RAISING is a high-performance method for identifying random transgene integration sites

Abstract

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

Diagnosis and early prediction of lymphoma using high-throughput clonality analysis of bovine leukemia virus-infected cells

“…1). The detection limit for RAISING targeting BLV was 0.012 copy/100 cells in PVL, which is comparable with that for RAISING targeting HTLV-1 (0.032% in HTLV-1 PVL) (14). These results suggest that RAISING targeting BLV is sensitive enough to detect proviral insertion sites, even in specimens with low PVL.…”

“…Recently, we developed R apid A mplification of Integration S ites without in terference by g enomic DNA contamination (RAISING) and a clonality analysis software (CLOVA), a highly sensitive, rapid, inexpensive, and high-throughput method to amplify and analyze random integration sites of transgenes in host genomes (14). RAISING and CLOVA were originally developed for the risk assessment of adult T cell leukemia/lymphoma (ATL) development in HTLV-1 carriers (14, 15). The clonality and proviral integration site of HTLV-1-infected cells can be examined by analyzing the sequence of the amplicon from RAISING using the CLOVA software.…”

“…The clonality and proviral integration site of HTLV-1-infected cells can be examined by analyzing the sequence of the amplicon from RAISING using the CLOVA software. The clonality value (Cv) of infected cells calculated by RAISING-CLOVA is an effective marker for the prediction of the risk of ATL onset (14).…”

“…BLV is considered to utilize similar mechanisms for proviral integration and tumorigenesis as HTLV-1 (9, 16). In our previous study, the proviral integration sites of BLV-infected cells in AL and EBL cattle was successfully amplified using RAISING (14). Sanger sequencing and HTS confirmed the clonal expansion of BLV-infected cells only in the EBL specimen, even though the number of tested specimens was quite limited ( n = 2) (14).…”

“…In our previous study, the proviral integration sites of BLV-infected cells in AL and EBL cattle was successfully amplified using RAISING (14). Sanger sequencing and HTS confirmed the clonal expansion of BLV-infected cells only in the EBL specimen, even though the number of tested specimens was quite limited ( n = 2) (14). The clonality analysis of BLV-infected cells by RAISING-CLOVA could be an effective method for the diagnosis and prediction of the EBL onset in cattle.…”