therapy

Optimizing CD45-Targeted Astatine-211-Radioimmunotherapy for Malignant and Non-Malignant Blood Disorders

ABSTRACT CD45 is expressed on almost all normal and neoplastic hematopoietic cells but not on non-blood cells and has, therefore, been pursued as a drug target. Initially centered on augmenting conditioning before hematopoietic cell transplantation (HCT) for blood cancers, there is increasing interest in expanding CD45-directed therapies into other settings, with radioimmunotherapy (RIT) being the major therapeutic modality so far. Investigators at our institution pioneered CD45 RIT with b-emitters such as iodine-131 (131I) using the murine monoclonal antibody (mAb), BC8. A phase 3 trial testing 131I-BC8 (131I-apamistamab [Iomab-B]) with allogeneic HCT in older adults with relapsed/refractory acute myeloid leukemia showed improved outcomes over conventional care, validating this approach. More recently, attention has shifted toward a-emitters that deliver substantially higher decay energies over much shorter distances than b-emitters, rendering them more suitable for precise and potent target cell killing. In our work, we focus on astatine-211 (211At) for its ideal half-life and decay without a-emitting daughters. For clinical application, mAbs are conjugated with the bifunctional boron cage molecule, isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10-NCS), to enable stable protein astatination. Three early-phase trials testing 211At-BC8-B10 as augmentation of HCT conditioning for patients with malignant and non-malignant blood disorders are ongoing, with emerging data indicating significant anti-tumor efficacy. Nonetheless, relapses still occur. Other important limitations include marked infusion toxicities and human antimouse antibody (HAMA) responses related to the murine nature of BC8 and dimer formation after 211At labeling of mAb-B10 conjugates with tissue residualization from 211At atom oxidation. The latter may contribute to the risk of liver cell injury, the dose limiting extramedullary toxicity of CD45 RIT. As a first step toward our goal of optimizing CD45 RIT, we have raised new, fully human CD45 mAbs as basis for novel therapeutics. In preliminary in vivo studies in immunodeficient mice, we found some of these mAbs to have greater anti-tumor efficacy than a humanized version of BC8 (HuBC8) we generated as a reference mAb. We will now conduct comparative in vivo CD45+ cell targeting (“biodistribution”) and anti-tumor efficacy studies to select a lead candidate mAb for clinical application and use protein engineering to maximize the selectivity and efficacy of targeted radiation delivery. We will use immunodeficient mice xenotransplanted with human leukemia cells for this purpose as no human approaches are available and in vitro testing is inadequate to measure both the targeting and biologic RIT effects on human leukemia cells. Mice provide the in vivo milieu needed for comprehensive evaluation. Development of improved mAb astatination methodologies to minimize off-target toxicities of 211At-RIT will further increase therapy specificity and reduce toxicity. In parallel, we will conduct genome-scale, unbiased target identification/validation studies to identify partner drugs for rational combination therapies aimed at enhancing the anti-tumor efficacy of 211At-CD45 RIT.

Short-wave infrared Cerenkov imaging to better visualize targeted radiotherapy and diagnostic radiotracers

SUMMARY. The problem: Cerenkov luminescence (CL) imaging (CLI) is a new imaging method that utilizes light emitted during decay of radiotracers. CLI merges optical and nuclear imaging by utilizing affordable yet highly sensitive optical cameras with clinical radiotracers. It provides fast and cheap clinical optical imaging to explore radiotracer distribution in patients. While not tomographic, CLI systems have a lower price, smaller footprint and higher resolution than nuclear imaging scanners. Yet, due to the very low signal intensity of CL its versatility remains limited since CLI requires strict exclusion of ambient light with an enclosure. Therefore, CLI requires novel approaches to make clinical imaging more feasible. We hypothesized that we could explore the short-wave infrared (SWIR) part of CL to enable CLI under ambient light without enclosure, providing improved and facile CLI, particularly of isotopes used for therapy that cannot be imaged otherwise. SWIR imaging (900- 1300 nm) has almost no autofluorescence, absorption or scatter but provides significantly higher depth penetration, yielding images with higher contrast and resolution compared to the visible range. Since typical LEDs do not emit light beyond 850 nm, they do not interfere with the SWIR camera. We can therefore perform CLI in the SWIR range (SWIR-CLI) without the limiting light-tight box and under ambient LED light and also achieve better signal penetration and accuracy. We will investigate if SWIR-CLI can be used to monitor distribution of therapeutic isotopes for targeted radiotherapy (TRT), a fast-expanding field as highlighted by Novartis’ acquisition of Lutathera and Pluvicto for the price of $6 bn. These agents are targeting 177Lu as therapy to neuroendocrine and prostate cancers. For TRT α-emitting isotopes are particularly attractive due to the α- particle’s short path length with high linear energy transfer. However, α-emitters are very difficult to image with conventional equipment. The α-emitter could be swapped with an imaging isotope, but this can alter the agent’s biodistribution. The α-particle itself does not have sufficient energy to produce CL but several daughters in the decay chains of most α-emitters produce electrons with sufficient energy to create CL. We have already imaged the α-emitter 223Ra in patients and have recently shown that CLI of α-emitters in the SWIR is possible. SWIR- CLI could therefore provide a facile imaging approach for α-emitters. We will answer with our three independent Aims the following questions: (1) Can we image diagnostic isotopes with SWIR-CLI? (2) Can we image therapeutic emitters with SWIR-CLI? (3) Can we use SWIR-CLI to image patients undergoing PET and/or TRT? Animal studies will employ established mouse cancer models to optimize imaging parameters and validate findings, directly informing the co-clinical Aim 3 trial. By eliminating the requirement for a light-tight enclosure and enabling CLI under ambient light, SWIR-CLI represents a significant shift in the practical deployment of CLI rather than an incremental improvement. Our study will broaden the reach of CLI by enabling imaging under ambient lighting, unlocking innovative new opportunities for CLI (monitoring TRT) in research & clinical settings.

Utilizing integrin-targeted PET imaging and therapeutics to predict and treat radiation-induced pulmonary fibrosis

Project Summary/Abstract. Lung cancer is the leading cause of cancer death in the US, with over 125,000 deaths annually. Radiation therapy (RT) is a critical component of curative lung cancer treatment for many patients. However, radiationinduced pulmonary fibrosis (RIPF) is a common side effect that carries a poor prognosis with limited treatment options. Up to 40% of patients with lung cancer who receive RT may experience RIPF. RIPF is a late effect of RT, typically occurring 3 or more months after treatment. The symptoms of RIPF can include shortness of breath, pleural effusions, decreased lung function, and respiratory failure. Cell surface integrin heterodimers play a key role in the pathogenesis of RIPF. In particular, the integrin αvβ6, which is expressed at a low level in the alveolar epithelium at baseline, is significantly upregulated upon RT damage. The key role of integrin αvβ6 in RIPF is illustrated by studies in which mice lacking integrin αvβ6, or treated with an αvβ6-blocking antibody, do not develop RIPF. Here, we propose to translate this mechanistic understanding of RIPF into novel approaches for monitoring and treating RIPF. We hypothesize that non-invasive αvβ6 PET imaging will be safe and can specifically bind to αvβ6 in patients with RIPF. Additionally, we hypothesize that a novel small-molecule integrin antagonist, IDL2965, can mitigate and treat RIPF in mice. In this project, we are utilizing mice to model RIPF, as mice develop RIPF that mimics human disease. In addition, cellular and in vitro models do not approximate the complex biology leading to the development of RIPF. Our data using [64Cu]Cu-DOTA-αvβ6-BP to detect early RIPF in mice are compelling in both single-fraction high-dose RT and lower dose-larger volume RT models (Lo et. al, IJROBP 2025). However, to progress to clinical trials in patients with cancer, we will obtain data to submit an Investigational New Drug (IND) application to the FDA. Importantly, we propose translating [64Cu]Cu-DOTA-αvβ6-BP PET imaging into patients with lung cancer, allowing us to better identify RIPF and develop a tool to determine the efficacy of IDL-2965 in future clinical studies. The specific aims of the proposal are: (1) Characterize the utility of [64Cu]Cu-DOTA-αvβ6-BP in mice with conventionally fractionated RT and identify circulating biomarkers of RIPF, and determine the in vivo toxicology of [64Cu]Cu-DOTA-αvβ6-BP to prepare and submit an exploratory Investigational New Drug (eIND) application to the FDA, (2) Conduct a first-in-human clinical trial of [64Cu]Cu-DOTA-αvβ6-BP to determine its safety and human dosimetry in patients with evidence of RIPF from computed tomography or in healthy controls, and (3) Determine the effect of integrin antagonism using IDL-2965 on mitigating RIPF in preclinical mouse models. The goals of this proposal are two-fold: (1) demonstrate safety and target specificity for [64Cu]Cu-DOTA-αvβ6-BP so that it can be used in future studies to identify RIPF and evaluate the efficacy of anti-fibrotic therapies, and 2) determine the ability of IDL-2965 to prevent RIPF in preclinical mouse models.

Urothelial Resurfacing with Irreversible Electroporation for Adjuvant Therapy of Bladder Cancer

PROJECT SUMMARY Over 70% of bladder cancer (BCa) patients are diagnosed with early-stage and localized non-muscle invasive disease (NMIBC), yet achieving durable cancer-free survival remains a significant challenge. Most of these patients will experience local tumor recurrence within five years following standard of care (SoC) transurethral resection of bladder tumor (TURBT) and intravesical adjuvant chemo- or immunotherapy. Recurrence is driven by microscopic tumors and premalignant lesions dispersed within the urothelial layer that survive and escape these treatments. As TURBT effectively treats tumors visible on imaging, current research has predominantly focused on drugs and biologics for improving intravesical adjuvant therapy. In this proposal we pose the provocative question whether a TURBT-like ablative technique can be extended to debulk malignancy in the entire bladder and investigate the synergy with intravesical adjuvant therapy in improving outcomes. Our objective is to address this technology and knowledge gap by developing and validating whole bladder urothelial resurfacing (WBUR) using irreversible electroporation (IRE). During IRE, microsecond-long pulsed electric fields (PEF) are used to induce rapid cell death by catastrophic permeabilization of the cell membrane, without affecting the extracellular matrix (ECM) within the treated tissue. In prior work, we designed devices that utilized this unique mechanism of IRE for performing penetrative ablation in the ureter, bile duct and bronchus of swine while preserving lumen function. Our findings provided strong rationale for IRE being an ideal candidate for WBUR as alternate techniques such as thermal ablation or ionizing radiation must be performed with extreme care in the bladder to avoid perforation or fistula formation. In subsequent preliminary work we developed technology to demonstrate the feasibility and safety of WBUR with IRE in a rat model of BCa and scalability in human-sized swine bladder. In Aim 1, we will investigate the cancer treatment efficacy of combination WBUR and intravesical adjuvant therapy. In Aim 2, validate WBUR derived liquid biopsy for monitoring cancer status. In Aim 3, engineer PEF delivery strategy to enhance the safety and specificity of WBUR. The innovation of our proposed work is defined by developing whole bladder ablation as a debulking strategy and examining its synergy with SOC adjuvant therapy (Aim 1), enabled by new electrode paradigm and PEF delivery strategy (Aim 3), monitoring by an unconventional liquid biopsy approach (Aim 2). Our work can immediately aid the management of NMIBC patients who cannot undergo radical cystectomy, with future application as a cancer prevention strategy in high-risk patients. Success of individual aims will result in major contributions to the topics of IRE, BCa treatment and diagnosis.

Targeting VIP–VPAC Signaling to Reverse Immune Exclusion and Enhance Immunotherapy Response in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that is largely unresponsive to chemotherapy and current immune checkpoint blockade drugs, highlighting a critical need for the development of innovative therapeutic strategies. This R01 proposal targets vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide overexpressed in PDAC, which signals through VIP receptors (VPAC) on cancer cells, T cells, and myeloid cells within the tumor microenvironment. Based on our recent success in developing selective and potent VPAC receptor antagonists, we hypothesize that blocking VPAC signaling will reverse immunosuppression in the PDAC TME by reducing immune checkpoint expression, enhancing chemokine-driven infiltration of cytotoxic T cells, and disrupting immunosuppressive interactions between T cells and myeloid cells, ultimately leading to durable anti-cancer immunity. We propose three specific aims to explore the immunosuppressive roles of VPAC signaling in PDAC. Aim 1 will identify the primary sources of VIP in PDAC tumors and characterize the effects of VPAC signaling on immune cell function and phenotype within the tumor microenvironment. Aim 2 will investigate how VPAC signaling influences immune cell migration into tumors by modulating chemokine receptors and directional signaling. Aim 3 will determine how VPAC signaling regulates interactions between T cells and immunosuppressive myeloid cells, particularly tumor-associated macrophages, and the resulting impact on anti-cancer immune responses and immunological memory. Our preliminary findings indicate that combined inhibition of VPAC signaling and PD-1 significantly enhances the regression of PDAC tumors in multiple mouse models, generating lasting protective immunity in cured mice without triggering autoimmune responses. We will use novel methods to pursue our aims, including inducible genetically engineered mouse models (GEMM) of PDAC, long-acting VPAC antagonists engineered with immunoglobulin Fc domains to improve their plasma half-life, and advanced microfluidics technologies to analyze immune cell movement within tumors. Animal experiments will be used to validate the translational potential of observations from in vitro organoids and microfluidic experiments. The GEMM and orthotopic mouse models of PDAC are necessary to provide critical insights into the 3-D structure of the TME and tumor regression in response to our novel immunotherapy. This research will be conducted by a multidisciplinary team with complementary expertise that will clarify the therapeutic potential of VPAC signaling inhibition in PDAC using sophisticated experimental tools and single-cell RNA sequencing. Ultimately, these findings could significantly improve the development of immunotherapeutic strategies for PDAC, potentially enhancing patient outcomes in pancreatic cancer and other malignancies expressing high VIP levels.

