The establishment of the Clinical Studies Area for the Development and Implementation of drugs, vaccines and medical devices for pediatric use has allowed the integration of the management system for the optimization and digitalization of clinical research processes and for the correct implementation and management of non-profit studies, with the further advantage of a Clinical Trials Management System (CTMS) to optimize the process of activation and continuation of Clinical Studies.

At the same time, thanks to the establishment of the Clinical Studies Quality Team Service (CTQT) on the basis of the requirements indicated by AIFA in the document “Project for the quality of trials for non-industrial purposes” (2010 revision), the researchers work in accordance with the Quality System of the Hospital, the applicable regulations on clinical trials (national and international), the Good Clinical Practice (GCP), with the aim of guaranteeing the quality requirements related to the conduct of non-profit clinical studies, with particular reference to Phase 1 studies as per AIFA Determination 809/2015.

Phase 1 studies in pediatric age are becoming increasingly numerous and fundamental for the treatment of rare diseases that begin in childhood. The Center is receiving numerous proposals, as it is the only Italian Center equipped with the organization required by the National Regulatory Body with specialized staff and the multidisciplinarity that characterizes it, with the exception of oncohaematological pathologies.

The types of studies concern: gene therapies, use of genetically modified organisms, use of antisense oligonucleotides or techniques using mRNA for the production of proteins missing in diseases with genetic deficiency. The project could be to train specialized research staff for each clinical area, both medical and nursing, capable of supporting the unit in the implementation phase of the studies by taking advantage of existing staff.

The studies coordinated and conducted in the “Oncohaematological Clinical Studies and Cellular Therapies” Area of ​​the Bambino Gesù Children’s Hospital are functional to the evaluation of the effectiveness and tolerability of new therapeutic options, in particular in the field of immunotherapy, cell and gene therapy, in the setting of the population of pediatric subjects and young adults suffering from onco-haematological pathologies refractory to conventional therapies.

The clinical trial of this specialist sector involves the timely integrated work of the clinical staff and the Hematological Oncology Trial Center and encourages the involvement of a significant network of academic and industrial collaboration, the implementation of new products – new drugs, new treatments and their stratification – clinically tested and therefore ensures a realistic benefit for patients, with an improved impact on the overall prognosis.

The establishment of the CTQT (Clinical Trial Quality Team), based on the requirements indicated in the document “Minimum requirements for participation in the AIFA project for the quality of trials for non-industrial (non-profit) purposes”, with particular reference to Phase 1 studies , has as its main objective the promotion and guarantee of adequate quality levels of non-industrial trials according to the principles of Good Clinical Practice (GCP) and Good Clinical Laboratory Practice (GCLP).

Over 150 single-center and multi-center clinical trials were conducted during 2022. For approximately 45% of the clinical trials, the Hospital acted as a coordinating center, at a national and international level. A third of the clinical studies were of a non-for-profit nature, largely of academic origin, while the remaining two-thirds were of a for-profit nature, by virtue of the profitable partnership with international pharmaceutical companies. 25% involved observational studies and 75% interventional, pharmacological or non-pharmacological studies.

The overall number of pediatric and young adult patients enrolled for the first time in a study protocol in 2022 is approximately 2,000, with reference to both interventional Clinical Studies and those of an observational nature (including pathology and studies on biological sampling). In particular, in 2022, 14 Clinical Studies were conducted on ATMP (Advanced Therapies Medicinal Products) in accordance with Good Clinical Practice and Good Manufacturing Practices, with the following fields of application:

• pediatric subjects and young adults suffering from haemoglobinopathies;
• pediatric and young adult subjects affected by highly refractory leukemia and lymphoma;
• pediatric and young adult subjects affected by neuroblastoma and other highly refractory solid tumors;
• pediatric subjects suffering from infectious complications following a hematopoietic stem cell transplant procedure;
• pediatric subjects suffering from metabolic diseases (adrenoleukodystrophy with involvement of the central nervous system).

The overall recruitment of these projects on ATMP in 2022 was approximately 50 patients, who were able to benefit from high-quality innovative treatments.

The Pharmaceutical Workshop of the Bambino Gesù Hospital is dedicated to the generation of cell and gene therapy products, configurable in the field of advanced therapies (ATMP – Advanced Therapy Medical Products) in accordance with Good Manufacturing Practices (GMPs).

