Team:ColumbiaNYC/Design

Proof of Concept

SilenshR was borne from an identified shortcoming in chemotherapy. Systemic administration of cytotoxic agents leads to death even in healthy cells causing diarrhea, vomiting, temporary sterility and hair loss(1). Additionally, our SilenshR innovation works in tandem with radiotherapy, the efficacy of which is diminished when the solid tumor microenvironment becomes hypoxic. Diatomic oxygen assists in radiotherapy by forming free radicals that damage DNA, causing apoptosis within solid tumor cancers. In fact, cells that are anoxic at the time of irradiation are 3 times more resistant to the radiotherapy than cells under normoxic conditions(2). However, when the cancer cells preferentially adopt an aerobic glycolysis metabolism over aerobic respiration, the intratumoral pH decreases along with the oxygen content of the cancer. This is one significant limitation of radiotherapy.

SilenshR is able to pick up the slack where radiotherapy is limited, as bacteria have been known to innately colonize and proliferate within the hypoxic and immune-privileged cores of tumors(3). Assuming SilenshR bacteria can grow within tumors, would this therapy be otherwise effective? Would the metabolic burden of shRNA production be too much for the bacteria, given the shRNA sequence is in a high-copy number pUC plasmid? Could the shRNA transcribed within the SilenshR vector quantifiably reduce gene expression in a host-mammalian cell? Will the quorum sensing invasiveness circuit reliably promote bacterial uptake by cancer cells?

Through characterization of the growth of SilenshR bacteria in a BL21(DE3) chassis, it was determined that the metabolic burden of shRNA production would not interfere with the growth and proliferation of the SilenshR bacteria. The shRNA was transcribed from the high copy number pUC plasmid under a T7 promoter. For these proof of concept experiments, the shRNA targeted expression of GFP and contains a sequence complementary to the mRNA of GFP in the CellBioLabs HeLa line. The SilenshR bacteria induced to express the T7 polymerase grew comparably to bacteria that were not induced to express T7 polymerase, reaching a similar stationary phase cell density in a similar amount of time. Both induced and non-induced populations grew at 37°C in a shaking incubator in LB media; cell densities and OD600 were evaluated every 30 minutes. At each time point, 5uL of culture was plated and the number of colony forming units (CFU) per mL was calculated.

Fig 1: Normal bacteria growth, used as a comparison to growth curve with IPTG induction.
Fig 2: The two graphs above are a comparison of bacteria growth without IPTG induction (without shRNA production) and with IPTG induction (with shRNA production). The shRNA produced is designed to inhibit eGFP. Since the two growth curves are virtually identical, this shows that production of the designed shRNA is not toxic to the bacteria.

Quorum Sensing

While current experiments are seeking to install the genes Invasin and HlyA under the quorum inducible Lux promoter from Aiivibrio fischeri, the quorum sensing circuit was evaluated using GFP as a reporter. The bacteria in an E. coli Nissle chassis were grown in M9 media (see protocols for precise formulation) and OD600 absorbance and fluorescence were measured every 10 minutes, as shown by the 2 graphs below. At an OD600 value of 0.418, sufficient cell density was achieved for expression of GFP.

Fig 3: Optical Density (OD) measurements for E.coli Nissle bacteria with quorum sensing circuit.

Fig 4: GFP Fluorescence of E.coli Nissle bacteria with quorum sensing circuit. The orange data points represent fluorescence over time in E.coli Nissle without quorum sensing circuit and the blue data points represent E.coli Nissle bacteria with the quorum inducible circuit.

Knockdown via Liposome Transfection

Finally, the GFP shRNA was isolated from the induced BL21 (DE3) cells with an miRNeasy Kit (Qiagen) and assessed by gel electrophoresis to verify presence of the shRNA transcript. Following, the shRNA was complexed with cationic liposomes and transfected into GFP-expressing HeLa cells (CellBioLabs) using the lipofectamine 2000 protocol. GFP expression from HeLa cells was quantified 48 hours following transfection of liposomes with PBS, positive control siRNA against GFP (CellBioLabs) and the induced SilenshR shRNA for GFP. The shRNA targeting GFP produced in the SilenshR bacteria showed a significant reduction in GFP expression within the HeLa cells, as quantified by flow cytometry.


Fig 5: Bar plot showing GFP knockdown in HeLa cells. The experiment was performed in triplicate. The error bars represent the standard deviation of the samples

References:

  1. Dunnill et. al. (2017). A Clinical and Biological Guide for Understanding Chemotherapy‐Induced Alopecia and Its Prevention.” Oncologist, doi: 10.1634/theoncologist.2017-0263
  2. Rockwell, S., Dobrucki, I. T., Kim, E. Y., Marrison, S. T., & Vu, V. T. (2009). Hypoxia and radiation therapy: Past history, ongoing research, and future promise. Current Molecular Medicine, 9(4), 442–458.
  3. Kathrin Westphal, Sara Leschner, Jadwiga Jablonska, Holger Loessner and Siegfried Weiss Cancer Res April 15 2008 (68) (8) 2952-2960; DOI: 10.1158/0008-5472.CAN-07-2984