Adrian P. Kells

Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington's disease

The presence of ongoing neurogenesis in the adult mammalian brain raises the exciting possibility that endogenous progenitor cells may be able to generate new neurons to replace cells lost through brain injury or neurodegenerative disease. We have recently demonstrated increased cell proliferation and the generation of new neurons in the Huntingtons disease human brain. In order to better understand the potential role of endogenous neuronal replacement in neurodegenerative disorders and extend our initial observations in the human Huntingtons disease brain, we examined the effect of striatal cell loss on neurogenesis in the subventricular zone (SVZ) of the adult rodent forebrain using the quinolinic acid (QA) lesion rat model of Huntingtons disease. Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunocytochemistry for cell type-specific markers. BrdU labeling demonstrated increased cell proliferation in the SVZ ipsilateral to the QA-lesioned striatum, resulting in expansion of the SVZ in the lesioned hemisphere. Quantification revealed that QA lesion-induced striatal cell loss produced a significant increase in the area of BrdU-immunoreactivity in the SVZ ipsilateral to the lesioned hemisphere between 1 and 14 days post-lesion compared with sham-lesioned animals, with the greatest increase observed at 7 days post-lesion. These changes were associated with an increase in cells in the anterior SVZ ipsilateral to the lesioned striatum expressing the antigenic marker for SVZ neuroblasts, doublecortin (Dcx). Importantly, we observed Dcx-positive cells extending from the SVZ into the QA-lesioned striatum where a subpopulation of newly generated cells expressed markers for immature and mature neurons. This study demonstrates that loss of GABAergic medium spiny projection neurons following QA striatal lesioning of the adult rat brain increases SVZ neurogenesis, leading to the putative migration of neuroblasts to damaged areas of the striatum and the formation of new neurons.

AAV-BDNF mediated attenuation of quinolinic acid-induced neuropathology and motor function impairment.

Maintenance and plasticity of striatal neurons is dependant on brain-derived neurotrophic factor (BDNF), which is depleted in the Huntingtons disease striatum due to reduced expression and disrupted corticostriatal transportation. In this study we demonstrate that overexpression of BDNF in the striatum attenuates motor impairment and reduces the extent of striatal damage following quinolinic acid lesioning. Transfer of the BDNF gene to striatal neurons using serotype 1/2 adeno-associated viral vectors enhanced BDNF protein levels in the striatum, but induced weight loss and seizure activity following long-term high-level expression. Lower concentration BDNF expression supported striatal neurons against excitotoxic insult, as demonstrated by enhanced krox-24 immunopositive neuron survival, reduction of striatal atrophy and maintenance of the patch/matrix organization. Additionally, BDNF expression attenuated motor impairment in the forelimb use cylinder test, sensorimotor neglect in the corridor food selection task and reversed apomorphine-induced rotational behaviour. Direct correlations were shown for the first time between BDNF-mediated attenuation of behavioural impairment and the integrity of the globus pallidus, seemingly independent from the severity of striatal lesioning. These results demonstrate that BDNF holds considerable therapeutic potential for alleviating both neuropathological and motor function deficits in the Huntingtons disease brain, and the critical role of pallidal neurons in facilitating motor performance.

Verification of functional AAV-mediated neurotrophic and anti-apoptotic factor expression.

The use of viral vectors for gene delivery offer many advantages for both basic research and therapeutic application through the continuous expression of a gene product within a target region. It is vital however that any gene product is correctly expressed in a biologically active form, and this should be confirmed prior to large scale in vivo studies. Using adeno-associated viral (AAV) vectors to direct the expression of either a neurotrophic factor or an anti-apoptotic protein, we have developed a range of in vitro assays to verify functional transgenic protein expression. Brain-derived neurotropic factor (BDNF) activity was confirmed by demonstrating enhanced generation of GABAergic neurons in embryonic (E15) striatal cultures and AAV-mediated glial-derived neurotrophic factor (GDNF) function using an assay for dopaminergic differentiation of embryonic (E14) ventral mesencephalic cultures. To assess functional anti-apoptotic factor expression we designed cell-survival assays, using embryonic cortical cultures to confirm Bcl-x(L) activity and the HT1080 cell-line for X-linked inhibitor of apoptosis protein (XIAP) activity following AAV-mediated expression. This study demonstrates that the use of functional assays provides valuable confirmation of desired biotherapeutic expression prior to extensive investigation with new gene delivery vectors.

AAV-mediated expression of Bcl-xL or XIAP fails to induce neuronal resistance against quinolinic acid-induced striatal lesioning.