Created within the research laboratories at the San Paolo Fuori le Mura headquarters, the structure has a surface area of ​​approximately 1,400 m2, which makes it the largest GMP facility present in an academic context in Italy.

In Italy there are currently 23 ATMP production sites authorized by AIFA, including the OPBG Pharmaceutical Workshop, authorized in 2017. During 2020, it was inspected by the AIFA Regulatory Agency, which approved the new remodeled structure of the production area, which now has eight manipulation rooms for products genetically modified with viral vectors, and a room for the extensive manipulation of cellular products. The OPBG Pharmaceutical Workshop also deals with the technological transfer of new CAR-T therapy products.

The Pharmaceutical Workshop Manager is the qualified person who coordinates the activities in compliance with the GMPs and the Quality System. In fact, the Pharmaceutical Workshop applies a Quality System for the management of all activities with an impact on the product. The application of this System guarantees compliance with the regulatory requirements and standards set by the GMPs, both nationally and internationally. The Quality System is managed, controlled and implemented by the Quality Assurance (QA) Unit through a procedure approved by the Qualified Person.

In addition to the advanced therapies already authorized for use and marketed in Italy, ATMPs can be used in clinical trials, for compassionate use or as non-repetitive use of advanced therapies.

Regarding compassionate use, the Ministerial Decree. 7 September 2017 “Therapeutic use of medicinal products subjected to clinical trials”, regulates in Italy the access to experimental pharmacological therapies, including advanced therapies, for use outside of clinical trials, for patients suffering from serious or rare diseases or who are in danger of life, when, in the opinion of the doctor, there are no further valid therapeutic alternatives. For access to advanced therapy medicines through compassionate use, the requirement of availability of results from at least phase II studies remains valid.

As regards non-repetitive use, it is possible to access advanced therapy medicines not yet authorized or not subject to specific clinical trials in Italy, subject to AIFA authorization for production and use, in the absence of a valid therapeutic alternative , in cases of urgency and emergency which place the patient in danger of life or serious damage to health. These cases (around 10 per year from 2021), not being covered by research funding, are borne by OPBG (cost: around 100k per treatment).

In other words, the attending physician can, under his responsibility, offer Italian pediatric patients who are not responding to traditional therapies, in danger of life or serious damage to their health and in the absence of therapeutic alternatives, the opportunity of “occasional access” to non-repetitive treatment. Furthermore, there is a concrete possibility that foreign patients, upon prescription from their doctor or supported by patient associations, can independently make requests for access directly to our facility.

The examination of the intestinal microbiota serves to have a map of the intestinal ecosystem: how it is composed, how it works, how it modifies and how it alters. The bacteria present in the intestine, in fact, change in relation to the state of health or disease.

There are many researchers who are studying the role of the microbiota in diseases and how to intervene for preventive or curative purposes. However, knowledge is still limited. Some studies have highlighted the positive or negative effects of certain microorganisms. Theoretically, by enriching the intestinal microbiota with “good” bacteria at the expense of “bad” bacteria, good health is promoted.

The analysis of the microbiota is based on the sequencing of microbial DNA. In the context of gastrointestinal infections, however, it is not only the presence of batteries that plays a role, but viruses and parasites such as protozoa and helmites also intervene. Therefore, within the microbiome, it is also important to characterize these agents.

Our laboratory is at the forefront internationally for the analysis of intestinal microbiota.

Unmanned aerial vehicles, or drones, are increasingly being used as a solution for transporting medical supplies, including blood bags, vaccines, medicines, diagnostic samples and even organs, in transplant cases. Some experimental tests indicate that the current technology can be considered suitable for carrying out safe transport, in compliance with the clinical and biological parameters required for the transported samples, including for example the maintenance of the cold chain.

However, this type of transport still requires accurate monitoring of the quality and safety of the sample, based on the evaluation of multiple biological parameters and the formulation of new standards for transport rules, in order to be considered as a valid alternative to current norm.

