{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:03:15Z","timestamp":1768521795451,"version":"3.49.0"},"posted":{"date-parts":[[2025,6,15]]},"group-title":"Cell Biology","reference-count":34,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,6,15]],"date-time":"2025-06-15T00:00:00Z","timestamp":1749945600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["R01HL130764"],"award-info":[{"award-number":["R01HL130764"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,6,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Thrombotic Thrombocytopenic Purpura (TTP) is caused by congenital or acquired deficiency of ADAMTS13, a metalloproteinase that cleaves von Willebrand Factor (vWF) multimers. Current treatments\u2014plasma exchange and immunosuppression\u2014are costly and associated with significant morbidity therefore, alternative strategies are needed.<\/jats:p>\n                <jats:p>We developed the kitJak2 platform for producing genetically engineered lab-grown red blood cells (lgRBCs) as drug delivery vectors. We hypothesized that membrane-bound ADAMTS13 displayed on lgRBCs could provide a durable treatment for TTP. To test this, we engineered erythroid cells expressing both wild-type and mutant variants MDTCS fragments of ADAMTS13, conferring resistance to autoantibodies. Flow cytometry and FRET-based assays confirmed robust membrane expression and enzymatic activity. Importantly, mutant MDTCS variants retained catalytic activity in the presence of plasma from TTP patients, whereas wild-type variants were inhibited.<\/jats:p>\n                <jats:p>For in vivo evaluation, we generated transgenic mice expressing MDTCS ADAMTS13 on their RBC membranes. These mice exhibited normal RBC half-lives and stable, catalytically active ADAMTS13 expression. Using a murine model of TTP\u2014where ADAMTS13 knockout mice challenged with recombinant human vWF (rhvWF) develop thrombocytopenia and schistocytes\u2014we demonstrated that transfusion of ADAMTS13-expressing RBCs significantly mitigated disease, preventing platelet loss and schistocyte formation. This confirms that membrane-bound MDTCS ADAMTS13 cleaves circulating rhvWF under physiological flow conditions in vivo.<\/jats:p>\n                <jats:p>Finally, employing our KitJak2 platform, we generated human enucleated lgRBCs expressing high levels of catalytically active ADAMTS13.<\/jats:p>\n                <jats:p>This novel work establishes proof-of-concept that membrane-anchored ADAMTS13-expressing lab- grown RBCs may offer a feasible and effective therapeutic approach for both congenital and acquired TTP.<\/jats:p>","DOI":"10.1101\/2025.06.12.659386","type":"posted-content","created":{"date-parts":[[2025,6,15]],"date-time":"2025-06-15T18:55:14Z","timestamp":1750013714000},"source":"Crossref","is-referenced-by-count":0,"title":["Proof-of-Concept in a Murine Model of Treatment of Thrombotic Thrombocytopenic Purpura Using Engineered Red Blood Cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3036-1005","authenticated-orcid":false,"given":"Karl S","family":"Roberts","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shouping","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Khulan","family":"Batbayar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zi","family":"Yan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5011-2420","authenticated-orcid":false,"given":"Joshua","family":"Muia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0692-4549","authenticated-orcid":false,"given":"J Justin","family":"Mulvey","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emmanuel","family":"Olivier","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James M","family":"Pullman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8977-6910","authenticated-orcid":false,"given":"Spero R","family":"Cataland","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1084-2135","authenticated-orcid":false,"given":"Eric E","family":"Bouhassira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025061716251314000_2025.06.12.659386v1.1","doi-asserted-by":"publisher","DOI":"10.1007\/s12185-009-0476-1"},{"key":"2025061716251314000_2025.06.12.659386v1.2","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2011-03-341131"},{"key":"2025061716251314000_2025.06.12.659386v1.3","doi-asserted-by":"crossref","unstructured":"Li J , Guo J , Yuan R , Xue L . 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