ERC FUNDED PROJECTS

Our sensory systems gather stimuli as elemental physical features yet we perceive a world made up of familiar objects, not wavelengths or vibrations. Perception occurs when the neuronal representation of physical parameters is transformed into the neuronal representation of meaningful objects. How does this recoding occur? An ideal platform for the inquiry is the rat whisker sensory system: it produces fast and accurate judgments of complex stimuli, yet can be broken down into accessible neuronal mechanisms. CONCEPT will examine the process that begins with whisker motion and ends with perception of the contacted object. Understanding the general principles for the construction of perception will help explain why we experience the world as we do. The main hypothesis is that graded neuronal representations at early processing stages are “fractured” to generate discrete object representations at late processing stages. Of particular interest is the emergence of object representations as the meaning of new stimuli is acquired. We will collect multi-site single-unit and local field potential signals simultaneously with precise behavioral indices, and will interpret data through advanced computational methods. We will begin by quantifying whisker motion as rats discriminate texture, thus defining the raw material on which the brain operates. Next, we will characterize the transformation of texture along an intracortical stream from sensory areas (where we expect that neurons encode whisker kinematics) to frontal and rhinal areas (where we expect that neurons encode objects extracted from the graded physical continuum) and hippocampus (where we expect that neurons encode objects in conjunction with context). We will test candidate processing schemes by manipulating perception on single trials using optogenetic methods.

2 500 000 €

Start date: 2012-06-01, End date: 2018-05-31

Sudden cardiac death (SCD) is a leading cause of death in western countries: coronary artery disease is the major cause of SCD in older subjects while inherited arrhythmogenic diseases are the leading cause of SCD in younger individuals. After 25 years dedicated to research of the molecular bases of heritable arrhythmias, the PI of this proposal now intends to pioneer gene therapy for prevention of SCD: a virtually unexplored field. The development of molecular therapies for rhythm disturbances is a high risk effort however, if successful, it will be highly rewarding. The PI has envisioned an ambitious and comprehensive project to target two severe inherited arrhythmogenic diseases: dominant catecholaminergic polymorphic ventricular tachycardia (CPVT) and Long QT syndrome type 8 (LQT8). The availability of a clinically relevant model is critical to ensure clinical translation of results: the team will exploit an existing CPVT model and will engineer a knock-in pig to model LQT8. The PI and her team will investigate innovative strategies of gene-delivery, gene-silencing and gene-editing to the heart comparing efficacy of different constructs and promoters. The team will also carefully engineer novel gene-therapy approaches to avoid the development of regional inhomogeneity in protein expression that may facilitate proarrhythmic events. Such a comprehensive approach will provide a most valuable core of knowledge on the comparative efficacy of a broad range of molecular strategies on the electrical milieu of the heart. It is expected that these results will not only benefit CPVT and LQT8 but rather they will foster development of gene therapy for other inherited and acquired arrhythmias.

2 314 029 €

Start date: 2015-11-01, End date: 2020-10-31

Friedreich’s Ataxia (FRDA) is a devastating degenerative disease with no specific therapy. It is passed by autosomal recessive inheritance and affects 1:30,000 individuals in Caucasian populations. Symptoms appear in the first decade of life and include progressive and unremitting lack of movement coordination, leading to complete inability, and dilated cardiomyopathy leading to congestive heart failure, the most common cause of premature death. FRDA is due to the insufficient transcription of the gene coding for the mitochondrial protein frataxin. Reduced cellular levels of frataxin cause impaired mitochondrial function and increased sensitivity to oxidative stress, leading to accelerated cell death in critical tissues. Severity of the disease critically depends on residual frataxin levels. Therapeutic efforts are mostly focused on increasing cellular frataxin . We found that frataxin is normally degraded by the ubiquitin-proteasome system. We identified the lysine responsible for the ubiquitination of frataxin and, by computational screening followed by experimental validation, we identified and validated a series of small molecules, called ubiquitin-competing molecules (UCM), that prevent frataxin ubiquitination and induce frataxin accumulation in cells derived from FRDA patients. Moreover, treatment with UCM partially rescues aconitase and ATP production defects in cells derived from FRDA patients. Our goal is two fold: 1) submit a set of leads we already identified, as well as their new and more complex derivatives, to preclinical testing in FRDA mice 2) identify the E3 ligase that is responsible for frataxin ubiquitination, and investigate the possibility to use it as a druggable target for small molecules to prevent frataxin degradation.

