Projects
MSCA Staff Exchanges 2023 HORIZON-MSCA-2023-SE-01
Programmable Bionanomaterials with Protein-Controlled Behavior - "Program-Material"
Role: Coordinator
Funding body: European Commission – REA – MSCA
Program-Material is a research and innovation staff exchange network dedicated to pushing the boundaries of nanomaterials and nanotechnologies by introducing integrated protein-controlled behaviors. The primary goal is to craft hybrid nanostructured materials featuring precise structural, functional, and spatiotemporal behaviors directly regulated by specific proteins. By incorporating synthetic biology components, these materials will demonstrate the ability to perform tailored chemical or biological functions, guided by specific proteins. This innovation holds tremendous potential for groundbreaking applications in biomedical and healthcare contexts. While traditional approaches focus on engineering nanomaterial properties to influence biological entities, Program-Material pioneers bio based control over nanomaterial properties, emphasizing programmable influences of biomolecules—particularly proteins. Program-Material seeks to cultivate a new generation of scientists at the intersection of materials science and biotechnology. This expertise will drive innovative solutions in advanced healthcare nanomaterials, fostering unprecedented interactions with biology, particularly in precision and nanomedicine applications. To achieve these ambitious goals, Program-Material united 9 academic partners and 1 non-academic partner in a collaborative network. This team spans European (Italy, The Netherlands, Estonia) and extra-EU institutions (USA, Australia, Argentina, Switzerland, Japan) and encompasses individuals ranging from PhD candidates to experienced researchers. This diverse composition ensures Program-Material’s robust interdisciplinary and intersectoral dimension, vital for a broad and effective impact. The program places a strong emphasis on actively involving young professors, providing pivotal support for their career progression. Knowledge transfer and associated training activities are crafted to equip scientists with specific competencies in materials science, bionanotechnology, and nanomedicine.
PNRR NextGenerationEU "THE - TUSCANY HEALTH ECOSYSTEM"
Biomarker-responsive DNA-based Organosilica Nanoparticles for Theranostics - "BioSilicaThera"
Role: Principal Investigator
Co-led by Prof. Francesco Ricci/Prof. Alessandro Porchetta, University of Rome Tor Vergata; Prof. Luisa De Cola, Mario Negri Institute for Pharmacological Research
Funding body: Italian Ministry for University and Research – PNRR – NextGenerationEU
Aptamers are single-stranded nucleic acids that bind to proteins and small molecules with high affinity by folding into a three-dimensional conformation. The clinical progression of therapeutic aptamers is hindered by several challenges, particularly concerning nuclease degradation, limited endosomal escape ability, and low effective intracellular delivery. BioSilicaThera aims to develop a biomarker-responsive hybrid Aptamer-Silica nanoparticle (Apt-SiNP) platform for theranostic applications. This platform will enable controlled degradation of silica NPs in response to overexpressed biomarkers (i.e. onco-miRNA), and consequent release of functional aptamers in the cytosol of cancer cells. Leveraging previous success from our network in supramolecular nucleic acid (NA)-based organosilica NPs, the project’s scientific goals include designing breakable Apt-SiNPs and delivering functional aptamers with therapeutic activity upon specific biomolecular interactions serving as a trigger input.
The challenges we wish to address are:
- a) Designing biomarker-responsive Apt-SiNPs using the molecular programmability of oligonucleotides.
- b) Delivering functional aptamers alongside Apt-SiNP degradation.
- c) Activating therapeutic functions in response to interactions with biomarkers inside the cells.
- d) Demonstrating the targeting capability and the therapeutic efficacy of the designed aptamers in human lung cancer cells and organoids in vitro.
The nanomaterials will be designed on one hand to incorporate the aptamer directly in the silica framework, but also, as an alternative, to decorate very small porous cage-like silica NPs. The small size of these NPs could potentially facilitate the development of intravenous delivery methods, whereas larger nanoparticles might be suitable for formulations administered via the intranasal route. BioSilicaThera anticipates significant advancements in smart nanomaterials for cancer theranostics, enhancing NA delivery in tumor models.