Integrins α4β7 in Leukocyte Rolling in Shear Flow, Firm Adhesion, and Therapy

Abstract. Integrin α4β7 facilitates leukocyte migration to sites of infection and autoimmune disease, making it an important therapeutic target for ulcerative colitis and Crohns disease. However, the currently approved antibody drug vedolizumab targeting α4β7 has limited efficacy. This proposal seeks mechanistic understanding of how α4β7 mediates rolling and firm adhesion of leukocytes during extravasation as well as how therapeutically relevant antibodies modulate α4β7 function to improve drug design. Unlike most integrins, α4β7 mediates rolling adhesion on its ligand MAdCAM. α4β7 can also mediate firm adhesion like α5β1. Integrins typically equilibrate between two low-affinity closed conformations and a high-affinity open conformation. Ligand binding is intimately coordinated with conformational change. During rolling adhesion, receptor-ligand bonds must rapidly form beneath rolling cells as cells are torqued by shear flow onto the substrate. Bonds must also rapidly dissociate at the upstream tethers to the substrate due to hydrodynamic force applied to the cell. To enable their function in rolling adhesion, we hypothesize that α4β7 ligand binding and dissociation and conformational change kinetics are faster than those of other integrins like α5β1 and that α4β7's pathways for conformational change may also differ. We propose that activation of the actin cytoskeleton in the transition from rolling to firm adhesion stabilizes α4β7 in a high-affinity state. Aim 1 will determine high-resolution structures of unliganded α4β7 and its complexes with MAdCAM or medically relevant antibodies using cryo- EM. These structures will reveal how these integrins recognize their ligands, the conformational changes due to ligand binding, and potential structural specializations that enable α4β7 to mediate rolling adhesion. The binding epitopes and conformational specificities of activating antibodies to the β7 subunit will also be defined. The structure of α4β7 bound to vedolizumab will resolve the contention around how it blocks MAdCAM binding. Aim 2 will quantitatively define the mechanisms by which α4β7 mediates both rolling and firm adhesion to improve therapies for inflammatory bowel diseases. Ligand affinity and binding kinetics of α4β7 stabilized in different conformations will be measured as well as single-molecule conformational change rates when bound and unbound to ligand. The effect of mutations that stabilize rolling or firm adhesion will be used to identify parameters important for each adhesion type. The tensile force and bond lifetimes during rolling and firm adhesion will be quantified at the single-molecule level. Together, our studies will enhance our structural, biochemical, and mechanical understanding of α4β7-mediated rolling and firm adhesion and will provide structural and functional information that can be utilized in the development of more effective therapies for inflammatory bowel diseases and multiple myeloma.

Cartilage targeting exosomes for OA gene therapy and pain treatment

Project Summary Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address this, we have developed surface-modified cartilage-targeting MSC exosomes as non-viral carriers for gene therapy. MSC derived exosomes have intrinsic therapeutic potential as they can induce cartilage repair and are non-immunogenic, making them desirable for gene delivery. We have engineered charge-reversed cationic exosomes by anchoring cartilage targeting optimally charged arginine-rich cationic peptide (CPC) motifs into the anionic exosome bilayer (Exo-CPC) by using buffer pH as a charge-reversal switch. Exo-CPC use charge interactions to penetrate through the full thickness of arthritic cartilage (close to tidemark) and deliver the packaged genetic material cargo to chondrocytes residing in the deep tissue layers while native anionic exosomes cannot. They can also bind within the synovial joint, making them effective for OA pain relief gene therapy. Here we will engineer charge-reversed Exo-CPC for delivery of IL-1RA (receptor antagonist of interleukin-1) mRNA and NaV1.8 (voltage gated sodium channel 1.8) inhibitor siRNA to stimulate both disease modifying response and long-term pain relief with a one-time intra-articular dose. IL-1RA mRNA targets are in the chondrocytes and synovium cells; Nav1.8 expressing nerves innervate into synovium and subchondral bone in OA – sites that Exo-CPC can readily target. Aim 1 will engineer cartilage targeting Exo-CPC for delivery of IL- 1RA mRNA and Nav1.8 inhibitor siRNA. Their ability to deliver IL-1RA mRNA to chondrocytes and IL-1RA protein translation efficiency will be evaluated in-vitro. Exo-CPC-Na v1.8’s ability to reduce NaV1.8 bioactivity of sensory nerves will also be evaluated. In Aim 2, their distribution intra-articular (proximity to NaV1.8-positive nerves), extra-articular, and DRG and spinal cord using partial meniscectomy NaV1.8-tdTomato reporter mice OA models will be evaluated. Additionally, their dose dependent reduction on MMP activity, neuronal excitability and pain- related behaviors, and any immunogenicity will be assessed. Aim 3 will use the determined functional doses to study the long-term disease modifying and pain-relief effects of mono and combination therapy with Exo-CPC- IL-1RA and Exo-CPC-Nav1.8 in rescuing injury induced tissue structural damage as well as in reducing pain (weight bearing asymmetry) for up to one month following IA administration in early vs. late stages (intervention at 2 vs 6 weeks) of MMT (medial meniscectomy) induced OA rats. The project paves way for utilizing the intrinsic therapeutic potential of MSC Exosomes as viral-free, non-immunogenic carriers for OA gene therapy by employing cartilage as a drug depot. Cationic exosomes can be used to deliver other OA gene targets, and can be widely used for targeting other negatively charged tissues like meniscus, ligaments, discs, fracture callus etc.

Targeting disulfidptosis in cancer: mechanisms and preclinical translation

Project Summary Studying regulated cell death is critical for our understanding of cellular homeostasis and tumor suppression. We recently discovered disulfidptosis as a new form of regulated cell death induced by disulfide stress under NADPH-depleting conditions in SLC7A11-high cancer cells. However, in contrast to our deep understanding of other cell death modalities such as apoptosis and ferroptosis, the molecular and metabolic underpinnings of disulfidptosis, along with its therapeutic implications, remain largely unexplored. The objectives of this application are to elucidate the mechanisms underlying disulfidptosis and to therapeutically target this form of cell death in SLC7A11-high cancers. The proposed studies will make extensive use of human cancer cell lines and integrated human cellbased molecular analyses, including metabolomics, proteomics, CRISPR screening, and biochemical studies, to define the metabolic and signaling mechanisms governing disulfidptosis. In addition, select in vivo studies are incorporated in the therapeutic validation components of the project, where tumor growth response, systemic drug exposure and tolerability, tumor microenvironmental influences, and host immune/stromal interactions must be evaluated in an organismal context to ensure translational rigor. Alternative in vitro systems such as organoids may provide useful complementary information on tumor-intrinsic responses, but they cannot fully recapitulate the systemic metabolic stress, pharmacologic exposure, and organism-level therapeutic efficacy required for these studies. It is expected that our proposed studies will reveal novel mechanisms underlying disulfidptosis and identify effective therapies to induce this form of cell death in SLC7A11-high cancers. Our proposal is highly innovative because it focuses on a previously unexplored cell death pathway in cancer therapy. Our proposed studies will have significant impact on both our understanding of the fundamental mechanisms of disulfidptosis and our ability to target this cell death pathway in cancer treatment.

Improving Disease-Modifying Therapy Uptake among Patients with Multiple Sclerosis

Project Summary/Abstract Recent advances in the epidemiology of multiple sclerosis (MS) indicate that its prevalence is similar among White (238 per 100,000) and Black (226 per 100,000) populations. These data challenge historic assumptions about individuals with northern European heritage having higher risk and prevalence of MS. Evidence also suggests that MS incidence may be higher than previously recognized in the United States and increasing over time with more individuals identified and diagnosed year over year. MS continues to impose significant and growing burden on patients, healthcare systems and society. These health differences in the diagnosis, treatment and symptom management of MS in light of the increasing prevalence of MS in the US are an important public health issue that requires broader urgent research and policy attention to reduce the overall disease burden. In this study, we will use real-world data derived from the electronic health records (EHR) from four large academic medical centers (University of Kentucky, University of Virginia, Virginia Commonwealth University, and University of Southern California). Extracted EHR data from these four medical centers will be deidentified, combined, and harmonized. We will use this combined data set to examine (1) whether there are any differences in the timely treatment of disease modifying therapy (DMT) among different MS populations, (2) any disparities in the management of symptoms and comorbidities, (3) how non-medical factors of health such as income, education, and health insurance status (patientlevel), linguistically appropriate care provision (provider-level), and neighborhood factors (system-level) affect these outcomes and influence disparities across populations, and (4) assess whether disparities exist in the risks of cardiovascular disease CVD and mortality in MS subgroups and examine if these disparities can be reduced with improved treatment of MS and vascular comorbidities. In pursuing these objectives, we will identify clinical solutions (e.g., optimal DMT sequences) and non-medical factors such as neighborhood factors such as poverty, educational achievement, crime rates, civic participation, and housing quality, access to care factors, and cultural and linguistic match between providers and patients that substantially contribute to health disparities. For actionable solutions, we will rank-order these factors by their relative importance in addressing disparities, which will guide decision-making at the policy, system, and provider level. Our long-term objective is to develop public health strategies and scalable solutions to reduce overall burden in the management of MS. This project is expected to help policy makers and health system administrators in prioritizing interventions and to have implications for clinical practice in improving care of all patients with MS in neurology clinics, at the healthcare system level, and for national health policy.

Development of an at-home weight-shifting balance game with musical biofeedback for older adults

Reducing fall risk is a dire societal need that requires interventions that over-prepare individuals to perform maneuvers important to daily mobility. Falling is often caused by improper weight shifting, and interventions that focus on developing weight-shifting abilities have shown improvements in clinical balance outcomes, including reduced fall incidence. Interventions that combine challenges to the cognitive and motor systems may be necessary to reduce fall-risk. Our central hypothesis is that leveraging gamification and “musical biofeedback” will improve balance abilities through practicing weight-shifting skills with increased cognitive and physical demands. Musical biofeedback conveys biological sensor data from the participant through specific musical sound parameters in real-time. Of particular interest in the proposal is the applicability to use musical biofeedback to train weight-shifting skills in a musical game. The goal is to develop a wearable sensor system that can be used at-home to practice and develop balance skills, while supporting cognitive engagement and motivation to adhere to exercise goals. To start, we are focusing on older adult end-users who typically have home exercise programs focused on weight-shifting. However, in the future, many other populations can benefit from this technology. In this Trailblazer award, the PI is leveraging her background in studying complex human maneuvers, developing musical biofeedback for older adults, and in algorithm development for mHealth sensors. The transdisciplinary team includes expertise in engineering, gamified rehabilitation technologies, home exercise programs, psychology of aging, and music. In the proposed research, our goals are to evaluate responses to the musical biofeedback game (Aim 1), validate the mHealth sensor system (Aim 2), and phenotype the gameplay behavior of fallers vs. non-fallers (Aim 3), relative to their baseline characteristics (Sub-Aim 3). Our long-term goal is for a variety of people to improve their balance control patterns while supporting and building their self-efficacy. We envision users, including older adults, training with musical biofeedback to safely (and enjoyably) prepare themselves to ambulate in their community – improving and preserving their mobility. The proposed research will pioneer using an emerging clinical technology – musical biofeedback – to train balance during weight-shifting tasks. The proposed research innovates how musical biofeedback, gamification, and focusing on weight-shifting and turns in balance training can be leveraged to challenge cognitive and physical body systems in fall-risk populations. By developing new therapy options and better understanding responses relative to baseline characteristics, this research improves clinical practices to reduce fall risk and deepens our understanding of dynamic balance control. Finally, the results of the proposed research will have translational impacts to help other fall-risk groups.

Multiplex single-cell chemical genomics to identify small molecule modulators of tumor cell-intrinsic immunogenicity in glioblastoma

PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme is the most common and aggressive primary brain cancer. Despite a multimodal treatment regimen of surgical resection, chemotherapy, radiotherapy, and tumor-treating fields, most patients succumb to the disease within two years of diagnosis. Cancer immunotherapy strategies have emerged as a powerful tool for treating aggressive solid tumors such as melanoma and non-small cell lung cancer. However, current strategies have led to low response rates in glioblastoma, resulting from its low immunogenicity. The proposed research program aims to identify small molecules capable of increasing the immunogenicity of glioblastoma cells, focusing on altering gene expression programs associated with recognition by the immune system and the ability of cytotoxic immune cells to target glioblastoma for destruction. We will use highly multiplex chemical transcriptomic profiling to determine the molecular consequence of exposing glioblastoma neurosphere models to 3,792 small molecules, targeting the majority of cellular activities and clinically relevant drug targets as well as a collection of previously identified immunomodulators. We will then determine how each exposure alters the expression of gene programs associated with tumor cell immunogenicity and response to therapy, including the expression of genes associated with the recognition by the immune system and those associated with immune checkpoints, as well as programs more broadly correlated with resistance to anti-cancer therapies. Chemical hits that meet specific criteria will be subjected to a medicinal chemistry review to further classify compounds by their suitability for treating malignancies in the brain. We will then screen chemical hits to determine their ability to modulate immune-mediated tumor cell killing using tumor- immune cell co-culture. Lastly, we will leverage gene editing and flow cytometry to validate hits based on on- target molecular effects and further refine the mechanism of action by inspecting the ability of drugs to modulate immunogenic programs at the protein level. Our chemical genomics screens aim to provide crucial information regarding the link between pathway activity and immunomodulation in GBM, a critical step to guide future efforts in GBM immunotherapy. More broadly, our study will establish single-cell chemical genomics as a scalable platform for phenotype-based screening for preclinical prioritization of chemical modulators of complex transcriptional phenotypes and provide a framework for hit prioritization, establishment of pipeline robustness and hit validation in the context of single- cell chemical genomics screens.

