Flanking-sequence exponential anchored–polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant–host–junction sequences

Abstract

Background-Retroviral vectors are regularly used to transduce stem cells and their derivatives for experimental and therapeutic purposes. Because these vectors integrate semi-randomly into the cellular genome, analysis of integranated retroviral DNA/host cell DNA junctions (IHJ) facilitates clonality studies of engrafted cells, allowing their differentiation, survival and fate to be tracked. In the case of any adverse events, IHJ analysis can allow the identification of potentially oncogenic integration sites. At present, most measures to assess IHJ are complex, insensitive and may be subject to IHJ selection bias inherent to the technology used.

Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients

“…After CTL infusion, the majority of patients had only a single integration site detected in each cell sample; however, repeat samplings of the same patient often revealed different integrants. We have extensively validated the accuracy and repeatability of the FLEA-PCR technique by analyzing individual clones and their mixtures, 21 resulting in the detection of the expected IHJ. Hence, the likeliest explanation for our detection of unique integrants at each sampling time is that a polyclonal population of transduced EBV-CTLs survives in vivo, so that individual samples of peripheral blood typically contain only a single integrant.…”

“…on April 28, 2019. by guest www.bloodjournal.org From because of modulation of a critical regulatory gene(s) after retroviral integration. We therefore used flanking sequence exponential anchored (FLEA)-PCR 21 to analyze integration sites before and after infusion. We subjected genomic DNA from 3 therapeutic products to FLEA-PCR and subcloned the PCR smears to generate a plasmid library.…”

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

“…In contrast, three pooled reactions, each with 100 ng of starting DNA, are required to attain up to 90% efficiency by regular LAM-PCR on the same clonal mixture. Several other approaches without restriction enzyme digest have been shown to access the integrome with a higher reported quantity of starting genomic DNA, for example, up to1 lg (Pule et al, 2008;Paruzynski et al, 2010). Re-free LAM-PCR allows for high genomic coverage and retrieval efficiency with a comparably low amount of starting genomic DNA.…”

“…Also, the labor-intensive and time-consuming nature of LAM-PCR provides opportunity for improvement if the method is to be widely adopted for longitudinal gene therapy studies. Several assays that do not employ restriction enzymes, flankingsequence exponential anchored PCR (FLEA-PCR) (Pule et al, 2008), non-restrictive linear amplification-mediated PCR (nrLAM-PCR) (Paruzynski et al, 2010), and transposase MuA based-PCR (Brady et al, 2011), have been developed. However, none of these methods have been proven to accurately quantitate clonal contributions in a polyclonal setting.…”

Development of Lentiviral Vectors for Targeted Integration and Protein Delivery

“…Methods that rely on the use of restriction enzymes to fragment genomic DNA are prone to so-called restriction bias that may result in unequal retrieval of IS from different parts of the genome [22,23]. Alternative Re-free methods include, for example, the nonrestrictive linear amplification-mediated PCR (nrLAM-PCR) [24], a MuA transposase-based PCR method [25], and the flankingsequence exponential anchored-PCR (FLEA-PCR) [26]. Instead of restriction enzymes, the genomic DNA can also be first sheared with sonication after which the process can continue similar to LM-or LAM-PCRs or their variants [27,28].…”