Abstract
Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies1,2,3,4. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology—linear amplification–mediated (LAM) PCR—for detecting and sequencing unknown DNA flanking sequences down to the single cell level5 (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies4,6,7,8. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette–specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5′ long terminal repeat (LTR) retroviral vector adjacent genomic sequences (Fig. 1 and Box 1).
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft, the German Ministry of Education and Research, the European Union and the US National Institutes of Health. We thank all present and former members and collaborators of our laboratory who have contributed work or materials to the many experiments, time and efforts toward establishing and optimizing this protocol.
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LAM-PCR is filed as a US patent 6514706 that is licensed to Cinciannati Childrens Hospital Medical Center (CCHMC), USA.
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Supplementary Table, Methods and Note
Supplementary Tables 1–2, Supplementary Methods, Supplementary Note (PDF 201 kb)
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Schmidt, M., Schwarzwaelder, K., Bartholomae, C. et al. High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR). Nat Methods 4, 1051–1057 (2007). https://doi.org/10.1038/nmeth1103
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DOI: https://doi.org/10.1038/nmeth1103
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