Publications by authors named "Liliane Tenenbaum"

30 Publications

  • Page 1 of 1

Selective targeting of striatal parvalbumin-expressing interneurons for transgene delivery.

J Neurosci Methods 2021 Apr 27;354:109105. Epub 2021 Feb 27.

Laboratory of Cellular and Molecular Neurotherapies, Center for Neuroscience Research, Clinical Neurosciences Department, Lausanne University Hospital, Switzerland. Electronic address:

PV mice--> combined with AAV-FLEX vectors allowed efficient and specific targeting of PV interneurons in the striatum. However, diffusion of viral particles to the globus pallidus caused massive transduction of PV projection neurons and subsequent anterograde transport of the transgene product to the subthalamic nucleus and the substantia nigra pars reticulata. Different AAV serotypes (1 and 9) and promoters (CBA and human synapsin) were evaluated. The combination of AAV1, a moderate expression level (human synapsin promoter) and a precise adjustment of the stereotaxic coordinates in the anterior and dorsolateral part of the striatum were necessary to avoid transduction of PV GP projection neurons. Even in the absence of direct transduction due to diffusion of viral particles, GP PV projection neurons could be retrogradely transduced via their terminals present in the dorsal striatum. However, in the absence of diffusion, GP-Str PV projection neurons were poorly or not transduced suggesting that retrograde transduction did not significantly impair the selective targeting of striatal PV neurons. Finally, a prominent reduction of the number of striatal PV interneurons (about 50 %) was evidenced in the presence of the Cre recombinase suggesting that functional effects of AAV-mediated transgene expression in PV striatal interneurons in PV mice should be analyzed with caution.
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http://dx.doi.org/10.1016/j.jneumeth.2021.109105DOI Listing
April 2021

Correction: MMP9/RAGE pathway overactivation mediates redox dysregulation and neuroinflammation, leading to inhibitory/excitatory imbalance: a reverse translation study in schizophrenia patients.

Mol Psychiatry 2020 Nov;25(11):3105

Center for Psychiatric Neuroscience, Department of Psychiatry, Centre Hospitalier Universitaire Vaudois and University of Lausanne (CHUV-UNIL), Prilly-Lausanne, Switzerland.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41380-020-0716-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962564PMC
November 2020

GDNF, A Neuron-Derived Factor Upregulated in Glial Cells during Disease.

J Clin Med 2020 Feb 7;9(2). Epub 2020 Feb 7.

Laboratory of Molecular Neurotherapies and NeuroModulation, Center for Neuroscience Research, Lausanne University Hospital, CHUV-Pavillon 3, av de Beaumont, CH-1010 Lausanne, Switzerland.

In a healthy adult brain, glial cell line-derived neurotrophic factor (GDNF) is exclusively expressed by neurons, and, in some instances, it has also been shown to derive from a single neuronal subpopulation. Secreted GDNF acts in a paracrine fashion by forming a complex with the GDNF family receptor α1 (GFRα1), which is mainly expressed by neurons and can act in as a membrane-bound factor or in as a soluble factor. The GDNF/GFRα1 complex signals through interactions with the "rearranged during transfection" (RET) receptor or via the neural cell adhesion molecule (NCAM) with a lower affinity. GDNF can also signal independently from GFRα1 by interacting with syndecan-3. RET, which is expressed by neurons involved in several pathways (nigro-striatal dopaminergic neurons, motor neurons, enteric neurons, sensory neurons, etc.), could be the main determinant of the specificity of GDNF's pro-survival effect. In an injured brain, de novo expression of GDNF occurs in glial cells. Neuroinflammation has been reported to induce GDNF expression in activated astrocytes and microglia, infiltrating macrophages, nestin-positive reactive astrocytes, and neuron/glia (NG2) positive microglia-like cells. This disease-related GDNF overexpression can be either beneficial or detrimental depending on the localization in the brain and the level and duration of glial cell activation. Some reports also describe the upregulation of RET and GFRα1 in glial cells, suggesting that GDNF could modulate neuroinflammation.
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http://dx.doi.org/10.3390/jcm9020456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073520PMC
February 2020

MMP9/RAGE pathway overactivation mediates redox dysregulation and neuroinflammation, leading to inhibitory/excitatory imbalance: a reverse translation study in schizophrenia patients.

