Abstract 5210: Ablation of an immunogenic human endogenous retrovirus in renal cell carcinoma cells through dual-guide CRISPR-Cas9 genome editing

Human endogenous retroviruses (HERVs) comprise 8% of the human genome and can be abnormally expressed in different tumor cells. Our lab discovered a unique and highly immunogenic HERV-E, CT-RCC HERV-E, that is selectively expressed in most clear cell renal cell carcinoma (ccRCC) cells. Although the function of select HERVs in some tumors has been defined, the role of CT-RCC HERV-E in ccRCC remains unclear. To characterize the impact of HERV-E expression on tumor oncogenesis in ccRCC, we developed a highly efficient strategy to ablate CT-RCC HERV-E through dual-guide CRISPR-Cas9. For isolation of edited cells, we knocked in a truncated CD19 (tCD19) into the same gene edited locus using adeno-associated virus (AAV) as donor templates. In order to ablate the entire 8.8 kb HERV-E region, we designed three single guide RNAs (sgRNAs) flanking the CT-RCC HERV-E genomic locus: one at the upstream site, sgRNA HERV-E Upst(1), and two downstream, sgRNA HERV-E Dwst(3) and sgRNA HERV-E Dwst(4). A single sgRNA targeting β2M was used as a negative control. HERV-E knockout was verified via real-time PCR, and the combination of sgRNAs Upst1-Dwst3 were chosen as the guides for all experiments, due to their higher knockout efficiency. We tested the MOIs of 2, 4, and 10 × 105 for AAV transduction following CRISPR knockout. Expression of CD19 was the highest at the MOI of 10 × 105, which was used for all experiments. Two RCC cell lines, RCC-TIU and RCC-UOK220, were used to validate this methodology. Edited ccRCC cells were enriched by CD19 magnetic microbeads, resulting in CD19 expression of over 96%. Genomic DNAs isolated from the two RCC cell lines confirmed simultaneous knockout of HERV-E and knock-in of tCD19. To isolate a population of cells that had homogenous HERV-E knockout, we single-cell sorted and then expanded CD19+ ccRCC cells in vitro. 252 and 145 clones were harvested from RCC-TIU and RCC-UOK220 respectively. PCR analysis of genomic DNA from 48 selected clones of each of the aforementioned cell lines unveiled both monoallelic and biallelic knockout of HERV-E. Eight biallelic knockout clones were harvested and further expanded, which gave rise to four clones from RCC-TIU and three clones from RCC-UOK220. Additional PCR screening confirmed that RCC-TIU A7, A9, and RCC-UOK220 D3 were biallelic knockout clones. Finally, we used digital PCR to examine the expression of two CT-RCC HERV-E transcripts, Env and RCC-8; No expression of these transcripts was found in the a