Individual Final Project

Abstract
Cold storage at 4 °C enhances platelet hemostatic function but induces a well-characterized storage lesion, including glycoprotein Ib (GPIb) clustering, desialylation, premature activation, and apoptosis. These alterations result in rapid clearance of transfused platelets, limiting their clinical utility despite improved function. Tardigrade-derived intrinsically disordered proteins (IDPs), such as CAHS, MAHS, and RAHS, form protective amorphous glass-like matrices under stress conditions, stabilizing proteins and membranes. This project aims to harness these properties to mitigate cold-induced platelet damage by engineering megakaryocytes and platelets to express IDPs. By incorporating optimized IDP gene constructs (synthesized via TWIST Bioscience), we seek to create platelets with enhanced structural resilience during cold storage, preserving both function and circulation time.

Specific Aim 1: Expression of Tardigrade IDPs in Mammalian Cells
Establish robust expression of CAHS, MAHS, and RAHS proteins in mammalian systems. Codon-optimized gene blocks will be synthesized and cloned into mammalian expression vectors under constitutive and inducible promoters. Expression will be validated using Western blotting, immunofluorescence, and subcellular localization analysis. Protein solubility and potential aggregation behavior will also be assessed to ensure compatibility with mammalian cellular environments.

Specific Aim 2: Optimization of IDP Constructs for Megakaryocyte and Platelet Expression
Refine IDP gene constructs for efficient expression in megakaryocytes and subsequent incorporation into platelets. This includes promoter selection (e.g., PF4 or CD41-specific promoters), transcript stability optimization, and evaluation of translational efficiency. Constructs will be tested in megakaryocytic cell lines (e.g., MEG-01) and primary differentiation systems. Functional assays will assess whether IDP expression alters baseline platelet activation, morphology, or viability.

Specific Aim 3: Generation of Engineered Megakaryocytes Producing IDP-Containing Platelets
Develop engineered megakaryocytes that stably express IDPs and produce modified platelets. Lentiviral or CRISPR-based integration strategies will be used to introduce IDP genes into hematopoietic progenitors or megakaryocyte precursors. Platelets derived from these cells will be analyzed for IDP retention, structural integrity, and resistance to cold-induced storage lesions. Functional assays will include aggregation, adhesion, and clearance studies in vitro and, if feasible, in vivo models.

Background and Significance
Platelet transfusion is critical in managing bleeding disorders, yet storage remains a major limitation. While cold storage enhances immediate hemostatic function, it accelerates clearance due to biochemical and structural changes collectively termed the storage lesion. These include receptor clustering (notably GPIb), loss of sialic acid residues, and activation of apoptotic pathways. Current strategies to mitigate these effects have had limited success.

Tardigrades, extremophiles capable of surviving desiccation and temperature extremes, utilize intrinsically disordered proteins (IDPs) to form reversible, glass-like matrices that stabilize cellular components. CAHS (cytosolic abundant heat-soluble), MAHS (mitochondrial), and RAHS (secretory) proteins are key mediators of this protection. Their ability to buffer macromolecular structures without requiring rigid folding makes them attractive candidates for preserving platelet integrity under cold stress. Introducing these proteins into platelets represents a novel bioengineering approach to extend platelet shelf life while maintaining function.

Experimental Design
The project will begin with synthesis of codon-optimized IDP gene blocks via TWIST Bioscience, followed by cloning into mammalian expression vectors. In Aim 1, transient transfection into HEK293 and other model cell lines will establish baseline expression, localization, and biophysical behavior of IDPs.

In Aim 2, constructs will be tailored for megakaryocyte-specific expression using lineage-restricted promoters and regulatory elements. These will be tested in megakaryocytic cell lines and stem cell-derived megakaryocytes, with evaluation of expression efficiency and cellular compatibility.

In Aim 3, stable integration strategies (lentiviral transduction or CRISPR/Cas9 knock-in) will be employed to generate engineered megakaryocytes. Platelets derived from these cells will be subjected to cold storage assays at 4 °C. Key readouts will include GPIb clustering (via microscopy), desialylation (lectin binding assays), activation markers (e.g., CD62P), apoptosis indicators, and functional assays such as aggregation and adhesion. Clearance potential may be assessed using macrophage uptake assays or animal models if available.

Together, these experiments will test whether IDP expression can act as a physical and molecular buffer against cold-induced platelet damage, providing a foundation for next-generation platelet storage technologies.

Group Final Project