**6.1 NK4 supplementation system via hydrodynamic gene delivery in mice**

Numerous clinical studies have indicated the apparent increases in serum HGF levels in patients during the progression of cancers (Wu *et al*., 1998; Osada *et al*., 2008). It is likely that HGF in blood protects cancer cell suspension from anoikis-like cell death (Zeng *et al*., 2002). Thus, we predict that over-production of NK4 in blood would overcome the HGF-mediated metastatic events seen in blood flow (and possibly in local sites). Hydrodynamic-based gene delivery is known to achieve an efficient expression of exogenous genes predominantly in the liver but much lesser in the kidney and spleen (Suda *et al*., 2007). Based on this background, we established a method for the induction and maintenance of higher levels of NK4 in blood through repeated injections of NK4 cDNA-containing plasmid.

For hydrodynamic-based gene delivery, 5 microgram of plasmid DNA (pCAGGS-NK4), or pCAGGS-empty (as a control), in saline was injected within 5 seconds into tail veins of mice at 2.4 ml per 30g body weight (Wen *et al*., 2004; 2007). As a result, exogenous NK4 was detected, and plasma NK4 reached a mean value of 49.5 ng/ml 24 hours post-bolus injection and decreased to 15.4 ng/ml on day 3. Following the second and third injections, the plasma NK4 level again reached approximately 70 and 130 ng/ml on days 8 and 15, respectively. Thus, plasma NK4 levels increased following additional administration of the expression plasmid, and were maintained at levels of > 8 ng/ml during 3 weeks post-treatment (**Fig. 5**).

Fig. 5. Hydrodynamics-mediated NK4 delivery system in mice. (A) An experimental protocol of NK4 gene administration. Five microgram of pCAGGS-NK4 was administered intravenously into mice on day 0, 7 and 14. (B) Changes in plasma NK4 levels following repetitive administration of expression plasmid for NK4. Arrows mean the time of plasmid administration. See reference (Wen *et al*., 2007) for further information.
