safety and potential benefits of the third-generation liver-specific lentiviral vectors in the treatment of familial hypercholesterolemia using direct intraportal liver gene therapy without the need for liver resection.

Fetal membranes act as a barrier for adenoviruses: gene transfer into exocoelomic cavity of rat fetuses does not affect cells in the fetus.

OBJECTIVES: In utero gene therapy has a potential to correct genetic disorders before the first clinical symptoms appear. Our aim was to examine whether the exocoelomic cavity between amniotic and chorionic membranes offers a minimally invasive route for gene transfer to the fetus during early pregnancy. STUDY DESIGN: We injected lacZ-adenovirus (4 x 10(9) pfu) during open surgery into the exocoelomic cavity of rat fetuses (n=50) and analyzed the fetuses and rat dams for transgene expression with X-gal staining and polymerase chain reaction. RESULTS: Giant cells around Reichert's membrane, the outermost extraembryonic membrane in rodents, were transduced; but no transduction was observed in the cells of the fetuses or rat dams. CONCLUSION: In rodents, the exocoelomic cavity does not offer a route for gene transfer into the fetus. It was concluded that fetal membranes act as a barrier that prevents adenoviral particles from passing between embryonic cavities.