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Insertion site pattern: global approach by linear amplification-mediated PCR and mass sequencing.
Methods Mol Biol. 2012;859:255-65
Authors: Bartholomae CC, Glimm H, von Kalle C, Schmidt M
Abstract
In gene therapy, viral or nonviral integrating vectors are used to deliver a corrected gene to replace the corresponding defective cellular gene. As vector delivery is (yet) commonly not targeted to a specific site in the host genome, and vector integration may lead to unwanted cellular gene deregulation, the comprehensive analysis of vector locations is a crucial approach to assess vector biosafety and to follow the fate of the gene corrected cells in vivo. The retrieved vector integration sites are unique for each transduced cell clone, thereby serving as a molecular marker and allowing to track distinct cell clones in various samples. Today, several PCR-based methods are available for the identification and characterization of unknown flanking DNA sequences (Mueller and Wold Science 246:780-786, 1989; Paruzynski et al. Nat Protoc 5:1379-1395, 2010; Schmidt et al. Nat Methods 4:1051-1057, 2007; Silver and Keerikatte J Virol 63:1924-1928, 1989). Thereof, the linear amplification-mediated PCR (LAM-PCR) proved to exhibit the highest sensitivity, allowing the detection of miscellaneous vector integration sites in one sample. The broad application spectrum and robustness of LAM-PCR has been approved by its application as a tool for the molecular follow up of gene-modified cells in preclinical and clinical gene therapy trials (Li et al. Science 296:497, 2002; Cartier et al. Science 326:818-823, 2009; Ott et al. Nat Med 12:401-409, 2006; Deichmann et al. J Clin Invest 117:2225-2232, 2007). The combination of LAM-PCR and next-generation sequencing (NGS) platforms offers the opportunity to study the clonal inventory and pharmacokinetics in clinical gene therapy studies.
PMID: 22367877 [PubMed - indexed for MEDLINE]
More...
Insertion site pattern: global approach by linear amplification-mediated PCR and mass sequencing.
Methods Mol Biol. 2012;859:255-65
Authors: Bartholomae CC, Glimm H, von Kalle C, Schmidt M
Abstract
In gene therapy, viral or nonviral integrating vectors are used to deliver a corrected gene to replace the corresponding defective cellular gene. As vector delivery is (yet) commonly not targeted to a specific site in the host genome, and vector integration may lead to unwanted cellular gene deregulation, the comprehensive analysis of vector locations is a crucial approach to assess vector biosafety and to follow the fate of the gene corrected cells in vivo. The retrieved vector integration sites are unique for each transduced cell clone, thereby serving as a molecular marker and allowing to track distinct cell clones in various samples. Today, several PCR-based methods are available for the identification and characterization of unknown flanking DNA sequences (Mueller and Wold Science 246:780-786, 1989; Paruzynski et al. Nat Protoc 5:1379-1395, 2010; Schmidt et al. Nat Methods 4:1051-1057, 2007; Silver and Keerikatte J Virol 63:1924-1928, 1989). Thereof, the linear amplification-mediated PCR (LAM-PCR) proved to exhibit the highest sensitivity, allowing the detection of miscellaneous vector integration sites in one sample. The broad application spectrum and robustness of LAM-PCR has been approved by its application as a tool for the molecular follow up of gene-modified cells in preclinical and clinical gene therapy trials (Li et al. Science 296:497, 2002; Cartier et al. Science 326:818-823, 2009; Ott et al. Nat Med 12:401-409, 2006; Deichmann et al. J Clin Invest 117:2225-2232, 2007). The combination of LAM-PCR and next-generation sequencing (NGS) platforms offers the opportunity to study the clonal inventory and pharmacokinetics in clinical gene therapy studies.
PMID: 22367877 [PubMed - indexed for MEDLINE]
More...