Howard E. Gendelman, M.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): We propose that protective CNS immunity can be generated by the interactions between brain mononuclear phagocytes (MP; macrophages and microglia) and CD8+ T lymphocytes. These occur in distinct manner, either through elimination of infected cells (the generation of cytotoxic T lymphocytes, CTL) or by T cell mediated neuroprotection. The proposal seeks to study how the brain is protected against a relentless attack by virus for many years and in most infected people. Furthermore, we suggest that the production of potent neurotrophic factors and the destruction of virus-related cells in the central nervous system (CNS) occurs as a consequences of MP activation. Based on these observations, we propose that the pathways of CNS immune activation regulate the destructive or trophic potential of the brain macrophages or microglia. To investigate mechanisms for immune-based neuroprotection during HIV infection, we will test T cell and nerve injury-mediated MP activation for the regulation of effector cell function in laboratory and animal model HIV CNS disease systems. For the latter, HIV-1 infected brain macrophages injected into the subcortex of immunodeficient mice induce potent HIV-1 specific CTL and neuroprotective activities resulting in the rapid elimination of infected cells and neuronal sparing. The role of specific HIV-1 strain in this response and the coordinate effects on inflammatory responses of the brain will be studied. The overall goal of these works is to determine the relative role of innate and acquired immunity to both control ongoing viral production and affect ways to protect neurons against injuries initiated by virus-infected MP. The central question being addressed are on defining the role of the peripheral immune system in protecting the brain against HIV-1 medicated injury for most of sub-clinical disease. This is a most important yet understudied question in HIV-1 neuropathogenesis with applicability to a whole range of neurodegenerative disorders. This project serves as an extension of our previous works seeking to determine how microglial immunity affects the pathogenesis, progression and the therapeutic options for HAD. [unreadable] [unreadable]

This Project will employ human monocyte-derived macrophages and primary microglia as indicator cells to test antiretroviral activities of several classes of anti-inflammatory and anti-oxidant compounds for treatments of HIV dementia. The Specific Aims are: 1) Analyze the anti-retroviral activities of novel compounds for treatment of HIV dementia. Based on the hypothesis that secretory products from HIV-infected and uninfected immune activated macrophages/microglia produce neurotoxic activities, compounds to be tested will include glucocorticoids and their mediators (including lipocortin-1), quinolinic acid inhibitors, lexipafant (a PAF antagonist), BB-1101) (a metalloproteinase and TNF release inhibitor) and antiretroviral compounds (with avid blood-brain barrier penetration). Using a panel of HIV-1 isolates (including neurotropic isolates) the investigators will analyze the viral life cycle after drug treatment and virus infection. The drugs listed above will be inoculated into macrophage cultures (in a dose dependent manner) then infected with HIV-1 obtained from brain, blood and/or lymphoid tissues. 2) Test compounds in Specific Aim 1 for their ability to alter macrophage effector cell functions. A variety of inflammatory molecules (including proinflammatory cytokines, chemokines, eicosanoids, PAF, quinolinic acid, and excitatory amino acids (EAA)) thought most relevant for HIV dementia will be assayed following HIV-1 infection of monocytes (with/out immune activation) and microglia following drug treatment. These results will be correlated to changes in neurotoxic activities (including, in select experiments, neuronal electrophysiologic testing). 3) Developmental therapeutics. Promising compounds uncovered in projects I and II will be subjected to the identical testing outlined in Specific Aims 1 and 2. In addition, RP-HPLC, immunoaffinity chromatography, radioimmunoassays and cytokine bioassays will be used to identify potentially novel neurotoxic molecules produced from HIV-1-infected macrophages and/or microglia that may serve as therapeutic targets. Neuronal viability assays will determine the relationships (if any) between induced specific macrophage secretory activities (toxin production) and neuronal survival. 4) Use a SCID animal model for HIV encephalitis to test therapeutics for HIV. Histopathological alterations in brain tissue of SCID mice after inoculation of HIV-1-infected monocytes or microglia into the brain will serve s therapeutic endpoints to assess interventions. Correlations will be made between histopathology and levels of neurotoxins in infected CNS tissue. The antioxidant, anti-inflammatory and/or anti-retroviral compounds (tested initially within Specific Aims 1 and 2) will be given to the mice prior to challenge with virus-infected cells. Histopathologic changes with/out drug treatments with virus-infected cells. Histopathologic changes with/out drug treatments will asses drug efficacy.

Neurologic manifestations of HIV infection show deficits in cognitive and motor functions. Central nervous system (CNS) pathology underlying these clinical events include myelin pallor, macrophage/microglial activation, profound reactive gliosis and neuronal dropout. Interestingly, a progressive neuronal loss occurs without (to any significant degree) direct neural cell infection. Our laboratory has dedicated itself towards understanding how secretory products from immune activated HIV- I-infected macrophage/microglia produce progressive viral-associated neuropathology. Our initial work uncovered a large number of macrophage secretory products (toxins) likely involved in such neuronal damage. These include, but are not limited to, eicosanoids, proinflammatory cytokines, platelet activating factor, free radicals and excitatory amino acids (including cysteine and quinolinic acid). The specific mechanism(s) responsible for neuronal loss have not as yet been identified. In this application, we will extend our works by investigating macrophage secretions and its associated neurotoxicity to: 1) utilize "state-of-the-art" electrophysiological methodologies to determine neuronal physiologic alterations following exposure to monocyte secretory products; 2) determine how pharmacological and/ or immunological manipulations of neurotoxic pathways can be prevented or reversed during HIV- I CNS infections; 3) characterize the electrophysiological basis for neuronal injury in our SCID mouse model of HIV encephalitis. In all works, the electrophysiological analyses will Include detailed patch clamp recording techniques to assess both synaptic and voltage-gated ion channels. Using this approach we intend to develop greater understanding for how secretory products from immune activated HIV- I- infected monocytes induce neuronal injury associated with progressive cognitive and motor dysfunctions seen in HIV-affected subjects.

DESCRIPTION (adapted from the Abstract): The proposed studies are based upon the observation that, although human immunodeficiency virus Type 1 (HIV) penetrates the brain early in disease, only in a subset of patients with advanced immunosuppression do inflammatory monocytes enter the brain in large numbers and result in neurological dysfunction. The underlying hypothesis is that brain microvascular endothelial cells (BMVECs) facilitate monocyte entry following direct HIV infection, up-regulation of adhesion molecules, or macrophage-mediated disruption of blood-brain barrier function (BBB). In previous studies the Investigator and his associates demonstrated that pro-inflammatory cytokines and other immunoregulatory molecules produced by macrophages disrupt BBB function and produce up-regulation of adhesion molecules on BMVECs. This provides one mechanism for transendothelial passage of virus and inflammatory cells across the BBB. The Investigator speculates that during advanced immunosuppression, immune competent macrophages can disrupt BBB function and permit passage of monocytes into brain. In addition, he proposes that specific viral strains facilitate neuroimmune activation and attendant monocyte recruitment/binding to endothelial cells, disruption of the BBB, and passage of inflammatory cells into the CNS. In this application the Investigator proposes to assay virus-endothelial cell infection and its relationship to BBB function. The researchers will use monocytes from HIV-infected patients with/without HIV dementia to determine if such cells have facilitated transendothelial migration. They will analyze several putative neurotoxins, as well as other potential agents isolated by RP-HPLC, to determine which factors might facilitate monocyte migration through BMVEC. Their recently-developed SCID mouse model of HIV encephalitis will be used to assess endothelial- and chemokine-mediated monocyte recruitment into the CNS. The combined efforts are designed to determine the viral and immune determinants for BBB dysfunction in AIDS, information that could be helpful in adapting strategies to prevent migration of monocytes into the brain during HIV dementia.