Engineering of a temperate Burkholderia cepacia complex phage to improve efficacy as a potential therapeutic

Project Summary Bacteria in the Burkholderia cepacia complex (Bcc) cause difficult to treat infections in patients with compromised respiratory systems, such as those with cystic fibrosis (CF). Alternative treatment options are needed, since antibiotics often fail these patients. Bacteriophage (phage) therapy is a promising strategy, yet therapeutically ideal phages are difficult to find and narrow in their range of use due to host specificity. In the proposed study, we continue development of a potential phage therapeutic sourced from Burkholderia itself. We have isolated a phage, called BCC02, that was present within the genome of a Burkholderia bacteria (a prophage) and have shown that it can kill other bacteria within the same genus. However, this phage still has the potential to integrate into other bacterial genomes, which is an undesirable trait for phage therapy. By engineering changes to the BCC02 genome using synthetic biology techniques, we hypothesize that we can increase its range of therapeutic potential by disabling its ability to integrate into the bacterial genome, and that this change will increase the number of bacteria that it can lyse. The specific aims of this project are to (1) engineer this phage to lose the ability to lysogenize (integrate into bacterial genomes) then test the effects of these modifications on bacterial host range and (2) test activity of our originally isolated phage, BCC02 as well as our engineered variant on a clinically relevant panel of patho-adapted isolates from patients with CF. We propose to use transformation-associated recombination (TAR) cloning methods to target the lysogeny control region of the BCC02 genome for removal. We hypothesize that loss of integration ability will force this phage into an obligately lytic lifestyle, where it will lyse all bacteria it is able to infect. Successful completion of this project will determine the feasibility of engineering obligately lytic Burkholderia-targeting phages from Burkholderia spp. prophages, shed light on the effects of lytic lifestyle on host range, and establish the utility of these phages for tackling particularly problematic clinical infections. In addition, this study may produce a Bcc- targeting phage that is primed for development to be used for phage therapy.

Host-pathogen-microbiome interactions in Mycoplasma genitalium pathology and treatment: experiments in a 3D organotypic cervical epithelium model to strengthen clinical guidelines

ABSTRACT Mycoplasma genitalium (MG) is an emerging sexually transmitted pathogen whose clinical outcomes in women are poorly understood. Unlike other bacterial sexually transmitted infections (STI), the CDC does not recommend MG screening for asymptomatic women because it is unclear how often asymptomatic MG leads to adverse reproductive outcomes like cervicitis, which can lead to further adverse outcomes, including pelvic inflammatory disease, infertility, and ectopic pregnancy. Epidemiologic data on MG and cervicitis are mixed, and mechanistic data primarily come from models that did not faithfully recapitulate in vivo cervical microphysiological conditions. Key elements they lacked are cervical mucus, which mediates host-pathogen interactions, and the cervicovaginal microbiota. The microbiota appears to contribute to MG outcomes, and our preliminary epidemiologic data indicate that MG and bacterial vaginosis (BV) may synergize to promote cervicitis. MG care is further complicated by its ongoing rise in antibiotic resistance. Resistance-guided therapy and novel antibiotics improve treatment outcomes, but these are not available in the US. Recent clinical and in vitro data indicate that metronidazole and tinidazole, two antibiotics that are available in the US and used to treat BV, may hold promise for improving MG treatment outcomes. The overall objective of this R21 is to generate robust experimental data to clarify MG pathology, evaluate potential therapies, and inform more thorough and actionable clinical recommendations. We developed an innovative in vitro 3D organotypic model of the cervical epithelium that is ideally suited for investigating MG pathology, host-MG-microbiota interactions, and potential therapies. The model uses primary human cervical cells and better recapitulates cervical epithelial structure and physiology (including cervical mucus production) than prior 2D models. It also allows for simultaneous STI infection and co- culture of live cervicovaginal microbiota. Using the 3D organotypic cervical epithelium model, we will determine if MG causes microbiota-dependent cervical epithelial damage, a hallmark of cervicitis (Aim 1), and we will test if metronidazole and tinidazole arrest MG infection (Aim 2). In both Aims, we will interrogate the potential mediating role of the microbiota by inoculating models with live representative cervicovaginal microbiota, and we will assess host-MG-microbiota interactions via transcriptomics. We hypothesize that a polymicrobial BV-like microbiota will exacerbate MG-induced cervical epithelial damage, and removal of a polymicrobial BV microbiota will partially mediate metronidazole’s and tinidazole’s anti-MG activity. The proposed Aims have high translational potential and will provide crucial pre-clinical evidence to inform more thorough and actionable MG testing and treatment guidelines and improve reproductive health outcomes. This R21 will generate some of the first experimental data on MG-host and MG-microbiota interactions, which we will use to support an R01 to validate these interactions during in vivo MG infection and identify novel therapeutic targets for MG.

Magnetic resonance true temperature imaging with high spatial and temporal resolution

ABSTRACT The knowledge of temperature and temperature distribution within the brain can be critical to understanding the healthy and diseased brain, its response to acute injury, and in monitoring critically important thermal interventions. There are several temperature sensitive properties such relaxation rates and the proton resonance frequency shift (PRFS) that can be measured with magnetic resonance imaging (MRI) methods but these methods can only measure temperature change. The PRFS method, which provides the most accurate measurement of temperature change can only measure true tissue temperature if the starting true temperature distribution is known. Fortunately, MR spectroscopy (MRS) methods have been developed that show great promise in the measurement of true temperature. These methods rely on the detection of a temperature independent spectral peak of protons bound to carbon atoms in high concentration metabolites, such as N- acetylaspartate (NAA), creatine (Cr) and choline (Cho) which can be used as a reference for the temperature dependent spectral peak of water protons. Both single voxel spectroscopy (SVS) methods and MRS imaging (MRSI) methods have been described but are slow because of the long readout time needed to achieve adequate spectral resolution and the need to perform multiple averages due to the low signal being measured. Echo-planar spectroscopic imaging (EPSI) speeds up MRSI by interleaving an oscillating imaging gradient to spatially encode one of the imaging dimensions simultaneously with spectral readout. Unfortunately, SVS, MRSI, and even EPSI are unsuitable for clinical applications because of the low spatial resolution (voxel size 1 cm3) and temporal resolution (multiple minutes). The goal of this project is to develop an MRI technique that can measure true temperature in the whole brain at spatial and temporal resolutions that enable clinical utility for acutely assessing and longitudinally monitoring healthy and diseased brain tissue, and real time monitoring of thermal interventional therapies. This innovative true temperature measurement technique combines EPSI, for low resolution background field measurements, with PRFS for high spatial and temporal resolution water proton measurements. While conventional EPSI methods interleave volumetric acquisitions with and without water suppression, we propose an innovative modification to take advantage of the very strong water signal to obtain a very high resolution, dynamic method for true temperature measurements. The MRI pulse sequence will be refined, validated (Aim 1), applied to healthy subjects and post-surgery patients at risk for infections (Aim 2), and applied to essential tremor (ET) patients during the required delay between repeated focused ultrasound sonications (Aim 3). Successful completion of the aims of this study will result in a clinically practical method to obtain true temperature measurements in the brain with a spatial and temporal resolution sufficiently high to meet the needs of monitoring focal thermal therapy treatments as well as to provide true temperature measurements over the entire brain for assessment of the state of the brain with disease, infection, and injury.

Targeting subtype specification as a driver of PDAC health disparities

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is refractory to current treatment strategies due in part to adaptive mechanisms of chemoresistance. Racial health disparities also confound the treatment and care of these patients. Blacks (people with African genetic ancestry) have significantly higher incidence rates of PDAC and decreased survival times compared to Caucasians (White genetic ancestry) even after socioeconomic status and tumor stages are controlled. Therefore, it is possible different racial groups exhibit unique molecular characteristics in PDAC tumors that contribute to these health disparities. The unique molecular characteristics that distinguish PDAC tumors between racial groups exhibiting disparities have the potential to identify new therapeutic targets. In a previous study, we identified 4 distinct subtypes of PDAC (Metabolic, Progenitor-like, Proliferative, and Inflammatory) that can be distinguished using multivariate analysis of quantitative proteomic data. While these PDAC subtypes are predictive of therapeutic response, this has not yet been analyzed in disparity factor balanced studies. We have examined the proteomes of primary PDAC tumors using quantitative mass spectrometry and identified unique protein signatures for Blacks and Whites. PDAC tumors from Black patients display features consistent with the Inflammatory subtype of PDAC, which is characterized by an inflamed microenvironment expressing complement proteins that can promote resistance to chemotherapy. Therefore, it is possible that race influences subtype and Blacks could preferentially develop the more aggressive and treatment refractory Inflammatory subtype. Strategies are needed to modulate subtype to improve response to chemotherapy. Toward this goal, our proteomic analysis identified polycomb repressor complex 1 (PRC1) protein RNF2 as being upregulated in PDACs from Blacks compared to Whites. We have also discovered that RNF2 regulates mRNA expression of the PDAC subtype specification factor GATA6 and inhibiting RNF2 promotes a molecular shift toward the more chemosensitive Classical subtype of PDAC. Therapeutic targeting can be achieved with Tazemetostat that inhibits the upstream PRC2 to prevent RNF2 binding the GATA6 promoter leading to its increased expression. Additionally, the Inflammatory subtype characterized by innate immune complement protein activation could be targeted with another FDA approved drug, Avacopan, which has not previously been studied in PDAC. Therefore, the Specific Aims of this proposal are designed to: 1) Evaluate the extent to which Tazemetostat treatment impacts chemotherapy-induced subtype plasticity in patient derived organoids; and 2) To determine the extent to which strategies targeting pathways associated with PDAC disparities affect progression and subtype characteristics in vivo. The successful completion of these aims has the potential to be moved quickly into phase I clinical trials since both Tazemetostat and Avacopan are FDA approved drugs. Furthermore, if successful, this project has the potential to mitigate health disparities in PDAC and broadly improve patient outcomes by implementing new precision interventions. The mouse models we propose faithfully recapitulate pancreatic cancer's clinical syndrome, histopathology and molecular properties, including the often-unique features of the stromal and immune responses that constitute the complex desmoplasia of this disease, which cannot be addressed using in vitro model systems

Targeted Prodrug Cytokines for Metastatic Breast Cancer Immunotherapy

Project Summary. Our approach directly addresses key limitations in targeting and treating metastatic breast cancer, where we propose the selective activation of modular immune-modulating cytokines within the hypoxic and ROS-active TME for delivery across the BBB, providing the necessary pre-clinical data for future clinical translation. The in vitro and in vivo investigations of this novel immunotherapeutic in immunocompetent models will allow our team to study the interplay between tumor-driven immune activation, cytokine signaling, and anti-tumor immunity in both primary and metastatic sites, and establish a robust groundwork for subsequent clinical validation within the OSUCCC. This proposal addresses two key challenges in developing a novel immunotherapy strategy for breast cancer by answering two hypotheses: (1) can a modular immunotherapy platform with tumor-selective activation of prodrug recombinant cytokines overcome these limitations in drug delivery, and (2) can the development of nanobody-cytokine fusions that can selectively target primary breast cancer tumors and cross the BBB to reach metastatic tumor sites? The first hypothesis focuses on achieving tumor environment-specific activation of prodrug-based recombinant cytokines. Protein cytokines are highly potent, and while others have tried to block their activity using a fused genetic linker to ‘mask’ functionality, no one has yet attempted to use a non-canonical-based chemical strategy to achieve this inhibition. Immune-modulating cytokines will be recombinantly expressed with integrated ncAAs that block cytokine activity until the function is regenerated in the breast cancer TME. Once the cytokine activity is controlled, our second hypothesis will be to achieve selective delivery of the cytokine via fusion to nanobodies. While success has been found in targeting primary tumors in drug and protein delivery, a key challenge remains in reaching secondary metastatic tumors in hard-to-reach sites (i.e., brain). Engineered nanobodies, with affinity for breast cancer tumors and the ability to bind to BBB transcytosis receptors, will enable selective delivery to metastatic breast-to-brain tumors, resulting in tumor- specific activation, immune responses, and improved therapeutic outcomes. This system can significantly improve therapeutic outcomes for patients with mBC by integrating selective activation and delivery mechanisms to reduce off-target effects and enhance tumor-specific immune responses in both primary and secondary metastatic tumor sites. Optimizing drug delivery systems to tune immune responses could offer more effective and less invasive treatment options when compared to traditional and engineered cell-based approaches. Our momentum towards precision medicine and targeted therapies holds significant promise for improving outcomes for mBC patients, and has the potential to serve as a pan-cancer treatment for aggressive metastatic cancers from the following aims: (1) generating a modular platform for tumor-specific activation of prodrug cytokines, (2) evaluating cytokine delivery and anti-cancer immune phenotypes in mBC.

