Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice

Abstract

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in Na_V1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between Na_V subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of Na_V1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of Na_V1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of Na_V1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of Na_V1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

Fig. 1.. Schematic of the overall strategy used for in situ Na_V1.7 repression using ZFP-KRAB and KRAB-dCas9 via the intrathecal route of administration (ROA).

Na_V1.7 is a DRG channel involved in the transduction of noxious stimuli into electric impulses at the peripheral terminals of DRG neurons. In situ repression of Na_V1.7 via AAV-ZFP-KRAB and AAV-KRAB-dCas9 is achieved through intrathecal injection, leading to disruption of the pain signal before reaching the brain.

Fig. 2.. Robust transduction of DRG via intrathecal delivery of AAVs.

(A) Representative three-dimensional maximum intensity projections from whole-mount DRGs along the neuraxis after intrathecal injections of AAV9-mCherry, illustrating distribution and transduction at different viral titers (1 × 10¹⁰, 1 × 10¹¹, or 1 × 10¹² vg per mouse). (B) Neuraxial distribution of small, medium, and large DRG neuronal soma as a function of their average soma fluorescent intensity (n = 4 mice per titer; cross-sectional area: small ≤ 300 μm², medium = 300 to 700 μm², large ≥ 700 μm²). (C and D) Representative 20× images of mice DRG transduced with 1 × 1012 vg per mouse of AAV9-mCherry (C) or AAV9-Zinc-Finger-4-KRAB (D) labeled with RNAscope in situ hybridization for Na_V1.7 (n = 3 for mCherry and n = 4 for Zinc-Finger-4-KRAB; scale bar, 50 μm). (E) Quantification of Na_V1.7 expression in AAV9-mCherry or AAV9-Zinc-Finger-4-KRAB treatment conditions: Individual RNAscope probes and cells were identified in each respective image and used to calculate the average number of probes per cell (dots represent individual biological replicates; n = 3 for mCherry and n = 4 for Zinc-Finger-4-KRAB; error bars are SEM; Student’s t test, *P = 0.0205).

Fig. 3.. In situ repression of Na_V1.7 leads to pain prevention in a carrageenan model of inflammatory pain.

(A) Schematic of the carrageenan-induced inflammatory pain model. (B) Time course of thermal hyperalgesia after the injection of carrageenan (solid lines) or saline (dotted lines) into the hind paw of mice 21 days after intrathecal (i.t.) injection with AAV9-mCherry and AAV9-Zinc-Finger-4-KRAB is plotted. Mean PWLs are shown (dots represent mean of individual biological replicates; n = 10; error bars are SEM). (C) Time course of thermal hyperalgesia after the injection of carrageenan (solid lines) or saline (dotted lines) into the hind paw of mice 21 days after intrathecal injection with AAV9-KRAB-dCas9-no-gRNA and AAV9-KRAB-dCas9-dual-gRNA is plotted. Mean PWLs are shown (n = 10; error bars are SEM). (D) The aggregate PWL was calculated as AUC for both carrageenan- and saline-injected paws (dots represent individual biological replicates; n = 10; error bars are SEM; Student’s t test, ****P < 0.0001). (E) In vivo Na_V1.7 repression efficiencies as determined by qPCR (dots represent individual biological replicates; qPCR was performed in technical triplicates; n = 5; error bars are SEM; values normalized to Gapdh; Student’s t test, ***P = 0.0008 and **P = 0.0033).

Fig. 4.. In vivo efficacy of ZFP-KRAB and KRAB-dCas9 in two neuropathic pain models.

(A) Schematic of the paclitaxel-induced neuropathic pain model. i.p., intraperitoneally. (B) In situ repression of Na_V1.7 via Zinc-Finger-4-KRAB and KRAB-dCas9-dual-gRNA reduces paclitaxel-induced tactile allodynia (dots represent individual biological replicates; n = 8; error bars are SEM; Student’s t test, ***P = 0.0007 and ***P = 0.0004). (C) In situ repression of Na_V1.7 via Zinc-Finger-4-KRAB and KRAB-dCas9-dual-gRNA reduces paclitaxel-induced cold allodynia (dots represent individual biological replicates; n = 8; error bars are SEM; Student’s t test, ****P < 0.0001 and **P = 0.008). (D) Schematic of the BzATP pain model. (E) In situ repression of Na_V1.7 via KRAB-dCas9-dual-gRNA reduces tactile allodynia in a BzATP model of neuropathic pain (dots represent mean of n = 5 biological replicates for KRAB-dCas9-no-gRNA and n = 6 biological replicates for the other groups; error bars are SEM; two-way ANOVA with Bonferroni post hoc test, ****P < 0.0001 and *P = 0.0469).

Fig. 5.. In situ repression of Na_V1.7 reverses chemotherapy-induced neuropathic pain.

(A) Schematic of the treatment for paclitaxel-induced chronic neuropathic pain model. (B) In situ repression of Na_V1.7 via Zinc-Finger-4-KRAB and KRAB-dCas9-gRNA reverses paclitaxel-induced tactile allodynia (dots represent individual biological replicates; n = 7 to 8; error bars are SEM; two-way ANOVA with Bonferroni post hoc test, ****P < 0.0001 and **P = 0.0027).

Fig. 6.. Long-term efficacy of ZFP-KRAB and KRAB-dCas9 in two independent pain models.

(A) Timeline of the carrageenan-induced inflammatory pain model. (B) The AUC of the aggregate PWL was calculated for both carrageenan- and saline-injected paws of mice 42, 84, and 308 days after intrathecal injection with AAV9-mCherry and AAV9-Zinc-Finger-4-KRAB. A significant increase in PWL is seen in the carrageenan-injected paws of mice injected with AAV9-Zinc-Finger-4-KRAB (dots represent individual biological replicates; n = 5 to 8; error bars are SEM; Student’s t test, ****P < 0.0001). (C) Schematic of the paclitaxel-induced neuropathic pain model. (D) In situ repression of Na_V1.7 via Zinc-Finger-4-KRAB and KRAB-dCas9-dual-gRNA reduces paclitaxel-induced tactile allodynia 105 days after last paclitaxel injection (dots represent individual biological replicates; n = 5 to 8; error bars are SEM; Student’s t test, ****P < 0.0001 and ***P = 0.0001). (E) In situ repression of Na_V1.7 via Zinc-Finger-4-KRAB and KRAB-dCas9-dual-gRNA reduces paclitaxel-induced cold allodynia (dots represent individual biological replicates; n = 5 to 8; error bars are SEM; Student’s t test, ****P < 0.0001).