Synthetic Anticancer Gene Medicine Exploits Intrinsic Antitumor Activity of Cationic Vector to Cure Established Tumors Christine Dufe `s, 1 W. Nicol Keith, 1 Alan Bilsland, 1 Irina Proutski, 1 Ijeoma F. Uchegbu, 2 and Andreas G. Scha ¨tzlein 1 1 Cancer Research UK Centre for Oncology and Applied Pharmacology, Beatson Laboratories, University of Glasgow, and 2 Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, United Kingdom Abstract The systemic delivery of genetic therapies required for the treatment of inaccessible tumors and metastases remains a challenge despite the development of various viral and synthetic vector systems. Here we show that a synthetic vector system based on polypropylenimine dendrimers has the desired properties of a systemic delivery vehicle and mediates efficient transgene expression in tumors after i.v. administra- tion. The systemic tumor necrosis factor A (TNFA) gene therapy was efficacious in the experimental treatment of established A431 epidermoid carcinoma, C33a cervix carcino- ma, and LS174T colorectal adenocarcinoma. Specifically, the systemic injection of dendrimer nanoparticles containing a TNFA expression plasmid regulated by telomerase gene promoters (hTR and hTERT) leads to transgene expression, regression of remote xenograft murine tumors, and long-term survival of up to 100% of the animals. Interestingly, these den- drimers and, to a lesser extent, other common polymeric trans- fection agents also exhibit plasmid-independent antitumor activity, ranging from pronounced growth retardation to complete tumor regression. The genetic therapy as well as treatment with dendrimer alone was well tolerated with no apparent signs of toxicity in the animals. The combination of intrinsic dendrimer activity and transcriptionally targeted TNFA when complexed was significantly more potent than either treatment alone or when both were administered in sequence. The combination of pharmacologically active synthetic transfection agent and transcriptionally targeted antitumor gene creates an efficacious gene medicine for the systemic treatment of experimental solid tumors. (Cancer Res 2005; 65(18): 8079-84) Introduction The clinical realisation of the potential benefits of gene therapy, in particular for cancer, remains limited, partly because of the challenges of systemic delivery (1, 2). To kill tumor cells at remote sites, a viral or synthetic vector has to travel in the vascular compartment to the tumor blood vessels, across the vessel wall, and through the interstitium to the cell (3). Nonviral delivery systems based on synthetic molecules such as cationic lipids or polymers are relatively simple compared with virus, but have been seen as being limited by low transfection efficiency, in particular in the tumor, after systemic administration. Here we show that systemic administration of such systems as highly potent gene medicines is feasible if the intrinsic antitumor activity of the dendrimer and its capability for efficient systemic delivery can be exploited. In particular, the PPI G3 dendrimer acts as a complexation and transfection agent as well as an active drug with intrinsic antitumor activity, thus creating a gene medicine with an efficacy comparable to or better than standard therapy (4). Materials and Methods Nanoparticles. The plasmid constructs were based on previously published sequences (5) and commercially available expression plasmids [pORF9-mTNFa and pORF-hTNFa (Invivogen, San Diego, CA); pCMV SPORThgal (Invitrogen, Renfrewshire, United Kingdom)] and were purified using an Endotoxin-free Giga Plasmid Kit (Qiagen, Hilden, Germany). Nanoparticles of plasmid DNA (50 Ag) and fractured PAMAM dendrimer (SuperFect, Qiagen, Hilden, Germany), linear polyethylenimine (ExGen 500, MW 22 kDa, Helena Biosciences, Sunderland, United Kingdom), or poly- propylenimine dendrimer generation 3 (PPI G3 ; DAB-Am16) was prepared in 5% dextrose according to the instructions of the manufacturer (100 AL SuperFect, 9 AL polyethylenimine) or as previously described (6). Animals. Female mice (CD1-nu; initial mean weight, 20 g) housed in groups of five at 19jC to 23jC with a 12-hour light-dark cycle were fed a conventional diet (Rat and Mouse Standard Expanded, B&K Universal, Grimston, United Kingdom) with mains water ad libitum. Experimental work was carried out in accordance with U.K. Home Office regulations and approved by the local ethics committee. Tumor cell suspensions [LS174T human colorectal adenocarcinoma (ATCC CCL-188), A431 epidermoid carcinoma (ATCC CRL-1555), and C33a human cervix carcinoma (ATCC HTB31)] in exponential growth were injected s.c. (10 6 cells per flank). Tumors typically reached diameters of 5 mm or more within 7 (LS174T) to 10 days (A431 and C33a). h-Galactosidase expression was evaluated 48 hours after i.v. administra- tion of lacZ plasmid nanoparticles; the whole tumor was incubated overnight in 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside (X-Gal) and histologic sections (paraffin, 20 Am) fixed in 10% formalin and counter- stained with eosin. Gene therapy treatment was administered by i.v. tail-vein injection (50 Ag of DNA in 200 AL) once daily on only one occasion (day 0) or on an alternative schedule of five occasions (treatment days 0, 2, 4, 6, and 8). Animals (n = 5) were weighed and volume was determined from daily caliper measurements (volume = d 3 Â p / 6). Results are expressed as means F SE and statistical significance was determined by one-way ANOVA followed by the Bonferroni post test (GraphPad Prism software). Results were expressed as relative tumor volume (rel.Vol tx = Vol tx / Vol t0 ) and responses classified analogous to Response Evaluation Criteria in Solid Tumors (RECIST; ref. 7). Progressive disease is defined as an increase in relative tumor volume >1.2-fold, stable disease as a relative volume between 0.7 and 1.2 of starting volume, partial response as measurable tumor with a reduction of more than 30% (0-0.7), and complete response as the complete absence of any measurable tumor. Differences were considered as significant when P < 0.05. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Andreas G. Scha ¨tzlein, Cancer Research UK Centre for Oncology and Applied Pharmacology, Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, United Kingdom. Phone: 44-141-330-4354; E-mail: A.Schatzlein@ beatson.gla.ac.uk. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-04-4402 www.aacrjournals.org 8079 Cancer Res 2005; 65: (18). September 15, 2005 Priority Report