Publications by authors named "Mariana Puntel"

34 Publications

Development of a platform process for the production and purification of single-domain antibodies.

Biotechnol Bioeng 2021 Feb 24. Epub 2021 Feb 24.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for single-domain antibodies in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two-step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as five weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in E. coli and purified using affinity tags. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/bit.27724DOI Listing
February 2021

Chronic Hippocampal Expression of Notch Intracellular Domain Induces Vascular Thickening, Reduces Glucose Availability, and Exacerbates Spatial Memory Deficits in a Rat Model of Early Alzheimer.

Mol Neurobiol 2018 Nov 26;55(11):8637-8650. Epub 2018 Mar 26.

Laboratory of Amyloidosis and Neurodegeneration, Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, C1405BWE, Ciudad Autónoma de Buenos Aires, Argentina.

The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aβ) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aβ in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aβ pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.
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http://dx.doi.org/10.1007/s12035-018-1002-3DOI Listing
November 2018

Iron Availability Compromises Not Only Oligodendrocytes But Also Astrocytes and Microglial Cells.

Mol Neurobiol 2018 02 14;55(2):1068-1081. Epub 2017 Jan 14.

Departamento de Química Biológica, Facultad de Farmacia y Bioquímica. IQUIFIB-CONICET, Universidad de Buenos Aires, Junín 956, C1113, Buenos Aires, Argentina.

When disrupted, iron homeostasis negatively impacts oligodendrocyte (OLG) differentiation and impairs myelination. To better understand myelin formation and OLG maturation, in vivo and in vitro studies were conducted to evaluate the effect of iron deficiency (ID) not only on OLG maturation but also on astrocytes (AST) and microglial cells (MG). In vivo experiments in an ID model were carried out to describe maturational events during OLG and AST development and the reactive profile of MG during myelination when iron availability is lower than normal. In turn, in vitro assays were conducted to explore proliferating and maturational states of each glial cell type derived from control or ID conditions. Studies targeted NG2, PDGFRα, CNPAse, CC1, and MBP expression in OLG, GFAP and S100 expression in AST, and CD11b, ED1, and cytokine expression in MG, as well as BrDU incorporation in the three cell types. Our results show that ID affected OLG development at early stages, not only reducing their maturation capacity but also increasing their proliferation and affecting their morphological complexity. AST ID proliferated more than control ones and were more immature, much like OLG. Cytokine expression in ID animals reflected an anti-inflammatory state which probably influenced OLG maturation. These results show that ID conditions alter all glial cells and may impact myelin formation, which could be regulated by a mechanism involving a cross talk between AST, MG, and oligodendrocyte progenitors (OPC).
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http://dx.doi.org/10.1007/s12035-016-0369-2DOI Listing
February 2018

Fibulin-2 is a key mediator of the pro-neurogenic effect of TGF-beta1 on adult neural stem cells.

Mol Cell Neurosci 2015 Jul 5;67:75-83. Epub 2015 Jun 5.

Leloir Institute Foundation, Institute for Biochemical Research of Buenos Aires - IIBBA CONICET, Patricias Argentinas 435, 1405 Buenos Aires, Argentina. Electronic address:

Transforming growth factor beta 1 (TGF-beta1), an anti-inflammatory cytokine, has been shown to have pro-neurogenic effects on adult Neural Stem Cells (aNSC) from the dentate gyrus and in vivo models. Here, we expanded the observation of the pro-neurogenic effect of TGF-beta1 on aNSC from the subventricular zone (SVZ) of adult rats and performed a functional genomic analysis to identify candidate genes to mediate its effect. 10 candidate genes were identified by microarray analysis and further validated by qRT-PCR. Of these, Fibulin-2 was increased 477-fold and its inhibition by siRNA blocks TGF-beta1 pro-neurogenic effect. Curiously, Fibulin-2 was not expressed by aNSC but by a GFAP-positive population in the culture, suggesting an indirect mechanism of action. TGF-beta1 also induced Fibulin-2 in the SVZ in vivo. Interestingly, 5 out of the 10 candidate genes identified are known to interact with integrins, paving the way for exploring their functional role in adult neurogenesis. In conclusion, we have identified 10 genes with putative pro-neurogenic effects, 5 of them related to integrins and provided proof that Fibulin-2 is a major mediator of the pro-neurogenic effects of TGF-beta1. These data should contribute to further exploring the molecular mechanism of adult neurogenesis of the genes identified and the involvement of the integrin pathway on adult neurogenesis.
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http://dx.doi.org/10.1016/j.mcn.2015.06.004DOI Listing
July 2015

Temozolomide does not impair gene therapy-mediated antitumor immunity in syngeneic brain tumor models.

