Corinna Burger

Recombinant adeno-associated viral vector (rAAV) delivery of GDNF provides protection against 6-OHDA lesion in the common marmoset monkey (Callithrix jacchus)

Glial cell line-derived neurotrophic factor (GDNF) has shown potential as a treatment for Parkinsons disease. Recombinant adeno-associated viral vectors expressing the GDNF protein (rAAV-GDNF) have been used in rodent models of Parkinsons disease to promote functional regeneration after 6-OHDA lesions of the nigrostriatal system. The goal of the present study was to assess the anatomical and functional efficacy of rAAV-GDNF in the common marmoset monkey (Callithrix jacchus). rAAV-GDNF was injected into the striatum and substantia nigra 4 weeks prior to a unilateral 6-OHDA lesion of the nigrostriatal bundle. Forty percent of the dopamine cells in the lesioned substantia nigra of the rAAV-GDNF-treated monkeys survived, compared with 21% in the untreated monkeys. Fine dopaminergic fibres were observed microscopically in the injected striatum of some rAAV-GDNF-treated monkeys, suggesting that rAAV-GDNF treatment may have prevented, at least in part, the loss of dopaminergic innervation of the striatum. Protection of dopamine cells and striatal fibre innervation was associated with amelioration of the lesion-induced behavioural deficits. rAAV-GDNF-treated monkeys showed partial or complete protection not only in the amphetamine and apomorphine rotation but also in head position and the parkinsonian disability rating scale. Therefore, our study provides evidence for the behavioural and anatomical efficacy of GDNF delivered via an rAAV vector as a possible treatment for Parkinsons disease.

rAAV-mediated nigral human parkin over-expression partially ameliorates motor deficits via enhanced dopamine neurotransmission in a rat model of Parkinson's disease

We hypothesized that over-expressing the E3 ligase, parkin, whose functional loss leads to Parkinsons disease, in the nigrostriatal tract might be protective in the unilateral 6-hydroxydopamine (6-OHDA) rat lesion model. Recombinant adeno-associated virus (rAAV) encoding human parkin or green fluorescent protein (GFP) was injected into the rat substantia nigra 6 weeks prior to a four-site striatal 6-OHDA lesion. Vector-mediated parkin over-expression significantly ameliorated motor deficits as measured by amphetamine-induced rotational behavior and spontaneous behavior in the cylinder test but forelimb akinesia as assessed by the stepping test was unaffected. rAAV-mediated human parkin was expressed in the nigrostriatal tract, the substantia pars reticulata, and the subthalamic nucleus. However, in lesioned animals, there was no difference between nigral parkin and GFP-transduction on lesion-induced striatal tyrosine hydroxylase (TH) innervation or nigral TH positive surviving neurons. A second lesion experiment was performed to determine if striatal dopamine (DA) neurotransmission was enhanced as measured biochemically. In this second group of parkin and GFP treated rats, behavioral improvement was again observed. In addition, striatal TH and DA levels were slightly increased in the parkin-transduced group. In a third experiment, we evaluated parkin and GFP transduced rats 6 weeks after vector injection without DA depletion. When challenged with amphetamine, parkin treated rats tended to display asymmetries biased away from the treated hemisphere. Nigral parkin over-expression induced increases in both striatal TH and DA levels. Therefore, while parkin over-expression exerted no protective effect on the nigrostriatal DA system, parkin appeared to enhance the efficiency of nigrostriatal DA transmission in intact nigral DA neurons likely due to the observed increases in TH.

Genome-wide analysis of aging and learning-related genes in the hippocampal dentate gyrus

