Kamel Khalili - US grants

JCV is a human polyoma virus which virus which has been isolated from the brain of patients with chronic demyelinating disease, progressive multifocal leukoencephalopathy (PML). This oncogenic virus is associated with the development of brain tumors including glioblastomas, medulloblastomas, neuroblastomas and other tumor of neural origin. JCV exhibits a highly specific host range and tissue specificity. In vitro, JCV grows exclusively in primary human fetal glial cells. Several studies have shown that the restricted host range of JCV to brain tissue in cell culture is determined at the transcriptional level. However, the mechanisms that govern tissue-specific transcription of this virus in brain cells remain unknown. The aim of the research outlined in this proposal is to define the mechanisms by which the JC virus genome is regulated transcriptionally in a tissue-specific manner. The experimental design includes: (1) identification of the cis-acting transcriptional control elements of the viral genome; (2) characterization and purification of the trans-acting regulatory proteins from permissive cells and tissue that by interacting with the cis-acting sequences that control transcription of the viral genome; (3) cloning of the gene encoding the regulatory proteins, and large- scale production of these proteins for detailed structural/functional studies. The information gained from these manipulations and analyses should increase understanding of the mechanisms that modulate transcription of the viral genes in neural cells and the tumorgenicity of this virus in brain.

The acquired immune deficiency syndrome (AIDS) is associated with a variety of clinical disorders involving the peripheral and central nervous system. Opportunistic infection of the central nervous systems and primary CNS lymphoma are often found in the later stages of the disease. In addition to inducing these secondary manifestations of immune suppression, HIV is thought to play a direct role in neuropathogenesis. The presence of HIV in brain appears to be associated with white matter changes, including vacuolar degeneration, enlarged astrocytes and demyelination. Quite similar histopathology is also observed in patients with progressive multifocal leukoencephalopathy (PML). While PML is a relatively infrequent disorder, latent infection with JCV appears to be fairly common. Virus reactivation and resulting neuropathology appears to be a consequence of immune suppression. The striking similarity between PML and AIDS leukoencephalopathy is suggestive of JCV reactivation as a consequence of HIV infection, either secondarily through the cell depletion, or directly by HIV encoded trans-acting factors. Since glial cells are productively infected by JCV and HIV, JCV reactivation through superinfections could be an in vivo mechanism of pathogenesis. Further support for this hypothesis stems from our observation which suggests that the HIV-1 encoded protein, tat, tranactivates expression of the JCV promoter in glial cells. The aim of the research outlined in this proposal is to define the mechanisms by which the JCV genome is activated transcriptionally by HIV-1 encoded protein in glial cells. The experimental designs include: (1) identification of the cis-acting responsive elements to the tat protein; (2) characterization and purification of the trans-acting regulatory proteins from tat producing glial cells, that interact with the cis-acting sequences and trans-activates JCV promoter. The information gained from these manipulations and analyses should increase our understanding about molecular mechanisms involved in the development of AIDS dementia and other neurological disorders prevalent in AIDS patients.

JCV is a ubiquitous human virus which is associated with the demyelinating syndrome progressive multifocal leukoencephalopathy and several neural tumors in humans such as medulloblastomas, glioblastomas, neuroblastomas, pineocytomas, and undifferentiated neuroectodermal tumors. Transgenic mice containing JCV genes develop, in addition to hypomyelination of the central nervous system, adrenal neuroblastomas, which metastasize to the pituitary glands and other tissues. JCV exhibits a highly specific host range and tissue tropism. This virus replicates exclusively in glial- origin cells in tissue culture. Several studies have established that the restricted host range of JCV to brain tissue is determined at the level of the viral gene transcription. However, the molecular mechanisms that confer the glial-specific activation of the viral gene expression remain unknown. Recent studies in our laboratory have indicated that the JCV control sequence contains a region, designated the B-domain, which by binding to a 45-kD glial-derived protein stimulates the viral early promoter in vitro. The goal of the research outlined in this proposal is to define the mechanism by which the JCV genome is regulated transcriptionally in a cell-specific manner. The experimental design includes: (1) extensive analysis of the JCV control region by linker scanning mutagenesis across the virus enhancer/promoter (2) purification of the participant glial-derived factors that interact with these regulatory elements to homogeneity and cloning the genes encoding these proteins; (3) identification of structural features of the regulatory proteins, i.e., defining DNA-binding domain and activation regions; (4) examination of the mode of expression of the regulatory proteins during brain development. The information obtained from these experiments should increase our current understanding of cell-type specific gene expression in the central nervous system and provide an initial step in devising strategies to impair JCV replication in the central nervous system.

This revised competing renewal program project grant application (PPG) seeks support for continued investigation of the molecular biology and genetics of viral-glial interaction in the central nervous system (CNS). This project remains focused on the molecular pathogenesis of two CNS disorders. One, Progressive Multifocal Leukoencephalopathy (PML), is induced by the human neurotropic virus, JCV, and the other, Acquired type 1 (HIV-1). The clinical similarity between PML and ADC, the association of PML with HIV-1 infection, and the utilization of common regulatory pathways by these two viruses, in the CNS underscores, the rationale for the simultaneous study of these diseases and their etiological agents at the molecular level. During the last funding period, through highly interactive and synergistic collaboration, the participants in this PPG have identified several regulatory proteins that modulate JCV gene expression and replication in CNS cells, and have molecular cloned genes responsible for expression of these proteins. Using in vitro cell culture and in vivo animal models, several proteins regulating myelin genes have been identified, molecularly cloned, and characterized to help unravel the mechanism whereby JCV T-antigen causes dysmyelination of the brain. Analysis of HIV-1 gene expression in the CNS has led to the identification of a novel regulatory pathway in astrocytic glial cells that modulates HIV-1 gene expression in these cells. Finally, we have developed and utilized highly sensitive in situ PCR methods to identify HIV-1 gene expression in these cells. Finally, we have developed and utilized highly sensitive in situ PCR methods to identify a HIV-1 gene expression in various CNS cells in clinical specimens and have employed these methods to examine viral replication. In the current PPG we shall maximally utilize the established collaboration among the leaders of this program to: (1) characterize the interaction of JCV and host regulatory factors, and their effects on viral gene expression/replication, and glial cell function; (ii) elucidate the molecular interactions between the HIV-1 regulatory protein, Tat, Tat- induced cytokines, and cell cycle regulatory factors from human microglial cells and astrocytes; iii) derived potent transcription factor, Tat, and the cellular regulatory protein Puralpha. These three highly integrated, yet independent, projects will benefit from the Neuropathology, and Tissue Culture Core which will provide a central source for reliable distribution of clinical samples and preparations of primary and established cells from fetal and adult brain cell cultures to all projects and preparation of virus stocks for studies in other components of this program. This program project brings together basic scientists and physicians with expertise in the areas of neurovirology, molecular, retrovirology, molecular and cellular biology, and neuropathology to perform the proposed studies.

The acquired immune deficiency syndrome (AIDS) is associated with a variety of clinical disorders involving the peripheral and central nervous system. Opportunistic infection of the central nervous systems and primary CNS lymphoma are often found in the later stages of the disease. In addition to inducing these secondary manifestations of immune suppression, HIV is thought to play a direct role in neuropathogenesis. The presence of HIV in brain appears to be associated with white matter changes, including vacuolar degeneration, enlarged astrocytes and demyelination. Quite similar histopathology is also observed in patients with progressive multifocal leukoencephalopathy (PML). While PML is a relatively infrequent disorder, latent infection with JCV appears to be fairly common. Virus reactivation and resulting neuropathology appears to be a consequence of immune suppression. The striking similarity between PML and AIDS leukoencephalopathy is suggestive of JCV reactivation as a consequence of HIV infection, either secondarily through the cell depletion, or directly by HIV encoded trans-acting factors. Since glial cells are productively infected by JCV and HIV, JCV reactivation through superinfections could be an in vivo mechanism of pathogenesis. Further support for this hypothesis stems from our observation which suggests that the HIV-1 encoded protein, tat, tranactivates expression of the JCV promoter in glial cells. The aim of the research outlined in this proposal is to define the mechanisms by which the JCV genome is activated transcriptionally by HIV-1 encoded protein in glial cells. The experimental designs include: (1) identification of the cis-acting responsive elements to the tat protein; (2) characterization and purification of the trans-acting regulatory proteins from tat producing glial cells, that interact with the cis-acting sequences and trans-activates JCV promoter. The information gained from these manipulations and analyses should increase our understanding about molecular mechanisms involved in the development of AIDS dementia and other neurological disorders prevalent in AIDS patients.

Progressive multifocal leukoencephalopathy (PML) is a subacute neurodegenerative disease of the central nervous system caused by the human polyomavirus, JCV. PML, once a relatively rare disease of middle- aged and elderly patients with a background of immunologic impairment is now much more common due to the high prevalence of AIDS. The surprisingly higher incidence of PML in AIDS patients than in other immunosuppressive disorders has suggested that the presence of Human Immunodeficiency Virus type 1 (HIV-1) in the brain may directly and indirectly contribute to the pathogenesis of this disease. It is hypothesized that cytokines, the peptide hormones which control the homeostasis of the immune system and have a fundamental role in inflammatory and immune-mediated reactions, participate in AIDS neuropathology by facilitating cross-communication between HIV-1 and other opportunistic infectious particles including JCV. Support for this hypothesis stems from results of several laboratories indicating that infection of microglia and astrocytes with HIV-1 stimulates production of some of these cytokines such as TGFbeta, TNF, and IL-1 both in vitro and in vivo. Furthermore, our studies revealed that several cytokines, including TGFbeta and IL-1, have the capacity to augment expression of the JCV genome in glial cells. The aim of the research outlined in this proposal is to define the molecular mechanism by which the JCV genome is transcriptionally activated by HIV-1-induced cytokines in glial cells. The experimental design includes: 1) Examination of JCV gene expression and viral DNA replication in lytically infected primary glial cultures treated with TNFalpha, IL-1, and TGFbeta; 2) Identification of the regulatory elements which participate in this induction by: (i) targeting DNA sequences within the viral promoter which are required for this induction; (ii) defining the cellular factor(s) which enhance JCV activity by interacting with the JCV genome; 3) Evaluation of the biological activities of cytokine-induced regulatory protein(s) on JCV gene transcription. The information gained from these studies should increase our understanding of the molecular mechanism involved in the development of PML and other disorders prevalent in AIDS patients, and provide critical information for devising effective and safe strategies for disease prevention.

This is a competing renewal program project grant application seeking support for resuming studies on the molecular events leading to the development of brain tumors. In the previous period of funding we utilized human neurotropic virus, JCV, to create experimental animals which developed tumors from external granular layer of the cerebellum modeling human medulloblastoma. In humans, medulloblastoma is the most common malignant brain tumor seen predominantly in children and is considered the prototypical embryonal neuroblastic neoplasm of the central nervous system (CNS). This program project, which is a confederation of three inter-related research projects with supporting scientific and administrative cores, each representing the natural evolution of the previously funded program, plans to launch a multidisciplinary approach utilizing experimental animals to decipher molecular events involved in the genesis of medulloblastoma. In Project #1, experiments are designed to investigate the regulation of gene expression involved in control of cell proliferation by the tumor suppressor protein, p53 and [g-catenin, a key component of the Wnt pathway which is involved in the oncogenesis and neurogenesis. By studying pl30/pRb2 pathway in Project #2, we will evaluate the the mechanism of regulation of this potent tumor suppressor and its well celebrated partner, p27 Kipl in medulloblastoma, determine the functional interaction of p130/pRb2 with JCV T-antigen, both in vitro and in pRb-knockout experimental animals. Finally, in Project #3, we will investigate the role of IGF-1 signal transduction pathway as our recent studies have provided compelling evidence for the involvement of major components of the IGF-1 receptor, IRS-1, which is induced by IGF-1 and triggers a cascade of cytoplasmid events involving AKT/PKB and MAP kinases in the development of medulloblastoma. These projects will be supported by the Experimental Animal Core (Core A) and the Neuropathology and Tissue Culture Core (Core B). The participants of this program project, who have worked synergistically to study CNS neoplasia, will convert the information from these molecular studies to translational research in devising therapeutic strategies toward brain tumors.