Clin Cancer Res 2014 Mar 5;20(6):1555-1565. Epub 2014 Feb 5.

Department of Neurosurgery, The University of Michigan, School of Medicine, 1150 West Medical Center Drive, MSRB II, Rm: 4570, Ann Arbor, MI 48109-0650.

Purpose: Glioblastoma multiforme is the most common primary brain cancer in adults. Chemotherapy with temozolomide (TMZ) significantly prolongs the survival of patients with glioblastoma multiforme. However, the three-year survival is still approximately 5%. Herein, we combined intratumoral administration of an adenoviral vector expressing Flt3L (Ad-Flt3L) with systemic temozolomide to assess its impact on therapeutic efficacy.

Experimental Design: Wild-type or immunodeficient mice bearing intracranial glioblastoma multiforme or metastatic melanoma were treated with an intratumoral injection of Ad-Flt3L alone or in combination with the conditionally cytotoxic enzyme thymidine kinase (Ad-TK), followed by systemic administration of ganciclovir and temozolomide. We monitored survival and measured the tumor-infiltrating immune cells.

Results: Although treatment with temozolomide alone led to a small improvement in median survival, when used in combination with gene therapy-mediated immunotherapy, it significantly increased the survival of tumor-bearing mice. The antitumor effect was further enhanced by concomitant intratumoral administration of Ad-TK, leading to 50% to 70% long-term survival in all tumor models. Although temozolomide reduced the content of T cells in the tumor, this did not affect the therapeutic efficacy. The antitumor effect of Ad-Flt3L+Ad-TK+TMZ required an intact immune system because the treatment failed when administered to knock out mice that lacked lymphocytes or dendritic cells.

Conclusions: Our results challenge the notion that chemotherapy leads to a state of immune-suppression which impairs the ability of the immune system to mount an effective antitumor response. Our work indicates that temozolomide does not inhibit antitumor immunity and supports its clinical implementation in combination with immune-mediated therapies.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-2140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959570PMC
March 2014

Protection induced by a glycoprotein E-deleted bovine herpesvirus type 1 marker strain used either as an inactivated or live attenuated vaccine in cattle.

BMC Vet Res 2014 Jan 8;10. Epub 2014 Jan 8.

Instituto de Virología Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias (CICV), Instituto Nacional de Tecnología Agropecuaria (INTA), Castelar, CC77, 1708 Morón, Argentina.

Background: Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections; causing a high economic loss in all continents. Use of marker vaccines in IBR eradication programs is widely accepted since it allows for protection of the animals against the disease while adding the possibility of differentiating vaccinated from infected animals.The aim of the present study was the development and evaluation of safety and efficacy of a glycoprotein E-deleted (gE-) BoHV-1 marker vaccine strain (BoHV-1ΔgEβgal) generated by homologous recombination, replacing the viral gE gene with the β-galactosidase (βgal) gene.

Results: In vitro growth kinetics of the BoHV-1ΔgEβgal virus was similar to BoHV-1 LA. The immune response triggered by the new recombinant strain in cattle was characterized both as live attenuated vaccine (LAV) and as an inactivated vaccine. BoHV-1ΔgEβgal was highly immunogenic in both formulations, inducing specific humoral and cellular immune responses. Antibody titers found in animals vaccinated with the inactivated vaccine based on BoHV-1ΔgEβgal was similar to the titers found for the control vaccine (BoHV-1 LA). In the same way, titers of inactivated vaccine groups were significantly higher than any of the LAV immunized groups, independently of the inoculation route (p < 0.001). Levels of IFN-γ were significantly higher (p < 0.001) in those animals that received the LAV compared to those that received the inactivated vaccine. BoHV-1ΔgEβgal exhibited an evident attenuation when administered as a LAV; no virus was detected in nasal secretions of vaccinated or sentinel animals during the post-vaccination period. BoHV-1ΔgEβgal, when used in either formulation, elicited an efficient immune response that protected animals against challenge with virulent wild-type BoHV-1. Also, the deletion of the gE gene served as an immunological marker to differentiate vaccinated animals from infected animals. All animals vaccinated with the BoHV-1ΔgE βgal strain were protected against disease after challenge and shed significantly less virus than control calves, regardless of the route and formulation they were inoculated.

