Developing an effective gene therapy for prostate cancer: New technologies with potential to translate from the laboratory into the clinic

Abstract

Prostate cancer is the second leading cause of cancer-related deaths in men in the U.S. At present, no single or combination therapy has shown efficacy in decreasing disease progression in patients with metastatic disease. A potentially viable approach for treating late-stage prostate cancer is gene therapy. Adenoviruses (Ad) are the most commonly used mode of gene delivery, but progress using this vector has been hampered by concerns over the safety and practicality of viruses including conditionally replicating Ads (CRAds), particularly for intravenous delivery, and the inefficiency of non-viral transfection techniques. Major challenges for effective gene therapy using Ads are the limited infectivity of regular Ad serotype 5 (Ad5) and the inability to specifically deliver the therapeutic directly into diseased tissue without trapping in the liver or elimination by the immune system. The shortcoming in using Ad5 is mostly attributed to a reduction in Coxsackie-adenovirus receptors (CAR) on the surface of cancer cells, which can be mitigated by generating tropism-modified Ads permitting CAR-independent infection of tumor cells. The limitations of systemic gene delivery can now be overcome by using a novel targeted-delivery approach such as ultrasound (US) contrast agents (microbubbles) to deliver effective therapeutic reagents, Ads, or recombinant proteins, combined with ultrasound-targeted microbubble destruction (UTMD), to develop a site-specific therapy in immune competent transgenic mouse models. These unique strategies for enhancing the efficacy of gene therapy provide a direct path to translation from the laboratory into the clinic for developing an effective gene therapy of prostate cancer.

Figure 1

Schematic representation of cancer terminator viruses (CTVs). In the CTVs the PEG-Prom drives the expression of E1A and E1B genes thus ensuring cancer-specific replication while the CMV-Prom regulates the expression of either mda-7/IL-24 or IFNγ in the E3 region of the Ad. These conditionally replication competent adenoviruses (CRCA) do not harm normal cells but induce oncolysis by Ad replication and diverse tumor-suppressor effects of the expressed transgene. (Reproduced with permission of the publisher, from Sarkar et al., 2005).

Figure 2

Tropism-modified adenovirus (Ad.5/3) shows enhanced infectivity in PC-3 low Coxsackie-adenovirus receptor (CAR) prostate cancer cells. (a) Schematic representation showing the construction of tropism-modified Ad for delivery of mda-7/IL-24. (b) Expression of CARs on the surface of DU-145 and PC-3 prostate carcinoma cells and P69 SV40 immortalized normal prostate epithelial cells. (c) P69, DU-145 and PC-3 cells were infected with the indicated p.f.u. per cell of Ad.5-Luc or Ad.5/3-Luc; and luciferase activity was determined 48 hours later. (d) P69, DU-145 and PC-3 cells were infected with the indicated p.f.u. per cell of Ad for 48 hours and total proteins were isolated. The expressions of MDA-7/IL-24 and EF-1a (as a loading control) proteins were analyzed by Western blotting analyses. (Reproduced with permission of the publisher, from Dash et al., 2010a).

Figure 3

Microbubble-assisted gene delivery. (a) Schematic representation of the microbubble delivery of Ad-GFP complexes and ultrasound (US) release in a tumor target site of the mouse. (b) Western blotting analysis of Ad-GFP/microbubble–transduced DU-145 tumor xenografts. Immunoblot showing the expression levels of green fluorescent protein (GFP) in DU-145 cells following ultrasound-targeted microbubble/Ad transduction of GFP at 96 hours. Only the tumor on the right flank was sonoporated for 10 min resulting in the delivery and expression of GFP. The left tumor, heart, lung, liver, and kidney were negative for GFP expression. GST–GFP was used as a positive control. Protein gel loading was normalized using β-actin as a control. (c) Ultrasound imaging and US contrast enhancement of in vivo transduced DU-145 tumor xenografts. B-mode US imaging of a tumor before MB contrast agent injection. (d) B-mode US imaging of the same tumor depicted in c following injection of microbubbles/Ad-GFP complexes. MBs cavitation within the targeted tumor dramatically enhances the tumor image within the US field of view. Ad, adenovirus; MB, microbubbles. (Reproduced with permission of the publisher, from Greco et al., 2010.)

Figure 4

Microbubble Encapsulated Ads Display Reduced Immunogenicity. The indicated vectors were injected systemically (i.v.) into the tail veins of C57B6 mice (n = 5). Complement (Sigma) was added to one set of the microbubble/Ad complex, whereas another microbubble/Ad complex was not treated with complement. 12 hours after the injections serum was collected from the mice. The indicated cytokines were tested using the Bio-Plex mouse cytokine 23-plexpanel kit with mouse serum samples as described by Bio-Rad Laboratories.