Generation of gene knockout cell models for formaldehyde-metabolizing enzymes in XP-D fibroblasts

• Grant number: 24/22449-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2025
• End date: February 28, 2026
• Field of knowledge: Biological Sciences - Genetics - Mutagenesis
• Principal Investigator: Carlos Frederico Martins Menck
• Grantee: Nicole Louise Belck
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 19/19435-3 - The role of DNA damage and mitochondrial function in vascular, immune and neurological ageing (DNA MoVINg), AP.TEM

Abstract

The ERCC2/XPD gene encodes a protein of the same name that takes part in the nucleotide excision DNA repair pathway (NER). Individuals with a mutation in this gene, such as patients with Xeroderma Pigmentosum group D, experience symptoms related to lesions on the genetic material caused by ultraviolet radiation from sunlight. In some cases, signs of neurodegeneration are also observed, which are not explained by this external agent, bringing suspicion that these cells are incapable of dealing with DNA lesions caused by an endogenous substance. In this sense, formaldehyde (FA), produced during metabolic cellular processes, is a carcinogen that in animal models of Cockayne Syndrome, a condition in which there are defects in proteins of a subpathway of NER, have been associated with the neurodegeneration phenotype by causing DNA damage. However, the enzymes encoded by the ADH5 and ALDH2 genes metabolize FA into formate, reducing its negative impact. Thus, in this project, using the CRISPR/Cas9 system, ADH5 knockouts, ALDH2 knockouts, and double ADH5/ALDH2 knockouts cellular models will be developed from fibroblasts with defects in XPD and fibroblasts of the same origin complemented with the functional XPD gene. Using cell viability, clonogenic survival, and cell cycle analysis assays, the sensitivity to FA will be evaluated. We expect to contribute to the understanding on the involvement of this aldehyde in the neurodegeneration of patients with dysfunctional XPD.