Gene therapy is a relatively new discipline of molecular medicine that will have a long-term impact on human health [1]. Its goal is to treat diseases through genome editing and gene expression modification. Gene therapy approaches are used to treat a variety of disorders, including neurological and infectious diseases, as well as monogenic and eye diseases [2–5]. Viral vectors, antisense oligonucleotides (ASO), RNA interference (RNAi), plasmids, deoxyribozymes (DZs), and CRISPR/Cas9 are some of the gene therapies under development [6-7]. However, studies have shown that such approaches have off-target effects, low affinity to folded RNA and are expensive (CRISPR and RNAi) (citation needed). Modern methods of addressing such issues include chemical modification of molecular tools and computer design. But such approaches are still being studied and do not guarantee high therapeutic effects [8]. DNAzyme, or deoxyribozyme (DZ), are single-stranded DNA molecular catalysts obtained through in-vitro screening technology [10] and can catalyze a variety of reactions, including RNA and DNA cleavage and ligation, as well as DNA phosphorylation [11]. For ion-dependent catalysis, they do not recruit enzymes in comparison with ASO and RNAi and Dzs are also simple in design, which makes them promising tools for RNA-cleaving gene therapy. In this work, to increase affinity for target RNA-cleaving DNAzymes, we developed bivalent DNAzymes (BDs), made of two Dz-ligands capable of RNA2 cleaving in several sites (Fig. 1). As in nature, the concept of multivalent DNAzyme consists of multiple ligands that bind to their target at multiple sites with high affinity and avidity resulting in effective inhibition or stimulation of biological responses [12]. Scientists have reported multivalency in biological systems as a powerful strategy for achieving high-affinity molecular recognition [13]. As original DNAzymes, BDs allow inhibition of expression of critical targeted genes via catalytic cleavage of mRNA, but their effectiveness is determined by avidity (which can be considered as the sum of the affinity of each active ligand containing the drug) instead of affinity, and we suppose that this parameter will increase drug-target interaction. We hope that such an approach can offer a promising future for improving gene therapy.

Our main goal was to improve the affinity and efficiency of DNAzymecleaving RNA by developing and optimizing DZ 10-23 base gene therapy molecules. We designed BDs that target folded mRNA and tested their efficiency in an in vitro physiological buffer to establish conditions necessary for effective gene knockdown. Next, we optimized DZ 10-23 by designing monovalent DZs with varied arm lengths (short arms with 1-2 nucleotides less and long arms with1-2 nucleotides more) to find the most efficient and stable construct. Finally, a larger RNA (STR-104) study was conducted to demonstrate the constructs' efficiency and stability with a larger or different target. BDs demonstrated higher cleavage efficiency (63.5%) in comparison with monovalent DZs and DZ associations of DZ1-DZ2 (9.2% and 23.4%, respectively). The most efficient multivalent design and the DZ1-DZ2 association were used to test the efficiency of both multivalent and monovalent designs at different concentrations (10nM, 25nM, 50nM, and 100nM). BDs at low concentration (25nM) was more efficient (33%) than DZ1-DZ2 (21%) at a higher concentration (100nM). Further investigation with different lengths (-/+1, -/+2 nucleotides) of binding arms and different melting temperatures of the linker tails influenced the efficiency of the designs. The result indicated that, as the length of the binding arms and the melting temperature of the designs increase, the efficiency also increases. This research has demonstrated that multivalent DNAzymes have great potential to increase DNAzyme-cleaving RNA affinity and efficiency as a therapeutic agent.

References

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