Kalyan C. Vinnakota

Gene therapy for Neurofibromatosis type 1 (NF1), defined as a collection of approaches that restore the quantity and function of neurofibromin, holds promise for treating various NF-associated clinical manifestations by addressing the root cause of the condition. The Gilbert Family Foundation’s Gene Therapy Initiative (GTI) launched one of the first focused research efforts to support a broad array of gene replacement, gene editing, and other strategies aimed at upregulating neurofibromin levels to mitigate symptoms and advance therapies for NF1. Efforts to discover and evaluate novel gene therapies to treat NF1 are hindered by foundational gaps in the field, the absence of quantitative assays, and a lack of robust preclinical models with reliable functional readouts. This perspective begins by shedding light on those key barriers that continue to hinder progress in NF1 gene therapy, outlines some of the strategies being explored to overcome them and offers pointers to additional strategies that merit further exploration. We then highlight several promising gene therapy strategies now in development. Successfully addressing the challenges described earlier is critical to realizing the full potential of these emerging and novel therapeutic approaches, setting the stage for effective and durable NF1 gene therapy interventions.

Research tools, such as model organisms, cell lines, and antibodies, are essential to designing and executing successful biological experiments. These resources are often shared or made commercially available to support scientific progress. Given the fast pace of research, it can be difficult to keep track of the large number of available tools. Moreover, for those new to a particular disease area, learning about the array of tools available can be a major impediment. Our experience in the neurofibromatosis field has shown that researchers struggle to identify the research tools available to them, determine where tools can be acquired, and understand what tools are most well-suited for which experiments. While a variety of databases exist to help researchers find useful research tools, these databases often a) are specific to one type of research tool while being disease-agnostic, b) provide only high-level information, c) do not contain information about in-development models, and d) do not contain observational data for the research tools. To address these limitations, we created the Neurofibromatosis Research Tools Database, a user-friendly, open-access database and companion portal designed to help the neurofibromatosis type 1 (NF1) research community easily find, obtain, and use NF1-relevant research tools. This prototype database catalogs a wide variety of NF1-relevant research tools using databases such as Cellosaurus, AntibodyRegistry, RRID Portal, among others, as well as information provided in literature and from the NF community. We aggregated and curated metadata for NF1-relevant animal models, cell lines, genetic reagents, antibodies, and biobanks. The database includes core metadata for all tools, e.g., name, type of tool, synonyms, developer, as well as tool type-specific metadata, e.g., for cell lines or animal models, the type of cancer that the model recapitulates. The database is also designed to store observational data contributed directly from the research community. Our companion web portal allows users to search and filter this database interactively and easily explore these tools. This website was built within the NF Data Portal (nf.synapse.org), and is available at tools.nf.synapse.org. Community members can actively contribute to the growth of the database and portal by submitting information about the reliability, biology, usage, and other observations on each research tool. By collating and curating this information and surfacing it in an open-access exploration portal, we anticipate that this database will serve as a valuable resource to help the NF community discover, understand, and use NF1 research tools. Citation Format: Brynn Zalmanek, James Goss, Mialy DeFelice, Jay Hodgson, Ashley Clayton, Stockard Simon, Marco Marasca, Julie Bletz, James A. Eddy, Milen Nikolov, Kevin Boske, Ljubomir Bradic, Jineta Banerjee, Kalyan Vinnakota, Caroline Morin, YooRi Kim, Robert J. Allaway. NF Research Tools Database: A knowledge base of experimental research tools for neurofibromatosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1675.

Identifying early indicators of toxicant-induced organ damage is critical to provide effective treatment. To discover such indicators and the underlying mechanisms of toxicity, we used gentamicin as an exemplar kidney toxicant and performed systematic perturbation studies in Sprague Dawley rats. We obtained high-throughput data 7 and 13 h after administration of a single dose of gentamicin (0.5 g/kg) and identified global changes in genes in the liver and kidneys, metabolites in the plasma and urine, and absolute fluxes in central carbon metabolism. We used these measured changes in genes in the liver and kidney as constraints to a rat multitissue genome-scale metabolic network model to investigate the mechanism of gentamicin-induced kidney toxicity and identify metabolites associated with changes in tissue gene expression. Our experimental analysis revealed that gentamicin-induced metabolic perturbations could be detected as early as 7 h postexposure. Our integrated systems-level analyses suggest that changes in kidney gene expression drive most of the significant metabolite alterations in the urine. The analyses thus allowed us to identify several significantly enriched injury-specific pathways in the kidney underlying gentamicin-induced toxicity, as well as metabolites in these pathways that could serve as potential early indicators of kidney damage.

Context-specific GEnome-scale metabolic Network REconstructions (GENREs) provide a means to understand cellular metabolism at a deeper level of physiological detail. Here, we use transcriptomics data from chemically-exposed rat hepatocytes to constrain a GENRE of rat hepatocyte metabolism and predict biomarkers of liver toxicity using the Transcriptionally Inferred Metabolic Biomarker Response algorithm. We profiled alterations in cellular hepatocyte metabolism following in vitro exposure to four toxicants (acetaminophen, carbon tetrachloride, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six hour. TIMBR predictions were compared with paired fresh and spent media metabolomics data from the same exposure conditions. Agreement between computational model predictions and experimental data led to the identification of specific metabolites and thus metabolic pathways associated with toxicant exposure. Here, we identified changes in the TCA metabolites citrate and alpha-ketoglutarate along with changes in carbohydrate metabolism and interruptions in ATP production and the TCA Cycle. Where predictions and experimental data disagreed, we identified testable hypotheses to reconcile differences between the model predictions and experimental data. The presented pipeline for using paired transcriptomics and metabolomics data provides a framework for interrogating multiple omics datasets to generate mechanistic insight of metabolic changes associated with toxicological responses.

