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. 2015 Feb;26(1):4-12.
doi: 10.1089/hgtb.2014.122. Epub 2015 Feb 5.

Quantitative shearing linear amplification polymerase chain reaction: an improved method for quantifying lentiviral vector insertion sites in transplanted hematopoietic cell systems

Sheng Zhou  1 , Melissa A Bonner, Yong-Dong Wang, Samuel Rapp, Suk See De Ravin, Harry L Malech, Brian P Sorrentino
Affiliations

Quantitative shearing linear amplification polymerase chain reaction: an improved method for quantifying lentiviral vector insertion sites in transplanted hematopoietic cell systems

Sheng Zhou et al. Hum Gene Ther Methods. 2015 Feb.

Abstract

In gene therapy trials targeting blood disorders, it is important to detect dominance of transduced hematopoietic stem cell (HSC) clones arising from vector insertion site (VIS) effects. Current methods for VIS analysis often do not have defined levels of quantitative accuracy and therefore can fail to detect early clonal dominance. We have developed a rapid and inexpensive method for measuring clone size based on random shearing of genomic DNA, minimal exponential PCR amplification, and shear site counts as a quantitative endpoint. This quantitative shearing linear amplification PCR (qsLAM PCR) assay utilizes an internal control sample containing 19 lentiviral insertion sites per cell that is mixed with polyclonal samples derived from transduced human CD34+ cells. Samples were analyzed from transplanted pigtail macaques and from a participant in our X-linked severe combined immunodeficiency (XSCID) lentiviral vector trial and yielded controlled and quantitative results in all cases. One case of early clonal dominance was detected in a monkey transplanted with limiting numbers of transduced HSCs, while the clinical samples from the XSCID trial participant showed highly diverse clonal representation. These studies demonstrate that qsLAM PCR is a facile and quantitative assay for measuring clonal repertoires in subjects enrolled in human gene therapy trials using lentiviral-transduced HSCs.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Strategy for qsLAM PCR assay. (A) Schematic of the qsLAM PCR method for VIS analysis. Vector sequences are depicted in red, genomic sequences in blue, and adapter sequences as black boxes. Three individual shear sites for a single VIS are schematically represented. The relative locations of primers for linear PCR (P1), and nested PCR (P2 and P3) are shown. (B) Primer binding sites (in red) and the −24 to −5 sequences (inside the rectangle) of the LTR-U5 of the lentiviral vector that is used for reads specificity assessment are indicated. The red line at the end of the compliment primer represents the sequence required for Mi-Seq. qsLAM, quantitative shearing linear amplification; VIS, vector insertion site.
<b>FIG. 2.</b>
FIG. 2.
Fluorescence in situ hybridization of the 19 vector-copy Jurkat clone. The full-length vector plasmid was used as a probe and hybridized to either the metaphase (A) or interphase (B) cells. Chr11 is enlarged to show the two insertion site signals present.
<b>FIG. 3.</b>
FIG. 3.
Quantitation of VIS frequency in the Jurkat control clone. Comparison of the use of shear site counts (black circles) and read counts (red squares) for quantification of each of the 19 VIS present in the transduced Jurkat control clone. The data for shear sites or read counts are expressed as a percentage of the total sites/reads for each of the 19 VIS shown on the X axis. The data show the average of four independent experiments (four sonications and four corresponding qsLAM PCR) with error bars showing the standard deviation for each VIS measurement. The blue line shows the expected value for each of the 19 VIS (5.3%).
<b>FIG. 4.</b>
FIG. 4.
Analysis of VIS in transduced human hematopoietic cells using qsLAM. (A) Relationship of shear site numbers with read count numbers in cultured human peripheral blood CD34+ cells transduced with a transiently produced lentiviral vector (CD34A), lentiviral vector produced in a stable cell line (CD34B), or in human peripheral blood samples from an XSCID gene therapy trial subject that were sorted for CD16 or CD14. Each dot represents an individual VIS present in the indicated sample. (B) Data from the test samples are here shown together with that from a 2% spike of DNA from the internal control Jurkat clone. This graph shows the percentage of total number of shear sites (Y axis) for each VIS present in the internal control sample (red dots) or from the polyclonal test samples (black dots). The X axis shows each of the VIS as individual columns ranked from most to least frequent going left to right as well as the total number of unique VIS.
<b>FIG. 5.</b>
FIG. 5.
Analysis of VIS in peripheral blood samples from transplanted pigtail macaques. (A) mCherry-expressing peripheral blood cells were sorted from two transplanted monkeys (A10WO17 and A10WO27) using the indicated sorting gates shown on this flow cytometry analysis. The percentages of mCherry+ cells are shown above each sorted population. (B) qsLAM PCR analysis of VIS from sorted peripheral blood samples. Black dots show individual VIS identified within the monkey samples and the number of VIS isolates from each animal is indicated. The red dots represent VIS from the internal control Jurkat clone, which was added to the monkey samples at a 2% dilution. The X axis shows the count of shear sites and the Y axis the read counts for each VIS. In animal A10WO16, a potentially dominant clone “A” was present and indicated by the blue circle.
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