Self-amplifying RNA as a medium for the treatment of diabetes type I & II

Design Brief

Alexandru Albu2, Sophia Bird4, Jayabhishek Chaudhary2, Grace Cofell3, Amanda Dang4, Morteza Faraji2, Noehil Ferolino1, Laura Keffer-Wilkes5, Marcel E. Koupantsis5, Marie Metz3, Samreet Mutti4, Aubrey Nilsson4, Enrique Zaki Olvis1, Karma Patel4, Priyanshi Patel3, Amber Quo4, Lisa Sallah2, Jessica Semmelrock5, Raiyana Shams4, Marataro Tatsuno4, Zitong Wu4, and Steven Yang4

1Catholic Central High School, 2Chinook High School, 3Lethbridge Collegiate Institution, 4Winston Churchill High School, & 5Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada

Reviewed on 7 May 2022; Accepted on 25 June 2022; Published on 15 October 2022

With help from the 2022 BioTreks Production Team.

AbstractVideoPDFCopyright

Diabetes is a disease associated with resistance to or a shortage of the body’s insulin supply. Individuals with diabetes suffer from elevated blood sugar levels that cause nausea and energy loss, among other symptoms. Affected individuals may undertake treatments such as diet and exercise regulation and insulin injections to maintain or control their blood sugar. However, these treatments are time-consuming and possibly expensive processes that can significantly impair their daily life. Our solution consists of a self-amplifying messenger RNA (sa-mRNA) that expresses insulin in a glucose-dependent manner. It will be injected into patients monthly, depending on the bioavailability, and take care of their insulin needs in a hands-free manner. One advantage of this solution is the decreased time spent controlling blood sugar levels. 

Furthermore, the costs for buying insulin and storing medically related supplies decrease. Potential side effects such as over-amplification of insulin leading to insufficient blood sugar levels in the body, which could result in additional symptoms, are of special concern. Patients would also still require regular injections of sa-mRNA, and it is unknown whether the proposed solution could produce satisfactory insulin levels. Thus, further testing is required to ensure the treatment’s safety and stability.

Keywords: Diabetes, self-amplifying messenger RNA (sa-mRNA), insulin

Authors are listed in alphabetical order. Laura Keffer-Wilkes, Marcel E. Koupantsis, and Jessica Semmelrock mentored the group. Please direct all correspondence to kefferwilkesl@uleth.ca.

This is an Open Access article, which was copyrighted by the authors and published by BioTreks in 2022. It is distributed under the terms of the Creative Commons Attribution License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background 

Three types of diabetes exist, all of which cause dangerous irregularities in blood sugar levels (Diabetes Around the World in 2021, n.d.). Diabetes was responsible for 6.7 million deaths in 2021, while 1 in 10 adults currently live with this health condition. Statistics specific to Canada are listed in Table 1. Diabetes insipidus, also known as insulin-dependent diabetes, juvenile diabetes, or, most commonly, type 1 diabetes, is a chronic autoimmune disease caused by a loss or malfunction of the insulin producing pancreatic beta cells. Damage to these cells results in the absence or insufficient production of insulin (Peyser et al., 2014; DiMeglio et al., 2018). As the hormone that increases the body’s ability to absorb glucose in the blood, insulin is vital. Without insulin, glucose builds up within the blood, inducing symptoms such as skin discoloration, polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger), blurred vision, unexplained weight loss, slow healing cuts and wounds, and heart, kidney, and nerve issues (Isley & Molitch, 2005). 

Type 1a diabetes occurs when beta cells are attacked by the immune system. Detected by a laboratory blood test, machines scan for biomarkers such as glutamic acid decarboxylase antibodies (GADA) or islet antibodies to membranous tyrosine phosphatase (ICA-512). Insulin replacement therapy is necessary to control blood sugar levels (Redondo et al., 2001). Type 1b diabetes differs in that there is no evidence of the immune system attacking beta cells. Also known as idiopathic diabetes, it is an unusual form of diabetes with almost complete insulin deficiency and a strong hereditary component (Kalyani, 2017, Transplant Surgery – Islet Transplant for Type 1 Diabetes, 2022). Although the system proposed herein may benefit other types of diabetes, type 1 is the main focus as it cannot be treated with lifestyle changes.