2-Deoxyglucose Therapy for Organophosphate Intoxication

Project Summary The main goal of this project is to determine the therapeutic potential of glycolysis inhibition as an adjunct to midazolam therapy in mitigating the long-term neurological effects from acute organophosphate pesticide and nerve agent (OPNA) exposure. Novel countermeasures are desperately needed for effective mitigation of morbidity and long-term effects of OPNAs. A variety of agents targeting glutamate, GABA and oxidative stress have been proposed, but glycolysis inhibitors have not been widely studied in OPNA intoxication. Dysregulated glucose metabolism plays a key role in seizures and neuronal injury following OPNA exposure. 2-Deoxyglucose (2-DG), a selective glycolysis inhibitor, has anticonvulsant and neuroprotection effects and hence can effectively mitigate acute and long-term OPNA neurotoxicity. In this project, we seek to identify the glycolysis inhibition as novel adjunct neuroprotection to midazolam therapy for OPNA exposure, with the goal of identifying 2-DG or related drugs as medical countermeasures. The glycolytic pathway represents a logical target for such intervention because glycolysis controls seizures and neuronal injury by regulating glucose utilization and activity in neurons and astrocytes in the brain. The proposed therapy is based on the hypothesis that acute OPNA neurotoxicity imparts sustained activation of the glycolysis pathway in the brain and therefore, 2- DG and selective glycolysis inhibitors prevents long-term neuronal damage neurological dysfunction. This hypothesis will be tested by using the FDA-approved (2-DG) or clinical-stage glycolytic inhibitors in two distinct OPNA models in rats: (Aim 1) To investigate the protective efficacy of 2-DG and novel glycolysis inhibitors against DFP-induced acute and long-term neuronal damage and neurological dysfunction. (Aim 2) Aim 2 (Year 2). To determine brain penetration, pilot toxicity and pharmacokinetic of 2-DG or other lead drug in naïve and DFP-exposed animals. Test drugs will be evaluated as per the NIH rigor criteria in a dose-related design in male and female rats and behavior/neuropathology will be checked for 3 months post-exposure. 2-DG and test drugs will be given starting 40-min after exposure to ONAs. Three primary outcome measures will be addressed for therapy effectiveness: (i) acute adjunct neuroprotection; (ii) chronic neuroprotectant efficacy; and (iii) prevention of neurological and behavioral deficits. The primary measures of neuroprotection include longitudinal MRI scanning, and extent of neurodegeneration, neuroinflammation, aberrant neurogenesis, and mossy fiber sprouting. Key neurological outcomes include memory deficits, depression, anxiety behavior, and neurological/motor deficits. The outcome of this project will provide “proof-of-efficacy” of a novel glycolytic therapy with FDA-approvable, repurposed drugs with promising potential to limit long-term effects of OPNAs in humans. Thus, the overall impact of the outcome is enormous for civilians, especially in developing a highly effective and safe post-exposure medical countermeasure for chemical nerve agents.

I3-BC: Image-Based Infiltrating Immune Cell Detection and Outcomes in Breast Cancer Clinical Trials

PROJECT SUMMARY Tumor infiltrating lymphocytes (TILs) represent an accessible biomarker of the tumor-immune microenvironment (TIME) in breast cancer, demonstrating consistent association with response to neoadjuvant chemotherapy and outcomes in HER2-positive and triple-negative breast cancer. Despite efforts to standardize TIL enumeration from hematoxylin and eosin stained tumor slides, TILs have not gained widespread adoption due to inter- observer variability, and time limitations in pathologic assessment, among others. Further, other key elements of the microenvironment, such as tumor-associated macrophages (TAMs), do not yet have standardized approaches for quantification or characterization. As a result, there is no assessment of the TIME for the vast majority of breast cancers diagnosed in the US and around the world. However, the rapid growth of digital pathology offers the potential to leverage computational approaches to overcome these limitations and democratize access to TIL and TAM enumeration. The overall goal of this project is to determine if computational approaches to TILs (existing) and TAMs (to be developed within this grant) are comparable to pathologist- enumerated TILs and TAMs and, further, associated with relevant patient outcomes from two phase III breast cancer clinical trials. Prior to project initiation, we have developed both a compute-intensive artificial intelligence- based TILs approach, an open source software (QuPath)-based TILs approach, and expertise in RNAseq-based immune quantification. We will first focus on TILs - benchmarking the two computational and RNAseq immune approaches against pathologist TIL counts (‘gold standard’) then evaluating association of each with event-free survival in two completed clinical trials (Aim 1). In parallel, we will develop a novel computational approache to enumerate and phenotype TAMs by using immunohistochemical staining for macrophage markers on the same slide with standard H&E, then apply in the same two clinical trials (Aim 2). Our approach is innovative because we will benchmark diverse approaches at scale in relevant clinical studies. The study is significant because we will determine if computational approaches to TILs/TAMs align with pathologist estimates and clinical outcomes, then ensure these algorithms are available to the community. Our long-term goal is to democratize computational TIL and TAM enumeration as pathology decision-support to facilitate integration of accessible tumor-immune microenvironment into clinical trials and care.

Overcoming Treatment Resistance by Targeting Polyploid Breast Cancer Cells with AI assisted Single-Cell Analysis

Therapy resistance remains a formidable challenge in breast cancer treatment, with emerging evidence identifying polyploid giant cancer cells (PGCCs) as key drivers. These cells, arising through whole-genome doubling (WGD) events, exhibit enhanced resistance to therapies, contributing to disease relapse. PGCCs are characterized by enlarged cell and nuclear sizes, increased DNA content, and greater resilience compared to non-PGCCs. Their prevalence escalates with disease progression and therapeutic stress, underscoring their critical role in treatment resistance. As such, we hypothesize that inhibiting polyploid cancer cells can effectively reduce therapeutic resistance. Despite this, effective strategies targeting PGCCs are limited, hindered by the lack of high-throughput methods to assess PGCC viability and abundance. Traditional screening assays lack the sensitivity to detect the elimination of small populations of PGCCs, while current detection methods, such as visual inspection and flow cytometry, are not suited for high-throughput compound screening. Our preliminary work has established a high-throughput single-cell morphological analysis pipeline capable of quantifying PGCCs, and we successfully screened 2,726 compounds for their efficacy on PGCCs. Based on the preliminary success, we aim to further improve its robustness and accuracy under diverse staining and imaging conditions, ensuring consistent performance across multiple labs for widespread use in PGCC/WGD studies, with deep learning to accelerate the discovery of therapeutic strategies targeting PGCCs. In addition to empirical screening, our scRNA-Seq analysis of PGCCs has revealed altered gene expression, particularly in genes associated with FOXM1, a transcription factor critical in cell cycle regulation and linked to poor outcomes in various cancers. PGCCs also show altered ferroptosis regulators and elevated reactive oxygen species (ROS), indicating susceptibility to ferroptosis. Here, we propose two independent and complementary aims. Aim 1: We will develop and validate a robust deep learning–based single-cell morphological analysis pipeline for accurate PGCC/non-PGCC discrimination across variable staining, imaging, and lab settings. The model will be benchmarked on independent datasets from external labs and released as open-source, version-controlled software with full documentation to support reproducibility and broad adoption in PGCC/WGD research. Aim 2: Leveraging our screen of 2,726 FDA-approved compounds and mechanistic studies of FOXM1 and ferroptosis, we will prioritize and validate therapies that eradicate PGCCs and reduce treatment resistance. Using patient- derived cells, 3D spheroids, and syngeneic/xenograft models, we will rigorously assess top candidates as monotherapy and in combination with standard-of-care agents. Successful completion of this project will accelerate PGCC/WGD research, advance therapeutic strategies to overcome breast cancer resistance, and especially deliver benefits to patients with high PGCC burden. Given the prevalence of WGD across solid tumors and its induction by standard therapies, our approach holds broad clinical relevance and translational impact.

Optimizing gamma-delta T cell receptor-mediated signaling to improve cancer immunotherapy

PROJECT SUMMARY The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell subpopulations, such as the gd T cells which are known to possess antitumor activities. Further, gd T cells are mostly independent of MHC-restriction, unconstrained by neoantigen burden, preferential homing to peripheral tissues and possess unique properties of T cells as well as natural killer cells making them an extremely attractive cancer immunotherapy target. One way of gd T cell activation involves the gd T cell receptor (gdTCR)-CD3 signaling pathway. gd T cell recognition of antigen by the gdTCR and the resulting proximal signaling through surrounding CD3 subunits are key steps of gd T cell activation. Even though the individual components of the gdTCR-CD3 and abTCR-CD3 complexes remain the same except for the TCRs, the complete gdTCR-CD3 complex extracellular structure is unknown. Identification of the specific extracellular interactions between the gdTCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate gdT cell immunity by targeting signaling through the gdTCR-CD3 complex. Our previous data showed that mutating residues in the constant domain of the abTCR resulted in altered ab T cell cytokine responses. Based on this data, our hypothesis is that gdTCR-CD3 signaling can also be modulated by targeting specific regions of the gdTCR by mutagenesis to improve gd T cell antitumor activities. To test our hypothesis, in Aim 1, we will use a novel photo-crosslinking and computational docking methodology to solve the complete extracellular structure of a gdTCR-CD3 complex. Further, we will use an in silico structure-based TCR design approach to identify gdTCR mutants that enhance signaling. In Aim 2, we will use an in vitro retroviral TCR display method using degenerate primers to create gdTCR mutant libraries at specific gdTCR sites such as Cg helix 3 and connecting peptide (CP) regions. In both instances, identified mutants will be tested for improved functionalities in an MHC-independent gd TCR (G115 Vg9Vd2 TCR) using in vitro cytokine and tumor-killing assays. Overall, the newly identified enhanced gd T cell clones could potentially lead to a new wave of effective cancer immunotherapy strategy by leaning into the largely untapped potential of gd T cells.

Personalized Spatial Regulatory Networks to Decode Breast Cancer Microenvironments

PROJECT SUMMARY Triple-negative breast cancer (TNBC) is an aggressive subtype with early recurrence, high metastatic burden, and limited treatment options. While genomic alterations contribute to its progression, epigenetic plasticity and spatial organization within the tumor microenvironment (TME) play critical roles in intra-tumor heterogeneity, immune evasion, and therapy resistance, yet remain poorly understood. To address this, we will develop a cost- effective and scalable methodology that integrates spatial ATAC-seq, spatial in situ transcriptomics (Xenium), and single-nucleus (sn) Epi Multiome sequencing (snRNA-seq + snATAC-seq) from core-needle biopsies, enabling high-resolution mapping of gene regulatory networks within the intact TME. Our preliminary data from six TNBC biopsies demonstrate that spatial in situ transcriptomics and spatial ATAC-seq provide critical insights into tissue architecture but suffer from data sparsity, necessitating the integration of single-nucleus Epi Multiome data to enhance cell-type annotation and impute missing genomic features. In Aim 1, we will establish a multi- modal workflow that maximizes molecular insights from limited biopsy material by optimizing tissue-preserving and multiplexed sequencing approaches. This includes leveraging patient-specific genetic variation to deconvolute nuclei-derived data and linking it to spatial transcriptomic and spatial chromatin accessibility profiles. In Aim 2, we will develop a computational framework to integrate these multi-layered datasets, enabling spatially resolved epigenomic-transcriptomic analysis that identifies key regulatory chromatin elements and transcriptional programs associated with TNBC progression, immune infiltration, and therapy resistance. This project will generate the first comprehensive, patient-specific spatial regulatory atlas of TNBC, providing fundamental insights into how chromatin accessibility and gene expression interact within the TME. Ultimately, this work will pave the way for novel precision oncology strategies, biomarker discovery, and the development of targeted therapies that address TNBC’s spatial and molecular heterogeneity.

Circulating and Mucosal Predictors and Effects of Therapeutic Interleukin-23 Blockade in Crohn's Disease

PROJECT SUMMARY/ABSTRACT Since its discovery 20 years ago, the cytokine interleukin (IL)-23 has increasingly been implicated in the pathogenesis of immune mediated diseases, such as Crohn’s disease (CD). Consequently, four monoclonal antibodies that block IL-23 are currently approved CD therapies, including risankizumab. Although suppression of pathogenic Th17 cells has been widely cited as the mechanism by which IL-23 blockade controls disease, there is a paucity of data to indicate that this is how such therapy works, and a few other immune cell populations expressing the IL-23 receptor could instead be its target. We therefore propose to study how risankizumab affects not only Th17 cells, but also mucosa-associate invariant T (MAIT) cells γδ T cells and (in the colon) type 3 innate lymphoid cells (ILC3s). In addition to quantifying these cells, we will study their gene expression to detect phenotypic differences in treated patients, and in the case of T cells, track their clonal expansion and deletion through their unique T cell receptor sequences. In colon samples, we will use a combination of single cell sequencing of sort-enriched immune cell populations and spatial transcriptomics to characterize cells in situ, at the site of disease, and determine how IL-23 blockade affects their microenvironment in vivo. By contrasting results in patients who do or do not respond therapeutically to IL-23 blockade, we will reveal valuable insights into how this treatment succeeds or fails in CD, in the process identifying predictive biomarkers to guide treatment decisions, and potentially identifying future molecular targets with which to prevent treatment failure.

