Natural and engineered mediators of desiccation tolerance stabilize Human Blood Clotting Factor VIII in a dry state
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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 sugarbased 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.
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The authors developed an assay to measure the efficacy of molecules that protect against environmental stress. They measured the ability of specific disaccharides and proteins to preserve the function of Human Blood Clotting Factor VIII in a clotting assay after repeated cycles of desiccation. As expected sucrose and trehalose stabilize FVIII after repeated cycles of desiccation (Figure 2). Full length CAHS D, derived from a tardigrade, does not protect the function of FVIII (Fig 3). However, the linker region alone of CAHS D protects FVIII even at low concentrations (Fig 3). They find that other intrinsically disordered proteins have a mixed effect on FVIII protection. LEA1 protects FVIII from desiccation but Hero9 does not (Fig 4). They go on to measure the protection of FVIII under thermal stress. They have an interesting result …
The authors developed an assay to measure the efficacy of molecules that protect against environmental stress. They measured the ability of specific disaccharides and proteins to preserve the function of Human Blood Clotting Factor VIII in a clotting assay after repeated cycles of desiccation. As expected sucrose and trehalose stabilize FVIII after repeated cycles of desiccation (Figure 2). Full length CAHS D, derived from a tardigrade, does not protect the function of FVIII (Fig 3). However, the linker region alone of CAHS D protects FVIII even at low concentrations (Fig 3). They find that other intrinsically disordered proteins have a mixed effect on FVIII protection. LEA1 protects FVIII from desiccation but Hero9 does not (Fig 4). They go on to measure the protection of FVIII under thermal stress. They have an interesting result whereby the CAHS D with a 2x linker protects FVIII from thermal stress (Fig 6) whereas the linker region alone of CAHSD had the most striking protection of FVIII under desiccation. I have some questions and suggestions for the authors.
Questions:
- Could you speculate about the biophysical/biochemical mechanism by which the different CAHS D constructs confer desiccation or heat resistance?
- Is it surprising that the construct that does not form a gel (Linker) is the one that confers the best desiccation resistance?
- Is there a biophysical or biochemical explanation for how a gel-forming protein might confer heat resistance but not desiccation resistance?
- If you compare the primary sequences or the domain structures of CAHS D, Hero9, and LEA1 are there any interesting similarities or differences that might explain the results you've obtained?
Comments:
- Are you using vector graphics in the figures? It is very hard to read much of the text in the figures. This often occurs when using images for line art instead of vector graphics.
- Figure 1 is not necessary or helpful. It would be more helpful to the reader to have a diagram depicting the actual assay being performed in the subsequent figures.
- It would be helpful to know how many times each experiment was repeated. This could be reported in the figure legends.
- It would also be helpful to readers to have a graphical summary of the results in a final figure that summarizes the protective effects of the various molecules tested.
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The authors developed an assay to measure the efficacy of molecules that protect against environmental stress. They measured the ability of specific disaccharides and proteins to preserve the function of Human Blood Clotting Factor VIII in a clotting assay after repeated cycles of desiccation. As expected sucrose and trehalose stabilize FVIII after repeated cycles of desiccation (Figure 2). Full length CAHS D, derived from a tardigrade, does not protect the function of FVIII (Fig 3). However, the linker region alone of CAHS D protects FVIII even at low concentrations (Fig 3). They find that other intrinsically disordered proteins have a mixed effect on FVIII protection. LEA1 protects FVIII from desiccation but Hero9 does not (Fig 4). They go on to measure the protection of FVIII under thermal stress. They have an interesting result …
The authors developed an assay to measure the efficacy of molecules that protect against environmental stress. They measured the ability of specific disaccharides and proteins to preserve the function of Human Blood Clotting Factor VIII in a clotting assay after repeated cycles of desiccation. As expected sucrose and trehalose stabilize FVIII after repeated cycles of desiccation (Figure 2). Full length CAHS D, derived from a tardigrade, does not protect the function of FVIII (Fig 3). However, the linker region alone of CAHS D protects FVIII even at low concentrations (Fig 3). They find that other intrinsically disordered proteins have a mixed effect on FVIII protection. LEA1 protects FVIII from desiccation but Hero9 does not (Fig 4). They go on to measure the protection of FVIII under thermal stress. They have an interesting result whereby the CAHS D with a 2x linker protects FVIII from thermal stress (Fig 6) whereas the linker region alone of CAHSD had the most striking protection of FVIII under desiccation. I have some questions and suggestions for the authors.
Questions:
- Could you speculate about the biophysical/biochemical mechanism by which the different CAHS D constructs confer desiccation or heat resistance?
- Is it surprising that the construct that does not form a gel (Linker) is the one that confers the best desiccation resistance?
- Is there a biophysical or biochemical explanation for how a gel-forming protein might confer heat resistance but not desiccation resistance?
- If you compare the primary sequences or the domain structures of CAHS D, Hero9, and LEA1 are there any interesting similarities or differences that might explain the results you've obtained?
Comments:
- Are you using vector graphics in the figures? It is very hard to read much of the text in the figures. This often occurs when using images for line art instead of vector graphics.
- Figure 1 is not necessary or helpful. It would be more helpful to the reader to have a diagram depicting the actual assay being performed in the subsequent figures.
- It would be helpful to know how many times each experiment was repeated. This could be reported in the figure legends.
- It would also be helpful to readers to have a graphical summary of the results in a final figure that summarizes the protective effects of the various molecules tested.
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