Natural and engineered mediators of desiccation tolerance stabilize Human Blood Clotting Factor VIII in a dry state

Abstract

Biologics, pharmaceuticals containing or derived from living organisms, such as vaccines, antibodies, stem cells, blood, and blood products are a cornerstone of modern medicine. However, nearly all biologics have a major deficiency: they are inherently unstable, requiring storage under constant cold conditions. The so-called "cold-chain", while effective, represents a serious economic and logistical hurdle for deploying biologics in remote, underdeveloped, or austere settings where access to cold-chain infrastructure ranging from refrigerators and freezers to stable electricity is limited. To address this issue, we explore the possibility of using anhydrobiosis, the ability of organisms such as tardigrades to enter a reversible state of suspended animation brought on by extreme drying, as a jumping off point in the development of dry storage technology that would allow biologics to be kept in a desiccated state under ambient or even elevated temperatures. Here we examine the ability of different protein and sugar-based mediators of anhydrobiosis derived from tardigrades and other anhydrobiotic organisms to stabilize Human Blood Clotting Factor VIII under repeated dehydration/rehydration cycles, thermal stress, and long-term dry storage conditions. We find that while both protein and sugar-based protectants can stabilize the biologic pharmaceutical Human Blood Clotting Factor FVIII under all these conditions, protein-based mediators offer more accessible avenues for engineering and thus tuning of protective function. Using classic protein engineering approaches, we fine tune the biophysical properties of a protein-based mediator of anhydrobiosis derived from a tardigrade, CAHS D. Modulating the ability of CAHS D to form hydrogels made the protein better or worse at providing protection to Human Blood Clotting Factor VIII under different conditions. This study demonstrates the effectiveness of tardigrade CAHS proteins and other mediators of desiccation tolerance at preserving the function of a biologic without the need for the cold-chain. In addition, our study demonstrates that engineering approaches can tune natural products to serve specific protective functions, such as coping with desiccation cycling versus thermal stress. Ultimately, these findings provide a proof of principle that our reliance on the cold-chain to stabilize life-saving pharmaceuticals can be broken using natural and engineered mediators of desiccation tolerance.

Labile assembly of a tardigrade protein induces biostasis

“…The observation that regardless of their condensed state CAHS proteins are protective in cells during osmotic shock intrigued us. This observation is in line with reports that in vitro, CAHS D provides protection in both its soluble and gelled form albeit via distinct mechanisms (Hesgrove et al, 2021;Packebush et al, 2023).…”

“…This variant does not slow metabolism, which suggests that CAHS D is capable of conferring protection against hyperosmotic stress at the cellular level in both in condensed and uncondensed form, likely through distinct mechanisms. This is in line with previous in vitro studies which show that gelling and non-gelling variants of CAHS D protect labile proteins to varying degrees during desiccation (Biswas et al, 2024;Hesgrove et al, 2021;Packebush et al, 2023). In its non-condensed form, CAHS D might confer protection to cells undergoing hyperosmotic stress through the prevention of protein aggregation via molecular shielding and/or stabilization of membranes via helical absorption (Hesgrove & Boothby, 2020).…”

“…CAHS D (also known as CAHS8_HYPEX; Uniprot: P0CU50) is a highly charged 227-residue protein that is disordered Eicher et al, 2022;Malki et al, 2022;Wang et al, 2023) and is required for the tardigrade H. exemplaris to survive desiccation robustly . Through the course of studying this protein, we and others have observed that CAHS D protein undergoes a solution-to-gel phase transition, which is in line with the behavior of other CAHS proteins (Figure 1a) (Eicher et al, 2022;Malki et al, 2022;Nguyen et al, 2022;Packebush et al, 2023;Sanchez-Martinez et al, 2023;Tanaka et al, 2022;Yagi-Utsumi et al, 2021).…”

Helicity of a tardigrade disordered protein contributes to its protective function during desiccation

“…To further assess the contribution of the helical linker in the context of the full‐length protein (e.g., with an N‐ and C‐termini), we utilized two variants, 2X LR and Ash1 LR (Figure 2A). 2X LR, is a variant of CAHS D generated by the tandem duplication of the helical linker region (Packebush et al, 2023). Ash1 LR is a variant generated by replacing the endogenous linker of CAHS D with a duplicated portion of the Ash1 domain of ASH1 (UniProt P34233), a protein involved in transcriptional regulation in S. cerevisiae with no connection to desiccation tolerance (Chandarlapaty & Errede, 1998).…”

“…The adoption of such higher‐order quaternary structure, either through helical interactions or other means, is likely to also play a role in promoting protection during drying (Belott et al, 2020; Eicher et al, 2022; Malki et al, 2022; Sanchez‐Martinez, Nguyen, et al, 2023; Tanaka et al, 2022; Yagi‐Utsumi et al, 2021). The formation of these higher‐order structures, at least for CAHS proteins, has been proposed to mediate desiccation tolerance at the protein level through several potential mechanisms including: coordination of water, providing a matrix for entrapment, or induction of a viscous state that slows unfolding/aggregation (Boothby, 2021; Boothby et al, 2017; Packebush et al, 2023; Piszkiewicz et al, 2019; Sanchez‐Martinez, Ramirez, et al, 2023). It should be noted that these mechanisms of protein protection are not mutually exclusive, and CAHS oligomerization may confer protection at additional scales, for example, at the cellular level via the formation of a cytoskeletal‐like network and/or induction of biostasis (Eicher et al, 2022; Sanchez‐Martinez, Nguyen, et al, 2023; Tanaka et al, 2022; Yagi‐Utsumi et al, 2021).…”