This application is a competitive renewal for "HIV-1, monocytes and the blood-brain barrier," R01NS36126-01. The major goals of the previous project were to: 1) assay virus-endothelial cell infection and its relationship to blood-brain barrier (BBB) function; 2) uncover putative glial neurotoxins (for example, stromal cell derived factor-one- nitric oxide, platelet activating factor, tumor necrosis factor alpha, among others) that influence monocyte migration into the brain; and 3) by utilizing a blood brain barrier (BBB) and severe combined immune deficiency disease (SCID) mouse model of HIV encephalitis (HIVE) to assess chemokine-mediated monocyte recruitment into the central nervous system. These goals were met. Our current focus is in elucidating the biophysical properties of blood-borne macrophages that regulate leukocyte entry into diseased brain during HIV- associated dementia (HAD). These events are pivotal to viral neuropathogenesis. Clearly, once inside the brain, mononuclear phagocytes (MP) (microglia, parenchymal and perivascular macrophages) serve as the Principal cellular reservoir for HIV. Moreover, following immune activation, MP secrete scores of immune "neurotoxic" factors that damage the BBB and neurophil. If macrophage chemotaxis and/or its transendothelial migration could be halted disease could be abrogated. Until now, little attention was paid to the spatial parameters of cell biophysiology, migration and the mechanisms underlying changes in cell shape and volume. Indeed, we have now demonstrated that HIV-1 infected MDM migrate faster than uninfected cells through organotypic brain cultures and Boyden chamber chemotaxis assays. Both infected and uninfected macrophages express ion channels. Thus, the focus of the current work is to decipher how ion channels effect macrophage cell volume and cytosolic calcium that effect the critical components of MP transendothelial migration. The processes that effect MP trafficking will be explored, in the context of macrophage differentiation, activation and viral infection. In particular, we will ascertain the exact ionic currents (independently and together), which are sensitive to cell volume, shape and movement. These may be measured, in part, by whole-cell and single-channel patch-clamp electrophysiological recording assays. Migration of MP will be performed on replicate cell suspensions through the use of artificial barriers (for example Boyden chemotaxis microchamber assays or a BBB model). Ion channel blockers will assess ways to halt the process of MP migration. To correlate these findings to what could occur in an infected human host, MP migration/invasion will be investigated in organotypic cultures of brain slices and in a SCID mouse model of HIVE. DiIC18 stained human monocytes (red) will be co-cultivated with DiOC18-stained organotypic brain slices (green). Cell migration will be evaluated by scoring DiIC18-stained monocytes under laser confocal microscopy (through serial optic sectioning of human or mouse brain tissue). The influence of cell activation and viral infection for the migratory process will also be assessed. Overall, we propose that MP can adjust their cell shape and volume to facilitate their invasion into narrow extracellular spaces of the brain. We hypothesize that these changes require alterations in ion fluxes resulting in water loss and cell shrinkage. Such studies are novel and to our knowledge have not been previously proposed for HAD. The data acquired could provide new insights and therapeutic intervention strategies for monocyte BBB trafficking during progressive HIV-1 infection in brain tissue.

DESCRIPTION: (Provided by applicant): The purpose of this core is to assist in the design of adjunctive therapeutic strategies for treatment and/or prevention of neurologic disease following human immunodeficiency virus type-one (HIV-1) infection. In this regard, purified human monocyte-derived macrophages (MDM) and MDM conditioned media (MCM) will be supplied, following HIV-1 infection and/or immune activation, to all investigators on request, within the Rochester Cooperative NeuroAIDS Drug Discovery Group (RCNDDG). In addition, promising anti-inflammatory and/or neuroprotective drugs, developed in laboratory assays or proposed, will be tested for therapeutic efficacy in a severe combined immune deficiency (SCID) mouse model of HIV-1 encephalitis (HIVE). Brain tissue and/or sera will be made available for measuring drug levels and pathology in the HIVE mice. Such works will support translational (bench to bedside) research efforts and directly effect the performance of subsequent clinical trials. The works, in toto, are based on the concept that HIV-1 associated dementia (HAD) is, in part, a reversible metabolic encephalopathy caused by defective immunity of virus-infected mononuclear phagocytes [(MP), microglia, perivascular and parenchymal macrophages]. These MPs serve both as reservoirs for productive HIV-1 infection and principal sources of neurotoxic activities within the central nervous system (CNS). The development of ways to inhibit toxic inflammatory activities in brain may serve to both ameliorate and prevent complications of persistent viral replication in brain serving as critical adjunctive therapies to ongoing potent anti-retroviral regimens, the principal goals of the RCNDDG.

DESCRIPTION (provided by applicant) The Neuroscience Research Training Program (NRTP) combines interdisciplinary training in Neurosciences with a focus on viral infection of the nervous system. The University of Nebraska Center for Neurovirology and Neurodegenerative Disorders (CNND), a focal point for the proposed training activities, provides research opportunities exploring the immune consequences of viral brain infection and its resultant alterations in neural function. The NRTP integrates programs in virology, neurotoxicology, molecular neuroscience, biophysiology, cellular neurobiology, biochemistry and neuroregeneration aimed at understanding how viral infections of the brain lead to cognitive, behavioral and motor dysfunction. Such a broad range of technical expertise under a single organizational structure, has provided high quality graduate education to both pre- and postdoctoral students. The CNND working with the newly established Nebraska Center for Virology has proven successful in providing unique research training opportunities, in recruitment of minority investigators and in measured training successes. Indeed, four of our listed trainers began as CNND postdoctoral fellows. Our faculty members were chosen for participation by their proven track records in pre- and/or post-doctoral education, their funding records and their abilities to enhance educational opportunities in the field of Neurovirology. The diversity and depth of the research endeavors combined with the backgrounds of the trainers should provide a unique research training/education environment for prospective students/fellows who seek a multidisciplinary training into studies of the neuroimmune consequences of viral infection of the nervous system. In support of the said goals, a unified neuroscience training and education program has now been established at the University of Nebraska Medical Center (UNMC). Monthly meetings/symposia, community-based initiatives, journal clubs, a lectureship series, and the Midwest Neuroscience Colloquium are regular fare for the UNMC Neuroscience Community. These are all designed to enhance the interactions between faculty, students and postdoctoral fellows. Administratively, the NRTP will consist of the program director, an executive committee and the trainers. This program permits a cross disciplinary experience while at the same time providing unique opportunities in an underdeveloped focused area of neuroscience, the consequences and therapeutic options for viral infections of the nervous system.

