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B - Gene Targeting and Gene Correction -> Gene Targeting and Gene Correction (Basic development of novel technologies for genome editing, with or without site-specific endonuclease. Abstracts focused on specific disease applications should be submitted to the suitable tissue/disease category)

1215: Durable Silencing of Pcsk9 by In Vivo Hit-and-Run Epigenome Editing

Type: Oral Abstract Session

Presentation Details
Session Title: Delivery Technologies and In Vivo Gene Editing
Location: Room 202
Start Time: 5/19/2022 10:15
End Time: 5/19/2022 10:30

Epigenome editing is emerging as a powerful new strategy to silence genes without altering the primary DNA sequence. In this regard, we and others have previously developed Engineered Transcriptional Repressors (ETRs), chimeric proteins containing a programmable DNA Binding Domain (DBD) fused to either one or more of the following epigenetic repressive effectors: KRAB, the catalytic domain of DNMT3A and DNMT3L. Of note, transient delivery of such ETRs, either as a triple combination or as all-in-one fusion proteins, was shown to induce efficient, long-term stable and specific epigenetic silencing of endogenous genes in both human and mouse cell lines, and primary human cells, including T lymphocytes and iPSCs. Here, we explored for the first time the ETR technology for silencing genes in vivo. To this end, we focused on Pcsk9, a gene involved in cholesterol homeostasis whose hepatic inactivation is under clinical testing for the treatment of hypercholesterolemia. First, we used an ad hoc developed Pcsk9 murine reporter cell line to select the best performing triple ETR combination for each of the following DBD platforms: catalytically deactivated Cas9 (dCas9; 8 target sites), Transcription Activator-Like Effectors (TALEs; 16 target sites) and Zinc Finger Proteins (ZFPs; 16 target sites). These in vitro studies identified ZFP-based reagents capable of inducing >90% long-term stable (up to 50 days) epigenetic silencing of Pcsk9. In parallel, we set-out to deliver the ETRs to the liver of mice. As the ETR technology entails the use of transient gene delivery modalities, we performed an in vivo screening to identify Lipid Nanoparticles (LNPs) compatible with efficient transfer of editing machinery to the liver, using here CRISPR-Cas9-mediated inactivation of Pcsk9 as a surrogate readout for LNP-mediated gene delivery efficiency. Among the 11 LNPs tested, 8 consistently induced robust reduction in circulating levels of Pcsk9 (up to 90%). Of these 8 candidates, one was selected for further studies, given its favourable toxicity and biodistribution profiles. We thus packaged the Pcsk9 ZFP-based ETRs into this LNP and first tested its performance on cultured primary murine hepatocytes. In this setting, ZFP-based ETRs nearly abrogated Pcsk9 expression, at levels comparable to those obtained with conventional gene editing. Prompted by these results, we then administered intravenously the LNP-ETRs in adult mice and followed the circulating levels of Pcsk9 and Low-Density Lipoproteins (LDLs) for up to 100 days. Early analyses of mice showed a rapid reduction of Pcsk9 which stabilized at ~50% of mock-untreated levels until the last time point analyzed. In line with these data, at day 30 post-LNP injection, LDL levels were reduced as compared to mock-treated mice (~35%). Comparable efficiencies and kinetics of Pcsk9 reduction were observed in mice treated with LNPs loaded with the catalytically active CRISPR-Cas9 programmed against Pcsk9. We are currently monitoring Pcsk9 in LNP-ETR treated mice as well as further optimizing ETR architecture. Overall, these data provide the first demonstration that transient ETR delivery can mediate durable epigenetic silencing in vivo, opening exciting new possibilities in the field of therapeutic gene silencing.

Martino Alfredo Cappelluti1, Valeria Mollica Poeta1, Angelo Lombardo1,2

1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy,2Vita-Salute San Raffaele University, Milan, Italy
 M. Cappelluti: None.

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