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Generation of X-CGD cells for vector evaluation from healthy donor CD34(+) HSCs by shRNA-mediated knock down of gp91(phox).

Brendel C, Kaufmann KB, Krattenmacher A, Pahujani S, Grez M - Mol Ther Methods Clin Dev (2014)

Bottom Line: Here, we describe a straightforward experimental strategy that circumvents this limitation.The knock down of gp91(phox) expression upon lentiviral delivery of shRNAs into CD34(+) cells from healthy donors generates sufficient amounts of X-CGD CD34(+) cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91(phox) transgene.We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.

View Article: PubMed Central - PubMed

Affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus , Frankfurt, Germany.

ABSTRACT
Innovative approaches for the treatment of rare inherited diseases are hampered by limited availability of patient derived samples for preclinical research. This also applies for the evaluation of novel vector systems for the gene therapy of monogenic hematological diseases like X-linked chronic granulomatous disease (X-CGD), a severe primary immunodeficiency caused by mutations in the gp91(phox) subunit of the phagocytic NADPH oxidase. Since current gene therapy protocols involve ex vivo gene modification of autologous CD34(+) hematopoietic stem cells (HSC), the ideal preclinical model should simulate faithfully this procedure. However, the low availability of patient-derived CD34(+) cells limits the feasibility of this approach. Here, we describe a straightforward experimental strategy that circumvents this limitation. The knock down of gp91(phox) expression upon lentiviral delivery of shRNAs into CD34(+) cells from healthy donors generates sufficient amounts of X-CGD CD34(+) cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91(phox) transgene. We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.

No MeSH data available.


Related in: MedlinePlus

NADPH oxidase activity upon gp91phox knock down in human CD34+ HSC derived cells. LV.sh88/91.Cer transduced CD34+ cells were subjected to in vitro granulocytic differentiation and FACS-sorted for Cerulean expression (purity: 90.0 ± 2.5%, n = 3) prior to oxidase activity measurements. (a) Oxidase activity was visualized in differentiated cells after PMA stimulation by the Nitro-Blue-Tetrazolium (NBT). Superoxide producing cells are visualized by a dark blue formazan deposit. Nontransduced cells were used as positive control while cells derived from a X-CGD sample served as negative control. Two magnifications of the same samples are shown (scale bar = 100 µm). (b,c) Cytochrome C assay was performed and values were corrected for purity after FACS sorting. (d) A dihydrorhodamine (DHR) assay was performed with unsorted populations after transduction and myeloid differentiation of CD34+ cells. Oxidation of dihydrorhodamine 123 to rhodamine 123 is shown for non-transduced wild type (wt), CD34+X-CGD patient sample derived cells (XCGD) and two Cerulean+ cell populations after transduction with LV.sh88/91.Cer (KD1, KD2). Included in the histograms are the fraction of superoxide producing (Rho+) cells, and the mean fluorescence intensity (MFI) of gp91phox protein expression in CD11b+ cells.
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fig3: NADPH oxidase activity upon gp91phox knock down in human CD34+ HSC derived cells. LV.sh88/91.Cer transduced CD34+ cells were subjected to in vitro granulocytic differentiation and FACS-sorted for Cerulean expression (purity: 90.0 ± 2.5%, n = 3) prior to oxidase activity measurements. (a) Oxidase activity was visualized in differentiated cells after PMA stimulation by the Nitro-Blue-Tetrazolium (NBT). Superoxide producing cells are visualized by a dark blue formazan deposit. Nontransduced cells were used as positive control while cells derived from a X-CGD sample served as negative control. Two magnifications of the same samples are shown (scale bar = 100 µm). (b,c) Cytochrome C assay was performed and values were corrected for purity after FACS sorting. (d) A dihydrorhodamine (DHR) assay was performed with unsorted populations after transduction and myeloid differentiation of CD34+ cells. Oxidation of dihydrorhodamine 123 to rhodamine 123 is shown for non-transduced wild type (wt), CD34+X-CGD patient sample derived cells (XCGD) and two Cerulean+ cell populations after transduction with LV.sh88/91.Cer (KD1, KD2). Included in the histograms are the fraction of superoxide producing (Rho+) cells, and the mean fluorescence intensity (MFI) of gp91phox protein expression in CD11b+ cells.

