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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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The Mu-based integration site recovery method. (A) Severe recovery biases in previous methods using restriction enzyme cleavage. A large collection of integration sites generated from SCID-X1 gene therapy (Supplementary Table S1) were analyzed and plotted to show the relative recovery frequency for different distances between the restriction enzyme site used in genomic DNA cleavage and the vector integration site. The graph summarizes data obtained using six different restriction enzymes. The sharp peak documents the recovery bias (the location of the peak differed modestly for the different restriction enzymes studied; data not shown). (B) Biased recovery efficiency for four restriction enzymes in a sample from an adverse event. The integration site within BMI1 was implicated in an adverse event in SCID-X1 patient 10 (3). Each bar indicates the percent of all integration sites from the leukemic cell sample deriving from the BMI1 site for each of the three restriction enzymes or three 6-cutter cocktail (Avr I, Spe I and Nhe I) used for isolation. (C) The engineered Mu DNA donor used in these studies. 5′ and 3′ DNA ends are as marked. The ‘N’ indicates the position of an amino-modifier that blocks the DNA 3′-end to prevent adaptor-to-adaptor amplification. The dark blue indicates binding sites for MuA transposase, light blue a spacer region and green the adaptor sequence for PCR amplification. (D) The Mu-mediated integration site recovery method. MuA transposition is used to install the engineered Mu DNA donor (top), allowing PCR amplification (middle). The PCR primers contain DNA barcodes (black segments) and primers for use in 454/Roche pyrosequencing (orange). PCR products can be used directly for pyrosequencing without cloning in bacterial plasmids.
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Figure 1: The Mu-based integration site recovery method. (A) Severe recovery biases in previous methods using restriction enzyme cleavage. A large collection of integration sites generated from SCID-X1 gene therapy (Supplementary Table S1) were analyzed and plotted to show the relative recovery frequency for different distances between the restriction enzyme site used in genomic DNA cleavage and the vector integration site. The graph summarizes data obtained using six different restriction enzymes. The sharp peak documents the recovery bias (the location of the peak differed modestly for the different restriction enzymes studied; data not shown). (B) Biased recovery efficiency for four restriction enzymes in a sample from an adverse event. The integration site within BMI1 was implicated in an adverse event in SCID-X1 patient 10 (3). Each bar indicates the percent of all integration sites from the leukemic cell sample deriving from the BMI1 site for each of the three restriction enzymes or three 6-cutter cocktail (Avr I, Spe I and Nhe I) used for isolation. (C) The engineered Mu DNA donor used in these studies. 5′ and 3′ DNA ends are as marked. The ‘N’ indicates the position of an amino-modifier that blocks the DNA 3′-end to prevent adaptor-to-adaptor amplification. The dark blue indicates binding sites for MuA transposase, light blue a spacer region and green the adaptor sequence for PCR amplification. (D) The Mu-mediated integration site recovery method. MuA transposition is used to install the engineered Mu DNA donor (top), allowing PCR amplification (middle). The PCR primers contain DNA barcodes (black segments) and primers for use in 454/Roche pyrosequencing (orange). PCR products can be used directly for pyrosequencing without cloning in bacterial plasmids.

Mentions: We first quantified biases in the detection of integrated DNA using protocols relying on restriction enzyme cleavage of genomic DNA. Analysis of the recoverability of an integration site based on its proximity to the nearest restriction site showed that integrated DNA ∼49 bp from a cleavage site was recovered most frequently, and frequency of recovery decreased sharply at longer or shorter distances (Figure 1A). The case of SCID-X1 patient 10 provides an example of complications due to this recovery bias (3,7). In this patient, an integrated vector activated expression of the nearby BMI1 proto-oncogene, which was associated with massive expansion of leukemic cells. When DNA from blood cells of patient 10 was cleaved and analyzed using four different restriction enzymes, only two enzymes allowed efficient recovery of the BMI1 integration site (Figure 1B). In an extreme effort to circumvent these limitations, a study of SCID-X1 gene-corrected patients used up to six different restriction enzymes to analyze each individual sample, but even with the difficulty and expense of this large scale effort, recovery was still significantly biased (7).Figure 1.


