Abstract
Using lentiviral technology, we recently demonstrated that incorporation of CD27 costimulation into CARs greatly improves antitumor activity and T cell persistence. Still, virus-mediated gene transfer is expensive, laborious and enables long-term persistence, creating therapies which cannot be easily discontinued if toxic. To address these concerns, we utilized a non-integrating RNA platform to engineer human T cells to express FRα-specific, CD27 CARs and tested their capacity to eliminate human FRα(+) cancer. Novel CARs comprised of human components were constructed, C4-27z and C4opt-27z, a codon-optimized variant created for efficient expression. Following RNA electroporation, C4-27z and C4opt-27z CAR expression is initially ubiquitous but progressively declines across T cell populations. In addition, C4-27z and C4opt-27z RNA CAR T cells secrete high levels of Th-1 cytokines and display strong cytolytic function against human FRα(+) cancers in a time- and antigen-dependent manner. Further, C4-27z and C4opt-27z CAR T cells exhibit significant proliferation in vivo, facilitate the complete regression of fully disseminated human ovarian cancer xenografts in mice and reduce the progression of solid ovarian cancer. These results advocate for rapid progression of C4opt-27z RNA CAR to the clinic and establish a new paradigm for preclinical optimization and validation of RNA CAR candidates destined for clinical translation.
Highlights
Due to difficulties associated with genetically modifying primary T lymphocytes using non-viral based systems, investigators have generally utilized retroviral and lentiviral vectors in experiments that required high levels of transgene expression and viability in human T cells [1,2,3]
A single open reading frames (ORFs) in the reverse complement strand at nucleotide position 1511 could not be removed as a switch from CAC to CAT (His at amino acid position 493) which would have created a new ORF in the antisense strand
CAR constructs were subcloned into a pD-A.lenti cloning site.2bg.150A vector (PDA) that was optimized for T cell transfection, CAR expression and RNA production [18]
Summary
Due to difficulties associated with genetically modifying primary T lymphocytes using non-viral based systems, investigators have generally utilized retroviral and lentiviral vectors in experiments that required high levels of transgene expression and viability in human T cells [1,2,3]. Adverse effects in patients related to administration of genetically-redirected T cells, including cytokine storm, cardiac arrhythmia, respiratory failure, seizures and even mortality have been reported using viral-based CAR T cell therapy [5,6,7,8,9,10,11]. In light of these potential events, a need exists for alternative, safe and effective gene transfer in T lymphocyte research, CAR development and clinical application. The transmission of CAR-based RNAs into T lymphocytes redirected these lymphocytes to recognize and destroy human leukemia in vivo, using various mouse models [17,18,19,20,21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