Conclusions: Based on its attenuation, immunogenicity and protective effect after challenge, BoHV-1ΔgEβgal virus is an efficient and safe vaccine candidate when used either as inactivated or as live attenuated forms.
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http://dx.doi.org/10.1186/1746-6148-10-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896737PMC
January 2014

Interleukin-1β and tumor necrosis factor-α: reliable targets for protective therapies in Parkinson's Disease?

Front Cell Neurosci 2013 29;7:53. Epub 2013 Apr 29.

Institute Leloir Fundation - IIBBA-CONICET Buenos Aires, Argentina.

Neuroinflammation has received increased attention as a target for putative neuroprotective therapies in Parkinson's Disease (PD). Two prototypic pro-inflammatory cytokines interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) have been implicated as main effectors of the functional consequences of neuroinflammation on neurodegeneration in PD models. In this review, we describe that the functional interaction between these cytokines in the brain differs from the periphery (e.g., their expression is not induced by each other) and present data showing predominantly a toxic effect of these cytokines when expressed at high doses and for a sustained period of time in the substantia nigra pars compacta (SN). In addition, we highlight opposite evidence showing protective effects of these two main cytokines when conditions of duration, amount of expression or state of activation of the target or neighboring cells are changed. Furthermore, we discuss these results in the frame of previous disappointing results from anti-TNF-α clinical trials against Multiple Sclerosis, another neurodegenerative disease with a clear neuroinflammatory component. In conclusion, we hypothesize that the available evidence suggests that the duration and dose of IL-1β or TNF-α expression is crucial to predict their functional effect on the SN. Since these parameters are not amenable for measurement in the SN of PD patients, we call for an in-depth analysis to identify downstream mediators that could be common to the toxic (and not the protective) effects of these cytokines in the SN. This strategy could spare the possible neuroprotective effect of these cytokines operative in the patient at the time of treatment, increasing the probability of efficacy in a clinical setting. Alternatively, receptor-specific agonists or antagonists could also provide a way to circumvent undesired effects of general anti-inflammatory or specific anti-IL-1β or TNF-α therapies against PD.
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http://dx.doi.org/10.3389/fncel.2013.00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638129PMC
May 2013

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma.

Toxicol Appl Pharmacol 2013 May 9;268(3):318-30. Epub 2013 Feb 9.

Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689, USA.

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression "on" or "off" according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1×10(8), 1×10(9), or 1×10(10) viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1×10(9) vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma.
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http://dx.doi.org/10.1016/j.taap.2013.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641940PMC
May 2013

Gene therapy-mediated reprogramming tumor infiltrating T cells using IL-2 and inhibiting NF-κB signaling improves the efficacy of immunotherapy in a brain cancer model.

Neurotherapeutics 2012 Oct;9(4):827-43

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90095, USA.

Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B [NF-κB] inhibitors) to suppress the function of Foxp3+ Tregs and enhanced effector T-cell functions. Anti-tumor immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immune-therapeutic strategies for glioblastoma, which are currently being tested in the clinic.
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http://dx.doi.org/10.1007/s13311-012-0144-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480576PMC
October 2012

Safety profile of gutless adenovirus vectors delivered into the normal brain parenchyma: implications for a glioma phase 1 clinical trial.

Hum Gene Ther Methods 2012 Aug 5;23(4):271-84. Epub 2012 Sep 5.

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

Adenoviral vectors (Ads) have been evaluated in clinical trials for glioma. However, systemic immunity against the vectors can hamper therapeutic efficacy. We demonstrated that combined immunostimulation and cytotoxic gene therapy provides long-term survival in preclinical glioma models. Because helper-dependent high-capacity Ads (HC-Ads) elicit sustained transgene expression, in the presence of antiadenoviral immunity, we engineered HC-Ads encoding conditional cytotoxic herpes simplex type 1 thymidine kinase and immunostimulatory cytokine Fms-like tyrosine kinase ligand-3 under the control of the TetOn system. Escalating doses of combined HC-Ads (1×10(8), 1×10(9), and 1×10(10) viral particles [VP]) were delivered into the rat brain. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points after vector delivery. Histopathological analysis did not reveal any evidence of toxicity or long-term inflammation at the lower doses tested. Vector genomes were restricted to the injection site. Serum chemistry did not uncover adverse systemic side effects at any of the doses tested. Taken together, our data indicate that doses of up to 1×10(9) VP of each HC-Ad can be safely administered into the normal brain. This comprehensive toxicity and biodistribution study will lay the foundations for implementation of a phase 1 clinical trial for GBM using HC-Ads.
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http://dx.doi.org/10.1089/hgtb.2012.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545520PMC
August 2012

