Chris Towne

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

BackgroundGene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice.ResultsDirect injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types.ConclusionWe have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Systemic AAV6 delivery mediating RNA interference against SOD1: neuromuscular transduction does not alter disease progression in fALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder arising from the selective death of motor neurons. Approximately 20% of familial ALS (fALS) cases are caused by toxic gain-of-function mutations in the superoxide dismutase 1 (SOD1) gene. We as well as others have provided proof-of-principle for the use of RNA interference (RNAi) against mutant SOD1 as a potential therapy for fALS. With the aim of maximizing the delivery of these silencing instructions, we explored the efficacy of intravenous delivery of recombinant adeno-associated virus (rAAV) serotype 6 expressing small hairpin RNAs targeting mutant SOD1 in the G93A SOD1 fALS mouse model. This approach resulted in a systemic transduction profile, corresponding to transduction of the entire skeletal musculature as well as heart and liver. In addition, motor neurons at all levels of the spinal cord and brain stem were transduced, amounting to 3-5% of the lower motor neuron pool. SOD1 protein levels were reduced by >50% in all the muscles that were examined. Crucially, this silencing profile did not alter the course of the disease in this fALS model, thereby providing compelling evidence that SOD1-mediated damage within skeletal muscles does not contribute to death of motor neurons in ALS. Further, this study demonstrates that motor neurons can be transduced across the length of the spinal cord through a single noninvasive delivery of rAAV.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Neuroprotection by Gene Therapy Targeting Mutant SOD1 in Individual Pools of Motor Neurons Does not Translate Into Therapeutic Benefit in fALS Mice

A major challenge in neurological gene therapy is delivery of the transgene to sufficient cell numbers in an atraumatic manner. This is particularly difficult for motor neuron (MN) diseases that have cells located across the entire spinal cord, brain stem, and cortex. We have used the familial mouse model of amyotrophic lateral sclerosis (ALS) to examine the feasibility of body-wide intramuscular injections of adeno-associated virus serotype 6 (AAV6), a vector capable of axonal retrograde transport, to deliver therapeutic genetic information across the lower MN axis. Neonatal muscle delivery of AAV expressing small hairpin RNAs (shRNAs) against the toxic transgene in this model, human mutant superoxide dismutase 1 (mSOD1), led to significant mSOD1 knockdown in the muscle as well as innervating MNs. This knockdown conferred neuroprotection and halted muscle atrophy in individually targeted MN pools. However, despite the vector being targeted to MNs that innervate muscle groups controlling eating, breathing, and locomotion, this approach was unable to therapeutically impact on disease progression in the ALS mouse model. These results stress the complexity of gene delivery for mSOD1 silencing and suggest that critical thresholds of protein knockdown and transduction across various cell types are required to translate local neuroprotective effects into functional improvements.

Lentiviral and Adeno-Associated Vector-Based Therapy for Motor Neuron Disease Through RNAi

RNAi holds promise for neurodegenerative disorders caused by gain-of-function mutations. We and others have demonstrated proof-of-principle for viral-mediated RNAi in a mouse model of motor neuron disease. Lentivirus and adeno-associated virus have been used to knockdown levels of mutated superoxide dismutase 1 (SOD1) in the G93A SOD1 mouse model of familial amyotrophic lateral sclerosis (fALS) to result in beneficial therapeutic outcomes. This chapter describes the design, production, and titration of lentivirus and adeno-associated virus capable of mediating SOD1 knockdown in vivo. The delivery of the virus to the spinal cord directly, through intraspinal injection, or indirectly, through intramuscular injection, is also described, as well as the methods pertaining to the analysis of spinal cord transduction, SOD1 silencing, and determination of motor neuron protection.

186. Translating an Optogenetic Gene Therapy Approach for Treatment of Neuropathic Pain in Humans

Optogenetics has been established as a powerful tool to study the central and peripheral nervous systems with its potential for directly treating human disease tantalizingly on the horizon. Optogenetic inhibition of pain in mice has been shown to be effective and provides an attractive initial application. We aim to translate this approach to treat neuropathic pain in humans. Firstly, we demonstrated that the inhibitory opsin, NpHR, delivered by intraneural injections of AAV6 could reduce pain (mechanical allodynia) in a clinically meaningful paradigm i.e. vector delivery after establishment of neuropathic pain in the chronic constriction injury (CCI) model. The inhibitory opsin, iC1C2 (a chloride channel requiring less light than NpHR to pass currents) could also reduce pain in this system. We next tested a surgical approach more amenable to patients than nerve injection. Lumbar puncture is a routine clinical practice and AAV8 delivery by this method results in efficient gene delivery to sensory neurons in rodents, dogs and pigs. We found that intrathecal administration of AAV8 expressing iC1C2 could decrease mechanical allodynia in the CCI mouse model. The magnitude of pain inhibition correlated with transduction levels and only 10% transduction of sensory neurons was required for pain relief. To further validate this approach we utilized a second rodent pain model with translational relevance. The mouse tibia fracture/cast immobilization model of Complex Regional Pain Syndrome (CRPS) presents extreme allodynia and recapitulates human disease effectively. We found that intrathecal delivery of AAV8 encoding iC1C2 following development of the CRPS model resulted in light-mediated pain inhibition. Taken together, our results confirm optogenetic therapy for neuropathic pain as an attractive clinical application of this technology.

229 EFFICACY AND SPECIFICITY OF RECOMBINANT ADENO‐ASSOCIATED VIRUS SEROTYPE 6 MEDIATED GENE TRANSFER TO DRG NEURONS THROUGH DIFFERENT ROUTES OF DELIVERY

with 100mM ATP. Total RNA was isolated using RNeasy Mini kit (Qiagen), reverse transcribed, and quantification was performed by qPCR (TaqMan, Applied Biosystems). The expression of gene modulation produced by ATP was calculated by the ddCt method and normalized to 18S as endogenous control. BDNF production in supernatants from ATP-stimulated cells was quantified using RayBio® Human BDNF ELISA kit (RayBiotech). ATP caused a time-dependent induction of BDNF gene expression in both groups of patients (up to 13 fold for OA and up to 8 fold for RA) and the healthy volunteer (up to 14 fold) as the induction reached its maximum at 2h. Extracellular release of BDNF was also induced and after 2h stimulation was 17.8–22 pg/ml for RA, 19.6–23 pg/ml for OA and 22 pg/ml for healthy control. BDNF is a neurotransmitter involved in nociceptive hypersensitivity in the central nervous system and we could show that its increased expression is a direct effect of ATP in synovial fibroblasts of both OA and RA patients thus BDNF might be a possible therapeutic target for better pain management of arthritic joint pain.