Mol Psychiatry 2020 11 25;25(11):2889-2904. Epub 2019 Mar 25.

Center for Psychiatric Neuroscience, Department of Psychiatry, Centre Hospitalier Universitaire Vaudois and University of Lausanne (CHUV-UNIL), Prilly-Lausanne, Switzerland.

Various mechanisms involved in schizophrenia pathophysiology, such as dopamine dysregulation, glutamate/NMDA receptor dysfunction, neuroinflammation or redox imbalance, all appear to converge towards an oxidative stress "hub" affecting parvalbumine interneurones (PVI) and their perineuronal nets (PNN) (Lancet Psychiatry. 2015;2:258-70); (Nat Rev Neurosci. 2016;17:125-34). We aim to investigate underlying mechanisms linking oxidative stress with neuroinflammatory and their long-lasting harmful consequences. In a transgenic mouse of redox dysregulation carrying a permanent deficit of glutathione synthesis (gclm), the anterior cingulate cortex presented early in the development increased oxidative stress which was prevented by the antioxidant N-acetylcysteine (Eur J Neurosci. 2000;12:3721-8). This oxidative stress induced microglia activation and redox-sensitive matrix metalloproteinase 9 (MMP9) stimulation, leading to the receptor for advanced glycation end-products (RAGE) shedding into soluble and nuclear forms, and subsequently to nuclear factor-kB (NF-kB) activation and secretion of various cytokines. Blocking MMP9 activation prevented this sequence of alterations and rescued the normal maturation of PVI/PNN, even if performed after an additional insult that exacerbated the long term PVI/PNN impairments. MMP9 inhibition thus appears to be able to interrupt the vicious circle that maintains the long-lasting deleterious effects of the reciprocal interaction between oxidative stress and neuroinflammation, impacting on PVI/PNN integrity. Translation of these experimental findings to first episode patients revealed an increase in plasma soluble RAGE relative to healthy controls. This increase was associated with low prefrontal GABA levels, potentially predicting a central inhibitory/excitatory imbalance linked to RAGE shedding. This study paves the way for mechanistically related biomarkers needed for early intervention and MMP9/RAGE pathway modulation may lead to promising drug targets.
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http://dx.doi.org/10.1038/s41380-019-0393-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577857PMC
November 2020

Combining Gene Transfer and Nonhuman Primates to Better Understand and Treat Parkinson's Disease.

Front Mol Neurosci 2019 11;12:10. Epub 2019 Feb 11.

MMDN, University of Montpellier, EPHE, INSERM, U1198, PSL University, Montpellier, France.

Parkinson's disease (PD) is a progressive CNS disorder that is primarily associated with impaired movement. PD develops over decades and is linked to the gradual loss of dopamine delivery to the striatum, the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). While the administration of L-dopa and deep brain stimulation are potent therapies, their costs, side effects and gradual loss of efficacy underlines the need to develop other approaches. Unfortunately, the lack of pertinent animal models that reproduce DA neuron loss and behavior deficits-in a timeline that mimics PD progression-has hindered the identification of alternative therapies. A complementary approach to transgenic animals is the use of nonhuman primates (NHPs) combined with the overexpression of disease-related genes using viral vectors. This approach may induce phenotypes that are not influenced by developmental compensation mechanisms, and that take into account the personality of animals. In this review article, we discuss the combination of gene transfer and NHPs to develop "genetic" models of PD that are suitable for testing therapeutic approaches.
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http://dx.doi.org/10.3389/fnmol.2019.00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378268PMC
February 2019

Regulated viral BDNF delivery in combination with Schwann cells promotes axonal regeneration through capillary alginate hydrogels after spinal cord injury.

Acta Biomater 2017 09 19;60:167-180. Epub 2017 Jul 19.