Table 1: Diabetes by age group within Canada, information from (Diabetes, by Age Group, 2021)

Age GroupNumber of PersonsPercentage
18 to 34 years93,7001.1
35 to 49 years271,8003.7
50 to 64 years734,2009.7
65 years and over1,180,30018.1
Total, 12 years and over2,285,2007.1

Insulin is a small protein hormone that is created in the pancreas (Figure 1). This important protein is responsible for regulating the amount of glucose in the bloodstream and the body’s metabolism of carbohydrates, fats, and proteins. Moreover, it assists the body in storing glucose in the liver, muscles, and fat (Figure 2). When a person eats, blood glucose levels rise, signaling the pancreas to release insulin and store sugar as energy for later use. Without this ability, a person’s blood sugar level may increase to a dangerous amount or drop too low. (Hess-Fischl & Jaffe, 2022).

Figure 1. Crystal structure of human insulin (PDB 1TRZ)
Figure 2. Insulin response pathway in humans. Insulin is produced in the pancreas. As blood glucose levels rise, insulin binds to receptors on tissues to store sugar as fat. Created with BioRender.

People with type 1 diabetes require insulin to maintain their blood glucose levels. Different types of insulin are available, including rapid-acting, short-acting (regular), intermediate-acting, and long-acting (Figure 3). Diabetic patients commonly inject a mixture of these types of insulin 4 or 5 times a day. These are injected with syringes and needles, insulin delivery pens, or insulin pumps. Insulin delivery pens and pumps store insulin for delivery into the patient’s body. The former is manual, while the latter is connected to a catheter, a thin, flexible tube that inserts or removes fluids into the body. Insulin also must be stored at 2–8°C throughout the day for at least 2.5 hours a day to be effective. Alternatively, for insulin delivered in a pen, the temperature must be 2–30°C (Collier & Weatherspoon, 2018).

Figure 3: Intensity of types of insulin over 24 Hours (Grey, 2019)

Artificial pancreas (closed-loop insulin delivery) is another approved system for patients above the age of 2. An artificial pancreas automatically monitors blood glucose levels, calculates the amount of insulin necessary throughout the day, and delivers this amount. Consisting of two indicators, an artificial pancreas typically uses a continuous glucose monitor (CGM) to track blood glucose levels automatically and a carbohydrate-measuring system. To use this system, the diabetic patient enters the amount of carbohydrates they consume during meals. The program calculates the amount of insulin needed and signals the insulin infusion pump. This device can deliver small doses of insulin into the body at times when blood glucose levels are not in the patient’s target range (Peyser et al., 2014).

Islet cell transplantation places purified and processed cells from an organ donor into the body of a type 1 diabetic patient. The beta cells in these islets are able to make and release sufficient amounts of insulin in people with type 1 diabetes, so they do not have to take daily injections of insulin. Often requiring two transplants to achieve insulin independence, a diabetic patient receives at least 10 000 islet “equivalents” per kilogram of body weight that is extracted from two donor pancreases. As the islets are slowly infused through a catheter into the portal vein of the liver, surgeons use local anesthetic and sedatives to ensure the safe reintegration of the beta cells (Transplant Surgery – Islet Transplant for Type 1 Diabetes, 2022).

The regular delivery of insulin to care for diabetes interrupts daily life. 45% of diabetic patients do not adhere to the self-care treatment of insulin injections or permanent diet changes and thus fail to complete them, resulting in failed treatment (Polonsky & Henry, 2016). Moreover, some diabetic patients who live in areas where insulin must be bought cannot afford the price of insulin or transplants. In the United States, where 37.3 million citizens are diagnosed with diabetes (National Diabetes Statistics Report | Diabetes, n.d.), a vial of insulin costs $332 (Rajkumar, 2019). Unfortunately, almost 800 million individuals worldwide spend a tenth of their savings on health-related expenses, which is worsened in economically-suffering areas of the world (Moucheraud et al., 2019).

Recent development in self-amplifying messenger RNA (sa-mRNA) research suggests that this platform can be used for protein replacement therapies, such as insulin replacement for diabetics. sa-mRNA is unique in replicating the original genomic RNA, creating many copies in vivo. Thus far, sa-mRNA has been shown to produce a protein of interest (Andries et al., 2015) (often an antigen), remain stable in the body for weeks, and have the translation of their encoded proteins controlled in a reversible manner (McCafferty et al., 2021). The ability to self-replicate is an essential aspect of this project as this increases the duration of protein expression. Like mRNA, sa-mRNA is transient but replenishes itself. The bioavailability of sa-mRNA varies depending on the sequence, so each new construct requires testing to determine how long the sa-mRNA would remain stable in mammalian cells. Most sa-RNAs are based on the genome of alphaviruses, such as the Sindbis virus, Semliki Forest virus, and the Venezuelan equine encephalitis virus (VEEV) (Rupp et al., 2015). This type of mRNA would allow for the target protein of insulin to be synthesized for a longer period of time, providing a better possible treatment for type 1 diabetes (Tews & Meyers, 2017). 

