668. Progress toward a Non-Viral Gene Therapy Protocol for the Treatment of Anemia
Authors: Magdolna G. Sebestyén, Julia O. Hegge, Mark A. Noble, David L. Lewis, Hans Herweijer, Jon A. Wolff
Journal: Molecular Therapy (2006)

Patients with progressive kidney disease develop anemia due to declining erythropoietin (EPO) production in the kidneys. The condition is currently treated by the frequent injection of recombinant human EPO. The expression of EPO following gene delivery to an ectopic site could provide a more convenient and more economical long-term solution. The major obstacles for the gene therapy of anemia have been: (1) finding a safe and efficient DNA delivery method to a tissue that can secrete EPO into the blood and (2) controlling the level of EPO expression by some means in order to prevent polycythemia. This study was initiated to evaluate the use of hydrodynamic limb vein (HLV) injections to overcome these obstacles. The procedure had been found safe and effective to deliver naked DNA into limb muscles and also made repeated injections possible. This offers a tool to control gene expression at the most fundamental level: the DNA dose. Using a rat anemia model, we are testing the additive effect of repeated small-dose injections to gain proof of concept for regulating hemopoiesis by the delivered DNA dose. We are also studying the effects of EPO expression in healthy rhesus monkeys to collect pre-clinical data about the extent and longevity of expression using various promoters and about dose-response correlations. We injected 14 monkeys with constructs containing the rhesus EPO coding region under the control of either the ubiquitous human cytomegalovirus (CMV) promoter, or the muscle specific mouse or human creatine kinase (MCK or hCK) promoters. Expression was assessed by measuring EPO concentration in the serum and by following the changes in the hematocrit (Hct). Changes in both parameters were promoter-dependent. The delivery of 6 mg/kg CMV-RhEPO resulted in a sharp increase in serum EPO levels that peaked on Day 7 post-injection. EPO levels then declined and by the end of 4 months they returned to the pre-injection baseline. As a result, the Hct increased substantially during the first 2–3 months, then gradually returned to pre-treatment levels. In contrast, delivery of the MCK-RhEPO construct (also at 6 mg/ kg) led to a lower and delayed peak of EPO expression on Day 14, followed by low, but above-baseline, stable expression sustaining elevated Hct levels for at least 9 months. EPO expression from the hCK promoter lacked the initial peak observed for the other two promoters. Instead, serum EPO levels slowly increased for 3 months leading to a slow but steady increase in the Hct. By 4 months, the physiological effect of 6 mg/kg hCK-RhEPO approached that of the MCK construct. Delivery of a 3mg/kg dose of the CMV and MCK plasmids resulted in EPO expression with similar kinetics at proportionally lower magnitude. Even though these rat and primate studies are still in progress without a final conclusion, they suggest that it will be possible to manage the serum EPO concentration and its physiological effect on hemopoiesis by administering repeated small-dose DNA injections until the desired effect is reached. These data will also help guide us in our choice of promoter depending on how long or short term EPO expression is indicated. Jon Wolff has a proprietory interest in Mirus Bio Corporation.