Introduction: RNA aptamers are small RNA oligonucleotides that bind to molecular targets in a manner analogous to antibodies. Theoretically, RNA aptamers have an advantage over antibodies in that large numbers of aptamers can be selected, sequenced and synthesized inexpensively in the laboratory. Here, we report a novel strategy for generating human T regulatory cell (Treg)-specific RNA aptamers.

Methods:

Human Treg-specific RNA aptamers were developed using whole cell systematic evolution of ligands by exponential enrichment (SELEX). Briefly, a diverse library of 2′fluoro-modified RNA sequences (∼1012) were transcribed from DNA templates containing random 20-mer regions flanked by SEL-2 specific constant sequences. Eight rounds of SELEX were conducted using mononuclear cells from a different donor for each round.

Cells were separated into Tregs (CD4+CD25high) and non-Treg CD4 cells (CD4+CD25low) by magnetic cell sorting using a CD4+CD25+ human Treg isolation kit. The aptamer library was pre-cleared using non-Treg CD4 cells. Unbound aptamers were incubated with Tregs from the same donor. Tregs were washed, lysed and Treg-associated aptamers amplified by RT-PCR with SEL2 primers, transcribed and prepared for the next round. Aptamers that bound to human Tregs were collected after each round and sequenced using Illumina-based high-throughput sequencing.

Results

SELEX was used to successfully enrich for Treg-specific RNA aptamers. Between 2,800,000 and 3,900,000 aptamers were sequenced per round. The number of unique reads decreased from 3,391,279 (initial library) to 594,206 (round 8). At the end of SELEX, 36 unique sequences were found in >1000 copies per million reads. The prevalence of most of these aptamers went up with each round of SELEX. For example, the aptamer designated as Tr-6 was not detected in the unselected pool of aptamers, but 220 copies were found after round 2, 1631 after round 4, and 4832 after round 8. After all 8 rounds, the top 36 sequences represented 25% of the total aptamer population.

Twenty-four aptamers were selected for further evaluation based on copy number, consistent enrichment after each round (indicating aptamers that bound to Tregs from each individual donor), bioinformatic properties that predict an aptamer with effective binding, and separation into different sequence and structure families, from which representative sequences were selected. Four aptamers have been tested for their ability to bind to Treg and CD4+ non-Tregs. All bind more extensively to Treg than to CD4+ non-Tregs.

Conclusion and future directions: A large number of human Treg-specific RNA aptamers was selected using whole cell SELEX. Ongoing studies are assessing the antigen specificity of the selected aptamers and whether they cross-react with Tregs from other species. The long term goal is to use these aptamers as in vitro diagnostic agents and therapeutically to reduce Treg activity, thereby enhancing the anti-tumor immune response.

Citation Format: Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner. Generation of T regulatory-cell specific RNA aptamers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2015-5022