JC virus is a common neurotropic polyoma virus. The oncogenicity of this virus has been amply demonstrated in hamsters and non-human primates and also in transgenic mice expressing JV virus large T-antigen. However, unlike the closely related BK virus and simian vacuolating virus, SV40, JCV exhibits a restricted tissue tropism which is reflected in the formation of neural tumors. The tissue-specific malignant transformation of neuroectodermal cells in animals as a result of JC virus infection and/or JCV T-antigen expression provides a unique opportunity to study the development and genesis of glioblastomas in the whole animal. The generation of these tumors is likely due to expression of the viral large T-antigen, due in part to its ability to bind and functionally inactivate the retinoblastoma tumor suppressor protein, pRb. Inactivation of pRb may also be accomplished by phosphorylation of this protein by a series of cyclins and their associated kinases including cyclin D:cdk4,6 and cyclin E:cdk2. Earlier studies have indicated an important role for transforming growth factor, TGFbeta, in inducing activities of these cyclin/cdk complexes, and thus converting pRb to its phosphorylated forms. Association of pRb with T-antigen or its phosphorylation results in liberation of the transcription factor, E2F from the Rb:E2F complex. E2F, in turn, stimulates expression of genes needed for transition through G0/G1 to S phase. Results from our and other laboratories have indicated that E2F and TGFbeta may mutually regulate each others' expression and that may alter the physiological balance required for the control of cell proliferation. Moreover, our recent studies have led to the identification of novel E2F related protein(s), GEAPs, in hamster glial cells which modulate transcription of S-phase specific gene. In this proposal we will focus on the interplay between cytokines (TGFbeta) and the cell cycle regulators, i.e., the E2F family of transcription factors to evaluate their mechanism of action in the pathogenesis of gliomas in a well-controlled in vivo system. The use of integrated MRI and neurohistopathological approaches will allow us to perform our molecular biological studies in highly characterized tumor tissue and cells during tumor development. We will perform parallel studies to evaluate the level of TGFbeta, E2F, and Rb gene expression and their activities during tumor formation, and by de-regulating their expression at various stages of cell cycle and tumor formation evaluate their importance in the cascade of events which leads to the genesis of cancer cells in brain. The results of these experiments, for the first time, will determine whether delicate balance in the expression of TGFbeta and E2F during the cell cycle phases play a role in the uncontrolled proliferation of cells and tumor formation in the experimental animal.

Progressive Multifocal Leukoencephalopathy (PML) is a subacute demyelinating disease of the human central nervous system (CNS). PML primarily afflicts individuals with defects in cell-mediated immunity and there is strong evidence suggesting that PML results from infection of glial-origin cells, i.e., oligodendrocytes and astrocytes with the human neurotropic virus, JCV. Evidently, these cells are destroyed or transformed during the course of this disease, resulting in demyelinating lesions in the brain. Previous observations by us and others have indicated that the neurotropism of JCV is determined primarily at the level of viral early and late gene transcription. Analysis of viral RNAs during the course of infection indicate differential activation of major viral early and late promoters during the course of infection. Since the early and late promoters are divergent and overlapping, it is of particular interest to determine the mechanism(s) responsible for the preferential use of the early and late promoters before and after DNA replication, respectively. since the transition from early to late occurs during/after DNA replication, when the viral early gene product, T-antigen, is present in the cells, we hypothesize that T-antigen, in concert with cellular factor(s), orchestrates differential expression of viral promoters. The central goal of this research proposal is to identify the mechanism(s) that mediates viral gene expression during the course of infection, and determines the regulatory components (cis- and trans- elements) that are participating in this process. The experimental design utilizes a variety of biochemistry, molecular virology and genetics techniques to identify the key regulatory component(s) that plays a critical role in the stimulation of viral RNA synthesis and viral DNA replication. The information obtained from these experiments should increase our current understanding of cell-type specific gene transcription in the CNS and should provide insight into the replication of viral and cellular DNA in the brain.

DESCRIPTION: This is a request to fund an International Symposium of Neurovirology, to be held at Thomas Jefferson University in Philadelphia. This will be a 2-1/2 day conference with nine formal sessions, a roundtable discussion, and a poster session. The PI will be assisted by an organizing committee of 10 individuals, all of whom have agreed to serve in this capacity. There will be 40 invited speakers and approximately 100 attendees. The meeting will be advertised in several journals. Attendees will be selected by the organizing committee based on their accomplishments in related areas, their backgrounds, and their areas of interest. Minorities, students and postdoctoral fellows will be encouraged to attend. Participants in NIH training programs will have their registration fees waived; all graduate students will receive reduced registration fees. The results of the symposium will be published in the Journal of Neurovirology.

In exploring this viral model of tumor pathogenesis in the CNS, animal experiments are an essential component. Toward that end, the goal of the Experimental Animal and Tissue Culture Core of this program is to support Projects, and to develop the Syrian hamster model of glial tumors derived from the intracerebral inoculation of JCV. In this core, we will focus on four goals. These are: (1) Developing primary brain tumors in newborn hamsters following the intracerebral injection of JCV; (2) Perform MR imaging and post process data analysis of hamster tumors in vivo to define the growth of tumor during the course of studies and obtain a correlative information with the histopathological analysis of the samples. (3) Preparation and inoculation of either JCV-infected Syrian hamster derived tumor cells into the flanks of Nude mice and brains of newborn hamster, to identify their growth characteristics in the whole animal system; (4) Harvesting of CNS tumor tissues which arise in JCV T-antigen transgenic mice; and, This core activity will help to synergize and integrate the program's three major projects and the other core activities.

DESCRIPTION (Adapted from applicant's abstract): Coordination of the immune response to viral infection and disease in the brain is believed to involve bi-directional discourse between the immune system and the central nervous system (CNS). Progressive multifocal leukoencephalopathy (PML) is a neurodegenerative disease associated with a wide range of neurological impairments and neurobehavioral dysfunction including subcortical dementia. The human polyomavirus, JCV, which infects greater than 70% of the adult population, is the etiological agent of this disease. Infection with JCV occurs during childhood and the virus remains at the latent state with no apparent clinical symptoms. However, under immunosuppressed conditions, the virus enters the lytic cycle, and upon cytolytic destruction of glial cells, causes PML. The investigators hypothesize that the bi-modal interaction of immune and nervous systems promotes a regulatory mechanism in the JCV-infected cells which suppresses viral replication and maintains the virus in the latent state. Thus, the absence of this negative regulator in the infected cells leads to the reactivation of JCV and the development of PML. In support of this hypothesis the investigators have demonstrated that secretory factor from immune cells derived from non-PML individuals and that stimulate expression of a cellular protein in glial cells which binds to the viral origin of DNA replication, inhibit JCV DNA replication in glial cells. In this research project, the investigators propose to: (i) determine the immune cell induced negative regulatory factors from glial cells which are responsible for suppression of JCV replication, (ii) evaluate the ability of immune cells from PML patients in suppressing JCV DNA replication and inducing the activity of the candidate suppressor protein; and (iii) molecularly clone the gene encoding the suppressor protein and assess its expression and function in PML and non-PML individuals.

The major thrust of this program project is to investigate the molecular biology of viral-glial gene interaction in the central nervous system (CNS), using a comprehensive and multi-disciplinary approach. Questions such as how viruses replicate in CNS-derived cells must be addressed through studies on the molecular biology of virus gene expression and replication and also in the context of how virus-encoded proteins may alter biological function of the infected cells. Therefore, the most pertinent information will be gathered by parallel studies of viruses with similar, but not identical neurohistopathology in brains from infected individuals. The clinical similarity between progressive multifocal leukoencephalopathy (PML) and AIDS encephalopathy, and the association of PML with HIV-1 infection underscores the rationale for simultaneous studies of the etiological agents of these diseases. We therefore propose to use two human viruses in our studies: (1) JCV, which causes the degenerative CNS demyelinating disease PML, in immunocompromised individuals and; (2) HIV-1, which is associated with a number of neurological disorders and encephalopathy, to investigate the molecular circuits that support viral replication in CNS-derived cells and result in a wide range of neurological disorders such as hypomyelination. The central goal of Project #1 is to understand the mode of JCV early and late gene regulation during the course of infection. Expression of the viral early protein, T-antigen, is critical for the transition from non-productive early phase to productive late phase. Moreover, previous studies using transgenic mice have demonstrated that the production of the JCV T-antigen in the CNS is associated with brain dysmyelination. Thus, experiments are proposed in Project #2 to examine the effect of T-antigen on expression of myelin- associated genes, such as myelin basic protein, proteolipid protein, and myelin-associated glycoprotein in a whole animal system. In Project #3 we propose to study transcriptional regulation of the HIV-1 LTR in glial cells, since our recent observations have indicated the involvement of a novel regulatory pathway in CNS cells that potentiates expression of the viral promotor. Finally, in Project #4, we propose experiments to identify the pattern of HIV-1 replication in astrocytic glial cells from brain of patients with AIDS and examine determinants of latency/productive infection by HIV-1. Together, the information gained from these studies will contribute to our understanding of the molecular pathways that are involved in regulation of viral gene transcription and replication in CNS-derived cells, viral-glial gene interaction, and represent an initial step in devising strategies to block viral replication in these cells.

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) affecting patients with immunosuppressive disorders, especially those infected with the human immunodeficiency virus type 1 (HIV-1). The human neurotropic polyomavirus, JCV, is the established etiologic agent of this disease which has the ability to productively infect and destroy oligodendrocytes, a subclass of glial cells that is responsible for production of myelin proteins and myelin sheaths in brain. The unique ability of JCV to replicate in oligodendrocytes rests on the activation of viral early gene transcription by a series of regulatory proteins present in glial cells. The product of the viral early gene, T-antigen, along with glial regulatory proteins, ensure subsequent events during the lytic cycle which include transcription of the viral late genes and replication of viral DNA. In addition to demyelination of white matter, histologic analysis of PML brain has revealed several morphological abnormalities including the appearance of enlarged oligodendrocytes with loss of normal chromatin, the presence of giant, bizarre astrocytes with pleiomorphic nuclei and mitotic figures in areas with no evidence for active viral replication. These observations suggest that expression of the viral early protein, T-antigen, in the absence of lytic infection, may interfere with host regulatory mechanisms, such as cell cycle circuitry pathways, to induce morphological alterations which are seen in pathological specimens of PML brain. In support of this concept, results from transgenic mice have indicated that expression of the JCV early protein, T-antigen, by a transgene containing the sequence for only the viral early genes induces dysmyelination of the CNS and several histological abnormalities similar to those seen in PML brain. According to our earlier results, the association of JCV T-antigen with myelin gene regulatory proteins and functional inactivation of these proteins may be responsible for the reduced levels of myelin gene expression in the brains of experimental animals. As such, in this research project we propose to: 1) investigate the molecular pathway whereby the JCV early protein, T-antigen, in the absence of viral lytic infection, may affect oligodendrocyte and astrocyte cell function; and 2) determine the molecular mechanism by which the JCV early protein, T-antigen, through its association with host regulatory proteins orchestrates viral gene expression and replication during lytic infection of glial cells. Such comprehensive studies of viral host interaction at the molecular level should enable us to understand the molecular pathogenesis of viral-induced CNS disorders and provide us with critical information and biological reagents for therapeutic intervention.

