Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

Zhou, Sheng; Ma, Bonner; Yd, Wang; Rapp, S; Ravin, Suk See De; Malech, Harry L.; Bp, Sorrentino

doi:10.1089/hgtb.2014.122

Cited by 13 publications

(17 citation statements)

References 38 publications

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“…Individual vector insertion sites were identified with the use of the quantitative shearing linear amplification (qsLAM) PCR method. 22…”

Section: Vector Copy Number and Insertion-site Analysismentioning

confidence: 99%

“…Vector integration site analysis of lineage-sorted blood cells obtained from Patients 1 through 7 at 6 to 21 months after infusion was performed with the use of a qsLAM PCR assay 22 ; integration site analysis of the 6-month sample from Patient 8 is in progress, and the results were not available at the time of this report. Unique vector-genome junctions provide clonal markers for each originally transduced hematopoietic progenitor, and the frequency of each unique junction sequence is a measure of the clonal proliferation.…”

Section: Vector Integration Site Analysismentioning

confidence: 99%

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Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1

et al. 2019

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BACKGROUND Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS We performed a dual-center, phase 1–2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four in-fants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, .)

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“…Individual vector insertion sites were identified with the use of the quantitative shearing linear amplification (qsLAM) PCR method. 22…”

Section: Vector Copy Number and Insertion-site Analysismentioning

confidence: 99%

Section: Vector Integration Site Analysismentioning

confidence: 99%

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1

et al. 2019

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“…Although some of the published protocols for retroviral integration site analysis use the LM-PCR approach, none of the available protocols was designed to accurately identify rare integration sites in samples from HIV infected individuals on therapy. For example, there are two published protocols for retroviral integration site analysis that were designed primarily for use with samples obtained from gene therapy patients [43,45]. Although both protocols are similar ours, there are, in both cases, important differences.…”

Section: Discussionmentioning

confidence: 99%

An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

et al. 2020

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Background: All retroviruses, including human immunodeficiency virus (HIV), must integrate a DNA copy of their genomes into the genome of the infected host cell to replicate. Although integrated retroviral DNA, known as a provirus, can be found at many sites in the host genome, integration is not random. The adaption of linker-mediated PCR (LM-PCR) protocols for high-throughput integration site mapping, using randomly-sheared genomic DNA and Illumina paired-end sequencing, has dramatically increased the number of mapped integration sites. Analysis of samples from human donors has shown that there is clonal expansion of HIV infected cells and that clonal expansion makes an important contribution to HIV persistence. However, analysis of HIV integration sites in samples taken from patients requires extensive PCR amplification and high-throughput sequencing, which makes the methodology prone to certain specific artifacts. Results: To address the problems with artifacts, we use a comprehensive approach involving experimental procedures linked to a bioinformatics analysis pipeline. Using this combined approach, we are able to reduce the number of PCR/ sequencing artifacts that arise and identify the ones that remain. Our streamlined workflow combines random cleavage of the DNA in the samples, end repair, and linker ligation in a single step. We provide guidance on primer and linker design that reduces some of the common artifacts. We also discuss how to identify and remove some of the common artifacts, including the products of PCR mispriming and PCR recombination, that have appeared in some published studies. Our improved bioinformatics pipeline rapidly parses the sequencing data and identifies bona fide integration sites in clonally expanded cells, producing an Excel-formatted report that can be used for additional data processing. Conclusions:We provide a detailed protocol that reduces the prevalence of artifacts that arise in the analysis of retroviral integration site data generated from in vivo samples and a bioinformatics pipeline that is able to remove the artifacts that remain.

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“…Historically, these data relied primarily on the identification of viral integration sites (VIS). Recently developed methods (Zhou, Bonner et al 2014) have shown all VIS in a sample can now be recovered, although previous percentages recovered were much lower (60-80%) (Schmidt, Schwarzwaelder et al 2007), (Kustikova, Baum et al 2008), (Gabriel, Eckenberg et al 2009). Nevertheless, quantification of clone numbers based on sequencing reads is inherently imprecise (Cornils, Bartholomae et al 2013), (Brugman, Suerth et al 2013).…”

Section: Human Blood Cell Production Barcoding Experiments: Making Thmentioning

confidence: 99%

A track of the clones: new developments in cellular barcoding

Lyne

Kent

Laurenti

et al. 2018

Experimental Hematology

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International experts from multiple disciplines gathered at Homerton College in Cambridge, UK from September 12-14, 2018 to consider recent advances and emerging opportunities in the clonal tracking of hematopoiesis in one of a series of StemCellMathLab workshops. The group included thirty-five participants with experience in the fields of theoretical and experimental aspects of clonal tracking, and ranged from PhD students to senior professors. Data from a variety of model systems as well as from clinical gene therapy trials were discussed alongside strategies for data analysis and sharing, as well as challenges arising due to underlying assumptions in data interpretation and communication. Recognizing the power of this technology underpinned a group consensus of a need for improved mechanisms for sharing data and analytical protocols to maintain reproducibility and rigor in its application to complex tissues.

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Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

Cited by 13 publications

References 38 publications

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1

An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

A track of the clones: new developments in cellular barcoding

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