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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

Consensus sequence at lentiviral integration sites. Base compositions of the top DNA strand at each position surrounding the integration sites were calculated. Integration occurs between positions −1 and +1 on the top strand. Base frequencies of nucleotides A (red circles), C (blue squares), T (green triangles), and G (black triangles) located 40 bases upstream and downstream of the integration site are plotted. The sequence and location of the flanking G-quadruplex-forming motifs are shown above the graph.
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fig4: Consensus sequence at lentiviral integration sites. Base compositions of the top DNA strand at each position surrounding the integration sites were calculated. Integration occurs between positions −1 and +1 on the top strand. Base frequencies of nucleotides A (red circles), C (blue squares), T (green triangles), and G (black triangles) located 40 bases upstream and downstream of the integration site are plotted. The sequence and location of the flanking G-quadruplex-forming motifs are shown above the graph.

Mentions: To investigate if the primary sequence at the integration target sites influenced target site selection, we extracted sequences flanking the LV-ARSA integration sites for further analysis. Forty bases upstream and downstream of each integration site (between −1 and +1) or the random control sites were aligned and the frequencies of A, C, G, and T at each position surrounding the integration sites were calculated. These values were compared with the expected value based on random control sites (Figure 4 and Supplementary Table S7). As expected, the base composition at each position surrounding the matched random control sites varied little from the expected values30 (Supplementary Table S7). The difference in frequency of each nucleotide at each position up to 40 bases upstream and downstream of the LV-ARSA integration sites was calculated (Figure 4). Inspection of the bases surrounding the LV-ARSA integration sites using QGRS Mapper (http://bioinformatics.ramapo.edu/QGRS/analyze.php) revealed two G-quad motifs flanking the integration site at positions −26 to −3 and +22 to +35.31 Of note, positions +1 to +5 comprise the duplicated target site sequence after integration of lentiviruses.


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)

Consensus sequence at lentiviral integration sites. Base compositions of the top DNA strand at each position surrounding the integration sites were calculated. Integration occurs between positions −1 and +1 on the top strand. Base frequencies of nucleotides A (red circles), C (blue squares), T (green triangles), and G (black triangles) located 40 bases upstream and downstream of the integration site are plotted. The sequence and location of the flanking G-quadruplex-forming motifs are shown above the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Consensus sequence at lentiviral integration sites. Base compositions of the top DNA strand at each position surrounding the integration sites were calculated. Integration occurs between positions −1 and +1 on the top strand. Base frequencies of nucleotides A (red circles), C (blue squares), T (green triangles), and G (black triangles) located 40 bases upstream and downstream of the integration site are plotted. The sequence and location of the flanking G-quadruplex-forming motifs are shown above the graph.
Mentions: To investigate if the primary sequence at the integration target sites influenced target site selection, we extracted sequences flanking the LV-ARSA integration sites for further analysis. Forty bases upstream and downstream of each integration site (between −1 and +1) or the random control sites were aligned and the frequencies of A, C, G, and T at each position surrounding the integration sites were calculated. These values were compared with the expected value based on random control sites (Figure 4 and Supplementary Table S7). As expected, the base composition at each position surrounding the matched random control sites varied little from the expected values30 (Supplementary Table S7). The difference in frequency of each nucleotide at each position up to 40 bases upstream and downstream of the LV-ARSA integration sites was calculated (Figure 4). Inspection of the bases surrounding the LV-ARSA integration sites using QGRS Mapper (http://bioinformatics.ramapo.edu/QGRS/analyze.php) revealed two G-quad motifs flanking the integration site at positions −26 to −3 and +22 to +35.31 Of note, positions +1 to +5 comprise the duplicated target site sequence after integration of lentiviruses.

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