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High-titer foamy virus vector transduction and integration sites of human CD34(+) cell-derived SCID-repopulating cells.

Nasimuzzaman M, Kim YS, Wang YD, Persons DA - Mol Ther Methods Clin Dev (2014)

Bottom Line: Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells.Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus.Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.

ABSTRACT
Foamy virus (FV) vectors are promising tools for gene therapy, but low titer is a major challenge for large-scale clinical trials. Here, we increased FV vector titer 50-fold by constructing novel vector plasmids and using polyethylenimine-mediated transfection. FV and lentiviral (LV) vectors were used separately to transduce human CD34(+) cells at multiplicities of infection of 25, and those cells were transplanted into immunodeficient mice. FV vector transduction frequencies of repopulating human cells were 37.1 ± 1.9% in unstimulated cells and 36.9 ± 2.2% in prestimulated cells, and engraftment frequencies were 40.9 ± 4.9% in unstimulated cells and 47.1 ± 3.3% in prestimulated cells. Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells. Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus. FV had an integration preference near transcriptional start sites and CpG islands of RefSeq genes but not within genes. Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations. Our new FV vector backbone may be a suitable candidate for developing therapeutic FV vectors for use in clinical trials.

No MeSH data available.


Related in: MedlinePlus

Vector transduction of bulk culture and clonogenic progenitors. (a) Percentages of GFP-expressing cells in bulk culture 1 week after vector transduction. (b) Percentages of GFP-expressing progenitor colonies on MethoCult medium 12 to 14 days after vector transduction. Unstimulated cells are designated FV-1st and LV-1st, and 24 h prestimulated cells are designated FV-2nd and LV-2nd. Both FV and LV vectors were transduced overnight on RetroNectin-coated plates at an MOI of 25. The experiments were done three times with mobilized human CD34+ cells from 3 donors. Data represent the mean and SEM.
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fig2: Vector transduction of bulk culture and clonogenic progenitors. (a) Percentages of GFP-expressing cells in bulk culture 1 week after vector transduction. (b) Percentages of GFP-expressing progenitor colonies on MethoCult medium 12 to 14 days after vector transduction. Unstimulated cells are designated FV-1st and LV-1st, and 24 h prestimulated cells are designated FV-2nd and LV-2nd. Both FV and LV vectors were transduced overnight on RetroNectin-coated plates at an MOI of 25. The experiments were done three times with mobilized human CD34+ cells from 3 donors. Data represent the mean and SEM.

Mentions: Granulocyte colony-stimulating factor–mobilized CD34+ cells from peripheral blood of three human donors were studied in three individual experiments. Unstimulated or prestimulated cells were transduced with either a FV or LV vector containing GFP at a multiplicity of infection (MOI) of 25 (see Supplementary Figure S2). MOI of 25 represents FV vectors titrated on HT1080 cells and LV vectors titrated on 293T cells. FV vector-transduced cells expressed 49.9 ± 5.9% of GFP in unstimulated bulk culture (Figure 2a, FV-1st) and 53.3 ± 3.3% of GFP in prestimulated bulk culture (Figure 2a, FV-2nd) 1 week after transduction. LV vector-transduced cells expressed 89.0 ± 10.2% of GFP in unstimulated bulk culture (Figure 2a, LV-1st) and 89.5 ± 8.3% of GFP in prestimulated bulk culture (Figure 2a, LV-2nd). The percentages of GFP+ progenitors in MethoCult cultured colonies using FV vector were 57.0 ± 6.0% in unstimulated cells (Figure 2b, FV-1st) and 63.7 ± 2.0% in the prestimulated cells (Figure 2b, FV-2nd). The percentages of GFP+ LV vector-transduced colonies were 80.5 ± 8.5% in unstimulated cells (Figure 2b, LV-1st) and 84.3 ± 3.4% in prestimulated cells (Figure 2b, LV-2nd). The total number of colonies for untransduced and FV vector-transduced groups were similar, but there were significantly fewer in LV vector-transduced groups.