Typically, insulin is expressed as a 110 amino acid-long protein. For the sa-mRNA design construct, the gene will only code for the sequences required for the mature, active form, which consists of only 51 amino acids, thus making post-translational processing unnecessary (Tokarz et al., 2018). Additionally, an RNA riboswitch with a glucose-aptamer will be used to control insulin gene transcription. Blood glucose levels should not be higher than 126 mg/dL after 8 hours of fasting or 200 mg/dL throughout the day (Hoffman, 2020). We hope to make the riboswitch sensitive to these levels of blood sugar. Furthermore, due to recent developments in mRNA for vaccines, there has been more research into making this a safer, longer-lasting procedure. This was mainly achieved through controlling factors such as immunogenicity, which are the types of immune responses from the body the RNA might trigger and extending the bioavailability, meaning the extent to which the RNA remains functional. 

Systems level

The insulin-encoding sa-mRNA will be injected into the body monthly. Once in the cytoplasm, it will produce basal levels of insulin, and has previously been reported as 15 ∓4.8 µU/ml  (Bagdade et al., 1967). When a glucose concentration of 6 mmol/L is surpassed in the bloodstream (a number between the healthy basal level of 5 mmol/L and the diabetes diagnosis threshold of 7 mmol/L), insulin production will be enhanced to compensate for the glucose spike. If glucose levels are high, more insulin will be produced, and vice versa. If glucose is not a sufficient trigger, we will use a cytosolic protein upregulated during hyperglycemia to trigger sa-mRNA replication. We aim to combine a sa-mRNA with a glucose-detecting aptamer to control insulin translation.

Device level

Alphaviruses create substantial amounts of subgenomic RNA encoding to create viral structural proteins, which is achieved by using their natural ability to self-replicate RNA (Alphavirus, sa-mRNA). Most sa-mRNA are based on the genome of alphaviruses and thus benefit from this ability by retaining their non-structural proteins, including the replication machinery. By replacing the viral structural proteins with the gene of interest (GOI), the production of viral particles ceases, and the desired GOI is amplified (sa-mRNA). 

During sa-mRNA replication, a complementary negative sense strand is produced, from which more positive sense genomic sa-mRNA and a subgenomic sa-mRNA are made. An RNA-dependent RNA polymerase complex is formed by the nsP1-4 genes in the positive sense genomic sa-mRNA (Bloom et al., 2020) and is responsible for the “self-amplifying” aspect of the sa-mRNA. In the later phase, each nsP is cleaved into individual nsPs. The separate nsPs use the negative-sense genomic RNA as a template to produce copies of the original sense mRNA. Within this newly replicated mRNA is a subgenomic promoter that triggers the production of subgenomic RNAs, which hold the gene for the protein of interest (Minnaert et al., 2021). These processes are illustrated in Figure 4. 

Figure 4: The functional cycle of sa-mRNA. The sa-mNRA arrives in the cell, and the non-structural proteins 1-4 (nsP) are translated into a negative-sense RNA strand. This early replication complex is later cleaved into individual nsPs. These form the cleaved replicase that uses the negative-sense strand as a template to generate new copies of the original genomic RNA. This leads to the production of the protein of interest. Figure adapted from Minnaert A. K., et al (2021).

The natural production of insulin is a three-step transformation (Figure 5). It begins as preproinsulin, which is subsequently converted into proinsulin via proteolytic action (breakdown). Following this, proinsulin is converted into the active polypeptide hormone anointed as insulin. The human insulin gene is a sequence composed of 110 amino acids that encode the protein known as insulin. However, this proinsulin protein is post-translationally processed to form the functional hormone. The mature protein consists of two peptide chains, an “A” chain and a “B” chain.  The “A” chain has a total of 21 amino acids, and the “B” chain has 30, making a much smaller 51 amino acid protein. By producing insulin directly in the patient’s body, we can avoid having to complete post-translational sequence modifications required by commercial insulin that normally assist in stability and purification, as the cellular machinery will do it for us.