Spinal Cord Injury Center, Heidelberg University Hospital, Heidelberg, Germany; Stark Neurosciences Research Institute, Indiana University School of Medicine, Dept. of Neurological Surgery and Goodman Campbell Brain and Spine, Indianapolis, USA. Electronic address:

Grafting of cell-seeded alginate capillary hydrogels into a spinal cord lesion site provides an axonal bridge while physically directing regenerating axonal growth in a linear pattern. However, without an additional growth stimulus, bridging axons fail to extend into the distal host spinal cord. Here we examined whether a combinatory strategy would support regeneration of descending axons across a cervical (C5) lateral hemisection lesion in the rat spinal cord. Following spinal cord transections, Schwann cell (SC)-seeded alginate hydrogels were grafted to the lesion site and AAV5 expressing brain-derived neurotrophic factor (BDNF) under control of a tetracycline-regulated promoter was injected caudally. In addition, we examined whether SC injection into the caudal spinal parenchyma would further enhance regeneration of descending axons to re-enter the host spinal cord. Our data show that both serotonergic and descending axons traced by biotinylated dextran amine (BDA) extend throughout the scaffolds. The number of regenerating axons is significantly increased when caudal BDNF expression is activated and transient BDNF delivery is able to sustain axons after gene expression is switched off. Descending axons are confined to the caudal graft/host interface even with continuous BDNF expression for 8weeks. Only with a caudal injection of SCs, a pathway facilitating axonal regeneration through the host/graft interface is generated allowing axons to successfully re-enter the caudal spinal cord.

Statement Of Significance: Recovery from spinal cord injury is poor due to the limited regeneration observed in the adult mammalian central nervous system. Biomaterials, cell transplantation and growth factors that can guide axons across a lesion site, provide a cellular substrate, stimulate axon growth and have shown some promise in increasing the growth distance of regenerating axons. In the present study, we combined an alginate biomaterial with linear channels with transplantation of Schwann cells within and beyond the lesion site and injection of a regulatable vector for the transient expression of brain-derived neurotrophic factor (BDNF). Our data show that only with the full combination axons extend across the lesion site and that expression of BDNF beyond 4weeks does not further increase the number of regenerating axons.
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http://dx.doi.org/10.1016/j.actbio.2017.07.024DOI Listing
September 2017

Glial Cell Line-Derived Neurotrophic Factor Gene Delivery in Parkinson's Disease: A Delicate Balance between Neuroprotection, Trophic Effects, and Unwanted Compensatory Mechanisms.

Front Neuroanat 2017 10;11:29. Epub 2017 Apr 10.

Laboratory of Cellular and Molecular Neurotherapies, Clinical Neuroscience Department, Center for Neuroscience Research, Lausanne University HospitalLausanne, Switzerland.

Glial cell line-derived neurotrophic factor (GDNF) and Neurturin (NRTN) bind to a receptor complex consisting of a member of the GDNF family receptor (GFR)-α and the Ret tyrosine kinase. Both factors were shown to protect nigro-striatal dopaminergic neurons and reduce motor symptoms when applied terminally in toxin-induced Parkinson's disease (PD) models. However, clinical trials based on intraputaminal GDNF protein administration or recombinant adeno-associated virus (rAAV)-mediated NRTN gene delivery have been disappointing. In this review, several factors that could have limited the clinical benefits are discussed. Retrograde transport of GDNF/NRTN to the dopaminergic neurons soma is thought to be necessary for NRTN/GFR-α/Ret signaling mediating the pro-survival effect. Therefore, the feasibility of treating advanced patients with neurotrophic factors is questioned by recent data showing that: (i) tyrosine hydroxylase-positive putaminal innervation has almost completely disappeared at 5 years post-diagnosis and (ii) in patients enrolled in the rAAV-NRTN trial more than 5 years post-diagnosis, NRTN was almost not transported to the substantia nigra pars compacta. In addition to its anti-apoptotic and neurotrophic properties, GDNF also interferes with dopamine homeostasis via time and dose-dependent effects such as: stimulation of dopamine neuron excitability, inhibition of dopamine transporter activity, tyrosine hydroxylase phosphorylation, and inhibition of tyrosine hydroxylase transcription. Depending on the delivery parameters, the net result of this intricate network of regulations could be either beneficial or deleterious. In conclusion, further unraveling of the mechanism of action of GDNF gene delivery in relevant animal models is still needed to optimize the clinical benefits of this new therapeutic approach. Recent developments in the design of regulated viral vectors will allow to finely adjust the GDNF dose and period of administration. Finally, new clinical studies in less advanced patients are warranted to evaluate the potential of AAV-mediated neurotrophic factors gene delivery in PD. These will be facilitated by the demonstration of the safety of rAAV administration into the human brain.
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http://dx.doi.org/10.3389/fnana.2017.00029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385337PMC
April 2017

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

Neurosci Lett 2016 Sep 29;630:209-215. Epub 2016 Jul 29.