High-titer foamy virus vector transduction and integration sites of human CD34(+) cell-derived SCID-repopulating cells.

Nasimuzzaman M, Kim YS, Wang YD, Persons DA - Mol Ther Methods Clin Dev (2014)

Vector transduction of bulk culture and clonogenic progenitors. (a) Percentages of GFP-expressing cells in bulk culture 1 week after vector transduction. (b) Percentages of GFP-expressing progenitor colonies on MethoCult medium 12 to 14 days after vector transduction. Unstimulated cells are designated FV-1st and LV-1st, and 24 h prestimulated cells are designated FV-2nd and LV-2nd. Both FV and LV vectors were transduced overnight on RetroNectin-coated plates at an MOI of 25. The experiments were done three times with mobilized human CD34+ cells from 3 donors. Data represent the mean and SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4441046&req=5

fig2: Vector transduction of bulk culture and clonogenic progenitors. (a) Percentages of GFP-expressing cells in bulk culture 1 week after vector transduction. (b) Percentages of GFP-expressing progenitor colonies on MethoCult medium 12 to 14 days after vector transduction. Unstimulated cells are designated FV-1st and LV-1st, and 24 h prestimulated cells are designated FV-2nd and LV-2nd. Both FV and LV vectors were transduced overnight on RetroNectin-coated plates at an MOI of 25. The experiments were done three times with mobilized human CD34+ cells from 3 donors. Data represent the mean and SEM.
Mentions: Granulocyte colony-stimulating factor–mobilized CD34+ cells from peripheral blood of three human donors were studied in three individual experiments. Unstimulated or prestimulated cells were transduced with either a FV or LV vector containing GFP at a multiplicity of infection (MOI) of 25 (see Supplementary Figure S2). MOI of 25 represents FV vectors titrated on HT1080 cells and LV vectors titrated on 293T cells. FV vector-transduced cells expressed 49.9 ± 5.9% of GFP in unstimulated bulk culture (Figure 2a, FV-1st) and 53.3 ± 3.3% of GFP in prestimulated bulk culture (Figure 2a, FV-2nd) 1 week after transduction. LV vector-transduced cells expressed 89.0 ± 10.2% of GFP in unstimulated bulk culture (Figure 2a, LV-1st) and 89.5 ± 8.3% of GFP in prestimulated bulk culture (Figure 2a, LV-2nd). The percentages of GFP+ progenitors in MethoCult cultured colonies using FV vector were 57.0 ± 6.0% in unstimulated cells (Figure 2b, FV-1st) and 63.7 ± 2.0% in the prestimulated cells (Figure 2b, FV-2nd). The percentages of GFP+ LV vector-transduced colonies were 80.5 ± 8.5% in unstimulated cells (Figure 2b, LV-1st) and 84.3 ± 3.4% in prestimulated cells (Figure 2b, LV-2nd). The total number of colonies for untransduced and FV vector-transduced groups were similar, but there were significantly fewer in LV vector-transduced groups.

Bottom Line: Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells.Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus.Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.

ABSTRACT
Foamy virus (FV) vectors are promising tools for gene therapy, but low titer is a major challenge for large-scale clinical trials. Here, we increased FV vector titer 50-fold by constructing novel vector plasmids and using polyethylenimine-mediated transfection. FV and lentiviral (LV) vectors were used separately to transduce human CD34(+) cells at multiplicities of infection of 25, and those cells were transplanted into immunodeficient mice. FV vector transduction frequencies of repopulating human cells were 37.1 ± 1.9% in unstimulated cells and 36.9 ± 2.2% in prestimulated cells, and engraftment frequencies were 40.9 ± 4.9% in unstimulated cells and 47.1 ± 3.3% in prestimulated cells. Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells. Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus. FV had an integration preference near transcriptional start sites and CpG islands of RefSeq genes but not within genes. Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations. Our new FV vector backbone may be a suitable candidate for developing therapeutic FV vectors for use in clinical trials.

No MeSH data available.


Related in: MedlinePlus