3.1. Cytotoxic Cancer Therapy
As mentioned above, PSMA is one of the first cancer cell-specific marker proteins used for siRNA delivery through aptamers. Other cancer cell-specific aptamer targets exploited for AsiC experiments or in combination with other drugs include receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR; detected on glioma, lung, and breast cancer) [51
], human epidermal growth factor receptor 2 (Erbb2 or HER2; particularly used for targeting breast cancer cells) [20
], and tyrosine-protein kinase-like 7 (PTK7; overexpressed in many different cancers) [53
]. For highly metastatic cancers, an aptamer recognizing alpha V and beta 3 (αVβ3) integrin was selected and joined to a siRNA against eukaryotic elongation factor 2, inhibiting proliferation and inducing apoptosis in target cells [54
]. Furthermore, also used as targets were an atypically glycosylated form of mucin (MUC-1) overexpressed on various human adenocarcinomas [55
], extracellular matrix protein Tenascin-C [56
], and nucleolin [57
], a protein almost universally present on cancer cell-surfaces and shuttling into the nucleus where it is found solely in benign cells. However, since the present paper focuses on more recent achievements and trends, we would like to point out other reviews covering earlier approaches for these aptamers [13
The nucleolin aptamer AS1411 has been used as a AsiC with an siRNA that exhibited two unprecedented features [60
]. First, this AsiC displayed an example of a DNA-aptamer conjugated to an siRNA, linked by non-cleavable maleimide chemistry. Secondly, since AS1411 is composed of two separate DNA-strands of the same G-rich sequence (each 25 nt) that together fold into a G-quadruplex structure [61
], the AsiC was able to carry two siRNA units, one on each of the two DNA strands (Figure 3
). This AsiC was specifically designed to suppress tumor invasion and angiogenesis by choosing siRNAs targeting the mRNAs of two genes involved in metastasis-associated epithelial-mesenchymal transition (EMT), snail family zinc finger 2 (SLUG) and neuropilin 1 (NRP1), which promote malignant transformation and activate key signaling pathways during different stages of metastasis.
Nucleolin is a multifunctional hnRNP present in the nucleus, but also in the cytoplasm and on the cell-surface, shuttling between these compartments. Additionally, nucleolin is upregulated in highly proliferating cells, including breast cancer, lymphocytic leukemia, and prostate carcinoma [67
]. To evaluate the efficiency of the AsiC, an in vivo lung cancer model was established by inoculation of non-obese-diabetic severe-combined-immunodeficiency (NOD SCID) mice with CL1-5 cells. These animals were then treated with each of the two AsiCs alone, or a combination of the two (a combination of 0.5 dose equivalents of the single AsiC treatment) intratumorally three times per week for 42 days. The tumor growth rate decreased by three-fold when single AsiCs or a combination of both AsiCs were administered. Additionally, the combination of the two AsiCs exhibited a synergistic effect, suppressing tumor invasion.
Recently, Wang et al. used AS1411 as a targeting decoration on cell-derived micelle-like vesicles (termed as extracellular vesicles) to deliver siRNAs and microRNAs (mRNAs) into MDA-MB-231 breast cancer cells [69
]. AS1411 potentially inhibits tumor growth [57
]. Thus, this aptamer has been used in three Phase II clinical trials and is one of the most promising candidates for approval by the U.S. Food and Drug Administration (FDA).
In another study, a co-delivery of two siRNAs in a bivalent PSMA aptamer (A10-3.2) AsiC was reported (see Figure 3
]. To tackle metastatic castration resistant prostate cancer (mCRPC), the authors chose EGFR and survivin as the targets for siRNA silencing. EGFR overexpression is associated with mCRPC and bone metastasis frequently occurring in advanced stages [72
]. Survivin is known as a member of the inhibitor of apoptosis protein (IAP) family and, thus, plays a pivotal role in the progression of PCa and other solid tumors [74
]. The AsiC was tested in a C4-2 PCa xenograft model, where it significantly suppressed tumor growth and angiogenesis. As confirmed by rapid amplification of cDNA ends (5′RACE) PCR, the inhibition of angiogenesis was mediated by an EGFR-dependent mechanism.
