Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture

<p>Abstract</p> <p>Background</p> <p>Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.</p> <p>Results</p> <p>Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.</p> <p>Conclusions</p> <p>The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.</p

AAV-mediated gene transfer to colon-innervating primary afferent neurons

Investigation of neural circuits underlying visceral pain is hampered by the difficulty in achieving selective manipulations of individual circuit components. In this study, we adapted a dual AAV approach, used for projection-specific transgene expression in the CNS, to explore the potential for targeted delivery of transgenes to primary afferent neurons innervating visceral organs. Focusing on the extrinsic sensory innervation of the mouse colon, we first characterized the extent of dual transduction following intrathecal delivery of one AAV9 vector and intracolonic delivery of a second AAV9 vector. We found that if the two AAV9 vectors were delivered one week apart, dorsal root ganglion (DRG) neuron transduction by the second vector was greatly diminished. Following delivery of the two viruses on the same day, we observed colocalization of the transgenes in DRG neurons, indicating dual transduction. Next, we delivered intrathecally an AAV9 vector encoding the inhibitory chemogenetic actuator hM4D(Gi) in a Cre-recombinase dependent manner, and on the same day injected an AAV9 vector carrying Cre-recombinase in the colon. DRG expression of hM4D(Gi) was demonstrated at the mRNA and protein level. However, we were unable to demonstrate selective inhibition of visceral nociception following hM4D(Gi) activation. Taken together, these results establish a foundation for development of strategies for targeted transduction of primary afferent neurons for neuromodulation of peripheral neural circuits

Compression trauma in the spinal cord and neural transplantation

Adult, Long Evans hooded rats received a compression trauma at the T11-T12 level of the spinal cord extending 50% in the dorsoventral plane. The central hemorrhagic mass was removed from the lesion, resulting in a cavity. Control animals received no further surgery, while experimental animals received transplants of either 15-d neocortical tissue or 15-d spinal cord tissue injected intraparenchymally into the lesion site. Qualitative analysis of control animals showed the spread of hemorrhage and edema into the spinal cord over time. It was shown quantitatively that the degeneration that follows spinal cord injury can be divided into early and late components. Quantitative analysis also revealed an intense glial reaction involving both astrocytes and oligodendrocytes. A glial scar was not seen in control animals using light microscopic techniques. A matrix of blood vessels, connective tissue, astrocytes, macrophages, and endothelial cells filled the lesion site, but this was different from a glial scar. Some fine axonal fibers were seen near the lesion site and were probably collateral sprouts. Transplants of embryonic neural tissue can survive, grow, differentiate, and become integrated in the spinal cord following compression trauma. Transplants of 15-d neocortical tissue grew very large and readily became integrated, but poor growth and integration of 15-d spinal cord tissue was seen. The pathological reaction and glial reaction was similar to that of control animals. More axon collaterals were seen in regions of good integration than in regions of poor integration or in control animals, possibly because the transplant provided an environment suitable for viewing these collaterals. A glial scar was seen in 11% of the sections from spinal cords with 15-d neocortical tissues, which is less than has been previously reported in studies using laceration lesion. In most instances, the growth of the transplant did not provide the compaction to cause the glial scar. Usually, the growing transplant pushed into the host spinal cord in such a way that the diffuse arrangement of glia cells remained. Only rarely, and only in very limited regions did the transplant exert a compacting force sufficient to cause a glial scar, and only then in regions of the spinal cord that were not well integrated

Targeting the somatosensory system with AAV9 and AAV2retro viral vectors.

Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products