Bacteria have been engineered recently as vectors for gene delivery. Compared to other vectors, they have several unique advantages, such as being capable of producing therapeutic agents without infecting host cells and active transport in tissues that may overcome physiological barriers to gene delivery. However, several key issues remain to be addressed in order to improve efficacy of bacterium-mediated gene therapy. For bacterial vectors delivered locally via intratumoral infusion, the first issue is bacterial dissemination from tumor to normal organs. It may significantly reduce gene delivery to tumor cells and cause adverse effects in normal tissues. Therefore, the Specific Aim 1 in the proposal is to investigate mechanisms of the dissemination using non-pathogenic Escherichia coli (E. coli). The study will quantify biodistribution of E. coli in mice after intratumoral infusion and use the data to determine kinetics and pathways of bacterial dissemination. Based on these results, the Specific Aim 2 is to develop novel strategies for blocking bacterial dissemination. A thermally sensitive polymer solution will be used to block the dissemination through tumor microvessels during intratumoral infusion and a novel synthetic gene circuit will be implemented into the E. coli for eliminating chronically disseminated bacteria at a few days after the infusion. The second issue is hindrance to bacterial transport in solid tumors, which may prohibit homogenous gene delivery. To improve the transport, the Specific Aim 3 is to genetically engineer E. coli to constitutively express invasin that can bind to several integrin receptors on tumor cells to facilitate transcellular transport. The study will quantify bacterial transport through transcellular and interstitial pathways in vitro, using both monolayer and multicellular layer (MCL) tumor models. Results from the study may provide important information on mechanisms of bacterial transport that can be used to improve bacterial delivery in vivo.
The objective of the proposed research is to address key issues in bacterium-mediated gene delivery. The study is innovative since systemic dissemination of bacteria has never been characterized in previous studies although intratumoral infusion has been a routine method for administration of all gene vectors. The proposed study will quantitatively investigate the dissemination and develop methods to block it. In addition, the study will explore synthetic biology, an emerging multidisciplinary field, for improving gene delivery. These innovative approaches to gene delivery are based on previous studies of the PI and Co-PI, funded by NSF and other agencies for improving delivery of macromolecules and viral vectors in solid tumors and developing novel synthetic gene circuits in bacteria. Results from the proposed study may reveal mechanisms of bacterium transport in tumors and facilitate discovery of novel design principles for bacterial vectors, which will likely be applicable to not only the E. coli used in this study but also other engineered bacteria.
Gene delivery is one of the main challenges in gene therapy. It has limited therapeutic efficacy in target tissues and caused adverse effects in normal organs. Results from the proposed research may be used to improve efficiency of gene delivery and safety in cancer gene therapy. In addition, the proposed research represents an important step in extending basic concepts and design methods in synthetic biology to address a pressing issue in cancer treatment. To further broaden the impact, the research projects will be integrated with biomedical engineering (BME) education at Duke University. Specifically, results from the research projects will be incorporated into three BME courses, which are currently taught by the PI and Co-PI. In addition, the research projects will involve undergraduate students through independent studies and the Pratt Research Fellow program at Duke University. Smaller research projects will also be developed for summer students recruited through an existing outreach program for underrepresented students and those with disabilities. These educational activities may stimulate students interests in science and engineering and encourage them to pursue careers in these fields. Finally, results from the proposed research will be presented at national and international conferences and published in scientific journals.
One of the challenges in cancer treatment is that therapeutic agents (e.g., drugs and genes) cannot reach the majority of tumor cells, because of various barriers in tumors that block transport and spreading of these agents. As a result, tumors often grow back after treatment. To develop strategies for overcoming the barriers, investigators in previous studies often need to use animal models of solid tumors. This requirement can make research complicated and inefficient. To solve the problem, we developed a new tumor model by growing tumor cells in a microfluidic channel to form three-dimensional tissue structures (see Figure 1). To demonstrate that the new model could mimic cellular barriers to small molecule drug delivery observed in animals, we investigated distribution of calcein-acetoxymethyl ester in microfluidic channels (see Figure 2). Both outer channels shown in Figure 1 were perfused with a solution containing this molecule and propidium iodide (red) at 12 hours post cell loading into the central channel. When calcein-acetoxymethyl ester was taken up by cells, the cells became green. The perfusion was performed for 20 min at 37oC. The image was taken immediately after the perfusion was stopped. The image shows that the penetration of calcein-acetoxymethyl ester into cells in the central channel is very limited. The depth of penetration was slightly improved after 3 hours but the pattern of calcein-acetoxymethyl ester distribution was still the same as that shown in Figure 2. To demonstrate that the new model could also be used for gene delivery studies, we investigated active transport of genetically engineered bacteria and distribution of E. coli cells co-expressing mCherry and invasin in the central channel containing B16.F10 skin tumor cells (see Figure 3). Stacked 2-µm optical slices of tumor and bacterial cells were obtained with confocal microscopy after the cells were loaded into the central channel and cultured for 12 hrs. Tumor cells pre-stained with calcein-acetoxymethyl ester are shown in green and E. coli cells are shown in red. The spreading of engineered bacteria in tumor tissues was more effective than that of calcein-acetoxymethyl ester shown in Figure 2. The results may inspire design of more efficient vehicles in the future for targeted delivery of drugs and genes in solid tumors. Overall, our study demonstrated that cellular structures in solid tumors are highly resistant to passive diffusion of molecules and that genetically engineered bacteria can spread effectively in tumors, suggesting that gene delivery vectors should be designed in such a way that they can move actively in target tissues. In future studies, the model will be used to facilitate development of new strategies for improving drug/gene distribution in tumor mass.