The main favorable aspects are the following:

1. Reduction of transportation times
2. Possibility of transporting different samples (blood – biological tissues – whole organs -pharmacy)
3. Cost reduction, variable depending on the type of material transported
4. Reduction of environmental pollution due to fuel consumption
5. Continuous improvement of technology related to unmanned flight

The main areas for improvement are the following:

1. Lack of reliable checks on the behavior of drones in meteorological conditions unfavorable
2. Limited flight range
3. Risk of falling to the ground
4. Difficulty in obtaining flight corridors
5. It is not yet demonstrated that all the analyzes that can be requested can be performed

In conclusion, it is likely that drones could represent a great opportunity in the field of public health. They can be used to transport blood, samples and biological substances, such as vaccines, reducing costs, personnel usage and travel time. Although there are still numerous weaknesses in the application of drones, everything suggests that they can be overcome with the advancement of technology and applied research.

Artificial intelligence technologies promise to become a formidable ally of the doctor and pediatrician for the possibility of achieving clinical objectives that were previously unthinkable in terms of quality of care and patient and family satisfaction. Interest in these systems has rapidly increased due to the availability of large computing power at low cost and the possibility of using large databases from different information sources.

This potential justifies the rapid proliferation of research projects involving the development of algorithms with clinical applications, and the related scientific publications that we are witnessing. Among others, New England Journal of Medicine has announced a new journal dedicated to artificial intelligence that will be published in 2024.

AI algorithms for pediatrics have the potential to reduce health inequalities, reduce healthcare costs and improve patient outcomes globally. For this technology to have an impact, algorithms need to be trained with the largest volume of data available. However, access to health data is problematic and currently represents the largest enforcement barrier.

The so-called “centralized architecture” approach (i.e., collecting large amounts of data in centralized repositories) has been successfully used to accelerate the development of consumer AI applications such as ChatGPT; however, this approach is not practicable in medicine and, in particular, in paediatrics. In fact, centralized architectures are not suitable for network collaboration, are not compatible with different applications, do not allow data to be managed in real time, do not preserve privacy and do not allow the application of local policies.

However, there is a so-called “federated learning” technology that guarantees support for collaborative networks while preserving privacy. Through this alternative approach it is possible to obtain accurate and high-quality assistance, even for children living far from reference centers or socioeconomically disadvantaged, through the implementation of real-time artificial intelligence applications to support diagnosis and treatments, which they preserve privacy at the point of care whatever the healthcare facility.

Recently, a spin-off from Stanford University, BevelCloud, developed a decentralized architecture for artificial intelligence in medicine that sits in the same environment as medical devices and patients to integrate data privacy assurance. The device generates a digital twin of the data source so as to standardize access to imaging data, laboratory data, laboratory tests, etc. without depending on the equipment that generates the data or the technological infrastructure. In this sense, the creation of an artificial intelligence laboratory for pediatrics in OPBG is planned, dedicated to the development of applications for any specialty with potential global scalability.

The functional validation of pathology mechanisms and the study of the efficacy and toxicity of molecules with therapeutic potential requires the targeted use of animal models, which provide the scientific solidity of the discoveries and optimize the path towards clinical trials.

For this purpose, the small freshwater fish zebrafish is highly versatile, informative and can effectively respond to various experimental needs (including pre-clinical ones) in the biomedical field, so much so that many pharmaceutical companies are adopting this model as an integral part of their experimental workflow.

Thanks to the numerous advantages and to basic and translational research of high scientific impact and in continuous growth, even the agencies evaluating the efficacy and safety of drugs at an international level (including EMA and FDA) will now be able to validate the results obtained in zebrafish patterns.

The key advantages of this model include:

1. high genetic homology with humans (>70%);
2. the speed with which large-scale experiments are conducted compared to the mouse model
3. the often immediate translational impact for the identification of biomarkers and molecules with pharmacological potential which allows a high predictive power, important for optimizing clinical trials, which are expensive and ethically burdensome
4. Breeding and testing, especially on small zebrafish embryos and larvae, is significantly more economical than other models
5. Finally, thanks to a delicate and non-invasive procedure possible in this model, zebrafish is today accepted as an “alternative” experimental system to classic large-sized animal models or for which highly invasive and ethically demanding procedures are often necessary

At the beginning of 2019, a facility dedicated to the zebrafish model was put into operation at our Research Laboratories which would allow, as a pilot phase and proof of concept, a targeted study aimed at functional genomics studies for the understanding of the molecular basis diagnosis of orphan diseases using gene editing and live microscopy approaches.

