Abstract 5659: In vivo efficiency and biodistribution of TNF-alpha expressing small-sized vector DNA after intratumoral nonviral jet-injection
Authors: Walther Wolfgang , Galling Nele , Kobelt Dennis , Aumann Jutta , Schmidt Manuel , Schlag Peter M. 
Published: August 2012

Abstract
Nonviral gene therapy is gaining growing importance and has been applied in numerous studies to treat cancer. Plasmid vectors are still used in the majority of cancer gene therapy approaches. However, it is broadly accepted, that plasmid-vectors need significant improvements to enhance their safety and efficiencies. In this context small-sized vectors are attractive alternatives. The MIDGE-platform (Mologen, Berlin, Germany) represents such small vector system harboring the essential expression cassette in a double-stranded linear DNA molecule and end-sealing oligonucleotide loops. We used the MIDGE-vector for the expression of the human TNF-alpha. Here we show, that this MIDGE-CMVhTNF system permits significantly improved in vivo TNF-alpha expression in the A375 and MeWo human melanoma xenotransplant model after intratumoral in vivo jet-injection gene transfer in nude mice. The comparison to the respective plasmid-vector revealed an up to 22-fold increase of the MIDGE-CMVhTNF mediated intratumoral TNF-alpha expression. Apart from these efficiency studies the safe use of such minimal-size vector is essential. We therefore analyzed the biodistribution and clearance of the MIDGE-CMVhTNF vector after jet-injection in A375 melanoma bearing nude mice. For this, the animals received 5 jet-injections through the skin into the tumor at a pressure of 2.8 Bar. Each jet-injection delivered 0.01 mL MIDGE-DNA at a concentration of 1mg/mL. The intratumoral MIDGE-DNA load was analyzed 5, 10, 20, 40 minutes and 3, 6, 24, 48, 72 hours as well as 7 days after jet-injection by quantitative real-time PCR. For the same time points the systemic dissemination of the MIDGE-DNA was quantitatively analyzed by real-time PCR in the blood, liver, lung, spleen, kidney, ovary, heart and brain. The analyses show high DNA-load of up to 12 ng vector-DNA / 250 ng tumor DNA and a clearance of this DNA in the tumors within 3 to 7 days. This is paralleled with the decline of TNF-alpha expression. Regarding systemic biodistribution MIDGE-DNA levels in blood samples are peaking at 30 minutes after jet-injection, followed by the rapid drop within 60 minutes after DNA application. The MDGE-vector DNA was also detected in liver, lung, spleen, kidney, brain, ovary and heart at varying pg-levels for up to 3 hours after jet-injection. This was followed by the clearance of this DNA within 6 to 24 hours after gene transfer. More importantly, in none of these organs TNF-alpha mRNA expression was detected by real-time RT-PCR at any time point. In summary these data indicate, that the MIDGE-CMVhTNF vector leads to improved in vivo transgene expression and shows only limited systemic biodistribution after intratumoral jet-injection, associated with its rapid clearance. These parameters are of importance for the potential clinical use of this vector.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5659. doi:1538-7445.AM2012-5659