A novel strategy for deliveringNiemann‐Pick typeC2proteins across the blood–brain barrier using the brain endothelial‐specificAAV‐BR1virus

Abstract

Treating central nervous system (CNS) diseases is complicated by the incapability of numerous therapeutics to cross the blood–brain barrier (BBB), mainly composed of brain endothelial cells (BECs). Genetically modifying BECs into protein factories that supply the CNS with recombinant proteins is a promising approach to overcome this hindrance, especially in genetic diseases, like Niemann Pick disease type C2 (NPC2), where both CNS and peripheral cells are affected. Here, we investigated the potential of the BEC‐specific adeno‐associated viral vector (AAV‐BR1) encoding NPC2 for expression and secretion from primary BECs cultured in an in vitro BBB model with mixed glial cells, and in healthy BALB/c mice. Transduced primary BECs had significantly increased NPC2 gene expression and secreted NPC2 after viral transduction, which significantly reversed cholesterol deposition in NPC2 deficient fibroblasts. Mice receiving an intravenous injection with AAV‐BR1‐NCP2‐eGFP were sacrificed 8 weeks later and examined for its biodistribution and transgene expression of eGFP and NPC2. AAV‐BR1‐NPC2‐eGFP was distributed mainly to the brain and lightly to the heart and lung, but did not label other organs including the liver. eGFP expression was primarily found in BECs throughout the brain but occasionally also in neurons suggesting transport of the vector across the BBB, a phenomenon also confirmed in vitro. NPC2 gene expression was up‐regulated in the brain, and recombinant NPC2 protein expression was observed in both transduced brain capillaries and neurons. Our findings show that AAV‐BR1 transduction of BECs is possible and that it may denote a promising strategy for future treatment of NPC2.

Endothelial and neuronal engagement by AAV-BR1 alleviates neurological symptoms and cholesterol deposition in a mouse model of Niemann-Pick type C2

“…The distribution pattern of NPC2 in the neurons was similar to our previous study investigating the AAV-BR1-NPC2 in healthy BALB/cJRj mice (9). Here the AAV-BR1 vector was designed to produce two separate proteins; enhanced green fluorescent protein (eGFP) and NPC2, where the eGFP accumulated intracellularly as an indicator of cellular transduction.…”

“…However, the mechanism of this BBB passage remains unknown. Loss of BBB integrity seen in pathological conditions was not observed in Npc2 -/- mice (9). The predominant appearance of NPC2-positive cells was in the hippocampus consistent with studies using healthy mice (9).…”

“…Loss of BBB integrity seen in pathological conditions was not observed in Npc2 -/- mice (9). The predominant appearance of NPC2-positive cells was in the hippocampus consistent with studies using healthy mice (9). Compared to evolutionary higher regions, the hippocampal vessels differ by lesser neurovascular coupling between vascular networks and functions of BECs and pericytes, which may facilitate viral vector binding and capillary migration.…”

“…Here the AAV-BR1 vector was designed to produce two separate proteins; enhanced green fluorescent protein (eGFP) and NPC2, where the eGFP accumulated intracellularly as an indicator of cellular transduction. However, also neuronal cells expressed eGFP, indicating that some of the systemically injected AAV-BR1 undergoes transport across the BBB leading to neuronal transduction (9). In our previous study in healthy mice (9), we further examined the possibility of AAV-BR1 vector transport across the blood-cerebrospinal fluid barrier (BCSFB) but found no evidence to support this.…”

“…However, also neuronal cells expressed eGFP, indicating that some of the systemically injected AAV-BR1 undergoes transport across the BBB leading to neuronal transduction (9). In our previous study in healthy mice (9), we further examined the possibility of AAV-BR1 vector transport across the blood-cerebrospinal fluid barrier (BCSFB) but found no evidence to support this. Furthermore, it seems unlikely that the widespread distribution of NPC2 throughout the brain, including the cerebral cortex, could be achieved through BCSFB transport.…”