1. ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice
- Author
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Lin Gan, Silvere Pagant, Robert J. Desnick, Makiko Yasuda, Scott Sproul, Michael C. Holmes, Kathleen Meyer, Marshall W. Huston, Susan St Martin, Luciana Moreira, and Thomas Wechsler
- Subjects
Signal peptide ,Transgene ,Genetic Vectors ,Globotriaosylceramide ,Gene Expression ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,In vivo ,Complementary DNA ,Drug Discovery ,Genetics ,medicine ,Animals ,Humans ,Transgenes ,Molecular Biology ,030304 developmental biology ,Pharmacology ,Gene Editing ,0303 health sciences ,Nuclease ,biology ,Chemistry ,Gene Transfer Techniques ,Genetic Therapy ,Dependovirus ,medicine.disease ,Fabry disease ,Fusion protein ,Zinc Finger Nucleases ,Cell biology ,Enzyme Activation ,Disease Models, Animal ,030220 oncology & carcinogenesis ,alpha-Galactosidase ,biology.protein ,Hepatocytes ,Molecular Medicine ,Fabry Disease ,Genetic Engineering - Abstract
Fabry disease, a lysosomal storage disorder resulting from the deficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted in vivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated in vivo genome editing has the potential to be an effective one-time therapy for Fabry disease.
- Published
- 2020