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) affecting patients with immunosuppressive disorders, especially those infected with the human immunodeficiency virus type 1 (HIV-1). The human neurotropic polyomavirus, JCV is the established etiologic agent of this disease which has the ability to productively infect and destroy oligodendrocytes, a subclass of glial cells that is responsible for production of myelin proteins and myelin sheaths in brain. The unique ability of JCV to replicate in oligodendrocytes rests on the activation of viral early gene transcription by a series of regulatory proteins present in glial cells. The product of the viral early gene, T-antigen, along with glial regulatory proteins, ensure subsequent events during the lytic cycle which include transcription of the viral late genes and replication to ensure subsequent events during the lytic cycle which include transcription of the viral late genes and replication of viral DNA. In addition to demyelination of white matter, histologic analysis of PML brain has revealed several morphological abnormalities including the appearance of enlarged oligodendrocytes with loss of normal chromatin, the presence of giant, bizarre astroyctes with pleiomorphic nuclei and mitotic figures in areas with no evidence for active viral replication. These observations suggest that expression of the viral early protein, T-evidence for active viral replication. These observations suggest that expression of the viral early protein, T-antigen, in the absence of lytic infection, may interfere with hot regulatory mechanisms, such as cell cycle circuitry pathways, to induce morphological alterations which are seen in pathological specimens of PML brain. In support of this concept, results from transgenic mice have indicated that expression of the JCV early protein, T-antigen, by a transgene containing the sequence for only the viral early genes induces dysmyelination of the CNS and several histological abnormalities similar to those seen in PML brain. According to our earlier results, the association of JCV T-antigen with myelin gene regulatory proteins and functional inactivation of these proteins may be responsible for the reduced levels of myelin gene expression in the brains of experimental animals. As such, in this research project we propose to: 1) investigate the molecular pathway whereby the JCV early protein, T-antigen, in the absence of viral lytic infection, may affect oligodendrocyte and astrocyte cell function; and 2) determine the molecular mechanism by which the JCV early protein, T-antigen, through its association with regulatory proteins orchestrates viral gene expression and replication during lytic infection of glial cells. Such comprehensive studies of viral host interaction at the molecular level should enable us to understand the molecular pathogenesis of viral-induced CNS disorders and provide us with critical information and biological regents for therapeutic intervention.

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) affecting patients with immunosuppressive disorders, especially those infected with the human immunodeficiency virus type 1 (HIV-1). The human neurotropic polyomavirus, JCV, is the established etiologic agent of this disease which has the ability to productively infect and destroy oligodendrocytes, a subclass of glial cells that is responsible for production of myelin proteins and myelin sheaths in brain. The unique ability of JCV to replicate in oligodendrocytes rests on the activation of viral early gene transcription by a series of regulatory proteins present in glial cells. The product of the viral early gene, T-antigen, along with glial regulatory proteins, ensure subsequent events during the lytic cycle which include transcription of the viral late genes and replication of viral DNA. In addition to demyelination of white matter, histologic analysis of PML brain has revealed several morphological abnormalities including the appearance of enlarged oligodendrocytes with loss of normal chromatin, the presence of giant, bizarre astrocytes with pleiomorphic nuclei and mitotic figures in areas with no evidence for active viral replication. These observations suggest that expression of the viral early protein, T-antigen, in the absence of lytic infection, may interfere with host regulatory mechanisms, such as cell cycle circuitry pathways, to induce morphological alterations which are seen in pathological specimens of PML brain. In support of this concept, results from transgenic mice have indicated that expression of the JCV early protein, T-antigen, by a transgene containing the sequence for only the viral early genes induces dysmyelination of the CNS and several histological abnormalities similar to those seen in PML brain. According to our earlier results, the association of JCV T-antigen with myelin gene regulatory proteins and functional inactivation of these proteins may be responsible for the reduced levels of myelin gene expression in the brains of experimental animals. As such, in this research project we propose to: 1) investigate the molecular pathway whereby the JCV early protein, T-antigen, in the absence of viral lytic infection, may affect oligodendrocyte and astrocyte cell function; and 2) determine the molecular mechanism by which the JCV early protein, T-antigen, through its association with host regulatory proteins orchestrates viral gene expression and replication during lytic infection of glial cells. Such comprehensive studies of viral host interaction at the molecular level should enable us to understand the molecular pathogenesis of viral-induced CNS disorders and provide us with critical information and biological reagents for therapeutic intervention.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the human central nervous system (CNS) that affects patients with impaired immune systems, including AIDS. A human papovavirus, JC virus, (JCV), has been implicated as the etiologic agent of this disease. JCV infects over 70 percent of the population at a very young age with no apparent clinical symptoms. In the brain of PML patients, viral particles are detected in cells of glial origin, i.e. oligodendrocytes and astrocytes. These cells are destroyed or transformed upon reactivation of the latent virus, resulting in demyelinating lesions in the brain. Reactivation of the virus and appearance of disease is associated with severe immunosuppression of cell- mediated immunity, yet the nature of control exerted by the immune system and mechanism that maintains JCV in its latent state is not understood. Results from a series of molecular and cellular studies revealed the involvement of several signaling pathways transmitted by immune cells via cytokines and immunomodulators that control expression and replication of the viral genome. In order to understand the underlying mechanism that controls replication of this neurotropic virus in glial cells, we have developed an in vitro cell culture system which allows us to investigate JCV DNA replication in the presence of immune cell secretory factors. Our results indicate that supernatant derived from activated immune cells is capable of suppressing replication of viral DNA in glial cells. Results from in vitro DNA-binding studies indicate that treatment of glial cells with T cell supernatant leads to elevated levels of nuclear protein in glial cells which binds specifically to the origin of viral DNA replication. Thus, we hypothesize that signals transmitted by functional immune cells to glial cells induce expression/activity of a suppressor protein which, by binding directly to the viral DNA sequence or indirectly via interaction with other regulatory proteins, prevents replication of the virus in these cells. In the absence of functional immune cells (i.e. immunosuppression), the lack of this barrier in glial cells permits viral replication in glial cells and the development of clinical disease in braom. To examine our hypothesis we propose to use immune cells from normal as well as immunosuppressed patients with PML to decipher the cross- communication between immune and nervous system and JCV gene expression. Furthermore, by employing nucleic acid array technology, we should gain important information on determining the portrait of cytokines and immunomodulators in healthy and diseased immune cells, as well as in glial cells exposed to immune cell factors from normal, and PML patients. The results of these experiments will provide unique information regarding the mechanisms involved in the reactivation of JCV in immunosuppressed individuals, and provide us with an intriguing biological tool to develop and use an antibody against the signaling factor/suppressor protein to determine its level in PML brain biopsy and normal brain tissue by immunohistochemical analysis, and to devise a strategy to block replication of the JCV genome.

The human neurotropic virus, JCV, is widespread in the human population and is the established etiology agent of the fatal demyelinating disease of the central nervous system (CNS), Progressive Multifocal Leukoencephalopathy (PML). Once a rare disease associated with lymphoproliferative and myeloproliferative disease, PML is now more frequently seen due to the AIDS epidemic. The JCV genome consists of circular double-stranded DNA that is separated into early and late coding sequences by the viral cell type-specific regulatory region. The viral early gene, T-antigen has important regulatory functions in orchestrating the viral lytic cycle and possesses the ability to interact with several important cellular proteins including the tumor suppressor protein, p53, pointing to the oncogenic potential of this virus. In support of this notion, earlier suppressor protein, p53, pointing to the oncogenic potential of this virus. In support of this notion, earlier studies have revealed that JCV has the ability to induce neural origin tumors in several animal models. By using the early genome of JCV, we have created transgenic animals that develop cerebellar primitive neuroectodermal tumors with extraordinary similarity to human medulloblastoma. Results from genetic and biochemical studies have revealed that while in some cells T-antigens is expressed and found in association with p53, there exists a population of tumor cells with extremely low, if any, levels of T-antigen production. Cell lines derived from several tumor tissues have allowed further characterization of T- antigen positive and T-antigen negative cells and are identified the presence of a novel mutant p53 with a deletion between residues 35 and 123. This observation is consistent with a hypothesis in which, at the early stage of the disease, expression of JCV T- antigen in primitive neuroectodermal cells and its association with p53 can functionally inactivate this tumor suppressor protein. As p53 can functionally inactivate this tumor suppressor protein. As p53 controls cell proliferation at the G1 and G2 stages of the cell cycle, and plays an important in genomic stability In this research project, we propose to: i) investigate p53 and its downstream effectors in T-antigen positive and T- antigen negative tumor cells at different phases of the cell cycle in vivo, and in tumor tissues during various stages of brain and tumor development; ii) conditionally knockout the function of p53 in transgenic mice at the desired time during brain development by expression of T- antigen in the rain utilizing an in vivo T-antigen tetracycline inducible system and evaluate p53-dependent cell cycle pathways; and iii) determine the oncogenic potential of mutant p053 as well as T-antigen in the development of medulloblastoma in the absence of wild-type p53 by expressing these proteins in p53 null cells and p53 knockout transgenic animals. In light of recent data pointing to the association of JCV with human medulloblastomas and the potential for the participation of p53 in these tumors, the study of JCV-T antigen transgenic animals should yield information involved in the development of pediatric medulloblastomas.

Project #1: Cell Cycle/Transcription Regulation in Medulloblastoma By using the early genome of the human neurotropic papovavirus, JCV, we have created transgenic animals that develop cerebellar primitive neuroectodermal tumors with extraordinary similarity to human medulloblastoma. Results from genetic and biochemical studies have revealed that while in some cells T-antigen is expressed and is found in association with p53, there exists a population of tumor cells with extremely low, if any, levels of T-antigen production. Cell lines derived from tumor tissue have allowed further characterization of T-antigen positive and T-antigen negative cells and have identified the presence of a novel mutant of p53 with a deletion in exon 4. This observation is consistent with a hypothesis in which, at the early stage of the disease, expression of JCV T-antigen in primitive neuroectodermal cells and its association with p53 can functionally inactivate this tumor suppressor protein. As p53 controls cell proliferation at the G1 and G2 stages of the cell cycle, and plays an important role in genomic stability, inactivation of p53 can cause deregulation of normal events of the cell cycle resulting in genomic instability. Evaluation of tumorigenecity of T-antigen positive and T-antigen negative cells revealed that unlike T-antigen negative cells, T-antigen positive cells are highly tumorigenic in nude mice suggesting that an additional pathway affected by T-antigen by T-antigen may be involved in this event. Examination of a Wnt signalling pathway which is implicated in neurogenesis and oncogenesis revealed stabilization of beta-catenin and its nuclear appearance in T-antigen positive, but not in T-antigen negative cells. The stabilized beta-catenin, upon association with the LEF transcription factor, can enter nuclei and by stimulating cyclin D 1 and cmye, deregulate the cell cycle and induce rapid cell proliferation. These observations led us to hypothesize that the evolution of medulloblastomas by T-antigen is mediated through distinct pathways such as inactivation of p53 and deregulation of Wnt. In this research project we propose to: i) Create and utilize an inducible animal model system that permits conditional expression of the JCV T-antigen in mouse brain during development and correlate T-antigen expression with histological markers and biochemical parameters; ii) Examine the ability of JCV T-antigen and mutant p53 (with a deletion in exon 4) in the induction of medulloblastoma in the absence of wild-type p53 b) creating JCV T-antigen and p53 mutant transgenic animals on the p53 null background; iii) Study the WNI signaling pathway in T-antigen transgenie mice which is programmed for developing PNETs during various stage, of brain development by evaluating the cytoplasmic and nuclear levels of beta-catenin and the level of LEF association with catenin in nuclei, and the activity of LEF-responsive genes including c-myc and cyclin D during the course of tumor formation. In light of recent data pointing to the association of JCV with human medulloblastomas and the potential for the participation of p53 mutant in these tumors, the study of JCV T-antigen transgenic animals should yield important information regarding the mechanism involved in the development of pediatric medulloblastoma.