FIL Giovani 2024 – Seed Grant for Young Researchers
Resurrecting ancestral actin-resistant DNase for cystic fibrosis lung disease therapy
Role: Partner
Principal Investigator: Dr. Giulia Mori, University of Parma.
Funding body: University of Parma.
Cystic fibrosis (CF) is a severe genetic disease associated with mutations in the CFTR gene, impairing ion transport across epithelial cell membranes. As a result, thick, sticky mucus accumulates in the airways, causing a permanent state of inflammation and progressive lung deterioration. The management of pulmonary symptoms in CF patients is a cornerstone of treatment, aimed at improving lung function, reducing exacerbations, and enhancing overall quality of life. Despite its recognized efficacy, the FDA-approved mucolytic drug based on recombinant human DNase1 (dornase alpha) encounters hurdles due to the inhibitory activity of actin, which coexists with DNA in mucus. This interaction compromises the effectiveness of DNase1, resulting in variable patient responses. An alternative to conventional DNase1
is found within the human genome, namely DNase1L2, which boasts natural resistance to actin inhibition. DNase1L2 has demonstrated heightened efficacy in reducing the viscosity of CF artificial mucus containing actin compared to dornase alpha. However, DNase1L2 faces challenges in production due to its insolubility in Escherichia coli and poor yield in Pichia pastoris. To address these challenges, this project proposes leveraging ancestral sequence reconstruction (ASR) to design more soluble and functionally active variants of DNase1L2, serving as improved therapeutics for CF. ASR is a computational method for inferring ancient protein sequences to probe the design space of modern proteins. By employing ASR-guided protein engineering, we aim to overcome the limitations posed by insolubility in traditional cell systems and enhance the pharmacological properties of DNase1L2. These engineered recombinant enzymes, demonstrating enhanced expression and solubility, will undergo comprehensive activity testing. This includes fluorescence-based assays using fluorophore-labeled single-stranded DNA to characterize DNase activity and rheological measurements with CF artificial mucus to evaluate mucolytic properties, ultimately facilitating the selection of the most promising candidate. PEGylation will be explored as a means to enhance stability and bioavailability. X-ray crystallography will provide insight into the structural determinants underlying the biochemical properties of engineered DNase1L2. This project will advance the development of actin-resistant DNase1L2 as an alternative mucolytic agent with enhanced efficacy and optimized production potentially suitable for CF lung disease therapy. By integrating expertise from bioinformatics, protein engineering, enzymology, analytical chemistry, and structural biology, this research represents a concerted effort towards the goal of expanding the treatment landscape for this rare and debilitating disease.
PRIN 2022
CRISPR-Cas-based sensing platforms for the monitoring of clinically relevant antibodies - "CRISPR-Abs"
Role: Principal Investigator.
Co-led by Prof. Alessandro Porchetta, University of Rome Tor Vergata.
Funding body: Italian Ministry for University and Research
Monitoring and quantifying clinically relevant antibodies as biomarkers for medical conditions and therapeutic regimens is essential for making informed clinical decisions. Current laboratory technologies, such as ELISA and CLIA, along with rapid point-of-care (POC) tests, have limitations that hinder their effectiveness. To address this issue, we aim to develop novel strategies for antibody detection that combine rapid POC analysis with high sensitivity and specificity, thereby paving the way for advanced serological testing platforms.
Our project revolves around leveraging the groundbreaking CRISPR-Cas technology, which has revolutionized genome editing and nucleic acid diagnostics. Our overarching objective is to reengineer and adapt CRISPR-Cas-based detection strategies for the specific detection of biomarker antibodies. These strategies will be seamlessly integrated into electrochemical and optical sensing platforms, enabling the rapid, ultrasensitive, and cost-effective detection of specific antibodies, particularly tumor-associated autoantibodies. These autoantibodies hold great promise as diagnostic and prognostic markers in the field of oncology.
