Tracking Engineered T Cell Therapies with an RNA-based Reporter Gene

• Published in: The Journal of nuclear medicine (1978) Vol. 60
• Main Authors: Chang, Bryan, Shang, Catherine, Lohith, Katheryn, Farwell, Michael
• Format: Journal Article
• Language: English
• Published: New York Society of Nuclear Medicine 01.05.2019
• Subjects: Affinity; Antigens; Aptamers; Binding; Biotechnology; Cell therapy; Chimeric antigen receptors; Design optimization; Expression vectors; Feasibility studies; Gene expression; In vivo methods and tests; Kinases; Lymphocytes; Lymphocytes T; Mammals; Nucleotide sequence; Nucleotides; Receptors; Reporter gene; Ribonucleic acid; RNA; Solid tumors; Substrate preferences; Substrates; Tracking; Tritium; Tumors
• ISSN: 0161-5505; 1535-5667

Purpose: T cells engineered with chimeric antigen receptors (CARs) have shown dramatic activity in a variety of cancers. However, a major obstacle in the development of CAR T cells directed against solid tumors is the difficulty in assessing treatment efficacy. Thus, in vivo cell-tracking methods are needed to noninvasively monitor the administered cells. A novel approach to in vivo cell tracking, which may overcome some of the limitations of the currently available protein-based reporter genes, involves the use of an RNA aptamer as a reporter gene. Here, we explored the feasibility of using a tetracycline-binding RNA aptamer, with one of the highest known aptamer/ligand substrate affinities (KD = 0.6 nM), as a reporter gene for cell-based therapies. Materials and Methods: Using the sequence of a previously described tetracycline-binding RNA aptamer, three expression vectors were generated: a vector encoding a single RNA aptamer; two additional vectors with 8 and 16 sequential tandem RNA aptamers, respectively, separated by short intervening stretches of 17 randomized nucleotides. These constructs were then stably transduced into a mammalian cell lines (HCT116 and HEK293). Cell uptake experiments were performed using commercially available tritium-labeled [3H]-tetracycline. Results: Initial uptake studies with commercially available 3H-tetracycline and the aptamer-expressing cell lines were compared to a negative control (wild type cell lines). Uptake was performed in triplicate in a 12-well plate with 1 million cells per well. Cells were incubated with 0.1 μCi 3H-tetracycline per well for 24 hours, then washed once with PBS. Blocking was performed with 10 μM cold tetracycline. The results reveal 57% increased uptake for the aptamer-expressing cell line when compared to control and successful blocking. Conclusions: Vectors containing tandem repeats of the high-affinity tetracycline-binding RNA aptamer were successfully transduced into two mammalian cell lines, and uptake studies with 3H-tetracycline demonstrated increased uptake in the RNA aptamer-containing cells compared to controls. Future directions will include optimization of the design and expression of the RNA aptamer, uptake studies with 123I-tetracycline, and in vivo imaging studies in mice.