Abstract
We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×109 viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.
Highlights
Chronic pain, such as that which follows nerve injury, is common and inadequately treated
In order to compare the efficacy and safety of AAV6-EGFP and AAV8-EGFP accurately, we initially aimed to ensure that the purified vectors were similar in quality so that differences between the two vectors would be solely attributable to the particle capsids rather than other differences in the preparations
The resultant titers of AAV6-EGFP and AAV8EGFP were 2.161013 genome copy number per ml (GC/ml) and 2.561013 GC/ml, respectively, with less than 5% of particles being empty for both vectors (Fig. 1A and 1B)
Summary
Chronic pain, such as that which follows nerve injury, is common and inadequately treated. Drug development for the treatment of chronic neuropathic pain has focused on agents that target specific biomolecules of interest in the sensory pathway. Many biological and pharmacological molecules have potential to modulate sensory neuron function in chronic pain models, there are major problems in delivering these agents into the relevant cell populations and sites. Disordered cellular mechanisms underlying chronic pain after peripheral nerve injury reside at diverse sites, including in receptive fields in peripheral tissues, in the somata of the injured sensory neurons, and in the dorsal horn (DH) of the spinal cord [1]. The dorsal root ganglia (DRGs), which harbor the somata of primary sensory neurons, are optimally situated as sites for pain pharmacotherapy. Direct injection into the DRG is well tolerated in both human and rodent subjects [2,3,4,5]
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