Matthew Wilson, Ph.D. - US grants

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) affects an estimated 7% of the US population and results in scarring and loss of peritubular fibroblasts which produce erythropoietin (EPO). EPO-deficient anemia of CKD is currently treated with recombinant EPO analog injections that have recently been associated with undesired side effects such as increased risk of stroke, heart attacks, and deep vein thrombosis which may preclude further use of this therapy. Although the mechanisms of these side effects are unclear, it is clear that bolus dosing of EPO analogs either weekly or monthly does not recapitulate the physiologic regulation of this important hormone and bolus dosing may alter EPO signaling pathways. Thus, there is a critical need to develop alternative therapies for anemia of CKD. Herein we describe an innovative experimental design using non-viral transposon-mediated gene transfer to develop a new strategy for therapy of anemia of CKD. Genetically modified T lymphocytes whose specificity is directed to persistent (latent) viruses such as Epstein-Barr virus (EBV) survive long-term (>8 years) in stable numbers in vivo due to chronic viral antigen stimulation. Moreover, preclinical and recent clinical studies have shown T cells can be readily induced to apoptose by activation of a co-transferred suicide gene, providing an additional layer of safety and control. We therefore hypothesize that virus specific T cells genetically modified to inducibly express EPO and a separately inducible suicide gene represent an ideal candidate cell population for sustained and safe treatment of anemia of CKD. In specific aim 1, we propose to modify virus specific murine T cells to inducibly express EPO and a suicide gene and we will infuse them into wild type and CKD mice to measure their effectiveness in regulating hematocrit levels in vivo. Specific aim 2 focuses on extending these genetic modifications to human T cells and testing them in vitro for their ability to be propagated long-term via chronic viral antigen stimulation, as well as inducibly express EPO and undergo selectively induced cell ablation if needed. In specific aim 3, we will evaluate the functionality of genetically modified human T cells from patients with CKD and determine the frequency of EBV-specific T cells and their response to EBV antigen in the presence and absence of transgenically expressed EPO ex vivo.

Abstract The pilot and feasibility (P&F) grant program represents an extremely valuable and effective opportunity for the Vanderbilt O'Brien Kidney Center to provide funds for the support of kidney disease related projects. Funding for the P&F program started in 2002 during a previous funding cycle for the O'Brien centers (2002-2007) and continued through the P30 Vanderbilt O'Brien Kidney Center (2008-2013). For this competitive application, the goal of the program continues to be to support i) small research projects by new investigators with little or no independent research support; ii) established investigators who are turning to kidney-related research for the first time; or iii) established investigators in kidney related research who are undertaking a strikingly new research direction. Applicants must hold a faculty appointment (Instructor or above) to submit a P&F proposal, and must meet the NIH Eligibility Guidelines for P&F support. In our previous funding cycles (2002-2013), the Vanderbilt O'Brien Kidney Center funded 19 applications, from a total of 53 applications. Summaries of the funded applications are detailed below. For this application, we are requesting funding for 9 applications for years 2017-2022, all of which will meet the guidelines of our P&F program and will extensively utilize our proposed biomedical cores.

Sleep is critical to memory and learning. During rapid eye movement (REM) or non-REM (NREM) sleep, subgroups of cell assemblies in hippocampal and sensory cortical circuits are reactivated in a temporally coordinated manner, forming a cortical-hippocampal-cortical loop of information processing during memory consolidation. Deciphering neural codes of hippocampal-neocortical memories during sleep would reveal important circuit mechanisms of memory consolidation. To date, a complete understanding of the mechanisms of hippocampal-neocortical memory processing and the interaction of their specific spatial/non­ spatial memory representations during sleep is lacking. Furthermore, little is known about the causal impact of the hippocampal-neocortical interactions on subsequent memory reactivation or post-sleep learning. In this proposal, we will dissect representations of spatial (where) and visual (what) memory in the rodent hippocampal CA1 and primary visual cortex (V1) during sleep. We will combine electrophysiology, population-decoding methods, optogenetics and closed-loop neural interface to decipher sleep-associated CA1-V1 population codes in memory coding. In Aim 1, we will identify visual cortical representations in a spatial navigation task and determine visual cortical neuronal firing dependency on space, experiences and visual cues. In Aim 2, we will uncover where (spatial) and what (visual) representations of CA1-V1 memory reactivations during sleep. In Aim 3, we will determine the causal role of the hippocampus in the V1-CA1-V1 loop of memory consolidation during sleep. Together, these results will enable us to casually dissect circuit mechanisms of hippocampal-neocortical memory coding during sleep, and to establish a new analysis paradigm to identify the contents of hippocampal-memory reactivations during sleep. Our project will provide further insight into memory-related neurological and psychiatric disorders and potential therapeutic treatment for targeted memory reactivation or enhancement.

A cell therapy capable of sustained therapeutic protein delivery in vivo has the potential to benefit both kidney disease and its complications. During our previous grant cycle, we developed and validated technology using transposon-modified antigen specific T cells for therapeutic protein delivery in vivo using erythropoietin (EPO) as a model system. We demonstrated delivery of murine EPO and therapy for anemia of chronic kidney disease in mice as a model system, and we demonstrated inducible human EPO expression from antigen-specific human T cells in vitro. We propose to significantly advance beyond our previous grant by extending our studies to human T cells in an in vivo context and testing an innovative approach to enhance long-term therapeutic enzyme delivery for Fabry disease that results from loss of ?-galactosidase A (?-gal A). ?-gal A -/- mice represent an animal model of human Fabry disease, which results from lack of an enzyme needed to metabolize fats leading systemic disease including kidney disease. In aim 1, we will test transposon-modified antigen-specific T cells for expression of ?-gal A in a Fabry disease model. We will extend our mouse studies with EPO to ?-gal A in a Fabry disease model using antigen-specific mouse T cells and vaccination. We will gene modify human T cells to express a chimeric antigen receptor (CAR) along with luciferase or human ?-gal A. Cells will be infused into NOD/SCID/Fabry mice to evaluate the ability of engineered antigen-specific human T cells to engraft, respond to vaccination, and short-term expression ?-gal A in an in vivo model. Although perforin is important in T cells for clearance of malignant cells, the perforin pathway also contributes to clearance of antigen expressing cells post vaccination. In aim 2, we will test perforin knockout in T cells as a way of enhancing long-term therapeutic protein delivery from antigen-specific T cells. We propose to test perforin knockout in the setting of antigen- specific T cells to determine if it will boost long-term persistence of cells delivering therapeutic proteins. In aim 3, we will test transposon-modified antigen-specific T cells for long-term expression of ?-gal A in a mouse model of Fabry disease. We propose to deliver optimized human CAR-T cells expressing human ?-gal A in a NOD/SCID/Fabry mouse model. We will evaluate for engraftment, vaccination response, ?-gal A activity, and globotriaosylceramide levels in tissues. The proposed studies will lead to the development of new cell therapies for kidney disease and its complications and have the potential for therapeutic impact well beyond the kidney.