Autoreactive T cells in lupus

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of adaptive immune tolerance in conjunction with innate immune system hyperactivity. Autoantibodies, produced by plasma cells derived from activated B cells, form proinflammatory immune complexes. These immune complexes drive feed forward loops that sustain a systemic inflammatory environment and deposit in tissues leading to potentially fatal organ damage. B cells receive help from T cells to produce antibodies. They also contribute to disease by shaping T cell responses and secreting cytokines. Recent case reports in which SLE patients were treated with anti-CD19 CAR-T cell therapy to deplete B cells highlight the pathogenic role of B cells in lupus and their value as a therapeutic target. However, a better understanding of how autoreactive B cells interact with autoreactive T cells may reveal more targeted points of therapeutic intervention that specifically block autoreactive responses while sparing protective ones. Antigen specific interactions between CD4+ T cells and B cells are required for the development of autoimmune disease in lupus. However, whether these critical interactions occur in germinal centers, where competition for CD4+ T cell help selects high affinity B cells, or in extrafollicular responses, where B cells may avoid peripheral tolerance checkpoints, is unclear. Gene expression profiles and pathways specific to autoreactive CD4+ T cells, and how they are shaped by their interaction with autoreactive B cells, are also ill defined. CD8+ T cells, which recognize antigen presented on MHC Class I, have also been suggested to modulate the fate of autoreactive B cells. They can directly kill autoreactive B cells as a means of tolerance, and a subset of CD8+ T cells has recently been shown to have B cell helper function. Whether and how such interactions between B and CD8+ T cells enhance or suppress the development of lupus is unknown. Here, we will use genetic and in vivo proximity labeling approaches to address these knowledge gaps. In Aim 1, we will test the hypothesis that antigen specific interactions between B and CD8+ T cells promote B cell activation and autoantibody production in lupus. We will prevent B cells, but not other cells, from undergoing cognate interactions with CD8+ T cells via B cell-specific deletion of B2M, a component of the MHC Class I complex, in two lupus models. In Aim 2, will use the uLIPSTIC in vivo proximity system to label all T cells interacting with B cells in lupus models compared to wild type controls. Features specific to these autoreactive T cells will be defined by flow cytometry, scRNA Seq, and scTCR-Seq. These studies will provide valuable molecular and cellular insight into the mutual activation of B and T cells in lupus. They will set the stage for future mechanistic studies defining the role of autoreactive T cell specific genes and pathways and potentially highlight new therapeutic targets specific to autoreactive B/T interactions.

Structure-Based Development of Nucleotide-Competing Inhibitors Against HIV-1 and LINE-1 Reverse Transcriptases

PROJECT SUMMARY Reverse transcriptases (RTs) from retroviruses and endogenous retroelements are essential polymerases that catalyze RNA- and DNA-dependent DNA synthesis. Nucleoside inhibitors (NIs) remain central to HIV-1 therapy and are also used against other viral infections and in cancer, but toxicity, limited selectivity, pharmacokinetic (PK) liabilities, and the emergence of drug resistance highlight the need for alternative RT inhibitor mechanisms. In contrast to NIs, nucleotide-competing inhibitors (NCIs) block the polymerase active site without requiring incorporation into nucleic acids. Structural studies by PI Ruiz have defined the NCI mechanism of action for HIV- 1 RT and revealed conserved binding modules shared across multiple polymerase families. These advances now enable rational discovery of improved NCIs. LINE-1 (L1) ORF2 RT is an emerging therapeutic target in cancer, autoimmunity, and aging, yet NIs are the only inhibitors known to act against L1 RT. Notably, the NCI-binding region is structurally similar between HIV-1 RT and L1 RT, suggesting that NCI recognition principles may extend across these two biologically distinct polymerases. This R21 seeks to establish proof-of-concept for NCI development against both enzymes. Aim 1 will discover and structurally optimize NCIs targeting HIV-1 RT by combining binding modules from known NCI chemotypes and determining their biochemical activity and co-crystal structures. Aim 2 will determine whether HIV-1 RT NCI principles translate to L1 RT by solving L1 RT/nucleic acid/NCI structures, evaluating enzymatic inhibition, and applying AI-based structure prediction and generative design to propose L1-specific NCI candidates. Cellular retrotransposition assays will test mechanism of action. Aim 3 will develop a fragment library tailored to protein–nucleic acid interfaces and perform fragment screening of HIV-1 and L1 RT/nucleic acid complexes to identify additional chemotypes that engage the NCI binding region. Successful completion will yield NCI scaffolds and mechanistic insights applicable to HIV-1 RT and L1 RT, define structural principles governing NCI recognition across two evolutionarily related polymerases, and establish new avenues for RT inhibitor development. The PI is highly qualified to lead this work, with extensive expertise in RT structural biology, drug design, and fragment-based discovery.

Dual mRNA Therapeutics for Liver Metastatic Uveal Melanoma

Abstract Uveal melanoma (UM) is the most common primary intraocular cancer in adults, accounting for approximately 70% of all ocular malignancies. Current treatments for primary UM include surgical tumor removal, transpupillary thermotherapy, and radiotherapy. Unfortunately, both surgical enucleation and brachytherapy have shown similar survival outcomes and carry an equivalent risk of metastasis. While the survival rate for patients with primary, non-metastatic UM is relatively high, metastatic uveal melanoma (MUM), especially when it spreads to the liver, remains universally fatal. The liver is the first site of metastasis in 80 to 90 percent of cases, and about 50 percent of UM patients develop liver metastases within 15 years of initial diagnosis. Median survival following liver metastasis is only 5 to 7 months, with an almost zero percent five-year survival rate. Currently, no available therapy significantly improves outcomes for patients with liver MUM. This R21 project addresses this urgent unmet need by developing liver-tropic mRNA therapeutics targeting two key drivers of MUM progression and metastasis: (1) constitutive activation of Gαq/11 caused by single-point mutations, and (2) loss-of-function mutations in BAP1. Both alterations occur in over 80 percent of UM patients and are associated with poor prognosis. We hypothesize that inhibition of constitutively active Gαq/11 and/or restoration of BAP1 tumor suppressor function will significantly suppress MUM progression and improve survival outcomes. Aim 1 focuses on delivering mRNA encoding a novel protein trap designed to specifically inhibit constitutively active Gαq/11 and its downstream oncogenic signaling pathways. Aim 2 seeks to restore wild-type BAP1, which is mutated or lost in approximately 84 percent of MUM cases, through liver-tropic mRNA delivery using a liver MUM model established via splenic inoculation. We will also evaluate the potential synergy between Gαq/11 inhibition and BAP1 restoration. The success of this project will not only advance our understanding of the disease mechanisms underlying MUM but also provide clinically viable strategies for treating liver metastases in uveal melanoma.

Neutralizing persistent IFN-I to improve HIV-specific CAR T cell therapy

PROJECT SUMMARY A critical hurdle to further improving the quality of life for people living with HIV (PLWH) is the need to resolve the residual immune activation and inflammation that persists even in those taking effective antiretroviral therapy (ART), which suppresses HIV replication. This unresolved and persistent immune activation is associated with increased type-I interferon (IFN-I) signaling, and increased incidence of comorbidities. Encouragingly, reports demonstrate that blocking IFN-I signaling in animal models of HIV infection can reduce HIV reservoirs and restore T cell immune function. We hypothesize that blocking IFN-I would likewise augment engineered T cell-based therapies against HIV, such as chimeric antigen receptor (CAR) T cells. Our prior work has demonstrated that when engineered to express both the 4-1BB and CD28 costimulatory domains and protected from HIV infection, HIV-specific CD4 ectodomain CAR T cells can reduce acute viremia, prevent CD4+ T cell loss, and reduce viral burden in the tissues of HIV-infected humanized mice. However, the reduction of plasma viral loads was ultimately transient, suggesting that the potency of HIV-specific CAR T cells should be further optimized for clinical translation. Our preliminary data highlights interferon-beta (IFNb) as a key immunosuppressive IFN-I negatively regulating CAR T cell proliferation, and we demonstrate that neutralizing IFNb in vivo enhanced the engraftment and persistence of HIV-specific CAR T cells adoptively transferred into HIV-infected ART- suppressed humanized mice. This proposal will interrogate whether IFNb neutralization augments CAR T cell therapy through 1) identifying the mechanism(s) by which chronic IFNb exposure mediates HIV-specific CAR T cell dysfunction, and 2) determining the effect of neutralizing IFNb on CAR T cell function and persistence in HIV infection in vivo. The proposed aims seek to develop the neutralization of IFNb as a novel immunotherapy approach to maximize the potency of HIV-specific CAR T cells aimed at achieving a functional HIV cure.

Continued HIV Production From Infected Macrophage In People On ART

PROJECT ABSTRACT After a few weeks of antiretroviral therapy (ART), HIV-1 RNA often decays to undetectable levels in blood. The initial decay is typically rapid due to the loss of short-lived, HIV-infected CD4+ T cells, but despite being adherent to ART, some people experience a subsequent period of slower decay and may require months to years to reach virologic suppression. The clinical significance of ‘slow decay’ of HIV-1 RNA after starting ART is currently unknown. Assessing the clinical significance of ‘slow decay virus’ requires identify the mechanisms generating it and exploring whether there is ongoing inflammation and neuronal damage in these people. There are three potential mechanisms that may generate ‘slow decay virus’ and they may have very different clinical implications. (1) Continued HIV-1 replication due to ineffective ART, poor ART adherence or drug- resistance. (2) Alternatively, ART could stop HIV-1 replication, but HIV-1 virions may continue to be produced by HIV-infected CD4+ T cells or (3) macrophage. Virus production without replication that emerges at the time of ART initiation is called primary nonsuppresible viremia (NSV) and is mechanistically distinct from secondary NSV observed in people who were previously suppressed. We recently examined four people who required approximately a year to become suppressed and found that ART stopped HIV-1 replication, but HIV-infected macrophage continued to produce substantial amounts of virus. These preliminary results are consistent with the long-held belief that after starting ART there is a period of rapid viral decay due to loss of HIV-infected CD4+ T cells, but some people have a subsequent period of slower decay due to continued virus production from long- lived, HIV-infected macrophage. The proposed work will expand on these observations and examine the mechanisms generating ‘slow decay virus’ in a much larger cohort of people on ART and explore the clinical implications of having ‘slow decay virus’ after starting ART (i.e. primary NSV). We will use existing, archived, longitudinal blood samples from 99 people in the MACS/WIHS Combined Cohort Study (MWCCS) who did not suppress HIV-1 RNA to undetectable levels by 6 months on ART (i.e. people with ‘slow decay virus’) and samples from 30 people who suppressed virus with typical, rapid kinetics. The proposed experiments will identify the mechanisms generating ‘slow decay virus’ during ART and the clinical implications of ‘slow decay virus’ (Aim 1). In our previous study, we also observed that ‘slow decay virus’ produced by macrophage often had nonsense/frameshift mutations in the HIV-1 vpr gene that may have promoted continued HIV-1 production from macrophage during ART. Specifically, we will explore whether ‘slow decay virus’ populations produced by macrophage have mutations in vpr or other genes that impact macrophage survival and/or HIV-1 production from infected macrophage (Aim 2). We will accomplish these aims using cutting-edge, but highly rigorous approaches. Accomplishing these aims will address clinical concerns about ‘slow decay virus’, the source of ‘slow decay virus’ as well as the role that Vpr plays in HIV-1 persistence and expression in macrophage during ART.

Development of a synthetic human centromere

PROJECT SUMMARY/ABSTRACT Human artificial chromosomes (HACs) are mini-chromosomes that can be stably inherited across many cellular generation. HACs are potentially powerful gene therapy vectors and extremely useful tools in biological research. The stability of HACs depends on the presence of a functional centromere. Centromeres are unique genomic loci that mediate the segregation of chromosomes during mitosis by forming kinetochores leading to microtubule attachment. These sites are specified by the incorporation of distinct nucleosomes in which histone H3 is replaced by CENP- A. Most centromeric nucleosomes are embedded in highly repetitive alpha-satellite DNA. The current versions of the HACs contain alpha-satellite centromeric DNA, are relatively inefficient and frequently recombine into the genome. Despite the presence of alpha-satellite DNA at centromeres, it is not absolutely required for centromere function. This is evidenced by the existence of neocentromeres in some people, and work from our lab and others that centromeres can be induced to form at non-centromeric sites. Deposition of centromeric nucleosomes is mediated by the CENP-A specific chaperone HJURP and the Mis18 complex. Previous work has shown that artificially targeting HJURP and Mis18 proteins to LacO arrays can create de novo centromeres at non-centromeric sites. This approach leads to the formation of a full centromere, recapitulating most of the characteristics of an endogenous centromere. Here we propose to develop a more versatile approach which can be re-programmed to target many different sequences. This powerful approach will provide new and exciting insight into the rules of centromere formation. The proposal will explore the practical application of de novo centromere formation in supporting the stability of human artificial chromosomes (HACs). We will test if these synthetic centromeres (SynCen) can lead to stable inheritance of a human artificial chromosome. More efficient stable non-repetitive synthetic centromere will greatly expand the potential use of HACs as gene therapy vectors.