DESCRIPTION (provided by applicant) Evidence abounds that inflammatory mononuclear phagocyte iMP; microglial cells, perivascular and brain macrophage) secretory processes are centrally implicated in the neuropathogenesis of HIV-1-associated dementia (HAD). Whether this can be harnessed to positively affect neurodegenerative processes is less apparent. Thus, this program project (PPG) seeks support for investigating the specific immunologic basis of HAD and linkages between it and other neurodegenerative disorders. The focal point of disordered immunity for HAD is the microglia, the cell that links all PPG research efforts. The studies will utilize, as its foundation, a well-developed research infrastructure within the Center for Neurovirology and Neurodegenerative Disorders (CNND) of the University of Nebraska Medical Center. The CNND consists of investigators of diverse expertise who maintain a unified focus for their research on how MP biology affects both neurodegeneration and neuroprotection. This group of scientists has significant expertise in areas of neurotoxicology, cellular immunology, neuropathology, neurophysiology, neuropharmacology and molecular biology. "State of the art" technologies in magnetic resonance imaging/spectroscopy, electrophysiology, gene arrays and proteomics are being developed and strengthen this proposal. Currently, ties between innate and acquired immunity for brain MP function serve to bridge all of the proposed projects. It is our hypothesis that inflammatory mechanisms cause or intensify tissue damage during neurodegenerative processes. Most importantly, we believe these can be reversed through drug or immune manipulations. We purport that the interplay between the peripheral immune system and the brain serves to counter neuronal death caused as a result of viral infection. The balance between MP trophic and toxic activities may, therefore, underlie the process of dementia. The strength of this proposal is its well-developed cross-disciplinary program of young, energetic investigators whose scientific expertise spans the gantlet of MP-virus-neural interactions. Working under one roof, in one organizational structure with a strong track record of collaboration and joint publications should serve the PPG well in its quest to merge research initiatives designed to address pathogenic and therapeutic aspects of HAD. Results of these works should have implications for monitoring disease and in developing novel therapeutics (for example, Alzheimer's disease) which now has few treatment options.

We propose that astrocyte dysfunction in HIV-1-associated dementia (HAD) occurs through its Interactions with infected brain mononuclear phagocytes (MP; perivascular and parenchymal macrophages and microglia). We will study how this occurs taking advantage of a newly developed proteomics and imaging facllity within our center. Magnetic resonance imaging (1HMRI), 1HMRS spectroscopy and single proton emission computed tomography will access cell movement and functlon in a rodent animal model of human HAD and HIV encephalitis (HIVE). The work will investigate how MP Infection and/or activation affects astrocyte neuroregulatory functions as well as explore primary infection of astrocytes de novo. It is proposed that secretion of MP pro-inflammatory factors (for example, tumor necrosis factor, quinolinlc acid and platelet activating factor) attenuates astrocyte signaling pathways leading to the down regulation of glial trophins including brain derived neurotrophic factor, glial derived neurotrophic factor, nerve growth factor, and/or transforming growth factor beta as well as attenuating glutamate transporter function, HIV-infected macrophages secretions (cellular or viral products) may also inclte the production of neurotoxins from astrocytes (for example, nitric oxide), glutamate, interleukin-1beta, MP-astrocyte interactions will be investigated for their abilities to affect autocrine and/or paracrine amplification of glial neurotoxins and chemotactic function. The relative role of virus and astrocyte secretory responses will be studied for its influence in the neuropathogenesis of HIV infection in a severe combined immunodeficient (SCID) mouse model of human disease. SCID mice will be injected intracerebrally with productively infected human astrocytes with vesicular stomatitis virus expressing HIV-1, with lymphotropic (LAI) or macrophage tropic (DVJ, ADA or JR-FL) viruses. The generation of cytotoxlc T lymphocytes from reconstituted HIVE mice will be used to eliminate infected macrophages from the brain and permit the assessment and evaluation of astrocyte repair processes. Lastly, a novel, state-of-art proteomics facility that includes on-line protein sequencing, will establish a comprehensive database of global changes of protein expression in human primary astrocytes as it relates to cell functlon. Altogether, these works will permit the determination of astrocyte and neuronal functions evaluated as a consequence of HIV-1 infection, MP function and chemotaxis in laboratory and animal models of human HAD. Most importantly, the project has strong collaborative ties with each of the other projects focusing on a unique but as yet understudied part of NeuroAIDS, namely the role played by astrocytes in the disease process.

This project is based on the notion that protective CNS immunity is generated by the interactions between brain mononuclear phagocytes (MP; macrophages and microglia)and CD8+ T lymphocytes. These occur in distinct manner, either through elimination of infected cells (the generation of cytotoxic T lymphocytes or CTL) or by T cell mediated neuroprotection. The end result of both processes is control of neuronal damage during progressive HIV-1 infection. This is a new field of investigation and is acknowledged as such. We hypothesize that the production of potent neurotrophic factors and the destruction of virus-infected cells in the central nervous system (CNS) occur as a consequence of MP activation. Based on these observations, we propose that the pathways of CNS immune activation regulate the destructive or trophic potential of the brain MP. To investigate this, we will test T cell and nerve injury-mediated MP activation for the regulation of effector cell function in a laboratory and animal model setting of HIV CNS disease. For the latter, HIV-1 infected brain macrophages injected into the subcortex of immunodeficient mice induce potent HIV-1 specific CTL and neuroprotective activities resulting in the rapid elimination of infected cells and neuronal sparing. The role of specific HIV-1 strains in this response and the coordinate effects on inflammatory responses of the brain will be studied. The overall goal is to determine the relative role of innate and acquired immune function to both control ongoing viral production and affect ways to protect neurons against metabolic injuries initiated by virus-infected MP. The central question being addressed are on defining the role of the peripheral immune system in protecting the brain against HIV-1 mediated injury for most of sub-clinical disease in most infected people. This is a most important, yet understudied question in HIV-1 neuropathogenesis with applicability to a whole range of neurodegenerative disorders. This project integrates with others in this program, in serving a common goal, to determine how microglial immunity effects the pathogenesis, progression and therapeutic options for HAD.

proteomics; biomedical facility; microglia; biomarker; macrophage; two dimensional gel electrophoresis; human tissue; protein purification; laboratory mouse; high performance liquid chromatography;

Abstract The interplay between the human immunodeficiency virus type one (HIV-1), CD4+ T cell subset numbers and function and central nervous system disease is the focus of this proposal now in its 24th year. During the prior investigative cycle systemic viral infection was not shown to influence nigrostriatal degeneration for Parkinson's disease (PD). However, effector immune responses for both innate and adaptive immunity were operative in PD, HIV-1, traumatic brain injury and Alzheimer's disease (AD). All can be ameliorated by immune transformation and can forestall neurodegenerative activities. While age dependent neurodegeneration in HIV/AIDS is linked to ?-synuclein neural inclusions and amyloid-? (A?) accumulation virus-induction of disease is less certain. What is rests in the interplay between immunity and misfolded proteins are serving as disease effectors. We posit that virus-induced neuroinflammation affect neurodegenerative disease. This is based on the notion that mononuclear phagocytes (MP: microglia and perivascular macrophages) and CD4+ T lymphocyte produce factors that affect the brain's microenvironment and can be transformed for therapeutic gains. T cell immunity drives the tempo of disease. To extend such observations we will determine the roles of adaptive immunity in affecting cognitive, behavior and neuropathobiology in the setting of HIV/AIDS and neurodegenerative diseases. We will generate HIV-1-infected and AD biogenesis rodents and use them to better understand disease events. We will elucidate the interplay between virus, immunity and brain subregion disease as well as A? biopathogenesis. These events will be linked to neuronal vitality. The interplay between CD4+ T cells and MP is a principal disease event while regulatory T cells are central to neuroprotective outcomes.

