Gutless Adenoviral Vectors Mediate Regulated Expression of Pseudomonas Exotoxin Fused to Mutated IL-13 in Human Glioma Cells

CANDOLFI; XIONG; KROEGER; PUNTEL; LIU; MONDKAR; LOWENSTEIN; CASTRO

Washington DC, USA

Congreso; Association of Neurological Surgeons; 2007

<!-- /* Font Definitions */ @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:1; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:0 0 0 0 0 0;} @font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-1610611985 1073750139 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin-top:0in; margin-right:0in; margin-bottom:10.0pt; margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} .MsoPapDefault {mso-style-type:export-only; margin-bottom:10.0pt; line-height:115%;} @page Section1 {size:8.5in 817.0pt; margin:97.0pt 54.25pt 1.0in 73.5pt; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;} --> Gutless Adenoviral Vectors Mediate Regulated Expression of Pseudomonas Exotoxin Fused to Mutated IL-13 in Human Glioma Cells Abstract No. 1816 Abstract Type: Poster Presentation 9:00 am to 5:29 pm, Sunday through Wednesday Exhibit HallPresenter: Marianela Candolfi, DVM Introduction: We developed a high capacity adenoviral (HC-Ad) plasmid expressing Pseudomonas exotoxin (PE) fused to a mutated IL-13 (muIL-13) with higher affinity for the glioma-associated IL-13-alpha-2R and negligible binding to the physiological IL13/IL4R. Methods: We constructed an HC-Ad plasmid, pSTK-mCMV-TetON-muIL-4-TRE-muIL-13-PE encoding domain II (cell entry and toxin activation) and domain III (induces cytotoxicity) of the PE linked to muIL-13. It also encodes a mutated IL-4 that blocks IL13/IL4R to further reduce the chance of muIL-13-PE binding to normal cells. Transgene expression is driven by the regulatable bidirectional TRE promoter, which is activated by a doxycycline-dependent transactivator (TetON). We also included a trans-repressor that inhibits TRE expression in the absence of Doxycycline. Transactivator and transrepressor expression is driven by the mCMV promoter. Results: Transfection of 293 and COS-7 cells with pSTK-mCMV-TetON-muIL-4-TRE-muIL-13-PE resulted in Doxycycline-dependent transgene expression, as determined by immunocytochemistry of muIL-4, muIL-13 and PE, and by IL-4 and IL-13 ELISA performed in their supernatant. Transgene expression was negligible without Doxycycline. Cell viability of 293 and COS-7 was unaffected by pSTK-mCMV-TetON-muIL-4-TRE-muIL-13-PE expression. However, their conditioned media induced cytotoxicity in IL-13-alpha-2R-expressing U251 and IN859 human glioma cells. Conclusion: Our results indicate that muIL-13-PE released from HC-Ad infected cells will be taken up by neighboring GBM cells that express the IL-13R-alpha-2, constituting a powerful regulated targeted approach. Additionally, because HC-Ad vectors are capable of sustaining long-term gene expression even in the presence of a systemic anti-Ad immune response as would be encountered in patients undergoing clinical trials, they are excellent candidates for implementing this approach in humans.