Della Valle et al. · Science Translational Medicine · 2022

LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes

Della Valle Francesco, Reddy Pradeep, Yamamoto Mako, Liu Peng, Saera-Vila Alfonso, Bensaddek Dalila, Zhang Huoming, Prieto Martinez Javier, Abassi Leila, Celii Mirko, Ocampo Alejandro, Nuñez Delicado Estrella, Mangiavacchi Arianna, Aiese Cigliano Riccardo, Rodriguez Esteban Concepcion, Horvath Steve, Izpisua Belmonte Juan Carlos, Orlando Valerio

Read the paper The product used

The study

What was asked, and what was found

Della Valle et al., Science Translational Medicine, 2022 asked why heterochromatin falls apart in premature ageing, and whether the RNA made by an awakened transposon is a cause rather than a consequence. The authors found that LINE-1 RNA builds up early in cells from patients with Hutchinson-Gilford progeria syndrome, Werner syndrome and several atypical progeroid syndromes, where LINE-1 expression ran four to seven times higher than in healthy cells. They then showed that this RNA binds and inhibits SUV39H1, the enzyme that writes the repressive histone 3 lysine 9 mark, which places the transposon transcript upstream of the chromatin loss rather than downstream of it.

The test was to take the RNA away. Self-delivering AUMsilence sdASOs designed against the first reading frame of the LINE-1 consensus were dissolved in the culture medium at 1 μM, with no transfection reagent, and refreshed every 4 days against a scramble control. In patient fibroblasts and in mesenchymal stem cells from progeria and Werner syndrome, the knockdown restored both histone 3 lysine 9 and histone 3 lysine 27 trimethylation, suppressed the senescence gene programme, raised proliferation, lowered the DNA damage marker, and reversed DNA methylation age on two separate epigenetic clocks.

The study then moved a mouse-specific oligonucleotide of the same design into an animal. LAKI progeria mice were given three intraperitoneal injections of 2 mg/kg, 10 days apart, from 8 weeks of age, against a scramble arm and a non-injected arm. Sequencing and qPCR confirmed LINE-1 knockdown in aorta, skin, spleen, kidney and skeletal muscle, and a dye-labelled version of the oligonucleotide was imaged in living animals to establish where it went. Median survival was longer in the treated arm than in the scramble arm, at p equal to 0.0010 by the Mantel-Cox test, and the histology of the worst affected tissues was improved. The Methods name no supplier, but the paper's own supplementary reagent table does: its ASO sheet has a column headed source, and five of its seven oligonucleotide rows name AUM there.

Key findings

Self-delivering AUMsilence sdASOs against LINE-1 RNA, dissolved in the culture medium of fibroblasts from patients with three different progeroid syndromes, restored the two repressive histone marks that these cells lose, reversed their DNA methylation age and suppressed the senescence gene programme.(Abstract)
A mouse-specific oligonucleotide of the same design, given systemically to a progeria mouse as three intraperitoneal injections of 2 mg/kg 10 days apart, lengthened median survival against the scramble arm, at p equal to 0.0010 by the Mantel-Cox test.(Results)
Treatment reached every organ examined and cut LINE-1 RNA in all of them, with knockdown confirmed by both sequencing and qPCR in the tissues themselves.(Results)
A dye-labelled version of the mouse oligonucleotide was injected and imaged in living animals to establish where it went and how long it lasted.(Results)
In cells, the knockdown also raised proliferation and lowered the DNA damage marker, so the effect went beyond histone marks to how the cells behaved.(Results)
The mechanism the study proposes puts the targeted RNA upstream of the chromatin defect, since LINE-1 RNA was shown to inhibit the histone methyltransferase whose product is the mark that goes missing.(Abstract)

Product: AUMsilence sdASO
Targets: LINE-1 · human · retrotransposon RNA, the L1-Ta and L1Hs full-length transcriptsLINE-1 · mouse · retrotransposon RNA, the L1-Tf, L1-Gf and L1-Af subfamilies
Models: human dermal fibroblasts from patients with Hutchinson-Gilford progeria syndrome, atypical progeroid syndromes and Werner syndrome, from the Coriell biobank · patient-derived cellshuman mesenchymal stem cells differentiated from progeria and Werner syndrome patient pluripotent stem cells · stem cellstail-tip fibroblasts from the LAKI progeria mouse and from wild-type mice · primary cellsLAKI mouse, a Lmna knock-in model of Hutchinson-Gilford progeria syndrome, dosed intraperitoneally · in vivo
Delivery: in cells, dissolved in the culture medium with no transfection reagent. In animals, three intraperitoneal injections at 2 mg/kg 10 days apart
Readouts: TaqMan qPCR, RNA sequencing, chromatin immunoprecipitation with qPCR, immuno-FISH, DNA methylation arrays and epigenetic clocks, mass spectrometry proteomics, senescence-associated beta-galactosidase assay, histology and immunohistochemistry, in vivo fluorescence imaging, survival analysis
Measured: concentration and schedule used on patient cells: 1 μM (dissolved in culture medium, redosed every 4 days, collected after two passages)dose and schedule used in the progeria mouse: 2 mg/kg (three intraperitoneal injections at 10 day intervals, starting at 8 weeks of age)survival difference between the targeting and scramble arms: 0.0010 by Mantel-Cox and 0.0056 by Gehan-Breslow for the targeting oligonucleotide against the scramble (LAKI mice, survival curves, with a non-injected arm as the third comparison)baseline elevation of LINE-1 expression in atypical progeroid syndrome fibroblasts, before any treatment: four to seven times higher (atypical progeroid syndrome patient fibroblasts against wild type)
Figures: Fig. 2 Senescence gene expression and heterochromatin marks in patient stem cells left untreated, scramble treated or treated with the oligonucleotides against LINE-1.Fig. 3A DNA methylation age measured on two epigenetic clocks in untreated, scramble treated and treated patient cells.Fig. 6B Survival curves for progeria mice given the targeting oligonucleotide, the scramble, or nothing.Fig. 6C Histology of aorta, skin, kidney and spleen from treated and control progeria mice.
Citation: Della Valle et al. LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes. Science Translational Medicine 2022;14:eabl6057. doi:10.1126/scitranslmed.abl6057

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