High Efficiency Restriction Enzyme–Free Linear Amplification-Mediated Polymerase Chain Reaction Approach for Tracking Lentiviral Integration Sites Does Not Abrogate Retrieval Bias

Abstract

Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.

Path to the Clinic: Assessment of iPSC-Based Cell Therapies In Vivo in a Nonhuman Primate Model

“…These clones had normal and stable karyotypes, with G-banding repeated just prior to selected in vivo autologous implantation experiments. The genomic location of the 303 bp non-expressed proviral vector fragment remaining in the genome following the transgene-excision was mapped by a modified restriction enzyme-free linear amplification-mediated polymerase chain reaction (Re-free LAM-PCR) (Wu et al, 2013) in the RhiPSC clones to be used for autologous transplantation (Table S1). The presence of this inert DNA tag allowed unequivocal assessment of the presence or absence of RhiPSCs following in vivo implantation.…”

Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model

“…12 Due to the lower sensitivity of these methods, this analysis was only performed in mice with high numbers of gene modified cells in the BM. Here, 31 insertions in six mice were recovered by the re-free LAM PCR and 55 insertions in three mice by the nrLAM-PCR method ( Supplementary Table S3 ).…”

Evaluating a Ligation-Mediated PCR and Pyrosequencing Method for the Detection of Clonal Contribution in Polyclonal Retrovirally Transduced Samples

“…(Kustikova et al, 2009b;Maetzig et al, 2011), we have established a method to analyze multiple samples in a single 454 pyrosequencing run, which allowed us to retrieve up to 2700 sequences per sample. The clonal repertoire reflected at this sequencing depth, however, is not exhaustive, partly because of the limitations imposed by the use of a given specific restriction enzyme (Harkey et al, 2007;Gabriel et al, 2009;Wu et al, 2013) and partly because of sampling issues. Moreover, the LM-PCR method can be skewed, as can any method that employs PCR amplification of intermediate products, toward amplification of smaller PCR products, among other limitations (Berry et al, 2012;Bystrykh et al, 2012), although we could not show that small amplicons had an effect on the extent to which the pyrosequencing reads correlate with the qPCR data ( Supplementary Fig.…”

“…Newly developed techniques, such as nonrestrictive LAM-PCR (Gabriel et al, 2009), a phage Mu transposition-based method (Brady et al, 2011), shear-splink (Koudijs et al, 2011), and Re-free LAM-PCR (Wu et al, 2013), eliminate the restriction enzyme bias and may thus also temper PCRassociated bias related to GC content, secondary structure, and amplicon length. As an alternative to the vector mark, vectors might be supplied with DNA barcodes (Gerrits et al, 2010;Lu et al, 2011).…”