Transduction of Craniofacial Motoneurons Following Intramuscular Injections of Canine Adenovirus Type-2 (CAV-2) in Rhesus Macaques

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.

Canine Adenovirus 2: A Natural Choice for Brain Circuit Dissection

“…Furthermore, CAV has been successfully used in a broad diversity of cortical and subcortical projection pathways (Table 1). Even though rodents were used as the animal models in most of CAV applications in Table 1, recently, CAV was also successfully used in non-human primates ( Table 1; Mestre-Francés et al, 2018;Bohlen et al, 2019;Dopeso-Reyes et al, 2019). These studies validate the applicability of CAV as a gene delivery tool in nonhuman primates, facilitating the investigation of neural circuits in a more human-relevant model.…”

“…Moreover, an ideal gene delivery vector should also avoid host immune responses and cytotoxicity. Indeed, CAV viruses are not human pathogens and do not induce significant cellular infiltration in rat brains (Soudais et al, 2004), nor in rhesus monkeys, unless abnormally high titers are used (Bohlen et al, 2019). Even a pre-existing immunity against human adenoviruses does not significantly affect CAV transduction (Klonjkowski et al, 1997;Kremer et al, 2000;Ibanes and Kremer, 2013).…”

“…For instance, the early region 1 (E1) gene, which is important for DNA replication, was deleted from CAV genome (Klonjkowski et al, 1997). The E1-deleted CAV is replication incompetent (Chartier et al, 1996;Fernandes et al, 2013) and thus causes negligible immune response in humans, non-human primates, and rodents at experimentally relevant titers (Kremer et al, 2000;Perreau and Kremer, 2005;Bohlen et al, 2019;Lau et al, 2019). Consequently, a large number of neurons can be transduced by E1-deleted CAV without being recognized and eliminated by immune cells (Kremer et al, 2000;Soudais et al, 2004;Perreau and Kremer, 2005;Lau et al, 2019).…”

“…For instance, Li et al (2016) revealed the existence of two separate neuronal populations in the rodent LC by injecting CAV carrying either red or green fluorophore in one of two known postsynaptic targets of LC neurons. Similarly, this straightforward experimental design was also used in non-human primates to express fluorescent proteins in neural pathways of interest, including motor neurons that innervate craniofacial muscles and midbrain neurons that project to the Cb (Bohlen et al, 2019;Dopeso-Reyes et al, 2019). This simple experimental design works well to tag projection neurons innervating a target of interest.…”

Intramuscular Delivery of Gene Therapy for Targeting the Nervous System

“…Furthermore, it is efficiently retrogradely transported along axons (Salinas et al, 2009 ), while a helper-dependent CAV-2 has been shown to drive transgene expression in the rodent CNS for over a year (Soudais et al, 2004 ). CAV-2 injection into craniofacial muscles of rhesus monkeys caused robust motor neuron transduction (Bohlen et al, 2019 ), while intramuscular administration in rats results in superior motor neuron uptake and transport compared to AdV serotype 5 (Soudais et al, 2001 ), which together highlight the potential of CAV-2 for motor neuron targeting via skeletal muscle.…”