Brain macrophage transplantation for research and therapy development

Rett syndrome, MECP2 and therapeutic strategies

The development of the iPS cell technology has revolutionized our ability to study development and diseases in defined in vitro cell culture systems. The talk will focus on Rett Syndrome and discuss two topics: (i) the use of gene editing as an approach to therapy and (ii) the role of MECP2 in gene expression (i) The mutation of the X-linked MECP2 gene is causative for the disease. In a female patient, every cell has a wt copy that is, however, in 50% of the cells located on the inactive X chromosome. We have used epigenetic gene editing tools to activate the wt MECP2 allele on the inactive X chromosome. (ii) MECP2 is thought to act as repressor of gene expression. I will present data which show that MECP2 binds to Pol II and acts as an activator for thousands of genes. The target genes are significantly enriched for Autism related genes. Our data challenge the established model of MECP2’s role in gene expression and suggest novel therapeutic approaches.

Traumatic brain injury and the visual sequela

Evolution of convulsive therapy from electroconvulsive therapy to Magnetic Seizure Therapy; Interventional Neuropsychiatry

In April, we will host Nolan Williams and Mustafa Husain. Be prepared to embark on a journey from early brain stimulation with ECT to state-of-the art TMS protocols and magnetic seizure therapy! The talks will be held on Thursday, April 25th at noon ET / 6PM CET. Nolan Williams, MD, is an associate professor of Psychiatry and Behavioral Science at Stanford University. He developed the SAINT protocol, which is the first FDA-cleared non-invasive, rapid-acting neuromodulation treatment for treatment-resistant depression. Mustafa Husain, MD, is an adjunct professor of Psychiatry and Behavioral Sciences at Duke University and a professor of Psychiatry and Neurology at UT Southwestern Medical Center, Dallas. He will tell us about “Evolution of convulsive therapy from electroconvulsive therapy to Magnetic Seizure Therapy”. As always, we will also get a glimpse at the “Person behind the science”. Please register va talks.stimulatingbrains.org to receive the (free) Zoom link, subscribe to our newsletter, or follow us on Twitter/X for further updates!

Gene therapy for hearing loss: where do we go from ear?

Use of brain imaging data to improve prescriptions of psychotropic drugs - Examples of ketamine in depression and antipsychotics in schizophrenia

The use of molecular imaging, particularly PET and SPECT, has significantly transformed the treatment of schizophrenia with antipsychotic drugs since the late 1980s. It has offered insights into the links between drug target engagement, clinical effects, and side effects. A therapeutic window for receptor occupancy is established for antipsychotics, yet there is a divergence of opinions regarding the importance of blood levels, with many downplaying their significance. As a result, the role of therapeutic drug monitoring (TDM) as a personalized therapy tool is often underrated. Since molecular imaging of antipsychotics has focused almost entirely on D2-like dopamine receptors and their potential to control positive symptoms, negative symptoms and cognitive deficits are hardly or not at all investigated. Alternative methods have been introduced, i.e. to investigate the correlation between approximated receptor occupancies from blood levels and cognitive measures. Within the domain of antidepressants, and specifically regarding ketamine's efficacy in depression treatment, there is limited comprehension of the association between plasma concentrations and target engagement. The measurement of AMPA receptors in the human brain has added a new level of comprehension regarding ketamine's antidepressant effects. To ensure precise prescription of psychotropic drugs, it is vital to have a nuanced understanding of how molecular and clinical effects interact. Clinician scientists are assigned with the task of integrating these indispensable pharmacological insights into practice, thereby ensuring a rational and effective approach to the treatment of mental health disorders, signaling a new era of personalized drug therapy mechanisms that promote neuronal plasticity not only under pathological conditions, but also in the healthy aging brain.

Therapy in the Digital Age: Learnings from a Swedish Study (n=2400) on CBT vs. PDT + thoughts about ChatGPT

Restoring function in advanced disease with photoreceptor cell replacement therapy

Mechanisms Underlying the Persistence of Cancer-Related Fatigue

Cancer-related fatigue is a prominent and debilitating side effect of cancer and its treatment. It can develop prior to diagnosis, generally peaks during cancer treatment, and can persist long after treatment completion. Its mechanisms are multifactorial, and its expression is highly variable. Unfortunately, treatment options are limited. Our research uses syngeneic murine models of cancer and cisplatin-based chemotherapy to better understand these mechanisms. Our data indicate that both peripherally and centrally processes may contribute to the developmental of fatigue. These processes include metabolic alterations, mitochondrial dysfunction, pre-cachexia, and inflammation. However, our data has revealed that behavioral fatigue can persist even after the toxicity associated with cancer and its treatment recover. For example, running during cancer treatment attenuates kidney toxicity while also delaying recovery from fatigue-like behavior. Additionally, administration of anesthetics known to disrupt memory consolidation at the time treatment can promote recovery, and treatment-related cues can re-instate fatigue after recovery. Cancer-related fatigue can also promote habitual behavioral patterns, as observed using a devaluation task. We interpret this data to suggest that limit metabolic resources during cancer promote the utilization of habit-based behavioral strategies that serve to maintain fatigue behavior into survivorship. This line of work is exciting as it points us toward novel interventional targets for the treatment of persistent cancer-related fatigue.

Aging promotes reactivation from metastatic melanoma dormancy

How does the primary tumor imprint a dormancy signature in disseminated tumor cells?

T cells specific for alpha-myosin drive immunotherapy-related myocarditis

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor

Harnessing mRNA metabolism for the development of precision gene therapy

Myelin Formation and Oligodendrocyte Biology in Epilepsy

Epilepsy is one of the most common neurological diseases according to the World Health Organization (WHO) affecting around 70 million people worldwide [WHO]. Patients who suffer from epilepsy also suffer from a variety of neuro-psychiatric co-morbidities, which they can experience as crippling as the seizure condition itself. Adequate organization of cerebral white matter is utterly important for cognitive development. The failure of integration of neurologic function with cognition is reflected in neuro-psychiatric disease, such as autism spectrum disorder (ASD). However, in epilepsy we know little about the importance of white matter abnormalities in epilepsy-associated co-morbidities. Epilepsy surgery is an important therapy strategy in patients where conventional anti-epileptic drug treatment fails . On histology of the resected brain samples, malformations of cortical development (MCD) are common among the epilepsy surgery population, especially focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC). Both pathologies are associated with constitutive activation of the mTOR pathway. Interestingly, some type of FCD is morphological similar to TSC cortical tubers including the abnormalities of the white matter. Hypomyelination with lack of myelin-producing cells, the oligodendrocytes, within the lesional area is a striking phenomenon. Impairment of the complex myelination process can have a major impact on brain function. In the worst case leading to distorted or interrupted neurotransmissions. It is still unclear whether the observed myelin pathology in epilepsy surgical specimens is primarily related to the underlying malformation process or is just a secondary phenomenon of recurrent epileptic seizures creating a toxic micro-environment which hampers myelin formation. Interestingly, mTORC1 has been implicated as key signal for myelination, thus, promoting the maturation of oligodendrocytes . These results, however, remain controversial. Regardless of the underlying pathophysiologic mechanism, alterations of myelin dynamics, depending on their severity, are known to be linked to various kinds of developmental disorders or neuropsychiatric manifestations.

Children-Agent Interaction For Assessment and Rehabilitation: From Linguistic Skills To Mental Well-being

Socially Assistive Robots (SARs) have shown great potential to help children in therapeutic and healthcare contexts. SARs have been used for companionship, learning enhancement, social and communication skills rehabilitation for children with special needs (e.g., autism), and mood improvement. Robots can be used as novel tools to assess and rehabilitate children’s communication skills and mental well-being by providing affordable and accessible therapeutic and mental health services. In this talk, I will present the various studies I have conducted during my PhD and at the Cambridge Affective Intelligence and Robotics Lab to explore how robots can help assess and rehabilitate children’s communication skills and mental well-being. More specifically, I will provide both quantitative and qualitative results and findings from (i) an exploratory study with children with autism and global developmental disorders to investigate the use of intelligent personal assistants in therapy; (ii) an empirical study involving children with and without language disorders interacting with a physical robot, a virtual agent, and a human counterpart to assess their linguistic skills; (iii) an 8-week longitudinal study involving children with autism and language disorders who interacted either with a physical or a virtual robot to rehabilitate their linguistic skills; and (iv) an empirical study to aid the assessment of mental well-being in children. These findings can inform and help the child-robot interaction community design and develop new adaptive robots to help assess and rehabilitate linguistic skills and mental well-being in children.

Programmed axon death: from animal models into human disease

Programmed axon death is a widespread and completely preventable mechanism in injury and disease. Mouse and Drosophila studies define a molecular pathway involving activation of SARM1 NA Dase and its prevention by NAD synthesising enzyme NMNAT2 . Loss of axonal NMNAT2 causes its substrate, NMN , to accumulate and activate SARM1 , driving loss of NAD and changes in ATP , ROS and calcium. Animal models caused by genetic mutation, toxins, viruses or metabolic defects can be alleviated by blocking programmed axon death, for example models of CMT1B , chemotherapy-induced peripheral neuropathy (CIPN), rabies and diabetic peripheral neuropathy (DPN). The perinatal lethality of NMNAT2 null mice is completely rescued, restoring a normal, healthy lifespan. Animal models lack the genetic and environmental diversity present in human populations and this is problematic for modelling gene-environment combinations, for example in CIPN and DPN , and identifying rare, pathogenic mutations. Instead, by testing human gene variants in WGS datasets for loss- and gain-of-function, we identified enrichment of rare SARM1 gain-of-function variants in sporadic ALS , despite previous negative findings in SOD1 transgenic mice. We have shown in mice that heterozygous SARM1 loss-of-function is protective from a range of axonal stresses and that naturally-occurring SARM1 loss-of-function alleles are present in human populations. This enables new approaches to identify disorders where blocking SARM1 may be therapeutically useful, and the existence of two dominant negative human variants in healthy adults is some of the best evidence available that drugs blocking SARM1 are likely to be safe. Further loss- and gain-of-function variants in SARM1 and NMNAT2 are being identified and used to extend and strengthen the evidence of association with neurological disorders. We aim to identify diseases, and specific patients, in whom SARM1 -blocking drugs are most likely to be effective.

When to stop immune checkpoint inhibitor for malignant melanoma? Challenges in emulating target trials

Observational data have become a popular source of evidence for causal effects when no randomized controlled trial exists, or to supplement information provided by those. In practice, a wide range of designs and analytical choices exist, and one recent approach relies on the target trial emulation framework. This framework is particularly well suited to mimic what could be obtained in a specific randomized controlled trial, while avoiding time-related selection biases. In this abstract, we present how this framework could be useful to emulate trials in malignant melanoma, and the challenges faced when planning such a study using longitudinal observational data from a cohort study. More specifically, two questions are envisaged: duration of immune checkpoint inhibitors, and trials comparing treatment strategies for BRAF V600-mutant patients (targeted therapy as 1st line, followed by immunotherapy as 2nd line, vs. immunotherapy as 2nd line followed by targeted therapy as 1st line). Using data from 1027 participants to the MELBASE cohort, we detail the results for the emulation of a trial where immune checkpoint inhibitor would be stopped at 6 months vs. continued, in patients in response or with stable disease.

Myelin Formation and Oligodendrocyte Biology in Epilepsy

Epilepsy is one of the most common neurological diseases according to the World Health Organization (WHO) affecting around 70 million people worldwide [WHO]. Patients who suffer from epilepsy also suffer from a variety of neuro-psychiatric co-morbidities, which they can experience as crippling as the seizure condition itself. Adequate organization of cerebral white matter is utterly important for cognitive development. The failure of integration of neurologic function with cognition is reflected in neuro-psychiatric disease, such as autism spectrum disorder (ASD). However, in epilepsy we know little about the importance of white matter abnormalities in epilepsy-associated co-morbidities. Epilepsy surgery is an important therapy strategy in patients where conventional anti-epileptic drug treatment fails . On histology of the resected brain samples, malformations of cortical development (MCD) are common among the epilepsy surgery population, especially focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC). Both pathologies are associated with constitutive activation of the mTOR pathway. Interestingly, some type of FCD is morphological similar to TSC cortical tubers including the abnormalities of the white matter. Hypomyelination with lack of myelin-producing cells, the oligodendrocytes, within the lesional area is a striking phenomenon. Impairment of the complex myelination process can have a major impact on brain function. In the worst case leading to distorted or interrupted neurotransmissions. It is still unclear whether the observed myelin pathology in epilepsy surgical specimens is primarily related to the underlying malformation process or is just a secondary phenomenon of recurrent epileptic seizures creating a toxic micro-environment which hampers myelin formation. Interestingly, mTORC1 has been implicated as key signal for myelination, thus, promoting the maturation of oligodendrocytes . These results, however, remain controversial. Regardless of the underlying pathophysiologic mechanism, alterations of myelin dynamics, depending on their severity, are known to be linked to various kinds of developmental disorders or neuropsychiatric manifestations.

