Recombinant pre-miR-29b for Alzheimer´s disease therapeutics

Abstract

MicroRNAs are arising as the next generation of diagnostic and therapeutic tools for gene silencing. Studies demonstrated that the miR-29 expression is decreased in Alzheimer's disease (AD) patients displaying high levels of human β-secretase (hBACE1). Recent advances toward an effective therapy for AD intend to employ miR-29 to suppress hBACE1 expression and subsequent Amyloid-β (Aβ) peptide. However, delivery of mature miRNA has demonstrated modest efficacy in vitro; therefore, the preparation of highly pure and biologically active pre-miRNA arises as one of the most important challenges in the development of these therapeutic strategies. Recently, we described a new strategy based arginine-affinity chromatography to specifically purify the recombinant pre-miR-29b. Following this strategy, the purified pre-miR-29b was successfully encapsulated into polyplexes that were further delivered in cytoplasm. It was verified that Chitosan/pre-miR-29b and Polyethylenimine/pre-miR-29b systems efficiently delivered pre-miR-29b to N2a695 cells, thus reducing the hBACE1 protein expression (around 78% and 86%, respectively) and Aβ42 levels (approximately 44% and 47%, respectively). Furthermore, pre-miR-29b downregulates the hBACE1 mRNA expression in 80%. Overall, it was demonstrated that the recombinant pre-miR-29b using polyplexes allowed to decrease the hBACE1 and Aβ42 expression levels, improving the currently available methodologies of miRNA-based therapeutics.

Figure 1. MTS assays conducted in CS/pre-miR-29b (A) and in PEI/pre-miR-29b (B), which were cultured in the presence of increasing concentrations of pre-miR-29b for 48 and 72 h.

Untreated cells and cells transfected with an unrelated RNA control were used as negative controls for cytotoxicity. Ethanol treated cells were used as positive control to induce toxicity. Mean percentage values relative to the untreated cells and standard error of the mean in 3 independent experiments are shown. ANOVA, mean ± SD.

Figure 2. Recombinant pre-miR-29b effect on BACE1 levels in N2a695 cells at different concentrations of the pre-miR-29b for 72 h.

(A) Representative confocal microscopy images of N2a695 cells treated with CS/pre-miR-29b, PEI/pre-miR-29b and Lipo/pre-miR-29b stained against BACE1. Quantification of fluorescence intensity for BACE1 protein expression in cells transfected with: (B) CS/pre-miR-29b, (C) PEI/pre-miR-29b, and (D) Lipo/pre-miR-29b. All results are expressed relatively to those in untreated cells and error bars represent standard deviations derived from three or more independent experiments performed in triplicate. ANOVA, mean ± SD.

Figure 3. Western blot analysis of endogenous hBACE1 and β-actin levels in the cell lysate of N2a695 cells treated with different concentrations of pre-miR-29b, at 24, 48 and 72 h.

(A–C): representative images of Western blot from N2a695 cells after 72 h transfection with CS/pre-miR-29b, PEI/pre-miR-29b and Synthetic miR-29b and Scrambled RNA, respectively. (D–F): densitometric quantifications of hBACE1 expression from Western blot images of CS/pre-miR-29b, PEI/pre-miR-29b and Synthetic miR-29b and Scrambled RNA, respectively. Error bars represent standard deviations derived from three or more independent experiments performed in duplicate. ANOVA, mean ± SD. (For simplicity purposes, the blots were cropped).

Figure 4. Effect of recombinant pre-miR-29b on hBACE1 mRNA levels in N2a695 cells following 24, 48 and 72 h treatment with: (A) CS/pre-miR-29b and (B) PEI/pre-miR-29b (C) Synthetic miR-29b and Scrambled RNA.

Values in the graphs are mean from triplicates of RT-qPCR threshold cycles for hBACE1 mRNA normalized to those of mRNA for GAPDH from 3 independent experiments and demonstrate significant differences across treatment conditions. ANOVA, mean ± SD.

Figure 5. Effect of recombinant pre-miR-29b on modulation of Aβ levels in N2a695 cells.

(A) Representative confocal microscopy images of untreated N2a695 cells and cells transfected with CS/pre-miR-29b and PEI/pre-miR-29b stained against Aβ_total peptide (scale bars 20 μm). (B) Aβ₄₂ ELISA in cell lysates collected 72 h after transfection with CS/pre-miR-29b and PEI/pre-miR-29b. All results are expressed relative to those in untreated cells and presented as mean values from at least 3 independent experiments. ANOVA, mean ± SD.