Abstract
While gene-directed enzyme prodrug therapy has shown potential as a cancer therapeutic in animal and clinical trials, concerns over the efficacy, selectivity, and safety of gene delivery vehicles have restricted its advance. In an attempt to relieve some of the demands on targeted gene delivery vehicles and achieve the full potential of enzyme prodrug therapy, cancer-targeted activity can be engineered into the enzyme itself. We previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells accumulating the cancer marker hypoxia-inducible factor-1α (HIF-1α). This HIF-1α-activated protein switch (Haps59) is designed to increase its ability to convert the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil in a HIF-1α-dependent manner. However, in cancer cell lines expressing Haps59 the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. In this work, we aimed to improve the cancer specificity of this switch via a directed evolution approach utilizing random mutagenesis, linker mutagenesis, and random insertion and circular permutation. We identified improved HIF-1α-activated protein switches that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. Additionally, the current bottleneck in the development of improved HIF-1α-activated protein switches is screening switch candidates in mammalian cells. To accommodate higher throughput and reduce experimental variability, we explored the use of Flp recombinase-mediated isogenic integration in 293 cells. These experiments raised the possibility that Haps59 can be activated by other interactors of the CH1 domain, and experiments in E. coli indicated that CITED2 can also activate Haps59. Although many CH1 binding partners are also oncogenes, CH1's promiscuous binding and subsequent off-target activation of Haps59 needs to be examined under normal physiological conditions to identify off-target activators. With aberrant activating molecules identified, further directed evolution can be performed to improve the cancer specificity of HIF-1α-activated protein switches.
Highlights
Despite the expansive development of targeted cancer therapies, traditional chemotherapeutics are still used during the course of treatment against most cancers
We created three types of libraries in an attempt to identify switches with properties superior to Haps59 via positive and negative genetic selections. These three libraries were 1) linker libraries in which the linker regions between the CH1 and yeast cytosine deaminase (yCD) domains of Haps59 were varied in length and amino acid sequence (Fig 2B), 2) a random mutagenesis library of Haps59 (Fig 2C), and 3) a library designed to consist of all possible circular permutations of the CH1 domain inserted at every position in yCD (Fig 2D)
We found that CITED2 activates Haps59 to a nearly identical extent as hypoxia-inducible factor-1a (HIF-1a), E1A does not (Fig 5C)
Summary
Despite the expansive development of targeted cancer therapies, traditional chemotherapeutics are still used during the course of treatment against most cancers Some chemotherapeutics, such as 5-fluorouracil, methotrexate, and taxol exert selective toxicity by exploiting the increased metabolism of cancer cells. The therapeutic window—the dosage range that can effectively kill cancer cells while leaving normal cells intact—is very narrow for chemotherapeutics. Even within this window side effects associated with toxicity to the digestive system, immune system, and hair follicles can be devastating to patients. Enzyme prodrug therapy (EPT) attempts to limit off-target toxicity by producing the active form of a chemotherapeutic selectively within cancer cells or the cancer microenvironment. An additional layer of tumor selectivity can be achieved via a tumor specific promoter, if the gene encoding the enzyme is delivered [8]
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