Figure 5: Visual of three-step transformation of insulin. Reprinted from “Insulin Post-translational Modification” (BioRender.com, 2022).

Parts level

The proposed sa-mRNA must contain human-specific translation sequences. For the initiation of translation of mammalian mRNA, the Kozak sequence acts as an enhancer. It is not necessary for the expression of proteins, but it has been found to significantly increase yield and assist with correct translation. The specific sequence that will be used is as listed: 5’ – GCCGCCACC – 3’. This was retrieved from The National Library of Medicine and has previously been used in a plasmid to transfect a human cell line and has been shown to function as predicted (McClements et al., 2021). 

sa-mRNA itself consists of a few sequences, the 5’ and 3’ conserved sequence elements (CSE) on both sides of the sa-mRNA strand, the nsP1-4 genes, a subgenomic promoter, and a gene of interest. For the purpose of this investigation, the subgenomic promoter and the non-structural proteins (nsP) 1-4 from Venezuelan equine encephalitis virus (VEEV) will be used. These sequences were acquired from a complete genome of a VEEV replicon vector YFV-C1 submitted to the National Library of Medicine by authors, Shustov, A.V. and Frolov, I.V (VEEV replicon vector, YFV-C1). 

Part of our proposed design includes an RNA riboswitch with a glucose aptamer that would allow for glucose concentration dependent control of transcription. A riboswitch can be used as a “switch”, controlling whether the ribosome binding site is accessible to initiate translation (Figure 6). There are a few structures an aptamer can take the form of, but the one that will be used in this investigation is a hairpin stem/loop with a high affinity and specificity to glucose. This affinity is due to its tertiary structure rather than a primary sequence (Pendergrast et al., 2005). The recognition and binding involve shape-dependent interactions, where a specific aptamer that fits glucose’s structure would be used. After glucose is bound to this aptamer, it unfolds the hairpin stem/loop, allowing the mRNA to bind to the ribosome and begin coding for the downstream target gene. This structure would allow this project to be glucose-dependent.

Figure 6. An example of an RNA aptamer binding a target molecule and undergoing a conformational change to allow for access to the ribosome binding site (RBS) and allow for downstream translation.

For our riboswitch design, in vitro testing will be completed to test for sensitivity and specificity. This construct will be a fusion of the riboswitch aptamer detection domain to a dye-binding fluorogenic aptamer to create an RNA-based fluorescent biosensor. The ligand (glucose) binds to its native riboswitch aptamer domain, which then allows for the fluorogenic aptamer (RNA Mango) to bind to the pro-fluorescent dye (thiazole orange) and activate its fluorescence. RNA Mango has been used extensively to label RNA for imaging, and its cousin Spinach 2 aptamer has been used to construct previous SAM-sensing RNA-based fluorescent biosensors (Autour et al., 2018; Filonov et al., 2014). Researchers from the Department of Biotechnology at the National Institute of Pharmaceutical Education and Research in Nagar, India, have developed an insulin RNA aptamer that we will utilize as part of our first steps to creating an insulin biosensor (Malik & Roy, 2013). This design could also be used as an insulin aptamer, where instead of turning on transcription of the downstream gene, binding of the target ligand (insulin) to the aptamer will stop transcription. Therefore, insulin protein expression will be controlled by blood insulin levels as opposed to blood glucose levels. This riboswitch will be tested similarly in vitro for specificity and efficiency.

Safety

As the project strives to work with diabetes and insulin, the focus will be primarily on producing insulin in human cells and producing insulin in a glucose-dependent manner. The 5th Edition of Biosafety in Microbiological and Biomedical Laboratories recommends that human cell lines should be handled in accordance with the Biosafety Level 2 (BSL-2) practices and containment. This requires personal protective equipment such as a lab coat and gloves and for all work to be performed within a biological safety cabinet. 

Before using human cell lines, Escherichia Coli will be used to store and amplify our DNA sequences. Risk 1 Groups such as E. coli laboratory require Biosafety Level 1 (BSL-1) laboratory practices and precautions as outlined by the 6th Edition of Biosafety in Microbial and Biomedical Laboratories (Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th edition, page 28).

Team members are required to complete a Workplace Hazardous Materials Information System “WHMIS” safety course before entering the lab. In BSL-1 Laboratory Standard Microbiology Practices, anyone entering the lab will be required to wear lab coats and gloves to avoid contaminating equipment or materials. Additionally, advisors from University staff will be present at all times to enforce that safety measures are followed. 

