Dataset Information

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR.

ABSTRACT: Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.

SUBMITTER: Hamada M

PROVIDER: S-EPMC6116345 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR.

Hamada Motoharu M Nishio Nobuhiro N Okuno Yusuke Y Suzuki Satoshi S Kawashima Nozomu N Muramatsu Hideki H Tsubota Shoma S Wilson Matthew H MH Morita Daisuke D Kataoka Shinsuke S Ichikawa Daisuke D Murakami Norihiro N Taniguchi Rieko R Suzuki Kyogo K Kojima Daiei D Sekiya Yuko Y Nishikawa Eri E Narita Atsushi A Hama Asahito A Kojima Seiji S Nakazawa Yozo Y Takahashi Yoshiyuki Y

EBioMedicine 20180803

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the in ...[more]

PMID: 30082227

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Project description:BackgroundPreviously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA.ResultsCompared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run.ConclusionTaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

Dataset Information

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR.

Publications

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR.

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