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DNA and RNA Nanotechnology 2015; 2: 42–52 Mini review Open Access Martin Panigaj*, Jakob Reiser Aptamer guided delivery of nucleic acid-based nanoparticles DOI 10.1515/rnan-2015-0005 Evolution of Ligands by Exponential enrichment) [4,5]. Received July 15, 2015; accepted October 3, 2015 Nucleic acid-based aptamers are especially well suited Abstract: Targeted delivery of bioactive compounds is a for the delivery of nucleic acid-based therapeutics. Any key part of successful therapies. In this context, nucleic nucleic acid with therapeutic potential can be linked acid and protein-based aptamers have been shown to to an aptamer sequence [6], resulting in a bivalent bind therapeutically relevant targets including receptors. molecule endowed with a targeting aptamer moiety and In the last decade, nucleic acid-based therapeutics a functional RNA/DNA moiety like a small interfering coupled to aptamers have emerged as a viable strategy for RNA (siRNA), a micro RNA (miRNA), a miRNA antagonist cell specific delivery. Additionally, recent developments (antimiR), deoxyribozymes (DNAzymes), etc. In addition in nucleic acid nanotechnology offer an abundance of to the specific binding, many aptamers upon receptor possibilities to rationally design aptamer targeted RNA recognition elicit antagonistic or agonistic responses that, or DNA nanoparticles involving combinatorial use of in combination with conjugated functional nucleic acids various intrinsic functionalities. Although a host of issues have the potential of synergism. Since the first report including stability, safety and intracellular trafficking describing an aptamer-siRNA delivery approach in 2006 remain to be addressed, aptamers as simple functional many functional RNAs and DNAs conjugated to aptamer chimeras or as parts of multifunctional self-assembled sequences have been tested in vitro and in vivo [7-9]. RNA/DNA nanostructures hold great potential for clinical Recent progress in the structure predictions of nucleic applications. acids has led to the implementation of protocols for self-assembly of RNA/DNA nanoparticles [10-14]. Such Keywords: Targeted delivery, aptamers, functional structures may provide scaffolds for simultaneous delivery chimeras, nucleic acid-based therapeutics, RNA/DNA of various functionalities, including aptamers as well as nanoparticles fluorescent dyes, chemotherapeutics and/ or proteins. This mini review highlights recent efforts on the use of nucleic acid aptamers as targeting moieties for nucleic 1 Introduction acid-based nanoparticles. Nucleic acid-based aptamers have gained prominence 2 Aptamers as functional chimeras as a promising platform for targeted drug delivery due to their ability to serve either as a therapeutic agent or as 2.1 Targeting the RNA interference (RNAi) an evolvable targeting modality [1-3]. Nucleic acid-based aptamers consist of in vitro selected oligonucleotides pathway mediated by nucleic acid aptamers involving libraries of up to 1016 randomly synthesized Knock down of gene expression provides a promising tool sequences through a method termed SELEX (Systematic in the context of nucleic-acid based therapies [15]. Uptake of silencing RNAs by cells is greatly facilitated by coupling them to aptamers. While cellular uptake of an siRNA linked *Corresponding author: Martin Panigaj, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, to an aptamer leads to silencing of one specific gene, miRNA Moyzesova 11, 040 01 Košice, Slovakia, e-mail: martin.panigaj@ allows simultaneous repression of multiple genes. upjs.sk In a recent proof-of-concept study for in vivo delivery Jakob Reiser, Division of Cellular and Gene Therapies, FDA/CBER, of aptamer (Apt) -miRNA conjugates the integration of 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA three functionalities in one simple RNA construct was © 2015 Martin Panigaj, Jakob Reiser, published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Aptamer guided delivery of nucleic acid-based nanoparticles 43 demonstrated. In this construct the antagonistic aptamer the complementary stick sequence was base paired with provided receptor specificity while the miRNA moiety the precursor of miR-126 (pre-miR-126). All three constructs resulted in the silencing of gene expression. In one such were internalized by endothelial cells, but only treatment approach Esposito et al. linked the GL21.T aptamer that is with aptamer-pre-miR-126 resulted in the reduction of specific to Axl, a receptor tyrosine kinase (an oncogene the VCAM-1 target and an increase in angiogenesis. Also, overexpressed in several human cancers), to the tumor there were differences in the response of tumor cell lines suppressor let-7g miRNA, resulting in the