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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

Screening of shRNAs for efficient knock down of gp91phox. (a) Schematic structure of lentiviral vectors tested for the knock down of gp91phox in PLB-985 cells. (b) Localization of the individual shRNA seeding sequences (colored in red and green) in the gp91phox coding region is shown aligned to the synthetic gp91s sequence. The best performers are highlighted in red. (c) PLB-985 cells were transduced with the shRNA vectors shown in a and 24 hours later subjected to puromycin selection for 4 days before gp91phox expression was analyzed in undifferentiated cells (gray) or in CD11b cells after granulocytic differentiation (>7 days, black bars). Gp91phox expression was normalized to non-transduced wild type cells (PLB-985). The gp91phox knock out cell line XCGD-PLB985 served as negative control (n ≥ 3). (d) Plasmid configuration of the gp91phox KD (LV.sh88/91.Cer), and respective control-vector (LV.Cer) used in this study. (e) Quantitative RT-PCR was performed on single cell clones derived from KD-vector transduced PLB-985 cells to assess gp91phox knock down efficiency at the mRNA level. Clones were grouped according to vector copy number (VCN) as determined by qPCR (VCN 1: 1.1 ± 0.1, n = 6; VCN 2: 1.7 ± 0.1, n = 3). Expression was normalized to gp91phox mRNA expression levels of non-transduced PLB-985 cells and cDNA derived from XCGD-PLB985 served as negative control. (f) Schematic structure of the internal cassette used to reconstitute gp91 expression (LV.gp91s.Crim)) in knock down cells and the respective control vector (LV.Crim). cPPT, central polypurine tract; endog., endogenous sequence; hPGK, human phosphoglycerate kinase promoter; IRES, internal ribosome entry site; LTR, self-inactivating long terminal repeat; RRE, rev-responsive element; SFFV, spleen focus forming virus promoter; wPRE, woodchuck hepatitis virus posttranscriptional regulating element. ψPackaging signal. Error bars = SD.
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fig1: Screening of shRNAs for efficient knock down of gp91phox. (a) Schematic structure of lentiviral vectors tested for the knock down of gp91phox in PLB-985 cells. (b) Localization of the individual shRNA seeding sequences (colored in red and green) in the gp91phox coding region is shown aligned to the synthetic gp91s sequence. The best performers are highlighted in red. (c) PLB-985 cells were transduced with the shRNA vectors shown in a and 24 hours later subjected to puromycin selection for 4 days before gp91phox expression was analyzed in undifferentiated cells (gray) or in CD11b cells after granulocytic differentiation (>7 days, black bars). Gp91phox expression was normalized to non-transduced wild type cells (PLB-985). The gp91phox knock out cell line XCGD-PLB985 served as negative control (n ≥ 3). (d) Plasmid configuration of the gp91phox KD (LV.sh88/91.Cer), and respective control-vector (LV.Cer) used in this study. (e) Quantitative RT-PCR was performed on single cell clones derived from KD-vector transduced PLB-985 cells to assess gp91phox knock down efficiency at the mRNA level. Clones were grouped according to vector copy number (VCN) as determined by qPCR (VCN 1: 1.1 ± 0.1, n = 6; VCN 2: 1.7 ± 0.1, n = 3). Expression was normalized to gp91phox mRNA expression levels of non-transduced PLB-985 cells and cDNA derived from XCGD-PLB985 served as negative control. (f) Schematic structure of the internal cassette used to reconstitute gp91 expression (LV.gp91s.Crim)) in knock down cells and the respective control vector (LV.Crim). cPPT, central polypurine tract; endog., endogenous sequence; hPGK, human phosphoglycerate kinase promoter; IRES, internal ribosome entry site; LTR, self-inactivating long terminal repeat; RRE, rev-responsive element; SFFV, spleen focus forming virus promoter; wPRE, woodchuck hepatitis virus posttranscriptional regulating element. ψPackaging signal. Error bars = SD.

Mentions: Initially, we tested five shRNAs individually in the context of a lentiviral vector for their activity to knock down endogenous gp91phox expression in the human myelomonocytic cell line PLB-985 (Figure 1a,b). Knock down efficiency of gp91phox expression was assessed in undifferentiated transduced cells and upon granulocytic differentiation by measuring gp91phox protein at the cell surface of transduced cells by flow cytometry using the human monoclonal anti-gp91phox antibody 7D519 (Figure 1c). Significant reduction of gp91phox positive cells was achieved with two shRNAs, sh88 and sh91 (P < 0.001 and P = 0.002, respectively), which were subsequently combined in a single lentiviral vector under the control of two distinct human DNA polymerase III promoters, namely U6 and H1. The insertion of the H1-sh91 sequence into the viral 3′ long terminal repeat (LTR) results in two transcription units per provirus upon reverse transcription (Figure 1d). This design led to the highest knock-down efficiency (88 ± 4%) in differentiated CD11b+ PLB-985 cells as estimated from gp91phox surface expression (Figure 1c). Clonal populations harboring 1–2 vector integrants confirmed gp91phox knock down at the mRNA level with a mean efficiency of 80 ± 12% (n = 9, Figure 1e). In this final knock down (KD) vector (LV.sh88/91.Cer, Figure 1d) a fluorescent marker gene, cerulean, allows the identification and sorting of KD-vector positive cells.


