EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo

Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these respon ses ran limit the therapeutic usefulness of the vector. In principle, the u tility of the vector can be improved by increasing its therapeutic index, t hat is, by either increasing its efficacy or decreasing its toxicity, A str ategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene exp ression, and thereby also reduce unwanted effects such as immune responses, Following up on our observation that treating polarized normal human bronc hial epithelial cells with calcium (Ca2+)-free medium or the calcium chelat or ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid ( EGTA) significantly enhanced the subsequent transfection of these cells wit h cationic lipid:pDNA complexes, me have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Trea ting polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an similar to 50-fold inc rease in transgene expression, This strategy was also tested in vitro and r esulted in substantial increases (up to 50-fold) in the ability of AdV vect ors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA, The enhancing effects of EGTA could not be duplicated with hypo- or hypero smotic treatments. Light microscopy of mouse trachea that had been EGTA tre ated and then infected,vith AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells, The apparent enhanced potency of AdV for airwa y cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likel ihood that it can be used for clinical indications requiring chronic admini stration of the vector.