Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo.

Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

Effects of in vivo treatment of mice with sulforaphane on repair of DNA pyridyloxylbutylation.

The phytochemical sulforaphane (SF) has gained interest for its apparent association with reduced cancer risk and other cytoprotective properties, at least some of which are attributed to activation of the transcription factor Nrf2. Repair of bulky DNA adducts is important for mitigating carcinogenesis from exogenous DNA damaging agents, but it is unknown whether in vivo treatment with SF affects adduct repair. At 12 h following a single oral dose of 100 mg/kg SF, an almost doubling in activity for repair of pyridyloxobutylated DNA was observed in CD-1 mouse liver nuclear extracts, but not in lung extracts. This change at 12 h in repair activity was preceded by the induction of Nrf2-regulated genes but not accompanied by changes in levels of the specific nucleotide excision repair (NER) proteins XPC, XPA, XPB and p53 or in binding of hepatic XPC, XPA and XPB to damaged DNA. SF also did not significantly alter histone deacetylase activity as measured by acetylated histone H3 levels, or stimulate formation of γ-H2A.X, a marker of DNA damage. A significant reduction in oxidative DNA damage, as measured by 8-OHdG (a biomarker of oxidative DNA damage), was observed only in DNA from the lungs of SF-treated mice 3 h post-dosing. These results suggest that the ability of SF to increase bulky adduct repair activity is organ-selective and is consistent with activation of the Nrf2 signaling pathway.

Sedative effects and pharmacokinetics of detomidine when administered intravenously and intravaginally as a gel in alpacas.

OBJECTIVES: To evaluate the sedative effects and pharmacokinetics of detomidine gel administered intravaginally to alpacas in comparison with intravenously (IV) administered detomidine. STUDY DESIGN: Randomized, crossover, blinded experiment. ANIMALS: A group of six healthy adult female Huacaya alpacas (70.3 ± 7.9 kg). METHODS: Alpacas were studied on two occasions separated by ≥5 days. Treatments were IV detomidine hydrochloride (70 µg kg-1; treatment DET-IV) or detomidine gel (200 µg kg-1; treatment DET-VAG) administered intravaginally. Sedation and heart rate (HR) were evaluated at intervals for 240 minutes. Venous blood was collected at intervals for 360 minutes after treatment for analysis of detomidine, carboxydetomidine and hydroxydetomidine using liquid chromatography-tandem mass spectrometry. Measured variables were compared between treatments and over time using mixed model analysis. Data are presented as the mean ± standard error of the mean, and a p value of <0.05 was considered significant. RESULTS: Onset of sedation was faster in treatment DET-IV (1.6 ± 0.2 minutes) than in treatment DET-VAG (13.0 ± 2.5 minutes). Time to maximum sedation was shorter in treatment DET-IV (8.3 ± 1.3 minutes) than in treatment DET-VAG (25 ± 4 minutes). Duration of sedation was not different between treatments. There was a significant linear relationship between sedation score and plasma detomidine concentration. HR was less than baseline for 60 and 125 minutes for treatments DET-IV and DET-VAG, respectively. The maximal decrease in HR occurred at 15 minutes for both treatments. The mean maximum plasma concentration of detomidine, time to maximum concentration and bioavailability for treatment DET-VAG were 39.6 ng mL-1, 19.9 minutes and 20%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Detomidine administration at the doses studied resulted in moderate sedation when administered IV or intravaginally to alpacas.