Jeffrey M. Zirger

Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses

ABSTRACT In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding β-galactosidase (β-gal) driven by a TetOn system containing the rtTASsM2 transactivator and the tTSKid repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-β-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-β-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-β-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-β-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.

Effects of ectopic decorin in modulating intracranial glioma progression in vivo, in a rat syngeneic model.

Given the failure of conventional treatments for glioblastoma, gene therapy has gained interest considerable in recent years. Gliomas are associated with a state of immunosuppression, which appears to be partially mediated by an increase in secretion of transforming growth factor-β (TGF-β) from glioma cells. Decorin, a small proteoglycan which can bind to and inactivate TGF-β, has been successfully used as an antitumor strategy on stably transfected tumor cells and has been shown to cause growth suppression in neoplastic cells of various histological origins. In this paper, we investigated the use of gene therapy to deliver the decorin transgene in a site-specific manner in an experimental model of intracranial gliomas. Our aim was to inhibit the glioma-associated immunosuppressive state, and prolong the survival of tumor-bearing rats.We studied the effects of decorin gene transfer in the rat CNS-1 glioma model. To assess the effect of ectopic expression of decorin on glioma progression in vivo, stably transfected CNS-1 cells expressing decorin were implanted into the brain parenchyma of syngeneic Lewis rats. The rats implanted with CNS-1 cells expressing decorin survived significantly longer than those in the control groups which received CNS-1 cells that did not express decorin (P<.0001). We then investigated whether the survival observed with decorin expressing cells could be mimicked in vivo, using recombinant adenoviruses (RAds) expressing the decorin gene under the control of two different promoters: the human immediate-early cytomegalovirus (h-IE-CMV) and the glial fibrillary acidic protein (GFAP). In vivo results showed that administration of RAd expressing the human decorin under the control of h-IE-CMV promoter has a small, but significant effect in prolonging the survival of experimental tumor bearing rats (P<.0001). Our data indicate that ectopic decorin expression has the potential to slow glioma progression in vivo. Our results also indicate that expression of decorin has to be present in all cells which constitute the intracranial tumor mass for the inhibition of tumor growth and prolongation of the life expectancy of tumor-bearing rats to be effective.

Rapid upregulation of interferon-regulated and chemokine mRNAs upon injection of 108 international units, but not lower doses, of adenoviral vectors into the brain.

ABSTRACT The innate immune response, characterized by the rapid induction of proinflammatory genes, plays an important role in immune responses to viral vectors utilized in gene therapy. We demonstrate that several innate proinflammatory mRNAs, i.e., those coding for the interferon (IFN)-regulated proteins interferon regulatory factor 1, 2′,5′-oligoadenylate synthetase, and double-stranded-RNA-dependent protein kinase as well as those coding for the chemokines RANTES, IFN-γ-inducible protein 10, and monocyte chemoattractant protein 1, were all increased in a statistically significant manner in response to 1 × 108 IU, but not lower doses, of a first-generation adenovirus injected into the naïve brain. This indicates the presence of a threshold dosage of adenovirus needed to elicit an acute innate inflammatory response.

Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFNγ, Perforin, and TNFα, and due to the Elimination of Transduced Cells

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4(+) and CD8(+) T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells.