A significant portion of cancer-related genes is regulated by miRNAs, and many reports demonstrated that miRNA expression is deregulated in human cancers [75
]. Thus, miRNA delivery has garnered attention within the past years, and the restoration of miRNA levels by specific delivery tools represents one strategy for cancer therapy. In 2014, Esposito et al. reported a multifunctional aptamer–miRNA construct for myeloid leukemia therapy [63
]. The AsiC construct (GL21.T–let) was composed of the RNA-aptamer GL21.T and the tumor-suppressing miRNA let-7g. GL21.T binds to oncogenic RTK Axl (Kd
= 12 nm) and led to abrogation of Axl-dependent signal transduction, such as extracellular-signal regulated kinase (ERK) and protein kinase B (PKB, Akt) phosphorylation [77
]. For Axl-positive cells in cell culture treatment with GL21.T–let, cancer cell survival and migration was strongly reduced. Also, inhibition of tumor growth was observed in a xenograft mouse model of human lung cancer. Recently, the GL21.T aptamer was used to deliver miR-212 into human non-small cell lung cancer (NSCLC) cells [64
]. This AsiC can inhibit the anti-apoptotic phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) implicated in a common treatment resistance against TNF-related apoptosis-inducing ligand (TRAIL). Using this approach, NSCLC cells were sensitized to TRAIL therapy and exhibited increased caspase activation. Recently, RNA-aptamer GL21.T was converted into a more stable DNA-aptamer with 2′-F-Py modifications and 5′-phosphorothioates at certain positions [78
]. This aptamer was examined for its ability to block Axl-phosphorylation in ovarian cancer using intraperitoneal animal models. Impairment of tumor growth and reduction of metastatic nodules was observed along with inhibition of migration and invasion of the cancer cells. Carla Esposito and Vittorio de Franciscis also identified an aptamer to the insulin receptor (IR), named GL56, using cell-internalization SELEX [79
]. Based on its ability to undergo efficient and rapid cell-uptake, this aptamer is a promising tool for the delivery of small RNAs into IR-dependent cancer cells.
3.2. Cancer Stem Cell Therapy
Cancer cells can be subdivided into different types based on their occurrence, morphology, behavior, and potential to evade natural defense or differentiate into another cell type. Cancer cells which exhibit the two lattermost abilities, are referred to as cancer stem cells (CSC), as they share capabilities like the ones ascribed to stem cells. They reproduce themselves and sustain the tissue or tumor. Targeting CSCs has therefore become a central interest in the development of new therapies. Moreover, new drugs are urgently needed as these cells show intrinsic resistance to conventional treatments [80
]. CSC specific biomarkers are required for targeted delivery using aptamers. Researchers in the field have been focusing on improving the cell-SELEX protocol to select aptamers predominantly for CSCs [81
]. Cell-surface markers such as the epithelial cell adhesion molecule (EpCAM), CD44, and CD133 have been tested for aptamer-mediated cell therapy [82
]. Other CSC-specific targets have been suggested, including CD34, a regulator of cell adhesion [83
]. Furthermore, CD38 is normally expressed on hematopoietic cells. Greater expression levels are found in bone marrow precursor cells protecting germline cells against apoptosis. While it is lost in mature B-cells, it is found in cells of chronic lymphocytic leukemia (CLL), where its expression signifies poor prognosis [84
]. Besides, CD38 was also shown to be expressed in skeletal and heart muscle, proximal convoluted tubules of kidney, and normal adult prostate [83
]. Whether cells of these tissue types would also be influenced by utilization of an aptamer targeting CD38 remains unclear. However, recent findings have shown, that deficiency or inhibition of CD38, which can degrade different nicotinamide dinucleotides, led to positive prognostic effects in cardiac tissues, such as protection against ischemia and reperfusion injury [86
] and endothelial dysfunction [87
]. Additionally, CD44 and CD24, normally expressed on B-cells, have been discussed as CSC markers for many carcinomas, with an emphasis on breast cancer [88
]. CD90 has been suggested as a marker of CSCs from the brain [90
], liver [91
], gastric [92
], and lung tumors [93
Shigdar et al. presented RNA-aptamers selected for CD133, out of which one (CD133-A15) was truncated to a 15mer [94
