Overview

Blood type incompatibility is one of the biggest barriers to emergency blood transfusions due to the lack of access to large blood banks or blood donors. The primary cause of incompatibility is the presence of antigens and extended chains on the surface of red blood cells. By enzymatically cleaving these groups, we aim to reduce incompatibilities, especially in emergency settings, where donor blood is scarce.

ABO System

Human blood types are categorized based on the presence of different antigen groups on the surface of red blood cells. Most common blood types of the ABO system have the H antigen, which serves as the precursor structure for the A and B antigens. In type A blood, the H antigen is enzymatically modified to form the A antigen; in type B blood, it is modified to form the B antigen; in type AB blood, both modifications occur, resulting in the presence of both A and B antigens; and in type O blood, the H antigen remains unmodified.

Extended Antigen Chains

Both the A and B antigens can also be extended by the addition of one or more monosaccharides, forming extended structures. These extended antigen epitopes are believed to further contribute to incompatibility problems between blood types [1]. By targeting not only the A and B antigens, but also their extended groups, we aim to create a more effective and safer way to increase the blood supply.

History of ECO RBC

Efforts to generate enzyme-converted O red blood cells (ECO RBC) date back to the 1960s, when researchers first demonstrated that certain exoglycosidases could cleave the terminal sugars of the A and B antigens to reveal the underlying H antigen [5]. The first clinically tested ECO-B RBCs were produced using an α-galactosidase from green coffee beans, which successfully removed the B antigens and proved safe in early transfusion trials [6]. However, these first generation systems required extremely high enzyme concentrations and acidic reaction conditions (pH 5 - 5.7), which are unsuitable for large-scale or clinical application [6]. Although the ECO cells circulated safely, crossmatch studies still showed unexpected agglutination reactions in 20 - 40% of recipient plasma samples, revealing that full immunological compatibility was not achieved [7].

Subsequent bacterial α-galactosidase and α-N-acetylgalactosaminidases with more neutral pH conditions were identified in the late 1990s, but these also required long incubation times (12 - 24 hours) and still failed to achieve complete antigen removal [8]. These limitations highlighted that the problem extended beyond enzyme efficiency or pH tolerance and crossmatch reactivity persisted even when canonical A and B epitopes were cleaved.

Modern metagenomic approaches uncovered new glycosidase families from the human gut microbiome that improved conversion [9]. However, in depth clinical validation with crossmatch hemagglutination analysis has not been conducted for these enzyme groups.

Recent work using Akkermansia muciniphila exoglycosidases advanced the ECO concept by targeting both the canonical and extended antigens simultaneously. This dual approach significantly reduced crossmatch reactivity and improved compatibility between ECO RBCs and recipient plasma [1]. These extended antigen active enzymes therefore overcome many of the shortfalls of earlier generations, providing a more clinically viable path toward universal donor blood and motivating the devlopment of our portable enzymatic conversion kit.

Our Goal

Our goal is to increase access to safe, compatible blood in places where it's needed most. This year, we have developed the UNIglobin Enzymatic Blood Conversion Kit to enzymatically cleave A and B antigens as well as their extended chains. This work builds upon previous iGEM teams and researchers who have identified strategies to convert A and B type blood [2-4]. Additionally, as our kit is compact and easy to use, it is specially optimized for emergency situations, where access to safe, compatible blood is inaccessible.

Enzymatic Conversion

We have identified five enzymes from Akkermansia muciniphila that target the extended antigen groups of red blood cells [1]. Used in succession, this enzymatic workflow is able to generate O type blood from A, B, or AB type blood.

Our Kit

1. Enzyme Packs: For storage and transport purposes, we provided lyophilized enzymes that cleave antigens and extended chains of each blood type. After rehydration in phosphate buffer solution, enzymes are ready to use.
2. Miniaturized Electronic Antigen Biosensor (MEAB): A wireless biosensor for detecting the concentration of ABO antigens on red blood cells.
3. Filtration Bag: Utilizing SCOBY, a kombucha precursor, we developed a built-in filtering system to filter leukocytes and separate plasma from red blood cells without the need for lab equipment.

References

[1] Jensen, M., Stenfelt, L., Ricci Hagman, J. et al. Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood. Nat Microbiol 9, 1176–1188 (2024).

[2] Lenny, L., & Goldstein, J. (1980). Enzymatic removal of blood group B antigen from gibbon erythrocytes. Transfusion, 20, 618.

[3] Kobayashi, T., Liu, D., Ogawa, H., Miwa, Y., Nagasaka, T., Maruyama, S., ... & Nakao, A. (2007). Alternative strategy for overcoming ABO incompatibility. Transplantation, 83(9), 1284-1286.

[4] TAS-Taipei 2021 iGEM Team. 2021.igem.org/Team:TAS_Taipei

[5] Fujisawa, K., Furukawa, K., Akabane, J. Studies on A and B blood group substances of human erythrocytes by blood group-specific enzymes. Kita Kanto Igatu, 13, 19-39 (1963).

[6] Goldstein, J., Siviglia, G., Hurst, R., Lenny, L. & Reich, L. Group B Erythrocytes Enzymatically Converted to Group O Survive Normally in A , B , and O Individuals. Science. 215, 168–170 (1982).

[7] Kruskall, M. S. et al. Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O. Transfusion 40, 1290–1298 (2000).

[8] Izumi, K., Yamamoto, K., Tochikura, T. & Hirabayashi, Y. Serological study using α-Nacetylgalactosaminidase from Acremonium sp. BBA - Gen. Subj. 1116, 72–74 (1992)

[9] Rahfeld P., Sim L., Moon H., Constantinescu I., Morgan-Lang C., Hallam S. J., Kizhakkedathu J. N., and Withers S. G. (2019) An enzymatic pathway in the human gut microbiome that converts A to universal O type blood. Nat. Microbiol. 4, 1475–1485 10.1038/s41564-019-0469-7