Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer.

Gene therapy is a potential route for the delivery of secreted therapeutic proteins, but pharmacologic control of expression will generally be required for optimal safety and efficacy. Previous attempts to achieve regulated expression in large animal models have been thwarted by transient expression or immune responses to regulatory proteins. We evaluated the ability of the dimerizer-regulated gene expression system to achieve controlled, long-term production of erythropoietin (Epo) following intramuscular administration of adeno-associated virus (AAV) vectors to 16 primates. All animals showed dose-responsive and completely reversible elevation of Epo and hematocrit in response to the dimerizer rapamycin, or analogs with reduced immunosuppressive activity, administered intravenously or orally. Animals that received optimized dual vectors showed persistent regulated expression for the duration of the study, with no apparent immune response to Epo or the regulatory proteins. Similar results were obtained with single vectors incorporating both the Epo and regulatory genes, including those packaged into serotype 1 AAV vectors to allow use of lower viral doses. For the longest-studied animal, regulated expression has persisted for more than 6 years and 26 induction cycles. These data indicate that one-time or infrequent gene transfer followed by dimerizer regulation is a promising approach for delivery of therapeutic proteins.

Effect of blood collection technique in mice on clinical pathology parameters.

A study was conducted in normal healthy C57BL/6 mice to determine the effect of method of blood collection on clinical pathology parameters and to provide value ranges for these parameters. Males and females were used and were randomly assigned to treatment groups based upon phlebotomy method. The blood was collected using one of four methods: intracardiac (IC), a single attempt at collection from the caudal vena cava (VC), collection from the caudal vena cava with collection of any extravasated blood from the peritoneum (MC), or retroorbital phlebotomy (RO). Evaluation of blood and serum samples was conducted for a number of serum biochemistries including liver function tests and complete blood count with differentials and platelet counts. Female mice demonstrated higher values for red blood cell number, hemoglobin (p < 0.002), hematocrit, alkaline phosphatase, albumin, total protein, and creatinine. Males demonstrated higher values for platelet counts, specific white blood cell numbers (total, neutrophil, lymphocyte, and eosinophil counts), globulin, amylase, and the BUN/creatinine ratio. Overall, the VC method was associated with the least variation in both sexes and appeared slightly better than the IC method for the parameters evaluated. The largest difference between groups was noted for the transaminase levels. While alanine aminotransferase (ALT) values were similar between the IC and VC groups, aspartate aminotransferase (AST) values were associated with less variation for the VC method. Transaminase levels for the MC and RO groups were associated with relatively large ranges and variation. ALT results from the RO method, the only method amenable to repetitive sample collection used in this evaluation, indicate that this is an acceptable method. The results demonstrate the substantial impact that phlebotomy method has on the assay results and that the VC or IC methods provide the most consistent results. The ranges by collection method and sex provided here can be used to select the preferred method of collection when designing a study and for comparison of data obtained with reference ranges. The authors recommend establishment of normal ranges based upon methods employed within an investigator's laboratory.

A New Method to Collect Bile and Access the Duodenum in Conscious Dogs.

A novel, totally implantable catheter system that allows complete bile collection and duodenal access in conscious, freely moving dogs is described. Bile collection catheters remained patent for an average of 417 days (range, 711010 days) in eight animals which were used on study. Three animals have been used to validate the models complete collection of bile via biliary recovery of an intravenous dose of 14C-glycocholic acid, and in selected animals, parameters potentially indicative of liver damage (serum alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, and total bilirubin levels) were within normal ranges for as many as 14 months after surgery. The eight study dogs have been used in a total of 29 studies, in which bile was successfully collected for 1248 h. The bile has been collected by using either a tethering system or a protected pouch arrangement. Compared to exteriorized catheter techniques, this system requires less maintenance and is better tolerated by the animals. The potential for a longer functional life span for individual animals, more normal liver enzymes, and the capability to selectively infuse towards the duodenum and flush the entire catheter and bile duct are other advantages of this model.