Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or phosphate-buffered saline. Piglets were euthanized three weeks post injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP-positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9-treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.

Gene delivery to the spinal cord using MRI-guided focused ultrasound.

Non-invasive gene delivery across the blood-spinal cord barrier (BSCB) remains a challenge for treatment of spinal cord injury and disease. Here, we demonstrate the use of magnetic resonance image-guided focused ultrasound (MRIgFUS) to mediate non-surgical gene delivery to the spinal cord using self-complementary adeno-associated virus serotype 9 (scAAV9). scAAV9 encoding green fluorescent protein (GFP) was injected intravenously in rats at three dosages: 4 × 10(8), 2 × 10(9) and 7 × 10(9) vector genomes per gram (VG g(-1)). MRIgFUS allowed for transient, targeted permeabilization of the BSCB through the interaction of focused ultrasound (FUS) with systemically injected Definity lipid-shelled microbubbles. Viral delivery at 2 × 10(9) and 7 × 10(9) VG g(-1) leads to robust GFP expression in FUS-targeted regions of the spinal cord. At a dose of 2 × 10(9) VG g(-1), GFP expression was found in 36% of oligodendrocytes, and in 87% of neurons in FUS-treated areas. FUS applications to the spinal cord could address a long-term goal of gene therapy: delivering vectors from the circulation to diseased areas in a non-invasive manner.

Glutamate Transporter GLT-1 Upregulation Attenuates Visceral Nociception and Hyperalgesia via Spinal Mechanisms Not Related to Anti-Inflammatory or Probiotic Effects.

Visceral pain is the most common reason for physician visits in US. Glutamate is the major excitatory neurotransmitter and mediates visceral nociceptive neuro-transmission and hypersensitivity. Removal of extracellular glutamate is predominantly mediated by glial glutamate transporter-1 (GLT-1). The pharmacological approach to up-regulate GLT-1 by 1 week administration of ceftriaxone (CTX) has been successful to mitigate visceral nociception. The present study shows that intrathecal delivery of selective GLT-1 antagonist dihydrokainate reversed CTX-blunted visceral nociceptive response, suggesting a spinal site of action. The role of GLT-1 up-regulation in animal models of colitis was studied. CTX treatment reversed TNBS-induced visceral hypersensitivity. In addition, CTX treatment initiated one week after the onset of DSS-induced visceral inflammation also attenuated visceral hypersensitivity, revealing a potential therapeutic effect. Cephalothin, a cephalosporin antibiotic lacking GLT-1 induction activity, failed to attenuate visceral nociception. CTX-induced changes in fecal microbiota do not support a role of probiotic effects in mitigating visceral nociception/hypersensitivity. Finally, adeno-associated virus serotype 9-mediated GLT-1 over-expression was effective to mitigate visceromotor response to 60 mmHg colo-rectal distension. These studies indicate that GLT-1 over-expression is a novel and effective method to attenuate visceral nociception, and is deserving of further study as a translationally relevant approach to treat visceral pain.