Neonatal Intraperitoneal or Intravenous Injections of Recombinant Adeno-Associated Virus Type 8 Transduce Dorsal Root Ganglia and Lower Motor Neurons

Abstract

Targeting lower motor neurons (LMNs) for gene delivery could be useful for disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. LMNs reside in the ventral gray matter of the spinal cord and send axonal projections to innervate skeletal muscle. Studies have used intramuscular injections of adeno-associated virus type 2 (AAV2) to deliver viral vectors to LMNs via retrograde transport. However, treating large areas of the spinal cord in a human would require numerous intramuscular injections, thereby increasing viral titer and risk of immune response. New AAV serotypes, such as AAV8, have a dispersed transduction pattern after intravenous or intraperitoneal injection in neonatal mice, and may transduce LMNs by retrograde transport or through entry into the nervous system. To test LMN transduction after systemic injection, we administered recombinant AAV8 (rAAV8) carrying the green fluorescent protein (GFP) gene by intravenous or intraperitoneal injection to neonatal mice on postnatal day 1. Tissues were harvested 5 and 14 days postinjection and analyzed by real-time polymerase chain reaction and GFP immunohistochemistry to assess the presence of AAV genomes and GFP expression, respectively. Spinal cords were positive for AAV genomes at both time points. GFP immunohistochemistry revealed infrequent labeling of LMNs across all time points and injection routes. Somewhat surprisingly, there was extensive labeling of fibers in the dorsal horns and columns, indicating dorsal root ganglion transduction across all time points and injection routes. Our data suggest that systemic injection of rAAV8 is not an effective delivery route to target lower motor neurons, but could be useful for targeting sensory pathways in chronic pain.

Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation

“…Axonal transport of AAV is serotype dependent in the CNS (30,31), and AAV9 transport following brain injection is driven by cytoplasmic dynein and kinesin proteins (32). AAV8 selectively transduces sensory neurons, but not motor neurons, in the peripheral nervous system (PNS) (23). It is likely that the vectors enter the neurons at nerve termini and are trafficked to cell bodies using the same transport machinery as HSV-1, and this conclusion is supported by our findings.…”

“…When certain capsid tyrosine residues were mutated to phenylalanine (Y733F), transduction efficiency was shown to be greatly increased relative to that seen with vectors with wild-type capsids (12)(13)(14)(15)(16)(17)(18)(19)(20). Most relevant to the current study, AAV is capable of transducing DRG sensory neurons via direct intraganglionic sciatic nerve injection (21,22) and intraperitoneal and intravenous injection (23), as well as via intrathecal injection following lumbar puncture (24)(25)(26)(27). Therefore, we sought to determine if rAAV vectors would be delivered to the same sensory neurons that HSV-1 infects when applied at an epithelial surface that has been treated to expose the underlying sensory nerve termini.…”

Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs

“…5,7,8,19 Viral vector remote delivery to the neonatal spinal cord has also been successfully reported in rodents. 19,33,34 However, the limited feasibility of these approaches for human therapy has been a major obstacle for clinical translation. Recently, efficacious transduction of spinal MNs in 3 kg African green monkeys following AAV6 intramuscular delivery, 10 and in neonatal and adult cats after AAV9 intravascular delivery 20 have brought considerable excitement to the field in terms of scalability of these systemic delivery approaches.…”

Intravenous Injections in Neonatal Mice

“…However, in rare instances dams can destroy entire litters. These injections can be successfully performed in mice as small as 0.8 g through 1.6 g. Alternative injection routes for neonate mice have been published by us and others including retro-orbital, intrajugular and intraperitoneal 10,15,22 . We prefer the injection route described here because it is clinically relevant, able to be performed by a single experimenter with no special equipment requirements and allows for a wide range of volumes to be administered.…”

“…Following injection of AAV, transduction of dorsal root ganglia, liver, heart, skeletal muscle, lung, and myenteric plexus of the gut has been observed 1,3,6,7,15 . Widespread transduction of the CNS and periphery makes this method of injection ideal for diseases requiring global expression of a transgene, such as Gaucher's disease 16 and other lysosomal storage diseases 17,18 , Batten's disease and related neuronal ceroid lipofuscinoses, 19 and Bardet-Biedl syndrome, a genetic multisystem disorder with onset of symptoms occurring in early childhood 20 .…”