Apoptotic mechanisms have been proposed to contribute to the selective loss of medium spiny striatal projection neurons in Huntingtons disease (HD). This raises the question as to whether enhancing the expression of anti-apoptotic factors in vulnerable striatal projection neurons can reduce their susceptibility to neurotoxic processes occurring in the HD brain. In this study AAV 1/2 vectors encoding either the anti-apoptotic factor Bcl-xL or XIAP were used to transduce striatal neurons prior to an intrastriatal injection of the excitotoxic glutamate analogue quinolinic acid (QA). AAV 1/2 vector treated rats were observed in behavioural tests undertaken to assess whether anti-apoptotic factor expression provided amelioration of motor function impairment following unilateral QA-induced striatal lesioning. AAV-XIAP treated rats displayed complete amelioration of an ipsilateral forelimb use bias relative to control animals. However, neither AAV-XIAP nor AAV-Bcl-xL treated rats demonstrated an improvement in sensorimotor neglect compared to control animals. Furthermore, we did not observe a significant reduction of QA-induced pathology in assessed neuronal populations of the basal ganglia. These results indicate that sole enhancement of XIAP or Bcl-xL is not sufficient to counteract QA-induced excitotoxic insult of striatal neurons.

86. Comparison of CNS Transduction by Different AAV Capsids in Mouse and Non-Human Primate

Adeno-associated viral (AAV) vectors are a platform of great potential for therapeutic gene delivery. One of the major challenges regarding AAV gene therapy is to deliver the transgene of interest to target cells at levels that result in expression that is both safe and effective. The AAV vector capsid is a strong determinant of gross biodistribution, cellular tropism, transduction efficiency and transgene expression. Neurodegenerative disorders such as Friedreichs ataxia present a significant challenge in developing an effective gene therapy that provides clinically relevant distribution and expression in the central nervous system (CNS). Friedreichs ataxia is caused by an expanded GAA trinucleotide repeat in the first intron of the frataxin gene which leads to reduced levels of frataxin, a key mitochondrial protein. Pathological features include progressive degeneration of dorsal root ganglia and sensory axons, dorsal columns, Clarkes column, and dentate nuclei of the cerebellum. Elevation of frataxin in these anatomic regions is a therapeutic approach that is likely to be beneficial in addressing the neurological components of disease. However, AAV-mediated gene delivery for therapeutic expression of frataxin in the CNS and distribution to Clarkes column and cerebellar dentate nuclei, have not been investigated to a significant extent. Furthermore, a direct head-to-head comparison of multiple capsids (AAV2, 5, 6, 9, DJ, DJ8, rh10) with intrathecal dosing in non-human primates, a key translational step for therapy, has not been reported previously. Here, we used these 7 capsids to package a single-stranded (ss) vector genome (vg) containing the human frataxin gene driven by the chicken β-actin promoter, and compared frataxin expression in mouse and then non-human primate CNS. In the mouse, dramatic differences in frataxin expression were observed after intracerebroventricular injection of these capsids. Intrastriatal injection in the mouse resulted in frataxin expression patterns that differed qualitatively from those observed after intracerebroventricular injection. In vivo data from both mouse and non-human primate studies will be reported. These results will not only guide the selection and optimization of a capsid for AAV gene therapy of Friedreichs ataxia, but also provide useful information for engineering capsids for other CNS disorders.

502. Optimization of Intrathecal Delivery of AAV for Targeting the Spinal Compartment

Adeno-associated viral (AAV) vectors are a platform of great potential for therapeutic gene delivery. One of the major challenges regarding AAV gene therapy is to deliver the transgene of interest to target cells at levels that result in expression that is both safe and effective. For diseases of the central nervous system (CNS) such as amyotrophic lateral sclerosis (ALS) and Friedreichs ataxia, it is important to identify a dosing paradigm that provides a relatively homogenous distribution of gene transfer along the rostral-caudal axis of the spinal column in the CNS and is translatable. Intrathecal (IT) administration is a delivery approach that has shown promise for providing such distribution. AAV dosing via the IT route has been reported in large mammals to provide greater CNS distribution, less exposure to peripheral organs and tissues, and reduced impact of immune responses than systemic dosing. However, to-date, IT dosing of AAV in large mammals has been investigated primarily by lumbar bolus administration, with a few studies assessing administration at more rostral sites either alone or in combination with a lumbar site. Parameters that are likely to have a major impact on CNS distribution such as volume, rate and duration of infusion have not been reported previously for IT dosing of AAV. Here, we studied the effects of these parameters as well as site of infusion on distribution of transgene expression in the non-human primate CNS, using AAVrh10 to package a vector genome (vg) containing the human frataxin gene driven by the chicken β-actin promoter. Four weeks after dosing, frataxin (FXN) expression was assessed in spinal cord and dorsal root ganglia (DRG) at multiple rostral-caudal levels, as well as in brain, cerebellum, and peripheral organs such as liver, spleen and heart. Quantitative assessments included measurements of vector genome copy number and mRNA and protein expression levels. Our results showed that the distribution of transgene expression in the spinal cord, DRG, cerebellum and brain were significantly impacted by the site(s) of IT delivery (i.e. cervical vs lumbar), and volume, rate and duration of infusion. As expected, there was transduction of peripheral organs, indicating systemic exposure of the AAV vector following IT delivery. In addition, FXN expression across different cell types is being studied by histological methods. These results will not only guide the optimization of IT delivery of AAV gene therapy for diseases such as ALS and Friedreichs ataxia, but also provide useful information for IT dosing for other CNS disorders.