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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

Knock down, re-expression of gp91phox and functional rescue of gp91phox activity in CD34+ HSC derived cells. (a) Mobilized peripheral blood CD34+ cells were transduced with the indicated vectors or vector combination further expanded for 6 days and subsequently subjected to granulocytic differentiation (2–3 weeks). The same gating strategy as described in Figure 2a was applied on viable CD11b+ myeloid cells. Upper panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue). Middle panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.Crim (red). Lower panels: Transduction and gp91phox re-expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.gp91s.Crimvector (red). The green fluorescence marker denotes the proportion of cells containing both vectors. The outcome of the experiment is summarized in ). (c) Oxidase activity according to the dihydrorhodamine (DHR) assay is shown for nontransduced (ntd), KD vector only and KD vector and LV.gp91s.Crim transduced cell populations and summarized in the lower right panel (n = 3). Percentages given refer to the Rho123+ cells within the CD11b+ cell fraction. ctrl, control, MFI, Mean fluorescence intensity, 7D5-FITC: FITC conjugated monoclonal murine antibody against human gp91phox. Error bars = SD, *P < 0.05, **P < 0.01, ***P < 0.001.
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fig4: Knock down, re-expression of gp91phox and functional rescue of gp91phox activity in CD34+ HSC derived cells. (a) Mobilized peripheral blood CD34+ cells were transduced with the indicated vectors or vector combination further expanded for 6 days and subsequently subjected to granulocytic differentiation (2–3 weeks). The same gating strategy as described in Figure 2a was applied on viable CD11b+ myeloid cells. Upper panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue). Middle panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.Crim (red). Lower panels: Transduction and gp91phox re-expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.gp91s.Crimvector (red). The green fluorescence marker denotes the proportion of cells containing both vectors. The outcome of the experiment is summarized in ). (c) Oxidase activity according to the dihydrorhodamine (DHR) assay is shown for nontransduced (ntd), KD vector only and KD vector and LV.gp91s.Crim transduced cell populations and summarized in the lower right panel (n = 3). Percentages given refer to the Rho123+ cells within the CD11b+ cell fraction. ctrl, control, MFI, Mean fluorescence intensity, 7D5-FITC: FITC conjugated monoclonal murine antibody against human gp91phox. Error bars = SD, *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: Next, we assessed if our KD-vector induced human model of X-CGD in HSCs allows for the functional evaluation of gene therapy vectors. Again, prestimulated CD34+ cells were transduced either with the KD- or LV.Cer vector and in addition with LV.gp91s.Crim or the respective control vector LV.Crim (Figure 1d,f). After transduction and granulocytic differentiation roughly 70% of the cells stained positive for the CD11b myeloid cell surface marker and from these only 3.4% of the LV.sh88/91.Cer transduced, Cerulean positive cells (KD-CD34+ cells) showed residual gp91phox expression at low levels when compared to gp91phox expression levels in Cerulean negative, wild type cells (Figure 4a, upper panels and Figure 4b). Thus, more than 95% of the KD-vector transduced cells lacked gp91phox expression. Transduction of the KD-CD34+ cells with LV.Crim did not alter the gp91phox expression pattern (Figure 4a, middle panels). In contrast, transduction of the KD-CD34+ cells with LV.gp91s.Crim resulted in 25–35% total transduction efficiency as determined by E2-Crimson expression by FACS with almost 100% of the E2-Crimson positive cells expressing gp91phox (Figure 4a, lower panels). The levels of re-expression even exceeded gp91phox expression by wild type cells (Figure 4b). Thus, gp91phox expression can be efficiently reduced (11.1-fold, P = 0.0099; Figure 4b) and rescued in myeloid (CD11b+) cells after gene transfer into primary human CD34+ cells using the strategy outlined here. As expected, re-expression of gp91phox also rescued the functional X-CGD phenotype of our model as demonstrated by ROS production in CD11b+ double transduced cells (KD- and LV.gp91s.Crim) according to the DHR assay (Figure 4c). Hence, the delivery of LV.gp91s.Crim into the KD-CD34+ cells resulted in re-expression of gp91phox and functional rescue of the X-CGD phenotype proving the applicability of our knock down strategy for the functional evaluation of gene therapy vectors.


