ERC FUNDED PROJECTS

"Catabolism of amino acids is an ancient survival strategy that also controls immune responses in mammals. Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions, including autoimmune diseases, in which it is often defective, and neoplasia, in which it promotes immune unresponsiveness. The PI’s group recently revealed that IDO does not merely degrade tryptophan and produce immunoregulatory kynurenines but also acts as a signal-transducing molecule independently of its enzyme activity. IDO’s signaling function relies on the presence of phosphorylable motifs in a region (small IDO domain) distant from the catalytic site (large IDO domain). Preliminary data indicate that IDO, depending on microenvironmental conditions, can move among distinct cellular compartments. Thus IDO may be considered a ‘moonligthing’ protein, i.e., an ancestral metabolic molecule that, during evolution, has acquired the DYNAMIC feature of moving intracellularly and switching among distinct functions by changing its conformational state. By means of computational studies, Macchiarulo’s group (team member) has identified distinct conformations of IDO, some of which are associated with optimal catalytic activity of the enzyme whereas others may favor tyrosine phosphorylation of IDO’s small domain. A switch between distinct conformations can be induced by the use of ligands that bind either the catalytic site or an accessory pocket outside the IDO catalytic site. The first aim of DIDO is to decipher the relationships between IDO conformations and multiple functions of the enzyme. A second aim is to identify small molecules with drug-like properties capable of modulating distinct IDO’s molecular conformations in order to either potentiate (a new therapeutic approach in autoimmune diseases) or inhibit (more efficient anti-tumor therapeutic strategy) immunoregulatory signaling ability of IDO."

2 442 078 €

Start date: 2014-02-01, End date: 2019-01-31

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

This proposal is aimed at identifying the molecular mechanisms that have brought the human Huntington Disease-causing Huntingtin (Htt) exon 1, with its pure and unstable CAG repeat, to be shaped the way it is today. Specifically, we intend to screen for genetic elements affecting Htt repeat length instability in dividing and postmitotic neuronal cells. The novelty of our approach relies on the construction of a human embryonic stem (hES) cell platform that couples highly efficient CRISPR/Cas9 technology with genome-wide screenings and third generation sequencing, to test the contribution of thousands of unequivocally barcoded cis and trans modifiers on Htt exon 1 repeats instability. In Aim 1, we will test the contribution of cis-modifiers to repeat instability during multiple mitotic divisions, by generating a hES cell platform where we will subsequently introduce a barcoded donor library of different Htt exon 1 constructs, with different CAG and flanking sequences, at the Htt locus. In Aim 2 our hES cell platform will be implemented with inducible Cas9 elements and sgRNAs libraries to perform genome-wide loss and gain of function (LOF, GOF) screenings of trans-acting modifiers of repeat sequence and size. The sgRNAs will act as barcodes for the modifier genes, allowing to test their causative role on repeat size changes. In Aim 3, we will exploit the neurogenic potential of hES cells in our LOF and GOF platforms to identify Htt exon 1 repeat modifiers in differentiating striatal neurons. Candidate modifier genes will be individually validated and tested for their functional impact on gene networks by transcriptome analysis. In all approaches, third generation sequencing and ad hoc computational pipelines will allow the simultaneous identification of the repeat changes and their association to the corresponding modifiers. Overall, this research proposal is expected to provide key molecular and genetic insights into the process of Htt repeat expansion in human

2 040 943 €

Start date: 2018-03-01, End date: 2023-02-28

"Knowing that the lysosomes contain a powerful cocktail of hydrolases capable of digesting cells and entire tissues, it is obvious that the maintenance of lysosomal membrane integrity is of utmost importance for all cells, and especially for cancer cells with dramatically increased lysosomal activity. Yet, the mechanisms that regulate lysosomal membrane stability have remained obscure, largely due to the lack of methods sensitive enough to detect partial lysosomal leakage and suitable for screening purposes. We have finally succeeded in developing such a method, which allows me to propose here a project whose major aim is to reveal how cells maintain the integrity of lysosomal membranes. Based on our emerging data that firmly connect heat shock protein 70 and sphingolipid metabolism to lysosomal membrane stability, we will devote a large part of the project to the molecular details of these connections and to the characterization of the effects of new and already approved (cationic amphiphilic drugs) sphingolipid-regulating drugs on lysosomal membrane stability and cell survival. Additionally, we will screen selected siRNA libraries to identify signaling networks and lysosome-associated proteins essential for lysosomal membrane integrity, and small molecule libraries to identify compounds that induce lysosomal cell death. The next step is to identify lysosome-stabilizing mechanisms that are especially important for cancer cell survival. And the ultimate goal is to validate corresponding drug targets and drugs (old and new) for the induction of lysosomal cell death in therapy resistant cancers. As a “by-product” we expect to identify putative drug targets for the treatment of degenerative diseases and lipid storage disorders, where the stabilization of the lysosomal membranes promotes cell survival."

