Kathy Tran's GOx Aptamer Selection Project (2019)

RNA Aptamer Against Glucose Oxidase to Initiate Glucose Starvation and Cell Death in Acute Myelogenous Leukemia

Introduction

Leukemia is a type of blood cancer that targets stem cells. Most often, leukemia starts in early forms of white blood cells. However, depending on the type of leukemia, it may start in other blood cell types. Specifically, acute myeloid leukemia (AML) is defined as a disease of older people seen in ages sixty and up. AML starts in the bone marrow which is known as the location where new blood cells are formed. Bone marrow consists of blood-forming cells, and a small fraction of the blood-forming cells are blood stem cells. While the blood stem cells develop into new blood cells, they may either become lymphocytes or other types of myeloid cells (Lowenberg, Downing, & Burnett, 1999). There have been many invasive treatments found to eliminate AML cells such as chemotherapy, radiation therapy, stem cell transplant, and/or surgery which can be very harmful towards these affected patients. In particular, chemotherapy kills fast-growing cancer cells; however, it also slows down the growth of healthy cells (Jordan, Guzman, & Noble, 2006). This may infer that current treatments for AML are not targeted to AML cells enough such that treatments are not directed to only cancer cell death. Therefore, with the use of aptamers, there is a chance for an effective therapeutic treatment through the oxidation of GOx.

Figure 2. The Enzymatic Action of Glucose Oxidase. (Bankar, Bule, Singhal, & Ananthanarayan, 2009)

The enzyme glucose oxidase, catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-1,5-lactone as seen in figure 2. The catalyzation of GOx may be utilized to induce apoptosis in cancerous cells. Based on the catalytic chemistry of GOx, the enzyme consumes glucose which would provide an alternative strategy for cancer-starvation therapy and the production of H2O2 (Golub, Freeman, Niazov, & Willner, 2011). GOx would interact with the cancer cell and initiate the depletion of glucose as well as produce hydrogen peroxide. The loss of glucose means the cell may not undergo glycolysis and respiration, which would result in no ATP production in cancer cells. With no ATP in the cells, the cancer cell would eventually perform apoptosis with the help of p53, a tumor suppressor. P53 is a protein that functions as a tumor suppressor because it would encode for a protein that would regulate the cell cycle. The tumor protein has the ability to conserve stability through preventing genome mutation in the cells (Kojima et al., 2005). In this case, the decrease in glucose in the cell may trigger the increase in p53 proteins which would result in one of the major functions of p53 - apoptosis. To conclude, cells heavily rely on glucose in order to produce ATP for cells. Therefore, with the help of glucose oxidase, the initiation of glucose starvation would ideally lead to apoptosis.

Through the use of aptamers, there could be a way to target cancer cells with glucose oxidase. An aptamer, a unique oligonucleotide, has a high binding affinity to a target such as a protein or a small molecule. The target for this application is glucose oxidase, which would serve as a therapeutic application. In specific, an aptamer with an affinity for glucose oxidase would bind to the KH1C12 aptamer against AML cells by the incorporation of a complementary sequence at the end of both aptamers (Sefah et al., 2009). The KH1C12 would then bind to receptor proteins on the surface of the AML cells which would initiate the cell to engulf the aptamer. Since the KH1C12 targeted acute myeloid cells, the glucose oxidase would only affect the cancer cells. The aptamer will gain entry to the cells to deliver the GOx through liposomal drug delivery; this will allow the delivery of therapeutic agents at the target site to the cell. Liposomes take effect on the tumor by implementing less amount of drug at the target site which minimizes toxic effect and increases therapeutic effect which leads to the enhancement of bioavailability (Jha & Malviya, 2016). This will provide the potential to maximize the effect of the drug concentration in AML cells. The GOx would initiate glucose starvation in the cancer cell which would then direct the cell to perform apoptosis as noted above.

Figure 1. In vitro selection of target-specific aptamers using SELEX technology. (Stoltenburg R; et al. 2007)

To find an aptamer with a high affinity for glucose oxidase, the SELEX method, shown in Figure 1, will be performed using an ​in vitro​ filter-based selection. This iterative process will decrease the species diversity of the pool. The filter-based method will be performed in order to immobilize glucose oxidase, which will then be introduced to the N71 RNA pool under certain conditions. Binding and selection will be performed in order to interact the nucleic acid pool and the target protein towards the binding equilibrium, and the weakly bound pool would be washed away. The selected species will be amplified and converted back into RNA in order to generate an enriched pool for utilization in subsequent rounds of selection. This iterative process will decrease the species diversity of the pool. The RNA will be put back into another subsequent round of selection. After several rounds of selection, the pool would be sequenced in order to find whether the pool has been enriched. The sequence of that possible aptamer can then be used in a binding assay to determine its effect on the target.

An RNA filter-based aptamer selection against glucose oxidase is underway. Currently, the SELEX process utilizing filter-based aptamer selection against glucose oxidase is underway. All procedures for round 2 have yielded successful results. Future work will include binding and selection of round 3. By the next reporting period, the goal is to finish the third round of selection against glucose oxidase. Once an aptamer has been selected against GOX, and a conjugate aptamer for KH1C12, an ideal therapeutic treatment may be developed for acute myeloid leukemia. This treatment would hold a prominent impact on the quality of life, patients’ prognosis, and cancer research.

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References

Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase — An Overview. ​Biotechnology Advances, 27​(4), 489–501. https://doi.org/10.1016/J.BIOTECHADV .2009.04.003

Golub, E., Freeman, R., Niazov, A., & Willner, I. (2011). Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer–thrombin complexes, and probing the activity of glucose oxidase. Analyst,136(​ 21), 4397–4401. https://doi.org/10.1039/C1AN15596B

Jha, S., & Malviya, P. K. S. and R. (2016). Liposomal Drug Delivery System for Cancer Therapy: Advancement and Patents. ​Recent Patents on Drug Delivery & Formulation, Vol. 10,​ pp.177–183.https://doi.org/http://dx.doi.org/10.2174/1872211310666161004155757

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Sefah, K., Tang, Z. W., Shangguan, D. H., Chen, H., Lopez-Colon, D., Li, Y., ... Tan, W. H. (2009).

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Stoltenburg R; ​et al​. SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. ​Biomol Eng,​ 2007, 24(​ 4):381-403.