DEVELOPMENT OF PHOTOLYASE ENZYME VARIANTS FUSED TO PEPTIDES FOR CELL PENETRATION AND NUCLEAR LOCALIZATION

• Grant number: 25/13399-6
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: October 01, 2025
• End date: September 30, 2030
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Carlota de Oliveira Rangel Yagui
• Grantee: Felipe Gobbi Gonçalves
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 23/17651-6 - STUDIES OF FORMULATION AND BIOLOGICAL EFFECT OF PHOTOLYASE IN IN VITRO SKIN MODELS AIMING AT PHOTOPROTECTION AND PREVENTION OF SKIN AGING, AP.R

Abstract

Ultraviolet (UV) radiation is primarily responsible for the genetic damage that leads to the development of non-melanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma. These tumors originate in keratinocytes that accumulate mutations, many of which are caused by photodamage, such as cyclobutane pyrimidine dimers (CPDs). The enzyme photolyase from Thermus thermophilus is capable of directly reversing this damage, representing a promising strategy to prevent the progression of lesions in epidermal cells to skin cancer. However, one of the greatest challenges for its use as a preventive strategy is the efficient delivery of the protein to the interior of the cells and its targeting to the nucleus, where the damaged DNA is found. Here we propose to develop recombinant variants of photolyase fused to cell-penetrating peptides (CPPs) that favor intracellular delivery and nuclear localization signal peptides (NLS) that target the nucleus, in addition to strategies that allow the control of the intracellular release of the enzyme through linkers sensitive to cleavage by cathepsin B. The constructs will be expressed in Escherichia coli and evaluated for their internalization capacity, subcellular localization, and efficiency in repairing CPDs in immortalized human keratinocytes. We expect to establish a technological basis for the development of more effective topical formulations for preventing UV-induced skin cancer. (AU)