Pedro R. Lowenstein - US grants

Parkinson's disease (PD) is a chronic neurodegenerative disorder, in which them is extensive degeneration of nigrostriatal dopaminergic neurons. Maintaining the heath of these cells using powerful neurotrophic factors has proven effective in a number of rodent and primate models of PD. The ultimate goal of this proposal is to develop novel high-capacity adenoviral systems for long-term, stable, non-cytotoxie, and non-immunogenic delivery of neurorestorative genes to the brain for the future treatment of chronic neurodegenerative diseases such as PD. Adenovirus-derived vectors serve as efficient gene transfer vehicles in the setting of the CNS. However, use of conventional adenoviral vectors has been limited due to diminished duration of transgene expression as a result of strong anti-viral immune and inflammatory responses that are elicited. Decreased vector-directed transcriptional activity typically leads investigators to administer higher vector doses that ultimately results in enhanced in vivo toxicity. In this proposal, we will utilize the novel design of helper-dependent, high-capacity adenoviral vectors for efficient, safe, and long-term transgene delivery to the brain for the treatment of PD. Specifically, high-capacity helper-dependent adenoviral vectors will be constructed and assessed in vivo. We will monitor their effectiveness and potential inflammatory and immune side effects in detail. We will also test the efficacy of the new vectors to express the potentially neurorestorative gene, glial cell line derived neurotrophic factor (GDNF), and evaluated its capacity to protect dopaminergic neurons in a rodent neurotoxic model of neurodegeneration. The reagents and principles established by this work will be of substantial value to the implementation of novel therapeutics for PD, facilitate the development of tools needed to achieve long-lived, safe, non-cytotoxic transgene expression, and may lead to the development of novel treatments for other chronic neurodegenerative diseases.

DESCRIPTION (provided by applicant): Gene therapy provides exciting new approaches to treat numerous incurable neurodegenerative disorders such as Parkinson's disease, multiple sclerosis, or brain cancer. Unfortunately the immune response to therapeutic vectors remains a major obstacle to the clinical realization of gene therapy. If primed, the immune system eliminates expression of therapeutic transgenes from the brain, curtailing gene therapy's efficacy. We will investigate immune regulation of transgene expression mediated by clinically effective first generation adenoviral vectors, and novel high capacity 'gutless'adenoviral vectors in a clinically relevant model. In this model animals will be pre-immunized to adenovirus to mimic immune status in the majority of human patients that were exposed to adenovirus before receiving gene therapy. In this proposal we will test the hypothesis that the immune system eliminates expression of therapeutic transgenes from the brain, primarily through cytotoxic, and secondarily, through non-cytotoxic mechanisms. To address these issues, we developed a specific method to differentiate immune system-induced brain cell death from selective down-regulation of vector-mediated transgene expression. This method is based on transgenic mice containing a floxed beta-gal construct, and viral vectors expressing Cre under pancellular and cell type specific promoters. Infected cells thus express a gene marker from their genomes, expression of which will be regulated independently of expression from the viral vector's genome. Preliminary experiments using our new method demonstrate that the immune system utilizes both mechanisms, namely it can eliminate expression of therapeutic transgenes from the brain by [1] direct cytotoxicity of transduced cells;and, [2] functional inhibition of transgene expression. Herein we will test specific hypothesis concerning the immune mechanisms that eliminate brain transgene expression, in both males and females, and in two mouse strains;one that displays TH1 biased immune responses (C57BI/6), and another one that displays a TH2 bias (DBA/2J). As a result of this work we will make available enhanced and safer gene therapy approaches, and more efficient clinical treatment paradigms. The results from this proposal will have a direct impact on experimental and clinical gene therapy, as well as make major contributions to understanding how the immune system eliminates or regulates gene expression in virally infected brain cells.