Cytotoxic immunological synapses do not restrict the action of interferon-γ to antigenic target cells.

Proc Natl Acad Sci U S A 2012 May 30;109(20):7835-40. Epub 2012 Apr 30.

Department of Neurosurgery, The University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Following antigen recognition on target cells, effector T cells establish immunological synapses and secrete cytokines. It is thought that T cells secrete cytokines in one of two modes: either synaptically (i.e., toward antigenic target cells) or multidirectionally, affecting a wider population of cells. This paradigm predicts that synaptically secreted cytokines such as IFN-γ will preferentially signal to antigenic target cells contacted by the T cell through an immunological synapse. Despite its physiological significance, this prediction has never been tested. We developed a live-cell imaging system to compare the responses of target cells and nonantigenic bystanders to IFN-γ secreted by CD8+, antigen-specific, cytotoxic T cells. Both target cells and surrounding nontarget cells respond robustly. This pattern of response was detected even at minimal antigenic T-cell stimulation using low doses of antigenic peptide, or altered peptide ligands. Although cytotoxic immunological synapses restrict killing to antigenic target cells, the effects of IFN-γ are more widespread.
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http://dx.doi.org/10.1073/pnas.1116058109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356634PMC
May 2012

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.

Mol Ther 2012 Apr 10;20(4):808-19. Epub 2012 Jan 10.

Cedars-Sinai Medical Center, Los Angeles, California, USA.

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.
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http://dx.doi.org/10.1038/mt.2011.243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321600PMC
April 2012

B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

Neoplasia 2011 Oct;13(10):947-60

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201571PMC
http://dx.doi.org/10.1593/neo.11024DOI Listing
October 2011

Identification and visualization of CD8+ T cell mediated IFN-γ signaling in target cells during an antiviral immune response in the brain.

PLoS One 2011 29;6(8):e23523. Epub 2011 Aug 29.

Board of Governors' Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

CD8(+) T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8(+) T cells recognize cells expressing target antigens, become activated, and secrete IFNγ. However, there are no methods to recognize individual cells that respond to IFNγ. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNγ. To identify individual mouse brain cells that respond to IFNγ we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNγ signaling, the gamma-activated site (GAS) promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8(+) T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNγ by anti-viral CD8(+) T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023523PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163574PMC
December 2011

Targeted toxins for glioblastoma multiforme: pre-clinical studies and clinical implementation.

Anticancer Agents Med Chem 2011 Oct;11(8):729-38

Department of Neurosurgery, Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI 48109-0650, USA.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. GBM is very aggressive due to its poor cellular differentiation and invasiveness, which makes complete surgical resection virtually impossible. Therefore, GBM's invasive nature as well as its intrinsic resistance to current treatment modalities makes it a unique therapeutic challenge. Extensive examination of human GBM specimens has uncovered that these tumors overexpress a variety of receptors that are virtually absent in the surrounding non-neoplastic brain. Human GBMs overexpress receptors for cytokines, growth factors, ephrins, urokinase-type plasminogen activator (uPA), and transferrin, which can be targeted with high specificity by linking their ligands with highly cytotoxic molecules, such as Diptheria toxin and Pseudomonas exotoxin A. We review the preclinical development and clinical translation of targeted toxins for GBM. In view of the clinical experience, we conclude that although these are very promising therapeutic modalities for GBM patients, efforts should be focused on improving the delivery systems utilized in order to achieve better distribution of the immuno-toxins in the tumor/resection cavity. Delivery of targeted toxins using viral vectors would also benefit enormously from improved strategies for local delivery.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364590PMC
http://dx.doi.org/10.2174/187152011797378689DOI Listing
October 2011

Gene therapy and targeted toxins for glioma.

