T2D patients inject themselves with medication three to four times a day and prick their skin to measure blood glucose an additional three to four times a day [1]. Subcutaneous administration of T2D medications host a variety of issues including needle supply, needle sterility, and bloodborne pathogens such as hepatitis B, hepatitis C, and HIV [2]. Unsafe injections are more prevalent in resource constrained communities, making this an especially pressing issue to address given such countries are disproportionately impacted by T2D [3]. We decided to reduce these potential hazards by eliminating needles and pursuing Helo as an orally deliverable T2D medication.
For S. cerevisiae to become an accessible and novel eukaryotic host for oral drug delivery, we must first modify its surface proteins [4]. Surface protein modification transforms S. cerevisiae cells into accurate therapeutic carriers, enhancing drug delivery efficiency [5]. By reinforcing S. cerevisiae's cell wall, otherwise known as bioencapsulation, we hypothesize Ex-4 will be able to bypass the gastrointestinal barrier and target epithelial M cells for final drug release. We plan to test various surface protein modifications to determine the ideal edit for our desired dosage.
To further implement our system, we plan to replace our galactose promoter for Ex-4 with an alternative lactose promoter (LAC9) [6]. As lactose is abundant in milk [7], it is it is an optimal Ex-4 protein expression inducer that is economical and widely accessible with over six billion people consuming milk globally [8]. Due to our genomic insert's current design, our yeast would only be able to produce Ex-4 in milk that contains lactase; the enzyme which breaks down lactose into glucose and galactose. This enzyme can be bought under the brand LACTAID® but adds an additional cost and assumes LACTAID® is accessible by our target users. By replacing our GAL1 promoter with a LAC9 promoter, we hypothesize our S. cerevisiae will express Ex-4 in milk, meaning global communities will be able to continuously produce their own T2D medication locally. Further, the genomic integration of Ex-4 in S. cerevisiae implies stable propagation of the protein for generations.
Lastly, an elastin-based polypeptide half-life extender sequence was added to our insert on the C-terminus of the S. cerevisiae gene fragment. The half-life extender is made up of a ten times repeat of the VPGVG amino acid sequence, and has been shown to increase the half-life of Ex-4 by 3.7 fold [9]. We will utilize this component once we reach in vivo trials. The optimized codon sequence will produce our functional protein when translated, and the half-life extender should not interfere with the binding between Ex-4 and the GLP-1 R.
In the event that reinforcing the cell wall of S. cerevisiae does not result in effective bioencapsulation, we considered Ex-4 expression in plant and algal hosts. Both plant and algal cell walls act as bioencapsulation mechanisms to protect protein drugs for oral delivery [10]. Additionally, recombinant chloroplasts yield high levels of protein expression and proper folding [11][12][13]. We also considered soybeans for their low glycemic index of 4.5, bioencapsulation ability, history of transformation, and well documented genome [14].
Furthermore, we looked into the algae, Chlamydomonas reinhardtii (C. reinhardtii), as a potential host. C. reinhardtii is known as a photosynthetic yeast due to its rapid growth and high photosynthetic efficiency [15]. Recombinant therapeutic proteins have been successfully expressed within chloroplasts and are therefore worth investigating for Ex-4 production [16]. Exploring alternative hosts will allow us to compare bioavailability and protein production, as well as give us a holistic understanding of potential delivery systems to increase Ex-4 accessibility.
After meeting with several endocrinologists, we discovered Ex-4 functions exclusively in the presence of elevated blood glucose, meaning that Ex-4 induced hypoglycemia is highly unlikely [17]. However, understanding the limits of our dosage remains an important component to the safe implementation of Helo. To ascertain what dose of Ex-4 we want S. cerevisiae to produce, our team coded a dosage-effect model. This model shows the correlation between a given dosage of Ex-4 and its pharmacological effect: insulin production within pancreatic beta cells. The formula used to model this effect falls under the umbrella of pharmacodynamic. We will collect data for this model by incubating recombinant Ex-4 produced by S. cerevisiae and E. coli with pancreatic mice cells. If Ex-4 binds to the GLP-1 R on those cells, we expect to see an influx of insulin secretion as measured by an Enzyme-Linked ImmunoSorbent Assay (ELISA).
A large aspect of our project was ensuring integration of our Ex-4 gene cassette into a safe and edible organism. S. cerevisiae is Generally Recognized as Safe (GRAS) by the U.S. FDA [18] and is commonly referred to as baker's yeast.
Cold chain storage describes the system of transporting and storing medications in refrigerated vehicles when en route to pharmacies. This system limits access to medication for many resource constrained communities. method of bypassing cold chain storage for our product would be to distribute our recombinant S. cerevisiae cultures as Active Dry Yeast (ADY). ADY has a shelf-life of around 12 to 18 months and does not require refrigerated storage [19]. Since Ex-4 is a heat stable protein at room temperature [20], we hypothesize that it could be contained within our dried yeast without experiencing denaturation.
By mixing our ADY with sugar and warm water, T2D patients can grow an S. cerevisiae starter culture. After the ADY is rehydrated, it can be stored in a refrigerator for up to two weeks [21]. S. cerevisiae can grow in the presence of sucrose or maltose [22] [23], two sugars that are abundantly present in most places around the world. For sucrose based growth media, communities can use water mixed with sugarcane, honey, maple syrup, sugar beets, molasses, agave, dates, and other fresh or canned fruits [24] to support culture growth. This culture may then be induced with milk to express Ex-4 via the LAC9 promoter we plan to implement within our system. We theorize that Ex-4 may be stored and propagated at local pharmacies and communities in less than one square meter.
Further, we plan to explore other strains of yeast such as Yarrowia lipolytica and Pichia pastoris as potential Ex-4 hosts given their low maintenance care, diverse set of growing conditions, and GRAS status [25][26]. Our newly established background in yeast engineering will allow us to test and choose the best Ex-4 producing strain of yeast. In addition, it is imperative the system we create prioritizes accessibility, low-cost, and safety. Our system will not be brought to market until we are confident that Helo is effective and safe to consume as confirmed by governmental bodies such as the U.S. FDA. We will perform a western blot analysis, binding affinity assay, and in vitro pancreatic mice cell culture test as steps to confirm the functionality and safety of our recombinant Ex-4.
Helo will be considered a genetically modified organism (GMO), which is a label that comes with many controversial aspects. However, Ex-4 is approved by the U.S. FDA and is currently marketed for subcutaneous injection [27]. We aim to show that using living systems to produce protein drugs is a cheaper and more accessible alternative to traditional drug manufacturing. Our locally grown system will be independent from large pharmaceutical companies while still maintaining efficacy and safety. Hence, we hope to reduce the stigmas associated with GMOs while simultaneously providing more accessible treatment for T2D patients.