Glial Promoter Selectivity following AAV-Delivery to the Immature Brain

Abstract

Recombinant adeno-associated virus (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP) expression driven by the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA)-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.

MOBP levels are regulated by Fyn kinase and affect the morphological differentiation of oligodendrocytes

“…S3A for cellular distribution). The used AAV vectors contain a shortened MBP promoter for efficient expression in oligodendrocytes (von Jonquieres et al, 2013). At 5 days after transduction, cell lysates were analyzed by western blotting and densitometric analyses to determine the relative protein levels of MOBP, MBP, CNP and PLP.…”

“…3) at a concentration of 1 µM in addition to DMSO controls. Wild-type (FynWT), kinase-inactive Fyn, MOBP and eGFP cDNA was cloned downstream of the expression cassette controlled by the shortened MBP promoter, and packaged into AAV vectors as described previously (von Jonquieres et al, 2013). Primary OPCs were transduced with 2×10 9 viral genomes (vg)/ml culture medium after 2 DIV.…”

Gene Therapy for the Nervous System: Challenges and New Strategies

“…This could considerably influence the pre-clinical development process of therapeutic interventions for neurodegenerative diseases, which determines the AAV vectors that will be tested in human clinical trials. As discussed below in "New Strategies", the apparent discrepancy in CNS transduction profiles of AAV vectors with the same capsid but carrying different CNS cell specific promoters or the CBA promoter [102,[110][111][112] suggests that the latter promoter may not mediate detectable transgene expression in all cell types in the brain.…”

“…Apparently, AAV vectors carrying the original 2.2 kb Gfap promoter [150], or a smaller truncated version (gfaABC 1 D) [151], transduce astrocytes in the mouse brain effectively and with a great degree of specificity [102,111]. Similarly, AAV vectors carrying the myelin basic protein (MBP) promoter transduce oligodendrocytes at high efficiency and with excellent cellular specificity [102,110,112]. These findings are important because oligodendrocytes are a key therapeutic target for demyelinating CNS diseases, such as Canavan disease, and they challenge the widely held perception that no presently available AAV is capable of mediating efficient gene transfer to this cell population.…”

“…These findings with cell-type specific promoters raise the possibility that our current knowledge on the CNS tropism of systemically delivered AAVs is most likely incomplete as the studies carried out thus far have used scAAV vectors encoding GFP under different forms of the CBA promoter [21,36,49,97]. For new AAVs that display strong CNS tropism after systemic delivery using some version of the CAG/CBA promoter, it may be useful to extend studies to include scAAV vectors encoding a marker gene (GFP most likely) under different cell specific promoters for neurons [152], astrocytes [102,110,111], microglia [153], and oligodendrocytes [102,110,112] to develop a more complete understanding of its potential for global gene therapy interventions.…”