DESCRIPTION (provided by applicant): We propose that the role played by mononuclear phagocytes (MP;microglia, perivascular and brain macrophage) in the neuropathogenesis of H1V-1 infection can be harnessed for therapeutic benefit. The work will be conducted in the Department of Pharmacology and Experimental Neuroscience at the University of Nebraska Medical Center (UNMC). The department is a result of the merger of the Center of Neurovirology and Neurodegenerative Disorders (CNND) and the UNMC Department of Pharmacology. The fusion of the two created a Neuroscience Program with one director, Howard E. Gendelman, significantly improved the program project scientific infrastructure. Indeed, the disciplines of neuroscience, immunology, and pharmacology are integrated as a result of the CNND's evolution since its formation in 1997. Ongoing collaborations in stem cell biology, neuropharmacology, virology, blood-brain barrier biology, proteomics, bioimaging, and molecular neuroscience are now operative and an integral part of this proposal. Links between intracellular processes, microglial activation, and neuronal degeneration provided opportunities to harness immune responses for therapeutic benefit. The proposal includes three projects that center around MP pathobiology including: neural progenitor stem cell mobility and function in laboratory and animal models of NeuroAIDS (project, 1, J. Zheng);a blood-borne monocyte derived-macrophage-based nanoparticle delivery of anti-retroviral drugs in NeuroAIDS (project 2, H. Gendelman);and studies of the molecular, biochemical, and cell biologic consequences to the blood-brain barrier and viral neuropathogenesis in laboratory and animal models of human disease (project 3, Y. Persidsky). The cores include: cell biology and brain tissue collections (core A, T. Ikezu), bioimaging (core B, M. Boska), proteomics (core C, P. Ciborowski), and an administrative oversight (core D, H. Gendelman). Each project/core will support, enhance, and provide

DESCRIPTION (provided by applicant): Drug toxicities, patient compliance and limited penetrance into viral reservoirs (notably the central nervous system (CNS), gut and lymphatic organs) have diminished long-term antiretroviral therapy (ART) efficacy for HIV infection. Eliminating residual infection despite ART affected suppression of plasma viremia is our project goal. Novel strategies are sought for improved ART delivery to mononuclear phagocytes (MP: monocytes, perivascular and tissue macrophages and microglia) to bring drugs to tissue viral reservoirs. During the past R01 NS36126 funding cycle, now in its 17th year, we pioneered the development of long-acting injectable nanoformulated ART (nanoART) with superior biodistribution and pharmacokinetics over native drugs. Monthly nanoART injection, to subcellular sites of viral replication is possible We posit that this Trojan Horse MP carriage of the drug formulations can facilitate long-lived storage depots, in liver and spleen, resulting in reduced viral mutation and sustained plasma ART levels. Our drug formulations can readily cross the blood- brain barrier (BBB), from monocytes to endothelial cells, and enable drug transfer to and from an infected CNS. The current proposal seeks to perfect nanoART technology and as such move it to human use. First, we seek to maximize viral clearance (measured reduction of integrated viral DNA) by developing a range of antiretroviral nanoformulated drugs used alone or in combination. These include ritonavir boosted atazanavir, maraviroc, lamivudine, dolutegravir and efavirenz. Second, synthesis of polymer drug packaging with attachments of specific ligand coating will direct the drug to specific virus' target cells, tissues and subcellular regions to optimize antiretroviral activities. We posit that such cell-targeted nanoART will facilitate cell entry, intracellular trafficking and drug secretion. Third, we will probe the best directed formulations in rodents using a newly discovered small magnetite ART (SMART) platform. This will readily permit evaluation of broad numbers of coating ligands. Fourth, we will evaluate the functional consequences to the cell as it is linked to intracellular particle trafficking and stability. Fifth SMART formulations that pass evaluation will be synthesized by crystalline particles and evaluated for drug pharmacokinetics (uptake, release, plasma, and tissue distribution). This will include measures of BBB penetrance and tissue drug levels in brain, spleen, lymph nodes, and liver. Histologic and imaging assays will evaluate potential drug toxicities. Last, the most promising drug polymer formulations will be tested for antiretroviral efficacy, immune and neuroprotection in a humanized mouse model of HIV infection and neuroAIDS. The overall premise is to develop the necessary state of the art tools to improve nanoART induced viral reduction in its CNS and other viral reservoirs.

The aims of the administrative core are to set and implement programmatic goals, scientific policies and operating procedures for the overall program as well as facilitate communication between projects and core leaders. In addition, the core will develop and set priorities for core usage and expand, enrich, and develop the scientific exchanges among all investigators. The infrastructure provided will monitor the scientific progress of projects and cores;maintain a system for fiscal accountability and resource allocation (for equipment and personnel);provide statistical support and assist in the planning, implementation of experiments and interpretation of data.

AIDS Dementia; AIDS Dementia Complex; AIDS Virus; AIDS neuropathy; AIDS with dementia; AIDS-related dementia; Acquired Immune Deficiency Syndrome Virus; Acquired Immune Deficiency Syndrome related dementia; Acquired Immunodeficiency Syndrome Virus; Active Oxygen; Acute; Affect; Animal Model; Animal Models and Related Studies; Animal Virology; Anti-Retroviral Agents; Antiretroviral Agents; Apoptotic; Arts; Astrocytes; Astrocytus; Astroglia; BALB/c; Benzeneethanamine, N,alpha-dimethyl-, (S)-; Biochemistry; Biologic Marker; Biological; Biological Markers; Biological Models; Biology; Blood monocyte; Body Tissues; Brain; Bypass; CD34; CD34 gene; Cell Death; Cell Function; Cell Process; Cell Surface Antigens; Cell physiology; Cells; Cellular Function; Cellular Matrix; Cellular Physiology; Cellular Process; Central Nervous System; Centrifugation, Density Gradient; Chair; Chairman; Chairperson; Chairwoman; Chemistry, Biological; Chronic; Clinical; Clinical Data; Co-culture; Cocultivation; Coculture; 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The elimination of the human immunodeficiency virus inside its central nervous system (CNS) sanctuary is affected by variable antiretroviral therapy (ART) penetrance across the blood-brain barrier (BBB), complex dosing regimens, costs, toxicities, biodistribution, and pharmacokinetic patterns of drug regimens. Despite advances in ART resulting in reductions in cerebrospinal fluid viral loads, neuroAIDS morbidities continue on the rise. One principal issue is achieving robust ART drug levels in affected brain subregions and maintaining the levels. To address this issue, we will develop nanoformulations of commonly used anti-retroviral and adjunctive drugs (for example, Celecoxib) with variable CNS entry profiles and use established or more novel means to deliver the drugs directly to diseased brain tissue captured inside blood-borne monocytes or macrophages. To this end, we will determine the means to maximize both the delivery and distribution of ART across the BBB. A three-step approach will be sought. First, comparative measures of nanoparticle (NP) drug formulations will be tested for entry and secretion into and from bone marrow-derived macrophages (BMM) and monocyte-derived macrophages (MDM). Here, viral protease and non-nucleoside reverse transcriptase inhibitor(s) will be packaged into phospholipids-coated NP. Cytotoxicity, anti-retroviral efficacy, mobility, and the functional consequences of macrophage carriage of the drug-laden particles will be measured. Second, pharmacokinetics (uptake, release, plasma, and tissue distribution) of the formulations will be investigated using BMM as a drug delivery system in mice. Third, ligand-formulated NP will be developed and tested in vitro then used to test direct intravenous administration in mice. Alternatively and to enhance NP entry into macrophages, formulations will be made with folate coatings and will be designed to specifically target macrophages, and as such, improve cell entry. Laboratory experiments reflecting immune activation of human monocytes and MDM will be developed to assess the optimal ways to enhance uptake of ligand-coated NP formulations. Thus, the abilities of drug to bypass the reticuloendothelial system and cross the BBB will be determined. High performance liquid chromatography analysis of spleen, lymph nodes, liver, and brain will provide confirmation of drug tissue penetrance and be used in tandem with histology and imaging assays. Lastly, the NP developed will be tested for anti-retroviral efficacy in affected brains of a primary and humanized mouse models of NeuroAIDS. All together, the goals are to enhance therapeutic efficacy and BBB migration of ART so that they can be translated for human use to improve disease outcomes in NeuroAIDS.

