Alzheimer’s disease (AD) is the world’s most common dementing illness, but no defensive treatments are available currently. A key reason is that AD drug development has been focused on symptomatic management without addressing basic cause of the disease. Hence, genetic intervention to suppress AD causative genes could represent an alternative to standard pharmacological approach. With the advent in therapeutic approach of RNA interference (RNAi), down-regulation of AD problematic genes including BACE1 has been extensively studied using viral vectors. Although viral vectors offered potential advantages for AD RNAi therapeutics, their clinical applications are limited by safety issue associated with viral-mediated carcinogenesis and immunogenicity, which led to the finding of nanotechnology-based non-viral vectors.
It is generally accepted that none of non-viral vectors is comparable to viral-vectors for delivery of genetic materials into host cells because non-viral vectors themselves are not equipped with modules for overcoming extracellular and intracellular barriers. Once delivered in body, therapeutic genes encounter extracellular barriers such as serum degradation, immune clearance, non-specific cell binding, and poor penetration of blood-brain barrier until they reach to target cells. After therapeutic genes are arrived in target cells, intracellular barriers inhibit RNAi activity which mainly includes cytotoxicity, poor endosomal escape and lysosomal degradation. The aim of the study is development of non-viral vector for AD RNAi therapeutics equipped with modules to conquer the biological barriers, which is divided into three parts; i) study 1: development of non-viral vector to overcome intracellular barriers, ii) study 2: surface functionalization of the non-viral vector to overcome extracellular barriers, and iii) study 3: verification of AD RNAi therapeutic potential of the developed non-viral vector in vivo.
Study 1 mainly focused on an approach of controlling cellular uptake mechanism and consequent intracellular route of complexes to overcome the intracellular barriers. Since it became clear that uptake mechanism by which complexes are internalized determines their intracellular fates, caveolar endocytosis has emerged as an important endocytic target because it provides avoidance of lysosomal degradation. Caveolae vesicles are generally immobilized at the plasma membrane by the actin cytoskeleton, and internalized by certain stimulation via ligand-receptor interaction or osmotic stress. With a view to generating osmotically active gene carrier which facilitates caveolar endocytosis, degradable poly(mannitol-co-PEI) gene transporter (PMT) was generated by crosslinking low molecular weight PEI providing proton sponge effect and mannitol diacrylate as an osmolyte linker. PMT/DNA showed lower cytotoxicity compared to PEI/DNA complexes due to easily degradable ester groups and partially negatively charged mannitol backbone which can shield the remaining cationic charges of PEI. The proton sponge effect of PEI backbone partially contributed to gene delivery of PMT. More importantly, selective stimulation of caveolar endocytosis by mannitol backbone provided enhanced transfection efficiency via successful avoidance of lysosomal degradation of cargo. The action mechanism of PMT/DNA complexes on stimulation of caveolae-mediated endocytosis was associated with the activation of Src-kinase by mannitol part of PMT.
In study 2, PMT was functionalized to overcome the extracellular barriers. Delivery of therapeutic siRNA to the brain is one of the biggest challenges for successful brain gene therapy because blood-brain barrier (BBB) permits selective entry of only few substances into the brain. In this study, ‘Trojan horse strategy’ was employed for ferrying PMT/siRNA complexes across the BBB. For this, PMT was conjugated with BBB-permeable rabies virus glycoprotein (RVG) via poly(ethylene glycol) (PEG) linker generating R-PEG-PMT. The PEG linker was expected to provide advantageous spatial arrangement of RVG peptide and stealth property on gene carrier. To determine the BBB penetration of complexes, in vitro BBB culture constructed by co-culture of bEnd.3 cells (mouse brain capillary endothelioma) and B23 cells (rat astrocytoma) on transwell system was utilized. The RVG ligand led to BBB penetration of PMT/siRNA complexes through receptor-mediated transcytosis via nicotinic acetylcholine receptors expressed on BBB. In mechanistic study, it was determined that improved BBB penetration of R-PEG-PMT/siRNA was achieved by stimulated receptor-mediated caveolar transcytosis. The enhanced accumulation in brain and biodistribution property of R-PEG-PMT/siRNA complexes was also demonstrated when systemically administrated.
In study 3, RNAi therapeutic potential of R-PEG-PMT/siBACE1 complexes was examined in vivo using normal C57/BL6 mice. Intravenous administration of R-PEG-PMT/siBACE1 for three times within 2 weeks has shown brain-targeted suppression of BACE1 without significant systemic toxicity. Furthermore, decreased BACE1 led to inhibition of beta-amyloid poduction, a marker of AD pathogenesis in hippocampus and cortex parts.
The overall results inferred that R-PEG-PMT is a promising tool for AD RNAi therapeutics. In aspect of overcoming intracellular barriers, i) degradable ester linkage of PMT reduced the cytotoxicity ii) PEI backbone of PMT has shown proton sponge effect, and iii) mannitol backbone provided the avoidance of lysosomal degradation via stimulation of caveolar endocytosis. In aspect of overcoming extracellular barriers, i) RVG offered neuronal cell targeting specificity, ii) stealth effect of PEG prevented the immune clearance in vivo, and iii) RVG directed the complexes across the BBB via receptor-mediated transcytosis and PMT facilitated trafficking of caveolae vesicle during transcytosis. The brain-targeted RNAi therapeutic effect of R-PEG-PMT/siBACE1 was demonstrated by suppression of BACE1 expression and beta-amyloid production. Consequently, R-PEG-PMT is a powerful gene carrier which can conquer the biological barriers itself, and possess the potential to be widely applicable in neurodegenerative diseases as a safe and efficient brain-targeted gene carrier.