Background & Aims
The voltage-gated sodium ion channel Nav1.7 is a key gate for transmission of noxious sensory input from peripheral neurons into the spinal cord and CNS. Human and rodent genetics show that constitutive genetic loss of Nav1.7 results in the loss of pain without motor, cognitive, or autonomic effects (1, 2, 3), suggesting that Nav1.7 inhibition could be a safe and effective therapy for pain. Key challenges for systemic Nav1.7 therapeutics include distribution to cellular sites of action, achieving a sufficiently high level of inhibition or receptor knockdown, and selectivity for Nav1.7 versus the sodium channel paralogs that govern excitability of brain, skeletal muscle, and heart. Branched, or divalent, small interfering RNA (di-siRNA) is a novel technology that knocks down target gene expression with high specificity, has broad distribution throughout the CNS, and has a months-long duration of action following a single dose directly into the cerebrospinal fluid (4). Here we describe and characterize the effects of ATL-301, a novel di-siRNA targeting Nav1.7.
Methods
ATL-301 was designed to target a sequence identical among human, rodent, and primate SCN9A mRNAs and not shared with any other sodium channel or human gene. Reduction of transcript in vitro by ATL-301 was measured in HEK cells overexpressing SCN9A via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and effects on functional Nav1.7 protein in vitro were determined using whole cell patch clamp electrophysiology. ATL-301 was formulated in saline and dosed in rats via intrathecal lumbar injection through implanted catheters. The evoked pain response was measured using the Hargreaves assay for sensitivity to painful thermal heat and with a pinprick assay for sensitivity to painful mechanical stimuli. Pain assessments were completed every two weeks following a single administration for a total duration of 12-18 weeks. Following takedown, transcript knockdown was assessed in sensory ganglia from the dorsal root by RT-qPCR and by RNAscope imaging, and protein knockdown was assessed via an in-house MesoScale Discovery ELISA.
Results
In vitro, ATL-301 reduced total cellular SCN9A mRNA expression by 60% and eliminated Nav1.7 currents, both with an IC50 of <10pM. There was no knockdown of SCN1A, SCN2A, SCN3A, SCN8A, SCN10A, or SCN11A in vitro at concentrations up to 10nM. ATL-301 administered to rats via intrathecal lumbar catheter resulted in dose-dependent analgesia, assayed as an increase in the time to paw withdrawal in the Hargreaves radiant heat test and by the number of withdrawals in the pinprick model of sharp mechanical pain. The analgesic effect paralleled effects reported upon complete genetic removal of Nav1.7 (2). In both pain models, analgesia from a single dose lasted approximately three months, and animals were bright, alert, and responsive throughout. ATL-301 reduced cytoplasmic but not nuclear SCN9A expression in L4-L6 ganglia and reduced protein expression in a dose-responsive manner. RNA-seq showed specific knockdown of SCN9A with no knockdown of other ion channels.
Conclusions
ATL-301 specifically knocked down SCN9A mRNA and Nav1.7 protein both in vitro and in vivo. A single intrathecal lumbar dose of ATL-301 resulted in sustained analgesia in rodents without apparent adverse effects. This effect was dose-dependent and durable for three months. These data suggest that ATL-301 may effectively target Nav1.7 and that di-siRNAs can address limitations to small and large molecule approaches such as CNS distribution, tolerability, and selectivity.
References
- Alsaloum et al. Nature Reviews Neurology 2020, 16:689-705
- Shields et al. J Neuroscience 2018, 38(47):10180-10201
- Goldberg et al. Clin Genet 2007, 71(4):311-319
- Alterman et al. Nature Biotechnology 2019, 37:884-894
Presenting Author
Corrie L Gallant-Behm
Poster Authors
Corrie Gallant-Behm
PhD
Atalanta Therapeutics
Lead Author
Qingmin Chen
PhD
Atalanta Therapeutics
Lead Author
Smita Jagtap
PhD
Atalanta Therapeutics
Lead Author
Taylor Lynch
Atalanta Therapeutics
Lead Author
Elizabeth Buck
Atalanta Therapeutics
Lead Author
Matthew Rook
PhD
Atalanta Therapeutics
Lead Author
Justin Siemian
PhD
Atalanta Therapeutics
Lead Author
David Tran
Atalanta Therapeutics
Lead Author
Chunhua Yang
Atalanta Therapeutics
Lead Author
Mingwei Li
Atalanta Therapeutics
Lead Author
Kelly Rogers
Atalanta Therapeutics
Lead Author
Alex Prinzen
PhD
Atalanta Therapeutics
Lead Author
Steven Mathieu
Atalanta Therapeutics
Lead Author
Greg Miglis
Atalanta Therapeutics
Lead Author
Guillermo Yudowski
phD
Atalanta Therapeutics
Lead Author
Garth Kinberger
PhD
Atalanta Therapeutics
Lead Author
Aimee Jackson
PhD
Atalanta Therapeutics
Lead Author
Stefan McDonough
PhD
Atalanta Therapeutics
Lead Author
Topics
- Treatment/Management: Pharmacology: Novel Targets