AAV2 and AAV8 viral vectors administered subretinally transduce retinal pigment epithelium and photoreceptors in monkey.

Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane, rather than fundamental differences in transport mechanisms among AAV serotypes.

Nerve growth factor (NGF) gene therapy rescues and stimulates cholinergic neurons, which degenerate in Alzheimer's disease (AD). In a recent clinical trial for AD, intraparenchymal adeno-associated virus serotype 2 (AAV2)-NGF delivery was safe but did not improve cognition. Before concluding that NGF gene therapy is ineffective, it must be shown that AAV2-NGF successfully engaged the target cholinergic neurons of the basal forebrain. In this study, patients with clinically diagnosed early- to middle-stage AD received a total dose of 2 × 10 ¹¹ vector genomes of AAV2-NGF by stereotactic injection of the nucleus basalis of Meynert. After a mean survival of 4.0 years, AAV2-NGF targeting, spread, and expression were assessed by immunolabeling of NGF and the low-affinity NGF receptor p75 at 15 delivery sites in 3 autopsied patients. NGF gene expression persisted for at least 7 years at sites of AAV2-NGF injection. However, the mean distance of AAV2-NGF spread was only 0.96 ± 0.34 mm. NGF did not directly reach cholinergic neurons at any of the 15 injection sites due to limited spread and inaccurate stereotactic targeting. Because AAV2-NGF did not directly engage the target cholinergic neurons, we cannot conclude that growth factor gene therapy is ineffective for AD. Upcoming clinical trials for AD will utilize real-time magnetic resonance imaging guidance and convection-enhanced delivery to improve the targeting and spread of growth factor gene delivery.

Adeno-associated virus (AAV) vectors can move along axonal pathways after brain injection, resulting in transduction of distal brain regions. This can enhance the spread of therapeutic gene transfer and improve treatment of neurogenetic disorders that require global correction. To better understand the underlying cellular mechanisms that drive AAV trafficking in neurons, we investigated the axonal transport of dye-conjugated AAV9, utilizing microfluidic primary neuron cultures that isolate cell bodies from axon termini and permit independent analysis of retrograde and anterograde axonal transport. After entry, AAV was trafficked into nonmotile early and recycling endosomes, exocytic vesicles, and a retrograde-directed late endosome/lysosome compartment. Rab7-positive late endosomes/lysosomes that contained AAV were highly motile, exhibiting faster retrograde velocities and less pausing than Rab7-positive endosomes without virus. Inhibitor experiments indicated that the retrograde transport of AAV within these endosomes is driven by cytoplasmic dynein and requires Rab7 function, whereas anterograde transport of AAV is driven by kinesin-2 and exhibits unusually rapid velocities. Furthermore, increasing AAV9 uptake by neuraminidase treatment significantly enhanced virus transport in both directions. These findings provide novel insights into AAV trafficking within neurons, which should enhance progress toward the utilization of AAV for improved distribution of transgene delivery within the brain.

An inverted position after intrathecal AAV9 infusion markedly enhances gene delivery to the brain.