I.R.I.B.H.M (Institute of Interdisciplinary Research), Université Libre De Bruxelles Campus Erasme, Brussels, Belgium; Department of Ophthalmology, CHU St-Pierre and Brugmann, Brussels, Belgium.

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression.
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http://dx.doi.org/10.1016/j.neulet.2016.07.054DOI Listing
September 2016

Tet-On Systems For Doxycycline-inducible Gene Expression.

Curr Gene Ther 2016 ;16(3):156-67

Laboratory of Experimental Virology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox. Since the initial design and construction of the original Tet-system, these bacterium-derived systems have been significantly improved for their function in eukaryotic cells. We here review how a dox-controlled HIV-1 variant was designed and used to greatly improve the activity and dox-sensitivity of the rtTA transcriptional activator component of the Tet-On system. These optimized rtTA variants require less dox for activation, which will reduce side effects and allow gene control in tissues where a relatively low dox level can be reached, such as the brain.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070417PMC
http://dx.doi.org/10.2174/1566523216666160524144041DOI Listing
October 2017

A regulatable AAV vector mediating GDNF biological effects at clinically-approved sub-antimicrobial doxycycline doses.

Mol Ther Methods Clin Dev 2016 30;5:16027. Epub 2016 Mar 30.

Laboratory of Cellular and Molecular Neurotherapies, Center for Neuroscience Research, Dept of Clinical Neuroscience, Lausanne University Hospital , Lausanne, Switzerland.

Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 10(9) viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses.
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http://dx.doi.org/10.1038/mtm.2016.27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813607PMC
April 2016

Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

Neurobiol Dis 2016 Apr 9;88:44-54. Epub 2016 Jan 9.

Departamento de Ciencias Médicas Básicas (Anatomía), Facultad de Ciencias de la Salud (Medicina), Instituto de Tecnologías Biomédicas (ITB, CIBICAN), Universidad de La Laguna, Tenerife, Spain; Centro de investigación Biomédica en Red sobre enfermedades neurodegenerativas, CIBERNED, Instituto de Salud Carlos III, Spain. Electronic address:

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.
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http://dx.doi.org/10.1016/j.nbd.2016.01.002DOI Listing
April 2016

Mild guanidinoacetate increase under partial guanidinoacetate methyltransferase deficiency strongly affects brain cell development.

Neurobiol Dis 2015 Jul 18;79:14-27. Epub 2015 Apr 18.

Neurometabolic Unit, Service of Biomedicine, Lausanne University Hospital, CH-1011 Lausanne, Switzerland. Electronic address:

Among cerebral creatine deficiency syndromes, guanidinoacetate methyltransferase (GAMT) deficiency can present the most severe symptoms, and is characterized by neurocognitive dysfunction due to creatine deficiency and accumulation of guanidinoacetate in the brain. So far, every patient was found with negligible GAMT activity. However, GAMT deficiency is thought under-diagnosed, in particular due to unforeseen mutations allowing sufficient residual activity avoiding creatine deficiency, but enough guanidinoacetate accumulation to be toxic. With poorly known GAA-specific neuropathological mechanisms, we developed an RNAi-induced partial GAMT deficiency in organotypic rat brain cell cultures. As expected, the 85% decrease of GAMT protein was insufficient to cause creatine deficiency, but generated guanidinoacetate accumulation causing axonal hypersprouting and decrease in natural apoptosis, followed by induction of non-apoptotic cell death. Specific guanidinoacetate-induced effects were completely prevented by creatine co-treatment. We show that guanidinoacetate accumulation without creatine deficiency is sufficient to affect CNS development, and suggest that additional partial GAMT deficiencies, which may not show the classical brain creatine deficiency, may be discovered through guanidinoacetate measurement.
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http://dx.doi.org/10.1016/j.nbd.2015.03.029DOI Listing
July 2015

Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

Front Mol Neurosci 2014 2;7:92. Epub 2014 Dec 2.

