miRNA-based therapies: strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents

Abstract

The discovery of miRNAs as important regulatory agents for gene expression has expanded the therapeutic opportunities for oligonucleotides. In contrast to siRNA, miRNA-targeted therapy is able to influence not only a single gene, but entire cellular pathways or processes. It is possible to supplement downregulated or non-functional miRNAs by synthetic oligonucleotides, as well as alleviating effects caused by overexpression of malignant miRNAs through artificial antagonists, either oligonucleotides or small molecules. Chemical oligonucleotide modifications together with an efficient delivery system seem to be mandatory for successful therapeutic application. While miRNA-based therapy benefits from the decades of research spent on other therapeutic oligonucleotides, there are some specific challenges associated with miRNA therapy, mainly caused by the short target sequence. The current status and recent progress of miRNA-targeted therapeutics is described and future challenges and potential applications in treatment of cancer and viral infections are discussed.

Figure 1. miRNA biogenesis, mechanisms of action and the use of therapeutic agents for modulation of miRNA functions

MicroRNA is transcribed initially as the pri-miRNA, which is processed by the nuclear nuclease Drosha to give the characteristic pre-miRNA stem loop structure. The pre-miRNA is transported from the nucleus to the cytoplama by exportin-5 and Ran-GTP. Further processing by Dicer with TRBP is essential for generation and selection of the guide strand, which is incorporated into the mature RISC, assembled by recruitment of the Ago2 nuclease. Depending on the binding sites (3′-UTR, ORF or 5′-UTR), and perfect or incomplete hybridization to the mRNA, the result is degradation of the target mRNA, storage in P-bodies, or translational repression. Therapeutic intervention is mainly focused on the use of oligonucleotides as either substitutes (mimics) of depleted endogenous miRNA, or as complementary strands with the aim of intercepting the miRNA guide strand (antagomirs, miRNA sponges). Other approaches with small molecules can inhibit the miRNA processing by inhibiting the binding of Drosha and Dicer, or unspecifically promote miRNA biogenesis by promotion of the interaction with TRBP.

Figure 2. Chemical oligonucleotide modification patterns for miRNA-based therapeutic oligonucleotides

For effective treatment with oligonucleotides, chemical modifications are essential to prevent rapid degradation and promote cellular uptake (A). For miRNA-based applications, phosphorothioates and 2′-O-methyl modified sequences are most commonly employed. While miRNA mimics only exhibit a small degree of modification, antagomirs can be fully modified. LNA nucleosides stabilize oligonucleotides and increase binding affinity, and are thus particularly useful for the inhibition of miRNA through seed sequence binding, and enable the simultaneous depletion of several miRNA with the same seed region motive. The derivatization usually does not cover the entire sequence, but is restricted to partial regions with the seed region often omitted. Besides structural modification, sequence length and format can also be tailored to show higher stability or selectivity (B). Possibilities include single or double strand formats, nicks, and connections by hairpins.

Figure 3. Small molecules for regulating miRNA expression and functions

The fluoroquinolone enoxacin has been identified as an enhancer of siRNA and miRNA effects. It promotes the interaction of nucleic acids with the HIV-1 TAR RNA binding protein (TRBP), an important co-factor of Dicer and integral component of the RNAi silencing complex (RISC). Several specific miRNA inhibitors have been described, and examples of structures (1 [129], 2, 3, and 4 [130]) are depicted.