1 496 200 €

Start date: 2012-03-01, End date: 2015-02-28

A foremost health problem stems from the burden of degenerative diseases, including heart failure, neurodegeneration, retinal degeneration and diabetes, essentially linked to the aging of the human population and the incapacity of post-mitotic tissues to undergo efficient repair. This is an ambitious, highly innovative project aimed at developing an in vivo selection procedure, based on gene transfer of two genetic libraries cloned into Adeno-Associated Virus (AAV)-based vectors, for the identification of novel secreted factors or microRNAs providing benefit against various degenerative diseases. Two arrayed libraries will be generated, one coding for ~1,300 cDNAs from the mouse secretome, the other for all known microRNAs (~800 genes). Pools of vectors from each library will be obtained with serotypes suitable for in vivo transduction of different organs. The vectors will be injected in a series of mouse models of degenerative disorders involving damage to cardiomyocytes,, neurodegeneration, retinal degeneration and loss of beta-cells in the pancreas. The degenerative conditions will drive the selection for secreted factors or miRNA putatively preventing cell apoptosis, enhancing residual cell function or, in the best possible scenario, promoting tissue regeneration. This in vivo selection approach, which is supported by very encouraging preliminary results, has never been attempted before and is rendered possible by the property of AAV vectors to be produced at high titers, infect tissues at high multiplicity, persist in the transduced cells for prolonged period of times and efficiently express their transgenes in vivo. In addition to its final goal of identifying novel biotherapeutics, the project entails the successful achievement of several intermediate objectives and is expected to extend both technology and knowledge beyond the state-of-the art.

1 824 000 €

Start date: 2010-04-01, End date: 2015-03-31

Background: CD4+ T lymphocyte subsets orchestrate immune responses in health and disease. Little is known on control of T cell differentiation exerted by microRNA that affect mRNA translation. The identification of microRNA and their targets that regulate differentiation of T cell subsets may provide new therapeutic targets for immune-mediated diseases. Since microRNA are released in exosomes and circulate in blood, activities of tissue-derived lymphocytes could be assessed by microRNA signatures in the serum. We have defined microRNAs present in resting lymphocyte subsets from peripheral blood and measured lymphocyte-derived microRNAs in the serum. We have also solved important challenges for the identification of microRNA targets, the definition of signatures of activated T cells and their monitoring in the serum, which form the key topics of this application. Advancing State-of-the-Art and objectives: We will identify microRNA of CD4+ T cell subsets purified from inflamed organs and investigate microRNA target network that regulates T cell differentiation. We will exploit this knowledge to profile signatures of in vivo activated T cells and to map genes that could improve understanding of T cell commitment. We will also develop quantitative assays to monitor microRNA signatures in the serum and provide functional evidence of key genes targeted by microRNA which could be targets of immunomodulatory drugs. Significance: This application addresses important challenges at the frontiers of immunology and could lead to significant advances in immunotherapies and diagnostic tools for patients with immune mediated diseases. New ways of identifying microRNA targets and techniques to quantify microRNA signatures in the serum, could be widely applicable in biomedical research.

2 496 000 €

Start date: 2011-06-01, End date: 2016-05-31

This proposal intends to apply Synthetic Biology to create a new bacterial species, Vaccinobacter, devoted to the production of multivalent, highly effective vaccines. The project originates from the evidence that Outer membrane Vesicles (OMVs) naturally produced by all Gram-negative bacteria can induce remarkable protective immunity, a property already exploited to develop anti-Neisseria vaccines now available for human use. OMV protection is mediated by the abundance of Pathogen-Associated-Molecular Patterns (PAMPs), known to play a key role in stimulating innate immunity. Moreover, OMVs can be engineered by delivering recombinant proteins to bacterial periplasm and outer membrane. Intrinsic adjuvanticity and propensity to be manipulated potentially make OMVs an ideal vaccine platform, particularly indicated when antigen combinations (for pathogens with genetic variability) and strong potentiation of immunity (for the elderly and cancer) are needed. However, full exploitation of OMVs as vaccines is prevented by: i) presence of potentially reactogenic compounds such as LPS, virulence factors, and toxins, ii) presence of several irrelevant proteins, which dilute immune responses, iii) lack of broadly applicable molecular tools to load OMVs with foreign antigens. Scope of the project is to provide novel solutions to solve these limitations and demonstrate the unique performance OMVs as vaccines by testing them on complex pathogens and cancer. Main project activities are: 1) remodelling of E. coli genome to create “Vaccinobacter”, a “living factory” of OMVs deprived of all unnecessary components but carrying the relevant immune potentiators, 2) characterization and optimization of the immune stimulatory properties of OMVs, 3) development of novel methods to incorporate foreign antigens into Vaccinobacter-derived OMVs, 4) loading of OMVs with selected pathogen- and cancer-derived antigens and demonstration of their protective efficacy in appropriate animal models.

