Research projects available at IIT:
Projects referent: Dafne Campigli Di Giammartino
Main research area: Deciphering RNA-based mechanism through advanced microscopy
1. LiveTrack - Visualization of RNA trafficking in human motor neurons through live cell imaging
LiveTrack (Wet). The aim of the project is to combine interdisciplinary approaches to identify the coding and non-coding RNAs that are transported in neurons, and to track their trafficking in live cells in the presence of several ALS-causative mutations, to study the impact of those mutations on the physiological RNA transport and local translation at synapses. Methodology: Cutting-edge super-resolution microscopy techniques and advanced molecular biology methods for RNA visualization, including single-molecule detection and single-particle tracking systems recently been used from our labs (Mariani D. et al., bioRxiv, 2023.09.11.557245 2023), Bucci A. et al., Nat. Commun., 2024). RNA detection will incorporate single-molecule FISH, aptamer-based live RNA imaging methods, CRISPR-based RNA imaging and methodologies for the visualization of localized (SunTag). Model: Human cell lines, motor neurons derived from human Induced Pluripotent Stem Cells (iPSCs).
PIs: Irene Bozzoni, Giuseppe Vicidomini.
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli
2. TransCond - Exploring transcriptional condensate dynamics in live cells
TransCond (Wet). Chromatin architecture plays a crucial role in gene expression regulation by allowing the 3D physical proximity between regulatory regions in the genome (Di Giammartino et al 2019, Di Giammartino et al 2020, Murphy et al 2020). Several studies suggest that transcriptional condensates could regulate gene expression by modulating the 3D genome structure. Within this framework RNA has been shown to be a potential important player in transcriptional condensates. Here, we aim at defining transcriptional condensate dynamics in live cells upon perturbation of target non-coding RNAs.
Methodology: A variety of CRISPR-based technologies will be applied to endogenously tag proteins involved in transcriptional condensate formation, tracking them in-vivo. CRISPR-Cas13 system will be used to perturb target RNAs of interest (using the CRISPR-Cas13 system). Global perturbation of epitranscriptomic marks will be achieved with an inducible degron system for epitranscript writers, available in-house.
The candidate will explore the molecular dynamics of transcriptional condensates formation/alterations using advanced microscopy techniques in live cells, such as super-resolution imaging, fluorescence fluctuation spectroscopy, and fluorescence lifetime analysis (Perego et al 2023). Model Systems. Pluripotent mouse embryonic stem cells (including lines genetically/epigenetically modified) and patient-derived glioblastoma stem cells.
PIs: Dafne Campigli di Giammartino, Giuseppe Vicidomini.
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli
3. Spatial - Spatial multi-omics to characterize non-coding RNA classes involved in 3D genome architecture
Spatial (Hybrid, Wet and Computational). Different types of RNAs have been suggested to play a role in organizing the cell nucleus during the interphase at various genomic scales (Farabella et al., 2021). Cutting-edge imaging technologies have risen to the challenge of simultaneously describing multiple components of the nuclear space at the single-cell level (spatial multi-omics), paving the way for a deeper understanding of the genome structure-function relationship (reviewed in Flores et al., 2023). This project will focus on developing computational and experimental approaches to optimize the design of Oligopaints probes for targeting newly identified RNA classes crucial for 3D genome organization. These will provide critical insights into the ncRNA-chromatin interactomes at the single cell level, identifying among all non-coding RNAs and DNA elements tested those that can play a functional role for nuclear organization and could be identified as potential targets for 3D genome modulation.
Methodology: application of advanced spatial genomics technologies, developed for the direct multi-component acquisition of specific genomic regions and RNAs. Our approaches incorporate oligoprobe design with the most recent high-resolution and single-molecule microscopy technologies (for example sequential OligoSTORM or ORCA) or testing alternative super-resolution approach as image scanning microscopy (ISM). Model Systems. Human primary cultures and 3D models.
PI: Irene Farabella, Giuseppe Vicidomini.