]. Endocytosis of the CD133-aptamers by HT29 colon cancer cells was confirmed by confocal microscopy, while for five different negative cell lines, no binding or internalization was observed, confirming the specificity of CD133-A15. Shortly after, Jiang et al. used this aptamer to deliver salinomycin-loaded nanoparticles into CD133+ hepatocellular carcinoma (HCC) cells, inducing apoptosis [95
]. Meanwhile, Chen et al. targeted dysfunctional epithelial progenitor cells (EPCs) instead of CSCs using CD133-A15 in a AsiC with a siRNA targeting adenosine kinase (ADK), showing the potential of this very small aptamer for AsiC delivery [96
EpCAM is overexpressed on tumor initiating CSCs, and thus aptamers binding to this molecule have been exploited for the delivery of siRNA AsiCs. In 2015, three groups reported on different approaches using EpCAM aptamers for delivery of siRNAs. Under the leadership of Wei Dun and Sarah Shigdar, a chemo-sensitizing approach using a Dicer substrate siRNA against survivin appended to an 18mer anti-EpCAM RNA-aptamer [97
] in a breast cancer xenograft mouse model was presented [98
]. Doxorubicin resistant MCF-7/ADR cells, in which survivin expression is 21-fold that of progenitor cells, were generated and subsequently used in the xenograft model. Administration of the AsiC to mice reversed tumor cell chemo-resistance, and a low dose of doxorubicin inhibited cell stemness as documented by the expression of different markers, eliminated cancer stem cells via apoptosis, and suppressed tumor growth, leading to prolonged survival of mice bearing chemo-resistant tumors.
The second paper published in 2015 described the use of an EpCAM aptamer linked to a PlK-1 siRNA for the treatment of breast cancer [99
]. In this study, tumor initiating triple-negative breast cancer (TNBC) cells (cells negative for estrogen receptors, progesterone receptors, and HER2) were targeted. The growth of Basal-A TNBCs (resembling basal-A TNBC primary tumors) was reduced upon subcutaneous treatment with the AsiC in a mouse model as well as in human breast cancer tissues in vitro. Growth of normal epithelial cells, basal-B TNBC cell lines (which resemble mesenchymal TNBC primary tumors), or normal human breast tissues was not inhibited by the AsiC. The knockdown was proportional to EpCAM expression. Moreover, the AsiC-induced knockdown of the mitosis regulator PlK1, suppressed tumor-initiating cells (TICs) of epithelial breast cancer cells as shown in in vitro functional assays (colony and mammosphere formation).
Finally, in 2015, Krishnakumar and colleagues described a two-pronged approached to EpCAM inhibition in cancer cells. This group constructed an AsiC composed of an EpCAM binding aptamer and a siRNA targeting EpCAM mRNA [100
], thereby creating a feedback loop in which the inhibition in the cancer cells was directly proportional to EpCAM expression. The anti-tumor activity was tested by using MCF7 cells in a mouse xenograft model. The AsiC induced EpCAM downregulation and inhibited cell proliferation. Different markers of pluripotency were downregulated upon this treatment: The transcription factors sex determining region Y box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and NANOG (not being an abbreviation but named after an old Celtic myth), as well as CD133. The same laboratory constructed polyethyleneimine (PEI)-based nanocomplexes (198 nm in diameter) bearing the same siRNA and aptamer against EpCAM [101
]. Specific binding and uptake were demonstrated in cultured MCF7 cells and a retinoblastoma cell line (WERI-Rb1), but the in vivo efficacy of this construct still needs to be examined.
Recently, de Franciscis and colleagues reported an aptamer-mediated combinatorial miRNA delivery approach, targeting glioblastoma stem-like cells (GSCs) [65
]. The authors constructed two RNA-aptamers with affinity to RTKs, one binding to Axl (GL21.T), which the authors had previously used to delivery microRNAs to cells, and another aptamer (Gint4.T) binding to Platelet-derived growth factor receptor beta (PDGFRβ). Both aptamers inherently act as inhibitors of their target RTKs, therefore augmenting the anti-tumorigenic effect. The combination of the tumor-suppressor, miR-137, and the antagonist of oncomiR, miR-10b, appended to the aptamers was tested and showed enhanced impact on tumor cell viability and migration. Furthermore, a reasonable amount of the aptamers and their respective AsiCs could cross the BBB. The authors speculated that this was a consequence of the transcytosis of the target RTKs. Interestingly, the Gint4.T aptamer has recently been conjugated to a mimetic peptide for targeted delivery to cardiac cells demonstrating the broad applicability of these aptamer ligands (personal communication).
