Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation.

Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain.

We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

Convection-enhanced delivery improves distribution and efficacy of tumor-selective retroviral replicating vectors in a rodent brain tumor model.

In the present study, we compared the therapeutic effect of tumor-selective retroviral replicating vectors (RRV) expressing the yeast cytosine deaminase (CD) delivered by convection-enhanced delivery (CED) or simple injection, followed by systemic administration of the pro-drug, 5-fluorocytosine (5-FC). Treatment with RRV-CD and systemic 5-FC significantly increased survival in rodent U87MG glioma model in comparison with controls (P<0.01). Interestingly, CED of RRV-CD followed by 5-FC further enhanced survival in this animal model in comparison with intra-tumoral injection of RRV-CD, followed by systemic 5-FC (P<0.05). High expression levels of Ki-67 were found in untreated tumors compared with treated. Untreated tumors were also much larger than treated. CED resulted in excellent distribution of RRV while only partial distribution of RRV was obtained after injection. Furthermore, RRV-CD and CD were also found in tumors from treated rats at study end points. These results demonstrated that RRV vectors may efficiently transduce and stably propagate in malignant human glioma, thereby achieving a significant in situ amplification effect after initial administration. We conclude that delivery of RRV into the glioma by CED provides much wider vector distribution than simple injection, and this correlated with better therapeutic outcomes.

Axonal transport of adeno-associated viral vectors is serotype-dependent.

We have previously shown that adeno-associated virus type 2 (AAV2) undergoes anterograde axonal transport in rat and non-human primate brain. We screened other AAV serotypes for axonal transport and found that AAV6 is transported almost exclusively in a retrograde direction and, in the same way as AAV2, it is also neuron-specific in rat brain. Our findings show that axonal transport of AAV is serotype dependent and this has implications for gene therapy of neurological diseases such as Huntington's disease.

Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders.

Gene replacement therapy for the neurological deficits caused by lysosomal storage disorders, such as in Niemann-Pick disease type A, will require widespread expression of efficacious levels of acid sphingomyelinase (ASM) in the infant human brain. At present there is no treatment available for this devastating pediatric condition. This is partly because of inherent constraints associated with the efficient delivery of therapeutic agents into the CNS of higher order models. In this study we used an adeno-associated virus type 2 (AAV2) vector encoding human acid sphingomyelinase tagged with a viral hemagglutinin epitope (AAV2-hASM-HA) to transduce highly interconnected CNS regions such as the brainstem and thalamus. On the basis of our data showing global cortical expression of a secreted reporter after thalamic delivery in nonhuman primates (NHPs), we set out to investigate whether such widespread expression could be enhanced after brainstem infusion. To maximize delivery of the therapeutic transgene throughout the CNS, we combined a single brainstem infusion with bilateral thalamic infusions in naive NHPs. We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites.

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

Maintenance and plasticity of striatal neurons is dependent on brain-derived neurotrophic factor (BDNF), which is depleted in the Huntington's 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 Huntington's disease brain, and the critical role of pallidal neurons in facilitating motor performance.

Temporal profile of subventricular zone progenitor cell migration following quinolinic acid-induced striatal cell loss.

A number of studies have demonstrated directed migration of neural progenitor cells to sites of brain injury and disease, however a detailed examination of when a cell is "born" in relation to injury induction and the migratory response of that cell has not previously been determined. This study therefore examined the temporal correlation between progenitor cell proliferation ("birth") and neuroblast migratory response into the damaged striatum following quinolinic acid (QA) lesioning of the adult rat striatum. Retroviral labeling of subventricular zone (SVZ)-derived progenitor cells demonstrated that cell loss in the QA-lesioned striatum increased progenitor cell migration through the rostral migratory stream (RMS) for up to 30 days. In addition, a population of dividing cells originating from the SVZ generated doublecortin positive neuroblasts that migrated into the damaged striatum in response to cell loss invoked by the QA lesion. Quantification of bromodeoxyuridine (BrdU)-labeled cells co-expressing doublecortin revealed that the majority of cells present in the damaged striatum were generated from progenitor cells dividing within 2 days either prior to or following the QA lesion. In contrast, cells dividing 2 or more days following QA lesioning, migrated into the striatum and exhibited a glial phenotype. These results demonstrate that directed migration of SVZ-derived cells and neuroblast differentiation in response to QA lesioning of the striatum is acute and transient. We propose this is predominantly due to a reduced capacity over time for newly generated neuroblasts to respond to the lesioned environment due to a loss or inhibition of migratory cues.

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 Huntington's 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 Huntington's 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 Huntington's 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.