ENGINEERING THE SECRETOME:MODIFYING CELLS WITH Cas13 TO STIMULATE ANGIOGENESIS

• Grant number: 24/00042-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2024
• End date: February 28, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Roberta Sessa Stilhano Yamaguchi
• Grantee: Sara Carolina Vieira Terra
• Host Institution: Faculdade de Ciências Médicas da Santa Casa de São Paulo (FCMSCSP). Fundação Arnaldo Vieira de Carvalho. São Paulo , SP, Brazil

Abstract

Peripheral Arterial Disease (PAD) is a clinical disorder that arises from stenosis or occlusion ofarteries in the upper or lower limbs. Current strategies and traditional pharmacotherapy for PAD treatment, without neovascularization, have limited evidence, especially in preventing amputations - the worst outcome of this disease. On the other hand, gene or cell therapy for angiogenesis stimulation studied so far has not shown consistent results in large randomized clinical trials.It is now known that hypoxia-inducible factor (HIF) is a key player in coordinating the cellular response to hypoxia, and HIF prolyl hydroxylase domain enzymes (PHDs) play a crucial role in HIF± degradation. Downregulation of PHDs via gene therapy aiming at stabilizing HIF emerges as a potential therapeutic target for PAD. Among various gene therapy tools, the CRISPRCas13d system stands out as the most promising, whose Cas13 effector protein is capable of regulating the expression of a target gene by cleaving its mRNA.Furthermore, conventional cell therapy used for PAD treatment has also shown limitations. It is known that adipose tissue-derived stem cells (ADSC) have significant angiogenic potential, mainly through their extracellular vesicles, which are proven to promote angiogenesis and reduce inflammation and cellular apoptosis.Thus, the present study aims to apply the Cas13 cleavage tool for the downregulation of the HIF prolyl 4-hydroxylase 2 enzyme (PHD2), a prominent agent in angiogenesis inhibition, to increase the expression of angiogenic genes in HUVEC endothelial cell line and ADSC stem cell line. After editing, the gene and protein expression, migration, and angiogenic capacity of modified HUVECs and modified ADSC's extracellular vesicles (exosomes) will be evaluated in vitro.