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.
Highlights
MicroRNAs are arising as the generation of diagnostic and therapeutic tools for gene silencing
BACE1 and Aβ are central players in the pathways implicated in Alzheimer’s disease (AD), it has been established that reducing BACE1 expression has been suggested as a potential strategy for mitigating the pathological processes underlying AD25,45
The miR-29a/b-1/c cluster is significantly decreased in the brains of AD patients, and this decrease is correlated with the increased level of BACE1 protein[16,21]
Summary
MicroRNAs are arising as the generation of diagnostic and therapeutic tools for gene silencing. 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. The miRNAs used for therapeutic purposes have mostly been produced by chemical synthesis (phosphoamidite chemistry, normally used for the generation of short oligoribonucleotides), enzymatic synthesis (longer RNAs can be produced by in vitro transcription) or via a plasmid (for miRNA expression into eukaryotic cell lines)[26] These methods can be very efficient in producing miRNA, in general, additional purification protocols to remove the impurities (failure sequences, impurities of pDNA template, enzymes, nucleotides, salts or buffer) from the production process have to be employed. Considering the rapidly growing interest on these novel biopharmaceuticals, as a result of its potential therapeutic application, novel technologies to improve their preparation are currently being pursued
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