title: Large volume flow electroporation of mRNA: clinical scale process.
abstract: Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3-7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency: >80% lysis at effector-target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery.
authors.affiliation: MaxCyte Inc., Gaithersburg, MD, USA.
authors.forename: Linhong
authors.initials: L
authors.lastname: Li
authors.forename: Cornell
authors.initials: C
authors.lastname: Allen
authors.forename: Rama
authors.initials: R
authors.lastname: Shivakumar
authors.forename: Madhusudan V
authors.initials: MV
authors.lastname: Peshwa
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D018274:Electroporation / Q000379:methods*; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011946:Receptors, Antigen / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014162:Transfection / Q000379:methods*
chemical_list: D018941:Antigens, CD19; D012333:RNA, Messenger; D011946:Receptors, Antigen