Background Patients with the genetic disorder Niemann-Pick type C2 disease (NP-C2) suffer from lysosomal accumulation of cholesterol causing both systemic and severe neurological symptoms. In a murine NP-C2 model, otherwise successful intravenous Niemann-Pick C2 protein (NPC2) replacement therapy fails to alleviate progressive neurodegeneration as infused NPC2 is unable to cross the blood-brain barrier (BBB). Genetic modification of brain endothelial cells (BECs) is thought to enable secretion of recombinant proteins thereby overcoming the restrictions of the BBB. We hypothesized that BBB-directed gene therapy using the AAV-BR1-NPC2 vector would transduce both BECs and neurons in a mouse model of NP-C2 ( Npc2 -/-). Methods Six weeks old Npc2 -/- mice were intravenously injected with the AAV-BR1-NPC2 vector. Post-mortem analyses included gene expression analyses, determination of NPC2 transduction in the CNS, and co-detection of cholesterol with NPC2 in neurons. Results The vector exerted tropism for BECs and neurons resulting in a widespread NPC2 distribution in the brain with a concomitant reduction of cholesterol in adjacent neurons, presumably not transduced by the vector. Conclusion The data suggests cross-correcting gene therapy to the brain via delivery of NPC2 from BECs and neurons.

INTRODUCTION:The treatment of neurological diseases is significantly hampered by the lack of available therapeutics. A major restraint for the development of drugs is denoted by the presence of the blood-brain barrier (BBB), which precludes the transfer of biotherapeutics to the brain due to size restraints.AREAS COVERED:Novel optimism for transfer of biotherapeutics to the brain has been generated via development of targeted therapeutics to nutrient transporters expressed by brain capillary endothelial cells (BCECs). Targeting approaches with antibodies acting as biological drug carriers allow for proteins and genetic material to enter the brain, and qualified therapy using targeted proteins for protein replacement has been observed in preclinical models and now emerging in the clinic. Viral vectors denote an alternative for protein delivery to the brain by uptake and transduction of BCECs, or by transport through the BBB leading to neuronal transduction.EXPERT OPINION:The breaching of the BBB to large molecules has opened for treatment of diseases in the brain. A sturdier understanding of how biotherapeutics undergo transport through the BBB and how successful transport into the brain can be monitored is required to further improve the translation from successful preclinical studies to the clinic.

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.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, meaning that only a few drugs can pass the BBB. Thus, for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify species-dependent differences that could explain the variations in TEER and aid to the selection of models for future BBB studies. The BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Additionally, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared. Expression and organization of tight junction proteins were in all three species influenced by co-culturing, supporting the findings, that TEER increases after co-culturing with astrocytes. All models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. The most interesting discovery was that even though the pBECs had higher TEER than rBECs and mBECs, the Papp did not show the same variation between species, which could be explained by a significantly larger cell size of pBECs. In conclusion, our results imply that the choice of species for a given BBB study should be defined from its purpose, instead of aiming to reach the highest TEER, as the models studied here revealed similar BBB properties.

Treatment of many diseases affecting the central nervous system (CNS) is complicated by the inability of several therapeutics to cross the blood–brain barrier (BBB). Genetically modifying brain capillary endothelial cells (BCECs) denotes an approach to overcome the limitations of the BBB by turning BCECs into recombinant protein factories. This will result in protein secretion toward both the brain and peripheral circulation, which is particularly relevant in genetic diseases, like lysosomal storage diseases (LSD), where cells are ubiquitously affected both in the CNS and the periphery. Here we investigated transfection of primary rat brain capillary endothelial cells (rBCECs) for synthesis and secretion of recombinant NPC2, the protein deficient in the lysosomal cholesterol storage disease Niemann Pick type C2. We demonstrate prominent NPC2 gene induction and protein secretion in 21% of BCECs in non‐mitotic monocultures with a biological effect on NPC2‐deficient fibroblasts as verified from changes in filipin III staining of cholesterol deposits. By comparison the transfection efficiency was 75% in HeLa‐cells, known to persist in a mitotic state. When co‐cultured with primary rat astrocytes in conditions with maintained BBB properties 7% BCECs were transfected, clearly suggesting that induction of BBB properties with polarized conditions of the non‐mitotic BCECs influences the transfection efficacy and secretion directionality. In conclusion, non‐viral gene therapy to rBCECs leads to protein secretion and signifies a method for NPC2 to target cells inside the CNS otherwise inaccessible because of the presence of the BBB. However, obtaining high transfection efficiencies is crucial in order to achieve sufficient therapeutic effects.