494. Optimizing the Mannitol-Facilitated CNS Entry of Peripherally-Delivered AAV2 Vector
Authors: Haiyan Fu, Julianne DiRosario, Douglas M. McCarty
Journal: Molecular Therapy (2006)

The blood-brain barrier (BBB) is a great obstacle to the development of therapies for many neurological diseases, especially those globally involving central nervous system (CNS). Mannitol is a known osmotic BBB disruption reagent, which can facilitate the temporary opening of the BBB and enable CNS entry of peripherally delivered therapeutic materials, including rAAV vectors, within a limited time frame. We have optimized the timing of vector infusion, following mannitol pretreatment, to achieve maximum entry of rAAV into the CNS of adult wildtype mice (6-8 months of age). At times ranging from 5-20 minutes (at 1 minute intervals, n≥4/group) after an IV infusion of mannitol (1-2mg/g bw), animals received an IV injection of 1x1011 genome-containing particles of self-complementary rAAV2 viral vector (scAAV2-CMV-GFP) expressing green fluorescent protein (GFP). Four weeks after the injection, vibratome sections (50mm) of whole brain and spinal cord were collected and evaluated using immunofluorescence to detected GFP expression. Vector transgene expression was observed in both neuronal and non-neuronal cells throughout the brain and spinal cord. We found that optimal transduction was observed in the CNS when the vector was IV injected 8 minutes after the administration of mannitol, with GFP expression detected in 1.2-1.5x106 brain cells. This was approximately 10-fold higher than the CNS transduction observed when the vector was injected at either 5 or 10 minutes after mannitol pretreatment, and 2000-fold higher than when the vector was given at 20 minutes post-mannitol infusion. We believe that this optimized injection regimen can be applied to therapeutic protocols for a broad range of CNS diseases, with the potential of greatly improving the global CNS delivery of viral vectors. The blood-brain barrier (BBB) is a great obstacle to the development of therapies for many neurological diseases, especially those globally involving central nervous system (CNS). Mannitol is a known osmotic BBB disruption reagent, which can facilitate the temporary opening of the BBB and enable CNS entry of peripherally delivered therapeutic materials, including rAAV vectors, within a limited time frame. We have optimized the timing of vector infusion, following mannitol pretreatment, to achieve maximum entry of rAAV into the CNS of adult wildtype mice (6-8 months of age). At times ranging from 5-20 minutes (at 1 minute intervals, n≥4/group) after an IV infusion of mannitol (1-2mg/g bw), animals received an IV injection of 1x1011 genome-containing particles of self-complementary rAAV2 viral vector (scAAV2-CMV-GFP) expressing green fluorescent protein (GFP). Four weeks after the injection, vibratome sections (50mm) of whole brain and spinal cord were collected and evaluated using immunofluorescence to detected GFP expression. Vector transgene expression was observed in both neuronal and non-neuronal cells throughout the brain and spinal cord. We found that optimal transduction was observed in the CNS when the vector was IV injected 8 minutes after the administration of mannitol, with GFP expression detected in 1.2-1.5x106 brain cells. This was approximately 10-fold higher than the CNS transduction observed when the vector was injected at either 5 or 10 minutes after mannitol pretreatment, and 2000-fold higher than when the vector was given at 20 minutes post-mannitol infusion. We believe that this optimized injection regimen can be applied to therapeutic protocols for a broad range of CNS diseases, with the potential of greatly improving the global CNS delivery of viral vectors.