Curr Gene Ther 2011 Jun;11(3):155-80

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of 15-18 months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208509PMC
http://dx.doi.org/10.2174/156652311795684722DOI Listing
June 2011

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics.

Proc Natl Acad Sci U S A 2010 Nov 28;107(46):20021-6. Epub 2010 Oct 28.

Gene Therapeutics Research Institute, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.
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http://dx.doi.org/10.1073/pnas.1008261107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993419PMC
November 2010

Human Flt3L generates dendritic cells from canine peripheral blood precursors: implications for a dog glioma clinical trial.

PLoS One 2010 Jun 11;5(6):e11074. Epub 2010 Jun 11.

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and Department of Medicine and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.

Background: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.

Methodology/principal Findings: Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-alpha and IFN-gamma. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.

Conclusions/significance: These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011074PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884015PMC
June 2010

A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

J Virol 2010 Jun 7;84(12):6007-17. Epub 2010 Apr 7.

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center/UCLA, 8700 Beverly Blvd., Davis Building Research Pavilion, Room 5090, Los Angeles, CA 90048, USA.

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.
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http://dx.doi.org/10.1128/JVI.00398-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876634PMC
June 2010

Gene transfer into rat brain using adenoviral vectors.

Curr Protoc Neurosci 2010 Jan;Chapter 4:Unit 4.24

Board of Governors Gene Therapeutics Research Institute, Departments of Medicine and Biomedical Sciences, Cedars-Sinai Medical Center and Departments of Medicine, and Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.

Viral vector-mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose-efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described.
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http://dx.doi.org/10.1002/0471142301.ns0424s50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883311PMC
January 2010

Adenovector GAD65 gene delivery into the rat trigeminal ganglion produces orofacial analgesia.

Mol Pain 2009 Aug 5;5:42. Epub 2009 Aug 5.

Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA.

Background: Our goal is to use gene therapy to alleviate pain by targeting glial cells. In an animal model of facial pain we tested the effect of transfecting the glutamic acid decarboxylase (GAD) gene into satellite glial cells (SGCs) of the trigeminal ganglion by using a serotype 5 adenovector with high tropisms for glial cells. We postulated that GABA produced from the expression of GAD would reduce pain behavior by acting on GABA receptors on neurons within the ganglion.

Results: Injection of adenoviral vectors (AdGAD65) directly into the trigeminal ganglion leads to sustained expression of the GAD65 isoform over the 4 weeks observation period. Immunohistochemical analysis showed that adenovirus-mediated GAD65 expression and GABA synthesis were mainly in SGCs. GABAA and GABAB receptors were both seen in sensory neurons, yet only GABAA receptors decorated the neuronal surface. GABA receptors were not found on SGCs. Six days after injection of AdGAD65 into the trigeminal ganglion, there was a statistically significant decrease of pain behavior in the orofacial formalin test, a model of inflammatory pain. Rats injected with control virus (AdGFP or AdLacZ) had no reduction in their pain behavior. AdGAD65-dependent analgesia was blocked by bicuculline, a selective GABAA receptor antagonist, but not by CGP46381, a selective GABAB receptor antagonist.

Conclusion: Transfection of glial cells in the trigeminal ganglion with the GAD gene blocks pain behavior by acting on GABAA receptors on neuronal perikarya.
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http://dx.doi.org/10.1186/1744-8069-5-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734545PMC
August 2009

Release of HMGB1 in response to proapoptotic glioma killing strategies: efficacy and neurotoxicity.

Clin Cancer Res 2009 Jul;15(13):4401-14

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Department of Medicine and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.

Purpose: In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches.

Experimental Design: The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-alpha, tumor necrosis factor-related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival.

Results: In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/-Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1 protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1.

Conclusions: Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-0155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2769255PMC
July 2009

T cells' immunological synapses induce polarization of brain astrocytes in vivo and in vitro: a novel astrocyte response mechanism to cellular injury.

PLoS One 2008 Aug 20;3(8):e2977. Epub 2008 Aug 20.

Board of Governors' Gene Therapeutics Research Institute, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

Background: Astrocytes usually respond to trauma, stroke, or neurodegeneration by undergoing cellular hypertrophy, yet, their response to a specific immune attack by T cells is poorly understood. Effector T cells establish specific contacts with target cells, known as immunological synapses, during clearance of virally infected cells from the brain. Immunological synapses mediate intercellular communication between T cells and target cells, both in vitro and in vivo. How target virally infected astrocytes respond to the formation of immunological synapses established by effector T cells is unknown.

