Limits...
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.

Show MeSH

Related in: MedlinePlus

Consensus sequences at points of adaptor addition. (A) Information content at junctions between the engineered Mu DNA and human DNA derived from vector integration site sequence reads. The x-axis shows the DNA sequence position, where the site of joining to Mu DNA is between positions 10 and 11 (arrow). Perfect sequence conservation has information content of 2 bits (y-axis). Note that some bases have little or no information content, so no letters are visible. (B) Information content at adaptor junctions from vector integration sites recovered after cleavage with the restriction enzyme Mse I, where the site of cleavage is between positions 10 and 11 (arrow).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113588&req=5

Figure 2: Consensus sequences at points of adaptor addition. (A) Information content at junctions between the engineered Mu DNA and human DNA derived from vector integration site sequence reads. The x-axis shows the DNA sequence position, where the site of joining to Mu DNA is between positions 10 and 11 (arrow). Perfect sequence conservation has information content of 2 bits (y-axis). Note that some bases have little or no information content, so no letters are visible. (B) Information content at adaptor junctions from vector integration sites recovered after cleavage with the restriction enzyme Mse I, where the site of cleavage is between positions 10 and 11 (arrow).

Mentions: We first compared recovery biases of the Mu and restriction enzyme-based methods. We determined the sequence preferences for Mu integration in human DNA in vitro for 5968 integration site sequence reads that included the Mu-end oligonucleotide DNA. Alignment of human sequences at Mu integration sites revealed a detectable consensus sequence closely resembling that reported previously for Mu transposition (37), but with much lower information content than cleavage sites for restriction enzymes (Figure 2A and B and Supplementary Report 2), indicating less bias in the cleaving/joining reactions.Figure 2.


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)

Consensus sequences at points of adaptor addition. (A) Information content at junctions between the engineered Mu DNA and human DNA derived from vector integration site sequence reads. The x-axis shows the DNA sequence position, where the site of joining to Mu DNA is between positions 10 and 11 (arrow). Perfect sequence conservation has information content of 2 bits (y-axis). Note that some bases have little or no information content, so no letters are visible. (B) Information content at adaptor junctions from vector integration sites recovered after cleavage with the restriction enzyme Mse I, where the site of cleavage is between positions 10 and 11 (arrow).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Consensus sequences at points of adaptor addition. (A) Information content at junctions between the engineered Mu DNA and human DNA derived from vector integration site sequence reads. The x-axis shows the DNA sequence position, where the site of joining to Mu DNA is between positions 10 and 11 (arrow). Perfect sequence conservation has information content of 2 bits (y-axis). Note that some bases have little or no information content, so no letters are visible. (B) Information content at adaptor junctions from vector integration sites recovered after cleavage with the restriction enzyme Mse I, where the site of cleavage is between positions 10 and 11 (arrow).
Mentions: We first compared recovery biases of the Mu and restriction enzyme-based methods. We determined the sequence preferences for Mu integration in human DNA in vitro for 5968 integration site sequence reads that included the Mu-end oligonucleotide DNA. Alignment of human sequences at Mu integration sites revealed a detectable consensus sequence closely resembling that reported previously for Mu transposition (37), but with much lower information content than cleavage sites for restriction enzymes (Figure 2A and B and Supplementary Report 2), indicating less bias in the cleaving/joining reactions.Figure 2.

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