DESCRIPTION (provided by applicant): Immunological synapses are the recently characterized microanatomical structures that underlie immune cellular interactions. Establishment of immunological synapses between CTLs and infected or malignant astrocytes precedes the elimination of these astrocytes from the brain. How individual CTL target cells respond to T cell attack remains poorly understood. We have evidence suggesting that infected astrocytes respond in an active manner to the T cell attack. Our data suggests that target infected astrocytes change from multipolar to unipolar cells, i.e. they adopt a novel polarized phenotype that appears to include a reorganization of the cytoskeleton and intracellular organelles. We will test whether this active cellular reorganization could influence the ultimate outcome of the T cell attack, e.g. death or survival of infected astrocytes. In this application we will test the hypothesis that both infected astrocytes and tumor glioma cells respond in an active manner to T cell attack, and that this response is induced by a T cell-dependent activation of a Rho-GTPase signaling pathway. We will study the astrocyte responses to T cell attack in vivo and in vitro, and analyze the molecular signaling pathways underlying these responses. We believe that understanding the cellular and molecular mechanisms by which infected and malignant astrocytes respond to T cell attack should lead to better ways to eliminate neurological viral infections and brain tumors, enhance therapeutic transgene expression from gene therapy viral vectors, or protect the brain from autoimmune attack. To do so, we propose to explore the cellular and molecular basis of glial cell responses to immune attack both in vivo and in vitro in three Specific Aims. Specific Aim 1 will test the hypothesis that formation of immunological synapses leads to the polarization of infected astrocytes and that this is dependent on the activation of a Rho-GTPase pathway;Specific Aim 2 will test whether CTL signaling at mature immunological synapses in vivo between anti-viral T cells and infected astrocytes effectively leads to the death of infected astrocytes, or whether astrocytes can withstand such an attack;and Specific Aim 3 will test the hypothesis that the effects of anti- tumor T cells on glioma cells are mediated through the formation of immunological synapses. PUBLIC HEALTH RELEVANCE: Immunological synapses form in vivo between antiviral CTLs, and virally infected or malignant astrocytes causing a reorganization of their cellular structure. We believe this response influences the ultimate outcome of the T cell attack, e.g. death or survival of infected or tumor glial cells. We believe that understanding the cellular and molecular mechanisms by which infected and malignant astrocytes respond to T cell attack should lead to better ways of eliminating neurological viral infections and brain tumors, enhance therapeutic transgene expression from gene therapy viral vectors, or protect the brain in cases of autoimmune attack.

DESCRIPTION (provided by applicant): Immunological synapses are the recently characterized microanatomical structures that underlie immune cellular interactions. Establishment of immunological synapses between CTLs and infected or malignant astrocytes precedes the elimination of these astrocytes from the brain. How individual CTL target cells respond to T cell attack remains poorly understood. We have evidence suggesting that infected astrocytes respond in an active manner to the T cell attack. Our data suggests that target infected astrocytes change from multipolar to unipolar cells, i.e. they adopt a novel polarized phenotype that appears to include a reorganization of the cytoskeleton and intracellular organelles. We will test whether this active cellular reorganization could influence the ultimate outcome of the T cell attack, e.g. death or survival of infected astrocytes. In this application we will test the hypothesis that both infected astrocytes and tumor glioma cells respond in an active manner to T cell attack, and that this response is induced by a T cell-dependent activation of a Rho-GTPase signaling pathway. We will study the astrocyte responses to T cell attack in vivo and in vitro, and analyze the molecular signaling pathways underlying these responses. We believe that understanding the cellular and molecular mechanisms by which infected and malignant astrocytes respond to T cell attack should lead to better ways to eliminate neurological viral infections and brain tumors, enhance therapeutic transgene expression from gene therapy viral vectors, or protect the brain from autoimmune attack. To do so, we propose to explore the cellular and molecular basis of glial cell responses to immune attack both in vivo and in vitro in three Specific Aims. Specific Aim 1 will test the hypothesis that formation of immunological synapses leads to the polarization of infected astrocytes and that this is dependent on the activation of a Rho-GTPase pathway; Specific Aim 2 will test whether CTL signaling at mature immunological synapses in vivo between anti-viral T cells and infected astrocytes effectively leads to the death of infected astrocytes, or whether astrocytes can withstand such an attack; and Specific Aim 3 will test the hypothesis that the effects of anti- tumor T cells on glioma cells are mediated through the formation of immunological synapses. PUBLIC HEALTH RELEVANCE: Immunological synapses form in vivo between antiviral CTLs, and virally infected or malignant astrocytes causing a reorganization of their cellular structure. We believe this response influences the ultimate outcome of the T cell attack, e.g. death or survival of infected or tumor glial cells. We believe that understanding the cellular and molecular mechanisms by which infected and malignant astrocytes respond to T cell attack should lead to better ways of eliminating neurological viral infections and brain tumors, enhance therapeutic transgene expression from gene therapy viral vectors, or protect the brain in cases of autoimmune attack.