Project 2 Abstract This project bridges long-acting antiretroviral therapeutic nanoformulation (nanoART) synthesis, viral tissue reservoir targeting and pharmacodynamic tests in rodents and large animal confirmatory studies when required. Three aims joint the project. The first, seeks to determine the effectiveness of the targeted drug formulations for sustained antiretroviral and immune responses in chronically HIV-1 infected humanized mice treated with decorated or targeted nanoART. Reductions in viral load and restored CD4+ T cell numbers will be assessed when compared against results seen with native drug, formulations containing untargeted ART and those animals treated with an irrelevant control drug (for example, fluconazole). Parallel tests for potential tissue and cellular toxicities and ?putative? drug-drug interactions, a special concern in the drug-abusing population, will be determined. The second seeks to evaluate further the most effective drug particles with the goal of quantitatively determining the subset of immunocytes in its tissue locale (gastrointestinal associated lymphoid tissue, bone marrow, lymph node and brain) that harbor residual virus. The, third will use combinations of antiretroviral and immune modulatory agents (for example, an indoleamine 2,3-dioxygenase inhibitor (IDIO, GSK3176181A) to induce HIV-1gag/pol-specific cytotoxic lymphocytes with the ?putative? goal of viral eradication. The most effective tested combinations for each of these aims will be cross-validated in SIV/SHIV infected rhesus macaques. The development of an injectable combination nanoART that can be dosed once every 6 months is realistic. Cabotegravir, is an already long-acting integrase inhibitor, currently in phase II trials. With a demonstrated half-life of 21?50 days following a single parenteral dose, it has the potential for an every three-month dose. Our own preliminary data offered in this project shows an up to 5-fold improvement in PK from what is being administered to eople with an even larger increase in drug reservoir targeting. This combined with the generation of polymer-encased pro-drugs of lamivudine and abacavir with markedly extended drug half-lives makes combination long-acting therapy realistic and without our grasp. On balance, we also acknowledge the obstacles. But balanced prior scientific successes, broad in-house technical experience and resources including trained personnel, equipment and infrastructure can meet the challenges. Further support includes access to developed and new antiretrovirals with support from GlaxoSmithKline and drug libraries. NanoART will include cell targeting to monocyte-macrophages and to subcellular organelles (late and recycling endosomes). We posit that modifications made of formulations in size, composition, coating, and charge will enhance nanoART cell uptake and potentiate subsequent drug delivery and release into viral reservoirs. These include susceptible CD4+ T lymphocytes. The establishment of the Nebraska Nanomedicine Production Plant, a good manufacturing production facility now housed at the medical center has enabled improved scale-up production of nanoART.

Core A Abstract: Core A, the administrative core, will implement programmatic goals and operating procedures for the overall program as well as facilitate communication between project and core leaders. The core will monitor and facilitate scientific exchanges among all investigators (projects and cores), the Internal and External Advisory Committees, GlaxoSmithKline investigators and the Nebraska Nanomedicine Production Plant. These will provide monitoring of scientific progress; maintain a system for fiscal accountability; oversee conflict of interests; provide broad statistical support; and assist in the planning, implementation, execution and interpretation of all experiments. Oversight will also be provided in assembling data for publications, submitting patent applications and ultimately for clinical product development.

While antiretroviral therapy (ART) leads to improved morbidity and reduced mortality for HlV-1 infected people, a major limitation rests in the need for lifelong daily regimens. Suboptimal adherence causes increased risk of treatment failure. Drug abuse disorders correlate with such sporadic adherences commonly resulting in accelerated HIV disease progression. Moreover, providers are often reluctant to prescribe ART to patients who abuse or are addicted to drugs because of concerns about the promotion of virologic resistance. Complicating matters further include common cognitive and motor disorders. These risk factors often result in poor treatment outcomes. The advent of slow release ART (ritonavir, indinavir, efavirenz, atazanovir and efavirenz) will positively impact these concerns. Thus, we propose to develop antiretroviral nanoparticles (nanoART) that are carried within circulating immunocytes and delivered to virus-target tissues. Cell-based nanoART theoretically would travel to sites of inflammation and release drug(s) slowly with limited tissue toxicities. Such a drug delivery system, if realized, can revolutionize ART treatment outcomes particularly those within the nervous system. This proposal builds on prior works conducted between our laboratories (Project 1 and 2, A. Kabanov and H. Gendelman). Preliminary investigations demonstrated "proof of concept" in that a single intravenous dose of the nanoART can elicit high-sustained tissue and plasma drug levels in the reticuloendothelial system and brain. NanoART can be taken up within minutes by circulating monocytes and released in tissues over a period of two weeks. Our partners in the University of Nebraska Medical Center College of Pharmacy (Project 1. A. Kabanov and Core C, C Fletcher) will be joined with our College of Medicine Departments of Radiology, Medicine, and Pharmacology and Experimental Neuroscience (Projects 2 and 3 and Core B, H. Gendelman, H. Fox, and M. Boska) to optimize nanoformulations for future human clinical use. This can now be facilitated through integrated cell biologic, pharmacologic, virologic, and molecular testing facilities within UNMC to move an "idea" from the laboratory bench through its translation to the bedside. First, nanoART will be manufactured then optimized in laboratory models of HIV infection. Second, the nanoART will be scaled for testing drug pharmacokinetics in rodents and rhesus macaques. Third, animal studies will be performed in virus-infected rhesus macaques infected with recombinant lentivirus hybrids of SIV and HIV (SHIV) for safety and efficacy investigations, respectively.

PR0JECT SUMMARY (See instructions): The goals of the Therapeutics core are to provide nanoformulations to invesfigators pursuing nanoformulated ntiretroviral and neuroprotective therapies for increased central nervous system (CNS) penetrance. The formulations developed address how monocytes and other immune cells may be harnessed for drug delivery. Before such novel therapies can be administered to people, we will determine, in well-validated laboratory and animal models, the optimal doses and formulation administration. The crux of the problem facing the Core, namely can nanoformulated antiretroviral therapy show sustained antiretroviral responses and slow release of drug in tissues, has now been addressed. The Core addresses a specific and important issue in the treatment of HIV and neuroAIDS, with broader implications for therapeutic interventions to other neurodegenerative diseases. Through this project, we will identify and manufacture candidate Nanoformulations of currently used efficacious antiretrovirals. These will be tested model systems of human disease, ranging from cultured monocytes to mice to monkeys, to examine pharmacokinefic, safety and efficacy. This Core, overall, represents work that may prove to be a major advance in the development of long-lasting therapeutic agents that can lead to real treatments both systemic and CNS human disease.