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)

Screening of shRNAs for efficient knock down of gp91phox. (a) Schematic structure of lentiviral vectors tested for the knock down of gp91phox in PLB-985 cells. (b) Localization of the individual shRNA seeding sequences (colored in red and green) in the gp91phox coding region is shown aligned to the synthetic gp91s sequence. The best performers are highlighted in red. (c) PLB-985 cells were transduced with the shRNA vectors shown in a and 24 hours later subjected to puromycin selection for 4 days before gp91phox expression was analyzed in undifferentiated cells (gray) or in CD11b cells after granulocytic differentiation (>7 days, black bars). Gp91phox expression was normalized to non-transduced wild type cells (PLB-985). The gp91phox knock out cell line XCGD-PLB985 served as negative control (n ≥ 3). (d) Plasmid configuration of the gp91phox KD (LV.sh88/91.Cer), and respective control-vector (LV.Cer) used in this study. (e) Quantitative RT-PCR was performed on single cell clones derived from KD-vector transduced PLB-985 cells to assess gp91phox knock down efficiency at the mRNA level. Clones were grouped according to vector copy number (VCN) as determined by qPCR (VCN 1: 1.1 ± 0.1, n = 6; VCN 2: 1.7 ± 0.1, n = 3). Expression was normalized to gp91phox mRNA expression levels of non-transduced PLB-985 cells and cDNA derived from XCGD-PLB985 served as negative control. (f) Schematic structure of the internal cassette used to reconstitute gp91 expression (LV.gp91s.Crim)) in knock down cells and the respective control vector (LV.Crim). cPPT, central polypurine tract; endog., endogenous sequence; hPGK, human phosphoglycerate kinase promoter; IRES, internal ribosome entry site; LTR, self-inactivating long terminal repeat; RRE, rev-responsive element; SFFV, spleen focus forming virus promoter; wPRE, woodchuck hepatitis virus posttranscriptional regulating element. ψPackaging signal. Error bars = SD.
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Related In: Results  -  Collection

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fig1: Screening of shRNAs for efficient knock down of gp91phox. (a) Schematic structure of lentiviral vectors tested for the knock down of gp91phox in PLB-985 cells. (b) Localization of the individual shRNA seeding sequences (colored in red and green) in the gp91phox coding region is shown aligned to the synthetic gp91s sequence. The best performers are highlighted in red. (c) PLB-985 cells were transduced with the shRNA vectors shown in a and 24 hours later subjected to puromycin selection for 4 days before gp91phox expression was analyzed in undifferentiated cells (gray) or in CD11b cells after granulocytic differentiation (>7 days, black bars). Gp91phox expression was normalized to non-transduced wild type cells (PLB-985). The gp91phox knock out cell line XCGD-PLB985 served as negative control (n ≥ 3). (d) Plasmid configuration of the gp91phox KD (LV.sh88/91.Cer), and respective control-vector (LV.Cer) used in this study. (e) Quantitative RT-PCR was performed on single cell clones derived from KD-vector transduced PLB-985 cells to assess gp91phox knock down efficiency at the mRNA level. Clones were grouped according to vector copy number (VCN) as determined by qPCR (VCN 1: 1.1 ± 0.1, n = 6; VCN 2: 1.7 ± 0.1, n = 3). Expression was normalized to gp91phox mRNA expression levels of non-transduced PLB-985 cells and cDNA derived from XCGD-PLB985 served as negative control. (f) Schematic structure of the internal cassette used to reconstitute gp91 expression (LV.gp91s.Crim)) in knock down cells and the respective control vector (LV.Crim). cPPT, central polypurine tract; endog., endogenous sequence; hPGK, human phosphoglycerate kinase promoter; IRES, internal ribosome entry site; LTR, self-inactivating long terminal repeat; RRE, rev-responsive element; SFFV, spleen focus forming virus promoter; wPRE, woodchuck hepatitis virus posttranscriptional regulating element. ψPackaging signal. Error bars = SD.
Mentions: Initially, we tested five shRNAs individually in the context of a lentiviral vector for their activity to knock down endogenous gp91phox expression in the human myelomonocytic cell line PLB-985 (Figure 1a,b). Knock down efficiency of gp91phox expression was assessed in undifferentiated transduced cells and upon granulocytic differentiation by measuring gp91phox protein at the cell surface of transduced cells by flow cytometry using the human monoclonal anti-gp91phox antibody 7D519 (Figure 1c). Significant reduction of gp91phox positive cells was achieved with two shRNAs, sh88 and sh91 (P < 0.001 and P = 0.002, respectively), which were subsequently combined in a single lentiviral vector under the control of two distinct human DNA polymerase III promoters, namely U6 and H1. The insertion of the H1-sh91 sequence into the viral 3′ long terminal repeat (LTR) results in two transcription units per provirus upon reverse transcription (Figure 1d). This design led to the highest knock-down efficiency (88 ± 4%) in differentiated CD11b+ PLB-985 cells as estimated from gp91phox surface expression (Figure 1c). Clonal populations harboring 1–2 vector integrants confirmed gp91phox knock down at the mRNA level with a mean efficiency of 80 ± 12% (n = 9, Figure 1e). In this final knock down (KD) vector (LV.sh88/91.Cer, Figure 1d) a fluorescent marker gene, cerulean, allows the identification and sorting of KD-vector positive cells.

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