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.
Generate disease models for rare genetic diseases in the laboratory
We generate induced pluripotent stem cells (iPSC) using somatic cells from patients affected by rare genetic diseases, including steroid-resistant nephrotic syndrome, atypical haemolytic uremic syndrome and polycystic kidney disease. Differentiating iPSC into renal progenitor cells, podocytes and endothelial and tubular cells allows us to identify the alterations to molecular mechanisms responsible for rare genetic diseases and to create in vitro renal structures for drug screening.
Correct or delete a gene using the CRISPR-Cas9 gene editing technique
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. Using this system, we generated induced pluripotent cell lines lacking the genes coding either for polycystin 1 or polycystin 2, two proteins known to be involved in autosomal dominant polycystic kidney disease. We also corrected a point mutation in the PAX2 gene in induced pluripotent cells derived from a patient with adult-onset focal segmental glomerulosclerosis. The edited cells have allowed us to understand the molecular mechanisms underlying the disease and will allow us to possibly design new, tailored drugs.
Development of new advanced therapy approaches to treat rare diseases
Using the CRISPR-Cas9 system we have modified healthy iPSC cells to make them into universal cells – also called hypoimmunogenic (hypo-iPSC) – that are no longer patient-specific and therefore usable as possible cell therapy for all patients. We will differentiate the hypo-iPSC into adult liver cells, with which we will create 3D structures capable, once transplanted into experimental models, of releasing therapeutic proteins into the circulation.
Characterisation of the morphological-structural lesions associated with kidney diseases
We analyse renal tissue from patients and experimental models in order to characterise the renal structural lesions in 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. Using SEM, we have demonstrated that the negligible renoprotection afforded by drugs, if started late in the course of the disease, is due to the loss of glomerular structural integrity, and specifically to the loss of the cells that are targeted by the drug, which therefore cannot exert its protective effect. 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. We also developed an innovative imaging method, called SE-SEM, based on SEM acquisition of secondary electrons emitted from a section of tissue placed on a carbon-coated glass slide. This method allows us to observe renal tissue and other organs with a resolution similar to that achieved by transmission electron microscopy, but in larger areas of interest and much more quickly than is possible with conventional electron microscopy methods.
International Consensus on Cardiopulmonary Resuscitation.