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli
Projects referent: Laura Cancedda
Main research area: Investigating RNA in Health and Neurological Diseases
1. SmallDown - Small noncoding RNAs and neuroinflammation as pathophysiological mechanisms underlying neural deficits in Down Syndrome
SmallDown (Wet). Neuroinflammation and RNA homeostasis are dysregulated in Down Syndrome - DS, therefore are regarded as possible targets for therapy in DS (Pinto B., et al., Neuron. 2020, Rastogi M, et al. Neuron. 2024), and other neural diseases. Building on previous data by the host labs, the project will investigate small ncRNAs, particularly microRNA, PIWI-interacting RNAs and tRNA fragments. The goal is to identify novel mechanisms underlying neural deficits as well as biomarkers and actionable targets to ameliorate neural and eventually cognitive impairments in DS.
Methodology: The project will investigate neurophysiology and behavior in murine and human models of DS, taking advantage of multipronged approaches including SOTA imaging, multi-omics and bioinformatics platforms available at IIT.
PIs: Laura Cancedda, Davide De Pietri Tonelli.
Locations: Center for Convergent Technologies (CCT-Morego) – Genoa – Istituto Italiano di Tecnologia (IIT) - https://www.iit.it/it/cct-morego
2. Cerebro - Primary cilia as epigenetic regulators of non-coding RNAs’ activity in adaptive behaviors
Cerebro (Wet). Identify upstream signals leading to non-coding RNA dependent transcriptional modifications in adaptive behavior including L1 RNAs, and their contribution to behavioral (in)flexibility (Nazzaro et al, 2012; Boender et al 2021; Mangoni et a., 2023). These represent new potential therapeutic substrates for neurodevelopmental and neuropsychiatric conditions.
Methodology: high-resolution approaches, like Nanopore single molecule sequencing, and state-of-the-art techniques to study chromatin modifications and accessibility (CUT&Tag and ATAC-seq). Pathways involved in the regulation of chromatin architecture will be modulated using AAV-mediated RNA interference. Functional impact investigated through large-scale in vivo imaging of neuronal populations, complemented by advanced behavioral analysis based on machine learning algorithms. Genomic variants in genes for neuronal primary cilia will be analyzed in WGS data of ASD and neurotypical individuals.
Model Systems. Transgenic murine models, primary samples, primary cultures and immortalized cell lines.
PI: Raffella Tonini, Stefano Gustincich.
Locations: Center for Convergent Technologies (CCT-Morego) – Genoa – Istituto Italiano di Tecnologia (IIT) - https://www.iit.it/it/cct-morego
3. SYNm6A - Role of synaptic RNA methylation in associative learning
SYNm6A (Wet). RNA methylation is an important determinant for mRNA stability, localization and translation. In the present project we will investigate whether mRNA m6A methylation at dendritic and synaptic level is involved in the regulation of synaptic plasticity We aim at identifying m6A methylation as a general organizer of local protein synthesis in activity-dependent synaptic remodeling and clarify the mechanisms of valence coding in limbic circuits.
Methodology: We will focus our investigation on synaptosomes using fibers from basolateral amygdala (BLA) to the CA1 region of the ventral hippocampus that have been associated with the processing of emotional valence. Synaptic RNA will undergo Nanopore sequencing to identify the mRNA m6A methylation profile at single isoform level. Next we will interfere with methylation and we will study the possible alterations in the formation (and maintenance) of fear memory.
Model Systems. Fos-TRAP mice, synaptosomes.
PI: Andrea Barberis, Luca Pandolfini.
Locations: Center for Convergent Technologies (CCT-Morego) – Genoa – Istituto Italiano di Tecnologia (IIT) - https://www.iit.it/it/cct-morego
Projects Referent: Marco De Vivo
Main research area: RNA-Targeted Drug Design for Cancer and Genetic Disorders
1. SplicINH - A Platform for Splicing Modulation
SplicINH (Wet) The project's scientific goal is to design new molecular entities targeting the active site of splicing machineries to modulate splicing. This involves identifying and characterizing nanomolar splicing modulators for potential development into antibiotics, anticancer agents, or modulators of congenital disorders. A longer-term objective is developing these hits into modulators of the human spliceosome with sequence-specific mechanisms.
