Abstract
Reliable viral vector-mediated transgene expression in primate motoneurons would improve our ability to anatomically and physiologically interrogate motor systems. We therefore investigated the efficacy of replication defective, early region 1-deleted canine adenovirus type-2 (CAV-2) vectors for mediating transgene expression of fluorescent proteins into brainstem motoneurons following craniofacial intramuscular injections in four rhesus monkeys (Macaca mulatta). Vector injections were placed into surgically identified and isolated craniofacial muscles. After a 1- to 2-month survival time, animals were sacrificed and transgene expression was assessed with immunohistochemistry in the corresponding motoneuronal populations. We found that injections of CAV-2 into individual craniofacial muscles at doses in the range of ∼1010 to 1011 physical particles/muscle resulted in robust motoneuronal transduction and expression of immunohistochemically identified fluorescent proteins across multiple animals. By using different titers in separate muscles, with the resulting transduction patterns tracked via fluorophore expression and labeled motoneuron location, we established qualitative dose-response relationships in two animals. In one animal that received an atypically high titer (5.7 × 1011 total CAV-2 physical particles) distributed across numerous injection sites, no transduction was detected, likely due to a retaliatory immune response. We conclude that CAV-2 vectors show promise for genetic modification of primate motoneurons following craniofacial intramuscular injections. Our findings warrant focused attention toward the use of CAV-2 vectors to deliver opsins, DREADDs, and other molecular probes to improve genetics-based methods for primate research. Further work is required to optimize CAV-2 transduction parameters. CAV-2 vectors encoding proteins could provide a new, reliable route for modifying activity in targeted neuronal populations of the primate central nervous system.
Highlights
Replication defective, early region 1-deleted canine adenovirus type 2 (CAV-2) vectors are an increasingly important tool for neurological research due to canine adenovirus type-2 (CAV-2)’s neuronal tropism and capacity to retrogradely label neurons projecting to the injection sites
Our histological assessments yielded three main conclusions: (1) The vector worked well to transduce genes into motoneurons of the ocular and facial cranial nuclei; (2) There was an effect of total viral load, with relatively small, single injections yielding the best labeling (M1801) and larger injections in 2–3 muscles working adequately (M18-03 and M19-01)
More extensive injections yielded no transgene expression, presumably due to an immune response triggered by the total viral load (M18-02); (3) With total load constrained to avoid counterproductive immune responses, we found an increasing dose-response relationship over the range of ∼1010 to 1011 viral particles injected
Summary
Replication defective, early region 1-deleted canine adenovirus type 2 (CAV-2) vectors are an increasingly important tool for neurological research due to CAV-2’s neuronal tropism and capacity to retrogradely label neurons projecting to the injection sites. CAV-2-GFP injected into the gastrocnemius of newborn mice produced motoneuronal labeling within the ventral horn of the lumbosacral spinal cord 24-days post-injection (Soudais et al, 2001). This and other work suggested that CAV-2 binds to the coxsackie and adenovirus receptor (CAR) (Soudais et al, 2000, 2001; Loustalot et al, 2016). This is likely because in mature, non-pathological (i.e., normal) muscles, CAR expression is confined to neuromuscular junctions (Shaw et al, 2004; Sinnreich et al, 2005)
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