Laboratory of Experimental Neurosurgery and Multidisciplinary Research Institute, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire - Université Libre de Bruxelles Bruxelles, Belgium.

We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.
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http://dx.doi.org/10.3389/fnmol.2014.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4251317PMC
December 2014

Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.

Hum Gene Ther 2014 Nov;25(11):977-87

1 Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona , 08193-Bellaterra, Spain .

Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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http://dx.doi.org/10.1089/hum.2014.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4236062PMC
November 2014

Biosafety of recombinant adeno-associated virus vectors.

Curr Gene Ther 2013 Dec;13(6):434-52

UNC Gene Therapy Center, University of North Carolina at Chapel Hill, 7119 Thurston Bowles Building, (104 Manning Drive) Campus Box 7352, Chapel Hill, NC 27599-7352, USA.

It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.
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http://dx.doi.org/10.2174/15665232113136660007DOI Listing
December 2013

A next step in adeno-associated virus-mediated gene therapy for neurological diseases: regulation and targeting.

Br J Clin Pharmacol 2013 Aug;76(2):217-32

Laboratory of Experimental Neurosurgery, Free University of Brussels (ULB), Brussels, Belgium.

Recombinant adeno-associated virus (rAAV) vectors mediating long term transgene expression are excellent gene therapy tools for chronic neurological diseases. While rAAV2 was the first serotype tested in the clinics, more efficient vectors derived from the rh10 serotype are currently being evaluated and other serotypes are likely to be tested in the near future. In addition, aside from the currently used stereotaxy-guided intraparenchymal delivery, new techniques for global brain transduction (by intravenous or intra-cerebrospinal injections) are very promising. Various strategies for therapeutic gene delivery to the central nervous system have been explored in human clinical trials in the past decade. Canavan disease, a genetic disease caused by an enzymatic deficiency, was the first to be approved. Three gene transfer paradigms for Parkinson's disease have been explored: converting L-dopa into dopamine through AADC gene delivery in the putamen; synthesizing GABA through GAD gene delivery in the overactive subthalamic nucleus and providing neurotrophic support through neurturin gene delivery in the nigro-striatal pathway. These pioneer clinical trials demonstrated the safety and tolerability of rAAV delivery in the human brain at moderate doses. Therapeutic effects however, were modest, emphasizing the need for higher doses of the therapeutic transgene product which could be achieved using more efficient vectors or expression cassettes. This will require re-addressing pharmacological aspects, with attention to which cases require either localized and cell-type specific expression or efficient brain-wide transgene expression, and when it is necessary to modulate or terminate the administration of transgene product. The ongoing development of targeted and regulated rAAV vectors is described.
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http://dx.doi.org/10.1111/bcp.12065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731597PMC
August 2013

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

PLoS One 2013 3;8(1):e53156. Epub 2013 Jan 3.

Laboratory of Experimental Neurosurgery, Université Libre de Bruxelles, Brussels, Belgium.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053156PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536800PMC
August 2013

Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

Hum Gene Ther 2012 Jul 5;23(7):742-53. Epub 2012 Jun 5.

Laboratory of Experimental Neurosurgery, Université Libre de Bruxelles, BE-1070 Brussels, Belgium.

The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.
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http://dx.doi.org/10.1089/hum.2011.216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404419PMC
July 2012

Recombinant AAV delivery to the central nervous system.

Methods Mol Biol 2011 ;807:159-77

Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium.

Recombinant AAV-mediated gene delivery to the CNS can be performed either by direct delivery at the target site or from the periphery, using intramuscular injections and retrograde transport along motor neuron projections or intravenous injections and blood-brain barrier crossing.In this chapter, we describe: 1. Methods for recombinant virus administration, including stereotactic surgery, intramuscular, and intravenous administration. 2. Methods to evaluate the number and biodistribution of brain and spinal cord cells expressing the transgene by immunohistochemisty as well as the amount of transgene product by ELISA in the target region. 3. Methods to characterize the cellular specificity of transgene expression by double immunofluorescence. 4. Methods to quantify the amounts of viral DNA as well as of transgene mRNA by quantitative PCR and RT-PCR, respectively.
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http://dx.doi.org/10.1007/978-1-61779-370-7_7DOI Listing
March 2012

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery.