We have previously described the transcriptional changes that occur in the hippocampal CA1 field of aged rats following a Morris Water Maze (MWM) training paradigm. In this report we proceed with the analysis of the dentate region from the same animals. Animals were first identified as age learning-impaired or age-superior learners when compared to young rats based on their performance in the MWM. Messenger RNA was isolated from the dentate gyrus of each animal to interrogate Affymetrix RAE 230A rat genome microarrays. Microarray profiling identified 1129 genes that were differentially expressed between aged and young rats as a result of aging, and independent of their behavioral training (p<0.005). We applied Ingenuity Pathway Analysis (IPA) algorithms to identify the significant biological processes underlying age-related changes in the dentate gyrus. The most significant functions, as calculated by IPA, included cell movement, cell growth and proliferation, nervous system development and function, cellular assembly and organization, cell morphology and cell death. These significant processes are consistent with age-related changes in neurogenesis, and the neurogenic markers were generally found to be downregulated in senescent animals. In addition, statistical analysis of the different experimental groups of aged animals recognized 85 genes (p<0.005) that were different in the dentate gyrus of aged rats that had learned the MWM when compared to learning impaired and a number of controls for stress, exercise and non-spatial learning. The list of learning-related genes expressed in the dentate adds to the set of genes we previously described in the CA1 region. This long list of genes constitutes a starting tool to elucidating the molecular pathways involved in learning and memory formation.

Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity

Intraventricular delivery of glial cell line-derived neurotrophic factor (GDNF) results in weight loss. We hypothesized that this effect of GDNF was likely mediated via its effects on dopaminergic neurons in the hypothalamus. Continuous rAAV-mediated GDNF expression in the hypothalamus of young and senescent rats resulted in weight loss compared to controls. However, GDNF-induced weight loss was unrelated to alterations in hypothalamic dopamine levels. The weight loss was associated with decreased food intake and increased energy expenditure, but these effects were not mediated by changes in hypothalamic NPY or POMC expression. Moreover, uncoupling protein 1 levels were unchanged in brown adipose tissue (BAT). The reduction in weight and adiposity were as great or greater in the aged rats even though aged rats are generally resistant to weight loss therapies. In summary, central GDNF gene delivery reduces weight and adiposity in young and aged rats through decreased food intake and increased energy expenditure. Our observations in aged rats suggest that GDNF may be especially effective in reducing obesity in aged obese rats.

Recombinant Adeno-Associated Viral Vectors for CNS Gene Therapy

Publisher Summary Recombinant Adeno-Associated Virus (rAAV) has become a promising vector for gene therapy and the treatment of neurological disorder. Gene therapy for disorders of the central nervous system (CNS) is a moderately invasive method for delivering large molecules inside the blood–brain barrier (BBB) as compared to mechanical devices such as pumps or reservoirs. The therapy focuses into localized delivery of a given transgene. In particular, disorders where it is beneficial to overexpress or reduce expression of a transgene in a particular anatomical region to the exclusion of global delivery are especially attractive targets. Other disorders that require transduction of the entire brain or spinal cord are at the moment challenges, for which optimization of diffusion of the vector and transduction efficiencies are in the developmental stage. The future of gene therapy will depend on addressing safety issues such as the immune responses to viral delivery and regulation of the transgene in the trial design Abstract Recombinant Adeno–Associated Virus has become a promising vector for gene therapy and the treatment of neurological disorders. The fact that AAV has not been associated with any pathological condition in humans and that the recombinant vector displays widespread cell tropism and stable expression in different tissues have made AAV a favored gene transfer system in recent years. This chapter discusses different aspects of the biology of AAV and the properties of this viral vector in the nervous system. Finally, factors that are important for the safe use of this gene delivery system into humans are discussed.

AAV for disorders of the CNS

Publisher Summary This chapter describes gene therapy of neurological disorders using mainly recombinant adeno-associated virus 2 (rAAV2) as a delivery vector. rAAV has proved to be an efficient and safe vector for gene delivery in the nervous system. Single injections into brain parenchyma cause little, if any, inflammatory response and only a modest humoral response to capsid antigen. This results in long-term gene expression that is believed to persist for the lifetime of the animal. AAV, therefore, is ideally suited for the task of gene transfer in the brain for all diseases that require long-term expression. Because vector injection in brain parenchyma results in limited diffusion, the logical candidates for rAAV gene therapy are the diseases that affect a discrete region of the brain, such as epilepsy or Parkinson disease. To effectively treat diseases that affect the entire CNS, better methods will have to be developed to distribute vector over the entire brain. In addition, many of the genes contemplated for therapy have associated toxicity when they are overexpressed.