In order to provide timely treatment for organ damage initiated by therapeutic drugs or exposure to environmental toxicants, we first need to identify markers that provide an early diagnosis of potential adverse effects before permanent damage occurs. Specifically, the liver, as a primary organ prone to toxicants-induced injuries, lacks diagnostic markers that are specific and sensitive to the early onset of injury. Here, to identify plasma metabolites as markers of early toxicant-induced injury, we used a constraint-based modeling approach with a genome-scale network reconstruction of rat liver metabolism to incorporate perturbations of gene expression induced by acetaminophen, a known hepatotoxicant. A comparison of the model results against the global metabolic profiling data revealed that our approach satisfactorily predicted altered plasma metabolite levels as early as 5 h after exposure to 2 g/kg of acetaminophen, and that 10 h after treatment the predictions significantly improved when we integrated measured central carbon fluxes. Our approach is solely driven by gene expression and physiological boundary conditions, and does not rely on any toxicant-specific model component. As such, it provides a mechanistic model that serves as a first step in identifying a list of putative plasma metabolites that could change due to toxicant-induced perturbations.

High capacity and low capacity running rats, HCR and LCR respectively, have been bred to represent two extremes of running endurance and have recently demonstrated disparities in fuel usage during transient aerobic exercise. HCR rats can maintain fatty acid (FA) utilization throughout the course of transient aerobic exercise whereas LCR rats rely predominantly on glucose utilization. We hypothesized that the difference between HCR and LCR fuel utilization could be explained by a difference in mitochondrial density. To test this hypothesis and to investigate mechanisms of fuel selection, we used a constraint-based kinetic analysis of whole-body metabolism to analyze transient exercise data from these rats. Our model analysis used a thermodynamically constrained kinetic framework that accounts for glycolysis, the TCA cycle, and mitochondrial FA transport and oxidation. The model can effectively match the observed relative rates of oxidation of glucose versus FA, as a function of ATP demand. In searching for the minimal differences required to explain metabolic function in HCR versus LCR rats, it was determined that the whole-body metabolic phenotype of LCR, compared to the HCR, could be explained by a ~50% reduction in total mitochondrial activity with an additional 5-fold reduction in mitochondrial FA transport activity. Finally, we postulate that over sustained periods of exercise that LCR can partly overcome the initial deficit in FA catabolic activity by upregulating FA transport and/or oxidation processes. Our bodies consume carbohydrates, fats, and amino acids as fuels, utilizing various catabolic pathways to transfer the energy required for normal physiological functions. The way these pathways function can have an important impact on overall health. While most catabolic pathways are known, we are still striving to understand how these pathways interact, are controlled, and change during exercise and in disease. Here, we have used computer modeling as a tool to understand fuel utilization differences during exercise for two animal models. High capacity running rats (HCR) were used as a healthy, fit cohort, and low capacity running rats (LCR) were used as a sedentary and disease-prone cohort. Our computer model results show that the HCRs are superior at fat utilization compared to LCRs because of their increased ability to transport and catabolize fatty acids. We postulate that these differences depend on exercise intensity and duration, such that longer acclimation periods may minimize fuel utilization differences between these rats.

The typical cause of death in pulmonary hypertension (PH) is right ventricular (RV) failure, with females showing better survival rates than males. Recently, metabolic shift and mitochondrial dysfunction have been demonstrated in RV failure secondary to PH. In light of evidence showing that estrogen protects mitochondrial function and biogenesis in noncardiovascular systems, we hypothesized that the mechanism by which estrogen preserves RV function is via protection of mitochondrial content and oxidative capacity in PH. We used a well‐established model of PH (Sugen+Hypoxia) in ovariectomized female rats with/without estrogen treatment. RV functional measures were derived from pressure–volume relationships measured via RV catheterization in live rats. Citrate synthase activity, a marker of mitochondrial density, was measured in both RV and LV tissues. Respiratory capacity of mitochondria isolated from RV was measured using oxygraphy. We found that RV ventricular‐vascular coupling efficiency decreased in the placebo‐treated SuHx rats (0.78 ± 0.10 vs. 1.50 ± 0.13 in control, P < 0.05), whereas estrogen restored it. Mitochondrial density decreased in placebo‐treated SuHx rats (0.12 ± 0.01 vs. 0.15 ± 0.01 U citrate synthase/mg in control, P < 0.05), and estrogen attenuated the decrease. Mitochondrial quality and oxidative capacity tended to be lower in placebo‐treated SuHx rats only. The changes in mitochondrial biogenesis and function paralleled the expression levels of PGC‐1α in RV. Our results suggest that estrogen protects RV function by preserving mitochondrial content and oxidative capacity. This provides a mechanism by which estrogen provides protection in female PH patients and paves the way to develop estrogen and its targets as a novel RV‐specific therapy for PH.