Implementation
Our project has been designed as a treatment for individuals with Type 1 Diabetes. After some engagement with community members, we learned about pancreatogenic diabetes or type 3c. This is a type of diabetes that follows the removal of parts of the pancreas due reasons such as cancer. Our project may be a viable treatment for this type of diabetes in addition to type 1. An extended investigation is being conducted to see if our project could be used in diabetic animals as well.
Sa-mRNA uses numerous parts to amplify its sequence. The specific parts used can be found in the parts section of our wiki. This amplification undergoes a few key steps to copy the sequences of the mRNA, illustrated below in figure 1. Initially, an RNA-dependent RNA polymerase complex is formed by the NSP1-4 genes in the positive sense of genomic sa-mRNA and is responsible for the “self-amplifying” aspect of the sa-mRNA (Bloom et al., 2020). 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 positive sense genomic sa-mRNA. A subgenomic promoter allows for the production of subgenomic RNAs, which only hold the gene for the protein of interest (Minnaert et al., 2021).
Figure 1. After the Dia-Beatable sa-mRNA is injected into the patient, the encapsulated sa-mRNA can safely enter cells adjacent to the bloodstream or interstitial space while avoiding detection from the immune system. In the cytoplasm of the cells are ribosomes, the cellular machinery that will use the Dia-Beatable sa-mRNA as instructions for making NSP1-4 (step 1). The NSP1-4 use that same sa-mRNA molecule as a template for making complimentary negative sense sa-mRNA (step 2). NSP1-4 autocatalytically cleaves into four NSPs that reform into a new cellular machine (step 3) that uses the negative sense sa-mRNA as a template for making new positive sense sa-mRNA, like the original that was injected (step 4). Using the subgenomic promoter, a subgenomic mRNA is also made, which contains the gene of insulin (step 4). The ribosomes within the cytoplasm will use either the sa-mRNA or the subgenomic mRNA as a template for making insulin (step 5). Both RNAs contain an insulin-binding aptamer that will prevent ribosomes binding, and thus translation if insulin concentrations are too high in the cell (step 6).
In order to implement Dia-Beatable in the real world, we would first patent our treatment. Our team connected with Ian Andrews who specializes in the intricate world of scientific patents. Please refer to the interview notes that can be found in the human practices section of our wiki (https://2022.igem.wiki/lethbridge-hs/interviews.html). He recommended we explore distribution and commercialization through Tech Connect, a local program in Lethbridge. Further collaboration with Ian Andrews could provide more insight into supplying products to health services across the globe.
For implementation into the healthcare system, our project could potentially be injected as a diabetes treatment at a hospital, pharmacy or vet clinic by trained professionals. Based on current research publications it is estimated that our sa-mRNA may be viable in the human body for 6 weeks. Re-use of the product is dependent on immune response evasion, RNA stability, and reliable delivery to cells. Encapsulating the sa-mRNA in something like an ionizable lipid platform like Acuitas “A9” product is important for achieving these goals. Lipid nanoparticles have been used for delivery of mRNA COVID vaccines (Hou, et al., 2021). By utilizing Dia-Beatable in a responsible, repeatable manner, the current mundane daily requirements of several insulin injections daily would be eliminated for people with type 1 diabetes.
Sa-RNA-based injections are different from conventional vaccine production in that they utilize DNA templates and low-temperature storage. Conventional injection productions only require cell-free systems. Sa-RNA-based injections are usually summarized with specific nanolipids, buffers, and filtrations to produce bulk drug products. Afterwards, the vaccine follows standard procedures of being stored in vials, labeled, and packaged for delivery. However, sa-RNA is advantageous in that it is able to produce doses more rapidly than conventional means.
Every sa-RNA treatment is different from the next. Thus, each production method must differ to best produce the treatment. In Dia-Beatable's case, our best option is to estimate how many doses a single factory or team can produce in a certain timeframe. And, based on how that estimate plays out in real-team, the team will have to adapt the estimate for future expansions to ascertain a reliable production plan. We will also have to plan sterilization procedures and storage measures for a production facility (Vickers et al., 2022).
With any genetic engineering project, there are safety aspects to consider, these concerns are amplified when any living system is directly impacted. Part of our designed project includes non-structural proteins 1-4 sequences from the Venezualan Equine Encephalitis virus. This is necessary for the project to be self-amplifying but inversely increases the virality of our construct. Other safety concerns involve injecting any foreign bodies into the body. Later experimental trials will provide further knowledge regarding the immune system and other systems response to injections. The backbone of our project could also be twisted for a number of ulterior motives. The designed system of producing proteins in the body could be switched with any protein. This could lead to harmful uses of our system (see Safety page for more detail).
However, there will also be individuals hesitant to accept Dia-Beatable as there is a stigma surrounding genetically engineered machines. For example, there are individuals who dislike taking injections due to a pseudo-belief that there are government bugs inside of the injection (Bertin, et al., 2020). This distrust often stems from a perception that scientific treatments and explanations go against personal moral beliefs, such as ones based in religious origins (Philipp-Muller et al., 2022). Our team has combated this issue of scientific distrust by going out into the community and explaining Dia-Beatable to community members. We also explain the science behind Dia-Beatable in our podcast, IGEP. Continuously explaining the facts behind our project will lessen the distrust around Dia-Beatable by clearing up the unknown.
References
[1] Bloom, K., van den Berg, F., & Arbuthnot, P. (2020, October 22). Self-amplifying RNA vaccines for infectious diseases. Gene Therapy, 28, 117-129. https://www.nature.com/articles/s41434-020-00204-y#:~:text=B%20Self%2Damplifying%20RNA%20encodes,and%20amplifies%20vaccine%2Dencoding%20transcripts
[2] Minnaert, A. M., Vanluchene, H. V., Verbeke, R. V., Lentacker, I. L., de Smedt, S. C. D., Raemdonck, K. R., Sanders, N. N. S., & Ramaut, K. R. (2021). Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across. Science Direct, 176. https://doi.org/10.1016/j.addr.2021.113900
[3] Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021). https://doi.org/10.1038/s41578-021-00358-0
[4] Vickers, J. V., Cramer, P. C., Eardley-Patel, R. E.-P., & Excell, O. E. (2022, August 1). Overcoming Engineering Challenges to Enable Commercial Scale mRNA Vaccine Manufacturing. BioPharm International, 35(8), 36–41. https://www.biopharminternational.com/view/overcoming-engineering-challenges-to-enable-commercial-scale-mrna-vaccine-manufacturing
[5] Bertin, P., Nera, K., & Delouvée, S. (2020, September 18). Conspiracy Beliefs, Rejection of Vaccination, and Support for hydroxychloroquine: A Conceptual Replication-Extension in the COVID-19 Pandemic Context. Frontiers in Psychology, 11. https://doi.org/10.3389/fpsyg.2020.565128