Editorial: Glial Cells: Managers of Neuro-Immunity

After many decades of study in the field of Neuroscience that were mostly centered on the neuron there is a mounting interest in the study of the function of the glial cells in many aspects and functions of the central nervous system. The involvement of glial cells in neuroimmunity is one of the critical pieces within this puzzle, and one that entails great complexity. An increasing number of publications shows that resident astroglia and microglia are the real managers of immune responses, orchestrating chemokine and cytokine release, blood cell infiltration, and promotion of angiogenesis, etc. Moreover, each disease and neuroinflammatory scenario seems to have its own distinct biochemical characteristics and glial phenotype. Classical definitions of resting and activated microglial cells or pro-inflammatory and anti-inflammatory phenotypes are recognized today as oversimplified models of glial cell functions and have since been surpassed by more defined and precise characterizations. The present Frontiers Research Topic (FRT) is a great example of this, since the study of different scenarios reflects diverse modes of glial activation and distinct complexities. We present here a selection of articles, both original research and reviews, solving clinical, and basic aspects of the biology of glial cells in neuro-inflammatory and neuro-immune scenarios. A good number of manuscripts in this FRT shows the importance of glial cell-derived inflammation on neurodegenerative diseases. Particularly, Ben Haim and colleagues, from Escartins lab, show a compelling review on the peculiar, and still poorly understood, roles of astrocytes in neurodegenerative diseases, unfolding the signaling pathways toward reactivity (Ben Haim et al.). Von Bernhardi et al., review the roles of glial cells in neurodegeneration, but focused on Alzheimers disease and particularly discussing the effects of the cytokine TGFβ (von Bernhardi et al.). Herrera et al. center their attention on another major neurodegenerative disorder, Parkinsons disease, and how stress and glucocorticoids may interact and play important roles in modulating microglial activation (Herrera et al.). Yuste and colleagues, give us an interesting view of the role of nitric oxide in neurodegenerative diseases from a glial-derived inflammatory perspective (Yuste et al.). Vieira et al., review the glial reaction triggered in multiple system atrophy (MSA) focused on the α-synuclein-mediated activation (Vieira et al.). Other papers are centered on Multiple Sclerosis. Crowley et al., for instance, present an original paper characterizing the roles of Baclofen, a well-known GABA B receptor agonist used clinically, for regulating TLR3 and TLR4 signaling in murine glial cells and peripheral monocytes obtained from Multiple Sclerosis patients (Crowley et al.). Huseby and colleagues review another aspect, focusing their manuscript on the amplification of the neuroinflammatory response due to glial cells-T cell interactions (Huseby et al.). Almolda et al. from Gonzalez and Castellanos lab, also review the topic of glia-lymphocyte crosstalk but compellingly covering other pathological scenarios, suggesting that microglial cells are able to acquire a phenotype of dendritic cells (Almolda et al.). We also include articles reflecting that inflammatory glial response is involved in mental and psychiatric alterations, which include frontotemporal dementia associated with amyotrophic lateral sclerosis (ALS), reviewed by Radford et al. and the pathogenesis of delirium, reviewed by Sfera et al. Due to its patent roles in neuro-immunity, microglial cells and brain macrophages are the main protagonists of many of the papers included in this FRT. We would like to highlight the appealing work on the in vivo characterization of microglial engulfment of dying neurons presented by Morsch et al., which represents a fine piece of basic science (Morsch et al.). From a clinical point of view, Spanos et al., review the roles of microglia in brain infection, particularly in CNS tuberculosis and how this may affect future therapeutic strategies (Spanos et al.). Following the focus on microglia, Perrotta et al., present an interesting perspective article on the importance of the microglia-glioma cells crosstalk in hormone and immune-derived response in glioma (Perrotta et al.). Particularly important for glioma is the modification of the phenotype, which varies from the classically activated to pro-tumoral phenotype. In this context of phenotype modulation, Kopitar-Jerala reviews the novel role of the cystatin, statin B, in modulating microglial cells toward a pro and anti-inflammatory response (Kopitar-Jerala). In a different scenario, Lopez-Pedrajas et al. report here that cocaine-treated rats show microglial activation in the cerebellum, suggesting that glial reaction may have important implications in motor control during drug addiction (Lopez-Pedrajas et al.). Importantly, for the field of the long-term maintenance of the neuro-immune response, an interesting original article, Cao et al., reports that prior activation of microglia during embryo development may have consequences in the susceptibility to inflammation in the life of the newborns (Cao et al.). This advocates for the importance of glial cells driving lasting immune responses. Finally, Bas et al., report that infiltrated macrophages participate in the repair of spiral ganglion cells and neurons forming the cochlear nerve, having important implications for successful cochlear implant surgery (Bas et al.). We believe that this comprehensive collection of articles contains valuable information that will contribute to the knowledge on how glial cells drive the management of neuro-immunity. As the editorial team, we hope you enjoy reading, Carlos Barcia, Gilles J Guillemin, James F. Curtin, Jeffrey M. Zirger.

143. Regulated, High Capacity Adenoviral Vectors Mediated Long-Term Gene Expression in the Brain Even in the Presence of a Peripheral Immune Response to Adenovirus

For gene therapy of chronic neurological disorders, vector systems should allow long-term transduction of brain cells in the complete absence of undesirable long-term side-effects. We have engineered a novel regulatable system consisting of a tetracycline-dependent reverse-transactivator, rtTA2S-M2, in combination with Tet-repressor, tTSKid, under the control of either the major immediate early human cytomegalovirus (hCMV) promoter or murine (mCMV) promoter, encoded within high capacity, helper-dependent adenovirus vectors (HC-Adv). We assessed inducibility, leakiness and time course of expression in-vitro and in-vivo within the central nervous system (CNS). We also assessed cell-type specific expression within neurons and glial cells, and the efficiency of the regulatable vectors in the presence of a systemic immune response to adenovirus. HC-Adv vectors, i.e., STK120mBM [pSTK120m (TRE-|[beta]|-Gal-pA)-(mCMV pr.-rtTA2S-M2-IRES-tTSKid-pA)-Kanamycin] and STK120hBM [pSTK120.1 (TRE-|[beta]|-Gal-pA)-(hCMV pr.-rtTA2S-M2-IRES-tTSKid-pA)-Kanamycin] were injected into the striatum of rat brains at 1|[times]|107 blue forming units. Twenty-four hours prior to injection, the rats were given drinking water with 2 mg/ml doxycycline (Dox) and 1% sucrose, or 1% sucrose only. At the appropriate time points, rats were perfused and brains were post-fixed in 4% PFA for 3 days and vibratome sectioned for immunohistochemistry. Assessment of transgene expression levels in vivo revealed higher potency of the mCMV driven TetOn switch when compared to the hCMV driven switch in the presence of Dox. Both mCMV and hCMV encoded regulatable switches produced negligible gene expression levels in the absence of Dox. Our results demonstrate that the rtTA2S-M2 transactivator in conjunction with IRES and tTSKid transcriptional silencer displays strong and stringent induction kinetics with negligible basal activity in the uninduced state; in vivo data show stringent regulation kinetics of |[beta]|-Galactosidase expression with co-localization of |[beta]|-Galactosidase within MAP-2 (neurons) and GFAP (astrocytes) immunoreactive cells in the striatum; expression of |[beta]|-Galactosidase was dependent on the concentration of Dox and the time after HC-Adv administration. Regulated, long-term transduction in the CNS was attained even in the presence of a systemic immune response to adenovirus as could be encountered in human patients, making HC-Adv vectors very attractive for CNS gene therapy.