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Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice

Abstract

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wild-type Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wild-type ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT), inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high-affinity receptor), and ITPR1-mediated cytoplasmic free calcium release in vitro. In addition, we show that gene transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT-mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

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Acknowledgements

A grant from the NIDCR R01DE022903 and funding from the Department of Anesthesiology, Perioperative Medicine, and Pain Management, University of Miami Miller School of Medicine, Miami, Florida, were used to perform this work.

Author contributions

RCL designed the overall study, setup collaborations, supervised these studies, analyzed data, primarily wrote, edited, and submitted this manuscript. GZZ performed the major portion of the experiments. GZZ assisted with experimental design, data collection, analyses, and manuscript preparation. XT, YK, UU, and DME contributed to experimental work included in this manuscript. KDS, UU, ESF, TW, ERM, LD, WM, and SBS contributed to the critical review and final editing of the manuscript.

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Conflict of interest

WM, LD, and SBS have financial holdings receive compensation from Algynomics, Inc. The authors declare that their affiliation(s) to Algynomics, Inc., including employment, consultancy, patents, products in development, or marketed products, does not alter our adherence to Gene Therapy policies on sharing data and/or materials. Algynomics, Inc. provided support in the form of salaries for authors SBS, WM, and LD, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “Author contributions” section. The remaining authors declare no conflicts of interest.

Correspondence to Roy C. Levitt.

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