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DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.

Wang GP, Garrigue A, Ciuffi A, Ronen K, Leipzig J, Berry C, Lagresle-Peyrou C, Benjelloun F, Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M, Bushman FD - Nucleic Acids Res. (2008)

Bottom Line: Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing.We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem.The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

View Article: PubMed Central - PubMed

Affiliation: University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.

ABSTRACT
Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160,232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy--in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

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Related in: MedlinePlus

The DNA bar coding strategy. Each LTR primer used in ligation-mediated PCR contained a unique DNA barcode that specified the mouse and tissue of origin. Each barcode consists of a unique 4-bp nucleotide sequence, inserted between the sequencing primer binding site and the LTR specific primer segment. Thus all sequencing reads begin with the 4-bp barcode identifiers. A sample primer with a bar code is shown at the bottom of the diagram.
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Figure 1: The DNA bar coding strategy. Each LTR primer used in ligation-mediated PCR contained a unique DNA barcode that specified the mouse and tissue of origin. Each barcode consists of a unique 4-bp nucleotide sequence, inserted between the sequencing primer binding site and the LTR specific primer segment. Thus all sequencing reads begin with the 4-bp barcode identifiers. A sample primer with a bar code is shown at the bottom of the diagram.

Mentions: To determine vector integration sites on the mouse genome, DNA fragments from host–vector junction were prepared using ligation-mediated PCR (6,17–20). Briefly, each DNA sample (1–1.5 μg) was digested with MseI. The digested samples were ligated to linkers, and then amplified by nested PCR. In order to sequence all the samples in a single sequencing experiment, primers that contain unique 4-bp barcodes were used in the second PCR step (Figure 1, Supplementary Table 1). The PCR products were gel purified, pooled, and then subjected to pyrosequencing as implemented by 454 Life Sciences (13). To minimize bar code ‘crossover’, the mouse samples were separated into four quadrants on a single picotiter sequencing plate. Inspection of the integration sites revealed 23 instances (<0.01% of total sequence reads) in which an identical integration site was observed in different mice in the same quadrant with barcodes of edit distance 1 (i.e. ‘barcode collisions’). These barcode collisions were resolved by removing all collision sites from the sample with the lower number of clones (which was usually one).Figure 1.


DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.

Wang GP, Garrigue A, Ciuffi A, Ronen K, Leipzig J, Berry C, Lagresle-Peyrou C, Benjelloun F, Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M, Bushman FD - Nucleic Acids Res. (2008)

The DNA bar coding strategy. Each LTR primer used in ligation-mediated PCR contained a unique DNA barcode that specified the mouse and tissue of origin. Each barcode consists of a unique 4-bp nucleotide sequence, inserted between the sequencing primer binding site and the LTR specific primer segment. Thus all sequencing reads begin with the 4-bp barcode identifiers. A sample primer with a bar code is shown at the bottom of the diagram.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2396413&req=5

Figure 1: The DNA bar coding strategy. Each LTR primer used in ligation-mediated PCR contained a unique DNA barcode that specified the mouse and tissue of origin. Each barcode consists of a unique 4-bp nucleotide sequence, inserted between the sequencing primer binding site and the LTR specific primer segment. Thus all sequencing reads begin with the 4-bp barcode identifiers. A sample primer with a bar code is shown at the bottom of the diagram.
Mentions: To determine vector integration sites on the mouse genome, DNA fragments from host–vector junction were prepared using ligation-mediated PCR (6,17–20). Briefly, each DNA sample (1–1.5 μg) was digested with MseI. The digested samples were ligated to linkers, and then amplified by nested PCR. In order to sequence all the samples in a single sequencing experiment, primers that contain unique 4-bp barcodes were used in the second PCR step (Figure 1, Supplementary Table 1). The PCR products were gel purified, pooled, and then subjected to pyrosequencing as implemented by 454 Life Sciences (13). To minimize bar code ‘crossover’, the mouse samples were separated into four quadrants on a single picotiter sequencing plate. Inspection of the integration sites revealed 23 instances (<0.01% of total sequence reads) in which an identical integration site was observed in different mice in the same quadrant with barcodes of edit distance 1 (i.e. ‘barcode collisions’). These barcode collisions were resolved by removing all collision sites from the sample with the lower number of clones (which was usually one).Figure 1.

Bottom Line: Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing.We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem.The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

View Article: PubMed Central - PubMed

Affiliation: University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.

ABSTRACT
Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160,232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy--in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

Show MeSH
Related in: MedlinePlus