The core of our innovation lies in engineering antibody-responsive nucleic acid-based devices, which will work in conjunction with tailored bioresponsive elements and various Cas enzymes. Our project’s key strategy involves employing rationally designed antibody-controlled nucleic acid systems as molecular translators. These systems convert the binding of a specific antibody into an arbitrary DNA/RNA input, thereby activating a CRISPR-Cas platform. This platform serves as an amplifier, providing an enhanced optical or electrochemical signal.
Our interdisciplinary approach draws from the fields of DNA nanotechnology, biomolecular engineering, and synthetic biology. By uniting these disciplines, we intend to deliver transformative technologies that enhance the efficacy and efficiency of antibody monitoring across a wide range of medical needs.
GUIDO BERLUCCHI FOUNDATION MINI-GRANT PROGRAMME 2022
DNA-Based Molecular Sensors for the Analysis of Oncogenic Zinc Fingers - “DNA-FINGERS”
Role: Principal Investigator.
Funding body: Guido Berlucchi Foundation
https://fondazioneberlucchi.org/it
Identifying molecular patterns and expression profiles associated with cancer is instrumental in translating biological insights into innovative molecular diagnostics and precision medicine therapeutics. Transcription factors (TFs) hold a pivotal role in gene regulation and various vital cellular processes. Their expression often goes awry in cancer cells, making them potential biomarkers for early diagnosis and promising targets in precision therapeutic approaches. Among these, Zinc Finger Transcription Factors (ZNFs) represent the largest TF family, known for their ability to bind specific double-stranded DNA sequences. Numerous studies have linked the abnormal expression of certain ZNFs to cancer onset and progression through diverse mechanisms.
Currently, the detection of ZNFs in biological samples relies on labor-intensive, time-consuming, and expensive laboratory techniques like Western Blot and ELISA. In contrast, in this project, we propose an innovative approach that harnesses the unique DNA-binding activity of ZNFs. Their precise binding to defined dsDNA motifs offers an opportunity to design DNA-based molecular sensors. This can be exploited to design synthetic DNA switches capable of changing conformation upon binding, enabling the engineering of sensing probes and TF-controlled nanomachines for synthetic biology applications. This binding activity-based strategy has recently inspired specific examples of DNA-based translators for TF-controlled DNA circuits yet has to be explored for designing ultra-sensitive technologies to analyze oncogenic ZNFs.
In this project, we propose to develop DNA-based systems that recognize specific oncogenic ZNFs and convert this binding event into an amplified optical or electrochemical signal. To achieve this, we plan to leverage tools and strategies from the field of DNA nanotechnology, where synthetic DNA serves as a programmable engineering material. Through the integration of complementary mechanisms, including new approaches involving cutting-edge CRISPR-Cas systems, we aim to create novel classes of molecular technologies that can be incorporated into optical and electrochemical devices. This innovation will facilitate the analysis of specific oncogenic ZNFs in biological samples, harnessing their inherent DNA-binding activity.
FIL Giovani 2022 – Seed Grant for Young Researchers
Theranostic nucleic acid-based nanodevices artificially regulated by proteolytic enzymes
Role: Principal Investigator.
Funding body: University of Parma and CARIPARMA Foundation.
Precision medicine aims to overcome the limitations of traditional diagnostics and therapeutics by targeting specific molecular markers associated with diseases. A promising category of precision medicine tools comprises dynamic molecular devices capable of translating biorecognition events into the activation of programmable molecular processes. These molecular devices offer precise spatiotemporal control and conditional activation, enabling the creation of innovative programmable theranostics with enhanced targeted and localized effects, combining both high specificity and sensitivity. DNA nanotechnology has made significant contributions to this endeavor. Synthetic DNA-based nanodevices, inherently programmable and versatile, can be tailored into customized diagnostics and therapeutics based on predictable base-pair interactions.
