Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May;17(5):844-50.
doi: 10.1038/mt.2009.16. Epub 2009 Mar 3.

Analysis of lentiviral vector integration in HIV+ study subjects receiving autologous infusions of gene modified CD4+ T cells

Gary P Wang  1 , Bruce L Levine, Gwendolyn K Binder, Charles C Berry, Nirav Malani, Gary McGarrity, Pablo Tebas, Carl H June, Frederic D Bushman
Affiliations

Analysis of lentiviral vector integration in HIV+ study subjects receiving autologous infusions of gene modified CD4+ T cells

Gary P Wang et al. Mol Ther. 2009 May.

Abstract

Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5'-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes.

PubMed Disclaimer

Figures

<b>Figure 1</b>
Figure 1
Diagram of integration site recovery strategy. (a) Use of a GTAG primer for first round amplification, followed by nested PCR for second round amplification. A detailed description of the VRX496 vector can be found in ref. 20. (b) Use of DNA bar coding to index amplification products during the second round PCR. LTR, long-terminal repeat.
<b>Figure 2</b>
Figure 2
Analysis of the information content in the local sequence at HIV integration sites. HIV integration sites in each data set were aligned and conserved bases identified. The y-axes indicate bits of information at each base; perfect conservation of a base would score as two bits. (a) Sequences from HIV infection of Jurkat cells.18 (b) Sequences from the VRX496 ex vivo samples. (c) Sequences from the VRX496 samples recovered from patients. The arrow indicates the location of the host virus DNA junction after integration. HIV, human immunodeficiency virus.
<b>Figure 3</b>
Figure 3
Comparison of in vivo and ex vivo lentiviral vector integration sites to the integration sites from control infections of Jurkat cells—analysis of proximity to genomic features. (a) Chromosomal distribution of integration sites in the ex vivo sample, the patient-derived sample, and the control infections of Jurkat cells.18 Random integration would correspond to the line at one. Favored integration is indicated by the bars above the line, disfavored by the bars below. Only every other chromosome is numbered. The right-most chromosome is Y. (b) Frequency of integration in RefGenes. (c) Frequency of integration in Giemsa dark and light bands. The Giemsa dark regions (left side) are higher in gene density. (d) Integration frequency in gene rich regions (scored over 8-Mb intervals surrounding integration sites). (e) Integration frequency in regions of differing transcriptional intensity (scored over 8-Mb intervals surrounding integration sites). The transcriptional intensity measure is similar to the gene density measure, but only genes scored as active using Affymetrix microarrays are counted. (f) Integration frequency in regions of differing CpG island density (scored over 8-Mb intervals surrounding integration sites). (g) Frequency of integration near proto-oncogene 5′-ends. Random integration would have bar heights of one on the y-axis.
<b>Figure 4</b>
Figure 4
Comparison of in vivo and ex vivo lentiviral vector integration sites to the integration sites from control infections of Jurkat cells—analysis of proximity to sites of histone methylation and bound DNA binding proteins. Values for each data set were compared to matched random controls. The direction and strength of each trend is quantified using the ROC area method described in ref. 14, the key to the left of the figure indicates the scale. Each row in the plot corresponds to a different form of histone post-translational modification or bound protein as scored in the Barski et al. “ChIP-seq ” data.32 Each column corresponds to an integration site data set. Comparisons were carried out over 1, 10, or 100 kb genomic intervals. ROC, receiver operating characteristic.
<b>Figure 5</b>
Figure 5
Ex vivo lentiviral vector integration favors the major grooves of DNA bound on nucleosomes. Positions of lentiviral integration sites on nucleosomes were predicted using the nucleosome prediction algorithm developed by Segal et al. (a) The percentage of total ex vivo lentiviral vector integration sites at each base pair (y-axis) is plotted relative to the dyad axis of nucleosome symmetry (position 0; the scale is in base pairs). (b) Fourier transformation of the data from a, showing the ~10.5 bp periodicity of integration frequency.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science. 2000;288:669–672. - PubMed
    1. Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med. 2002;346:1185–1193. - PubMed
    1. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415–419. - PubMed
    1. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2003;348:255–256. - PubMed
    1. Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008;118:3132–3142. - PMC - PubMed

Publication types