The laboratory deals with creating in vitro models of rare genetic diseases with the aim of studying the molecular mechanisms involved in the progression of the disease and to identify targeted therapies.
In the laboratory the following techniques are used:
- cell biology, including the generation of induced pluripotent stemcells (iPSCs) from patients suffering from rare genetic diseases and the development of protocols to differentiate iPSC cells into adult cells
- molecular biology, including the use of the CRISPR/Cas9 technique to correct gene mutations in patient cells or to eliminate the expression of a gene
- microscopy, including optical, fluorescence, confocal, transmission and scanning electron microscopy (which is equipped with a focused ion beam (FIB) for the 3D reconstruction of biological samples) and laser microdissection with subsequent DNA, RNA, microRNA and protein analysis.
In vitro disease modelling for rare genetic diseases
We generate induced pluripotent stem cells (iPSC) from somatic cells of patients affected by rare genetic diseases, including steroid-resistant nephrotic syndrome, the atypical hemolytic uremic syndrome and the polycystic kidney disease. Differentiating iPSC into renal progenitor cells, podocytes, endothelial and tubular cells allow us to clarify the molecular mechanisms underlying the genetic rare diseases affecting the kidney, and create in vitro renal structures for drug screening.
Gene editing by CRISPR/Cas9 to correct or delete a gene
The CRISPR-Cas9 system is based on a protein, Cas9, which can be easily programmed to cut DNA at specific points in the genome. This system can be used both to eliminate specific genes and as a genomic corrector. In particular, we have generated induced pluripotent cell lines lacking the genes coding either for polycystin 1 or polycystin 2, two proteins known to be involved in the autosomal dominant polycystic kidney disease. We have also corrected a point mutation in the PAX2 gene in induced pluripotent cells derived from a patient with adult-onset focal segmental glomerulosclerosis. In both cases, the edited cells will allow us to understand the molecular mechanisms underlying the disease and possibly design new tailored drugs.
Characterization of the morphological-structural lesions associated with kidney diseases
We analyse renal tissue from patients and experimental models in order to characterise the constellation of renal structural lesions induced by renal pathologies and to evaluate the efficacy of pharmacological treatments, with a particular focus on the ultrastructure and function of the glomerular filter. With the aim of creating the first scanning electron microscopy (SEM) atlas of renal pathology, we have analysed patients with severe diabetic nephropathy, the single leading cause of end-stage renal disease in the industrialised world and have demonstrated that the negligible renoprotection afforded by drugs, if started late in the course of the disease, is due to loss of glomerular structural integrity. Using a specific SEM application, we have also documented how the glomerular filtration pores are really organised and assessed a digital morphometrical method for quantifying filtration pore dimensions, a crucial tool for evaluating glomerular filter integrity.
International Consensus on Cardiopulmonary Resuscitation.