Cellular heterogeneity is a fundamental property of glioblastoma (GBM) tumors and presents a major barrier for therapeutics. During the past six years we characterized this heterogeneity in glioblastoma, and in other types of glioma, using single cell RNA-seq (scRNA-seq). We found consistent patterns of heterogeneity across patients, such that each tumor harbors multiple subpopulations of cells that resemble neurodevelopmental cell types. The consistency of these cellular states across patients highlights their potential clinical significance. Yet, our understanding of how these common states are regulated and of how distinct states respond, individually or in combinations, to potential treatments, remain poorly understood. Furthermore, it is unclear if additional states of clinical significance remain to be uncovered. To address these challenges, we will perform extensive studies of GBM patient samples, animal models and gliomasphere culture models. First, we will dissect the regulation of the common cellular states (Aim 1). Single cell ATAC-seq of patient samples will be used to infer transcriptional regulators, Spatial Transcriptomics will be used to infer environmental interactions, and resulting predictions will be tested further in established model systems. Second, we will search for novel cellular states, including rare states or those specifically associated with invasion to the brain parenchyma (Aim 2). Third, we will examine strategies for treatment of heterogenous tumors that are composed of multiple states (Aim 3). We will screen for state-specific drug sensitivities, as well as for drugs that induce state transitions, and develop rational combinations to eliminate multiple co-existing states, while considering interactions among states. Taken together, these studies will considerably expand our understanding of cancer heterogeneity and develop strategies to target heterogeneous tumors.

Neuropsychiatric disorders are a leading cause of global disability-adjusted life years, and solutions are lacking. Can digital twins be useful? At least in some cases, we hold they will be central to progress. Recent findings suggest that non-invasive brain stimulation may be a valuable option in conditions such as epilepsy or Alzheimer's (AD). Still, a better understanding of mechanisms and patient-specific factors is needed. Personalized hybrid brain models uniting the physics of electromagnetism with physiology – neurotwins or NeTs – are poised to play a fundamental role in understanding and optimizing the effects of stimulation at the individual level. We ambition to deliver disruptive solutions through model-driven, individualized therapy. We will build a computational framework – weaved and validated across scales and levels of detail– to represent the mechanisms of interaction of electric fields with brain networks and assimilate neuroimaging data. This will allow us to characterize the dynamical landscape of the individual brain and define strategies to restore healthy dynamics. Benefitting from existing databases of healthy and AD individuals, we will deliver the first human and rodent NeTs predicting the effects of stimulation on dynamics. We will then collect detailed multimodal measurements in mice and humans to improve the predictive power of local and whole-brain models under the effects of electrical stimulation, and translate these findings into a technology pipeline for the design of new personalized neuromodulation protocols which we will test in a cohort of AD patients and healthy controls in randomized double-blinded studies. With research at the intersecting frontier of nonlinear dynamics, network theory, biophysics, engineering, neuroscience, clinical research, and ethics, Neurotwin will deliver model-driven breakthroughs in basic and clinical neuroscience, with patients ultimately benefiting from safe, individualized therapy solutions.

Gene therapy has recently shown remarkable potential to offer definitive treatments for otherwise incurable diseases. Currently, seven gene therapy products have reached the market and many more are entering clinical testing for selected indications. Moreover, emerging technologies for targeted gene editing are complementing the scope of conventional gene transfer, opening the way to precise gene correction and making possible to silence, activate or recode any sequence of interest in the genome. However, in order to realize the full potential of these strategies and broaden application of gene therapy, the field has to solve several major hurdles, including: risk of insertional mutagenesis by gene transfer vectors, limited efficiency and durability of some gene correction strategies, off target effects of editing tools, poor tissue targeting and immune response to editing components and delivery vehicles. UPGRADE will offer radical new solutions to overcome these hurdles. We will exploit and further develop disruptive new technologies for precision gene and epigenome editing and for site-specific transgene insertion, and stringently characterize their specificity and cellular responses. We will combine these improved technologies with advanced viral and non-viral vectors enabling cell/tissue targeting and immune evasion, to generate prototypes of advanced medicinal products (AMP). The safety and efficacy profile of each AMP will be stringently validated in tissues (hematopoiesis, heart and skeletal muscle, liver, retina) and disease models (muscle wasting, storage and blood disorders, hypercholesterolemia) paradigmatic for unmet medical need and potential long-term cure, if the limitations of current gene therapy strategies are overcome. These AMPs represent versatile products portable to the treatment of several other diseases because of related pathogenesis or correction strategies, thus providing the basis for tackling diseases affecting large patient groups.

Up to 70% of cardiovascular events are not prevented by current therapeutic regimens. In search for additional, innovative strategies, immune cells have been recognized as key players contributing to atherosclerotic plaque progression and destabilization. Particularly the role of innate immune cells is of major interest, following the recent paradigm shift that innate immunity, considered to be incapable of learning ability, does exhibit a memory feature transduced via epigenetic modulation. Compelling evidence shows that atherosclerotic factors promote immune cell migration by pre-activation of innate immune cells. In this project called REPROGRAM, we aim to prove that innate immune cell activation via epigenetic reprogramming perpetuates the upheld systemic inflammatory state in cardiovascular disease which is common in other chronic inflammatory diseases. This opens a new therapeutic area in which epigenetic modulation of innate immune cells effectively decreases systemic inflammation impacting on chronic inflammation as well as the development of co-morbidities. The integrated use of in vitro, ex vivo and in vivo studies, including cells, mice and patients, allows translation from in vitro mechanisms to diseases (molecule-to-man) and extrapolation to cohorts (man-to-mass), enabling us to demonstrate relevance and therapeutic potential of targeting trained immunity in cardiovascular and chronic inflammatory diseases. Enforced by the promising data in oncology, the future prospects for epigenetic interventions in cardiovascular and chronic inflammatory diseases are eminent, attested by the large residual cardiovascular disease burden and the huge societal impact of other chronic inflammatory diseases. The REPROGRAM consortium consisting of key opinion leaders in the field of cardiovascular (systems) biology, immunology, epigenetic therapies and rheumatoid arthritis, with a large intersectoral network, guarantees rapid translation of early mechanistic discoveries