Trafficking of adeno‐associated virus vectors across a model of the blood–brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells

Abstract

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult due to the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells (BMVEC) as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates BMVEC barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that 1) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and 2) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in-vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity.

The human brain endothelial barrier: transcytosis of AAV9, transduction by AAV2

“…Merkel et al . () strengthen their findings by addressing whether AAV9 transcytosis was energy‐dependant. Using cell culture inserts plated with human BMVECs, the authors demonstrated that the movement of AAV9 across the endothelial barrier is impeded at 4⁰C, indicative of metabolic (active) transport, congruent with transcytosis.…”

“…In the current issue of J Neurochem , Merkel et al . () investigated trafficking mechanisms of AAV2 and AAV9 across a barrier composed of endothelial cells. Brain microvascular endothelial cells (BMVECs) were isolated from the temporal lobe or hippocampal formation of human subjects seeking operative treatment for epilepsy.…”

“…(b) Results from Merkel et al . () indicate that AAV 9 utilizes transcytosis, an active transport mechanism, to shuttle through BMVEC s and transduce astrocytes, without compromising the endothelial barrier. AAV 2 in contrast, transduces BMVEC s with very limited passage through the barrier.…”

“…The experimental model used by Merkel et al . () investigates the human brain endothelial barrier, and offers the possibility to screen new recombinant AAVs for transcytosis and subsequent transduction of CNS‐derived cells. The use of human BMVECs in this model provides a platform through which the fundamental mechanisms and molecular components of AAV attachment and transport through the endothelial cell layer may be elucidated.…”

“…In vitro models relevant to the human BBB, such as the one described by Merkel et al . () can be used to accelerate discoveries relevant to AAV receptors, internalization and cellular traffcking mechanisms.…”