RNAi applications in biology and medicine

The field of oligonucleotide-based therapy experienced a revival with the discovery of RNA interference (RNAi) in 1998₁. RNAi is a conserved endogenous mechanism, which is triggered by double-stranded (ds) RNAs leading to target-specific inhibition of gene expression by promoting mRNA degradation or translational repression. There are two RNAi pathways that are guided either by small interfering RNAs (siRNAs), which are perfectly complementary to the mRNA or by microRNAs (miRNAs), which bind imperfectly to their target mRNA₂. SiRNAs can also induce direct transcriptional gene silencing (TGS) in the nucleus, although the mechanisms underlying this are well understood in mammalian systems_3,4.

In lower eukaryotes, such as nematodes, insects and plants, RNAi represents an anti-viral defense mechanism, in which viral dsRNA molecules are processed by the RNase III enzyme Dicer into small 19-23 nucleotides long double-stranded siRNAs, which contain 3’-end overhangs and 5’-end phosphate groups. These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where the passenger or sense strand of the siRNA duplex is degraded and the guide or antisense strand leads with full complementarity to the targeted mRNA sequence. The endonuclease Argonaute 2 (Ago2), a member of the RISC complex, cleaves the guide strand – mRNA duplex leading to subsequent degradation of the mRNA. Once activated, RISC can mediate multiple rounds of mRNA cleavage causing a potent knock-down effect. A breakthrough in the field of siRNA therapeutic agents was achieved by Elbashir and colleagues (2001) who demonstrated that synthetic, exogenously applied dsRNAs of 21 nucleotides in length can induce silencing in mammalian cells5. In addition to the siRNA design of 21mer duplex with 3’-overhangs at both sides, Dicer-substrate formats such as 27mers or short hairpin (sh) RNAs have been developed that elicit a more potent gene-silencing effect at lower concentrations as compared to conventional 21mer siRNAs6-8.

Small Interfering RNAs (siRNAs) as therapeutic agents

It is remarkable how quickly after its discovery RNAi has been established as the method of choice for targeted inhibition of gene expression in mammalian systems. Because RNAi uses a natural pathway for gene silencing, it generally results in a greater potency of knockdown than AONs or ribozymes. Preclinical results have confirmed the effectiveness of RNAi and have generated serious optimism about the potential for siRNA drugs. As with the other oligonucleotide-based approaches, the applications of siRNAs as a therapeutic agents face most of the above mentioned challenges. Some of these challenges however, have already been addressed in the course of AON and ribozyme development.

The general rules for the rational choice of target sequences for AONs described above also apply siRNA designs. In recent years, many algorithms and tools have been developed to predict and design specific siRNA sequences (reviewed in9,10). Two siRNA specific limitations will be discussed in more detail: off-target effects and immunogenic toxicity.

Off-target effects can be most likely attributed to the function of siRNAs as microRNAs, if they contain seed sequences that match 3’-untranslated regions in non-targeted mRNAs. Careful selection of the sequence can minimise off-target effects and chemical modifications at the second position of the seed sequence in the guide strand can even further reduce the unwanted transcript silencing11,12. Another concern with siRNA-mediated therapy is the possible interactions of siRNAs with Toll-Like receptors or internal dsRNA receptors such as retinoic acid inducible protein I (RIG I) and dsRNA-dependent protein kinase (PKR)12-15. Selective 2’-O-Me or LNA modifications of sense or antisense strands can abolish the induction of the interferon response by evading TLR detection15.

Cell-specific delivery in vivo is one of the crucial steps in development of oligonucleotide-based therapeutic agents and remains a key challenge of siRNA therapeutic application. Systemic delivery of siRNAs to specific cells via cell-surface receptors would provide the maximal therapeutic benefit by decreasing the amount of drug and avoiding non-specific silencing or toxicity in healthy cells. A liposome-based complex that included an anti-transferrin receptor single-chain antibody fragment as the targeting moiety specifically and efficiently delivered siRNAs to primary and metastatic tumors when systemically administered16. Moreover, the surface of nanoparticles can be coated with cell-type specific ligands that might expand their properties towards specific delivery17. In a Ewing sarcoma tumor mouse model, the self-assembled nanoparticles composed of cyclodextrin-containing polycations (CDPs), transferrin ligands, PEG and siRNAs were able to bind to the transferrin receptor and carry siRNAs into the tumor cells to inhibit tumor formation18. Another cell-specific delivery strategy includes the usage of cell-type specific ligands such as antibodies or aptamers. Heavy-chain antibody fragments (Fabs) specific for the HIV-1 envelope glycoprotein gp120 were fused to protamine, a nucleic-acid binding protein, to bind the siRNAs19. Injection of this complex into mice only targeted HIV envelope-expressing B16 melanoma cells and inhibited tumor growth. Similarly, antibody-protamine fusion proteins targeting the human integrin lymphocyte function-associated antigen-1 efficiently delivered siRNAs in primary lymphocytes, monocytes, and dendritic cells20.

