Adenovirus- and cationic-lipid-mediated intratumoral gene transfer and natural killer cell activity in a SCID mouse melanoma tumor model

摘要

Three distinct efforts were undertaken to evaluate various aspects of intratumoral transgene expression in a xenogenic melanoma SCID mouse model. First, we evaluated the ability of different polycationic substances to improve replication-deficient adenovirus expression and transduction efficiency of a β-galactosidase (β-Gal) transgene. We demonstrated increased transgene expression after mixing an E1-deleted adenovirus vector with cationic lipids, polybrene, protamine sulfate, and CaCl₂ prior to transduction in vitro. We were unable to demonstrate any benefit when adenovirus was admixed with protamine sulfate prior to intratumoral injection into subcutaneous tumors in the SCID mouse. Next, we investigated factors affecting intratumoral gene transfer with cationic lipid-DNA complexes. Preparations containing VR-1103, a DNA plasmid encoding the gene for human IL-2, either alone or complexed with the cationic lipid, DMRIE/DOPE were injected into subcutaneous (s.c.) human melanoma tumors in SCID mice. Our studies revealed that cationic lipid addition was highly effective for intratumoral gene transfer compared to naked DNA and IL-2 transgene secretion was consistently higher when lipid:DNA complexes were formulated at higher lipid:DNA ratios (w/w). Various intratumoral injection techniques tested did not yield statistical improvements in gene transfer levels. These results indicate that formulation and dosage of cationic lipid:DNA complexes, but not injection technique, play a key role in determining the level of intratumoral transgene expression. Lastly, Natural Killer (NK) cells play an important role in combating infectious and oncogenic diseases, and IL-2 has been shown to promote proliferation and activation of NK cells in vitro and in vivo. We investigated the consequences of local IL-2 transgene secretion in a subcutaneous melanoma tumor model using cationic lipid-mediated gene transfer. Intratumoral injection of DMRIE/DOPE alone or DMRIE/DOPE:VR-1103 complexes had no effect on the percentage of tumor-associated NK cells (asGM1⁺) after 1 or 2 days; however, by day 6, the percentage of intratumoral NK cells and intratumoral granzyme A levels were elevated and growth of UM449 tumors was slowed in IL-2 transfected tumors. The total number of NK cells per tumor was unchanged in IL-2 transfected vs. non-transfected tumors. Total granzyme A activity was significantly elevated in IL-2 expressing tumors, indicating that local IL-2 expression leads to NK activation and tumor growth inhibition.