Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

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Alzheimer's disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution. These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD.

Gene transfer to the brain is a promising therapeutic strategy for a variety of neurodegenerative disorders including Parkinson‟s disease (PD). PD is the second most common neurodegenerative disease. Although many drugs have been developed and introduced into the market to provide symptomatic treatment, there is still no cure for PD. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for injured nigrostriatal dopamine neurons and is currently being evaluated as a potential treatment for PD. Gene therapy allows localized, long-term and stable transgene expression after a single intervention to obtain a therapeutic effect. Regulatable promoters for transgene expression furthermore allow optimizing GDNF concentration to avoid undesirable biological activity and clinical side effects. In the first part of the study, an autoregulatory tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTetbidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Eight weeks after a single intrastriatal injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids (rAAV2/1), the GDNF protein level was respectively 15 fold higherand undistinguishable from the endogenous level in doxycycline(Dox) treated and untreated animals. However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV2/1-rtTAM2-GDNF, the rAAV2/1-rtTAM2-WPRE-GDNF vector harboring a woodchuck hepatitis post-transcriptional regulatory element, which increases and stabilizes the transgene transcript, expressed a similar concentration of GDNF in the induced state but a basal level ~2.5-fold higher than the endogenous striatal level. However, the distribution of GDNF in the striatum in induced state was more widespread using the rAAV2/1-rtTAM2-WPRE-GDNF vector as compared to rAAV2/1-rtTAM2- GDNF. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase (TH) was evidenced in dopaminergic terminals of Dox-treated but not untreated animals. In the second part of my study, functional (behavioural) and neurochemical changes mediated by delayed intrastriatal GDNF gene delivery in the partial Parkinson‟s disease rat model were investigated. The rAAV2/1-rtTAM2-WPRE-GDNF vector (3.5 108 viral genomes) was administered unilaterally in the rat striatum 5 weeks after intrastriatal injection of 6-hydroxydopamine (6-OHDA) which produces a partial and progressive lesion of the nigro-striatal dopaminergic pathway. Rats were treated with Dox or untreated from the day of vector injection until sacrifice at 4 or 14 weeks (continuous treatment). A sub-group was Dox-treated for 7 weeks (temporary treatment) then untreated until 14 weeks. In the absence of Dox, the GDNF tissue concentration was found to be equivalent to the endogenous level in 6-OHDA-lesioned rats. In the presence of Dox, it was ~10-fold higher. 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. Moreover 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 third part of my study consisted in the evaluation of the respective dose-range of therapeutical and undesirable effects of GDNF. Functional effects appeared after delivery of 3.5 108 viral particles which produced 200-300 pg/mg protein of GDNF in the lesioned rat striatum (see above). In order to evaluate the viral dose producing undesirable effects, we compared two different doses of vector: 3.5x108 and 4.4x109 viral genome. In the low dose group, the GDNF concentration in the striatum was ~300 pg/mg protein in the Dox-treated animals and equivalent to the endogenous level in untreated animals (~20 pg/mg protein). In contrast, in the high dose group, GDNF levels reached ~1200 pg/mg protein in induced animals but up to ~300 pg/mg protein in uniduced animals. In the low dose group, Dox-dependent downregulation of TH but no asymetrical behaviour was evidenced. In the high dose group, TH downregulation was observed in both Dox+ and Dox-rats. In addition, amphetamine-induced rotational behaviour was evidenced in Dox+ but not in Dox-rats. These data suggest that low doses of virus are sufficient to induce therapeutically-relevant but not undesirable functional effects of GDNF. Nevertheless,a neurochemical effect of GDNF (TH down-regulation) did appear at low dose. In order to understand the GDNF-induced motor asymmetry, we investigated the anatomical pattern of TH down regulation in striatum. Strikingly, there was a greater loss of TH labeling in striosomes than in the surrounding matrix. Receptors which are known to be differentially expressed in the striosomes i.e. µ-opioid receptor(MOR-1) and N-methyl-D-aspartic acid (NMDA) receptor 1 (NR1) as compared to the matrix were analyzed in the high-dose group of animals. MOR-1 was not affected by GDNF gene delivery. In contrast, NR1 was down regulated. The potential relationship between TH and NR1 down-regulation as well as other previously described neurochemical effects of GDNF (as enhancement of DA release and metabolism, of DA neurons excitability or of TH phosphorylation) and behavioural asymmetry remains to be clarified. As summary, our data suggest that behavioural and neurochemical effects of striatal delivery of GDNF can be controlled by Dox by using the autoregulatory rAAV2/1-TetON- GDNF vector, provided the dose range of gene delivery is carefully adjusted. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished