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Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration.

McAllister RG, Liu J, Woods MW, Tom SK, Rupar CA, Barr SD - Mol Ther Nucleic Acids (2014)

Bottom Line: LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA.In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells.Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Center for Human Immunology, Western University, London, Ontario, Canada.

ABSTRACT
The blood-brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.

No MeSH data available.


Related in: MedlinePlus

Distribution of unique HIV-1 integration sites in non-B DNA-forming motifs. Heat map illustrating the distribution of unique HIV-1 integration sites in and near non-B DNA-forming motifs. The ratios of HIV-1 integration sites to matched random control sites are shown for the following previously published datasets: 293T, PBMCs, Jurkat, HOS, macrophage, H9, HeLa, SupT1, and IMR90. Darker shades represent higher fold-changes in the ratio of integration sites to matched random control sites. Bin 0 = number of integration sites (N) located within features; Bin 1= 0 < N ≤ 0.5 kb; Bin 2 = 0.5 kb < N ≤ 5 kb; Bin 3 = 5 kb < N ≤ 50 kb; Bin 4 = 50 kb < N ≤ 500 kb. Significant differences are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) (χ2 test).
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fig6: Distribution of unique HIV-1 integration sites in non-B DNA-forming motifs. Heat map illustrating the distribution of unique HIV-1 integration sites in and near non-B DNA-forming motifs. The ratios of HIV-1 integration sites to matched random control sites are shown for the following previously published datasets: 293T, PBMCs, Jurkat, HOS, macrophage, H9, HeLa, SupT1, and IMR90. Darker shades represent higher fold-changes in the ratio of integration sites to matched random control sites. Bin 0 = number of integration sites (N) located within features; Bin 1= 0 < N ≤ 0.5 kb; Bin 2 = 0.5 kb < N ≤ 5 kb; Bin 3 = 5 kb < N ≤ 50 kb; Bin 4 = 50 kb < N ≤ 500 kb. Significant differences are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) (χ2 test).

Mentions: Next, we analyzed a variety of HIV-1 integration site datasets in diverse human cell types 293T,37 Jurkat,37,38 macrophage,16 HeLa,20,39 IMR90 (refs. 40,41), PBMC,41 HOS,37 H9 (ref. 20), and SupT1 (refs. 29,42) to determine if integration sites were enriched in non-B DNA-forming motifs (Supplementary Table S9). Integration in or near multiple non-B DNA-forming motifs was highly enriched in each of the datasets (Figure 6 and Supplementary Table S10). Integration in or near several non-B DNA motifs was strongly favored among the different datasets and preference for specific types of non-B DNA-forming motifs varied for each cell type, possibly indicating cell-type-specific effects.


Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration.

McAllister RG, Liu J, Woods MW, Tom SK, Rupar CA, Barr SD - Mol Ther Nucleic Acids (2014)

Distribution of unique HIV-1 integration sites in non-B DNA-forming motifs. Heat map illustrating the distribution of unique HIV-1 integration sites in and near non-B DNA-forming motifs. The ratios of HIV-1 integration sites to matched random control sites are shown for the following previously published datasets: 293T, PBMCs, Jurkat, HOS, macrophage, H9, HeLa, SupT1, and IMR90. Darker shades represent higher fold-changes in the ratio of integration sites to matched random control sites. Bin 0 = number of integration sites (N) located within features; Bin 1= 0 < N ≤ 0.5 kb; Bin 2 = 0.5 kb < N ≤ 5 kb; Bin 3 = 5 kb < N ≤ 50 kb; Bin 4 = 50 kb < N ≤ 500 kb. Significant differences are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) (χ2 test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig6: Distribution of unique HIV-1 integration sites in non-B DNA-forming motifs. Heat map illustrating the distribution of unique HIV-1 integration sites in and near non-B DNA-forming motifs. The ratios of HIV-1 integration sites to matched random control sites are shown for the following previously published datasets: 293T, PBMCs, Jurkat, HOS, macrophage, H9, HeLa, SupT1, and IMR90. Darker shades represent higher fold-changes in the ratio of integration sites to matched random control sites. Bin 0 = number of integration sites (N) located within features; Bin 1= 0 < N ≤ 0.5 kb; Bin 2 = 0.5 kb < N ≤ 5 kb; Bin 3 = 5 kb < N ≤ 50 kb; Bin 4 = 50 kb < N ≤ 500 kb. Significant differences are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) (χ2 test).
Mentions: Next, we analyzed a variety of HIV-1 integration site datasets in diverse human cell types 293T,37 Jurkat,37,38 macrophage,16 HeLa,20,39 IMR90 (refs. 40,41), PBMC,41 HOS,37 H9 (ref. 20), and SupT1 (refs. 29,42) to determine if integration sites were enriched in non-B DNA-forming motifs (Supplementary Table S9). Integration in or near multiple non-B DNA-forming motifs was highly enriched in each of the datasets (Figure 6 and Supplementary Table S10). Integration in or near several non-B DNA motifs was strongly favored among the different datasets and preference for specific types of non-B DNA-forming motifs varied for each cell type, possibly indicating cell-type-specific effects.

Bottom Line: LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA.In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells.Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Center for Human Immunology, Western University, London, Ontario, Canada.

ABSTRACT
The blood-brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.

No MeSH data available.


Related in: MedlinePlus