Kajal Singhal's CIAP Aptamer Project (2019)

Potential Aptamer Against Calf Intestinal Alkaline Phosphatase (CIAP) to create an Improved Therapeutic for Ulcerative Colitis as an Alternative to Traditional Medications

Ulcerative colitis is an inflammatory bowel disease (IBD) that is currently affecting more than 900,000 Americans (Ng et al., 2018). Those who are affected by ulcerative colitis are put at an increased risk of developing cancer (Crohn’s & Colitis Foundation of America, 2014). Ulcerative colitis is a chronic disease that induces the inflammation of the colorectal mucosa of the large intestine (My Virtual Medical Center, 2018). The disease is idiopathic --meaning that the cause of it is unknown-- but there is evidence linking ulcerative colitis to an imbalance of endotoxins called lipopolysaccharides (LPS), which are produced by gram-negative bacteria that live in the human gut (Ramasamy et al., 2011). Intestinal alkaline phosphatases (IAP), which can be found in the liver and kidneys of humans, dephosphorylates molecules that induce inflammation in the body and are thus crucial for gut homeostasis (Bilski et al., 2017). In addition, IAPs are related to the detoxification of LPS (Bilski et al., 2017). Diminished levels of IAP are correlated with increased LPS toxicity, which leads to gut inflammation (Bilski et al., 2017). Moreover, decreased levels of IAP are associated with IBDs such as celiac disease and metabolic syndrome (Bilski et al., 2017).

Although ulcerative colitis is incurable, current treatment includes the use of anti-inflammatory aminosalicylates. However, this medication potentially induces headaches, nausea, and gastrointestinal pain in the patient. To get around this, an aptamer can be utilized. A characteristic of aptamers which make them a more appealing alternative to traditional medications is that they solely act on the target for which they have a high binding affinity for and do not result in side effects. Finding an aptamer against calf intestinal alkaline phosphatase (CIAP) is one step closer towards discovering an aid that is more efficient in treating ulcerative colitis. By delivering an oral pill to the large intestine containing large amounts of CIAP, I can reduce the amounts of proinflammatory LPS and theoretically lessen inflammation in that area.

Aptamers are composed of single-stranded nucleic acids, such as ssDNA or ssRNA, that have a high binding affinity for a specific target. Aptamers have a diverse set of applications, such as functioning as a diagnostic tool in order to test for a disease or being a therapeutic. One benefit of aptamer technology as opposed to antibodies is their characteristic of being chighly stable, thus withstanding denaturation for a greater period of time. To execute my therapeutic application against ulcerative colitis, an enteric-coated pill will be orally administered. This pill will contain CIAP target, an anti-CIAP aptamer and an anti-LPS aptamer. The anti-CIAP aptamer will be inhibitory and the anti-LPS aptamer will have no other function other than binding to LPS. Once the pill reaches the intestines, the anti-LPS aptamer will bind to LPS and enable the inhibitory anti-CIAP aptamer to release CIAP. I currently have not worked out the logistics of how CIAP will be released after the anti-LPS aptamer binds to LPS, but I am planning to utilize natural metabolic processes in the body to separate the covalent bonds between the two aptamers. CIAP will execute its role in the dephosphorylation of LPS in the intestines in order to reduce inflammation associated with inflammatory bowel diseases such as ulcerative colitis. This process can be visually summarized in Figure 1.

In order to find an aptamer to be comprised in the therapeutic pill, the SELEX method will be performed. This cyclic process begins with a diverse pool of nucleic acids and involves the procedure of washing unbound species from those that are target-bound, ultimately leaving the aptamer with the highest specificity to the target to remain. An aptamer against CIAP, called VDH2.14, has been developed by Vincent Huynh of the University of Texas at Austin and has an inhibitory effect on CIAP (Huynh et al., 2014). Since CIAP has a negative charge at a neutral pH of 7.0, there is a diminished ability for effective binding of negatively charged nucleic acids to it (Embl- Ebi, n.d.). However, a selection buffer that incorporates divalent salts --which functions like an ionic bridge connecting nucleic acids to the target-- would aid in the binding. A buffer with a pH that is similar to that of the human body --such as PBS buffer which has a pH of 7.4-- would be utilized for this aptamer selection because the application deals with a therapeutic in the body. Since the pill will be enteric-coated, it will not be broken down by stomach acids and will be thus be broken down in the intestines. The structure of human intestinal alkaline phosphatase can be visualized in Figure 2.

Therapeutic applications of an aptamer against CIAP involve the treatment of LPS-mediated diseases, such as sepsis and Parkinson’s disease, by detoxifying LPS levels. Moreover, CIAP has been utilized as a reporter enzyme for assays that incorporate substrates. An example of this substrate is 1,2 dioxetane, which luminesces when CIAP dephosphorylates phosphate groups (Drolet et al., 1999). A few laboratories conducting research on CIAP are the Medical College of Wisconsin, the Institute of Risk Assessment Sciences at Utrecht University, and the Massachusetts General Hospital in coordination with Harvard Medical School (Beumer et al., 2003).

The objective of aptamer selection against CIAP is to find an aptamer that can be utilized to help create a therapeutic that functions better than traditional medication, which result in unwanted side effects. There are currently no results of the aptamer selection against CIAP, as the first round of selection is underway. Reverse transcription was performed to form more E1 ssDNA for lsPCR, and thus lsPCR will soon be conducted.

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References

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