Establishing a reference method to illuminate Tdarks with clone-aware single cell transcriptional perturbome in iPSC models: a pilot study for rare congenital heart defects using cardiac organoids

This project has been funded thanks to the Joint Call Fondazione Cariplo and Fondazione Telethon 2025.

There are over 5,000 genes, known as Tdarks, that are not well understood but could play a role in rare genetic diseases. Identifying how these genes contribute to diseases is crucial for developing new treatments. To help with this, we have created a method called iPS2-seq, which allows us to study these genes in human cells. This method combines two powerful techniques: switching off specific genes in cells and analyzing the effects of these changes at a very detailed level. It also tracks how different groups of related cells behave, improving the accuracy of the results. In this study, we will use iPS2-seq to focus on 10 Tdarks linked to congenital heart diseases (CHDs). Collectively, CHDs are the most common birth defect, affecting around 1 in 100 newborns, and its impact is growing due to better medical care. While we know that genetics plays a big part in CHD, many of the genes responsible are still unknown. Some Tdarks have been suggested as possible causes, but their roles are still unclear. We will explore the function of these Tdarks by creating 3D models of heart tissue from human stem cells. By switching off each of the 10 Tdarks in these models, we will observe how they affect heart development and which biological processes are involved. This will help us understand which genes are essential for normal heart function and which ones contribute to the disease. Our findings will not only provide insight into the genetic causes of CHD but also help us establish iPS2-seq as a tool that can be applied to study many other diseases. This method will offer a faster, more cost-effective way to investigate the role of Tdarks in various conditions, leading to new possibilities for future treatments.