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Integrating viral gene transfer vectors are usually used as gene delivery tools in clinical gene therapy trials. They can provide stable integration and continuous gene expression of the transgene in the treated host cell. But the integration of the reverse-transcribed vector DNA into the host genome is a potentially mutagenic event that may directly contribute to unwanted side effects. A comprehensive and accurate analysis of the integration site (IS) repertoire is indispensable to study clonality in transduced cells obtained from patients undergoing gene therapy and to identify potential in vivo selection of affected cell clones.
Up to now, next-generation sequencing (NGS) of vector-genome junctions allows sophisticated studies on the integration repertoire in vivo and in vitro. Some researchers have explored the use of the Illumina MiSeq Personal Sequencer platform to sequence vector ISs amplified by linear-amplification mediated PCR (LAM-PCR) and non-restrictive linear amplification-mediated PCR (nrLAM-PCR). MiSeq-based high-quality IS sequence retrieval is accomplished by the introduction of a double-barcode strategy that substantially minimizes the frequency of IS sequence collisions compared to the conventionally used single-barcode protocol.
An overview of LAM-PCR
LAM-PCR allows identifying and characterizing unknown flanking DNA adjacent to known DNA of any origin. More specifically, LAM-PCR has been developed to localize viral vector IS within the host genome. Genetic elements like transposons or retroviruses integrate their genome into the host genome in a (semi-) random manner. In many cases, it is decisive to know exactly the position where these vectors integrated. LAM-PCR has been proven to be superior to alternative techniques like ligation-mediated PCR and its variants or inverse PCR. The sensitivity and robustness of the method arises from the initial preamplification of the vector-genome junctions and magnetic selection of amplified PCR products. Like the alternative methods mentioned, LAM-PCR relies on the use of restriction enzymes, introducing a bias into retrieval capacity of IS. Thus, only a subset of the IS repertoire (the integrome) can be detected in one reaction. This bias is minimized through the parallel analysis of a given sample using optimal combinations of restriction enzymes.
Because of the high sensitivity resulting from preamplification of the junctions with specific primers hybridizing in the known DNA sequence, it is possible to amplify and detect even rare junctions down to the single cell level. Contrary, in a polyclonal situation LAM-PCR can amplify thousands of different junctions in one single reaction. Nevertheless, due to the use of restriction enzymes, only a subfraction of the integrome can be analyzed by LAM-PCR for the presence of junctions with every particular restriction enzyme. Therefore, repeated analysis of the same sample with different enzymes is recommended. If no LAM-PCR amplicons are present on the gel, most likely the distance between the location of the known DNA fragment and the closest recognition site of chosen restriction enzyme is too large to result in LAM-PCR products. In this case, other enzymes can be used to amplify the junction. Lately, a variant of the technology termed nrLAM-PCR has been developed that circumvents the use of restriction enzymes. Moreover, nrLAM-PCR allows unbiased genome-wide analysis of a sample in a single reaction.
Figure 1. LAM-PCR and nrLAM-PCR
For LAM-PCR, flanking sequences are amplified by linear PCR using biotinylated primers hybridizing to vector sequences. Subsequent steps involve magnetic capture of the biotinylated PCR products, hexanucleotide priming by Klenow polymerase for double-strand DNA synthesis and restriction digest. And after digestion, a double-stranded sequence adaptor carrying a molecular barcode is ligated to the restricted DNA. Also for nrLAM-PCR, two linear PCR amplification steps with a vector specific biotinylated primer are used. And subsequent steps involve magnetic capture of the biotinylated PCR products and ligation of a single-stranded linker cassette carrying a molecular barcode.
The outline of LAM-PCR
A new combination of linear amplification of target DNA with solid-phase second strand synthesis, followed by ligation of an oligonucleotide cassette and then nested exponential PCR is devised for the detection and direct genomic sequencing of unknown retroviral vector integration sites.
Figure 2. Outline of LAM-PCR
(A) Linear PCR with a long terminal repeat (LTR)–specific biotinylated primer is performed by repeated primer extension. Subsequently, the amplified fragments of target DNA are enriched by magnetic tag selection of extension primers.
(B) A second DNA strand of each enriched target sequence is synthesized by random hexanucleotide priming.
(C) Resulting double-stranded DNA is specifically digested with the restriction enzyme Sse9I (Sse), which cuts within genomic DNA approximately every 256 bp. Thus, the length of each fragment is dependent on the distance of the vector insertion site from the next Sse 9I recognition sequence.
(D) An asymmetric oligonucleotide ligation cassette (LC) is ligated to the end of the Sse9I-digested fragments.
(E) Nested exponential PCR amplifications are performed with LC-specific forward primers (LC 1 followed by LC 2) and LTR-specific reverse primers (LTR II followed by LTR III).
The applications of LAM-PCR
LAM-PCR has been adapted to identify IS from other integrating vectors (transposons, lentiviral vectors) and also to identify integration patterns of passively integrating vectors like adeno-associated vectors (AAV) or integrase-defective lentiviral vectors (IDLV). Applications of LAM-PCR are widespread: traditionally, the technique is widely used to study the clonal composition of gene-modified cells in patients that have undergone gene therapy or to assess the biosafety of novel vector systems by unraveling their integration behavior. Lately, LAM-PCR enabled determining specificity and off-target activity of designer nucleases by an IDLV trapping assay.
In addition, LAM-PCR allows to easily follow the fate of a transduced cell over time in an organism. That allows to identify proto-oncogenes as well as tumor suppressor genes and also to study hematopoiesis or cancer stem cell biology. Last but not least, LAM-PCR is adapted to study T-cell receptor diversity in humans. The intrinsic power of the technology is strengthened by linking the method to deep sequencing technologies that allow characterizing millions of unknown flanking DNA with single nucleotide resolution in whole genomes.