Cancer Immunol Immunother 2011 Feb 13;60(2):273-81. Epub 2010 Nov 13.

Interdisciplinary Research Institute (IRIBHM), Université Libre de Bruxelles, Faculty of Medicine, route de Lennik 808, Brussels, Belgium.

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an "all in vivo" strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful "all in vivo" vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.
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http://dx.doi.org/10.1007/s00262-010-0941-yDOI Listing
February 2011

Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.

Hum Gene Ther 2010 Oct;21(10):1273-85

Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
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http://dx.doi.org/10.1089/hum.2009.223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957240PMC
October 2010

Differential transgene expression profiles in rat brain, using rAAV2/1 vectors with tetracycline-inducible and cytomegalovirus promoters.

Hum Gene Ther 2008 Nov;19(11):1293-305

Multidisciplinary Research Institute (IRIBHM), Université Libre de Bruxelles, Hôpital Erasme, Brussels, Belgium.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for the safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors (recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1) using the tetracycline-inducible (TetON) expression cassette in comparison with the cytomegalovirus (CMV) promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although green fluorescent protein (GFP) was expressed mainly in neurons with both vectors, the relative proportions of DARPP-32-positive projection neurons and parvalbumin-positive interneurons were, respectively, 13:1 and 2:1 for the CMV and TetON vectors. DARP32-positive neurons projecting to the globus pallidus were strongly GFP positive with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV vector but poorly by the TetON vector. Numerous GFP-positive cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP-positive neurons were observed with the CMV vector but not the TetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-TetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase-positive neurons by the TetON vector whereas with the CMV vector, GFP-positive cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-TetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.
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http://dx.doi.org/10.1089/hum.2008.099DOI Listing
November 2008

Reversible neurochemical changes mediated by delayed intrastriatal glial cell line-derived neurotrophic factor gene delivery in a partial Parkinson's disease rat model.

J Gene Med 2009 Oct;11(10):899-912

Laboratory of Experimental Neurosurgery, Université Libre de Bruxelles, Brussels, Belgium.

Background: Efficient protection of dopaminergic neurons against a subsequent 6-hydroxydopamine lesion by glial cell line-derived neurotrophic factor (GDNF) gene delivery has been demonstrated. By contrast, the neurorestorative effects of GDNF administered several weeks after the toxin have been less characterized. In particular, whether these were permanent or dependent on the continuous presence of GDNF remains elusive.

Methods: A tetracycline-inducible adeno-associated virus (AAV)-1 vector expressing human GDNF cDNA was administered unilaterally in the rat striatum 5 weeks after 6-hydroxydopamine. Rats were treated with doxycycline (dox) or untreated from the day of vector injection until sacrifice (4 or 14 weeks). A sub-group was dox-treated for 7 weeks then untreated until 14 weeks. The motor behavior was assessed by amphetamine-induced rotations and spontaneous forelimb asymmetry. The amounts of tyrosine hydroxylase (TH), serine-40-phosphorylated TH (S40-TH) and aromatic amino acid decarboxylase (AADC) proteins were compared by western blotting and the dopamine levels quantified by high-performance liquid chromatography.

Results: Dox-dependent behavioral improvements were demonstrated 4 weeks post-vector injection. At later time points, spontaneous partial recovery was observed in all rats, but no further improvement was found in dox-treated animals. TH levels were significantly increased in dox-treated rats at all time points. By contrast, striatal dopamine and S40-TH were increased at 4 weeks, but not 14 weeks, and AADC remained unchanged. Dox withdrawal after 7 weeks, resulted in TH levels comparable to the controls at 14 weeks.