To ensure the development of the experimental workflow, highly specialized personnel with international certification and a documented track record on the animal model and with adequate training and experience were recruited to allow the experimentation and generation of the body responsible for the Animal welfare (OPBA) for the zebrafish model.

Despite the logistical difficulties associated with the COVID-19 pandemic, the first activities of the exploitation facility have allowed the achievement of important milestones in the field of translational research.

Some examples:

1. description of a new family of genetic diseases (Nature Communications 2022, PMID: 36369169)
2. identification of SPRED2 as a new gene implicated in Noonan syndrome (Am J Hum Genet 2021, 108:2112-2129)
3. efficacy studies of molecules with therapeutic potential (J Med Chem 2021, 64:15973-15990. PMID: 34714648)
4. development of innovative experimental models for the analysis of the effect of molecules in vivo and of targeted and innovative protocols to guarantee animal welfare and rapid genetic screening of CRISPR-Cas mutants (Sci Rep 2022, 12:22597).

The teleost fish breeding laboratory at the OPBG, in addition to the zebrafish use facility already in operation, would make it possible to considerably streamline the authorization process and the bureaucratic burden necessary for the breeding and cultivation of specific transgenic lines that mark different populations cells, of mutants of interest without severe phenotype and for experimentation on embryos.

This would therefore allow, also thanks to the presence of an expert and dedicated core staff, to offer a portfolio of dedicated transversal services which include the breeding, management and use of teleost models for functional genomic studies of pediatric diseases and examination of examination of high-throughput efficacy of pharmacological molecules in a preclinical setting.

Overall, the zebrafish laboratory would offer the scientific community inside and outside OPBG direct access to this model system, with dedicated advice, support and collaboration on research projects.

The Research Laboratories of the Bambino Gesù Children’s Hospital are structures for translational research equipped with advanced technologies, capable of supporting scientific activities aimed at studying the biological bases of diseases and their therapy.

The subdivision of the spaces dedicated to the laboratories reflects the organizational model of the scientific activity, divided into Research Areas, within which the various Units with specific objectives and purposes are distributed, which guarantee accessibility to the data and scientific processes both to internal staff and to those who work in the scientific networks with which the Hospital collaborates.

The laboratories are distributed over approximately 5,000 m2 of the San Paolo Fuori le Mura Research Center and other affiliated sites: in particular, we recall the OPBG “Clinical Immunology and Vaccinology” laboratories located at the Tor Vergata Polyclinic.

The laboratories are equipped with common technological platforms that offer transversal services to the various research activities, characterized by cutting-edge instruments and staff dedicated to their management, to support researchers in setting up and conducting experimental activities. A substantial part of the equipment is made up of latest generation equipment, dedicated primarily to research in the field of molecular and cellular biology.

We also have IT platforms for processing and processing data that facilitate communication and sharing of information, and an area dedicated to the isolation and maintenance of primary and secondary cell cultures, stem cells, pluripotent stem cells induced, organoids, and finally animal cell lines (murine and rat).

The Quality Management System of the Research Laboratories is certified compliant with the ISO 9001:2015 standard by the DNV certification body. Below is a detail of the technologies present in the Research Laboratories.

Flow cytometry

• 4 flow cytometers for the simultaneous analysis of up to 20 parameters and 2 flow cytometers for the simultaneous analysis of up to 50 parameters with high sensitivity and high definition resolution for the analysis of extremely small cell populations (microvesicles and exosomes)
• 2 flow cytometer sorters for the simultaneous analysis of up to 20 parameters, capable of discriminating and separating particles of sub-micron size
• Imaging mass cytometry (IMC) platform with up to 135 acquisition channels, for the analysis of biomarkers and interactions at the subcellular level from tissues, preserving the architecture and morphology of the initial sample

Chromatography and Mass Spectrometry

• Single quadrupole UHPLC/MS system for high-throughput qualitative and quantitative analysis of target molecules in complex mixtures, screening of specific compounds and confirmation of molecular weights of assays of interest.
• GC/MS-SPME system, for the metabolomic analysis of complex matrices.
• Two UHPLC/MS/MS QTRAP/LIT platforms, for quantitative and qualitative analysis with high linear sensitivity, dedicated to metabolomic and lipidomics investigations.
• A TTOF nano-LC/MS/MS platform and a QTRAP/LIT micro-LC/MS/MS platform, for qualitative explorations, fast profiling of specific analyzes of interest, and high-resolution quantitative workflows for metabolomics and proteomics applications .
• Three mass spectrometry platforms with triple architecture (quadrupole, ion trap and OrbitrapTM) and equipped with nano-LC, of which two with multiple fragmentation technologies (CID, HCD, ETD and EThCD), for in-depth discovery analysis , for the characterization of post-translational protein biosynthesis phases and for the qualitative and quantitative understanding of molecular flows and processes

