The Present and Future -
2011 -
As of 2011, SELEX development has shifted to focus on a so-called "Multiple-Target High Throughput SELEX," in which multiple aptamers are selected for different targets, all the while reducing selection times by innovatively incorporating and combining technologies and techniques. Nanotechnology also allows miniaturized work areas into small devices (microchips), which removes the possibility of cross-contamination.
Multiple aptamer-based drugs remain in various stages of development, with some in several phases of clinical development all over the world. A few are listed at right, although there are many more of these drugs that are not included in that particular listing. A variety of aptamer applications have yet to be realized, although they are all equally promising and exciting, and hold the potential to alter the world.
A History of Aptamers
The concept of joining nucleic acids with proteins emerged in the 1980s from research on human immunodeficiency virus (HIV) and adenoviruses. Since the emergence of the Systematic Evolution of Ligands by Exponential enrichment (SELEX) process, aptamer development has focused mostly on perfecting this SELEX process to develop optimized aptamers that can compete with antibodies in the pharmaceutical market. It has now been over 20 years since then, and 25+ different SELEX processes have been developed. Now, the aptamer world is on the cusp of increased therapeutic application.
Genesis of SELEX -
1990
Professor Larry Gold, and then graduate student Craig Tuerk, developed the SELEX process during an experiment at the University of Colorado. A randomized pool of around 65,500 nucleotide sequences underwent the process, and two of these sequences were isolated. The experiment's results led to the concept that nucleic acids could be used to recognize proteins.
Gold and Tuerk would be awarded the patent for SELEX technology in 1993 - this patent would be one of several that would slow development until well into the latter half of the 2000s. The two would go on to win the European Inventors of the Year 2006 award in the Non-Europeans category for their work in developing SELEX.
Andy Ellington and Jack Szostak, working independently of Gold and Tuerk, devised the same process simultaneously. Ellington coined the term "aptamer."
Gerald Joyce at the Scripps Institute in La Jolla, California, also developed SELEX around the same time.
The First Aptamer-based Drug: Macugen -
2004
Pegaptanib Sodium (trade name Macugen) was approved by the FDA in December 2004, the first aptamer-based drug to be approved for therapeutic use. Macugen targets VEGF-growth factor, to treat Age-Related Macular Degeneration (AMD).
Although initially selling well, Macugen eventually lost out to the mAb-based drug Lucentis.
Improving SELEX Libraries -
1995-1996
Scientists then set their sights on preparing SELEX for clinical in vivo applications. Smith et al. incorporated non-nucleic functional units to produce blended SELEX pools. In this experiment, a functional unit conjugated to an aptamer allowd for a covalent bond to be formed between the unit and a site on the target. These findings increased the number of structures and interactions available for the ligands.
The same year, 1995, Dobbelstein et al. developed a method called cDNA-SELEX that used natural, instead of synthetic, sources. These researchers used human cells and proteins to uncover binding sites.
In 1996, Spiegelmer Technology was developed. Spiegelmers are aptamers that are L-ribose based, and as such are highly resistant to nuclease degradation. L-ribose is an artificial "mirror image" of the naturally occurring D-ribose, hence the name - "Spiegel" is German for "mirror." Noxxon Pharma focuses on the development of Spiegelmer-based drugs, utilizing these characteristics that favor in vivo applications.
Covering the Basics -
1990-1994
Although the Classic (original) SELEX method set the stage for aptamer development, there were still many areas that required perfection.
The development of aptamers continued in Negative-SELEX developed by Ellington and Szostak in 1992. This was done to address problems in which aptamers would bind to environmental components (i.e. agarose matrix) in non-specific interactions. The aptamer library would be incubated on the matrix alone before being introduced to the target, thus eliminating these non-specific aptamers. This increased aptamer affinity by ten times.
Counter-SELEX was created in 1994 and increased aptamer specificity. Aptamers were incubated with target analogues, which are molecules with uncanny similarity to the actual targets, before proceeding to be introduced to the target - eliminating aptamers that bound to the wrong targets (the analogues).This method was developed by Jenison et al.
Professor Larry Gold (left), Professor Craig Tuerk (right), developers of SELEX
Professor Larry Gold (left), Professor Craig Tuerk (right), developers of SELEX
Timeline of SELEX development (Click to Expand)
Timeline of SELEX development (Click to Expand)
The Magic Bullet Concept -
End of 19th century - Early 20th century
German scientist Paul Ehrlich envisioned a "magic bullet" of medicine - a treatment that would target only the disease-causing agent with high specificity and affinity and leave healthy tissue untouched.
