# AAV2retro Enters Axons of Passage and Extensively Transduces Corticospinal Neurons After Injection into Spinal White Matter

## TL;DR

A new gene therapy delivery method targets spinal cord neurons to promote regeneration after injury.

## Contribution

AAV2retro injected into spinal white matter efficiently transduces both forelimb and hindlimb corticospinal neurons in mice.

## Key findings

- AAV2retro injected into cervical dorsal columns transduces corticospinal neurons in both forelimb and hindlimb regions.
- Dorsal column injections do not damage white matter beyond a narrow injection track.
- Gray matter injections result in minimal labeling of hindlimb sensorimotor cortex neurons.

## Abstract

Background: Adult neurons in the central nervous system often fail to regenerate after spinal cord injury (SCI). Regenerative gene therapies could potentially promote corticospinal axon regeneration, restoration of motor circuitry, and functional improvement after SCI, but translational methods for targeted gene delivery to corticospinal neurons are needed. AAV2retro is an engineered variant of the adeno-associated virus 2 (AAV2) capsid that demonstrates greatly enhanced retrograde transduction of projection neurons. When injected into spinal gray matter, AAV2retro retrogradely transduces neurons in the sensorimotor cortex that project to the injected spinal level. Methods: We initially hypothesized that injection of AAV2retro into the dorsal column white matter immediately rostral of a mouse cervical spinal injury would target transected axons and broadly transduce both forelimb and hindlimb corticospinal neurons. We tested this hypothesis by comparing four groups of mice treated with AAV2retro carrying the tdTomato reporter gene by (1) injection into intact C4 gray matter, (2) injection into intact C4 dorsal column white matter, (3) injection into C4 gray matter bordering a C5 dorsal column lesion, and (4) injection into C4 dorsal column white matter bordering a C5 dorsal column lesion. Results: After injection of AAV2retro into intact C4 dorsal column white matter, we observed extensive transduction of corticospinal neurons throughout both the forelimb and hindlimb sensorimotor cortical regions, and large numbers of transduced hindlimb corticospinal axons in the lumbar spinal cord. Dorsal column injections did not detectably damage the white matter beyond a narrow injection track. In contrast, after injection of intact C4 gray matter, we observed minimal labeling of neurons in the hindlimb sensorimotor cortex or corticospinal axons in the lumbar spinal cord. Conclusions: We conclude that AAV2retro can enter axons of passage in the dorsal column white matter of the spinal cord, and that injecting the cervical dorsal columns can efficiently target both forelimb and hindlimb corticospinal neurons in mice. This new approach for targeted gene delivery to corticospinal neurons could improve the safety and specificity of regenerative gene therapies for spinal cord injury.