DESCRIPTION (provided by applicant): Neuronanomedicine is a new research field defined as the development and translation of nanotechnology for diagnosis and treatment of degenerative, inflammatory, infectious, vascular, addictive, behavioral and metabolic disorders of the nervous system. The research discipline is interdisciplinary and in its infancy. Hurdles need be overcome for success to be realized that improve known blood brain barrier restrictions, nanotoxicologies, inefficient drug targeting to diseased brain regions and in reaching specific neural subcellular compartments. These all pose significant challenges to the field. To facilitate successful research outcomes we plan to bring together scientists from divergent backgrounds engaged in neuronanomedicine to present research findings that will help define the field while also facilitating opportunities for trainees to directly engage in currnt and future discussions. The American Society for Nanomedicine (ASNM) has taken a leadership role by focusing its 2013 meeting to the multifaceted discipline of neuronanomedicine. The ASNM is well positioned to assume this role as the society's goals since its 2008 inception has rested in clinical translation of nanotechnologies. Such goals will be fostered in an open forum discussion that will include, but is not limited to, bioethics, safety an toxicity, intellectual property, and product commercialization. A major focus for the conference is in bringing early career investigators into the field of translational neuronanomedicine. ASNM's emphasis on innovation and practicality of approaches for speedy translation parallels the conference goals on merging divergent disciplines and approaches to facilitate collaborative research from product development to laboratory and animal testing and ultimately to human clinical trials. The proposal while led by an experienced physician-scientist, Dr. Howard Gendelman, takes advantage of a highly seasoned broad-disciplined board. Overall, the focus on the nervous system represents an evolution of ASNM leadership and a further change in direction for the meeting. It will enable new directives in the field to keep abreast with needs an research scope in nanotechnology.

DESCRIPTION (provided by applicant): This shared investigator R01 is an extension of 5 P01 NS043985-09. The current work builds on prior successes in developing long-acting nanoformulated antiretroviral therapies (nanoART) to improve drug delivery and therapeutic outcomes for aged virus-infected people. Elucidating the interplay between aging, HIV disease and ART is the overarching project goal. The tools are available to address this interplay which can simplify drug regimens and improve disease outcomes. First, nanoART is available for long- term testing of antiretroviral responses together with central nervous system (CNS) and broad metabolic functions. Second, interdisciplinary bioimaging, behavior, and nanopharmaceutics can evaluate virus, drug, immune, and age-related toxicities. Third, drug pharmacokinetic, pharmacodynamics, drug-drug interactions can be measured by employing PXR humanization (the androstane receptor with replacement of the mouse Cyp3a with human CYP3A4) in rodents. This can be used to evaluate long-term immune and antiviral responses. Fourth, a novel tool designed to improve ART delivery to viral reservoirs was discovered for theranostics (simultaneous diagnostics and therapeutics). This system is called small magnetite ART (or SMART) and permits assay of drug biodistribution by imaging tests. Such outcome measures would improve ART CNS and lymphoid delivery and thus combat persistent HIV-1 infection. A group of investigators with productive histories of working together was assembled. They include neuroscientists (H. Gendelman, Co- PI), immunopathologists (L. Poluektova, Co-PI), aging and cognitive behavior researchers (S. Bonasera), bioimaging experts (M. Boska) those with expertise in neurodegenerative diseases (R. L. Mosley), and experts in nanomedicine and drug delivery (X. Liu). The work is timely and relevant. Although ART has profoundly reduced morbidities and mortality for HIV infection coincident with virus reductions and immune preservation, the prevalence of neurocognitive impairments and drug toxicities remain common. Indeed, the doubling of virus-infected patients > 50 years of age is upon us. New co-morbid conditions now include cerebrovascular disease, non-AIDS malignancies, insulin resistance, hyperlipidemia, dementia, and liver, renal and bone disorders. This demands new model systems for disease studies relevant to current HIV/AIDS trends. Indeed, the work reflects the changing epidemiologic patterns of human disease. [We acknowledge that the prior submission lacked preliminary data and details about our humanized brain model and nanoformulations and response of the animals to treatments. A number of recent publications and significant new preliminary data are now included that addresses each of these concerns in a thorough and reasoned manner.] The tools that are needed to tackle relevant questions in HIV and aging are also available for study.

Abstract The elimination of the human immunodeficiency virus (HIV) from its central nervous system (CNS) and peripheral reservoirs is a requirement for a disease-cure. To our knowledge, we are the first to have achieved this goal in tests performed in a limited number of infected humanized mice. To validate such early successes we propose to build a four-step ladder with a final crest of latent virus eradication. First, a newly developed humanized mouse brain-lymphoid model of neuroAIDS will identify productively infected perivascular and meningeal macrophages and restricted infection in parenchymal cells. This rodent model most closely reflects human brain disease as demonstrated by robust molecular, virologic and neuroimmunologic tests. Second, long acting slow effective release antiretroviral therapy (LASER ART) also now fully developed in our laboratories can now facilitate a pinpoint localization of the latently infected viral brain reservoir. Third, viral excision strategies will be employed to eliminate residual virus and preclude HIV reactivation. The gene editing CRISPR/Cas9 system developed by Temple University Medical Center investigators including CCR5 and viral excision strategies will reduce then eliminate any ongoing infection and integrated proviral DNA from infected cells. The CRISPR/Cas9 constructs will deliver its cargo to brain and peripheral tissue sites using specific serotypes of adeno-associated virus. This will enable permanent HIV eradication in humanized mice without viral reactivation and as such preclude any ongoing brain infection and subsequent neural damage. Fourth, in order to prove the therapeutic strategy effective both for brain and peripheral lymphoid tissue virus (including the gut-associated lymphoid tissue, lymph node, spleen and genitourinary system) we will cease ART administrations and following time periods measured in months to provide cross validating evidence for viral eradication by measure rebound. Given the risks associated with HIV reactivation in the CNS this approach must show effectiveness for its abilities to target latent virus. Taken together, the proposal seeks support to employ combination LASER ART and potent molecular viral and immune-based regimens for elimination of viral depots. The overall premise is to develop the ?state of the art? tools required to permanently eliminate virus detected in the CNS and peripheral infectious reservoirs.

Therapeutics Core Abstract Antiretroviral therapy remains the gold standard for the treatment of human immunodeficiency virus infection. While having dramatic effects on disease morbidity and mortality, it demands life-long adherence. Secondary toxicities, viral mutation, co-morbid diseases, constitutional drug-associated symptoms, adherence of complex regimens ?pill burdens?, and drug-drug interactions are further complicating limitations. Nanoformulated long- acting antiretroviral therapy with half-lives measured in month(s) require infrequent administration but with equivalent or superior therapeutic efficacy compared to standard drug regiments. These have the potential to revolutionize current antiretroviral therapy. Cell-targeted nanoformulations provide yet another boost by improving drug delivery to viral reservoirs and by providing novel opportunities for viral eradication. This Core seeks funds to bring together and be a resource for investigators skilled in cell biology, virology, pharmaceutical science, immunology, animal science, clinical medicine, and good manufacturing practice to move laboratory discoveries to the patient?s bedside. The Core is innovative in research design and translation. While providing unique collaborative opportunities, the science is high impact with true discovery potential. ! !