Mentions: We used granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood from healthy donors to establish a surrogate model for primary human X-CGD cells. Thus, we first analyzed whether the introduction of the knock down vector in CD34+ cells reduces or even abrogates ROS production in their in vitro differentiated myeloid progeny (CD11b+) upon PMA stimulation. After CD34+ cell isolation cells were pre-stimulated for 1 day before transduction with the LV.sh88/91.Cer. After transduction prolonged in vitro culture (2–3 weeks) in the presence of human G-CSF was required for full granulocytic maturation and optimal gp91phox expression. After sorting for Cerulean+ cells CD11b+ cells derived from KD transduced CD34+ cells revealed only background staining in the nitroblue-tetrazolium (NBT)-reduction assay, similar to the staining obtained with myeloid progeny derived from X-CGD CD34+ cells (Figure 3a). In contrast, wild type CD11b+ cells demonstrated oxidase activity as indicated by the reduction of NBT to insoluble formazan in the majority of the cells as documented by a dark blue deposit on the cells (Figure 3a). This observation was further confirmed by the quantitative cytochrome C assay, in which only residual superoxide production (12.7% of wt) could be measured in the CD11b+ KD-cells (Figure 3b,c) roughly mimicking the complete absence of superoxide production we had observed previously in XCGD-CD34+ derived cells.18,22 We also used the dihydrorhodamine123 (DHR123) assay to detect ROS produced by KD-, X-CGD and normal phagocytes after PMA stimulation. Cerulean expressing cells showed only residual rhodamine123 (Rho123) staining suggesting minimal superoxide production by these cells, similar to samples derived from X-CGD patients (Figure 3d). Thus, the LV.sh88/91.Cer vector efficiently induced the X-CGD phenotype in primary CD34+ cells proving the applicability of our knock down strategy to generate a surrogate human model of X-CGD.


Generation of X-CGD cells for vector evaluation from healthy donor CD34(+) HSCs by shRNA-mediated knock down of gp91(phox).

Brendel C, Kaufmann KB, Krattenmacher A, Pahujani S, Grez M - Mol Ther Methods Clin Dev (2014)

NADPH oxidase activity upon gp91phox knock down in human CD34+ HSC derived cells. LV.sh88/91.Cer transduced CD34+ cells were subjected to in vitro granulocytic differentiation and FACS-sorted for Cerulean expression (purity: 90.0 ± 2.5%, n = 3) prior to oxidase activity measurements. (a) Oxidase activity was visualized in differentiated cells after PMA stimulation by the Nitro-Blue-Tetrazolium (NBT). Superoxide producing cells are visualized by a dark blue formazan deposit. Nontransduced cells were used as positive control while cells derived from a X-CGD sample served as negative control. Two magnifications of the same samples are shown (scale bar = 100 µm). (b,c) Cytochrome C assay was performed and values were corrected for purity after FACS sorting. (d) A dihydrorhodamine (DHR) assay was performed with unsorted populations after transduction and myeloid differentiation of CD34+ cells. Oxidation of dihydrorhodamine 123 to rhodamine 123 is shown for non-transduced wild type (wt), CD34+X-CGD patient sample derived cells (XCGD) and two Cerulean+ cell populations after transduction with LV.sh88/91.Cer (KD1, KD2). Included in the histograms are the fraction of superoxide producing (Rho+) cells, and the mean fluorescence intensity (MFI) of gp91phox protein expression in CD11b+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362359&req=5

