Pelisch et al. · eNeuro · 2021

Use of a Self-Delivering Anti-CCL3 FANA Oligonucleotide as an Innovative Approach to Target Inflammation after Spinal Cord Injury

Pelisch Nicolas, Rosas Almanza Jose, Stehlik Kyle E., Aperi Brandy V., Kroner Antje

The study

What was asked, and what was found

Pelisch et al., eNeuro, 2021 asked whether a single inflammatory molecule could be shut down inside an injured spinal cord, and whether that would change the course of the secondary damage that follows the initial trauma. The target was the chemokine CCL3, which rises after injury, and which the same group had already shown matters by studying mice that lack it. The question here was whether an oligonucleotide could do the same job in a way that could be given.

AUM BioTech designed and synthesised four AUMsilence sdASOs against mouse Ccl3 from the reference transcript, and the paper prints all four sequences. Screened in primary mouse bone marrow derived macrophages stimulated with lipopolysaccharide, three of the four did nothing measurable and the fourth cut CCL3 messenger RNA by about 80% at 10 μM added straight to the medium with no transfection reagent. Labelled versions of the same molecule entered every macrophage in the dish within 2 hours. That fourth sequence carried the rest of the study.

In vivo the oligonucleotide was given by intrathecal lumbar puncture at 10 mg/kg into mice with a moderate thoracic contusion injury. Seven days after a single injection the labelled molecule was still at the lesion, inside microglia, monocyte-derived macrophages and astrocytes, though not inside neurons. One dose or two suppressed CCL3, TNF and interleukin 1 beta only to day 3, while three doses given a day apart held CCL3 down by about 60% out to day 7 and pulled TNF and interleukin 1 beta down by about 50%. Fewer macrophages and microglia sat next to the lesion at day 5, while astrocyte numbers and markers of axonal damage were unchanged. The behavioural and structural endpoints were mixed, and the authors report them plainly: open field locomotor scores did not separate from control and lesion volume at day 28 was no smaller, while a significantly higher share of treated animals achieved plantar placement in the first week. The value of the paper for a reader choosing a reagent is the delivery: a self-delivering oligonucleotide reached cells inside the injured central nervous system after a lumbar injection and held a target down for a week.

Key findings

AUM BioTech designed and made four AUMsilence sdASOs against mouse Ccl3 from the reference transcript, and the paper prints all four sequences.(Materials and Methods, first subsection, on the oligonucleotides and how they were given)
Screening the four in lipopolysaccharide-stimulated primary macrophages found one that cut CCL3 messenger RNA by about 80% at 10 μM with no transfection reagent, and the other three did not move it, so that one carried the rest of the study.(Results, first section)
A single intrathecal lumbar injection put the labelled oligonucleotide inside microglia, monocyte-derived macrophages and astrocytes at the spinal cord lesion, and it was still detectable there 7 days later.(Results, second section, on detection at the lesion site after intrathecal injection)
Three intrathecal doses of 10 mg/kg, one a day for 3 days, held CCL3 down by about 60% in the injured cord out to day 7, and pulled TNF and interleukin 1 beta down by about 50% with it. One dose or two lasted only to day 3.(Results, third section, on the reduced inflammatory response after injection)
Fewer macrophages and microglia sat next to the lesion in treated animals at day 5, while astrocyte numbers and the markers of axonal damage were unchanged.(Results, fifth section, on macrophage numbers at the lesion site)
The functional and structural endpoints were mixed and the authors report both: open field locomotor scores did not separate from control and lesion size was unchanged, while more treated animals reached plantar placement in the first week.(Results, sixth and final section, on tissue damage after injury)

Product: AUMsilence sdASO
Targets: Ccl3 · mouse · mRNA
Models: mouse bone marrow derived macrophages, C57BL/6, stimulated with lipopolysaccharide · primary cellsmouse thoracic contusion spinal cord injury, intrathecal lumbar puncture at L5 and L6 · in vivo
Delivery: added directly to the culture medium at 10 μM in vitro with no transfection reagent, and injected intrathecally by lumbar puncture at 10 mg/kg in vivo
Readouts: RT-qPCR, ELISA, flow cytometry, immunofluorescence and confocal microscopy, Basso mouse scale locomotor scoring, lesion volume and myelin quantification
Measured: knockdown of CCL3 messenger RNA in lipopolysaccharide-stimulated macrophages by the fourth of four candidate oligonucleotides: about 80% (10 μM added directly to the medium for 24 h, n = 3 per group, p < 0.05 against scrambled control)sustained knockdown of CCL3 in the injured spinal cord after three intrathecal doses: about 60% (three injections of 10 mg/kg every 24 h for 3 days, measured at day 7 after injury, n = 5 per group)knockdown of the downstream inflammatory transcripts TNF and interleukin 1 beta in the injured spinal cord: about 50% (three intrathecal injections, day 7 after injury)uptake of the fluorescently labelled oligonucleotide in cultured macrophages: all cells showed fluorescence after 2 h (10 μM added directly to the medium, no transfection reagent)which cells at the spinal cord lesion took up the labelled oligonucleotide 7 days after a single intrathecal injection: microglia, monocyte-derived macrophages and astrocytes took it up; neurons did not (10 mg/kg intrathecal, injured and naive mice, n = 4 to 5 per group)macrophage and microglial numbers next to the lesion 5 days after injury: significantly reduced in treated animals, p = 0.01, while astrocytes and axonal damage markers were unchanged (n = 5 per group, day 5 after injury)locomotor outcome: open field scores were not significantly different, while more treated mice achieved plantar placement (n = 10 per group in the pretreatment paradigm, n = 12 per group in the post-injury paradigm, chi-squared test, p < 0.05)lesion size and preserved myelin at day 28: no significant difference (n = 9 per group, pretreatment paradigm)
Figures: Fig. 1 Uptake of the labelled oligonucleotide by primary macrophages within 2 h with no transfection reagent, and the screen of four candidate sequences in which one cut CCL3 by about 80%.Fig. 2 The labelled oligonucleotide at the spinal cord lesion 7 days after one intrathecal injection, inside microglia, monocyte-derived macrophages and astrocytes but not neurons.Fig. 3 How long the knockdown lasts against how many doses were given, at transcript level and at protein level by ELISA, for CCL3, TNF and interleukin 1 beta.
Citation: Pelisch et al. Use of a Self-Delivering Anti-CCL3 FANA Oligonucleotide as an Innovative Approach to Target Inflammation after Spinal Cord Injury. eNeuro 2021;8:ENEURO.0338-20.2021. doi:10.1523/ENEURO.0338-20.2021

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