Methodology: The methodology includes RNA structure-based drug design, medicinal chemistry, synthesis of novel entities, biochemistry, cell biology, and structural biology. Advanced technologies like RNA crystallography, cryoEM, RNA molecular dynamics, and AI-powered drug design strategies are employed.
PIs: Marco De Vivo and Marco Marcia
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli
2. DDSSL - RNA-Targeted Drug Discovery Leveraging Synthetic Lethality in Cancer Therapy
DDSSL (Wet) The project aims to advance small-molecule RNA drug design using computational, structural, and biophysical approaches targeting RNAs identified as synthetic lethality (SL) partners from genomic screenings of cancer patients.
Methodology: Techniques such as SAXS, NMR, and biophysical data will provide kinetic and thermodynamic information on RNA-small molecule interactions. The small-molecule drugs identified through rational design will be validated in 2D and 3D models of human cancer cells, establishing a proof of concept for RNA-targeted drug discovery. This approach offers a selective and efficient mechanism to target cancer cells.
PIs: Andrea Cavalli, Stefania Girotto, Francesco Nicassio.
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli
Projects Referent: Irene Farabella
Main research area: Investigating the role of non-coding RNA in Human Cancer
1. 3DHubs (CHT-Erzelli, Genoa) - Dissecting RNA-dependent modulation of 3D hubs heterogeneity in cancer
3DHubs (Computational). Complex topological assemblies, in which multiple genes and regulatory elements are frequently interacting, are called 3D hubs and are considered a pivotal gene regulatory layer (Mendieta-Esteban et al., 2021; Di Giammartino et al., 2020;Di Giammartino et al., 2019). We focused on hyperconnected 3D enhancer-promoter hubs and demonstrated that hub-interacting genes exhibit high and coordinated expression at the single-cell level and strong association with oncogenic pathways (manuscript under revision). Based on this premises, this project aims at (i) providing key insights into the physical and functional communication within 3D hub, (ii) identify key RNAs drivers, and (iii) quantify the 3D hub connectivity and heterogeneity.
Methodology: the candidate will use existing computational tools (Mendieta-Esteban et al., 2021; Farabella et al., 2021) and develop novel methods to analyze an extensive collection of genomic information from cancer samples (3D genome, epigenome, RNA-interactome), aimed at acquiring a multi-omics view of the 3D hub interactome and focusing on RNAs element as drivers.
Model System: Human cancer cell lines such as patient-derived glioma stem cells.
Project hosted at CHT-Erzelli in Genoa.
PI: Irene Farabella, Dafne Campigli di Giammartino.
2. SMART-C (CCT-Morego, Genoa) - Small Molecules Acting on RNA to Treat Cancer
SMART-C (Wet). The project is specifically focused on the identification and biological evaluation of small molecules targeting non-coding RNAs involved in cancer cell survival and chemoresistance.
Methodology: The identification of small molecules targeting RNA will be achieved by integrated innovative screening methodologies, such as fragment-based screening using 19F-Nuclear Magnetic Resonance (NMR) and catalytic enzyme-linked click chemistry assays (cat-ELCCA). For the biological evaluation of the compounds, our approaches integrate diverse drug discovery technologies developed in house for monitoring different molecular pathways and for validating the activity and selectivity of compounds, such as fluorescence and luminescence cell-based reporter assays for in vitro real-time microscopy and in vivo imaging studies.
Model Systems. Human cancer cell lines, primary cultures, human organ cultures, preclinical animal models.
Project hosted at CCT-Morego in Genoa.
PI: Benedetto Grimaldi, Tiziano Bandiera.
Projects Referent: Mattia Pelizzola
Main research area: New Technologies for RNA Diagnostics and Therapeutic Strategies
1. ModsInBetween - Shaping the coding transcriptome through the epitranscriptional regulation of the non-coding transcriptome in human cancer
ModsInBetween. Epitranscriptional RNA modifications are emerging as determinant of gene expression programs of coding and non-coding transcripts. We aim to study how the epitranscriptome influences the ability of non-coding transcripts to regulate the coding transcriptome.