[unreadable] DESCRIPTION (provided by applicant): This application seeks support for 5th International Symposium on Neurovirology that will be held as a 2.5 day meeting scheduled for September 25-27, 2003 in Baltimore, Maryland. The 5th Symposium will be preceded by a 2-day Workshop on HIV Molecular & Clinical Neuroscience to be held in the same location. Specifically, the application requests funding to cover the costs of (1) plenary session speakers, (2) travel grants for graduate students, post-doctoral fellows, and clinical fellows, (3) publication costs associated with the preparation of the program and abstracts in the Journal of Neurovirology, the official journal of the International Society of Neurovirology, (4) preparation of on-site registration packet materials, (5) poster session costs, (6) rental of audiovisual equipment, (7) rental of rooms for workshop and poster sessions, (8) secretarial effort association with registration, program preparation, abstract processing, and general meeting preparation activities, and (9) web-based management of all meeting activities. The Specific Aims of the 5th International Symposium on Neurovirology are to provide a forum for the dissemination of new information related to (1) the impact of other neurologic diseases and the aging process on HIV-associated CNS disease, (2) the impact of the innate immune responses on virus infection of the CNS, (3) homeostasis in the CNS and the effects of viral infections in astrocytes, (4) protein folding diseases, including prions and cellular proteins, (5) bioterrorism and emerging infections, including the assessment of risks posed by neurotropic viruses, (6) host genetics and the impact of viral infection of the CNS, (7) gene therapy and neural stem cell approaches to CNS infections, and (8) neuroimmune responses and CNS trafficking. Overall, the 5th Symposium will seek to update investigators working the field of neurovirology and related disciplines with leading edge information so that important gaps in knowledge can continue to be identified. Armed with this information, attendees will work toward formulating new questions and experimental directions to enhance the development of new strategies to prevent and treat neurologic disease associated with prions, HIV, and other viral pathogens. The application also seeks an additional two-years of funding to establish the Symposium Series as an annual event to continue to develop this important area of investigation. It is anticipated that approximately 350 individuals attending the 5th Symposium. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This application seeks support for 6th International Symposium on NeuroVirology that will be held as a 2.5 day meeting scheduled for September 10-14, 2004 in Sardinia, Italy. The 6th Symposium will be preceded by a 2-day Workshop on Neuroprotection and HIV-1 CNS Diseases to be held in the same location. Specifically, the application requests funding to cover the costs of (1) plenary session speakers, (2) travel grants for graduate students, post-doctoral fellows, and clinical fellows, (3) publication costs associated with the preparation of the program and abstracts in the Journal of NeuroVirology, the official journal of the International Society of NeuroVirology, (4) preparation of on-site registration packet materials, (5) poster session costs, (6) rental of audiovisual equipment, (7) rental of rooms for workshop and poster sessions, (8) secretarial effort association with registration, program preparation, abstract processing, and general meeting preparation activities, and (9) web-based management of all meeting activities. The Specific Aims of the 6th International Symposium on NeuroVirology are to provide a forum for the dissemination of new information related to (1) emerging infections and biodefense, (2) virus-host cell receptor interaction, (3) regulation of viral gene expression and life cycle, (4) multiple sclerosis, (5) prion detection and transmissible encephalopathy, (6) viral neuropathogenesis, (7) latency and reactivation, (8) demyelinating disorders, and (9) tumor virology. Overall, the 6th Symposium will seek to update investigators working the field of neurovirology and related disciplines with leading edge information so that important gaps in knowledge can continue to be identified. Armed with this information, attendees will work toward formulating new questions and experimental directions to enhance the development of new strategies to prevent and treat neurologic disease associated with prions, HIV, and other viral pathogens. It is anticipated that approximately 350 individuals attending the 6th Symposium. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a competing continuation of a program project seeking support to further our investigation of the molecular biology and genetics of the interaction between viruses and host cells in the central nervous system. The major thrust of this program remains the study of the mechanism of two viral-induced diseases of the CNS: i) Progressive multifocal leukoencephalopathy (PML), which is caused by replication of the human neurotropic JCV in glial cells; and ii) the Acquired Immune Deficiency Syndrome (AIDS) associated dementia and encephalopathy which is induced by the presence of HIV-1 in brain. The clinical ties between PML and AIDS, the association of PML with HIV-1 infection, and the utilization of common regulatory pathways by these two viruses in the CNS underscores the rationale for the simultaneous investigation of the molecular biology of these two viruses and the mechanism of the diseases. During the first period of funding (1993 to 1999), we focused our attention on deciphering the molecular pathways involved in the control of viral gene expression in brain. In the second period of funding (1999 to 2004), our emphasis has been on the interaction of viral proteins with the host cell cycle and cross-communication between infected and uninfected CNS cells via cytokines and viral proteins. Through highly interactive and synergistic collaboration, the participants of this program have discovered several cellular proteins including Pur-alpha that modulate JCV and HIV-1 gene expression by associating with T-antigen and Tat, respectively; and uncovered several regulatory pathways that modulate HIV-1 gene expression in glial cells, such as TAR-independent pathway that enables Tat to augment expression of TAR-negative genes such as cytokines and JCV in CNS cells. In this current program, our emphasis on studying viral-host interaction rests on the impact of viral infection and viral proteins involved in the maintenance of host cells genomic stability, and the effect of factors involved in the control of gene integrity on regulation of viral gene expression and replication. This novel avenue of research was chosen based on the most current discoveries pointing to the cross-talk between viruses and DNA repair machinery that contributes to dysregulation of pathways involved in the integrity of the host genome and activation of viral gene expression and regulation. In Project #1 (K. Khalili et al.), we will investigate the effects of T-antigen and Agnoprotein of JCV which are produced at the early and late phases of infection, respectively, on homologous recombination and non-homologous end-joining. In Project #2 (S. Amini et al.), we will investigate the molecular interaction between HIV-1 Tat and RadSl, a key component of homologous recombination pathway, and their impact on HIV-1 gene transcription and replication via several cellular proteins including C/EBPbeta and NF- kappaB whose function is implicated in AIDS CNS disease. In Project #3 (J. Rappaport), we will investigate the physical and functional communication of the regulatory proteins of HIV-1 (Tat) and JCV (T-antigen and Agnoprotein) with Pur-alpha and their impact on DNA repair and genomic stability in CNS cells. These three highly integrated but independent projects will utilize a central Neuropathology and Tissue Culture Core which will provide a reliable source for distribution of clinical samples and preparation of primary and established cells from the CNS. This program project brings together basic scientists and physicians with expertise in neurovirology, molecular retrovirology, CNS cell biology, and pathology to perform the studies.

The human polyomavirus, JCV, is the etiologic agent of Progressive Multifocal Leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS) that usually affects immunosuppressed patients. JCV has an unusually narrow tissue tropism which restricts its productive replication to oligodendrocytes, a subclass of glial cells in the CNS which are responsible for production of the myelin sheath. Earlier studies by us and others have indicated that the cell type-specific tropism of JCV to glial cells is determined at the level of viral early gene transcription, which is responsible for the production of T-antigen. In collaboration with host regulatory proteins, T-antigen orchestrates subsequent events during the viral lytic cycle including viral DNA replication and late gene transcription and leads to the destruction of oligodendrocytes and demyelination of the brain. Histological analysis of PML brains has revealed that in addition to demyelination of white matter, a number of abnormalities are observed in both surviving oligodendrocytes and the other subclass of glial cells, astrocytes, suggesting dyregulation of pathways responsible for host cell homeostasis including control of the cell cycle and DNA repair. In support of this notion, results from immunohistochemical staining of clinical samples revealed the presence of double-strand breaks, indicative of dysfunctionality of DNA repair, an unusual expression of proteins which are in control of cellular proliferation, and apoptosis. Accordingly, results from in vitro cell culture infection revealed that the JCV early protein, T-antigen, and the viral non-structural protein, Agnoprotein, by dysregulating the level of expression and activities of factors involved in cell cycle control and DNA repair may affect a process that leads to the development of some of the pathological features that are seen in oligodendrocytes and astrocytes of PML. In this research project we aim to: (i) investigate the effect of JCV early protein on process involved in genomic stability at the early stage of viral infection by analyzing its interaction with IRS-1, p53, and Rad51, three critical proteins that, by communicating with T-antigen, can influence the process of homologous recombination; (ii) determine the molecular mechanisms by which the JCV late protein, Agnoprotein, through its association with p53 and Ku70 dysregulates non-homologous end joining during the course of infection. Such comprehensive studies of viral -host interaction at the molecular level should enable us to understand the molecular pathogenesis of JCV-CNS disorders and provide us with pivotal information for therapeutic intervention.

Project #3: In the central nervous system, HIV-1 induced disorders are coupled with dysregulation of cytokines as a result of their abnormal pattern of expression. Among these cytokines, Tumor Necrosis Factor a (TNFa), has captured much attention due to the positive feedback interplay between the HIV-1 regulatory protein, Tat, and the downstream executors of the TNFa signaling pathway, including NFKB transcription factors. Expression of TNFa can also be dysregulated by opiates suggesting that cooperativity between HIV-1 and opiates at the molecular level may influence the neuropathogenesis of AIDS in drug-addicted patients. However, the molecular mechanism involved in the activation of TNFa upon HIV-1 infection in the context of drugs of abuse such as morphine remains unknown. Our preliminary results support the possible involvement of the p65 subunit of NFKB and p38 MARK signaling in this event. Activation of TNFa can promote oxidative stress in cells which, in turn, initiates a positive feedback cascade of events that further enhances TNFa production. Morphine has been shown to induce oxidative stress via an unknown pathway leading us to envision a role for TNFa in morphine-induced oxidative stress during the course of viral infection. Together with activated TNFa, the HIV-1 regulatory protein (Tat) and the envelope protein (gp120) can exert a toxic effect on neurons causing neuronal injury and death and morphine can accelerate this by affecting several key regulatory events that control neuronal cell survival. We plan to identify the mechanism by which morphine and HIV-1 infection upregulate TNFa gene expression and to determine the signaling events that are involved in Tat, gp120, and TNFa induced neuronal cell dysfunction. In the context of this program project, we will be able to develop and utilize, in parallel, cell culture models from HIV-1 and SIV-1 infected macrophages from human and monkey to unravel the various regulatory pathways that are affected upon drug treatment and viral infection, and cross examine the biological relevance of the in vitro cell culture findings to the results from the experimental SIV/macaque model that is the central focus of this program. Through such an integrated synergism with the other participants of this program, the outcome of these molecular studies will provide critical information that can be translated toward the development and use of therapeutic approaches in addicted AIDS patients.