2 612 828 €

Start date: 2014-06-01, End date: 2019-05-31

The main goal of this proposal is to introduce innovative devices and protocols (based on nano- manipulation, the response of micro-(nano-)mechanical oscillators and nano-fluidics) to carry out, precise, high throughput, high sensitivity, and low cost interactomic measurements. We aim at measuring, in parallel, the concentration of up to several proteins or non-coding RNA molecules, in samples down to the single cell level, following the real time concentrations of several biomarkers in patient-derived samples down to femto-molar concentrations, and single-circulating-tumor-cell resolution. We plan to develop our program a) by applying the principles and practice of intrinsically differential measurements, e.g. by building a self-assembled nano-devices that provide robust outputs measurable with topographic AFM imaging, electrochemical measurements, or gel electrophoresis, and b) by using the vertical equivalent of cantilever oscillators (pillars) that we plan to use as quartz “microbalances” that are 10,000 time more sensitive than cantilevers w. r. t. measurable min. mass and 100 time more sensitive w. r. t. dilution. The proposal’s core strategy is to exploit the PI’s expertise in innovative instrumentation and his integrated physical chemistry know-how, leading a highly multi-disciplinary staff to closely interact with first class medical staff in hospital settings to solve, and validate the solution of, relevant medical problems by generating innovative and versatile sensing devices. For instance, the sensitivity of our sensors will allow protein or miRNA analysis from very small and homogeneous samples of tumor cells, as well as the ease identification circulating tumor cells (CTCs) for, in addition to improved cancer diagnostics, also the prediction of patient response to treatment. The convergence between chemistry and biology, through nanotechnology, physics and computational approaches, with medical diagnostics will enable our team to come up with more versatile and reliable diagnostic tools, while stimulating fundamental research in fields as diverse as stem cells differentiation and the study of cell physiopathology.

2 979 700 €

Start date: 2011-07-01, End date: 2016-06-30

For chronic kidney diseases there is little chance that the vast majority of the world¿s population will have access to renal replacement therapy with dialysis. There is yet no adequate alternative with curative intent than allo-transplantation. This approach is seriously impaired by eventually limited graft survival and by the scarce availability of donors. We propose an innovative strategy that would help to simultaneously overwhelm the reduced access to dialysis, the need for organs for transplantation, the avoidance of patient exposure to immunosuppressants. The overall focus is based on the idea of generating new kidneys by tissue engineering technologies starting from a whole-kidney scaffold with intact three-dimensional geometry and vasculature created by de-cellularization of a kidney harvested from the patient with progressive chronic kidney disease (CKD). Repopulation of the kidney scaffold could be achieved with patient-specific induced pluripotent stem (iPS) cells generated by reprogramming to a pluripotent status of somatic cells. After regeneration, the kidney will be transplanted in the same patient. Thereafter, the same procedures will be repeated with the contralateral native injured kidney, after in vivo assessment of the proper functional performance of the newly generated transplanted organ. This goal will be pursued through the three steps introductory to the development of the best strategy for translating the proposed frontier research to patients with CKD: 1.Create an intact whole-kidney scaffold by de-cellularization of a rodent kidney and validate the procedure with a human kidney. 2. Attempt to reconstruct the organ by reseeding the rodent and human whole-kidney scaffolds with iPS cells generated by reprogramming adult dermal fibroblasts to a pluripotent status. 3. Deeply characterize the function of the new rodent and human kidneys by in vivo and in vitro techniques.

2 493 000 €

Start date: 2011-05-01, End date: 2016-04-30

Hematopoietic stem cell gene therapy has a tremendous potential to treat human disease. Yet, in conjunction with the first successful results in the clinic, severe adverse events linked to the gene transfer protocol were reported. Recently, we provided proof-of-principle of two new powerful strategies to improve the efficacy and safety of gene transfer: 1) regulating transgene expression by exploiting cellular microRNAs; 2) targeting integration at predetermined sites of the genome by forcing homologous recombination with designer Zinc finger nucleases. Here we will investigate the microRNA network regulating hematopoiesis and exploit the new knowledge to develop vectors with stringently controlled expression throughout the hematopoietic lineages. We will develop Zinc finger nuclease-based vectors that insert the transgene with high efficiency and specificity either downstream to its own endogenous promoter or into a safe genomic harbor that allows for robust expression without interference on the neighboring genes. By combining these strategies we will provide radically improved gene transfer platforms. Furthermore, we will exploit these technologies for the generation and genetic correction of induced pluripotent stem cells, providing a potentially unlimited source of patient-derived vector free gene corrected multipotent stem cells for future applications of regenerative medicine. The new gene therapy strategies will be tested in pre-clinical models of leukodystrophies and immunodeficiencies, for which we have extensive experience, and should enter a clinical trial for at least one such disease by the end of the proposed funding period. If successfully validated, the new strategies may eventually broaden the scope of gene therapy in medicine.

2 500 000 €

Start date: 2010-05-01, End date: 2016-01-31