- Cell-Specific Aptamer-Functionalized RNAi: A New Prospect for Targeted siRNA Delivery (2013)

In recent years, nucleic acid based therapeutics has progressed rapidly. In addition to their utility as a stand-alone strategy, these therapeutic nucleic acids that include small interfering (si)RNAs, microRNAs, aptamers, antisense DNAs, DNAzymes, and ribozymes can potentially be used as adjuvants for multipronged drug treatment. In particular, the highly specific mechanism of RNA interference (RNAi) which inhibits the expression of disease genes is extremely attractive for the treatment of a wide variety of human maladies. However, one of the most formidable impediments to clinical translation of RNAi is effective delivery of the small interfering RNAs (siRNAs) to specific cells or tissues at therapeutic doses. Currently, extensive efforts to develop various internalizing molecules have been made for target-specific siRNA delivery. Among them, cellspecific aptamers represent an exciting approach for targeted siRNA delivery. In this chapter, we review the current advances of cell-specific aptamers in cell recognition and targeted delivery, with a particular focus on the development of the aptamer-functionalized siRNA or nanocarrier for targeted gene silencing. 5.1 IntroductionAlthough the concept of nucleic acid based therapeutics is not a new one, it has captured the wide attention of scientists for a long time [1, 2]. Over the past few decades, a wide variety of nucleic acids such as siRNAs [1, 2], microRNAs [3, 4], aptamers [5, 6], antisense DNA and RNA [7, 8], mRNAs [9, 10], plasmid DNAs [11], DNAzymes, and ribozymes [12] have been proposed as possible drugs. Some of these nucleic acid candidates have been put on the back burner or are still in development because of current technical challenges; however, some nucleic acids have entered clinic trails, and a few have even successfully made it to market; e.g., Vitravene [13], the antisense oligonucleotide inhibiting cytomegalovirus (CMV)-induced retinitis, and Pegaptanib (or Macugen) [14], the first therapeutic aptamer approved for the treatment of neovascular agerelated macular degeneration (AMD). Since the discovery of RNAi in 1998 [15], the use of RNAi-based therapeutics (for example, siRNAs [16]) to silence target genes associated with human diseases has progressed tremendously due to their high specificity and potency. The research on RNAi has fuelled excitement for their potential clinical application [1]. So far, there are over 20 RNAi-based drugs in early-or mid-stage clinical trials, several of which are indicating strong promise for further drug development [3]. For example, in 2004 Acuity Pharmaceuticals (now Opko Health) announced the first siRNA-related clinical trial, in which Bevasiranib, an unmodified siRNA targeting vascular endothelial growth factor (VEGF), was given in patients with wet AMD [17]. Additionally, the first in-human Phase I clinical trial using a targeted nanoparticle system to deliver siRNA to patients was conducted and showed the direct evidence for siRNA-mediated gene silencing via the RNAi pathway [18]. However, like any other drugs in development, some impediments have caused failures in the clinical translation of RNAi-based therapeutics. In March 2009, Opko decided to terminate the Phase III clinical trial of bevasiranib, since it was unlikely to achieve its primary endpoint of reducing visionloss. In this case, as an unmodified siRNA, bevasiranib was given by intravitreal injection without a particular formulation that could enhance its silencing performance. Since negatively charged nucleic acids do not readily traverse cellular membranes and are vulnerable to degradation without some protective covering or/and appropriate chemical modifications, such direct administration ultimately resulted in poor pharmacokinetics and indirect gene silencing. In this regard, some optimized strategies should be considered, which could include appropriate chemical backbone modifications, an effective delivery vehicle, a new dosing schedule and a combinatorial formulation with other therapeutic nucleic acids, and so on. While this frustration tempered the early excitement in RNAi technology, it triggered extensive efforts to surmount these key hurdles to the widespread use of RNAi as a therapy. Among these hurdles, one of the most important is delivery of RNAi agent (such as siRNAs) to specific cells or tissues at therapeutic doses [19]. With the intent of developing a targeted intracellular delivery system, numerous strategies have been reported, in which various internalizing molecules with high specificity and affinity to a cellular receptor were assembled or complexed with the siRNA. Based on specific interaction between the targeting ligand and its cellular receptor, the recognition and internalization of the therapeutic siRNAs by the target tissue should be capable of dramatic improvement. Most recently, another type of therapeutic nucleic acids termed aptamers show promise as potential candidates for targeted siRNA delivery. These cell-specific aptamers can actively target a distinct cell population or tissue in a cell-type-specific manner [20, 21]. By functionalizing the cell-specific aptamers to therapeutic agents or delivery vehicles, the cellular uptake is enhanced and the local concentration of the drug in the targeted cells or tissues is increased, thereby improving the therapeutic efficacy. Currently, a number of aptamer-functionalized therapeutics has been successfully developed for targeted delivery [22, 23]. In particular, with the technological maturation and increasing knowledge of RNAi as well as aptamers and their mechanism of action, it seems natural to partner the two therapeutic nucleic acids for expanding the options for targeted RNAi (Fig. 5.1). In this chapter, we review recent progress of cell-specific aptamers that mediate targeted therapy of nucleic acid drugs placing particular attention to development of aptamer-functionalized siRNAs or nanocarrier for targeted gene silencing.