364. Impact of Injection Volume on Hydrodynamic Delivery To the Liver in Mice

Hydrodynamic gene delivery is a widely prevailed method for gene delivery to the liver especially in rodents, because of its efficiency and simplicity. A transient enlargement of the liver derived from physical force, which is generated by a rapid injection of a large amount of solution through the tail vein of a mouse, plays an important role for the gene transfer.We previously demonstrated that the expansion speed of the liver is a primary determinant for gene transfer efficiency, because the liver volume at the end of the injection (final volume) was not significantly different between hydrodynamic (5 sec) and slow (60 sec) injections, as long as the same amount of volume of 9% of body weight (BW) was injected (kanefuji, et al. Mol Ther Methods Clin Deve 1;14029). However, the relationship between the injection volume and final volume has not been clarified.The present study aimed to evaluate volume-dependent physical impacts on the liver in hydrodynamic injection. Physical impacts to the liver were quantified in mice by measuring the final volume using a cone beam computed tomography (CBCT) and serum concentration of alanine aminotransferase (ALT). Hydrodynamic (9% of BW/5 sec), half-hydrodynamic (5% of BW/5 sec), and half-slow (5% of BW/60 sec) injections were performed with contrast medium including 300 mg/ml of iodine through the tail vein of mice. Just after the injections, CBCT studies were performed without any surgical intervention to collect volume data of the liver, and the final volumes were shown as the relative volume of that of control mice. Blood samples were collected for the assay of serum concentration of ALT at the time points of 1, 4, 24, 48, and 168 hours after the injections.The average final volumes were 125.8±11.5 and 119.7±4.8% in half-hydrodynamic and half-slow injections, and were not significantly different from each other, while they were significantly lower than that of 173.1±10.4% in hydrodynamic injection (p>0.99 and <0.05, respectively, Kruskal-Wallis followed by Dunn's Multiple Comparison test). The average levels of ALT 4 hours after the injections were 82.6±72.4 U/L and 1582±701.5 U/L in half-hydrodynamic and hydrodynamic injections, which were significantly different from each other (p<0.01, Mann-Whitney test), and returned to the normal level within 48 hours after the injections.These results clearly indicate that the final volume evidently depends on the injection volume but not speed in both hydrodynamic and half-hydrodynamic injections. Furthermore, the reduction of the injection volume markedly suppressed the elevation of liver enzyme in serum after the injection.From a safety viewpoint, there is no doubt that an injection with less volume is advisable as far as sufficient gene delivery is guaranteed. A further study is on going to make the efficacy of half-hydrodynamic injection comparable with that of hydrodynamic injection.