Immunotherapy is a rapidly expanding field in targeted cancer treatment. Instead of targeting the cancer cell itself, the agents used target immune cells. Historically, the idea of boosting the immune system to fight tumor growth goes back more than hundred years ago when, in the late 19th century, a New York surgeon, William Coley, attempted to stimulate immune responses by injecting bacteria into tumor sites of his patients.
Checkpoint blockade is one strategy of cancer immunotherapy. In 2003, the Gilboa group published the first aptamer acting as a checkpoint blocker by binding to cytotoxic T-lymphocyte associated protein 4 (CTLA-4), also known as CD152 [102
]. CTLA-4 expressed on the surface of T-cells downregulates responses to stimuli such as the ones mediated by CD28 activation to prevent immunological overreaction [103
]. The antagonistic RNA aptamer to CTLA-4 was arranged as a tetrameric construct which, due to multivalence-related effects, had enhanced potency in inhibiting CTLA-4 in vitro and tumor immunity in mice. Recently, a siRNA-AsiC using a CTLA-4 aptamer has been reported for the delivery of anti-STAT3 siRNAs to malignant T lymphocytes. STAT3 supports tumor survival, proliferation, and invasion and can lead to immunosuppression. An earlier study by Kortylewski et al. using CpG ONTs bound to Toll-like receptor-9 (TLR9) to deliver siRNAs indicated that STAT3 inhibition leads to antitumor immune response [104
]. In addition, Herrmann et al., using the CTLA-4 aptamer-STAT3-siRNA AsiC in CD4+ T regulatory cells, showed that knocking down STAT3 along with the blockade of CTLA-4 caused an increase of CD8+ T effector cell response (and therefore increasing the impact of T lymphocytes against tumor cells) in an in vivo model [105
Most cancer immunotherapies work by modulation of lymphocyte co-receptors, which can be inhibitory or stimulatory. 4-1BB (CD137/TNFSF9) is a stimulatory receptor found on various types of immune cells [106
]. One of 4-1BB’s functions is the activation of CD8+ T-cells. An aptamer binding to 4-1BB acts as an artificial ligand inducing oligomerization of the receptor and, hence, initiates stimulatory signals, leading to increased T-cell survival [107
The same 4-1BB aptamer was used by Berezhnoy et al. to deliver a siRNA against mTOR complex 1 (mTORC1) into CD8+ T-cells [108
]. The dual function of this AsiC ensured that activation of the T-cells and suppression of mTORC1 proceeded in parallel. Consequently, differentiation of the targeted T-cells into memory T-cells was more efficient and immunosuppressive side effects from mTORC1 inhibition in other cell types were avoided, as they occur by non-selective treatment with rapamycin. The enhanced antitumor immunity was shown in a mouse model, in which animals were immunized with irradiated B16 melanoma cells, treated with the AsiC (i.v.) or rapamycin (i.p.) the next day, and finally were subjected to an additional xenotransplant of the melanoma tumor cells 50 days later.
Recently, Rajagopalan et al. reported a monovalent 4-1BB aptamer, which is not activated by itself, for siRNA delivery into already activated CD8+ T-cells [109
] to modulate the differentiation of these cells towards memory precursor effector cells (MPECs) rather than towards short-lived effector cells (SLECs), which would undergo apoptotic death after a short life span. This strengthening of the immunological memory was induced by using an anti-CD25 siRNA. Downregulation of CD25, which is expressed on the target cells, prevented interleukin-2 (IL-2) binding to CD25 and the subsequent differentiation into SLECs.
3.4. Anti-Viral Therapy
Besides cancer treatment, AsiCs have been designed and tested for the treatment of viral infections, mainly against infections by the human immunodeficiency virus (HIV). RNA-aptamers binding to the virion protein HIV-1 gp120 [47
] as well as cluster of differentiation 4 (CD4) on the surface of HIV-infected T-cells [112
] have been selected to hinder the interaction of these two proteins during the entry of HIV-1 into its target cells. Moreover, these aptamers have successfully been combined with siRNAs targeting viral genes. The region in the 9.2 kB RNA genome of HIV preferentially targeted with siRNAs is an exon that encodes two essential regulatory elements of HIV, the HIV trans-activator (tat) and the regulator of expression of virion proteins (rev).