GL21.T-let chimera to treatment with Ch3. It is unclear why no biological (Fig. 1A) [16]. Application of the GL21.T-let chimera to mice function was detected with the other two chimeras. bearing A549 (Axl+) tumors downregulated let-7g target Zhou et al. selected an aptamer targeting human genes leading to apoptosis, decreased cell proliferation, B-cell activating factor receptor (BAFF-R) to deliver siRNAs and a reduction in tumor size. Using different conjugation to non-Hodgkin’s lymphoma cells [22]. Activation of strategies and a combination of additional aptamers BAFF-R upregulates pathways leading to enhanced B-cell (e.g., a prostate specific membrane antigen aptamer) and proliferation, but neither aptamers R1 or R14 activated miRNA sequences (e.g., miR-16) the authors demonstrated BAFF-R. Down-regulation of STAT3 was previously the versatility of their approach. demonstrated to inhibit growth of lymphoma tumor cells. The aberrant regulation of certain miRNAs during To deliver siRNAs corresponding to STAT3 two pairs of Apt- malignant transformation makes them potential siRNA chimeras were designed. In the first pair, the sense therapeutic targets for inhibition using antimiRs [17]. or antisense strands were linked to an aptamer involving AntimiRs are synthetic oligonucleotides that base pair an eight-nucleotide linker (Fig. 1D). In the second pair with corresponding miRNAs. Such miRNAs complexed of chimeras, the siRNAs were connected to the aptamer with antimiRs do not bind target mRNAs resulting in an through stick sequences (Fig. 1D). All chimeras led to the increase in the expression of the target gene repressed silencing of STAT3 expression, albeit down-regulation by the miRNA [18]. Recently, Pofahl et al. reported on the mediated by Apt-linker-siRNAs was more profound than delivery of an antimiR, targeting the miR-21, through the using Apt-stick-siRNA chimeras. nucleolin-binding aptamer (AS1411) [19]. Blocking of a cellular receptor with an aptamer In a follow up study the de Franciscis’ group used the represents a plausible strategy to prevent binding and entry GL21.T aptamer to deliver RNA-based antimiRs inhibiting of viruses into host cell. Similarly, binding of aptamers to the cancer progression-associated miRNA miR-222 (Fig. viral glycoproteins decreases virus attachment to target 1B) [20]. Treatment of Axl or platelet-derived growth factor membrane proteins [23,24]. The first approach also offers receptor β expressing cells with the GL21.T-222 or Gin4.T-222 the potential to bolster antiviral effects through aptamer conjugates resulted in the decrease of target miR-222 levels delivery of siRNAs targeting viral and virus-required host and an increase of the miR-222 target proteins. Again, as in genes. previous work, the authors observed a synergistic effect of In two recent studies Zhou et al. investigated functional the GL21.T-222 conjugate on the reduction of cell migration in vivo delivery of anti-HIV-1 siRNAs via RNA aptamers and an increase in the sensitivity to temozolomide induced targeting HIV-1 glycoprotein gp120 or human CCR5 (C-C cell death. Interestingly, when the authors intended to chemokine receptor type 5). In the first study, an aptamer enhance the antagonizing potential using two antimiR- targeting HIV-1 glycoprotein gp120 and a siRNA were joined 222 moieties connected in tandem, no additive effect was via a “sticky bridge” [25]. Complementary sticky bridge detected. In contrast, employing an aptamer with two strands were connected to the 3′-end of the aptamer and different antimiRs (antimiR-222 and antimiR-10b) led to to one of the antisense or sense siRNA sequences using the reduction of the two respective miRNAs and increased a carbon linker. To prevent the creation of viral escape target protein levels. mutants, both virus-encoded and cellular transcripts In a related study three different strategies for were targeted including the viral transcripts encoding conjugating miRNAs (miR-126) to a transferrin receptor HIV-1 tat and rev, and transcripts encoding CD4 that is aptamer (TfRA) were tested (Fig. 1C) [21]. In the first study, required for HIV-1 entry and transportin 3 (TNPO3) that is a chimera (Ch1) consisting of the mature miR-126-5p required for viral integration. The intravenous injection covalently linked to TfRA and base-paired to miR-126-3p of an anti-gp120 Apt-siRNAs mixture to humanized mice was used. For the Ch2 chimera used in the second study, downregulated expression of all three genes targeted. the TfRA contained a “stick” sequence complementary to Most of the mice treated had undetectable viral loads up a stick part linked to the mature miR-126-5p annealed to to three weeks after the final treatment. Moreover,

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