VALIDATION OF CANDIDATE ENHANCERS OF CARDIAC SPECIFIC GENES

• Grant number: 23/15481-6
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: April 22, 2024
• End date: April 21, 2025
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: José Eduardo Krieger
• Grantee: Nikolas Dresch Ferreira
• Supervisor: Sean M Wu
• Host Institution: Instituto do Coração Professor Euryclides de Jesus Zerbini (INCOR). Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Institution abroad: Stanford University, United States
• Associated to the scholarship: 23/05709-0 - Effect of Notch1 signaling in hiPSC-derived cardiomyocytes differentiation, BP.DD

Abstract

Current differentiation protocols for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) yield a mixture of immature cardiac subtypes, limiting their therapeutic application. Unraveling the specific mechanisms regulating cardiac specialization is fundamental for refining hiPSC-CM differentiation and comprehending the complexities of the cardiac system. We have explored the roles of key molecules in cardiac specialization. Depletion of IRX3 transcription factor revealed its role in early heart development by enhancing hiPSC commitment to cardiac differentiation. Additionally, inhibiting the Notch1 pathway during hiPSC-CM differentiation led to significant changes resembling a ventricle-like working phenotype. Despite these advances, the precise mechanisms governing these specializations remain elusive. We want to address these challenges focusing on the validation of candidate enhancers of cardiac genes recently identified by Dr. Sean Wu's lab at Stanford University. Using CRISPR technology, we want to silence enhancers associated with MYL2, a ventricle-specific gene, following its characterization in differentiated cardiomyocytes. This approach will be conducted in a hiPSCs-line featuring a MYL2-tdTomato reporter. The impact of enhancer silencing on MYL2 expression will then be assessed through fluorescence intensity quantification. The enhancers that have affected MYL2 expression will then be selected for further characterization. This strategy has the potential to provide novel insights about the target gene and the key enhancers underlying cardiac-specific differentiation.This international internship application represents a collaborative effort between Dr. José Eduardo Krieger and Dr. Sean Wu's lab (Division of Cardiovascular Medicine - Stanford University School of Medicine). Our primary goal is to validate candidate enhancers of genes related to cardiac development, capitalizing on the unique expertise developed in Dr. Sean Wu's group. This proposal is feasible within the proposed 12 months and will advance the overall theme of my thesis. It will also provide me with expertise in methodologies that can be incorporated into our Lab at InCor upon my return.