DESCRIPTION (provided by applicant): HIV-1 infection of the central nervous system (CNS) induces conditions of stress that trigger an array of cellular responses with the capacity to affect HIV-1 gene expression, alter the homeostatic state of the host cell, and stimulate co-existing opportunistic pathogens. Recent results from evaluating brain tissue obtained from AIDS patients with neurologic disorders have revealed enhanced expression of BAG3 in astrocytes and perivascular microglial cells. BAG3 is an anti-apoptotic/pro-survival protein that associates with a key stress response chaperone protein, HSP70, and modulates its activity on re-folding of damaged proteins and delivery of its cargo to proteasomes. These events influence several pathways involved in cell survival and apoptosis including mitochondrial membrane depolarization, caspase activation, DNA damage, cell cycle progression, and others. Activation of BAG3 can also be observed during the course of HIV-1 infection of microglia, a cell type that supports viral replication in the brain. Interestingly, activation of BAG3 appears to augment cell survival as silencing of BAG3 by siRNA increases the rate of apoptosis in HIV-1 infected microglial cells. Induction of BAG3 may have a negative impact on HIV-1 replication as BAG3 possesses the ability to suppress HIV-1 gene transcription in both microglia and astrocytes. Indeed these events may be reversed once the level of BAG3 is reduced in the cells. In glial cells this can be accomplished upon activation of the human opportunistic virus, JCV, whose early protein suppresses BAG3 transcription and may alleviate the negative effect of BAG3 on HIV-1 and promote apoptotic pathways. These observations are relevant to the neuropathogenesis of AIDS as replication of JCV, which results in the development of progressive multifocal leukoencephalopthy (PML), is frequently seen in AIDS patients. All of these observations have led us to hypothesize that BAG3, by assisting cells to survive the initial infection with HIV-1, can play a critical role in converting cells to become a long-term reservoir for the virus. With this notion, we plan to investigate the molecular events involved in the differential regulation of BAG3 in CNS cells, identify the pathway by which BAG3 suppresses HIV-1 expression and replication, investigate the impact of JCV via suppression of BAG3 upon HIV-1 expression, and determine the mechanism involved in BAG3 mediated cell survival in HIV-1 infected cells. We will employ molecular, cellular, and virological approaches to address these questions and examine the biological relevance of our findings by immunohistochemical evaluation of clinical samples from patients with HIV-1 CNS disease. PUBLIC HEALTH RELEVANCE: The ability of certain cell types to survive for long periods following HIV-1 infection and thereby to function as long-term viral reservoirs is a critical consideration in HIV-1 therapy and appears to be a major obstacle to eradicating the virus from infected hosts particularly from brain. The studies proposed in this application explore the involvement of one of the key cellular proteins, BAG3, in this event.

DESCRIPTION (provided by applicant): This application seeks support for an animal neuroimaging package which will allow us to adapt a brand new state-of-the-art human 3 Tesla MRI scanner for small animal neuroimaging. Funds are requested to obtain customized rat and mouse coils fully compatible with our new 3 Tesla human scanner that are designed for high resolution brain and spinal cord imaging. The 3 Tesla human MRI will be devoted exclusively to research. The machine and coils will form the basis for a "Small Animal MR Imaging Core Facility" at Temple University School of Medicine which will primarily serve the needs of NIH funded researchers from the Departments of Microbiology, Neuroscience, Pharmacology, and Radiology at Temple University School of Medicine, the Department of Computer Science at Temple University's College of Science and Technology, and the Department of Psychology at Temple University's College of Liberal Arts. Additionally, access will be granted to secondary users from the Departments of Neurology, Neurosurgery, and Physiology in the School of Medicine. The primary and secondary users of this core facility focus their research efforts on a broad range of paradigms including pharmacotherapeutic, behavioral, and physiological changes in neurological disorders as well as molecular pathogenesis of neuronal dysfunction, demyelination, CNS neoplasia, and the development of novel molecular imaging agents and post-process techniques. The Small Animal MR Imaging Core Facility will have priority usage of the human 3 Tesla MRI and allocation of core resources will be made by an Advisory Committee. Institutional commitment for the Small Animal MR Imaging Core Facility has been secured insuring its maximum usage and efficiency for in vivo translational research. The ability to perform high resolution neuroimaging and longitudinal studies on a human 3 Tesla magnet will give us the ability to directly translate our findings in animal models to non-invasive diagnostic, molecular, and functional neuroimaging in the clinic. PUBLIC HEALTH RELEVANCE: The proposed magnetic resonance imaging (MRI) equipment will allow us to use a human MRI to scan our small animal models of neurological disorders at high power. In vivo imaging of these models will allow us to test and validate non-invasive novel diagnostic and therapeutic strategies that can be directly used in the clinic.

The main objective of this application is to establish a Comprehensive NeuroAIDS Core Center (CNACC) at Temple University with the goal to improve and expand research related to HIV-1/AIDS and its associated neurological and mental disorders. The infrastructure built through this core facility will provide local and regional investigators with broad, reliable and efficient services including establishing primary mammalian cell culture from brain and virus infection, gene expression and proteomics analysis at the tissue, cellular, and molecular levels to unravel the mechanisms of disease and discoveries of new biomarkers, developing new experimental animals to investigate the molecular biology, pathogenesis, and cognitive aspects of HIV-1 CNS disease, and clinical and neurobehavioral examination at both domestic and international levels. The Administrative Core will provide the infrastructure for coordinating and overseeing all activities of the CNACC including delineation of the strategic plan, identification of short and long term objectives, execution of detailed plans to be implemented, carrying out internal and external reviews and assessment tools based on established criteria to ensure productive and efficient use of resources to achieve the overall objectives of CNACC and offer biostatistical support for data analysis and interpretation obtained from basic science and clinical cores.

BASIC SCIENCE I CORE: CELL CULTURE/NEUROTROPIC VIRUS, NEUROSCIENCE, AND PROTEOMICS The overall objective of the Basic Science Core I (BSCI) is to provide in vitro shared resources and training to basic and clinical researchers to study neurological disorders at the molecular level, related to Human Immunodeficiency Virus (HIV) infection. During HIV central nervous system (CNS) disease, disparities between virus-associated insults and host-mediated attempts at rescue and repair are responsible for cognitive deficits. To protect cells of the CNS, and to promote beneficial responses, key elements in the signaling crosstalk between cells of the CNS and virus must be understood. Information regarding cell-virus biochemical signaling interactions must be addressed, in large part, by in vitro studies. The BSCI will provide investigators with reliable access to high quality CNS cell cultures, virus and viral products, and other factors that contribute to HIV CNS disease which are critical to delineate signaling pathways important in disease progression. BSCI will also provide assistance and expertise in classical neuroscience techniques and tools to support investigation of cellular/molecular interactions between cells of the CNS and HIV that underlie neurocognitive dysfunction. The BSCI will also provide state-of-the-art services for discovering the molecular mechanisms involved in the development of AIDS-associated CNS dysfunction through proteomics and data analysis. Novel methods for discovering of biomarkers, innovative differential expression profiling and bioinformatics along with interactomics and phosphoproteomics are among the services that will be reliably and efficiently offered to the investigators. Our core will work closely with the other cores to promote a comprehensive multidisciplinary collaborative center program. This synergistic approach will ensure the success of both recipients of CNACC developmental awards and other members of the Center in conducting productive high impact research into HIV associated neurological disorders.

DESCRIPTION (provided by applicant): Infection of the central nervous system with HIV-1 can trigger a cascade of defense mechanisms that are aimed at blocking expression of the viral genome at various stages of its life cycle. In turn, HIV-1 has evolved several regulatory events via its accessory proteins, more notably Tat, to overcome the cellular defense pathways and through a series of diverse modulatory event induces maximum damage to the cells while ensuring a productive viral life cycle in the infected cells. Recently, much attention has been focused on the impact of HIV-1 infection on host cell homeostasis, more specifically the interaction of HIV-1 with cellular pathways that control chromosomal integrity via homologous and non-homologous DNA repair. While the role of HIV-1 Vpr in affecting DNA damage is well documented, recent observations (shown here) point to the ability of Tat in the induction of Rad51 expression in primary microglia and astrocytes, the two cell types that play an important role in neuropathogenesis of AIDS. This observation corroborates the results from infection studies showing increased levels of Rad51 in HIV-1 infected microglia and astrocytes in culture and in AIDS brain with HIV encephalitis. Rad51 is the major regulator of the homologous recombination repair pathway that in coordination with other cellular proteins ensures chromosomal integrity. Evidently, unscheduled activation of Rad51 may have an adverse impact on several cellular pathways and in some instances even compromise chromosomal integrity. Interestingly, induction of Rad51 by Tat protein may have a positive feedback effect on HIV-1 promoter activity in microglia and astrocytes. In this respect, our preliminary data point to the possible recruitment of NF-B and cyclin T1, the two regulators of HIV-1, by Rad51 for stimulation of the LTR in CNS cells. All these observations provide a rationale for us to hypothesize that the reciprocal interaction of HIV-1 through its transactivator, Tat, with the host recombination repair regulator, Rad51 creates a condition that leads to activation of the HIV-1 promoter in microglia, astrocytes and possibly macrophages, and alters host recombination repair pathways in favor of HIV-1. In this application, we seek support to launch a series of well-integrated molecular, virological, and cellular studies to decipher the molecular events associated with cross-communication of HIV-1 and host DNA repair machinery and develop a strategy, based on our observations, to inhibit HIV-1 gene expression and activation in cells that support viral infection. Throughout our studies the relevance of our molecular discoveries to the neuropathogenesis of HIV-1 will be verified at every stage through the use of a unique collection of brain specimens from HIVE patients (provided by the Manhattan Brain Bank). Thus, through this novel integrated approach, our studies will provide important information relevant to HIV-1/CNS diseases that can be used to improve the current method for treatment of AIDS patients with neurological disorders.

This revised application seeks support to establish a Comprehensive NeuroAIDS Core Center (CNACC) at the Temple University School of Medicine in order to bring highly needed infrastructure for basic scientists and clinicians involved in HIV-1/AIDS research and neurological, neurodegenerative, and neurobehavioral disorders. The central theme of CNACC is structured based on our hypothesis that understanding the mechanisms of HIV-1/CNS interactions at the molecular, cellular, and experimental animal model levels and bidirectional communication of laboratory findings and clinical observations to validate basic science discoveries are prerequisites for the development of effective, safe, and reliable approaches for early diagnostics and therapeutics for AIDS-associated neurological dysfunctions. The CNACC will provide unprecedented infrastructure to a large group of neuroAIDS investigators who plan to pursue their objectives using multidisciplinary approaches in cell culture, small animal models, and in the clinical setting for assessing gene expression and biomarker identification at the cellular and molecular levels. Further, through the Developmental Core, the CNACC will provide a unique opportunity for training and mentoring of junior and clinical investigators and attract and develop physician scientists in the field of neuroAIDS. Through CNACC, we will provide start-up funds for new and innovative pilot projects of newly recruited, independent investigators and will support feasibility studies for more established neuroAIDS investigators. The funding through this center will create a unique infrastructure that will serve to enhance and extend the effectiveness of ongoing HIV-1 investigations and promote translational research in neuroAIDS at Temple and other medical institutions in the greater Philadelphia area that are involved in basic science and clinical AIDS research. With its comprehensive structural organization encompassing broadly based cores ranging from molecular biology to experimental animals to the clinical arena directed by skilled and highly competent investigators from various disciplines, CNACC will support research in a variety of areas such as virology, basic and behavioral neuroscience, and clinical science, all of which are aimed toward the discovery of better diagnostics and effective therapeutic agents toward AIDS/CNS disorders.

