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Portoles et al. · International Journal of Molecular Sciences · 2024

Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice

Portoles Irene, Ribera Jordi, Fernandez-Galan Esther, Lecue Elena, Casals Gregori, Melgar-Lesmes Pedro, Fernandez-Varo Guillermo, Boix Loreto, Sanduzzi Marco, Aishwarya Veenu, Reig Maria, Jimenez Wladimiro, Morales-Ruiz Manuel

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The study

What was asked, and what was found

Portoles et al., International Journal of Molecular Sciences, 2024 asked what the RNA helicase DHX15 does in the liver, a question nobody had put directly despite the enzyme being repeatedly linked to liver cancer. The team came at it three ways: a CRISPR knockout in zebrafish, a heterozygous mouse made by TALEN, and silencing in wild type mice. The genetic arms established the ground. Zebrafish embryos lacking the gene had no liver at all and died at 8 days after fertilisation. Heterozygous mice had thinner hepatic vessels, regenerated poorly after two thirds partial hepatectomy, could not restore glycogen, and grew fewer and smaller liver metastases after Hepa 1-6 cells were implanted.

The silencing arm used AUMsilence sdASO against Dhx15, designed and supplied by AUM BioTech, with a matched scramble control from the same source. The oligonucleotide was injected into the tail vein at 10 mg/kg/day every third day. A single dose gave 80% silencing in the liver, measured by immunoblot, and it held to 72 hours after injection. Silencing was milder in lung and spleen. The authors describe the chemistry as reaching cells without any delivery reagent or formulation.

In the Hepa 1-6 hepatocellular carcinoma model, mice pre dosed with the Dhx15 sdASO grew primary tumours of 179.6 plus or minus 80.06 cubic millimetres at 5 weeks against 1085 plus or minus 277.1 in the scramble group. Tumours in the silenced group also had a smaller blood vessel perimeter and lumen and fewer lymphatic vessels, and the silenced livers showed lower Vegf-a, Vegf-d, Vegfr1 and Vegfr3. The clinical side of the paper measured DHX15 in serum: 300.3 plus or minus 88.2 picograms per millilitre in 62 patients with hepatocellular carcinoma against 32.4 plus or minus 27.7 in 24 healthy controls, with 35 patients with cirrhosis sitting between the two. Veenu Aishwarya of AUM BioTech is a co-author, credited with designing and contributing the oligonucleotides.

Key findings

  • A single intravenous dose of AUMsilence sdASO against Dhx15 silenced the gene by 80% in mouse liver, and the knockdown was still there 72 hours later.(Results, 2.6. “AUMsilence ASO Mediated Dhx15 Silencing in Mice Reduces Primary Tumor Volume in an HCC Mouse Model”)
  • In a syngeneic Hepa 1-6 liver cancer model, mice dosed with the Dhx15 sdASO grew tumours of 179.6 plus or minus 80.06 cubic millimetres against 1085 plus or minus 277.1 in the matched scramble group at 5 weeks.(Results, 2.6. “AUMsilence ASO Mediated Dhx15 Silencing in Mice Reduces Primary Tumor Volume in an HCC Mouse Model”)
  • Tumours in the silenced animals also had a smaller blood vessel perimeter and lumen and fewer lymphatic vessels, so the effect reached the tumour vasculature and not only its size.(Results, 2.6. “AUMsilence ASO Mediated Dhx15 Silencing in Mice Reduces Primary Tumor Volume in an HCC Mouse Model”)
  • The silenced livers showed lower expression of the vascular genes Vegf-a, Vegf-d, Vegfr1 and Vegfr3, matching what the authors saw in genetically deficient animals.(Results, 2.6. “AUMsilence ASO Mediated Dhx15 Silencing in Mice Reduces Primary Tumor Volume in an HCC Mouse Model”)
  • The authors describe the oligonucleotides as reaching cells without any delivery reagent or formulation, and report optimal delivery to the liver.(Discussion)
  • Losing Dhx15 entirely left zebrafish embryos with no liver at all, which is how the authors arrived at the gene in the first place.(Results, 2.1. Impaired Liver Development in Dhx15-Deficient Zebrafish Embryos)
  • Circulating DHX15 was about nine times higher in the serum of patients with hepatocellular carcinoma than in healthy subjects, at 300.3 plus or minus 88.2 against 32.4 plus or minus 27.7 picograms per millilitre.(Results, 2.6. “AUMsilence ASO Mediated Dhx15 Silencing in Mice Reduces Primary Tumor Volume in an HCC Mouse Model”)

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