Findings: Herein we demonstrate that, as a consequence of T cell attack, infected astrocytes undergo dramatic morphological changes. From normally multipolar cells, they become unipolar, extending a major protrusion towards the immunological synapse formed by the effector T cells, and withdrawing most of their finer processes. Thus, target astrocytes become polarized towards the contacting T cells. The MTOC, the organizer of cell polarity, is localized to the base of the protrusion, and Golgi stacks are distributed throughout the protrusion, reaching distally towards the immunological synapse. Thus, rather than causing astrocyte hypertrophy, antiviral T cells cause a major structural reorganization of target virally infected astrocytes.

Conclusions: Astrocyte polarization, as opposed to hypertrophy, in response to T cell attack may be due to T cells providing a very focused attack, and thus, astrocytes responding in a polarized manner. A similar polarization of Golgi stacks towards contacting T cells was also detected using an in vitro allogeneic model. Thus, different T cells are able to induce polarization of target astrocytes. Polarization of target astrocytes in response to immunological synapses may play an important role in regulating the outcome of the response of astrocytes to attacking effector T cells, whether during antiviral (e.g. infected during HIV, HTLV-1, HSV-1 or LCMV infection), anti-transplant, autoimmune, or anti-tumor immune responses in vivo and in vitro.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002977PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2496894PMC
August 2008

Regulated expression of adenoviral vectors-based gene therapies: therapeutic expression of toxins and immune-modulators.

Methods Mol Biol 2008 ;434:239-66

The Board of Governors' Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and Department of Molecular, Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.

Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the "OFF" state, and expression should quickly reach therapeutic levels in the "ON" state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA(2)S-M2 inducer and tTS(Kid) silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo.
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http://dx.doi.org/10.1007/978-1-60327-248-3_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633597PMC
September 2008

High-capacity adenovirus vector-mediated anti-glioma gene therapy in the presence of systemic antiadenovirus immunity.

J Virol 2008 May 20;82(9):4680-4. Epub 2008 Feb 20.

Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Davis Bldg., Research Pavilion, Rm. 5090, Los Angeles, CA 90048, USA.

Gene therapy is proposed as a novel therapeutic strategy for treating glioblastoma multiforme (GBM), a devastating brain cancer. In the clinic, antivector immune responses pose formidable challenges. Herein we demonstrate that high-capacity adenovirus vectors (HC-Ads) carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase (TK) induce tumor regression and long-term survival in an intracranial glioma model, even in the presence of systemic antiadenovirus immunity, as could be encountered in patients. First-generation Ad-TK failed to elicit tumor regression in this model. These results pave the way for implementing HC-Ad-TK-mediated gene therapy as a powerful adjuvant for treating GBM.
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http://dx.doi.org/10.1128/JVI.00232-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293067PMC
May 2008

Immunization against the transgene but not the TetON switch reduces expression from gutless adenoviral vectors in the brain.

Mol Ther 2008 Feb 8;16(2):343-51. Epub 2008 Jan 8.

Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.

Immune responses against vectors or encoded transgenes can impose limitations on gene therapy. We demonstrated that tetracycline-regulated high-capacity adenoviral vectors (HC-Ads) sustain regulated transgene expression in the brain even in the presence of systemic pre-existing immune responses against adenoviruses. In this study we assessed whether systemic pre-existing immune responses against the transgene products, i.e., beta-Gal or the tetracycline-dependent (TetON) regulatory transcription factors (rtTA2(S)M2 and the tTS(Kid)), affect transgene expression levels and the safety profile of HC-Ads in the brain. We pre-immunized mice with plasmids encoding the TetON switch expressing rtTA2(S)M2 and the tTS(Kid) or beta-Gal. HC-Ads expressing beta-Gal under the control of the TetON switch were then injected into the striatum. We assessed levels and distribution of beta-Gal expression, and evaluated local inflammation and neuropathological changes. We found that systemic immunity against beta-Gal, but not against the TetON switch, led to inflammation and reduction of transgene expression in the striatum. Therefore, the regulatory TetON switch appears to be safe to use, and capable of sustaining transgene expression in the brain even in the presence of an immune response against its components. Systemic immunity against the transgene had the effect of curtailing its expression, thereby affecting the efficacy and safety of gene delivery to the brain. This factor should be considered when developing gene therapies for neurological use.
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http://dx.doi.org/10.1038/sj.mt.6300375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703652PMC
February 2008

Flt3L and TK gene therapy eradicate multifocal glioma in a syngeneic glioblastoma model.