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)

The Mu-based integration site recovery method. (A) Severe recovery biases in previous methods using restriction enzyme cleavage. A large collection of integration sites generated from SCID-X1 gene therapy (Supplementary Table S1) were analyzed and plotted to show the relative recovery frequency for different distances between the restriction enzyme site used in genomic DNA cleavage and the vector integration site. The graph summarizes data obtained using six different restriction enzymes. The sharp peak documents the recovery bias (the location of the peak differed modestly for the different restriction enzymes studied; data not shown). (B) Biased recovery efficiency for four restriction enzymes in a sample from an adverse event. The integration site within BMI1 was implicated in an adverse event in SCID-X1 patient 10 (3). Each bar indicates the percent of all integration sites from the leukemic cell sample deriving from the BMI1 site for each of the three restriction enzymes or three 6-cutter cocktail (Avr I, Spe I and Nhe I) used for isolation. (C) The engineered Mu DNA donor used in these studies. 5′ and 3′ DNA ends are as marked. The ‘N’ indicates the position of an amino-modifier that blocks the DNA 3′-end to prevent adaptor-to-adaptor amplification. The dark blue indicates binding sites for MuA transposase, light blue a spacer region and green the adaptor sequence for PCR amplification. (D) The Mu-mediated integration site recovery method. MuA transposition is used to install the engineered Mu DNA donor (top), allowing PCR amplification (middle). The PCR primers contain DNA barcodes (black segments) and primers for use in 454/Roche pyrosequencing (orange). PCR products can be used directly for pyrosequencing without cloning in bacterial plasmids.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113588&req=5

Figure 1: The Mu-based integration site recovery method. (A) Severe recovery biases in previous methods using restriction enzyme cleavage. A large collection of integration sites generated from SCID-X1 gene therapy (Supplementary Table S1) were analyzed and plotted to show the relative recovery frequency for different distances between the restriction enzyme site used in genomic DNA cleavage and the vector integration site. The graph summarizes data obtained using six different restriction enzymes. The sharp peak documents the recovery bias (the location of the peak differed modestly for the different restriction enzymes studied; data not shown). (B) Biased recovery efficiency for four restriction enzymes in a sample from an adverse event. The integration site within BMI1 was implicated in an adverse event in SCID-X1 patient 10 (3). Each bar indicates the percent of all integration sites from the leukemic cell sample deriving from the BMI1 site for each of the three restriction enzymes or three 6-cutter cocktail (Avr I, Spe I and Nhe I) used for isolation. (C) The engineered Mu DNA donor used in these studies. 5′ and 3′ DNA ends are as marked. The ‘N’ indicates the position of an amino-modifier that blocks the DNA 3′-end to prevent adaptor-to-adaptor amplification. The dark blue indicates binding sites for MuA transposase, light blue a spacer region and green the adaptor sequence for PCR amplification. (D) The Mu-mediated integration site recovery method. MuA transposition is used to install the engineered Mu DNA donor (top), allowing PCR amplification (middle). The PCR primers contain DNA barcodes (black segments) and primers for use in 454/Roche pyrosequencing (orange). PCR products can be used directly for pyrosequencing without cloning in bacterial plasmids.
Mentions: We first quantified biases in the detection of integrated DNA using protocols relying on restriction enzyme cleavage of genomic DNA. Analysis of the recoverability of an integration site based on its proximity to the nearest restriction site showed that integrated DNA ∼49 bp from a cleavage site was recovered most frequently, and frequency of recovery decreased sharply at longer or shorter distances (Figure 1A). The case of SCID-X1 patient 10 provides an example of complications due to this recovery bias (3,7). In this patient, an integrated vector activated expression of the nearby BMI1 proto-oncogene, which was associated with massive expansion of leukemic cells. When DNA from blood cells of patient 10 was cleaved and analyzed using four different restriction enzymes, only two enzymes allowed efficient recovery of the BMI1 integration site (Figure 1B). In an extreme effort to circumvent these limitations, a study of SCID-X1 gene-corrected patients used up to six different restriction enzymes to analyze each individual sample, but even with the difficulty and expense of this large scale effort, recovery was still significantly biased (7).Figure 1.

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