Aptamers have also been shown to be useful for siRNA delivery. An aptamer selected against the prostate-specific membrane antigen (PSMA), a cell surface receptor that is over expressed in prostate cancer cells and vascular endothelium. McNamara and colleagues coupled the siRNA covalently to the aptamer via a nucleic acid linker21. The aptamer-siRNA chimeras recognised specifically LNCaP, a prostate cancer cell line expressing PSMA, and induced cell death; in contrast, no effect was determined in PSMA-negative cell lines. These chimeras also facilitated inhibition of tumor growth and tumor regression in a xenograft mouse model of prostate cancer after intratumoral injection. A different strategy used an anti-HIV envelope (gp120) aptamer fused to an anti-HIV 27mer siRNA targeting HIV tat / rev common exon. Only cells infected with HIV were targeted with this chimeric construct, and several fold reduction in viral p24 antigen production was observed over a one week period of treatment. In this setting both the aptamer and siRNAs have inhibitory functions making them a dual inhibitory system22.

Preclinical studies have demonstrated the safe use and the potential for therapeutic benefit of RNAi-mediated gene silencing23-25. SiRNAs are in early stage clinical trials for the treatment of viral infections, cancer and ocular diseases. Phase I studies are planned for numerous other diseases including neurodegenerative diseases, asthma / allergies, and inflammatory diseases. The most advanced stage testing for a siRNA-based drug is for the treatment of viral infection and was developed by Alnylam Pharmaceuticals (Cambridge, MA, USA). The siRNA ALN-RSV01 was designed against the respiratory syncytial virus (RSV), which causes severe respiratory illness, primarily in infants. The unmodified siRNAs, administered by inhalation, showed significant viral reduction in experimentally infected adult volunteers compared to the placebo group in a Phase II GEMINI study and is now being tested in RSV patients with naturally acquired infection.

MicroRNAS (miRNAs) as therapeutic targets and agents

Since the first microRNA (miRNA) lin-4 was discovered in Caenorhabditis elegans in 199326, several hundred miRNAs have been identified in animals, plants and viruses (see miRNA database: miRBase27-29). MiRNAs are endogenous, often highly conserved RNA molecules of ~22 nucleotides in length that are involved in the regulation of major events in the cell including differentiation, proliferation and apoptosis30. Estimates suggest that about 30% of the human genome may be under the control of miRNAs which regulate expression of multiple gene targets31. When incorporated into a silencing machinery similar to the siRNA complex, mature miRNAs bind within the 3’-untranslated region (3’-UTR) of the target mRNA forming mismatched duplexes and repress translation through different mechanisms32.

In recent years, it has been discovered that altered expression of specific miRNA genes contribute to an increasing number of human diseases such as cancer, neurological diseases, metabolic disorders or cardiac diseases (reviewed in 33-35). In cancer, miRNAs that can function as tumor suppressors or as oncogenes (referred to as oncomirs) have been identified by high throughput and cell-based assays36-38. The reduction or deletion of a miRNA that normally regulates tumor suppressors leads to tumor formation and proliferation. On the other hand, the amplification or over expression of miRNAs that have oncogenic function can also promote tumor formation. The role of miRNAs in cancer is further supported by the fact that about half of the annotated human miRNAs are encoded within fragile regions of chromosomes, which are associated with various human cancers39. Analysis of SNP databases for humans and mice have revealed that mutations in miRNA sequences or their target site might be important in diseases as well suggesting interesting new therapeutic targets.

Expression profiling of miRNAs has revealed that miRNAs are promising diagnostic and prognostic biomarkers as well as for classification, staging and progression of disease40. The widespread role in diseases makes miRNAs potentially interesting targets for therapeutic intervention.

Inhibition of miRNA function was successfully accomplished by an antisense approach via the use of anti-miRNA AONs in cell culture and in vivo41,42. Modified antisense oligonucleotides were named e.g. antagomirs, miRNA ASOs or antimiRs when applied against miRNA targets. 2’-O-Me, 2’-O-MOE, morpholino and LNA modifications were demonstrated to be most efficient in terms of binding affinity and silencing efficacy.

Progress in the functional use of miRNAs as biomarkers in diagnosis and prognosis of diseases as well as exploiting potential therapeutic strategies against miRNAs is remarkable keeping in mind the short period of time since miRNA discovery. Nevertheless, this field is still in its infancy and major hurdles remain the same as for siRNA and AON technology such as the systemic delivery to specific tissues and cell-types, stability and toxicity. Targeting miRNAs as a therapeutic strategy is also complicated by the fact that on the one hand one miRNA can regulate several different mRNAs and on the other hand the 3’-UTRs of mRNAs contain multiple binding sites and can be modulated by diverse miRNAs. The biology of miRNAs and the complex regulation network is far from understood and future investigations remain to provide new insights in this promising field.

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