Aptamers as targeted therapeutics: current potential and challenges

Abstract

Nucleic acid aptamers, often termed 'chemical antibodies', are functionally comparable to traditional antibodies, but offer several advantages, including their relatively small physical size, flexible structure, quick chemical production, versatile chemical modification, high stability and lack of immunogenicity. In addition, many aptamers are internalized upon binding to cellular receptors, making them useful targeted delivery agents for small interfering RNAs (siRNAs), microRNAs and conventional drugs. However, several crucial factors have delayed the clinical translation of therapeutic aptamers, such as their inherent physicochemical characteristics and lack of safety data. This Review discusses these challenges, highlighting recent clinical developments and technological advances that have revived the impetus for this promising class of therapeutics.

Figure 1. The generation of RNA aptamers

A commercially obtained ssDNA pool is used as an initial template for generating a dsDNA library by PCR, and is subsequently converted into a corresponding RNA library via in vitro transcription for the first selection cycle. A) Purified protein-based SELEX uses four key steps: (1) the RNA library is incubated with the target protein; (2) the bound species are isolated from the unbound sequences through various partitioning strategies; (3) target-bound sequences are recovered and (4) subjected to re-amplification (reverse transcription, PCR, and in vitro transcription) into a new RNA library for the next selection cycle. Through the iterative rounds, specific aptamers are enriched and identified by sequencing analysis. B) Whole cell-based SELEX, consisting of four main steps: (1) counter selection by incubating RNA library with negative cells that do not express the target protein; (2) positive selection by incubating recovered unbound sequences with positive cells expressing the target protein; (3) recovery of target-bound sequences; and (4) re-amplification of recovered species and generation of a new RNA pool for the next selection round. C) Live animal-based SELEX. After IV administration and circulation of an RNA library in the animal model, the tissue or organ of pathological interest is harvested and the bound sequences are extracted. Subsequently, the recovered RNA sequences are re-amplified to make a new RNA library for the next selection cycle.

Figure 2. Schematics of bivalent RNA aptamers used as agonists

A) The bivalent 4-1BB aptamer. The 3′-terminus of two 4-1BB aptamers are attached to a 21-nt complementary linker sequence and subsequently annealed together. B) The bivalent OX40 aptamer. The 3′-end linker sequences (20 nt in length) of OX40 aptamers were annealed to a flexible DNA scaffold. A tandem repeat of 20 nt DNA oligos was connected by a flexible polyethylene spacer.

Figure 3. Schematics of cell type-specific RNA aptamers used as delivery agents

A) An aptamer-siRNA/miRNA chimera. The chimera is synthesized as two pieces followed by an annealing step to make the chimeric RNA molecule. B) An aptamer-shRNA chimera synthesized as one piece. C) An aptamer-streptavidin-siRNA conjugate. The siRNA and PSMA RNA aptamers are chemically conjugated with a biotin group. Then the two biotinylated siRNAs and two aptamers are non-covalently assembled via a streptavidin connector. D) The bispecific PSMA-4-1BB aptamer conjugate. The PSMA RNA aptamer and a bivalent 4-1BB RNA aptamer are tethered to complementary linker sequences and hybridized through Watson-Crick base pairing. E) An aptamer-sticky bridge-siRNA/miRNA conjugate. The aptamer and siRNA/miRNA are appended to complementary GC-rich bridge sequences and annealed by simple mixing that allows Watson-Crick base pairing. F) An aptamer-sticky bridge-antimiR conjugate. The single or multiple antimiR oligonucleotides are hybridized with the aptamers via a GC-rich sticky bridge. G) The 3WJ-aptamer/antimiR RNA conjugate. It contains an epidermal growth factor receptor (EGFR) aptamer as a targeting agent, an anti-miR-21 sequence as a therapeutic agent, a fluorescent dye (Alexa647) as an imaging agent, and a three-way junction (3WJ) motif as a molecular scaffold. H) An aptamer-protein conjugate. Chemical synthesis incorporates a primary amino group at the 5′-end of the PSMA aptamer, allowing chemical modification with a cross-linker agent (SPDP, N-Succinimidyl 3-[2-pyridyldithio]-propionate), and subsequent conjugation with the cysteine residue of gelonin toxin through a disulfide linkage. I) An aptamer-anticancer drug conjugate. It is made by using an acid-labile acylhydrazone linkage or formaldehyde linkage. J) Physical conjugation between an aptamer and anthracycline drug (dox) through intercalation.

Figure 4. Cell type-specific aptamer-functionalized nanocarriers for targeted therapy

Multiple components such as therapeutics (therapeutic oligonucleotides, chemotherapy agents), actively targeting agents (cell type-specific aptamers), and imaging agents (fluorescent dyes or radioactivity agents) are rationally assembled in one nanoscale carrier to achieve multifunctional nanomedicine. (1) Upon binding of the aptamer portion of nanocarrier conjugate to the target receptor on the cell surface, (2) the conjugate is internalized into cells, probably through a receptor-mediated endocytosis pathway. (3) It is presumed that the conjugate shuttles into the endosome; subsequently, the therapeutic agents dissociate from the complex and escape the endosome. The released therapeutic agents mediated therapeutic function.