Histology and traditional and confocal microscopy

• Semi-automated preparation of histological and cytological microscopic preparations (sampling, fixation, inclusion, staining) and support and assistance for the preparation of fluorescence markings, signal detection and interpretation of fluorescence data, image analysis techniques and training in the use of applications for conventional and advanced microscopy
• Laser microdissection system for isolating areas of tissue, single cells, subcellular structures, such as chromosomes, used for the selection of samples for genomics, transcriptomics, proteomics and metabolomics studies, as well as for the investigation of in vivo cell cultures
• Microscopy platform for cellular investigations, with live-cell imaging technology and software for qualitative and quantitative image analysis
• Digital platform for automatic high-capacity scanning and archiving of histological, brightfield and fluorescence slides
• Integrated bright field and fluorescence cytometry and live cell imaging system for tracking growth of adherent cell populations; suspended cell count; morphological analysis and growth monitoring of embryoids, colonies, spheroids, wound healing, post-transfection/transduction cellular analysis; iPSC reprogramming monitoring (fiblobast doubling, colony counting and embrodial formation, immunostaining for differentiation); morphological and morphometric analysis of 3D models also for small animals; differentiation of cell populations and co-cultures; chemotaxis; migration and invasion in 2D and 3D; cytotoxicity analysis without the use of radioactive substances; cell viability analysis, internalization and phagocytosis, apoptosis, cell secretion
• Microscopy platform for high-content screening applications (confocal spinning-disk, transmission and fluorescence technology), for 2D and 3D acquisitions for assays of fixed and live cells, spheroids and organoids, co-cultures for interaction analysis protein-protein and conformational changes, phenotypic fingerprints, live-imaging even of small animals
• Three confocal microscopy stations, operating in galvo and resonant mode (resonant scanning at 8 MHz speed, super-resolution by deconvolution), one of which is also equipped with a white laser integrated into a hardware-software system for the analysis of fluorescence lifetime (FLIM), energy transfer by resonance between molecules, for acquisitions and analysis of fixed samples and vital samples in live imaging and time-lapse, multi-color acquisitions, 3D rendering, multidimensional acquisitions (4D/5D) in long-term vital conditions, acquisitions of fast molecular dynamics (ionic flow imaging) using the resonant scanner associated with the confocal microscopy stations
• A light sheet microscopy station for minimizing the phototoxicity of the fluorescent sample (spheroid or organoid, organs or entire specimens of animal models e.g. mouse, zebrafish) induced by the excitation of laser sources

Genomics

• New systems for fast real time qPCR analysis, equipped with array technology for high-throughput gene expression analysis and genotyping
• New Sanger sequencing technologies (up to 24 capillaries), with in-lane standardization and normalization of the results, associated with real-time data quality assessment systems
• New technologies for high-throughput single-cell gene expression preparation and analysis, parallel automation of molecular separation and barcoding at the single-cell level, detection of genomic heterogeneity, clonal evolution, profiling of lymphocyte transcripts, regulatory mechanisms and epigenetic heterogeneity (Assay for Transposase-Accessible Chromatin), libraries for exome and genome-wide scale sequencing
• Next Generation Sequencing systems for the study and analysis of the exome, the transcriptome and the entire human genome, for studies on the functional and clinical relevance of mutations affecting non-coding regions of the human genome, for the identification of new disease genes and for the recognition of new molecular mechanisms underlying genetic diseases, as well as for metagenomics applications
• State-of-the-art high-resolution microarray scanner for SNP genotyping, structural variant analysis, genome-wideassociation studies (GWAS), quantitative analysis of methylation sites within the human genome
• Platform for genetic expression analysis (mRNA and miRNA, DNA Copy Number Variation) of up to 800 targets, simultaneously per sample, completely automatically and without the need for signal amplification or the execution of a PCR step

Manages the collection, process, conservation and distribution of biological material, and related associated data, of patients assisted at the Hospital, respecting the rights of the participants involved and according to proven quality standards to guarantee complete traceability of the activities carried out.

