Nonviral RNA Transfection to Transiently Modify T Cells with Chimeric Antigen Receptors for Adoptive Therapy

Abstract

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.

CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia

“…Short-term expression may also be desirable for CARs directed against antigens expressed on normal cells when sustained on-target toxicity is a concern. T cells engineered by RNA insertion through electroporation 36 have marked replicative capacity and can produce substantial tumor responses. 37,38 However, expression typically lasts 7 days or less, so long-term disease control, although still possible, would require multiple infusions with this approach.…”

Mechanisms of resistance to CAR T cell therapy

“…Certain aspects of vector design are likely to affect the efficiency of gene transfer and, potentially, the activity of the resulting CAR T cell product, although these aspects have not been systematically evaluated. RNA transfection has also been used to deliver the CAR gene construct, resulting in transient CAR expression (because the CAR gene is not inserted into the genome of the T cells 50 ). Such transient CAR-expressing T cell products have only short-lived benefits in preclinical models, with no durable responses observed 51 .…”

Acute lymphoblastic leukemia: a comprehensive review and 2017 update

“…148 Another method of gene delivery involves direct transfer of an mRNA construct through electroporation. 149 As no DNA is inserted into the genome of the T-cell, this eliminates the risk of malignant transformation. Given the high replicative potential of these T cells, this methods also offers the advantage of a profound antitumor response.…”