RAISING: 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 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 next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate 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

“…The products of the second PCR were purified using AMPure XP (Beckman Coulter, Fullerton, CA, USA) and analyzed using Sanger sequencing with a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on a 3130Xl or 3730Xl DNA Analyzer (Thermo Fisher Scientific). Clonality analysis of BLV-infected cells was performed using the sequencing signal data of each sample by a CLOVA software (14), an R program that automatically analyzes the clonality value (Cv) of transgene-integrated cells by dividing the average of signal peak area values of 20 nucleotides at 5' terminal of host genome sequence of the dominant clone by that at 3' terminal of BLV proviral sequence.…”

“…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.…”