ABSTRACT Despite advances in basic and translational neuroscience research, the development of new therapeutics remains in want. The National Institutes of Health has recognized the need to translate bench research to therapies that improve human disease outcomes and initiated programs that train researchers who can effectively conceptualize neurological disease processes. One critical ?in need? area is in the discipline of neuroimmunity. This research area remains understudied despite its close linkage to the pathobiology of degenerative, infectious, developmental, and psychiatric disorders. To these ends, our training goal is to provide talented students with a fundamental understanding of peripheral and central immunology as it affects neuronal injury, differentiation, regeneration, and protection. The program is designed to provide the student with broad exposure to research methods that facilitate technical proficiency. The program ensures that the student will acquire broad knowledge in neuroimmunity. This would allow critical thinking for how inflammation affects the pathogenesis and treatment of neurological disorders. Several approaches are proposed to achieve this goal. First, is the use of our newly published textbook Neuroimmune Pharmacology (2nd Ed.) designed specifically as a coursework guide in neuroscience, immunology, and pharmacology. Second, is in developing a cross discipline mentorship training to provide the student with opportunities to intersect studies of immunity and neural function. Third, are ?unique? research experiences in systems biology, cell signaling, glial and neuronal biology, human disease models and synaptic physiology. These opportunities serve to complement research in neural genetics, development, repair, and pharmacology. Fourth, are formal student presentations to interdisciplinary basic neuroscientists and supervisory committees to acquire research feedback in design, interpretation and conceptualization of ongoing research activities. This serves to challenge existing paradigms and existing student perceptions. Fifth, are uniquely offered cross-discipline team mentoring, teaching, and clinical neurological experiences. Sixth, are cross-disciplinary internships where students will complete thesis component(s) in another laboratory using a different research approach and mentor. Seventh, are sustained community, university and logistical support. By coordinating the training efforts of divergent research groups linked by common interests in neuroimmunity trainees will develop deeper understandings of innate and adaptive immunity in relationship to neurologic disease. Such trainees will be better prepared to develop successful careers in studies of disease pathobiology and therapeutic interventions for human nervous system disorders.

Abstract This proposal seeks funds to translate existing antiretroviral drugs (ARVs) into long acting scalable medicines. A tripartite approach is offered. The first is the conversion of ?partially? hydrophobic ARVs into lipophilic prodrug crystals encased in polymers for efficient transfer across cell and tissue barriers. The second is deployment of mononuclear phagocyte (MP; monocyte, macrophage and dendritic cells) as drug depots by housing the crystalline ARV particles into autophagosomes and releasing drug inside the cell through hydrolysis. The third decorates the particles with ligands serving to facilitate entry into cell and tissue lymphoid viral reservoirs. The overarching goal is to make long-acting slow effective release antiretroviral therapy (LASER ART) with dosing intervals of once every six months in order to maximize the effectiveness of any treatment or pre-exposure prophylaxis regimen. Complete control of viral replication in the central nervous system, lymph nodes and gut associated lymphoid tissues is the desired endpoint. To accomplish this multidisciplinary research, a partnership was forged between a medicinal and polymer chemist (B Edagwa) and a virologist, cell biologist and immunologist (H Gendelman). This merger will continue to speed the transformation of short to long acting ARVs (for example entry, nucleoside and nonnucleoside reverse transcriptase and integrase inhibitors) in combination therapy. Biological testing will follow product development in relevant cell and animal models of human disease. Formulation safety will be realized by first testing replicate short acting ARV formulations. The pathway for drug/nanocrystal development will move forward by a series of now carefully crafted Go No Go criteria. Drug choices, formulation stability, drug combinations, tissue and cell targeting, toxicology, pharmacokinetics and pharmacodynamics (PD) profiles follow a carefully organized and safe action plan based on drug performance. Several have ?reached? nonhuman primates through drug manufacture by a now operational good laboratory practice facility. These new LASER ART formulations can be joined with an autophagy medicine to further extend cell drug depots (HA Gelbard). The successful outcome of these experiments can circumvent drug toxicities, improve regimen adherence, provide more crafted viral prevention measures and easily enter viral reservoirs such as the brain. Maximal reduction in residual infection is the project goal. The research builds on an already strong track record in macrophage-targeted nanomedicines. Antiretroviral responses and endosomal trafficking will be tested by computation (TA Wysocki). PD screens in humanized mice (S Gorantla) will validate the findings. The overall premise is to develop the necessary ?state of the art? tools to positively transform existing antiretroviral treatment regimens by bench to bedside approaches.

Abstract This proposal seeks funds to translate existing antiretroviral drugs (ARVs) into scalable long acting medicines. A step-wise approach is offered with defined ?go no go? criteria. The first is conversion of nucleoside reverse transcriptase and an integrase inhibitor(s) into lipophilic prodrug nanocrystals. These conversions are designed for simple, safe and scalable productions under current good manufacturing practices. Following the production of ARV nanocrystals, the second step will optimize CD4+ T cell uptake while sustaining drug depots in macrophages. ARV release from prodrugs will occur by controlled hydrolysis enabling sustained antiretroviral potency. The third will facilitate distribution of prodrug nanocrystals into lymphoid, gut associated lymphoid tissue, genitourinary and central nervous system tissues. The fourth, will complete tests designed to limit off target toxicity (S Cohen) by screening pro-, native- and nanocrystal- drug formulations in cell and tissue assays. These extended toxicology tests will serves to investigate any or no adverse events that follow increased ARV levels in cells and tissues. Our goal is the production of safe well-tolerated long acting slow effective release antiretroviral therapies with ?putative? dosing intervals of up to once every six months. The design will offer maximal effectiveness for pre-exposure prophylaxis and treatment regimens. The specific aims are supported by extensive published data sets. To accomplish the proposed research, a partnership was made between a medicinal and polymer chemist (B Edagwa) and a virologist, cell biologist, and immunologist (H Gendelman). This collaboration will accelerate transformation of short- to long-acting ARVs. Biological testing will follow product development in cell and animal models. Formulation safety will be realized by continuous testing of replicate short-acting native ARV formulations in good laboratory and current good manufacturing practice facilities. The pathway for ARV nanocrystal development will move forward by sequential Go No Go criteria. Drug choices, formulation stability, drug combinations, tissue and cell targeting, toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) profiles follow the action plan. Several have ?reached? drug manufacture. The outcome of these experiments can reduce drug toxicities, improve regimen adherence, and provide enhanced viral prevention into tissue reservoirs. The research builds on an already strong track record in cell-targeted nanomedicines. Antiretroviral responses and endosomal trafficking will be tested. PK and PD mouse screens (S Gorantla) will validate the findings. Overall, our long-term goal is to transform existing antiretroviral treatment regimens into long acting therapies.

Abstract Our laboratories birthed the field of human immunodeficiency virus (HIV) theranostics. The new field allows simultaneous detection (diagnostics) and treatment (therapeutic) for the identification and ultimate elimination of viral tissue compartments and cellular reservoir sites with a focus on the central nervous system. By employing theranostics viral entry sites in lymph nodes, gut and brain can be tracked during antiretroviral therapy (ART). Cellular viral targets including CD4+ T cell populations and mononuclear phagocytes (MP; monocytes, macrophages, microglia and dendritic cells) subcellular endosomal structures can now be targeted for drug delivery in sites of active viral growth. The advantage of theranostics rests in that any steps towards improved HIV therapeutics and elimination strategies that requires precise targeted delivery of antiviral drugs. Bringing virus-combating agents to anatomically privileged tissues of latent viral infection can be defined through magnetic resonance and single photon emission computed tomography imaging facilitated by multimodal antiretroviral drug (ARV) probes. To deploy such technologies, as virus detectors we have successfully mirrored HIV infection in both the human brain and in lymphoid tissue by creating a first in kind completely humanized ?microglial? mouse. The animal is populated by human CD4+ T cells and MPs and as such contains the principal ?human? HIV-1 target cells in a murine model background. Thus, in the current proposal we plan to advance a theranostic nanosystem through improvements in the physical and chemical properties of particles that resemble a complete HIV-1 virion. The realization of the projects? goals can result in the accurate assessment of viral biodistribution and optimal antiretroviral responses. To achieve this outcome we will employ two different nanoparticle formats. The first is bismuth sulfur nanorods and the second is a pseudovirus. Each of the made particles will be detector-tagged and ARV loaded. The combinations of a bioimaging detector and payload deliverer defines our multimodality system that enables unique insights into virus compartmentalization, drug biodistribution and hidden viral reservoirs. The long-term goal is to improve current therapeutic regimens with an emphasis on those that target the nervous system. The research brings together a group of chemists, biologists, pharmacologists, virologists, radiologists and immunologists with a long successful track record of working effectively as a team with singular goals to develop products that facilitate HIV-1 control.

