Design of an efficient enzyme in the activation of the chemotherapeutic drug cyclophosphamide for use in gene therapy of cancer

Ecuador reports an incidence of more than twenty -eight thousand cases of cancer per year, of which the prostate prevail with 47.5 cases per hundred thousand inhabitants and follows the breast with 34.4 cases per hundred thousand inhabitants. In the case of women, in 2017, breast cancer occupied the eleventh place in the list of general causes of death. This makes it one of the main health order problems in the country. According to Solca (2021), eight out of every hundred women in the city of Quito will develop breast cancer throughout their lives; Figure that has been increasing over the years. The treatment of breast cancer consists mainly of elimination of malignant cells through methods that include surgery, chemotherapy, radiation, immunotherapy and hormone therapy. However, many of these therapies do not discriminate normal tumor cells causing collateral damage to the patient. An alternative to reduce cytotoxicity to normal tissues and to treat cancer cells selectively is the gen-directed profármaco enzyme therapy (GDEPT). This therapy consists of i) Introduction of a coding gene for a non -endogenous enzyme, ii) intracellular expression and production of the enzyme and iii) activation of the profármaco after its administration in white cells. Cyclophosphamide (Profármaco) is a molecule widely used in breast cancer chemotherapy, however, its active form is highly toxic to normal tissues such as: for example: gastrointestinal tract, capillary follicles and spinal cord. Through GDEPT, strategies such as the activation of cyclophosphamide can be used only when it is in the affected tissue (using a vehicle as a retrovirus), avoiding its activation by liver enzymes and systemic distribution. It is known that enzymes such as Human P450 cytochromes (CYP2B and CYP3A) convert cyclophosphamide to their toxic metabolite that attacks and damages the DNA of rapid division cells. However, these enzymes are not efficient enough (k m = 2 mm), which limits its use in GDEPT. Fortunately, through the use of molecular techniques such as ancestral sequences reconstruction (ASR) and evolution directed in this project, its robustness, efficiency and affinity of the enzyme by cyclophosphamide can be increased. Consequently, with this project it is expected: i) Promote the activation of cyclophosphamide to its anti -cancer form thanks to the improved kinetic characteristics of the P450 enzyme; I) reduce the dose of administered cyclophosphamide required to generate a cytotoxic effect compared to the enzyme control; Iii) favor the Bystander effect "and therefore cell death and elimination of the in vitro tumor. All these results will contribute, to the future, to improve the efficiency of breast cancer treatment mediated by cyclophosphamide and reduce the side effects and costs associated with it."

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