title: Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes.
abstract: Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene expression (>90%) in vivo. Moreover, cy5-MAG2.2-GFP delivery to the neonate CNS resulted in selective GFP expression in oligodendrocytes for at least 8 months. Broadly, the characterization of the extremely short yet oligodendrocyte-specific human MAG promoter may facilitate modeling neurological diseases caused by oligodendrocyte pathology and has translational relevance for leukodystrophy gene therapy.
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Georg
authors.initials: G
authors.lastname: von Jonquieres
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Dominik
authors.initials: D
authors.lastname: Fröhlich
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Claudia B
authors.initials: CB
authors.lastname: Klugmann
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Xin
authors.initials: X
authors.lastname: Wen
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Harasta
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Roshini
authors.initials: R
authors.lastname: Ramkumar
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Ziggy H T
authors.initials: ZH
authors.lastname: Spencer
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Housley
authors.affiliation: Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, UNSW Australia Sydney, NSW, Australia.
authors.forename: Matthias
authors.initials: M
authors.lastname: Klugmann
keywords: AAV; gene therapy; leukodystrophy; myelin-associated glycoprotein; oligodendroglia; white matter disorders