Neuro Oncol 2008 Feb 13;10(1):19-31. Epub 2007 Dec 13.

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Department of Molecular and Medical Pharmacology, University of California Los Angeles 90048, USA.

The disseminated characteristics of human glioblastoma multiforme (GBM) make it a particularly difficult tumor to treat with long-term efficacy. Most preclinical models of GBM involve treatment of a single tumor mass. For therapeutic outcomes to translate from the preclinical to the clinical setting, induction of an antitumor response capable of eliminating multifocal disease is essential. We tested the hypothesis that expression of Flt3L (human soluble FMS-like tyrosine kinase 3 ligand) and TK (herpes simplex virus type 1-thymidine kinase) within brain gliomas would mediate regression of the primary, treated tumor mass and a secondary, untreated tumor growing at a distant site from the primary tumor and the site of therapeutic vector injection. In both the single-GBM and multifocal-GBM models used, all saline-treated control animals succumbed to tumors by day 22. Around 70% of the animals bearing a single GBM mass treated with an adenovirus expressing Flt3L (AdFlt3L) and an adenovirus expressing TK (AdTK + GCV) survived long term. Approximately 50% of animals bearing a large primary GBM that were implanted with a second GBM in the contralateral hemisphere at the same time the primary tumors were being treated with AdFlt3L and AdTK also survived long term. A second multifocal GBM model, in which bilateral GBMs were implanted simultaneously and only the right tumor mass was treated with AdFlt3L and AdTK, also demonstrated long-term survival. While no significant difference in survival was found between unifocal and multifocal GBM-bearing animals treated with AdFlt3L and AdTK, both treatments were statistically different from the saline-treated control group (p < 0.05). Our results demonstrate that combination therapy with AdFlt3L and AdTK can eradicate multifocal brain tumor disease in a syngeneic, intracranial GBM model.
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http://dx.doi.org/10.1215/15228517-2007-045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2600834PMC
February 2008

One-year expression from high-capacity adenoviral vectors in the brains of animals with pre-existing anti-adenoviral immunity: clinical implications.

Mol Ther 2007 Dec 25;15(12):2154-63. Epub 2007 Sep 25.

Board of Governors' Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.

The main challenge of gene therapy is to provide long-term, efficient transgene expression. Long-term transgene expression from first generation adenoviral vectors (Advs) delivered to the central nervous system (CNS) is elicited in animals not previously exposed to adenovirus (Ad). However, upon systemic immunization against Ad, transgene expression from a first generation Adv is abolished. High-capacity Advs (HC-Advs) provide sustained very long-term transgene expression in the brain, even in animals pre-immunized against Ad. In this study, we tested the hypothesis that a HC-Adv in the brain would allow for long-term transgene expression, for up to 1 year, in the brain of mice immunized against Ad prior to delivery of the vector to the striatum. In naïve animals, the expression of beta-galactosidase from Adv or HC-Adv was sustained for 1 year. In animals immunized prior to vector delivery, expression from a first generation Adv was abolished. These results point to a very long-term HC-Adv-mediated transgene expression in the brain, even in animals that had been immunized systemically against Ad before the delivery of HC-Adv into the brain. This study therefore indicates the utility of HC-Adv as a powerful gene therapy vector for chronic neurological disorders, even in patients who had been pre-exposed to Ad prior to gene therapy.
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http://dx.doi.org/10.1038/sj.mt.6300305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268647PMC
December 2007

Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro.

Neuro Oncol 2007 Jul 23;9(3):245-58. Epub 2007 May 23.

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90069, USA.

Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model.
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http://dx.doi.org/10.1215/15228517-2007-012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1907414PMC
July 2007

Adenoviral-mediated gene transfer into the canine brain in vivo.

Neurosurgery 2007 Jan;60(1):167-77; discussion 178

Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

Objective: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding beta-galactosidase (betaGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo.

Methods: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding betaGal (Ad-betaGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, betaGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration.

Results: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and betaGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed.

Conclusion: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials.
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http://dx.doi.org/10.1227/01.NEU.0000249210.89096.6CDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095776PMC
January 2007