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Assarnia, Sogand

University of British Columbia

Lipoprotein Lipase (LPL) is responsible for the clearance of triglyceride-rich lipoproteins from the blood. LPL Deficiency is an autosomal recessive genetic disease caused by mutations in the LPL gene that disrupt normal LPL enzyme function resulting in severe hypertriglyceridemia and pancreatitis. Previously, our lab developed an AAV-based gene therapy to treat LPL deficiency by delivering the LPL gene into patients demonstrating proof of concept for viral-based gene therapy. This treatment became the first gene-augmentation therapy to receive regulatory approval (Glybera®). One limitation of this approach, however, is the dysregulated expression of the delivered therapeutic transgene. Gene editing may overcome some limitations of gene augmentation gene therapies, such as dysregulated transgene expression. We hypothesize that CRISPR/Cas9 base editing delivered via lipid nanoparticles can repair the mutant LPL gene and demonstrate a proof of concept for this novel therapeutic approach. We investigated the use of CRISPR/Cas9 based editing composed of a partially deactivated Cas9 (nCas9) protein with an adenine deaminase to directly repair the common P207L mutation in the LPL gene. We generated Flp-In T-RExTM 293 cell lines stably expressing either LPLP207L and wildtype LPL as model systems to explore the gene editing repair of LPLP207L. The effectiveness of base editing was measured both by Sanger DNA sequencing and measuring the restoration of LPL enzyme activity. After optimization of this process, we observed an approximately 50% correction of the LPLP207L mutation by Sanger DNA sequencing, which corresponded with a 52% restoration of LPL enzyme activity compared to wildtype LPL, using an NG-ABE8e base editor. These results demonstrate a proof-of-concept for DNA base editing as a novel treatment strategy to directly repair the LPLP207L mutation that causes LPL deficiency.

Text

2022-09-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/79674

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/