Radiopharmaceutical evaluation of novel bifunctional chelators and bioconjugates for tumour imaging and therapy

Bispidines (3,7-diazabicyclo[3.3.1]nonane) and their derivatives act as bifunctional chelators (BFC), combining the advantages of multidentate macrocyclic and acyclic ligands e.g. high kinetic inertness, rapid radiolabelling under mild conditions. This bicyclic chelator system shows a great diversity in terms of its denticity and type of functional groups, yielding a wide range of multidentate ligands that can bind a variety of different metal ions. In addition, they allow a facile functionalisation of targeting molecules such as peptides, peptidomimetics, and bispeci􀄀c antibodies. Herein, examples of various bispidine complexes labelled with [64Cu]Cu2+, [111In]In3+, [ 177Lu]Lu3+ or [ 225Ac]Ac3+ will be presented which provide a picture of how different substituents in􀄁uence the coordination mode. Target-speci􀄀c radiolabelled bispidine-based conjugates (e.g. peptides, antibody fragments, antibodies) investigated in vivo by positron emission or single-photon emission computed tomography will be presented and discussed in terms of their suitability for nuclear medicine applications.

Faking emotions and a therapeutic role for robots and chatbots: Ethics of using AI in psychotherapy

In recent years, there has been a proliferation of social robots and chatbots that are designed so that users make an emotional attachment with them. This talk will start by presenting the first such chatbot, a program called Eliza designed by Joseph Weizenbaum in the mid 1960s. Then we will look at some recent robots and chatbots with Eliza-like interfaces and examine their benefits as well as various ethical issues raised by deploying such systems.

Reconstructing inhibitory circuits in a damaged brain

Inhibitory interneurons govern the sparse activation of principal cells that permits appropriate behaviors, but they among the most vulnerable to brain damage. Our recent work has demonstrated important roles for inhibitory neurons in disorders of brain development, injury and epilepsy. These studies have motivated our ongoing efforts to understand how these cells operate at the synaptic, circuit and behavioral levels and in designing new technologies targeting specific populations of interneurons for therapy. I will discuss our recent efforts examining the role of interneurons in traumatic brain injury and in designing cell transplantation strategies - based on the generation of new inhibitory interneurons - that enable precise manipulation of inhibitory circuits in the injured brain. I will also discuss our ongoing efforts using monosynaptic virus tracing and whole-brain clearing methods to generate brain-wide maps of inhibitory circuits in the rodent brain. By comprehensively mapping the wiring of individual cell types on a global scale, we have uncovered a fundamental strategy to sustain and optimize inhibition following traumatic brain injury that involves spatial reorganization of local and long-range inputs to inhibitory neurons. These recent findings suggest that brain damage, even when focally restricted, likely has a far broader affect on brain-wide neural function than previously appreciated.

The Synaptome Architecture of the Brain: Lifespan, disease, evolution and behavior

The overall aim of my research is to understand how the organisation of the synapse, with particular reference to the postsynaptic proteome (PSP) of excitatory synapses in the brain, informs the fundamental mechanisms of learning, memory and behaviour and how these mechanisms go awry in neurological dysfunction. The PSP indeed bears a remarkable burden of disease, with components being disrupted in disorders (synaptopathies) including schizophrenia, depression, autism and intellectual disability. Our work has been fundamental in revealing and then characterising the unprecedented complexity (>1000 highly conserved proteins) of the PSP in terms of the subsynaptic architecture of postsynaptic proteins such as PSD95 and how these proteins assemble into complexes and supercomplexes in different neurons and regions of the brain. Characterising the PSPs in multiple species, including human and mouse, has revealed differences in key sets of functionally important proteins, correlates with brain imaging and connectome data, and a differential distribution of disease-relevant proteins and pathways. Such studies have also provided important insight into synapse evolution, establishing that vertebrate behavioural complexity is a product of the evolutionary expansion in synapse proteomes that occurred ~500 million years ago. My lab has identified many mutations causing cognitive impairments in mice before they were found to cause human disorders. Our proteomic studies revealed that >130 brain diseases are caused by mutations affecting postsynaptic proteins. We uncovered mechanisms that explain the polygenic basis and age of onset of schizophrenia, with postsynaptic proteins, including PSD95 supercomplexes, carrying much of the polygenic burden. We discovered the “Genetic Lifespan Calendar”, a genomic programme controlling when genes are regulated. We showed that this could explain how schizophrenia susceptibility genes are timed to exert their effects in young adults. The Genes to Cognition programme is the largest genetic study so far undertaken into the synaptic molecular mechanisms underlying behaviour and physiology. We made important conceptual advances that inform how the repertoire of both innate and learned behaviours is built from unique combinations of postsynaptic proteins that either amplify or attenuate the behavioural response. This constitutes a key advance in understanding how the brain decodes information inherent in patterns of nerve impulses, and provides insight into why the PSP has evolved to be so complex, and consequently why the phenotypes of synaptopathies are so diverse. Our most recent work has opened a new phase, and scale, in understanding synapses with the first synaptome maps of the brain. We have developed next-generation methods (SYNMAP) that enable single-synapse resolution molecular mapping across the whole mouse brain and extensive regions of the human brain, revealing the molecular and morphological features of a billion synapses. This has already uncovered unprecedented spatiotemporal synapse diversity organised into an architecture that correlates with the structural and functional connectomes, and shown how mutations that cause cognitive disorders reorganise these synaptome maps; for example, by detecting vulnerable synapse subtypes and synapse loss in Alzheimer’s disease. This innovative synaptome mapping technology has huge potential to help characterise how the brain changes during normal development, including in specific cell types, and with degeneration, facilitating novel pathways to diagnosis and therapy.

Genetic-based brain machine interfaces for visual restoration

Visual restoration is certainly the greatest challenge for brain-machine interfaces with the high pixel number and high refreshing rate. In the recent year, we brought retinal prostheses and optogenetic therapy up to successful clinical trials. Concerning visual restoration at the cortical level, prostheses have shown efficacy for limited periods of time and limited pixel numbers. We are investigating the potential of sonogenetics to develop a non-contact brain machine interface allowing long-lasting activation of the visual cortex. The presentation will introduce our genetic-based brain machine interfaces for visual restoration at the retinal and cortical levels.

Antisense oligonucleotide mediated exon skipping therapy development for Duchenne muscular dystrophy takes more than an oligonucleotide

Mutation targeted gene therapy approaches to alter rod degeneration and retain cones

My research uses electrophysiological techniques to evaluate normal retinal function, dysfunction caused by blinding retinal diseases and the restoration of function using a variety of therapeutic strategies. We can use our understanding or normal retinal function and disease-related changes to construct optimal therapeutic strategies and evaluate how they ameliorate the effects of disease. Retinitis pigmentosa (RP) is a family of blinding eye diseases caused by photoreceptor degeneration. The absence of the cells that for this primary signal leads to blindness. My interest in RP involves the evaluation of therapies to restore vision: replacing degenerated photoreceptors either with: (1) new stem or other embryonic cells, manipulated to become photoreceptors or (2) prosthetics devices that replace the photoreceptor signal with an electronic signal to light. Glaucoma is caused by increased intraocular pressure and leads to ganglion cell death, which eliminates the link between the retinal output and central visual processing. We are parsing out of the effects of increased intraocular pressure and aging on ganglion cells. Congenital Stationary Night Blindness (CSNB) is a family of diseases in which signaling is eliminated between rod photoreceptors and their postsynaptic targets, rod bipolar cells. This deafferents the retinal circuit that is responsible for vision under dim lighting. My interest in CSNB involves understanding the basic interplay between excitation and inhibition in the retinal circuit and its normal development. Because of the targeted nature of this disease, we are hopeful that a gene therapy approach can be developed to restore night vision. My work utilizes rodent disease models whose mutations mimic those found in human patients. While molecular manipulation of rodents is a fairly common approach, we have recently developed a mutant NIH miniature swine model of a common form of autosomal dominant RP (Pro23His rhodopsin mutation) in collaboration with the National Swine Resource Research Center at University of Missouri. More genetically modified mini-swine models are in the pipeline to examine other retinal diseases.

Chemogenetic therapies for epilepsy: promises and challenges

Expression of Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) on excitatory hippocampal neurons in the hippocampus represents a potential new therapeutic strategy for drug-resistant epilepsy. During my talk I will demonstrate that we obtained potent suppression of spontaneous epileptic seizures in mouse and a rat models for temporal lobe epilepsy using different DREADD ligands, up to one year after viral vector expression. The chemogenetic approach clearly outperforms the seizure-suppressing efficacy of currently existing anti-epileptic drugs. Besides the promises, I will also present some of the challenges associated with a potential chemogenetic therapy, including constitutive DREADD activity, tolerance effects, risk for toxicity, paradoxical excitatory effects in non-epileptic hippocampal tissue.

fMRI of cognitive reappraisal, acceptance, and suppression emotion regulation strategies in basic and clinically applied contexts

The ability to effectively regulate emotions is a fundamental skill related to physical and psychological health. In this talk, I will present behavioral and fMRI data from several different studies that examined cognitive reappraisal, acceptance, and suppression emotion regulation strategies in healthy controls participants and in the context of randomized trials of cognitive behavioral therapy, mindfulness- based stress reduction, and aerobic exercise as interventions for adults with anxiety disorders. We will also examine the implementation of different types of functional connectivity analytic approaches to probe intervention-related brain mechanism changes.

Gene Therapy in Epilepsy

Activity-dependent Gene Therapy for Epilepsy

Personalized Psychotherapy

Mechanisms of CACNA1A-associated developmental epileptic encephalopathies

Developmental epileptic encephalopathies are early-onset epilepsies, often refractory to therapy, with developmental delay or regression. These disorders carry poor neurodevelopmental prognosis, with long-term refractory epilepsy and persistent cognitive, behavioral and motor deficits. Mutations in the CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 voltage-gated calcium channels, result in a spectrum of neurological disorders, including severe, early-onset epileptic encephalopathies. Recent work from the Rossignol lab helped characterize the phenotypic spectrum of CACNA1A-related epilepsies in humans. Using conditional genetics and novel animal models, the Rossignol lab unveiled some of the underlying pathophysiological mechanisms, including critical deficits in cortical inhibition, resulting in seizures and a range of cognitive-behavioral deficits. Importantly, Dr. Rossignol’s team demonstrated that the targeted activation of specific GABAergic interneuron populations in selected cortical regions prevents motor seizures and reverts attention deficits and cognitive rigidity in mouse models of the disorder. These recent findings open novel avenues for the treatment of these severe CACNA1A-associated neurodevelopmental disorders.

Understanding the Assessment of Spatial Neglect and its Treatment Using Prism Adaptation Training

Spatial neglect is a syndrome that is most frequently associated with damage to the right hemisphere, although damage to the left hemisphere can also result in signs of spatial neglect. It is characterised by absent or deficient awareness of the contralesional side of space. The screening and diagnosis of spatial neglect lacks a universal gold standard, but is usually achieved by using various modes of assessment. Spatial neglect is also difficult to treat, although prism adaptation training (PAT) has in the past reportedly showed some promise. This seminar will include highlights from a series of studies designed to identify knowledge gaps, and will suggest ways in which these can be bridged. The first study was conducted to identify and quantify clinicians’ use of assessment tools for spatial neglect, finding that several different tools are in use, but that there is an emerging consensus and appetite for harmonisation. The second study included PAT, and sought to uncover whether PAT can improve engagement in recommended therapy in order to improve the outcomes of stroke survivors with spatial neglect. The final study, a systematic review and meta-analysis, sought to investigate the scientific efficacy (rather than clinical effectiveness) of PAT, identifying several knowledge gaps in the existing literature and a need for a new approach in the study of PAT in the clinical setting.

Brain-Machine Interfaces: Beyond Decoding

A brain-machine interface (BMI) is a system that enables users to interact with computers and robots through the voluntary modulation of their brain activity. Such a BMI is particularly relevant as an aid for patients with severe neuromuscular disabilities, although it also opens up new possibilities in human-machine interaction for able-bodied people. Real-time signal processing and decoding of brain signals are certainly at the heart of a BMI. Yet, this does not suffice for subjects to operate a brain-controlled device. In the first part of my talk I will review some of our recent studies, most involving participants with severe motor disabilities, that illustrate additional principles of a reliable BMI that enable users to operate different devices. In particular, I will show how an exclusive focus on machine learning is not necessarily the solution as it may not promote subject learning. This highlights the need for a comprehensive mutual learning methodology that foster learning at the three critical levels of the machine, subject and application. To further illustrate that BMI is more than just decoding, I will discuss how to enhance subject learning and BMI performance through appropriate feedback modalities. Finally, I will show how these principles translate to motor rehabilitation, where in a controlled trial chronic stroke patients achieved a significant functional recovery after the intervention, which was retained 6-12 months after the end of therapy.

Cluster Headache: Improving Therapy for the Worst Pain Experienced by Humans

Cluster headache is a brain disorder dominated clinically by dreadful episodes of excruciating pain with a circadian pattern and most often focused in bouts with circannual periodicity. As we have understood its neurobiology new therapies, including those directed at calcitonin gene-related peptide, are helpful improve the lives of sufferers.

