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

Abstract

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.

Figure 1. Anterograde and Retrograde transport after AAV infusion

Diagram illustrates anterograde and retrograde transport of AAV vectors from the site of delivery to a second distally located brain region. (a) Axonal anterograde transport requires the transport of viral particles via an axon projecting from the site of vector injection to a distal area with subsequent transduction of cells located within the brain region where the axon ends. The presence of fibers only in the distal area is not classified as anterograde transportation of the AAV vector. (b) Retrograde transport of AAV vectors occurs when viral particles are taken up by axonal terminals in the injection site and are then transported back to the neuronal cell soma where they subsequently transduce the neuron.

Figure 2. Cellular specificity of AAV6-GFP

Neuron-specific transduction after parenchymal infusion of AAV6-GFP (a, c and e) with no transgene expression detected in glia within regions of the cortex (b), striatum (d) and/or thalamus (f). Inserts show high power magnification images of GFP⁺ cells. Scale bar 20 μm.

Figure 3. Thalamo-cortical transport of AAV6 and AAV2

GFP expression in neuronal cell bodies of the cortex 3 weeks after thalamic infusion was found only in AAV6-GFP animals (a), and not seen after AAV2-GFP delivery (b). Transduction of cortical cell bodies after AAV2-GFP infusion was detected 6 weeks after thalamic injection (c). Scale bar for low 500 μm and high magnification 10 μm.

Figure 4. Axonal transport of AAV6 and AAV2 after thalamic infusion

(a) Wiring diagram of the most representative connections in the basal ganglia organization. To simplify the diagram, regions colored in white as well as dashed axonal projections were not analyzed in the present study, but their presence and connectivity to other regions is acknowledged. Regions in colored green represent GFP⁺ areas after thalamic infusion of AAV6-GFP. Comparative infusion of AAV6-GFP at 3 weeks and AAV2-GFP at 6 weeks into the thalamus region revealed the presence of GFP⁺ cell bodies within the substantia nigra pars reticulata only after AAV6-GFP administration (b); this was not observed in AAV2-GFP animals (c). Note that tyrosine hydroxylase staining (red) reveals the sharp demarcation between compacta and reticulata compartments of the substantia nigra. Str: striatum, GP: globus pallidus, STN: subthalamic nucleus, Ctx: cortex, Th: thalamus, SNc/r: substantia nigra pars compacta/reticulata. Scale bar 500 μm.

Figure 5. Thalamo-striatal projections corroborated the absence of anterograde transport of AAV6 vector 6 weeks after surgery

Striatal analysis after thalamic AAV6 infusion reveals the absence of GFP+ cells (a) suggesting the lack of anterograde transport. In contrast, GFP-transduced cells were found in the striatum of animals that received AAV2 (b) demonstrating the anterograde transport of the vector. Scale bar 50 μm.

Figure 6. Retrograde axonal transport of AAV6 after striatal infusion

(a) Wiring diagram of the most representative connections in the basal ganglia organization. Transport of AAV6-GFP after striatal infusion is depicted in the diagram in green. Close examination of brain regions with known projections to the striatum demonstrated the presence of GFP⁺ cell bodies within the cortex (b), thalamus (c) and substantia nigra pars compacta (d, Green, GFP; Red, tyrosine hydroxylase), indicating a retrograde axonal transport of the vector through these projections. Note that green-colored regions in the diagram are representative of GFP⁺ cell bodies. Scale bar 500 μm.