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.

Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform

“…We have developed our clustering protocol to err on the side of over-clustering because a key function of vector integration site analysis is to detect clonal dominance, which can sometimes indicate autonomous growth or insertional mutagenesis, and it is therefore important to avoid splitting single integration sites into multiple artificial clones. The number of unique integration sites mapped in our study was consistent with those using LAM-PCR followed by next generation short-read sequencing, which typically detects around 200 to 8,000 unique integration sites from 10 3 to 10 6 transduced T cells [3,5,6]. However, in our analysis of polyclonal clinical samples using inverse PCR and cassette ligation PCR in parallel, only 7% of unique vector integration sites could be detected by both methods.…”

“…A majority (61 -68%) of integration sites were intragenic, with 55 -58% within the introns and 6 -12% within the exons; with the remaining 32 -39% of integration sites being intergenic (Fig.6B). There was a predilection for vector integration near transcription start sites (TSS) (Fig.6C), which was consistent with other reports [3,5,6].…”

“…The length of the flanking DNA sequence is predetermined by the position of the restriction site and hence integration sites that are very close to the restriction sites will always be very difficult to align. However, it may be possible to perform parallel analysis using different sets of restriction enzymes or tagmentation without restriction enzymes to increase the proportion of aligned reads [5,6].…”

“…Although targeted transgene integration using CRISPR/cas9 and other genome editing techniques hold great promise and may well be the path of the future [1,2], the vast majority of current gene-modified cellular therapeutics use gammaretroviral, lentiviral or non-viral vectors that are non-targeted and can integrate at multiple sites, with some predilection for open chromatin and transcriptionally active regions [3][4][5][6]. Analysis of vector integration sites can provide critical information on the clonality of gene-modified cells and potential biological impacts of specific transgene insertion sites, including the potential for insertional mutagenesis through the inactivation of tumor suppressor genes or activation of proto-oncogenes, such as LMO2 [7,8] and EVI1 [9]; or alternatively, enhanced therapeutic efficacy, such as enhanced chimeric antigen receptor (CAR) T cell function through transgene disruption of TET2 [10].…”

Overhauling CAR T Cells to Improve Efficacy, Safety and Cost

“…Since these components may be encoded by plasmids, their manipulation and clinical production are easier and less costly than that of viral vectors [ 122 , 123 , 124 ]. In addition, the integration pattern of transposons is more random than seen with lenti or retroviral vectors, thus reducing risks associated with insertional mutagenesis [ 125 ]. Even so, methods are being explored for the targeted integration of transposons in ‘safe havens’ within the genome [ 122 ].…”