DESCRIPTION (provided by applicant): Inhibiting glioma invasion using targeted nanoparticles High grade gliomas are uniformly lethal, even following surgery, temozolomide chemotherapy and radiotherapy. Tumor recurrence is caused by regrowth of glioma cells which infiltrate large distances throughout the normal brain. Glioma-like stem cells are thought to initiate tumor recurrence as they can remain quiescent for a long time; this allows them to resist cytotoxic agents and therapies that rely on cell division (i.e., chemotherapy, radiotherapy). Examination of neuropathological samples of human glioma tumors (representing advanced symptomatic tumors) suggest that glioma cells migrate along blood vessels, white matter tracts, the extracellular space, and subpially. However, it has been difficult to characterize in molecular and cellular detail the individual migration paths in either human tumors or in experimental gliomas. To understand the cellular basis of initial glioma cell invasion we are characterizing the anatomical, biochemical and molecular basis for glioma growth and invasion. We have recently discovered that many glioma cells and glioma stem cells can grow preferentially along the network provided by the tumoral and peritumoral vasculature. As centrifugal glioma invasion occurs along tumoral and peritumoral vessels we now aim to target the blood vessels that sustain glioma cell invasion throughout the brain. Our preliminary data indicate that F3-targeted hydrogel nanoparticles target the tumoral blood vessels that support glioma cell growth, and glioma cell invasion, as well as glioma cells. In this R21 application we propose to test if biocompatible and bio-degradable, F3-targeted hydrogel nanoparticles loaded with therapeutic drugs (i.e., cisplatin, temozolomide) will kill those vessels that sustain glioma dispersion from the central tumor mass into normal brain parenchyma, as well as the main glioma tumors. The peptide F3 binds to nucleolin, a protein overexpressed by tumor vasculature and by glioma tumors, but not by normal brain. We hypothesize that selective killing of tumor blood vessels (utilizing F3-targeted nanoparticles loaded with cisplatin) will inhibit glioma invasion, in combination with F3-targeted nanoparticles loaded with temozolomide to kill the main glioma mass. This proposal will test the hypothesis that combined F3-nanoparticle mediated killing of tumor blood vessels providing the substrate for glioma invasion, and of glioma cells, will reduce glioma growth and tumor recurrence. Our previous experience in the translation of basic science advances into early phase clinical trials for the treatment of human patients suffering from malignant glioma (FDA IND-14574), supports our assertion that, should experiments support our proposed hypothesis, we will be able to efficiently translate such results into Phase I clinica trials for GBM patients.

DESCRIPTION (provided by applicant): High grade gliomas are uniformly lethal, and resistant to surgery, chemotherapy and radiotherapy. The precise cellular and molecular mechanisms by which glioma cells disperse through the brain and grow to form macroscopic symptomatic tumor masses remains poorly understood. Herein we propose to test novel cellular, molecular and mechanistic hypotheses concerning glioma growth, and how to translate this knowledge into new anti-glioma therapeutics. Preliminary work from my laboratory, using confocal, electron and multiphoton microscopy has shown that glioma cells and human glioma stem cells disperse through the brain in vivo by traveling preferentially along the perivascular compartment, a potential migration network surrounding the brain microvasculature. As glioma cells move throughout the perivascular network they dislodge glial endfeet from blood vessels and compromise adjacent brain tissue; this is later replaced by tumor cells. We have also generated preliminary data that a glycan binding protein, galectin-1, is essential for this growth mechanism. Down regulation of galectin-1 abolishes glioma growth in the brain in vivo, without affecting growth in vitro. These new data have several clinical consequences: (i) lymph drains from the brain through the perivascular compartment; its obstruction by gliomas would contribute to glioma-induced edema; (ii) human glioma tumors grow to large size before causing symptoms; glioma cell replacement of atrophied brain tissue could explain protracted and indolent tumor growth, and the delayed changes in total brain volume; (iii) inhibition of galectin-1 could represent a novel treatment of human gliomas. This proposal will (I) test the hypothesis that rodent and human glioma cells, and glioma stem cells grow preferentially along the perivascular space; (II) test the hypothesis that galectin-1 mediates glioma perivascular invasion and growth, and that inhibition of galectin-1 can be used as a novel therapeutic strategy; and (III) test the hypothesis that inhibition of galectin-1 will enhance specific anti-glioma immune responses. By progressing from glioma pathophysiology to molecular mechanisms of glioma migration to experimental therapeutics, we aim for our work to lead to novel early phase clinical translational trials for the treatment of human gliomas. Of note, our first clinical trial for gene therapy of human gliomas is approaching the start of patient recruitment (it was approved by FDA on 4/7/11 [IND 14574] and very recently by the University of Michigan IBC and IRB). Therefore, our laboratory is in a strong and realistic position to guide our research towards the translational implementation of novel clinical trials for this currently deadly human cancer.