A gp120 aptamer AsiC designed by Zhou et al. combined blocking of viral adhesion through gp120 to CD4, and knockdown of the viral regulatory proteins [47
]. It specifically suppressed the replication of HIV-1 in a humanized mouse model. The AsiC construct used in this study can be described as the original 2′-F-RNA-aptamer 3′-terminally extended by the sense strand of the siRNA. A 2–4 nt linker between these two sequences was applied for flexibility and enhanced Dicer processing. The siRNAs’ antisense strand was simply annealed to this construct. Zhou et al. also investigated the efficiency of AsiCs with different length of the siRNA. When a 27 nt Dicer substrate siRNA was implemented instead of a classical 21 nt siRNA, the silencing effect was improved by 20%. The authors speculated that Dicer-generated 21–23 nt siRNAs might be incorporated more efficiently into RISC. In a following study, the efficacy of the chimer was further evaluated [111
]. In HIV-infected humanized mice, the intravenously administered gp120-aptamer-siRNA AsiC led to a reduction in viral loads by several orders of magnitude. The aptamer itself was already capable of inhibiting HIV-1 infection, but, when used as an aptamer-siRNA AsiC, the duration of the inhibition was enhanced, due to target mRNA degradation that was validated by 5′RACE PCR. In these experiments, AsiCs of non-binding aptamer variants and siRNAs lacking the aptamer served as negative controls.
The other prominent target for aptamer-mediated HIV treatment is human CD4 on T-helper cells. In 2011, Wheeler et al. published an aptamer-siRNA AsiC with an aptamer selected earlier for staining CD4 positive cells [112
]. In this AsiC study, a 21 nt siRNA that targeted the mRNA of C-C-motive Chemokine Receptor type 5 (CCR5), which is used by HIV-1 as a co-receptor for its entry into the host cell, was chosen. In addition, HIV-1 gag
genes were used as siRNA targets. Specific gene knockdown was not only observed in CD4+ T cells and macrophages, but the AsiCs were also tested on human cervico-vaginal tissue explants. The authors also administered gels containing the aptamer siRNA AsiC intravaginally to humanized mice. In this model of topical application, vaginal transmission of HIV to both the mice and the cervico-vaginal explants was significantly prevented. Moreover, this study showed that locally and topically applied AsiCs were less subjected to degradation than those systemically delivered as in the gp120-aptamer AsiCs, resulting in enhanced efficacy.
A year later, Kai and colleagues successfully converted this CD4 targeting 39mer RNA-aptamer into a DNA-aptamer and used it for a siRNA-AsiC to downregulate HIV-1 protease [114
]. This is noteworthy because the secondary structure of RNA- and DNA-analogs are likely to differ significantly, due to their different ribose puckering. Furthermore, molecular interactions might further be impacted in the case of a DNA-analog because of missing hydroxyl- and fluoro-substituents (compared to 2′-F-Py modified RNA that is usually employed). In this study, binding and uptake of the DNA-aptamer-AsiC into CD4+ T cells was confirmed by microscopy using the fluorescently labeled AsiC. The inhibitory effect on the HIV-1 protease was examined by using quantitative RT-PCR (qRT-PCR) on T cells that had been transfected with the mammalian expression vector plasmid pcDNA-HIR-PR. Interestingly, the DNA-aptamer AsiC was even more potent than the RNA-aptamer counterpart. While the reason for this finding was not elucidated, investigations with other RNA-aptamers should be pursued, as it might lead to a better understanding of AsiC applications in general.
Rossi and colleagues, who are responsible for the biggest innovations in the field of anti-viral aptamer-siRNA-AsiCs, also added bioconjugation strategies to the AsiC field [13
]. One objective when designing AsiCs is to simplify conjugation, while preserving siRNA efficacy [42
]. The sticky bridge approach, mentioned above, is a convenient method of combining the two RNAs in a non-covalent manner, providing opportunities for testing various combinations of each RNA at a reasonable cost [47
]. A general example of these sticky bridge AsiCs is given in Figure 3
. The sticky bridge comprises a GC-rich complementary annealing sequence that is appended to both RNAs enabling the annealing of the strands to each other. Additionally, a three-carbon linker provides the flexibility to the bridged RNAs, ensuring Dicer processing of the siRNA.