EVALUATION COMMITTEE

The Faculty Council has appointed the following adjudication committee to evaluate the thesis and the associated lecture: 

• Prof. DI Dr. Winfred Neuhaus, Principal Scientist, Molecular Diagnostics, Austrian Institute of Technology
• Professor Thomas Corydon, Institut for Biomedicin – Forskning og uddannelse, Aarhus University, Denmark
• Associate professor Emil Kofod-Olsen, Department of Health Science and Technology (HST), Aalborg University, Denmark (Chairman)

Moderator: Professor Torben Moos, Department of Health Science and Technology (HST), Aalborg University
 

ABSTRACT

The blood-brain barrier (BBB) presents a significant challenge for treating brain diseases. Composed of tightly interconnected brain capillary endothelial cells (BCECs), the BBB protects the brain from potentially harmful substances circulating in the bloodstream. Unfortunately, the BBB also prevents most drugs from reaching the brain parenchyma, making drug delivery to the central nervous system significantly difficult. Despite decades of research into strategies to circumvent the BBB, a highly specific and efficient drug delivery method is still needed. BBB-directed gene therapy offers a promising avenue for delivering molecules directly to the brain, by genetically modifying the BCECs, to produce and secrete therapeutic proteins, thereby turning them into protein factories delivering relevant proteins to the brain and the bloodstream. This is particularly relevant for genetic diseases caused by a global protein deficiency, such as the lysosomal storage disorder, Niemann-Pick disease type C2 (NPC2). NPC2 is characterized by both visceral and neurological symptoms due to a deficiency of the secretory lysosomal protein NPC2. Beyond protein delivery, BBB-targeted gene therapy could also be used to alter the expression of existing transporters within the BCECs and thereby modify the transport of molecules across the BBB. This could potentially be used to improve the glucose uptake in the brain by increasing the expression of glucose transporters, which could impact neurodegenerative diseases presenting with glucose hypometabolism like Alzheimer's disease (AD). This dissertation investigates the potential of employing BBB-directed gene therapy using viral and non-viral vectors for the delivery of NPC2 protein, as well as the relevance of increasing glucose transporter expression at the BBB in AD.

In Study I, the delivery of the NPC2 gene to BCECs and the subsequent biological effect on protein secretion were investigated using an in vitro BBB model based on primary rat cells. A non-viral vector was used to deliver the NPC2 gene. The study demonstrated that primary BCECs could be modified to produce and secrete NPC2 protein, with a resulting therapeutic effect in NPC2-deficient fibroblasts. However, the transfection efficiency using the non-viral vector was too low in post-mitotic, polarized BCECs, demonstrating the need for a more efficient gene therapy vector to ensure sufficient gene delivery to the BCECs.

Study II was therefore conducted using an adeno-associated virus vector, referred to as AAV-BR1, as it had previously demonstrated high specificity towards BCECs following systemic injections in mice. AAV-BR1 mediated NPC2 gene delivery was studied using an in vitro BBB model based on primary cells from mice and in BALB/cJRj mice following intravenous injections. Widespread transduction of BCECs was observed in the brains of healthy mice, leading to an increased Npc2 gene expression, however, not sufficient to detect a corresponding rise in NPC2 protein levels in the brain or the blood. Using the in vitro BBB model, transduction resulted in both a luminal and abluminal secretion of recombinant NPC2 proteins into the cell culture media on both sides of the model simulating the blood and the brain compartments. Secreted NPC2 protein reversed abnormal cholesterol accumulation in NPC2-deficient fibroblasts.

To explore the significance of BBB-directed gene therapy in altering glucose transporter expression, Study III comprised a systematic review. This review summarized glucose transporter expression in post-mortem brains from AD patients and in several rodent models of AD, supplemented by original analysis of the glucose transporter 1 (GLUT1) expression in TgSwDI and 5xFAD transgenic AD mouse models. The review revealed that studies based on samples from AD patients have consistently shown reductions in GLUT1 and GLUT3, two crucial transporters responsible for brain glucose uptake. Moreover, these changes were linked to AD pathology and cognitive decline in multiple studies. While most rodent models replicate the human findings of reduced GLUT1 and GLUT3, some studies exhibited varying results, emphasizing the complexity of AD modeling. Results from several studies suggested that alterations in glucose transporter expression may play a role in the development of AD, highlighting the potential of targeting glucose transporters as therapeutic strategies for this disease.

Finally, the effect of AAV-BR1-mediated GLUT1 gene therapy was tested using the in vitro BBB model based on primary mouse cells in Study IV. This resulted in significant transduction of BCECs comparable to previous observations but without a subsequent increase in the GLUT1 transgene expression, thus no alterations in the GLUT1 protein expression or glucose transport were observed. Therefore, further studies are needed to investigate this phenomenon and the potential of using BBB-directed gene therapy to increase GLUT1 expression and thus glucose transport to the brain.

In conclusion, BBB-directed gene therapy seems to have the potential to treat neurological diseases through transduction and protein production within the brain. Furthermore, it can be implied that normalization of glucose transporter expression within the brain is a relevant therapeutic strategy in AD, but more studies are needed to determine the optimal application of BBB-directed gene therapy for this approach.