? DESCRIPTION (provided by applicant): Innate immune responses against glioma (GBM) are poorly understood. Most studies have focused on adaptive T cell immune responses. Innate immune responses are thought to be needed primarily, to activate T cell responses, rather than mediate direct cytotoxicity against tumors. Recently we showed that NK cells inhibit GBM progression, and exert powerful anti- GBM cytotoxicity. In turn, to evade NK- killing GBMs produce potent inhibitors of NK cells. Having established that NK cells inhibit GBM growth and invasion, we will evaluate the complex network of innate immune cells and signaling pathways responsible for this powerful anti-GBM response. Our data support the hypothesis that other innate immune cells, besides NK cells, are necessary for the powerful NK-mediated anti-GBM responses, as GR1 depletion abolishes NK- mediated GBM killing. In AIM 1 will identify the network of innate immune cells required to inhibit GBM progression. Our preliminary data show that Myd88 signaling is necessary for trafficking of innate immune cells to the tumor microenvironment and control tumor growth. In AIM 2 we will test the hypothesis that Myd88 transduces cellular responses to TLR9, IL18, and/or IL33 signaling in cells of the myeloid lineage within the tumor microenvironment. We will assess in which cells Myd88 signaling is needed for NK cells to kill GBM cells. Preliminary data suggest that the cGAS-STING-IFN? pathway is also necessary for NK-mediated GBM killing. In AIM 3 we will test the hypothesis that signaling via the cGAS-STING-IRF3-IFN? pathway on pDCs -or other myeloid cells- is necessary for full cytotoxic NK activation. We propose to test whether both pathways (Myd88 and STING) are necessary for innate immune-mediated inhibition of GBM progression. In summary, our proposal will ascertain the network of innate immune cells and signaling pathways that jointly inhibit GBM progression. In addition, the work proposed will also establish if the two innate signaling pathways (Myd88 and STING) converge to stimulate malignant GBM killing. The complex innate immune network and its signaling through Myd88 and STING to inhibit brain tumor progression solely via innate immunity have not yet been elucidated. Finally, we will test therapeutic combinations of a conditional cytotoxic-immune stimulatory approach (Ad-TK Ad-Flt3L) with the activation of innate immune signaling pathways (Myd88 and STING) in genetically engineered mouse models of GBM. In the long term, we aim to develop novel translational clinical trials, as we achieved earlier for gene/immune-therapeutic treatment of human gliomas using Ad-TK and Ad-Flt3L (NCT01811992).

? DESCRIPTION (provided by applicant): Innate immune responses against glioma (GBM) are poorly understood. Most studies have focused on adaptive T cell immune responses. Innate immune responses are thought to be needed primarily, to activate T cell responses, rather than mediate direct cytotoxicity against tumors. Recently we showed that NK cells inhibit GBM progression, and exert powerful anti- GBM cytotoxicity. In turn, to evade NK- killing GBMs produce potent inhibitors of NK cells. Having established that NK cells inhibit GBM growth and invasion, we will evaluate the complex network of innate immune cells and signaling pathways responsible for this powerful anti-GBM response. Our data support the hypothesis that other innate immune cells, besides NK cells, are necessary for the powerful NK-mediated anti-GBM responses, as GR1 depletion abolishes NK- mediated GBM killing. In AIM 1 will identify the network of innate immune cells required to inhibit GBM progression. Our preliminary data show that Myd88 signaling is necessary for trafficking of innate immune cells to the tumor microenvironment and control tumor growth. In AIM 2 we will test the hypothesis that Myd88 transduces cellular responses to TLR9, IL18, and/or IL33 signaling in cells of the myeloid lineage within the tumor microenvironment. We will assess in which cells Myd88 signaling is needed for NK cells to kill GBM cells. Preliminary data suggest that the cGAS-STING-IFN? pathway is also necessary for NK-mediated GBM killing. In AIM 3 we will test the hypothesis that signaling via the cGAS-STING-IRF3-IFN? pathway on pDCs -or other myeloid cells- is necessary for full cytotoxic NK activation. We propose to test whether both pathways (Myd88 and STING) are necessary for innate immune-mediated inhibition of GBM progression. In summary, our proposal will ascertain the network of innate immune cells and signaling pathways that jointly inhibit GBM progression. In addition, the work proposed will also establish if the two innate signaling pathways (Myd88 and STING) converge to stimulate malignant GBM killing. The complex innate immune network and its signaling through Myd88 and STING to inhibit brain tumor progression solely via innate immunity have not yet been elucidated. Finally, we will test therapeutic combinations of a conditional cytotoxic-immune stimulatory approach (Ad-TK Ad-Flt3L) with the activation of innate immune signaling pathways (Myd88 and STING) in genetically engineered mouse models of GBM. In the long term, we aim to develop novel translational clinical trials, as we achieved earlier for gene/immune-therapeutic treatment of human gliomas using Ad-TK and Ad-Flt3L (NCT01811992).