Mohamad Abouelnaaj's SOD1 Aptamer Project (2017)

Anti-SOD1 Aptamer Complex: A Potential Therapeutic for Familial ALS

Introduction & Background

Amyotrophic Lateral Sclerosis (ALS) involves the degeneration of motor neurons exclusively. With no existing treatment option and affecting millions in the US alone, the disease presents itself as both an economic and social burden. Recent research into the identification of the causes of ALS has identified SOD1 as an enzyme of interest in cases of the disease that are familial in nature. In healthy individuals, SOD1 acts as a powerful antioxidizing enzyme within cells (Li & Zhou 2011). The enzyme converts radicals into both hydrogen peroxide and less reactive oxygen species. Thus, SOD1 plays a rather important role in protecting the cell from the damages of oxidative stress along with other functions (Bowler, 1992). In ALS patients with the mutant form of SOD1, the mutant has been shown to adopt aberrant properties resulting in the generation of an overall toxic within the cell (Wong et al., 1995). It is not known exactly as to why a toxic effect is generated by the mutant but it is believed that toxicity arises due to the wide-ranging effects the mutant has on various cellular pathways as shown in Figure 2 (Dion et al., 2009).

The diverse set of functions involving SOD1 and the complex pathways affected by SOD1 mutation are believed to result in the overall toxicity of the misfolded enzyme (Dion et al., 2009). As such, targeting of SOD1 presents the possibility of not only better understanding the role of the enzyme in familial ALS but also the possibility of developing of therapeutic medicine.

Current research into ALS treatment has focused heavily on targeting mutant SOD1 in patients with the familial form of the disease. Recently, siRNAs inhibiting the catalytic activity of SOD1 have been identified and delivered into rats engineered with a mutant SOD1 gene (Ralph et al., 2005). The siRNAs were delivered via a lentivirus vector that randomly inserts the cDNA sequence into the rat’s genome (Ralph et al., 2005). Upon incorporation into the genome, the siRNA is produced by the host cell and inhibits mutant SOD1 function. The study had shown that engineered rats injected with the siRNA sequence experienced nearly an 80% increase in life expectancy (Ralph et al., 2005). Such findings indicate that inhibition of mutant SOD1 activity can serve as a means of improving the outcomes of ALS patients. However, it is important to note that delivery via a lentivirus maintains a slight chance of oncogene activation due to the random insertion of the siRNA sequence. As such, the development of an aptamer that binds to SOD1 could provide a means of more direct and targeted inhibition of mutant SOD1 activity in patients suffering from familial ALS.

Once an aptamer is developed for SOD1, a possible delivery mechanism of the RNA sequence could involve the use of an anti-SOD1 aptamer complex involving nickel beads and anti-transferrin aptamers as shown in Figure 3. Transferrin receptors serve as potential targets for aptamer delivery for two reasons. Firstly, transferrin receptors are expressed ubiquitously across cells including those involved in the blood-brain barrier. Secondly, an aptamer that binds to transferrin receptors has been developed and has been shown to induce internalization of attached siRNAs once bound (Chu et al. 2006) As such, it is proposed that a complex involving the developed anti-SOD1 aptamer, appropriate beads and anti-transferrin aptamer could serve as a means of delivery of the anti-SOD1 aptamer across the blood brain barrier in therapeutic doses.

The aptamer development process involves a methodology referred to as systematic evolution of ligands by exponential enrichment, or SELEX, in which a pool of RNA is repeatedly enriched as to include RNA sequences which have been shown to bind well to the target protein. Vital steps in the process include target immobilization and selection in which the His-tagged SOD1 is bound to a corresponding bead as to allow for separation of bound and unbound RNA, cycle course PCR (ccPCR) in which the optimal number of cycles required for amplification of the resulting DNA is identified, large scale PCR (lsPCR), and transcription in which the resulting DNA is then transcribed back into RNA allowing for the start of another round of selection. Since the application of the aptamer involves injection into the bloodstream, the wash buffer used in the selection process was 5.7 mM phosphate-buffered saline (PBS) due to its pH value (7.4) that matches closely physiological conditions.

Thus far in the selection process, round 1 has been completed and round 2 has been started with reverse transcription completed. After successful completion of 5-6 rounds, the RNA pool will be sequenced in an attempt to identify any motifs. This will then be followed by a binding assay. It is hoped that the development of an anti-SOD1 aptamer will provide a therapeutic medicine capable of improving outcomes in patients suffering from ALS.

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References

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