Therapeutic aptamers

In order to apply DNA and RNA aptamers in the clinic their pharmacokinetic and phar-macodynamic characteristics have to be optimized. There are three major obstacles that need to be overcome in order to generate a therapeutic aptamer: Metabolic instabil-ity, fast renal clearance and fast biodistribution from the plasma compartment to the tissues.⁵ Aptamers can be optimized by truncation or chemical modifications to make them easier to synthesize or to increase the stability and resistance to nucleases. Usually, an aptamer is minimized to its smallest entity that still sustains the binding affinity to the target. This allows to minimize costs that are associated with the production of the aptamer as well as eases the downstream solid-phase synthesis.⁵ A lot of work has been done to stabilize antisenses oligodeoxynucleotides (ODNs)^11–13or siRNA¹⁴that can also be applied to aptamers. In the case of RNA aptamers the inclusion of 2’- fluoro pyrimidines or 2-O-Me purines is a common method to increase the aptamers stability

and half-lifein vivo. Unfortunately these nucleotides are not efficiently incorporated by standard RNA polymerases, which had to be engineered to be able to incorporate these nucleotides more efficiently.^{15, 16} Another method to increase the stability of oligonu-cleotidesin vivois to include locked nucleic acids (LNAs) or the use of spiegelmers. The use of LNAs has been described by Schmidt and colleagues;¹⁷ in short, the 2-O and the 4-C of locked nucleic acids are connected by a methylene bridge thus by including LNAs into the oligonucleotide sequences the base pairing stability is increased which results in a higher stability and reduced accessibility to nucleases. Spiegelmers, in contrast, are enantiomers of nucleic acids. The D-nucleic acids are replaced by their L-form. This modification prevents the recognition of ODNs by nucleases leading to an increased stabilityin vivo. Also, post-synthesis modifications can be used, for example the fusion to polyethyleneglycol (PEG) polymers changes the pharmokinetics of aptamers. Small molecules are, in general, characterized by a fast clearance through the renal system which can be prevented by increasing the molecular weight of an aptamer.^{18, 19} The most common method is the fusion of a polyethyleneglycole (PEG) moiety to the ap-tamer. Pegaptanib, an aptamer approved by the FDA for the treatment of age-related macular degeneration, is conjugated to a 40 kDa PEG that decreases the renal clearance.²⁰ After optimization of the pharmacokinetics an aptamer can be therapeutically applied by several delivery strategies. Four possible approaches are known by which aptamers can be used as treatment decoys. Firstly, a siRNA sequence can be directly linked to the oligonucleotide sequence of the aptamer. It has been shown by McNamara and colleagues that it is possible to deliver siRNA against pro-survival genes (PLK1 and BCL2) with a PSMA specific RNA aptamer. This approach resulted in the silencing of the target gene and promoted prostate cancer cell deathin vitroandin vivo.²¹ Secondly, siRNA or other therapeutic substances can be biotinylated and conjugated to strepta-vidin. Next, biotinylated aptamers are linked to streptavidin as well and mediate the delivery to target cells. Chu et al. showed that anti-Lamin A/C and anti-GAPDH siRNA conjugated to the PSMA RNA aptamer with biotin:streptavidin were delivered success-fully to LNCaP cellsin vitro.²² Another attempt is to modify an aptamer with a toxin by

a Succinimidyl (3-[2-pyridyldithio]-propionate) crosslinker which reacts with its N-hydroxysuccinimide (NHS) ester with primary amines of the modified RNA sequence.

Subsequently, the pyridinyldisulfide reacts with -NH2groups of the toxin which yields in a reversible disulfide bond. With this approach Chu et al. showed that they were able to crosslink the PSMA aptamer with the toxin gelonin and successfully delivered the toxin into PSMA expressing cells in vitro.²³ Finally, it is also possible to conjugate aptamers to nanoparticles which encapsulate toxins for the targeted delivery to tumor cells. Furthermore, proteins can be encapsulated in nanoparicles for the delivery of anti-gens to antigen presenting cells. It has been shown that encapsulating doxetacel into PLGA nanoparticles and conjugating these to a PSMA aptamer succeeded in prostate cancer cell death in vitro as well as in tumor regression in a LNCaP xenograft mouse model of prostate cancer.²⁴

In this study we planned to generate DNA aptamers against the murine proteins PSMA, DC-SIGN as well as DEC-205. We chose PSMA as the prostate tumor antigen because its expression is mainly restricted to prostate cells and highly upregulated during prostate cancer.²⁵ DC-SIGN and DEC-205 are both expressed on dendritic cells. In the case of PSMA we thought to deliver siRNAs against pro-survival genes (PLK1 and BCL2) as well as toxins to prostate cancer cells to facilitate a targeted therapy against PCa. Ad-ditionally we wanted to present encapsulated prostate cancer antigens to DCs by DC specific aptamers to induce an immune reponse against PCa. We cloned the extracellular domains of our target proteins into mammalian expression vectors and were able to pu-rify sufficient amounts of recombinant murine PSMA, DC-SIGN and DEC-205. We were successful in performing twelve SELEX rounds for two targets (DC-SIGN and PSMA), but we could not identify clones that showed high affinity to the target proteins.

4.3 Materials

4.3.1 Biological Materials