LONG-ACTING ANTIPSYCHOTICS: OPTION DOWN THE ROCKY ROAD, NICE TO HAVE OR ESSENTIAL CHOICE?

Time and again we are faced with the question at what point in the treatment of schizophrenia a depot formulation should be used. The data on the so-called LAIs (Long-Acting Injectables) has steadily increased in recent years. Today, we have very good evidence for the early use of depot therapies. However, the willingness and consent of the patient for this form of pharmacotherapy remains central to the successful use of LAIs. In his lecture, Prof. Correll will talk about the current evidence for the use of LAIs summarizing the latest studies.

Gene therapy for Optic Neuropathies

How inclusive and diverse is non-invasive brain stimulation in the treatment of psychiatric disorders?

How inclusive and diverse is non-invasive brain stimulation in the treatment of psychiatric disorders?Indira Tendolkar, Donders Institute for Brain, Cognition and Behavior, Department of Psychiatry. Mental illness is associated with a huge socioeconomic burden worldwide, with annual costs only in the Netherlands of €22 billion. Over two decades of cognitive and affective neuroscience research with modern tools of neuroimaging and neurophysiology in humans have given us a wealth of information about neural circuits underlying the main symptom domains of psychiatric disorders and their remediation. Neuromodulation entails the alteration of these neural circuits through invasive (e.g., DBS) or non-invasive (e.g., TMS) techniques with the aim of improving symptoms and/or functions and enhancing neuroplasticity. In my talk, I will focus on neuromodulation studies using repetitive transcranial magnetic stimulation (rTMS) as a relatively safe, noninvasive method, which can be performed simultaneously with neurocognitive interventions. Using the examples of two chronifying mental illnesses, namely obsessive compulsive disorders and major depressive disorder (MDD), I will review the concept of "state dependent" effects of rTMS and highlight how simultaneous or sequential cognitive interventions could help optimize rTMS therapy by providing further control of ongoing neural activity in targeted neural networks. Hardly any attention has been paid to diversity aspects in the studies. By including studies from low- and middle income countries, I will discuss the potential of non-invasive brain stimulation from a transcultural perspective.

Developing metal-based radiopharmaceuticals for imaging and therapy

Personalised medicine will be greatly enhanced with the introduction of new radiopharmaceuticals for the diagnosis and treatment of various cancers, as well as cardiovascular disease and brain disorders. The unprecedented interest in developing theranostic radiopharmaceuticals is mainly due to the recent clinical successes of radiometal-based products including: • 177LuDOTA-TATE (trade name Lutathera, FDA approved in 2018), a peptide-based tracer that is used for treating metastatic neuroendocrine tumours • Ga 68 PSMA-11 (FDA approved in 2020), a positron emission tomography agent for imaging prostate-specific membrane antigen positive lesions in men with prostate cancer. In this webinar, Dr Brett Paterson and PhD candidate Mr Cormac Kelderman will present their research on developing the chemistry and radiochemistry to produce new radiometal-based imaging and therapy agents. They will discuss the synthesis of new molecules, the optimisation of the radiochemistry, and results from preclinical evaluations. Dr Brett Paterson is a National Imaging Facility Fellow at Monash Biomedical Imaging and academic group leader in the School of Chemistry, Monash University. His research focuses on the development of radiochemistry and new radiopharmaceuticals. Cormac Kelderman is a PhD candidate under the supervision of Dr Brett Paterson in the School of Chemistry, Monash University. His research focuses on developing new bis(thiosemicarbazone) chelators for technetium-99m SPECT imaging.

Mechanisms and precision therapies in genetic epilepsies

Large scale genetic studies and associated functional investigations have tremendously augmented our knowledge about the mechanisms underlying epileptic seizures, and sometimes also accompanying developmental problems. Pharmacotherapy of the epilepsies is routinely guided by trial and error, since predictors for a response to specific antiepileptic drugs are largely missing. The recent advances in the field of genetic epilepsies now offer an increasing amount of either well fitting established or new re-purposing therapies for genetic epilepsy syndromes based on understanding of the pathophysiological principles. Examples are provided by variants in ion channel or transporter encoding genes which cause a broad spectrum of epilepsy syndromes of variable severity and onset, (1) the ketogenic diet for glucose transporter defects of the blood-brain barrier, (2) Na+ channel blockers (e.g. carbamazepine) for gain-of-function Na+ channel mutations and avoidance of those drugs for loss-of-function mutations, and (3) specific K+ channel blockers for mutations with a gain-of-function defect in respective K+ channels. I will focus in my talk on the latter two including the underlying mechanisms, their relation to clinical phenotypes and possible therapeutic implications. In conclusion, genetic and mechanistic studies offer promising tools to predict therapeutic effects in rare epilepsies.

Exploring and targeting CNS inflammation in brain metastases

Making spinal sensory interneurons from stem cells for regenerative therapies

Dr. Gupta is carrying out his post doctoral studies in the Buter Laboratory in UCLA. He is applying his his knowledge of embryology to stem cells for developing regenerative therapies to treat spinal cord injuries. In this talk, he will discuss how understanding the logic for spinal cord development led us to derive diverse sensory neuronal classes from embryonic stem cells. The spinal sensory neurons enableus to perceive our environment through modalities that are lost in spinal injury patients. These ESC derived senory neurons can help regain sensation in spina cord injury patients through regenerative therapies.

Visual restoration from prosthesis to optogenetic therapy

Regenerative Neuroimmunology - a stem cell perspective

There are currently no approved therapies to slow down the accumulation of neurological disability that occurs independently of relapses in multiple sclerosis (MS). International agencies are engaging to expedite the development of novel strategies capable of modifying disease progression, abrogating persistent CNS inflammation, and support degenerating axons in people with progressive MS. Understanding why regeneration fails in the progressive MS brain and developing new regenerative approaches is a key priority for the Pluchino Lab. In particular, we aim to elucidate how the immune system, in particular its cells called myeloid cells, affects brain structure and function under normal healthy conditions and in disease. Our objective is to find how myeloid cells communicate with the central nervous system and affect tissue healing and functional recovery by stimulating mechanisms of brain plasticity mechanisms such as the generation of new nerve cells and the reduction of scar formation. Applying combination of state-of-the-art omic technologies, and molecular approaches to study murine and human disease models of inflammation and neurodegeneration, we aim to develop experimental molecular medicines, including those with stem cells and gene therapy vectors, which slow down the accumulation of irreversible disabilities and improve functional recovery after progressive multiple sclerosis, stroke and traumatic injuries. By understanding the mechanisms of intercellular (neuro-immune) signalling, diseases of the brain and spinal cord may be treated more effectively, and significant neuroprotection may be achieved with new tailored molecular therapeutics.

Genetics and Therapy of Inherited Retinal Diseases

New Frontiers in Understanding and Treating Migraine Headaches

In this presentation I will describe how the CGRP project started and culminated in the development of gepants and mAbs for successful therapy. The outstanding question regarding the preponderance of female migraineurs also remains. I will present views on the reason behind this and suggest that understanding the hormonal influence will pave the way to alleviating hormone related migraine.

Towards targeted therapies for the treatment of Dravet Syndrome

Dravet syndrome is a severe epileptic encephalopathy that begins during the first year of life and leads to severe cognitive and social interaction deficits. It is mostly caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes for the alpha-subunit of the voltage-gated sodium channel (Nav1.1) and is responsible mainly of GABAergic interneuron excitability. While different therapies based on the upregulation of the healthy allele of the gene are being developed, the dynamics of reversibility of the pathology are still unclear. In fact, whether and to which extent the pathology is reversible after symptom onset and if it is sufficient to ensure physiological levels of Scn1a during a specific critical period of time are open questions in the field and their answers are required for proper development of effective therapies. We generated a novel Scn1a conditional knock-in mouse model (Scn1aSTOP) in which the endogenous Scn1a gene is silenced by the insertion of a floxed STOP cassette in an intron of Scn1a gene; upon Cre recombinase expression, the STOP cassette is removed, and the mutant allele can be reconstituted as a functional Scn1a allele. In this model we can reactivate the expression of Scn1a exactly in the neuronal subtypes in which it is expressed and at its physiological level. Those aspects are crucial to obtain a final answer on the reversibility of DS after symptom onset. We exploited this model to demonstrate that global brain re-expression of the Scn1a gene when symptoms are already developed (P30) led to a complete rescue of both spontaneous and thermic inducible seizures and amelioration of behavioral abnormalities characteristic of this model. We also highlighted dramatic gene expression alterations associated with astrogliosis and inflammation that, accordingly, were rescued by Scn1a gene expression normalization at P30. Moreover, employing a conditional knock-out mouse model of DS we reported that ensuring physiological levels of Scn1a during the critical period of symptom appearance (until P30) is not sufficient to prevent the DS, conversely, mice start to die of SUDEP and develop spontaneous seizures. These results offer promising insights in the reversibility of DS and can help to accelerate therapeutic translation, providing important information on the timing for gene therapy delivery to Dravet patients.

Gene therapy in neuromuscular and mitochondrial disorders

Can we repair the Parkinsonian brain?

Hyperbaric Oxygen and the Brain: Concussions to COVID

Hyperbaric oxygen [HBO] treatments are an underappreciated way to get oxygen to injured tissue. Concussions, and now post-COVID neuropsychiatric issues have become a major cause of disability. Data from objective testing will be presented to discuss our clinic experience TREATING these conditions.

Anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome

Anti-PD1 immunotherapy exacerbates cognitive deficits induced by immunogenic cancer in mice

Autobiographical memory and reminiscence therapy in healthy older adults : an fMRI study

Autologous haematopoietic stem cell gene therapy for people with Friedreich’s ataxia

Brain Tumor Treatment Using Tunable Local Chemotherapy

Cannabidiol as an add-on therapy to overcome the slow-onset and – possibly – resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex

Cognitive effects in non-CNS cancer survivors before and 1-year after standard anthracycline-based chemotherapy treatment

Combination therapy of donepezil and environmental enrichment on memory deficits in amyloid-beta-induced Alzheimer's disease rats

Co-Targeting Tumor Microenvironment-Instigated Adaptation To Hypoxia Renders Glioblastoma More Susceptible To Oncolytic Virus Immunotherapy

Deep Learning of Brain Spacetime to Predict Outcome of Vision Restoration Therapy using Non-invasive Brain Stimulation

Development of a new drug screening system for Rett syndrome therapy

Development of folate receptor alpha-functionalized gene-therapy vectors for targeted brain delivery

Dual PI3Kδ/γ inhibitor Duvelisib prevents development of chemotherapy induced neuropathic pain

E2F4DN-based gene therapy recovers long-term potentiation and hippocampal-dependent memory in homozygous 5xFAD mice

Electrophysiological and behavioral characterization of murine model exposed to acute sarin sublethal doses and antidote therapy evaluation

Exploring the Mechanism of Action of the Novel Remyelination Therapy Nefiracetam

Exposing microvascular endothelial cells to low energy accelerated protons and its relevance for hadrontherapy applications

Fatigue and Sleepiness Measures in Myotonic Dystrophy Type 1 Patients Treated with Cognitive Behavioral Therapy and Graded Exercise Therapy

Folate receptor α positive hybridosomes as vehicles for non-invasive brain-targeted gene therapy

Is GDNF dose essential for Parkinson ’s disease gene therapy success?

AAV gene therapy delivering recombinant dimeric peptides targeting PICK1 fully relieve chronic neuropathic pain

Gene Therapy Research and the Brain: Is Africa Ethically, Legally and Socially Ready?

Gene therapy targeting the blood-brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency

Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain

IGF-1 gene therapy on dopaminergic neurons and glial cells interaction in early cognitive deficits in a neurodegenerative animal model

The interaction of Reminiscence Therapy plus walking interventions on cognitive performance and well-being of older adults with early stage dementia of Alzheimer type

Intra-arterial mesenchymal stem cell therapy regulates Aquaporin 4 to alleviate perifocal vasogenic edema post-stroke in animal model of ischemic stroke

Intravenous gene therapy using AAVPHP.eB for metachromatic leukodystrophy

Investigating the Effects of Flotation Restricted Environment Stimulation Therapy on Neural Networks in Chronic Pain Patients via Functional Magnetic Resonance Imaging

Isolation and characterization of hPSC-derived striatal progenitor subpopulations for cell therapy in Huntington’s Disease

NIR Laser Photobiomodulation as promising therapy for Brain Hypoxia/Ischemia: in vitro study in organotypic hippocampal slices

Matrix therapy combined with mesenchymal stem cells-based approach in the context of brain ischemia

Meditation: A potential therapy targeting amygdala for the prevention & reversal of Alzheimer's Disease

Metformin as a potential anti-aging therapy: targeting the tripartite synapse

miR-135 as a New Target for Antidepressant Therapy: Preclinical Study

Nanoparticle-mediated enzyme replacement therapy for Krabbe disease

Neural precursor/stem cell-based therapy for Rett syndrome

Ozone Therapy as a Minimally-invasive Alternative in patients with Acute Lumbar Disc Herniation: A Randomized Clinical Trial

Preclinical development of a therapy for Chronic Traumatic Spinal Cord Injury using Wharton's Jelly Mesenchymal Stromal Cells: proof of concept and regulatory compliance

AAV-mediated gene therapy for sensory regeneration after spinal cord injury