Methodology: Cutting-edge methods developed in the hosting labs for the study of: the dynamics of RNA metabolism, RNA modifications and gene expression programs at single-molecule resolution via Nanopore sequencing. Complementary expertise related to non-coding RNAs (expression, regulation, functional abrogation by CRISPR or ASOs) and gene expression control (at bulk and at single-cell resolution) are provided by Nicassio lab; gene circuits in gene expression control provided by Siciliano lab.
Model System: Human cancer cell lines.
Project: Hosted at CGS in Milan.
PIs: Mattia Pelizzola, Francesco Nicassio and Velia Siciliano.
2. PersisT - Engineering persistent T cell therapy regulated by T cell exhaustion-associated RNA Binding Proteins
PersisT. The goal is to set foundations for the engineering of a novel class of T cell-based therapeutics that harness the endogenous changes emerging during exhaustion. We will focus on a class of post-transcriptional regulators, the RNA Binding Proteins (RBP) whose contribution in exhaustion has not been established yet.
Methodology: approaches for the rapid prototyping of DNA-and RNA encoded circuits regulated by RBPs have been developed in Siciliano’s lab, using high-resolution technologies, including single-cell multi-omics coupled with bioinformatics tools. Dedicated microfluidic devices allow for the assessment of the proliferative capacity of the engineered T-cell, the analysis of the produced cytokines, and, in turn, the accurate spatio-temporal monitoring of the interaction between the engineered immune cells and the target cancer cells. High-resolution imaging technologies, including two-photon laser scanning fluorescence microscopy and super resolution will be also used to follow the cell dynamics.
Model Systems. Primary human T cells. Target cancer cell lines.
Project: Hosted at CABCH in Naples and CNLS in Rome.
PI: Velia Siciliano, Giancarlo Ruocco.
Projects Referent: Gian Gaetano Tartaglia
Main research area: Aptamer-Based Modulation and Imaging of Protein Aggregates
1. DAATAM! (CHT-Erzelli, Genoa) - Deciphering Aptamer Actions through Advanced Microscopy
DAATAM! (Wet) The project aims to understand how RNA aptamers inhibit protein aggregation, a key factor in neurodegenerative diseases like ALS (PMID: 35739092) . By using advanced microscopy techniques in live cells, the project seeks to identify aptamers with anti-aggregation properties and elucidate their mechanisms.
Methodology: Techniques such as super-resolution microscopy, fluorescence fluctuation spectroscopy, and fluorescence lifetime analysis will be employed. Starting with TDP-43 aggregation, the project will expand to other proteins related to neurodegenerative diseases, advancing the development of aptamer-based therapeutics.
PI: Elsa Zacco, Gian Gaetano Tartaglia, Giuseppe Vicidomini.
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli ; Center for Convergent Technologies (CCT) – Genoa – Istituto Italiano di Tecnologia (IIT) - https://www.iit.it/it/cct-morego
2. SYNGA (CCT-Morego, Genoa) - Dissecting the Role(s) of RNA in the Modulation of α-Synuclein Aggregates
SYNGA (Wet and Computational) The project aims to elucidate the role of non-coding RNAs in the formation, growth, and morphology of protein aggregates, specifically focusing on α-synuclein, which is implicated in neurodegenerative diseases like Parkinson’s, Lewy body dementia, and multiple system atrophy (PMID: 37870427). The goals include investigating the structural and temporal details of protein/RNA interactions and designing RNA-based aptamers for targeted intracellular action.
Methodology: Using techniques such as time-resolved static and dynamic light scattering, TEM, and confocal microscopy, combined with computational approaches like catRAPID, the project seeks to clarify RNA's mechanistic role in amyloid aggregation and develop therapeutically actionable RNA constructs.
PI: Nicola Tirelli, Gian Gaetano Tartaglia
Locations: Center for Human Technology (CHT) – Genoa (Erzelli) https://www.iit.it/it/cht-erzelli ; Center for Convergent Technologies (CCT) – Genoa – Istituto Italiano di Tecnologia (IIT) - https://www.iit.it/it/cct-morego