A more substantial challenge lies in developing dynamic DNA-based theranostics responsive to target proteins. Achieving this necessitates the establishment of artificial protein-DNA communication and the implementation of non-trivial binding-induced mechanisms. Current methods, relying on proximity effects or conformational changes, only address a fraction of clinically relevant proteins with DNA-based devices, leaving behind other crucial protein families, including proteolytic enzymes. These enzymes could potentially serve as specific endogenous triggers for the controlled activation of DNA-based nanodevices. Of particular interest are metalloproteinases, essential markers in cancer research due to their aberrant expression in various types of cancer. Enabling these proteins to interact with nucleic acid-based systems could open new avenues for precision medicine strategies.
In response to this challenge, this project proposes the creation of novel nucleic acid-based molecular devices capable of communication with specific proteases, regulated by their natural enzymatic activity. The overarching objective is to develop molecular tools that can convert protease-dependent cleavage of a peptide substrate into controlled signal processing and/or the release of biotherapeutics. These incorporate protease-responsive peptide nucleic acid (PNA)-peptide hybrids, which can be integrated into modular DNA-based systems for protease-dependent signal processing. This endeavor will yield new programmable molecular devices with the potential for applications in analytical chemistry, precision medicine, and synthetic biology.
2022 Excellent Science for Horizon Europe
Hybrid healthcare bionanomaterials actively controlled by biology
Role: Principal Investigator.
Funding body: Italian Ministry for University and Research and University of Parma.
The primary objective of this project is to pioneer the development of programmable nanotechnologies capable of precise activation, regulation, and in situ control by specific target biomolecules. Specifically, this project supports the creation of innovative biomedical technologies that can integrate sensing, processing, and response functionalities. These smart nanodevices leverage nanostructured materials in conjunction with synthetic biology components to execute customized chemical or biological functions in response to the recognition of specific biomolecules. These include nanoparticle formulations of nucleic acid nanomachines engineered for target-induced theragnostics, nanomaterials incorporating biological components into their bulk structure, enabling programmable bio-responsive properties, and nanomotors responsive to biomolecular inputs, facilitating controlled spatiotemporal manipulation within cellular environments.
This project plays a pivotal role in addressing critical challenges within the Health research and innovation domain of the PNR, aligning with the strategic plan “HealthTech for Society.” The activities undertaken in this project are central to the PNR Technologies for Health intervention area, focusing on the articulation “Bio-hybrid systems for new frontiers in biotechnology research and in precision and personalized medicine.” The aim of this project is to introduce programmable bioresponsive technologies that integrate an array of functionalities. In doing so, the sensing of specific biomolecules can be translated into chemical or biological actions with diagnostic or therapeutic implications.
FIL Giovani 2020 – Seed Grant for Young Researchers
Programmable chem-bio chimera translators as dynamic, functional probes for molecular sensing of informative protein markers
Role: Principal Investigator.
Funding body: University of Parma and CARIPARMA Foundation.
This project’s primary objective is to harness the expression of specific proteins, widely recognized as disease hallmarks, as potent indicators for screening, early diagnosis, and prognosis. Essential classes of proteins, including proteases, transcription factors, and other functional biomolecules, play pivotal roles in disease development and progression. In this context, protein-based molecular diagnostics play a fundamental role in enabling timely medical interventions. Therefore, there is an urgent need for strategies that enable sensitive, rapid, cost-effective, and unequivocal biomarker detection, particularly those capable of providing insights into the functional activity of the target protein.
Our approach capitalizes on the intrinsic ligand-binding and enzymatic activity inherent in specific marker proteins. We aim to utilize this functionality as the initial input of a sense-and-process detection system, enabling the engineering of protein-responsive molecular systems. In this innovative approach, the analyte of interest is not the protein itself, but rather its functional activity. DNA nanotechnology provides the basis for designing the proposed molecular devices. This project integrates elements of organic synthesis, analytical chemistry, and synthetic biology, synergistically combining these disciplines to create programmable biomolecular sensors specifically tailored for protein markers.