2 499 960 €

Start date: 2014-02-01, End date: 2019-01-31

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

Acute myeloid leukemia (AML) is the most common acute leukemia in adults accounting for approximately 15,000 new cases/year in Europe and 20,000 new cases/year in US. Currently, 40-50% of AML patients (age 18-60 years) and only 5-10% of older patients (who are usually more frequently affected by the disease) can be cured using conventional chemotherapy +/- allogeneic hematopoietic stem cell transplantation. Thus, AML still remains an urgent medical need which calls for new forms of molecular targeted therapies (similarly to those available for acute promyelocytic leukemia). The P.I. previously discovered the nucleophosmin (NPM1) mutations, the most common genetic lesion in AML (about one-third of cases) and gave fundamental contributions in the translation of this seminal discovery into the clinic (improved classification of myeloid neoplasms according to WHO, genetic-based risk- stratification of AML patients, monitoring of minimal residual disease and first demonstration of the anti-leukemic activity of actinomycin D). The present research proposal is focused on improving therapy of NPM1-mutated AML. Specifically, it is aimed to: i) identify novel chemical tools interfering with NPM1 functions by interacting with the N-terminal portion of the protein (objective 1); ii) conduct a clinical trial (AML-PG02; Eudract 2014-003490-41) with actinomycin D in older untreated and/or unfit patients with NPM1-mutated AML and to better understand in vitro and in mice models the mechanisms of action of this drug, used alone or in combination with other agents (objective 2); iii) develop compound mouse models aimed to investigate how NPM1 mutations cooperate with FLT3-ITD or DNMT3A mutations in promoting AML with the goal to better understand the characteristics of relapsed cases and to design new therapeutic strategies (objectives 3 and 4): and iv) generate a murine model for testing the feasibility of “in situ” vaccination in AML, especially in NPM1-mutated AML (objective 5).

2 895 836 €

Start date: 2017-11-01, End date: 2022-10-31

For practice of personalized medicine in cancer, non-invasive tools for diagnosing at the molecular level are needed. Molecular imaging methods are capable of this while at the same time circumventing sampling error as the whole tumor burden is evaluated. We recently developed and performed the first-ever clinical PET scan of uPAR, a proteolytic system known to be strongly associated with metastatic potential in most cancer forms. We believe this new concept of uPAR-PET is a major breakthrough and has the potential to become one of the most used PET tracers as it fulfils unmet needs in prostate and breast cancer. Based on this, together with additional proof-of-concept data we obtained on targeting uPAR for optical imaging and radionuclide therapy, we now plan to develop and take into patients these new technologies for improved outcome. Specific aims are to develop and translate into human use: 1. A PET uPAR imaging ligand platform for visualization of the aggressive phenotype and risk-stratification to be used in tailoring therapy, e.g. in prostate cancer to decide whether prostatectomy is necessary. 2. uPAR-PET combined with simultaneous 13C-hyperpolarized pyruvate MRSI (Warburg effect). This will increase prognostic power, refine tumor phenotyping and thereby allow for better tailoring of therapy and early prediction of treatment response. 3. A uPAR optical imaging technology for guiding removal of cancer tissue during surgery. This will help delineate cancer tissue for removal while leaving healthy tissue behind. We expect better outcome with removal of less tissue. 4. Selective radionuclide therapies targeting uPAR positive, invasive cancer cells using β or α emitters. Dose planning will be performed using uPAR-PET imaging. This image-therapy pairing is also known as theranostics. Our endeavour is ambitious, yet realistic considering our competencies and track-record. We expect cancer patients to benefit from our new methods within the 5 year time-frame.

2 072 000 €

Start date: 2015-10-01, End date: 2020-09-30