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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.

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.

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.

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., (© 2013 The Authors. British Journal of Clinical Pharmacology © 2013 The British Pharmacological Society.)

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.

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.

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.

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.

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.

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.

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.

Parkinson's disease is a neurodegenerative disorder affecting the dopaminergic neurons in the substantia nigra. Aggregation of alpha-synuclein appears to play a central role in the pathogenesis. Novel animal models for neurodegeneration have been generated by lentiviral vector-mediated locoregional overexpression of disease-associated genes in the adult brain. We have used lentiviral vectors to overexpress a clinical mutant of alpha-synuclein, A30P, in the rat substantia nigra. This overexpression induced time-dependent cytoplasmic and neuritic accumulation of alpha-synuclein and neurodegeneration. A subgroup of the rats developed asymmetric rotational behavior after administration of amphetamine. In addition, these animals displayed reduced dopamine transporter binding visualized by 123I-FP-CIT microSPECT imaging. The behavioral and microSPECT data were validated by histological analysis. There was a strong correlation between the reduction of dopaminergic neurons in the substantia nigra and the reduction of dopamine transporter binding in the striatum. MicroSPECT imaging enables non-invasive imaging of the neurodegeneration allowing longitudinal follow-up in this new animal model for Parkinson's disease and the evaluation of neuroprotective drugs.