	US 12,378,523 B2
	Platelets as delivery agents
Keith Andrew Moskowitz, Westfield, IN (US); Amber Nicole Lee, Montgomery Village, MD (US); and Rafael Jorda, Merignac (FR)
Assigned to Cellphire, Inc., Rockville, MD (US)
Filed by Cellphire, Inc., Rockville, MD (US)
Filed on Jul. 28, 2023, as Appl. No. 18/361,772.
Application 18/361,772 is a continuation of application No. 16/697,401, filed on Nov. 27, 2019, granted, now 11,767,511.
Claims priority of provisional application 62/828,043, filed on Apr. 2, 2019.
Claims priority of provisional application 62/775,141, filed on Dec. 4, 2018.
Claims priority of provisional application 62/773,931, filed on Nov. 30, 2018.
Prior Publication US 2023/0383258 A1, Nov. 30, 2023
Int. Cl. C07H 21/02 (2006.01); C12N 5/078 (2010.01); C12N 15/113 (2010.01); C12N 15/85 (2006.01)

CPC C12N 5/0644 (2013.01) [C12N 15/113 (2013.01); C12N 15/85 (2013.01); C12N 2310/14 (2013.01); C12N 2310/141 (2013.01); C12N 2510/00 (2013.01)]

20 Claims

1. A method of preparing RNA agent-loaded cryopreserved platelets, comprising:

providing platelets;

treating the platelets with a RNA agent that is a siRNA and/or a miRNA in the presence of a cationic transfection reagent, and a loading buffer comprising a salt, a base, and a loading agent comprising one or more saccharides selected from the group consisting of trehalose, sucrose, maltose, mannose, dextrose, and xylose, for a time period in the range of 5 minutes to 4 hours, and at a temperature in the range of 18-42° C., to form RNA agent-loaded platelets; and

cryopreserving the RNA agent-loaded platelets in the presence of the loading buffer, dimethyl sulfoxide (DMSO), and polysucrose to form the RNA agent-loaded cryopreserved platelets,

wherein the RNA agent-loaded cryopreserved platelets after storage at a temperature of about −80° C., upon thawing:

retain at least 90% of the RNA agent, and

retain hemostatic function by displaying the property of occluding a capillary coated with collagen and tissue factor in an in vitro total thrombus-formation analysis system (T-TAS).