BioProject

Virus and viral vector integration into a host genome have risks associated with various diseases, including cancers. More...

Virus and viral vector integration into a host genome have risks associated with various diseases, including cancers. Currently, RAIS (Rapid Amp lification of Integration Site) and the other next generation sequencing (NGS)-based detection methods for the integration sites have been developed, and reported to be useful but still remain several concerns particularly about the sensitivity, versatility, cost, and time. Herein, we show the improved version of RAIS, RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) that amplify Human T-cell leukemia virus-1 (HTLV-1)-integrated fragments even in samples with low proviral load (PVL)(>0.032%) within approximately 3.5 h. RAISING with Sanger sequencing and NGS analysis discriminated between non-malignant and the malignant cells in HTLV-1-infected human specimens and bovine leukemia virus (BLV)-infected cattle specimens, respectively. Furthermore, RAISING simultaneously could amplify multiple transgene-integrated fragments (HTLV-1, HIV-1; human immunodeficiency virus, SIV; simian immuno-deficiency virus, HBV; hepatitis B virus, ADV; adeno virus, and low-density lipoprotein receptor knock-in mice; Ldlr-mLO-4 and Ldlr-mLO-5) simply by changing each of the transgene-specific primer sets. Importantly, RAISING with NGS analysis also successfully discriminated between on- and off-targets integrating the Loxp donor sequence into an unpredictable position in the genome-edited Ldlr-knock-in mice. Taken together, these results strongly suggest that RAISING is a high-performance detection method that can be used for the assessment of multiple-transgene integration events, especially in the emerging gene therapy sector. Less...