Abstract 2166 RNA Aptamer Selection Against SOD1 to Create a Therapeutic for ALS Patients

Diagnoses are on the rise for the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) and current treatments do little to extend life, creating a dire forecast that punctuates the need for effective ALS treatments. ALS affects approximately 200,000 people globally (ALS Society of Canada) and cases are predicted to increase by 69% between 2015 and 2040 (Nature Communications). There are only 7 drugs that treat ALS, most of which only improve life expectancy by a few months (ALS Association). One of these drugs, Toferson, attacks the mutated form of a protein whose overexpression in the brain is linked to ALS: Superoxide Dismutase 1 (SOD1). In the disease state, mutant SOD1 leads to accumulations or inclusion bodies of proteins in the brain. Although originally only aggregates of mutated SOD1 were associated with ALS, it is now understood that aggregates of wild-type SOD1 can also accelerate its progression. This work seeks to develop an ALS treatment that dispels overexpressed wild-type SOD1, dismantling its aggregates, by introducing anti-SOD1 aptamers. Aptamers are short, single-stranded RNA molecules that can selectively bind to a specific target, such as SOD1, and are identified through a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). SELEX involves iterative rounds of selection, amplification, and purification. These steps narrow a large pool of random RNA sequences to those that bind SOD1. An aptamer that binds wild-type SOD1 protein will be used to create an oligonucleotide complex which will act as a delivery mechanism for the enzyme ubiquitin. The complex will form a bridge between the aptamer and ubiquitin. Because the complex connects them, when the aptamer binds to an aggregate of wild-type SOD1 protein, ubiquitin will be delivered to that aggregate. The ubiquitin coming into contact with the aggregates will initiate aggrephagy, a type of autophagy that degrades protein aggregates. Because ubiquitin tags proteins in the body for degradation, the use of this complex will result in the body degrading the aggregates of wild-type SOD1 protein in the brain. In order to prevent the aptamer from binding to SOD1 monomers, additional rounds of SELEX will be performed to increase the aptamers' binding affinity specifically to SOD1 aggregates as opposed to SOD1 monomers. Currently, 3 full rounds of SELEX have been completed, as well as the early phases of round 4. The results of selection will be cloned and sequenced after the fifth or sixth round of selection in order to determine if a viable aptamer against SOD1 has been found. If an appropriately stringent aptamer against SOD1 is found, progress can begin on building the oligonucleotide complex to create the proposed therapeutic for ALS. The main objective of this research is to utilize SELEX to find an aptamer against SOD1 for the purpose of creating a ubiquitin-delivery system to combat the negative effects of excess wild-type SOD1 in the brains of ALS patients. Because this method would target aggregates of wild-type SOD1, when used in combination with a drug like Toferson, which limits the harmful effects of aggregates of mutated SOD1, it could decrease the rate of progression of ALS by attacking ALS symptoms from two fronts. Finding this anti-SOD1 aptamer and creating an oligonucleotide complex to use in a ubiquitin delivery-system may advance ALS treatment significantly and improve the health outcomes of the hundreds of thousands of patients afflicted with this disease.