A polymer library approach to suicide gene therapy for cancer

Abstract

Optimal gene therapy for cancer must (i) deliver DNA to tumor cells with high efficiency, (ii) induce minimal toxicity, and (iii) avoid gene expression in healthy tissues. To this end, we generated a library of >500 degradable, poly(beta-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally approximately 4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer/promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.

Fig. 1.

Amino (6–94) and acrylate (B–LL) monomers used to create poly(β-amino ester) library.

Fig. 2.

In vitro transfection potential of poly(β-amino esters). The transfection potential of polymers synthesized at the optimal amine/acrylate ratio and at the optimal polymer/DNA ratio is shown. Polymers were synthesized at 6 amine/acrylate ratios (1, 1.025, 1.05, 1.1, 1.2, and 1.3) unless marked with a red arrow, in which case they were synthesized at 12 amine/acrylate ratios (0.6, 0.8, 0.9, 0.95, 0.975, 1, 1.025, 1.05, 1.1, 1.2, 1.3, and 1.4). Polymers were synthesized at 95°C in the absence of solvent (blue bars) or at 60°C in the presence of 2 ml of DMSO (red bars). The amine/acrylate ratio of the optimal polymer is listed next to the monomer composition.

Fig. 3.

Tumor transfection in vivo. Xenografts of PC3 human prostate tumor cells were injected with C32 (1.2:1 amine/acrylate ratio) complexed to pCAG/luc DNA at a 30:1 polymer/DNA ratio; in vivo jetPEI complexed to pCAG/luc DNA according to the manufacturer's instructions; and naked pCAG/luc DNA. Two days after transfection, mice were imaged and bioluminescence was quantified. (Left) Pseudocolor images representing light emitted from tumors superimposed over grayscale reference image of representative mice from each group of five. (Right) Quantification of the emitted photons from each tumor. Horizontal bars indicate the mean value for each treatment group.

Fig. 4.

Muscle transfection in vivo. Healthy muscle was injected with C32 (1.2:1 amine/acrylate ratio) complexed to pCAG/luc DNA at a 30:1 polymer/DNA ratio; in vivo jetPEI complexed to pCAG/luc DNA according to the manufacturer's instructions; and naked pCAG/luc DNA. Two, 6, and 20 days after transfection, mice were imaged and bioluminescence was quantified. (Upper) Pseudocolor images representing light emitted from muscle superimposed over grayscale reference image of representative mice from each group of five. (Lower) Quantification of the emitted photons from each injected muscle. Horizontal bars indicate the mean value for each treatment group.

Fig. 5.

Histological analysis of muscle and tumors after transfection. Photomicrographs of hematoxylin and eosin-stained sections of muscle (A and B) and tumor (C and D) taken with a 10× objective. (A) Muscle injected with PEI/DNA shows damaged myocytes with calcifications, indicated with arrows. (B) Muscle injected with C32/DNA shows no pathology. (C) Uninjected tumor control. (D) Tumor injected with C32/DNA shows no histological differences from control tumor.

Fig. 6.

Tumor growth after i.t. injection of C32-pRSV/FRT2PSA.FLP/DT-A or C32/salmon sperm DNA nanoparticles. Nanoparticles were injected on day 0 and then every other day for a total of six injections (50 mg of DNA per injection, 30:1 polymer/DNA ratio). Tumor volume was measured with calipers on day 0 and day 11. Fold increase in tumor volume is the ratio of these two measurements. Horizontal bars indicate the mean value for each treatment group.