Conclusions: Delayed GDNF gene delivery only transiently improved dopaminergic function. Over the long term, TH was more abundant, but not functional, and the increase was lost when GDNF gene expression was switched off.
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http://dx.doi.org/10.1002/jgm.1377DOI Listing
October 2009

Recombinant AAV viral vectors serotype 1, 2, and 5 mediate differential gene transfer efficiency in rat striatal fetal grafts.

Cell Transplant 2008 ;16(10):1013-20

Laboratory of Experimental Neurosurgery, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium.

Intrastriatal grafts of fetal ganglionic eminences (GE) can reverse symptoms of striatal lesions in animal models of Huntington's disease. On the other hand, neurotrophic factors have been shown to protect host striatal neurons from ongoing degeneration. Neurotrophic gene transfer into GE prior to grafting could combine the benefits of striatal neuron replacement and in situ delivery of neurotrophic factors. Here we evaluate the potency of recombinant adeno-associated viruses (rAAV) as vectors for gene delivery into rat embryonic (E15) GE using the eGFP reporter gene under the control of the strong cytomegalovirus (CMV) promoter. We observed a very efficient expression of the eGFP reporter gene in organotypic cultures of GE infected with rAAV serotype 1 from 4 days until at least 4 weeks postinfection. In contrast, transduction was low and absent when using serotype 2 and serotype 5 rAAV, respectively. Two months after transplantation of rAAV2/1-infected embryonic GE in adult rat striatum, more than 20% of grafted cells expressed eGFP. The majority of transduced cells in the graft were neurons as indicated by colabeling of GFP-immunoreactive cells with the NeuN marker. Our study suggests that GE transduced by rAAV-serotype 1 vectors could be an interesting tool to mediate efficient expression of a gene coding a neurotrophic factor in Huntington's disease.
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July 2008

Recombinant AAV Viral Vectors Serotype 1, 2, and 5 Mediate Differential Gene Transfer Efficiency in Rat Striatal Fetal Grafts.

Cell Transplant 2007 Nov;16(10):1013-1020

Laboratory of Experimental Neurosurgery, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium.

Intrastriatal grafts of fetal ganglionic eminences (GE) can reverse symptoms of striatal lesions in animal models of Huntington's disease. On the other hand, neurotrophic factors have been shown to protect host striatal neurons from ongoing degeneration. Neurotrophic gene transfer into GE prior to grafting could combine the benefits of striatal neuron replacement and in situ delivery of neurotrophic factors. Here we evaluate the potency of recombinant adeno-associated viruses (rAAV) as vectors for gene delivery into rat embryonic (E15) GE using the eGFP reporter gene under the control of the strong cytomegalovirus (CMV) promoter. We observed a very efficient expression of the eGFP reporter gene in organotypic cultures of GE infected with rAAV serotype 1 from 4 days until at least 4 weeks postinfection. In contrast, transduction was low and absent when using serotype 2 and serotype 5 rAAV, respectively. Two months after transplantation of rAAV2/1-infected embryonic GE in adult rat striatum, more than 20% of grafted cells expressed eGFP. The majority of transduced cells in the graft were neurons as indicated by colabeling of GFP-immunoreactive cells with the NeuN marker. Our study suggests that GE transduced by rAAV-serotype 1 vectors could be an interesting tool to mediate efficient expression of a gene coding a neurotrophic factor in Huntington's disease.
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http://dx.doi.org/10.3727/000000007783472372DOI Listing
November 2007

Efficiency of adeno-associated virus type-2 vectors in non-human primate Schwann cells.

Neuroreport 2005 Nov;16(16):1757-62

Institut National de la Santé et de la Recherche Medicale, Myelin and Ion Muscular Channels Diseases Laboratory, Institut Federatif des Neurosciences (IFRNS), University Pierre and Marie Curie, Paris, France.