? DESCRIPTION (provided by applicant): Despite the effectiveness of antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) that affect HIV infected individuals continue to increase. The prevalence of HAND and the incomplete reversal of neurocognitive dysfunctions after antiretroviral therapy have called for novel therapeutic approaches. Among the various pathophysiology of HAND, synaptic dysfunction likely underlies cognitive impairments. Interestingly, Tat, an essential HIV-1 viral protein, is present in the cerebrospinal fluid of individuals virologically controlled on cART. Furthermore, Brain-specific HIV protein Tat expression in mice mimics key aspects of HAND pathology in the post-cART era, suggesting that Tat may be responsible for the sustained central nervous system complications in patients receiving cART. Tat is known to cause neuronal injury via excitotoxic mechanisms. Furthermore, HIV-1-infected patients have significantly higher concentrations of glutamate in their plasma and cerebrospinal fluid compared to uninfected controls. Elevated levels of glutamate disrupt normal neural transmission in the brain, contributing to the neuropathogenesis of HIV-1 infection. In the past decade we have established that blocking the activity of glutaminase (GLS), a primary enzyme for the production of glutamate, could alleviate macrophages and microglia neuroinflammatory and neurotoxic response. We have demonstrated causal effects of innate immune activation and proinflammatory on the GLS function in macrophages, microglia, and neurons. Furthermore, we have observed an intriguing release of GLS by macrophages, microglia, and neurons, through unidentified mechanism(s) that could cause neuronal injury. Extracellular vesicles (EVs), which include microvesicles and exosomes, have emerged as an important cellular mechanism for GLS release. Therefore, in the current proposal, we hypothesize that the release of GLS-containing EVs is a critical pathogenic event in HIV-1-mediated neuronal injury and hippocampal synaptic dysfunction. Moreover, we hypothesize that blocking aberrantly upregulated/released GLS through GLS inhibitors could have therapeutic effects in HAND. Information will be provided as whether brain-specific overexpression of GLS is sufficient to induce brain inflammation, impair synaptic integrity and cognition in mice, and whether macrophage-specific conditional knockout of GLS gene and blocking of GLS-containing microvesicles release could protect neuronal function in a Tat transgenic mouse model of HAND. Furthermore, novel water-soluble GLS inhibitors will be evaluated for their therapeutic potentials in HAND relevant animal models. The elucidation of the GLS dysregulation and its contribution to pathophysiology of HAND will aid in developing potential novel agents for the treatment of HAND and other neurodegenerative disorders.

Abstract This project contains a highly collaborative investigative team with interdisciplinary expertise with significant potential impact for HIV/AIDS prevention. The proposal includes pharmacologic, virologic, animal and product development studies designed to halt disease transmission through novel long-acting (LA) antiretrovirals. These are named LA slow effective release antiretroviral therapies (LASER ART) designed to facilitate HIV-1 prevention by intense bench to the clinic translational studies. Innovative interdisciplinary approaches contain a detailed research plan, extensive preliminary and broadly published supportive data. Creativity and innovativeness are offered placed at higher risk than a conventional research project. The work builds on the development of parenteral nanoformulations of chemically modified antiretroviral drugs designed to improve adherence. The drugs are currently offered once/day in pill form but will be converted to up to once a year administration. Support from expert pharmacologists and pharmaceutical scientists with University researchers are operative. The drugs include dolutegravir (DTG), emtricitabine (FTC) and tenofovir (TFV) created to extend their apparent drug half-life, efficacy and abilities to target viral reservoirs. They are, in measure, DTG and FTC and TFV prodrug + nucleotide (ProTides) designated ?N? for nanoformulation, ?M? for esterification and ?P? for ProTides. The created NPFTC and NPTFV and NM2DTG demonstrate sustained plasma and tissue drug concentrations of > 90% inhibitory concentration from months to a year. Based on encouraging results, we seek funds to facilitate large scale development that would facilitate future human studies. The final formulations would be characterized by sustained prodrug hydrolysis with reduced injection volumes. The pathway forward follows established partnership with the Clinton Health Access Initiative and oversight by ViiV Healthcare and Gilead Sciences. The overarching goal is safety, reproducibility and ?scale-up? that follows US Food and Drug Administration-approved current good manufacturing practices (cGMP). The specific aims are each supported by extensive published data sets forged through the multidisciplinary research. Creation and characterization focus on prodrug formulations, toxicology, and pharmacokinetics profiles follow a safe developmental action plan. The work is facilitated by a fully operational cGMP facility and rhesus macaque validations. The lead formulation will be developed with our CHAI partners. We posit that the creation of LASER ART DTG or FTC and TFV will have a profound impact on HIV prevention.

ABSTRACT Mononuclear phagocytes (MP; monocytes, macrophages, dendritic cells. and microglia) serve as human immunodeficiency virus type one (HIV-1) reservoirs, sites of viral persistence and latency, and inducers of end- organ disease. All are commonly linked to HIV-1 pathobiology. However, the key relevance of the MP viral reservoir rests in the central nervous system (CNS) of those people living with HIV (PLWH). In those PLWH and receiving antiretroviral therapy (ART), the evidence for the size, scope, and disease relevance of the CNS viral reservoir remains under appreciated. The discordance between laboratory MP infection and tissue persistence in an infected human host is also not yet known. MP can have an extended life span and possess self-renewing potential, and as such, are likely more relevant in disease than currently appreciated. Evaluation of the significance of MPs, in general, and the microglia specifically will help define the importance of MPs during natural infection. For the CNS specifically, HIV-1 enters the brain soon after infection and replicates in perivascular macrophages and MGL along with limited numbers of astrocytes. Viral set point and timing of ART initiation determines the latent reservoir size; each affects the efficiency of any eradication strategy. Knowledge of the viral dynamics, CNS viral invasion, susceptibility to MP infection, and composition of CNS HIV reservoir will facilitate effective therapeutic interventions. To each of these ends, we will employ novel techniques to study the MP HIV-1 reservoir in laboratory systems and in a newly developed human microglial mouse model of human disease. We will use basic and applied MP biology, theranostics, novel ART nanoformulations, molecular and cellular biology, and our unique animal model to study the means to eliminate viral infection at the subcellular, cellular, and tissue level with newly designed and novel therapeutic methods that include gene therapy strategies. Our aims are to determine the efficiency of viral suppression by native and nanoformulated ART (at subcellular level), assess the breadth of the CNS viral reservoirs against viral set points (defined by the initiation of ART), and to explore combination strategies for HIV-1 elimination in a new developed humanized microglial mouse.