(T1130-04-21) Plasmid ApoE2 Gene Delivery Across the Blood Brain Barrier for the Treatment of Alzheimer's Disease

Purpose: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that disrupts cognitive function, eventually leading to memory loss. This disease is caused by misfolded proteins (amyloid beta) and phosphorylated TAU protein accumulation in the brain, blocking electrical signals to neuronal cells; hence, brain cells begin to die. The ApoE gene provides instruction for making apolipoprotein E, a major lipid transporter possessing three (3) isoforms: ApoE2- the protective, ApoE3- the neutral, and ApoE4- the primary genetic risk factor of AD. Studies have shown that elevated levels of ApoE2 reduce the risk of AD progression. Gene therapy has been identified to possess immense potential for the treatment of numerous neurological diseases. Liposomes have been used as a vehicle for drug and gene delivery because of their biocompatibility and the possibility of incorporating hydrophilic and hydrophobic molecules. My ongoing research is focused on delivering encapsulated chitosan- pApoE2 across the blood-brain barrier (BBB) using liposomes functionalized with brain-specific cell-penetrating peptides and targeting ligands for ApoE2 protein expression in brain cells, specifically neurons, for the treatment of AD.

Methods: Liposomes were prepared using a thin film hydration technique. Briefly, the DOTAP 45mol%, DOPE 45mol%, DSPE-PEG 8mol%, and Cholesterol 2% were dissolved in chloroform (2): methanol (1) followed by evaporation using a Rota-vapor to form a thin film. The thin film was rehydrated using HEPES buffer PH 7.4 and sonicated for 15 minutes. Cell-penetrating peptides and targeting ligands were coupled with DSPE-PEG (2000)-NHS by reacting the N-hydroxysuccinimide ester of the PEGylated lipid with the primary amines of the cell-penetrating peptides and targeting ligand (Transferrin) under alkaline conditions to form stable amide bonds. The characteristics of the liposomal formulations were determined by dynamic light scattering using a zeta sizer for their particle size, zeta potential, and poly-dispersity index. The encapsulation efficiency of pApoE2 complexed with chitosan was further determined. Cytotoxicity assay and blood compatibility studies were conducted in brain endothelial cells and erythrocytes to determine non-toxic liposomal concentration for in-vitro and in-vivo studies. Cell uptake studies at varying time points were performed using Lissamine-rhodamine-labeled liposomes in brain endothelial cells, primary astrocytes, and neurons. Quantitative and Qualitative analysis of transfection efficiency using pApoE2 and green fluorescent protein (GFP) respectively, were also determined in these cells. In addition, an In-vitro blood-brain barrier (BBB) model supplemented with hydrocortisone was developed using primary astrocytes and brain endothelial cells to assess the transport efficacy of the liposomes.

Results: The characterized liposomes had particle size between 156.4 ± 7.71 considering mean ± SD (n =4), poly-diversity index obtained had values between 0.19 ± 0.02 considering mean ± SD (n =4), zeta potential value was obtained to be 19.78 ± 0.2 considering mean ± SD (n =4) and Encapsulation efficiency 86.2 ± 1.2% considering mean ± SD (n =4). Cytotoxicity assay (MTT) and hemolysis assay showed a concentration of 100 nmoles of the liposomal formulation to be non-toxic and non-hemolytic, respectively. Quantitative analysis of cellular uptake of lissamine-rhodamine labeled liposome showed a statistically significant difference p < 0.05 between the single functionalized liposomes and dual functionalized liposomes with the highest uptake at 4 h. Dual functionalized liposomes have exerted significantly higher transfection in brain endothelial cells, primary astrocytes, and neurons compared to plain and single-functionalized liposomes. Trans-endothelial electrical resistance and Permeability coefficient of different in-vitro blood-brain barrier models constructed using bEnd3 as a monolayer, bEnd3/Astrocytes as co-culture showed the TEER of bEnd3/Astrocytes treated with hydrocortisone to be statistically significantly higher (p  < 0.05) than bEnd3/Astrocytes without HC treatment and bEnd3 cells only. The permeability coefficient showed a statistically significant difference (p  < 0.05) between the groups. Additionally, quantitative analysis of apoe2 levels conducted on 3-month-old C57/BL6 mice after treatment showed statistically significant (p  < 0.05) higher ApoE2 expression levels in mice treated with dual functionalized liposomes compared to plain or single functionalized liposomes treatment groups.

Conclusion:

Liposomes were successfully prepared, with cationic zeta potential and uniform size distribution. The developed dual functionalized liposome has shown higher in-vitro and in-vivo transfection efficiency, showing a potential for successfully transporting ApoE2 gene across the BBB to the brain, elevating ApoE2 levels in neurons to treat Alzheimer's disease. From the ongoing research, we expect our gene delivery dual functionalized liposomal formulation to increase ApoE2 levels in ApoE Knockout mice, validating our gene delivering cargo for the potential treatment of AD.

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants R01 AG051574, RF1 AG068034, and 3RFI AG0680

Characterization of Different Liposomal Formulations


ApoE2 Expression in bEND.3 cells, Primary astrocytes and Primary neurons 48hrs Post Treatment

In-Vitro BBB Model Studies