Due to the size of sa-RNA, it is difficult to deliver without causing an immune response. Developments are being made to optimize the RNA modifications (Andries et al., 2015) and purification processes (Zhong et al., 2021) for reducing innate immunogenicity. Another concern is that too much insulin will be produced, causing hypoglycemia. The incorporation of an insulin-sensitive switch to toggle off translation of more insulin may be useful, but there is no guarantee the local accumulation of insulin would not interfere. Furthermore, the sa-mRNA will reside in the cells, while the insulin will be exported into the blood, meaning there will be a disconnect between the two molecule populations. More consideration has to be made to avoid such problems before testing moves past cultured cell lines.

Discussion

The advantage of a sa-mRNA method for producing insulin is that the time a diabetic needs to spend caring for their blood sugar levels will be greatly reduced. Patients will have a greater capacity to break down sugar and thus less worry about consuming too much. Depending on the bioavailability of treatment, we hope to reduce the frequency of the patient’s insulin injections resulting in easier use and less interference with daily life. We also hope to achieve a cost-effective alternative to current insulin injections. Currently, procuring a standard unit (100mL) of injectable insulin can range from $2.00 to $100, and patients use anywhere from one to six units per month (table 2). With our solution, we hope to provide an alternative to branded medicine that has been a significant factor in increased medical expenses (Cost of Insulin by Country 2022).

Table 2: Average price of insulin for type 1 and type 2 diabetes per standard unit (Rand Corporation, 2018)

CountryInsulin Type
Rapid
ShortIntermediateLong
US$58.00$57.50$73.00$47.00
Canada$9.88$7.10$7.02$15.77
Mexico$14.30$12.21$8.57$20.54
Chile$15.01$16.57$17.92$26.19
Australia$5.92$5.79$5.17$8.63

Some immediate disadvantages of our solution are that we cannot test it on humans due to iGEM safety rules. Hence, we cannot know how effective our solution will be in humans and related organisms. The proposed sa-mRNA treatment is also a temporary solution in the sense that it will run out and require replenishment. This solution is a treatment for the diabetes condition, which is ideally better than current solutions. However, it is not a permanent cure. Nonetheless, we hope this treatment will offer people with diabetes the opportunity to significantly reduce the need for constant needles and medication or eliminate it entirely.

Next steps  

Testing in the lab will begin in June 2022. The tests require our team to first design and order DNA constructs. One will encode our self-amplifying mRNA for human insulin after approval from the iGEM Safety Committee for the use of the self-amplifying sequences. Additionally, we will combine computational modeling with systematic evolution of ligands by exponential enrichment (SELEX) to design and optimize an RNA aptamer that binds glucose. The optimized sequence will be encoded alongside green fluorescent protein (GFP) in a DNA construct. These DNA constructs will be transformed into DH5α E.coli for storage and amplification. Extracting the DNA from the E.coli through a mini-prep or maxi-prep procedure will provide template material for in vitro transcription (IVT). RNA production will be confirmed through urea-PAGE analysis. Testing for glucose sensitivity will be completed in vivo using the translation of GFP. Flow cytometry will be utilized to measure the intensity of GFP production, and those RNA sequences will be further characterized for glucose binding ability.

After these experiments have succeeded, our hope is to transfect the mammalian cells with the RNA that encodes for insulin. Following this, we will lyse the cells and perform a western blot to make sure that a cell pathway is being activated by our insulin, meaning the insulin is present, folded properly, and active. These tests will also allow us to determine the bioavailability in mammalian cells, thus informing ideal treatment regimes to test. Testing will also map out the regular expression level of insulin from the RNA and the effectiveness of our glucose-binding switch, allowing for further optimization. This project is currently in the screening and lead optimization portion of a typical drug production pipeline.  ADMET (absorption, distribution, metabolism, elimination, and toxicity) studies come next, followed by development, which involves the first stages of clinical testing.

Author contributions

Video was produced by AD, AQ, and SM. Background section was written by PP, EZO, GC, LS, MM, RS, NF, AD, and SB. Systems section was written by MF, AN, and SY. Device section was written by JC, and SB. Parts section was written by SB, AN, and EZO. Safety section was written by EZO, PP, AD, NF, and RS. Discussion section was written by ZW, KP, JC, AD, and MT. Next steps section was written by AD, SY, RS, and AQ. Article was edited by LKW, JS, and MEK.

Acknowledgments

The authors would like to thank their mentors for their help in preparing this manuscript.

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