Adult macaque Schwann cells were infected using adeno-associated virus type-2-derived vectors expressing the green fluorescent protein reporter gene under the control of the cytomegalovirus, the hybrid cytomegalovirus-betaactin, the myelin basic protein or the tetracycline-inducible promoters. On the basis of green fluorescent protein expression, gene transfer efficiency was compared in resting and dividing conditions following or not following hydroxyurea or etoposide treatment. Hydroxyurea allowed promoter-dependent expression of green fluorescent protein in infected Schwann cells. Etoposide treatment led to a high percentage of green fluorescent protein expressing cells (over 50%) with all promoters tested. When infected cells were grafted into demyelinated nude mice spinal cord, green fluorescent protein expression was only observed with the cytomegalovirus-betaactin and tetracycline-inducible promoters. In addition, adeno-associated virus type-2 infection reduced the grafted cell survival but increased their differentiation.
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http://dx.doi.org/10.1097/01.wnr.0000185960.11465.16DOI Listing
November 2005

Minocycline in phenotypic models of Huntington's disease.

Neurobiol Dis 2005 Feb;18(1):206-17

Laboratory of Neurophysiology, ULB-Erasme, Brussels, Belgium.

Minocycline has been shown to be neuroprotective in various models of neurodegenerative diseases. However, its potential in Huntington's disease (HD) models characterized by calpain-dependent degeneration and inflammation has not been investigated. Here, we have tested minocycline in phenotypic models of HD using 3-nitropropionic acid (3NP) intoxication and quinolinic acid (QA) injections. In the 3NP rat model, where the development of striatal lesions involves calpain, we found that minocycline was not protective, although it attenuated the development of inflammation induced after the onset of striatal degeneration. The lack of minocycline activity on calpain-dependent cell death was also confirmed in vitro using primary striatal cells. Conversely, we found that minocycline reduced lesions and inflammation induced by QA. In cultured cells, minocycline protected against mutated huntingtin and staurosporine, stimulations known to promote caspase-dependent cell death. Altogether, these data suggested that, in HD, minocycline may counteract the development of caspase-dependent neurodegeneration, inflammation, but not calpain-dependent neuronal death.
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http://dx.doi.org/10.1016/j.nbd.2004.09.017DOI Listing
February 2005

Clinical potential of minocycline for neurodegenerative disorders.

Neurobiol Dis 2004 Dec;17(3):359-66

Laboratory of Experimental Neurosurgery, ULB-Erasme, 1070 Brussels, Belgium.

Minocycline, an antibiotic of the tetracycline family, has been shown to display neurorestorative or neuroprotective properties in various models of neurodegenerative diseases. In particular, it has been shown to delay motor alterations, inflammation and apoptosis in models of Huntington's disease, amyotrophic lateral sclerosis and Parkinson's disease. Despite controversies about its efficacy, the relative safety and tolerability of minocycline have led to the launching of various clinical trials. The present review summarizes the available data supporting the clinical testing of minocycline for these neurodegenerative disorders. In addition, we extend our discussion to the potential applications of minocycline for combining this treatment with cellular and molecular therapy.
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http://dx.doi.org/10.1016/j.nbd.2004.07.012DOI Listing
December 2004

Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vector-mediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor.

Exp Neurol 2004 Oct;189(2):303-16

Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, 1105 AZ Amsterdam, The Netherlands.

Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.
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http://dx.doi.org/10.1016/j.expneurol.2004.05.014DOI Listing
October 2004

Minocycline-induced activation of tetracycline-responsive promoter.

Neurosci Lett 2003 Dec;352(3):155-8

Laboratory of Experimental Neurosurgery, CP602, U.L.B.-Erasme, Batiment C, niveau 6, 808 route de Lennik, 1070, Brussels, Belgium.

Minocycline has been suggested to be an anti-apoptotic compound and an anti-inflammatory agent in various models of neurodegeneration. In the present study, using a stable cell line expressing green fluorescent protein under the control of a tetracycline-responsive promoter, we demonstrate that minocycline is able to promote tetracycline-controlled gene expression although it needs longer time and higher concentration to reach the effect obtained with the classical inducer doxycycline. Furthermore, the extinction of the system after antibiotics removal is faster when using minocycline. Interestingly, minocycline displays lower cytotoxicity than doxycycline. It is thus tempting to speculate that combining the intrinsic neuroprotective activity of minocycline with its ability to induce tetracycline-regulatable promoters would be greatly beneficial for neuroprotective/neurorestaurative gene therapy.
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http://dx.doi.org/10.1016/j.neulet.2003.08.067DOI Listing
December 2003