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)

Knock down, re-expression of gp91phox and functional rescue of gp91phox activity in CD34+ HSC derived cells. (a) Mobilized peripheral blood CD34+ cells were transduced with the indicated vectors or vector combination further expanded for 6 days and subsequently subjected to granulocytic differentiation (2–3 weeks). The same gating strategy as described in Figure 2a was applied on viable CD11b+ myeloid cells. Upper panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue). Middle panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.Crim (red). Lower panels: Transduction and gp91phox re-expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.gp91s.Crimvector (red). The green fluorescence marker denotes the proportion of cells containing both vectors. The outcome of the experiment is summarized in ). (c) Oxidase activity according to the dihydrorhodamine (DHR) assay is shown for nontransduced (ntd), KD vector only and KD vector and LV.gp91s.Crim transduced cell populations and summarized in the lower right panel (n = 3). Percentages given refer to the Rho123+ cells within the CD11b+ cell fraction. ctrl, control, MFI, Mean fluorescence intensity, 7D5-FITC: FITC conjugated monoclonal murine antibody against human gp91phox. Error bars = SD, *P < 0.05, **P < 0.01, ***P < 0.001.
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fig4: Knock down, re-expression of gp91phox and functional rescue of gp91phox activity in CD34+ HSC derived cells. (a) Mobilized peripheral blood CD34+ cells were transduced with the indicated vectors or vector combination further expanded for 6 days and subsequently subjected to granulocytic differentiation (2–3 weeks). The same gating strategy as described in Figure 2a was applied on viable CD11b+ myeloid cells. Upper panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue). Middle panels: Transduction and gp91phox expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.Crim (red). Lower panels: Transduction and gp91phox re-expression in CD34+ cells transduced with LV.sh88/91.Cer (blue) and LV.gp91s.Crimvector (red). The green fluorescence marker denotes the proportion of cells containing both vectors. The outcome of the experiment is summarized in ). (c) Oxidase activity according to the dihydrorhodamine (DHR) assay is shown for nontransduced (ntd), KD vector only and KD vector and LV.gp91s.Crim transduced cell populations and summarized in the lower right panel (n = 3). Percentages given refer to the Rho123+ cells within the CD11b+ cell fraction. ctrl, control, MFI, Mean fluorescence intensity, 7D5-FITC: FITC conjugated monoclonal murine antibody against human gp91phox. Error bars = SD, *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: Next, we assessed if our KD-vector induced human model of X-CGD in HSCs allows for the functional evaluation of gene therapy vectors. Again, prestimulated CD34+ cells were transduced either with the KD- or LV.Cer vector and in addition with LV.gp91s.Crim or the respective control vector LV.Crim (Figure 1d,f). After transduction and granulocytic differentiation roughly 70% of the cells stained positive for the CD11b myeloid cell surface marker and from these only 3.4% of the LV.sh88/91.Cer transduced, Cerulean positive cells (KD-CD34+ cells) showed residual gp91phox expression at low levels when compared to gp91phox expression levels in Cerulean negative, wild type cells (Figure 4a, upper panels and Figure 4b). Thus, more than 95% of the KD-vector transduced cells lacked gp91phox expression. Transduction of the KD-CD34+ cells with LV.Crim did not alter the gp91phox expression pattern (Figure 4a, middle panels). In contrast, transduction of the KD-CD34+ cells with LV.gp91s.Crim resulted in 25–35% total transduction efficiency as determined by E2-Crimson expression by FACS with almost 100% of the E2-Crimson positive cells expressing gp91phox (Figure 4a, lower panels). The levels of re-expression even exceeded gp91phox expression by wild type cells (Figure 4b). Thus, gp91phox expression can be efficiently reduced (11.1-fold, P = 0.0099; Figure 4b) and rescued in myeloid (CD11b+) cells after gene transfer into primary human CD34+ cells using the strategy outlined here. As expected, re-expression of gp91phox also rescued the functional X-CGD phenotype of our model as demonstrated by ROS production in CD11b+ double transduced cells (KD- and LV.gp91s.Crim) according to the DHR assay (Figure 4c). Hence, the delivery of LV.gp91s.Crim into the KD-CD34+ cells resulted in re-expression of gp91phox and functional rescue of the X-CGD phenotype proving the applicability of our knock down strategy for the functional evaluation of gene therapy vectors.

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