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Background: The efficacy and safety of intracerebral gene therapy for brain disorders like Parkinson's disease depends on the appropriate distribution of gene expression., Objectives: To assess whether the distribution of gene expression is affected by vector titer and protein type., Methods: Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received a 30-µl inoculation of a high- or a low-titer suspension of AAV5 encoding glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP) in the right and left ventral postcommissural putamen. The inoculations were conducted using convection-enhanced delivery and intraoperative MRI (IMRI)., Results: IMRI confirmed targeting and infusion cloud irradiation from the catheter tip into the surrounding area. A postmortem analysis 6 weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection site that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the substantia nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many neurons of the SNpc and SNpr., Conclusions: After controlling for target and infusate volume, the intracerebral distribution of the gene product was affected by the vector titer and product biology.

Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.

Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.

For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.

Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP-positive cells, performed using immunohistochemistry and confocal microscopy, showed that most of the GFP-positive cells by either serotype were NeuN-positive neuronal profiles. The rAAV5 vector further displayed the ability to transduce non-neuronal cell types in both rats and pigs, albeit at a low frequency. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species., (Copyright 2009 Elsevier Inc. All rights reserved.)

During the last decade, identification of the genes involved in familial forms of Parkinson's disease (PD) has advanced our understanding of the mechanisms underlying the development of different aspects of PD. However the available animal models still remain as the main limiting factor for the development of neuroprotective therapies that can halt the progression of the disease, through which we wish to provide a better quality of life for the PD patients. Here, we review the recently developed animal models based on overexpression of PD-associated genes using recombinant viral vectors. Recombinant adeno-associated viral vectors, in particular, have been very useful in targeting the nigral dopamine neurons both in the rodent and the primate brain. In order to provide insights into the establishment of these models in the laboratory, we will not only give an overview of the results from these studies but also cover practical issues related to the production and handling of the viral vectors, which are critical for the successful application of this approach.

Adenovirus-based (Ad) vectors are used widely for experimental gene transfer to the CNS. Ad transduce many cell types including postmitotic neurons. However, their use for CNS gene transfer is limited due to the host immune response elicited. Furthermore, the extensive distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows adenoviral vectors to infect a broad range of host cells which may be disadvantageous in tissues with various different cell types, like the CNS. The use of tissue-specific promoters allows for neuron-restricted gene expression, even though gene expression driven by these promoters is often very weak. Accordingly, increased transgene expression levels from viral transcription units are needed in order to improve the overall performance of Ad vectors. We designed a high-capacity Ad vector (HC-Ad) that allows for high-level, neuron-restricted transgene expression and shows no obvious signs of immunogenicity or toxicity in the mouse brain.

Adenoviruses are widely used as gene transfer vehicles because they can be produced at high titers, they have a large transgene capacity, and can transduce both dividing and non-dividing cells. One disadvantage of adenoviral vectors is the narrow therapeutic window due to a dose-dependent humoral as well as a T-cell dependent host immune response directed against the transduced cells, that leads to a reduction of transgene expression with time. By increasing the levels of protein expression from transcription units, vector titres may be decreased without a significant loss of transgene expression. Introns are required for efficient expression of many protein coding genes. In addition, splicing signals are required for some genes in order to be translated. Therefore, a chimeric intron sequence was introduced at the 3' end of the transgenes to study its effect on protein expression from adenoviral vector constructs. Transfection of 293 cells with the adenoviral shuttle plasmids pMH4-EGFP-Int, pMH4-E314.7-Int, pMH4-BclX(L)-Int and pMH4-Luc-Int lead to a 1.8-20-fold increase in protein expression as compared to constructs lacking an intron. Injection of Ad-CMV-Luc-Int into the brain of C57Bl/6 mice results in an approximately three-fold increase of luciferase activity as compared to Ad-CMV-Luc. In conclusion, insertion of an intron sequence leads to a significant increase in transgene expression both in vitro and in vivo, thus allowing for reduction of the adenoviral vector dose used.

Unraveling the causes of regeneration failure in the adult injured CNS has remained a challenge in neurobiology. The notion that CNS neurons lose their regenerative potential during development has been challenged by the identification of several promoters of axon growth. Novel methods are required that allow to study and quantify interactions of molecular determinants, and to envisage future treatment applications. Here we report a novel, highly reproducible method for monitoring axonal regeneration of mature retinal ganglion cells (RGCs) in vitro. In contrast to earlier explantation methods, primary cultures derived from adult rodent retina are kept viable without growth factor supplements. Further, since intraretinal RGC axons remain unmyelinated, regeneration can be followed independently of non-permissive white matter compounds. Applying tracing techniques prior to retinal explantation, cell survival can be correlated to outgrowth activity on the single cell level. Following intervention with pharmacological, growth factor, or gene transfer treatments, retinal explants, and partially RGC neurites, can be processed for protein and gene expression analysis. This novel procedure will prove useful to get insight into complex cell survival and regeneration promoting cascades, and will complement in vivo strategies such as transgenic and knock out mouse models., (Copyright 2004 Elsevier B.V.)

Tumor necrosis factor-alpha (TNF-alpha) represents a central distal mediator of inflammation. It is a protein released upon infections, traumatic, lesions or autoimmune disorders from immunocompetent cells and thus maintains or enhances the inflammatory reaction. The determination of such mediators in experimentally challenged animals is a mean for testing the efficacy of putative drugs. Alternative attempts for the assessment of mediator release from cell cultures are limited by profound differences in the time course of mediator release in vitro. We checked the hypothesis that these kinetic differences are due to the lack of elimination of mediators formed and released in vitro. We used the release of TNF-alpha from liver macrophage cultures stimulated with the bacterial cell wall component endotoxin as a model. The discrepancy between the in vivo release of the cytokines during endotoxic shock in the rat and the in vitro release from Kupffer cells was confirmed. By using a continuous open perfusion system instead of a static culture, the simulation of an elimination resulted in a mediator release that closely resembled the kinetics seen in vivo. Perfusion cultures appear to be suitable for relevant in vitro screening models in drug development and testing.