Maternally Inherited Leigh Syndrome is an incurable pediatric mitochondrial disorder with progressive loss of mental and movement abilities, caused by mutations in the ATP6 gene. It encodes for the subunit a of Complex V, which results in its instability, reduced ATP production rate, and elevated ROS levels. Patient-derived neural progenitor cells (NPCs) with a mutation in ATP6 display abnormally high mitochondrial membrane potential (MMP), which has been exploited to identify compounds of the class of PDE5 inhibitors (PDE5i) as possible candidates able to normalize it. However, the action mechanism is still unknown. This project aims to: i) establish a novel preclinical model for MILS to be exploited for a large (> 5500 compounds) high-throughput screening of repurposable drugs; ii) validate the phosphodiesterase 5 inhibitor (PDE5i) Sildenafil as an effective therapeutic option for MILS. Methods: Through a reprogramming-based strategy, human MILS-NPCs carrying homoplasmic mutations on the ATP6 gene were generated.

In my lab, biochemical techniques were used to evaluate CV assembly and enzymatic activity. The MMP of MILS-NPCs carrying different mutations was measured with flow cytometry. Cells were treated overnight with Sildenafil 10 µM, and the pharmacological effect and action mechanism were evaluated. Concomitantly, the MMP of Leigh Syndrome (LS)-NPCs with mutations in complex I and complex IV was investigated with high-content screening (HCS) imaging. Results: MMP hyperpolarization is a constant feature of MILS-NPCs, regardless of the pathogenic mutation, making it an ideal hallmark for phenotypic drug screening. All mutations affected protein stability, impaired assembly of complex V, and reduced OXPHOS-driven ATP content. Sildenafil normalized hyperpolarized mitochondria in MILS-NPCs. LS-NPCs presented depolarized MMP; when treated with Sildenafil, a partial re-polarization was observed. Preliminary data suggest that the action mechanism may involve a cGMP-dependent pathway. Conclusions: Sildenafil normalizes the hyperpolarized MMP and partially rescues depolarized MMP, making it a plausible therapeutic option in LS.

State of the art The blood vessels in most solid tumors have structural and functional abnormalities (1), which facilitate cancer cell intravasation and dissemination to distant organs. This abnormal vasculature favors resistance to anticancer drugs and radiotherapy, causes chaotic blood flow, and creates local hypoxia that triggers invasive genetic programs (1). Tumor blood vessel defects arise from altered regulation of endothelial cell adhesion to the extracellular matrix (ECM), primarily mediated by integrins. Currently, there is no direct integrin activation tool in live cells able to identify key modulators for therapeutic targeting.

Project’s goals The aim of the project is to unveil novel mechanisms of α5β1 integrin activation in endothelial cells (ECs) by means of: - the generation of a β1 integrin activation conformational sensor (β1IAS) in ECs by CRISPR-Cas9 technology - the identification of β1 integrin activation modulators through siRNA library-based high throughput screening (HTS) and Enrichment Analysis Project The β1 sensor was generated by exploiting the luminescence-based NanoBiT split luciferase tool (by Promega), able to directly and quantitatively detect β1 integrin activation. The two subunits of the NanoBiT probe were cloned into a flexible β1 integrin loop, whose conformation is affected by the hybrid domain swingout that happens when integrin gets activated. Then, a constitutive knock-in (KI) human immortalized Telo Aortic Endothelial Cell (TeloHAEC) clone, stably expressing the luminescent β1IAS, was created by CRISPR-Cas9 technology. The clone expressing β1IAS was used in an endothelial-specific siRNA library HTS, in collaboration with Misvik Biology. The rationale of the screening was to quantify the luminescent signal of the sensor to assess the impact of silenced genes on β1 integrin activation. Genes causing a decrease (z-score<-2) or an increase (z-score>2) in IAS luminescence were identified as, respectively, major positive (111 genes) and negative (44 genes) regulators of β1 integrin activation in ECs. The 155 selected candidates were enriched in pathways related to integrin-based focal adhesion, angiogenesis, and axon guidance. I am currently focused on the functional characterization of the top candidate hits to understand the mechanisms by which they influence integrin activation. This understanding is crucial for leveraging these candidates as therapeutic targets to normalize abnormal cancer vascularization. Recently, a new class of reticular adhesion complexes (RAs), based on clathrin machinery elements, has been reported (2). Since αvβ5 integrin is the primary adhesive receptor involved, future project perspectives aim at the generation of a β5 integrin activation conformational sensor. Methods - NanoBiT is a tool based on a molecularly engineered split luciferase formed by two subunits able to bright luminescence when brought in proximity and after furimazine substrate giving - CRISPR-Cas9 technology has been exploited to insert the NanoBiT gene (555 bp) in human β1 integrin gene - siRNA library-based HTS has been performed on 384-well plates and by lipid-mediated siRNA transfection in β1IAS KI TeloHAECs. Bioinformatic analysis were performed using R language and EnrichR was carried out for enrichment analyses - Immunofluorescence, adhesion/migration assays performed with an electrical impedance-based system (xCELLigence), and protein-protein interaction assays will be employed for candidates functional characterization

The aim of the project is to develop a 3D model based on biocompatible polymeric scaffolds engineered with human cells for skin regenerative medicine. At present, there is no an equivalent cutaneous able to satisfactorily mimic the native skin, nor a bio-engineered model that is also capable of facilitating the process of skin repair, especially if characterized by a profound loss of substance. In these experimental models, human mesenchymal stromal cells (hMSCs), fibroblast and or keratinocytes can be employed.

