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. 2025 Mar:88:40-49.
doi: 10.1016/j.ymben.2024.12.003. Epub 2024 Dec 7.

Metabolic engineering improves transduction efficiency and downstream vector isolation by altering the lipid composition of extracellular vesicle-enclosed AAV

Paula Espinoza  1 , Ming Cheng  1 , Carrie Ng  1 , Demitri de la Cruz  1 , Elizabeth D Wasson  2 , Deirdre M McCarthy  2 , Pradeep G Bhide  2 , Casey A Maguire  3 , Miguel C Santoscoy  4
Affiliations

Metabolic engineering improves transduction efficiency and downstream vector isolation by altering the lipid composition of extracellular vesicle-enclosed AAV

Paula Espinoza et al. Metab Eng. 2025 Mar.

Abstract

Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-ΔPTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust in-vivo transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors.

Keywords: Downstream; EV-AAV; Membrane engineering; Upstream.

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Conflict of interest statement

Conflict of interest statement C.A.M. has a financial interest in Sphere Gene Therapeutics, Inc., Chameleon Biosciences, Inc., and Skylark Bio, Inc., companies that are developing gene therapy platforms. C.A.M.‘s interests were reviewed and are managed by MGH and Mass General Brigham in accordance with their conflict-of-interest policies. M.C.S. has filed a patent application with claims involving the ΔPTDSS1 strategy and composition of the mEV-AAV9 vector.

Update of

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