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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

Analysis of integration site spacing on the murine genome. Integration site clustering was assessed by comparing the spacing between lentiviral vector encoding human arylsulfatase A (LV-ARSA) integration sites to the spacing between the same number of random sites. The lengths (L) of bases between integration sites were calculated and distributed into seven intersite length “bins,” with the shortest intersite lengths to the left and the longest to the right. Significant differences are denoted by asterisks (****P < 0.0001, *P < 0.05; χ2 test).
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fig3: Analysis of integration site spacing on the murine genome. Integration site clustering was assessed by comparing the spacing between lentiviral vector encoding human arylsulfatase A (LV-ARSA) integration sites to the spacing between the same number of random sites. The lengths (L) of bases between integration sites were calculated and distributed into seven intersite length “bins,” with the shortest intersite lengths to the left and the longest to the right. Significant differences are denoted by asterisks (****P < 0.0001, *P < 0.05; χ2 test).

Mentions: Regional genomic “hotspots” for retroviral vector integration have been reported in in vitro and in vivo datasets, including in HIV-1-infected individuals.21,27,28,29 To determine if clustering of integration sites was evident in the dataset, the distribution of lengths of DNA segments between integration sites was compared with the distribution expected with random integration (Figure 3). Significantly more short intersegment distances were observed with the LV-ARSA sites compared with the random control sites, indicative of clustering (P < 0.0001, Fisher's exact test). Further inspection of the integration sites revealed that 66 genomic positions hosted two or more independent integration events, representing 42.9% of all integration sites (Supplementary Table S5). Thirty-seven of these 66 genomic regions shared the same integration site in two or more mice. Nineteen regions hosted 3 or more integration sites within 20 bases of each other. The genomic region (chr10: 9832769-9832909) located in an intron of the stxbp5 gene was highly favored for integration. Eleven of the 434 (2.5%) independent integration sites were located in this region within 140 bases of each other. Closer inspection of the target DNA sequence for these 11 integration sites revealed that all 11 sites were located in or within 100 bases of a direct repeat sequence located at position chr10:9832707-9832930.


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)

Analysis of integration site spacing on the murine genome. Integration site clustering was assessed by comparing the spacing between lentiviral vector encoding human arylsulfatase A (LV-ARSA) integration sites to the spacing between the same number of random sites. The lengths (L) of bases between integration sites were calculated and distributed into seven intersite length “bins,” with the shortest intersite lengths to the left and the longest to the right. Significant differences are denoted by asterisks (****P < 0.0001, *P < 0.05; χ2 test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221599&req=5

fig3: Analysis of integration site spacing on the murine genome. Integration site clustering was assessed by comparing the spacing between lentiviral vector encoding human arylsulfatase A (LV-ARSA) integration sites to the spacing between the same number of random sites. The lengths (L) of bases between integration sites were calculated and distributed into seven intersite length “bins,” with the shortest intersite lengths to the left and the longest to the right. Significant differences are denoted by asterisks (****P < 0.0001, *P < 0.05; χ2 test).
Mentions: Regional genomic “hotspots” for retroviral vector integration have been reported in in vitro and in vivo datasets, including in HIV-1-infected individuals.21,27,28,29 To determine if clustering of integration sites was evident in the dataset, the distribution of lengths of DNA segments between integration sites was compared with the distribution expected with random integration (Figure 3). Significantly more short intersegment distances were observed with the LV-ARSA sites compared with the random control sites, indicative of clustering (P < 0.0001, Fisher's exact test). Further inspection of the integration sites revealed that 66 genomic positions hosted two or more independent integration events, representing 42.9% of all integration sites (Supplementary Table S5). Thirty-seven of these 66 genomic regions shared the same integration site in two or more mice. Nineteen regions hosted 3 or more integration sites within 20 bases of each other. The genomic region (chr10: 9832769-9832909) located in an intron of the stxbp5 gene was highly favored for integration. Eleven of the 434 (2.5%) independent integration sites were located in this region within 140 bases of each other. Closer inspection of the target DNA sequence for these 11 integration sites revealed that all 11 sites were located in or within 100 bases of a direct repeat sequence located at position chr10:9832707-9832930.

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