The purposes of the PhD project are both the improvement of the scaffolds for cutaneous applications and the optimization and standardization of the cell culture procedures for skin regeneration. Particularly, the 3D polymeric scaffolds will be modified and ameliorated in order to better reply to the clinical needing and these materials will be characterized in terms of porosity, physico-mechanical and thermal properties. Then, the study aims to improve the expansion of hMSCs, from different tissue sources (bone marrow, adipose tissue, umbilical cord), and induce their differentiation for skin regeneration. In fact, the protocols of cells differentiation has to be optimized and standardized, compared to the previously experience, in order to better define the timing for cells differentiation in vitro. In addition, a protocol of fibroblasts and keratinocytes expansion from donor skin biopsy to scaffolds will be settled. Cells will be grown and functionally characterized by comparing the effects of human Platelet Lysate (hPL) versus Fetal Bovine Serum (FBS) addition to culture media into the polymeric scaffolds for skin regeneration. The topical application of hPL can stimulate naturally the tissue regeneration and accelerate the healing processes through the local enrichment of growth factors. Moreover, cell culture procedure using hPL instead of FBS promotes cell differentiation and tissue repair, avoiding the risks of transmitting animal diseases thanks to the crucial mitogenic and chemotactic molecules contained in hPL. Analysis of cells viability, proliferation, differentiation will be done both in the 2d culture, as control, and 3d model in the novel scaffolds.

The methylation of RNA adenosines into N6-methyladenosine (m6A) is the most abundant internal mRNA modification in mammalian cells. Its deposition and removal are mediated by writer and eraser proteins respectively, while readers target m6A modified RNAs towards different biological processes (e.g., translation and decay). Consequently, m6A affects multiple cellular functions, including cell proliferation. Indeed, CRISPR- knock-out (KO) screenings identified m6A effectors as essential in cancer cell lines, e.g., colorectal cancer (CRC) and acute myeloid leukaemia (AML). This opens new questions regarding the mechanisms by which writers, readers and erasers contribute to the function of m6A in cancer, which may be investigated with CRISPR-cytosine base editing (CBE). In this technology, cytidine deaminase fused to a nicking Cas9 nuclease performs C->T transitions, allowing programmable nucleotide changes. This enables to edit m6A effectors at increased resolution and precision, if compared to classic genetic knockouts.

The aim of this project is to study m6A in cancer cell proliferation by means of a pooled CRISPR screening. By applying CRISPR-CBE and KO, enrichment analysis of the sgRNAs will determine the association between specific single-nucleotide mutations and gene-loss, and the proliferation phenotype. In details, by collaborating with bioinformatic expertise, we prepared the in-silico sgRNA library targeting m6A and control genes. We applied a tiling strategy (i.e., multiple sgRNAs targeting different gene locus) to perform a high-density mutagenesis of target genes. Afterwards, we purchased the synthesis of the library as an oligo pool and its cloning in a lentiviral vector. In parallel, we cloned the CBE and Cas9 sequences into lentiviral vectors that confer doxy-inducibility to their expression. We used these vectors for lentivirus preparation in HEK293TN cells. Following viral isolation, we infected and selected HT-29 CRC lines (HT-29 iBE and HT-29 iCas9). Upon induction, we confirmed the expression at gene (RT-PCR) and at protein level (Fluorescence microscopy and flow cytometry). The next steps include tests on the activity of the editors in selected cells by transfection of a set of sgRNAs targeting test sites. We also planned to prepare the lentivirus from the sgRNA plasmid library and use it to infect HT-29 iBE/iCas9 cells at MOI 0.3 (i.e., one sgRNA per cell). We will select sgRNA+ cells, and, upon induction of the editor (T0), the cells will proliferate for 2 weeks (T1). The Next Generation Sequencing of sgRNAs at T1 versus T0 will result in significantly depleted guides which identify gene domains essential for cell proliferation. Furthermore, we will perform the same experiments in AML cell lines in which m6A pathway proved to be essential for proliferation. Our screening will identify m6A genes important for cancer cell proliferation, and that might represent druggable sites. In addition, we will create a dataset of gene variants associated with the proliferative phenotype, which may be useful for assessing the function of variants of uncertain significance. The study will investigate the role of m6A writers, erasers and readers in CRC and AML proliferation and expect to provide molecular insights for the development of novel drug treatments.