DESCRIPTION: While long term combined antiretroviral therapy (cART) has greatly improved survival rates among AIDS patients, a substantial proportion of HIV-1 infected individuals continue to develop comorbidities including heart failure (HF) secondary to left ventricular dysfunction at higher rates than non-HIV-1 individuals. The underlying mechanisms whereby HIV-1 increases susceptibility to HF remain poorly understood. In this respect, HIV-1 Tat, which is produced and released by the latent viral reservoir and upon its circulation can be taken up by uninfected cells, has received special attention due to its ability to induce an array of dysregulatory events that perturb cell and organ function. HIV-1 Tat has the capacity to induce injury and promote death in a broad range of cells including myocytes, and its expression promotes cardiac disease in laboratory animals. Our recent studies demonstrate that HIV-1 Tat physically associates with BAG3, a stress induced protein which is involved in protein quality control, and modulates autophagy and apoptosis. BAG3 is critical for normal cardiac development and maintenance as BAG3 knockout mice develop left ventricular (LV) dysfunction and have a shortened lifespan. Furthermore, recent studies have identified a potential role for BAG3 in patients with HF as heterozygous mutations that decrease the level of BAG3 have been identified in patients with HF within families who develop a heritable form of dilated cardiomyopathy. Moreover, ventricular myocardium isolated from failing human hearts examined at the time of heart transplantation exhibit nearly 50% reduction in BAG3 levels in cardiac tissue, all of which supports the importance of BAG3 for healthy heart function. At the subcellular level, the association of BAG3 with the actin capping protein, CapZ?1, promotes its complexation with Hsp70, events that stabilize CapZ?1and facilitate its proper subcellular distribution in cardiomyocytes. In addition, BAG3 is an important regulator of filamin and myopodin and regulates protein turnover by autophagy in cardiomyocytes. Our preliminary results suggest that the interplay between BAG3 and HIV-1 Tat impacts the ability of BAG3 to regulate several pathways involved in the structural and functional integrity of cardiomyocytes. Thus, one can envision a model in which the inactivation of BAG3, upon its association with HIV-1 Tat, recapitulates the clinical manifestations seen in patients with heart failure associated with low/dysfunctional BAG3. By capitalizing on the expertise of two groups of accomplished investigators in cardiovascular research and in HIV-1 pathogenesis and BAG3 biology, we will investigate the cellular and molecular mechanisms involved in HIV-1 induced cardiomyopathy in cell cultures, animal models, and clinical samples. The outcome of these studies will provide a unique set of information aimed at developing prognostic biomarkers of disease severity and therapeutic strategies for treating HIV-1 associated cardiomyopathy.

DESCRIPTION (provided by applicant): According to the CDC, greater than 1.1 million people in the United States and more than 35 million people worldwide are infected with HIV-1. While the introduction of combined antiretroviral therapy, cART, has greatly improved survival rates among AIDS patients, a substantial portion of HIV-1 infected individuals remain at risk for the development of full blown AIDS as a result of reactivation of latently infected cells, partly due t nonadherence to medication and emergence of drug resistant viruses. Moreover, HIV-1 positive long term survivors continue to develop comorbidities including an accelerated aging process, neurocognitive disorders, heart failure, and others. From the virological point of view, as none of the current treatments suppress viral gene transcription, it is suspected that low, yet continuous, levels of viral early proteins with regulatory and pathogenic activities may contribute to the development of these quality of life threating illnesses. Sadly, none of the efforts toward the development of vaccines against HIV-1 have shown promising outcomes. Thus, curing of AIDS by eradicating the HIV-1 genome in infected subjects requires a novel strategy that is specific, highly effective, sustained, and irreversible. Recently, we have adapted a genetic approach using the clustered regulatory interspaced short palindromic repeat-assisted system (Cas) and a short complementary single-stranded RNA, called guide RNA or gRNA, which specifically targets the U3 region of the HIV-1 LTR promoter and precisely excises a segment of the viral regulatory sequence required for its expression. In addition, the employment of single and multiplex gRNA in our Cas system show promising results that include eradication of the entire HIV-1 genome in latently infected microglial cells, thus abrogating viral gene expression and transcription. Based on this preliminary observation, we propose to develop an RNA-guided Cas9 that acts as molecular scissors and, by disrupting various regions of the LTR and/or removing the entire viral genome, abrogates reactivation of the virus in macrophages, microglia and astrocytes which serve as the viral reservoir in the brain. Furthermore, we will explore the feasibility of our single and multiplex Cas9 system for use as a prophylactic compound in in vitro HIV-1 infection culture models. The outcome of this molecular genetic and virological approach will provide a solid platform for developing preclinical and clinical studies toward the treatment f AIDS and its associated neurological and neurobehavioral disorders.

SUMMARY The Administrative Core is an integral component of this Program Project Grant. The Core will have primary responsibility for all financial management of the projects and cores. The Administrative Core will act as an extension of the Department of Neuroscience Administrative Office to maintain all accounting records. The Administrative Core will manage purchasing of supplies, major and minor equipment, and all necessary materials for each project and core. In addition to its financial responsibilities, the core will also have various administrative responsibilities. The administrative coordinator will work closely with Dr. Khalili on tasks such as scheduling the monthly Executive Committee meetings to review progress. This Core will also organize the annual External Advisory Committee meeting making all necessary travel arrangements and hotel accommodations for committee members, and the annual retreat for core participants. The Core will prepare all manuscripts and maintain records of correspondence, etc. The Core will prepare the annual non-competing continuation progress reports. The Administrative Core will also maintain records on all institutional protocols regarding the use of human subjects, vertebrate animals, biological materials, technology transfer and patent usage, etc. This Program Project will require a significant exchange of biologically active reagents and samples. The Administrative Core will closely collaborate with the Project Leaders on a regular basis to facilitate the receipt and exchange of all biologically active reagents utilized within the individual laboratories.

? DESCRIPTION (provided by applicant): More than 20% of the glucose used by the body is consumed by the brain, thus, any alteration in the physiological process associated with glucose metabolism and energy regulation can have deleterious effects on brain functioning. Emerging data indicate that acute and chronic exposure to cocaine alters metabolic and bioenergetic pathways in the brain as well as in other organs and affects mitochondria function and ATP production. Cocaine abuse is a significant risk factor for becoming infected with HIV-1 and studies show that in combination, cocaine and HIV-1 lead to significantly greater damage to the CNS. In this context, our recent studies point to the negative impact of HIV-1 infection on glycolytic pathways in macrophages, which is beneficial for viral replication, and the ability of HIV-1 Tat released by the infected macrophages and microglia to disturb the integrity of mitochondrial DNA bioenergetic pathways and mitochondria turnover in neuronal cells. All of these observations along with previous reports on the ability of cocaine and HIV-1 Tat to induce production of mitochondrial reactive oxygen species (ROS) provides a rationale for us to hypothesize that cocaine and Tat synergistically disrupts metabolic and bioenergetic pathways in the brain, disturbing neuronal cell function, affecting oligodendrocyte status and their communication with neurons, thereby promoting neurologic/neurocognitive disorders in cocaine/HIV-1 cohorts. Here, we have developed a comprehensive, integrated multidisciplinary research program composed of three projects led by experts in neuroscience of HIV-1, cocaine research, metabolic pathways and mitochondria biology to investigate, in Project #1, the reciprocal interactions of cocaine/HIV-1 on glucose metabolism in macrophages and microglia, the cells that are primarily responsible for latency and productive HIV-1 infection in the brain an the release of viral and cellular toxic factors including HIV-1 Tat. In Project #2, we will investigate the impact of cocaine and HIV-1 Tat, released by infected cells on mitochondrial homeostasis in neurons by concentrating on genetics of mitochondrial DNA on mitochondrial bioenergetic pathways and ATP production, and biogenesis of mitochondria. In Project #3, we will focus our attention on the effects of cocaine and HIV-1 Tat on oligodendrocytes and the cross-talk between oligodendrocytes and neurons in relation to glucose metabolism, lipid biosynthesis and retinoic acid signaling. This program will be supported by a highly specialized, state-of- the art core facility: Mitochondria Physiology and Imaging Core (Core B) and will greatly benefit from the unique infrastructure offered by two NIH-funded centers, i.e. Center for Substance Abuse Research (CSAR) and Comprehensive NeuroAIDS Center (CNAC) at our institution. The outcome of this highly integrated, innovative and interactive research program will provide a panoramic view of the molecular and cellular events involving metabolic and bioenergetic pathway in cocaine and HIV-1 induced CNS disease, advance the field in and area that remains unexplored, and offer fundamental information toward the development of therapeutic molecules against HIV-1/cocaine induced CNS disease.

SUMMARY Survival of neuronal cells in response to various acute and chronic environmental stresses including infection, inflammation, and substance abuse depends on a cascade of biochemical reactions and signaling events to maintain energy production for cellular survival. Similar to HIV-1 infection, cocaine targets the brain and elicits a range of toxicities in multiple cell types, especially neurons. Previous findings suggest that chronic cocaine exposure and HIV-1 infection may synergize to exacerbate neuronal cell injury and death to a greater extent than either agent alone. As survival of neurons is highly dependent on the integrity and functionality of mitochondria, it is hypothesized that dysregulation of oxidative phosphorylation and impaired mitochondria function resulting from chronic cocaine use in the context of HIV-1 plays a critical role in the events leading to brain cell damage. Supporting this notion, we found that treating neuronal cells with cocaine decreases mitochondrial ATP production, an effect that was remarkably enhanced in the presence of Tat, a toxic HIV-1 protein found in blood and cerebrospinal fluid of HIV-1+ patients. Also, cocaine and HIV-1 Tat cooperatively suppressed mitochondrial function, seen as a decrease in membrane potential, and enhanced mitochondrial- dependent cell death. In addition, we found that Tat damages neuronal mitochondrial DNA (mDNA) by elevating ROS production, inhibiting antioxidant defenses, and impairing the base-excising mDNA repair enzyme OGG1, causing the accumulation of somatic mutations in mDNA. Such combinatorial action of HIV-1 Tat and cocaine also affected mitochondrial content/biogenesis by modulating mitophagy, via pathways involving BAG3 and the PINK1, parkin/HSP70 signaling axis. Together, these findings support our novel working model, in which cocaine and HIV-1 Tat act synergistically to promote mitochondrial dysfunction, diminishing ATP production and enhancing neuronal cell death. This project will leverage the expertise of all participants of this program project in cocaine research, mitochondrial biology, and HIV-1 neuropathogenesis, though concerted in vitro and in vivo studies to decipher the underlying molecular mechanisms responsible for the augmented neuropathological effects of cocaine abuse in the setting of HIV-1 infection.

SUMMARY The Mitochondrial Physiology and Imaging Core (Core B) will be a focal point of the program by providing the infrastructure to support numerous end-point assays to assess cellular pathology, mitochondrial function, and the detailed analysis of cellular bioenergetics. Utilizing the vast experience and expertise of the Core Directors, and the cutting edge equipment at their disposal, the Core will not only perform assays but will further participate in experimental design, prioritization of resources, and the interpretation of data. In addition, Core B will identify additional areas for collaboration across all projects as the program matures and thus be central to the interactive environment and comprehensive analysis of the overall platform as data is generated. The scientific core will provide a wide-range of services pertinent to the overall success of the program including: the assessment of cellular bioenergetics and mitochondrial function, live-cell imaging to assess cellular function and structure, histological imaging to assess neuropathology and ex vivo imaging to assess ROS generation in situ in real-time. These services will provide the backbone for the major focus of this Program Project Grant to determine the impact of cocaine abuse on the molecular neuropathogenesis of HIV-1 infection in the brain.