fig3: NADPH oxidase activity upon gp91phox knock down in human CD34+ HSC derived cells. LV.sh88/91.Cer transduced CD34+ cells were subjected to in vitro granulocytic differentiation and FACS-sorted for Cerulean expression (purity: 90.0 ± 2.5%, n = 3) prior to oxidase activity measurements. (a) Oxidase activity was visualized in differentiated cells after PMA stimulation by the Nitro-Blue-Tetrazolium (NBT). Superoxide producing cells are visualized by a dark blue formazan deposit. Nontransduced cells were used as positive control while cells derived from a X-CGD sample served as negative control. Two magnifications of the same samples are shown (scale bar = 100 µm). (b,c) Cytochrome C assay was performed and values were corrected for purity after FACS sorting. (d) A dihydrorhodamine (DHR) assay was performed with unsorted populations after transduction and myeloid differentiation of CD34+ cells. Oxidation of dihydrorhodamine 123 to rhodamine 123 is shown for non-transduced wild type (wt), CD34+X-CGD patient sample derived cells (XCGD) and two Cerulean+ cell populations after transduction with LV.sh88/91.Cer (KD1, KD2). Included in the histograms are the fraction of superoxide producing (Rho+) cells, and the mean fluorescence intensity (MFI) of gp91phox protein expression in CD11b+ cells.
Mentions: We used granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood from healthy donors to establish a surrogate model for primary human X-CGD cells. Thus, we first analyzed whether the introduction of the knock down vector in CD34+ cells reduces or even abrogates ROS production in their in vitro differentiated myeloid progeny (CD11b+) upon PMA stimulation. After CD34+ cell isolation cells were pre-stimulated for 1 day before transduction with the LV.sh88/91.Cer. After transduction prolonged in vitro culture (2–3 weeks) in the presence of human G-CSF was required for full granulocytic maturation and optimal gp91phox expression. After sorting for Cerulean+ cells CD11b+ cells derived from KD transduced CD34+ cells revealed only background staining in the nitroblue-tetrazolium (NBT)-reduction assay, similar to the staining obtained with myeloid progeny derived from X-CGD CD34+ cells (Figure 3a). In contrast, wild type CD11b+ cells demonstrated oxidase activity as indicated by the reduction of NBT to insoluble formazan in the majority of the cells as documented by a dark blue deposit on the cells (Figure 3a). This observation was further confirmed by the quantitative cytochrome C assay, in which only residual superoxide production (12.7% of wt) could be measured in the CD11b+ KD-cells (Figure 3b,c) roughly mimicking the complete absence of superoxide production we had observed previously in XCGD-CD34+ derived cells.18,22 We also used the dihydrorhodamine123 (DHR123) assay to detect ROS produced by KD-, X-CGD and normal phagocytes after PMA stimulation. Cerulean expressing cells showed only residual rhodamine123 (Rho123) staining suggesting minimal superoxide production by these cells, similar to samples derived from X-CGD patients (Figure 3d). Thus, the LV.sh88/91.Cer vector efficiently induced the X-CGD phenotype in primary CD34+ cells proving the applicability of our knock down strategy to generate a surrogate human model of X-CGD.

Bottom Line: Here, we describe a straightforward experimental strategy that circumvents this limitation.The knock down of gp91(phox) expression upon lentiviral delivery of shRNAs into CD34(+) cells from healthy donors generates sufficient amounts of X-CGD CD34(+) cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91(phox) transgene.We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.

View Article: PubMed Central - PubMed

Affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus , Frankfurt, Germany.

ABSTRACT
Innovative approaches for the treatment of rare inherited diseases are hampered by limited availability of patient derived samples for preclinical research. This also applies for the evaluation of novel vector systems for the gene therapy of monogenic hematological diseases like X-linked chronic granulomatous disease (X-CGD), a severe primary immunodeficiency caused by mutations in the gp91(phox) subunit of the phagocytic NADPH oxidase. Since current gene therapy protocols involve ex vivo gene modification of autologous CD34(+) hematopoietic stem cells (HSC), the ideal preclinical model should simulate faithfully this procedure. However, the low availability of patient-derived CD34(+) cells limits the feasibility of this approach. Here, we describe a straightforward experimental strategy that circumvents this limitation. The knock down of gp91(phox) expression upon lentiviral delivery of shRNAs into CD34(+) cells from healthy donors generates sufficient amounts of X-CGD CD34(+) cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91(phox) transgene. We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.

No MeSH data available.


Related in: MedlinePlus