Ehrlich worked to develop chemotherapy and other methods to fight cancer. He was a pioneer in leading medical research towards the causes of diseases, and not just in treating symptoms.
His dream of a "magic bullet" would be realized in Monoclonal Antibodies, and eventually epitomized in Aptamers. Both seek out disease-causing agents with high affinity and specificity; discriminating between disease and healthy tissue.
Paul Ehrlich in a 100+ year old lab.
Paul Ehrlich in a 100+ year old lab.
Monoclonal Antibodies -
1975
Using theories made by scientist Niels Jerne, researchers Georges Köhler and César Milstein collaboratively developed a process that successfully created Monoclonal Antibody-producing hybridomas. This made high affinity and specificity custom targeted antibodies a feasibly possibility for disease treatment.
The three were awarded the Nobel Prize in Physiology or Medicine in 1984.
The First mAb-based Drug -
1985
Orthoclone OKT3 (Trade name: Muromonab) was the first Food
and Drug Administration approved Monoclonal Antibody-based drug. Muromonab was used as an antirejection drug for kidney transplants.
Orthoclone OKT3 - Trade name Muromonab
Orthoclone OKT3 - Trade name Muromonab
Macugen with administration device
Macugen with administration device
Image Citation 11: Adenovirus
Image Citation 11: Adenovirus
The concept of joining nucleic acids with proteins emerged in the 1980s from research on human immunodeficiency virus (HIV) and adenoviruses. This research showed that small structured RNAs are encoded by these viruses to bind to cellular proteins with high affinity and specificity - by definition what would come to be
known as aptamers.
Origins of Aptamer Develpment -
1980s
L-ribose and D-ribose mirror image concept.
L-ribose and D-ribose mirror image concept.
Spiegelmer structure
Spiegelmer structure
1997-1999
Nearing a decade of development, aptamers were now more easily selected and were ready for new functions rather then just recognition.
In 1997, Coulter et al. made the first selection of an aptamer inside a mammalian cell. Modeled on in vitro SELEX, this version used the living cell's biological processes to perform the selection and was named In Vivo SELEX.
Burke and Willis pioneered the development of aptamers with complex functions in 1998. Aptamers were fused together and used to perform multiple functions - this was named Chimeric SELEX.
Multi Stage SELEX, a variant of Chimeric SELEX, was developed by Wu et al. in 1999 and simplified several steps in the process.
Several aptamer chimeras.
Several aptamer chimeras.
Toggle and Tailored SELEX -
2000-2003
Toggle-SELEX was envisioned in 2001 to address problems in which aptamers lost affinity for larger targets. Bianchini et al. used cloned aptamers that were separated into stronger individual aptamers, a simplified process that selected very specific aptamers with increased binding affinity.
White et al. developed this method further, in which "pre-selected" libraries were used. These libraries are mixtures of both desired target proteins were mixed with aptamers. One of the target proteins was then incubated, and then alternated with the other, so that aptamers with affinity for both remained.
Tailored-SELEX was developed by Vater et al. in 2003 to obtain shortened sequences.
Entering the Cell & Complex
Aptamers -
Modern Updates -
2004-2011
Aptamers were finally at a point where they could be feasibly obtained and could survive inside living organisms. Now, additional and specialized technologies would be incorporated into basic SELEX processes for desired effects.
Medonsa et al. developed Capillary Electrophoresis (CE-SELEX) in 2004, which uses electrophoretic migration to separate bound and unbound aptamers - this works because aptamer-target complexes are heavier then either while separated. An overview of how electrophoresis works can be found below.
Stoltenberg et al. developed FluMag-SELEX in 2005, a process in which fluorescent labeling and magnetic beads are used to more readily eliminate unspecific interactions and allow smaller concentration of the target.
TECS-SELEX (Target Expressed on Cell Surface), designed by Ohuchi et al. in 2006 allowed aptamers, for the first time, to bind to cell surface proteins - this would eventually expand to other cell surface molecules.
Next Generation SELEX in 2009 implemented microarray technology to detect aptamer-target interactions.
The status of several aptamer-based drugs' development (Click to Expand).
The status of several aptamer-based drugs' development (Click to Expand).
Classic SELEX process showing stages at which modified variants deviate (Click to Expand).
Classic SELEX process showing stages at which modified variants deviate (Click to Expand).
Image Citation 38: Gerald Joyce
Text Citation 65: Jack Szostak
Text Citation 64: Craig Tuerk
Image Citation 38: Gerald Joyce