Aptamers - the genesis of nanogenetics for neuro-ailments

An inherent barrier of brain encumbers both diagnosis and treatment of nervous system ailments, making them more vulnerable and severe. Owing to the unmet need for efficient management of these maladies, brain targeted nanodelivery approaches are being extensively explored. Amongst these, aptamers are emerging as cogent targeting tools, attributing to their high specificity, selectivity, affinity (KD value: 1 nM - 1 pM), small size (10-50 nm), stability, biomimetic nature and fail-safety.

Structurally, aptamers (‘aptus’: to fit, ‘meros’: particle) are 3-D conformational D-oligonucleotides (15-70 bases) of single stranded DNA or RNA, which are synthesized chemically by systematic evolution of ligands by exponential enrichment (SELEX) process, The principal here is based on affinity based selection of DNA/RNA oligonucleotides from a pool of about 1012-15 sequence motifs against the target through a reiterative five step process viz. binding, partition, elution, amplification, and conditioning.

Pharmacodynamically, aptamers can be visualised as the nucleic acid chemical variants of antibodies that bind to the cognate target with a comparable affinity that blocks/interrupts its function, with additional advantages of non-immunogenicity, heat stability and unlimited shelf life. The only concern of probable instability of aptamers in vivo due to their oligonucleotide construct is also overruled by chemical modifications of sugar backbone at 2’ position (2’-fluro, 2’-O-methyl, phosphorothionate), which in a nutshell makes them the most promising and lucrative avenue for targeted delivery.

Application of aptamers
High selectivity and affinity based application of aptamers as in vitro diagnostic tools is now well conceived and the current research is directed towards their application as complete therapeutic packages, especially in neurological arena.  The current state of art in aptamers research for neurological conditions viz. neurodegenerative disorders, brain carcinomas, multiple sclerosis, stroke, mood disorders etc. reveal their potential role at two levels:

1. As therapeutic agents interrupting functions of over-expressed proteins underlying disease pathogenesis
2. As active brain targeting ligands facilitating entry of drug or drug carrying nanocarriers across brain and/ or cells. In this scenario, the conjugation of aptamers with therapeutic agents or vehicles can be achieved via both covalent and non-covalent linkages.

These multifaceted applications of aptamers are being comprehensively investigated for development of efficient treatment modalities and are summarized here to give an insight in their role in brain delivery.

Alzheimer’s disease
It is one of the most common form of neurodegenerative diseases, clinically characterized by the extracellular deposition of ß-amyloid (Aß) plaques resulting from the abnormal cleavage of amyloid precursor protein by ß-secretase (BACE1) that disrupts the neuronal activity. The condition is further worsened by precipitation of intracellular hyperphosphorylated neurofibrillary tau tangles. Disease pathogenesis indicate selective silencing of BACE1 and/or inhibition of Aß fibril and tau tangle formation as a convincing treatment modality. Considering this, highly selective RNA aptamers against the aforementioned targets were developed and proven to be very efficient in vitro. Further, in a comparative study between and antibody and RNA aptamer against tau phosphatase inhibitor (I1PP2A), aptamer indicated greater enhancement in tau phosphatase (PP2A) activity (almost 22%) confirming its potential in treatment of taupathy and underlying neurodegeneration.

Prions diseases
These are a group of conditions (Kuru and Creutzfeldt–Jakob disease in humans and mad cow disease in animals) clinically characterized by alteration of cellular soluble prion protein (PrPC) to insoluble and infectious aberrant scrapie form PrPSC resulting in underlying neurodegeneration. Arresting this conversion of PrPC to PrPSC was reported by 2’-fluoro modified and 2’-fluoropyrimidine modified RNA aptamers against ß-sheet rich aberrant isoforms of PrPSC which not only prevented aggregation of PrPSC but also retarded its formation in vitro. The point of concern here is that developed aptamers possess therapeutic potential but do not play any role in cellular transport and thus need potent delivery systems for aptamer transport across brain.

Another well thought target involves the neurotrophin receptor which is a cell surface receptor associated with synaptic and neuronal function and plasticity which is generally downregulated during neurodegenerative disorders. A RNA aptamer agonist of this receptor was thus investigated indicating enhancement of neurotrophin mimetic activity, in turn restoring normal brain function. Thus, considering diversity in pathogenesis of neurodegeneration, aptamers can be designed against respective targets as therapeutic agents.

Brain tumour
Glioblastoma is the most severe form of brain tumour having worst prognosis with average life expectancy of 14 months, which is ascribed to the lack of intracellular targeting treatment regimen. Herein, aptamer could be visualised as an active targeting ligand, owing to which a 75 nucleotide long DNA aptamer (GMT8) against glioblastoma cells (U87) was developed and functionalized on docetaxel-loaded poly(ethyleneglycol)-poly(e-caprolactone) (PEG-PCL) nanoparticles that indicated significant increase in uptake and inhibition of glioblastoma and interestingly did not show any increase in uptake across normal neuronal cell-lines, proving their selectivity. The therapy efficiency was further improved by functionalization of the same nanodelivery system with dual targeting approach viz. phage-displayed TGN peptide against blood brain barrier and an AS1411 aptamer against brain carcinoma cells. This ensured both penetration and targeting across endothelial and tumour cells, opening a wide arena for aptamer research against over-expressed proteins in various forms of brain tumours.

Nicotinic receptors
Nicotinic acetylcholine receptor (AChR) is one of the most scrutinized target, which upon autoimmunization leads to myasthenia gravis - a disorder characterized by muscular weakness and is also a binding site for inhibitory effects of the most abused drugs viz. cocaine, phencyclidine leading to addiction and other side effects. The research here is directed towards development of aptamers against the antibody generated as an autoimmune response or as inhibitors against the protein that selectively and competitively overrides the binding of antibody and/ or abused drugs.
Moreover, aptamers are now being researched for further improvement in their stability and selectivity wherein a degradation resistant enantiomeric L-oligonucleotide form of aptamers known as spiegelmers are being developed and have shown promising results against the opioid receptors that regulates nervous system function via endogenous neuropeptides viz. nociceptin/ orphanin etc.

In sum, during the past couple of decades, aptamers have emerged from a concept to a reality finding widespread applications in the area of diagnostics and therapeutics. Being an efficient and a superior alternative to monoclonal antibody technology, a market share of approximately $1.9 billion by 2014 has been forecast .

Here, one must understand that ‘design and delivery’ are the two key aspects for successful emergence of this technology in management of neurological ailments. Further, with thorough understanding of the molecular mechanics associated with brain disorders a two-level strategy of aptamers as therapeutic and/ or targeting ligand needs to be coherently explored so as to achieve selective and specific onsite efficacy. With many impending applications of aptamers, currently in pipeline and budding developmental stages, soon we can expect an aptamer based product to enter the pharmaceutical market for neurological disease management.