? DESCRIPTION (provided by applicant): The entry of HIV-1 into the brain during the early and/or late stages of infection leads to the development of a viral reservoir in the vast majority f individuals despite effective combination antiretroviral therapy (cART). In this regard, HIV-1 proviral DNA integration into the chromosomes of brain perivascular macrophages, microglial cells, astrocytes, and perhaps brain endothelial cells leads to the establishment of viral latency/persistence within these important cellular compartments as well as resting memory T cells and cells of the monocyte-macrophage lineage in the peripheral circulation and likely other tissues during the course of HIV disease. The extent of viral gene activation and expression in the periphery and following viral brain penetration may be dependent on therapeutic efficacy in a given reservoir, the diversity of the viral quasispecies, host immune activation profiles, and a number of comorbidity factors such as substance abuse, aging, and other chronic infections or cancers. To varying degrees, these factors are integrally involved in modulating the production of full-length and truncated viral RNA, toxic viral proteins (Tat, Nef, Vpr, and gp120), and infectious virus within and outside the brain. Consequently, there is a critical need for new strategies to eliminate all forms of integrated provirus from latently/persistently infected cells thereby preventing infectious production as well as the production of neurotoxic viral proteins that could also be produced from defective genomes not eliminated by currently available therapeutic strategies. To this end, a team experienced investigators has been assembled with complementary expertise in viral diversity and molecular architecture of the HIV-1 genome (B. Wigdahl, Drexel University), viral latency (J. Karn, Case Western Reserve University), and gene excision technology (K. Khalili, Temple University) to examine the Hypothesis that the CRISPR/Cas9 gene editing platform can be tailored to develop precision-guided gene editing strategies to eliminate HIV-1 from the latently infected resting memory CD4+ T-cell reservoir and reservoir cells with the brain. To address this hypothesis, three specific aims are proposed. In Aim 1 sequence and bioinformatic information from viral genetic studies performed with HIV-1-infected patients will be utilized to design gRNAs to precisely guide the HIV-1 excision process (Drexel University). In Aim 2 we will develop and test in vitro HIV-1- specific gene editing systems (Temple University), combined with expertise in the molecular biology of HIV-1 latency (Case Western Reserve University), which will culminate in ex vivo experimentation in Aim 3 on HIV-1- infected samples. These studies will set the stage for future in vivo animal studies for validating the approach toward clinical application. The overarching goal is to develop a robust experimental procedure which can be employed through various gene delivery platforms, such as nanomolecules, lentivirus, or stem/progenitor cell engineering, to provide a cure for AIDS.

SUMMARY Survival of neuronal cells in response to various acute and chronic environmental stresses including infection, inflammation, and substance abuse depends on a cascade of biochemical reactions and signaling events to maintain energy production for cellular survival. Similar to HIV-1 infection, cocaine targets the brain and elicits a range of toxicities in multiple cell types, especially neurons. Previous findings suggest that chronic cocaine exposure and HIV-1 infection may synergize to exacerbate neuronal cell injury and death to a greater extent than either agent alone. As survival of neurons is highly dependent on the integrity and functionality of mitochondria, it is hypothesized that dysregulation of oxidative phosphorylation and impaired mitochondria function resulting from chronic cocaine use in the context of HIV-1 plays a critical role in the events leading to brain cell damage. Supporting this notion, we found that treating neuronal cells with cocaine decreases mitochondrial ATP production, an effect that was remarkably enhanced in the presence of Tat, a toxic HIV-1 protein found in blood and cerebrospinal fluid of HIV-1+ patients. Also, cocaine and HIV-1 Tat cooperatively suppressed mitochondrial function, seen as a decrease in membrane potential, and enhanced mitochondrial- dependent cell death. In addition, we found that Tat damages neuronal mitochondrial DNA (mDNA) by elevating ROS production, inhibiting antioxidant defenses, and impairing the base-excising mDNA repair enzyme OGG1, causing the accumulation of somatic mutations in mDNA. Such combinatorial action of HIV-1 Tat and cocaine also affected mitochondrial content/biogenesis by modulating mitophagy, via pathways involving BAG3 and the PINK1, parkin/HSP70 signaling axis. Together, these findings support our novel working model, in which cocaine and HIV-1 Tat act synergistically to promote mitochondrial dysfunction, diminishing ATP production and enhancing neuronal cell death. This project will leverage the expertise of all participants of this program project in cocaine research, mitochondrial biology, and HIV-1 neuropathogenesis, though concerted in vitro and in vivo studies to decipher the underlying molecular mechanisms responsible for the augmented neuropathological effects of cocaine abuse in the setting of HIV-1 infection.

SUMMARY Opiate abuse is a significant risk factor for HIV-1 infection and several studies have shown that, in combination, opiates and HIV-1 lead to significantly greater damage to the brain. Thus, a new combinatory strategy is needed to impede HIV-1 infection and mitigate opiate effects on the CNS. In spite of significant advances in anti-retroviral therapy (ART), the elimination of HIV-1 CNS reservoirs remains a formidable task. This is mainly attributed to the integration of the HIV-1 proviral DNA into the host genome causing viral latency in the reservoirs, including the brain. Further, the inability of ART to penetrate the BBB after systemic administration makes the brain one of the dominant HIV reservoirs. Thus, elimination of HIV-1 from the brain remains a clinically daunting and key task in the cure of HIV-1/opioid CNS disease. Most recently, we (Dr. Khalili's lab at Temple University) developed an RNA directed gene-editing strategy using Cas9/gRNA that successfully eliminates entire integrated copies of the HIV-1 genome from the host chromosome. However, delivery of this powerful Cas9/gRNA complex across the BBB is limited and an effective method for delivery and release of Cas9/gRNA is critically required to eliminate the HIV reservoir in the brain. Our laboratory (Dr. Nair's team at Florida International University) has recently patented (US patent: US20130317279 A1 and WO patent: PCT/US2013/068698) technology involving novel magneto-electro nanoparticle (MENP) based drug delivery system, which offers capability of on-demand drug release across the BBB. The collaboration of these two laboratories provided preliminary evidence that Cas9/gRNA binds to MENP, navigated across the BBB by magnetic force, and on-demand release of functionally active Cas9/gRNA by external AC stimulation. We provide evidence that morphine induced activation of HIV infection could be mitigated by methylnaltrexone (MTNX) (µ receptor antagonist). In this multi-PI application we hypothesize that efficient nanoformulations (NFs) containing Cas9/gRNA and MTNX can serve as an effective carrier to deliver Cas9/gRNA targeting HIV-1 across the BBB for the recognition and complete eradication of the HIV reservoir in brain and to treat/prevent neurological deficits observed in morphine-using HIV infected subjects. To test our hypothesis, we propose to refine our design method, and develop, characterize, and evaluate the delivery and on-demand release of Cas9/gRNA using an in vitro BBB-HIV infection model (Aim #1). Next, we will evaluate and pre-screen the in vivo efficacy of the developed NFs in excising integrated copies of HIV DNA in Tg26 transgenic mice harboring the entire viral genome (Aim #2). In Aim #3 we will develop and use BLT mouse model to validate and assess the in vivo efficacy of the MENP-Cas9/gRNA NFs to recognize and eradicate latently infected HIV-1 reservoirs. Finally, in Aim #4 we will examine the in vivo efficacy of the most pre-screened NFs in a BLT morphine mouse model to assess the potential excision of HIV-1 proviral DNA and morphine induced reactivation of latent HIV infection and to reverse neurological deficits by NFs containing MNTX.

? DESCRIPTION (provided by applicant): Neurologic complications and related diseases, including HIV-1 associated neurocognitive disorders or HAND, remain one of the most devastating clinical manifestations of HIV-1 infection even after treatment with antiretroviral therapy. Accordingly, it is now well recognized that a novel strategy is needed to eradicate AIDS and its associated co-morbidities as current antiretroviral therapies have failed to eliminate HIV-1 from the infected host. Here, we seek support to continue operation of a highly needed Comprehensive NeuroAIDS Core Center (CNAC) that was established in 2011 at the Temple University School of Medicine in partnership with Drexel University College of Medicine in Philadelphia to provide infrastructure for performing innovative research in the neuroscience of HIV-1/AIDS. The central theme of CNAC, in the second round of funding, will be to foster an environment for performing translational research in neuroAIDS by offering expertise and service in molecular and cellular biology as well as the genetics and virology of HIV-1 infection of the CNS using in vitro, in vivo, and ex vivo systems provided by the various cores. It is our goal that CNAC promotes research toward understanding mechanisms responsible for HAND and provide new paths for the eradication of infected cells and viruses and the treatment of AIDS/CNS disease. Accordingly, more emphasis will be on cell- and viral-based methodologies and the assessment of the impact of HIV-1 on cell function pertaining to CNS diseases. To accomplish our goal, the CNAC will provide expertise in neural cell cultures, viral reagents, microelectrode array, gene editing strategies, and high end proteomics/metabolomics through its Basic Science Core I (BSC I). Basic Science Core II (BSC II) will provide expertise and facilities in the employment of animal models for studying HIV-1 brain interaction and behavioral testing/training and histological training and related services. The Clinical and Translational Research Support Core (CTRSC) of the CNAC, with greater than 2,000 patients participating in two well-characterized/controlled cohorts at Temple University Hospital (North Philadelphia) and Drexel's Hahnemann Hospital (Center City Philadelphia) will support development and maintenance of cohesive IRB protocols, offer expertise in cognitive performance and neuropsychology, virology/immunology and immunoactivation, and viral genetics/genomics and bioinformatics. Through the Developmental Core, the CNAC will offer a unique opportunity for training and mentoring of junior investigators and clinical investigators, and attract and develop physicians/scientists in the field of neuroAIDS. The CNAC will provide infrastructure by i) providing start-up funds through its Developmental Core, ii) offering intellectual support and technical services through its state-of-the-art basic science cores and iii) access to an expansive well-operating clinical core, for conducting innovative research toward the development of intelligent, effective, and safe genetic and cellular/immunologic strategies toward a cure of AIDS. Further, the funding through CNAC will create an environment for the development of collaborative research in neuroAIDS at Temple, Drexel and other medical institutions in the greater Philadelphia area, and extend its collaboration with other AIDS research centers in Philadelphia and the nation.

DESCRIPTION (provided by applicant): This revised competing renewal application provides a plan for the continuation and further development of the Interdisciplinary and Translational Research Training Program (ITRTP) in NeuroAIDS for predoctoral students studying central nervous system complications of HIV infection and related areas of research. This is a joint program from two institutions, Temple University and Drexel University, located in close proximity in Philadelphia, Pennsylvania. This program also integrates training activities and research resources available in the Comprehensive Center for NeuroAIDS (CNAC) involving both Temple and Drexel University as well as long-standing MD/PhD and clinical research training programs in AIDS and Neurovirology based in the Department of Neurology, and Center for AIDS Research (CFAR) at the University of Pennsylvania. Our ongoing program further develops a citywide interdisciplinary and translational research training program in NeuroAIDS through shared resources, joint seminars, workshops, symposia, invited speakers, as well as thesis mentoring and educational activities at both institutions. The graduate curriculum at both institutions is designed to provide a broad based scientific foundation in biomedical sciences, including neuroscience, immunology, microbiology, and pharmacology and physiology. This curriculum includes responsible conduct of research, scientific communication, statistics, as well as advanced courses with in- depth training in molecular and cellular neurobiology, neuropathogenesis, and translational neuroscience. This program brings together multiple biomedical basic science departments and integrates joint training activities at two institutions with nearby University of Pennsylvania. With the inclusion of clinical AIDS investigators, as well as CNAC Core facilities, our training program is not only interdisciplinary, but exposes students to basic sciences and clinical perspectives focusing on HIV/AIDS/NeuroAIDS. Our joint training program, together with our joint infrastructural resources available through our CNAC and the nearby University of Pennsylvania CFAR, provides a strong, interactive, and highly successful training environment in neurovirology and NeuroAIDS within the greater Philadelphia region. In order to support and enhance our under-represented minority recruitment within this program, this application now includes the provision for summer laboratory training of undergraduates and medical students that would provide pathways for entry into our PhD and MD/PhD training program in NeuroAIDS.

