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A method to sequence and quantify DNA integration for monitoring outcome in gene therapy.

Brady T, Roth SL, Malani N, Wang GP, Berry CC, Leboulch P, Hacein-Bey-Abina S, Cavazzana-Calvo M, Papapetrou EP, Sadelain M, Savilahti H, Bushman FD - Nucleic Acids Res. (2011)

Bottom Line: Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia.Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing.The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

View Article: PubMed Central - PubMed

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

ABSTRACT
Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

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Reduced recovery bias using the Mu-mediated integration site isolation method. (A) Relative recovery rates compared at the LMO2 promoter for (i) the Mu-mediated method; (ii) six tests with single restrictions enzymes or pools or (iii) a mixture of all six. The recovery rates were calculated from data on the placement of integration sites relative to restriction enzyme cleavage sites or Mu transposase sites used in their isolation, using the data in Supplementary Table S1 and statistical methods described in Supplementary Report 2. Calculated integration site recovery rates were then used to annotate each base over 10 kb at the LMO2 promoter (chr11, bases 33770412–33780411). ‘Six enzymes’ indicates pooled data for the six sets below. For a perfectly unbiased method, all such rates equal 1.0. (B) Statistical analysis of biases in recovery of integration sites. The x-axis plots each base of the LMO2 promoter analyzed above, treating each as a potential integration target. Sites were ranked by expected ease of isolation, with the easiest to isolate to the right. The y-axis shows the calculated proportion of sequences recovered given the measured recovery biases. Perfect unbiased recovery would follow a line from lower left to upper right. Statistical methods and P values for pair-wise comparisons are summarized in Supplementary Report 2.
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Figure 3: Reduced recovery bias using the Mu-mediated integration site isolation method. (A) Relative recovery rates compared at the LMO2 promoter for (i) the Mu-mediated method; (ii) six tests with single restrictions enzymes or pools or (iii) a mixture of all six. The recovery rates were calculated from data on the placement of integration sites relative to restriction enzyme cleavage sites or Mu transposase sites used in their isolation, using the data in Supplementary Table S1 and statistical methods described in Supplementary Report 2. Calculated integration site recovery rates were then used to annotate each base over 10 kb at the LMO2 promoter (chr11, bases 33770412–33780411). ‘Six enzymes’ indicates pooled data for the six sets below. For a perfectly unbiased method, all such rates equal 1.0. (B) Statistical analysis of biases in recovery of integration sites. The x-axis plots each base of the LMO2 promoter analyzed above, treating each as a potential integration target. Sites were ranked by expected ease of isolation, with the easiest to isolate to the right. The y-axis shows the calculated proportion of sequences recovered given the measured recovery biases. Perfect unbiased recovery would follow a line from lower left to upper right. Statistical methods and P values for pair-wise comparisons are summarized in Supplementary Report 2.

Mentions: Figure 3A illustrates the relative recovery frequencies using the LMO2 promoter as an example, which was chosen because the gene has been involved in several adverse events in SCID-X1 gene therapy (3,38). Note that although Figure 3A summarizes results on a single region of the genome, the biases were measured genome-wide from integration site data for each method and are shown at LMO2 for purposes of illustration. Recovery was relatively consistent for the Mu-mediated method over all sites, whereas the restriction enzyme methods show sharp peaks and valleys, where valleys indicate locations where an integration event would be difficult or impossible to isolate and the peaks frequently recovered sites that would mask more rare sites. Figure 3B shows the data plotted as the cumulative recovery frequency, where perfectly unbiased recovery would be indicated by a curve that followed the diagonal from lower left to upper right. The Mu-mediated recovery method most closely approaches the diagonal and is significantly closer than even the method using six restriction enzymes (P < 0.001; see Supplementary Report 2 for statistical methods), documenting that the Mu-mediated method is the least biased.Figure 3.


A method to sequence and quantify DNA integration for monitoring outcome in gene therapy.

Brady T, Roth SL, Malani N, Wang GP, Berry CC, Leboulch P, Hacein-Bey-Abina S, Cavazzana-Calvo M, Papapetrou EP, Sadelain M, Savilahti H, Bushman FD - Nucleic Acids Res. (2011)

Reduced recovery bias using the Mu-mediated integration site isolation method. (A) Relative recovery rates compared at the LMO2 promoter for (i) the Mu-mediated method; (ii) six tests with single restrictions enzymes or pools or (iii) a mixture of all six. The recovery rates were calculated from data on the placement of integration sites relative to restriction enzyme cleavage sites or Mu transposase sites used in their isolation, using the data in Supplementary Table S1 and statistical methods described in Supplementary Report 2. Calculated integration site recovery rates were then used to annotate each base over 10 kb at the LMO2 promoter (chr11, bases 33770412–33780411). ‘Six enzymes’ indicates pooled data for the six sets below. For a perfectly unbiased method, all such rates equal 1.0. (B) Statistical analysis of biases in recovery of integration sites. The x-axis plots each base of the LMO2 promoter analyzed above, treating each as a potential integration target. Sites were ranked by expected ease of isolation, with the easiest to isolate to the right. The y-axis shows the calculated proportion of sequences recovered given the measured recovery biases. Perfect unbiased recovery would follow a line from lower left to upper right. Statistical methods and P values for pair-wise comparisons are summarized in Supplementary Report 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Reduced recovery bias using the Mu-mediated integration site isolation method. (A) Relative recovery rates compared at the LMO2 promoter for (i) the Mu-mediated method; (ii) six tests with single restrictions enzymes or pools or (iii) a mixture of all six. The recovery rates were calculated from data on the placement of integration sites relative to restriction enzyme cleavage sites or Mu transposase sites used in their isolation, using the data in Supplementary Table S1 and statistical methods described in Supplementary Report 2. Calculated integration site recovery rates were then used to annotate each base over 10 kb at the LMO2 promoter (chr11, bases 33770412–33780411). ‘Six enzymes’ indicates pooled data for the six sets below. For a perfectly unbiased method, all such rates equal 1.0. (B) Statistical analysis of biases in recovery of integration sites. The x-axis plots each base of the LMO2 promoter analyzed above, treating each as a potential integration target. Sites were ranked by expected ease of isolation, with the easiest to isolate to the right. The y-axis shows the calculated proportion of sequences recovered given the measured recovery biases. Perfect unbiased recovery would follow a line from lower left to upper right. Statistical methods and P values for pair-wise comparisons are summarized in Supplementary Report 2.
Mentions: Figure 3A illustrates the relative recovery frequencies using the LMO2 promoter as an example, which was chosen because the gene has been involved in several adverse events in SCID-X1 gene therapy (3,38). Note that although Figure 3A summarizes results on a single region of the genome, the biases were measured genome-wide from integration site data for each method and are shown at LMO2 for purposes of illustration. Recovery was relatively consistent for the Mu-mediated method over all sites, whereas the restriction enzyme methods show sharp peaks and valleys, where valleys indicate locations where an integration event would be difficult or impossible to isolate and the peaks frequently recovered sites that would mask more rare sites. Figure 3B shows the data plotted as the cumulative recovery frequency, where perfectly unbiased recovery would be indicated by a curve that followed the diagonal from lower left to upper right. The Mu-mediated recovery method most closely approaches the diagonal and is significantly closer than even the method using six restriction enzymes (P < 0.001; see Supplementary Report 2 for statistical methods), documenting that the Mu-mediated method is the least biased.Figure 3.

Bottom Line: Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia.Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing.The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

View Article: PubMed Central - PubMed

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

ABSTRACT
Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

Show MeSH
Related in: MedlinePlus