Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus

Abstract

In this study, a rAAV vector carrying a reporter gene,Infusion of approximately 6 × 10 9 particles (2 × 10 5 infec-'humanized' green fluorescent protein (GFP), linked to the tious units) of rAAV-MBP-GFP into mouse brains resulted transcriptional control region from the myelin basic protein in the GFP expression specifically in white matter. The (MBP) gene (a myelin-forming cell-specific gene) was con-GFP protein was detected 15 days later by immunostainstructed. Transduction of oligodendrocytes was carried out ing, specifically in the oligodendrocytes. No astrocytes both in vitro and in vivo. The GFP expression was detected were transduced. Our studies suggested that cell types for at least 3 weeks in both transduced oligodendrocyte cell other than neurons in the central nervous system can also line (MOCH-1 cells) and primary cultures of rat oligodendbe transduced by rAAV using a cell-type-specific transcriprocytes. Preferential GFP expression in oligodendrocytes tional control region or promoter. The MBP transcriptional was observed in the primary cultures. In contrast, transduccontrol region might be suitable for gene therapy and other tion with rAAV carrying the CMV promoter produced neurobiology studies requiring direct targeting to the myelistronger GFP fluorescence in various cell types, with the nating cells. majority of GFP-expressing cells being the astrocytes.

Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo

“…However, this assay is biased toward cells in which the CMV promoter is active, and may favor neurons over other cell types such as oligodendrocytes. 12,13 We have also used the scAAV vectors to investigate the limits of rAAV transduction in the liver. Previous studies have shown that hepatocytes are not efficiently transduced by rAAV, the limiting factor apparently being the conversion of the single-stranded vector DNA to duplex by the liver cells.…”

Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

“…16 In addition, pathological responses may alter the expression profile of neurons or glia, for instance, reactive astrocytes often exhibit an upregulation of GFAP and other cellular factors, which may affect AAV expression using a glial promoter.…”

“…One advantage of AAV-2 virus for in vivo functional genomic studies is its lack of toxicity, 31 which is related to this paucity of viral elements; infection with AAV-2 in the brain is not known to elicit a systemic immune response, nor is there inflammation or injury in brain regions where the vector has been injected. 5,7,9,16,31 Despite the safety advantage related to minimal native AAV elements, the small packaging capacity of AAV vectors has limited gene transfer applications. Previous studies suggested that up to 6.0 kb can be packaged into virus, but these 'over-sized' viruses were not infectious, 32 hence the common assumption is that the packaged genome must be limited to just 110% of wild-type AAV or no greater than ෂ5.1 kb.…”

“…30 However, other work supports a role for 'promoter targeting' with AAV-2; for example, the myelin basic protein (MBP) promoter has been reported to drive expression in oligodendrocytes using AAV-2. 16 Therefore, in addition to testing promoter effects on gene expression in neurons, we wished to determine if expression would occur in glia using a strong glial promoter, despite the clear tendency of AAV-2 to bind to neurons. Once promoter effects were tested, we reasoned that it would be possible to modify the AAV-2 capsid to increase expression in glia, for instance using the pseudotyped AAV-2/5 capsid which shows increased glial uptake (unpublished data).…”

“…[1][2][3][4] To date, most studies on brain gene transfer have focused on vectors carrying viral promoters, [5][6][7][8][9][10] although some have looked at mammalian cell type-specific promoters. [11][12][13][14][15][16][17] In this study, we compared the strength and stability of expression, as well as cellular specificity, of AAV-2 vectors expressing reporter genes under the control of constitutive, viral, or other cellular promoters.…”