Even with increased restore Virus persists in tissues with the potential to become reactivated when ART is discontinued underscoring a major challenge of HIV eradication. The possibility of curative treatment for HIV-1 infection has been energized by the ?Berlin patient?, who received stem cell transplants from a CCR5?32-homozygous donor after radiation and chemotherapy for his acute myelocytic leukemia and has been undetectable for the virus in his blood and tissues for at least 10 years since stopping antiretroviral therapy (ART) after the transplants. In our previous studies, we successfully applied CRISPR/Cas9 gene editing to target and completely eradicate integrated HIV sequences in in vitro cell culture models, ex vivo cultured HIV+ patient derived cells and in vivo using HIV transgenic mice and rats and most recently in close collaboration with Dr. Howard Gendelman's team (UNMC) in ART treated HIV-infected humanized mice. In up to a third of infected treated humanized mice, virus was not detected in blood, spleen, lung, kidney, liver, gut- associated lymphoid tissue and brain by ultrasensitive nested and digital droplet PCR and RNAscope tests. No viral rebound was demonstrated after ART cessation. In this application, we will employ a gene editing strategy for targeting SIV and CCR5 in SIV-infected non-human primate model. In light of our preliminary data and published studies, our central hypothesis is that employment of a combined CRISPR/Cas9 gene editing platform can effectively excise SIV proviral DNA from the latent viral reservoir and by editing CCR5 gene prevent spread of the virus and reinfection in the animal leading to a reduction in the functional viral reservoir contemporary anti-retroviral therapy (ART) regimens the rate of survival, HIV remain an enormous health immune health and is not curative. that suppress viral replication burden . Unfortunately, ART does and have not fully and/or sterile cure. To in in vivo SIV animal eradicating virus from host genomes inducible viral RNA reservoir In this application, we will establish SIV and CCR5 gene editing platforms in SIV-infected non-human primates (Aim 1) and determine the immunological and virological effects and the underlying mechanism of cure or delayed viral rebound after gene editing and antiretroviral therapy interruption (ATI) (Aim 2). We will use single and then a sequential dual editing that targets the cellular gene (CCR5) followed by the proviral DNA (SIV). Studies in this application will this end, a team of experienced investigators has been assembled with unique expertise models and immunology (T. Burdo, Temple University), (K. Khalili, Temple University), and (J. Karn, Case Western Reserve University). gene editing technologies for state of the art assays measuring the allow us to gain valuable insight into the ability of CRISPR/Cas9 gene editing for cure strategies. These studies infection, in vivo are highly translatable and will provide knowledge toward the eradication or functional cure of HIV off ART without detectable plasma viremia orthe ultimate goal being to achieve a prolonged period otherevidence of active infection.

ABSTRACT: This competing renewal application provides a plan for the continuation and further development of the ?Interdisciplinary and Translational Research Training Program (ITRTP) in NeuroHIV? for predoctoral students studying central nervous system (CNS) complications of HIV infection and related areas of research. This is a joint program from two institutions, Temple University and Drexel University, located in close proximity in Philadelphia, Pennsylvania. This program also integrates training activities and research resources available in the Comprehensive Center for NeuroAIDS (CNAC) involving both Temple and Drexel University and a relationship with the clinical research training programs in HIV and Neurovirology based in the Departments of Medicine, Microbiology, and Neurology as well as other clinical and basic science departments and the Center for AIDS Research (CFAR) at the University of Pennsylvania. Our ongoing program will continue to develop our citywide interdisciplinary and translational research training program in NeuroHIV through shared resources, joint seminars, workshops, symposia, invited speakers, as well as thesis mentoring and educational activities available at both Temple and Drexel. The graduate curriculum at both institutions is designed to provide a broad-based scientific foundation in biomedical sciences, including neuroscience, immunology, microbiology, pharmacology and physiology. This curriculum includes responsible conduct of research, scientific communication, statistics, as well as advanced courses with in-depth training in molecular and cellular neurobiology, neuropathogenesis, and translational neuroscience. This program brings together multiple biomedical basic science departments and integrates joint training activities at two neighboring institutions (Temple and Drexel) with unique and long-standing relationships between investigators located at the University of Pennsylvania positioned immediately adjacent to Drexel. Inclusion of access to clinical HIV investigators in translational student research projects, as well as Temple/Drexel CNAC basic science and clinical core facilities greatly enhances our training program with respect to its interdisciplinary nature. It also exposes students to the interrelatedness of basic sciences and clinical perspectives focusing on HIV CNS dysfunction, co-morbid conditions (i.e. aging, substance abuse, co-infecting pathogens, cancer), HIV vaccine and prevention strategies, and novel neuroprotective and therapeutic strategies to treat and cure HIV infection in the periphery and reservoirs including the brain. Our joint training program, together with our joint infrastructural resources available through our CNAC, provides a strong, interactive, and highly successful training environment in neurovirology and neuroHIV within the Philadelphia region. In order to support and enhance our under-represented minority recruitment within this program we will continue to provide summer laboratory training of medical students, and students in health professional programs including post- baccalaureate students that would provide pathways for entry into our Ph.D. and M.D./Ph.D. training program in NeuroHIV.

Even increased the with contemporary anti-retroviral therapy (ART) regimens that the survival rate , HIV remain s an enormous health burden disease by eliminating proviral DNA, which is present in the genome of infected host cells. suppress viral . Unfortunately, ART has replication and have failed to cure The persistence of the viral genome in tissues with the potential to become reactivated, when ART is discontinued, underscores the importance of alternative, perhaps companion, strategies for eradication of the HIV genome from the latently infected reservoirs. The possibility of a curative treatment for HIV infection has been energized by the ?Berlin patient? and more recently the ?London patient?, who both received stem cell transplants from a CCR5?32-homozygous donor after radiation and chemotherapy for leukemia and have been undetectable for the virus in his blood for 12 and 2 years since stopping ART after the transplants. CCR5 is a cellular protein that serves a co-receptor for HIV infection, therefore its genetic inactivation prevents viral infection and its spread throughout the body of the infected individual. In previous studies, we successfully applied CRISPR- Cas9 gene editing strategy to target and eradicate integrated HIV sequences in in vitro cell culture models, ex vivo cultured HIV+ patient derived and in vivo in HIV-infected humanized mice model. We presented data at CROI 2019 and here of in vivo editing of the SIV genome in blood of SIV-infected macaques after i.v. injection of AAV9/CRISPR-Cas9. In this multi Principal investigator (mPi) grant application, we will test the hypothesis that employment of a combined gene editing platform can effectively excise SIV proviral DNA and further editing of the CCR5 gene will remove viral targets in the animal leading to a reduction in the functional viral reservoir and/or complete elimination of replication competent virus in the treated animals. To this end, we will employ CRISPR-Cas9 technology for sequential targeting and permanent inactivation of both SIV and CCR5 in an SIV-infected non-human primate model. To achieve our goals, we investigators have formed a team of experienced with extensive experience in in vivo SIV animal models and immunology (T. Burdo, mPi, Temple University), gene editing technologies for eradicating virus from host genomes in in vitro cell cultures and in vivo animal models novel assays In this application, we will establish the CCR5 and dual SIV and CCR5 gene editing platforms in SIV-infected non-human primates and determine the immunological and virological effects and the underlying mechanism of cure or delayed viral rebound after gene editing and antiretroviral therapy interruption (ATI). We will use single and then a sequential dual editing targeting the cellular gene (CCR5) followed by the SIV proviral DNA. The outcome of these studies will offer valuable insight into the potential of CRISPR-Cas9 gene editing for consideration in the HIV cure strategies. (K. Khalili, mPi, Temple University), and (J. Karn, co-I, Case Western Reserve University). measuring the inducible viral RNA reservoir using

PROJECT SUMMARY It is estimated that more than 10% of the 1.1 million HIV positive patients in United States will be over 65 years old in less than ten years, a critical stage in life where the the risk for developing Alzheimer?s disease (AD) increases. Earlier results from clinical studies showed several abnormalities including difficulties with memory, thinking, and reasoning become more common in older HIV positive patients, implying that their risk factors may be elevated when compared with their HIV negative counterparts. This notion brings up the concept that HIV infection, even in patients whose virus is well controlled by antiretroviral therapy (ART), may be more prone to developing neurodegenerative disorders including AD. Considering the inability of ART to effectively suppress expression of viral proteins, including HIV Tat, which is a profoundly neurotoxic protein, we envision a scenario in which, by dysregulating the protein quality control (PQC) pathway, Tat may perturb homeostasis of key proteins associated with the pathogenesis of AD and set the stage for the development of disease. Several data from our and other laboratories support this concept. First, we demonstrated that Tat inhibits expression of BAG3, a co-chaperone/partner of HSP70 that is involved in the removal of dysfunctional and obsolete organelles including mitochondria through a process called mitophagy, and participates in autophagy and clearance of damaged and misfolded proteins by the protein quality control (PQC) pathway. Second, activation of BAG3 is concurrent with a decrease in the level of phosph-tau and an increase in the clearance of tau in neuronal cells. Third, soon after destabilization of microtubules, tau associates with Hsp70/Hsc70, a key partner of BAG3. Fourth, induction of Tat in the brains of transgenic animals increases accumulation of phospho-tau. Thus, Tat- mediated reduction in the level of BAG3 may have a damaging effect on neuronal cells by interrupting the process that ensures intracellular removal of the toxic from of tau, yet maintains the healthy species of tau that is critical for neuronal cell function. Hyper-phosphorylation of tau, which contributes to its misfolding and toxicity, may be attributed to Tat via induction of ROS, ER stress, and mitochondrial dysfunctionality. The latter is of particular interest as truncated tau has been implicated in dysregulation of mitochondrial dynamics and healty energy metabolism. These observations prompted us to hypothesize that, on one hand, Tat impacts the quality and concentration of proper levels of functional tau and the clearance of its toxic form by suppressing expression of BAG3, and on the other hand, Tat contributes to the generation of the toxic tau and dysfunctionality of bioenergetic pathways and mitochondria by inducing stress conditions in cells. To examine this model, we will employ in vitro primary neuronal cultures, ex vivo animal brain tissue, and an in vivo animal model to unravel the underlying molecular basis of HIV-1/Tat-induced perturbation of PQC and homeostasis of tau in neuronal cell function.

While antiretroviral therapy (ART) has dramatically reduced HIV disease morbidity and mortality, it has failed to eliminate viral reservoirs. Interruption of treatment leads to activation of latent virus and rebound viremia within weeks. Novel strategies are urgently needed to eradicate latent infections and enhance the immune system leading to sustained, durable control of viral rebound following the cessation of ART. In response to RFA-AI- 20-035 Martin Delaney Collaboratories for HIV Cure Research, we now submit the application entitled CRISPR for Cure. The overarching goal of this program is to use genome editing mediated by CRISPR to enhance immune responses and directly ablate HIV proviruses. We have assembled a collaborative team of highly accomplished basic and translational scientists working in tandem with community stakeholders and a small biotechnology company to develop CRISPR-based therapies to directly target the HIV provirus and to enhance immunological responses. The research program is comprised of three highly interactive research foci (RF) that will utilize interdisciplinary, innovative and collaborative research approaches with community and government input. RF1 will use next generation sequencing and novel barcoded viruses to define the HIV reservoir and the impact of epigenetic mechanisms on proviral rebound. In RF2, we will enhance effector NK and CTL cell function and killing and limit viral spread by target cells using innovative genome editing strategies. RF3 will create and test the next generation of inducible, multiplex CRISPR with increased specificity, potency and safety for delivery by CD4 tropic lymphoid AAV9 for eradication of HIV-1 proviral DNA in animal models whose immune cells are modified in RF2 and assess the possibility of both a universal and personalized CRISPR in eliminating replication competent virus in vivo. In addition to the shared focus on CRISPRs technology, the Collaboratory will undertake a highly integrated experimental agenda through the shared use of barcoded viruses in humanized mice and unique support MISTRG humanized mice that differentially human hematopoietic stem and progenitor cell maintenance and myelopoiesis;rhesus macaques infected with a novel SIV barcoded virus; ex vivo clinical samples from a well characterized cohort and the use of adenoviruses to efficiently deliver CRISPRs to an in vivo humanized animal model carrying cells from patient-derived PBMCs. The outcome of this comprehensive and multidisciplinary program by the ?CRISPR for Cure? the Collaboratory, will accelerate the use of gene editing strategies towards eradication of HIV infection from body or sustained viral remission following cessation of antiretroviral therapy.With resources available from our private sector partner, we will be well positioned for further GMP manufacturing development, and future initial clinical investigations.