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.

Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques

“…Once inside, recombinant AAV genomes largely persist as stable episomes that can produce transgene expression for the life of the cell( 51 ). In the CNS, AAV vectors infect a variety of cells including neurons, astrocytes, oligodendrocytes, endothelial cells and ependymal cells( 41 , 52 , 53 ). We previously reported extensive CNS astrocyte transduction in mice and non-human primates using the identical AAV9 construct as this study( 22 , 41 ).…”

Adeno‐associated virus‐based Alzheimer's disease mouse models and potential new therapeutic avenues

“…Interestingly, the cytomegalovirus (CMV) promoter or the CMV/chicken β‐actin hybrid promoter have been reported to exert activity in neurons. However, swapping CMV‐based promoters for glia‐specific promoters achieved cell lineage‐selective green fluorescence protein (GFP) expression in astrocytes and oligodendrocytes in mice (Georgiou et al, ; von Jonquieres et al, ; von Jonquieres et al, ; von Jonquieres et al, ; Watakabe et al, ). Similar promoter selectivity has been confirmed in mouse models of neurodegenerative diseases (Georgiou et al, ; von Jonquieres et al, ) and also in rat and NHP (Bassil et al, ; Lawlor et al, ; Mudannayake, Mouravlev, Fong, & Young, ).…”

“…However, swapping CMV-based promoters for glia-specific promoters achieved cell lineage-selective green fluorescence protein (GFP) expression in astrocytes and oligodendrocytes in mice (Georgiou et al, 2017;von Jonquieres et al, 2013;von Jonquieres et al, 2016;von Jonquieres et al, 2018;Watakabe et al, 2015).…”

Evaluation of Gene Therapy as an Intervention Strategy to Treat Brain Injury from Stroke

“…Each have their own innate attributes and deficiencies, which must be considered in relation to the size of the gene sequence to be inserted, the target cell population, and the protein expression profile. In addition to the innate variations between viral vector system, viral serotypes will also affect the target cell specificity and protein expression ( Davidson et al, 2000 ; von Jonquieres et al, 2013 , 2016 ). As noted below, HSV vector-mediated expression has been reported with a few hours ( Hoehn et al, 2003 ), whereas other commonly used viral vectors exhibit expression profiles that take days or weeks to establish ( Mason et al, 2010 ).…”

“…As noted below, HSV vector-mediated expression has been reported with a few hours ( Hoehn et al, 2003 ), whereas other commonly used viral vectors exhibit expression profiles that take days or weeks to establish ( Mason et al, 2010 ). Further alterations in the expression profile of the protein of interest can be driven with capsid modifications, as well as the promoter used to drive gene expression, which can bias glial versus neuronal expression, and the potential to incorporate gene cassette control elements ( von Jonquieres et al, 2013 , 2016 ). The broad consideration of technical development of gene therapy platforms for clinical applications, including non-viral modalities, and use of gene regulatory strategies such as shRNA are outside of the scope of this review, which is a perspective on the opportunity and exemplar prospective gene targets.…”