The ultimate aim is to guarantee translational research, aimed at understanding the pathogenetic mechanisms and improving the health and well-being of the child.

The Biobank is certified compliant with ISO 9001:2015 and is a member of the national biobank network of the BBMRI-ERIC Research Infrastructure (Biobanking and BioMolecular Resources Research Infrastructure – European Research Infrastructure Consortium).

The Biobank Service is made up of:

• a laboratory (approximately 28 m2) intended for the manipulation and execution of quality controls, with related laboratory equipment necessary for biochemical analyses, molecular and cellular biology investigations;
• a room (approximately 180 m2) for the conservation of biological samples and built according to the UNI 11827:2021 standard. There are two nitrogen lines connected to two separate tanks (5000 L each) for the storage of liquid nitrogen, in order to guarantee the redundancy of the system and the tapping for the feeding of cryocontainers with liquid nitrogen vapors (n 11 cryocontainers at T -196°C and 10 at T -80°C). The room is sized to install additional n. 13 cryocontainers and is currently occupied by mechanical freezers (9 freezers at -80°C) where almost all of the historical collections of biological material are preserved (more than 100,000 samples of 8 different types of biological material, relating to approximately 40,000 pediatric patients). The liquid nitrogen system also supplies the conservation area (accredited by the National Transplant Center and AIFA) intended for the products of the Pharmaceutical Workshop, where no. 11 liquid nitrogen vapor cryocontainers for storage at -196°C. The cryopreservation room and related systems are managed through a monitoring system, which can also be controlled remotely, for the recording and traceability of all events relating to the functioning of the infrastructure.

14 different types of biological material are currently stored, from approximately 7,000 pediatric patients (including perinatal) classified in 18 different ICD10 and with more than 1,400 different genetic defects.

All the Biobank’s activities – from document conservation to sample traceability – have been computerized and made traceable through the choice of dedicated applications integrated into the Documentum D2 platform. In this mode, it was possible to make sample information accessible to researchers and clinicians through the same healthcare applications (OBG Clinic and W Hospital) used for healthcare activities.

The Biobank regularly collaborates with OPBG Information Systems for the dematerialization and digitalization of the informed consent collection process for the collection and conservation of biological samples for research purposes. These activities are configured in the projects of the Italian node of BBMRI-ERIC, aimed at promoting a multidisciplinary participatory process with institutional approval for the implementation of federated research and the acceleration of translational research at an international level.

Furthermore, thanks to the collaboration with healthcare personnel, a project has been launched to stratify the patient data entered into the Biobank database by pathology, in order to support clinicians in defining cohorts for planning clinical research activities.

As a member of the BBMRI-ERIC Infrastructure, the Biobank was involved in the Quality Management Service activities of optimizing protocols relating to the conservation and quality control of biological material.

As part of technological advancement and process digitalization, the Biobank has participated in the design of IT solutions for the integration and interoperability of data, to simplify the sampling and access process. The collaboration of the Biobank with the MOLGENIS team – supporting the IT activities of some European Reference Networks of which OPBG is the reference center – made it possible to define the project requirements to automate the transfer of data to “meta-registries” of patients between disease reference networks.

It was a first step to establish the possibility of data integration and interchangeability and to encourage a data management system for federated biobanks, to improve integrated access to FAIR data through multi-criteria searching of samples and datasets, in compliance with the requirements of the GDPR 2016/679 and respecting the degree of control required by each individual biobank participating in the process.

The Biobank was selected by BBMRI-ERIC as a pilot for various projects aimed at the digitalisation of processes: in particular, in 2022 it participated in the feasibility study of the integration of pediatric biobanks into the European Joint Program on Rare Diseases (EJP-RD) Virtual Platform (project Del 11.6-Virtual platform of RD resources annotated with EJP ontological model, H2020-SC1-2018-Single-Stage-RTD SC1-BHC-04-2018) and the implementation activities of a shared profile at European level of Digital Use Conditions/Common Conditions of use Elements (project Del 12.2- First Report on core set of FAIR software tools and on extended set of unified FAIR data standards, applied in EJP RD).