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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

Transient Delivery of Epigenome Editors Stably Represses PCSK9 and Lowers LDL Cholesterol in Non-Human Primates

Type: Oral Abstract Session

Presentation Details
Session Title: Late-Breaking Abstracts 1
Location: Room 515 AB
Start Time: 5/19/2023 9:00
End Time: 5/19/2023 9:15

Precise epigenetic modulation at targeted DNA sequences using CRISPR/dCas9, TALE, or zinc finger DNA-binding domains has tremendous promise as a next-generation therapy for repressing gene targets associated with disease in a variety of cell types and tissues. This strategy allows for more accurate control over gene expression and avoids the risk of cutting or permanently modifying DNA. Mechanistically, epigenetic repressors can deposit methylation around the DNA target region and induce long-term repression of gene targets when delivered either transiently or permanently. To test the efficacy of transiently expressed epigenetic repressors in vivo in a large animal model, we delivered a lipid nanoparticle (LNP) carrying RNA encoding an epi-repressor targeted specifically to the cynomolgus macaque PCSK9 promoter to non-human primates (NHPs). Silencing PCSK9 results in reduction of LDL-cholesterol and is a therapeutic target for the prevention of cardiovascular disease. While currently approved PCSK9 inhibitors require repeat administration to maintain efficacy, epigenome editing offers the potential to durably silence PCSK9 without altering the genetic sequence. First, we tested the efficacy and durability of epigenetic repression in human liver cell lines. We observed stable repression of PCSK9 in a human liver cell line for six months after a single transient dose of epigenome editor, as well as stable methylation around the target site. We then tested the same epi-editor in primary hepatocytes isolated from cynomolgus macaques. After a single administration, we observed 93% PCSK9 mRNA repression and a drop in protein expression to below the limit of assay detection 14 days after transfection, which is the approximate lifespan of primary hepatocytes in culture. We then packaged this epi-repressor targeting the PCSK9 promoter into an optimized LNP formulation and delivered a single dose intravenously into three NHPs. Two additional NHPs were dosed with PBS as a negative control. Upon LNP delivery, a transient spike in ALT and AST levels was observed but normalized within 1-2 weeks. Otherwise, the animals were observed to be healthy and normal without any adverse clinical symptoms. Within 3 days of the epi-repressor administration, we observed a sharp decline in serum PCSK9, and sustained repression of ~80% has been observed out to 9 weeks, with readings ongoing weekly. This reduction in PCSK9 is accompanied by ~50% stable reduction in LDL-C. To confirm the mechanism of action of the epi-repressor, liver biopsies were collected post LNP administration and DNA was extracted for targeted methylation sequencing. The region surrounding the gRNA target site was sequenced and differential CpG methylation was quantified. In animals treated with the epi-repressor, peak methylation occurred in the region surrounding the gRNA binding site, with up to 68% CpG methylation at certain residues compared to ~1% in PBS controls. We observed the spread of methylation was limited to a ~2.6kb region surrounding the PCSK9 CpG island. In summary, our findings show that transient delivery of this epi-repressor to NHPs led to epigenetic remodeling of target loci and support epigenome editing as a therapeutic modality for robust and durable gene silencing.

Jennifer B. Kwon1, Alec Knapp2, Andrew Hill2, Kristen J. Browoleit1, Sai An1, Stuart Sundseth1, Tyler Goodwin1, Michael Hefferan1, Kendra Congdon1, Charles A. Gersbach1,3, Blythe D. Sather2

1Tune Therapeutics, Durham, NC,2Tune Therapeutics, Seattle, WA,3Duke University, Durham, NC
  J.B. Kwon: 1; Commercial Interest i.e. Company X; Tune Therapeutics. 1; What was received? i.e. Honorarium; Salary, stock. 1; For what role? i.e. Speaker; Employed.

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