Wenyan Miao1, Daniel Rios1, Mehrnaz Gharaee-Kermani2, Craig Dobry2, Artur Jaroszewicz1, Cedric Arisdakessian1, Enrique Garcia-Rivera1, Nafeeza Hafeez1, Brian Desrosiers1, Jess Floro1, Jared Steranka1, Menachem Fromer1, Dennis Zaller1 and J. Michelle Kahlenberg2, 1Rome Therapeutics, Boston, MA, 2University of Michigan, Ann Arbor, MI
Background/Purpose: Long Interspersed Element-1 (LINE-1) are retrotransposable DNA elements that make up ~17% of the human genome, and their role in health and disease is still being evaluated. Published studies have suggested that LINE-1 may contribute to the development and progression of autoimmune diseases such as lupus by triggering the type I interferon (IFN) production via activation of the innate immune system through nucleic acid sensing pathways. Additionally, studies have shown that lupus patients have higher level of LINE-1 in kidneys and blood compared to healthy controls, but expression of LINE-1 in SLE skin, where type I IFN signatures are dominant, has not been studied. We thus examined LINE-1 RNA and protein expression in lupus patient skin biopsies, and the impact on inflammatory signals upon a LINE-1 reverse transcriptase (RT) inhibitor in cellular and murine models.
Methods: All human subjects gave informed, written consent for the study. Formalin-fixed, paraffin embedded skin sections from non-lesional, non-sun exposed and UVB-treated skin were examined for changes in interferon-stimulated gene (ISG) expression via RNA-seq, LINE-1 ORF1p and phospho-STING via immunohistochemistry, and for LINE-1 ORF1 and ORF2 transcripts by RNA-scope. A novel, potent LINE-1 RT inhibitor, RPT-A, was characterized using LINE-1 RT enzyme assay, cellular LINE-1 retrotransposition assay, a cellular model of Aicardi-Goutières syndrome, and a UV-irradiated keratinocyte model. To investigate its impact on type I interferon pathway in lupus settings, we assessed its activity in regulating ISGs in UV-irradiated keratinocytes from lupus patients and healthy controls by RNA-seq. We also studied the impact of RPT-A on disease in a TREX1 knockout interferonopathy mouse model.
Results: Non-lesional biopsies from SLE skin exhibit increased ORF1p protein and increased ORF2 transcript staining and a concurrent increase in phospho-STING staining, suggestive of activation of nucleic acid sensing pathways. UV stimulation increased expression of LINE-1 transcript and protein in both healthy control and SLE skin. Tri-phosphorylated form of RPT-A inhibited enzymatic activity of LINE-1 RT with an IC50 of 0.03 µM. RPT-A inhibited cellular LINE-1 retrotransposition, decitabine-induced interferon response in TREX1 deficient THP-1 cells, and UV-induced phospho-TBK1 in human HaCaT keratinocytes. LINE-1 knockdown with shRNA and siRNA in the THP1 and HaCaT cells mimicked the inhibitory effect of RPT-A. Keratinocyte cell lines derived from lupus patients pre-treated with RPT-A exhibit reduced ISGs upon UV irradiation in a dose-dependent fashion. Further, 5-6 week old TREX1 knockout mice dosed with RPT-A orally once a day for 28 days exhibited reduced serum anti-dsDNA antibodies, reduced myocardial ISGs and reduced immune infiltrates in the heart and kidney.
Conclusion: LINE-1 RNA and protein are increased in SLE skin and is induced by UV exposure. Inhibition of LINE-1 RT activity results in decreased ISG expression and improved disease activity in a murine interferonopathy model. Inhibition of LINE-1 reverse transcriptase holds promise as a novel therapy for diseases in which type I IFNs drive disease.
W. Miao: Rome Therapeutics, 3; D. Rios: Rome Therapeutics, 3; M. Gharaee-Kermani: Rome Therapeutics, 5; C. Dobry: Rome Therapeutics, 5; A. Jaroszewicz: Rome Therapeutics, 3; C. Arisdakessian: Rome Therapeutics, 3; E. Garcia-Rivera: Rome Therapeutics, 3; N. Hafeez: Rome Therapeutics, 3; B. Desrosiers: Rome Therapeutics, 3; J. Floro: Rome Therapeutics, 3; J. Steranka: Rome Therapeutics, 3; M. Fromer: Rome Therapeutics, 3; D. Zaller: Rome Therapeutics, 3; J. Kahlenberg: AstraZeneca, 1, Bristol-Myers Squibb(BMS), 2, 5, EMD Serano, 2, exo therapeutics, 2, Gilead, 2, GlaxoSmithKlein(GSK), 1, horizon Therapeutics, 2, Janssen, 5, Pfizer, 2, ROME Therapeutics, 2, 5, Rome Therapeutics, 5, Ventus Therapeutics, 2, 5.
