The application of viral vectors in gene therapy is rapidly developing and the therapeutic results are highly promising. Actively and passively integrating (viral) vectors may fail to deliver, or trigger severe side effects by undirected or unintended integration into the genome of the target cell. Studies aiming to define and improve the efficiency and safety of the vectors should be conducted, according to regulatory recommendations, ahead of and during a clinical trial. Integration site analyses for many European but also worldwide gene therapy trials have been established and performed by the founders and the employees of the start-up company GeneWerk at the German Cancer Research Center (DKFZ) in Heidelberg, Germany. You as a customer can now access this unique expertise via GeneWerk GmbH.
For the determination of integration sites and all other fusion sequences we offer 2 methods, the (nr)LAM-PCR as well as the enrichment of the wanted sequences with a target enrichment system (e.g. Agilent Sure Select).
Integration site analysis of viral vectors with (nr)LAM-PCR
The identification of viral vector flanking genomic sequences is performed with linear amplification mediated PCR (LAM-PCR) as described in Schmidt et. al 2007. Optionally, non-restrictive variant of LAM-PCR (nrLAM-PCR) is applied (Gabriel et al. 2009, Paruzynski et al. 2010).
For LAM-PCR, flanking sequences are amplified by linear PCR using biotinylated primers hybridizing to vector sequences (e.g. 3-prime region of the long terminal repeat (LTR) of the vector). Subsequent steps involve magnetic capture of the biotinylated PCR products, hexanucleotide priming by Klenow polymerase for double strand DNA synthesis and restriction digest using e.g. MluCI and MseI. After digestion, a double-stranded sequence adaptor (linker cassette) 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 is used. Subsequent steps involve magnetic capture of the biotinylated PCR products and ligation of a single stranded linker cassette carrying a molecular barcode. For both methods, LAM-PCR and nrLAM-PCR, the ligated PCR product is used as template in an exponential PCR using biotinylated vector- and adaptor-specific primers. Magnetic capture of the biotinylated PCR-products is performed before reamplification with nested vector- and adaptor-specific primers in a second exponential PCR step.
LAM-PCR amplicons are regularly sequenced on the MiSeq instrument (Illumina) after sample preparation for high-throughput sequencing. Therefore, an additional PCR with special fusion-primers carrying MiSeq specific sequencing adaptors is performed. DNA barcoding is used to allow parallel sequencing of multiple samples in a single sequencing run.
Raw sequence data are trimmed according to sequence quality (Phred 30). Only sequences carrying correct barcodes (linker cassette barcode, sequencing adapter barcodes) are further analyzed. Our (semi-) automated bioinformatical data mining pipeline is used to analyze the data according to your needs (Arens et al. 2012 and unpublished).
- Schmidt M, Schwarzwaelder K, Bartholomae CC, Zaoui K, Ball C, Pilz I, Braun S, Glimm H, von Kalle C: High-Resolution Insertion Site Analysis by Linear Amplification-Mediated PCR (LAM-PCR). Nature Methods. 4, 1051-1057, 2007.
- Gabriel R, Eckenberg R, Paruzynski A, Bartholomae C, Nowrouzi A, Arens A, Howe SJ, Recchia A, Cattoglio C, Wang W, Faber K, Schwarzwaelder K, Kirsten R, Deichmann A, Ball CR, Balaggan KS, Yáñez-Muñoz RJ, Ali RR, Gaspar HB, Biasco L, Aiuti A, Cesana D, Montini E, Naldini L, Cohen-Haguenauer O, Mavilio F, Thrasher AJ, Glimm H, von Kalle C, Saurin W, Schmidt M. Comprehensive genomic access to vector integration in clinical gene therapy. Nature Medicine. 15, 1431-1436, 2009.
- Paruzynski A, Arens A, Gabriel R, Bartholomae CC, Scholz S, Wang W, Wolf S, Glimm H, Schmidt M, von Kalle C: Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nature Protocols. 5, 1379-1395, 2010.
- Arens A, Appelt JU, Bartholomae CC, Gabriel R, Paruzynski A, Gustafson D, Cartier N, Aubourg P, Deichmann A, Glimm H, von Kalle C, Schmidt M: Bioinformatic clonality analysis of next-generation sequencing-derived viral vector integration sites. Human Gene Therapy Methods. 23, 111-118, 2012.
With the advent of next generation sequencing (NGS) technologies, new approaches are promising to identify vector IS in a more comprehensive way, qualitatively and quantitatively. In line with cancer genomics studies where all exon studies are used, we offer partial or complete vector genome target enrichment sequencing (TES).
The results of vector genome TES yield information on clonality (integration sites), quantitative individual clonal contributions and vector genome stability. The additional enrichment of cellular subgenomic DNA regions in parallel to vector sequences further allows to define the vector copy number (VCN) in the analyzed DNA samples (comparable to a seperate qPCR) .